DEVELOPMENTAL CHANGES IN ASSIMILATIQN AND

musmcmon '0? PHOTOSYNTHATE m '- _- g i g’ * : : [ff
BLACK WAlNUT (‘J‘UGLANS mam Li)._AND. ‘ . f _ f . _ : 5 1
HONEvmcuST (‘GLEDITSSAWRIACANTHOS L )2 ~ '
SEEDLINGS -

Thesis for the Degree. offinD.

MlCHIGAN STATE UNIVERSITY

STANLEY BARTON CARPENTER
1971

“rs-.3

This is to certify that the

thesis entitled

Developmental changes in assimilation and
translocation of Photosynthate in black
walnut (Juglans Nigra L.) and honeylocust
(Gleditsia Triacanthos L.) seedlings

presented by

 

 

Stanley Barton Carpenter

has been accepted towards fulfillment
of the requirements for

Ph.D. Forestry

degree in

 

 

James W. Hanover

L- ‘
ILUIIIVCISity‘ -7 "
“Jr.” ,‘I .—~.. r Y J

 

 

Major professor

Date March 24 1971

0-7639

a- . “ml

ABSTRACT

DEVELOPMENTAL CHANGES IN ASSIMILATION AND TRANSLOCATION
OF PHOTOSYNTHATE IN BLACK WALNUT (JUGLANS NIGRA L.) AND
HONEYLOCUST (GLEDITSIA TRIACANTHOS L.) SEEDLINGS

 

 

BY

Stanley Barton Carpenter

Growth patterns, rates of photosynthesis and
respiration, and the incorporation of photoassimilated
14CO2 into major fractions of the leaves, stem, and roots
were studied in black walnut and honeylocust seedlings
during the first growing season. Black walnut is typical
of those deciduous trees with the preformed shoot growth
habit whereas honeylocust has the sympodial growth habit.
Other differences between the two species include seed
size, leaf dimorphism, and nastic leaf movement.

Analyses of cumulative height growth, leaf area
accretion, and dry matter production showed height growth
in black walnut was of short duration and was completed
early in the growing season. In contrast, honeylocust
seedlings grew continuously in height through the summer
to September 1. Similar trends were observed in leaf

area accretion and dry matter production. However, root

growth in both species occurred to September 21.

Stanley Barton Carpenter

Rates of photosynthesis and reSpiration were deter-
mined in the laboratory with an infrared-gas analyzer in a
closed system. Honeylocust seedlings exhibited a remark-
able superiority over black walnut in rates of net photo-

synthesis computed on a leaf area basis. Net photosynthesis

ranged as high as 14 mg CO2 dm-2 hr_1 for black walnut and
as high as 20 mg CO2 dm—2 hr-l for honeylocust. However,

when net or total photosynthesis was calculated on a whole
seedling basis, black walnut because of its larger leaf
area showed much higher rates of CO2 uptake. Net photosyn-

thesis increased gradually in honeylocust seedlings and
reached a peak incorporation of 15.5 mg CO2 dm-2 hr.l on
August 10 and remained at a high level through September 21

as leaf abscission began. Net photosynthesis in black

walnut reached a peak level of incorporation of 7.8

mg CO2 dm-2 hr-1 on July 27 and then declined sharply to a
low level of 1.3 mg CO2 dm-2 hr-1 on September 7. There

was considerable variation in rates of net photosynthesis,
dark respiration, and photorespiration among individual

trees of both species. Rates of CO2 evolution for dark
respiration were as high as 10 mg CO2 dm-2 hr-l for black
walnut and 13 mg CO2 dm”2 hr-l for honeylocust. Photo-

respiration rates ranged as high as 9 mg CO2 dm_2 hr-1 in

2

black walnut and 22 mg CO dm- hr.l for honeylocust.

2
Distinct differences were observed in the pattern

14 O O O I O I I
of C incorporation into amino ac1d, organic ac1d, sugar,

non-water soluble, and ethanol-insoluble fractions extracted

 

Stanley Barton Carpenter

from the leaves, stem, and roots. In black walnut seed-
lings there was a gradual shifting of metabolic activity
from the leaves to the stem and finally to the roots as
the growing season progressed. In contrast the pattern
of metabolism and translocation in honeylocust seedlings
remained static. The leaves of walnut seedlings labeled
early in the growth cycle showed reduced levels of radio-
activity as leaf abscission began in the fall indicating
redistribution of metabolites when leaf abscission occurs.

14

In contrast high levels of C remained in honeylocust

leaves on September 21 as leaf abscission began. The

study of the incorporation of 14CO2 into specific sugars of
black walnut showed distinct differences when a period of
maximum photosynthetic activity was compared with a period
of low photosynthetic activity. Sucrose accounted for

66 per cent of the total radioactivity recovered on June 15.
Smaller amounts of radioactivity were found in glucose,
fructose, raffinose, and stachyose. On September 7 the
sucrose fraction contained only 44 per cent of the total
14C recovered from walnut seedlings. Increased amounts

of radioactivity were found in glucose, fructose, raffinose,

and stachyose.

 

DEVELOPMENTAL CHANGES IN ASSIMILATION AND TRANSLOCATION

OF PHOTOSYNTHATE IN BLACK WALNUT (JUGLANS NIGRA L.) AND

 

HONEYLOCUST (GLEDITSIA TRIACANTHOS L.) SEEDLINGS

 

BY

Stanley Barton Carpenter

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Forestry

1971

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to
Dr. James W. Hanover (Chairman) whose generous encourage-
ment, suggestions, and assistance made this study possible.
Other members of my guidance committee--Drs. D. P. Penner,
G. Schneider, J. A. D. Zeevaart, and F. W. Snyder--also
made valuable contributions. I am further grateful to
Mr. R. Comstock whose assistance in the laboratory helped
bring this study to its conclusion. Finally, I wish to
thank my wife, Jane, for her encouragement and typing
assistance.

The study was completed while the author was on a
National Defense Education Act Fellowship. Additional
financial support was provided by the McIntire-Stennis

Cooperative Forestry Research Program.

ii

TABLE OF CONTENTS

ACKNOWLEDGMENTS . . . . . . . . . . .

LIST OF

LIST OF

TABLES O O O O O O O O O O O

FIGURES O O I O O O O O C O 0

INTRODUCTION . . . . . . . . . . . .

Chapter

I.

II.

III.

HEIGHT GROWTH, LEAF AREA ACCRETION, AND
WEIGHT PRODUCTION . . . . . . .

Introduction . . . . . . .
Materials and Methods . . . . .
Results and Discussion . . . . .
Height Growth . . . . . . .
Leaf Area Accretion . . . . .
Dry Weight Production . . . .
Comparison of Species . . . .

PHOTOSYNTHESIS AND RESPIRATION . . .

Introduction . . . . . . .
Materials and Methods . . . . .
Results and Discussion . . . .

Photosynthetic Efficiency . . .
Effect of Light Intensity . . .
Photorespiration . . . . . .
C02 Compensation Point . . . .

TRANSLOCATION OF LABELED PHOTOSYNTHATE

Introduction . . . . . .
Materials and Methods . . . . .

iii

Page

ii

vii

15

15
l6
19
19
27
31
34

37

37
38

 

Chapter
Results and Discussion . . . . . .
Short Term Translocation of 14C
in Black Walnut . . . .
Short Term Translocation of 14C
in Honeylocust . . . . . . .
Redistribution of 14C in Black Walnut
Redistribution of 14C in Honeylocust
The Incorporation of 14C into the
Sugars of Black Walnut . . . .

IV. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS
LIST OF REFERENCES . . . . . . . . . . .

APPENDIX 0 O O O O O O O O O O O C 0

iv

Page
41
41
46
50
51
51
55
6O

65

Table

1.

LIST OF TABLES

Seasonal changes in cumulative height,
leaf area, and dry weight of black
walnut and honeylocust seedlings . . . .

Analysis of variance of net photosynthesis
of black walnut seedlings at four dates
and three light intensities . . . . .

Analysis of variance of net photosynthesis
of honeylocust seedlings at four dates
and three light intensities . . . . .

Seasonal changes in total photosynthesis of
developing black walnut and honeylocust
seedlings at 10,000 ft-c . . . . . .

Effect of developmental stages on net
photosynthesis at different light
intensities in black walnut and
honeylocust seedlings . . . . . . .

Effect of developmental stages on photo-
respiration at different light intensities
in young black walnut and honeylocust
seedlings . . . . . . . . . . .

Developmental changes in the C02 compensation
point in young black walnut and honeylocust
seedlings at 25°C . . . . . . . . .

Redistribution of 14C between the leaves,
stems, and roots of black walnut and
honeylocust seedlings . . . . . . .
Changes in the distribution of 14C among
the sugars of leaves, stem, and roots of
black walnut seedlings on two dates during
the first growing season . . . . . .

Page

22

26

28

29

33

36

44

53

Table Page

10. Variation in net photosynthesis between
individual seedlings of black walnut
and honeylocust on four dates during
the first growing season . . . . . . . 65

11. Variation in dark respiration between
individual seedlings of black walnut
and honeylocust on four dates in the
first growing season . . . . . . . . . 56

12. Variation in photorespiration between
individual seedlings of black walnut
and honeylocust at four dates during

the first growing season . . . . . . . 67
13. Variation in the CO compensation point
between individuaI seedlings of black

walnut and honeylocust seedlings on

four dates during the first growing

season . . . . . . . . . . . . . 68
14. Seasonal effect on incorporation of 14C

into major fractions extracted from

leaves, stem, and roots of black walnut

seedlings after one day and at the end

of the first growing season. Three

seedlings were labeled on each of four

dates during the growing season . . . . . 69
15. Seasonal effect on incorporation of 14C

into major fractions extracted from

leaves, stem, and roots of honeylocust

seedlings after one day and at the end

of the first growing season. Three

seedlings were labeled on each of four

dates during the growing season . . . . . 70

vi

Figure

1.

LIST OF FIGURES

Page

Developmental changes in net photosynthesis,
photorespiration and dark respiration in
black walnut seedlings . . . . . . . . 20

Developmental changes in net photosynthesis,
photorespiration and dark respiration in

honeylocust seedlings . . . . . . . . 23
Extraction procedure . . . . . . . . . 40
Seasonal effect on incorporation of 14C into

major fractions extracted from leaves, stem,

and roots of black walnut seedlings one day

after treatment . . . . . . . . . . 42
Seasonal effect on incorporation of 14C into

major fractions extracted from leaves, stem,

and roots of honeylocust one day after

treatment . . . . . . . . . . . . 47

vii

INTRODUCTION

Deciduous hardwood species dominate the eastern
half of the United States yet surprisingly little is known
of their growth and metabolism. Important physiological
processes in most valuable hardwood species are virtually
unstudied. More research has probably gone into the
elimination of hardwoods and their subsequent replacement
with conifers than towards increasing our knowledge of
their basic physiology.

Despite their large size, forest trees character-
istically exhibit low rates of photosynthesis when compared
with crop plants (Jarvis and Jarvis, 1964; Larcher, 1969).
Comparative photosynthetic rates of deciduous and coni-
ferous trees have seldom been measured under similar con-
ditions. Deciduous trees would appear to be at a distinct
disadvantage in terms of growth because of their deciduous
habit. Photosynthesis in deciduous hardwoods is of course
limited to the time that the leaves are present and per-
haps only a portion of this period. Maximum rates of
photosynthesis are achieved only after the leaves reach

full size (Shiroya, gt_al., 1961). Leaf abscission is

preceded by a period of gradually increasing senescence
of the leaves during which photosynthesis has been shown
to be greatly reduced (Rhoads and Wedding, 1953). On the
other hand photosynthesis in coniferous species occurs
throughout the year during favorable conditions. Photo-
synthesis has been observed by Freeland (1945) in Pinus

sylvestris L., Picea mariana (Mill.) B.S.P., and Pinus

 

nigra Arnold during the winter when temperatures ranged
as low as -6°C. Others have also detected photosynthesis
and reSpiration at temperatures below freezing (Brown,
1970; Parker, 1953).

Available data indicate that deciduous hardwoods
are not disadvantaged by the limitations of a leafless
period. They may grow and photosynthesize at greater
rates than conifers allowing for greater dry matter accu-
mulation over the period of a year. Verduin (1953) pro-

jected photosynthetic rates of Picea pungens Engelm. to

 

be only one-seventh the rates reported for apple. Jarvis
and Jarvis (1964) observed that maximum rates of net
photosynthesis for temperate zone conifers were in the

range of 5 to 10 mg CO2 dm-2 hr 1 compared to rates of

10 to 20 mg CO2 dm-2 hr.1 for broad-leaved trees and
shrubs. Kramer (1953) reported (work of Polster in Ger-
many) that the average rate of photosynthesis of evergreen
conifers for an entire season is considerably lower than
that of broad-leaved species, and the rate of deciduous

larch is intermediate.

Kramer and Decker (1944) in comparative studies of
hardwoods and pines attributed the lower rates of photosyn-
thesis of the pines to mutual shading of the needles.
Krueger and Ferrell (1965) found maximum rates of net
photosynthesis in young Douglas-fir seedlings to be as .
high as 18 mg co2 dm‘2 hr-l. Brix (1967) also reported i”

similar high rates for this species. Many studies have

shown that broadleaf species attain maximum photosynthesis ,-

 

at lower light intensities than conifers.

It is clear that more studies on the factors limit-
ing growth and photosynthesis in woody plants are needed.
Since most available information indicates that deciduous
trees are photosynthetically more efficient than conifers,
their study seems a logical first step. Ledig (1969) has
developed a mathematical model for relating growth and
photosynthesis in tree seedlings. He includes rates of
net photosynthesis, distribution of assimilate between the
leaves and other organs, and seasonal aspects of photo-
synthesis. This type of approach appears to have consider-
able merit for evaluating the photosynthetic efficiency of
a Species.

Several workers have followed the seasonal course
of photosynthesis, respiration, and metabolism in coni-
fers (Gordon and Larson, 1968; Neish, 1958; Nelson, 1964;
Schier, 1970; and Shiroya, gt $1., 1966). Few studies of
this type have been undertaken with deciduous forest tree

species. The study reported here was done to learn more

about the seasonal and developmental patterns of photosyn-
l4

thesis, respiration, and metabolism of labeled CO2 in
deciduous broad-leaved trees. The experimental plants
were first-year potted seedlings of black walnut (Juglans
nigra L.) and honeylocust (Gleditsia triancanthos L.)
grown outdoors near East Lansing, Michigan. The following
growth characteristics were studied at four dates from
June to September, 1970: total height growth, leaf area
accretion, and dry matter production of leaves, roots, and
stem. The purpose of these measurements was to serve as
an aid in the interpretation of measurements of photosyn-

l4C labeled

thesis, respiration, and translocation of
photosynthate.

Photosynthesis, dark respiration, and photorespira-
tion also were measured at four dates from June to Sept-
ember, 1970 in order to study the seasonal development and
efficiency of the photosynthetic processes.

Finally, the fate of labeled 14CO2 was determined
at four dates from June to September, 1970 in leaves,
stem,and roots 24 hours after exposure and again at the

time of leaf abscission to determine seasonal trends in

metabolism of the two species.

 

 

CHAPTER I

HEIGHT GROWTH, LEAF AREA ACCRETION,

AND DRY WEIGHT PRODUCTION

Introduction

 

A thorough understanding of growth and development
patterns of a species is basic to the study of other physio-
logical processes in that species. The course of seasonal
height growth has been defined for several deciduous
species (Kienholz, 1941; Kozlowski and Ward, 1957). Such
studies have contributed to our understanding of the sym-
podial and preformed shoot growth habits of deciduous trees
and shrubs. These studies also suggest possible transloca-
tion patterns and priority of stored foods metabolism.

Other studies of seasonal cambial activity have also
revealed general patterns for lateral growth of woody plants
(Fritts, 1958; Reimer, 1949). Fayle (1968) has reviewed
much of the literature on root growth.

Growth in one part of a tree is influenced by the
growth in other parts. Such correlative growth responses
are thought to reflect competition for food, water,

minerals and hormones. An example is the ability of

reproductive structures to mobilize metabolic products
often at the expense of vegetative organs. Little is
known about the specific interactions of growth between
the various organs of woody plants. Bey and Phares (1968)
determined the seasonal growth patterns of the shoot and
roots for five seed sources of one-year old black walnut
and reported cumulative dry matter production of the roots
of only about 10 grams. These low values coupled with an
abrupt rather than sigmoid cumulative height growth curve
seem to indicate that the seedlings in their study were
grown under conditions of moisture stress.

' The first objective of my study was to define
normal seasonal development of first-year, potted black
walnut and honeylocust seedlings grown outdoors under
' optimal conditions of moisture and mineral nutrition.

Data are presented for cumulative height growth, leaf

area accretion, and dry matter production of leaves, stem,

and roots.

Materials and Methods
First-year black walnut (Juglans nig£a_L.) and
honeylocust (Gleditsia triacanthos L.) seedlings were used
as experimental material. Black walnuts were collected
from several individual trees in the vicinity of Michigan
State University in September and October, 1969. They

were immediately husked, sown in buried 11% liter pots

and left outdoors to overwinter at the M.S.U. Tree

Research Center. Honeylocust seeds were collected from

a single tree on the Michigan State University Campus in
April, 1970 and sown in 4-liter pots on May 23, 1970
following treatment with concentrated sulfuric acid for

40 minutes. Germination of the black walnuts began on

May 18, 1970 and continued sporadically through June 1970
until about 57 per cent of the seeds had germinated. Honey-
locust began to germinate on June 10 and 99 per cent had
germinated within a period of one week.

Pots containing germinated seeds were buried at a
spacing of 61 x 61 cm behind a wind barrier at the Tree
Research Center where they remained during the 1970 grow-
ing season. Each seedling was irrigated daily to maintain
the soil in the pots at or near field capacity. At three-
week intervals each pot was heavily fertilized with an
aqueous solution containing major and minor nutrients.
Frequent weeding kept the seedlings free of competition.

The foliage of the walnut seedlings was sprayed several

times with a 0.5 per cent aqueous solution of zineb to mini-
mize damage from the walnut anthracnose [Gnomonia leptostyla
(Fru.) Ces. and DeN.]. Temperature and solar radiation dur-

ing the study period were not unusual for southern Michigan.

One month after germination 12 trees were selected
from each species for height, leaf area, and dry weight
determinations. Sample trees were selected on the basis

of uniformity of height, appearance of the foliage, and

germination date. An additional 24 sample trees were
selected on the same basis and were set aside for the
photosynthesis and translocation portions of this study.
At four dates, approximately one month apart,
three trees were selected at random from within the 12
sample trees and taken into the laboratory for analysis.
Measurement dates for black walnut were June 15, July 27,
August 17, and September 7 and for honeylocust July 13,
August 10, September 1, and September 21. Total height
measurements were recorded to the nearest 0.1 cm. Leaf
areas were planimetered to the nearest 0.5 cm2 and totaled
for the whole seedling. Dry weights of leaves, stem,
roots, and whole plant were determined by weighing to
nearest 0.001 9 after drying in an oven at 60°C for 24

hours.

Results and Discussion

 

Table 1 shows the seasonal changes in cumulative
height, leaf area, and dry weight for each species during
the 1970 growing season. Black walnut is typical of those
deciduous Species with the preformed shoot growth habit.
Honeylocust shows a growth pattern characteristic of
deciduous species with the sympodial growth habit where
persistent terminal buds are not formed and the shoot tip

dies and abscises.

 

 

 

 

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10

Height Growth

Black walnut completed about 80 per cent of its
height growth before June 15. Growth then slowed between
June 15 and July 27. By July 27 height growth had ceased.
Five black walnut sources grown at Ames, Iowa terminated

height growth in early July (Bey and Phares, 1968).

I L"

Honeylocust seedlings showed a pattern of height
growth characteristic of the sympodial growth habit. Only
60 per cent of its height growth had been attained by
July 13. Growth continued at a reduced rate between
July 13 and August 10 but was essentially complete by
August 10. A small but significant amount of height growth

continued through September 21.

Leaf Area Accretion

 

Leaf area increased very rapidly in the developing
black walnut seedlings. It reached a level of 52.6 cm2
by June 15 (Table 1). Between June 15 and July 27 the
leaf surface area doubled and reached a peak of 124.4 cmz.
Between July 27 and August 17 leaf area decreased as older
leaves of the lower crown began to senesce and abscise.
Leaf abscission accelerated after August 17 and by Sept-
ember 27 only 52.5 cm2 of leaf area remained.

Honeylocust seedlings showed a different pattern
of leaf area accretion. Rapid leaf area buildup occurred

during the first two months after germination. After

August 10 leaf area accretion ceased for a short period.

11

Between September 1 and 21 a second increase in leaf area
occurred and a maximum for the season of 35.8 cm2 was
reached. Leaves of lower crown began to abscise shortly

before September 21.

Dgy Weight Production

Trends in dry weight of the leaves were similar to
those for leaf area accretion (Table 1). However, leaf
dry weight decreased after September 1 while leaf area
continued to increase. It is possible that development
and enlargement of new leaves masked the abscission of
some leaves in the lower crown.

Stem growth of black walnut occurred only in the
first two months after germination. The most rapid
increase occurred between June 15 and July 27. After
July 27 stem growth ceased. Stem growth of honeylocust
seedlings was greatest in early summer and fall. The
most rapid rise occurred between July 13 and August 10.
After September 1 a second peak period of stem growth
increase was recorded.

Both species showed two periods of root growth
separated by a period of relative inactivity (Table 1).
In walnut seedlings the first peak of activity occurred
between June 15 and July 27. Between July 27 and August
17 little root growth occurred. A second period of root
growth occurred between August 17 and September 7. Most

of the root growth in developing honeylocust seedlings

12

occurred between July 13 and August 10. As in walnut this
was followed by a period of little growth. Between Sept-
ember l and September 27 a second period of root growth
occurred.

Total plant dry matter reflected the activity of
the root fractions (Table 1). Both species showed a steep
rise in total dry matter followed by a period of inacti-
vity. This in turn was followed by a second period of

growth in the fall.

Comparison of Species

Developing black walnut and honeylocust seedlings
showed height growth patterns characteristic of the pre-
formed shoot and sympodial growth habits respectively.
The height growth of black walnut occurred over a rela-
tively short time period in comparison with honeylocust.
The latter showed slight but continued height growth in
the fall.

Both Species have a compound leaf structure.
Black walnut leaves are of the odd-pinnate type and honey-
locust leaves are of the 2-pinnate decompound type. The
pattern of leaf area accretion and the total leaf area
prOduced in a single season were remarkably different
between the two Species. Walnut seedlings reached a maxi-
mum leaf area of 124.4 cm2 within two months after germi-
nation and then leaf area declined. Walnut characteris-
tically looses its leaves early in comparison to other

deciduous Species (Kramer, 1943). Most leaf area expansion

13

in honeylocust seedlings occurred within two months of
germination. This was followed by a long period of little
increase. A second period of leaf area buildup occurred
in the fall. Perhaps the most remarkable difference
between the two Species was the tremendous difference in
total leaf area produced. At its peak, relatively early
in the growing season, the average black walnut seedling
had a total leaf area of 124.4 cmz. The maximum leaf

area for honeylocust seedlings was 35.8 cm2 and this was
not obtained until September 21.

Much of the variation in early growth patterns and
total dry matter between the two Species may be due to the
large difference in seed size. Black walnut is among the
largest seeds of the plant kingdom, 40 seeds per pound while
honeylocust seeds are considerably smaller, 2,800 seeds per
pound (U. S. Forest Service, 1948).

Data on leaf dry matter generally followed the
trends of leaf area accretion. Dry matter data for the
stem and roots were further indicative of the preformed
shoot and sympodial growth habits. Walnut stem growth
ceased sometime prior to July 27 while honeylocust stem
growth continued throughout the study period. Growth of
the roots was maximal in early summer and fall. The
results here agree partly with those of Lyr and Hoffmann
(1967) who reported that maximum growth of roots of

deciduous trees occurred in early summer compared with

 

l4

coniferous species which grow more uniformly through the

whole vegetative period.

P}.

CHAPTER II

PHOTOSYNTHESIS AND RESPIRATION

Introduction

 

Studies of photosynthesis of forest trees have
dealt largely with Single measures of photosynthesis under
certain "standard” conditions. Other studies have been
aimed at determining the effects of specific environ-
mental factors on the photosynthetic process. Much of
this work has been summarized by Larcher (1969) and Kramer
(1958). Considerable effort has gone into the study of
specific environmental factors which facilitate the opti-
mum rate of photosynthesis in the species studied. Less
attention has been given to the measurement of photosyn-
thesis during developmental states within the annual cycles
of growth and ontogeny of the species. Often age in years
and plant size have been the only factors considered. It
is usually not apparent to the reader just where within
the annual cycle of growth and development the measurements
were made.

Several studies have been made on seasonal fluctua-

tions of photosynthesis in evergreen trees (Bourdeau, 1959;

15

16

Helms, 1965; McGregor and Kramer, 1963). Fewer studies
have been made in deciduous trees (Heinicke and Childers,
1937; Saeki and Nomoto, 1958). Most of these studies
were conducted outdoors and seasonal fluctuations in
environmental factors obscure or complicate the study of
photosynthetic efficiency.

In the work reported here photosynthetic efficiency
for developing black walnut and honeylocust seedlings grown
outdoors in southern Michigan was measured at intervals

during the first growing season.

Materials and Methods

 

First-year black walnut and honeylocust seedlings
grown outdoors under optimum conditions of soil moisture
and mineral nutrition were used. See Chapter I for details
of the experimental material.

On four dates, approximately one month apart from
June to September 1970, different groups of five seedlings
of each Species were brought into the laboratory for
determinations of photosynthesis and respiration rates
under standard conditions of light intensity and tempera-
ture. Relative humidity was monitored but not controlled.
Due to the mechanics of the translocation portion of the
study, it was not possible to follow photosynthesis and
respiration within the same group of five seedlings
throughout the growing season. The sampling dates for
walnut were June 15, July 27, August 17, and September 7.

Honeylocust seedlings were sampled on July 13, August 10,

17

September 1, and September 21. The last date for each
species coincided with the start of leaf abscission.

Photosynthesis and respiration measurements were
made in a closed system using a controlled environment
chamber constructed of acrylic plastic. The chamber was
about 50 liters in volume and was surrounded by a water
jacket for temperature stabilization. Four SOC-watt
weather resistant flood lamps were immersed in an acrylic
plastic water bath above the chamber. Additional tempera-
ture and light control was achieved by placing the chamber
into a large Sherer-Gillett controlled environment room.
Light intensity was controlled with a Powerstat Type
3PN1368 variable transformer. Temperature was stabilized
at 25°C :4 by varying the water levels in the water bath
and chamber water jacket and manipulation of the tempera-
ture control of the controlled environment room. Rela-
tive humidity varied between 45 and 65 per cent.

A Beckman infrared gas analyzer model 215 and a
Sevoriter recorder were used to detect and record CO2 con-
centration changes. Small Rotron fans circulated air in
the chamber and a Masterflex tubing pump maintained a flow
rate of 900 ml/min. in the closed system. A "Drierite"
(Ca 804) dessicant column was used to remove water vapor
from the air stream flowing into the gas analyzer. Slight

CO absorption by the Ca SO4 drying column did not signi-

2
ficantly affect the results.

18

Individual seedlings including their pot container
were placed into the acrylic chamber and equilibrated at
25°C and 10,000 ft-c of light intensity. Polyethylene
bags were sealed around the pots to eliminate the effects
of soil respiration and evaporation from the soil surface.
After a lS-minute conditioning period the CO2 concentra-

tion in the measurement chamber was raised to a level

slightly in excess of 400 ppm, the chamber was sealed,

 

and the CO2 uptake was recorded until the chamber CO2 con-
centration had decreased to around 200 ppm. The chamber
was then opened to ambient air and the light intensity was
lowered to 7,000 ft-c for honeylocust and 3,000 ft-c for
black walnut. After a 15-minute conditioning period at
the new light intensity the chamber was sealed and photo-
synthesis was again recorded from 400 ppm to 200 ppm.

Upon reaching a C02 concentration of 200 ppm, the system

was again opened to the ambient air and the light inten-
sity was lowered a second time to 4,000 ft-c for honey-
locust and 1,800 ft-c for black walnut. After a lS-minute

conditioning period at the new light intensity the CO2

uptake was recorded from 400 ppm to the CO compensation

2

point. Upon reaching the CO compensation point the

2
lights were turned off and dark respiration was recorded

for two hours following the light period.

The rate of CO depletion from 330 to 270 ppm was

2

used as the measure of net photosynthesis. Rates were

19

computed from slopes of lines drawn tangent to the recorder

tracings.

Results and Discussion

 

Photosynthetic Efficiency

 

Net photosynthesis in developing black walnut seed-

lings reached a peak incorporation of 7.8 mg CO2 dm-2 hr-l

on July 27, the second measurement date (Figure 1). By

August 17 net photosynthesis had declined to 7.1 mg CO

2
dm”2 hr-l. After August 17 net photosynthesis decreased
Sharply to a low level of 1.3 mg C02 qu2 hr-l as rapid

abscission began. A factorial analysis of net photosynthesis
at the different measurement dates and light intensities
revealed dates to be highly Significant (Table 2). Dark
respiration followed an irregular pattern during the period
of study (Figure l). The highest rates of dark respiration
were found during the period of rapid leaf expansion and
when leaf abscission began. The high rate on September 7
may reflect the dying of the leaves as they began to
abscise.

Net photosynthesis in developing honeylocust seed-
lings showed a much different pattern (Figure 2). Net

photosynthesis on July 13 was 10.6 mg CO2 dm-2 hr-l. By

2 -1

August 10 it had increased to 15.5 mg CO dm- hr and

2
remained near this level through September 1. After

September 1 net photosynthesis began to decline and by

September 21 it was 12.2 mg CO2 dm-2 hr-l. The relatively

20

Figure l.--Developmental changes in net photosynthesis,
photorespiration and dark respiration in
black walnut seedlings.

21

     

  

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N

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22

TABLE 2.--Analysis of variance of net photosynthesis of
black walnut seedlings at four dates1 and
three light intensities.2

 

 

 

 

Source of Variance Percent of Total Variance
Date 37**

Light intensity 8**

Date x light intensity 1

Error 4

1June 15, July 27, August 17, and September 7

2

1,800; 3,000; and 10,000 ft-c

**Significant at the 1% level

23

Figure 2.--Developmenta1 changes in net photosynthesis,
photorespiration, and dark respiration in
honeylocust seedlings.

 

 

 

 

_ — N
(D N m 0
n 1 n 141

C02 UPTAKE mg C02 dm'z hr"
A

=' “assesses
HONEYLOCUST MPHOTORESPIRATION

- DARK RESPIRATION

Fl

 

 

 

 

 

 

co2 EVOLUTION mg C02 am" hr"

 

 

 

 

JULY l3

    

AUG IO | SEPT 2|

 

&
Z
7
4

25

high rate of net photosynthesis on September 21 may be
characteristic of the sympodial growth habit. The develOp-
ment of new leaves may have kept the rate of net photosyn-
thesis at a high level even though leaves of the lower
crown had begun to abscise. A factorial analysis of net
photosynthesis of the different measurement dates and

light intensities Showed date to be highly significant

(Table 3). Dark respiration was highest, 9.5 mg CO2 dm-2

hr-l, in honeylocust seedlings on July 13 as rapid leaf

expansion occurred (Figure 2). On August 10 it declined

to approximately 8 mg CO2 dm“2 hr.1 and remained near this

level through September 1. In sharp contrast to black
walnut dark respiration in honeylocust was lowest as leaf
abscission began.

There was considerable variation in rates of net
photosynthesis and dark respiration among individual trees

of both species. Net photosynthesis ranged as high as

14 mg CO2 dm"2 hr.l for black walnut and as high as 20
mg CO2 dru-2 hr.l for honeylocust (see Appendix, Table 10).
Rates of CO2 evolution for dark respiration were as high

as 10 mg CO dm-2 hr"l for black walnut and 13 mg CO

2
dm-2 hr—l for honeylocust (see Appendix, Table 11).

2

Both black walnut and honeylocust are considered
shade intolerant and reproduction is limited to openings
in the forest canopy (Baker, 1949). The low rate of net
photosynthesis of black walnut in comparison with honey-

locust was unexpected. The growth of walnut seedlings is

26

TABLE 3.--Analysis of variance of net photosynthesis of
honeylocust seedlings at four dates1 and
three light intensities.2

 

 

 

Source of Variance Percent of Total Variance
Date 65**

Light intensity 25**

Date x light intensity 1

Error 9

1July 13, August 10, September 1, and September 21

2

4,000; 7,000; and 10,000 ft-c

**Significant at 1% level

 

27

remarkably rapid considering its early leaf abscission

in comparison with other hardwoods. There is no available
information on the early growth rates of honeylocust seed-
lings. To further investigate the differences between the
two species, rates of total photosynthesis were determined
on a leaf area and whole plant basis (Table 4). On a leaf
area basis honeylocust seedlings exhibited a remarkable
superiority in carbon dioxide uptake over walnut. However,
when total photosynthesis was expressed on a whole plant
basis the Situation was reversed. Black walnut presumably
because of its greater leaf surface area surpassed honey-

locust in total CO uptake. It is possible other leaf

2
factors also may be involved. It seems reasonable that
differences in the number, distribution, and size of stomata
as well as the thickness of the leaf and its mesophyll may
further contribute to differences in photosynthesis between
the two species. In walnut stomates are restricted to the
upper epidermis and average 46,000 per cm2 (Meyer and
Anderson, 1952). Observations by the author indicate that
the stomates of honeylocust also are restricted to the

upper epidermis. Data on stomatal size and relative dis-

tribution are not available for either species.

Effect of Light Intensity
The limited objectives of this study did not permit
a detailed examination of the effects of light intensity on

the photosynthetic apparatus. Table 5 shows the effects of

 

28

TABLE 4.--Seasonal changes in total photosynthesisl of
developing black walnut and honeylocust seed-
lings at 10,000 ft-c.

 

 

Total Photosynthesis
Species and

 

Dates mg CO2 dm_2 hr.l mg CO2 Seedling"1 hr.-1

Black Walnut

June 15 10.42 53.8

July 27 12.8 164.5

August 17 12.7 108.0

September 7 8.4 34.1
Honeylocust

July 13 20.0 25.6

August 10 23.0 55.6

September 1 23.9 60.2

September 21 19.3 65.4

 

1Total photosynthesis = net photosynthesis + dark respiration

2Mean of 5 trees

29

TABLE 5.--Developmental changes in net photosynthesis at
different light intensities in black
walnut and honeylocust seedlings.

 

Species and

Net Photosynthesis2

l'

 

 

 

 

 

Date
Black Walnut 1,800 ft-c 3,000 ft-c 10,000 ft-c j
mg CO2 dm-2 hr.l L
June 15 4.4 a 5.0 a b 4.0 a b
July 27 5.9 a 8.8 a 7.8 a
August 17 5.0.a 6.8.a 7.1 a
September 7 1.4 a 1.6 b 1.3 b
Honeylocust 4,000 ft-c 7,000 ft-c 10,000 ft-c
mg €02 am"2 hr'l
July 13 10.9 a 12.4 a 10.6 a
August 10 14.3 a 16.6 a 15.5 a
September 1 14.1 a 16.8 a 15.8 a
September 21 10.3 a 13.4 a 12.2 a

 

1

ent at 5 per cent level (Tukey's weprocedure)

2Mean of 5 trees

Means not followed by same letter are significantly differ-

30

three light intensities on net photosynthesis in walnut and
honeylocust. Factorial analyses showed highly significant
differences in the effects of light intensity (Tables 2 and
3). In both species there was an increase in net photosyn-
thesis between the low and intermediate light intensities.
The inability of honeylocust to carry on net photosynthesis
at light intensities below 4,000 ft-c early in the annual

growth cycle, did not permit the study of the same light

f,‘

intensities for both species. There was a decline in net
photosynthesis at full sunlight, 10,000 ft-c, for both
species. Other workers have found similar solarization
effects in other deciduous hardwoods (Borman, 1953;
Kozlowski, 1949). Solarization in conifers occurs at much
higher light intensities (Ronco, 1961).

The results of this study permit only some very
general statements concerning light saturation for each
species. Light saturation for black walnut lies somewhere
between 3,000 and 10,000 ft-c. It is probably closer to
3,000 ft-c than 10,000 ft-c because light saturation for
other hardwoods with similar growth habits lies in this
range. Honeylocust has an unexpectedly high light satura-
tion. It lies somewhere between 7,000 and 10,000 ft-c.
Early in the growing season no measurable net photosyn-
thesis occurred in this species below 4,000 ft-c. Later

net photosynthesis was detected at lower light intensities.

31

Photorespiration

 

The question has often been asked, do plants

‘ respire in the light or is "dark" respiration turned off
during active photosynthesis? From measurements of the
rates of O2 and CO2 exchange in illuminated plants it has
been determined that most plants do respire in the light .1

while they are carrying on photosynthesis. This photo-

respiration is separate from mitochondrial respiration

 

since it is not sensitive to inhibitors of mitochondrial
respiration. PhotoreSpiration consumes reducing power
generated by photosynthesis and uses it to reduce molecular
oxygen. Photorespiration thus shortcircuits photosynthesis
by diverting the normal flow of light-induced reducing

It

power from the reduction of CO to the reduction of O

2 2'
has been estimated that in some plants photorespiration
utilizes up to 50 per cent of the reducing power generated
by photosynthesis (Lehninger, 1970). Some tropical plants
including maize and sugar cane Show no apparent photo-
respiration.

Photorespiration is difficult to measure since it

involves simultaneous CO exchange with photosynthesis.

2
Photorespiration has seldom been measured in forest trees.
Townsend (1969) found rates of photorespiration in young
western white pine seedlings approaching those of dark
reSpiration. In certain crop plants the release of CO2

in photorespiration may be three to five times greater than

the rate of release in darkness (Zelitch, 1969). No data

_.'~4»..u

32

is available on photorespiration measurement in deciduous
hardwoods.

Photorespiration in this study was estimated by
extrapolating photosynthetic response curves to zero CO2
concentration (Brix, 1968). The accuracy of this technique
has been challenged (Bravdo, 1968; Brix, 1968; Zelitch, a.
1966). However, its use seems justified since little

photoreSpiration data are available for forest trees and i

 

since all current techniques for estimating photorespira-
tion have been questioned.

Seasonal trends in photorespiration derived by the
extrapolation method are shown in Figures 1 and 2. It is
evident that photorespiration does change substantially
within the annual growth cycle of plants. Rates of photo-
respiration were generally related to photosynthetic acti-
vity. High rates of photoresPiration usually accompanied
high rates of net photosynthesis. (Rates of photorespira-
tion were considerably higher in honeylocust than black
walnut.

There was considerable variation in rates of photo-
respiration among individual trees of both Species. Photo—

respiration ranged as high as 9 mg CO2 dm-2 hr-1 in black

walnut and 22 mg CO2 dm_2 hr-1 for honeylocust (see Appen-
dix, Table 12).
The rate of photorespiration has been shown to

increase with increasing light intensity. This occurred

in black walnut on three dates (Table 6). However,

33

TABLE 6.--Effect of developmental stages on photorespiration
at different light intensities in black walnut and
honeylocust seedlings.

 

 

 

 

 

 

Species and Date Photorespiration
Black Walnut 3,000 ft-c 10,000 ft-c
mg co2 dm'2 hr-l
June 15 6.41 8.4
July 27 7.6 e 9.2
August 17 7.9 7.3
September 7 4.0 7.6
Honeylocust 4,000 ft-c 10,000 ft-c
mg CO2 dm-2 hr.-1
July 13 8.8 4.8
August 10 10.1 8.4
September 1 20.1 21.5
September 21 4.7 0.6
1

Mean of 5 trees

 

34

increasing the light intensity from 4,000 to 10,000 ft-c
caused a decrease in the rate of photorespiration in
honeylocust on three dates. The reason for this is
unknown. There was a measurable decrease in the rate of
net photosynthesis at 10,000 ft-c. Perhaps this decrease
resulting from solarization would also Show up in decreased
photorespiration since photosynthesis and photorespiration ”

are biochemically linked or because certain cellular com-

‘. ,a-x.

ponents of the photorespiratory process are affected. A

 

similar decrease in photosynthetic activity in black walnut

did not result in decreased photorespiration.

CO Compensation Point

2
Another means of evaluating the photosynthetic

efficiency of plants is the determination and comparison

of CO

compensation points. The CO compensation point

2 2

may be defined as that concentration of CO2 at which the

evolution of CO by reSpiration from illuminated leaves

2
equals photosynthetic CO2 uptake. Low compensation points
are thought to be correlated with high photosynthetic
efficiency because plants lacking photorespiration have
compensation points near 0 ppm C02.

Few determinations of the CO2 compensation point
have been made for forest trees. Townsend (1969) found
extremely high CO2 compensation points in young western

white pine seedlings. The CO2 compensation points deter-

‘ mined in this study were similar for the two species

'H

35

(Table 7). The compensation points were generally indica-
tive of photosynthetic activity. For both Species the CO2
compensation points were lowest when rates of net photo-
synthesis were highest. Later, as photosynthetic activity
decreased the compensation points increased.

There was considerable variation in CO2 compensa-

"'l

tion points among individual trees of both species. Com-

pensation points ranged as low as 95 ppm for black walnut i

 

and 105 ppm for honeylocust during the period when rates

of photosynthesis were highest (see Appendix, Table 13).

TABLE 7.--Effect of developmental stages on the CO
compensation point in black walnut and

36

honeylocust seedlings at 25°C.

2

 

 

Species and Date CO2 Compensation Point
PPm
Black Walnut
June 15 1601
July 27 118
August 17 145
September 7 232
Honeylocust
July 13 155
August 10 114
September 1 156
2

September 21

 

1Mean of 5 trees

2Data not obtained

“1 ii

1.3:;

CHAPTER III

TRANSLOCATION OF LABELED PHOTOSYNTHATE

Introduction

 

Many of the great advances in biochemistry during
the last decade were made possible by the use of radio-
isotopes. A major contribution of the radiotracer tech-
nique was the determination of the path of carbon in
photosynthesis by Calvin and Benson (1948). Radioactive
carbon has been used to study seasonal photosynthate pro-
duction and translocation in conifers (Gordon and Larson,
1968; Schier, 1970; Shiroya gt 31., 1966). Fewer studies
have followed the rate of 14CO2 in deciduous hardwoods
(Hanson, 1964). Roberts (1964) used l4CO2 to study the
effect of water stress on the translocation of photosyn-
thate in yellow-poplar (Liriodendron tulipifera L.). More
recently Larson and Gordon (1969) used 14CO2 to study the
translocation of photosynthate from selected leaves of
young cottonwood (Populus deltoides Bartr.) trees.

Most metabolic studies of deciduous trees have been

limited in that they have been of short duration and

usually have not attempted to follow the incorporation of

37

 

38

14C into specific compounds extracted from various plant

organs. In this study developing black walnut and honey-
locust seedlings grown outdoors were photosynthetically
labeled at several dates during the growing season. At
two time intervals following labeling the radioactivity
in Specific compounds of leaf, stem, and root fractions I! f

was determined.

Materials and Methods L

 

 

First-year black walnut and honeylocust seedlings
grown outdoors under optimum conditions of soil moisture
and mineral nutrition were used as experimental material.
See Chapter I for details about the experimental material.

On four dates, approximately one month apart from
June to September 1970, six seedlings of each species
were brought into the laboratory and exposed to 14CO2
under defined conditions of light intensity and tempera-
ture. Walnut seedlings were labeled on June 15, July 27,
August 17, and September 7. Honeylocust seedlings were
labeled on July 13, August 10, September 1, and September 21.

Whole seedlings were labeled in the same type of
closed system used for the determination of rates of
photosynthesis and respiration described in Chapter II.

Individual seedlings including the pot container
were placed in the acrylic chamber at a temperature of 25°C
and a light intensity of 10,000 ft-c. Polyethylene bags

were used to seal off the pots. After a lS-minute

39

conditioning period the chamber was sealed off and the

plant was exposed to about 500 no of 14CO by reacting

2
Ba14CO with perchloric acid. All plants were exposed to

14

3

CO2 for a period of two hours. On most dates this was

sufficient time to reach the CO2 compensation point.
Immediately after labeling the plants were returned to >-
the outdoors.

One day after photoassimilation three of the .

 

labeled plants were separated from the soil by washing and
divided into leaves, stem, and roots. The remaining three
seedlings were harvested in Similar fashion on September 21.
This date will hence be referred to as the end of the grow-
ing season as it coincided with the start of rapid leaf
abscission. Each plant part was cut into small pieces and
separated into five fractions, amino acids, organic acids,
sugars, non-water soluble, and ethanol insoluble, by means of
extraction procedure shown in Figure 3. Ion exchange

columns were of the type used by Romberger (1960).

The non-water soluble and ethanol insoluble frac-
tions were taken to dryness. Sugar, amino acid, and
organic acid fractions were reduced to about 5 ml. To
determine radioactivity, each fraction was wet-combusted

(Van Slyke gt 31., 1951) and the evolved 14CO was trapped

2
in a solution of ethanolamine and glycol monomethyl ether
(1:2 v/v). The scintillation solution was prepared accord-

ing to the method of Jaffay and Alvarez (1961). Radio-

activity was determined in a Packard Tricarb Liquid

4O

Leaves, Stem, or Roots
I

l

Homogenized in 200 m1
hot 80% ethanol

 

Filtered, washed pad with
hot 90% ethanol then
hot absolute ethanol

l

I 7

Filtrate Residue
l

 

l
Concentrated in rotary Extracted with hot

vacuum evaporator, ethanol/benzene

under 40° C, to 10 ml. (1:2), filtered, washed

Slurried residue with with same solvent,

Celite, added hot water washed with acetone

to form paste, filtered,

washed pad with hot

water r’ l
Non-water soluble Residue
fraction

 

 

Ethanol Insoluble

Filtrate Residue ‘
Fraction

Concentrated to I
near dryness and

made to 50 ml

with water

Ion Exchange

J

Effluent Anion column Cation column
(neutral (AG 3-X4A) (AG 50l-X8)
fraction)

Sugar Organic acid Amino Acid
Fraction Fraction Fraction

Figure 3.--Extraction procedure.

41

Scintillation Spectrometer. After quench correction by the
internal standard method, radioactivity of all fractions in
each plant part was expressed as a percentage of the total
DPM in the whole tree at the date of harvest. Quantitive
determinations of the various fractions were not made and

therefore specific activity could not be calculated.

Results and Discussion

 

14

Short Term Translocation of C in Black Walnut

 

Black walnut seedlings harvested one day after

14

photoassimilation of CO at several growth stages

2
revealed a pattern of metabolism and translocation which
may be characteristic of trees with the preformed shoot
growth habit. Developing organs and meristematic areas
are known to be sinks for photosynthates. See Appendix,
Table 14 for a complete listing of the incorporation of
14C into the various compounds studied.

Between June 15 and July 27 there was a very large
increase in the leaf area of the walnut seedlings. This
growth pattern was reflected in the high levels of radio-
activity recovered from compounds extracted from the
leaves (Figure 4). Approximately 79 per cent of the total
activity recovered on June 15 was found in the leaves
(Table 8). A high proportion of this activity occurred in
the non-water soluble fraction, probably in chlorophyll and
other pigments (Figure 4). The other ethanol—soluble com-

pounds, sugars and amino acids, were at relatively high

 

42

Figure 4.--Seasonal effect on incorporation of 14C into
major fractions extracted from leaves, stem,
and roots of black walnut seedlings one day
after treatment.

PERCENT OF TOTAL ACTIVITY
RECOVERED FROM WHOLE PLANT

PERCENT OF TOTAL ACTIVITY

PERCENT OF TOTAL ACTIVITY
RECOVERED FROM WHOLE PLANT RECOVERED FROM WHOLE PLANT

43

 

 

 

 

 

4" \ LEAVES -- BLACK WALNUT
44 - \
I’ X
- X
28 \ / \
‘I \ \
24- \ / \
. \ / \
20 4 \ /"‘~ / \
/ .X ‘y \
I6j . /. \\ / “\ \
/' n
_. \J x’ “X \
'2 ‘ *7. “~.\
‘I‘ K I” ‘\\‘ \‘U
8: \ \:‘ ...... «d, \\\
4 A 0' ...... '\‘ I
-I \6.\ . x /
JUNE I5 1qu 27 AUG. I7 SEPT. 7
'6} STEM
I 2 " 4’ ‘, I ‘ " ’ ‘\‘
8 4 \
41 1.5. \x
. o- 44.4“" ~\. ““~--+
J' ~ ~ ..—-" _ «-
JUNE l5 JULY 27 AUG. I7 SEPT. 7
72 q
.. ’/
68 « '
‘I ROOTS X
28 - ’/
.I /§
24 ~ ,
. / —-<> AMINO ACIDS
20. '1 «no ORGANIC ACIDS
. / _-_. SUGARS
.6 . x -— - NON-WATER SOLUBLE
/ —---+ ETHANOL INSOLUBLE

 

 

 

44

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45

levels on this date also. Radioactivity in the ethanol-
insoluble fraction was considerably higher than that found
in conifers after similar short term periods of assimila-
tion. This was probably due to the incorporation of 14C
into the construction of cell walls and other structural
compounds in leaf development. Further evidence that the RI

leaves were the principal center of metabolic activity on

June 15 was obtained by comparing them with levels of

 

radioactivity extracted from the stem and roots (Figure 4).
Radioactivity in the stem and root fractions were rela-
tively low on June 15. A significant amount of radio-
activity was found in the ethanol-insoluble fraction of

the stem. This was undoubtedly due to the rapid primary
and secondary growth which took place at this time. Radio-
activity in the ethanol soluble and insoluble fractions of
the roots was at a low level on June 15.

By July 27 metabolic activity in the stem and roots
increased. Leaf area reached its maximum by July 27 and
this was reflected in the increased radioactivity of the
ethanol-insoluble fraction which peaked at this time. The
non-water soluble fraction of the leaves had decreased by
July 27, probably indicative of reduced pigment synthesis.
The level of radioactivity in most fractions of the stem
reached a peak on July 27 indicative of the cessation of
primary growth. The level of radioactivity present in the
stem after July 27 probably was due to lateral growth and

lignification.

46

After July 27 the center of metabolic activity
moved from the stem to the roots. The amount of radio-
activity recovered from the ethanol-insoluble fraction of
the roots during the growing season increased almost
linearly (Figure 4). On June 15 only 6 per cent of the
total radioactivity was recovered from the roots (Table
8). By September 7 this fraction accounted for 72 per
cent of the activity. Other root extracts reached a peak
between July 27 and September 7. By September 7 all root
fractions except the ethanol-insoluble one had decreased

to low levels similar to those found on June 15.

Short Term Translocation of 14C in Honeylocust

Honeylocust seedlings harvested one day after l4CO2
photoassimilation did not show a distinct pattern of
shifting metabolic activity from the leaves to the stem and
finally to the roots (Figure 5). This indicates that meta-
bolic and translocation patterns in deciduous plants differ
between the sympodial and preformed shoot habits. A com-
plete listing of the incorporation of 14C into the various
compounds studied is given in the Appendix, Table 15.

There was considerable radioactivity in the leaves
of honeylocust on July 13 but measurable amounts of radio-
activity also were extracted from the stem and roots
(Table 8). Much of the radioactivity extracted from the

leaves on July 13 was found in the ethanol-insoluble

fraction probably reflecting leaf expansion. The

—__— I

47

Figure 5.--Seasona1 effect of incorporation of 14C into
major fractions extracted from leaves, stem,
and roots of honeylocust one day after treatment.

P40”
). -I
tile
2 1
Bass
“ I
43
< 20‘
82 .

o
"O: l6'I
ILLI.
Co ‘

.I

“51% l2.

8% 8‘

mo *

(Lu: 4.
IE

48

LEAVES -- HONEYLOCUST

 

 

 

 

 

28

.b
1

JULY l3 AUG. IO SEPT: SEPT. 2|

. f
STEM A /
.I x I
/ . /
/' \l
. ‘(
/ I.
\g /' / \
‘\‘ I I ‘\

 

PERCENT OF TOTAL ACTIVITY
RECOVERED FROM WHOLE PLANT
5

54I

.I

e

9.3

l

i?)
l

 
 

 

JULYI3 AUG. I0 SEPTI SEPT. 2I

ROOTS f
I 4
'/
\ /'
\\ /'
‘\ , —o AMINO ACIOS
\ / -----o ORGANIC ACIOS
\ ,I —-—A SUGARS
\ J — _. NON-WATER SOLUBLE
\ \.--" —---+ ETHANOL INSOLUBLE
\ \ .45.
A2.o.‘\-\‘ _’-a

 

 

PERCENT OF TOTAL ACTIVITY
RECOVERED FROM WHOLE PLANT
5's

 

 

JULY I3 AUGIO SEPTI SEPT. 2I

49

ethanol-insoluble fraction also accounted for most of the
radioactivity recovered from the stem and roots, probably
indicative of the rapid primary growth which occurred at
this time.

After July 13 much of the radioactivity in the leaf
and stem portions was concentrated in the several ethanol-
soluble fractions. These fractions in the leaves remained
at high levels through September 1. This indicated that
metabolic activity continues at relatively high levels
going into the fall months. Between July 13 and August 10
there was a decrease in the amount of radioactivity
recovered from the ethanol-insoluble fraction of the leaves.
After August 10 the large portion of the radioactivity in
the ethanol-insoluble fraction of the stem was probably
indicative of secondary growth.

The roots of honeylocust had two periods of active
growth. The first occurred at the start of the growing
season and was followed by a period of relative inactivity.
A second surge of growth occurred near the end of the grow-
ing season. Metabolic activity showed a similar pattern
as evidenced from the radioactivity recovered from the
ethanol-insoluble fractions of the roots (Figure 5).

l4 .
C was incorpor-

Between August 10 and September 1 little
ated in the ethanol-insoluble fraction of honeylocust

roots.

50

Redistribution of 14C in Black Walnut

 

The harvesting of plants at the end of the growing
season after labeling at several points during the period
of growth and development provided some valuable insight
on the redistribution of metabolic compounds within decidu-
ous species as the leafless period begins.

As the end of the growing season approached, radio-
activity in the roots accounted for about 65 to 70 per
cent of the total radioactivity recovered from walnut seed-
lings (Table 8). Much of this activity was found in the
ethanol-insoluble fraction. Apparently this reflects the
conversion of metabolities into storage compounds and the
continuation of root growth into the fall months.

Only 5 to 10 per cent of the total radioactivity
was recovered from the leaves as leaf abscission began
(Table 8). This is in contrast to higher levels found a
short time after labeling. Thus it appears that walnut
was able to transport many of the labeled metabolities out
of the leaves as abscission began.

The stem contained about 10 per cent of the
recovered radioactivity at the end of the growing season.
Similar levels were found during the growth season after
Short term assimilation. A high proportion of this
activity was found in ethanol insoluble compounds and

14

probably reflects the incorporation of C into non-mobile

structural compounds.

 

51

Redistribution of 14C in Honeylocust

l4

 

Less redistribution of C occurred in honeylocust

as the growing season ended than in walnut. A large

amount of 14C, 34 per cent, remained in the leaves on
September 21 (Table 8). Much of this activity was located
in ethanol-soluble compounds indicating that high metabolic
activity continues in the sympodial growth habit as leaf
abscission begins. The extremely low activity, 3 per cent,
in the ethanol-insoluble fraction of the leaves on Septem-

ber 21 was due to a shorter time interval between labeling

and harvesting.

The Incorporation of 14C into the

Sugars of Black Walnut

Subsamples were taken from the sugar extracts of
black walnut seedlings labeled on July 27 and September 7
and harvested the following day to study the incorporation
of 14C into specific sugars. These dates were chosen to
compare periods of high and low photosynthetic activity.

The neutral fraction from ion exchange (Figure 3)
containing sugars was reduced to 1 ml and chromotographed
on Whatman # 1 paper with butanol-water-acetic acid
(4:5:1) in the descending direction for 48 hours. Auto-
radiography was used to locate the sugars. Tentative
identification was established by comparing RG'S with
those obtained in other species under similar chromato-

graphic conditions. Standard sized discs containing the

various labeled sugars were cut from the chromatograms

52

and placed in vials containing 15 m1 of the scintillation
medium used in other parts of this study. After a 24-hour
equilibration period the vial containing the chromatograph
disc was placed in a Packard Tricarb Scintillation Spectro-
meter for determination of radioactivity. Previous work
has shown that counting efficiencies on the order of 85
per cent can be obtained by this technique (Davidson, 1962).

Table 9 shows the results of this analysis. A
period of maximum photosynthesis, June 15, is compared with
September 7, a period of low photosynthetic activity.
Sucrose accounted for 66 per cent of the total radioactivity
recovered on June 15. This was expected as sucrose is
believed to be the principal translocated form of carbo-
hydrate. Much of the sucrose was recovered from the roots
indicating rapid translocation of photosynthates. Smaller
amounts were found in the leaves and stem. Some glucose
and fructose were labeled. Very small amounts of 14C were
found in raffinose and stachyose in the leaves only.
Little 14C was recovered from walnut stems.

On September 7 the sucrose fraction contained only
44 per cent of the total 14C recovered from walnut seed-
lings. Increased amounts of radioactivity were found in
the glucose and fructose fractions. Most of this activity
was found in the leaves probably indicating that most

14

CO fixed on September 7 had remained in the leaves and

2
translocation had slowed. Zimmermann (1968) found little

phloem translocation and increased levels of sucrose in

 

TABLE 9.--Changes in the distribution of

53

14C among the

sugars of leaves, stem, and roots of black
walnut seedlings on two dates during the

first growing season.

 

Percent of total 14C in the
sugar fraction after 24
hours of assimilation

 

 

 

 

 

 

 

 

 

 

 

 

June 15 September 7
Sucrose
Leaves 15.6 16.5
Stem 6.2 3.2
Roots 44.1 24.6
-Glucose
Leaves 9.1 19.2
Stem 0 0.5
Roots 9.6 6.7
Fructose
Leaves 7.6 18.7
Stem 0 0.5
Roots 6.5 6.5
Raffinose
Leaves 0.8 0.9
Stem 0 0
Roots 0 1.6
Stachyose
Leaves 0.5 0.4
Stem. 0 0
Roots 0 0.7
All
Leaves 33.6 55.7
Stem 6.2 4.2
Roots 60.2 40.1

 

1Mean of 3 trees

 

54

the leaves of white ash (Fraxinus americana L.) trees
following leaf senescence. Increased radioactivity was
recovered in the stachyose and raffinose fractions on
September 7, particularly from the roots. Raffinose and
stachyose are common in later phases of the growing season
and in perennial plants and their buildup may be asso-

ciated with cold-hardiness (Axelrod, 1965).

 

CHAPTER IV

SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

Terminal buds are formed by many but not all
deciduous hardwoods. In the preformed shoot type of growth
the foliage leaf primordia and their unextended internodes
constitute the preformed shoot which will be extended dur-
ing the next growth period. Some deciduous species do not
form terminal buds. These species are said to exhibit the
sympodial growth habit. In these species the shoot tip
aborts in summer and the function of the terminal bud is
assumed by the uppermost laterals.

An important contribution of this study was the
comparison of growth patterns and photosynthetic effi-
ciency between these two contrasting types of growth.

Black walnut is typical of many species which exhibit
the preformed shoot growth habit while honeylocust is
characteristic of those with the sympodial growth habit.

Height growth of black walnut was of short duration
and was completed early in the growing season. In contrast
honeylocust seedlings grew continuously to September 1-

Similar trends were found in the dry matter production

55

 

56

of the leaves and stem. However, root growth in both
species continued through the summer to September 21.
The patterns of leaf area accretion and the total leaf
area produced in a single season were remarkably differ-
ent between the two species. Walnut seedlings reached a
maximum leaf area of about 124 cm2 within two months of
germination and then leaf area rapidly declined due to
abscission. Most leaf area accretion in honeylocust
seedlings also occurred within two months of germination,
but some new leaves were added continuously through the
growing season.

Honeylocust seedlings exhibited a remarkable

superiority in CO uptake over black walnut seedlings on

2

a leaf area basis. However, black walnut because of its
larger leaf area had much higher rates of CO2 uptake on
a whole seedling basis. Net photosynthesis increased

gradually during the summer in honeylocust seedlings and
reached a peak incorporation of 15.5 mg CO2 dm_2 hr-l on
August 10 and remained at a high level through September

21 as leaf abscission began. Net photosynthesis in black

walnut reached a peak of 7.8 mg CO2 dm_2 hr.1 on July 27

and then declined sharply to a low level of 1.3 mg CO2

dm-2 hr.1 on September 7. There was considerable varia-

tion in rates of net photosynthesis, dark respiration, and

photorespiration among individual trees of both species.

Net photosynthesis ranged as high as 14 mg CO2 dmm2 hr-1

in black walnut. A.high of 20 mg CO dm-2 hr-l was

2

 

57

recorded for honeylocust. Rates of dark respiration were

2 -1

as high as 10 mg CO dm- hr for black walnut and

2
13 mg CO2 dm-2 hr.l for honeylocust. Photorespiration

ranged as high as 9 mg CO2 dm-2 hr-1 in black walnut and
a high of 22 mg CO2 dm-2 hr.1 was recorded for honeylocust.
Limited data indicated that the light saturation point for
honeylocust was near 7,000 ft-c while that for black walnut
was substantially lower, being approximately 3,000 ft-c.

Rates of photorespiration and the CO compensation points

2
differed greatly during the annual cycle of growth and
were indicative of photosynthetic activity. For both
species the CO2 compensation points were lowest when rates

of net photosynthesis were highest. Later, as photosyn-

thesis activity decreased the compensation points increased.

There were clear differences between the two species
in patterns of metabolism and translocation of photosynthate.
Black walnut seedlings shifted their metabolic activity
from the leaves to the stem and finally to the roots as
the growing season progressed. In the fall practically
all metabolic activity was found in the roots. In contrast
to black walnut patterns of metabolism and translocation in
honeylocust seedlings were less distinct. Zimmermann (1964)
reported that the translocation pattern in continuously
growing shoots is somewhat complicated because movement from
mature leaves is both upward into shoot tips and downward
towards the roots. Thus phloem transport in internodes

between mature leaves is bidirectional in nature. In aging

 

58

cottonwood leaves there is a shifting of the translocation
pattern from upward to bidirectional and finally to a

predominantly downward direction (Larson and Gordon, 1969).

Relatively high rates of metabolic activity continued in
all parts of honeylocust seedlings as leaf abscissiOn began.
The leaves of walnut seedlings labeled early in the growing
season Showed reduced levels of radioactivity in the fall
as leaf abScission began. In these plants the roots
accounted for 65 to 70 per cent of the total radioactivity.
It appears that walnut redistributes many metabolities from
the leaves as leaf abscission begins. Less redistribution
of 14C was observed in honeylocust seedlings as the growing
season ended. High levels of 14C remained in honeylocust
leaves on September 21 as leaf abscission began.

In developing black walnut seedlings sucrose
accounted for most of the radioactivity recovered when
specific sugars were studied on July 27, a period of high
photosynthetic activity. On September 7, a period of low
photosynthetic activity, increased amounts of radioactivity
were found in glucose, fructose, raffinose, and stachyose.

Light saturation points for photosynthesis deter-
mined in this study were based on very limited data. These
results need to be verified and refined. There was sub-
stantial variation in rates of photosynthesis and respira-
tion between individual trees of both species. A closer
examination of the genetic variation in photosynthetic and

respiratory rates is of great interest to genetcists as a

 

59

basis for tree improvement. There was also considerable
variation in rates of photorespiration between individuals

of each species that deserves further study.

The incorporation of 14CO2 into ethanol-insoluble

cellular components by black walnut and honeylocust was

much more rapid than that previously found in conifers. .

These results may be useful in comparing photosynthetic r

efficiency between these two broad groups. Short term

 

labeling studies would be of value in this area. Except
for the limited examination of sugars, the present study

did not attempt to study the seasonal incorporation of

14

CO into specific metabolities. Further study in this

2
area is essential before the deciduous growth habit can be

completely understood and effectively manipulated by man.

 

LIST OF REFERENCES

 

 

LIST OF REFERENCES

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Bey, C. F. and R. E. Phares. 1968. Seasonal growth pattern
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60

 

61

Fayle, D. C. F. 1968. Radial growth in tree roots.
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Heinicke, A. J. and N. F. Childers. 1937. The daily rate
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62

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63

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64

Verduin, J. 1953. A table of photosynthetic rates under
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M. H. Zimmerman (ed.). The formation of wood in
forest trees. Academic Press, New York.

 

 

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65

 

 

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66

TABLE ll.--Variation in dark respiration between individual
seedlings of black walnut and honeylocust on
four dates in the first growing season.

 

 

 

 

Observation Black Walnut
NIIter June 15 July 27 August 17 September 7
---------------- mg CO2 am"2 hr-1----------------
l. 6.2 4.2 7.6 9.2
2. 4.8 5.6 2.7 6.0
3. 5.8 8.1 5.0 5.7
4. 802 3.6 6.9 6'6
5. 6.6 3.3 5.8 10.1
Mean 6.3 6.2 5.6 7.6
Honeylocust

July 13 August 10 September 1 September 21

 

1. 12.9 6.4 12.8 4.6
2. 8.4 6.0 4.9 1
3. 9.4 4.6 6.5 12.6
4. 7.6 10.0 7.7 3.7
5. 8.6 10.7 8.6 2.0
Mean 9.4 7.5 8.1 5.7

 

lInsufficient Data

 

67

 

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68

TABLE 13.--Variation in the CO compensation point between
individual seedlings of black walnut and honey-
locust seedlings on four dates during the first
growing season.

 

 

 

 

Observation Black Walnut
Number June 15 July 27 August 17 September 7
--------------------- ppm----------------------
1. 160 110 1 235
2. 140 1 100 235
3. 130 130 165 1
4. 195 95 170 225
5. 175 135 145 1
Mean 160 118 145 232
Observation Honeylocust
Number July 13 August 10 September 1 September 212
--------------------- ppm----------------------
1, 210 100 180
2. 140 110 l
3. 145 105 160
4. 130 135 140
5. 150 120 145
Mean 155 114 156

 

1Insufficient Data

2Data not obtained because of low rate of net photosynthesis

 

69

 

 

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70

 

 

 

 

 

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