THE EFFECT OF HIGH TEMPERATURE

ON THE FREE AMINO ACIDS OF COMMON PEA

{PISUM SJTIVUM L.)

By

ESMAIL HOSSEINI SHOKRAII

A DISSERTATION PRESENTED TO THE GRADUATE COUNQL OF

THE UNIVERSITY OF FLORIDA

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
April, 1965

ACiti-

culturaI

LIJRARV

UNIVERSITY OF FLORIDA

3 1262 08552 5664

ACKNOWLEDGMENTS

The author wishes to express his best gratitude and
thanks to Dr. David S. Anthony, Chairman of the Committee,
for his kindness and continuous supervision during the
pursuit of this degree, especially during the course of
research and preparation of this dissertation.

The assistance of the members of the committee,
Dr. G. Ray Noggle, Dr. R. H. Biggs, Dr. A. D. Conger and
Dr. G. J. Fritz is also gratefully acknowledged.

Appreciation is also extended to the Eulhright
Commission, the American Friends of the Middle East,
University of Florida and the Department of Botany for
their very generous fellowships and assistantships which
made it possible for the author to extend his graduate
studies at the University of Florida.

XX

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ii

LIST OF TABLES V

LIST OF FIGURES vi

INTRODUCTION 1

LITERATURE REVIEW 9

A. Effects of high temperature on,
microorganisms 9

B. Higher plants 11

MATERIALS AND METHODS 22

A. Plant material and growing conditions 22

B. Sampling 25

C. Preparation of extracts for amino

acid analysis 2^■

D. Purification of extracts by ion-

exchange resin 2^■

E. Qualitative identification of amino

acids in the extracts 25

F. Quantitative determination of soluble

amino acids in pea extracts. ... 26

G. Protein determination 57

H. Chemical treatment techniques .... ^0

SAMPLING AND PHYSIOLOGICAL AGE ^3

RESULTS AND DISCUSSION ^7

I. Growth Characteristics of Plants Under

Optimum and High Temperature Conditions. . ^7

II. Amino Acid Analyses of Peas Under Optimum

and High Temperature Conditions 55

III. Protein Estimation 88

iii.

Page

lY. The Effects of Added Metabolites at High

Temperature 105

SUMMARY AND CONCJLUSIONS 109

LITERATURE CITED 115

BIOGRAPHICAL SKETCH 122

iv

LIST OF TABLES

Table Page

1. Growing Conditions in Growth. Ch.amber 2$

2. Preparation of Buffers 30

3. Percentage Recovery of Amino Acids from tlie

Long and Short Column 33

4. Composition of Metabolite Solutions Sprayed on
Leaves 42

5. Amino Acid Content of Leaves - First Period of
Sampling 70

5. Amino Acid Content of Leaves - Second Period

of Sampling 73

7. Amino Acid Content of Root - Second Period of
Sampling 76

8. Amino Acid Content of Leaves - Third Period of
Sampling 81

9. Amino Acid Content of Leaves - Fourth Period

of Sampling 85

10. Amino Acid Content of Leaves - Fifth Period of
Sampling 89

11. Protein Content of Leaves 102

LIST OF FIGURES
Figure Page

1. Daily heiglit increase of pea plants under two
temperature regimes ^■^

2. Growth. (heighLt increase) of the pea plants
under high and optimum temperature conditions
as compared with the groups of plants which
were depistillated (in order to stop fruit
formation) at the optimum temperature condition 51

3. Daily height increase at optimum condition
(pistil removed) 5^

^, Total fresh weight increase and the weekly
increase of fresh weight at high and optimum
temperature conditions 58

5. Total dry weight increase and the weekly
increase of dry weight at high and optimum
temperature conditions 51

6. Increase in percent dry weight at different
stages of growth at optimum and high
temperature conditions 5^

7. Shoot-root weight ratio versus time 6^

8. Chromatograms comparing amino acid constituents
of leaves of pea plants under optimum and high
temperature conditions 75

9. Chromatograms of amino acid constituents of
roots at optimum and high temperature
conditions 78

10, Chromatograms of amino acid constituents of
leaves at optimum and high temperature 83

11. Chromatograms of amino acid constituents of
leaves at optimum and high temperature 87

VI

Figure Page

12, Chromatograms of amino acid constituents of
leaves at optimum and h.igli temperature,

(Fifth period of sampling) 91

13. Histograms comparing the changes in the amount
of aspartic acid, asparagine, glut amine and
glutamic acid in the leaves of plants grown
under optimum and high temperature during five
successive weeks of growth 9^

1^. Histograms comparing the changes in the amount
of alanine, serine, glycine and threonine in
the leaves of plants grown under optimum and
high temperature during five successive weeks
of growth 96

15. Histograms comparing the changes in the amount
of homoserine, valine, leucine and isoleucine
in the leaves of plants grown under optimum
and high temperature during five successive
weeks of growth 98

15, Histograms comparing the changes in the amount
of -aminobutyric acid, lysine, histidine and
phenylalanine plus tyrosine in the leaves of
plants grown under optimum and high
temperature during five successive weeks of
growth 100

17. Total soluble protein content of leaves of the
plants grown under optimum and high tempera-
ture during five successive weeks of growth. , 10^

18, Total amino acid content of leaves of plants
grown under optimum and high temperature
conditions during five successive weeks of

growth 10^

VI X.

INTRODUCTIOiT

In spite of the large amount of descriptive and
morphological information available concerning the effects
of high temperature on plant growth and development,
especially with the invention of modern climatic control
facilities such as growth chambers and the phytotron (78),
little is known of the biochemical effects of high tempera-
ture on plants. Further research in this area should be
quite interesting and hopefully fruitful from agricultural
and physiological points of viev;. There are many economic
plants that are restricted to certain areas mainly because
of their requirement for a certain temperature range.

Further studies in this field seem to be quite
necessary to develop the fundamental information needed to
establish rational approaches to such problems as treatment
of climatic lesions and the adaptation of different plants
of temperature zones to tropical and sub-tropical areas or
vice versa. Such research would provide basic information
about one of the most important variables of growth in
plants — a variable which has not been thoroughly investi-
gated (28,29).

There is little information concerning the major

factors tliat limit plant growth, and development at high
temperatures, hut in general one can cite the hjrpothesis
that plant growth or stage of development may be altered
through limitation of the velocity of a single reaction.
Thus, limitation of a single or a few necessary critical
substances may cause the changes in growth and development.

According to the present concepts of molecular
biology, the destruction of proteins, DNA or RNA might cause
limitation of growth at high temperature. (High temperature
might more correctly be called supra-optimal temperature,
but is seldom so described in the literature or in the
present paper.) Thermal studies on soluble RNA in vitro
revealed the maximum temperature limit for acceptance of
amino acids is around 75° C. The "melting apart" of the
two strands of DNA at an elevated temperature is also widely
reported, and it is believed that the temperature at which
strand separation of DNA could occur is in the range of 70° C,
to 90° C. (18).

The greatest range of adaptation to temperature is
found in microorganisms, where some species are found that
are able to grow at --4-° C. and others to +70° C. (50).
Several fungi may also grow below 0° C, but their upper
limit of growth is ^0° C. to 50*> C. Of course, duration of
temperature is important since, for a short period of time,
some spores can tolerate temperatures as high as 150** C.

(The temperature limits of growth, and development in
poikilothermic organism are tabulated in the "Handbook of
Biological Data" by Spector (58),)

The highest temperature at which organisms grow on
the earth's surface are found in hot spring areas in the
Yellowstone region of the United States, in Japan, New
Zealand and Iceland (10), There are numerous reports of
algae and bacteria found in these places where the highest
temperature is 73" C Studies by Koffler (33) on life in
hot springs indicate that the thermophilic organisms have
evolved some kind of protein that is much more thermostable
than that of mesophilic species,

Flowering plants appear to have temperature limits .
similar to those of fungi, as they can grow at several
degrees below zero and at temperatures above 50** C. The
temperature limits of desertic plants have been discussed
briefly by Kurtz (35) and the maximum is found in desert
areas .of the United States, Among the members of the cactus
family there are species like Opuntia that grow at tempera-
tures as high as 58** C In Australian deserts, some species
of Atriplex, e.g, A. vesicarium, exhibit a maximum rate of
photosynthesis at ^O" C, to 50° C, Most of these desertic
plants are obligate thermophiles. The lethal high tempera-
ture according to Lorenz (48) varies with: 1) species of
plant; 2) duration and method of ■ application of temperature.

since gradual increase of temperature lias a much, less harmful
effect; 3) age of tissue and organ; ^) time of exposure to
high temperature (day or night); 5) part of plant (root or
shoot), tops of plants are more resistant than roots. This
last mentioned phenomenon may be due to the presence of more
protective tissue aro\ind the stem and leaves rather than to
difference in heat tolerance of protoplasm.

The damage caused by higher than optimum temperature
on flowering plants and lower plants seems to be mediated in
large part by an effect on some chemical reactions within
the plants. However, only in a very few cases has the
biochemistry of the plants been studied in relation to high
temperature as was mentioned before ($6,^2),

Although there is little information on reactions
affected, there are observations of biochemical differences
which have been attributed to high temperature such as:
change of amount of aromatic compounds in flowers and fruits
(especially in the tea plant), decrease of soluble sugar,
formation of pigments, and changes in pigment concentration
of leaves, flowers and fruits. The red and pink colors
often become more pronounced at lower temperatures since
anthocyanin pigment formation actually is responsive to
sugar content which in turn is. affected by temperature.

Growth, as determined by fresh weight and dry weight,
reduces at high temperatures. The cause of this reduction

is not really understood, although several explanations are
offered. Many investigators believe that decrease in growth
of plants at temperatures above optimum is partly due to
limitation of translocation. Temperature affects trans-
location (77). Maximum translocation occurs at about 10** C.
in many plants. Thus, indirectly, root weight might change
with high temperatures as root weight is quite responsive to
rate of translocation. High temperature causes an increase
in respiration greater than an increase in photosynthesis,
and this would cause a decrease of growth mostly due to
reduction in sugar and other reserve materials (4,20,26,4-7).
Nightingale (59) reports that orchard trees are able to
accumulate more carbohydrate at moderate rather than at high
temperatures. In" sugar beets there is an inverse correlation
between the amount of sugar and the temperature of the
environment. Finally, among the other effects of high
temperature that could affect growth are mentioned suscepti-
bility of plants to disease and also the increased phyto-
toxicity of toxic compounds, e.g. 2,4- D is more toxic at
high temperature than at low temperature.

An interesting and complex interaction between plajits
and temperature is diurnal and annual thermoperiodicity
which are very common among the higher plants. In general,
the optimum temperature for night is lower than for day.
Spring annuals die when subjected to a high night temperature

of about 25* C, al-fchough tiiey will grow well at a day
temperature of 25° C.

It has been observed (3^,78,79) that in certain
plants resistance to cold can be increased by growing tbem
under a condition of alternating ligbt and dark in which the
daily rhythm is not a 2-4- hour cycle, but somewhat longer.
On the other hand, the same plant can be made resistant to
high temperature damage by growing it under a daily cycle
of less than 2^ hours.

All these facts suggest that most plants do have a
kind of timing mechanism or a diurnal rhythm which seems to
coincide with a 2^ hour period (8) at optimum temperature.
At low temperature the cycle of diurnal rhythm is slower,
aind there is a failure of synchrony between the plant and
outside environment. This in turn can cause metabolic im-
balance and possible visible disturbances.

In general, the factors that have been suggested as
responsible for the effects of higher than optimum tempera-
ture on plants could be summarized as follows (^2):

Change (decrease) in availability of gases. -In some
microorganisms it has been shown that high temperature has
a direct effect only on the availability and amount of
gases dissolved in the culture medium.

Acceleration of breakdown of enzymes, vitamins and
other me t ab elites. -Again , extensive experiments with

microorganisms in controlled culture media iiave shown that
the requirement of the organism for a growth factor or enzyme
will rapidly increase with elevation of temperature.

Changes in the "balance of interconnected reactions. -
Temperature extremes thus might cause accumulation of some
toxic products, or reduction of necessary factors. In
either case, the regulation of metabolism would change.
Consequently, growth would be affected.

Enzyme inactivati on. -Studies with microorganisms
support the idea that simple inactivati on of a single
thermolabile enzyme could result in a lesion due to higher
than optimum temperature, e.g., the high heat sensitivity
of nicotinamide adenine dinucleotide (UADHo) oxidase has
been demonstrated in microorganisms, A similar effect of
high temperature on enzymes of higher plants could exist.

Inhibition of enzyme formation, -The effect of high
temperature inhibition of the formation of an enzyme can be
due to either inactivation and destruction of RITA, or the
inhibition of activity of the operator gene for enzyme
formation.

Enzyme activation. -Several experiments show that heat
activation of an enzyme might occur, since heat can destroy
a thermolabile inhibitor of an enzyme and thus cause indi-
rectly an activation of an enzyme reaction.

8

In biocliemical studies of tiiglier than optimum tempera-
ture it is important to select a plant that has the following
qualifications :

It should be sensitive to high temperature.
It must have a rather short life span to make large
numbers of replicates possible in a reasonable time.
It should be rather small in size to be carried and
handled easily in controlled conditions.
It- should be able to grow well under controlled
conditions and also in artificial media.
There are other important points to consider in addition to
choice of plant material. The method of application of
high temperature must be considered. Is high temperature
going to be given only at the beginning of germination or
during matiu?ity? Another question to consider is the
duration and intensity of the high temperature stress.

LITER/.TURE REVIEW

A. Effects of high, temperature on micro orp;anisms

The largest body of work on the hiochemical effects
of temperature considered to be higher than optimum has been
done using microorganisms.

It has been shown in a number of microorganisms that
the reduction in growth rate which occurs at higher than
optimum temperatures could be prevented or maximum growth
could be restored by supplying a specific chemical (meta-
bolite) in the growth medium. If the temperature was much
higher than optimum, addition of that specific metabolite
had to be greatly increased or a combination of two or
three different metabolites was required to restore the
growth of the microorganism to or toward normal (^2),

If the temperature was dropped back from supra-
optimum to optimum, maximum growth was restored without the
further addition of any of the metabolites.

The so-called temperature sensitive mutants of
Neurospora crassa and Escherichia coli have been extensively
studied (2,51,52,57). It has been shown that ITeurospora
crassa can grow quite well at temperatures up to 35° C. to
40*» C, however, there is a mutant of Neurospora which grows

9

10

well at or below 25° C. Its groxirth. rate decreases markedly
with further increase of temperature until at 28° C. growth.

stops completely. Growth can he restored at 28° C. in this

-4-
microorganism hy addition of 2,5 x 10 gm of riboflavin to

each liter of culture solution. There seems to be a perfect

riboflavin-temperature-growth response interaction (57).

Other mutants have been found that can be protected
against the development of high temperature lesions by
simultaneous supplementation with adenine, pyrimidine and
methionine in their media (55). A temperature sensitive
uracil-requiring mutant has also been isolated (25) where
there is a complete block for uracil synthesis at high
temperature. Addition of uracil to the growth medium of
this organism causes normal growth even at temperatures
higher than optimum. Temperature-dependent tyrosine re-
quirement in Neurospora also has been reported (2^) and was
interpreted as a result of lower thermostability of the
mutant enzyme. Addition of typrosine prevented the defect.

In E, coli, panthothenate-requiring temperature-
sensitive mutants have been isolated. In this microorganism,
the enzyme responsible for the condensation of 3-alanine and
pantoic acid to form pantothenic acid is apparently more heat
labile than other enzymes. Thus, the addition of pantho-
thenic acid to cultures of this temperature-sensitive mutant
allows them to grow at elevated temperatures in a manner

11

identical with, normal strains. Among the organic compounds
wtLich. have been found to reduce high temperature damage in
one or more of a large group of microorganisms and fungi by
several investigators (3,9,1^,61,65) one may mention:
methionine, glutamic acid, thiamin, hiotin, a number of
other amino acids, and other members of the vitamin B complex.

In some instances there is no inhibition of growth
because of high temperature, but, on the other hand, Eertman
(19) has seen the induction of impairment of some bio-
chemical processes. For instance, in Pasteurella pestis at
37" G. (optimum temperature is 27° C.) the formation of
pesticin stops. At high temperature, addition of amino
acids such as leucine and/or isoleucine relieves the high
temperature effect, and pesticin can be produced the same
as under optimum temperature.

B. Higher plants

Some of the earliest v/ork on the biochemical effects
of abnormal temperature was by James Bonner (7) who proposed
the term "climatic lesion" for the first time. Kis concept
was that at higher or lower than optimum temperature one or
several biochemical events cannot take place simply because
the enzymes responsible for those biochemical syntheses are
more thermolabile than other enzymes. Therefore, after a
short time there would be a shortage of those particular
metabolites. Bonner proposed that the shortage of such

12

metabolites is tlie cause of tlie "climatic lesion" and th.at
the lesion might be cured by external supplementation of the
particular compound or compounds which were absent. In other
words, under unfavorable temperature conditions, green plants
become heterotrophic for one or more specific metabolites.
Bonner tested his theories by experiments with Cosmor (6).
However, his published data are all concerned with the
effects of low temperature. He found the addition of thiamin
in the nutrition medium of the Cosmos caused an increase of
growth as measured by fresh weight at temperatures lower
than optimum. His results were most significant where dry
weight was as low as 5 per cent of that under optimum
conditions. Under these circumstances, thiamin caused a
40 per cent increase in dry weight. At a temperature where
dry weight was half of maximum, the increase due to thiamin
addition was 20 per cent. Addition of thiamin at the optimum
temperature had no stimulatory effect on grov/th. The con-
clusion was made that at low temperature, inhibition of
growth of the Cosmos plant can be overcome by an external
source of thiamin. This is the first recorded successful
chemical prevention of a climatic lesion. Later work on
prevention of low temperature damage on eggplant ( s ol anum
melongens) (32) has shown a low temperature (14° C. night;
20* C. day) inhibition of growth can be overcome with a
mixture of ribosides sprayed on the leaves. While at optimum

13

temperature (23* C. nigat; 30° C. day) ribosides have little
or no effect. With tomato plants it has been shown that
soaking of sseds in a solution of nicotinic acid caused
significant increase in tolerance of seedlings to low
temperature inhibition of growth (32),

The alleviation of a high temperature lesion appears
to have been accomplished for the first time by Galston and
Hand (15). They showed that, in etoilated pea epicotyl
sections and also in excised etoilated leaves, the addition
of adenine to the growth medium decreased the high tempera-
ture inhibition of growth. Adenine stimulation of grov/th
at optimum tempera"cure was only half as much at high tempera-
ture (35° C). They concluded that inhibition of growth at
high temperature in the pea tissue was partly due to adenine
destruction or inhibition of its synthesis, S'urther work
by Galston (17) on growth of intact pea plants (var, Alaska)
in the Earhart Phytotron Laboratory did not show a complete
reversal of high temperature-induced decrease of growth by
adenine supplementation where plants were kept at high night
temperature and optimum day temperature. Possibly the
failure of successful cure of high temperature lesions with
adenine in this case was due to the use of high night tempera-
ture as the experimental condition, whereas later it was
shown that the pea is more sensitive to high day temperatures
(15). Later work of Lockhart (^6) with the same variety of

1^

pea indicated tliat adenine had no significant effect on stem
elongation and growth or on onset of maturity of the whole
pea plant kept at 30° C. However, it should be noted that
the plants in this study were kept at a constant high
temperature (day and night).

Despite the inconsistencies with respect to adenine
supplementation cited above, it is possible that adenine may
have some importance; because the adenine level in high
temperature-resistant pea plants increased with high tempera-
ture conditions while in a temperature-sensitive strain,
adenine decreased (22),

Additional evidence that adenine deficiency may be a
cause of high temperature injury in plants was obtained by
McCune (5^) with the common duckweed (Lemna minor). He
showed that this plant could be protected against high
temperature damage by the addition of adenine in the form
of adenosine to its culture medium. Guanosine also was
found to be effective.

Additional work, showing climatic lesions and their
chemical treatment in peas, was reporued by Ketellapper and
Bonner (31). They found that mixtures of essential metabo-
lites such as a vitamin B mixture, or a riboside mixture,
psirtially or completely prevented the reduction of growth
at high temperatures. Sucrose and vitamin C also were found
to be effective. Ketellapper (32) found that inhibition of

15

growth at a temperature a few degrees iiigher than optimum
could "be completely stopped "by a spray of sucrose on leaves
while for conditions several degrees higher than optimum
there was no significant effect for sucrose. This indicated
that sucrose and other metabolite effects are temperature
specific. It seems probable that at least part of the high
temperature response is mediated through the chemical
machinery of the plant, although the experimental evidence
is often confusing.

The study and interpretation of climatic lesions due
to high temperature in plants is complicated by several
factors. For example, within a given genus and species
there may be strains or varieties showing both quantitative
and qualitative differences in response to temperatures.
Langridge (59,^0), working with a wide range of strains of
Arabidopsis thai i ana, showed that the inhibition of growth
at elevated temperatures varied markedly from strain to
strain. Further, he showed that the high temperature
inhibition cf growth was prevented in certain Arabidopsis
strains by the addition of biotin, in another strain by
cytidine, in another by choline, while in still other strains
he was unable to prevent the high temperature effect by
chemical means. Ketellapper (32) also warned that the
effective substances for preventing high temperature effects
may vary from species to species, and even from variety to
variety within a species.

16

Interpretation of negative results in experiments
involving attempted cure or reversal of climatic lesions
by chemical means is difficult since one may not "be sure
whether the added compound actually reached the affected
tissue (32).

Another surprising complication even within a given
genus, species and variety in the study of high temperature
effects is the variation due to seed source. In research
with peas (var, Unica) , Highkin (21,22) found that the
conditions iinder which the seeds had been grown altered the
effects of subsequent temperature treatment. He found that
constant day and night temperatures maintained over several
generations successively reduced the vigor of progeny in a
cumulative sense. Further, seeds from plants that had been
maintained for a few generations at constant temperature did
not produce plants of normal vigor when planted at optimal
fluctuating temperatures until the second or third generation
under the optimum conditions. Subsequently, Ketellapper (52),
with the same pea variety, showed that the effect of high
temperature on growth is counteracted either by sucrose or
by a vitamin B mixture and a riboside mixture, depending on
seed source (i.e. temperature conditions under which seed
had been grown). These results suggested that the phenotype
is a combination of its genetic heritage, its present environ-
ment, and the environmental conditions under which its
parent grew (23,^1).

17

Langridge (^2) believes a number of these growth
stimulations by chemicals reported above for higher plants
is of poor reproducibility and of doubtful statistical
significance , thus is in contrast to the high temperature
lesions that have been observed among microorganisms. In
the work with Arabidopsis thai i ana already described,
Langridge and Griff ing (^0) did obtain clearcut, significant
responses to chemical additions at high temperatures in
aseptic culture.

Another interesting aspect of high temperature effects
on plants from the biochemical point of view is the effect
of high temperature on the response to auxin and gibberellin.
Galston (16) and Lockhaxt (^6) studied responses of plants
to hormones under high temperature conditions. A silkless
mutant variety of corn at 20** C. day and 1^° C. night
temperature showed a 50 per cent elongation response to
applied indole acetic acid (lAA), whereas a normal variety
gave no response at this temperature regime. However, at a
higher temperature, 25° C. day and 20° C. night, the silkless
variety showed no response to lAA while the normal variety
gave a 16 per cent elongation response. Lockhart observed
that applied gibberellin on leaves of Alaska peas grovm at
high temperature had a very significant effect on delaying
the high temperature acceleration of senescence which
usually accompanies the onset of maturity. Later, Lang
(37,38) showed that failure of Hyocyamus in flower formation

18

at high temperature is apparently because of gihberellin
deficiency rather than anything else since Hyocyamus needs
a large amount of gibherellin to elongate the flov/er stalk.
It was sho\\ni that gibberellin sprayed on leaves of Hyocyamus
and some other plants can replace the need of low night
temperature which otherwise was necessary for flower for-
mation. Among the other works that can be considered
related to this problem the following papers need to be
mentioned. Yarwood (80,81) has studied acquired tolerance
of leaves to heat (acquired heat tolerance of microorganism
has been known for some time (62)) and also has observed
translocation of heat injury from one leaf to another.
When leaves of a number of a species of plants were pre-
treated at a temperature of 50° C. , they subsequently (12,
1^ hours later) tolerated a temperature of 55° C. up to
three times as long as did the control leaves which were not
previously heated. Also, a temperature of 65° C. applied
unilaterally to plsoits killed the heated leaves, but at the
same time the leaves in the other unheated side or branches
became injured without any possible direct heat transfer (81),
The above mentioned reports constitute a review of the avail-
able literature dealing with the problem of metabolic or
biochemical effects of high temperature on plants. From the
scattered but divergent results already in the literature,
it seems likely that there is not a single general chemical

19

explanaticn for heat injury, but rather there may be many
explanations in many plants. Obviously, much more informa-
tion is needed before any general or specific theory of
heat injury, backed by solid evidence, can be elaborated.

One omission in studies of high temperature injury
in plants became apparent from the literature review, To
our knowledge, with the partial exception of one paper in
the Russian literature (60), there is no published evidence
that anyone has attempted to make quantitative biochemical
analyses comparing the composition of plants grown at high
temperature with that of the same plant grown at optimum
temperatures. That is, there is no direct quantitative
measure of the biochemical effects of high temperature on
higher plants.

Thus, since technological advancement has recently
been made in isolating and identifying most of the bio-
chemical constituents of plants, it seemed reasonable that
an organized investigation of the quantitative effects of
heat on the biochemistry of plants might advance our under-
standing more rapidly than spraying or adding to the plant
in "shotgun" fashion a variety of metabolites with the hope
that this sprayed mixture contained the substance which was
deficient under the high temperature condition. In such
experiments, there must be the further hope that the critical
substance (or substances) can be taken up and translocated
to the site of need.

20

However, it was very difficult to decide whicli groups
of compounds should "be studied in preference to other groups.
The final decision was to study amino acids and proteins.
This decision was made for the following reasons.

Davern (11) extended the work of Ketellapper in a
study of nhe effects of amino acids on the growth and
development of clover (Trifolium suhterraneum) , a cold
temperature requiring plant. It has been shown that spray
of casein hydrohysate on the plant under high temperature
has quite a significant effect on the growth, while the
effect on plants under optimum conditions is not significant.
Davern did not study the effect of each single amino acid
but he used three groups of amino acids; protein amino acids,
non-protein amino acid and ring-structure amino acids. The
first and second groups were found to be much more effective
(especially the first group).

Petinov (60), in a research with leaf tissue culture,
has shown that high temperature increased the amount of free
amino acids. Ee was not looking for a lesion, and his
analytical technique was only roughly quantitative (one-
dimensional paper chromatography) .

Earlier work of F. C. Stev/ard (70) has shown the
effects of various nutritional conditions as well as light
and dark period and high night temperature on the free amino
acids of Mentha piperita (mint plant). Ee has shown that

21

amino acids change drastically with changes in the environ-
mental and nutritional variables.

Research on a large group of microorganisms and lower
plants have shown that high temperature lesion and inhibition
of growth can be prevented or reduced by a number of amino
acids (5,27,66).

Levitt (^^,^5) suggested a possible correlation be-
tween frost hardiness and high temperature hardiness. He
postulated that both are concerned with protein structure.

In view of the available evidence, it seemed
reasonable, as a first quantitative approach, to examine
the free amino acids and proteins of plants at optimum and
supra-optimum temperature, hereafter usually referred to as
high temperature conditions.

MATERIALS AM) METHODS

A. Plant material and grov/inp; cor.ditions

Pea seeds (Pi sum sativum variety Greater Progress and
Vanda)* were hand-graded for uniformity, soaked five hours
in tap water at room temperature and then sown in plastic
flats (15" X 12" X 6.5"). The planting medium was a mixture
of equal volumes of washed sand and vermiculite. The flats
were watered (2,000 ml) and were transferred to growth
chambers (Percival Growth Chamber Model E-57). Every day
prior to germination of seeds each flat received 500 ml of
distilled water. After the seeds germinated, extra seedlings
and abnormally small or large seedlings were discarded in
order to have a uniform group of seedlings. All flats were
thinned to 12 seedlings with approximately equal spacing.
Ten days after planting, plants were watered with 500 ml of
Hoagland's solution per flat every other day. The plants
under high temperature conditions were given additional dis-
tilled water occasionally as needed to eliminate water
stress. The temperature of nutrient solution v/as not ad-
justed beforehand to the temperature of growing conditions
(usually it was less, especially at the high temperature
condition). The light source was a combination of fluorescent

*Purchased from Joseph Harris Co., Inc., Moreton Farm,
Rochester 11, New York.

22

lei-nperature
condition

23

TABLE 1
Growing Conditions in Growth Chamber

Photo temparature Nyctotemperature

C16 hr.) (8 hr.) LiQ:ht intensity

Optimum

High temperature

23 + 1
30 -!- 1

17 j; 1
23 -:- 1

1200 ± 200 f .c.
1200 + 200 f .c.

(cool white) and incandescent bulbs (Sylvania lamps). For
each, experimental condition te&'.ed 60 plants were used. Ten
plants were selected at random after the first week oi
growth for measurement of daily height increase.

B. Sam-pling

Six plants were selected randomly for each extract.
First, they were washed free of rooting medium and weighed
immediately. The number of leaves, height of shoot, total
and shoot fresh v/eight, root v;eight, shoot-root ratio, dry
weight and later number of buds^ flowers and fruits were
measured. Alcoholic extracts were prepared from leaves,
roots, and stem. The plant tissues v;ere cut into small
(0,5 cm) pieces and dropped into about 150 ml of 80 per cent
ethyl alcohol at room temperature. The alcohol v/as allov;ed
to remain in contact v;ith the plant tissue during storage at
0* C, prior to analysis, (This storage period was from one
to three months,) The time of original sampling was the

2A-

sane (8:.00 P.K.) in tlie "trwo temperature conditions and
usually th.e tine of sampling was 12 hours after watering.
Samples were taken once a v;eek.

C. Preng^ration of extracts for amino acid analysis

Th.e stored alcoholic extracts and the tissue residue
were homogenized thoroughly in a glass homogenizer and
vacuum filtered through Whatman #1 filter paper, xhe fil-
trate was concentrated in a Rinco flash evaporator under
vacuum at room temperature. The concentrated (now aqueous)
extract was centrifuged 15 minutes at maximum speed in a
clinical centrifuge (approximately lOOOxg). The precipitate
was discarded. An equal volume of chloroform was added to
the supernatant with vigorous shaking to remove fats and
pigments. The chloroform layer was discarded. The defatted
aqueous extract prepared in this v:ay vxas further concentrated
to 1 nl/1 gm wet weight of original plant sample.

D. Purification of extracts b:;" ion-exchan?2;e resin

An ion-exchange column approximately 15 cm in length,
containing 10 gms Dov/ex 50., was washed several times with
distilled v/ater, the Dov/ex 50 v/as regenerated hy passing
80 ml 3 IT EGl through the column. Since the volume of the
15 cm resin column v;as 15 nl, the 80 ml HCl rinse v/as about
six columnL volumes (67). The ECl vras washed out by passing
200 or more ml of v;ater through the column until the column
was chloride free (checked \;ith AgNO^). The plant extracts

25

were passed tlirough. th.e washed coluua in ice cold condition
(column was placed in ice and v;,-.ter) at a maximum flow rate
of ten drops/minute. At tliis state, th.e amino acids v'ere
tiglitly held near the top of the column. The column was
rinsed with 100 ml distilled water to v;ash out the last
traces of anionic and neutral materials. The amino acids
were then eluted with Iffl^OE (72). The first elution was
with 0.15 N KH^OH (50 ml/ml of resin bed) and then with 2 N
NH^OH (25 ml/ml of resin). Elution was also carried out in
the cold (column was placed in ice and water). The NH^OH
elute was collected and the column was washed with distilled
water until the eluate was ammonia free (phenolphthalein
test). The combined eluates were collected and concentrated
undei- vacuum to an appropriate volume (usually 1 ml /I gm
fresh tissue). The purified amino acids obtained in this
way v;ere adjusted to pE 2.25 and were stored in a freezer
(-15" C). The resin column was regenerated for the next
run by washing with 5 '^ ECl (6 ml/ECl/1 ml resin bed).

E. Qualitative identification cf amino acids in the extracts

Two-dimensional paper chromatography was used to
separate and identify the amino acids. A sample of 100 ul
of plant extract prepared as described in section D was
placed as a single small spot in the corner (2.5 cm from
each of the two edges) of a pO x ^0 cm piece of Whatman #3
paper. The spotted paper was developed in a chromatographic

26

chamber, which, was maintained at approximately 25° C. , in an
air-conditioned rcum. The solvent used were: butanol,
acetic acid and H2O for the first phase and phenol-water,
for the second phase (71,75). Preparation of solvents: 1)
125 ml butanol plus 125 ml water plus 30 ml acetic acid
(lower phase discarded) upper phase was used for paper
chromatography, I50 ml of solvent were used in one trough
for two papers; 2) 160 ml of pure phenol and ^0 ml of dis-
tilled water plus 0.5 nil of ammonia. Equilibration time of
papers and solvents in chromatographic chamber was alv;ays
two hours. A 1 per cent solution of ninhydrin in acetone
was used to stain amino acid spots. Paper chromatograms of
known amino acids were first prepared, and then they were
used to compare with those of the plant extract chromatogram
to identify the kind and number of the soluble amino acids
present in the extract. The colored spots were eluted with
10 ml 50 per cent ETOH and the optical density was deter-
mined spectrophotometrically (72,73j7^).

F. Quantitative determination of soluble amino acids in pea
extracts

After repeated tests, we rejected paper chromatography
as a quantitative method for amino acids because the method
did not yield consistent results. The equipment and tech-
nique available to us allowed only a qua!l,itative or rough
quantitative measure of the amino acids.

27

For quantitative separation and measurement of the
amino acids in our extracts, we used an adaptation of tb.e
ion-exchange separation method of Moore, Spademan and Stein
(58).

1. Preparation of ion-exchange columns, -The acidic
and neutral amino acids were separated in a column of cation
exchange resin Aminex-MS IR-120 Fraction D, particle size
56+9 u.* The resin bed was 150 cm long. The basic amino
acids were separated on another column of Aminex-MS,
Fraction C, particle size 40 + 7 u. * The resin bed was 15 cm
long. The temperature in both columns was kept constant by
the presence of a water jacket around the columns through
which water of the desired temperature was circulated.
Elution was accomplished by passing solutions of sodium
citrate buffer of appropriate pH values through the columns.

For separation of the acid and neutral amino acids,
a thick walled chromatographic column (obtained from Sci-
entific Glass Apparatus Company) 0.9 cm inside diameter and
165 cm in length was used. In the bottom of the column was
a sintered plate to retain the resin. The resin was washed
with four times its volume of distlled water, then washed
twice with 0.2 IT pH 3.25 citrate buffer. The resin was kept
in slurry form in the buffer until used. Packing of the long

Piirchased from BIO-Rad Laboratories, Richmond,
California,

28

coluain was done in. five sections. Before the first section
of resin was poured the outlet of the colui^n v;as closed.
(Then a slurry containing one-fifth of the total resin in
buffer was added to the column. The amount used in this, and
in four subsequent additions > was 19.6 gm of resin in 60 nl
of buffer. After the slurry had been added, the column
•outlet v;as opened, and after 1 cm of resin bed was settled
under gravity flow, then air pressure (5.8 psi = 50 cm
mercury) was applied at the top of the column. After the
resin settled to constant height, the supernatant extra
buffer v:as withdrav;n and the next section was poured on top
of the firm surface of the first section, The procedure was
then repeated for each additional section until all five
sections had 'oeer^ added and packed. The final height of
resin must be several centimeters more than 150 cm to allow
for further packing of resin during operation. (It v;as
161 cm in the beginning.) An extension tube was attached to
the top of the chromatographic column by means of rubber
tubing, and this in turn v;as connected to a separatory funnel
containing buffer. A pinch clamp was installed below the
funnel. Before the column was used it v/as treated v;ith 100
ml of 0.2 N NaOH and then was equilibrated with buffer of
pH 5.25. The rate of flow was 18 ml/hr. This flow rate was
maintained by applying an air pressure of 5.2 psi to the top
of the buffer reservoir (separatory funnel), during column

29

operation (temperature during operation of long column was

For separation of tlie basic amino acids, a slaort
column (15 cm) was prepared with. 12 gm of Aminex-KS Resin
(Fraction C, size of particle 4-0 + 7 u) , The resin was
washed carefully several times with distilled water and then
three times with citrate "buffer of pH 4.25. The column was
packed under five pounds pressure in two sections, in a
manner similar to the packing of the long column. During
operation, the column was usually under two pounds of air
pressure at 30° C, and it had a flow rate of 22 ml/hr.
This column was used only for the basic amino acids, and it
took only nine hours for one extract to be analyzed. Citrate
buffers of different pH were prepared as shown in Table 2.

Thiodiglycol (TG) vras added to the buffers before
use. Brij 35 detergent v;as used only once every four to
five v;eeks since it caused problems in work with the drop
counter. (Brij 35 reduces surface tension and thus the
volume of drops change.) It was necessary to wash the column
after Brij 35 addition for two to three days to eliminate
Brij 35 effect. In all the citrate buffers, phenol protected
the buffer against microbiological activity. A final adjust-
ment of pH of the buffers was necessary, usually 1 ml of 50
per cent IfaOH or 2 ml of concentrated ECl caused a change of
about 0.01 unit in pH value of the buffers (1 liter).

30

TABLE 2
Preparation of Buffers

Na

Citric

NaOH

HCl

Water to a .

Cone.

acid

97 %

(cone.)

final Vol.

Phenol

TG*

Brij"*"

pH

(normal)

m

-m

ml

of (::;er)

^m

ml

35 ml

2.25

0.20

105

42

80

5

5

3.25

0.20

840

330

426

40

40

10

5

4.25

0.20

840

330

118

40

40

10

5

5.28

0.35

491

288

136

20

20

5

4.26

0.38

532

312

307

20

20

5

*TG (Thiodiglyeol) .

"^rij 35 detergent solution--prepared by dissolving 50 gm of Brij 35 in
150 ml hot H2O.

31

To prevent bublDle forciation wh-en th.e temperature was
raised to 50° G, in the column, it was necessary to boil tlie
buffer. (Volume of buffer should be checked after boiling,
in order to keep Na concentration constant.) Hot buffer was
transferred to the separatory funnel (buffer reservoir) via
a long-necked funnel to introduce the boiled buffer below
the level of a paraffin oil layer constantly maintained in
the buffer reservoir. (There must always be a layer of oil
over the buffer to prevent air mixture with buffer.) Addi-
tional buffer for immediate use was kept under a layer of
paraffin oil. The buffer reservoir was connected to the
top of the column via two male and female ball Joints (a
small amount of vacuum grease was alviays used to make tight
connection).

2. Operation of the long column (for neutral and
basic amino acids). -The load of the column should not be
very much more than the amount that v/as used for paper
chromatography, usually about 1.0 umole of each amino acid
was satisfactory. A sample of the purified plant extract
was applied on top of the resin bed with a bent tip pipette
(that is, it v;as carefully run down the glass wall of the
column in order not to disturb the surface of the resin bed).
The temperature of the column was brought to 50° C. before
the addition of sample, then $.2 pounds pressure was applied
until the top surface of the extract layer was brought to

52

the resin surface in tlie column. Then, 1 ml of buffer at
pH 2.25 was added and after this, buffer also was brought
level with the top of the resin bed. Then the column was
connected to the separatory funnel containing pH 3.25 buffer.
Air pressure of 5.25 psi v:as applied. Effluent was collected
in 1 ml fractions v/ith a fraction collector (Research
Specialties Co., Mod, 1205). After 260 tubes v/ere collected,
buffer at pH 4.25 (0.2 IT Na) was substituted for the buffer
at pH 5.25 to elute the neutral amino acids. Usually, in
the long column the time for a single extract analysis was
about 24- hours (up to the isoleucine and leucine peak).

To determine the location of different amino acids
in the effluent, a series of known amino acids was tried.
After the location of each amino acid v;as knox'jn, several
tests on synthetic mixtures were made to make sure of repro-
ductibility and also the percentages of recovery of amino
acids from the column. The order of appearance and degree
of separation of the peaks was completely reproducible. Re-
covery percentages for each amino acid were determined as
shown in Table $. The column must be renovated after each
run, since otherwise all basic amino acids vri.ll be retained
in the column. Renovation is accomplished simply by passing
100 ml of 0.2 N HaOH through the column. Subsequently, the
column must be washed thoroughly with the appropriate buffer
to become readjusted before being reused. There is no need

53

TABLE 3
Percentage Recovery of Amino Acids from the Long and Short Column

Per Cent

1. Aspartic acid 105

2 . Alanine 95

3. Arginine 89

4. Glutamic acid 92

5. Glycine 79

6. Hiotidine 92

7. Isoleucine 92

8. Leucine 93

9. Lysine 90

10. Proline 86

11. Phenylalanine 98

12. Serine 103

13. Homoserine 95

14. Threonine 102

15. Tyrosine 98

16. Valine 98

5^

for pressure dxiring renovation of the column and readjustment,
It is good to add 1 ml Brij 35 solution to th.e 100 ml NaOH
to help clean the column. (Hov/ever, this does not need to
he done more than once a month.)

3. Operation of short column (for basic amino acids).-
The general procedure is the saune as that with the long
column, but column loading with amino acids should be less
than with the long column. Air pressure of two pounds is
enough to produce a 25 ml/hr flow rate. The temperature
used was 32 +1 C. Elution was with citrate buffers at pH
^.25 and 5.28 (.35 1^ Na) (100 tube wii^h pH ^.25 then pH
changed to 5.28). The short column also has excellent repro-
ductibility, and recovery is more than 95 per cent for all
amino acids.

^. Colorinetric analysis for the amino acids (53).-
The reagents necessary for color development are prepared
as follows:, 1) stock NaCN 0.01 M (^9 lag per cent); 2)
acetate buffer 3 N pH 5.3 270 gm ITa acetate (NaCH^COO, 3 H2O)
plus 50 ml concentrated glacial acetic acid and 200 ml dis-
tilled water brought to 750 ml with distilled water. This
buffer is very concentrated (3 j^) aiid does not need any
microbial protecting agent; 3) acetate cyanide, 2 ml of
solution one and 98 ml acetate buffer; A-) ninhydrin solution,
3 per cent ninhydrin in methyl cellosolye (ethylene glycol
monomethyl ether). Methyl cellosolve was checked each time

55

"before use to make sure it was very low in peroxides, To
2 ml of metliyl cellosolve in a test tube, 1 ml A- per cent KI
was added, if it was colorless or a very light straw color,
the methyl cellosolve v/as regarded as satisfactory, other-
wise it needed to be distilled; 5) diluent: 50 per cent
isopropyl or ethyl alcohol in HoO.

Test tubes collected in the fraction collector were
tested for amino acids by addition of 0.5 nl acetate-cyanide
(reagent 5) and 0.5 ml ninhydrin (reagent ^) to each -cube.
The contents of the tubes were thoroughly mixed by shaking
and swirling. The tubes then were placed in boiling water
bath (in a rack of 50 tubes) and were allowed to remain
there for 15 minutes. The heat supply to the water bath
was such that boiling started again shortly after the rack
of tubes was placed in the water bath. After 15 minutes,
the tubes were taken out of the water bath, and the contents
of each were diluted immediately with 5 ml of 50 per cent
EtOH (at room temperature). The tubes were then shaken to
mix the solutions. After half an hour, allowed for color
development, the optical density (O.D.) was read at 570 mu
in a Spectronic 20 colorimeter for all amino acids. For
proline, the tubes were also read at ^^0 mu. All readings
were against a blank of 50 per cent alcohol. An additional
5 or 10 ml of diluent were added to tubes with color in-
tensity above 0.80 O.D. In such cases the resulting O.D.

36

values were multiplied ^j 1*7 (,7 ^ + ^ nil/7 ml) or 2,^
(7 ail + 10 ml/7 ml) for correct amino acid concentration.
The reagents should not be mixed together unless they are
going to he used immediately, since the resultant solution
is unstable and is useless after tv;o hours. The reaction
mixture taken from hot v/ater was immediately diluted because
pausing for cooling at this step before addition cf diluent
alcohol produced high background values. The stream of
diluent was always directed at the center of the test tube,
to introduce a maximum of air. The diluted reaction mixture
should be allov/ed to cool to room temperature before reading
in the Spectronic 20. The color produced is quite stable.
After 12 hours there v/as only a 10 per cent decrease in the
color intensities. A standard solution of leucine was always
tested v;ith each run to make sure of the efficiency of the
reagents.

Color intensities of individual amino acids were
plotted against tube number. The area enclosed by the re-
sulting curves v;as a direct measure of the total amount of
the amino acid.

Test tubes for this work must be vrell matched for
light transmission. However, commercially matched tubes
were not available so ordinary tubes were examined, and a
large matched set was collected.

37

Washing of test tubes is important. Used tubes were
first rinsed with, hot water and then were washed with Fisher
Sparkleen detergent in hot water. They v/ere again rinsed in
hot water and finally v/ere rinsed two tines in distilled H2O
and were dried in an oven at 110° C. Occasionally the tubes
were also washed in a dilute (0.2 IT) solution of hydrochloric
acid and were then rinsed in distilled water.

G. Protein determination

For the measurement of total soluble protein, the
handling of the plant material for sampling was the same as
for the amino acids. Samples for protein determination were
prepared in the following steps:

1. A weighed amount of leaves (1 to 5 S'^)' were rinsed
with cold distilled water for a short time.

2. These leaves were homogenized in ice cold condition
with a hand tissue homogenizer (in 25 ml distilled
EoO/gm leaves) for five minutes.

5. The vjell homogenized tissue v;as quickly passed

through cheesecloth (ice cold condition). Filtrate
was brought to a measured volume at about 25 ml/gm
tissue.

^. Equal amounts of ice cold 5 per cent trichloroacetic
acid were added to the filtrate in number 3 above,
with shaking, to obtain complete precipitation of
the protein.

38

5. Tlie sample was centrifuged for ten minutes at
1000-^G.

6. Th.e precipitate was promptly washed with, acetone to
remove chlorophyll and other pigments (20 ml acetone
per gm of tissue),

7. After another ten minutes centrifugation at 1000 G,
the supernatant pigment was removed and saved for
total chlorophyll determination,

8. The precipitate, which was mainly protein, was dis-
solved in 0,91 N NaOH (40 ml NaOH/per 1 gm tissue)
for protein estimation by the phenol reagent (^9).

9. A measured volume of the protein solution (usually
0.25 to 0,5 ml) vxas placed in a graduated 10 ml test
tube, and 5 ml of a mixture of the following reagent
was added to the tube :

Reagent A - 100 ml 2 per cent UaCO^ in 1 IT ITaOH
Reagent B - 1 ml 4- per cent ITa tartrate in H2O
Reagent C - 1 ml 2 per cent CuSO^, 5 £20 in H2O.
(The three reagents must be mixed together shortly
before use,) After addition of the reagent the tube
was shaken vigorously and v.-as left at room temperature
for ten minutes,
10, A volume of .5 ml of Phenol reagent (Folin-Ciocalteu)
was added to the sample tube; the tube was shaken
well and was allov:ed to stand 50 minutes at room
temperature.

39

11. Preparation oi standard curve:

A standard curve was prepared using a solution of
bovine serum albumin (amounts ranged from 50 ugm
through 500 ugm). 'The standard curve obtained did
not follow Beer's law for high concentrations, so
the sample must be low in concentration in order to
get an accurate reading. It is very important to be
careful not to denature protein during handling;
dilute bovine solution denatures usually very fast.
The colored solutions obtained were read in a
Beckman D.U. spectrophotometer or Spectronic 20 at
wavelength of 750 mu. The values presented are
usually an average of two or three determinations.
For determination of total protein we used the method
of Lowry (50). This .method is based on the final color
formation due to both the Biuret reaction and the Folin
reagent (reduction of the phosphomolyhdic-phosphotungstic
reagent by the tyrosine and tryptophan in the treated pro-
tein). There are two disadvantages to this method: 1) the
amount of color yield varies with different proteins. There-
fore, it is good only for comparison of the quantity of two
proteins of the same nature; 2) the color is not strictly
proportional to concentration since for high concentration
it does not completely obey Beer's law; thus dilute solutions
of protein should be used. Since the color intensity per
unit of protein varies with different proteins, there should

^0

"be a correction factor to calculate tlie absolute amount of
tlie protein in comparison to a standard such, as bovine serum
albumin. This is \!h.en the absolute quantity of a protein
needed to be determined, othervrise for determination of the
relative protein content of the same plant variety under
two different growth conditions there is no need for such a
factor to be applied. In the research reported here we
were more interested in differences (relative values)
rather than the absolute quantity. Despite the disadvantages
of the Lowry method, there were several advantages, which led
us to adopt it. The method is more sensitive and convenient
than is the digestion and Nesslerization method. Also, it
is 10-20 times more sensitive than the U. V. absorption
method of protein determination. It is much less liable to
inaccuracy due to turbidity, while it is almost 100 times
more sensitive than the Biuret method for low concentrations
of proteins,

H. Chemical treatment techniques

Metabolites v;ere applied as a leaf spray to plants
under different temperature conditions. Plants were sprayed
every other day until dripping wet. Eor a better wetting of
leaves, surfactant (Tween 20 -0.01 per cent) was added to
the metabolite solution. The different metabolite solutions
which were the same as those employed by Ketellapper (32),
included:

^1

1. Vitamin B mixture.

2. Vitamin C.

3. Nucleosides,

4. Sucrose.

The composition of chemicals (metabolites) sprayed on leaves
is shown in Table ^. The control plants were sprayed with
water plus surfactant. Chemical treatment continued for
several weeks. Plants under both optimum and high tempera-
ture condition were treated with these metabolites.
Measurement of height, dry vzeight, fresh weight and finally
flov/er and fruit formation was carried out to examine the
effect of these metabolites.

^2

TABLE 4
Composition of Metabolite Solutions Sprayed on Leaves

Mixture Concentration

1) Vitamin B Mixture

Thiamin 20 mg/1

Riboflavin 5 mg/1

Ca-pentathenate 20 mg/1

Biotin 1 mg/1

Pyridaxin 20 mg/1

Nicotinic acid 40 mg/l

Inositol 200 mg/1

Folic acid 5 mg/1

2) Riboside Mixture

Adenosine 3 x 10"^ M

Guanos ine 3 x 10"^ M

3) Ascorbic Acid 1 g/1

4) Sucrose Solution

Sucrose 1 g/1

Sulfanilamide 125 mg/1

SAMPLING MD PHISICLOG-ICAL AGE

Sampling from plants under different environmental
conditions according to th.eir chronological age is not
advisable when studying ttie biochemical effects of one of
the environmental variables. Differences in physiological
age at a given chronological age soon develop, introducing
a second variable which may have pronounced int erf erring
effect on the study of the first variable.

In the experiments with high and low temperature
effects, after a short time plants of the same chronologi-
cal age would not have the same physiological age, since
tine of flowering and fruit formation would be profoundly
affected by the temperature. Thus, there are biochemical
changes associated v/ith the stage of maturity v;hile it is
biochemical changes due to temperature, at equivalent
physiological ages, which we v;ished to study. Accordingly,
we took the following steps to obtain maximum uniformity of
plant material and to insure sampling of plants at physio-
logically equivalent age.

Seeds, and then the young seedlings, were hand-graded
to obtain uniform size plants by discarding unusually small
or large plants. Samples for extraction v;ere taken from at

A-3

4^

least five plants. The five plants were ciiosen from a rather
large number of plants (usually 60) in a random way (67).
To eliminate tlie problem of physiological age dif-
ferenoes, a numerical index of vegetative development, the
plastochron index of Erickson (12), v;as determined in each
case and samples were taken for analysis at equivalent
indices. The period between the initiation of successive
leaves as well as the size of leaves are features considered
in the plastochron concept. A plastochron is defined by
Erickson as the time between corresponding stages of develop-
ment of successive leaves. (For a corresponding stage of
development, it is possible to choose either the initiation,
maturation or attainment of a standard length of leaves.)
In our research, which was carried out in climatic controlled
chambers, the physiological ages of plants grov;n at high
and optimum temperatures were calculated according to
Erickson' s formula (12, 13 ,53? 56) .

P.I. = n +

log ^(a) " ^°^20

log L(.) - log L^^^3_^

where :.

n = the serial number of leaves which are equal to
or just larger than an arbitrary value, which
is 20 mm in our e:^eriment,
L^ N = is the length of the highest leaf vvrith length
equal to or larger than 20 mm,

A-5

L/ -I N = is tlie length of the next higher leaf smaller
i.a+i;

than 20 mm in. length,
P.I. = plastochron index.
Usually the P.I. is a good index of the developmental age for
a plant which has stahle spiral phylotaxy (12). Luring the
vegetative period of grov/th, the P.I. is linearly related to
time. Plotting of P.I. values of each day against time after
germination gives almost a straight line and can be used as
a good substitute for chronological age. If the length of
the leaf a is exactly equal to the arbitrary length value,
the plant v;ould have a P.I. equal to n, but this does not
happen frequently; usually L^ n is more or less than that
value. Por example, if L/'^n equals 25 mm and Lr^ l^ equals
17 mm at a given time, it is clear that the P.I. of the plant
is more than n and less than (n+1). In practice, the P.I.
calculation is carried out by counting all the leaves that
are equal to or larger than 20 mm. Then measurements are
made of the length of two successive leaves which respectively
are less '^(g^.j_\ and just equal to or more than 20 mm (L, >, ) .
Therefore, in our example, if n = 15, L/ n = 25 mm, and
■^Ca+1") "^ '^'^ ^^^^^ '^'^^ P^I. value would be:

loe

25

PI - 15 + log 25 - lop; 20 ^^° 20 , , ^^
^'^' - ^5 + log 25 - log 17 = "77725 ' ^^'^^

^6

All samples taken for extraction from plants grown
in the two temperature conditions v;ere taken at such times
as to have the same P.I. value j^ 0,2 P.I. units.

After the plants passed the vegetative period and
reached the reproductive stage of life the plastochron index
could not he used any more. For the extracts taken after
floxvering started, the time after flower initiation was
considered only as the chronological time. Three extracts
were taken during the vegetative period, one in the flowering
stage and one seven days after flowering when small fruits
are formed. In most cases, plants under high temperature
conditions did not produce any fruit or at least not any
perfect fruit, so they remain for a long time in the vegeta-
tive stage and ultimately start rosette type of growth. It
is evident that hiochemical comparison of this group v/ith
the optim\im temperature group (which have fruit and are in
a perfect reproductive stage) is of little value. That is
why further samples were not taken, Pruit formation in one
series of plants under optimum condition v;as inhibited simply
hy the removal of pistil or ovary with a sharp needle to
produce similar physiological conditions in the optimum and
high temperature groups. Grov/th characteristics of this
group v;ere compared with that of the high temperature group.

RESULTS AND DISCUSSION

I« Grov;tli Gliaracteristj,cs of Plants Under Optiinun
and High. Temperature Conditions

A. Slon°;ation (increase in total height)

The pattern of grov/th (height increase) in the rela-
tively heat-sensitive pea variety Greater Progress was quite
different at high and optiaum temperature conditions (figures
1 and 2), The plants grown at high temperature grew very
rapidly during the first weeks after germination (lA- to 19
days after seeds were planted). At optimum temperature, the
rate of elongation gradually rose to a maximum at about 19
days after seeds v;ere planted. The sudden and sharp drop
in the rate of elongation at the high and optimum temperature
condition coincided approximately v;ith the time that the
reserve materials of the seed were exhausted. However, the
drop in growth at this stage v;as greater at high than at
optimum temperature. Flower bud formation usually started
about 25 days after planting at the optimum condition and
occurred about three days earlier under the high temperature
regime. 5'lowering caused a decline in the rate of stem
elongation, and by the time fruit set started, the elongation
stopped altogether. At the high temperature condition there

^7

Fig. 1. Daily height increase of pea plants under two teir.pera-
ture regimes.

(a) early drop in elongation

(b) flowering period

(c) fruit formation occurring

(d) abscission of flowers and immature fruits occurring at
the high temperature condition

(e) second flowering period at high temperature condition

(f) second absicssion of fruits and flowers

HEIGHT INCREASE (MM. /DAY)

C>4

_1_

O

I

ru

— 1—

^9

M

C5^

O"

o

ro

0^

O
CO

>

Tl

-!

m

R

in

rn o
n

04
CD '

o

O

o

i

t

—

o

<r>

-0
H

-A.

^^

H

F

iM

:^

-0

m

33

-i

t>

i.

H

■■<j

r;

i'-i

Ti

3j

m

^

c

:o

m

4^

o<..-

F16.

Fig. 2. Growth (height increase) of the pea plants under high
and optimum temperature conditions as compared with the groups of plants
which were depistillated (in order to stop fruit formation) at the
optimum temperature condition.

51

cm 25-

20'

15

-O OPTIMUM TEMPERATURE

O H16M TEMPERATURE
-A- OPTIMUM (PISTIL REMOVED)

o-o

0-

0 5

DAYS

10

20 25 30 35 40 45

FIG. 2

52

was a decline in the rate of lieight increase during tlie
flowering period similar to the optimum temperature condi-
tion, but tlie elongation never ceased altogetlier because
there was no perfect fruit set at this condition. At high
temperature, abscission of the flov/ers usually occurred;
if not, any young fruit that did form abscissed very early
in the developmental stage. After the first flov/ering period
stopped without any fruit being formed, the rate of stem
elongation of the plant under the high temperature regime
again increased. IThis increase in elongation continued until
a second flowering period started. Since we were interested
in studying the effects of temperature per se rather than a
possible secondary effect of flower initiation and abscission,
we attempted to produce more comparable plant material by
removing the pistils of flowers as they formed in a group of
plants at optimum temperature.

The pattern of. stem elongation seen after flower
initiation in the high temperature series could be obtained
also under the optimum temperature condition v/hen the flowers
were depistillated in order to stop fruit formation (figure 3)
The main difference in the pattern of growth between the two
conditions seen in this experiment v;as that flowering
occurred more often and much faster \>rhen the pistil was re-
moved by hand at optimum temperature than when the flower
and fruit drop v;as caused by high temperature. The increase

Fig. 3. Daily height increase at optimum temperature condi-
tions (pistil removed) .

(a) early drop in elongation

(b) flowering period

(c) fruit set

(d) pistil (or very small fruit) removed

(e) flowering started for second time

(f) removal of pistil or young fruit

(g) flowering started for third time
(h) removal of pistil or young fruit

HEIGHT INCREASE (MM. /DAY)

5^

o

_1_

Oi

I

o

L_

O

1

(J\

ro

o>

o

ro

O

o

>,

ro

1N">

a>

>

--)

ro

^

m

-n

^^

ro

cy)

o>

rn

n

o

ro

w

o

«=r

rn

w

:o

o

m

-D

Ol

i

1\J

o

CO

o

ro"

^

•j^'

FIG.

o

55

and decrease in growtti rate was also sharper and nore
frequent in the dcpistillatod, optinum-teraperature series.
The life span of plants under optinum conditions usually
was about two months, vrhile at high temperature it usually
exceeded three months.

To test whether the inhibition of fruit formation,
and hence longer period of growth might be due to a failure
of pollination, groups of plants grov/n under the high
temperature condition were brought to optimum- temperature
for one weelc during the flowering period. There was no
significant improvement as far as formation of perfect fruit
was concerned. Kov/ever, if they were kept for a period
longer than one v;eek at optimum- xiemperature, the fruit set
occurred in a normal way. These observations eliminate the
possibility that inhibition of fruit formation was due to
the simple failure of pollination at high temperature. How-
ever, this does not indicate whether pollen was viable or
not at high temperature. Of course,, high temperatures might
have affected the rate of maturation of flower pistils and
stamen so that they did not mature at the same time, a
process that is likely to happen in many self-pollinating
plants under certain environmental conditions. If the
failure of fruit set at high temperature was due to deranged
maturation of flower parts directly caused by the elevated
temperature, it would seem obvious that the initiation of
this derangement occurred more than a week prior to pollina-
tion.

56

A number of tlie growth studies vrere repeated with, a
more heat-resistant variety of pea (var. Wanda) at the same
time and under the same growing condition. In contrast to
the results with the heat-sensitive variety (var. Greater
Progress), there was not very much difference in the growth
characteristics of the var. Wanda under the two conditions.
Although the number of flowers and total yield was less at
high temperature, there was no problem of flowering and
fruit formation as observed in the case of the heat-sensi-
tive variety. (The data for this part are not presented
in this dissertation.)

B. Fresh weight increase at hig:h and optimum temperature

During the first three weeks after planting the seeds,
there was little difference in amount or rate of increase in
fresh weight at the tv;o temperature conditions (Figure 4).
Plowever, three v/eeks after the seeds were planted the rate
of fresh v/eight increase dipped sharply in the high tempera-
ture group but not until the fourth week in the plants which
were at optimum- temperature. Later on, after the fifth week
of growth, there was an increase in the rate of fresh weight
accumulation at the high temperature condition. This in-
crease occurred after the first abscission of flowers, at
the time that rate of growth and elongation was increasing
at high temperature, while at optimum-temperature the rate
continued to decrease. However, increase in fresh weight vs.

Fig. 4. Total fresh weight increase and the weekly increase
of fresh weight at high and optimum temperature conditions.

15-

58

■o- OPTIMUM TEMPERATURE
O- HIGH TEMPERATURE

^ 0
LlI

□ OPTIMUM TEMPERATURE

□ HIGH TEMPERATURE

-b— ' — L.

1

n

^

2 3 4 5

V;£EKS AFTER SEEDS V/ERE PLANTED

FIG.4

59

time had a lower rate at higli temperature, and the total
fresh weight was much lower at high temperature; part of
this decrease rate of fresh weight accumulation at high
temperature seems to be due to the abscission of flowers and
young fruits which could cause a lowered rate of increase in
total fresh weight.

C. Increase in total dry weight at high and optimum-
temperature

Total dry weight per plant as well as weekly increase
in dry weight were almost the same at the tv;o temperature
conditions during the first three weeks of growth, but later
the rate of dry weight increase was much faster at optimum
conditions (Figure 5). After the fifth week of growth, the
rate of dry weight increase dropped at high and optimum
conditions simultaneously. However, the rate of increase
of dry weight continued to drop at optimum conditions while
at high temperature it started to increase again after the
sixth week of growth. Here again, this increase was mainly
due to rapid vegetative growth that started after the first
abscission of flowers and fruits occurred at high temperature
conditions. The final dry weight per plant (after seven
weeks of growth) was almost twice as much at optimum-
temperature conditions as at high temperature.

Fig, 5. Total dry weight increase and the weekly increase
of dry weight at high and optimum temperature conditions.

61

2.5

2.25-

2.0-

. 1.75-

CD

H 15 -
X
CD

UJ .
5 I.25H

>

Q 1.0

.75-

5 -

.25-

CD

LlI
CO

<c

u
or
o

.4-

3-

.2-

>, .'■

liJ

0 4-

— o— OPTIMUM TEMPERATURE
-O- HIGH TEMPERATURE

[n OPTIMUM TEMPERATURE
□ HIGH TEMPERATURE

n

Pn

JZuL

r
2

r
3

ii

1

: :

4 5 6

WEEKS AFTER SEEDS WERE PLANTED

1

7

F16.5

62

D, Increase in percent of dry weight

The percent of dry weight was almost the same at the
two conditions, and there was a continuous rise in the
percentage of dry weight as the plants became older. After
fruit set, however, there was a very rapid rise in the
percent dry weight at optimum-temperature conditions, v;hile
at high temperature there was a slow rise in percent dry
weight.

E. Shoot-root ratio

The ratio of shoot v;eight to root weight was almost
the same through three weeks of growth at the two tempera-
ture conditions (Pigure 7). Later the ratio increased at
the optimum condition (as fruit formation started) and be-
came much more than at the high temperature condition. The
low ratio of shoot to root at this period, in plants grown
at high temperature, v;as partly due to abscission of flowers
and fruits. But, after the fifth week of growth, the ratio
in the high temperature group started to increase; and
finally, by the sixth week the ratio became equal to that
of optimum conditions. This increase in shoot-root ratio
probably was a result of the spurt in vegetative growth
observed at this time in the plants exposed to high
temperatures.

Fig. 6. Increase in per cent dry weight at different stages
of growth at optimum and high temperature conditions.

(a) Fruit set occurred in plants grown at optimum temperature
conditions, but not in plants grown at high temperature.

Fig. 7. Shoot-root weight ratio versus time.

(a) Ratio increases faster at optimum condition during
flowering and early fruit formation period.

(b) Ratio became higher because of fruit formation at the
optimum condition. Finally, fast vegetative growth after
the first abscission of flowers and fruit at the high
temperature condition increases the ratio.

20-1

X 18-

LJ

^ 16H

Q

CO

14-

2-

LiJ

.'T

O

10

2

0^

8

-O- OPTIMUM TEMPERATURE
O- HIGH TEMPERATURE

64

O -. _

5-

4-

O

f,

'<

8 3.

o
o

t/5

2-

1-^

. OPTIMUM TEMPERATURE
O - HIGH TEMPERATURE

3

5

7

WEEKS AFTER SEEDS WERE PLANTED

FIG. 6 end 7

65

II, Amino Acid Analyses of Peas Under Optimum
and High Temperature Conditions

A. General

The results of the quantitative analyses with ion-
exchange column chromatography of the plant extract for
their amino acids are shown in Figure 18 and in Tables 5
through 10. Results are expressed in terms of micromoles
of amino acid per gram fresh weight. Values for the amino
acid quantity present were calculated on the "basis of the
color yield with ninhydrin of a standard solution of
leucine, observed in frequently repeated standard curve
determinations. The results in most cases v/ere repeated
two or three times to make sure of the reproducibility and
consistency of the location of the peaks for each amino
acid in the effluent. After reproducibility and consistency
of the chromatograms were well established, further analyses
of the extracts were carried out only once. After several
analyses, it was found that elaborate purification of the
plant extracts was not necessary in order to make quantita-
tive determination of amino acids. (Efficiency of the column
for analyses of amino acids in the partially purified extracts
was almost the same as for well-purified extracts.)

66

B. Problems in separation, and identification of individual
amino acids

There ^^ras some uncertainty concerning the location
or adequacy of resolution of certain peaks in the chromato-
grams. Careful control of conditions, or additional steps
resolved a number of these problems. For example, the
location of valine, isoleucine and leucine peaks depended
on the time when citrate buffer of pH 3.25 was replaced
with the citrate buffer of pH ^.25.

The asparagine and glutamine peaks normally v;ere
combined with each other and serine, making it impossible
to determine the quantity of these amino acids. Therefore,
determination of the quantity of asparagine, glutamine and
serine was carried out by the hydrolysis of the asparagine
and glutamine in the extract. For this purpose, the puri-
fied plant extracts were hydrolyzed in a solution of 0.1 N
HCl at 100° C. for three hours (20 ml KCl for 0.5 ml extract
equivalent of 0.5 gm fresh tissue); the hydrolyzed extracts
were chromato graphed, and the increase in quantity of
aspartic and glutamic acid as compared with that of a non-
hydrolyzed extract was determined and calculated as
asparagine and glutamine. Serine determination after
hydrolysis was also carried out. In all cases, the amount
of asparagine was much more than glutamine. Synthetic
mixtures of a known quantity of serine, asparagine and

67

glutamine were hydrolyzed in the same v/ay and th.en ctiromato-
graplied; tlie results obtained showed that there was an 8
per cent loss due to hydrolysis and handling of the sample.
Therefore, values in the hydrolyzed extracts were corrected
for this loss. In analyses of extracts, it was observed
that a number of peaks appeared early before the aspartic
acid, which, on the basis of its acidity, one would expect
to find as the first peak. These early peaks were found to
be mainly peptides, soluble in 80 per cent ethanol. Treat-
ment of extracts with 0.1 N HGl caused the disappearance of
all of these peaks except one. The peak immediately before
the aspartic acid (the one that did not disappear on
hydrolysis) appeared to be methionine sulfoxide, according
to the location of a knox'Wi sample of methionine sulfoxide
in a chromatogram of a synthetic mixture of amino acids.
Assignment of methionine sulfoxide to this peak is consistent
with the results of Lawrence (-+3) in similar analyses.
Methionine is well known as an unstable amino acid that
oxidizes readily to methionine sulfoxide during handling and
storage of plant extract. A hydrolysis method was also
carried out for exact determination of the quantity of
methionine sulfoxide, because the peak of methionine sulf-
oxide in unhydrolyzed samples coincided with one of the most
abundant alcohol-soluble peptides of pea seedlings, known as
-glutamyl alanine (74). Finally, the total amount of

68

peptides was calculated by summing all the peaks appearing
before aspartic acid (minus netbionine sulfoxide). Proline
determination was not satisfactory in most cases. Usually
tbe large size of the glutamic acid peak, v/hich appeared
just before the proline peak, caused the two of them to
overlap. The low optical density yield of proline per
umole (even at ^^0 mu wavelength) v;ith ninhydrin reagent
and also the low content of proline in the extract, made
proline contamination an insignificant factor in the glutamic
acid analysis. However, in most cases these same factors
made it impossible to obtain quantitative data for proline
in the extracts. There was one peak (designated as unknown
A) which emerged immediately after proline and before
glycine. This peak became larger in the high temperature
condition during the flowering period. Ve did not have
sufficient material to carry out tests to establish the
identity of the substance giving this peak. Hov;ever, the
peak is in the position to be expected for a-aminoadipic acid,
known to be a constituent of pea plants (^3,7^) • The glycine
peak usually was low and coincided in part with some very
small interfering peaks. The values presented for glycine
are the average of two determinations on each sample. It
was necessary to use a certain amount of judgment as to what
constituted the true shape of the glycine curve. Accordingly,
the precision and accuracy is not as good for glycine as for
most of the other amino acids.

69

C. Veekly chanp-es in amino acid content: at hlafn and
ootirau" tor:.r)oratures

-• ?ix'5t period of sar-pl inn;. -Results are presented in
Table 5 and Figure 1.8 for the total alcohol soluble (free)
amino acids content of leaves (um/gm f. v/t.) of peas, one
week after seeds were planted (approximately three days
after germination, when no fully expanded leaves v;ere present)
The amount of total free amino acid in leaves of the high
temperature plants was much greater (1.7 times) than in the
optimum temperature plants. The amount of each individual
amino acid is shovra. in Table 5. This early time in the life
of the pea plant, one should remember, is a period of active
hydrolysis and utilization of reserve proteins of the seed,
as v;ell as a period of synthesis of nev; proteins in the
developing seedling. ITot unexpectedly, amides were present
in very high amounts at both temperature conditions. How-
ever, the total amides (asparagine plus glutamine) at high
temperature v;ere almost 2,5 times higher than at the optimum
temperature condition. More amino acid and especially more
amide formation at high temperature could possibly be due to
more rapid protein breakdovrn and faster growth at high
temperature during the first week of grov;th. The total pep-
tides content, homoserine and also methionine sulfoxide, in
both extracts v;ere higher in the first week than at any other
time. In contrast, the amount of isoleucine and leucine v:as

70

TABLE 5
Amino Acid Content of Leaves - First Period of Sampling

Amino acid

pM per gram fresh weight

Temperature
Optimum Hipih

Ratio

Methionine sulfoxide

Aspartic acid

Serine

Asparagine

Glutamine

Threonine

Homo serine

Glutamic acid

Unknown A

Glycine

Alanine

Valine

Leuc ine

Isoleucine

Peptide

2.72

2.96

2.S5

5.00

2.10

0.34

1.43

3.27

0.61

0.96

1.82

0.77

Trace

Trace

5.60

3.24
2.04
3.26
15.9
6.34
0.47
4.23
4.95
0.82
0.48
2.12
0.98
Trace
Trace
6.20

1.20
0.69
1.14
3.18
3.01
1.38
2.95
1.51
1.34
0.50
1.16
1.27

1.11

71

low in the first v;eek of growth.. There v;as not a sufficient
amount of extract from these young and small plants to permit
measurements of the basic amino acids.

2. Second period of sampling. -Samples v;ere taken from
a group of plants 1^ days after planting at optimum and 13
days at high temperature (with P.I. values very close to
each other),

a. Amino acid of leaves. The amount of amino
acid in the extract of high temperature plants v;as almost
three times higher than optimum temperature plants. However,
at both conditions the amount of amino acids was less than
the previous sample which was taken about a week earlier.

Comparison of the individual amino acids in the
second sampling period showed that the most outstanding dif-
ference between the two extracts was the presence of a
rather large amount of amides in the high temperature
plants, v;hile the amide content of the optimum temperature
plants v;as quite low. If amides v;ere not included in the
total amino acid content of the high temperature plants,
then the total amount of amino acids in the high temperature
plants would be two times higher than at optimum temperature
conditions. The high amide content at the high temperature
condition could have been due to further proteolysis and
metabolism of seed proteins (which caused amide formation as
an ammonia detoxifying mechanism). On the other hand, the

72

high, amount of amides and total amino acids could have re-
sulted from a lower protein synthesis at the high tempera-
ture condition, resulting in a larger amino acid pool (1).

Methionine sulfoxide and peptide content at high
temperature was also much higher than at the optimum
temperature. In general, all the other amino acids at high
temperature were in a substantially higher amount. The peak
of unknown A was very lov; (trace) in extracts of plants
grown under optimum conditions while it was high in the high
temperature extract. The results of analyses for this
period of sampling are shown in Tahle 6 and Figure 8,

h. Amino acid content of roots. Since there was
a very large difference in the total amount of amino acids
of leaves at the two conditions in the second period samples,
an extract from the roots at this stage of growth was
analysed, to study the differences in amino acids in the
other parts of the plants as compared with leaves (Table 7
and Figure 9). Like the leaves, total amino acid content
of roots was higher (about 2.5 times) at high temperature
than at optimum temperature. In general, however, the
pattern of amino acids in roots was somewhat different from
that observed in leaves of the same plants. At both tempera-
tures the amount of homoserine v;as much higher in roots,
although it was almost three times higher in the extract from
plants grown at high than at optimum temperature. Homoserine

TABLE 6
Amino Acid Content of Leaves - Second Period of Sampling

73

Amino acid

pM per gram fresh weight
Temperature

Optimum liish

Ratio

Methionine sulfoxide

Aspartic acid

Threonine

Serine

Asparagine

Glutamine

Komo serine

Glutamic acid

Proline

Unknown A

Glycine

Alanine

Valine

Isoleucine

Leucine

Tyrosine plus
phenylalanine

^-arainobutyric acid

Lysine

Histidine

Arginine

Peptides

0.08
2.22
0.25
0.80
1.10
0.55
0.81
1.33
Present
Trace
0.13
0.90
0.27
0.23
0.13

0.65
0.33
0.48
0.96
0.33
Trace

2.05

25.60

3.52

1.58

0.49

1.96

2.30

2.37

6.54

5.94

2.82

5.12

1.57

1.93

3.83

2.88

Trace

—

0.29

—

0.14

1.08

1.90

2.11

0.63

2.33

0.32

1.39

0.48

3.69

0.51

0.78

0.97

2.93

0.24

0.50

1.37

1.42

0.84

2.57

1.50

~M

Fig. 8. Chromatograms comparing amino acid constituents of
leaves of pea plants under optimum and high temperature conditions.
(Second period of sampling.) The upper chromatogram is from 0.5 gm
of leaves at optimum temperature, but the lower one is from 0.25 gm
of leaves at high temperature.

le

16

1.2-
10-

i^

2-

fAsporlic
' Acid (]

Glutomic Acid

A
?

VAH

■ 2N No Ciifote Buffer. pH 3.25-

Alonine

75

Optimum Temperature

Vnline

Glycine

Leucine

isoieucme /^

-gN No Citfote Buffer pH 4 ?5-

'°E°f,uen/(°u "'° '^° '®° '°° ''° 2"° '^° 280 300 320 340 360 380 400

1.8

L6-

1.4-

12-

lO

Methionine Aspcrtic
Sulfoxide, A Acid

II

Peptides '

^

Threonine

'V

i\j^

Serine*Glutamine
♦Asporogine

Glutomic Acid

Aionine

Glycine

High Temperature

Voline

■.2N NoCifrole Suffer. oH 3.25-

I awe WW" '« J.

■.2N No' CitroTe Buffer. pH 4.25 ■

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

Effluent (ml.)

FIG. S

76

TABLE 7
Amino Acid Content of Root - Second Period of Sampling

Amino acid

pM per gram fresh weight

Temperature
Optimum High

Pvatio

Kethionine sulfoxide

Aspartic acid

Threonine

Serine

Asparagine

Glutamine

Eomo serine

Glutamic acid

Proline

Unknown A

Glycine

Alanine

Valine

Isoleucine

Leucine

Tyrosine plus
phenylalanine

2J-aminobutyric acid

Lysine

Histidine

1.17
0.69
0.37
0.58
1.47
1.01

11.37
0.77

Trace
0.41

Trace
0.56
0.45

Trace

Trace

Trace
0.16

Trace
0.25

2.05
1.13
0.57
1.12
3.20
2.05

30.12
1.07

Trace
0.73
0.48
1.03
0.50

Trace

Trace

0.11

0.41

Trace

0.51

1.75
1.63
1.54
1.93
2.17
2.02
2.66
1.39

1.78

1.82
1.11

2.56

2.04

Fig. 9. Chromatograms of amino acid constituents of roots at
optimum and high temperature conditions. (Second period of sampling.)

The upper chromatogram is from 0.5 gm of root at optimum temperature,

but the lower one is from 0.3 gm of root at high temperature.

78

Homoierina

Optimum Temperature

Leucina

I.8-,

1.6-

1.4

1.2-'

.5.

1.0

.8-

.2-

Homoterina

Glutomic Acid

Alanine

V/V^;W^W^

High Tonperature

Vaiina

•,2N No Citrale Buffer, pH 3.25-

-.2N No Citrate Buffer, pH 4.25 -

100 120 140 160 130 200 220 240 260 280 300 320 340 360 380 400

Effluent (ml.)

FIG. 9

79

is not a protein amino acid althougli it is knov;n to be an
intermediate between aspartic acid and threonine. The high
homoserine content in roots of Leguminosae has been a sub;ject
of considerable research and discussion by Virtanen and
Sasaoka (64,76). They showed homoserine is absent in seeds
but that it is synthesized during germination. The amide
content in roots also was much more (three times) at high
temperature than" at the optimum temperature. Methionine
sulfoxide also was about two times higher than at optimum
temperature. It will be recalled that similar results for
amide and methionine sulfoxide content were observed in
leaves.

3. Third period of samplin::^.-This sample v;as taken
from plants 20 days after planting at high temperature and
22 days after planting at the optimum temperature condition
(with P.I. values very close to each other). The plants
were almost in their ma:cimum vegetative growth and a few
days away from flowering. The total amino acids of leaves
from plants grown at high temperature were two-thirds of the
previous (13 days) sample, while at optimum conditions there
was a rise (by 1.'4- times) in total amino acids. In this
sample, the total amino acids at high temperature were a
little more than at the optimum temperature (the ratio was
19:16). This situation, v;hich v/as quite different from that
observed in the previous (second) sampling period, can be

80

interpreted as being due largely to two processes: 1) a
faster rate of respiration at tlie high temperature, which
would deplete the seed reserves more rapidly than at the
optimum temperature; 2) a faster rate of organic matter
production by photosynthesis at the optimum condition. The
photosynthetic system is probably more efficient in the
plants grown at optimum temperature (as the leaves are
larger and they have more chlorophyll). These factors
could have caused the increase in amino acids of the opti-
mum temperature plants, while at the same time, caused a
decrease since the previous sampling period in amino acid
content of plants at the high temperature condition. The
pattern of amino acids was more similar at the two tempera-
ture conditions in this sampling period than at any other
time. However, the unknown A was absent at the optimum
condition while it was quite a large peak at the high
temperature condition. Total amide content, also, was very
similar at the two temperature conditions. The results are
shown in Table 8 and Figure 10.

^. Fourth period of sampling. -Samples were taken when
the plants were in the full flowering stage (three days after
first flov;er bud formation). The high temperature plants
v;ere 2? days post-planting while plants at the optimum con-
dition were 29 days. The P.I. values were nearly identical
for the two groups of plants. Total amino acids of leaves

81

TABLE 8
Amino Acid Content of Leaves - Third Period of Sampling

Amino acid

pl4 per gram fresh weight
Temperature

Ootimum Hich

Ratio

Methionine sulfoxide

Aspartic acid

Threonine

Serine

Asparagine

Glutamine

Homoserine

Glutamic acid

Proline

Liiknown A

Glycine

Alanine

Valine

Isoleucine

Leucine

Tyrosine plus
phenylalanine

^aminobutyric acid

Lysine

Kistidine

Arginine

Peptides

0.45

1.02

2.26

1.94

2.53

1.30

0.41

0.50

1.22

2.00

1.66

0.83

2.09

2.21

1.05

0.94

1.19

1.26

0.97

1.34

1.38

3.17

4.13

1.30

Present

Present

—

—

0.31

—

Trace

0.18

—

2.24

1.58

0.70

0.37

0.50

1.35

0.23

0.11

0.47

0.27

0.13

0.48

0.20

0.35

1.75

0.49

0.51

1.04

0.21

0.23

1.09

0.10

0.20

2.00

Trace

0.35

—

0.69

0.88

1.27

Fig. 10. Chromatograms of amino acid constituents of leaves
at optimum and high temperature. (Third period of sampling.)

I e-i

P Serine
; ' +GlulQmine
-fAsporogine

Glutomic Acid

2N No Citrote Buffer, pH 3 25-

Alonine

Glycine

83

optimum Temperature

Valin*

'N-V.VW' V-

-.2N No Citrote Buffer, pH 4 25 -

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

Effluent (ml)

Glutomic Acid

Alanine

A M Glycine

High Temperature

Valine

■.2N No Citrote Buffer, pH 4.25 -

100 120 140 160 ISO 200 220 240 260 280 300 320 340 360 380 400

Effluent (mL)

FIG. 10

at the optimum condition showed a significant decrease while
at the high temperature condition the total amino acids did
not change since the previous sampling period. The ratio of
total amino acids of high to optimum temperature v;as 21:13 =
1,6. The amounts of methionine sulfoxide and also total
amide content at the optimum condition showed a sharp de-
crease since the last sampling period. Total amide in the
plants grown at optimum temperature was one-sixth of those
at the high temperature (Table 9 and Figure 11). The un-
known A increased at high temperature, while it was absent
at the optimum temperature condition. There was a substantial
increase in homoserine content at both conditions. Lysine
was very low (trace) at high temperature while histidine v/as
very lov; at the optimum temperature condition.

5, Fifth period of sampling. -Samples were taken from
plants one week after their full flowering period (one v:eek
after the previous sample). Plants under optimum temperature
had set small fruits while very fev/ if any fruit could be
observed on the high temperature plants. There was little
change in total amino acid content of the plants under high
temperature conditions, while there was a considerable de-
crease at the optimum temperature condition. Total amino
acid content in the high temperature plants was twice that
under optimum conditions. There v;as a decrease of amide,
such that just a trace was left at the optimum condition

85

1ABIJL 9
Amino Acid Content of Leaves - Fourth Period of Sampling

pl>I per gram fresh weight
Temperature
Amino acid Cotimui-', Kigh

Ratio

Methionine sulfoxide

Aspartic acid

Threonine

Serine

Asparagine

Glutamine

Homoserine

Glutamic acid

Proline

Unknown A

Glycine

Alanine

Valine

Iso leucine

Leuc ine

Tyrosine plus
phenylalanine

ZT-aminobutyric acid

Lysine

Hi St id ine

Arginine

0.4G
1.C5
0.41
1.20
0.29
0.10
2.63
2.53
Trace

0.14
1.S5
0.57
0.13
0.12

0.27

0.63

0.20

Trace

0.38

0.91

1.96

0.34

1.43

2.05

0.50

4.87

3.27

Trace

0.32

0.11

2.42

0.57

0.28

0.22

0.44

1.14

Trace

0.33

0.16

2.27
1.86
0.82
1.19
7.06
5.00
1.85
1.29

0.78
1.31
1.00
2.15
1.83

1.62
2.80

0.42

Fig. 11. Chromatograms of amino acid constituents of leaves
at optimum and high temperature. (Fourth period of sampling.)

I8n

16

12-

10

■^ 8

Homos«rin«

Methionine
Sulfoxide

S?

Optimum Temperature

-.2N No Citrote Suffer, pH 3.25-

-.2N Na Citrate Buffer, pH4,25-

i^ ^ I'^O ieo"^ 180 200 220 240 260 280 300 320 340 360 360 400

Effluent (mO

1-8-1

1.6-

1.2

1.0-

•S 8-

.6-

.4-

.2-

Alonine

Glycine

High Temperature

Vo.ine

V^AaA'/-/' ^JJ^^^ ^yh'^'

Isoleucine

Leucine

'^^•^

.2N No Citrate Buffer, pH 3.25-

-»"J .2NNQCitrote Buffer, pH4.25-

100 120 140 160 ISO 200 220 240 260 280 300 320 340 360 380 400

Effluent (ml.)

FIG. II

88

.while under th.e high, temperature condition there was a large
amount of amide. Methionine sulfoxide v/as also decreased in
the optimum condition, while it v;as still high at high
temperature conditions (Table 10 and Figure 12). The only
amino acid v;hich was increased at both temperatures was
homoserine. At the optimum temperature condition, the amount
of aspartic acid was very low while glutamic acid and alanine
were not decreased. At the high temperature there was only
a little decrease in aspartic acid. The unknown A was very
high at the high temperature (higher than in any other
sampling period), while at the optimum temperature it was
absent the same as in the previous sample. At high tempera-
ture, lysine, which was absent in the previous sample, was
again present while histidine became very lov/ at both
temperature conditions.

III. Protein Estimation

The results of protein measurements in leaves of the
plants under high and optimuu ueiaperature conditions, for
five different periods of sampling, are given in Table 11
and in Figure 1?.

The results are presented in terms of mg equivalent
of bovine serum albumin per gm of fresh v;eight. Protein
determinations were not from the same plant materials that
were used for amino acid studies but rather from the same

TABLE 10
Amino Acid Content of Leaves - Fifth Period of Sampling

89

Araino acid

-ji'I per g-ram fresh weight
Temperature

O"otir.um Hic-h

Ratio

Methionine sulfoxide

Aspartic acid

Threonine

Serine

Asparagine

Glutamine

Homo serine

Glutamic acid

Proline

Unknown A

Glycine

Alanine

Valine

Isoleucine

Leucine

Tyrosine plus
phenylalanine

^-aminobutyric acid

Lysine

Histidine

Arginine

0.16

0.34

0.36

0.92

0.14

Trace

3.45

1.63

Trace

0.43
1.55
0.23
0.12
0.06

0.19

0.72

0.24

Trace

0.12

0.60

3.75

1.30

3.82

0.58

1.61

1.80

1.95

1.14

8.14

0.34

—

2.97

8.60

2.73

1.67

Present

—

0.65

~

0.24

0.55

2.92

1.88

0.52

2.26

0.23

1.91

0.07

1.16

0.81

4.20

1.20

1.56

0.14

0.58

Trace

—

Trace

M..

Fig. 12. Chromatograms of amino acid constituents of leaves
at optimum and high temperature. (Fifth period of sampling.)

18-,

16

12

,1.0-

§

.6-

4-

Asporfic ^.

AcKJ Threonine

Methionine
Sulfoxide / \

'k ,\

-V. 'j

W L,rV/

f Glutomic Acid

L Alanine

Glycine

n

91

Optimum Temperature

Voline

--2N No Cilfole Buffer, pH 3.25 -

.2N No Cilrote Buffer. pH 4 25-

'°Effiuen,",°n„., '"° ''° '^° 2°° 220 240 280 280 300 320 340 360 380 45o

High Temperature

340 360 360

FIG. 12

92

variety of plants v/liich v;ei*e grcvm later under (as nearly
as possible) the same conditions. In the first sample,
which v:as taken seven days after the seeds were planted,
protein content was high in plants under "both temperature
regimes but it was a bit higher in the optimum temperature.
High protein content at this sample period could result
from solublization and translocation of proteins from the
cotyledons to the young, yet not completely expanded leaves.
At the second sample v^-hich v.'as taken two weeks after planting,
there was a sharp decrease in protein concentration of plants
grown at the two temperature conditions, when compared with
the first sample. Again, the protein concentration was
higher under the optimum than at the high temperature con-
dition.

The third sampling was made 21 days after planting at
high temperature and 23 days after planting at optimum con-
ditions in order to sample at the same plastochron index
(P,I,), At this sampling period there v/as a sharp increase
in protein content of the plants at the two conditions.
Once again the total protein of the plants at the optimum
temperature condition was higher than that of the plants at
the high temperature regime. The great increase in protein
content over the previous sampling may have resulted from:
increased photosynthetic activity; a decrease in the rate
of growth (under both temperature conditions) ; and finally,

Fig. 13. Histograms comparing the changes in the amount of
aspartic acid, asparagine, glutamine and glutamic acid in the leaves
of plants grown under optimum and high temperature during five succes-
sive weeks of growth.

12
'o
<

o
c

£
<

4-

3H

o~

0-

15'

4

Aspartic Acid

. !

Asparagins

0'

e_G lute mine

3-

D

I*" ■//
fe" "'

4-

3-
2-

Glutamine
Acid

%

y..J

L optimum i emp.
DHigh Temp.

J_L

Jl

: i

I 2 3

- V/eekly Period of Sampling

FIG. 13

4

9^

.n

..ii

Fig. 14. Histograms comparing the changes in the amount of
alanine, serine, glycine and threonine in the leaves of plants grown
under optimum and high temperature during five successive weeks of
growth.

Alanins

n

0-

Seriae

Glycine

2 0.75-
o

<

g 0.5-

'i

*^ 0.25-
=1 0-

L

Threonine

0.6-

0.4-

0.2-

LJ optimum lamp,
D High Temp.

I I

i: 1

n

L^ L

D m.

h

12 3 4

Weekly Period of Sampling

FIG. 14

56

Fig. 15. Histograms comparing the changes in the amount of
homoserine, valine, leucine and isoleucine in the leaves of plants
grown under optimum and high temperature during five successive weeks
of growth.

4-

2-i

0
0.75
0.5-
0.25-

2
'o
<

o

c

E O.oH

£
<

0.4-

0.2-

Homoserir.e

n

Vaiinv

5

Leucine

0

0.6-
.0.4-

02-
0-

.1^ L

isoleuclns

HZi

G optimum temp.
DHigh Temp.

r

f

I

1
! ' 3

I !

n

r

VVeekiy Period of Sampling

?IG. 15

i t

r

98

Fig. 16. Histograms comparing the changes in the amount of
iT-aninobutyric acid, lysine, histidine and phenylalanine plus tyrosine
in the leaves of plants grown under optimum and high temperature during
five successive weeks of growth.

I.O
0.75-

0.5-
0.25-

0
045-
0.3-
0.15-

^-Aminobutyric Acid

Lysine

o

<

o

c

£
<

1.2-J

0.8-

04-

0-

0.75-
0.5-
0.25-
0

ii

Histidine

l"] Optimum Temp.
D High Temp.

100

rQ

Phenylalanine * Tyrosine

* 1

^■'1

J !_

^.

n

rn

/eekly Period of Scmpiing

FIG. 16

101

an increase in percent dry \veigb.t of leaves. The data on
protein concentration and ttie amino acid content at this
same stage indicated that leaves of this age not only have
a high capacity for producing or storing amino acids but
also a high capacity for incorporating the amino acids into
proteins.

The fourth sampling v:as made during flowering and
approximately four days after the first flower "bud formation
(28 days and 30 days from planting at high and optimum
temperature, respectively). With the fourth sampling there
was a sharp decrease over the previous sampling period in
protein content of plants at the optimum temperature condi-
tion while the protein content of plants at high temperature
was not changed since the previous sampling. Probably the
greater reproductive activity of the plants at the optimum
temperature resulted in a mobilization of protein to the
reproductive tissue and away from the leaves.

The last sampling was made a week after the fourth
sampling period; plants under the optimum temperature con-
dition had small fruits by this time. Eov.^ever, there was
only a small decrease of protein content of plants growing
under both temperature regimes. Results are shov/n in Figure
17 and Table 11.

102

TABLE 11
Protein Content of Leaves*

Sample age

Protein content

Protein content

(weeks after

mg/gm

mg/gm

planting)

optimum temperature

high temperature

1

50.4

42.1

2

26.5

18.0

3

40.5

28.4

4

25.8

30.1

5

24.8

26.4

*Values are given in terms of mg equivalent of bovine serum albumin.

Fig. 17. Total soluble protein content of leaves of the plants
grown under optimum and high teir.perature during five successive weeks of
growth. Amount of protein is given in terms of mg equivalent of bovine
serum albumin.

Fig. 18. Total amino acid content of leaves of plants grown
under optimum and high temperature conditions during five successive
weeks of growth. Amount of amino acid is given in terms of jir^iole per
gm fresh weight of leaves.

50n

40-

iD

I-
O

q:

20-

O- OPTlWlUI/i TEMPERATURE
O-HIGH TE.ViPERATURE

10^

iO"

50-

8

40-

\

\

\

\

\

\

<,— OPTIMUM TEMPERATURE
O - HIGH TEMPERATURE

\

CO

<
o

so-

lo-

\

-1

12 3 4

WEEKS AFTER SEEDS Vv'ERE PLANTED

FIGS. 17 and 18

105

IV. The Effects of Added Keta"bolites at Hip;b. Temperature

The purpose of experimentation in this area was to
examine the effects of various chemicals (metabolites) v/hich
have heen used by others (51,52) to inhibit or decrease the
injuries in plants caused by high temperatures. Identical
conditions v/ere used for these tests as had been used in the
previous amino acid studies. All experiments v/ith added
metabolites v;ere done in duplicate Vw^ith ^0 to 50 plants in
each test.

A. Sucrose effect

Treatment of the plants v.dth sucrose did not prevent
the high temperature injuries according to the data obtained.
Addition of sucrose for three weeks to pea plants under high
temperature conditions did not increase the height of plant,
fresh weight, dry weight, flowering or fruit formation. In
fact, the 10 per cent concentration of sucrose used as a
spray (a concentration used by others) (52), was observed
to be quite an inhibitor of growth. After a short tine of
treatment with that concentration, ■ the entire group of test
plants- dried up,. Later a dilute (1 per cent) solution of
sucrose was tried on the plants for several weeks under high
and optimum temperature conditions, the plants remained
healthy but the results obtained were never clearcut. The
observations v;ere consistent v;ith earlier work in this field

106

v;ith. other varieties of peas whicli sTaov/ed tliat the beneficial
effect of sucrose was limited to the condition where the high
temperature was only a few degrees more than optimum (32)
while in this experiment the temperature v;as about 8'=* C.
higher than the optimum. Possibly in these experiments,
the detrimental effects of high temperature was not simply a
decrease of carbohydrates,

B. Effects of vitamin B complex

Pour weeks of treatments with vitamin B complex as a
spray on plants under high temperature conditions caused an
increase over the control plants of approximately 12 per
cent in height and 11 per cent in fresh weight. No marked
effects on dry weight and fruit formation were observed,
although the number of flowers was somewhat greater on the
treated than on the untreated plants, These results did not
show as great an effect of vitamin B as those obtained by
other v/orkers v;ith a different variety of peas (32) or with
other plants at sub-optimal temperatures (^0).

C, Effects of other treatments

ITo beneficial effects were obtained by treatment of
plants v;ith a solution of nucleosides or with vitamin C as
a leaf spray.

107

D. G-eneral comments on metabolite treatments

a?iie failure of several metabolites to reduce or pre-
vent lesions in one variety of pea plant grovrn under high'
temperature conditions seems to indicate tliat there may not
be a single lesion due to high temperature that is universal
among different varieties of a species of plant. Kox\rever,
complications exist which attach reservations to this
statement. It is possible that one reason for the insigni-
ficant result in our experiments and the inconsistencies in
results of others (32,40,42) could be the inaccessibility
of the metabolites to the point of need because of lov; rate
of absorption through the epidermal tissue of leaves, of
destruction of the metabolite on or in the plant, or of
failure of adequate translocation v;ithin the tissues. An-
other problem in interpretation of experiments of this type
can be the variation of effect with varying degree of high
temperature stress. As the temperature increases, the
number of critical reactions damaged by high temperature may
increase, and so the metabolites needed to be supplemented
under different temperature conditions may not alv/ays be the
same. Such qualifying observations were also stated in the
case of research on different strains of Arabidopsis by
Langridge (40) where different metabolites (biotin or nucleo-
tides) v;ere needed for different strains of Arabidopsis.
Combinations of several different metabolites at different

108

temperature regimes may "be needed to stop or reduce the
injuries caused by supra-optimal temperature, and tliis lias
^oeen stiovm to "be true in tlie case of many microorganisms

Tlie purpc33 o^ tliis irrvesti^atior: was "co ncaGuro so::rio
of tho "bioclieziical effects accosipaii^/ins "clinatic Icsicr:"
(?), specifically, the "bicclienical effects of supra-optiz.al
teiiperatures* 1'rie plant naterial ch-osen v:as the pea (Pis'jg
sativu" L. var. Greater Progress), Data v.-ere ootained v/hich.
showed that the tenperatiire chosen for high or supra-
optiiial (25'* C. night and 30° C. day) did produce sone
clisiatic lesions without killing the plant. Grov/th, as
ncasured "by increase in height and hy increase in v/et and
dry vroight, v;as less (af"jer tv:o v:eeks) in the planijs gro"i-;n.

CV "^ /", "*^ .''^ v-.ViTT* ": '"• /"N-^ ■'" ci "-^ "^ T.r"^ .'-1 '. -Ti c c

scL/ '.;c;>t> c~xs o v^^vCu.. V— i^c*r_yj j-^juc^io 1— d-i^urxcij. v.'cib ocwc:— nc;a
v.'hich v;as suitable for the assess^ient of the oiocheinical
effects of heat injnrj^, Qhe changes in free (alcohol-
soluhle) anino acids and ootal solnhla pro"cein v:ere selected
for investigation. Data are presented to shov; the v:eelcly
changes in ohe concentration of each of 13 individual aiaino
acids in extraccs of leaves tr-r-cughout the vegetative stage
of the pea plant and into the reproductive stage for plants
grov;n at high and at optimum temperature (1?'' C. night and

2p« C. day).

109

110

A great deal of caution v.'as exercised to obtain ex-
tracts from tlie t^;o groups of plants at the same physiologi-
cal stage in order to he ahle to study the effects of high
temperature "oer se rather than a secondary effect. This
precaution was achieved by sampling from (at optimum and
high temperature) plants with very similar plastochron
indices at any sampling period. That is, physiological age,
rather than chronological age, was the criterion for
sampling time.

Amino acids were found to be responsive both quanti-
tatively and qualitatively to the temperatures at which the
plants v/ere grown.

At all stages of growth, the total free amino acid
content of leaves of pea plants grown at high temperature
vjas higher than that of plants gro\;n a'c optimum temperature.
There were, hovjever, changes in concentration at various
stages of growth in both the high temperature and optimum
temperature plant material. The total amino acid in leaves
dropped after two weeks of growth under both temperature
conditions; however, the drop in the plants grown at optimum
temperature was greater than in the plants grovm at high
temperature. At later times, the free amino acid content
of leaves of plants grown at high temperature remained al-
most constant while continuing to decrease in the plants
grown under optimum temperature conditions. Since fruit set

Ill

was observed under optiin.uirL conditions by about the fiftli
week of growth, but never at high, temperature, it was con-
sidered that at least part of the difference in amino acid
content in the last sanipiings might have been due to changes
in metabolism in passing from a vegetative to a reproductive
stage in the plants grovm at optimum temperature.

Although most individual amino acids v/hich v;ere
quantitatively measured were at least somewhat higher in
the plants grown at high temperature, some were more markedly
increased than others. Glutamic and aspartic acids and, to
a greater extent, their amides, glutamine and asparagine,
were at a very much higher concentration at the high
temperature regime, especially during the first week or two
of the life of the plants. Alcohol-soluble nitrogenous
constituents which appear to bo small peptides, and also an
amino acid tentatively identified as methionine sulfoxide,
were greatly elevated in the plants growing under the high
temperature regime. An amino acid provisionally identified
as a-amino adipic acid appeared in extracts of leaves of
young plants grovm at high temperature and increased with
increasing age of the plants until it became one of the
major components of the free amino acids of older plants.
On the other hand, the amount of this amino acid in leaf
extracts of plants grovm at optimum temperature was very
low after a week of growth, and after tv/o weeks it dis-
appeared completely.

112

Less extensive analyses of roots (exarained only at
t\\ro weeks after planting) also indicated similar effects of
high temperature on the free amino acid content of roots.
That is, the amino acid content was higher in roots of
plants grown at high temperature than in roots of plants of
the same plastochron index grown at optimum temperatures.
However., with respect to certain individual amino acids
there v/ere some quantitative differences from the effects
ohserved in leaves.

Total protein content of leaves also vxas determined
at v;eekly intervals throughout the vegetative stage and into
the reproductive stage in two groups of plants grown at two
temperature regimes. As in the case of amino acids, there
were marked differences "between the two groups. However,
in contrast to the results on free amino acids, there was
less protein in the leaves of the high temperature plants
throughout the vegetative stages. I-Jhen fruit set occurred
in the optimum temperature series, the protein content
dropped until it \iras equal to that of the high temperature
plants (the latter did not set complete fruit).

The increase of free amino acids and decrease in
protein content ohserved at high temperature suggests that
the free amino acids were produced at the expense of the
protein, that is, hy increased protein degradation. This
would he- in agreement with the hypothesis of Petinov (60).

115

Petinov showed, in limited semi-quantitative studies, that
amino acids remained at higher concentration in excised
leaves held at high temperature even when inhihitors oi the
Krehs cycle v;ere applied to the tissues; that is, when amino
acid synthesis from carbohydrate was supposedly blocked by
the inhibitors.

Certain features of these data do not support the
protein degradation hypothesis as the only reason for' amino
acid increase at high temperature, but rather, tend to
support the notion that the increase in free amino acids
resulting under the stress of high temperature v/as the re-
sult of increased synthesis as suggested ''oj Steward (70).
She work of Steward also involved environmental sti-ess (in
the mint plant, Mentha piperita) other than temperature,
such as photoperiod and nutritional variables. It was shov/n
that there was an increase in Krebs cycle acids and a-keto
acids at the same time that an increase in amino acids
occurred. V^Tiile no data were obtained on the Krebs cycle
acids and/or a-keto acids in this present study, an eleva-
tion of non-protein amino acids at high temperature was
observed. In fact, this increase even in some cases exceeded
the increase in protein amino acids. Further, the changes
in amino acid content with stage of maturity were not re-
flected in inversely proportional changes in protein content.

114

Attesipts v;ere made to prevent or cure by daenical
means tlie "climatic lesions" induced by high temperature.
Leaf sprays of various metabolites such as sucrose, a
vitamin B complex mixture, vitcimin C and a mixture of
nucleosides did not appreciably alter the effects of high
temperature.

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BIOGRAPHICAL SKETCH

The author, Esmail Hosseini Shokraii, was horn in
Tehran, Iran, May 1, 1935. He received his elementary
education in Tehran and graduated from Alhorz High School,
June, 195^.

He entered Tehran University, Tehran, Iran, in
September, 195^, and received the degree of Licentiate of
Biological Sciences in July, 1957. He was employed as
Instructor of Plant Physiology at the Faculty of Science,
Tehran University and at the same time continued graduate
studies and received the degree of Post Licentiate of
Botany in June, I960, and since then worked as assistant
professor of plant physiology until July, 1962. In July,
1962, he received a Pulhright Travel Award, a Fellowship
from the American Friends of the Middle East and a non-
resident tuition scholarship from the University of Florida
for graduate studies in the Botany Department of the
University of Florida, He received a Research Assistant-
ship from the Agricultural Experiment Station during the
second and third years of graduate studies. He worked ■
toward the degree of Doctor of Philosophy in Plant Physiology
in the Botany Department which he received in April, 1965.
He is a member of Alpha Zeta.

122

This dissertation was prepared under tlie direction of
the chairman of the candidate's supervisory committee and
has been approved by all members of that committee. It has
been submitted to the Dean of the College of Agriculture and
to the Graduate Council , and was approved as partial ful-
fillment of the requirements for the degree of Doctor of
Philosophy.

April 2^, 1965

^

^^jI^.^

an, College of Agriculture

Dean, Graduate School

SUPERVISORY COMMITTEE:

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