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This is to certify that the
dissertation entitled

CARBON PARTITIONING IN SWEET CHERRY (Prunus avium L.)
ON DWARFING PRECOCIOUS ROOTSTOCKS DURING FRUIT
DEVELOPMENT

presented by
Marlene Ayala

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CARBON PARTITIONING IN SWEET CHERRY (Prunus avium L.) ON
DWARFING PRECOCIOUS ROOTSTOCKS DURING FRUIT DEVELOPMENT

By

Marlene Ayala

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Horticulture

2004

ABSTRACT

CARBON PARTITIONING IN SWEET CHERRY (Prunus avium L.) ON
DWARF ING PRECOCIOUS ROOTSTOCKS DURING FRUIT DEVELOPMENT

By

Marlene Ayala

Understanding carbon (C) partitioning is important for development of better
management strategies to improve sweet cherry fruit quality on high-yielding,
vigor-reducing rootstocks. To study the relative importance or temporal
relationships of the primary leaf populations (i.e., fruiting spur, non-fruiting spur
and current season shoot leaves) as sources of C for sweet cherry fruit and shoot
development, a series of partitioning experiments using girdling, defoliation,
fruit thinning and 13C-labeling was established with sweet cherry trees on
dwarfing/ semidwarfing Gisela (GI) rootstocks. A preliminary girdling and
defoliation experiment isolated fruit of ’Hedelfinger’ / GIS and ’Ulster’ /GI6 from
different leaf sources and indicated that leaf populations on both fruiting and
non-fruiting branch segments were required for optimum fruit development.
There was not a sufficient compensatory effect when one of the main leaf
populations was eliminated. A second experiment used 13COz to label non-
fruiting spur leaves on ’Sam’ / G15 limbs with three different crop loads
(quantified as leaf area to fruit ratios LA / F= 140, 75, or 40 cm2 / fruit), which
indicated that fruit were stronger sinks than current season shoots during stage

III of fruit development. A third experiment quantified the relative C

contribution of each leaf population to fruit and shoot development during key
points throughout fruit development. Leaves on fruiting spurs, non-fruiting
spurs and the new terminal shoot were exposed to 13’COz labeling on five
representative phenological dates during fruit development. Spur and shoot
leaves were significant sources of C for fruit and vegetative growth. Fruits were a
priority sink vs. new shoot growth, in terms of C allocation, during the entire
period of fruit development. The highest fruit sink strength was during stages I
and III. Current season shoot growth provided a C source for fruit as early as
stage 1. Finally, a fourth experiment on ’Regina’/ G1 6 labeled with 13C02 after
terminal bud set determined the extent that storage reserves were used for
spring growth, particularly fruit, and defined the transition phase during which
current photoassimilates become the primary C source. In fall, the major storage
organs were roots, older wood in the trunk and branches, and buds. During
spring, 13C-reserves were remobilized and partitioned to flowers, fruits and
young leaves from before budbreak until 14 days after full bloom (DAFB). The
highest 13C levels in growing sinks were detected between bloom and fruit set.
Reproductive organs had the strongest sink activity until 14 DAFB. Overall, these
results provide a physiological foundation for canopy relationships that may
help to develop specific orchard management strategies to promote a more
sustainable balance between vegetative and reproductive growth in high density

sweet cherry orchards on vigor-limiting rootstocks.

Copyright by
MARLENE AYALA
2004

DEDICATION

To my parents, my brother and my husband with love

ACKNOWLEDGMENTS

I would like to express my gratitude to my major professor, Dr. Greg Lang
for his guidance and support during the course of my graduate studies at
Michigan State University. Thank you for the opportunity to learn more about
sweet cherry and ways to do research. I am deeply grateful to my committee
professors: Dr. Jack Preiss, Dr. Jim Flore, Dr. Suzanne Lang and Dr. Ken Poff for
their insights and advice during my graduate studies. Each of you gave one skill
to be a better professional and person.

I will always be thankful to Dr. Ken Poff for giving me the opportunity to
learn beyond academics. Your friendship, advices and way of seeing life will
never be forgotten. You changed my way of thinking. Thank you.

Thank you to all my friends at MSU: David Mota, Karen Cichy, Sonali
Padhye, Marcus Duck, Elzette van Royen, Conrad Schutte, Giovambattista
Sorrenti, Costanza Zavalloni, Dario Stefanelli, Daniele Trebbi, Elizabeth Landa,
and Mohamed Tawfik. Your friendship and company were a great support
during all these years and a precious gift that I received in the United States. I
would like to give special thanks to my officemate, Randy Vos, who will be an
unforgettable friend.

I would especially like to thank to Universidad Catélica de Chile for
giving me the opportunity to come to the United States to get a Ph. D. and

develop as a future faculty.

vi

I would like to thank Anne and Ron Perry and Becky and Randy Beaudry
for their friendship to my husband and I during these years. I appreciate that you
included these foreigners in your family traditions.

Finally, I would like to thank my parents (Fernando and Sergia), my
brother (Cristian) and my husband Mauricio, who were the columns that
supported me during good and tough times. Thank you all for your love,

encouragement and strength.

vii

TABLE OF CONTENT

LIST OF TABLES ............................................................................................................. xi
LIST OF FIGURES ......................................................................................................... xiv
CHAPTER I
LITERATURE REVIEW .......................................................................................................... 1
Sweet Cherry Description and Production Trends ..................................................... 2
Sweet Cherry Rootstocks and the Gisela Series ........................................................... 3
Reproductive and Vegetative Habits of Sweet Cherry Trees .................................... 5
The Importance of Carbon Economy and Partitioning in Fruit Trees ..................... 8
Carbohydrate Metabolism in Rosaceae Species ........................................................ 12
Storage Reserves ............................................................................................................. 14
Definition and importance ................................................................................ 14
Type of storage reserves .................................................................................... 16
CHzO reserves ........................................................................................ 16
N itrogenous reserves and other minerals .......................................... 17
Storage organs .................................................................................................... 17
Seasonal pattern of storage reserves in woody perennials .......................... 18
Seasonal pattern of storage reserves in sweet cherry ................................... 21
The Use of Labeled Carbon to Study Carbon Fluxes and Partitioning .................. 23
Source-Sink Relationships ............................................................................................ 26
Sink strength ....................................................................................................... 26
Sink and source limitation ................................................................................ 27
Sink and source manipulation ......................................................................... 29
Fruit as sink and shoot as sink and source ..................................................... 32
Fruit as a sink .......................................................................................... 32
Extension shoots as sink and source ................................................... 33
Carbon translocation patterns: orthostichy and distance ............................ 35
Photosynthesis and Sink Activity ................................................................................ 37
Respiration and Sink Activity ...................................................................................... 39
Rationale and Objectives ............................................................................................... 41
LITERATURE CITED .................................................................................................... 44
CHAPTER II
EXAMINING THE INFLUENCE OF DIFFERENT LEAF POPULATIONS ON SWEET CHERRY
FRUIT QUALITY ................................................................................................................. 62
Abstract ........................................................................................................................... 63
Introduction .................................................................................................................... 65
Material and Methods ................................................................................................... 68

viii

Statistical Analysis ......................................................................................................... 70

Results .............................................................................................................................. 70
Discussion ....................................................................................................................... 72
Acknowledgements ....................................................................................................... 76
LITERATURE CITED .................................................................................................... 77
CHAPTER III
THE EFFECT OF CROP LOAD ON 13C-PHOTOASSIMILATE PARTTTIONING IN SWEET
CHERRY DURING STAGE 111 OF FRUIT DEVELOPMENT .................................................... 84
Abstract. ........................................................................................................................... 85
Introduction .................................................................................................................... 87
Materials and Methods ................................................................................................. 90
Plant material ...................................................................................................... 90
13C Pulse-labeling ............................................................................................... 91
Sampling and analysis ....................................................................................... 93
Statistical Analysis ......................................................................................................... 94
Results .............................................................................................................................. 95
Phenological characterization at pulsing ........................................................ 95
Translocation patterns ....................................................................................... 95
13C Partitioning ................................................................................................... 96
Relative carbon allocation ................................................................................. 98
Discussion ....................................................................................................................... 99
LITERATURE CITED .................................................................................................. 106
CHAPTER IV
13C-PHOTOASSIMILATE PARTITIONING IN SWEET CHERRY (PRUNNUS AVIUM L.)
DURING FRUIT DEVELOPMENT ...................................................................................... 114
Abstract ......................................................................................................................... 115
Introduction .................................................................................................................. 117
Materials and Methods ............................................................................................... 120
Plant material .................................................................................................... 120
Phenological characterization before 13COz pulsing ................................... 121
13C Pulse-labeling ............................................................................................. 12
Sampling and analysis ..................................................................................... 123
Selection of representative pulse labeling dates for 13C analysis .............. 125
Climatic data ..................................................................................................... 125
Statistical Analysis ....................................................................................................... 126
Results ............................................................................................................................ 126
Growth in two-year-old branches ................................................................. 126
Leaf area ................................................................................................ 126
Fruits ...................................................................................................... 127
Current season shoots ......................................................................... 127
Relative FW and DW partitioning ................................................................. 128

ix

Translocation patterns in two-year-old branches ........................................ 129

Total 13C in leaves and fruit immediately after pulsing ............................. 129
Total 13C recoveries 48 hours after pulsing .................................................. 130
Absolute and relative partitioning of 13C 48 hours after pulsing .............. 131
Fruit spur leaves as 13'C source ........................................................... 131
Non-fruiting spur leaves as 13C source ............................................. 133
Terminal shoot leaves as 13C source .................................................. 134
Partitioning of 13C from distal leaf populations .......................................... 136
13C Partitioning in individual fruits .............................................................. 136
Discussion ..................................................................................................................... 137
LITERATURE CITED .................................................................................................. 148
CHAPT ER V
13C-PHOTOASSIMILATE PARTITIONING IN SWEET CHERRY (PRUNNUS AVIUM L.)
DURING EARLY SPRING .................................................................................................. 176
Abstract. ......................................................................................................................... 177
Introduction .................................................................................................................. 179
Materials and Methods ............................................................................................... 182
Plant material .................................................................................................... 182
13C Labeling ....................................................................................................... 182
Growth measurements .................................................................................... 183
13C Sampling and analysis .............................................................................. 184
Statistical Analysis ....................................................................................................... 186
Results ............................................................................................................................ 186
Phenological characterization ........................................................................ 186
13C-Labeled storage reserves at leaf abscission ............................................ 187
13C-Reserve partitioning at budbreak ........................................................... 188
13C-Reserve partitioning during early spring .............................................. 189
Relative 13C-reserve partitioning throughout the canopy during spring 191
Discussion ..................................................................................................................... 192
LITERATURE CITED .................................................................................................. 202
CHAPTER VI
DISSERTATION PROJECT SUMMARY ................................................................................ 215
Summary ....................................................................................................................... 216
APPENDD< A ................................................................................................................ 222
APPENDD< B ................................................................................................................ 225
APPENDD< C ................................................................................................................ 244

LIST OF TABLES

CHAPTER II

Table 1. Morphological features of ’Ulster’/ G16 and ’Hedelfinger’ / G15
branches. Means from 60 branches per combination. Measurements were

recorded prior to treatment imposition and late in fruit development (65 and
67 days after full bloom). .............................................................................................. 80

Table 2. Diameter, weight, soluble solids (SS), color and final fruit

number/ branch of ’Ulster’/ G16 and ‘Hedelfinger’ /G15 at harvest (June and
July, 2001, respectively). C - Untreated limb (control); T1 - Branch girdled at
its base; T2 - Branch girdled at both sides of the wood bearing newly fruiting
spurs, i.e., source leaves are those associated with the branch segment that
grew in 1999; T3 - Branch girdled at its base and at the junction of the
previous season growth and the current season growth, i.e., source leaves are
those associated with the branch segments that grew in both 1999 and 2000;
T4 - Branch girdled as in T3 plus removal of all spur and lateral leaves on the
fruiting branch segment, i.e., source leaves are those associated with the
branch segment that grew in 2000; T5 - Branch girdled as in T3 plus removal
of all spur and lateral leaves on the non-fruiting branch segment, i.e., source
leaves are those associated with the branch segment that grew in 1999. .............. 81

CHAPTER III

Table 1. Morphological features of 2-year-old ’Sam’/ Gisela 5 sweet cherry
branches before 13C pulse-labeling. Mean i SE, n=220. ......................................... 110

Table 2. 'Sam’ sweet cherry fruit quality parameters measured at each pulse-
labeling (52, 59 and 63 days after full bloom, DAF B) and at commercial
harvest (67 DAF B). Mean i SE, n=50. ....................................................................... 110

Table 3. Growth and morphological measurements of current season
’Sam’ / Gisela 5 sweet cherry growth at each pulse-labeling date (52, 59 and 63
DAFB) and harvest (67 days after full bloom, DAFB). Mean i SE, n=25 ............. 111

Table 4. Relative 13C enrichment for fruit and current season leaves. Mean 1-
SE, n=15. Calculations based on total 13C -absolute (pg 13'C / g DW) recoveries
for the four organs. ...................................................................................................... 111

CHAPTER IV

Table 1. Fruit quality parameters measured weekly between stages I and III
on 'Ulster’ / Gisela 6 sweet cherry branches (19 May to 4 Aug, 2003). Fruit
remained on the tree until 96 DAF B. Mean i SE, n= 30. ........................................ 153

Table 2. Length and leaf number of current season shoots on 2-year-old
’Ulster’/ Gisela 6 sweet cherry branches between full bloom and terminal bud
set (30 Apr to 4 Aug, 2003). Mean t SE (n=170). ..................................................... 154

Table 3. 13C content in different organs of a 2-year-old ’Ulster’ / Gisela 6 sweet
cherry branch during stages I and II of fruit development (25 DAF B, 25 May
and 40 DAFB, 9 Jun). Mean 1: SE, n=5. ...................................................................... 155

Table 4. 13C content in different organs of a 2-year-old ’Ulster’ / Gisela 6 sweet
cherry branch during stage III of fruit development (44 DAF B, 13 June; 56
DAFB, 25 Jun; 75 DAFB, 14 Jul). Mean :1: SE, n=5. ................................................... 156

Table 5. Total 13C content (mg 13C) in pulsed fruiting spur, non-fruiting spur

and terminal shoot leaves at O h after each 13C pulse-labeling, and total 13C
content recovered for the whole branch at 48 h after each 13C pulse-labeling.
Calculations are based on total DW of organs. Mean :l: SE, n=5. .......................... 157

Table 6. 13C content in fruit sampled immediately (0 h) after labeling of the
fruiting spur leaves at each pulse-labeling date. Mean i SE, n=5. ....................... 158

Table 7. 13C content in different organs of a 2-year-old ’Ulster’/ Gisela 6 sweet
cherry branch 48 h after pulsing of the fruiting leaf population with 13C02.
Mean :l: SE, n=5. ............................................................................................................ 159

Table 8. 13C content in different organs of a 2-year-old ’Ulster’ / Gisela 6 sweet
cherry branch 48 h after pulsing of the non-fruiting spur with 13C02. Mean :1:
SE, n=5. .......................................................................................................................... 160

Table 9. 13C content in different organs of a 2-year-old ’Ulster’ / Gisela 6 sweet
cherry branch 48 h after pulsing of the current season shoots. Mean t SE,
n=5. ................................................................................................................................ 161

Table 10. 13C content in fruit, wood and fruiting spur leaves on 2-year-old
’Ulster’/ Gisela 6 sweet cherry branches 48 h after pulsing the fruiting spur,
non-fruiting spur and terminal shoot leaf populations with 13C02. Mean :l: SE,
n=5. ................................................................................................................................. 162

Table 11. 13C content measured in non-fruiting spur leaves on 2-year-old
’Ulster’/ Gisela 6 sweet cherry branches 48 h pulsing the fruiting spur, non-
fruiting spur and terminal shoot leaf populations with 13C02. Mean :t SE,

n=5. ................................................................................................................................. 163

Table 12. 13C content measured in basal, medial and apical leaves and wood

of terminal shoots on 2-year—old ’Ulster’/ Gisela 6 sweet cherry branches 48 h
after pulsing the fruiting spur, non-fruiting spur and terminal shoot leaf
populations with 13C02. Mean t SE, n=5. ................................................................. 164

Table 13. 13C content in pericarp (flesh) and endocarp (pit) of ’Ulster’ / Gisela 6
sweet cherry fruit 48 h after labeling to fruiting spur leaves at each date.

Mean + SE, n=5. ............................................................................................................ 165
CHAPTER V

Table 1. Current season growth (Shoot) measurements of ’Regina’ / Gisela 6

sweet cherry trees between bloom and terminal bud set (2003). Mean i SE,

n=40. ............................................................................................................................... 207

Table 2. ’Regina’/ Gisela 6 sweet cherry fruit growth measurements from fruit
set through stage 111. Mean i SE, n= 25 .................................................................... 207

xiii

LIST OF FIGURES

CHAPTER I

Figure 1. Leaf populations on a typical 2—year-old sweet cherry branch ............... 61

CHAPTER II

Figure 1. Sites of girdling treatments T1 to T5 imposed on 2-year-old sweet
cherry branches. Black arrows indicate sites of girdling for all the treatments.
Black dots indicate defoliated sections for T4 and T5. ............................................. 82

Figure 2. Relative current season growth on ’Ulster’/ G16 with (C, T1 and T2)
and without girdling (T3, T4 and T5) at the junction of the previous season

(2000) growth and the current season (2001) growth. Calculations were based

on shoot length measured weekly for each treatment. ............................................ 83

Figure 3. Relative current season growth on ’Hedelfinger’ / G15 with (C, T1

and T2) and without girdling (T3, T4 and T5) at the junction of the previous
season (2000) growth and the current season (2001) growth. Calculations were
based on shoot length measured weekly for each treatment ................................. 83

CHAPTER III

Figure 1. 13C content (expressed as atom %) in fruits and current season

growth leaves during stage III (52, 59 and 63 DAF B). Means t SE are

represented in colored bars for each organ within a certain treatment. Means

for a certain organ followed by the same letter are not significantly different

at a=0.05. ....................................................................................................................... 112

Figure 2. 13'C -Relative partitioning (%) in distal and proximal fruits during

stage III (52, 59 and 63 DAFB). Means :t SE are represented in colored bars

and vertical lines, respectively. Each treatment is represented by a different
color. Colored bars within the same organ followed by the same letter are not
significantly different at a = 0.05 and or = 0.01, respectively. ............................... 113

xiv

CHAPTER IV

Figure 1. Relative cumulative growth of the terminal shoot and fruit on 2-
year—old 'Ulster’/ Gisela 6 sweet cherry branches. Calculations are based on
weekly measurements of shoot length (cm) and fruit diameter (mm). Mean :l:

SE, n=30. SI: Stage 1, SH: Stage II, 5111: Stage III, Post 5111: Post Stage III. ............ 166

Figure 2. Cumulative leaf area of individual spurs, non-fruiting spurs and
terminal shoots of 2-year-old ’Ulster’ / Gisela 6 sweet cherry branches. Mean :l:
SE, n=30. ........................................................................................................................ 167

Figure 3. Relative fresh weight partitioning on 2-year-old ’Ulster’ / Gisela 6

sweet cherry branches at each pulse-labeling date (May to Jul, 2003).

Calculations are based on absolute total FW for each organ (Appendix 3.10).
Mean :l: SE, n=30. FS: fruiting spur; NFS: non-fruiting spur. ................................. 168

Figure 4. Relative dry weight partitioning in 2-year-old ’Ulster’/ Gisela 6

sweet cherry branches at each pulse-labeling date (May to Jul, 2003).

Calculations are based on absolute total DW for each organ (Appendix 8.12).
Mean i SE, n=30. FS: fruiting spur; NFS: non-fruiting spur .................................. 169

Figure 5. 13C -Re1ative partitioning among different organs on 2-year-old
’Ulster’/ Gisela 6 sweet cherry branches. Calculations are based on absolute
amounts of 13C recovered for each organ 48 h after each 13COz pulse of the
fruiting spur leaves. For statistics, see Table 7. Mean :l: SE, n=5. FS: fruiting

spurs; NFS: non-fruiting spurs; TS: terminal shoot. ............................................... 170

Figure 6. 13C -Re1ative partitioning among different organs on 2-year-old
’Ulster’/ Gisela 6 sweet cherry branches. Calculations are based on absolute
amounts of 13C recovered for each organ 48 h after each 13COz pulse of the
non-fruiting spur leaves. For statistics, see Table 8. Mean :t SE, n=5. FS:

fruiting spurs; NFS: non-fruiting spurs; TS: terminal shoot. ................................. 171

Figure 7. 13C -Relative partitioning among different organs on 2-year-old
’Ulster’/ Gisela 6 sweet cherry branches. Calculations are based on absolute
amounts of 13C recovered for each organ 48 h after each 13COz pulse-labeling

to terminal shoots. For statistics see Table 9.Mean t SE, n=5. FS: fruiting

spurs; N FS: non-fruiting spurs; TS: terminal shoot. ............................................... 172

Figure 8. 13C -Relative partitioning among leaves and wood of current season
shoots on 2-year-old ’Ulster’ / Gisela 6 sweet cherry branches. Calculations are
based on absolute amounts of 13C recovered in shoots pulsed directly with

13C02 at each date. Mean :t SE, n=5. FS: fruiting spurs; NFS: non-fruiting

spurs; TS: terminal shoot. ........................................................................................... 173

Figure 9. 13C -Relative partitioning among leaves and wood of terminal shoots

on 2-year-old ’Ulster’ / Gisela 6 sweet cherry branches. Calculations are based

on absolute amounts of 13C recovered in shoots pulsed directly with 13C02 at
each date. Mean i SE, n=5. ......................................................................................... 174

Figure 10. Relative 13C partitioning between pericarp and endocarp of fruit

from 2-year-old ’Ulster’/ Gisela 6 sweet cherry branches 48 h after 13C02

pulsing of fruiting spur leaves. Calculations are based on absolute amounts of
13C recovered in both tissues at five dates. For statistics, see Table 13. Mean 3:

SE, n=5. .......................................................................................................................... 175

CHAPTER V

Figure 1. 13C atom % excess in different organs of ’Regina’ / Gisela 6 sweet

cherry at leaf abscission (Nov, 2002). Colored bars indicate mean for each

organ. Vertical lines indicate SE, n=5. Means followed by the same small

letter are not significantly different at a = 0.05 and or = 0.01. Obtained p-value

< 0.0001. ......................................................................................................................... 208

Figure 2. 13C atom % excess in different organs of ’Regina’ / Gisela 6 sweet

cherry at budbreak (Apr, 2003). Colored bars indicate mean for each organ.
Vertical lines indicate SE, n=5. Means followed by the same small letter are

not significantly different at or = 0.05 and on = 0.01. Obtained p-value < 0.0001. 209

Figure 3. 13C atom % excess in different organs of ’Regina’ / Gisela 6 sweet

cherry at leaf abscission (Nov, 2002) and budbreak (Apr, 2003). Colored bars
indicate mean for each organ. Vertical lines indicate SE, n=5. Asterisk and NS
indicate presence or absence of significant differences between sampling

dates, respectively at a = 0.05. Obtained p-value < 0.0001. ................................... 210

Figure 4. . 13‘C atom % excess detected in aerial organs of ’Regina’/ Gisela 6

sweet cherry during spring (May to Jun, 2003). Asterisks indicate presence of
significant differences among organs at each developmental stage at a = 0.05.
FB1: first bloom; FB2: full bloom; FS: fruit set; SI: stage I; SII: stage II. ................ 211

Figure 5. 13C atom % excess for different aerial organs of ’Regina’ / Gisela 6

sweet cherry during spring (May to Jun, 2003). Colored bars indicate mean

for each organ. Vertical lines indicate SE, n=5. Means within a given sampling
period followed by the same small letter are not significantly different at or =
0.05. FS: fruiting spur, NFS: non-fruiting spur; CSG: current season growth ..... 212

Figure 6. 13C atom % excess values in reproductive and vegetative tissues of
fall-pulsed ’Regina’/ Gisela 6 sweet cherry trees during fall (Nov, 2002) and
spring (Apr to Jun, 2003). Colored lines indicate mean for each organ. Vertical
lines indicate SE, n=5. Means within a given stage period followed by the

same small letter are not significantly different at or = 0.05. LA: leaf abscission;
SG: side green; FB1: first bloom; F B2: full bloom; FS: fruit set; SI: stage I; SII:

stage II ............................................................................................................................ 213

Figure 7. Total 13C content (pg 13C) for different organs of ’Regina’ / Gisela 6
sweet cherry trees during spring (May to June, 2003). Colored bars indicate

mean for each organ. Vertical lines indicate SE, n=5. Means within a given

stage followed by the same letter are not significantly different at at = 0.05. FS:
fruiting spur, NFS: non-fruiting spur; CSG: current season growth. ................... 214

CHAPTER I

LITERATURE REVIEW

Sweet Cherry Description and Production Trends

Sweet cherry (Prunus avium L.), a member of the Rosaceae family, is a
temperate deciduous tree that is thought to have originated in forests close to the
Caspian and Black Seas of Eastern Europe and Western Asia (Webster, 1996).
Sweet cherry trees are characterized for large stature (>10 m) and strong apical
dominance in natural environments (Webster, 1996; Lang, 2000). More recently,
sweet cherry has become one of the most profitable tree fruits due to
improvements in germoplasm breeding and selection (i.e., introduction of new
varieties and rootstocks), management practices in the field, and storage and
transportation.

Consequently, the international commercial production of sweet cherry is
increasing. Europe and Asia are the most productive continents. In North
America, >90% of the total sweet cherry production is from the United States
(US) (Whiting, 2001). In 2003, the US produced 215,000 mt on 30,712 ha, with
Michigan being the fourth largest US sweet cherry producer (FAOSTAT data,
2004). Among fruit commodities, sweet cherry is one of the most highly prized
(Lang, 2000). A short postharvest life and limited climatic adaptability foster a
profitable niche in world markets and reduce competition (Maguylo, 2003).

Recently, US sweet cherry production is in transition to high density, early
fruiting orchards (Lang, 1998). Profitable orchard management of sweet cherry
on vigorous rootstocks such as Mazzard (Prunus avium L.), Mahaleb (Prunus

mahaleb L.) or Colt (P.pseud0cerasus x P.avium) is being challenged by

inefficiencies associated with large tree size and a long establishment period
before fruiting (Lang, 2000). These characteristics are undesirable due to low
initial yields, delayed return on capital investment and inefficiency of orchard
operations (e.g. pruning and harvest labor, pest control, etc.) (Whiting, 2001). In
addition, labor costs have increased and labor availability has decreased
considerably in recent years. As a consequence, sweet cherry growers are
adopting dwarfing and semi-dwarfing rootstocks, which are characterized by
small canopies and positive effects on precocity and yield (Weber, 2001). High
density systems using dwarfing precocious rootstocks are more labor efficient
and economically viable. These modern orchards are more uniform, have high
and early yields, require lower production and harvest inputs, and are easier to

protect against rain or bird damage.

Sweet Cherry Rootstocks and the Gisela Series

Historically, the most popular rootstocks used in commercial sweet cherry
orchards, in North America and around the world, have been seedlings of
Mazzard and Mahaleb, or clones of ’Colt’ (Webster and Schmidt, 1996; Perry,
1987; Lang, 1998). Unfortunately, sweet cherries on these rootstocks are not
convenient for highly intensive systems since trees are too vigorous and do not
flower until the 6th or 7th year (Lang, 2000). However, the introduction of a new
generation of more dwarfing rootstocks has great potential to promote precocity,

high productivity and reduced vigor. In the 1980 5, several of the more

promising international rootstock selections were imported into the US and
established under different climatic and soil conditions throughout the country
(Perry et a1., 1996). So far, the most promising dwarfing and semi-dwarfing
rootstocks belong to the Gisela (Giessen, GI) series, particularly G15 (148/ 2) and
GI6 (148 / 1), both hybrids of P. cerasus x P. canescens (Webster and Lucas, 1997;
Andersen et a1., 1999; Lang, 2000; Webster, 2001). The GI rootstocks were
developed in a breeding program initiated in 1965 at Justus Liebig University in
Giessen, Germany (Franken-Bembenek, 1996). These rootstocks induce flowering
several years earlier than usual (from the 3rd to the 5th year) and provide a size
control that ranges from 50% (G15, dwarfing) to 80% (G16, semidwarfing) of
similar trees on standard Mazzard (Lang, 2000).

Before commercial adoption of GI rootstocks by American growers
becomes routine, many physiological questions must be elucidated. The
extensive experience with dwarfing apple rootstocks provides some clues for
intensive sweet cherry management However, the seasonal growth and fruiting
habit of sweet cherry differ from those of apple, implying that the physiological
consequences of similar orchard practices might not have the same results.
Despite the advantages of GI rootstocks to induce precocity and higher yield
efficiency (Webster, 2001), such trees have the tendency to crop excessively
beginning about the 4th or 5th year, resulting in small sized fruit (Andersen et a1.,
1999). In addition, GI rootstocks might have a reduced capacity for CH20 storage

due to smaller root systems and trunk and branch tissues (Lang, 2001a).

Precocious sweet cherry trees on GI rootstocks have the potential to quickly
reach and imbalance between early vegetative and fruiting growth, leading a
prolonged period of ’runting out’ (Lang, 2001a,b). Thus, the balance between leaf
area, storage reserves and crop load capacity becomes more critical in achieving
high quality fruit (Lang, 2000). To overcome this situation, more precise canopy
development, i.e., greater precision in management of crop loads and the
different leaf populations within the canopy, is required to optimize the balance

between vegetative growth and fruit quality (Lang, 2001 a,b).

Reproductive and Vegetative Habits of Sweet Cherry Trees

Sweet cherry produces simple buds, which can be reproductive or
vegetative. Normally, reproductive buds are initiated in the leaf axis of new or
existing non-fruiting spurs; however, a few flowers also can be initiated in the
leaf axis of single buds near the base of new shoots. A reproductive spur may
have 1 to 6 buds, each of which may contain multiple inflorescences (Thompson,
1996). Vegetative buds form in the leaf axis on current season new shoots; in the
subsequent season, each vegetative bud will become a non-fruiting spur, which
in turn will initiate flowers to bloom the following year. Vegetative growth
consists of extension shoots (either lateral or terminal) and spurs (short shoots
with minimal internode length). Extension shoots and spurs generally emerge
concomitant with bloom. In vigorous sweet cherry trees, spur leaves (fruiting

and non-fruiting spurs) complete their development early in the season (~3

weeks after bloom); however, lateral and terminal shoot growth continues
through harvest. During this initial post-bloom period, spur leaves constitute the
primary source of C for fruit growth (Roper et a1., 1987). The reproductive effort
(i.e., the proportion of total biomass allocated in reproductive structures) of
dwarfing trees on G1 rootstocks is different from those on more standard
vigorous rootstocks such as Mazzard and Mahaleb. Flowers per unit branch-size
often are much more numerous on trees on G1 rootstocks (Webster, 1996).
Recently, Maguylo (2003) found that the number of reproductive buds and
flowers of ’Hedelfinger’ on either G15 or G16 spurs was ~4 and 3, respectively; on
Mazzard, these were 0.4 and 0.8, respectively.

Healthy and abundant leaf populations, producing a continuous supply of
photosynthates throughout the season, are essential for growth and storage. In
many species of the Rosaceae family, including sweet cherry, spurs and
extension shoot leaves are the main sources of current photosynthates for
vegetative and reproductive growth (Roper et a1., 1987; Corelli-Grappadelli et a1.,
1994; Teng et a1., 2001). In most species, photoassimilate production by spur
leaves is not sufficient to support optimal fruit growth and import of assimilates
synthesized by leaves external to bearing spurs is required (Roper et a1., 1987;
Lakso and Corelli-Grappadelli, 1992; Tustin et a1., 1992; Corelli Grappadelli et a1.,

1994; Teng et a1., 1998, 2001).

In a typical two-year-old sweet cherry branch, current photoassimilates

for fruit and shoot growth are provided by three distinct leaf populations. These

are described below (See Figure 1):

a. Fruiting spur leaves: these are located on the 2-year-old section of the

branch. There are ~7 to 9 leaves at each spur. Fruit are borne on these
spurs. The primary purpose of this leaf population is thought to supply

CHzO to adjacent fruit and nearby shoots (Lang, 2001b).

b. Non-fruiting spur leaves: these are located acropetally to the spur fruit on

C.

1-year-old section of the branch. There are ~6 to 8 leaves at each spur.
Lang (2001b) suggested that this leaf population is a ’power house of
CHzO production’ due to two reasons: (1) there is a 6- to 8-fold increase in
leaf number compared to the same node during its formation the year
before; (2) this segment does not have fruit to support directly.
Accordingly, this leaf population might help to supply CH20 to nearby
current season shoots, developing leaves, and developing fruit on older
wood.

Leaves on current season shoots: these leaves are located acropetal to the
fruit There is one leaf at each node or single bud. Lang (2001b) suggested
that these leaves may be sinks for CHzO during active shoot extension
early in spring; however, at maturity they may constitute a source of

CHzO for nearby sinks and probably for distant fruit.

If we consider summer 2004 as a reference in time, fruiting spur leaves
would be located on shoot growth that was formed during 2002, non-fruiting
spur leaves would be on shoot growth formed in 2003, and current season shoot
leaves would be formed during 2004.

Little is known about the relative importance or temporal relationships of
different leaf populations as sources of current photosynthates for fruit and shoot
development in dwarfing sweet cherry trees. As indicated above, it is believed
that, as in other Rosaceae species, fruiting and non-fruiting spur leaves support
fruit growth from early developmental stages onwards but there is not direct
evidence for this assumption. In addition, the contribution of leaves on current
season shoots, as a potential C source during fruit development, has not been

documented.

The Importance of Carbon Economy and Partitioning in Fruit Trees

The CHzO economy of plants has received considerable attention during
recent years. The carbon economy of a tree includes the acquisition of C by
photosynthesis and subsequent utilization for biomass synthesis and
maintenance (Buwalda, 1991). In this process, C has been considered a ’common
currency’ to asses C allocation patterns and costs in plants (Reekie and Bazzaz,
1987a,b). So far, the understanding of these processes in fruit trees is limited and

only a few studies have focused on sweet cherry. Currently, one of the most

important challenges in crop physiology is to determine the mechanism
governing the partitioning to and dry matter accumulation of individual sink
organs (Wardlaw, 1990). Crop production is dependent not only on the ability of
the plant to intercept light for C fixation, but also on the partitioning of CHzO
into economically important organs (Minchin et a1., 1997). Generally, it is
accepted that the majority of the historical increase in crop yield has been
possible due to shifts in partitioning patterns rather than changes in
photosynthetic rates or respiration (Gifford and Evans, 1981; Patrick, 1988).
Therefore, future insights regarding assimilate partitioning may contribute to
improvements in crop productivity by increasing total biomass production and
by favoring assimilate transfer to the harvestable portion of the crop (Patrick,
1988). Indeed, this is one consequence of using dwarfing rootstocks in sweet
cherry trees; fruit production is increased and vegetative growth is decreased.
However, fruit quality is also of critical importance, not just biomass production.
Dry matter partitioning is the end result of a coordinated set of transport
and metabolic processes governing the flux and distribution of C from source
organs via a transport path to the sink organs (Patrick, 1988; Marcelis, 1996;
Daudet et a1., 2002). Partitioning of assimilates within the sites of synthesis
(source) and between sources and various competing Sites of utilization (sink), is
under genetic and environmental regulation (Daie, 1989). In deciduous fruit
trees, CHzO partitioning is affected by several factors, which include: assimilate

supply from photosynthesis, availability of storage reserves, canopy structure,

light interception, organ development, respiration, crop load, rootstock, cultural
practices and environmental conditions (McCammant, 1988; Keller and Loescher,
1989; DeJong 1999). All of these factors must be integrated to understand the
whole CHzO economy of sweet cherry (Flore and Layne, 1999).

The balance between vegetative growth and fruiting is manipulated
through horticultural practices to increase yield and / or quality and reduce
management costs. Many studies have focused on the consequences of
competition between organs and how this affects fruit development and quality.
Fruit play a major role in biomass allocation, as they are major Sinks for
assimilates (Heuvelink, 1997). Biomass allocation to fruit strongly affects total
fruit production, the weight of individual fruits and their quality components,
which are all important determinants of the economic yield. In dwarfing sweet
cherry trees, excessive flowering produces excessive crop loads, which result in
small fruit (Andersen et a1., 1999; Lang, 2000; Lang, 2001a,b). For fresh
consumption, it is often desirable to have a smaller number of larger fruits rather
than a large number of small ones, because the value per unit is much lower for
small fruit than for large ones (Jackson, 1989; Stover, 2000). A high fruit yield is
desirable and high biomass allocation to fruit is important; however, as the
allocation to fruit is at the expense of vegetative growth, which is needed for the
formation of leaf area, and hence light interception for photosynthesis, too high
of an allocation of biomass to fruit will affect future production capacity

negatively (Heuvelink, 1997). Enhanced fruit growth at the expense of vegetative

10

growth has been reported for several species (Forshey and Elfving, 1989; Kappel,
1991; Grossman and DeJong, 1995), including sweet cherry trees on dwarfing
precocious rootstocks (Whiting, 2001). On the other hand, while a certain level of
vigor is essential, excessive vegetative vigor reduces flowering and fruit set
(Forshey and Elfving, 1989).

As indicated above, yield improvement in fruit trees involves dry matter
production by various leaves populations and its partitioning and accumulation
in harvested organs, i.e., fruits. A better yield is achieved by successful
regulation of source-sink relationships, which influence the production and
utilization of C of the whole tree (Ho, 1988; DeJong and Grossman, 1995).
Minchin et a1. (1997) indicate that C source-sink relationships are important in
controlling fruit growth, and may ultimately determine crop yield. Assuming the
competition among sink organs for CHzO is dependent on the intrinsic ability of
sink organs to control C partitioning based on their sink strength, studies on
determination of sink strength may provide better strategies to improve crop
productivity (Ho, 1988; Marcelis, 1993; Grossman and DeJong, 1994).

In sweet cherry trees, interactions between vegetative and reproductive
sweet cherry growth change during the growing season. During early stages of
development, fruits and vegetative organs compete for storage reserves
(Loescher et a1., 1990), while later in the season, mature leaves provide fruit and
shoots with photoassimilates (Roper et a1., 1987). Most of the research studies on

sweet cherry C partitioning are based on the study of trees on vigorous

11

rootstocks; few experiments have focused on the dynamics of CHzO partitioning
on dwarfing or semi-dwarfing rootstocks, which might differ from more
Vigorous trees due to their reduced aerial woody structures, smaller root systems
and higher harvest index. Flore and Sams (1986) indicate that in sour cherry
(Prunus cerasus L.), photosynthesis may limit yield when crop loads are high and
foliage development is low (i.e., LA / F ratios < 2). This might be the case in

dwarfing sweet cherry trees, which have reduced LA / F ratios.

Carbohydrate Metabolism in Rosaceae Species

In sweet cherry, total nonstructural carbohydrates (TNC) consist mainly of
starch, sorbitol, sucrose, fructose, glucose and raffinose (Keller, 1986;
McCammant, 1988; Keller and Loescher, 1989). Sorbitol and sucrose are the major
translocated CHzO in several species of the Rosaceae family, especially in the
subfamilies Pomoidae and Prunoidae (Gao et a1., 2003). Sorbitol, a sugar alcohol,
is synthesized in mature sweet cherry leaves (Keller and Loescher, 1989 ) and
transported through the phloem to various sink tissues, where is metabolized
and converted into other CHzO (Bieleski and Redgwell, 1985). Sucrose, also
considered storage CHzO, accounts for a fourth of the soluble CHzO in sweet
cherry (Keller, 1986). Sucrose is synthesized and utilized by leaves of different
ages (Loescher et a1., 1982, Bieleski and Redgwell, 1985). Glucose, fructose and

sorbitol are the major TNC in sweet cherry fruit (Keller, 1986).

12

The relative abundance of TNC in sweet cherry tissues changes
qualitatively and quantitatively during the season (Keller and Loescher, 1989).
The seasonal TNC changes have been described by Keller (1986) and
McCammant (1988) as follows: TNC decrease before budbreak in all perennial
tissues except spurs. At budbreak, fructose and glucose predominate in buds.
Sorbitol is the most abundant soluble CHzO at this time. During fruit
development TNC accumulate slowly in all tissues. After harvest, TNC are
accumulated at a higher rate, reaching their highest level at leaf abscission.
Starch is the most abundant storage material. At the onset of dormancy,
raffinose, fructose and glucose are abundant. During dormancy, interconversion
of starch and soluble CHzO occurs, with sucrose as the most predominant
soluble CHzO.

Specific enzymes are involved in synthesis or degradation of CHzO in
rosaceous species. The enzyme NADPH-dependent aldose 6-phosphate
reductase (A6PR) is responsible for sorbitol synthesis in green tissues (Loescher
et a1., 1982; Bieleski and Redwell, 1985; Loescher and Everard, 1996; Sashanishi et
a1., 1998). In sink tissues, sorbitol is catabolized by the enzymes NAD-dependent
sorbitol dehydrogenase (SDH), which converts sorbitol to fructose (Negm and
Loescher, 1981 ; Loescher et a1., 1982; Lo Bianco and Rieger, 2002a,b) and sorbitol
oxidase (SOX), which converts sorbitol to glucose (Y amaki, 1980). On the other
hand, sucrose catabolism in sink tissues occurs via sucrose synthase (SS), soluble

acid invertase (AI) and neutral invertase (N1) activities (Lo Bianco et al., 1999b;

13

Lo Bianco and Rieger, 2002a,b). SDH, SOX and AI activity correlate positively
with fruit sink strength and growth rate (Lo Bianco and Rieger, 2002b). SDH

activity correlates with shoot elongation (Lo Bianco et al., 1999a)

Storage Reserves
Definition and importance

In most deciduous woody perennials, the immediate sources of CH20 are
recently synthesized photoassimilates and accumulated reserves (Oliveira and
Priestley, 1988). Storage reserves are materials produced in excess of current
requirements and which later may be used to support metabolism and growth
(Priestley, 1960). These ’substances’ (organic compounds and nutrients) are not
used directly in growth and respiration but stored in the tree until required
(Glerum, 1980). The use of CHzO reserves is subject to a temporal and spatial
distribution Since the contents of storage reserves fluctuates, and major sites of
storage may be remote from the sites of utilization (Oliveira and Priestley, 1988).

Storage reserves are important for several life processes. Reserves are used
for winter survival, metabolism, respiration, defense, healing, vegetative and
reproductive growth, fruit development and new growth in spring (Kandiah,
1979a,b; Oliveira and Priestley, 1988; Loescher et a1., 1990; Kozlowski and
Pallardy, 1997). Increased cold hardiness has been attributed to CHzO
accumulation during fall (Johnson and Howell, 1981). More vigorous trees are

able to accumulate more CHzO to heal injuries due to pathogen or insect attacks,

14

synthesize defensive chemicals and tolerate various environmental stresses
(Kozlowski and Pallardy, 1996). Reserves also are important for ’regrowth’ after
pruning, premature defoliation and early season frost (McCammant, 1988;
Kozlowski et a1., 1991). In pecan (Caryn illinoensis Koch), grape (Vitis vinifera L.)
and sweet cherry, premature defoliation reduced the accumulation of CHzO
reserves in fall (W orley, 1979; Smith et a1., 1986; McCammant, 1988; Candolfi-
Vasconcelos et a1., 1994). Alternate bearing also has been attributed to the
availability of stored reserves. In pistachio (Pistacia vera L. Pistah.) and pecan,
increased CHzO reserves have been observed after an ’off’ year (Crane et a1.,
1976; Smith et a1., 1986; Wood, 1995). A decrease in alternate bearing might be
due to more time for the tree to accumulate CHzO reserves before leaf fall
(Stevenson and Shackel, 1998).

Several authors indicate that the initial stages of spring growth in
deciduous fruit trees must depend upon mobilization of reserves accumulated
the previous season, until new leaves become photosynthetically competent to
provide current photosynthates (Priestley, 1960; Hansen, 1967b; Quinlan, 1969;
Oliveira and Priestley, 1988). Reserves are essential for new growth because they
provide energy and structural resources before root N uptake and

photosynthesis occurs in spring (Cheng and Fuchigami, 2002).

15

Types of storage reserves
a. CHzO reserves

Quantitatively, CHzO constitute the predominant components of storage
reserves; however, qualitatively, N and other minerals such as P, Ca, K and Mg
are equally important (Tromp, 1983; Oliveira and Priestley, 1988). CHzO reserves
include soluble and insoluble forms. Starch is the main insoluble storage form in
woody organs and is synthesized whenever a high level of sugars accumulates
(Tromp, 1983; Kozlowski and Pallardy, 1996). In sweet cherry, starch is the most
common storage material (Keller and Loescher, 1989). Small amounts of
hemicelluloses also are found in storage organs (Taylor et a1., 1975), but their
function is primarily structural as a component of cell walls (Oliveira and
Priestley, 1988). Hemicellulose is used during maturation of current season
growth (Priestley, 1960). Among soluble CHzO, sorbitol, mannitol, sucrose,
glucose, fructose and raffinose have been reported as important for storage in
various woody perennials (Crane et a1., 1976; Loescher et a1., 1990). In some
species of the Rosaceae family, such as apple (Malus domestica Borkh.) and sweet
cherry, sorbitol is the principal soluble storage CH20 in non-photosynthetic cells
(T romp, 1983; Oliveira and Priestley, 1988; Loescher and Everard, 1996). In sweet
cherry leaves, sorbitol accumulates more than starch (Roper et a1., 1988), and
raffinose accumulates during dormancy (Keller, 1986; Keller and Loescher, 1989).

Other soluble CHzO found in small amounts in storage organs include inositol,

16

xylose, rhamnose, maltose, trehalose, arabinose, ribose, mannose, galactose and

stachyose (Loescher et a1., 1990).

b. Nitrogenous reserves and other minerals

N reserves are also composed of soluble and insoluble fractions. Amino
acids and amides, mainly arginine and asparagine, are the major soluble
compounds, while proteins correspond to the insoluble fraction (Oliveira and
Priestley, 1988). Mobilization and recycling of N reserves in spring is critical to
support new growth shortly after budbreak, since at this time conditions for root
uptake are not optimal (Habib et a1., 1989). In apple, N reserves become available
for new growth in spring through hydrolysis of bark and wood protein
(Kennedy et a1., 1975). In sweet cherry, remobilization of N reserves from roots

occurs during the first 35 to 50 days after budbreak (Grassi et a1., 2003).

Storage Organs

The whole perennial structure of a tree can be considered as a storage
organ (Kandiah, 1979a,b; Loescher et a1., 1990). In most angiosperm trees or
’hardwoods’, CHzO reserves are accumulated predominantly in living ray and
axial pachenchyma cells of woody axes (i.e., branches and trunk) and roots
(Oliveira and Priestley, 1988). The importance of woody axes and roots as storage
organs vary among species (Tromp, 1983; Priestley, 1960; Loescher et a1., 1990).

Some studies indicate that there is no difference in the potential value of reserves

17

from different regions of the tree since no specific regions for CHzO storage exist
due to a similar distribution of CHzO reserves above or below ground parts
(Priestley 1960; Tromp, 1983; Araujo and Williams, 1988; Kandiah, 1979a,b).
However, a preferential accumulation of CH20 reserves seems to occur in roots
of some woody perennials (Hansen, 1967b; Quinlan, 1969; Kandiah, 1979a,b;
Keller, 1986; Loescher et a1., 1990). In sweet cherry, CH20 and N reserves in roots
were higher than in other storage organs such as trunk and shoots (Loescher and
Keller, 1989; Grassi et a1., 2003). Roots might be the most important storage organ

in sweet cherry because of their high starch content (Keller, 1986).

Seasonal pattern of storage reserves in woody perennials

Seasonal fluxes of storage reserves, mainly CHzO, have been studied
extensively in apple (Hansen, 1967b; Quinlan, 1969; Hansen and Grauslund,
1973; Hansen, 1971; Priestley, 1960; Kandiah, 1979a,b), sweet cherry
(McCammant, 1988; Keller, 1986; Keller and Loescher, 1989), peach (Prunus
persica (L.) Batsch) (Gaudillere et a1., 1992; Moing and Gaudillere, 1992; Caruso et
a1., 1997; Inglese et a1., 2002), pecan (Davis and Sparks, 1974; Worley, 1979;
Lockwood and Sparks, 1978 a,b; Smith et a1., 1986), grape (Vitis vinifera L.)
(Winkler and Williams, 1945; Scholefield et a1., 1978; Bains et a1., 1981; McArtney
and Ferree, 1999), kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R.

Ferguson) (Buwalda et a1., 1990; Buwalda, 1991 ; Greaves et a1., 1999), cranberry

18

( Vaccim'um macrocarpon Ait) (Birrenkott et a1., 1991 ; Hagidimitriou and Roper,
1994) and blueberry (Vaccinium corymbosum L.) (Maust et a1., 2000).

The production, partitioning and utilization of CHzO reserves follow
specific seasonal patterns in deciduous fruit trees. Levels of reserves in perennial
organs have a similar pattern of initial deposition, followed by depletion in early
spring and subsequent replenishment later in summer and fall (Tromp, 1983). In
Spring, growing sinks attract nutrients from sources, i.e., storage organs,
elsewhere in the tree. However, later in the season, new leaves become self-
sufficient and sink demand changes to other organs. In late summer, shoot
growth slows or ceases and nutrient accumulation in perennial tissues increases
in importance, predominating in fall before leaf senescence.

Early stages of development in spring depend on reserves accumulated in
the tree during the previous season (Oliveira and Priestley, 1988). Depletion of
CHzO reserves in shoots and roots of several species usually begins before
budbreak and continues after bloom during early shoot growth (Priestley, 1960;
Hansen, 1967b; Hansen and Grauslund, 1973; Gaudillere et a1., 1992; Moing and
Gaudillere, 1992; Caruso et a1., 1997; Inglese et a1., 2002; Scholefield et a1., 1978;
Bains et a1., 1981; Buwalda, 1991 ; Lockwood and Sparks, 1978a,b; Birrenkott et a1.,
1991; Hagidimitriou and Roper, 1994; Teng et a1., 1999). In apple, early CHzO
reserve depletion was due mainly to respiration with only a small portion (<
20%) used as building material for new growth (Hansen and Grauslund, 1973;

Kandiah, 1979a,b). labeled C fixed during the previous fall has been detected

19

during early spring growth of leaves, flowers, fruit and shoots of apple, grape,
japanese pear (Pyrus pyrifolia Nakai) and pecan (Hansen, 1967b; Hansen, 1971;
Scholefield et a1., 1978; Teng et a1., 1999; Lockwood and Sparks, 1978a,b). During
early spring, root activity increases and significant amounts of CHzO are used in
metabolism, respiration, structural growth and are incorporated into amino acids
(Oliveira and Priestley, 1988). Storage reserves also are used in cambial activity
and phloem formation (Oliveira and Priestley, 1988). Cambial activity begins
before budbreak and phloem differentiation precedes xylem formation (Evert,
1963). After reaching the lowest CHzO levels, most species begin to accumulate
storage reserves immediately. However, during fruit development and ripening,
this process is slow or absent (Roper et a1., 1988; Caruso et a1., 1997; Inglese et a1.,
2002). Higher accumulation rates in permanent structures are detected after
shoot extension has ceased in summer, when vegetative growth slows down and
storage exceeds consumption (Chong, 1971; Kandiah, 1979a,b; Gaudillere et a1.,
1992; Oliveira and Priestley, 1988; Jordan and Habib, 1996; Caruso et a1., 1997 ;
Bains et a1., 1981; Buwalda, 1991 ; Smith et a1., 1986; Birrenkott et a1., 1991;
Hagidimitriou and Roper, 1994). After terminal bud set in late summer and
before leaf fall, CHzO reserves (mainly starch hydrolyzed to soluble transport
sugars) are translocated basipetally to perennial storage organs (Priestley, 1960;
Hansen, 1967b; Quinlan, 1969; Hansen and Grauslund, 1973; Kandiah, 1979a,b;
Hale and Weaver, 1962; Araujo and Williams, 1988; Lokwood and Sparks, 1978;

Davis and Sparks, 1974) to become part of structural growth or storage reserves,

20

mainly starch (Oliveira and Priestley, 1988; Loescher et a1., 1990). At the
beginning of dormancy, starch contents are highest (Caruso et a1., 1997 ; Bains et
a1., 1981 ; Smith et a1., 1986; Jordan and Habib, 1996; Birrenkott et a1., 1991 ;
Hagidimitriou and Roper, 1994). During winter, conversion of starch to soluble

sugars occurs (Bains et a1., 1981).

Seasonal pattern of storage reserves in sweet cherry

In sweet cherry, flowering often occurs before leaves are fully expanded
and early stages of reproductive (flowers and fruits) and (spurs, extension shoots
and roots) vegetative growth are dependent on the storage reserves accumulated
the previous season (McCammant, 1988; Keller and Loescher, 1989). Other
deciduous trees are less dependent on stored reserves since canopies are nearly
fully expanded before anthesis (Keller and Loescher, 1989).

Seasonal nonstructural carbohydrate partitioning in sweet cherry trees on
standard (vigorous) rootstocks has been studied previously (Keller, 1986; Keller
and Loescher, 1989; McCammant, 1988). TNC in perennial organs of ’Bing’ sweet
cherry on standard rootstocks changed both qualitatively and quantitatively
during the year (Keller, 1986; Keller and Loescher, 1989; McCammant, 1988).
TNC declined in 1- and 2-year-old shoots and roots, beginning in mid-April and
reaching a minimum in early May (McCammant, 1988; Roper et a1., 1988).
Shortly before budbreak, TNC decreased in all perennial organs except spurs

(Keller, 1986; Roper et a1., 1988; Keller and Loescher, 1989). After bloom, TNC

21

increased slowly until fruit harvest (Keller, 1986; Keller and Loescher, 1989;
McCammant, 1988).However, the rate of accumulation slowed down during the
last 4 to 6 weeks of fruit growth (Keller, 1986; Keller and Loescher, 1989). After
fruit ripening and cessation of shoot extension, CHzO reserves accumulated in
different sweet cherry organs reaching a maximum at leaf abscission (Keller,
1986; Keller and Loescher, 1989; McCammant, 1988). Starch levels in current
season growth, older Shoots, trunk (1- to 3—year-old growth rings and bark) and
roots were the greatest in fall (Roper et al., 1988). At the onset of dormancy, all
soluble CHzO increased, especially sorbitol (McCammant, 1988). During winter,
interconversion of starch and soluble CH20 in the wood of trunk and 1- and 2-
year old shoots occurred. By February, sorbitol declined, while fructose and
glucose began to peak in mid-April, a week before bloom (McCammant, 1988).
Radioactive labeling of storage reserves in sweet cherry indicated that, at
budbreak, buds had the highest 14C recoveries compared to surrounding wood
and bark. Shortly after leaf expansion, leaves were highly radioactive but the
amount of label decreased as the shoot increased in length. When shoots were 20
to 30 cm long, expanding leaves were less radioactive than fully expanded leaves
in the middle and base of the same shoot, indicating a reduced use of storage
reserves.

Flow of C during early Spring growth of sweet cherry trees is dependent
on both storage reserves and current photosynthates. Currently, there is no

information regarding the relative importance of these two components on the

22

dynamics of remobilization and partitioning of CHzO reserves during spring in
trees on more dwarfing rootstocks. It would be valuable to characterize the
transition phase, in which storage reserves are depleted and current
photosynthates become the primary source for vegetative and reproductive

growth.

The Use of Labeled Carbon to Study Carbon Fluxes and Partitioning

The use radioactive carbon (14C), supplied as 14COz pulses, to study
translocation patterns, carbon fluxes and partitioning of assimilates has been
reported for several woody species. Traditional experiments in apple, peach,
apricot (Prunus armeniaca L.), sour cherry, grape and pecan differ depending on
whether the 14C02 was applied to whole trees, individual branches, shoots or
single leaves of a shoot (Quinlan, 1969; Hansen, 1969; Corelli-Grappadelli et al.,
1994; Kappes and Flore, 1989; Toldam-Andersen, 1998; Hale and Weaver, 1962;
Davis and Sparks, 1974). Labeling methods vary from simple to highly
sophisticated (Farrar, 1993). Few 14C partitioning studies have been carried out
using whole trees in full production; most considered young non-bearing trees
(Quinlan, 1965; Hansen, 1967a,b; Wang et al., 1996; Wang and Quebedeaux, 1997,
1998; Bieleski and Redgwell, 1985; Kappes and Flore, 1989; Kandiah, 1979a,b).
However, the use of uniform individual shoots, either attached to or excised
from mature trees, has allowed a more practical study of reproductive (flowers

and fruit) effects on C fluxes (Hansen, 1970, 1971 ; Lakso and Corelli-Grappadelli,

23

1992; Corelli-Grappadelli et al., 1994; Corelli-Grappadelli et al., 1996; Davis and
Sparks, 1974; Génard et al., 1998; Johnson and Lakso, 1986a,b; Bepete and Lakso,
1998)

Recently, labeling with non-radioactive 13C, supplied as 13C02,, has
provided a useful and environmentally friendly tool to monitor respiration and
carbon fluxes in enriched sour cherry, peach, japanese pear, kiwifruit, walnut
(Iuglans regia L.) and persimmon (Diospyros kaki Linn. Ebenaceae) trees
(Lombardini et al., 2001; Moing and Gaudillere, 1992; Teng et al., 1998; Teng et
al., 1999; Teng et al., 2001; Amano et al., 1998; Maillard et al., 1994; Nakano et al.,
1998)

In nature, there are two stable isotopes of carbon, 12C and 13C (Griffiths,
1993; Brugnoli and Farquhar, 2000). 12C is the lighter and most abundant isotope,
with ~98.89% of the global carbon pool, while 13C is the heavier isotope in a
proportion of ~1.11% (Griffiths, 1993). During photosynthetic C02 fixation,
fractionation of stable carbon isotopes occurs, and as consequence plants are
depleted in the heavier isotope 13‘C (Brugnoli and Farquhar, 2000). In C3 plants,
fractionation occurs during diffusion of gaseous C02, through the boundary
layer and stomata to the intercellular space. Additional fractionation steps occur
during the liquid phase at the sites of carboxylation and during enzymatic
reactions associated with carboxylation by ribulose-1,5-biphosphate carboxylase-
oxygenase (Rubisco), dark respiration and photorespiration (Brugnoli and

Farquhar, 2000).

24

The isotopic composition of plant inorganic material is measured by
isotope ratio mass spectrometers (Griffiths, 1993). Plant tissues are converted to
C02 by combustion and mass spectrometry analysis gives the abundance ratio R,
which is defined as R=13COz/12COz. Results are traditionally expressed as 513C,
which is defined as 613C=Rp/ (Rs-1); where Rp is the isotope ratio in plant
samples and Rs is the ratio of the internationally accepted standard, Cretaceous
belemnite from the Pee Dee formation in South Carolina (PDB=0.01124).13C

enrichment for different plant tissues has been calculated as follows (Boutton,

1991 ; Vivin et al., 1996):

813C (%o) = [(Rsample-Rstandardfl Rstandard] x 1000 Eq (1)
Rsampxe = 13'C/ 12C = [813C/ (1000 + 1)] x RPDB Eq (2)
F=13C/(13C+12C)=R/<R+1) Eq<3>
Atom% excess = (Fpostdose-Fbasenne) x 100 Eq (4)
New 13C content = (Atom% excess / 100) x Dry Matter x [C] Eq (5)

where the 513C (%o) value is calculated from the measured C isotope ratios of the
sample and standard gases (Eq.1). The absolute ratio (R) of a sample is defined
by Eq. 2, where RPDB = 0.0112372. Atom % excess is used as an index to
determine the enrichment level of a sample following the administration of the

13C tracer in excess of the 13C baseline prior to the 13C02 pulse (Eq.3 and 4). The

25

new 13C pool is calculated for the different branch components according to dry

mass and C concentrations (Eq. 5).

Source-Sink Relationships
Sink strength

A plant can be considered as a collection of individual sinks (reproductive
and vegetative), which compete with each other (Wright, 1989; Flore and Layne,
1999). Carbon moves between sources and Sinks as a function of source supply,
Sink demand and distance between sources and sinks (DeJong and Grossman,
1995). Sink organs are net importers of assimilates (Ho, 1988). Meristem tissues,
such as developing leaves or root tips, are considered ’utilization sinks” since
most of the C is used for growth and respiration. Storage organs, such as fruit,
stems or roots, are considered ’storage sinks’ because a substantial amount of C
is stored in different forms and the storage process may be the controlling step
for C imports (Ho, 1988). Sinks change their competitive ability with growth,
leading to the diversion of CHzO towards stronger sinks (Ho, 1988; Flore and
Layne, 1999). The ’sink strength’ of a sink organ has been defined as the ability to
import assimilates and it often is measured as the product of sink size and sink
activity (Ho, 1988; Zamski, 1996; Hansen, 1989). Some authors propose that the
sink strength is the driving force for C transport and dry matter partitioning
among sinks is regulated by the sinks themselves (Gifford and Evans, 1981 ;

Hansen, 1989; Marcelis, 1996). However, others suggest that the term sink

26

strength is misleading since the distribution of assimilates is organized and
coordinated at different levels by the entire source-pathway-sink plant system
and is not a property of sinks alone (Minchin and Thorpe, 1993; Thornley, 1993;
Stitt, 1993; Farrar, 1993; Farrar, 1996; Minchin et al., 1997). Some considerations to
study sink strength in fruit trees include: (1) the distance between source leaves
and active sinks since certain leaves supply CHzO for particular sinks; (2) the
sink strengths for reproductive and vegetative parts of the plant differ spatially
and temporally throughout the season; (3) the direction of CH20 translocation is
dependent on phyllotaxy; and (4) the priority of fruit over vegetative growth
during CH20 distribution (Kappes, 1985; Flore and Layne, 1999). A hierarchy of
sink strength in trees has been proposed by Kramer and Kowslozki (1979):
fruits>young leaves and stem tips>mature leaves>cambia>roots>storage tissue.
Recently, Whiting and Lang (2004) proposed a hierarchy of developmental
sensitivity to low LA / F ratio for aerial organs of dwarfing sweet cherry trees
(’Bing’/ G15): trunk>fruit soluble solids (stage III)>fruit growth (stage

III)>LA/ spur>shoot elongation>fruit growth (stages I and II)>LA/ shoot.

Sink and source limitation

The C available to support maintenance and growth of Sink organs
depends on photoassimilates supplied by different leaf populations and storage
reserves (Farrar and Williams, 1991 ; Grossman and DeJong, 1995; Flore and

Layne, 1999; Basile et al., 2002). However, the allocation of assimilates is different

27

from one sink organ to another and the priority of C partitioning changes with
the developmental stage (Ho, 1988). As indicated above, an order of priority
exists, with developing fruits and seeds being the strongest sinks (Wright, 1989).
DeJong (1999) indicates that organ growth is a consequence of the genetic
potential for growth (which interacts with environmental conditions), the CHzO
availability and the inter-organ competition for resources. The growth of
reproductive and vegetative sinks may be restricted by C availability, which is
considered a ’source limitation’, or by the inherent ability of the organ to utilize
assimilates, which is a ’sink limitation’ (Patrick, 1988; Basile et al., 2002). Growth
and yield will be optimized when both the C source and sink activities increase
Simultaneously (Gifford et al., 1984). DeJong and Grossman (1995) suggest that
source limitation results from insufficient C availability and / or the inability of
the translocation system (’ transport limitation’) to deliver C to sinks. The last
situation may be the result of long distance transport, high translocation
resistance or competition from other sinks (’competition limitation’).

Source limitations during early fruit growth may decrease cell division,
while limitations during late fruit development may reduce cell enlargement.
Partitioning studies in peach, plum (Prunus salicina L.) and blueberry indicate
that stages I (mainly fruit cell division) and HI (mainly fruit cell elongation) of
fruit development are periods of source limitation, while stage 11 (during
endocarp lignification) is considered as a period of Sink limitation (Pavel and

DeJong, 1993; DeJong and Grossman, 1995; Basile et al., 2002; Swain and Darnell,

28

2002; Berman and DeJong, 2003). In sweet cherry, reproductive and vegetative
growth occurs simultaneously during fruit development (Roper et al., 1987). This
situation might generate competition between actively growing aerial sinks, i.e.,
fruits and extension shoots, for the available C provided by different leaf
populations and storage reserves. Little information about periods of sink or
source limitation during fruit development is available for sweet cherry,
particularly in scion/ rootstock combinations using dwarfing GI rootstocks.
Source limitation affecting fruit size and vegetative growth may occur in
dwarfing and semi dwarfing trees due to their lower LA / F ratios and higher
harvest index. Too much fruit depresses the productivity of the whole tree since
as crop load increases the fraction of dry matter partitioned to other organs

decreases (Lakso et al., 1999).

Sink and source manipulation

Interactions between sink organs have several effects on trees: (1)
reduction of vegetative growth by developing fruit, (2) reduction of fruit growth
by developing vegetative sinks, and (3) competition between individual fruit
(Wright, 1989). In several species, sink-source ratios have been manipulated
experimentally by increasing or decreasing sink strength (i.e., the demand for C)
or source strength (i.e., the availability of C). Reductions in sink strength by
reducing crop loads (i.e., increasing LA/ F ratios) have been shown to increase

the C supply to other fruit and / or vegetative growth due to a reduction in sink

29

competition (Gucci and Flore, 1989; Grossman and DeJong, 1995; Maage, 1994).
In peach and plum, fruit removal changed C distribution, which in turn
increased fruit Size due to a reduction in source limitation. Trees with higher
fruit number had a stronger sink demand and showed limitations in C supply
(Pavel and DeJong, 1993; DeJong and Grossman, 1995; Basile et al., 2002; Marsal
et al., 2003). On the other hand, fruit removal in apple, peach, and blueberry
increased vegetative growth indicating a source limitation to vegetative
development of leaves, wood and roots (Maggs, 1963; Swain and Darnell, 2002;
Grossman and DeJong, 1995; Berman and DeJong, 2003; Forshey and Elfving,
1989)

Reductions of source strength, to reduce C availability for fruit and
vegetative growth, have been studied by using girdling (i.e., interruption of
phloem translocation), partial defoliation and shading of vegetative and
reproductive sections. Results varied depending on the timing at which source
manipulation was carried out. In peach and nectarine, trunk and branch girdling
induced CH20 accumulation above the girdling (Jordan and Habib, 1996) and
increased fruit size and sugar content (Allan et al., 1993), although shoot growth
was decreased (Di Vaio et al., 2001). In the same species, shading reduced C
export from lateral shoots to fruit (Corelli-Grappadelli et al., 1996). In apple,
experiments using shading demonstrated that shoot growth was a priority over
fruit growth for C partitioning since export to fruit from shoots was reduced

(Corelli-Grappadelli et al., 1994; Bepete and Lakso, 1998). In raspberry (Rubus

30

idaeus L.), girdling and leaf removal resulted in lower dry weights of
reproductive components (Privé et al., 1994). Similarly, in cranberry and
kiwifruit, partial defoliation (i.e., removal of the new and older leaves) and
girdling reduced fruit weight and number (Roper and Klueh, 1994; Buwalda and
Smith, 1990; Piller et a1., 1998). In japanese pear, girdling and defoliation of
different age spurs indicated that fruit on young spurs import CHzO from older
spurs, while fruit on older spurs depend on their own leaves (Teng et al., 1998).
Girdling of grape canes at veraison stimulated shoot growth and increased leaf
area at the expense of fruit production (Novello et al., 1999). Finally, girdling of
sweet cherry spurs to isolate fruit from the major sources of photoassimilates
showed the deleterious effects on fruit quality; fruiting spur leaves were not the
only C source to support fruit growth, and import of assimilates synthesized by
leaves distal to the bearing spurs was required for optimal fruit development
(Roper et al., 1987).

Clearly, manipulation of sink and source relationships constitutes a
practical approach to obtain more information about the contribution of various
leaf populations and storage reserves in fruit and vegetative growth during the
growing season. An optimal LA / F ratio is a key factor to assure an adequate
balance between fruit quality and vegetative growth, as indicated for sour cherry
(> 21eaves/fruit)( Iayne and Flore, 1993), plum (6 to 10 leaves / fruit) (Maage,
1994), and peach (120-220 cmP- LA / F) (M. Génard, personal communication)

(Famiani et al., 2000), and sweet cherry (200 to 300 cm2 LA / fruit) (Whiting, 2001).

31

In dwarfing sweet cherry trees, low LA/ F ratios had a negative effect on fruit
quality (Roper et al., 1987; Whiting, 2001) and vegetative growth; however crop

load reductions improved fruit characteristics (Whiting, 2001).

Fruit as sink and shoot as sink and source
a. Fruit as a sink

In Prunus sp., fruit development follows a double sigmoidal pattern,
which has been divided into three stages (Tukey and Young, 1939; Labreque et
al., 1985; Flore, 1994; Costes et al., 1995; Berman and DeJong, 1996). Following
pollination and fruit set, stage I is characterized by active cell division and rapid
initial growth. Stage II or ’pit hardening’ is associated with endocarp
lignification, slower growth of the pericarp and rapid embryo development.
Stage III or ’final swell’ is a period of rapid fruit growth characterized by
mesocarp cell enlargement and dry matter accumulation. Although a major
period of cell division occurs early during fruit development, and cell
enlargement is important during ’final swell’, cell division and cell expansion are
not exclusive during these stages (Tukey and Young, 1939; Scorza et a1., 1991).
Final fruit size depends on cell number and size. Although there is not detailed
histological information for sweet cherry fruit, in sour cherry fruit, cells of the
mesocarp increase in number during the pre-bloom stage and stage I, which is
the period of maximum division (Tukey and Young, 1939). In addition, 50 to

80% of cherry fruit growth occurs during this stage and at maturity the largest

32

cells increase 25 times in diameter compared to their size in stage I (Tukey and
Young 1939; Flore, 1994). In peach, differences between small and large- fruited
cultivars are apparent in the ovary as early as 175 days pre-bloom (Scorza et al.,
1991). The competitive ability of stone fruit and their CHzO demand change
through these three phases of sink activity (Basile et al., 2002; DeJong, 1999). In
plum, stage I often is source-limited, while in peach stages I and III of fruit
growth are source-limited as a result of competition from other fruit and
vegetative sinks (Grossman and DeJong, 1995; Basile et a1., 2002). On the other
hand, stage II is usually sink-limited due to genetic factors (Berman and DeJong,

2003).

b. Extension shoots as sink and source

In several fruit tree species, vegetative development of extension shoots
competes with, and seems to have a priority for CHzO over, reproductive
development early in the season during fruit cell division (Corelli-Grappadelli et
al., 1994; Bepete and Lakso, 1998). However, later in the season, shoots develop
enough leaf area and have the potential to support not only their own growth
but also other sinks such as fruit (Johnson and Lakso, 1986a,b; Roper et al., 1987;
Corelli-Grappadelli et al., 1994). Most of the information about the role of
extension shoots in the C balance of whole trees or limbs has been reported in
apple. Several studies indicate that an apple shoot becomes self-supporting after

5 or 6 leaves develop, with ~20% of the CHzO used in shoot growth coming from

33

storage reserves (Hansen, 1967a,b; 1971). Export of current photosynthates from
extension shoots began ~21 days after full bloom (DAFB) with ~10 to 16
unfolded leaves, and it increased considerably ~35 DAF B, when shoots had ~17
unfolded leaves (Johnson and Lakso, 1986a,b; Lakso and Corelli-Grappadelli,
1992; Corelli-Grappadelli et al., 1994). The upper 8 and 9 leaves exported C to the
shoot tip, while mid leaves exported bidirectionally and basal leaves exported
basipetally (Quinlan, 1965). In this Species, short shoots, with more mature leaf
area, exported more total CHzO than long shoots during the early period of
growth (Johnson and Lakso, 1986a,b). Short shoots contribute CHzO during early
fruit growth because of the reduced C investment in the supporting axis (Lauri
and Kelner, 2001). It has been hypothesized that in apricot shoots, cambial
growth occurring after leaf expansion is probably responsible for early shoot
growth cessation leading to spur formation; crop loads seem not to affect this
process (Costes et al., 2000). In sour cherry, extension shoots became net CHzO
exporters at 27% expansion, which was ~17 days after leaf emergence (Kappes
and Flore, 1989; Flore and Layne, 1999). In peach, extension shoots were stronger
sinks ~15 DAFB, but began exporting C to fruit ~28 DAF B (Corelli-Grappadelli et
al., 1996). These authors suggested that over time, a shift in priority occurs
between vegetative sinks that is related to shoot maturation. In the same species,
initial shoot size has been suggested as an important determinant of final shoot
growth, since as for fruit, a larger initial shoot contains more dividing cells.

Longer shoots with higher leaf areas (i.e., bigger source size) have a higher

34

potential as source of C for fruit growth (Génard et al., 1998). Currently, there is
not enough information about the impact of extension shoots on fruit and
vegetative growth in less vigorous sweet cherry trees. It might be interesting to
determine the timing for the shifting of extension shoots from sink status to

source status for other sinks such as fruit and secondary growth.

Carbon translocation patterns: orthostichy and distance

Assimilate translocation to sink organs can be acropetal or basipetal from
the source. Unidirectional and bidirectional transport from different leaf
populations to different sinks have been reported for apple, sour cherry, peach,
pecan, grape, cranberry and red raspberry (Rubus idaeus L.) among others
(Quinlan, 1965; Hansen, 1969; Corelli Grapadelli et al., 1994; Kappes and Flore,
1989; Toldam-Andersen, 1998; Corelli-Grappadelli et al., 1996; Davis and Sparks,
1974; Hale and Weaver, 1962; Roper and Klueh, 1994; Privé et al., 1994). The
transport of assimilates is suggested to follow a rule similar to the Miinch
hypothesis (Daudet et al., 2002). This mechanism assumes a viscous flow of
phloem sap in response to the hydraulic pressure (turgor) gradient which is due
to both the concentration in the source and the concentration gradient between
regions of phloem loading (sources) and regions of phloem unloading (sinks)
across transport-resistance pathways (Thornley and Johnson, 1990; Daudet et al.,

2002)

35

Several studies using radioactive C indicate that the leaf orthostichy (i.e.,
’phyllotaxy’) and vascular connections between source leaves and sink organs
are two factors responsible for the patterns of assimilate distribution (Ho, 1988).
That is, certain leaves feed particular sinks (Flore and Lakso, 1989). In sour
cherry, a 2 / 5 phyllotaxy influenced the direction and the onset of CHzO export
from shoot leaves. Leaves with angular distances of 144° had separate
translocation paths, while leaves with distances < 72° shared some of their
translocation paths (Kappes and Flore, 1986; 1989). In peach indicate that in
peach, a 2 / 5 phyllotaxy might influence C translocation from different leaf
sources (Corelli-Grappadelli et al., 1996). In apple, clear effects of phyllotaxy on
C distribution and partitioning to fruit have been documented (Corelli-
Grappadelli et al., 1994; Hansen, 1969). In grape, translocation patterns between
leaves and fruit clusters also have been attributed to vascular connections (Hale
and Weaver, 1962). Similarly in raspberry, C translocation was related to leaf
phyllotaxy 75% of the time (Privé et al., 1994).

Location of sink and sources and temporal separation of growth activities
seem to influence transport patterns and assimilate partitioning (Bruchou and
Génard, 1999; DeJong, 1999). In several species, assimilate partitioning to fruit
depends on their position relative to the leaves rather than their distance from
the source (Bruchou and Génard, 1999).The importance of sink proximity to
source leaves has been demonstrated in peach and kiwifruit by using girdling,

partial defoliation and LA / F adjustment (Ben Mimoun et al., 1995; Buwalda and

36

Smith, 1990; Bruchou and Génard, 1999). In addition to positional effects on
translocation, temporal separation of sink activities has been proposed. As
example, in peach rapid leaf and shoot expansion occurs early in the growing

season and rapid fruit enlargement during stage III occurs later in the season

(DeJong, 1999).

Photosynthesis and Sink Activity

Photosynthesis is a fundamental process for plant productivity. During
photosynthesis, C02 is converted to CHzOs, which are transported within the
tree for fruit and vegetative growth. Flore (1994) indicated that the
photosynthetic potential of a fruit crop is controlled by the environment and by
the sink demand of various organs. The environment influences: (a) physical and
biochemical reactions, (b) leaf morphology, and (c) manufacture of the
photosynthetic machinery. 0n the other hand, sink demand might control
photosynthetic rate through a feedback signal from the sink itself.

The presence of fruit and / or increased vegetative sink strength has been
associated with an increase in photosynthetic rate (A) in several fruit crops (Flore
and Lakso, 1989). Reekie and Bazzaz (1987a,b) refer to this increase as
’reproductive photosynthesis’. Plants with low source : sink ratios (i.e., limiting
leaf number) increase A more than plants with high sourcezsink ratio (i.e.,
limiting fruit number) (Farrar and Williams, 1991). Traditionally, the effect of

crop load on A has been studied by comparing fruiting and non-fruiting plants.

37

In several species, the presence of fruit has been shown to have a positive effect
on A. However, there are cases in which fruit had little or no effect on A.
Increases in A during the period of fruit development have been reported for
peach, plum, apple and sweet cherry (Fujii and Kennedy, 1985; DeJong, 1986;
Gucci et al., 1991a,b; Ben Mimoun et al., 1996; Gucci et al., 1994; Wiinsche and
Palmer, 1997 ; Palmer et al., 1997). Partial defoliation has been shown to affect A
similarly. In sour cherry, leaf removal resulted in A enhancement due to
photosynthetic compensation (Layne, 1992). In general, the detection of a fruit
sink effect on A requires a source-limiting condition, such as low LA/ F ratios or
severe defoliation. Source limitation to A occurs when the capacity of the
reaction involved in photosynthate supply is not optimal for sink demands,
while a sink limitation occurs when the rate of use of photosynthates is less than
the rate of photosynthesis (Layne and Flore, 1993). Studies that have not found
the fruit Sink effect on A include reports on sweet cherry, sour cherry and apple
(Sams and Flore, 1983; Roper et al., 1988; Giuliani et al., 1997; Flore and Layne,
1999; Whiting, 2001).

The mechanism by which fruits regulate A are unclear. High crop loads
might affect A due to an increase in sink strength (Giuliani et al., 1997). On the
other hand, the lack of a relationship between crop load and A , in some cases,
has been associated with the presence of alternative sinks such as strong shoot
growth (Gucci et al., 1991b; Giuliani et al., 1997; Palmer et al., 1997). The decline

in A following fruit removal has been attributed to stomata] and non-stomatal

38

limitations (DeJong, 1986; Gucci et al., 1991a,b) and / or end-product inhibition.
The presence of fruit increases stomatal conductance and accelerates physical
and biochemical processes in leaves (Forshey and Elfving, 1989). In addition, the
excessive accumulation of TNC, particularly starch, due to lack of a sink strength
or excessive CH20 supply might regulate A via end-product inhibition in leaves
(Herold, 1980; Flore and Lakso, 1989; Gucci et al., 1991a; Wiinsche and Palmer,
1997). Plant hormones and low orthophosphate (Pi) concentrations in the cytosol
and stroma of the chloroplast also have been proposed to influence A in source
leaves (Herold, 1980).

Fruits are able to photosynthesize during early stages of development and
it has been suggested that the C fixed directly by fruit can impact in the C budget
of individual fruits (Hansen 1970; DeJong and Walton, 1989; Kappes, 1985). In
sour cherry, fruit gross photosynthesis contributed ~19%, 30% and 1.5% of the
CH20 used during stages I, II and III of fruit development, respectively; ~70% of
the fixed C was incorporated into fruit dry matter, while the rest was used in
dark respiration (Flore and Layne, 1999). In apple, fruit photosynthesis was <
15% of the total C supply during the season (Jones, 1981), although it may

contribute to fruit growth early in the season (Lakso et al., 1999).

Respiration and Sink Activity
DeJong and Grossman (1994) indicate that the two major components of

CH20 demand in trees are growth and respiration. Two major components of

39

plant respiration have been described: (1) growth ’construction’ respiration,
which is defined as the C02 evolution directly related to the production of new
cellular materials and (2) maintenance respiration, which supplies energy for
subsistence of existing tissue (Amthor, 1984; Amthor, 1989). Maintenance
respiration is assumed to have priority over vegetative and reproductive growth
(Marcelis et al., 1998; Lescourret et al., 1998). C is partitioned first for
maintenance of existing biomass, and the remaining C is partitioned for growth
of various organs according to their respective sink strength, which depend on
their relative growth rates (Buwalda, 1991). Respiration costs vary with growth
rate, temperature (Q10 of ~15 to 2) and plant size or biomass (Ho, 1988; Amthor,
1984; DeJong and Grossman, 1994; Flore and Layne, 1999; DeJong, 1999). There is
no information regarding respiratory costs in sweet cherry; however, respiratory
demands of peach and apple trees have been documented. In peach, growth
simulations indicated that daily maintenance respiration increased during the
season due to increases in biomass and temperature. 0f the total fixed CO2, ~33%
was utilized in maintenance respiration, while ~66% was used for growth and
growth respiration (Grossman and DeJong, 1994). Fruit respiration accounted for
~16 to 20% of the total fruit CH20 requirements, while the rest was fixed as
biomass. The highest specific respiration rates in fruit were detected during early
development (DeJong and Walton, 1989). In general, total CH20 cost (dry matter
plus growth respiration) of fruit growth was ~35% greater than total respiratory

costs of leaf, stem and trunk growth (Grossman and DeJong, 1994). In apple,

40

dark respiration costs ranged between 27 to 30% of the fixed C02 for a full year
(Lakso et al., 1999). The highest specific respiration rates occurred during spring
when new leaves, shoots, fruit, stems and roots are growing most actively. Later
in the season, maintenance respiration of leaves and the main perennial
structures of the tree are low. In the case of fruit, respiration-rates were high
during cell division (~1 month) but declined during cell expansion (Lakso et al.,
1999). Jones (1981) estimated that ~15% of the C imported by fruit was used in
respiration, while the other 85% was accumulated as dry matter in the fruit.
Clearly, the respiratory activity of fruit trees is both qualitatively and
quantitatively important in the C balance equation (Lakso, 1994). However, more
information about whole-plant respiration is required for many woody species,

including sweet cherry.

Rationale and objectives

Partitioning studies in sweet cherry trees on traditional vigorous
rootstocks have provided insight for orchard management decisiOns regarding
appropriate pruning, crop load regulation and other practices. However, with
the move toward high-density orchards by US sweet cherry growers, additional
research is required to understand the role of fruit sink strength and CH20
partitioning when trees are grown on dwarfing and semi-dwarfing rootstocks
such as the GI series. GI rootstocks are interspecific hybrids that have the

potential to promote precocious reproductive bud formation, high yield

41

efficiency and reduced vegetative vigor. So far, the implementation of standard
sweet cherry management practices for trees on G1 rootstocks has resulted in
high yields but small fruit, which is a critical problem since top quality fruit
provides the best returns to growers.

Little is known about the relative importance or temporal relationships of
different sweet cherry leaf populations within the canopy as sources of C for fruit
and Shoot development in dwarfing trees. Moreover, the partitioning of C and
the effect on sink strength of fruit and shoots during fruit development has not
been characterized in detail. Previous data and increasing grower experience
indicates that reproductive and vegetative growth often become unbalanced after
the 4th year of production on dwarfing rootstocks if the natural canopy leaf-area-
to-fruit (LAzF) ratios are not altered in some way. Thus, manipulation of the
reproductive and vegetative sinks may be a tool to regulate sink strength and
competition among sinks during periods of resource limitation, particularly
during fruit development Adjustments in LA/ F ratios through practices such as
pruning to remove or stimulate leaf area or fruit and flower thinning and/ or
spur extinction might help to overcome the problem of overcropping and small
fruit size. In this study, dwarfing and semi—dwarfing trees on G1 rootstocks were
used to investigate partitioning during fruit development Results of this
research provide a physiological foundation for canopy relationships that may

help to develop specific orchard management strategies to promote a more

42

sustainable balance between vegetative and reproductive growth in high density

sweet cherry orchards.

The main objectives of this study were to:

1. define the temporal importance of various leaf populations as sources of C
for fruit and shoot growth during the whole period of fruit development.

2. determine the effect of reproductive and vegetative sink strengths on C
partitioning during fruit development.

3. determine the importance of storage reserves as a source of C for initial
fruit growth.

4. define the transition phase during which the dependence of new growth
on storage reserves shifts to current photosynthate assimilation as the

primary source for subsequent vegetative and reproductive development.

43

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60

 

Year: 2002 2003 2004

Leaf Fruiting spurs Non-fruiting Current season
Population: spurs shoot

 

 

Figure 1. Leaf populations on a typical 2-year-old sweet cherry branch.

Images in this dissertation are presented in color.

61

CHAPTER II

EXAMINING THE INFLUENCE OF DIFFERENT LEAF POPULATIONS ON

SWEET CHERRY FRUIT QUALITY

62

Examining the Influence of Different Leaf Populations on

Sweet Cherry Fruit Quality

M. Ayala and G. Lang. 2004. Acta Horticulturae 636: 481-488.

Keywords: Prunus avium L., fruit development, shoot, carbohydrates, defoliation,

girdling, source, sink, Gisela rootstock.

Abstract

Understanding sweet cherry (Prunus avium L.) carbohydrate (CH20)
partitioning and source-sink relationships might lead to better management
strategies for sweet cherry fruit‘quality on high-yielding, vigor-reducing
rootstocks. Six limb treatments were established on fruiting branches of
‘Hedelfinger’/ Gisela 5 and ‘Ulster’ / Gisela 6 to isolate or combine two of the
main leaf populations that serve as CH20 sources for developing fruit: the leaves
on the branch segment of newly-formed spurs (previous season growth having
non-fruiting spurs and some new lateral shoots) and the leaves on the branch
segment of previously-formed spurs (two-year-old growth having fruiting spurs,
plus some previous season and new lateral shoots). At harvest, fruit weight,
diameter, and soluble solids (SS) were evaluated for each treatment. For both
scion/ rootstock combinations, fruits from the branch treatment isolated from the

rest of the tree by girdling and having a full complement of leaf populations

63

were larger and had higher SS than the partial leaf population treatments or the
branch having a full set of leaf populations but not isolated by girdling. Fruits
supplied exclusively by the leaf populations on either the fruiting spur branch
segment or the non-fruiting spur branch segment were significantly smaller and
had decreased SS levels. It was evident that the leaf populations most closely
associated with the fruiting spur branch segment were insufficient sources of
CH20 for optimal fruit development However, these populations were clearly
important, as Similar sub-optimal results were also found when only the leaf
populations on the non-fruiting spur branch segment were present Leaf
populations on both fruiting and non-fruiting branch segments were required for
full fruit development and there was not a sufficient compensatory effect when

one of the main leaf populations was eliminated.

Introduction

Sweet cherry (Prunus avium L.) fruit quality, primarily size and sweetness,
is highly dependent on CH20 availability and partitioning, which in turn are
dependent on the number and strength of competing sinks. Within a sweet
cherry branch, the major sinks that might be considered to be competitors of
developing fruit include vegetative growth (current season growth of shoots),
other fruits and developing spur leaves.

Sweet cherry fruit size is dependent on cell division and enlargement.
Fruit cell division occurs before anthesis and continues through the initial phase
of stage I (Flore, 1994; Flore and Layne, 1999). During this period fruit constitute
important sinks attracting assimilates (Ho, 1988). Later in fruit development,
fruit sink strength changes to reach a maximum during stage III (or ’final swell’),
when cells are actively elongating (Flore, 1994; Flore and Layne, 1999). In sour
cherry (Prunus cerasus L.), early stage fruits act as sinks by removing
photoassimilates from the translocation system. In this species, dry matter
partitioning is dependent on the fruit growth stage, with the higher
accumulation of carbon (C) in the fruits during final swell (Toldam-Andersen,
1998)

Loescher et a1. (1986) suggested that in sweet cherry, spur (fully expanded
3 weeks after bloom) and current season shoot leaves were the primary source of
CH20 for fruit growth. Similarly, Roper and Loescher (1987) found a positive

correlation between fruit quality and leaf area per fruit These authors reported

65

that spur leaves alone were not able to support fruit growth during stage HI and
CH20 import from other sources (i.e. non-fruiting spurs and current season
growth leaves) was required. In sour cherry, shoots became net exporters of
photosynthates 15 days after budbreak (DABB), while apple shoots began net
export 20 to 25 DABB (Kappes and Flore, 1986; Johnson and Lakso, 1986). Thus,
current season growth has the potential to provide at least some photoassimilates

for fruit growth in these two species.

In sweet cherry, vegetative and reproductive growth occur
simultaneously, and this can result in a strong intra-plant competition for
available assimilates (Roper et al., 1987). After the previous season CH20
reserves have been depleted in early spring, thus the sweet cherry canopy must
produce current photoassimilates for the rest of the seasonal growth and a new
pool of storage reserves for initial growth the next spring. Healthy and abundant
leaf populations, producing a continuous supply of photosynthates throughout
the season, are essential for growth and storage. Both girdling (eliminating
transport via phloem tissue and importation of stored CH20), or defoliation
(eliminating photosynthetic tissue and the availability of current
photoassimilates), change source-sink relationships and the utilization of CH20
for growth. Girdling effects have been reported for several stone fruits including
sweet cherry, peach (Prunus persica L.) and nectarine (Roper et al., 1987; Allan et
al., 1993; Jordan and Habib, 1996). In addition, the physiological responses to

selective or partial defoliation have also been studied in species such as sour

66

cherry, cranberry (Vaccinium macrocarpon Ait) and pecan (Carya illinoinensis
(W angenh.) K. Koch.) (Layne and Flore, 1992; Layne and Flore, 1993; Roper and

Klueh, 1994; Worley, 1979).

The understanding of CH20 partitioning and sink-source relationships
during fruit growth might lead to better management strategies to improve
sweet cherry fruit quality on high-yielding, vigor-reducing Gisela rootstocks.
Scion/ rootstock combinations using Gisela (GI) stocks tend to overcrop early in
the orchard life and, consequently, leaf area to fruit (LA / F) ratios become
unbalanced, resulting in smaller fruits (Andersen et al., 1999). The objective of
this experiment was to study the role of different leaf populations on fruit
growth and development in combinations using GI stocks. We hypothesized that
leaf populations on current season growth, previous season growth, and 2-year-
old wood are all important to support optimal fruit growth and development. To
test this hypothesis, we manipulated CH20 availability by girdling and
defoliating a 2-year-old sweet cherry branch during Stage I of fruit development.
In this way, we created an artificial redistIibution of non-structural CH20 to sink
organs (fruit and shoots). This was an initial approach to study the dynamics of
partitioning in a sweet cherry fruiting branch on a dwarfing scion / rootstock

combination.

67

Materials and Methods

In May 2001, at the Clarksville Horticultural Experiment Station (CHES)
of Michigan State University, an experiment on 2-year-old (first shoot growth
occurred in 1999) fruiting branches of ’Hedelfinger’/ Gisela 5 (G15) and
’Ulster’ / Gisela 6 (G16) sweet cherry trees was established. Six limb treatments
(T) were created by using girdling and defoliation to isolate the various leaf
populations that serve as CH20 sources for developing fruits. In this way, CH20
that should have been translocated to sink organs, such as fruits and current
season growth, were restricted artificially. Limbs were either girdled and / or
defoliated at different sections of the branch, depending on the treatment (See
Figure 1). The treatments included:

0 C - Untreated limb (control).

0 T1 - Branch girdled at its base, i.e., isolation from the rest of the tree.

0 T2 - Branch girdled at both sides of the wood bearing newly fruiting Spurs
(growth that occurred in 1999), i.e., the CH20 source for developing fruit
was limited to only the leaves of fruiting spurs and any nearby lateral
shoots.

0 T3 - Branch girdled at its base and at the junction of the previous season
(2000) growth and the current season (2001) growth, i.e., CH20 source for
developing fruit included both the fruiting and non-fruiting spur leaves,

plus any lateral shoot leaves on these two segments of growth.

68

0 T4 - Branch girdled as in T3 plus removal of all leaves associated with the
branch segment containing fruiting spurs, i.e., CH20 source was limited to
leaves of non-fruiting spurs (growth that occurred in 2000) and any
nearby lateral shoots.

- T5 - Branch girdled as in T3 plus removal of all leaves assOciated with the
branch segment containing the non-fruiting spurs, i.e., CH20 source was
limited to leaves of fruiting spurs (growth that occurred in 1999) and any

nearby lateral shoots.

Before imposing the treatments, we identified a population of 200
branches having similar vigor, crop load, length and diameter, and from these,
10 branches per treatment were selected randomly. A total of 60 branches per
scion/ rootstock combination were used. Initial morphological measurements for
each combination included: diameter / branch, length / branch, initial fruit
number / branch, fruiting and non-fruiting spur number / branch, lateral
Shoots/ branch. Average leaf area (LA) cmz/ leaf population and average LA
cmz/ branch were measured late in fruit development (Table 1). Girdling (1 cm in
width at the base of the limb) and defoliation were carried out 12 days after full
bloom (DAF B) early in Stage I of fruit development. Tissue removed at the
girdled section included periderm, phloem and cambium. Later in fruit
development (stage III), the re-growth on the girdled area was further removed

to avoid phloem translocation. In the case of T4 and T5, we eliminated ~35% of

69

the total leaf area / branch by removing all leaves (spur and lateral shoot) from
the branch segment that contained either fruiting or non-fruiting spurs,
depending on the treatment.

Weight, diameter, soluble solids (SS), color and final fruit number were
recorded for each branch after commercial harvest Harvest was 65 DAFB (27

Jun) for ’Ulster’/ G16 and 67 DAF B (5 Jul) for ’Hedelfinger/ G15 .

Statistical Analysis
The data were analyzed using proc mixed SAS 8e for Windows (SAS Institute,

Cary, NC.)

Results

There were significant morphological differences between ’Ulster’/ G16
and ’Hedelfinger’/ G15. ’Ulster' / G16 had more spurs per branch and higher total
LA/ branch than ’Hedelfinger’/ G15 (Table 1). In addition, initial LA / F ratios of
’Ulster’ / G16 were higher than those of ’Hedelfinger’/ G15. Although the length
of branches was similar, ’Ulster’ / G16 had more fruiting and non-fruiting fruit
spurs compared with those in ’Hedelfinger’/ G15. The number of lateral shoots
per branch also differed significantly between combinations, being greater in
’Hedelfinger’/ G15. The fruiting and non-fruiting spur leaf populations
contributed the greatest component LA to overall branch LA, with 72 and 63%,

for Ulster’ / G16 and ’Hedelfinger’ / G15, respectively.

70

There were significant differences among treatments in both ’Ulster’ / G16
and ’Hedelfinger’ / G15 for fruit diameter, weight and SS of fruits at harvest
Differences in final fruit number occurred only in ’Hedelfinger’ / G15; however,
there was high variability within treatments for this parameter. For both
scion/ rootstock combinations, fruits from branches that were isolated from the
rest of the tree (T1) were larger and had higher SS than the other treatments,
including the controls (C). Conversely, fruits for which the CH20 source was
limited to only those leaves on the branch segment having fruiting Spurs (T2) or
non-fruiting spurs (T4), were significantly smaller and had decreased SS levels
compared to C and T1 (Table 2). ’Ulster’ / G16 fruits for which the CH20 source
was limited to only the leaves on the fruiting spur branch segment (T5) or to both
the fruiting and non-fruiting spur branch segment (T3) had a reduced diameter,
weight and SS, as compared to the intact control branch (C) or the otherwise
intact branch that was isolated from the rest of the tree (T1). However, the same
trend was not observed in ’Hedelfinger’ / G15, for which T3 (fruiting and non-
fruiting spur and lateral shoot leaves) and T5 (only leaves on the fruiting spur
branch segment) did not affect fruit quality as much as T2 and T4 did. Fruit color
was affected positively in both rootstock / scion combinations when the entire
branch was isolated by girdling (T1).

There were no Significant differences in relative current season growth for
either scion / rootstock combination (See Figures 2 and 3). However, current

season growth on ’Ulster’/ G16 increased when girdled (T 3-T5; see Figure 2),

71

4‘

while current season growth on ’Hedelfinger’ / G15 decreased when girdled (C

and T1-T2; see Figure 3).

Discussion

Branches girdled only at their base, i.e., isolated from the rest of the tree,
had fruit with greater diameter, weight, SS content and color compared to the
rest of the treatments, including the untreated branches for which potential
CH20 to support developing fruit included multiple tissues throughout the tree.
This suggests that the isolation of the branch from the rest of the tree prevented
CH20 export to other sink organs of the tree, thereby benefiting local fruit
growth. These agree with the positive effects of girdling on peach fruit quality
have been reported (De Villiers et al., 1990; Allan et al., 1993).

Fruit quality of ’Ulster’/ G16 and ’Hedelfinger’/ G15 was affected
negatively as various restrictions of CH20 sources were imposed. Thus, when
branches were either girdled at both ends of the wood bearing fruiting spurs (i.e.,
CH20 sources for fruit were those leaves associated with Spurs and laterals on
the fruiting segment of the branch), or at the base and at the junction between the
previous and current season growth, with the fruiting spur (and associated
lateral) leaves then removed (i.e., CH20 sources were those leaves on non-
fruiting spurs and lateral shoots), there was a detrimental effect on the fruit size
and SS levels. Several previous reports illustrate the deleterious effects of

girdling and defoliation on assimilate supply to sink organs, which in turn

72

impact final fruit growth and quality in different species including sweet cherry,
apple (Malus x domestica Borkh.), japanese pear (Pyms pyrifolia Nakai), kiwifruit
(Actinidia deliciosa var.deliciosa cv. ’Hayward’) and grape (Vitis vinifera L.)
(Roper et al., 1987; Atkinson et al., 2001; Ferree and Palmer, 1982; Teng et al.,
1998; Buwalda and Smith, 1990; Harrell and Williams, 1987). Restriction in C
budget induces reductions in fruit size, number, weight, SS and color. Moreover,
a delay in fruit maturity time has been detected (Harrell and Williams, 1987).
When the leaf populations on fruiting branch segments were the sole
supply of CH20 and import from (or export to) the rest of the tree was
prevented, good fruit quality was not achieved. Thus, leaves on the 2-year-old
wood were not able to support optimal development of their own fruit. These
data support the observations of Roper et a1. (1987) who found that in sweet
cherry the isolation of fruiting spurs from other leaf populations during stage II
and 111 had a negative impact on fruit weight, SS and color. These authors
concluded that in sweet cherry, fruiting spur leaves are not the only carbon
source to support fruit growth and import of assimilates synthesized by leaves
external to the bearing spurs is required. In the same species, Atkinson et al.
(2001) demonstrated that the isolation of each fruiting spur from the rest of the
tree by using girdling reduced fruit weight These authors concluded that
individual spurs were under a source limiting situation, which in turn influenced

final fruit size but not fruit number per cluster.

73

Similarly, when leaves on the non-fruiting branch segment were the sole
CH20 supply, optimal fruit development was not achieved either. Therefore,
leaves from the non-fruiting spurs and associated laterals could not compensate
for the lack of sufficient CH20 originating from fruiting spur and lateral and
terminal LA. In apple, fruit growth is supported by CH20 produced by extension
shoots and non-fruiting and fruiting spurs. Although, the contribution of these
leaf population varied depending on position of source and sink organs within
the canopy and their developmental stage (Hansen, 1969; Tustin et al., 1992;
Corelli-Grapadelli et al., 1994).

When the branch was girdled at its base and at the junctions of the
previous and current season growth (i.e., CH20 for developing fruits was both
the fruiting and non-fruiting spur leaves, plus all laterals) in ’Ulster’ / G16, fruit
size, SS and color were affected negatively. This was the opposite for
’Hedelfinger’/ G15, which had good quality fruit compared with fruits of
’Ulster’ / G16. Two possibilities might be proposed to explain this difference
between scion/ rootstock combinations. First, Hedelfinger’/ G15 had more lateral
shoots in fruiting and non-fruiting wood (i.e. more LA to supply with CH20) in
comparison to ’Ulster’/ G16. Second, it is likely that the isolation of the terminal
current season growth from the rest of the branch had different effects on the C
partitioning between fruit and terminal shoots depending on the grafted
combination. In the case of ’Ulster’ / G16, terminal shoots seem to be an important

source of photoassimilates for fruit, while for ’Hedelfinger’ /G15 terminal shoots

74

might constitute a sink competing with developing fruit. When new shoot
growth was girdled at its base, ’Ulster’ / G16 shoot growth increased compared to
the ungirdled branches, suggesting that it was able to support its own growth
and probably export some CH20 to the rest of the branch. The opposite effect
occurred in ’Hedelfinger’ / G15, which suggests that either the lower scion vigor
of ’Hedelfinger’ (vs. ’Ulster’), or that imposed by the more dwarfing rootstock
G15 (vs. G16), established a physiological condition in which terminal current
season shoots were insufficient to support their optimal vegetative growth, much
less to contribute significantly to fruit growth. Consequently, terminal current
season growth in ’Hedelfinger’/ G15 might import current photosynthates from
other sources during elongation. In comparison, sour cherry shoots became net
exporters of photosynthates 15 days after budbreak (DABB), while in apple
shoots began net export 20 to 25 DABB (Kappes and Flore, 1986; Johnson and
Lakso, 1986). Thus, sweet cherry current season growth might have the potential
to provide at least some photoassimilates for fruit growth. The temporal role of
current season growth, as a CH20 source, for sweet cherry fruit development is
unknown. We presume that current season growth undergoes a transition from
being a competitor sink with the fruit in early stages of development to a source

of CH20 for the fruit at late stages of fruit development.

We conclude that leaf populations of either fruiting or non-fruiting branch
segments alone are insufficient sources of CH20 for optimal fruit development in

sweet cherry. Both fruiting and non-fruiting spurs and lateral LA are required to

75

maximize fruit growth. There is not a significant compensatory effect when one
of these populations is reduced or eliminated, and the contribution of current
season growth to fruit development appears to vary by rootstock vigor and / or
scion variety.

The use of girdling and defoliation is the first step to elucidate the fate of
current photosynthates produced by the three distinct leaf populations within a
2-year-old sweet cherry branch. We demonstrated that the lack of LA reduces the
availability of CH20 for fruit growth and, as a consequence, quality is affected
negatively. Questions to be answered next include the role of storage reserves in
early fruit growth, the fate of CH20 synthesized by non-fruiting sections of a 2-
year-old branch, and the importance of the current season shoot growth for fruit

growth and development.

Acknowledgements

Appreciation is expressed to the International Dwarf Fruit Tree
Association and the Pontificia Universidad CatOIica de Chile for financial
support of this project, to Professor James Flore for technical and professional
advice, and to Mauricio Canoles and Dario Stefanelli for their help in the

orchard.

76

LITERATURE CITED

Allan, P., A.P. George, R.) Nissen, T.S. Rasmussen. 1993. Effects of girdling on
growth, yield and fruit maturity of the low chill peach cultivar
Flordaprince. Austral. J. Exp. Agr. 33: 781-785.

Andersen, R.L., T. Robinson, G.A. Lang. 1999. New York fruit quarterly 7: 1-4.

Atkinson, C.J., M.A. Else, A.P. Stankiewicz, A.D. Webster. 2001. Limited
availability of assimilates: effects on the abscission of sweet cherries. Acta
Hort 557 : 457-463.

Buwalda, J.G., G.S. Smith. 1990. Effects of partial defoliation at various stages of
the growing season on fruit yields, root growth and return bloom of
kiwifruit vines. Sci. Hort. 42: 29-44.

Corelli-Grapadelli, L., A.N. Lakso, J.A. Flore. 1994. Early season pattern of
carbohydrate partitioning in exposed and shaded apple branches. J. Amer.
Soc. Hort. Sci. 119: 596-603.

De Villiers, H., J.G.M. Cutting, G. Jacobs, D.K. Strydom. 1990. The effect of
girdling on fruit growth and internal quality of ’Culemborg’ peach. J.
Hort. Sci. 65: 151-5.

Ferree, D.C., J.W. Palmer. 1982. Effect of spur defoliation and girdling during
bloom on fruiting, fruit mineral level and net photosynthesis of ’Golden
Delicious’ apple. J. Amer. Soc. Hort. Sci. 107: 1182-1186.

Flore, J. 1994. Stone fruit. pp. 233-270 In: B. Schaffer and P. Andersen (eds.),
Handbook of environmental physiology of fruit crops, Vol. I: Temperate
crops, CRC Press, Boca Raton, Fla.

Flore, J., D. Layne. 1999. Photoassimilate production and distribution in cherry.
HortScience 34: 1015-1018.

Harrell D.C., L.E. Williams. 1987. Net CO2 assimilation, water potential and non-
structural carbohydrates of Thompson Seedless. p. 142-146. In: J.M. Rantz

(Ed.): Proc. Intern. Symp. On table grape production, June 28-29,
Anaheim.

Hansen, P. 1969. IV. Photosynthate comsumption in fruits in relation to the leaf-
fruit ratio and to leaf-fruit position. PhysiolPlant 22: 186-198.

77

Ho, LC. 1988. Sink strength and sugar metabolism. Ann. Rev. Plant. Physiol.
Plant. Mol. Biol. 39: 355-378.

Johnson, R.S., A.N. Lakso. 1986. Carbon balance model of a growing apple shoot:
11. Simulated effects of light and temperature on long and short shoots. J.
Amer. Soc. Hort. Sci. 111: 164-169.

Jordan, M.0., R. Habib. 1996. Mobilizable carbon reserves in young peach trees
as evidenced by trunk girdling experiments. J. Exp. Bot. 47: 79-87.

Kappes, E.M., J.A. Flore. 1986. Carbohydrate balance models for ’Montmorency’
sour cherry leaves, shoots and fruit during development Acta Hort. 184:
123-127.

Layne, D.R., J.A. Flore. 1992. Photosynthetic compensation to partial leaf area
reduction in sour cherry. J. Amer. Soc. Hort Sci. 117: 279-286.

Layne, D.R., J.A. Flore. 1993. Physiological responses of Prunus cerasus to whole-
plant source manipulation. Leaf gas exchange, chlorophyll fluorescence,
water relations and carbohydrate concentrations. Physiol. Plant. 88: 44-51.

Loescher, W.H., T.R. Roper, J. Keller. 1986. Carbohydrate partitioning in sweet
cherry. Proc. Wash. State Hort. Assn. 81: 240-248.

Roper, T.R., J.S. Klueh. 1994. Removing new growth reduces fruiting in
cranberry. HortScience 29: 199-201.

Roper, T.R., W.H. Loescher. 1987. Relationship between leaf area per fruit and
fruit quality in ’Bing’ sweet cherry. HortScience 22: 1273-1276.

Roper, T.R., W.H. Loescher, J. Keller, C. Rom. 1987 . Sources of photosynthates
for fruit growth in ’Bing’ sweet cherry. J. Amer. Soc. Hort Sci 112: 808-
812.

Teng, Y., K. Tanabe, F. Tamura, A. Itai. 1998. Effects of spur defoliation and
girdling on fruit growth, fruit quality, and leaf and shoot carbohydrate
levels on the spurs of different ages in ’Nijisseiki’ pear. J. Japan. Soc. Hor.
Sci. 67: 643-650.

TOldam-Andersen, T.B. 1998. The seasonal distribution of 14C-labelled
photosynthates in sour cherry (Prunus cerasus). Acta Hort. 468: 531-540.

78

Tustin, S.L., L. Corelli-Grapadelli, G. Ravaglia. 1992. Effect of previous-season
and current light environments on early-season spur development and
assimilate translocation in ’Golden Delicious’ apple. J. Hort Sci. 67: 351-
360.

Worley, RE. 1979. Fall defoliation date and seasonl carbohydrate concentration
of pecan wood tissue. J.Amer. Soc. Hort. Sci. 104: 195-199.

79

Table 1. Morphological features of ’Ulster’/ G16 and ’Hedelfinger’/ G15 branches.
Means from 60 branches per combination. Measurements were recorded prior to
treatment imposition and late in fruit development (65 and 67 days after full
bloom).

 

 

Parameter ’Ulster’/GI6 ’Hedelfinger’lGIS
Branch length (cm) 99.0 d: 1.2 a2 101.0 d: 1.2 a
Branch diameter (mm) 20.0 i 0.3 a 17.0 i 0.3 b
Initial fruit number 54.8 :l: 3.0 b 77.9 i 3.9 a
Total spur number 37.5 i: 0.6 a 28.5 i 0.4 b
Fruiting spur number 15.6 i 0.4 a 13.3 i 0.4 b
Non-fruiting spur number 21.9 i 0.4 a 15.2 :l: 0.4 b
Lateral shoot number 7.8 i 0.3 b 11.5 i 0.4 a
Intemode length (cm) 2.2 :l: 0.1 b 3.2 i 0.1 a
Fruiting spur leaf area (cmZ) 4,623.5 :l: 120.9 a 3,272.9 :t 108.5 b
Non-fruiting spur leaf area (cm?) 4,657.9 :t 78.6 a 3,220.9 i 85.9 b
Lateral shoot leaf area (cmZ) 3,464.5 i 114.4 b 3,850.6 is 133.5 a
Total leaf area per branch (cm?) 12,7459 :1: 181.0 a 10,3444 1: 180.6 b
Leaf area (cm2)/ fruit ratio 202.4 1: 21.1 a 164.7 3: 33.6 b

 

2 Means within a row followed by the same small letter are not significantly different
at or = 0.05.

80

Table 2. Diameter, weight, soluble solids (SS), color and final fruit

number/ branch of ’Ulster’/ G16 and ‘Hedelfinger’/ G15 at harvest (June and July,
2001, respectively). C - Untreated limb (control); T1 - Branch girdled at its base;
T2 - Branch girdled at both sides of the wood bearing newly fruiting spurs, i.e.,
source leaves are those associated with the branch segment that grew in 1999; T3
- Branch girdled at its base and at the junction of the previous season growth and
the current season growth, i.e., source leaves are those associated with the branch
segments that grew in both 1999 and 2000; T4 - Branch girdled as in T3 plus
removal of all spur and lateral leaves on the fruiting branch segment, i.e., source
leaves are those associated with the branch segment that grew in 2000; T5 -
Branch girdled as in T3 plus removal of all spur and lateral leaves on the non-
fruiting branch segment, i.e., source leaves are those associated with the branch
segment that grew in 1999.

 

 

 

 

 

Ulster Diameter Weight SS Final Fruit Color
/ G16 (mm) (g/ fruit) (°Brix) number (H°)
C 21.1 i 0.2 b 6.1 i 0.2 c 18.4 i 0.1 bc 47.0 i 6.3 a 13.0 i 0.4 b
T1 21.9 i 0.2 a 7.1 i 0.2 a 21.8 i 0.1 a 38.0 :t 7.3 a 8.8 :l: 0.2 d
T2 19.8 i 0.2 c 5.4 :t 0.2 d 15.2 i 0.2 d 54.0 i 9.2 a 15.3 d: 0.3 a
T3 19.3 :t 0.2 c 6.6 i 0.2 b 18.6 i 0.2 b 50.0 d: 10.3 a 12.9 :l: 0.6 b
T4 19.2 :t 0.2 c 5.1 i 0.3 d 17.9 i 0.2 c 42.0 i: 4.8 a 10.8 i 0.3 c
T5 20.9 i 0.2 b 6.2 d: 0.2 c 18.4 i 0.1 bc 43.0 :l: 5.6 a 13.9 i 0.4 b
Hedelfinger/ Diameter Weight SS Final Fruit Color
G15 (mm) (g / fruit) (°Brix) number (H°)
C 21.1 i 0.2 b 7.0 :t 0.2 a 17.2 i 0.1 b 43.0 i 12.4 b 14.6 :l: 0.5 a
T1 21.9 :t: 0.1 a 6.8 i 0.2 a 18.8 i 0.1 a 46.0 i 8.6 ab 11.6 i: 0.6 b
T2 20.9 :t 0.2 b 6.2 :t 0.3 b 15.0 i: 0.2 c 74.0 i 7.9 a 15.7 :l: 0.4 a
T3 21.7 :t 0.1 a 7.0 :t 0.2 a 18.3 i 0.1 a 55.0 i 6.6 a 10.9 i 0.5 b
T4 20.3 i 0.3 c 5.9 i 0.2 b 16.7 i 0.2 b 52.0 :t 6.6 ab 11.7 i: 0.4 b
T5 21.4 i 0.2 ab 6.7 :t 0.2 a 15.4 :t 0.1 c 45.0 :t 6.8 b 14.9 d: 0.5 a

2 Means within a row followed by the same small letter are not significantly different at
Of- = 0.05.

 

81

Year 1999 2000 2001

Type Fruiting spurs Non-fruiting Current season
spurs growth

 

   

Control
T 1

 

 

T2
T3

 

 

 

T 4 defoliated

T5

 

 

 

 

1::

 

defoliated i?

Figure 1. Sites of girdling treatments T1 to T5 imposed on 2-year-old sweet
cherry branches. Black arrows indicate sites of girdling for all the treatments.
Black dots indicate defoliated sections for T4 and T5.

82

 

 

j -9-Unglrdled top shoot (C,T1,T2) I

I

j -B-Glrdled top shoot (T3. T4. T5) j

 

I“
o

 

1.0 '

Relative current season growth

 

 

’1' 'Ir ’1' “iv '9 ‘Ir '9
*9 49$ 0’99 9'9 «$89 $9 4‘39

Date
Figure 2. Relative current season growth on ’Ulster’ / G16 with (C, T1 and T2) and
without girdling (T3, T4 and T5) at the junction of the previous season (2000)
growth and the current season (2001) growth. Calculations were based on shoot
length measured weekly for each treatment.

 

 

 

 

 

 
 

 

3.0 I l
l I -9-Unglrdled top shoot (C,T1. T2) 1 I
I l.
g i . -E-Grrdled top shoot (T3, T4, T5) . I
a I n I
c 2.0 + u __ ....... , ,. - ___, a
2 . a A at
8 I "’ l
:3 . I
g I
o l
31.0 ~~v~v77 —H——i
fi .
3 l
a .
0.0 LL A . . .4
’lv ’1' '1' ’1' '1' ‘\r “to
a“? 0’99 9'99 9’9 6’9 a”? «if?

Date
Figure 3. Relative current season growth on ’Hedelfinger’ / G15 with (C, T1 and
T2) and without girdling (T3, T4 and T5) at the junction of the previous season
(2000) growth and the current season (2001) growth. Calculations were based on
Shoot length measured weekly for each treatment.

83

CHAPTER III

THE EFFECT OF CROP LOAD ON 1:‘IC-PHOTOASSIMILATE PARTITIONING

IN SWEET CHERRY DURING STAGE 111 OF FRUIT DEVELOPMENT

The Effect of Crop Load on 13C-Photoassimilate Partitioning from
Non-Fruiting Spur Leaves in Sweet Cherry

During Stage III of Fruit Development

Keywords: Prunus avium L., fruit growth, current season growth, carbohydrates,

translocation, source, sink, partitioning, carbon, Gisela rootstock.

Abstract

Fruit quality and productivity are influenced by photoassimilate
partitioning among different sink organs. In sweet cherry, 50 to 80% of fruit
growth occurs during stage III of the double sigmoidal growth curve, when new
shoots are still extending, likely in competition with developing fruits for current
photosynthates. To study the role of the non-fruiting spur leaves as a source of
assimilates for fruits and developing shoots during stage 111, an experiment using
Z-year-old fruiting branches of ’Sam’ sweet cherry on the dwarfing rootstock,

Gisela 5, was established. Three crop load treatments, based on leaf area-to-fruit
ratio (LA: F) were imposed: LA : F = 140, 75, or 40 cmz/fruit. 0f the three leaf
Populations on the fruiting branch (fruiting spur, non-fruiting spur and new
tel‘IIIinal shoot leaves), non-fruiting spur leaves were exposed to 13C02 labeling
on tI‘tree different dates during stage III (52, 59 and 63 DAF B). Fruits and leaves

f r Om the terminal shoot (both located in distal and proximal positions from the

85

labeled leaves) were sampled one and two days after labeling for analysis by gas
chromatography mass spectrometry (GC-MS). 13C fixed by non-fruiting spur
leaves was translocated both acropetally and basipetally. For all 3 pulsing dates,
fruits were more highly enriched in 13C (i.e., had higher atom %) than were
young leaves, and proportional enrichment ranged between ~87% to 96% of
recovered 13C, indicative of the stronger sink activity of fruit compared to that of
shoots. There was not a consistent or significant crop load effect on 13C-
partitioning between fruit and shoots. However, differences in translocation
between organs of the same branch, for a given treatment, were significant, as
the fruits in closest proximity to the branch segment of non-fruiting spurs
generally had the highest relative 13C content (up to 64%, compared to more
distal fruits which ranged from 26% to 40% of recovered 13C). Shoot leaves had

considerably lower 13C contents, ranging between 1.6% and 11 % of the 13C

recovered.

86

Introduction

Fruits are major sinks for assimilates in fruit trees (Wright, 1989; DeJong
and Walton, 1989; Basile et al., 2002). During fruit growth, dry matter (mainly
carbon, C) and water accumulate. Dry matter accumulates in fruit as a result of C
assimilation by different leaf populations and the subsequent distribution among
reproductive and vegetative sinks (Teng et al., 2001). Stone fruit growth follows a
double-sigmoidal curve, which can be divided into three stages (Flore, 1994;
Berman and DeJong, 1996). Stage I is associated with initial growth and rapid cell
division. Stage II or ’pit hardening’ coincides with endocarp development and
slow growth of the pericarp. Stage III or ’final swell’ is characterized by rapid cell
enlargement and dry matter accumulation. Sweet cherry (Prunus avium L.) fruit
achieves between 50 to 80% of its final size during final swell (Flore, 1994).

A plant can be considered as a collection of individual sinks (reproductive
and vegetative) which compete with each other (Wright, 1989; Flore and Layne,
1999). Sink strength, defined as the sink size multiplied by sink activity, is the
driving force for C transport and competition between sink organs (Gifford and
Evans, 1981 ; Hansen, 1989; Ho, 1996). The C available to support maintenance
and growth of sink organs depends on photoassimilates supplied by different
leaf populations and storage reserves (Grossman and DeJong, 1995; Flore and
Layne, 1999; Basile et al., 2002). In sweet cherry, reproductive and vegetative
growth occurs simultaneously during fruit development (Roper et al., 1987).

Leaves on fruiting and non-fruiting spurs complete development early in the

87

season (~3 weeks after bloom). However, current season shoot growth continues
through harvest in well-managed trees and during this time spur leaves
constitute the primary source of C for fruit growth (Roper et al., 1987). This
suggests that fruits might compete with each other and with current season
shoots for available C coming from different sources. Competition between
reproductive and vegetative growth, under source limiting conditions, has been
reported in partitioning studies in peach (Prunus persica L. Batsch.) (Grossman
and DeJong, 1995; DeJong and Grossman, 1995; Corelli-Grappadelli et al., 1996;
Berman and DeJong, 2003).

Fruit growth potential and C availability limit final crop yield in trees
(Pavel and DeJong, 1993). Source limitation results in insufficient C availability to
support potential organ growth (DeJong and Grossman, 1995). Swain and
Darnell (2002) indicate that periods when sources are limiting to reproductive
and vegetative growth can be studied by manipulating sources (i.e., the
availability of C) or sinks (i.e. the demand for C). In peach and blueberry
(Vaccinium corymbosum L.), stages I and III of fruit development are periods of
source limitation, while stage II is considered as a period of sink limitation (Pavel
and DeJong, 1993; Swain and Darnell, 2002). Reductions in sink demand by
reducing sink loads have been shown to increase the C supply to fruit and / or
vegetative growth (Gucci and Flore, 1989; Grossman and DeJong, 1995). In peach,

fruit removal increases the leaf area to fruit ratio (LA / F), which in turn increases

88

fruit size due to a reduction in source limitation (Pavel and DeJong, 1993; DeJong
and Grossman, 1995).

In many species of the Rosaceae family including sweet cherry,
photoassimilate production by spur leaves is not sufficient for optimal fruit
growth, and import of assimilates synthesized by leaves external to the bearing
spurs is required (Roper et al., 1987; Lakso and Corelli-Grappadelli, 1992; Corelli
Grappadelli et al., 1994; Tustin et al., 1992; Teng et al., 1998, 2001; Chapter 2). In a
2-year-old sweet cherry branch, current photoassimilates for fruit and shoot
growth can be provided by three distinct leaf populations: non-fruiting spur
leaves (acropetal to the fruit), fruiting spur leaves where fruit are borne, and
single leaves on new shoots (acropetal or basipetal to the fruit).

There are few experimental data on the movement and partitioning of
assimilates in sweet cherry branches, particularly precocious and dwarfing
rootstocks. So far, little is known about the importance of the non—fruiting spur
leaves as a C source for fruit growth. Lang (2001) suggested that this population
of leaves can help supply C to nearby new shoots or to the fruits developing
farther down the branch. In the case of a high number of fruit per fruiting spur,
as is common for combinations using Gisela rootstocks, C supplied by non-
fruiting spurs might become extremely important for fruit growth, especially
during periods of source limitation.

We hypothesized that the crop load, quantified by LA / F ratio of a whole

fruiting branch unit, influences the fate and amount of C partitioned from non-

89

fruiting spurs to fruit and shoots during stage 111. To test this hypothesis, we
used sweet cherry trees on the dwarfing rootstock Gisela 5 (G15, Prunus cerasus x
P.canescens), which is precocious, productive (often 5 to 15 kg / tree in the 4th to 7th
year) and generally achieves ~ 30 to 50% of the canopy volume of standard trees
(F ranken-Bembenek, 1996). Two-year old sweet cherry branches were labeled
three separate times with 13C, as 13CO2, during stage HI of fruit development. 13C
labeling has been used previously in other tree fruit partitioning studies
(Lombardini, 1999; Nakano et al., 1998). Therefore, the main objectives of this
study were to: (1) evaluate the use of 13C as a non-radioactive pulse-labeling
technique directly in the orchard; (2) elucidate the importance of the non-fruiting
spur population as a source of C for fruit and current season growth during final
fruit swell and, (3) determine whether different LA / F ratios alter C translocation

patterns.

Materials and Methods
Plant material

The experiment was conducted during summer 2002 in a commercial
orchard near Sparta, Michigan. Two-year-old fruiting branches on four-year-old
trees of sweet cherry ’Sam’ on the dwarfing the rootstock G15 were selected. Two
hundred and twenty branches having similar vigor, length and diameter were
identified (Table 1). Most the branches were located in the medium and upper

sections of the canopy and had comparable light exposures. Three LA / F ratio

90

treatments were imposed during stage III of fruit development Branches were
grouped by their natural LA / F ratios and, from these groups, 7 branches per
treatment were selected randomly for each 13C labeling date. The crop loads of
the selected branches were adjusted to the following LA / F (in cm2/ fruit): ~140
(treatment 1, T1), ~ 75 (treatment 2, T2) and ~ 40 (treatment 3, T3). These ratios
corresponded to 13:3, 25i4 and 48:5 fruits / branch, respectively. Lateral current
season shoots were eliminated to leave only the new terminal growth. A total of
thirty branches (including three for natural baseline 13C abundances for each
date) were used for each pulse-labeling date. Fifty fruits were sampled to
measure weight (fresh and dry), diameter, soluble solids (SS) and color. Twenty
five shoots were used to measure weight (fresh and dry), length and leaf
number. Fruits and shoots were sampled at each pulse-labeling date (see below

for details).

13C pulse-labeling

On three dates (52, 59 and 63 days after full bloom, DAFB) during stage III
of fruit development, the branch section bearing non-fruiting spur leaves was
enclosed in 3.7 L transparent Mylar® balloon-chambers and pulsed for 15 min
with 13C02. A total of 1.3 mmol of 13C02 was injected into the chamber. 13C02
was generated by injecting 0.25 ml of 80% lactic acid into a 1 L plastic wash bottle

containing 0.25 g of barium carbonate (98 atom% 13C). The bottle was squeezed

91

every 2 min to pump 13C02 into the chamber. The labeling was carried out on
sunny days between 10:00 AM and 12:30 PM.

Single leaf gas exchange of non-fruiting spur leaves on selected branches
was measured prior to and during the pulse-labeling, using a CIRAS-2 infrared
gas analyzer (PP-Systems Inc, Haverhill, Massachusetts, USA). The objective was
to calculate the average rate of C02 uptake (between 9:00 AM and 12:00 PM) and
carry out the pulse-labeling when assimilate rate values were positive. Net
assimilation rate (A) of non-fruiting spurs varied among branches ranging
between 5.0 and 18.0 umol In:2 5‘1. Considering this natural variability, it was
estimated that 15 min exposures would allow 13CO2 uptake for all variations.

Labeling conditions were similar among labeling dates; however, light
and temperature levels varied with ambient conditions and branch position
within the canopy. Leaf temperature during the labeling period ranged from
23°C (9:00 AM) to 32°C (12:30 PM). Light levels were more variable and
depended on the position of the branch within the canopy and the presence of
clouds during labeling period. The photosynthetically active radiation (PAR) of
the non-fruiting spur leaves ranged from 200 (shaded leaves) to 2705 umol In'2 5'1

(well exposed leaves).

92

Sampling and analysis

At 24 and 48 h after labeling, two 0.2 cm2 discs per leaf and whole fruits
were collected from the labeled branches and frozen immediately in liquid
nitrogen for subsequent determination of13C enrichment by using gas
chromatography mass spectrometry (GC-MS, model Europa Integra, PDZ
Europa, United Kingdom). Additional samples were collected from unlabeled
branches for natural abundance calculations. Fruit samples were collected from
fruiting spurs located in the upper and lower portions of the two-year section of
the branch. Leaf samples were collected from fully expanded and developing
leaves on current season growth. The plant material was oven-dried at 70°C for
72 to 96 h and subsequently ground using a Wiley mill (20 and 40 mesh). 13C

enrichment was calculated according to Boutton (1991) and Vivin et a1. (1996) as

follows:

813C (%0) = [Rampt-Rsmdard/Rstandard] x 1000 Eq (1)
Rsample = 13C/12C = [813C/ (1000 + 1)] x Rpm; Eq (2)
F = 13C/ (”012(3) = R/<R+1) Eq (3)
Atom% excess = (Fpostdose-Fbaseune) x 100 Eq (4)
New 13C content = (Atom% excess / 100) x dry matter x [C] Eq (5)

Relative Partitioning (%) = (New 13C content in the organ) /

(New 13C in all the sampled organs) x 100 Eq (6)

93

where the 813C (%o) value is calculated from the measured carbon isotope ratios
of the sample and standard gases (Eq. 1). The absolute ratio (R) of a sample is
defined by Eq. 2, where Rpm; = 0.0112372. Atom % excess is used as an index to
determine the enrichment level of a sample following the administration of the
13C tracer in excess of the 13C baseline prior to the 13C02 pulse (Eq. 3 and 4).The
new 13C pool is calculated for the different branch components according to dry
mass and C concentration (Eq. 6). As the sampling of leaves and fruits was
carried out without removing the whole branch, the partitioning of new 13C was
expressed as a percentage of the total 13C input for the sampled branch

components.

Statistical Analysis

Analysis of variance was conducted by using PROC MIXED procedures of
the SAS statistical analysis program (SAS Institute Inc, Cary, North Carolina).
Covariance analysis indicated that there was not a significant covariate effect of
total 13C fixation on the 13C partitioning among organs. There was not a
significant effect of the sampling date on 13C recoveries; therefore, statistical

analyses considered the total number of observations for both sampling dates.

94

Results
Phenological characterization at pulsing

Most of the branches had an average of 5 non-fruiting and 11 fruiting
spurs, with 97 .1:3.0 and 86.9i3.7 cm2 LA / spur, respectively. Leaf number (~7
per spur) did not differ between spur types. The three 13C02 pulses were
imposed when fruit development was 34% (52 DAFB), 58% (59 DAFB) and 81 %
(63 DAF B) of final fresh weight (FW) at commercial harvest (Table 2). Between
the first and the third pulse-labeling, SS and fruit diameter increased 30% and
17%, respectively. In contrast, fruit hue angle (H°) and firmness decreased 410 %
and 210% respectively (Table 2). At these dates, current season shoots were 82, 89
and 98% of their final length (Table 3). At 52 DAF B, shoots had ~16 leaves (3
folded leaves) and ~ 43% of the leaves were still expanding. At the third pulse-
labeling, shoots had their final leaf number present (zero folded leaves) and ~

25% of the leaves at the tip were still expanding.

Translocation Patterns

Non-fruiting spur leaves were a source of photoassimilates for fruit and
vegetative growth during stage III of fruit development. In most of the branches,
13C fixed by this leaf population was translocated both basipetally (fruit) and
acropetally (shoot). However, there were some branches with unidirectional

translocation. In these branches, 13C was only translocated basipetally to the fruit.

95

For the three pulse dates, most of the branches (96%) had 13C enrichments in both
distal and proximal fruits. In some branches (3%), 13C was translocated to fruits
proximal to the labeled source but no label was found in more distal fruits. The
opposite was also observed in few branches (1 %) for which distal fruits were
labeled but proximal fruits had no label.

Most of the branches (76%) had 13C enrichment in mature and developing
leaves of the terminal shoot. A few branches (6%) showed no 13C enrichment for
either mature or developing leaves. Most of these occurred for T2 and T3 at the
last pulse-labeling (63 DAFB). There were some branches (10%) in which mature
leaves were 13C enriched, but no label was found in developing leaves. The
opposite was also observed; a similar number of branches (10%) had 13C label in

developing leaves but not in mature leaves.

13C Partitioning

The total 13C recovered (as ug13C/ g DW) at 24 and 48 h after the pulse for
the four sampled organs, ranged between 18 and 209 (52 DAF B), 50 and 450 (59
DAFB) and 31 and 350 (63 DAF B) ug13C/ g DW/ branch. Branches in which all
the sampled organs had levels of enrichment close to natural abundance values
(Atom % S 1.082, leaves and 5 1.084, fruit) were discarded for lack of successful
labeling.

There was not a significant LA / F ratio effect on the level of 13C

enrichment (atom %) of fruits and shoot leaves (Figures 1A, B and C; see

96

Appendix A1 for statistics). However, the data showed two general trends
across stage 111. First, the highest atom % values were detected in fruits as
compared to shoots. Second, the lower the LA / F ratio (i.e., the higher the crop
load) the higher the atom % measured in proximal fruit compared to distal fruit.
Paradoxically, there were some branches in which distal fruits had higher atom
% than proximal fruits. This was particularly evident in the lowest crop load (140
cm2 LA/ fruit), for which between 33 and 86% (depending on the pulse date) of
the analyzed branches had higher atom % in distal fruits than in proximal fruits.
Despite some of the branches in T2 (75 cm2 LA / F) and T3 (40 cm2 LA / F) having
distal fruits more 13C enriched than proximal fruits, most had proximal fruit with
higher 13C contents.

Differences in partitioning of 13C among sampled organs of the same
branch, for a given treatment, were highly significant (Table 4). Fruit in close
proximity to pulse-labeled source leaves had the highest relative 13C enrichment
(55 to 64%) compared to more distant fruits (28 and 40%). Mature leaves at the
base of the terminal shoot, which were closer to the labeled source, had lower
13C contents than developing leaves at the upper section of the shoot, but they
were not significantly different. Comparisons between the total 13C recovered for
fruit (distal plus proximal) and shoot leaves (proximal plus distal) indicate that
fruit growth is a much stronger sink than shoot growth during stage III.
Considering all the labeling dates, between 87% and 96% of the recovered 13C

was partitioned to fruit, while between only 4% and 13% was partitioned to

97

current season growth. A general analysis of the data indicated that in the
lowest crop load (140 cm2 LA/ F), 13C was distributed more evenly between distal
and proximal fruit as compared to the higher crop loads (75 and 40 cm2 LA / F),
which showed a greater difference between 13C enrichments of distal and

proximal fruits.

Relative carbon allocation

Initial measurements (prior to LA / F ratio adjustments) indicated that 70
to 75% of the fruit was concentrated in the medial and proximal section of the 2-
year-old wood, a characteristic of sweet cherry on numerous rootstocks
(Maguylo et al., 2003). Fruiting Spurs on these sections had more fruits than those
in the distal bottom section. After the treatments were imposed, fruiting density
(fruit number / spur) decreased for T1 (140 cm2 LA / fruit) and T2 (75 cm:2
LA/ fruit). In these two treatments, fruit were distributed more evenly with an
average 1.0 and 2.5 fruits / spur, respectively. The highest LA / F ratios (T 3, 40 cm2
LA / fruit) represented the natural crop load and fruiting density (averaging 4.5
fruits / Spur) for ’Sam’/ G15 trees, with fruit distribution being more concentrated
in the medial and proximal portions of the fruiting section. Considering the total
crop load, its distribution, the 13C gain/ organ and the DW/ organ at each
labeling date, the 13C relative partitioning between fruit and shoot leaves was
estimated for each treatment (see Appendix A2 for statistics). At 52 and 59

DAF B, LA / F ratios and the distribution of the crop load on the fruiting wood

98

significantly affected 13C partitioning between distal and proximal fruit (Figures
2A and B). However, this effect was not significant at the last 13C pulse-labeling
(63 DAFB, Figure 2C). For the first two labeling dates, the higher the crop load
(i.e., the lower the LA / fruit ratio), the more C was translocated to proximal fruit
and less to distal fruit. At the end of stage 111, though this trend was similar to the

other labeling dates, the differences were not significant.

Discussion

Distribution of newly-fixed C was studied in 2-year-old sweet cherry
branches on ’Sam’ / G15, a dwarfing scion/ rootstock combination. The
manipulau'on of sink-source relationships by changing the LA/ F ratios of
individual branches and the use of stable isotope 13C were used to characterize
and quantify the relative partitioning of C, fixed by non-fruiting spur leaves,
between rapidly growing fruit and the elongating terminal shoot during stage III.

Non-fruiting spur leaves provided a C source for both fruit and shoots
during stage III. In the majority of the branches, regardless of the treatment, non-
fruiting spurs exported 13C basipetally to the fruit and acropetally to the terminal
shoot Bidirectional transport from non-fruiting spurs and other leaf populations
has been reported for apple (Malus domestica Borkh.), sour cherry (Prunus cerasus
L.), pecan (Carya illinoensis Koch.) and grape (Vitis vinifera L.), among others
(Hansen, 1969; Corelli Grapadelli et al., 1994; Kappes and Flore, 1989; Toldam-

Andersen, 1988; Davis and Sparks, 1974; Hale and Weaver, 1962). There were

99

branches in which unidirectional translocation to fruit also was observed.
Unidirectional translocation has been observed in sour cherry, apple and
cranberry (Vaccinium macrocarpon Ait.) (Kappes and Flore, 1989; Hansen, 1969;
Roper and Klueh, 1996). A general analysis of the data indicated a distinct effect
of orthostichy on 13C translocation patterns. Phyllotactic patterns might explain
the large variation between distal and proximal fruit 13C enrichments on
branches of a particular treatment. For example, two branches in T1 (140 cm2

LA / F) showed a very different pattern of C partitioning between fruits. In one,
83% of 13C was recovered in the distal fruit and only 10% in the proximal fruit
The opposite was observed in an otherwise similar branch, for which no 13C was
recovered in distal fruit but 90% was recovered in proximal fruit The influence
of vascular connections on partitioning between different organs has been
described for apple, grapes, sour cherry and red raspberry (Rubus idaeus L.)
(Hansen, 1969; Corelli Grapadelli et al., 1994; Kappes and Flore, 1986; Fernandez
and Pritts, 1993; Privé et al., 1994).

LA / F ratios can affect C partitioning between fruit and vegetative growth
in apple and peach (Giuliani et al., 1997; DeJong and Grossman, 1995; Berman
and DeJong, 2003). In our experiment, different LA / F ratios did not affect
differences in 13’C values recovered between fruit and shoot leaves, relative to
different treatment LA / F ratios, were not significant, probably due to the shoot
to shoot variability described above. However, comparisons of the relative

partitioning of 13C among the four analyzed organs, for a particular LA / F ratio,

100

indicated that rapidly enlarging fruit attracted 13C more strongly than did
elongating shoots (Figures 1A, B and C). On average, fruit attracted between 88
and 97% of the recovered 13C vs. 4 to 12% in shoots. This strong sink demand
was constant during stage III. In sour cherry and peach, fruit have priority over
vegetative growth when leaves and fruit are in competition for resources (Flore
and Layne, 1999; DeJong and Grossman (1995). Caruso et al. (1997), using a less
vigorous rootstock in peach (’Flordaprince’ on MrS 2/ 5), found that the reduced
vegetative growth during fruit development resulted in higher dry matter
partitioned to the fruit than to vegetative structures. 0n the other hand, Kappel
(1991), using ’Lambert’ sweet cherry on the vigorous seedling rootstock
Mazzard (Prunus avium L.), found that current season growth was a greater sink
for photosynthates than was fruit, suggesting that fruit on such sweet cherry
trees may not be a predominant sink for C. Our results suggest that sweet cherry
fruit are stronger sinks than shoots during stage III in combinations using the
dwarfing rootstocks. Less Vigorous vegetative growth with G1 rootstocks results
in shorter shoots, with earlier terminal bud set (Franken-Bembenek, 1996). In
shorter shoots of apple, the onset of C export occurs earlier than in a longer
shoots due to an early cessation in the sink demand of the shoot tip (Lakso and
Corelli-Grappadelli, 1992). Our experiment was carried out when shoots had
achieved between ~80% (~9 fully expanded leaves) and 100% (~13 fully
expanded leaves) of their final length. It is highly likely that the shoots were

already net exporters and, therefore, competition between fruit and shoots was

101

minimal. We suggest that in dwarfing sweet cherry combinations, terminal
current season growth constitutes a C source for fruit growth during stage III. In
apple, 35 DAFB, shoots had become a significant C source fruit growth (Lakso
and Corelli Grapadelli, 1992). Similarly, in sour cherry, net export from terminal
shoots began 17 days after leaf emergence, when shoots have accumulated ~27%
of their final DW (Kappes, 1985).

As has been reported previously in apple and peach (Hansen, 1969; Ben
Mimoun et al., 1995; Bruchou and Génard, 1999), we detected a positional effect
on translocation to fruit in which those in close proximity to the labeled leaves
had higher relative 13C enrichments than more distal fruits. Competition between
developing fruits has been reported by Wright (1989). It was not the aim of this
paper to evaluate differences in fruit size at different positions of the fruiting
section, but differences in fruit growth rate and size might explain this
differential demand for C (Hansen, 1969). Unpublished data (G.A. Lang,
personal communication) indicates that fruit size tends to vary across position,
and when the LA / F increases, the variation in fruit size due to position
decreases. It seems that when C is not limiting, fruit sizes tend to be similar. In
contrast, under source limiting conditions, C distribution appears to be unequal
and fruit size less uniform. Interestingly, the highest LA / F ratio (lowest crop
load) resulted in a more even 13C distribution between proximal and distal fruit.
In this treatment, a greater number of branches had distal fruit with higher or

similar 13C enrichments compared to proximal fruit. Factors influencing the

102

translocation of photosynthates from certain leaf populations include: (1)
vascular connections between organs; (2) LA / F ratios; (3) vigorous growth of
fruit; (4) position and size of the spur within a branch; and (5) other unknown
factors (Hansen, 1969).

When fruit distribution and total crop load per branch were included in
the partitioning analysis, there were significant differences among different crop
load treatments in terms of relative 13C partitioning. At 52 and 59 DAFB, in the
lower LA / F ratio treatment (i.e., ~75% of the high cr0p load was in the medial
and proximal sections of the 2-year-old wood), most of the 13C was found in
proximal fruit with lower amounts partitioned to distal fruit. These higher crop
loads might have imposed a source limiting condition, which generated
competition between fruit at different positions. The other two treatments, which
had spurs with a fairly uniform number of fruits and higher LA / F ratios, did not
differ, indicating a higher C availability. According Grossman and DeJong
(1995), as these treatments were thinned, fruit growth should have been sink-
lirnited. At 63 DAF B, despite that the trend was similar to the other two pulses,
fruit distribution did not significantly affect the relative partitioning among
organs. It seems that in late stage III, fruit located more acropetally decrease their
C demand earlier than those fruit located more basipetally. This might a
consequence of the decrease in the growth rate of proximal fruit, which would

promote a greater translocation to distal fruit. More information about

103

competition between sweet cherry fruits at different positions within a branch
remains to be elucidated.

General implications of these results assist in developing concepts for
precise branching and cropping in sweet cherry on dwarfing rootstocks. Greater
precision in canopy development, i.e., more precise management of crop loads
and the different source leaf populations, may help to optimize the balance
between vegetative growth and fruit quality (Lang, 2001). It has been reported
that combinations using G15 have a serious problem with overcropping and
lower quality fruit beginning about the 4th or 5th year. To overcome this situation
and manage LA / F ratios more precisely, it is critical to have a good
understanding of sink-source relationships. More knowledge about the role of
the various leaf populations as sources of C for developing fruit will help to
develop pruning and training strategies to manage high-density orchards.

In summary, our findings indicate that the hierarchy for C distribution
within a typical sweet cherry branch on a dwarfing rootstock, during stage III,
would be proximal fruit>distal fruit>mature or developing leaves on a shoot. We
conclude that in 2-year-old branches: (1) non-fruiting spur leaves constitute an
important C source for fruit growth during stage III; (2) fruits are a C
partitioning priority over current season growth during stage III and this is
relatively constant during stage III; and (3) fruits at different positions compete

for C fixed in non-fruiting spur sections. These results provide a better

104

understanding about the link between vegetative growth and fruit development

in dwarfing sweet cherry trees.

105

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109

Table 1. Morphological features of 2-year-old ’Sam’/ Gisela 5 sweet cherry
branches before 13C pulse-labeling. Mean :t SE, n=220.

 

 

Parameter Average or Range

Branch length (cm) 86.2 i 2.7
Branch diameter (mm) 12.2 i 0.5
Initial fruit number/ branch 2 10 to 65
Total spur number 16.4 i 1.2
Fruiting spur number / branch 10.9 i 0.9
Non-fruiting spur number / branch 5.5 i 0.5
Total fruiting spur leaf area (cmz) 951.9 i 72.9
Total non-fruiting spur leaf area (cm2) 534.2 i 58.4

 

7- Range in fruit number before thinning.

Table 2. ’Sam’ sweet cherry fruit quality parameters measured at each pulse-
labeling (52, 59 and 63 days after full bloom, DAFB) and at commercial harvest
(67 DAFB). Mean i SE, n=50.

 

 

DAF B Fresh Dry 8.5 Diameter Color Firmness
weight weight (°Brix) (mm) (Hue°) (g / mm)
(8) (g)
52 2.2 i 0.52 0.5 i 0.1 10.0 i 0.3 16.5 i 0.2 73.5 i 1.8 497.7 i 24.2
59 3.8 d: 0.5 0.7 i 0.1 11.1 i 0.6 17.7 i- 0.6 30.6 i 0.5 281.5 i 5.9
63 5.3 i 0.6 0.8 i 0.1 13.7 i 0.5 19.9 i 0.9 17.8 i 0.3 230.8 i 5.4
67 6.5 i 0.5 0.9 i 0.1 16.4 i 0.8 20.4 i 0.4 8.5 i 0.1 169.9 i 1.2

 

1 Commercial harvest (2 Jul, 2002).

110

Table 3. Growth and morphological measurements of current season
’Sam’ / Gisela 5 sweet cherry growth at each pulse—labeling date (52, 59 and 63
DAFB) and harvest (67 days after full bloom, DAF B). Mean i SE, n=25.

 

 

 

Measurement DAF B
52 59 63 67W

Total fresh weight (g) 10.6 :t 1.0 13.0 i 1.1 14.4 i 0.6 15.1 i 0.4
Total dry weight (g) 4.9 1r 0.3 5.0 i 0.4 6.1 i 0.5 6.2 i 0.8
Length (cm) 20.9 i 1.1 22.6 i 2.3 24.9 i 1.2 25.4 i 1.4
Total leavesz 16.0 i 1.3 17.1 :t 1.1 18.0 i 1.1 18.4 d: 0.7
Fully expanded leaves 9.1 i 1.1 10.7 i 0.9 12.5 i 1.2 13.7 i 1.3
Developing leaves." 6.9 i 1.1 6.4 i 1.4 5.5 i 0.9 4.69 i 0.7
Folded leaves 3.2 i 0.5 2.4 i- 0.4 0.2 i 0.2 0.0 :t 0.0
Leaf area (cm?) 288.0 .1: 2.0 334.1 i 15.2 365.8 i 4.0 406.5 i 7.6

 

2 Total leaves include fully expanded and developing leaves.
Y Developing leaves include folded and unfolded not fully expanded leaves.
W Commercial harvest

Table 4. Relative 13C enrichment for fruit and current season leaves. Mean i SE,
n=15. Calculations based on total 13C-absolute (pg 13C/ g DW) recoveries for the
four organs.

 

 

 

 

 

 

DAF B 2 Relative 13C Proportion (%)
Fruit Leaves Obtained
Distal Proximal Proximal Distal p-value
52 30.5 i 7.0 by 61.3 i 6.8 a 1.8 i 0.7 c 6.6 i 2.6 c < 0.0001
59 34.4 i 4.0 b 59.4 i 8.3 a 2.3 i 0.5 c 3.9 i 1.2 c < 0.0001
63 36.8 i 4.2 b 57.7 i 5.3 a 2.1 i 0.6 c 3.6 i 1.3 c < 0.0001

 

Z DAFB: days after full bloom.
y Means in given row followed by the same letter are not significantly different
at 0: =0.05.

111

1.110

 

    

1.105 1 a a a I 40 LA cmZ/Fruit
I 75 LA cm2/Fruit
1,100 A I 140 LA cm2/Fruit

1.095 a

 

Atom %

 

 

    

 

 

Distal Fruit Proximal Fruit Proximal Shoot Distal Shoot
Organ by Position

Figure 1. 13C content (expressed as atom %) in fruits and current season growth
leaves during stage III (52, 59 and 63 DAFB). Means :1: SE are represented in
colored bars for each organ within a certain treatment Means for a certain organ
followed by the same letter are not significantly different at a=0.05.

112

 

100 1 I 40 cmZ/Fruit a
90 _ I 75 cm2/Fruit
30 u El 140 cm2/Fruit

 

l3C Partitioned to Fruit

 

 

 

 

Distal Fruit Proximal Fruit

Figure 2. 13C-Relative partitioning (%) in distal and proximal fruits during stage
III (52, 59 and 63 DAF B). Means :t SE are represented in colored bars and vertical
lines, respectively. Each treatment is represented by a different color. Colored
bars within the same organ followed by the same letter are not significantly
different at a = 0.05 and a = 0.01, respectively.

113

CHAPTER IV
13C- PHOTOASSIMILATE PARTITIONING IN SWEET CHERRY

DURING FRUIT DEVELOPMENT

114

13C- Photoassimilate Partitioning in Sweet Cherry

During Fruit Development

Keywords: Prunus avium L., stage 1, stage 11, stage III, shoot growth, leaf area,
sink strength, fruit quality, carbohydrates, translocation, source, carbon, Gisela,

rootstock.

Abstract

Fruit size, quality and yield are influenced by photoassimilate synthesis
and subsequent partitioning among different sink organs. Little is known about
the relative importance or temporal relationships of different leaf populations as
sources of carbon (C) for sweet cherry fruit and shoot development, particularly
on vigor-reducing rootstocks. It was hypothesized that the partitioning of C
fixed by different leaf types in semi-dwarfing sweet cherry trees is influenced by
reproductive and vegetative sink demands during fruit development, with the
sink strength of fruit being considerably greater than that of shoots as a
consequence of the higher harvest index in this type of tree. To study the
contributions of different leaf populations to fruit and shoot development during
stages I, II and 111, an experiment using 2-year-old fruiting branches of ’Ulster’
sweet cherry on the semi-dwarfing rootstock, Gisela 6 (G16), was established. The

three leaf populations on the fruiting branch, i.e., fruiting spur, non-fruiting spur

115

and new terminal shoot leaves, were exposed to 13C02 labeling on five different
dates (25, 40, 44, 56, 75 days after full bloom, DAFB) during fruit development.
Two days after labeling, whole branches were removed and different organs
were prepared for analysis by gas-chromatography mass-spectrometry (GC-MS).
Spur and shoot leaves were significant sources of C for fruit and vegetative
growth. In terms of C allocation, fruits were a priority sink vs. new shoot growth
during the entire period of fruit development. The highest fruit sink strength was
during stages I and 111. Current season (terminal) shoot growth provided a C
source for fruit as early as stage I. It seems that resource limitations during stages
I and III of fruit development affect final fruit size in sweet cherry on Gisela (GI)
rootstocks. The source-sink relationships elucidated in this study provide a
physiological foundation for the development of specific orchard management
strategies to promote a more sustainable balance between vegetative and

reproductive growth in high density sweet cherry orchards.

116

Introduction

In many tree fruits of the Rosaceae family, spur and extension shoot
leaves are the main sources of current photosynthates for reproductive and
reproductive growth (Teng et al., 2001; Roper et al., 1987 ; Corelli-Grappadelli et
al., 1994). In stone fruit trees, fruit are major sinks for assimilates (DeJong and
Walton, 1989). During fruit development, dry matter (mainly carbohydrates,
CH20) and water content increase. Flore (1985) indicate that ~90% of the fruit
dry weight (DW) is comprised of CH20. Dry matter accumulates in fruit as a
result of carbon (C) assimilation by different leaf populations within the canopy
and subsequent partitioning among different sinks (Teng et al., 2001). In Prunus
sp., fruit development follows a double sigmoidal pattern, which is divided into
three stages (Labreque et al., 1985; Flore 1994; Costes et al., 1995; Berman and
DeJong, 1996). Following pollination and fruit set, stage I is characterized by
active cell division and rapid initial growth. Stage II or ’pit hardening’ is
associated with endocarp lignification and slower growth of the pericarp. Stage
III or ’final swell’ is a period of rapid fruit growth characterized by mesocarp cell
enlargement and dry matter accumulation. Fifty to 80% of cherry fruit growth
occurs during this stage (Flore, 1994; Chapters 3).

A tree can be considered as a collection of individual sinks (reproductive
and vegetative) that compete with each other (Wright, 1989). Reproductive and
vegetative growth occurs simultaneously during sweet cherry (Prunus avium L.)

fruit development (Roper et al., 1987). Leaves on fruiting and non-fruiting spurs

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reach full leaf area early in the season. However, current season shoot growth
continues developing leaf area through harvest in well managed trees. This
situation might generate competition between actively growmg aerial sinks, i.e.,
fruits and extension shoots, for the available C provided by different leaf
populations. In peach (Prunus persica (L.) Batsch), periods of resource limitation
lead to competition for photoassimilate between reproductive and vegetative
organs (Grossman and DeJong, 1995). The sink demand of an organ varies with
the time of year since its developmental demands change during the season
(Flore and Layne, 1999). Thus, during plant growth, sinks may change in
competitive ability to attract assimilates (Wright, 1989). Roper et al. (1988)
indicate that since sweet cherry fruit development occurs during a short period
(60 to 70 days), fruit sink effects might be highly prioritized.

The primary sink activities of fruits are growth and respiration (DeJong
and Goudriaan, 1989). The C available to individual organs depends on the
supply of photoassimilates from sources (leaves or storage reserves) and the
demand for resources by sink organs (Basile et al., 2002). Farrar (1996) and
Michin et a1. (1997) suggest that the distribution of assimilates is controlled by
the entire source- sink-pathway plant system and is not a property of sinks alone.
In contrast, Marcelis (1996) proposed that dry matter partitioning among sinks is
regulated by the sinks themselves and the effect of sources is indirect via the

formation of sink organs.

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Photosynthesis determines the amount of C available for plant growth
(Farrar and Williams, 1991). In a 2-year-old sweet cherry branch, current
photoassimilates for fruit growth can be provided by fruiting spur leaves where
fruit are borne, non-fruiting spur leaves and from single leaves on new shoots
(Roper et al., 1987). Fruiting spur leaves are not the only C source to support fruit
growth, and import of assimilates synthesized by leaves distal to the bearing
spurs is required for optimal fruit development (Roper et a1. 1987; Chapter 2 and
3). Previous branch girdling and defoliation studies demonstrated the
deleterious effects on fruit quality when fruit were isolated from the major
sources of photoassimilates (Roper et a1., 1987 ; Chapter 1). Leaf populations on
both fruiting and non-fruiting branch segments were required for optimal fruit
development (Chapter 1).

Little is known about the relative importance or temporal relationships of
these different leaf populations as sources of C for sweet cherry fruit and shoot
development In this study, 2-year-old sweet cherry branches on ’Ulster’ / Gisela
6 (G16) in which fruiting spur, non-fruiting spur and current season shoot leaf
populations were labeled with 13C by pulsing with 13C02 five times during stages
I, II and III of fruit development. It was hypothesized that the partitioning of C
fixed by different leaf populations would be strongly and differentially
influenced by reproductive and vegetative sink demands during fruit
development. Accordingly, the main objectives of this study were to: (1)

elucidate the temporal importance of each leaf population as a source of

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assimilates for fruit and shoot growth during the whole fruit development
period; (2) determine predominant translocation patterns depending on the stage
of fruit development; and (3) quantify differences in the amount of C exported
from different leaf sources to fruit and shoot growth at various intervals during

the growing season.

Materials and Methods
Plant material

The experiment was conducted during summer 2003 at Michigan State
University’s Clarksville Horticultural Experimental Station (CHES), Clarksville,
Michigan. Two-year-old fruiting branches on 7-year-old trees of ’Ulster’ sweet
cherry on the semi-dwarfing rootstock G16 (Prunus cerasus L. x Prunus canescens
L.) were selected for 13C pulse-labeling during stages I, II and III of fruit
development. A population of six hundred branches having similar vigor, crop
load, length and diameter was identified. Ten branches per treatment were
selected randomly from this population for each 13C pulse-labeling date. Most
the branches were located in the middle and upper sections of the canopy. 13C-
labeling treatments corresponded to the fruiting spur (FSP-Lf1), non-fruiting
spur (NFSP-Lf2) and current season shoot (SH-Lf3) leaf populations at different
stages of fruit development. A total of thirty branches (including three for

natural baseline 13C abundances for each date) were used for each pulse-labeling

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date. Lateral current season shoots were eliminated to leave the new terminal
shoot growth as the sole current season shoot source and sink.

Fruits, spurs and shoots were measured weekly. Thirty fruits were
measured for weight (fresh and dry), diameter and soluble solids (SS). Thirty
fruiting spurs, non-fruiting spurs and current season shoots were measured for

weight (fresh and dry), leaf number and leaf area (LA).

Phenological characterization before I3C02 pulsing

Individual branches were measured and characterized morphologically
prior to the beginning of the sequential 13C labeling. Measurements included:
branch length, branch diameter, fruiting and non—fruiting spur number, shoot
length, shoot leaf number and fruit number. Terminal shoot growth (current
season extension growth) was measured weekly from budbreak until terminal
bud set for the entire population of branches.

The experimental branches were similar in vigor and morphology, but
differed by position within the canopy. Branch length and diameter was similar
among branches selected for a specific pulse-labeling date (Appendix B.1). At the
final pulse-labeling date (75 days after full bloom, DAFB), the average diameter
was greater compared to that measured at the first pulse-labeling date (25
DAF B). Most of these branches had a similar number of fruiting and non-fruiting
spurs, which ranged between 12 and 14 spurs per section, respectively

(Appendix B.1). Despite the similarity in the number of fruiting and non-fruiting

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spurs, the length of the fruiting spur section (2001 shoot growth) was higher than

that of the non-fruiting section (2002 shoot growth; Appendix B.1).

13C pulse-labeling

On eight dates during fruit development, 25 DAF B (25 May), 33 DAFB (2
Jun), 40 DAF B (9 Jun), 44 DAFB (13 Jun), 51 DAFB (20 Jun), 56 DAFB (25 Jun), 63
DAF B (2 Jul) and 75 DAF B (14 Jul), fruiting spurs leaves, non-fruiting spur leaves
and current season shoot leaves were enclosed as separate populations in
transparent Mylar® balloon chambers of different volumes and pulsed for 15 to
20 minutes with 13COz. A total of 3.9 mmol of 13CO; was pumped into the
chambers. 13’COz was generated by injecting 0.75 ml of 80% lactic acid into a 1 L
wash bottle containing 0.75 g of Barium carbonate (98 atom% 13C). The plastic
bottle was agitated and squeezed every 2 minutes to pump 13COz into the
chamber. The labeling was carried out during sunny days between 10:00 AM and
12:30 PM.

Climatic conditions were similar among labeling dates; however localized
light and temperature levels varied somewhat with ambient conditions and
branch position within the canopy. The photosynthetically active radiation (PAR)
varied among leaf populations depending on branch orientation, within a
specific pulse-labeling date and among dates (Appendix B.2).

Single leaf gas exchange of fruiting, non-fruiting spur and current season

growth leaves on selected branches was measured prior to and during the pulse-

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labeling with a CIRAS-2 infrared gas analyzer (PP-Systems Inc, Haverhill,
Massachusetts, USA). The average rate of C02 uptake for each date (between 9:00
AM and 12:30 PM) was calculated so that the pulse-labeling was carried out
when assimilation rate values were positive. Net assimilation rate (A) varied
among the three leaf populations and dates (Appendix B.3). Considering this
natural variability, it was estimated that 15 min exposures for the three
treatments would allow optimal 13C02 uptake among all orientations of branches

and young developing shoots.

Sampling and analysis

Immediately after labeling, 1 or 3 fully expanded leaves were sampled
from each treatment to estimate the initial total 13C fixed by each leaf population. ,
When fruiting spurs were the labeled population, fruit were also sampled to
estimate the 13C fixed due to fruit photosynthesis.

At 48 h after each pulse-labeling, whole branches were harvested
destructively to measure fresh and dry weight of different organs. Measured
organs included: fruiting spur leaves, non-fruiting spur leaves, current season
shoot leaves, and fruit plus wood and bark of the fruiting, non-fruiting and
current season shoot sections. Current season shoot growth was divided further
into mature fully expanded leaves (leaves at the base of the shoot), developing
leaves (leaves along the middle of the shoot), young leaves (leaves at the tip of

the shoot) and wood. Fruit size and number per branch also was measured.

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Branches were then prepared for 13C-analysis. Analyzed organs included
those indicated above. In addition, five single fruit from the fruiting spur branch,
that was labeled directly, were divided into pericarp and endocarp. All plant
materials were oven-dried at 70°C for 72 to 96 h and ground using a Wiley mill
(20 and 40 mesh). Additional samples were prepared from unlabeled branches
for natural abundance calculations. 13C enrichment was measured by using gas
chromatography mass spectrometry (GC-MS, PDZ Europa 20-20 mass
spectrometer and ANCA-GSL sample combustion unit, PDZ Europa, Sandbach,
Cheshire, United Kingdom). 13C enrichment for different organs was calculated

according to Boutton (1991) and Vivin et al. (1996) as follows:

813C (%o) = [(Rsample-Rstandmy Rstandard] x 1000 Eq (1)
Rsample = 13C/12C = [813C/ (1000 + 1)] x Rpm; Eq (2)
F = IBC/(13C+12C) = R/<R+1> Eq (3)
Atom% excess = (Fpostdose-Fbasenne) x 100 Eq (4)
New 13C content = (Atom% excess / 100) x dry matter x [C] Eq (5)

Relative Partitioning (%) = (New 13C content in the organ) / (New 13C in all the

sampled organs) x 100 Eq (6)

where the 813C (%o) value is calculated from the measured C isotope ratios of the
sample and standard gases (Eq.1). The absolute ratio (R) of a sample is defined

by Eq.2, where RpDB = 0.0112372. Atom % excess is used as an index to determine

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the enrichment level of a sample following the administration of the 13C tracer in
excess of the 13C baseline prior to the 13C02 pulse (Eq.3 and 4).The new 13C pool is
calculated for the different branch components according to dry mass and C
concentrations (Eq. 5). The relative partitioning of new 13C was expressed as a

percentage of the total 13C input for the branch components (Eq. 6).

’ Selection of representative pulse-labeling dates for 13C analysis

The eight 13COz pulses were imposed when fruit dry weight (DW) was
21% (25 DAFB), 23% (33 DAFB), 24% (40 DAFB), 25% (44 DAFB), 41% (51 DAFB),
57% (56 DAFB), 78% (63 DAFB) and 100% (75 DAFB) of its final value measured
at 75 DAFB (Appendix B.4). At these dates, current season shoot DW was 18%,
24%, 29%, 39%, 54%, 62%, 82% and 100% of its final value.

After analyzing the relative growth curves (Figure 1) for fruit and current
season shoots for the 2003 growing season, five representative pulse-labeling
dates were selected for 13C analysis. Thus, stages I and II were evaluated at 25
and 40 DAF B, respectively. Stage III was evaluated at three different pulse-
labeling dates at 44, 56 and 75 DAFB. The last 13C-pulse was carried out late in

stage III, which was 12 days after commercial harvest (2 Jul).

Climatic Data
Climatic parameters such as air temperature, PAR and growing degree

days (GDD, base 4.4 0C) were recorded at CHES during the period of the

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experiment (May to Aug, 2003) and were obtained from the Michigan

Automated Weather Network (MAWN).

Statistical Analysis

Analysis of variance was conducted by using PROC MIXED procedures of
the SAS statistical analysis program (SAS Institute Inc, Cary, N .C.). The statistical
model for the overall experiment was a three way factorial design with three
factors: treatment (T=3), date (D=5) and organ (O=8). As extremely high levels of
13C enrichment were expected in directly labeled leaves, these were not

considered for statistical analysis.

Results
Growth in Two-Year-Old Branches
a. Leaf area

The LA per branch increased from budbreak to 96 DAF B. LA of individual
fruiting and non-fruiting spurs increased until 54 DAF B, while shoots continued
developing leaves until 96 DAFB (Figure 2; Appendix B.5). Individual non-
fruiting spurs had a greater final LA than that of fruiting spurs, with 134.8 $4.2
and 119.1:50 cm2 LA / spur, respectively. Leaf number of fruiting and non-
fruiting spurs did not differ. Shoot leaf area increased rapidly between 26 and 54
DAFB. At terminal bud set (96 DAFB), shoots had an average of ~852 cm2 LA.

The average leaf size by population was ~20 cm2 for fruiting spur leaves, ~22 cm?-

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for non-fruiting spur leaves and ~44 cm2 for shoot leaves. Total LA / branch for a
certain leaf population and LA / fruit (F) ratios for branches used at the different

labeling dates are provided in Appendix B6 and C7, respectively.

b. Fruits

Fruit set occurred between 5 and 12 DAFB (256 to 312 GDD). Stage I began
13 DAF B (319 GDD) and continued until 32 DAF B (483 GDD). Stage II occurred
between 33 (492 GDD) and 46 DAFB (604 GDD). Stage III began 47 DAFB (618
GDD) and finished 75 DAFB (1135 GDD). Fruit remained on the tree and was
measured from 75 DAFB until terminal bud set (96 DAF B, 1477 GDD). The total
GDD accumulated during each fruit developmental stage are provided in
Appendix B.8.

Sixty percent of the final DW and 50% of the SS were accumulated during
stage III fruit development (Table 1). Commercial harvest was carried out 63
DAFB. At that date, fruit fresh weight (FW) and SS were 5.5 g and 17 °Brix,
respectively. Between commercial harvest and terminal bud set, fruit FW and
DW increased 2.7 g and 1.0 g, respectively. During the same period, SS increased

from 17 to 25 °Brix,

c. Current season shoots
Current season terminal shoots began extension growth around full bloom

(Figure 1). Shoots elongated rapidly between 12 and 54 DAFB (Table 2). Their

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growth rate decreased when fruit began growing rapidly during stage III. Final
shoot length was ~35 cm at terminal bud set. Leaf number increased until 75
DAF B. At terminal bud set, the final leaf number was ~22 per shoot. Given that
sweet cherry has a 5-leaf phyllotaxy, the 2003 growth represents slightly more
than 4 full phyllotaxic repetitions. Fifty percent of the final shoot DW was

accumulated between 40 and 63 DAF B.

Relative PW and DW partitioning

FW relative partitioning was similar between stage I (25 DAF B) and the
beginning of stage III (44 DAF B). Fruit and wood constituted most of the FW of
the branch, followed by spur leaves and shoot (Figures 3; Appendix B9 and
B10). Later in stage III the FW distribution changed, with 60% to 70% of the total
FW partitioned to the fruit. FW partitioned to the shoot fluctuated between 3
and 6% of the total FW. As in the case of FW partitioning, DW was partitioned
mostly to fruit and wood (Figures 4; Appendix B11 and B12). However,
between stage I and beginning of stage III, the relative partitioning favored
wood. This situation changed during stage 111 since most (40% to 52%) of the DW
of the branch was partitioned to fruit Partitioning to extension growth of
terminal shoots fluctuated between 4 and 7% of the total DW per branch.

Total FW and DW of 2-year-old fruiting branches increased 61% and 64%,
respectively, between 25 and 75 DAF B. The greatest change in DW was detected

in the fruit and shoots, which increased DW by 84% and 82%, respectively,

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between 25 and 75 DAF B. Non-fruiting wood DW increased more than that of
fruiting wood, 46% vs. 30%, respectively. Fruiting and non-fruiting spur leaf DW
increased with time, but such changes were minor compared to those detected in

other organs of the branch.

Translocation patterns in two-year-old branches

By 48 h after 13C labeling of fruiting spur, non—fruiting spur and terminal
shoot leaves, there were differences in the 13C translocation patterns depending
on leaf population (Tables 3 and 4). A large proportion of the labeled C remained
in the pulsed leaves. In addition, significant amounts of 13C were found in wood
of different sections, indicating active 13C translocation to different organs at the

moment of branch removal.

Total 13C in leaves and fruit immediately after pulsing

All (100%) branches, for all leaf populations and pulse-labeling dates,
were labeled successfully. The total 13C content in fruiting, non-fruiting and
current season leaves immediately after the labeling did not differ significantly
(Table 5). Moreover, the amount of 13C fixed by these three leaf populations did
not show significant differences along labeling dates (Table 5). Despite the lack of
differences in total 13C among leaf populations, it is important to note that at 25

DAFB, the amount of 13C in terminal shoot leaves was considerably lower than

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that detected for the fruiting and non-fruiting spur leaves. At this date, shoots
were 9 cm in length and had only 10 leaves (Table 2).

Fruit from fruiting spur leaves (FSP-Lfl) were directly exposed to 13C02.
Individual fruit fixed 13C at all the pulse-labeling dates; however, 13C content
varied significantly among labeling dates (Table 6). At 25 DAFB, fruit fixed the
highest amounts of 13C. At 40 and 44 DAFB, fruit continued fixing 13C but in
lower quantities. No significant differences were detected among these two
dates. At 56 and 75 DAFB, the amounts of 13C recovered in fruit were lowest and

no significant differences were detected between these dates.

Total 13C recoveries 48 hours after pulsing

Forty-eight h after 13C labeling of fruiting spur, non-fruiting spur and
terminal shoot leaves, there were differences in the 13C translocation patterns
depending on leaf population (Tables 3 and 4). A large proportion of the labeled
C remained in the pulsed leaves. In addition, significant amounts of 13C were
found in wood of different sections, indicating active 13C translocation to
different organs at the moment of branch removal.

Across all labeling dates, the total amount of 13C recovered in the branches
48 h after labeling was lower than the amount of 13C fixed initially (Table 5); due
to unexplained tree variability, 4% of the branches were an exception to this
result The lowest 13C values per branch were recovered when shoot leaves were

pulsed. The largest differences were measured 25 DAF B, when total 13C

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recoveries from pulsed shoot leaves, were ~60% lower than those measured in
either of the pulsed spur leaf treatments, which were similar during fruit
development. At 75 DAF B, 13C recoveries after 48 h were similar for all the

treatments.

Absolute and relative partitioning of 13C 48 hours after pulsing

By 48 h after pulsing, there were significant differences in 13C content
among different organs of the branch, depending on pulsed leaf population and
labeling date. For all three source leaf populations, the greatest proportion of 13C
was detected in fruit. This predominant partitioning to fruit was constant for
stages I, II and III. However, there were significant differences among treatments
regarding the amount of 13C partitioned to fruit at each pulse-labeling date. The
highest 13C levels in fruit were detected when fruiting spur leaves were the
labeled source, and this was evident on all the labeling dates. The second most
important source for fruit was the non-fruiting spur leaves. The lowest 13C
recoveries in fruit were found when extension shoot leaves were the labeled
source. However, for the last pulse labeling (75 DAF B), shoot leaves were equally

as important as non-fruiting spur leaves for C partitioning to the fruit.

a. Fruiting spur leaves as 13C source
Fruiting leaves were a source of current photoassimilates for fruit and

vegetative growth during stages I, II and III of fruit development. In all the

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pulse-labeling dates, 13C fixed by this leaf population was translocated
predominantly to fruit and wood subtending the labeled leaves (Tables 3 and 4).
In most (86%) of the branches, acropetal translocation to non-fruiting spur leaves,
non-fruiting wood, and current season wood and leaves was detected. A few
branches (13%) did not show translocation to either non-fruiting leaves or
different sections of the shoot This was particularly evident 44 DAFB, when 100
% of the branches did not translocate 13C to any of the organs located acropetally
to fruiting spur leaves.

There were significant differences in the absolute amount of 13’C recovered
for each organ (Tables 3 and 4). Most of the translocated 13C was partitioned to
fruit, followed by fruiting spur wood. Lower 13C contents were detected in the
rest of the organs. The highest 13C recoveries in fruit were detected 56 DAF B
(stage III), followed 75 (stage III), 44 (stage III) and 25 (stage I) DAFB (Table 7).
The lowest 13C levels were recovered at 40 DAFB (stage 11). At 25 and 56 DAF B,
the amounts of 13C recovered in 2-year-old-wood were considerably lower
compared to those found on the rest of the labeling dates. When fruit were
exposed directly to 13CO; labeling, they constituted an additional source of C as
indicated previously (Table 6).

The relative partitioning indicated that between 18% and 36% of the 13C
recovered was 48 h later in fruiting spur leaves (Figure 5; Appendix B.13). The
rest of the 13C was recovered in different organs in the following order: fruit (57

to 79%), fruiting spur wood (3 to 9%), non-fruiting spur wood (0 to 1 %), non-

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fruiting spur leaves (0 to 1%) and shoot (0 to 0.1%). The highest percentage of 13C
partitioned to fruit occurred at 56 DAF B, followed by the 25 DAFB pulse. The
lowest was detected 75 DAF B. The lowest 13C recovery in fruiting spur leaves
was at 56 DAF B. Greater detail for the minimal 13C partitioning in shoots is

shown in Appendix B.14.

b. N on-fruiting spur leaves as 13C source

Non-fruiting leaves also were a source of current photoassimilates for
fruit and vegetative growth during stages I, II and III of fruit development In all
the pulse-labeling dates, 13C fixed by this leaf population was translocated
predominantly basipetally to fruit and fruiting wood (Tables 3 and 4). Significant
amounts of 13C were detected in the wood subtending the labeled leaves.
Acropetal 13C translocation to current season wood and leaves also was
observed. A few branches (12%) did not show translocation to either fruiting
leaves or different sections of the shoot. The majority of these branches were
from the 44 DAF B pulse.

There were significant differences in the absolute amount of 13C recovered
for each organ (Tables 3 and 4). Most of the translocated 13C was partitioned to
fruit, followed by fruiting spur and non-fruiting spur wood. Lower 13C
recoveries were found in fruiting spur leaves and the terminal shoot. The highest
13C recoveries in fruit were detected at 56 DAFB, followed by 25, 75 and 44 DAF B

(Table 8). The lowest 13C recovery was found at 40 DAFB. Partitioning of 13C to

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non-fruiting and fruiting wood followed an opposite pattern to that of fruit, with
wood 13C contents lowest on those dates in which recoveries in fruit were the
highest, i.e., 56 and 25 DAFB.

The relative partitioning indicated that between 20% and 50% of the 13C
recovered 48 h later was in non-fruiting spur leaves (Figure 6; Appendix B.15).
The rest of the 13C was recovered in different organs in the following order: fruit
(31 to 71%), fruiting spur wood (5 to 17%), non-fruiting spur wood (3 to 8%),
shoot (0 to 0.2%) and fruiting spur leaves (0 to 1%). The highest percentage of 13C
partitioned to fruit occurred at 56 DAFB, followed by the 25 DAFB pulse. The
rest of the pulse-labeling dates showed a similar partitioning. The lowest 13C
recovery in non-fruiting spur leaves was at 56 DAFB. Greater detail for the

minimal 13C partitioning to shoots is shown in Appendix B.16.

c. Terminal shoot leaves as 13C source

Current season shoot leaves were an additional source of photoassimilates
for fruit and vegetative growth during stages I, II and III of fruit development In
all the pulse-labeling dates, 13C fixed by this leaf population was translocated
basipetally to non-fruiting spur leaves, non-fruiting spur wood, fruiting spur
leaves, fruiting spur wood and fruit (Tables 3 and 4). Several branches (16%) did
not translocate 13C to either fruiting or non-fruiting leaves. Significant amounts

of 13C were detected in leaves and wood of current season shoots. However, a

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preferential translocation to fully expanded leaves and developing leaves in the
basal and medial position of the shoot was detected at all labeling dates.

There were significant differences in the absolute amount of 13C recovered
for each organ (Tables 3 and 4). Most of the translocated 13C was partitioned to
fruit, followed by either non-fruiting spur wood or fruiting spur wood (Tables 3
and 4). Lower 13C recoveries were found in fruiting spur and non-fruiting spur
leaves. The highest 13C contents in fruits were found at 56 DAF B, followed by 75
DAFB (T able 9). At these dates, shoots were ~30 cm (~20 leaves) and ~34 cm (~22
leaves) in length (Table 2). The lowest recovery was detected at 25 DAFB, when
shoots were ~10 cm in length and had only ~10 leaves. 13C recoveries in fruiting
and non-fruiting wood were lowest at 56 DAF B.

The relative partitioning indicated that between 31 % and 69% of the 13C
recovered 48 h later was in terminal shoot leaves (Figure 7; Appendix B.17). The
rest of the 13'C was found in different organs in the following order: fruit (18 to
59%), non-fruiting spur wood (4% to 16%), fruiting spur wood (5 to 11 %),
fruiting spur leaves (0 to 1%) and non-fruiting spur leaves (0 to 1%). The highest
percentage of 13C partitioned to fruit occurred at 56 DAF B, followed by 75 and 25
DAFB pulses. The lowest was measured 44 DAFB. The lowest 13C recoveries in
shoot leaves and wood were found at 56 DAFB. More detailed relative 13C

partitioning in shoots is shown in Figures 8 and 9.

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Partitioning of 13C from distal leaf populations

In most of the organs and tissues analyzed for 13C content, translocation
from the leaf populations distal from the organ of interest did not differ
significantly (Tables 10, 11 and 12). Two exceptions were fruit and non-fruiting
spur wood. As indicated above, fruit attracted more 13C translocated from
fruiting spur leaves (Table 10). However, at 56 DAF B, non-fruiting spur
contributed as much as fruiting spur leaves to fruit growth. In addition, non-
fruiting spur and terminal shoot leaves contributed similar amounts of 13C at 75
DAF B. With regard to non-fruiting spur wood tissues, their 13'C content varied
significantly depending on the pulsed leaf population and fruit developmental
stage (Table 11). When fruiting spur leaves were pulsed, the amount of 13’C
recovered in non-fruiting spur wood was lower than when non-fruiting or
terminal shoot leaves were pulsed. For the latter treatments, the highest 13C
levels recovered in non-fruiting wood were at 40 and 75 DAFB, while the lowest

were measured at 25 and 56 DAFB.

13C partitioning in individual fruit

At 48 h after 13C labeling, fruit pulsed directly with 13C02 were highly 13C
enriched at all pulse-labeling dates. The highest 13C contents in individual fruits
were at 25 and 56 DAFB, with 218 and 221 pg 13C / fruit (Table 13). The lowest

contents were at 40 and 75 DAFB, with 116 and 119 pg 13C/fruit.

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The absolute partitioning between pericarp (flesh) and pit (seed) changed
significantly between dates (Table 13). At 25 and 40 DAFB, 75 and 82% of the
total 13C in the fruit was partitioned to the pit (Figure 10; Appendix B.18). At the
beginning of stage III (44 DAFB), the partitioning between fruit tissues was
similar, although more 13C was partitioned to the pit At 56 and 75 DAF B, 78%
and 92% of the total 13C recovered was in the pericarp. Across pulse-labeling
dates, the highest 13C content in the pericarp was detected at 56 DAFB (stage III),
while the lowest occurred at 40 DAFB (stage II). In the case of the pit, the highest

13C content was found at 25 DAFB, while the lowest occurred at 75 DAFB.

Discussion

The partitioning of newly-fixed C during fruit development was studied
in 2-year-old sweet cherry branches of ’Ulster’/ G16, a semidwarfing
scion/ rootstock combination. 13C was used as a tracer to characterize and
quantify the relative partitioning of 13C, fixed as 13COz, by the three major
photosynthetic sources of assimilates within a 2-year-old limb. Branch sections
containing the fruiting spur, non-fruiting spur, and terminal shoot leaf
populations were labeled with 13C during stages I, II and III of fruit development.
Our objective was to elucidate the contribution of each leaf population as a
source of current photoassimilates for fruit and shoot growth during the entire

period of fruit development.

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Translocation patterns differed depending on the leaf population that was
pulsed. Fruiting spur, non-fruiting spur and shoot leaves exported 13C-
photoassimilates to both fruit and shoot growth during stages I, II and III.
Fruiting spur leaves exported 13C to their own fruit and wood. These leaves also
translocated 13C to non-fruiting spurs and terminal shoots. However, a few
branches did not translocate 13C out of the fruiting section. Non-fruiting spur
leaves exported 13C bidirectionally to fruit and terminal shoots. The predominant
translocation of these leaves was basipetally to the fruit A few branches did not
translocate 13C to the shoot. These results are in agreement with our prior study
carried out on ’Sam’ / Gisela 5 limbs in which non-fruiting spur leaves
translocated most of the labeled C to fruit during final swell (Chapter 2). Current
season growth leaves translocated 13C basipetally to the non-fruiting and fruiting
sections. As in the case of spur leaves, the predominant 13C export was towards
fruit When either non-fruiting spur or terminal shoot leaves were labeled, the
wood located basipetally from these sources was highly enriched in 13C.
Unidirectional and bidirectional transport from different leaf populations have
been reported for apple (Malus domestica Borkh.) (Hansen, 1969; Corelli
Grappadelli et al., 1994), sour cherry (Prunus cerasus L.) (Kappes and Flore, 1986;
Toldam-Andersen, 1998), pecan (Carya illinoensis Koch.) (Davis and Sparks, 1974),
grape (Vitis vinifera L.) (Hale and Weaver, 1962), cranberry (Vaccinium

macrocarpon Ait.) (Roper and Klueh, 1996), and red raspberry (Rubus idaeus L.)

138

(Privé et al., 1994) among others. In these species the predominant translocation
is either to fruit or to current season shoots.

13C distribution among different organs varied, depending on the labeled
source (leaf population) and the developmental stage. The 13C relative
partitioning indicated a predominant 13C distribution to fruit regardless of the
photoassimilate source and the stage of fruit deve10pment Fruit were a stronger
sink than current season shoots and had the highest 13C enrichments. The
highest fruit sink strength was detected 56 DAFB (stage III). In peach (Prunus
persica (L.) Batsch), fruit were a stronger sink for photoassimilates than were
stems (Grossman and DeJong, 1995). In sour cherry, the highest fruit sink
strength was during stage III (Flore and Layne, 1999). During stage I, sink
activity of small fruit was an important factor to attract 13C assimilates for cell
division since the highest 13C atom % excess per unit basis was detected at this
time. Increased sink activity of fruit promotes the uptake of assimilates, which in
turn accelerates its growth rate (Hansen, 1987). As in peach (DeJong and
Grossman, 1995), the competitive ability of sweet cherry fruit varies during
development by changing individual sink activity and / or total sink strength. In
sour cherry, small fruit act strongly as sinks by removing C from the
translocation system, which is explained by their high specific growth rate in
young fruit (Toldam-Andersen, 1998). Similarly in grape, sink activity of the
immature small berry was important for DW accumulation during the first week

of growth when cell expansion is slow (Coombe, 1989).

139

There were significant differences among 13C sources regarding the
amount of 13C partitioned to fruit. The more distant the 13C source, the lower the
amount of 13C that was detected in fruit On average, fruiting spur leaves
contributed more 13C (60 to 80%) to fruit than did non-fruiting spur (30 to 70%)
and shoot leaves (18 to 60%). This was the trend for stage I, stage II and the
beginning and end of stage III. The exception was at 56 DAFB (mid-stage III,
~812 GDD), during rapid cell enlargement and dry matter accumulation, when
the amounts of 13C partitioned to fruit were significantly higher than those
detected from any of the 13C-sources at other pulse labeling dates. The lowest 13C
contents in pulsed leaves of all sources were found in stages I (25 DAFB) and III
(56 DAF B). In addition, all wood of different sections had reduced 13C levels
during mid-stage III. The presence of fruit actively demanding photoassimilates
has been reported to reduce CHzO levels in sweet cherry leaves on shoot, non-
fruiting spur and fruiting spur wood (Roper et al., 1988). Similarly, in Japanese
pear (Pyrus pyrifolia Nakai) the 13’C content of spur leaves decreased during the
period of rapid fruit growth (Teng et al., 2001).

At 48 h after pulsing, the amount of 13C recovered per branch was 11 %
and 44% lower than the initial 13’C fixation across all leaf sources. The highest
recoveries (62% to 89% of the 13C fixed initially) were at 25 and 56 DAF B.
Differences in recoveries may have been a consequence of either export out of the
limb or respiratory costs of different sink organs. In a previous experiment using

the same scion / rootstock combination (Chapter 2), girdling at the base of 2-year-

140

old branches increased fruit size and shoot length, indicating CH20 export out of
the limb. Thus, examiningboth studies, it appears some 13C was exported out of
the branch during fruit development In peach, branch autonomy with respect to
C partitioning during stage III of fruit development was not absolute, indicating
C translocation from other sources (Marsal et al., 2003). In addition, some 13C
must have been used in respiration. Respiration costs are greatest for growth of
new organs such as developing fruit and shoots and roots (Lakso et al., 1999).
Loescher et al. (1986) estimated that 16 to 23% of the total CHzO requirements for
sweet cherry fruit growth were used in respiration, while in peach 16 to 20% of
the seasonal CHzO requirements were used by developing fruit (DeJong and
Walton, 1989). A high respiration rate in persimmon (Diospyrus khaki L.) fruit was
important for maintaining sink strength during final swell (Nakano et al., 1998).
Fruit were able to photosynthesize 13COz during stages I, II and III. Fruit
photosynthesis has been reported for sour cherry (Kappes and Flore, 1986; 1989).
The highest 13C fixations were detected during stages I and II. It is likely that the
13C fixed directly by fruit had some impact in the C budget of individual fruits
(Hansen, 1970; DeJong and Walton, 1989; Kappes, 1985). In sour cherry, fruit
gross photosynthesis contributed 19%, 30% and 1.5% of the CH20 used during
stages I, II and III of fruit development, respectively; ~70% of the CHzO was
incorporated into fruit dry matter, while the rest was used in dark respiration
(Flore and Layne, 1999). In apple, fruit photosynthesis is < 15% of the total C

supply during the season (Jones, 1981), although it may contribute to fruit

141

growth early in the season (Lakso et al., 1999). In this study, 2 days after labeling,
13C partitioning between pericarp (flesh) and pit (seed) varied significantly
depending on the fruit developmental stage. The highest 13C contents in single
fruits were detected during stage I (25 DAFB) and the peak of final swell (56
DAFB), while the lowest 13C contents were measured during stage I and at the
end of stage III (75 DAFB). During stages I and II, more 13C (74 to 80%) was
partitioned to the pit However, late in stage III (56 DAFB and 75 DAFB), most of
the total 13C (77% to 83%) was recovered in the pericarp. Teng et al. (2001)
reported that Japanese pear fruit accumulated most of the 13C in its flesh during
the period of active growth. Similar results have been reported for peach fruit
(Corelli-Grappadelli et al., 1996).

Current season growth was not a strong sink for assimilates during sweet
cherry fruit development Minimal amounts of 13C (< 1 %) were found in shoots
when fruiting and non-fruiting spur leaves were labeled. This trend was similar
among the 3 fruit developmental stages. Kappel (1991), using ’Lambert’ sweet
cherry on Prunus avium L. seedling, a vigorous rootstock, reported that current
season growth had a greater sink strength for photosynthates than fruit since
more DW accumulated in shoots. This would imply that differences in source-
sink relationships and relative partitioning might depend on the genotype of the
rootstock (Moing and Gaudillere, 1992; Caruso et al., 1997). In the current study,
when terminal shoots were labeled directly, their mature basal leaves and

developing medial leaves had higher 13C enrichments than those of young apical

142

leaves and wood. Young apical leaves imported minimal amounts of 13C from
spur leaves. More mature leaves at the base of the terminal shoot must have
synthesized adequate CH20 to be partitioned between the shoot tip and fruit.
These results agree with those found in a prior experiment using ’Sam’ on Gisela
5, for which young leaves on current season growth did not constitute a strong
sink for 13*C photoassimilates during stage III of fruit development (Chapter 3).
Interestingly, shoots exported 13C to fruit, even during very early stages of
development (i.e., with only ~10 cm in length and ~10 leaves). In sour cherry,
shoots become net CHzO exporters 17 days after budbreak (Kappes, 1985).
Apple extension shoots begin C export with ~9 to 17 leaves (Lakso and Corelli-
Grappadelli, 1992; Corelli-Grappadelli et al., 1994), while peach extension shoots
begin exporting to fruit 30 DAFB (Corelli-Grappadelli et al., 1996).

The highest 13C export from terminal shoots (60% of the total C recovered
per branch) to other organs was detected at 56 DAF B. At this time, fruit were
rapidly accumulating dry matter and the terminal shoot (~ 30 cm in length and
20 leaves) began decreasing its growth rate. The lowest 13C export from shoots
was detected at the beginning of stage III (44 DAFB, 570 GDD), when shoots
were elongating rapidly. According to these results, the terminal shoot not only
supported its own growth, but was a C source for fruit growth as well. Roper et
a1. (1987) proposed that during stage III, part of the photoassimilates used for
fruit growth might come from single leaves on shoots since spur leaves were not

able to support optimal fruit growth. As mentioned above, fruit was always the

143

strongest aerial sink for current photoassimmilates translocated from different
leaf sources. The priority of the fruit growth over vegetative growth is further
supported by the fact that current season shoots began exporting 13C to fruit as
early as 25 DAFB. It is likely that fruit growth had a detrimental effect on shoot
development in sweet cherry combinations on dwarfing and semi-dwarfing
rootstocks. Grossman and DeJong (1995) reported that the presence of fruit
decreased stem length and DW accumulation in a peach cultivar with a short
fruit growth period, suggesting competition for C between vegetative growth
and fruit.

Significant amounts of 13‘C were found in structural wood of directly
labeled and unlabeled sections, indicating active translocation at the moment of
branch removal. A portion of this 13C must have been utilized for primary
growth of shoots and secondary growth of older wood as observed in apricot
(Costes et al., 2000). When fruiting and non-fruiting spur leaves were 13’C labeled,
wood subtending fruit was the most 13C enriched. When shoot leaves were
labeled, non-fruiting spur wood had higher 13C levels than terminal shoot wood.
For this leaf population, the lowest 13C partitioning to non-fruiting wood was at
56 DAF B, when levels were considerably lower than at other pulse-labeling
dates. During stage II (40 DAFB) and late stage III (75 DAF B), translocation from
the shoot to fruit was reduced since 13C content in non-fruiting spur wood
increased. These observations might suggest the presence of sink limiting

condition at stage II and late during fruit development Sink limitations due to

144

resource restrictions and decreased transport and phloem unloading capacities
have been documented for late maturing peach and plum (Prunus salicina L.)
cultivars (Pavel and DeJong, 1993; DeJong and Grossman, 1995). In sweet cherry,
reductions in translocation might influence C partitioning to fruit in the short
term (Michin et al., 1997), although the vascular system responds is able to
generate a higher translocation rates to keep the fruit growth rate constant
(Bustan et al., 1995; Heuvelink, 1996).

In this study, LA / F ratios were low and varied between 33 and 60 cm2
LA / F (< 1 to 3 leaves / fruit), which indicates a persistent source limitation
during fruit development. Stage I (319-483 GDD) and mid-stage 111 (753-874
GDD) were periods characterized by stronger resource restrictions in C
availability. At stage I, the canopy was still developing and leaves of spurs and
extension shoots were competing for CHzO with young developing fruit
(Chapter 5), while at mid-stage III fruit were accumulating dry matter rapidly.
Failure to grow to full size potential is assumed to be a consequence of source
limitations, i.e., insufficient CH20 for dry matter accumulation (Berman and
DeJong, 1996). However, growth limitations also seem to be determined by
processes in the sink (fruit) itself and by genetic constraints (Starck, 1983;
Marcelis, 1996; Basile et al., 2002). In highbush blueberry (Vaccinium coryrnbosusm
L.), high crop loads during stage I and III imposed source limitations, which
affected fruit and vegetative development (Swain and Darnell, 2002). Reduction

in CH20 availability to support potential growth might lead to competition

145

among individual organs (DeJong and Grossman, 1994; DeJong, 1999). In peach
and apple the maximum potential DW accumulation of an individual fruit is
achieved when most of the fruit are removed early during development
(Grossman and DeJong, 1995; Basile et al., 2002; Palmer, 1992). On the other
hand, in kiwifruit (Actinidia deliciosa (A.Chev.) C.F. Liang et A.R. Ferguson),
shoot elongation during fruit growth had a negative effect on partitioning of 13C
into fruit (Amano et al., 1998). In sour cherry, LA / F ratios < 2 are indicative of
source limitation, which is especially important during stage III, the period of
maximum sink strength (Flore, 1985; Layne and Flore, 1993). In sweet cherry, a
higher LA / F ratio has been postulated to be necessary for fresh market fruit
quality (Whiting and Lang, 2004). In our study, a low fruit DW at commercial
harvest reflected a restriction in photosyntathes to support optimal fruit growth.
Fruit loads accounted for more than 50% of the total DW for an individual
branch at commercial harvest, indicating high biomass allocation to fruit It is
likely that fruit vs. fruit and fruit vs. shoot competition resulted in smaller fruit
and reduced shoot growth, which is a common characteristic of trees on GI
roostocks.

The results provide additional information about the contribution of the
various leaf populations in sweet cherry canopies as sources of C for developing
fruit and vegetative growth. Clearly, the natural balance between reproductive
and vegetative growth is not optimal for production of premium quality fruit in

high-density sweet cherry orchards with dwarfing and semi-dwarfing GI

146

rootstocks. To overcome this situation, a more precise management of LA / F
ratios is critical to achieve not only an optimal crop load, but also an optimal
development of leaf area during the productive years of the orchard (Lang, 2001
a,b).

In summary, in 2-year-old sweet cherry branches, on a semi-dwarfing
scion / rootstock combination: (1) spur and shoot leaves constitute a significant
sources of C for fruit and vegetative growth; (2) current season (terminal) shoot
growth provides a C source for fruit as early as stage I; (3) fruits are a priority
sink vs. new shoot growth, in terms of C allocation during the entire period of
fruit development; (4) the highest fruit sink strength is during stage III; and (5)
resource limitations during fruit development affect final fruit size in semi-
dwarfing combinations (i.e., with rootstocks of the GI series). These results
provide a physiological foundation for canopy relationships that may help to
develop specific orchard management strategies to promote a more sustainable
long-term balance between vegetative and reproductive growth in high density

sweet cherry orchards.

147

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152

Table 1. Fruit quality parameters measured weekly between stages I and III on
’Ulster’/ Gisela 6 sweet cherry branches (19 May to 4 Aug, 2003). Fruit remained
on the tree until 96 DAF B. Mean $ SE, n= 30.

 

 

Developmental DAFBz Fresh Dry Diameter SS
stage Weight Weight (mm) (°Brix)
(g) (23)
Stage I 19 0.6$0.1 0.06 $ 0.02 9.7 $ 0.3 3.4 $ 0.2
26 1.1 $0.1 0.1 $0.02 11.7 $ 0.1 6.6 :t 0.4
Stage II 33 1.2 $0.1 0.2 $ 0.03 12.6 $0.1 8.6 $ 0.1
40 1.4 $0.1 0.3 $ 0.02 13.2 $ 0.1 7.9 $ 0.1
Stage III 47 2.2 $0.1 0.5 $ 0.01 15.1 $0.3 10.6 $ 0.3
54 2.8 $0.1 0.6 $ 0.01 16.0 $ 0.3 12.5 $ 0.3
61 5.4 $ 0.2 1.3 $ 0.3 20.4 $0.3 17.4 $ 0.3
68 6.4 $ 0.2 1.4 $ 0.2 21.3 $0.3 19.0 $ 0.3
Terminal bud set 75 7.3 $ 0.2 1.8 $ 0.1 22.4 $ 0.2 22.1 $ 0.3
82 7.9 $ 0.1 1.8 $ 0.2 23.1 $0.1 23.2 :t 0.4
89 7.9 $ 0.2 2.2 $ 0.5 22.0 $0.2 25.0 :t 0.5
96 8.2 $ 0.2 2.3 $ 0.3 23.2 $0.2 25.0 $ 0.3

 

153

z DAF B: days after full bloom. Full bloom occurred on 30 Apr, 2003.

Table 2. Length and leaf number of current season shoots on 2-year-old
’Ulster’/ Gisela 6 sweet cherry branches between full bloom and terminal bud set
(30 Apr to 4 Aug, 2003). Mean $ SE (n=170).

 

 

 

Developmental DAFBz Shoot Length Leaf Number
Stage (cm)
Total Unfolded

Full Bloom 0 0.5 $ 0.1V 3.1 $ 0.2 0.7 $ 0.2
Fruit set 5 0.7 $ 0.1V 8.5 $ 0.2 6.8 $ 0.3

12 2.5 $ 0.1 9.7 $ 0.2 7.2 $ 0.2

Stage I 19 6.2 :l: 0.1 10.8 :t 0.1 9.3 $ 0.1

26 9.4 :l: 0.2 11.6 $ 0.1 10.0 :I: 0.1

Stage II 33 13.9 :t 0.2 13.1 $ 0.1 11.2 $ 0.1

40 17.9 :t 0.3 15.1 $ 0.2 13.3 :t 0.1

Stage III 47 23.7 :t 0.4 17.3 $ 0.2 15.4 :t: 0.1

54 28.2 $ 0.5 19.1 :I: 0.2 17.6 $ 0.2

61 31.7 $ 0.7 20.4 $ 0.3 19.6 $ 0.2

68 33.2 $ 0.8 21.2 $ 0.3 20.7 :t: 0.3

Terminal bud set 75 33.9 :t: 0.9 21.7 $ 0.4 21.8 $ 0.4
82 34.5 $ 0.9 21.8 :t 0.4 21.8 $ 0.4
89 34.6 $ 0.9 21.9 $ 0.4 21.9 $ 0.4

96 34.7 $ 0.9 21.9 $ 0.4 21.9 $ 0.4

 

Z DAF B: days after full bloom. Full bloom occurred on 30 Apr, 2003.

Y At these dates, a few rudimentary shoots were emerging from apical buds.

154

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157

Table 6. 13C content in fruit sampled immediately (0 h) after labeling of the
fruiting spur leaves at each pulse-labeling date. Mean 3: SE, n=5.

 

Developmental Stage DAFBz pg 13C/ g DW

 

 

Stage I 25 188.3 i 64.8 ay
Stage II 40 69.3 :I: 12.5 b
Stage III 44 98.0 i: 17.6 b
56 8.4 i 2.7 c
75 11.6 :t 3.7 c
I DAF B: days after full bloom. Full bloom occurred
on 30 Apr, 2003.

y Means within a column followed by the same
small letter are not significantly different at a =
0.05. Obtained p-value < 0.0001.

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Table 10. 13C content in fruit, wood and fruiting spur leaves on 2-year-old
’Ulster’/ Gisela 6 sweet cherry branches 48 h after pulsing the fruiting spur, non-
fruiting spur and terminal shoot leaf populations with 13C0; Mean 1 SE, n=5.

 

 

 

 

 

 

 

 

pg 13C/ organ
Pulsed Leaf
Population: Fruiting spurs Non-fruiting spurs Shoot
DAFBz

FSY FRUIT
25 13253.0 i 1457.4 ax 10977.0 :t 1218.1 b 2621.7 :1: 501.7 c
40 10422.0 3: 683.2 a 6274.3 1 1134.8 b 3951.9 :t 640.6 c
44 12506.0 :t 1234.6 a 7241.6 i 1562.4 b 3475.8 :1: 533.4 c
56 28531.0 1 969.0 a 24451.0 :1: 932.0 b 12073.0 :1: 3368.9 c
75 14685.0 :1: 2007.3 a 8949.5 i 1923.2 b 7916.1 :l: 1782.7 b

F5 WOOD
25 802.0 i 199.0 ax 2055.3 :1: 472.7 a 746.3 :1: 129.7 a
40 1789.4 :1: 385.6 a 3355.2 :1: 534.8 a 1766.0 i 405.3 a
44 1825.8 1 391.2 a 3585.7 i 1415.1 a 927.9 :l: 103.2 a
56 935.3 i 242.6 a 1767.5 :1: 290.3 a 887.7 :l: 101.0 a
75 1469.2 i 339.4 a 3017.5 1: 783.7 a 2969.0 i 745.6 a

PS LEA V55
25 8484.5 :t 496.3 W 33.9 i 10.4 ax 22.1 i 15.4 a
40 6404.8 i 762.7 W 138.4 1: 78.7 a 3.8 i 0.4 a
44 9123.7 1: 527.2 W 0.0 i 0.0 a 9.6 i 1.0 a
56 6581.6 :1: 327.9 W 187.3 1 128.2 a 69.2 i 19.0 a
75 10259.0 :t 1689.6 W 48.4 i: 25.8 a 12.2 i: 4.5 a

 

z DAFB: days after full bloom. Full bloom occurred on 30 Apr, 2003.

y PS: fruiting spur.

X Means within a row followed by the same small letter are not significantly
different at a = 0.05.

W Fruiting spur leaves were pulsed directly with 13C02. Leaves were not
considered in statistical analysis

162

Table 11. 13C content measured in non-fruiting spur leaves on 2-year-old
’Ulster’ / Gisela 6 sweet cherry branches 48 h pulsing the fruiting spur, non-
fruiting spur and terminal shoot leaf populations with 13C02. Mean i SE, n=5.

 

 

 

 

 

 

pg 13C/ organ
Pulsed Leaf
Population: Fruiting spurs Non-fruiting spurs Shoot
DAFBz
NFSy WOOD
25 138.4 .+. 47.8 b)( 999.1 i 166.7 a 752.3 1 149.9 ab
40 121.2 i 87.2 c 1519.0 :I: 106.3 b 3019.3 i 761.9 a
44 0.0 i 0.0 b 1747.2 :t 366.9 a 1853.0 i 366.6 a
56 54.8 i 9.1 b 1136.3 1 198.7 a 660.6 :I: 87.4 ab
75 110.0 1 11.2 c 1297.9 :t 213.0 b 2587.4 1 229.8 a
NFS LEAVES
25 165.7 3: 87.6 ay 9992.6 i 1204.7W 35.9 i 13.1 a
40 47.2 i 4.7 a 8405.4 i 735.8W 21.9 i 9.4 a
44 0.0 i 0.0 a 10684.0 1 1034.4W 0.0 i 0.0 a
56 116.5 :t 74.5 a 6892.3 i 13092.0W 71.4 i: 31.2 a
75 55.1 i 8.2 a 13092.0 i 1371.8W 43.5 i 10.3 a

 

Z DAF B: days after full bloom. Full bloom occurred on 30 Apr, 2003.

Y NFS: non-fruiting spur.

x Means within a row followed by the same small letter are not significantly
different at a = 0.05.

W Non-fruiting spur leaves were pulsed directly with 13C02. Leaves were not
considered in the statistical analysis.

163

Table 12. 13C content measured in basal, medial and apical leaves and wood of
terminal shoots on 2-year-old ’Ulster’/ Gisela 6 sweet cherry branches 48 h after
pulsing the fruiting spur, non-fruiting spur and terminal shoot leaf populations
with 13C02. Mean 1 SE, n=5.

 

pg 13C/ organ

 

 

 

 

 

 

 

 

 

Pulsed Leaf
Population: Fruiting spurs N on-fruiting spurs Shoot!
DAFBz
TSY BASAL LEAVES
25 10.1 i 7.1 ax 16.6 i 5.6 a 1568.7 1: 294.5W
40 5.0 i: 2.4 a 17.4 :t 10.7 a 2952.7 i 343.9W
44 0.0 i 0.0 a 1.6 i 1.5 a 4960.6 :t 761.9W
56 17.1 :t 3.8 a 29.2 i 8.4 a 1774.0 :1: 380.0W
75 19.4 d: 2.7 a 51.1 i 22.1 a 5073.5 :1: 832.3W
TS MEDIAL LEA VES
25 15.1 i 11.5 a 13.9 i 3.7 a 2368.3 i 301.0W
40 0.8 i 0.4 a 3.5 i 1.3 a 4169.8 :1: 603.3w
44 0.0 i: 0.0 a 29.1 i 25.3 a 4247.1 3: 585.6W
56 7.6 i 2.1 a 24.4 :t 5.9 a 1733.2 1 105.3W
75 24.3 i 9.2 a 36.8 :t 13.4 a 4494.7 1: 559.8"
TS APICAL LEAVES
25 1.3 i 0.4 a 2.5 i 0.6 a 784.4 i 218.7W
40 0.6 i 0.4 a 8.6 :t 6.2 a 1330.4 :1: 259.6W
44 0.0 i 0.0 a 39.7 i 3.5 a 2724.2 1: 368.7W
56 5.4 :t 1.1 a 13.0 :l: 3.7 a 1335.1 1 321.6W
75 7.6 i 2.3 a 17.4 i 6.2 a 2653.7 1 774.1W
TS WOOD
25 3.6 :t 1.7 a 6.7 i 2.9 a 493.5 1 146.5W
40 1.5 i: 0.7 a 10.4 :1: 4.3 a 1078.3 :1: 307.2W
44 0.3 i 0.3 a 46.2 :t 34.0 a 1764.8 1: 327.9w
56 10.0 i 3.2 a 26.3 :t 4.3 a 512.9 d: 59.4W
75 22.5 i 7.7 a 42.7 :t 16.8 a 1518.1 1 175.9W

 

zDAFB: days after full bloom. Full bloom occurred on 30 Apr, 2003.

Y TS: terminal shoot.

x Means within a row followed by the same small letter are not significantly different at a =

0.05.

W Leaves were pulsed directly with 13C02. Leaves were not considered in statistical analysis.

164

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175

CHAPTER V

13C- PHOTOASSIMILATE PARTITIONING IN SWEET CHERRY
DURING EARLY SPRING

176

13C- Photoassimilate Partitioning in Sweet Cherry

During Early Spring

Keywords: Prunus avium L., storage reserves, transition, spring remobilization,

sink, Gisela rootstock, stage 1, fruit set.

Abstract

In deciduous fruit trees, storage reserves accumulate during fall and are
used for early spring growth. In sweet cherry (Prunus avium L.), stored reserves
are critical for early growth and there is a transition phase during which current
photoassimilates become the primary source for support of reproductive and
vegetative sinks. As little is known about this transition, an experiment using 4-
year-old ’Regina’ sweet cherry on the semidwarfing rootstock, Gisela 6, was
established. Using whole canopy exposure chambers, five trees were pulse-
labeled with high levels of ”€02 three times during fall (Aug-Sep). At leaf drop,
leaves, buds, wood, bark and roots were sampled for gas chromatography-mass
spectrometry (CC-MS) analysis of pre-winter storage reserves. The major
storage organs (i.e., those with the highest 13C atom % excess) were roots, older
wood in the trunk and branches and buds. During spring, newly developing
organs (flowers, fruits and leaves) were sampled weekly from bloom to stage III

of fruit development for additional GC-MS analysis. The 13C-reserves were

177

remobilized and partitioned to flowers, fruits and young leaves from before
budbreak (side green) until 14 days after full bloom (DAFB). The highest 13C
levels in growing sinks were detected between bloom and fruit set The isotopic
composition of new organs differed significantly among organs and phenological
stages. Reproductive organs had the strongest sink activity until 14 DAFB, but in

terms of total dry matter, non-fruiting spurs had the highest sink strength.

178

Introduction

In most deciduous woody perennials, the primary sources of assimilates
are newly-synthesized photosynthates and accumulated reserves (Oliveira and
Priestley, 1988). Storage reserves have been defined as materials or substances
(organic compounds and nutrients) produced in excess of current requirements
and which may be used later in support of metabolism and growth (Priestley,
1960; Glerum, 1980). Carbohydrates (CH20), in the form of starch and soluble
sugars, are the major component of reserve materials in the tree, but nitrogen
(N), in the form of proteins and amino acids, and other minerals also are
important (Tromp, 1983). Reserves accumulate in various organs including buds,
leaves, branches, stems, roots, seeds and fruits (Kozlowski and Pallardy, 1997).

Storage reserves are important for several life processes such as winter
survival, metabolism, respiration, defense, healing, vegetative and reproductive
growth, fruit development and new growth in spring (Kandiah, 1979a,b; Flore et
al., 1983; Oliveira and Priestley, 1988; Loescher et al., 1990; Kozlowski and
Pallardy, 1997). Several authors indicate that the initial stages of spring growth of
deciduous fruit trees must depend upon mobilization of reserves accumulated
the previous season, until new leaves become photosynthetically competent to
provide new assimilates (Priestley, 1960; Quinlan, 1969; Hansen, 1967; Hansen,
1971 ; Oliveira and Priestley, 1988). Storage reserves are utilized in new growth

and respiration to provide energy and cellular structure materials before root N

179

uptake and photosynthesis occurs in spring (Hansen 1967; Cheng and
Fuchigami, 2002).

In sweet cherry (Prunus avium L.), flowering usually occurs before leaves
are fully expanded, and early stages of reproductive (flower and fruit) and
vegetative (spur, extension shoot and root) growth are dependent on the storage
reserves accumulated the previous fall (McCammant, 1988; Keller and Loescher,
1989). Thus, early fruit growth seems to be solely dependent upon stored CH20
reserves (Lang, 2001 a). Other deciduous trees such as apple are less dependent
on stored reserves since canopies are developed more fully before bloom (Keller
and Loescher, 1989; Hansen, 1971). An intermediate situation has been described
for Japanese pear (Pyms pyrifolia N akai), which usually has ~30% of the final leaf
area at full bloom (Teng et al., 1999).

The major accumulation of reserves in perennial structures begins after
terminal bud set (Oliveira and Priestley, 1988). In sweet cherry, storage reserves,
mainly starch, accumulate in different organs after fruit ripening and cessation of
shoot extension, reaching a maximum concentration at leaf abscission (Keller,
1986; Keller and Loescher, 1989; McCammant, 1988). In spring, activated
meristems draw upon assimilates from storage organs throughout the tree
(Tromp, 1983). This continues until new leaves become competent sources of
photoassimilates and other parts of the tree require nutrients for metabolism
(I romp, 1983). Premature leaf abscission might result in CH20 storage limitation;

therefore, any type of biological stress (e. g., leaf damage due to pests and

180

diseases) should be avoided since it might reduce the amount of storage CH20
available for new growth the next year (Flore, 1994). In sweet cherry and pecan
(Carya illinoensis Koch), premature defoliation reduced the accumulation of
storage reserves in fall (Worley, 1979; McCammant, 1988). In grapes (Vitis vinifera
L.), premature defoliation altered the natural translocation pattern of storage
reserves and dry matter partitioning (Candolfi-Vasconcelos et al., 1994).

The hypothesis that storage reserves are a source of carbon (C) for initial
fruit growth during stage I before current photoassimilates become the major C
source was tested in sweet cherry. C partitioning in fall and remobilization of
reserves in spring were studied in young, fruiting ’Regina’ sweet cherry on the
semi-dwarfing rootstock, Gisela 6 (Prunus cerasus x P. canescens). 13C was used as
a tracer to distinguish between the two main sources of assimilates for early
spring growth, those synthesized and accumulated the previous fall (i.e., storage
reserves) and current photosynthates produced during the following spring by
newly expanded leaves. 13C constitutes a useful physiological technique since C-
3 plants discriminate against 13C02 during photosynthesis and it has been used
to study the fate of C in other species (Farquhar et al., 1982; Boutton, 1991 ; Teng
etaL,1999)

The main objectives of this study were: (1) to study the distribution of 13C-
reserves among organs during early spring following 13C02 assimilation the
previous fall; (2) to elucidate whether storage reserves constitute a C source for

initial fruit growth during stage I; and (3) to define the C source transition phase,

181

during which the dependence of new growth on storage reserves shifts to current
photosynthate assimilation as the primary source for vegetative and

reproductive development.

Materials and Methods

Plant material

The experiment was conducted at Michigan State University’s Clarksville
Horticultural Experimental Station, Clarksville, Michigan. Five orchard grown 4-
year-old sweet cherry trees of ’Regina’ on the semi-dwarfing rootstock Gisela 6
(G16) were selected for pulse—labeling with high 13C02 levels in fall 2002. Trees
were trained to a central leader and had similar height, trunk cross-sectional area
(TCSA) and leaf area (LA, ~11.3i0.5 m2). During spring 2003, 2-year-old limbs on
2— and 3-year-old trunk sections bloomed for the first time. Trees were not
pruned during the experiment. Trees were fertilized and microsprinkler irrigated

following standard commercial practices.

13C labeling

After terminal bud set in 2002, each tree was enclosed in a transparent
polyethylene balloon (volume 6.3 m3) and pulsed for 20 min with 13C02. A total
of 5.1 mmol of 13C02 was injected into each balloon. 13COz was generated by
adding 5.0 ml of 80% lactic acid to each of two 1 L plastic bottles containing 5 g of

barium carbonate (98 atom% 13C). As the reaction generated 13C02, the plastic

182

bottles were pumped manually into the balloon via plastic tubing. The labeling
was carried out during the morning of sunny days between 10:00 AM and 12:00
PM. The first pulsing was done on 12 Sep and was repeated on 25 Sep and 12 Oct
to assure adequate labeling of reserves. Labeling conditions were similar
between labeling dates; however, light and temperature levels varied with
ambient conditions. Net assimilation rate (A) was measured at each labeling date
with a CIRAS—2 infrared gas analyzer (PP-Systems Inc, Haverhill, Massachusetts,
USA) and ranged from 3.0 to 14.1 umol m-2 s-1 on 12 Sep, 2.2 to 9.3 umol m'2 5‘1

on 25 Sep, and 0 to 8.6 umol rn'2 s-l- on 12 Oct

Growth measurements

Growth of target sinks (flowers, fruits, spurs and current season shoots)
was quantified weekly during spring 2003. Four representative shoots per tree
were measured for extension growth and leaf number (total folded and unfolded
leaves). A sample of 25 fruits was measured for fresh weight (FW), diameter and
soluble solids (SS). A sample of 10 fruiting spurs, 1O non-fruiting spurs, 1O
shoots, 50 flowers and 50 developing fruit, were collected weekly for FW and dry
weight (DW) determinations.

The total number of apical and lateral meristems (fruiting spurs, non-
fruiting spurs, and single buds) were counted soon after budbreak (Apr, 2003)
and at subsequent terminal bud set (Aug, 2003). This included potential fruits

counted during bloom and at fruit set (see Appendix C1).

183

13C Sampling and analysis

Two sets of plant tissues were sampled from the labeled trees. The first
consisted of trunk, branch and root sections (Appendix C.2), which were
collected soon after leaf abscission (2 Nov, 2002) and at budbreak (’side green’
stage) of bud development (19 Apr, 2003). Small (5x5 cm) patches of wood and
bark, from 2002 growth, were removed from each tree at different locations along
the trunk and branches. Wood samples consisted of the xylem tissue. Bark
samples included periderm, phloem and cambium. Fruiting spurs and single
buds were collected randomly throughout the canopy. Roots were collected in
the first 60 cm of the root zone below the surface by excavating at four points
around the trunk between and within rows. Roots then were separated according
to size into fine (< 1mm), medium (1-5 mm) and coarse (> 5 mm).

The second set of plant tissues were from actively growing vegetative and
reproductive aerial organs sampled during spring and summer (May to Jul) of
2003. These included fruiting spurs, non-fruiting spurs, single buds, single
flowers, spur flowers, young leaves at the tip of current season growth and fruits
(Appendix C3). Additional samples of the same organs were collected from
three unlabeled trees for natural abundance calculations.

Samples were frozen immediately in liquid nitrogen for subsequent 13C
enrichment determination by gas chromatography mass spectrometry (GC—MS).

The plant material was oven-dried at 70°C for 72 h and subsequently ground

184

using a Wiley mill (20 and 40 mesh). 13C enrichment was calculated according to

Boutton (1991) and Vivin et al. (1996) as follows:

513C (%o) = [(Rsample-Rstandardv Rstandard] x 1000 Eq (1)
Rsample = 13C/12 = [(513C/1000) + 1] x Rpm; Eq (2)
F =13C/(13C + 12C) = R/(R + 1) Eq (3)
Atom% excess = (Fpostdose- F baseline) x 100 Eq (4)
New 13C content = (Atom% excess / 100) x dry matter x [C] Eq (5)

where the 813C ( %o) is calculated from the measured carbon isotope ratios of the
sample and standard gases Eq. (1). The absolute ratio (R) of a sample is defined
by Eq. 2, where Rpm; = 0.0112372. 13C abundance in the sample is expressed as 13C
atom% excess. This value is used as an index to determine the enrichment level
of a sample following the administration of the 13C tracer in excess of the 13C
baseline (atom % approx. 1.108%) prior to the 13CO: pulse (Eq. 3 and 4). The new
13C content is calculated for the different organs according to dry mass and 13C
concentrations. The absolute amount of recovered 13C for each organ was

expressed as ug 13C (Eq. 5).

185

Statistical Analysis
Analysis of variance was conducted by using PROC MIXED procedures of
the SAS statistical analysis program (SAS Institute Inc, Cary, NC). Covariance

analyses were conducted for repeated measurements during spring.

Results
Phenological characterization

During spring 2003, 2-year-old limbs (growth of 2001) on 2- and 3-year-
old trunk sections bloomed for the first time. The rest of the canopy was
comprised of vegetative growth, which included 1-year-old shoots (growth of
2002) with non-fruiting spurs and current season growth (growth of 2003) with
single leaves.

The first visual sign of budbreak (’side green’ stage) was observed 15 days
before full bloom (DBF B), after an accumulation of 148 growing degree-days
(GDD, base 44°C). At first or early bloom (~ 4 to 6 DBFB; 239-263 GDD), only
single and spur flowers and non-fruiting spurs were grong actively. Single
flower buds at the upper section of the limb (at the base of 2002 growth) bloomed
earlier than spur flower buds (those on 2001 growth). At full bloom (264-287
GDD), growth was evident for the organs described above plus fruiting spurs
and young leaves of growing shoots. Fruit set occurred (~ 4 to 7 days after full

bloom (DAFB) 288-342 GDD). Stages I, II and III of fruit development occurred

186

from ~ 8 to 26 DAF B (2343-522 GDD), 27 to 38 DAF B (523-680 GDD) and 39 to 63
DAFB (681-1102 GDD), respectively (see Appendix C4).

The sequential order in which organs began exhibiting visual signs of
growth was: single flower, spur flower and non-fruiting spur meristems,
terminal shoot meristems, and finally fruiting spur meristems. Dry matter
increased for all organs until 35 DAFB. At that point, foliar growth of fruiting
and non-fruiting spurs ceased, but shoots and fruits continued accumulating DW
(Appendix C5 and C6).

Current season shoot length and leaf number increased rapidly from 14
DAFB until 49 DAF B (Table 1). On the other hand, fruit showed a rapid increase
in growth beginning 42 DAFB, with 40% of final size achieved during stage III
(Table 2). The sigmoidal and double sigmoidal growth curves of current season

shoots and fruits, respectively, are shown in Appendix C.7.

13C-labeled storage reserves at leaf abscission

The levels of 13C in all organs at leaf abscission (N ov, 2002) were above
natural abundance. However, 13C varied significantly among organs (Figure 1;
Appendix OS for statistics). The highest 13C levels, expressed as higher 13C atom
% excess, were detected in 2- and 3-year-old wood (grown in 1999 and 2000) of
the trunk, roots (coarse and medium) and vegetative buds. Significant 13C levels
also were found in younger wood (2001) of the trunk, as well as branches,

fruiting buds and fine roots. Current season growth (2002) and bark from

187

sections of various age had significantly lower 13C atom % excess. At leaf
abscission, 13C content in leaves was ~74% lower than that measured in leaves
immediately after labeling. The 13C loss due to leaf abscission was ~14% of the

13C fixed in fall.

13C-Reserve partitioning at budbreak

The levels of 13C in all organs collected at side green (Apr, 2003) remained
above natural abundance (Figure 2; Appendix 8 for statistics). However, 13C-
reserves were either remobilized or utilized during the period between leaf
abscission and budbreak. The highest 13C atom % excess values at budbreak were
detected in fruiting buds, non-fruiting buds and coarse roots. Significant 13C
levels also were detected in medium roots, fine roots and wood grown in 2001
(trunk and branch). The rest of the organs had lower 13C atom % excess values
that were not different from each other.

In most of the organs, 13C atom % excess detected at budbreak was lower
or similar to those values measured at leaf abscission (Figure 3; Appendix 8 for
statistics). The only exceptions were fruiting and non-fruiting buds, which had
higher 13C atom % excess at budbreak than at leaf abscission. The greatest
increase (~45%) in 13C atom % excess values was detected in fruiting buds. The
greatest reductions in 13C atom % excess were detected in 2- and 3-year-old wood

of the trunk, followed by those of most of the bark sections. 13C levels of roots of

188

all sizes and 1-year-old wood (grown in 2001) did not show a significant change

between leaf abscission and budbreak.

13C-Reserve partitioning durin g early spring

13C levels significantly higher than the natural abundance were detected
from first bloom (~ 6 DBFB) until the beginning of stage I (~14 DAFB). Significant
differences in 13C atom % excess were detected among aerial organs within
specific developmental stages, indicating differences in the level of dependency
on storage reserves (Figure 4; Appendix 9 for statistics). Moreover, 13C levels
decreased in all organs with time and there were significant differences between
stages. From 21 to 35 DAFB, 13C contents were relatively constant for all organs,
indicating either a decline in dependence on, or a steady depletion of, 13’C storage
reserves.

13C atom % excess values were highest during first and full bloom (Figure
5; Appendix 9 for statistics). During first bloom, spur flowers had the highest 13C
levels followed by single flowers and non-fruiting spur leaves. At this stage,
fruiting spur leaves and shoot leaves had not yet begun to grow. At full bloom,
similar 13C levels were detected in flowers (single and spur clusters) and young
shoot leaves, followed by non-fruiting spur leaves. Fruiting spurs had the lowest
13C content. At this stage, shoots were 0.3 cm in length and had 3 small

developing leaves (Table 1).

189

Between full bloom and fruit set, a dramatically lower 13C atom % excess
(i.e., lower 13C enrichments) was observed in all organs. However, 13C levels
were still higher than natural abundance values. At fruit set (~7 DAF B), fruiting
spur leaves and tiny fruits (0.3 g FW) had the highest 13C contents, followed by
non-fruiting spur leaves. In contrast to full bloom, fruiting spur leaves had the
highest, and shoots the lowest, 13C gains. At this stage, shoots were 1.9 cm in
length with 9 developing leaves (Table 1).

At the beginning of stage I (~14 DAFB), fruits had the highest 13C levels,
followed by fruiting spur leaves. Non-fruiting spur leaves and shoots had the
lowest 13C levels. Later in stage I (~21 DAFB), 13C levels reached their lowest
point. For the first time, non-fruiting spurs had the highest 13C atom % excess
compared to the rest of the organs. The 13*C levels in shoots (4.8 cm in length and
10 leaves) were closest to natural abundance values. After this, relatively
constant 13C levels for all organs, especially for shoots, indicated minimal
additional contributions from 13C-reserves.

As indicated above, 13C content varied not only among organs but also
among developmental stages. Reproductive and vegetative tissues, collected at
leaf abscission and at and after budbreak, had a distinct 13C seasonal fluctuation
pattern (Figure 6). At leaf abscission, vegetative meristems were even more
highly enriched with 13C than reproductive buds. However, this was reversed at
budbreak through bloom, indicating remobilization of 13C-reserves from other

storage organs to flower buds. 13C contents of reproductive organs (flowers and

190

fruits) also were higher than those of vegetative buds at 14 DAFB. During later
stages of fruit development, this situation was again reversed, with higher 13C

contents in vegetative buds.

Relative 13C-reserve partitioning throughout the canopy during spring

Considering the 13C gain and the total number of units (i.e., total DW) for
a particular organ at each developmental stage, the total 13C partitioning
(expressed as pg 13C) was calculated for each of the aerial organs sampled
through 28 DAFB. After that, 13C levels of all organs remained close to natural
abundance levels and did not vary considerably. These calculations provide a
better understanding of the absolute amount of 13C that was partitioned to all
units of a particular organ at specific stages. Clearly, spurs, flowers and shoots
compete for 13C reserves during bloom. However, the greatest relative
partitioning was to non-fruiting spurs (Figure 7; Appendix 10 for statistics). The
highest recovery for this organ type occurred at full bloom, when spur leaves
were actively growing. Single and spur flowers used 13C reserves in low amounts
relative to those partitioned to non-fruiting spurs. In terms of flower types, spur
flowers had a greater relative 13C demand than single flowers.

Between fruit set and 28 DAF B, the pattern of partitioning among organs
was consistent. Non-fruiting spur leaves attracted most of the labeled reserves,
while partitioning to fruit, fruiting spur leaves and shoot leaves resulted in low

and similar 13C contents. Fruit did not attract an important amount of 13C and the

191

highest gains were detected at 7 and 14 DAFB, when fruit DW were only 33 and

150 mg, respectively.

Discussion

Carbon partitioning in fall and remobilization of reserves in spring were
studied in 4-year-old sweet cherry trees on a semi-dwarfing rootstock. 13C was
used as a tracer to distinguish between the two main sources of assimilates for
early spring growth, those synthesized and accumulated the previous fall (i.e.,
storage reserves) and current photosynthates produced during the following
spring by newly expanded leaves. The main objective was to study the
partitioning of CHzO reserves among organs in spring, and to characterize the
transition phase, in which storage reserves are depleted and current
photosynthates become the primary source for vegetative and reproductive
growth.

Two types of information are reported here, the 13C abundance (as 13C
atom % excess) and the absolute amount of 13C recovered for each organ (as
ug13C). The first value is indicative of the 13C gain per individual organ with
respect to its 13C natural abundance level, which we suggest to be an index of
sink activity. The second value considers the amount of 13C partitioned to a
specific organ in terms of dry matter (i.e. total number of units) and provides an

estimate of the sink strength of that organ.

192

At leaf abscission, the 13C content in leaves was ~74% lower than that
measured immediately after labeling, indicating either 13C translocation to other
organs, respiratory loss or both. 13C loss due to leaf abscission was ~14% of the
13‘C fixed in fall. Basipetal translocation of CH20 and other nutrients from leaves
to perennial storage organs, after terminal bud set and prior to leaf drop, have
been reported in apple (Kandiah, 1979a,b; Quinlan, 1969), Japanese pear (Teng et
al. 1999), grape (Hale and Weaver, 1962; Araujo and Williams, 1988), pecan
(Lockwood and Sparks, 1978a,b; Davis and Sparks, 1974) and sweet cherry
(Loescher et al., 1990) to become part of structural growth or storage reserves
(Oliveira and Priestley, 1988). The higher 13C accumulations were detected in
older wood of the trunk (1999 and 2000), coarse roots and vegetative buds. Less
important storage organs were younger wood in trunk and branches, fruiting
buds and fine roots. Bark from different sections did not store much 13C
compared with the other organs. This was in contrast to high CH20
accumulation in wood of the trunk and older branches has been found in sweet
cherry and apple (Keller and Loescher, 1989; Greer et al., 2002).

At budbreak, the pattern of 13C distribution throughout the tree was
different from that at leaf drop. The 13’C content of wood and bark from older
sections (1999 and 2000) had decreased significantly. However, fruiting buds had
13C contents that were dramatically higher than those at leaf drop, indicating that
13C-reserves were remobilized from other storage organs during the period

between dormancy and budbreak. It is likely that 13C-reserves were translocated,

193

prior to bloom, from wood and bark of the trunk to the reproductive meristems,
based on fluctuations in 13C levels between leaf drop and budbreak.

Reproductive meristems had the strongest sink activity for the
remobilized 13C-assimilates even before budbreak, and these continued being a
priority for 13C partitioning until 14 DAFB. Remobilization and utilization of
storage reserves for metabolism during dormancy as been reported previously
(Tromp, 1983; Priestley, 1981 ; Oliveira and Priestley, 1988). It was not the aim of
this research to develop a comprehensive accounting of 13C-reserve use, as by
non-cropping-related sink activities (such as those of roots, phloem and cambial
growth), but it is possible that before budbreak, some of the 13C reserves already
had been used for these additional sink activities as reported by Oliveira and
Priestley (1988). Keller and Loescher (1989) indicate that aboveground sweet
cherry tissues begin to utilize CH20 in late winter and interconversions of starch
to soluble sugars in wood and bark occur during dormancy. Similar reductions
in the amount of reserves and remobilization from roots and stems to
meristematic regions over winter have been reported for apple (Hansen, 1967;
Quinlan, 1969; Priestley 1981). In this species, the first indication of phloem
differentiation appears in early April, preceding xylem development by ~6
weeks (Evert, 1963). Certainly, some of the 13C-reserves were used for respiration
over the course of fall and winter as well. CH20 reserve depletion and

concentration gradients during dormancy have been attributed to maintenance

194

respiration and bud development, which is related to temperature (Oliveira and
Priestley, 1988; Ogrén, 2000).

Interestingly enough, 13’C-content of roots, considered the major storage
organ in sweet cherry (Loescher et al., 1990), did not vary at least until budbreak.
These results are in agreement with those reported by Keller and Loescher (1989)
which that indicate that in sweet cherry, root CHzO reserves do not decrease
until budbreak. It is possible that in sweet cherry, 13C-reserves in roots may
constitute a source of C for aerial sinks later in spring; that is, the first reserves
used may be those closest to the sites of sink activity, with distant storage sites
such as roots being remobilized only as more localized reserves are depleted,
forming a gradient of sorts as reported in kiwifruit (Actinidia deliciosa (A.Chev.)
C.F. Liang et A.R. Ferguson) and peach (Prunus persica (L.) Batsch) (Greaves et
al., 1999; Jordan and Habib, 1996). The utilization of sweet cherry and apple root
CH20 reserves is soil temperature dependent, with little depletion at
temperatures <10°C (McCammant, 1988; Greer et al., 2002). However, it may be
possible that after leaf abscission, 13C continued being translocated to roots from
aerial storage organs, thereby, offseting any losses to respiration and / or root
growth. If so, the utilization or remobilization of 13C reserves in roots may not be
noticed in early spring. It is also interesting that in fine roots 13C contents did not
decreased between leaf abscission and budbreak. Fine roots of sweet cherry have
been suggested as storage organs by Keller and Loescher (1989). The contribution

of root reserves to new spring growth is unclear and might depend on species,

195

tree age (i.e., root to shoot ratio), cultivar and rootstock (Loescher et al., 1990;
Priestley, 1981). In peach, rootstock vigor, crop load and the time of ripening
affected the extent of CHzO reserve utilization among cultivars (Inglese et al.,
2002). In prune (Prunus domestica L.), the rootstock genotype modified the
kinetics of CHzO mobilization and interconversion in the dwarfing rootstock
’Pixy’ (Gaudillere et al., 1992). Additional information is required to elucidate the
role of roots as a storage organ for more dwarfing combinations.

Various studies have reported that storage reserves are important to
support early spring growth (flowers, leaves, shoots and fruits) in deciduous
species (Quinlan, 1969; Hansen and Grauslund, 1973; Lockwood and Sparks,
1978a, b; Tromp, 1983; Oliveira and Priestley, 1988; Loescher et al., 1990;
McArtney and Ferree, 1999). In our study, we confirmed that early spring growth
of sweet cherry flowers, fruits, spur leaves and shoots was supported by reserves
accumulated the previous fall. Mobilization of stored 13C to new aerial growth
was detected before budbreak and continued until 14 DAF B, when spur and
shoot leaves were not yet fully developed. Therefore, reproductive and
vegetative growth competed strongly for remobilized storage reserves during
bloom and initial fruit growth. The use and competition for storage reserves in
early stages have been studied in apple (Hansen, 1967; Quinlan, 1969; Hansen,
1971 ; Kandiah, 1979a,b), pecan (Lockwood and Sparks, 1978a, b) grape
(Scholefield et al., 1978), apricot (Prunus armeniaca L.) (Costes et al., 1995) and

Japanese pear (Teng et al., 1999). The level of dependence on, and competition

196

for, reserves may be influenced by the order in which organs begin growing in
spring. The importance of budbreak phenologies for potential partitioning effects
among cultivars has been reported previously for two Southern highbush
blueberry (Vaccinium corymbosum L.) cultivars, ’Misty’ and ’Sharpblue’ (Maust et
al., 2000). In ’Sharpblue’, floral and vegetative budbreak occurs simultaneously,
while vegetative budbreak in ’Misty’ occurs several weeks after floral budbreak.
Root starch concentrations decreased ~65% between dormancy and bloom in
’Sharpblue’, indicating a strong mobilization of reserves before budbreak. In
contrast ’Misty’ root starch concentrations decreased only ~35%. The increased
rate of starch depletion in 'Sharpblue’ during the period prior to bloom resulted
in a greater rate of leaf development relative to ’Misty’, which in turn resulted in
an increase in newly-synthesized CH20 to supply developing fruit and replenish
root CH20. In japanese pear, initial growth of leaves and shoots is more
dependent on storage reserves than are organs that develop later (Teng et a1.,
1999). Similarly, in apple extension shoots, leaves developed earlier in spring
were more dependent on storage reserves than were upper (later developing)
leaves (Quinlan, 1969).

The greatest dependence of sweet cherry ’Regina’ on storage reserves was
at bloom; after this, utilization of 13C-reserves declined. At fruit set, 13C levels of
different growing organs were lower but the competition continued. The
demand of individual organs varied during this period, which was reflected in

their 13C concentrations. Accordingly, the highest sink activity was detected in

197

reproductive organs (flowers and fruit). However, considering the total number
of units for a particular organs type or tissue, vegetative structures had the
highest sink strength for 13C-reserves. At 14 DAFB, the 13C concentration in all
organs decreased, indicating a decline in dependence or depletion of 13C-
reserves. Keller and Loescher (1989) demonstrated that CHzO in sweet cherry
roots, wood and bark decline rapidly during full bloom. Similar reductions in
storage reserves during spring, especially in roots, have been reported for apple
(Priestley, 1960; Quinlan, 1969; Hansen, 1967; Kandiah, 1979a,b), pecan
(Lockwood and Sparks, 1978 a,b) and japanese pear (Teng et al., 1999). Depletion
of storage reserves after bloom has been attributed to the abscission of floral
tissues and unfertilized flowers (Hansen, 1971 ; Teng et al., 1999). Other reports
indicate that reserves decrease after budbreak primarily due to respiratory loss,
with a small portion used for new reproductive and vegetative growth (Hansen,
1967; Hansen and Grauslund, 1973; Kandiah, 1979a,b). In apple, most of the fruit
growth depends on current photosynthates produced by newly formed leaves
and only a small portion (<20 to 25%) of the reserves is used for new growth
(Hansen, 1967; Hansen and Grauslund, 1973; Kandiah, 1979b; Johnson and
Lakso, 1986). Hansen (1971) suggested that 50 to 75% of the structural materials
of flowers and shoots come from storage reserves until flowers show color and
shoots have developed 5 to 6 leaves (i.e., ~200 mg DW/ spur and 500-1000 mg
DW/ extension shoot). This seems to be the case in sweet cherry as well since the

current results indicate that of total 13C fixed, only between 3 to 11% was

198

partitioned to new aerial organs until 14 DAF B, when extension shoots had ~ 5
leaves and fruit were 12 mm in diameter.

In sweet cherry, final fruit size is dependent on cell division (stage I) and
subsequent cell elongation at final swell (stage III). There is not detailed
histological information for sweet cherry fruit, but in sour cherry (Prunus cerasus
L.) fruit, cells of the mesocarp increase in number during the pre-bloom stage
and stage I, which is the period of maximum division (Tukey and Young, 1939).
Scorza et al. (1991) indicate that in peach, cell number in the mesocarp and not
cell size is the major difference between small- and large-fruited peach cultivars
and this difference is detected early during the growth of the ovary (~175 days
before full bloom). Lang (2001a) proposed that N and CH20 reserves are critical
for final flower development, bloom and fruit set in sweet cherry. At this time,
cell division is taking place rapidly in young shoots and fruits, defining final fruit
potential size and spur leaf area. These findings confirm that storage reserves are
the major C source for reproductive organs during bloom and early stages of
fruit cell division. At these early stages, competing sinks are source limited
because the canopy is not fully expanded. Source limitation results in insufficient
C availability to support potential organ growth (DeJong and Grossman, 1995). A
period of extreme source limitation might occur in sweet cherry using dwarfing
and semi-dwarfing rootstocks, which bloom heavily and begin cropping
excessively about the 4th or 5th leaf, resulting in reduced final fruit size. The

timing of reserve utilization by reproductive meristems, in competition with

199

other sinks through 7 to 14 DAF B, suggests that reserve levels may be a potential
determinant for variation in fruit set and final fruit size in less vigorous sweet
cherry trees.

It is important to point out that reserve partitioning in 4-year-old trees
was evaluated, which bloomed for the first time during the experiment
Therefore, the crop load was the same as would be for a tree in full production
since most of the tree was in a vegetative stage. This may explain the fact that in
terms of sink strength, more 13C-reserves were partitioned to vegetative growth,
especially non-fruiting spurs. This partitioning pattern cannot be extrapolated to
mature trees; however, unpublished data (M. Ayala, personal observation)
indicate that the same trees bloomed extensively one year later and the
vegetative growth (expressed as shorter current shoots) was reduced. Such a
situation may be more likely to promote the partitioning of most of the storage
reserves to reproductive organs, which would compete strongly among
vegetative sinks and other growth early in the season.

In summary, this study indicates that the hierarchy for stored C
distribution among aerial organs of a 4-year-old sweet cherry tree is dynamic
from budbreak through stage I of fruit growth. During this period, reproductive
organs have the highest sink activity for storage reserves until 14 DAFB;
however, a strong competition between flowers, fruits and different leaf
populations occurred. Late in stage I, with shoots of ~5 cm in length and 10

leaves and fruit of ~0.2 mg DW, storage reserves do not constitute the main

200

source of assimilate and new expanded leaves become the major source of C for
fruit and shoot growth.

These results advance the understanding of the importance of storage
reserves for early spring growth in sweet cherry using dwarfing and
semidwarfing rootstocks, which are known for increased precocity and high
yields. Practical implications of this research include: (1) the maintenance of
healthy photosynthetic sources during the previous fall to promote optimal
reserve accumulation in storage sites, (2) a more precise manipulation of aerial
growing centers to achieve a more balanced partitioning of reserves during early
spring, (3) the selection of scion/ rootstock genotypes for optimal CH20
accumulation and distribution during the postharvest period, and (4) the
avoidance of late summer stresses such as drought or defoliation due to diseases
or insects. Good and coordinated horticultural practices after harvest (i.e., timing
of N fertilization, pest and disease control, irrigation and appropriate summer
pruning) will promote an optimum CH20 supply for storage and subsequent use

in new growth during early spring.

201

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205

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206

Table 1. Current season growth (shoot) measurements of ’Regina’ / Gisela 6 sweet
cherry trees between bloom and terminal bud set (2003). Mean : SE, n=40.

 

 

 

Developmental Stage Days Shoot Length Leaf Number
relative (cm)
to full
bloom
Total Folded Unfolded
First white/ first bloomZ -6 0.3 : 0.01x 3.1 : 0.2 2.5 : 0.1 0.6 : 0.1
Full bloom 0 0.5 :l: 0.01 8.0 : 0.2 4.3 : 0.2 3.7 : 0.2
Fruit set 7 1.9 : 0.1 9.1 : 0.2 2.1 : 0.1 7.0 : 0.2
Stage I 14 4.8 : 0.2 9.6 : 0.2 1.6 : 0.1 8.0 : 0.2
Stage I 21 9.1 : 0.4 10.5 : 0.2 1.8 : 0.1 8.7 : 0.2
Stage I 28 14.6 : 0.5 12.0 : 0.2 2.0 : 0.1 10.0 : 0.2
Stage II 35 24.2 : 0.9 14.8 : 0.2 2.5 : 0.1 12.3 : 0.2
Stage II 42 30.6 : 1.1 16.9 : 0.3 2.7 : 0.2 14.2 : 0.2
Stage III 49 38.5 : 1.3 18.9 : 0.4 1.9 : 0.2 17.0 : 0.3
Stage III 56 42.9 : 1.6 19.6 : 0.4 1.3 : 0.2 18.3 : 0.4
Stage III 63.V 45.6 : 1.9 20.2 : 0.5 1.0 : 0.1 19.2 : 0.5
Stage III 70 46.6 : 2.0 20.7 : 0.5 0.5 : 0.2 20.2 : 0.5
Terminal bud set 77 47.0 : 2.0 20.8 : 0.6 0.3 : 0.2 20.5 : 0.5

 

2 Developmental stages overlapped during this week.
Y Fruit was kept on the tree after commercial harvest.
X At this date a few rudimentary shoots emerging from terminal buds.

Table 2. ’Regina’ / Gisela 6 sweet cherry fruit growth measurements from fruit set
through stage III. Mean : SE, n= 25.

 

 

Developmental DAFBz GDDY Fresh Dry Diameter

Stage Weight Weight (mm)
(s) (3)

Fruit set 7 342 0.3 :002 0.03 : 6.2 : 0.2

Stage I 14 405 1.2 :01 0.15 : 11.5 : 0.5

21 469 1.5 :01 0.20 : 13.0 : 0.2

28 533 1.7 :01 0.31 : 13.6 : 0.2

Stage II 35 618 1.9 :01 0.48 : 14.6 : 0.2

42 722 2.5 :01 0.70 : 16.2 : 0.2

Stage III 49 843 4.8 :03 1.04 : 19.8 : 0.4

56 968 8.8 :05 1.98 : 25.4 : 0.6

63 1087 11.2 :07 2.73 :t 26.0 : 4.9

 

Z DAFB: days after full bloom.
y GDD: growing degree days.

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214

CHAPTER VI

DISSERTATION PROJECT SUMMARY

215

Summary

Interest is high among US sweet cherry growers to adopt dwarfing and
semi-dwarfing precocious rootstocks, such as the Gisela (GI) series, which are
characterized by small canopies and positive effects on precocity and yield. High
density systems using dwarfing precocious rootstocks are more labor efficient
and economically viable when yields and fruit quality can be maintained or
improved. These modern orchards are more uniform, have high and early yields
and require lower production and harvest inputs. Before commercial adoption of
GI rootstocks by American growers becomes routine, however, many
physiological questions must be elucidated. Additional research is required to
understand the role of fruit sink strength and carbohydrate (CH20) partitioning
when trees are grown on dwarfing and semi-dwarfing precocious rootstocks,
particularly of the GI series selections that are currently available. So far, the
implementation of standard sweet cherry management practices for trees on GI
rootstocks has resulted in high yields but small fruit, which is a critical problem
since top quality fruit provides the best returns to growers.

As little was known about the relative importance of different sweet
cherry leaf populations (i.e., fruiting and non-fruiting spurs and current season
shoot leaves) and storage reserves as sources of carbon (C) for fruit and Shoot
development in dwarfing trees, this study focused on the following objectives:

(1) to define the temporal importance of various leaf populations as sources of C

216

for fruit and shoot growth during the whole period of fruit development, (2) to
determine the effect of reproductive and vegetative sink strengths on C
partitioning during fruit development, (3) to determine the importance of storage
reserves as a source of C for initial fruit growth, and (4) to define the transition
phase during which the dependence of new growth on storage reserves shifts to
current photosynthate assimilation as the primary source for subsequent
vegetative and reproductive development. Accordingly, a series of partitioning
experiments using girdling, defoliation, fruit thinning and 13C-isotopic labeling
of different leaf populations and storage reserves was established with sweet
cherry trees on dwarfing (Gisela 5, G15) and semidwarfing (Gisela 6, G16) GI
rootstocks.

A preliminary girdling and defoliation experiment isolated fruit of
’Hedelfinger’/ G15 and ’Ulster’ / G16 from different leaf sources. Results indicated
that fruits supplied exclusively by the leaf populations on either the fruiting spur
branch segment or the non-fruiting Spur branch segment were significantly
smaller and had decreased SS levels. Leaf populations on both fruiting and non-
fruiting branch segments were required for full fruit development and there was
not a sufficient compensatory effect when one of the main leaf populations was
eliminated.

A second experiment used 13C02 to label non-fruiting spur leaves on
’Sam’ / G15 limbs with three different crop loads quantified as leaf area (LA) to

fruit (F) ratio (LA / F = 140, 75, or 40 cm2/ fruit) 3 times during stage III of fruit

217

development Results indicated that 13C fixed by non-fruiting Spur leaves was
translocated both acropetally and basipetally. For all 3 pulsing dates, fruits were
more highly enriched in 13C than were young leaves, suggesting that the Sink
activity of fruit was stronger compared to that of shoots. There was not a
consistent or significant crop load effect on 13C-partitioning between fruit and
shoots. However, differences in translocation between organs of the same
branch, for a given treatment, were significant, as the fruits in closest proximity
to the branch segment of non-fruiting spurs generally had the highest relative 13C
content (up to 64%, compared to more distal fruits which ranged from 26% to
40% of recovered 13C). As crop load increased, this trend for preferential
partitioning became more pronounced. Shoot leaves had considerably lower 13C
contents (ranging between 1.6% and 11 % of the 13C recovered).

A third experiment quantified the relative C contributions of different leaf
populations on ’Ulster’ / G16 limbs to fruit and shoot development during stages
I, II and III of fruit development. The three leaf populations on the fruiting
branch, i.e., fruiting spur, non-fruiting spur and new terminal shoot leaves, were
exposed to 13C02 labeling on five representative phenological dates (25, 40, 44, 56,
75 days after full bloom, DAFB) during fruit development Results indicated that
spur and shoot leaves were significant sources of C for fruit and vegetative
growth. 13C fixed by different leaf sources was translocated acropetally,
basipetally or both. In terms of C allocation, fruits were a priority Sink vs. new

shoot growth during the entire period of fruit development. However, the more

218

distant was the 13C source, the lower the amount of 13C detected in fruit Fruit
photosynthesized some 13C in early stages of development (stages I and H). The
highest fruit sink strength was during stages I and III, while the highest shoot
sink strength was during rapid elongation. The terminal current season shoot
provided a C source for fruit as early as stage 1.

Finally, a fourth experiment on ’Regina’/ G16 trees labeled with 13C02 after
terminal bud set determined the extent of storage reserve use of for spring
growth, particularly fruit, and defined the transition phase during which current
photoassimilates become the primary C source. In fall, the major storage organs
were roots, older wood in the trunk and branches, and buds. During spring, 13C-
reserves were remobilized and partitioned to flowers, fruits and young leaves
from before budbreak (Side green) until 14 DAFB. The highest 13C levels in
grong sinks were detected between side green and fruit set. The isotopic
composition of new organs differed significantly among organs and phenological
stages. Reproductive organs had the strongest sink activity until 14 DAFB, but in
terms of total dry matter, non-fruiting spurs had the highest sink strength as a
function of being the predominant aerial tissue type in the 4—year-old tree.

Previous data and increasing grower experience indicates that
reproductive and vegetative growth often become unbalanced after the 4th or 5th
year of production on dwarfing and semidwarfing G1 rootstocks if the natural
canopy LA / F ratios are not altered in some way. Thus, manipulation of the

reproductive and vegetative sinks may be a tool to regulate sink strength and

219

competition among sinks during periods of resource limitation, particularly
during fruit development. So far, chemical thinning of flowers or fruit is not a
common practice in sweet cherry. Therefore, adjustments in LA/ F ratios through
practices such as pruning to remove or stimulate leaf area, or fruit and flower
thinning and / or spur extinction might help to overcome the problem of
overcropping and small fruit Size. The appropriate timing for each of these
practices will depend on the final objective. For instance, removal of some of the
current season growth after leaf abscission (once storage reserves have been
accumulated) or during winter might be a possible strategy to control excessive
crop load 2 years later, and additionally stimulate more vigorous vegetative
growth (i.e., more leaf area to support fruit growth) the year after pruning.
Growers should be encouraged to begin this type of management soon after
establishment, by 4.th or 5th leaf or as crop load become a significant sink, to
achieve and maintain more balanced trees, in terms of crop load and LA. Pruning
current season shoots during the period of fruit development might be
detrimental if it is not done precisely since, as demonstrated in this study, this
leaf population constitutes a C source for fruit as early as stage 1, and its removal
could negatively affect fruit quality.

Another alternative to control excessive crop loads might be spur
extinction (i.e., selective spur removal) during summer or after terminal bud set.
Selective removal of reproductive spurs every season would reduce future crop

loads. However, a disadvantage of this technique is that it does not promote

220

additional LA development on current season shoots. Thus, because spur leaf
area is not sufficient for optimal fruit development, extinction would not
promote the supplemental LA contributed by extension shoots, which might
become a limitation to keep optimal LA/ F ratios in more dwarfing
scion/rootstocks combinations. In the future, it would be interesting to explore
how the mix of shoot pruning and spur extinction may shift source-sink
relationships depending on the inherent vigor and precocity of the

scion/ rootstock combination. For instance, scion / rootstocks combinations using
(315, a dwarfing rootstock, might require more pruning to promote extension
shoot growth than combinations using (316, a semidwarfing rootstock. Extension
programs should emphasize the importance of distinct leaf populations as C
sources for fruit and shoot development. Sweet cherry growers using dwarfing
and semidwarfing rootstocks can use the results of this dissertation to promote
more leaf area development and protect leaves not only during fruit
development, but also after harvest when storage reserves are being
accumulated. Overall, results of this study provide a physiological foundation
for the canopy relationships that may guide to develop specific orchard
management strategies to promote a more sustainable balance between
vegetative and reproductive growth in high density sweet cherry orchards on

vigor-limiting rootstocks.

221

APPENDIX A

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Appendix 32. Ranges of air temperature and photosynthetically active radiation
(PAR) measured between 9:00 AM and 13:00 PM during each pulse-labeling date
(25, 40, 44, 56 and 75 days after full bloom, DAF B). Data obtained from the
Michigan Automated Weather Network (MAWN).

 

 

DAF B Air temperature PAR
(°C) (KI/m2)
25 8.8 - 13.4 1735-16208
40 11.9 - 18.3 327.9-2559.4
44 14.1 - 18.0 291.4-1367.4
56 24.3 - 29.9 10727-27740
75 20.0 - 26.3 10804-23666

 

227

Appendix B.3. Ranges of net photosynthesis (A), photosynthetically active
radiation (PAR) and leaf temperature ("I“) of each leaf population within the

canopy at each pulse-labeling (25, 40, 44, 56 and 75 days after full bloom, DAFB).

 

 

 

 

 

 

n=5.
DAFB leaf population PAR Leaf T° A
(pmol m-Zs-l) (°C) (pmol C02 m-25'1)

25 Fruiting spurs 80 - 282 21.3 - 22.5 2.9 - 5.9
Non-fruiting spurs 216 - 780 20.8 - 27.7 7.4 - 12.7
Terminal shoot basal 183 - 403 22.1 - 23.5 4.6 - 8.4
Terminal shoot medial 117 - 296 21.5 - 23.2 0.9 - 5.5
Terminal shoot apical 76 - 212 21.2 - 23.5 -0.7 - -8.2

40 Fruiting spurs 1096 - 1832 25.1 - 27.7 8.7 - 11.6
Non-fruiting spurs 923 - 1480 25.1 - 27.8 8.6 - 16.9
Terminal shoot basal 109 - 1440 23.6 - 26.9 1.0 - 16.3
Terminal shoot medial 106 - 1301 21.8 - 27.0 -0.8 - 15.0
Terminal shoot apical 87 - 1235 23.1 - 28.7 -0.4 - 2.8

44 Fruiting spurs 183 - 307 24.6 - 25.9 5.9 - 9.8
Non-fruiting spurs 535 - 707 26.1 - 27.6 7.4 - 12.8
Terminal shoot basal 447 - 524 25.8 — 26.3 7.9 — 11.3
Terminal shoot medial 557 - 681 26.0 - 26.9 7.2 - 7.5
Terminal shoot apical 568 - 641 27.3 - 28.1 -3.0 - -6.3

56 Fruiting spurs 447 - 1396 31.8 - 33.6 8.0 - 19.7
Non-fruiting spurs 923 - 1708 33.3 - 34.9 8.2 - 19.2
Terminal shoot basal 905 - 1565 34.0 - 35.5 9.1 - 15.0
Terminal shoot medial 773 - 1352 34.5 - 35.4 10.4 - 12.6
Terminal shoot apical 894 - 1425 36.3 - 36.8 -2.7 - 2.2

75 Fruiting spurs 183 - 1700 27.5 - 30.5 7.1 - 19.9
Non-fruiting spurs 76 - 1444 26.7 - 32.8 1.4 - 13.9
Terminal shoot basal 846 - 1788 30.0 - 33.3 14.1 - 22.5
Terminal shoot medial 417 - 1612 30.7 - 32.9 11.0 - 20.4
Terminal shoot apical 527 - 1656 31.9 - 32-8 -8.9 - 18.9

 

228

Appendix B.4. Fruit and terminal current season shoot dry weight (DW)
measured on 2-year-old ’Ulster’ / Gisela 6 sweet cherry branches at each labeling
date. Mean t SE, n=30.

 

 

 

Developmental DAFBZ Dry weight (g)
stage
Fruit Terminal shoot
I 25 0.35 :t 0.07 2.5 i 0.1
II 33 0.37 i 0.01 3.3 i 0.1
40 0.39 :l: 0.01 4.0 :l: 0.2
III 44 0.40 i 0.01 5.4 :t 0.2
51 0.67 :t 0.01 7.4 i 0.4
56 0.94 i 0.02 8.6 i 0.4
63 1.28 i 0.04 11.3 i 0.6
Terminal bud set 75 1.64 i 0.05 13.7 i 0.9

 

Z DAF B: days after full bloom. Full bloom occurred on 30 Apr, 2003.

229

 

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230

Appendix 36. Total leaf areas for the fruiting, non-fruiting and terminal shoot
sections of 2-year-old ’Ulster’ / Gisela 6 sweet cherry branches at each pulse-
labeling date. Mean i SE, n=30.

 

 

 

Developmental DAFBz Total Leaf Area per Leaf Population
Stage (cm?)
Fruit Non-fruiting Terminal
Spurs spurs shoot

I 25 1322.3 1 41.1 1443.5 1: 60.1 248.7 :t 12.9
II 33 1427.7 i 45.2 1467.5 i 63.7 357.2 i 20.2
40 1456.7 1 51.4 1411.4 :1: 55.1 423.6 1: 13.4
111 44 1385.2 i 38.2 1551.2 1: 55.5 509.4 1: 55.2
51 1503.5 i 54.4 1776.1 :1: 64.7 614.3 :t 25.8
56 1623.5 :t 62.9 1610.3 i 73.5 668.9 i 28.3
63 1537.2 1 40.2 1756.9 i 95.3 729.6 i 34.1
Terminal bud set 75 1523.6 1 78.3 1600.3 :l: 82.7 844.5 i 54.2

 

Z DAF B: days after full bloom. Full bloom occurred on 30 Apr, 2003.

231

Appendix 37. Total leaf area, fruit number and leaf area to fruit (LA / F) ratios for
the 2-year-old ’Ulster’/ Gisela 6 sweet cherry branches measured at each pulse-
labeling. Mean :l: SE, n=30.

 

 

Developmental DAFBz Leaf area / branch Fruits / branch LA / F ratio
stage (cm?) (cm2/ fruit)

Stage I 25 2984.3 :1: 67.0 94.5 i: 3.3 32.6 :1: 1.3

Stage II 33 3252.4 1: 82.2 79.8 d: 3.3 42.7 :t 2.0

40 3291.6 :t 82.8 79.8 d: 3.4 43.5 :t 2.2

Stage III 44 3445.8 :t 61.9 67.1 :|: 3.6 54.9 i 2.6

51 3908.3 i 97.4 77.1 i 3.0 53.1 i 2.2

56 3899.6 :t 80.0 68.7 :t 3.0 60.0 i 2.6

63 3980.4 :1: 101.5 68.3 i 3.3 60.7 i: 2.9

Terminal bud set 75 3994.4 i 126.0 66.1 i3.2 60.9 i 3.0

 

Z DAF B: Days after full bloom on 30 Apr, 2003.

232

Appendix B.8. Growing degree day (GDD, base 44°C) accumulation for stages I,
II and III of fruit development at CHES (May 1 to Aug 4, 2003).

 

 

Developmental Stage DAFBz GDD
Fruit Set 5 to 12 256.0 - 312.4
Stage I 13 to 32 319.4 - 483.2
Stage II 33 to 43 492.1 - 604.7
Stage III 44 to 75 618.5 - 1134.8
Post Stage III 76 to 96 1152.9 - 1476.6

 

DAF B: days after full bloom. Full bloom occurred on 30 Apr, 2003.

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Appendix B.13. Relative 13C partitioning among different organs on 2-year-old
’Ulster’ / Gisela 6 sweet cherry branches at 48 h after 13C02 pulsing of fruiting
spur leaves. Calculations are based on absolute amounts of 13'C recovered for
each organ at each pulse-labeling (See Materials and Methods). Mean i SE, n=5.

 

 

 

 

Organ Relative 13C Partitioning (%)
DAFBx
25 40 44 56 75
PSI leavesY 32.5 :t 2.1 30.4 i 1.7 34.1 :t 2.2 17.9 i: 0.9 36.4 :t 5.5
NFSZ leaves 0.7 i 0.2 0.2 i 0.2 0.0 :l: 0.0 0.3 :l: 0.2 0.2 i 0.0

TS2 basal leaves 0.0 i 0.0 0.0 i 0.0 0.0 d: 0.0 0.0 :1: 0.0 0.1 i 0.0
TS medial leaves 0.1 i 0.0 0.0 i 0.0 0.0 :l: 0.0 0.0 i 0.0 0.1 i 0.0
TS apical leaves 0.0 i: 0.0 0.0 i 0.0 0.0 i 0.0 0.0 :t 0.0 0.0 i 0.0

TS wood 0.0 i 0.0 0.0 i 0.0 0.0 :l: 0.0 0.0 :l: 0.0 0.1 :l: 0.0
FS wood 3.0 :l: 0.7 8.8 i 2.0 7.0 :t 1.6 2.5 :l: 0.6 5.4 :l: 1.4
NFS wood 0.5 i 0.2 0.6 i 0.4 0.0 :l: 0.0 0.1 i 0.0 0.4 :t 0.0
Fruit 63.2 :t 1.9 59.9 i 2.2 58.9 i 2.8 79.1 i 1.1 57.3 :1: 6.7

 

2 PS: fruiting spur; NFS: non-fruiting spur; TS: terminal shoot.
Y Fruiting spur leaves were pulsed directly with 13COz.
X DAFB: days after full bloom. Full bloom occurred on 30 Apr, 2003.

238

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239

Appendix B.15. Relative 13C partitioning among different organs on 2-year-old
’Ulster' / Gisela 6 sweet cherry branches measured at 48 h after 13C02 pulsing of
the non-fruiting spur leaves. Calculations are based on absolute amounts of 13C
recovered for each organ at each pulse-labeling. Mean i SE, n=5.

 

 

 

 

Organ Relative 13C Partitioning (%)
DAFBx
25 40 44 56 75
FSZ leaves 0.2 i 0.0 0.7 i 0.4 0.0 d: 0.0 0.5 i 0.4 0.2 :t 0.1
NFSz leavesY 41.3 i 0.6 42.7 :t 2.7 46.1 i 1.7 19.9 i 2.1 49.4 i 4.8

TSZ basal leaves 0.1 i 0.0 0.1 i 0.1 0.0 :t 0.0 0.1 d: 0.0 0.2 i 0.1
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TS apical leaves 0.0 i 0.0 0.0 i 0.0 0.2 :t 0.1 0.0 :l: 0.0 0.1 :t 0.0

TS wood 0.0 i 0.0 0.1 :t 0.0 0.2 :t 0.1 0.1 i 0.0 0.2 i 0.1
FS wood 8.4 :t 1.5 16.9 i- 2.5 14.5 :1: 5.1 5.1 i: 0.7 12.1 :1: 3.9
NFS wood 4.2 i 0.5 7.8 i 0.7 7.6 i 1.6 3.3 :l: 0.5 5.0 :l: 1.1
Fruit 45.8 i 1.5 31.7 i 5.3 31.3 :t 5.3 70.9 i 2.6 32.7 :I: 5.2

 

2 FS: fruiting spur; NFS: non-fruiting spur; TS: terminal shoot
y Non—fruiting spur leaves were pulsed directly with 13'C02.
x DAFB: days after full bloom. Full bloom occurred on 30 Apr, 2003.

240

 

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241

Appendix B.17. Relative 13C partitioning in 2-year-old ’Ulster’ / Gisela 6 sweet
cherry branches at 48 h after 13C02 pulsing of terminal shoots. Calculations are
based on absolute amounts of 13C recovered for each organ at each pulse-
labeling. Mean :t SE, n=5.

 

 

 

Organ Relative 13C Partitioning (%)
DAFBx
25 40 44 56 75
F82 leaves 0.2 i 0.2 0.0 i: 0.0 0.1 i 0.1 0.5 :t 0.2 0.0 :t 0.0
NFSZ leaves 0.4 :t 0.2 0.1 i 0.1 0.0 i 0.0 0.4 i 0.2 0.2 i 0.0

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FS wood 8.8 i 1.9 10.1 i 2.9 4.8 i 0.6 5.0 i 0.6 10.7 i: 2.1
NFS wood 8.1 :t 1.6 15.7 i 2.4 9.1 i 1.3 3.7 i 0.4 10.0 :t 1.6
Fruit 27.2 i 1.5 22.3 i 4.1 17.5 i 2.2 59.2 :t 6.2 28.3 i 6.0

 

2 FS: Fruiting spur; NFS: N on-fruiting spur; TS: terminal shoot.
Y Current season leaves and wood were pulsed directly with 13C02.
x DAF B: days after full bloom. Full bloom occurred on 30 Apr, 2003

242

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243

APPENDIX C

244

Appendix C.1. Total number of aerial organs on 4—year-old ’Regina’/ Gisela 6
sweet cherry trees at budbreak and post— budset (2003).

 

 

 

 

 

 

 

Tree Side green bud stage Terminal bud set
Fruiting spursz Non fruiting spursY Shootsx Single budw
1 173 860 95 1554
2 46 959 135 1749
3 74 759 77 992
4 94 782 100 1260
5 77 597 66 773
Average 93 791 95 1266

 

2 Number of fruiting spurs on 2—year-old limb sections (2001 growth).

Y Number of non-fruiting spurs on 1-year-old limb sections (2002 growth).
x Number of extension shoots that grew in 2003.

W Total number of single buds on extension shoots grown in 2003.

245

Appendix C.2. Tissues collected from ’Regina'/ Gisela 6 sweet cherry tree at leaf
abscission (Nov, 2002) and at budbreak (side green) stage (Apr, 2003) for 13C
analysis.

 

 

Tree section Organ / Tissue Year of growth
Trunk Bark 19992, 2000)’, 2001x
Wood 1999, 2000, 2001
Branch Bark 2001, 2002W
Wood 2001, 2002
Spur buds 2001
Single buds 2002
Root Coarse (> 5 mm) -

Medium (1-5 mm) -
Fine (< 1mm) -
2 Growth of 1999 corresponds to 3—year-old-wood in fall 2002.
Y Growth of 2000 corresponds to 2-year-old-wood in fall 2002.
x Growth of 2001 corresponds to 1-year—old-wood in fall 2002.
W Growth of 2002 corresponds to current season growth in fall 2002.

 

246

Appendix C.3. Aerial organs of ’Regina’ / Gisela 6 sweet cherry trees sampled at
different developmental stages during spring and summer (2003) for 13C
analysis.

 

Stage Organs

 

Buds,v Single Spur Fruiting Non- Shootsx Fruits
flowers flowers spurs fruiting

 

spurs
Side green x
First bloomZ x x x
Full bloom x x x x x

Fruit set x x x x
Stage I x x x x
Stage II x x x x
Stage III x x x x

 

Z ’First white’ and ’first bloom’ stages overlapped during the same week.
y Includes vegetative and reproductive buds.
x Current season growth (2003).

247

Appendix C4. Growing degree days (GDD) accumulated at each developmental
stage and sampling substages for ’Regina’ / Gisela 6 sweet cherry trees. Base
temperature: 4.4 °C.

 

 

 

 

 

 

 

 

Developmental Stage Days relative to Accumulated GDD
full bloom

Side green - 15 to -12)’ 148-166
-13x 163

First bloomZ - 4 to - 6 239 - 264
-5 245

Full bloomZ O 264 - 319
1 287

Fruit set 4 to 7 320 - 342
7 342

Stage I 8 to 26 343- 533
14 405
21 469
28 533

Stage II 29 to 42 534 - 722
35 618
42 72

Stage III 43- 64 723 - 1102

49 843
56 968
63 1087

 

2 Overall bloom period lasted ~ 10 to 12 days.

3' Number of days at a specific developmental stage considering full bloom
(9 May, 2003) as reference date.

W Sub-stage at which organs were sampled within each main
developmental stage.

248

 

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Days From Full Bloom

Appendix C.5. Accumulation of dry weight (DW) matter in ’Regina’/ Gisela 6
sweet cherry spurs, shoots and fruit. Period between first bloom and terminal
bud set (May to Jul, 2003). Weekly measurements, n=10. Fruit were eaten by
raccoons 63 DFFB.

249

Appendix C.6. Dry weight (DW) of different organs at each developmental stage
for ’Regina’/ Gisela 6 sweet cherry trees, n=10.

 

 

 

Stage Days DW
relative (mg)
to full
bloom
Bud Single Flower Spur Fruiting Non— Shoot Fruit
flower cluster flower spurs fruiting
spurs
Side Green -15 add
First bloom -6 - Y 4.6 228.3 0.0 0.0 66.7 21.0 -
Full bloom 0 - 24.4 - 25.0 123.0 208.8 129.0 -
Fruit set 7 - - - - 149.6 269.0 . 232.2 33.3
Stage I 14 - - - - 315.5 425.9 316.5 148.2
21 - - - - 482.3 582.7 1249.3 196.6
28 - - - - 573.0 608.7 1726.0 307.4
Stage II 35 - - - - 533.4 885.0 2847.9 478.0
42 - - - - 493.7 934.3 3588.4 696.2
Stage III 49 - - - - 543.3 891.5 4951.9 1042.0
56 - - - - 738.0 978.8 5496.6 1984.8
63 - - - - 766.6 1492 5826.4 2731.8

 

Z DFFB: days from full bloom.
Y dash indicates that the organ was not sampled at a certain date.

250

 

+ Shoot

90 q +Fruit

Relative Growth (%)

 

 

 

 

60714212835424956637077
Day from Full Bloom

Appendix C.7. Cumulative relative growth of ’Regina’ / Gisela 6 sweet cherry
shoots and fruits between first bloom and terminal bud set (May to Jul, 2003). n=
25 (fruit) and n=15 (shoots). Fruit was eaten by raccoons 63 DFF B.

251

 

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