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31293 01050

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LIBRARY

Michigan State
University

 

 

 

This is to certify that the

dissertation entitled

GIBBERELLIN METABOLISM AND APPLE FLOWERING

presented by

JUN BAN

has been accepted towards fulfillment
of the requirements for

Jh- D- degree in WIRE

MA

Major professor

Date October 26, 1996

 

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

PLACE II RETURN BOX to roman thb checkout from your rooord.
TO AVOID FINES return on or baton dot. duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU It An Afflnnntlvo ActlorVEmol Opportunlty Institution
mm

GIBBERELLIN METABOLISM AND APPLE FLOWERING
BY

JUN BAN

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture Science

1996

ABSTRACT
GIBBERELLIN METAHBOLISM AND APPLE FLOWERING
BY
Jun Ban

The presence of seeds in 'Spencer Seedless’ apple
inhibited flower initiation. Fruit removal experiments
showed that A critical time for inhibition began around 35
days after full bloom (DAFB). Seeded fruits were
significantly larger than seedless ones at harvest. However,
fruit weight did not differ significantly until 36 or 37
DAFB in both years.

“C-Ghu was injected into seeds and apices of ’Spencer
Seedless' and 'Spartan' apple to investigate the metabolism
of Gh(s) in relation to apple flowering. No qualitative or
quantitative differences were found between metabolites in
apices of bourse shoots on spurs bearing seeded vs. seedless
fruits, or between cultivars ('Spencer Seedless’ vs.
'Spartan’). However, the rate of metabolism was higher in
apices of bourse shoots in the presence of seedless fruits.
Seven major metabolites were detected in apices, six in
seeds. Tissues external to the site of injection were

oxidized to determine the transport of metabolites. Only a

small percentage of the 1"C injected (about 1.47 to 3.22% in
1992, 0.09 to 0.13% in 1993) was transported from seeds to
other tissues, and only ca. 0.1 to 0.4% (1992) and 0.01 to
0.04% (1993) was detected in the tissues outside the fruit.
With but one exception, no radioactivity occurred in the
apex when “C-GAQ was injected into seeds, but one polar
metabolite was found in the cluster base and two metabolites
in the fruit flesh. When apices were injected, very low
amounts of radioactivity (0.2 to 6%) were recovered from
outside the injection site: one polar metabolites in cluster

bases was found in the cluster bases.

ACKNOILIDGHENTB

I would like to express my sincere gratitude and
appreciation to Dr. Frank G. Dennis, Jr., my major
professor, for his guidance, advice and support. He has
shown me how to be a better scientist and more importantly,
how to be a better person.

Many thanks to Drs. Martin J. Bukovac, James A. Flore,
Kenneth L. Poff, Jan A.D. Zeevaart for serving on my
guidance committee and for their valuable suggestions and
reviews of my thesis.

Gratitude is extended to Dr. John D. Everard for his
suggestions and technical advice during my research, and for
his willingness to be as the substitute committee member for
Dr. Flore.

Thanks to my colleagues, John Neilsen, Mario Mandujano
and other helpful friends, for their support.

A special thanks to Beverly Chamberlin for her exellent
help in analysis of samples using GC-MS.

iv

I am most deeply grateful to my wife Xiaoyang Xia and
my son Dajun Ban, for their love, patience and moral

support.

TABLE OF CONTENTS

LIST OF Tums 0.0.0.... ..... O OOOOOOOOOOOOOOOOOOOOOOOOOO ..ix

LIST OF FIGURES ... ....... ..... ......................... xiii

LITERATURE REVIEW ... .......................... . .......... 1
Introduction .................... ................... 1
Biennial bearing in fruit production ............... 1
Effects of defoliation and defruiting .............. 4

Effects of growth regulators on flowering .......... 7
The effects of GAs on flowering of herbaceous plants
........................ ...... . ..... . ........ . 8
The effects of GAs on flowering of conifers......... 8
The effects of GAs on flowering of fruit trees. ..... 9
Effects of growth retardants and/or GA synthesis
inhibitors on flowering of tree fruits..............12
Endogenous factors affecting flowering . ...... ......13
Gibberellins in apple tissues ..................... 17
Seasonal changes of gibberellins in apple seeds

vi

............................................. 21
The changes of GAs in vegetative tissues Of apple
............................................. 21
The role Of gibberellins in flower initiation ..... 22
Summary ........................................... 26
SECTION I. EFFECT OF TIME OF FRUIT REMOVAL ON FLOWER
INITIATION IN ’SPERCER SEEDLESS' APPLE, AND EFFECT OF
CUTTING FRUITS ON RETENTION AND GROWTH OF ’SPERNCER
SEEDLESS’AND ’PAULARED’ FRUITS.
Abstract .......................................... 28
Introduction .......................................29
Materials and methods ..............................30
Results ............................................32
Discussion .........................................42

Literature cited ....................;.. ..... .......44

SECTION II. METABOLISM OF “C-GAR AND CHARACTERIZATION OF
METABOLITES IN APPLE SEEDS AND APICES IN RELATION TO FLOWER
INDUCTION.
Abstract ...........................................47
Introduction .......................................48
Materials and Methods ..............................49
Results ............................................69
Discussion ........................................118

Literature cited ..................................121

vfi

SECTION III. TRANSPORT OF “C-GA12 METABOLITES FROM APPLE
SEEDS AND APICES TO THE FRUIT AND OTHER TISSUES, AND
CHARACTERIZATIONOF THE METABOLITES.
Abstract ..........................................127
Introduction ......................................128
Materials and methods .............................130
Results ...........................................131
Discussion ........................................159
Literature cited ..................................160
Summary 164
Suggestion for further research..........................167

References CitedOQOOOO......OOOOOOOOO.....OOOOOOOOOOOOOI 168

fifi

LIST OF TABLES

LITERATURE REVIEW
Table 1 - Families and species Of plants in which
alternate bearing has been reported ..................2
Table 2 - Gibberellin effects on flowering Of tree

fruit 0.0.0.0000...0.000.000.0000.........OOOOOOOOOOOIO

Table 3 - Gibberellins identified in apple tissues...18

SECTION I
Table 1 - Diameter and weight Of fruit, and length of
seed and embryo Of 'Spencer Seedless’ at the time Of
sampling or wounding and diameter Of wounded fruit at
harvest (170 DATE) in 1992 and 1993 (Ten fruits per
sample). ...........................................36
Table 2 - Effect Of wounding at various times on
abscission Of 'Spencer Seedless' apple fruit in 1992
and 1993 and ’Paulared' fruit in 1993. Untreated fruits
(CK) were marked for comparison on the first date Of

treatmentOCOOOOIOOOOOOOOO......OOOOOOOO00.0.0000000039

Section II

Table 1 - No. of Spurs and fruits (seed treatment) Of

ix

'Spencer Seedless' treated 1992-1995...... ........ .62
Table 2 - Rf values Of some standard gibberellins on
TLC with developing solvent ethyl acetate : chloroform
: acetic acid (15:5:1)..............................68
Table 3 - Retention times of some standard gibberellins
on HPLC, using a methanol/water gradient (see text for
conditions).........................................74
Table 4 - Rf values of metabolites of “C-GAQ in
extracts of apple seeds and apices........... ..... ...80
Table 5 - Relative amounts Of metabolites of 1"C-GAW in
seed of 'Spencer Seedless’ in 1992 and 1993 and
'Spartan' in 1992 as a percentage Of the total
radioactivity recovered by HPLC. Only one sample
processed per cultivar and date in 1992, 3 samples in
1993.................................................96
Table 6 - Effects Of seeds and time Of treatment on
levels Of metabolites of 1"C-GA12 recovered from apices
Of 'Spencer Seedless' in 1992. Means for 3 replicate
samples as a percentage of total DPM recovered
following HPLC......................................100
Table 7 - Effects Of seeds and time of treatment on
levels of metabolites of 1"C-GAW recovered from apices
of 'Spencer Seedless’ in 1993. Means for 3 replicate
samples as a percentage Of total DPM recovered
following HPLC......................................106

SECTION III

Table l - Effect Of time of treatment on total
radioactivity recovered in different tissues, and on
distribution Of total radioactivity, following
injection of 1"C-GAQ into seed of 'Spencer Seedless'
apples in 1992. All samples (about 100 mg DW, one per
treatment) oxidized and C02 collected. . . . . . . . . . . . . . . 133
Table 2 - Effect Of time Of treatment on percentage of
total radioactivity recovered (DPM/100 mg DW), and
distribution of total radioactivity recovered, in other
tissues following injection Of 1‘C-GAu, into seed of
'Spencer Seedless’ apple in 1993. All samples (about
100 mg DW, 3 per treatment) oxidized and CO;
collected...........................................136
Table 3 — Percentage of total recovered radioactivity
and distribution Of radioactivity 72 hr after injection
of “c-GAQ into seeds Of 'Spencer Seedless' in yitrg.
l994................................. .......... .....140
Table 4 - Effects Of time Of treatment and presence Of
seeds on total radioactivity recovered in different
tissues, and on distribution Of radioactivity following
injection of “C-GAQ into bourse shoots of 'Spencer
Seedless' apple in 1992. All samples (about 100 mg DW,
3 per treatment) oxidized and (:02 collected. . . . . . . . . 142
Table 5 - Effects of time of treatment and presence Of
seeds on total radioactivity recovered in different

tissues, and on distribution of radioactivity following

injection Of “C-GAu into bourse shoots of 'Spencer
Seedless' apple in 1993. All samples (about 100 mg DW)
oxidized and co; collected 148
Table 6 - Effects of seeds and time of treatment of
total radioactivity and distribution among tissues 72
hr after injection Of 1"(t-6A1; into bourse shoots Of

'Spencer Seedless’ apple in yitgg in 1994 .........151

xfi

LIST OF FIGURES

LITERATURE REVIEW

Figure 1 - Cycle Of biennial bearing Of apple trees.
.......................................5

SECTION I
Figure 1 - The effect of removal Of seeded vs. seedless
fruit on flowering of 'Spencer Seedless’ spurs the
following year. NP-not pollinated (seedless) fruits.
P-pollinated (seeded) fruits. Fruits removed in 1992
and 1993: flowering recorded in 1993 and 1994. ......35
Figure 2 - Effects Of seeds on growth Of ’Spencer
Seedless' apple in 1992 (A) and 1993 (B). ...........38
Figure 3 - Diameters of seeded 'Spencer Seedless' apple
fruits at time Of wounding (initial) and at maturity
(170 DAFB), and diameters Of non-treated controls at
maturity, in 1992 and 1993. Time Of treatment (DAFB) is
indicated on the ordinate ............. ........ ......41

SECTION II
Figure 1 - HPLC profile in methanol/water system for
1‘C-(iA12 biosynthesized from R,S-4,5-“C-mevalonate (MVA)
(see text for the conditions of biosynthesis and

Chromatograth)oooooooooooooooooooooo.0.0.00.0000000053

)tiii

Figure 2 - Abundance of selected ions in mass spectrum
of Me-TMS derivative of 1"C-GAQ synthesized from 1"C-MVA
following GC-MS (see text for conditions)............55
Figure 3 - Mass spectrum of Me-TMS GAR synthesized
from 1"(t-MVA. Ions Characteristic Of GA” are 360 (M+),
328, 300 (base peak-X) and 241. Additional ions are
evident at x+2, x+4, X+6, and X+8, indicating presence
of radioactive forms.................................57
Figure 4 - Expanded mass spectrum Of Me-TMS derivative
of “C-GAu (see Fig. 3), showing evidence of
radioactive forms....................................59
Figure 5 - Mass spectrum of Me-TMS ether of chemically
synthesized 1"c-GAQ obtained from L. Mander. Relative
heights Of base peaks at 300/302 indicate that
approximately 80% is 1"C-GAu. with one labelled
carbon...............................................61
Figure 6 - HPLC profile Of metabolites Of 1"C-GAngin
extract Of 'Spencer Seedless' apple seeds treated 36
DAFB in 1993. Numbers indicate approximate retention
times (min)..........................................71
Figure 7 - HPLC profiles of metabolites Of “C'GAQ in
extracts of seed of 'Spencer Seedless' on branches held
in xitzg in 1994. Tissues sampled 72 hr after
treatment. A. Treated 40 DAFB: B. Treated 45 DAFB.

......OOOOCOOCOOOOOO......OOOOOOOOOOOO0.073

Figure 8 - Autoradiogram Of TLC plate following

flw

chromatography of metabolites of 1"CnGAu, from 'Spencer
Seedless’ apple seeds in ethyl acetate : chloroform :
acetic acid (15:5:1). Numbers indicate elution times on
HPLC. Non-hydrolyzed metabolites (left): acid
hydrolyzed metabolites (right).......................79
Figure 9 - Comparison Of Chromatographic properties of
metabolites of 1‘C-GA12 in seeds (A) and apices (B) Of
apple with those of known GAs (see Table 2,3,7). -
Metabolites in extracts; - Known GAs: Known GAs,
based upon data Of Lin and Stafford (1991)..........82
Figure 10 - HPLC profiles of metabolites of 1"C-GAw in
extracts of apices Of ’Spencer Seedless' apple treated
36 DAFB (A) and 48 DAFB (B) in 1993 ................84
Figure 11 - Abundance of selected ions in mass spectrum
of Me-TMS derivative of residual 1"C-GA12 (AP37) in
apices of ’Spencer Seedless’ in 1995 (TIC, ions 241,
300, 328)...........................................86
Figure 12 - Mass spectrum of the Me-TMS derivative of
the residual 1‘C-(§A12 (AP37) in extract Of apices Of
'Spencer Seedless' in 1993..........................88
Figure 13 - Upper portion of mass spectrum of the Me-
TMS derivative Of residual 1"C-(iAu, (AP37) in extracts
of apices of 'Spencer Seedless’ in 1993 ............90
Figure 14 - Autoradiogram of TLC plate following
chromatography of metabolites Of 1"CI-GAmin extracts of

apices of 'Spencer Seedless' in ethyl acetate :

XV

chloroform : acetic acid (15:5:1). Numbers indicate
elution times on HPLC. Non-hydrolyzed metabolites
(left): acid hydrolyzed metabolites (right).........95
Figure 15 - Effects of time Of treatment on relative
levels Of metabolites of 1"C4313“... recovered from seeds
of 'Spencer Seedless’ and 'Spartan' apple in 1992-93.
Data points indicate radioactivity as a percent of
total radioactivity recovered following HPLC. Values
for 1 sample (1992) or means for 6 ( 2 times Of
sampling x 3 replicates) samples (1993). Vertical
bar-lsd (p80.05) for 'Spencer Seedless' in 1993.....99
Figure 16 - Effects Of time of treatment and Of
incubation (24, 48 hr) on content Of 1"C-GA12 (% of
total recovered) in apices of spurs bearing pollinated
(P) vs. non-pollinated (NP) flowers Of 'Spencer
Seedless’ in 1992. Vertical bars: 3 standard deviation.
................................103
Figure 17 - Effects Of time Of treatment and Of
incubation (24, 48 hr) on content Of 1"C-GAu (% of
total recovered) in apices of spurs bearing pollinated
(P) vs. non-pollinated (NP) flowers Of ’Spencer
Seedless’ in 1993. Vertical bars: 1 standard deviation.
..............................105
Figure 18 - “C-GAQ remaining in apices on spurs with
seedless and seeded fruit of ’Spencer Seedless' and on

spurs of 'Spartan' 24 hr after treatment 1992. Vertical

xfi

bars: t standard deviation.

SSN=Spencer Seedless, not pollinated

SSP=Spencer Seedless, pollinated

SP=Spartan, pollinated

.......................................109

Figure 19 - Effects Of time Of treatment on relative
levels of metabolites of 1‘C-GA12 recovered from apices
of 'Spencer Seedless’ and 'Spartan' apple in 1992-93.
Data points indicate radioactivity as a percent of
total radioactivity recovered following HPLC. Values
are means for 6 (2 times Of sampling X 3 replicates)
samples (1992 and 1993). Vertical bar = lsd (p=0.05)
for 'Spencer Seedless’ in 1993 ....................111
Figure 20 - HPLC profiles Of metabolites of 1"C--(.'.A12 in
extracts Of apices of 'Spencer Seedless' on branches
held in xitzg in 1994. Tissues sampled 72 hr after
treatment.

A. and C. Treated 26 June (40 DAFB)

B. and D. Treated 1 July (45 DAFB)

A. and B. Spurs with seeded fruits

C. and D. Spurs with seedless fruits.

.......................................113

Figure 21 - 1‘C-GAr, remaining (%) in seeds (A), and in
apices of spurs with seedless (B) or seeded fruits (C)
of ’Spencer Seedless' after 24 (1992 and 1993) or 72 hr

(1994). (Treatments in 1994 were applied to fruits and

xfii

apices on severed branches). Vertical bars: 1 standard
deviation..................................... ..... 115
Figure 22 - Effects of time Of treatment and Of
incubation time on content of “C-GAQ (% of total
recovered) in apices of spurs of 'Spartan' in 1992.
Vertical bars: 1 standard deviation... ...... .. ..... 117
SECTION III
Figure 1 - Percentage Of total radioactivity recovered
from ’Spencer Seedless' apple tissues after injection
of “C-sA,z into seeds in 1992 (A) and 1993 (B). Note
that scales on vertical axes differ for the two years.
Values for BS, BL and apex in 1992 are multiplied by
50................................... .......... ....135
Figure 2 - Radioactivity recovered (DPM/loo mg DW) from
inner part Of fruits Of 'Spencer Seedless’ apple 24 and
48 hr after injection of “-GA12 into seeds at different
dates in 1993.......................................139
Figure 3 - Effects Of time Of treatment and presence of
seeds on total radioactivity recovered (DPM/loo mg DW)
in inner part Of fruit following injection of 1"C-GAu
into bourse shoots Of 'Spencer Seedless' apple in 1992.
...... ..... .. ........ ..........l45
Figure 4 - Chromatographic characteristics of a polar
metabolite in cluster base following injection of 1"C-
GA“ into the seeds or apices of 'Spencer Seedless'

apple. A: HPLC profile, using methanol/water gradient

xvfii

as described in the text. B: Radioautogram Of this
metabolite following TLC in ethyl acetate : chloroform
: acetic acid (15:5:1). 1-before hydrolysis; 2-after
acid hydrolysis....................................154
Figure 5 - HPLC traces (see text for conditions) Of
metabolites extracted from fruit flesh following
injection Of 1‘C-GA12 into seeds in .1992 (A) and 1993
(B)................................................156
Figure 6 - TLC Of metabolites Of 1‘C-GAW extracted from
fruit flesh following injection Of “C-GAn into seeds.
1-metabolite eluted at 29 min on HPLC (see Fig. 2); 2-
metabolite eluted at 33 min. Left - before acid

hydrolysis; right - after acid hydrolysis .........158

flm

LITERATURE REVIEW

Introduction

The main objectives of growing fruit trees are to get
reasonable yields and good quality fruit and to maintain the
tree's productive life. How to control flowering is one Of
the key issues in reaching these goals. A common problem is
biennial bearing -- cropping every other year--which reduces
yield and quality. Many species from different families
exhibit this tendency (Table 1).

MW

Biennial bearing affects fruit size, color, and
quality. In general, half Of the total yield for an "on
year" plus an "off year" is less than the yield for one "on
year" Of a regular bearing cultivar. More branches are
broken in the ”on year" because of the excessive weight Of
fruits. By depletion of the reserve products in the ”on
year”, the tree can become more susceptible to winter injury
than regular bearing trees (Bomeke, 1955).

Much research has been done on biennial bearing, but
the problem still exists. A better understanding of the
mechanisms controlling flowering is essential to solving

1

Table 1. Families and species of plants in which alternate

bearing has been reported.

 

Family Species Common name Reference
Anacardiaceae Mangifgra indie; mango Singh,
1971
2153331; yer; pistachio Crane and
Nelson,
1971
Corylaceae $911135 gygllgna hazel Gardner,
1966
Ericaceae Veggininm
mggzgggngn cranberry Lenhardt,
1976
Eaton,
1978
Euphorbiaceae Aleuzitgs forgii tung Potter, et
al., 1947
Juglandaceae Cary; illinggngig pecan Davis and
Sparks,
1974
Oleaceae glee gnzgpgga Olive Stutte and
Martin,
1986

Table 1 (cont'd)

Rutaceae Cums sinensis
91m reticular;
Cums mama.

Sapindaceae 1.1.5.9111 emanate

Rosaceae Malus 5112253115

Erasmus

magmatic:

mm

orange

tangerine

satsuma

litchi

apple

pear

plum,prune

apricot

Maggs &
Alexander,
1969 Moss,
1969: West
and
Barnard,
1935
Jones, et
al.,1975
Iwasaki,
et al.,
1962
Chandler,
1950
Jonkers,
1979
Jonkers,
1979
Couranjou,
1970
Fisher,

1951

this problem.

Trees usually bear on alternate years because fruit set
is excessive during the "on year". When the quantity Of
fruit on the tree in relation to the amount of foliage is
excessive, flower bud formation is reduced or entirely
prevented. Thus in the season following the " on year", the
reduction in bloom results in a short crop: in the " Off
year ", too many fruit buds form. Once begun, such a
fruiting pattern tends to continue. Practically all
cultivars bear more heavily on alternate years, but the
tendency is more pronounced in certain cultivars than in
others. A typical cycle of biennial bearing is shown in
Fig. 1.

Individual limbs or whole trees may exhibit this cyclic
bearing pattern, depending upon the previous cropping and
weather conditions.

In most cases, large crops inhibit flowering. However,
in pistachio heavy cropping does not inhibit flower
initiation, but induces abscission Of the flower buds

(Crane,et al., 1976).

MW
Early defoliation and defruiting experiments with

apple, pear, and prune revealed that leaves promoted flower
bud formation but subsequent setting Of seeded fruit

inhibited it. Davis (1957) removed prune flowers and

”Snow Ball” ___' HOW! initial

(100% full bloom) fruit set
Many flowers initiated No resting spurs
Many rewng spurs No flowers initiated

/

No fruilset

Fig.1. Cycle Of biennial bearing Of apple trees. (Williams,

1974).

fruitlets at 10-day intervals after full bloom. As
treatment was delayed return bloom the following spring fell
to nearly zero after 50 days and thereafter. When leaves on
apple spurs were removed at weekly intervals after full
bloom, no flower buds formed on those spurs that were
defoliated within 6 to 10 weeks after full bloom, but
flowering increased as treatment was delayed (Harley, et
al., 1942).
Defoliation of rapidly growing shoots Of 'Chico', a

highly fruitful walnut cultivar, did not inhibit pistillate
i flower bud formation (Ryugo and Ramos, 1979). The flowering
stimulus in this cultivar is seemingly omnipresent, being
translocated to the dormant buds in the defoliated zone from
older leaves below or new ones being formed above this zone.
When shoots were pruned back tO the fourth, eighth, or
twelfth nodes from the apex in July, buds that developed
into shoots from the fourth node bore some pistillate
flowers while those at lower nodes had proportionately more
flowers. Examination Of comparable buds on unpruned shoots
revealed only sepal primordia. Flower differentiation
proceeded rapidly when buds were forced to grow.

Alternate bearing has been investigated in apples
longer and more extensively than in any other fruit trees.
Alternation has been and still is a problem of horticultural
importance in many countries (Williams and Edgerton, 1974).

In the United States, alternate bearing is less important

for apples than it used to be. This is due partly to
selection of regularly bearing cultivars, but much more to
the development of chemical thinning programs, which
indirectly regulate flower production (Jonkers, 1979:
Williams and Edgerton, 1974; Williams, 1979). However,
thinning response is cultivar dependent: some cultivars are
very difficult to thin, and thinning effectiveness varies
with weather conditions. Moreover, synthetic chemicals
represent a potential pollution problem. Therefore a better
understanding of the flowering process is needed in order to

control flowering naturally.

W

The flowering response in some plants can be influenced
by specific stimuli such as daylength or vernalization, but
in others flowering is not controlled specifically by
external factors. Most woody plants, including apple, fall
into the second group and for this reason have not been
examined in detail by workers involved in research on
flowering. Exogenous factors include light (Tramp, 1984),
pruning (Schupp, et al., 1992; Barden, 1989: Gao et al.,
1992), shoot orientation (Jindal, 1990, 1992), ringing and
scoring ( Iwahori, 1990), rootstock (Schupp, 1992; Gao, et
al., 1992), nitrogen nutrition (Grasmanis and Edwards, 1974:
Gao, et al., 1992), phosphorus supply (Neilsen, et al.,

1990: Taylor and Nichols, 1990), water supply (Jones, 1987),

temperature (Tromp, 1984: Osanai, et al., 1990) and growth
regulators/grOwth substances (Tromp, 1973: McLaughlin and
Greene, 1984 ). Given my research problem, I will confine
my discussion to effects of natural and synthetic growth

regulators on flowering, with emphasis on gibberellins.

W.

The most intensive research relating GA with flowering
has been done with long-day herbaceous plants, as GA can
induce such plants tO flower under short-day. However, in
some cases GA just induces bolting, i.e., stem elongation,

without inducing flowering.

WWW-

For woody plants, there is increasing evidence that

specific GAs and their metabolites are involved in flowering
(Moriz, 1989; Pharis, 1991, cited by Bonnet-Masimbert and
Webber, 1995). A mixture Of the less polar GAs--GA‘ and
GAr-induces flowering in £13m, whereas GA; is commonly
used for species Of Cypressaggae and Iaxggiaggge. Doumas,
et al. (unpublished results, cited by Pharis 1991) Observed
higher concentrations Of GA, and GA7 in shoots and primordia
Of flowering Douglas-fir as compared to vegetative plants.
Primordia with a high potential to flower had'50 to 80 times
more GA, than primordia on nonflowering Douglas-fir trees.

Root pruning applied in the absence Of an exogenous

application of GA,” increased the amount of GA7 present in
the primordia. When both root pruning and GA“? treatments
were applied, a strong synergistic increase was observed.

Therefore GA appears to have a direct influence on

flowering in Home. We and mm species-

WWW-

GAs inhibit flowering of most fruit trees (Table 2).
However, GA, reportedly promotes and GA-, inhibits flowering
Of apple (Looney, et al., 1985). Depending on the species,
the sites and modes of GA action may differ, and this may
account for the contradictory responses Observed between and
within species. Nevertheless, there is no explanation at
present why fruit trees respond differently from conifers to
application Of GAs.

Oliveira and Browning (1993) reported that GA4,<HM and
(Mg inhibited floral initiation in Erunug ayium by 9-17%,

CA, by 43%, GA; by 65-71% and 2,2-dimethyl GA, by 78%. 6A9
and GAzo were inactive. Thus activity of GAs with a C-3
hydroxyl was increased markedly by a double bond in the C-
1,2 or C-2,3 position, and activity increased with
increasing hydroxylation. They found that juvenile and
mature shoots differed in sensitivity to a C-1,2 or C-2,3
double bond and that phase change altered the GA complement,
GA receptor or transduction mechanisms of 2‘ gyigm. Floral

initiation and growth probably have different requirements

Table 2. GA effects on flowering of tree fruits

 

 

Species GA Flower Resp. Reference
Apple GA; Hull and Lewis, 1959: Buban
and Faust, 1982; Guttridge,
1962; Marcelle and Sironval,
1963: Dennis and Edgerton,
1966.
GAuy Marina and Greene, 1981;
Wertheim, 1973.
GA, Looney, et al., 1978; Looney,
et al., 1985.
GA7 Hoad,1984: Tromp,1982.
Pear GA; Griggs and Iwakiri, 1961.
Cherry,sweet GA; Bradley and Crane, 1959.
Cherry,sour GA; Bukovac, et al., 1986.
Plum GA; Bradley and Crane, 1959.
Almond GA; Bradley and Crane, 1959.
Peach GA; Edgerton, 1966: Hull and
Lewis, 1959; Gur, et al.,
1993.
Citrus GA; Guardiola et al., 1982;

Goldschmidt and Goren, 1985.

 

10

in terms of GA transport and/or action. They proposed that
flower initiation in fruit trees is related to the
metabolism of GAs. Whether this control is primary or
secondary is still not clear.

In most polycarpic angiosperms, applied gibberellin
(GA;) is associated with floral inhibition (Table 2). In
contrast, applying growth retardants such as daminozide
(SADH) increases flower initiation.

(Hg inhibits or prevents flower bud formation if it is
applied to shoots during the flower bud induction stage
(Hull and Lewis, 1959: Griggs and Iwakiri, 1961). Dennis
and Edgerton (1966) showed that GA sprays to apple trees at
or before flower initiation inhibited flowering. In rare
instances applied gibberellin induces or promotes flowering
( See Table 2). Gibberellin promotes extension growth in
many plants, and Jackson and Sweet (1972) suggested that in
apple, flower initiation is prevented because GA stimulates
vegetative growth. However, application of GAUV, known to
be endogenous in apple seeds, inhibited flower initiation,
yet shoot growth was not increased significantly (Wertheim,
1973). Merino and Greene (1981) noted that gibberellin
treatment did not increase vegetative growth, hence the
inhibition of flowering could not be attributed to
gibberellin-induced growth. Rather, inhibition must have
been a direct result of the gibberellin sprays. These
results cast doubt on the idea that GA(s) inhibit flowering

11

by increasing shoot growth.

GA “q inhibited flowering in ’Empire' apple (Greene,
1989). Repeat applications 19 and 34 days after full bloom
were only slightly more inhibitory to flowering than one
application of 50, 100, or 150 mg.liter'1 made 10 days after
full bloom. McArtney (1994) showed that a single spray of
either GA; or CA,” at full bloom in the ”Off" year reduced
the severity of the biennial bearing cycle Of 'Braeburn'
apples. The proportion Of flowering spurs one and two years
after application was linearly related to the concentration

of GA applied -— negatively and positively, respectively.

Flower bud formation in many fruit tree species is
enhanced by application Of growth retardants. Both
daminozide (SADH) and cycocel (CCC) inhibit GA biosynthesis
and favor flower bud initiation. Increased flowering has
been reported in apple (Williams, 1972; Greenhalgh and
Edgerton, 1965; Edgerton and Hoffman, 1966: Luckwill, 1970),
pear (Griggs and Iwakiri, 1968), peach (Edgerton, 1966),
plum (Couranjou, 1968), citrus (Monselise and Goren, 1977;
Nir, et al., 1972), mango (Maiti, et al., 1971) and other
species. Ryugo et a1. (1972) found that SADH reduced the GA
content of apices Of upright-growing cherry branches. The

GA content also decreased and flowering was promoted when

12

shoots were bent towards the horizontal position. Head and
Monselise (1976) sprayed M 26 rootstocks with daminozide and
measured gibberellin and ABA content of the stem tips.
Within 2 days of spraying, ABA content of the tissue
increased and 5 days after spraying a decrease in
extractable GA-like substances was observed.
Thus,daminozide could be affecting growth by altering the
hormone balance in the stem tips. Marangoni, et al.
(unpublished -- cited by Ryugo, 1986) reported that a
flower cluster Of 'Muscat Of Alexandria' grape formed
several weeks later on the terminal shoot when rapidly
growing shoots were headed back to the eighth node and
sprayed with CCC, whereas heading back alone did not induce
flowering. Young SADH-treated cherry and pear trees bloomed
at an earlier age than comparable untreated ones (Ryugo,et
al., 1972).

Hence, any treatment that inhibits GA synthesis and

limits shoot elongation apparently favors flower formation.

A

Iades1n2nI_fastere_affesting_f12!!rinsi
glxhgn!‘:.§ll‘ Harley et al., (1942) concluded that

an ”intimate association between starch content and flower
bud differentiation" existed in apple spurs. Following this
report, a tremendous amount of work on utilization of
carbohydrate reserves was carried out (Priestley, 1970;

Jonkers, 1979), but its practical implications for the

13

understanding of flowering and alternation in apple were
very meager. Jackson and Sweet (1972) found no unequivocal
evidence to support the belief that carbohydrates play a
direct, or even supportive, role in flower initiation in
woody plants. The results Of Stutte and Martin's experiment
(1986) showed few and inconsistent differences in sucrose,
fructose and mannitol concentrations in shoots of Olive
trees exposed to 850 umols"m'2 vs. 150 umols"m'2 PAR. The
high level of starch in the former treatment had no effect
on subsequent flowering of bearing or nonbearing trees.
They concluded that an insufficient supply Of carbohydrate
reserves was not the primary factor limiting flower
induction. Other reports on pistachio (Crane et al., 1976)
and citrus (Jones et al., 1974) also showed that
carbohydrates were not a limiting factor for flower bud
differentiation. For citrus, however, contradictory results
were also reported (Hilgeman et al., 1967; Goldschmidt and
Golomb, 1982: Schaffer et al., 1985). Data from Goldschmidt
et al. (1985) showed that autumn girdling and GA;‘treatment
were both effective and additive in increasing starch
contents of leaves and twigs Of ’Shamouti’ orange trees.
However, 6%; inhibited flowering whereas girdling promoted
it.

Even studies of photosynthetic efficiency and
carbohydrate utilization with intact and partly defoliated

regular and alternating apple cultivars (Avery et al., 1979)

14

did not provide evidence that carbohydrate reserves were
limiting, but rather that meristems differ in their capacity
for carbohydrate mobilization. Thus reserve mobilization
and flowering may be closely regulated, possibly by hormonal
factors.

£1.11; In apples, strong inhibition of flower bud
initiation occurs during the.early stages of fruit
development. The physiological processes responsible for
this effect are unknown (Buban and Faust, 1982). After
successful pollination fruits usually contain many
developing seeds. In certain cases seeds inhibit flowering
(apples) or induce flower bud abscission (pistachio).

Chan and Cain (1967) showed that seeded ’Spencer
Seedless' fruits inhibited flower initiation whereas
seedless ones did not. This suggested that presence of seeds
was the main factor controlling biennial bearing, rather
than depletion of nutrients by fruits. Seeds produce
relatively large amounts of gibberellin that presumably
inhibit flower initiation. Further confirmation Of the role
of seeds was provided by Huet (1972) using seeded and
seedless fruits of 'Williams' pear. He demonstrated that
seeded fruits were more inhibitory to flowering than were
seedless ones. However, neither seeded nor seedless fruits
inhibited flowering in 'Bartlett' pear trees in California
(Griggs et al., 1970). Huet (1972) reported that spurs of

'Williams' pear bearing seeded fruits initiated flowers if

15

their leaf surface were sufficiently large. In California,
growth was very vigorous and leaf surface may have been
sufficient to overcome the inhibitory effects of the seeds.
Chan and Cain (1967) reported that seeded fruits did not
inhibit flowering when 'Spencer Seedless' apple trees were
grown in sand culture in a greenhouse; under those
conditions tree growth was vigorouS. Neilsen (personal
communication) evaluated flowering response in ' Spencer
Seedless' apple vs. fruit weight, seed number, cropload, and
bourse shoot length. Across all seed number categories
greater than 4, shoots less than 2 mm never flowered and
shoots longer than 16 mm almost always flowered. In shoots
2 to 4 mm long flowering decreased sharply as seed number
increased. Flowering in shoots 5 to 9 mm long appeared to
be inhibited as seed number increased, but this was less
pronounced than in short shoots. Thus shoot length (leaf
area?) overrode the seed's inhibition Of flower initiation,
confirming Huet's result. Neilsen also showed that fruit
weight per spur and fruit density did not affect return
bloom if fruit were seedless. This finding does not support
the hypothesis that competition for carbohydrates is the
mechanism whereby the fruit inhibits flowering.

Grochowska (1968a) found that surgical removal of apple
seeds after 'June drop' ( fruit size 1.7 to 2.0 cm in
diameter) had no effect upon return bloom unless growth

regulators were substituted for the seeds, GA; inhibiting,

16

IAA promoting flowering. Stutte and Martin (1986) did a
series of experiments to determine the role of the seed and
crop load on flower induction in Olive. Killing the seed
with a needle prior to endocarp sclerification promoted
flower formation in two cultivars. Surprisingly, however,
fruit removal did not stimulate flowering. Dry matter
accumulation and partitioning in both seeded and seed-killed
fruit were similar. Thus, carbohydrate demand did not appear
to be a primary factor in flower formation. They postulated
three hypotheses to explain why killing the seed promoted
flowering while fruit removal did not. 1) The seed is a
source of flower inhibiting compounds and the fruit is a
source of flower promoting substances. 2) The fruit
competes with potential flower buds for flower promotive
compounds.» 3) The seed sequesters flower promotive
compounds originating from the fruit. Timing is critical
here, for flowering of alive is favored if fruits are

removed early in the season.

Wilma.
Nitsch (1958) first demonstrated GA-like activity, and

Dennis and Nitsch (1966) tentatively identified.Gm; and 6A7
in methanol extracts of immature apple seeds. The
identification was confirmed by MacMillan (1968) and
Luckwill, et al. (1969) using gas chromatography-mass

spectrometry (GC-MS); GA, was also found. Many more GAs

17

Table 3. Gibberellins identified in apple tissues

 

Tissue Gibberellinisi Reference—
Immature seeds A4,: A7 Dennis and Nitsch,

 

1966: MacMillan,
1968: Luckwill, et

al., 1969; Road,

1978, 1980

A1; 393A123A153A173320; Hoad, 1978, 1980

M3As133a313-OH-A123

15-3-0H'A13 9,11-dehydro-

:iso-A;

A61; A62 Kirkwood and
MacMillan,1982

A6; Avanzi, et al., 1988

‘ A1:A;:A.:A7;A.:A9; Lin, et al., 1991

315731773193520:AZ43

Ag3A3stAuiAs13A53i

ihuihmiaafibafi

A1:A;:A4,:A7:A53A9:A12: Steffens,et al.,1991

A1533173A193A203A2‘3A3‘3

18

Table 3 (cont'd)

Immature fruit

Vege.tissue

AniMihsiilssihwkfi
AarAarAa:

A13AI3A93A123AmA-m: :

A53 3 iSO'GAy

A1133! A‘IA7IA9IA‘IZIA15I

A173 AjofhzoiAzsf Au33353

M33453 A533A543A613A623

Aesthetimeo: Au: 3-ePi-

4MK3ANI
A73; 9 , 15-cyclo-GA9;

9,15-cyclo-GA9.

A3

Aw

A123 A173 A193 A203 A293

1%33

19

16,17-dihydro-17-

Ramirez, 1993

Hedden, et al., 1993

Ovama, et al., 1996

i13,2£,3£,3o and llfi-hydroxy-

Hayashi, et al.
1968; Dennis,

unpublished

Dennis, unpublished

Koshioka, et., 1983b

Saavedra, et al.

1989

Table 3 (cont'd)
Oil-A”; 2-OH-A53: mono-

Midi-bathe

20

have since been identified in extracts of seeds and other
tissues of apple (Table 3).

The GAs identified in immature apple seeds represent GAs in
the early l3-hydroxylation pathway and early non-
hydroxylation pathway.

WW
Luckwill, et al., (1969) measured seasonal levels of

GA-like substances - presumably a mixture of GA‘ and 6A7 -
in seeds of 3 apple cultivars, using the lettuce hypocotyl
assay. Maximum activity occurred 9 weeks after full bloom,
when the embryo was approaching full length and the
secondary endosperm had attained maximum volume. Two
promoters of Aygng mesocotyl section elongation, which were
not GA-like, were also characterized and quantified, maximum
activity occurring at 6 and 13 weeks, respectively. Dennis
(1976) reported that levels of gibberellin-like substances
in apple seeds and fruit flesh were at a maximum in early
July, shortly after June drop. Bioassay of extracts prepared
at this time indicated that over 95% of the activity
occurred in the endosperm. Extracts of fruit flesh
contained two GA-like components, both more polar than GAH7.
Activity per unit fresh weight was approximately 3000-fold

higher in seeds than in fruit tissue.

21

WWW-

six GAs were found in extracts of shoots of standard
and dwarf apple trees (Steffens and Hedden, 1992). They
reported that levels of GA“ GA; and GA. were similar in mid-
April when both standards and dwarts started to develop. On
June 2, standard shoots contained higher concentrations of
GA”, can and GA”. In both stardard and dwarf trees the
content of GA; and GA, on May 12 and June 2 were similar to
those on April 20, whereas the GA”, GAzo, GA1 and 6A2, levels
had declined. GA levels continued to decline between June 2
and 22 in standard shoots. During that time, shoot
elongation stopped. No qualitative differences of
gibberellins were found between standard and dwarf apples.
However, GA”.was higher in dwarf apple trees than the

standard ones during summer time.

mm.

The role of endogenous hormones in flower initiation in
woody plants and the effects of applied growth regulators on
this process have been studied extensively (Luckwill et a1,
1978: Road 1978; Ramirez and Road, 1978; Ramirez, 1979).

The evidence that gibberellins inhibit flower
initiation is both direct and indirect. Luckwill, et al.,
(1969) showed that the concentration of GA-like substances
in seeds increased considerably about 5 weeks after full

bloom -- the period when seeded fruitlets became inhibitory

22

to flower initiation (Luckwill 1970). Proof that seed
gibberellins are responsible for inhibition requires
demonstration that they can move from the fruit into the
spur where flowers are initiated. Luckwill (1974) found
more GA-like activity in spurs with fruits than in those
without fruits, but there was no evidence that the compounds
were moving from the fruit through the pedicel into the spur
tissues. However, compounds, including GAs, injected into
seeds or locules of apples can move into the bourse shoot
(Grochowska, 1974). Higher concentrations of endogenous
gibberellin-like substances have been observed in diffusates
from pedicels of biennial-bearing cultivars of apple than in
those from annual cultivars (Hoad, 1978). Gil (1973)
reported greater GA-like activity in diffusates from pear
fruit of the biennial cultivar 'Winter Nelis’ than in those
from fruit of the annual cultivar 'Bartlett'. Hoad (1978)
obtained more gibberellin-like activity in diffusates from
seeded 'Spencer Seedless' fruitlets than from seedless ones.
As GA content of the diffusate was very low, the compounds
could not be identified by GC-HS. However, their Rfs on
thin layer chromatography resembled those of GAuq. He also
(1978) investigated the diffusate collected from apple
fruitlets during the period prior to flower initiation.

More hormones (oat mesocotyl assay) moved out of the
biennial flowering cultivar 'Laxton's Superb' than from the

annual cv: 'Cox's Orange Pippin’. Ebert and Bangerth

23

(1981) investigated the levels of diffusible and extractable
gibberellin-like substances from fruits of the alternate-
bearing 'King of the Pippins’ and the regularly fruiting
'Golden Dolicious' by using the barley endosperm bioassay
(Note: Golden Delicous is annual in Germany, but biennual in
the 0.8.). In 'King of the Pippins' a very steep increase
in the level of activity occurred in both years during the
second week after full bloom, reaching a maximum about 3
weeks after full bloom. Maximum levels in diffusates from
control fruits were approximately 40 to 45 times higher than
corresponding values from chemically thinned fruits. In
later stages of fruit development, the contents of
diffusible GA-like substances again increased, but this time
no marked difference existed between control and treated
fruits, with the exception of those treated with
naphthaleneacetamide, in which the second peak occurred
earlier. A different pattern was found in the regularly
fruiting 'Golden Delicious'. With this cultivar, no
increase in diffusible GAs from untreated fruits was
observed until 4 weeks after full bloom, and peak values
occurred during the 5th week. The thinning treatments
caused no significant change in the amount of extractable
GAs, which was very similar in fruits from both cultivars.
Harino and Greene (1981) noted that gibberellin-like
activity in diffusates of flowers or fruits of ’Empire' was

detected at bloom and continued to increase until 45 DAFB.

24

Hore GA-like activity was obtained from fruit-bearing spurs
than from vegetative spurs. However, peak gibberellin
activity in seeds and diffusates was not paralleled by
elevated activity in fruiting spurs. Thus, inhibition of
flowering may not be related solely to diffusion of
gibberellin to the spurs during one short time period. In
preliminary experiments, Pierson (personal communication,
1996) found more GA; in bearing shoots compared with non-
bearing shoots.

Another way in which transport has been investigated is
by introducing radiolabelled hormones into the seeds, and
following their movement from the fruit. Several papers
(Knight and Webster, 1986: Hoad, 1978; Green, 1987)
indicated movement of GAs; however only a very small
proportion of the labelled GAs moved out of the fruit.
Knight and Webster (1986) applied GA; to a single flower per
cluster of 'Conference’ pear and 'Early Rivers' cherry at
full bloom and 50% petal-fall, respectively, and to a single
fruitlet per cluster of 'Victoria' plum four weeks after 50%
petal-fall. They measured the distribution of label to
flowers or fruitlets, either on the same spur or on adjacent
spurs, but little or no radioactivity was detected outside
the treated organ. When Hoad (1978) applied 3H-GA9 to seeds
of intact fruit, no activity was detected in the spur tissue
72 hr later. However, he found that more radioactivity was

detected in the spur tissue of the strongly biennial

25

cultivar 'Laxton' than the regular cultivar 'Cox' after
applying 3H-GA3 to seeds. Green (1987) continued these
experiments; transport was limited for all compounds used
(“c-6A,, aldehyde, 3mm“ 1‘C-ZIIAA). She found that there was
no difference in GA content of seeds of the biennial
cultivar 'Tremlett's Bitter’ vs. the annual bearing one
'Dabinett"in terms of quantity and quality. She could not
detect GA‘ or GA-, in fruit diffusates, and only a small
amount of GA, was identified by GC-HS.

Another hypothesis, proposed by Looney et al., (1978),
is that seeds affect metabolism of GAs in the bourse shoot
and thereby inhibit flowering. Looney, et al., (1985)
demonstrated that GA‘ promotes flowering and GA7 inhibits
flowering of apple. They also sprayed §+4Hu on apple
trees, and measured the rate of metabolism. Trees destined
to flower metabolized GA faster than those in which

flowering did not occur.

Ell-II!

Some commercial apple cultivars ('Paulared’, 'Golden
Delicious', etc.) are strongly biennial, flowering and
bearing fruits every other year. This problem was once
attributed to a reduction in carbohydrate reserves in the
bearing year, few flowers being formed when many fruits
were competing with apical meristems for carbohydrates. But

Chan and Cain (1967) reported that seedless fruit of the

26

facultatively parthenocarpic cultivar 'Spencer Seedless' did
not inhibit flowering, whereas seeded fruit did. This
finding cannot be explained by the carbohydrate hypothesis.
Since apple seeds contain high concentrations of GAs, and
exogenous application of GAs can inhibit flower formation in
apple, GAs originating in the seeds may inhibit flower
initiation. How GAs might influence flowering is not yet
clear. One hypothesis proposes that gibberellins from the
seeds move to the buds and inhibit flowering. Another
hypothesis is that the seeds affect metabolism of GAs in the
bourse sheet, resulting in an inhibition of flowering
(Looney, et al. 1978). The purposes of my research were to
determine if:

1. GAs move from apple seeds to the apex of the bourse
shoot; .

2. The presence of seeds in the fruit affects metabolism of

GAs in the apex.

27

DICTIOH I lffect of Time of fruit Removal on flower
Initiation in 'Spencer Seedless' Apple, and
lffeot of cutting Fruits on Retention and
Growth of 'Bpencer Seedless' and 'Peulered'

fruits.
ABSTRACT

Defruiting experiments confirmed that seeded fruit of
'Spencer Seedless' apple inhibited flower initiation and
seedless ones did not. Significant inhibition began around
35 days after full bloom (DAFB). At that time, seed length
was 7.6 mm (75% of final length) in 1992, and 7.2 mm in 1993
(64* of the final length). Embryos had not begun rapid
growth at that time. Seeded fruits were significantly
larger than seedless ones at harvest, but diameters did not
differ significantly until 52 DAFB in 1992 and 41 DAFB in
1993, and fruit weight did not differ significantly until 36
or 37 DAFB in both years. Cutting fruits to expose the
locules and seeds, then replacing the apical portion, had no
effect on abscission of fruit of 'Spencer Seedless'.
However, wounded fruits were smaller than controls at
maturity. In contrast, seeded 'Paulared’ fruits all

abscised within 2 weeks when similarly treated.

28

INTRODUCTION

Biennial bearing is a common problem in fruit
production. The presence of a heavy fruit crop inhibits
flower initiation in the adjacent shoot buds (Greenhalgh and
Edgerton, 1967: Parry, 1974; Ludders, 1978). Alternate
bearing was once thought to be the result of nutritional
deficiencies. However, there is no unequivocal evidence to
support the belief that carbohydrates play a direct, or even
supportive, role in flower initiation in woody plants
(Jackson and Sweet, 1972). The level of carbohydrates had
no effect on the flowering pattern of olive trees (Stutte
and Martin, 1986). Chan and Cain (1967) found that seeded
fruit of 'Spencer Seedless' and 'Ohio 3', both facultatively
parthenocarpic cultivars, inhibited return bloom, whereas
seedless fruits did not. Removal of seeded fruits at
intervals after bloom indicated that 65% of the inhibition
occurred within the first 3 to 5 weeks after pollination.
Huet and Lemoine (1972) also found seeded pears to be more
inhibitory to flower initiation than seedless ones. These
results clearly show that seeds, rather than fruits, inhibit
flower initiation.

Apple fruit retention is dependent upon seed
development for several weeks following anthesis. Abbott

(1958) demonstrated that early seed removal 4 weeks after

29

petal fall induced abscission of 'Cox's Orange Pippin’
fruits. Replacement of the seeds with a lanolin paste
containing naphthaleneacetic acid (NAA) permitted continued
development. However, NAA did not prevent abscission of
deseeded 'Crawley Beauty' fruits. After 7 weeks, seeds
could be removed with little or no effect upon abscission.
Southwick, et al. (1962) reported that growth of 'Golden
Delicious' fruits was independent of seed development after
'June' drop, although ’McIntosh' fruit tended to abscise
following‘seed removal. Gucci, et al., (1991) observed
that 'Paulared' also had a high abscission potential
following seed removal, even when fruits were half grown.
The purpose of this research was to determine the effects of
time of removal of seeded vs. seedless fruit of 'Spencer
Seedless' on flower initiation, and to evaluate the effects
of cutting the fruits (wounding) on fruit retention and

growth of both seeded and seedless fruits.

MATERIALS AND METHODS
21§n;_|gtg:131§1 Two mature trees of 'Spencer Seedless'
apple, an apetalous and facultatively parthenocarpic
cultivar, and two mature 'Paulared' trees were used at the
Horticultural Research and Teaching Center, Michigan State
University, I: . Lansing .
Clusters of flowers on individual branch units of

'Spencer Seedless' were pollinated with a mixture of

30

'McIntosh' and 'Delicious' pollen in 1992 and 1993 to obtain
seeded fruits: flowers on the remaining branches were not

pollinated, and served as a source of seedless fruits.

. . . .. ‘
L3:gngg;_ggg§1111;; Fruits were removed from limbs bearing
pollinated and non-pollinated flowers (25 spurs each at each
date) at intervals between 25 and 120 days after full bloom
(DAFB) in 1992 and 1993, and spurs were tagged to evaluate
return bloom.

Diameter and weight of fruits were recorded, and seed and

embryo length were measured.

WW- An
additional 10 fruits (one per spur) of both seeded and
seedless 'Spencer Seedless' and 10 fruits of 'Paulared' (all
seeded) were cut in half with a sharp knife on each date,
leaving the seeds intact. All non-treated fruits were
removed fom the spurs. The apical half was then replaced,
and parafilm used to cover the wound. An additional 10 non-
treated fruits of both cultivars were also tagged on the
first date of treatment (30 to 37 DAFB). The abscission
rate and the diameter of the wounded fruit were recorded at

harvest.

31

RESULTS

WWW
Langnggx_figg§1ggg;; Seedless fruits had essentially no

effect on flowering, even when left until harvest (Fig. 1).
However, flowering of spurs bearing seeded fruit declined
dramatically within about 25 DAFB, although approximately 50
(1992) and 25% (1993) of the spurs flowered even when fruits
remained until harvest (Fig. 1). These results confirm
those of Chan and Cain, except that they observed 6 to 27%
flowering, depending upon year, when seeded fruits were left
until harvest. The relatively high rate of return bloom
when seeded fruits were removed late in 1992 was probably
the result of the use on these dates of spurs with
relatively long bourse shoots, which have a greater

flowering potential (Neilsen, unpublished data).

eve o ' nce e I.

Seeded fruit were larger at harvest than seedless fruit.
Differences in diameter did not become significant until 7
to 8 weeks after full bloom, but differences in weight were
significant at 6 weeks after full bloom (Table 1, Fig. 2).

Seed had reached almost 70 percent of full length by 37
days after full bloom in 1992, and 50 percent of full length
at 30 days in 1993 (Table 1, Fig. 2). The embryo became

visible at 45 DAFB in 1992, 41 DAFB in 1993. Embryo lengths

32

were only 2 to 3 percent of mature length at this time

(Table 1).

WWW

Abscission of 'Spencer Seedless' fruits following wounding
ranged from 10 to 30 percent -- similar to the untreated
control, whereas all wounded ’Paulared' fruits abscised
within 10 to 14 days after cutting (Table 2). Wounding
strongly inhibited fruit enlargement of both seeded and
seedless fruits. Fruits treated 4 to 5 weeks after full
bloom were considerably smaller than control fruits, and
the earlier the fruits were wounded, the greater the effect
( Table 1, Fig. 3). Seeded fruit did not grow appreciably
more than seedless ones following treatment. Average fruit

diameter increased only 3 to 8 mm following early treatment.

33

Figure l - The effect of removal of seeded vs. seedless
fruit on flowering of 'Spencer Seedless’ spurs the following
year. NP-not pollinated (seedless) fruits. P-pollinated
(seeded) fruits. Fruits removed in 1992 and 1993: flowering

recorded in 1993 and 1994.

34

200

 

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125

octoBoE 3.5 .o anecdotes

Days After Full Bloom

35

Table 1. Diameter and weight of fruit, and length of seed and embryo of
‘Spencer Seedless’ at the time of sampling or wounding and diameter of
wounded fruit at harvest (170 DAFB) in 1992 and 1993 (ten fruits per
sample).

 

DAFB Seed Fruit diam. Fruit weight Seed length Embryo length Final diam".

 

 

 

 

(mm) (9) (mm) (mm) (mm)
1992

37 + 30 15 8 0 39

- 29 n.s. 13 n.s. 37 n.s.
45 + 32 20 8 0.3 44

- 29 n.s. 15* 4O n.s.
52 + 41 34 8 2 53

- 38 n.s. 27' 49 n.s.
59 + 57 57 10 4 73

- 49* 45* 63*
170 + 77 172 12 10 77

- 68" 164* 68*

1993

30 + 25 8 6 0 29

- 21 n.s. 6 n.s. 24 n.s.
36 + 28 12 7 O 36

- 27 n.s. 8 n.s. 34 n.s.
41 + 34 21 8 0.2 45

- 30 n.s. 14* 41 n.s.
48 + 40 30 8 0.8 55

- 36* 22" 49 n.s.
170 + 74 168 11 10 74

- 68’ 158* 68*

 

*Significantly less than seeded control , based upon standard deviation of the
mean.

"F’Inal diameters at harvest of fruits cut at the same times, but not treated.

36

Figure 2 - Effects of seeds on growth of 'Spencer Seedless'
apple in 1992 (A) and 1993 (B).

37

Frult diameter (mm)

 

401

 

 

 

 

 

 

 

50 100

Days after full bloom

38

 

 

 

 

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co 0 o o o om om om cm on om om om cm 00 +
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new «mm? 5 =2. made .3283 .858. .o someones co mos: 95:9, am @5950; .o 82m. .w 29m...

39

Figure 3 - Diameters of seeded 'Spencer Seedless' apple
fruits at time of wounding (initial) and at maturity (17o
DAFB), and diameters of non-treated controls at maturity, in

1992 and 1993. Time of treatment (DAFB) is indicated on the

ordinate.

40

 

 

 

 

A 80 - Non-treated Control
1992
E ....................... 1993
2' I Mature 92
§ 30.. 0 Initial 92
El Mature 93
0 Initial 93 I
E 40 d I I I
20 I l l
20 30 40 50

Days after full bloom

41

 

DISCUSSION

Fruit removal indicated that seeded fruits of 'Spencer
Seedless' had inhibited return bloom by 50 to 70% five weeks
after full bloom, whereas seedless fruits had no effect.

The critical time for flower inhibition therefore was before
35 DAFB. My results parallel those of Chan and Cain (1967)
except that they observed 6 to 27% flowering, depending on
the year, when seeded fruits were left until harvest.
Neilsen.(personal communication) found similar results with
'Spencer Seedless'. Again, variation in degree of
inhibition occurred from year to year, and seeds did not
inhibit flower initiation completely. Even when fruits were
left until harvest, about 30% of the bourse shoots flowered.
These results imply that seeds are not the only factors
involved in controlling flower initiation. Neilsen
(unpublished data) found that both seed number and shoot
length had quantitative effects on flowering response of
'Spencer Seedless', with flowering declining with seed
number and increasing with bourse shoot length. Shoots 16
mm or longer flowered regardless of seed number. Chan and
Cain (1967) observed that seeds had less effect on return
bloom of 'Spencer Seedless' when trees were grown in a
greenhouse, where bourse shoots grew vigorously, and had

greater leaf area. Further confirmation was provided by

42

Huet and Lemoine (1972) with seeded and seedless fruits of
'Williams' pear. They reported that spurs bearing seeded
fruit could flower if their leaf surface were sufficiently
large. Thus some factor(s) coming from leaves apparently
overcomes the inhibitory effects of the seed.

The critical time of inhibition was within five weeks
after full bloom. At that stage, the embryos were very
small, implying that the endosperm or nucellus is
responsible for inhibition. Auxin levels in apple seeds
rise within 35 DAFB (Luckwill, 1959); these compounds may be
involved in controlling flower initiation, although
gibberellins are believed to play a more prominent role
(Luckwill, 1978).

Seeded fruit differed little in size from seedless
fruit during the critical time for inhibition of flower
initiation, and Neilsen (unpublished data) found no
relationship between weight of fruits per spur and
subsequent flowering of bourse shoots. Thus competition
for carbohydrates cannot explain the effect.

Although wounding caused all 'Paulared’ fruits to abscise,
it did not affect fruit retention of 'Spencer Seedless’ even
when the fruits were relatively small: however, fruit size
was reduced. The effect on size lessened as treatment was
delayed. The data of Abbott (1958), Southwick, et al.
(1962) and Gucci,et al. (1991) indicate that wounding
reduced fruit size considerably, but that fruit grew much

43

more than did 'Spencer Seedless' fruits following wounding.
Previous workers used lanolin pastes, rather than Parafilm
to cover the wounds, and Southwick, et al. (1962) noted that
cut fruits treated with lanolin grow more than those left
open to the air.

Seed and fruit conditions were good after cutting and
injecting 1 ul of 10% ethanol into the seeds (data not
shown). Thus the experimental system could be used to study
the metabolism of 14C-GA12 1n_yiyg in subsequent research
(Sections II, III).

No treatments were applied prior to 5 weeks after full
bloom, therefore one cannot conclude that seedless fruits
are less subject to abscission following wounding prior to

this time than are seeded ones.

LITERATURE CITED

Abbott, D.L. 1958. The effects of seed removal on the growth

of apple fruitlets. Annual report, Long Ashton Research

Station. p. 52-56.

Chan, B.C. and J.C. Cain. 1967. The effect of seed

formation on subsequent flowering in apple. Proc. Amer. Soc.

Hort. Sci. 91:63-67.

44

Greenhalgh, W.J. and L.J. Edgerton, 1967. Interaction of
Alar and gibberellin on growth and flowering of the apple.
Proc. Amer. Soc. Hort. Sci. 91:9-17.

Grochowska, M.J. 1968. The influence of growth regulators
inserted into apple fruitlets on flower bud initiation.

Gucci, R., S. Hazzoleni, and F.G. Dennis, Jr. 1991. Effect
of fruit wounding and seed removal on abscission of apple
fruit between June drop and harvest. New Zealand J. Crop

Hort. Sci. 19: 79-85.

Huet, J. and J. Lemoine, 1972. Etude des effets des
feuilles et de fruits sur l'induction florale des

brachyblastes du poirier. Physiol. Veg. 10:529-545.

Jackson, D.I. and G.B. Sweet. 1972. Flower initiation in

temperate woody plants. Hort. Abstr. 42:9-24.

Luckwill, L.C. 1959. Fruit growth in relation to internal
and external chemical stimuli. In: Cell, Organism and

Milieu. Rudnick, D. (ed.) Pub. Ronald Press Co. pp 223-251.

Luckwill, L.C., and R.D. Child. Part tree thinning of apples

with area. 1978. Acta Hort. 80:271-271.

45

Ludders, P. 1978. Effect of Alar 85 and TIEA on apple trees

with different fruit loads. Acta Hort. 80:133-137.

Parry, H.S. 1974. The control of biennial bearing of

Laxton's Superb apple trees. J. Hort. Sci. 49:123-130.

Southwick, F.W., W.K. Weeks, E. Sawada, and J.F. Anderson.
1962. The influence of chemical thinners and seeds on the

growth rate of apples. Proc. Amer. Soc. Hort. Sci. 80:33-42.
Stutte, G.W. and G.C. Martin, 1986. Effect of light

intensity and carbohydrate reserves on flowering in olive.

J. Amer. Soc. Hort. Sci. 111:27-31.

46

sscuow n Hetabolism of “c411,; and Characterisation of
Hetabolites in Apple seeds and Apioes in

Relation to Flower Induction.

ABSTRACT

Radioactive metabolites were analyzed in apple seeds and
apices after injection of 1"C-GAmduring the flower
induction period. No qualitative or quantitative
differences were found between metabolites in apices of
bourse shoots on spurs bearing seeded vs. seedless fruits,
or between cultivars (’Spencer Seedless’ vs. ’Spartan').
However, the rate of metabolism was higher in apices of
bourse shoots in the presence of seedless fruits. Seven
major metabolites were detected in apices, six in seeds.
Four of the metabolites in seeds may be conjugated, as they
were less polar after acid hydrolysis. [Abbreviations used:
ATP-adenosine triphosphate: PEPaphosphoenolpyruvate:
NADPH-nicotinamide adenine dinucleotide phosphate (reduced
form): GA-gibberellin]

47

INTRODUCTION

Apple seeds inhibit flower initiation (Chan and Cain,1967:
Neilsen , 1994: Ban, 1996). Because seeds contain high
concentrations of gibberellins (GAs) and/or GA-like
compounds during early phases of fruit growth (Luckwill, et
al., 1969: Dennis, 1976) and because several GAs can inhibit
flowering of apple (Buban and Faust, 1982: Guttridge, 1962:
Marcelle and Sironval, 1963: Dennis and Edgerton, 1966:
Marino and Greene, 1981: Wertheim, 1973: Looney et al.,
1978, 1985: Hoad, 1984: Tromp, 1982), Luckwill (1970)
suggested that seed GAs may inhibit flowering. More auxin
and/or GA-like activity was observed in diffusates from
seeds or pedicels of biennial vs. annual cultivars of apple
(Grochowska and Karasewska, 1976: Hoad, 1978). However,
Ebert and Bangerth (1981) found no differences in hormone
profiles in diffusates from biennial vs. annual cultivars
over several years. Injection of 1"C-GAu, resulted in very
low radioactivity in tissues outside the fruit, and trace
amounts or no radioactivity were detected in bourse shoot
apices, where flower initiation occurs (Green, 1987).
These results cast doubt on the hypothesis that GAs move
from seeds to apices and there inhibit flower initiation.
Looney, et al.,(1978) suggested on alternative
hypothesis. They reported a positive relationship between

rate of 3H-GA‘ metabolism in shoots and return bloom in

48

apple. They proposed that spurs initiating flowers have a
greater capacity to metabolize GAs. Thus seeds might
inhibit flower initiation by affecting GA metabolism in the
apices. My objective was to investigate both of these
hypotheses, as well as to determine how GA“ is metabolized

by apple seeds and bourse shoots.

MATERIALS AND NETEODS

21.n§_-g;g:ialpl Two mature trees of 'Spencer Seedless'
apple, which is apetalous and facultatively parthenocarpic,
and one annual bearing cultivar, ’Spartan', were used at the
Horticultural Research and Teaching Center, Michigan State
University, E. Lansing. Although 'Spartan' bears annual
crops of fruit, the spurs alternate (personal observation).
The inhibitory effect of seeds becomes evident 20 DAFB,
based upon flowering response of defruited spurs (Ban,
1996), and reaches a maximum after 40 to 50 DAFB.

Clusters of flowers on individual branch units of
’Spencer Seedless' were pollinated with a mixture of
'McIntosh' and 'Delicious' pollen in 1992 and 1993 to obtain
seeded fruits: flowers on the remaining branches were not
pollinated, and served as a source of seedless fruits. One

’Spartan’ tree was open-pollinated.

49

W123. The methods used by Bimberg and
Brenner (1986), as modified by J.A.D.Zeevaart (personal

communication), were used to prepare 1"C-GA12, the precursor
of all the known gibberellins. Briefly, R,S-4,5-“C-
mevalonate lactone , specific activity 54 mCi/mmol
(purchased from Amersham, Buckinghamshire, England) was
hydrolyzed to mevalonic acid (“C-MVA) with 0.5 M NaOH.
Liquid endosperm from pumpkin (gngnrbita maxing) seeds was
collected on an ice bath approximately 1 month prior to
fruit maturity. The endosperm was homogenized in a hand-
held glass homogenizer, and the homogenate was dialyzed
against potassium phosphate buffer [1 M KZHPO‘ : 1 M 101sz :
0.5 n MgC12.6HZO (47.5 ml : 2.5 ml : 5.0 ml, diluted to 10
L)]. The dialysate was frozen as droplets in liquid
nitrogen, and stored at -80'C. The enzyme preparation was
thawed as needed, and incubated with 1‘C-MVA for 3 hr at
30'C in 30 ml centrifuge tubes on a shaker. The reaction
mixture contained: “c-nva, 300 ul = 20 uCi: 0.5 M ngc12, 25
ul: 0.1 H MnC12,.25 ul: 0.5 M ATP, 25 ul: 0.5 M PEP, 25 ul:
0.05 M NADPH, 25 ul: 0.2 M 2,2'dipyridyl, 25 ul: and enzyme
preparation, 2.075 ml. Acetone was added to give 50%
acetone to stop the reaction, the solution was centrifuged
to precipitate proteins, and the supernatant was partitioned
6 times against ethyl acetate. The aqueous layer was
discarded. The ethyl acetate fraction was chromatographed

on aluminum-backed silica gel TLC plates in chloroform :

50

ethyl acetate : acetic acid (75 : 25 : 1) and “C-GAu was
localized by autoradiography. The “C-GAQ band was eluted
with acetone : methanol (1 : 1). HPLC was used for final
purification (Fig. l), the “C-GAQ being detected with a
flow-through detector (B-Ram, IN/US System, Inc.). Specific
activity of purified “C-GAQ was determined by GC-MS (Figs.
2-4). The final product contained a mixture of unlabelled
GAulplus GAu with 1,2,3, or 4 labelled carbons. The
specific activity of 1"C-GAu was calculated according to the
method of Browen and MacMillan (1972) (95 uCi/umol for 1‘C-
can. synthesized in 1992, 115 uCi/umol in 1993) . “C-GA12 used
in 1995 was bought from Dr. L. Mander (Research School of
Chemistry, Australian National University, Canberra, ACT
0200, Australia) , with specific activity 56 mCi/mmol: this

was labelled in only one carbon (Fig. 5).

We... “C-GAaz was applied to seeds and
bourse shoot tips in_yiyg (Table 1). On each date in 1992,

fruits from hand-pollinated flowers were reduced to 2 per
spur, and cut in half equatorially to expose the seeds in
the 5 main locules. A total of 0.3 uCi of 1"C-GAu, was
injected into 3 to 4 seeds of each fruit using a hypodermic
syringe. The top half of the fruit was then replaced and
held in place by wrapping the wound with a strip of
Parafilm.

“C-GAu'was also injected into bourse shoot tips on

51

Figure 1 - HPLC profile in methanol/water system for “C-GAu
biosynthesized from R,S-4,5-“C-mevalonate (see text for the

conditions of biosynthesis and chromatography)

52

 

 

1700'
.1

(woo) Aiiniioeowea

53

50

30

20

10

Time (mln)

Figure 2 - Abundance of selected ions in mass spectrum of
Me-TMS derivative of 1"C-GAmsynthesized from 1"C-MVA

following GC-MS (see text for conditions).

54

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55

Figure 3 - Mass spectrum of Me-TMS GA” synthesized from
“c-iiva. Ions characteristic of can are 360 (11+), 328, 300
(base peak-X) and 241. Additional ions are evident at x+2,

x+4, X+6, and X+8, indicating presence of radioactive forms.

56

 

 

 

 

 

 

 

 

 

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57

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Figure 4 - Expanded mass spectrum of Me-TMS derivative of

1‘C-GAu (see Fig. 3), showing evidence of radioactive forms.

58

 

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59

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Figure 5 - Mass spectrum of Me-TMS ether of chemically
synthesized 1“C-GAmobtained from L. Mander. Relative
heights of base peaks at 300/302 indicate that approximately
80% is l4C-GA12 with one labelled carbon.

60

 

 

 

 

 

 

 

 

 

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61

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Table 1. No. of spurs and fruits (seed treatment ) of ‘Spencer Seedless’ treated.
1992-1995

 

Point of application of 14C-GA12

 

Seed Bourse Shootz

 

 

Seed present: Yes No Yes
Year Date DAFB
1992 June 16 37 1 6 6
June 24 45 1 6 6
July 1 52 1 6 6
July 8 59 1 6 6
1993 June 16 30 8 6 6
June 22 36 8 6 6
June 27 41 8 6 6
July 4 48 8 6 6
1994* June 28 40 4 3
July 1 45 4 3 3
1995 July 5 51 - 80 85

 

2 0.3 uCi/spur, 0.1 uCi/apex (0.05 uCi/apex in 1995)
* Treatments applied to seeds and bourse shoots on excised branches

62

spurs bearing either seeded or seedless fruits, 0.1 uCi
being injected with a hypodermic syringe approximately 5 mm
below the apex. Transparent plastic sleeves, open at the
end, were placed around all treated spurs and bourse shoots
to prevent loss of radioactivity in case of rain.

An in_y1§:g experiment was performed at 40 and 45 DAFB
in 1994 to provide greater quantities of metabolites of
'Spencer Seedless’ for qualitative analysis (Table 1).
Branches ( 30 cm in length ) bearing fruiting spurs were cut
from the trees and their bases placed in water. On transfer
to the laboratory, 1‘C-GAu was injected into the seeds or
apices, using the same quantities as in 1992-1993 (0.3 uCi
per fruit and 0.1 uCi per apex), and the branches were
placed in a growth chamber (16 h light / 8 h dark) at 25'C
(light) and 16'C (dark) for 72 hr.

In 1995, 80 bourse shoots on spurs bearing seedless
fruits and 65 bourse shoots bearing seeded fruits were
selected, then treated with “C-GAn in_yiyg in order to
obtain larger quantities of metabolites. Samples of tissues

were collected and extracted as described below.

§g|p11_gg11gg§1gny Spurs with treated apices were collected
24 and 48 hr after treatment in both 1992 and 1993. Spurs

with treated fruits (seeds) were collected after 48 hr in
1992, and after 24 and 48 hr in 1993. Samples were

collected 72 hr after treatment in both 1994 and 1995.

63

Samples were immediately dissected into treated seeds,
remaining fruit tissue (inner and outer parts separately),
pedicel, cluster base, bourse shoot(s), and leaves. When
bourse shoots were treated, the apices (1 cm) were separated
from the remainder of the shoot. The separated tissues were
frozen at -20'C and lyophilized, and the dry tissues were

stored at -20'C with a desiccant to prevent rehydration.

Wine W-
Lyophilized tissues were extracted with cold 80% methanol
in a cold room (4'C) using a Polytron (PT-MR3000,
Kinematica AG, Littau-Switzerland, Brinkmann Instruments
Inc., Cantiague Rd., Westbury, New York) homogenizer. The
extract was centrifuged at low speed to precipitate the
tissue, the solvent decanted and the precipitate re-
extracted with the same solvent. This was repeated three
times for each extraction. After removal of an aliquot for
radiocounting, the tissue was discarded, the combined
extracts filtered, and the filtrate partially evaporated in
a 'Speedvac' (Savant SC 200, Forma Scientific, Inc.
Marietta, Ohio) centrifugal vacuum evaporator. The residual
solvent was filtered by centrifugation using 400 ul
microspin filters (Life Science Products, Inc.), and 0.2 to
0.5 ml of filtrate was injected into the HPLC. When more
than one sample was available for each date, up to three

separate samples were extracted and chromatographed.

64

Zi_LAI§l_ISIR1!£_1122§1; Apex tissues were extracted

in the same manner as for small samples, but the extracts
were fractioned on a silicic acid column prior to HPLC.
Columns were prepared as described by Powell and Tautvydas
(1967). After stirring and shaking with distilled water, the
suspension of silicic acid (100 mesh) was allowed to settle
and the finer particles poured off. This was repeated until
the silicic acid settled within a few minutes. The particles
were then dried to constant weight at 100'C.

The silicic acid (20 g) was mixed thoroughly with 12.5
ml 0.5 N HCOOH, resulting in a free-flowing powder. This was
slurried with hexane saturated with 0.5 N HCOOH and poured
into a glass column 22 mm in diameter to form a column 20 cm
in height. Additional hexane/HCOOH was forced through with
air pressure to pack the column. I

Extracts were dried on glass wool and the sample placed
on the top of the column, which was then eluted stepwise
with increasing concentations of ethyl acetate in hexane,
both solvents having been saturated with 0.5 N HCOOH. The
sequence used was: 0%: 10%: 30%: 50%: 80% and 100% ethyl
acetate, and finally, 25% n-butanol in ethyl acetate. Thirty
ml of each solvent were used except for 100% hexane (100
ml). The first two fractions contained little or no
radioactivity, and were discarded. The remaining fractions
were evaporated to dryness and the residues dissolved in 30%

methanol for HPLC, as described for small samples.

65

n2ng. Samples were chromatographed on a C18 Bondapak
reverse phase column (250 x 4 mm), using a Waters (Waters
600) HPLC. A discontinuous gradient of methanol in water
was used, both solvents containing 0.1 M acetic acid or
trifluoroacetic acid (TFA). The following protocol was
used: 30% MeOH, 5 min., 30% to 100% in 25 min: 100%, 10 min,
then 100% to 30% in 2 min. to complete the cycle. Flow rate
was 1 ml/min, and pump pressure ranged from 400 to 2500 psi,
depending upon the solvent being pumped. The radioactivity
was measured by the flow-through radioactive detector (8-
RAM). Retention times of available reference GAs and of

additional GAs are given in Table 2.

W- Data were quantified by
calculating both the DPM associated with each metabolite,

and the same value as a percentage of the total
radioactivity recovered. Analysis of variance was used on
the data obtained in 1993 to determine the significance of
quantitative differences due to time of treatment, time of
sample collection (24 vs 48 hr), and the presence of seeds.
Duncan's multiple range test at p < 0.05 was used when more

than two means were compared..

1h13_1.1g:_ghzglatggxgph11 Metabolites were dissolved in

methanol,_and chromatographed (ascending) on ’Eastman

Chromagram' thin layer silica gel plates (20 x 20 cm), using

66

ethyl acetate / chloroform / acetic acid (15:5:1) as the
developing solvent. The plates were exposed to x-ray film
in darkness for 10 to 14 days, the film was processed, and
Rf values were calculated for radioactive zones. Several
standard GAs were also chromatographed for comparison of Rf

values (Table 2).

WWW Metabolites were

transferred to 100 ul conical vials, reduced to dryness by
vacuum centrifugation, and then dissolved in a small volume
of methanol for methylation. Diazomethane in ether (ca. 50
ul) was added and the vials were sealed and left for 30
minutes. The ether was evaporated under nitrogen, and all
samples were again dried by vacuum centrifugation.
Methylated samples were further derivatized by adding 5 ul
of TMS solution (trimethylsilyl reagent in pyridine, Sigma
Sil.A, Sigma Chemical Company, St.Louis, MO), sealing the
vials and heating them in an oven at 80'C for 10 minutes.

Samples were injected into a Hewlett-Packard 5890 A gas
chromatograph attached to a JEOL JMS-AX 505H mass
spectrometer. A DB-5 ms capillary column (30 m x 0.25 mm x
0.25 um film thickness) was used. Helium was the carrier gas
at a flow rate of about 1 ml.min'1 (at head pressure of 70
KPa). The samples were injected on-column with the initial
column temperature at 50'C, followed by temperature

programming at 40'C.min4 to 220'C and then 6'C.min'1 to

67

Table 2. Rf values of some standard gibberellins on TLC with developing

solvent ethyl acetate : chloroform : acetic acid (15:5:1)

 

 

 

 

GA Rixalue
Observed Publishedz

GA1 0.37
GA2 0.22 0.19
GA3 0.39 0.37
GA4 0.58 0.61
GAS 0.59
GA6 0.57
GA7 0.59 0.52
GA8 0.24
GA9 0.74 0.78
GA10 0.33
GA1 1 0.74
GA12 0.77 0.78
GA13 0.46
GA14 0.63
GA15 0.78
GA18 0.35
GA19 0.42

 

ZTakahashl (1983)

68

300'C. The column was connected directly to the ion source
via a heated interface. The ion source temperature was
280°C, the electron ionization energy was 70 ev, and the

emission current was 0.25 mA.

a1!;g11111_g1_.g§gp911;31: Metabolites were dissolved in
methanol (ca. 200 ul), and the volume adjusted to 1 ml with

water. Hydrochloric acid (0.1 M, 5 ml) was added, and the

solution was incubated in a water bath for at least 6 hr at
80'C. After incubation, the sample was allowed to cool to
room temperature, then extracted 3 times with ethyl acetate

(about 20 ml in total).

RESULTS

Characterisation of metabolites.

£11‘_..-n11£& Six major metabolites of i‘C-GAu, in both
'Spencer Seedless' and 'Spartan' seeds were eluted at 23,
26,28, 31, 34, and 36 min. (Fig. 6). For convenience these
will be referred to as $23, 826, etc. All of the “C-GA12
(retention time 37 min) was metabolized within 24 hr.

A slightly different pattern of metabolites was
observed when branches were held in 21319 in 1994 and seeds
were sampled 72 hr after treatment. On 26 June, some 1"C-

GA“ (S37) remained in the tissues, and metabolites were

69

Figure 6 - HPLC profile of metabolites of 1"C-GAu. in extract
of 'Spencer Seedless’ apple seeds treated 36 DAFB in 1993.

Numbers indicate approximate retention times (min).

70

 

36

 

 

 

 

 

 

 

 

 

ll

 

 

 

 

. I W L
‘ 6M. ......lxhijw ..................... WWW

0 30

Time (min)

71

Figure 7 - HPLC profiles of metabolites of “C-GAugin
extracts of seed of ’Spencer Seedless' on branches held in
yitng in 1994. Tissues sampled 72 hr after treatment. A.

Treated 40 DAFB: B. Treated 45 DAFB.

72

. Radioactivity (DPM)

175 w

 

H
Di
3

 

 

.‘ ...-co

_ Mm-AN A“ 8.43m

 

 

 

 

 

 

 

 

 

 

 

 

Time (min)
73

Table 3. Retention times of some standard gibberellins 0n HPLC, using
a methanol/water gradient (see text for conditions).

 

 

 

GA Bgtgmigg time (min)
Observed PublishedZ
GAs 5
GA8 5.1
GAE, 10.1 6.3
6A,, 6.5
GA33 7
GA30 7.5
GAz, 7.9
6A,, 8.2
GA.1B 8.4
GA41 9.2
GA3 15.6 9.4
GA2 16.0 15
GA1 18.2 10.5
GAS 26.1 17.9
GA10 18.1
GAz, 28.0 19
GA“ 21.4
GAB 21.5
GA19 29.0 22.4
GAS, 22.6
6A,, . 23.5
GA7 30.5 24.9

74

Table 3 (cont'd)

GA, . 31.6
GAs,
6A2,
6A9 32.8
GA15
GA12 37.0

26.1
27.8
28.9
29.4
29.8
35

 

2 Lin and Stafford (1991 )

75

evident at retention times of 5, 22, 31, and 35 min (Fig 7).
On 1 July, these metabolites were again evident, as well as
a large quantity of an additional component at 25 min. The
compound(s) at 5 min was low in quantity and poorly
resolved: although not observed in other seed samples, it
occurred in several extracts of treated apices (see below).

Available standards with similar retention times (Table
3) were GA, (26.1 min), and GAzo (27.95 min). The
metabolites eluted at 34 and 36 min appeared to be less
polar than GA.) on HPLC.

The metabolites (combined from all samples of seeds
based on the retention time on HPLC) were subjected to thin
layer chromatography (Fig. 8), using ethyl acetate:
chloroform: acetic acid (15:5:1) as the solvent.

Radioactive zones were visualized by autoradiography. 823,
826, 828, and S34 were almost immobile in this solvent (Fig.
8), with Rfs of 0.05 to 0.1. 831 contained at least 8
components varying in Rf from 0 to 0.73. S36 was resolved
into 4 compounds (Rfs 0.2, 0.27, 0.35, and 0.41).

828, S34, and S36 were not affected by acid hydrolysis
(Fig. 8), and $31 was affected little, if at all. However,
many additional components were evident in S23 and S26,
suggesting that these were conjugates. Several of the
compounds in the hydrolysate of 823 had Rfs similar to, if
not identical with, the components in S31, suggesting that

$31 had been conjugated to give 823. Several additional,

76

less polar, compounds not observed in other samples were
also evident.

Comparison of the Rf values (Table 4) and HPLC retention
times of metabolites with those of standard GAs (Tables 2,3:
Fig. 9) indicated that 826 could contain GAun although this
has not been identified in apple tissue to date. No
gibberellins or gibberellin derivatives could be identified
in the extracts by GC-MS.

Apgx_§gnp1g§L Eight major metabolites were observed
following HPLC of extracts of apices treated with “C-GAu
(Fig. 10). These had retention times of 37, 36, 34, 32, 30,
29, 28, 27 and 5 min (hereafter referred to as AP37, AP36,
etc.). AP5 occurred only in small amounts. AP27 was found
only in samples collected relatively late (59 DAFB in 1992,
41 and 48 DAFB in 1993). AP37 was identified by GC-MS as
the parent compound, “C-GAu (Figs. 11-13 ). Comparison of
retention times on HPLC (Table 3) indicated that AP28 could
be GAzo, AP29 GA”, and AP32 GA‘. Both AP34 and AP36 were
apparently less polar than available GAs other than GAun

When silicic gel column fractions from large samples
collected in 1995 were subjected to HPLC, AP37 was found in
the 30% ethyl acetate fraction: AP36 and AP34 in 50%: AP32
and AP30 in 80%: and AP29, AP28 and AP27 in 100%.

The metabolites were chromatographed on TLC using ethyl
acetate: chloroform: acetic acid (15:5:1) as the developing

solvent (Fig. 13). The most polar metabolite (AP27)

77

I!

8‘:

uld

in

732

all

3%

Figure 8 - Autoradiogram of TLC plate following
chromatography of metabolites of 1"C-GA12 from 'Spencer
Seedless' apple seeds in ethyl acetate : chloroform : acetic
acid (15:5:1). Numbers indicate elution times on HPLC. Non-
hydrolyzed metabolites (left): acid hydrolyzed metabolites
(right).

78

Solvent front

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...: .. .. .. . . . ... . . .... .... .

 

 

 

34

31

Hydrolyzed

 

232628

31

28
Not hydrolyzed

 

2326

79

Table 4. Fit values of metabolites of 1“C-GA12 in extracts of apple seeds

 

 

 

 

 

 

 

and apices.

Metabolite Rf_val_ue

(min) Non-hydrolyzed Hydrolyzed
Seeds

23 0, 0.05 0, 0.1 , 0.2, 0.3, 0.35, 0.49

26 0, 0.1 0, 0.07, 0.15, 0.29, 0.37

28 0, 0.1 0.1

31 0, 0.05, 0.08, 0.18, 0.23 0.1, 0.25

34 0, 0.1 0.1

36 0.2, 0.27, 0.33, 0.4 0.2, 0.27, 0.33, 0.4
Anise:

27 0, 0.08, 0.13 0, 0.08, 0.13, 0.31

28 0, 0.15, 0.22 0, 0.15, 0.22

29 0.29 0.29

30 0.27 0.27

32 0.31 0.31

34 0.61 0.61

36 0.03, 0.69 0.03, 0.69

37 0.77 0.77

 

80

Figure 9 - Comparison of chromatographic properties of
metabolites of “C-GA12 in seeds (A) and apices (B) of apple
with those of known GAs (see Table 2,3,7) . .0 - Metabolites
in extracts: I - Known GAs: i2 Known GAs, based upon data of

Lin and Stafford (1991), adjusted for retention time.

81

' Rf value (TLC)

1.0

 

 

 

 

 

 

 

 

 

 

@Seeds
0.8- A? A:
A9
0.6- ABS A: A7. Iii A14
A4
0 A13
A3
A
0'“ . I1 I2 0 g A19 0
A10 0
A18 0 O O
0.2- ’2 o o o o
O O O O
0.0 i O_I_C T
1.0
e Apices
0.8- .612
A14 A9 0
0.6- “E Q5 ‘7. .9 o
A1'3 M
A3
0 4' I AI [2 ME A19
A18 0 O O O
021 I 0
A2 00
O
0.0 . . cc . °
15 20 25 30 40
Retention time (HPLC)

82

Figure 10 - HPLC profiles of metabolites of "C-Ga‘z in
extracts of apices of 'Spencer Seedless' apple treated 36

DAFB (A) and 48 DAPB (B) in 1993.

83

Radloactlvlty (DPM)

 

 

34
36

29
30 37
32

28

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1T_1L‘ 4 A :34 QM} 1- q L My .1 103le

 

 

 

 

 

 

 

 

. B
36
. 28
. 37
q 32
30
‘ 7M 34
* r I I ‘1 ‘7 l 1 W

O 10 20 30 4O 50

Time (min)

84

Figure 11 - Abundance of selected ions in mass spectrun of
Me-TMS derivative of residual “C-Ghu (AP37) in apices of

'Spencer Seedless' in 1993 (TIC, ions 241, 300, 328).

85

vmhm.ofl

homo.wv

wmvm.mH

o.me~w~.

.chnm

cmom

own

38:5: 58

 

 

 

 

d

we
2:5 8.: eczema

d u d d 1 1 ‘ I

d

omw
mwm
w
com n
U
r n?
D.
U
0
9
fivm
UHF
‘ J (1
oH

boom.mv xm:

86

Figure 12 - Mass spectrum of the He-THS derivative of the
residual ‘“C-GAu:(AP37) in extract of apices of 'Spencer

Seedless' in 1993.

87

N\_2

 

 

 

 

 

 

 

ace cmm can omm com om“ com
...1...:....................._ .. ._. ... ._.... ...... ... _....._......_......11...._._1 _ __....__ _._...1_ _..... ._.... 1......1L11.1-_.__1...o
A ..._____. j __ a N .....____..._ ___... ... ___—._._. ___. _ L:
.... ... .s
u N

m w .o~

scn . .

ocm H z
. .oe
- -om
2.. ...n
-oa
2;

sea

 

 

 

 

eouepunqe eAgeneu

Figure 13 - Upper portion of mass spectrum of the He-TMS
derivative of residual “C-Ghu.(AP37) in extracts of apices

of 'Spencer Seedless’ in 1993.

89

NE

 

 

o m m o o m o m N o o N
u p 1 ._.- ._...._ .__._. p . . 3:1.-- p .2. .L. ._-.1 . __. ....u. ___: - _. __ .1 ._ _w..__1_ r_._._:u —_ D
_ ___ _ ... ___ ___ .
m N m N
m m m N o N H N fl .
m N m m .o N
m c m .

 

 

com

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9O

contained 2 to 4 components, the least polar of which had
anRF of 0.12. AP28 was less polar, and two components
appeared to be identical with those in AP27. The solubility
of the remaining metabolites paralleled that observed on
HPLC. None of the metabolites matched the standard GAs both
in HPLC retention times and in Rf values on TLC (Fig.9).
Acid hydrolysis had little effect on Rfs (Fig. 14), except
that the least polar component in AP27 moved farther up the
plate. Therefore, these metabolites, unlike those found in
seeds, are probably not conjugated; if conjugated, they are
not acid hydrolyzable. Quantification of metabolites.

51191., 'Spencer Seedless'. Only one sample was
chromatographed on each sampling date in 1992, but 3 samples
were analyzed on each date in 1993.

As noted above, no 1‘C-GA12 remained after 24 (1993) or
48 hr (1992 and 1993). The data were quantified by
determining the DPH represented by each metabolite and
expressing this as a percentage of the total radioactivity
recorded for each sample, after subtraction of background.
This resulted in values (Table 5) that did not total 100
as radioactivity not included in peaks was included as part
of the total.

In 1992 activity in $36 fell continuously from 37 to 59
DAFB (Table 5, Fig. 15), whereas levels of the other
metabolites fluctuated, but showed no particular trends.

The quantity of 834 was relatively high on all sampling

91

dates.

Values for $36 in 1993 tended to parallel those
observed in 1992 (Fig. 15), although the initial level (30
DAFB) rose prior to dropping. Several differences between
sampling dates were significant (836, 834, $31, 823), but
seldom paralleled values observed in 1992. Levels of both
836 and 834 were significantly lower after 48 hr than after
24 hr (Table 5), but these differeces were small, and were
not accompanied by increases in more polar components.

Lflpgxggniy One sample of ’Spartan' was chromatographed
on each sampling date in 1992. The only noticeable parallel
between data for ’Spartan’ and those for 'Spencer Seedless'
was the steady decline in 836 from 37 to 60 DAFB (Table 5,
Fig. 15).

33191;. Lfipgnggz_figgglgggi. Three replicates were
extracted for each individual treatment in 1992 and 1993.
Rate of metabolism of AP37 (“C-GA“) consistently declined
with treatment date, thus more was recovered in both years
as treatment was delayed (Table 6, Figs. 16, 17). In 1992,
metabolism was signifiantly more rapid in bourse shoots of
spurs bearing seedless fruit than in those of spurs bearing
seeded fruits (Fig. 16). This was also true for the 24 hour
sample in 1993 (Fig. 17), but differences at 48 hr were
small and non-significant in 1993. Rate of metabolism (24
vs. 48 hr) appeared to be affected by the presence of seeds

(Tables 6, 7, Fig. 18), but these differences were not

92

consistent from year to year, and interactions between time
of treatment and the presence of seeds were also apparent.
However, AP28 declined significantly with sampling time in
both years (Fig. 19). More metabolites were found in the
apex of spurs with seedless fruits than in those with seeded
fruits in the in yitrg experiment (Fig. 20), but comparison
with samples taken in yiyg indicated fewer metabolites
overall (compare Figs. 10 vs. 20). The rate of metabolism
of “C-GAn in the apex was higher in 2129 than in gitrg (Fig
21).

LfipgztgnL; Three samples of 'Spartan' were taken at
each date in 1992. Rate of metabolism of 1"CnGAn again
declined with time (Table 4, Fig. 22), although differences
were non-significant until 59 DAFB. Little difference
between 24 and 48 hr was evident in the quantity remaining.
Levels of AP29, AP32, and AP36 paralleled those for 'Spencer
Seedless' sampled the same year, but changes in other

metabolites differed.

93

Figure 14 - Autoradiogram of TLC plate following
chromatography of metabolites of “C-GAu in extracts of
apices of 'Spencer Seedless' in ethyl acetate : chloroform :
acetic acid (15:5:1). Numbers indicate elution times on
HPLC. Non-hydrolyzed metabolites (left): acid hydrolyzed
metabolites (right).

94

Solvent front

 

95

so

 

28 29 30 32 34 36
Hydrolyzed

27

Not hydrolyzed

Table 5. Relative amounts of metabolites of ”C-GA12 in seed of ‘Spencer
Seedless’ in 1992 and 1993 and ‘Spartan’ in 1992 as a percentage of
the total radioactivity recovered by HPLC. Only one sample processed
per culture and date in 1992, 3 samples in 1993.

 

Treat WEL—
QAFE timem) 36 34 31 28 25 23

 

 

 

 

 

M. 1992
37 48 1 1 20 8 14 2 17
45 48 4 23 20 16 3 5
52 48 3 19 15 10 3 20
59 48 __0 _29 __6 _5 _14; .9
Mean 5 21 12 12 13 10
spenger §§§§l§§§. 1993
30 24 11 13 12 10 8 15
48 5 6 11 13 13 18

36 24 1O 11 16 17 11 17
48 11 10 21 17 1O 16
41 24 15 13 16 17 12 12

 

48 6 14 12 10 12 6
48 24 1 6 32 26 13 1
48 0 3 30 32 12 1
Mean30 8b 10a 11 c 120 10a 17a
36 10 ab 11 8 19b 17b 10a 17a
41 11 a 138 14c 14bc 138 8b
48 10 5b 308 288 14a 10
24 9 11 18 17 12 11
48 6*" 8* 19 1.8 11 1O

96

l «L'

Table 5 (cont’d)

 

 

 

 

 

 

Treat. Retentien time (min)
_QELJImelH) 36 34 31 28 26 23
w

37 48 1 2 24 6 16 7 6

45 48 1 O 18 1 O 8 9 9

52 48 8 1 4 8 1 3 8 1 4

59 48 __‘1 .29 _2 .38 _.‘1 _1.
Mean 9 1 9 8 1 9 7 8

 

97

Figure 15 - Effects of time of treatment on relative levels
of metabolites of “C-GAnrrecovered from seeds of 'Spencer
Seedless' and 'Spartan' apple in 1992-93. Data points
indicate radioactivity as a percent of total radioactivity
recovered following HPLC. Values for 1 sample (1992) or
means for 6 ( 2 times of sampling x 3 replicates) samples
(1993). Vertical bar-lsd (p-0.05) for 'Spencer Seedless' in

1993.

98

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  
  

 

 

 

 

 

 

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101

Figure 16 - Effects of time of treatment and of incubation

(24, 48 hr) on content of 1‘C--G.Au1 (% of total recovered) in
apices of spurs bearing seeded (P) vs. seedless (NP) fruits
of 'Spencer Seedless' in 1992. Vertical bars: 1 standard

deviation.

102

 

 

 

 

 

33 3.52.5. up <66 3

5'9

5'2

 

 

 

 

 

33 95.382 N w <06 3

TIME (DAFB)

103

Figure 17 - Effects of time of treatment and of incubation
(24, 48 hr) on content of 1"C-GAQ (% of total recovered) in
apices of spurs bearing seeded (P) vs. seedless (NP) fruits
of 'Spencer Seedless' in 1993. Vertical bars: 1 standard

deviation.

104

 

 

24 HR

 

 

 

 

 

3C aEEsEo. up <66 3

 

 

48HR

 

 

 

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10~
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3.; assoc—2 up (0-0 3

TIME (DAFB)

105

 

 

 

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107

Figure 18 - 1"C--Ca'1ll1zremaining in apices on spurs with
seedless and seeded fruit of 'Spencer Seedless' and on spurs
of 'Spartan’ 24 hr after treatment 1992. Vertical bars: 1
standard deviation.

SSH-Spencer Seedless, not pollinated

SSP-Spencer Seedless, pollinated

SP-Spartan, pollinated

108

 

11%//////////////////,

SSN

24 hr

 

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO
OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO
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SP

 

 

 

 

 

 

 

 

 

 

 

 

Days after full bloom

 

 

 

 

 

 

 

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3L uEEoEo. «€0.04.

40-

109

Figure 19 - Effects of time of treatment on relative levels
of metabolites of 1‘C--G.A1z recovered from apices of 'Spencer
Seedless' and 'Spartan' apple in 1992-93. Data points
indicate radioactivity as a percent of total radioactivity
recovered following HPLC. Values are means for 6 (2 times
of sampling x 3 replicates) samples (1992 and 1993).

Vertical bar - lsd (p-o.05) for 'Spencer Seedless' in 1993.

110

Radloactlvlty (% of total recovered)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Days after full bloom

111

Figure 20 - HPLC profiles of metabolites of “C-GAu in
extracts of apices of 'Spencer Seedless' on branches held in
yitre in 1994. Tissues sampled 72 hr after treatment.

A. and C. Treated 26 June (40 DAFB)

8. and D. Treated 1 July (45 DAFB)

A. and 8. Spurs with seeded fruits

C. and D. Spurs with seedless fruits.

112

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113

Figure 21 - "C-GAu remaining (4) in seeds (A), and in
apices of spurs with seedless (B) or seeded fruits (C) of
'Spencer Seedless' after 24 (1992 and 1993) or 72 hr (1994).
(Treatments in 1994 were applied to fruits and apices on

severed branches). Vertical bars: 2 standard deviation.

114

remalnlng (°/o)

14C-GA12

 

 

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Seeds 593
I94

 

 

80

 

 

 

 

60-

 

Apex. Seedless

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Days after full bloom

115

 

Figure 22 - Effects of time of treatment and of incubation
time on content of 1‘C-cmup (% of total recovered) in apices

of spurs of 'Spartan' in 1992. Vertical bars: 1 standard

deviation.

116

 

 

 

 

Spartan 92

 

 

82.5454. 4. <06 ..

55

50

45

40

35

DAFB

117

DISCUSSION

Several hypotheses have been proposed as to how seeds
inhibit flowering in apple. The most popular hypothesis is
that gibberellins produced (or accumulated ?) in the seeds
are transported to the apex of the bourse shoot where they
inhibit flowering (Luckwill, 1970). The second is that seeds
somehow control metabolism of GAs in the bourse shoot,
favoring the accumulation of those that inhibit flowering
and/or reducing the concentrations of those that favor
flowering (Looney, et al., 1978). A third possibility is
that seeds compete for flowering hormones (florigen ?)
produced by the leaves, thereby reducing the quantity
available for the bourse buds.

my objective was to examine the metabolism of “C-GAn in
both (a) seeds and (b) bourse shoots bearing either seeded
(inhibit flowering) or seedless (do not inhibit flowering)
fruits. The most important findings were that 1‘C-GAm was
metabolized more slowly in apices bearing seeded fruits than
in those bearing seedless ones, and that the rate of
metabolism declined during the period of treatment (June-
July). Unfortunately, the quantities of metabolites
available were insufficient for identification by GC-NS.
Nevertheless, these results support the hypothesis of

Looney, et al., (1978) that flower induction is associated

118

with a more rapid rate of metabolism of GAs by vegetative
tissues. Whether the relatively small difference in rate
observed (ca. 15 8) is physiologically significant cannot be
determined at this writing.

The rate of metabolism of “C-GAu was much more rapid in
seeds than in apices, none remaining after 24 hours in seeds
in experiments performed in_yiye: however, a significant
quantity remained even after 72 hr when excised branches,
rather than trees, were used. The number of metabolites
formed was also reduced 1n_xi§:e. The slower rate in_yi§ze
may indicate that conditions were not optimum for
metabolism, or that root factors (cytokinins ?) may affect
metabolism of GAs in the aerial parts of the tree. Note
that cytokinins can promote flowering in apple (Ramirez and
Road, 1981).

No consistent pattern was evident in seeds or apices to
indicate a step-wise conversion of one metabolite to
another, as observed in the metabolism of “C-GAu-aldehyde
by mature apple embryos (Chilembwe, 1992). Although
quantitative differences were evident over time in some
instances, these were rare, and year-to-year and cultivar-
to-cultivar variations were high. The most consistent
change was the decline in the quantity of 836 with time
(Fig. 15).

Little 1‘C moved out of the fruit following treatment of

seeds with “C-GAu (Ban, 1996), confirming the observations

119

of Green (1987). However, both Green (1987) and Bangerth
(personal communication) found that radio-labelled IAA was
transported out of the fruit, although in small quantities.
Conceivably, then, auxin produced by the seed could be the
signal that controls GA metabolism in the apex.

A parallel situation exists in the control of growth of the
pericarp by seeds of pea. Huizen, et al. (1995) studied the
conversion of “C-GA", to "C-GAzo in pea pericarp with or
without seeds, and in deseeded pericarp treated with 4-
chloroindole-3-acetic acid (4-Cl-IAA), which is endogenous
in pea seeds (Narumo, et al., 1968). Seed removal
essentially prevented conversion, whereas deseeded pods
treated with the auxin continued to produce GAap The
authors postulated that seeds produce 4-Cl-IAA that moves to
the pericarp and there controls the metabolism of GAs.

The pea seed is attached directly to the pericarp, where
growth occurs, whereas hormones from apple seeds must pass
through the pedicel, the cluster base and the bourse shoot
in order to reach the meristem, where flowering occurs.
Nevertheless, the parallel is suggestive.

Four of the 6 major metabolites found in apple seeds were
strongly polar on TLC, even though they were eluted from the
HPLC column at retention times similar to those of ”free"
GAs. These are probably glycosides, although Rfs of 3 of
the 4 did not change following acid hydrolysis. In

contrast, 6 of the 8 metabolites in apices behaved as ”free"

120

GAs on TLC (Fig. 14). However, none matched the standard GAs
available in both retention time on HPLC and Rf value on
TLC (Fig. 9).

Some seed and apex samples contained a polar metabolite
which also occurred in the cluster base, although the amount

of this metabolite was very low.

LITERATURE CITED

Ban, J. 1956. Effect of time of fruit removal on flower
initiation in 'Spencer Seedless' apple, and effect of
cutting fruits on fruit retention and growth of 'Spencer
Seedless' and 'Paulared' fruits. Ph.D. Thesis (Part I).

Michigan State Univ., E. Lansing, MI.

Birnberg, P.R., M.L. Brenner, G.C. Davis, and H.G. Carnes.
1986. An improved enzymatic synthesis of labeled gibberellin

A12-aldehyde and gibberellin A12. Anal. Biochem. 153:1-8.

Browen, D.H., and J. MacMillan, 1972. Determination of

specific radioactivity of 14C compounds by mass

121

spectrometry. Phytochemistry 11:2253-2257.

Buban, T. and H. Faust. 1982. Internal control and
differentiation of flower bud induction in apple trees.
p.174-203 In: J. Janick (ed.) Horticultural Reviews, Vol. 4.

AVI Publishing, Nestport, Conn.

Chan, B.C. and J.C. Cain. 1967. The effect of seed formation
on subsequent flowering in apple. Proc. Amer. Soc. Hort.

Sci. 91:63-67.

Chilembwe, E.C. and F.G. Dennis, Jr. 1992. Metabolism of
1"C-GA12-aldehyde by apple embryos in relation to dormancy

release. Proc. Plant Growth Reg. Soc. Amer. pp.49-63.

Davis, L.D. 1957. Flowering and alternate bearing. Proc.

Amer. SOC. Hort. 861. 70:545-556.

Dennis, F.G. Jr. 1976. Gibberellin-like substances in apple

seeds and fruit flesh. J. Amer. Soc. Hort. Sci. 101:629-633.

Dennis, F.G. Jr. and L.J. Edgerton, 1966. Effect of
gibberellin and ringing upon apple fruit development and
flower bud formation. Proc. Amer. Soc. Hort. Sci. 88:14-24.

Ebert, A. and F. Bangerth, 1981. Relations between the

122

concentration of diffusible and extractable gibberellin-like
substances and the alternate-bearing behavior in apple as
affected by chemical fruit thinning. Scientia Hort. 15:45-

52.

Green, J. 1987. The hormone control of biennial bearing in

cider apples. Ph. D. thesis, University of Bristol, UK.

Grochowska, H.J. and Karaszewska, A. 1976. The production of
growth promoting hormones and their active diffusion from
immature developing seeds of apple cultivars. Fruit Science

Rept. Skierniewice 3:5-16.

Guttridge, C.G. 1962. Inhibition of fruit-bud formation in

apple with gibberellic acid. Nature 196:1008.

Hoad, G.V. 1978. The role of seed-derived hormones in the

control of flowering in apple. Acta Hort. 80:93-103.

Huizen, R.V. J.A. Ozga, D.H. Reinecke, B. Twitchin, and L.N.
Hander. 1995. Seed and 4-chloroindole-3-acetic acid
regulation of gibberellin metabolism in pea pericarp. Plant

Physiol. 109:1213-1217.

Lin, J.T. and A. E. Stafford. 1991. Gradient Cu,high-

'performance liquid chromatography of gibberellins. J.

123

Chromatography 543:471-474.

Looney, N.E., A. Damienska, R.L. Regge, and R.P. Pharis.
1978. Metabolism of 3H-gibberellin A4 in relation to flower

initiation in apple. Acta Hort. 80:105-114.

Looney, n.s., R.P. Pharis, and n. Noma. 1985. Promotion of
flowering in apple trees with GA4 and c-3 epi gibberellin

A4. Planta 165:292-294.

Luckwill, L.C. 1970. The control of growth and fruitfulness

in apple trees. pp.237-254 In: L.C. Luckwill and G.V.
Cutting. eds. _Ihe_nhxsieleg¥_ef_tree_crons. Academic Press.

New York.

Luckwill, L.C., P. Weaver, and J. Macmillan. 1969.
Gibberellins and other growth hormones in apple seeds. J.

Hort. Sci. 44:413-424.

Marcelle, R. and C. Sironval. 1963. Effect of GA3 on

flowering of apple trees. Nature 198: 405.
Harino, F. and D.w. Greene. 1981. Involvement of GAs in

biennial bearing of 'Early McIntosh' apples. J. Amer. Soc.

Hort. Sci. 106:593-596.

124

Harumo, 8., H. Hattori, H. Abe, and K. Hunakata. 1968.
Isolation of 4-chloroindolyl-3-acetic acid from immature

seeds of Elena aetiynm. Nature 219:959-960.

McLaughlin, J.H. and D.W. Greene. 1981. Effects of 6-8AP,
GA“q and SADH on fruit set, fruit quality, vegetative
growth, flower initiation and flower quality of Golden

Delicious apples. HortScience 16:34-39.

Neilsen, J.C. 1994. Effects of seed number, time of
defruiting, and bourse shoot length on flowering in apple

(Abstract) HortScience 29:472.

Powell, L.E. and K.J. Tautvydas. 1967. Chromatography of
gibberellins on silica gel partition columns. Nature
213:292-293.

Ramirez, H. and G.V. Hoad. 1981. The effects of growth
substances on fruit bud initiation in apple. Acta Hort.

120:131-136.

Tomaszewski, H., L. Chvojka., R. Bulgakov., and L. Hejmova.
1967. Auxin-kinetin interaction in the differentiation of
flowers in apple buds. Hath-Naturwiss 16:653-654.

Tromp, J. 1982. Flower bud formation in apple as affected by

125

various gibberellins. J. Hort. Sci. 57:277-282.

Wertheim, S.J. 1973. Fruit set and June drop in 'Cox's
Orange Pippin' apple as affected by pollination and
treatment with a mixture of GA‘ and GA7. Scientia Hort.
(80th.) 1385-105.

126

SECTION III Transport of “c-GAW Hetabolites from Apple
Seeds and Apices to the Fruit and Other
Tissues, and Characterisation of the

Metabolites.
ABSTRACT

1"C -GAu1was injected into apple seeds and bourse shoots in
2129, and radioactivity was detected by oxidizing tissues
external to the site of injection. Only a small percentage
of the “C injected (about 1.47 to 3.22% in 1992, 0.09 to
0.13% in 1993) was transported from seeds to other tissues,
and only ca. 0.1 to 0.4% (1992) and 0.01 to 0.04% (1993) was
detected in the tissues outside the fruit. Even lower
transport was found following injection in an in 21:22
experiment. With one exception, no radioactivity occurred in
the apex when “C-GAn was injected into seeds, but one
polar metabolite was found in the cluster base and two
metabolites in the fruit flesh. When apices were injected,
from 0.2 to 6% of the total radioactivity was recovered from
tissue outside the point of injection: a major part of this
occurred in leaves but one metabolite was found in the

cluster bases. Metabolites in cluster bases and fruits

127

differed in chromatographic properties, but were more polar

than1Gmg on TLC.

INTRODUCTION

Some commercial apple cultivars have serious alternate
bearing problems. In 'Spencer Seedless' apple, a
facultatively parthenocarpic cultivar, seeds inhibit flower
initiation (Chan and Cain, 1967: Neilsen, unpublished data:
Ban, 1996). Nitsch (1958) first demonstrated GA-like
activity in immature apple seeds, and Dennis and Nitsch
(1966) tentatively identified GA‘ and GA7 in methanol
extracts. Many GAs have since been identified in extracts of
apple seeds ( Luckwill, et al., 1969: Head, 1978: Road and
Ramirez, 1980: Kirkwood and MacMillan, 1982: Steffens, et
al., 1991: Ramirez, 1993: Hedden, et al., 1993). Luckwill,
et al. (1969) showed that the concentration of GA-like
substances in seeds increased during early stages of fruit
development. He also reported that more GA-like activity
occurred in spurs with fruits that in those without fruits,
and proposed that GAs synthesized in seeds were transported
to the bourse shoot, and there inhibited flower initiation.
In support of this hypothesis, more GA-like activity was
found in diffusates from pollinated 'Spencer Seedless'

fruitlets than from non-pollinated ones (Head, 1978).

128

Marine and Greene (1981) detected gibberellin activity in
diffusates of flowers or fruits of 'Empire' at bloom: this
continued to increase until 45 DAFB. Fruit bearing spurs
contained more GA-like activity than vegetative spurs.
However, the changes in gibberellin activity in the seeds
did not match those in fruiting spurs. Thus diffusion of
gibberellin may not be the sole factor related to inhibition
of flowering. .

Looney, et al. (1978) proposed an alternative
hypothesis: the presence of seeded fruits reduces the rate
of metabolism of GAs in the bourse shoot, resulting in a
higher concentration and therefore inhibition of flowering.

Head (1978) injected 3H-GA‘, -GA; and -GA.;, as well as
1"C-IAA and “C-sucrose, into apple seeds in 2129, and
measured the amount of radioactivity recovered from the
bourse shoots on the same spurs. Following injection of
sucrose and IAA, more “C was recovered from bourse shoots
of a biennial cv. (Laxton's Superb) than from those of an
annual cultivar (Cox's Orange Pippin): however, the
differences between cultivars in amount of 3H transported
following application of GAs to seeds were not significant,
and 3H from GA, was not detectable in the bourse shoot.

Green (1987) continued these experiments with these and
other compounds, but transport was very limited, regardless
of the form of GA injected (“c-cm12 aldehyde, 311-81., “c-

IAA). She reported that GA contents of seeds of a biennial

129

bearing cultivar (Tremlett's Bitter) and an annual bearing
one (Dabinett) were similar qualitatively and
quantitatively. In diffusates from fruits, no GA‘ or GA-, was
found, and only a small amount of GA. was identified by GC-
US.

My purpose in this research was to investigate
transportation of the GAs and /or their metabolites

following injection of 1‘C-G.A1z:into the seeds and apices.

IETERIRLS AND METHODS

21.nt_|.§ezielle Plant materials and procedures were
identical to those used in section II of this thesis, with
few exceptions. 'Spencer Seedless' was again used.

9 a .. . y . . ,. ,- , 1‘
Weighed quantities of lyophilized samples were oxidized in
an automatic oxidizer (Biological Oxidizer-OX 400, R.J.
Harvey Instrument Corporation, Hillsdale, N.J.) and the C02
produced was collected in scintillation fluid. Radioactivity
was recorded on a liquid scintillation analyzer (1500 TRI-
CARB, Downers Grove, Illinois) and corrected for background.
Percentage recovery of radioactivity in various tissues was

calculated on the basis of the total recovered from all

130

tissues, including the portions injected (seeds or apex).

fi}.§1e§1enl_englzeie: For seed samples, one sample was used
per treatment in 1992, 3 in 1993 and 1994: for apex samples,

2 in 1993 and 1994, 3 in 1992. The data obtained in 1993
were subjected to analysis of variance, and significant
differences determined by F value (2 treatments) or Duncan's

Multiple Range Test (3 or more treatments).

RESULTS

WW1:
1n;e_eeegee 1122; Oxidation of tissues other than seed
tissues following injection of 1‘C--G.A1z into the seeds
indicated that most of the activity remained in the inner
portion of the flesh, adjacent to the seeds (primarily ovary
tissue), with much less activity in the outer portion (Table
1, Fig. 1). Only 1.2 to 3.2% of the radioactivity was
transported from the seeds, and 80 to 96% of this remained
in the fruit. Most of remaining activity was in the pedicel
and cluster base, with less than 0.01% of the total injected
being recovered from the bourse shoot. Radioactivities were
found in the apex only on one date, 59 DAFB. Less
radioactivity (based on total radioactivity in the tissues)

was transported from seeds as fruit enlarged.

131

1123‘, In 1993, much less radioactivity was found outside
the seeds (about 0.1% of the total injected), and only about
0.03% outside of the fruits (Table 2, Fig. 2). Transport to
the inner portion of the fruits declined significantly as
the season progressed , but differences in other tissues
were non-significant. No radioactivity was found in the
bourse shoots, bourse shoot leaves, or apex. Transport to
the fruit, but not other tissues, increased as incubation
time was prolonged from 24 to 48 hr.

1121; Again, very low percentages of the total
radioactivity injected into seeds were translocated to
adjacent tissues (0.13%, with only 0.04% being transported
outside the fruits) (Table 3). None was recovered from

vegetative tissues.

WW1M
epieeee_ 1222, When the 1‘C--GA12 was injected into the apex,
1.9 to 6.0% of the radioactivity was transported: a major
part of this (0.55 to 3.88%, or 40% of the total recovered)
was found in the leaves (Table 4). More radioactivity was
transported to bourse shoot leaves, cluster bases, and inner
portions of fruits on spurs with seedless fruits than on
those with seeded fruits. Data for percent recovery
paralleled these data except that differences in fruit
tissues were not significant (Fig. 3). With the longer

treatment time (48 hr) less radioactivity was recovered in

132

Table 1. Effect of time of treatment on total radioactivity recovered in different
tissues , and on distribution of total radioactivity ,following injection of “C-GAu,
into seed of ‘Spencer Seedless' apples in 1992. All samples (about 100 mg DW,

one per treatment) oxidized and 002 collected.

 

Apex

 

 

 

DAFBZ Inner Outer Ped. CB CL es BL Total

Radioactivity recovered ( DPM/100 mg DW)
37 9204 655 653 567 531 58 0 0
45 3984 286 51 1 151 162 25 0 0
52 1 680 1 75 421 242 506 45 0 O
59 2916 .32 .32 .23 _43 4.0 16 1.28
Mean 4236 301 404 247 31 0 42 - 1 9 32

Per cent of total radioactivity recovered'

37 2.23 0.61 0.05 0.25 0.09 0.01 0 0 3.22
45 1.25 0.27 0.04 0.06 0.03 <0.01 0 0 1.65
52 0.73 0.23 0.04 0.11 0.09 0.01 0 0 1.21
59 1.25 9.116 59.91 1).-m SLOJ. 2.9.1 ELQE 2.0.1. M1
Mean 1.37 0.32 0.03 0.11 0.05 <0.01 <0.01 <0.01 1.89

 

Y- °/. of total injected
ZAbbreviations:

DAFB - Days after full bloom CL -- Cluster base leaves

Inner -- Inner part of treated fruit as - Bourse shoot

Outer - Outer part of treated fruit BL - Bourse shoot leaves

Pad. - Pedicel CB -- Cluster base

133

Figure 1 - Percentage of total radioactivity recovered from
'Spencer Seedless' apple tissues after injection of 1‘C-GAu
into seeds in 1992 (A) and 1993 (8). Note that scales on
vertical axes differ for the two years. Values for BS, BL

and apex in 1992 are multiplied by 50.

134

 

 

 

 

 

 

 

 

0.15
O 1 ‘

Hex... cv.—0.600.— 533.8268 Bach.

0.05 “

 

135

Table 2. Effect of time of treatment on percentage of total radioactivity

recovered (DPM/100 mg DW) , and distribution of total radioactivity recovered,

in other tissues following injection of "C-GA,2 into seed of ‘Spencer Seedless’

apple in 1993. All samples (about 100 mg DW, 3 per treatment) oxidized and

CO2 collected.

 

 

 

 

DAFB Tlme(l1) Total Inner Outer Ped. CB CL '1
R l iv‘ r
30 24 224 31 1 51 42 O
48 305 25 2.1. m. 2.2. '""
Mean 2648 298 1 1 18 388 1 18
36 24 141 24 93 31 1 4
48 1.9.1 3.1 2.91 26 9.
Mean 1 66b 288 1508 308 7a
41 24 89 29 121 1 5 1 1
48 12.3 2.1 12 25 3.1.
Mean 1060 258 97a 21 a 21 a
48 24 71 1 6 91 21 20
48 9.6 .9 9.3 12 11
Mean 84c 13a 878 178 198
Mean 24 123 25 1 14 27 1 1
48 179" 22 1 08 25 1 8

136

Table 2 (cont’d)

 

Per cent of total radioactivgy‘ recoveredY

 

30 24 0.09 0.03 0.02 0.01 0.02 0
48 0-08 m_ an 59.21. 929.1. 0-_01.
Mean 'o.04a 0.02a 0.01a 0.01a 0.018
36 24 0.06 0.03 0.02 0.01 0.01 0.01
48 0.10 M 0_.Cfi 9&1 Q_._0_1_ _0
Mean 0.048 0.038 0.018 0.018 <0.01a
41 24 0.08 0.03 0.03 0.01 0.01 0.01
48 0.08 0.53; M 0.01 Q._Q_1_ L0;
Mean 0.048 0.028 0.01a 0.01a 0.018
48 24 0.08 0.03 0.02 0.01 0.01 0.01
48 0.08 0.04 0.01 0.01 0.01 0.01
Mean 0.038 0.028 0.01a 0.01a 0.01a
mean 24 0.03 0.02 0.01 0.01 0.01
48 0.04" 0.02 0.01 0.01 ' 0.01
Abbreviations:

Inner - inner part of treated fruits
Outer - Outer part of treated fruits
BS - Bourse shoot
Fed - pedicel

Y % of total injected

CB - Cluster base
CL -Cluster base leaf
BL -Bourse shoot leaf

abc Mean separation within columns and sets by DMRT p < 0.05
* Significantly different from 24 hr. sample by ANOVA p < 0.05

137

Figure 2 - Radioactivity recovered (Dru/100 mg DW) from
inner part of fruits of 'Spencer Seedless' apple 24 and 48

hr after injection of “-6812 into seeds at different dates
in 1993.

138

 

24hr
48m

IIOIII

Days after full bloom

 

 

 

25

 

m. m m m m m m

2 1 1.

95 we. 2: 22.5.
:3.— 35: 5 cv.—2500.. 33:23:33—

139

Table 3. Percentage of total recovered radioactivity and distribution of

radioactivity 72 hr after injection of 14C-GA12 into seeds of ‘Spencer Seedless' i_n

 

 

 

 

vitro. 1994
DAFBZ Inner Outer ' Ped
Fl dio ctivi r ver d DPM/1 m DW
40 188 43 23
45 1.5L. 1.5.— 2.2...
Mean 172 30 25
Per cent of total radioactivityY
40 0.08 0.06 <0.01
45 0.09 0.02 <0.01
Mean 0.09 0.04 <0.01

 

zAbbreviation: DAFB— Days after full bloom

Inner - Inner part of fruit
Outer — Outer part of fruit
Ped -- Pedicel

Y °/o of total injected

140

Table 4. Effects of time of treatment and presence of seeds on total radioactivity
recovered in different tissues2 , and on distribution of.radioactivity following
injection of “C-GA12 into bourse shoots of ‘Spencer Seedless’ apple in 1992.
All samples (about 100 mg DW , 3 per treatment) oxidized and “002 collected.

 

 

 

 

DAFB Seed Time(h) asz BL ca F Total
'v' P
37 — 24 4120 3410 670 260
48 281 0 4050 1 080 420
+ 24 3260 1 080 880 180
48 118d 1149 11;; gm
Mean 23938 24208 8358 2738
45 - 24 1610 2130 660 100
48 1 450 2530 1 670 230
+ 24 3320 1 060 380 180
48 E10 89.11— 4.3_Q_ 3.19
Mean 2273b 1 653b 535b 205b
52 - 24 1 050 800 350 130
48 2630 850 850 120
+ 24 2140 1 220 460 90
48 1.122 65.0.— 5.6.0__ fl
Mean 1735c 879C 555b 108C
59 - 24 1 450 970 680 70
48 1 1 50 320 680 80
+ 24 1 670 780 520 80
48 980 47g 820 _65
Mean 1 31 39 6359 546:; 7451
Mean - 2034 1883 703 176
+ 21 22 91 1"" 533*" 153‘
24 2327 1431 575 136
48 1 829*" 1362 660 1 93""
Interaction " ns ** ns

141

Table 4 (cont’d)

Per cent of total radioactivity recoveredx

 

 

 

 

37 - 24 1.35 3.47 0.73 0.20 5.76
48 0.75 3.88 1.10 0.28 6.01
+ 24 0.93 1.48 0.99 0.13 3.53
48 0&2 L33 051 0011 3.12
Mean 0.898 2.558 0.898 0.198
45 - 24 0.51 2.53 0.81 0.09 3.97
48 0.44 3.54 0.71 0.17 4.87
+ 24 1.12 1.77 0.44 0.15 3.48
48 0.33 1.33 0._19__ 0,23 3.25
Mean 0.73b 2.368 0.60b 0.16b
52 - 24 0.37 1.48 0.36 0.15 2.37
48 0.97 1.41 1.01 0.13 3.51
+ 24 0.64 2.06 0.52 0.10 3.32
48 0.33 1,13 0L 0,03 2.21
Mean 0.58c 1.50b 0.62b 0.120
59 - 24 0.60 1.88 0.83 0.12 3.43
48 0.40 0.55 0.82 0.13 1.91
+ 24 0.64 1.47 0.64 0.15 2.90
48 0.33 0.33 033_ 012 1.89
M 0.439 1.1 .71 .1
Mean - 0.66 2.29 0.78 0.16
+ 0.68 1.48"" 0.63“" 0.14
24 0.76 2.00 0.65 0.14
48 0.59'” 1 .78 0.76’ 0.16“"
Interaction ' ns "'

2- Excluding point of injection

ns

Abbreviations: BS - bourse shoot
BL - bourse leaf

142

C8 '- cluster base
F - Fruit inner part

Table 4 (cont’d)

x % of total injected

abc (see table 3)
* Significantly different from spurs with seedless fruits or from 24 hour
sample at p < 0.05("), p < 0.01(**) or p < 0.001 (...) by ANOVA

143

Figure 3 - Effects of time of treatment and presence of
seeds on total radioactivity recovered (DEM/100 mg DW) in
inner part of fruit following injection of “C-GAu into

bourse shoots of 'Spencer Seedless' apple in 1992.

144

Radioactivity (DPM/ 100 mg DW)

 

 

"U- Seedless

I e OI e r Seeded

 

 

Days after full bloom

145

 

the bourse shoots and more in the cluster base and fruits.
Both the total amount and the percentage of radioactivity
recovered from bourse shoots, bourse shoot leaves and fruit
flesh declined as fruit enlarged, while recovery from the
cluster base varied little with time (Table 4).

13033 Radioactivity was again transported from the apex to
other parts, although the amounts were much lower than in
1992 (Table 5). Less than 0.2% was found outside of the
point of injection; of this about 70% remained in the
bourse. None was recovered from fruit or pedicel tissue.
Neither the presence of seeds nor sampling time had a
significant effect on transport, but the amount of
radioactivity in the bourse shoot again fell with time.

1021 Transport was again very low in 1994: approximately
0.13% of tne radioactivity was transported, of which 80-90%
remained within the bourse. Bourse shoots on spurs bearing
seeded fruits contained less radioactivity than did those on
spurs bearing seedless fruits, as was observed for bourse
shoot leaves in 1992. Data for bourse shoot leaves
paralleled those for bourse shoots, but differences were not

significant. No activity was found in the fruit (table 6).

. o, o 1‘! __ e o ‘ o .- .7“vo e ' 2 ' L ..! . A .
In 1992, one polar metabolite was found in the cluster base
following injection of “C-GAQ into the seeds or apices on

37, 45 and 52 DAFB (Fig. 4). Its retention time, 6 minutes,

146

suggested that it was a conjugate, although acid hydrolysis
of this polar metabolite did not change its Rf value (0.12)
on TLC (Fig. 4). In 1993 although radioactivity was
detectable following oxidation of tissue samples (Table 2),
no metabolites were evident using HPLC with the flow-through
detector. Two metabolites present in fruit flesh following
injection of “c-can into seeds had retention times of 29
and 34 min on HPLC on all sampling dates in 1992 and at 30
oars in 1993 (Fig. 5).

When examined by TLC, these metabolites had Rf values
of 0.04 and 0.17, respectively (Fig. 6). The elution time of
the more polar compound on HPLC suggested that it might be
6119, but it was much more polar than 6A9 on TLC. The less
polar compound was eluted after GA, on HPLC, but it, too,

had a much lower Rf on TLC.

147

Table 5. Effects of time of treatment and presence of seeds on total
radioactivity recovered in different tissues, and on distribution of radioactivity

following injection of 1"'C-GAw into bourse shoots of ‘Spencer Seedless’ apple

in 1993. All samples (about 100 mg dw, ) oxidized and “C02 collected.

 

 

 

 

 

 

 

DAFB e Tlm n 8" 31. B c T t I
Radioa iv‘ r v re DPM/100 m DW
30 - 24 116 51 30 0
48 90 32 35 16
+ 24 97 32 25 24
48 1.0L. 45 510—. 1.2
Mean 1028 408 358 158
36 - 24 66 29 18 0
48 81 19 40 12
+ 24 45 23 13 37
48 IL. L 8.9.— 9__
Mean 67b 238 288 1 58
41 - 24 71 21 29 0
48 ‘ 96 32 29 o
+ 24 53 31 23 0
48 76 1_6__ 2.8—. .0_
Mean 74ab 258 278 0b
48 - 24 42 17 17 14
48 38 18 11 0
+ 24 31 18 17 0
48 37 1_4_ 32__ L
M 37c 13 173 L
Mean - 75 27 26 5
+ 65 25 27 12'
24 65 28 -22 10
48 74 32 8

25

Table 5 (cont)

Per cent oft t Ir di ’vity recoveredx

 

 

30 - 24 0.04 0.08 0.03 0 0.14
48 0.03 0.05 0.03 0.02 0.14
+ 24 0.03 0.05 0.03 0.03 0.14
48 999_ 9.9.7— 9.9L 9.99 0.19
Mean 0.03 0.06 0.04 0.02
36 - 24 0.02 0.04 0.02 0 0.08
48 0.03 0.03 0.04 0.02 0.12
+ 24 0.01 0.04 0.02 0.02 0.09
48 993— 994— 994_ 992 0.12
Mean 0.02 0.04 0.03 0.01
41 - 24 0.03 0.04 0.03 0 0.09
48 0.04 0.06 0.04 0 0.1 1
48 ML 9.99_ 9L .L 0.09
Mean 0.03 0.05 0.03 0
48 - 24 0.01 0.03 0.02 0.02 0.09
48 0.02 0.04 0.01 0 0.07
+ ‘ 24 0.01 0.03 0.02 0 0.07
48 9L 99L DLQL 9.91 0.08
Mean 0.02 0.03 0.02 0.01
Mean - 0.02 0.05 0.03 0.01
+ 0.02 0.04 0.03 0.01
24 0.02 0.04 0.03 0.01
48 0.03 0.04 0.04 0.01
Interaction _n; LI§ & 3;
Abbreviations: BS - bourse shoot BL - bourse leaf

CB - cluster base CL - cluster leaf

149

Table 5 (cont’d)
Y Excluding point of injection
x °/o of total injected

abc (see table 3)
* Significantly different from seedless fruits at p < 0.05 by ANOVA

150

Table 6. Effects of seeds and time of treatment on total radioactivity and
distribution among tissues 72 hr after injection of 1“C-GA,2 into bourse shoots of
‘Spencer Seedless’ apple 10 vitrg in 1994

 

 

 

 

 

DAFBZ Seed BSY BL CB
R i " r v r PM/l ' m o
40 - 130 84 17
+ 114 _4_7__ *1
Mean 122 65 18
45 - 190 66 31
+ .82— 2.L_ 2L.
Mean 138 46 30 L
Mean - 160 74 24
+ 101 36 23

 

 

40 - 0.04 0.15 0.02

+ 9_0.3_ & 9.09—
Mean 0.04 0.12 0.03
45 - ML 0-12 19L

+ 0 03 0.05 0 04
Mean - 0.06 0.13 0.03

+ 0.03" 0.07 0.03

2 Abbreviations DAFB— Days after full bloom
BS - Bourse shoot
BL - Bourse shoot leaves
CB - Cluster base

Y Excluding point of injection
151

Table 6 (cont’d)
x % of total injected
"' Significantly different from seedless fruits at p < 0.01 by ANOVA

 

152

Figure 4 - Chromatographic characteristics of a polar
metabolite in cluster base following injection of “C'Ghuf
into the seeds or apices of 'Spencer Seedless’ apple. A:
HPLC profile, using methanol/water gradient as described in
the text. 8: Radioautogram of this metabolite following TLC
in ethyl acetate : chloroform : acetic acid (15:5:1). 1-

before hydrolysis: 2-after acid hydrolysis.

153

 

Badloactlvlty (DPM)

 

 

A Solvent front

0
1

”0‘

mmiwewuwtmia

0

 

 

 

 

 

 

Time (min)

154

 

Figure 5 - HPLC traces (see text for conditions) of
metabolites extracted from fruit flesh following injection

of “c-cau into seeds in 1992 (A) and 1993 (B).

155

 

 
 

 

W B
_.a

2283 3.26.8.3:

 

 

10

Time (min)

156

Figure 6 - TLC of metabolites of “C-Ghu extracted from
fruit flesh following injection of “C-Gauginto seeds. 1-
metabolite eluted at 33 min on HPLC (see Fig. 2): 2-
metabolite eluted at 29 min. Left - before acid hydrolysis:

right - after acid hydrolysis.

157

Solvent front

d fro:
eeds. 1-
2.

hydrolysis;

 

0 e
__ _ -_ ' ___-{.3 _ __..._ I
1 2 1 2
Nm'hydrolyzed Hydrolyzed

158

 

DISCUSSION

Following injection of “c-cau into seeds, only low amounts
of radioactivity were detected in other tissues. In only one
sample was radioactivity found in the apex, where flower
initiation occurs, when “C.GAQ was injected into the
seeds. Green (1987), working with 2 biennial cider apple E‘
cultivars, obtained similar results. She observed very low
amounts of “C-GA1z-aldehyde (0.005 to 0.006% of total

injected) in diffusates from fruit pedicels following

 

injection of 1"'C-GAn-ald into seeds, and similar results
were obtained with 311-68... However, radioactive compounds in
diffusates from fruits injected with 311-68,, co-
chromatographed with standard 68‘. Green (1987) also
analyzed endogenous gibberellins by GC-MS, but no
qualitative or quantitative differences were found in
bearing vs. non-bearing trees of either cultivar. Thus she
obtained no evidence that seed GAs are transported to the

apex and there inhibit flower initiation.

Several reasons could be suggested for my failure to detect
transport from seeds to apices and 2190-23153: 1) transport
does not occur; 2) the “C-Ghufinjected did not enter the

proper pool, and was conjugated to give inactive compounds

or artifacts: 3) incubation time (24 to 72 hr) was too brief

159

for measurable transport to occur. Although all “c-cau was
metabolized by the seeds within the first 24 hours (Ban,
1996, Part II), transport of metabolites may require a
longer period of time. Recovery of radioactivity was low
(15 to 25%): had recovery been better, greater transport
might have been evident.

Transport from apices to fruits was observed only in 1992.
One may argue that incubation time was not long enough.
Endogenous gibberellins might accumulate in apices after
several days, even weeks.

The Rf values (0.12 from cluster base, 0.04 and 0.16 from
flesh) of transported metabolites indicated that they were
not CA. or say, which are thought to be the GAs affecting
flower initiation in apple. The Rf value of the transported
metabolites did not match those of known gibberellins under
the conditions I used. These metabolites could be
conjugates, given their polarity, but acid hydrolysis did
not change their Rf values. More work is needed to identify

and investigate the function of these metabolites.

160

 

LITIRLIURI CITED

Ben, J. 1996. Effect of time of fruit removal on flower
initiation in 'Spencer Seedless' apple, and effect of
cutting fruits on retention and growth of 'Spencer Seedless'
and ’Paulared' fruits. Ph.D. thesis (Part I). Michigan State

University.

Chan, B.C. and J.C. Cain. 1967. The effect of seed

formation on subsequent flowering in apple. Proc. Amer. Soc.

 

Hort. Sci. 91:63-67.

Dennis, F.G., Jr. and J.P. Nitsch. 1966. Identification of
gibberellin A‘ and A7 in immature apple seeds. Nature

211:781-782.

Green, J.R. 1987. The hormonal control of biennial bearing
in cider apples. Ph.D. Dissertation. Univ. of Bristol,

England.

Grochowska, R.J. 1968. The influence of growth regulators
inserted into apple fruitlets on flower bud initiation.

Bull. Acad. Polon. Sci. CIV 16:581-584.

Hedden, P., G.V. Head, P. Gaskin, N.J. Lewis, J.R. Green, M.

Furber, and L.N. Mander, 1993. Kaurenoids and gibberellins,

161

including the newly characterized gibberellin A“, in
developing apple seeds. Phytochemistry 32:231-237.

Head, G.V. 1978. The role of seed-derived hormones in the

control of flowering in apple. Acta Hort. 80:93-103.

Head, G.V. and H. Ramirez. 1980. La funcion de las
giberelinas sintetizadas en las semillas del fruto para el “—1

control de la floracion en manzanos. Turrialba 30:284-288.

 

Kirkwood, P.S., and J. Macmillan. 1982. Gibberellin.mw, A“
and.mg: Partial synthesis and natural occurrence. J. Chem.

Soc. Perkins Trans. 1:689-697.

Looney, N.E., A. Damienska, R.L. Regge, and R.P. Pharis.
1978. Metabolism of 3I-l-gibberellin A‘ in relation to flower

initiation in apple. Acta Hort. 80:105-114.

Luckwill, L.C. 1970. The control of growth and fruitfulness
of apple trees. p. 237-254 In: L.C. Luckwill and C.V.
Cutting (eds.). Physiology of tree crops. Academic Press,

New York.

Luckwill,L.C., P. Weaver, and J. Macmillan. 1969.
Gibberellins and other growth hormones in apple seeds. J.

Hort. Sci. 44:413-424.

162

Marina, F. and D.W. Greene. 1981. Involvement of GAs in
biennial bearing of 'Early NcIntosh’ apples. J. Amer. Soc.

Hort. Sci. 106:593-596.

Nitsch, J.P. 1958. Presence de gibberellines dans l'albumen

immature du pommier. Bull. Soc. Bot. de France 105:479-482.

Ramirez, H. 1993. Identification of gibberellins in Golden 5"

Delicious apple seeds. Acta Hort. 329: 95-97.

Steffens, G.L., J.T. Lin, A.E. Stafford, J.D. Metzger, and

 

J.P. Hazebroek. 1992. Gibberellin content of immature
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seasons. J. Plant Growth Regulation. 11:165-170.

163

 

 

SUIIIIY

Defruiting experiments indicated that seeds inhibit
flowering of 'Spencer Seedless' apple. There are two main
hypotheses to explain this phenomenon. 1) GAs synthesized in
seeds move to the apex, and there inhibit flowering. 2)The
presence of seeds controls the metabolism of GAs in the *5
apex, then and this affects flower initiation there. ’
Following injection of 1"C-GA12 into seeds, only a very small

amount of radioactivity was transported, and most of this

 

remained in the fruit flesh. With but one exception no
radioactivity was found in the apex. These observations
provide little support for the first hypothesis. However,
several reasons may be offered for the failure to detect
transport from seeds to apices. 1) the 1‘C-Gllui injected did
not enter the proper pool or overloaded the system, and was
conjugated to give inactive compounds or artifacts. Most of
the metabolites in seeds were conjugated, possibly because
the amount of 1"C-sz injected was excessive and/or the GA“
did not enter the proper pool. 2) The incubation time (24
to 72 hr) was too short for measurable transport to occur.
Although, most of the 1"C-GAui was metabolized within 24 hr,
more time may be required for transport. However, th amount
of transport increaseed only slightly when incubation time

was extended to 48 hr. 3) The recovery of radioactivity was

164

low (15 to 25%)of the total injected. Had recovery been
better, greater transport might have been evident. Host of
the radioactivity was lost (about 70 to 80%) between the
time of injection and the time of sampling. The GA may have
been metabolized and converted to gaseous “C'COQ.

One polar metabolites was found in the cluster base after
injecting 1‘C-GAnginto seeds or apices: two were found in
the flesh after injecting 1‘C-GAminto seeds. Rf value of re
the metabolite found in the cluster base was 0.12, those of
metabolites in the flesh were 0.04 and 0.16. Neither acid

nor enzymatic (glycosidase, cellulase) hydrolysis changed

 

their Rf values.

1"!

The metabolism of “C-GAQ was slower in apices bearing
seeded fruits than in those bearing seedless ones, and the
rate of metabolism declined during the period of treatment
(June - July). There were seven major metabolites found in
the apices, six in the seeds. Unfortunately, the quantities
of metabolites available were insufficient for
identification by GC-HS. However, these results support the
second hypothesis proposed by Looney, et al. which is that
the presence of seeds reduce the rate of metabolism of GAs
in the apices, thereby reduce the flower initiation.

The rate of metabolism of 1"C-GAmwas much more rapid in
seeds than in apices, none remaining after 24 hr. in
experiments performed in 1100: however, a significant

quantity remained even after 72 hr when excised branches

165

were used. The number of metabolites formed was also reduced
111nm.

Four of the six major metabolites found in apple seeds were
strongly polar on TLC, even though they were eluted from the
HPLC column at retention times similar to those of 'free'
GAs. In contrast, 6 of the 8 metabolites in apices behaved
as 'free' GAs on TLC. However, none matched the standard GAs
available in both retention time on HPLC and Rf value on
TLC. Some seed and apex samples contained a polar metabolite
which also occurred in the cluster base, although the amount

of this metabolite was very low.

166

 

Suggestions for further research

1. Obtain sufficient quantities of metabolites for
identification by GC-HS.

2. Select spurs with bourse shoots less than 2 cm in length
for comparison the metabolism of “C-GAu.in apices of
bearing vs. non-bearing spurs. A length of 2 cm is critical
based on J. Neilsen's results (personal communication), It
longer shoots tending to flower whether seeds are present or

not.

 

3. Apply an auxin such as NAA or 4-Cl-IAA to spurs bearing

seedless fruits to determine if it stimulates GA metabolism,

h:

as has been reported for pea fruits. Or apply an auxin
inhibitor antagonist such as TIBA to spurs bearing seeded
fruits to determine the rate of metabolism of 1"C-GAui is

reduced.

167

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