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The effect of supraoptimal temperatures on the growth and Kreds' cycle acid contents of Arabidopsis thaliana

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Title:
The effect of supraoptimal temperatures on the growth and Kreds' cycle acid contents of Arabidopsis thaliana
Creator:
Shiralipour Soltanabadi, Azizollah, 1935-
Publication Date:
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English
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67 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Plant physiology ( lcsh )
Plants, Effect of temperature on ( lcsh )
Growth (Plants) ( lcsh )
Botany thesis Ph. D
Dissertations, Academic -- Botany -- UF

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1966.
Bibliography:
Includes bibliographical references (leaves 61-64).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Azizollah Shiralipour Soltanabadi.

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Full Text
THE EFFECT OF SUPRAOPTIMAL
TEMPERATURES ON THE GROWTH AND
KREBS' CYCLE ACID CONTENTS OF
ARABJID OPSIS THALIANA
By
AZIZOLLAH SHIRALIPOUR SOLTANABADI
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA December, 1966




ACKNOWLEDGMENTS
The author wishes to express his deep gratitude to Professor D. S. Anthony., chairman of the supervisory committee, for his close and continuous supervision during the course of this research and the preparation of this dissertation.
He also would like to extend his appreciation to Dr. T. E.
Humphreys for his valuable suggestions and for the use of his equipment.
The writer is also grateful to Dr. H. H. Luke and Dr. W. L.
Pritchett', members of his committee,, for their advice and suggestions during the course of his research.
Special appreciation is extended to Dr. L. A. Garrard for his suggestions made during the course of this research and his guidance in-preparing this manuscript.
The author acknowledges with much gratitude the patience, encouragement and help of his wife, Joan, without whom this degree could not have been completed.




TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ......................... ii
LIST OF TABLES ......... ........................... iv
LIST OF FIGURES ........ ............................ v
INTRODUCTION ............. .......................... 1
MATERIALS AND METHODS ........ ...................... l.11
RESULTS AND DISCUSSION ....... ...................... ... 23
Effect of Supraoptimal Temperature
on Growth Characteristics .... .............. ... 23
Chemical Cure of the "Lesions" Caused
by Supraoptimal Temperatures .... ............ .. 32
Effect of Supraoptimal Temperature on the Contents of
the Krebs' Cycle Acids ............... 42
S...................................... 58
LITERATURE CITED .......... ......................... 61
BIOGRAPHICAL SKETCH ........... ...................... 65
iii




LIST OF TABLES
Table Page
1. GROWING CONDITIONS IN GROWTH CHAMBER . . . . . 12
2. PER CENT RECOVERY OF ORGANIC ACIDS . . . . . 20
3. EFFECT OF TEMPERATURE ON LENGTH OF THE MAIN ROOT OF
ARABIDOPSIS THALIANA . . . . . . . . . 26
4. EFFECT OF TEMPERATURE ON NUMBER OF SECONDARY ROOTS OF
ARABIDOPSIS THALIANA . . . . . . . . . 28
5. EFFECT OF TEMPERATURE ON LENGTH OF THE STEM OF
ARABIDOPSIS THALIANA . . . . . . . . . 29
6. EFFECT OF TEMPERATURE AND ADDED METABOLITES ON NUMBER
OF SEED PODS AND SEEDS OF ARABIDOPSIS THALIANA . . . 31
7. EFFECT OF TEMPERATURE AND VARYING CONCENTRATIONS OF
SUCROSE ON FRESH WEIGHT OF ARABIDOPSIS THALIANA . . 39
8. EFFECT OF TEMPERATURE AND VARYING CONCENTRATIONS OF
SUCROSE ON DRY WEIGHT OF ARABIDOPSIS THALIANA . . . 40
9. EFFECT OF TEMPERATURE ON ORGANIC ACID CONTENT OF THE
SHOOT OF ARABIDOPSIS THALIANA 2 WEEKS AFTER PLANTING . h44 10. EFFECT OF TEMPERATURE ON ORGANIC ACID CONTENT OF THE SHOOT OF ARABIDOPSIS THALIANA 3 WEEKS AFTER PLANTING . 45 11. EFFECT OF TEMPERATURE ON ORGANIC ACID CONTENT OF THE SHOOT OF ARABIDOPSIS THALIANA 4 WEEKS AFTER PLANTING . 47
iv




LIST OF FIGURES
Figure Page
1. Loss of organic acids on storage ...... .......... 18
2. Effect of varying temperatures on fresh weight of
the plant .......... ........................ 25
3A. Effect of added metabolites on fresh weight of plants
grown at optimal temperature ....... ............... 34
3B. Effect of added metabolites on fresh weight of plants
grown at supraoptimal temperature ...... ............ 34
4A. Effect of added metabolites on dry weight of plants
grown at optimal temperature ... ........... ..36
4B. Effect of added metabolites on dry weight of plants grown at supraoptimal temperature ... ............ 36
5. Effect of temperature on citric acid content at
different ages ........... ...................... 49
6. Effect of temperature on malic acid content at
different ages ......... ...................... 51
7. Effect of temperature on Succinic acid content at
different ages ......... ...................... 53
8. Effect of temperature on fumaric acid content at
different ages ......... ...................... 55
9A. Effect of optimal temperature on organic acid content at different ages .................... 57
9B. Effect of supraoptimal temperature on organic acid content at different ages ....... ... ... 57
v




INTRODUCTION
Many of the environmental variables affecting the growth of plants have been brought under control, but temperature is still a major limiting factor (Davern, 1959). Although a large amount of information concerning the effects of supraoptimal temperatures on plant growth and development is available, knowledge of the biochemical effects of such temperatures is limited. Since much of the data concerning the effects of temperature on the chemistry of growth processes of plants were derived from experiments with bacteria and algae, further biochemical studies on higher plants are necessary.
It was suggested that, under some extreme temperatures, plant growth is depressed by inactivation of one or more reactions which are sensitive to such temperatures (Langridge and Griffing, 1959). This depression in growth caused by extreme temperatures was termed "climatic lesions" by Bonner (1957). The causes of lesions produced in plants by supraoptimal temperatures are not yet known, although several theories have been proposed by different workers.
Some of these theories have been reviewed by Langridge (1963). Levitt (1951, 1962) believes that frost, heat and drought injuries are related and are all caused by denaturation of the protoplasmic proteins. The thiol or sulfhydryl groups in adjacent protein molecules in the cells of the plants grown in optimal temperatures are
1




2
sufficiently far apart to preclude formation of disulfide bridges between adjacent molecules. Adverse climatic conditions supposedly result in intracellular rearrangement in which free thiol groups of protein constituents closely approach each other with the subsequent formation of disulfide bonds. When optimum conditions again prevail, protein denaturation results from the tensions created by this intermolecular bonding.
It has been shown that the elevation of temperature from optimal to supraoptimal has a pronounced effect on the respiration of higher plants (Went, 1957; Petinov and Molotkovsky, 1957; Geromino and Beevers, 1964). The decrease in respiratory activity as a result of supraoptimal temperatures is believed to result from protein denaturation (Went, 1957).
Self-toxification by ammonia resulting from intensified proteolysis is one of the earliest views explaining the cause of "plant destruction" ("lesions" in Bonner's terminology)induced by supraoptimal temperatures. The toxic action of ammonia, formed endogenously in the plant was reflected by impaired respiration, wilting and eventual death of the plant (Petinov and Molotkovsky, 1957).
Decrease in availability of gases is another hypothesis for the cause of heat injury. Borek and Waelsch (1951) have shown that, when the bacterium Lactobacillus arabinosus was transferred from optimal to supraoptimal temperatures, addition of phenylalanine, tyrosine and aspartic acid became necessary for growth unless CO2 tension was increased. These amino acids were also required at optimal temperatures,




3
if the bacteria was grown under reduced CO2 tension. These observations suggest that "lesions" may result from decreased CO2 availability.
Pollock (1945) and Knox (1953) were among the first who showed that the formation of a particular enzyme was inhibited by supraoptimal temperatures, although the enzyme itself had maximum stability at such temperatures. This particular effect of supraoptimal temperatures, in inhibiting enzyme formation has been observed also by other workers (Pollock, 1950; Knox, 1951; Bernheim, 1955; Halpern, 1961). According to Bernheim (1955) inhibition of enzyme formation results from the destruction of RNA during exposure to supraoptimal temperatures.
Inactivation of enzymes at supraoptimal temperatures is another hypothesis under consideration. In this regard,Rahn and Schroeder (1941) found that treating Bacillus cereus at 460 for 10 minutes reduced the activity of peroxidase by 14 per cent and catalase activity by 20 per cent.
Supraoptimal temperatures can accelerate the breakdown of
enzymes and other metabolites. Organisms which can function at such temperatures must synthesize protein at rates sufficiently high to replace the enzyme protein broken down by heat (Allen, 1950).
The uncoupling of respiration and oxidative phosphorylation
caused by supraoptimal temperatures is another theory for the explanation of heat injury. It has been shown (Molotkovsky, 1961) that the activity of ATP-.ase decreased with increasing temperatures, but the amount of inorganic phosphate increased continuously in excised leaves.




4
Since exposure of plant tissues growing at optimal temperatures to 2,4-dinitrophenol also resulted in the accumulation of inorganic phosphate, it was concluded that the observed accumulation of inorganic phosphate in plants grown at supraoptimal temperatures resulted from the formation of uncoupling agents at such temperatures.
It has been shown that the growth of many organisms ceases at temperatures only slightly above optimal, but that normal growth can be restored by the addition of only a single organic substance. However, by raising the temperature a little higher an additional substance becomes necessary to counteract the temperature inhibition of growth. Thus, as the temperature is increased, these additive requirements to restore growth become progressively more numerous. Apparently, higher temperatures do not affect the genes because the growth rate can be fully restored by transferring the plants to optimal temperatures (Langridge, 1963).
Bonner's proposal of the possibility of .,chemical cure of climatic lesions" is of particular interest because of its considerable potential economic importance. Although the higher plant is considered an autotrophic organism, it has been possible to cure temperatureinduced lesions in a few varieties of a few species by external supplies of organic compounds.
The first so-called temperature-sensitive mutants Neurospora crassa and Escherichiacoli have been extensively studied (Atwood and Mukai, 1953; McElroy and Mitchell, 1946; Maas, 1950; Maas and Davis, 1952; Mitchell and Houlahan, 1945). It has been shown that a mutant




5
of Neurospora could grow at or below 250, but that growth decreased at temperatures above 25 and was completely arrested at 280. The addition of adenine to the medium partially restored growth; however,
normal growth was obtained only by the addition of adenine, histidine and methionine. Histidine and methionine were ineffective when supplied singularly or together in the absence of adenine. Other mutants with different requirements for protection against heat injury have been found. In this regard a riboflavin-requiring, temperature-sensitive mutant (Mitchell and Houlahan, 1945), a uracil-requiring mutant of Neurospora(Houlahan and Mitchell, 1947) and a pantothenate-requiring, temperature-sensitive mutant in E. coli (Maas, 1950; Maas and Davis, 1952) should be mentioned.
The removal of supraoptimal temperature inhibition of growth in excised pea stem sections by adenine has been studied by Galston and Hand (1949). They showed that high temperature lesions in Pisum sativum can be greatly reduced by the application of adenine as a foliar spray. A temperature-adenine growth response interaction was obtained. The amount of growth obtained by application of adenine at 300 was half the increment obtained at 350. These results indicate that high temperature growth inhibition in pea plants could be in part due to an adenine deficiency. However, additional studies (Galston, 1957) in the temperature-controlled facilities of Earhart Laboratory gave a little, if any, indication that adenine was effective in reducing supraoptimal temperature lesions. In this experiment, the day temperature was kept constant and the night temperature was varied from




6
low to high levels. This could indicate that the adenine limitation was the result of high temperatures during the day period.
In another experiment (Galston, 1959), with a different temperature regime but with the same variety of pea (night temperature was 60 lower than the day temperature), the investigator was unable to show any growth response to adenine at any temperature tested. Despite these negative results from studies with whole plants, the adenine effect may have some significance in that the total adenine level of a high temperature-tolerant pea strain increases with increasing temperature, while the total adenine level of a supraoptimal temperature-sensitive strain decreases with increasing temperatures (Highkin, 1958). These changes in adenine level may reflect changes in RNA status associated with meristematic activity, which are the results and not the cause of primary lesions. In the whole plant other supraoptimal temperature-induced lesions could be occurring which would preclude the detection of an adenine effect unless these other lesions were also removed. The results of Galston and Hand (1949) were confirmed by Highkin (1958). On the other hand, other workers did not find the. application of adenine to be effective for the removal of the "lesions" caused by supraoptimal temperatures in pea plant (Lockhart, 1958; Ketellapper, 1963). However, it was found that a vitamin B mixture or ribosides increased the dry weight up to 40 per cent at supraoptimal temperatures (Ketellapper and Bonner, 1961). Sucrose was found to be effective at temperatures only slightly above optimal but not at still higher temperatures (Ketellapper, 1963). This suggests that the effect of sucrose was temperature specific.




7
Adenosine and guanine were added to Lemna minor cultured at
supraoptimal temperatures by McCune (1956) who observed that they prevented the death of the plants at such temperatures.
A genotype-dependent auxin limitation in two varieties of corn was investigated and was shown to be affected by temperature (Galston, 1957). The "silkiness" variety gave a 50 per cent elongation response
to addition of indoleacetic acid at 200 day and 150 night temperatures where the "normal" variety gave a 16 per cent elongation response to the
application of auxin while the "silkless" variety showed no response.
The application of gibberellin to Alaska peas which were growing under a supraoptimal temperature regime gave a striking effect in delaying the supraoptimal temperature-accelerated senescence which accompanies the onset of maturity (Lockhart, 1958).
Spraying casein hydrolysate on clover plants Trifolium subterraneum grown under supraoptimal temperatures, caused a significant alleviation of supraoptimal temperature lesions whereas the plants did not respond to hydrolysate under optimal conditions (Davern, 1959).
Petinov and Molotkovsky (1957) reduced the supraoptimal temperature lesions in sunflower, corn, pumpkin and oats by spraying the leaves with organic acids. Citric and malic acids were particularly effective in this regard.
In working with Arabidopsis thaliana a very clear-cut case of effective chemotherapy of high temperature growth and developmental inhibition has been obtained by Langridge and Griffing (1959). Plants were grown with aseptic culture techniques and under controlled




8
environmental conditions. Forty-three different races were grown under three different temperature regimes, 250, 300 and 31.5. Five races (PI, BLA, DI, HI, and LS) showed particular sensitivity (depressed in growth and morphologically abnormal) to the 31.-5 temperature regime; three of these (PI, LA, and BLA) responded to chemical supplements. Further study showed that in two races (PI and BLA) addition of 6 or
3 pg/plant of biotin to the culture medium completely prevented the supraoptimal temperature lesions. The third race (LS) partially responded to cytidine applied at the rate of 0.25 mg/plant. Two other races (DI and HI) did not respond to any applied supplements. It is possible that the metabolites required by nonresponding races were not present in the supplements which were tested, although the yeast extract used might be expected to contain most of the diffusible substances needed by plants. Of course, it is also possible that certain applied supplements were not taken up by the plants.
The "lesions" caused by supraoptimal temperatures may be completely eliminated by providing the plant with one or more metabolites which have been destroyed by such temperatures. Since the replacement of temperature-sensitive cultivated plants by their temperatureresistant alternate varieties is a slow and limited process and because of the great economic importance of this problem, it seems reasonable to direct more attention to the "chemical cure of climatic lesion.1 The ideal compounds are those, which do not affect the plant growth under optimal temperatures but improve its growth under supraoptimal temperatures (Langridge and Griffing, 1959).




9
In general, very little is known about specific biochemical compounds or kinds of metabolism which are particularly sensitive to supraoptimal temperatures in higher plants. There are relatively few published reports showing the quantitative effects of supraoptimal temperatures on specific biochemical constituents of higher plants. In this respect, the effect of day length, mineral nutrition, and night temperature on nitrogenous compounds (Rabson and Steward, 1959), the effect of supraoptimal temperatures on free amino acids (Petinov and Molotkovsky, 1960; Shokraii, 1965), some fragmentary data on organic acids (Geromino and Beevers, 1964; Kliewer, 1964) and a recent study on RNA content (Ying, 1965; Brown, 1965) should be mentioned. In the biochemical work reported in this paper, attention will be confined to the effect of supraoptimal temperatures on the organic acids of the Krebs' cycle. In order to understand the biochemical effects of temperature on plants the effect of such temperatures on the individual life and growth processes must also be known. Growth, especially elongation of cells, has a high Q105 indicating a chemically rather than physically controlled phenomenon (Chao and Loomis, 1947). Due to this fact, a variety of growth measurements as well as chemical analyses were made.
There are numerous reports concerning the effect of temperature on growth and development of plants. Some of these have been reviewed by Went (1953) and Langridge (1963).
For the kind of work presented here it is imperative to use aseptic methods, since this eliminates at least one variable, the




10
microorganisms. It is also important to select a. small plant which has
a short life cycle and which is sensitive to supraoptimal temperatures. The ideal test plant should be able to be grown aseptically on an agar medium in test tubes. This permits easy application of additives for the "chemical cure of climatic lesions" to the agar medium, and allows many replications in a small space.
A plant belonging to the tribe Arabideae of the Cruciferae
family, Arabidopsis thaliana (L.) Heynh (Variety PI, mouse-ear kress), was selected as meeting all of the requirements as a desirable test
plant for these studies. It is a very small plant with a short life cycle (24 1 days), is sensitive to supraoptimal temperatures, and can be grown aseptically on agar medium in test tubes.




MATERIALS AND METHODS
A. Plant Materials and Growing Conditions
Seeds of Arabidopsis thaliana were soaked in sterilized, distilled water between two filter papers in a petri dish. The petri dish was placed in a refrigerator for 24 hours, at 40. This cold treatment insured more uniform germination of the seeds
(Langridge, 1957). Following their cold treatment, the seeds were washed for 3 minutes in 10 per cent Chlorox solution after which they were washed several times with distilled water. The seeds were then transferred to sterile agar in 150 X 25 mm (pyrex) test tubes.
The agar medium was prepared by a method modified from that of Langridge (1957). A 0.8 per cent agar solution was prepared by adding 8.0 g of agar (Bacto-Agar, Difco Laboratories, Detroit, Michigan) to one liter of Hoagland solution (Hoagland and Arnon, 1938).
The pH of the resulting agar suspension was adjusted to pH 6.0 with concentrated KOH. The agar suspension was liquified by placing in an autoclave at 125O and 15 psi for 20 minutes. Aliquots of approximately 10 ml were placed in the test tubes, and the tubes were
plugged with nonabsorbent cotton. The culture tubes were then sterilized in an autoclave at 1250 and 15 psi for 15 minutes After removal from the autoclave and solidification of the agar, the seeds were transferred to the surface of the medium under aseptic conditions
11




12
in a transfer box. The culture tubes were then placed in growth chambers set to provide either a supraoptimal temperature or an optimum
temperature regime. The temperatures were those actually observed for the agar medium (Table 1).
TABLE 1
GROWING CONDITIONS IN GROWTH CHAMBER
Temperature Phototemperature Nyctotemperature Light
Condition (16 hr) (8 hr) Intensity
Optimum 250 1 180 1 1100 200 f.c.
Supraoptimal 320 1 25o 1. 1100 200 f.c.
The source of light was a combination of fluorescent (cool white) and incandescent bulbs.
B. Growth Measurements
The seedlings were observed daily for a 5-week growth period
during which a number of growth measurements were made. These measurements included: length of the primary root and number of secondary roots, length of main stem, number of leaves, number of seed pods and seeds, and the fresh and dry weights of the shoots at 2, 3 and h weeks of age. In order to determine the effects of supraoptimal temperatures on the growth of the plants and the effect of various chemicals in curing "climatic lesions" resulting from supraoptimal temperatures the




13
following procedure was followed: groups of ho seedlings each were
grown under both supraoptimal and optimal temperature regimes with or without chemical additives in the agar medium. On the first day following germination five more uniform plants were selected from each treatment to be used for daily growth measurements. Additional five seedling samples were selected at random at 1, 2, 3 and 4 weeks
after planting for the determination of fresh and dry weights. Each experiment was replicated 2 or 3 times.
C. Chemical Analysis
Preparation of Tissue Extracts. Alcoholic extrac ts were prepared from the shoots of plants at 2,, 3 and h weeks of age for the determination of the organic acids of the Krebs' cycle. Whole *shoots (1-2 g) were cut in small pieces and extracted for 5 minutes in 50-100 ml of boiling 80 per cent ethanol. The extract was cooled to room temperature and filtered through Whatman No. 1 filter paper. The plant residue was homogenized in a glass homogenizer with an additional 40 ml of hot 80 per cent ethanol. This extract was again filtered through Whatman No. 1 filter paper and was combined with the
first filtrate. The volume of the combined filtrate was reduced under vacuum in a rotary evaporator at room temperature. The concentrated
0
filtrate was centrifuged at 10300 X g for 20 minutes at 0 Te supernatant fraction was collected by decanting and the pellet was taken
up in a smalll amount of distilled water. The suspended pellet was. again centrifuged and the supernatant fraction combined with that from




14
the first centrifugation. The volume of the supernatant was reduced
to 10 ml.
The concentrated supernatant was passed through a Dowex 50 X 8 (Hydrogen form) cation-exchange column (150 X 12.7 mm) and the column was rinsed by subsequent passage of 100 ml of distilled water. The
rate of flow of the column was controlled to give 10 drops/minute or less. The amino acids of the extract remained on this column while
the eluate contained the organic acids of the Krebs' cycle. The volume of the eluate was reduced to 10 ml before further separation.
Separation of Organic Acids. The organic acids of the eluate from the Dowex 50 column were then separated on a Dowex 1 X 8 anionexchange column in the formate form (Luke and Freeman, 1965). Dowex
1 X 8 resin in the chloride form was washed successively with portions of 2 N formic acid until chloride could no longer be detected. The resin was then freed of excess acid by washing with distilled water. The resin (now in the formate form) was oven-dried at 50-600. A 150 X 6.5 mm column was prepared by placing 3 g of the dried resin in a 500 X 6.5 mm glass tube. The tip of the tube was tapered and plugged with a small piece of glass wool. The resin bed was capped with another glass wool plug. The column was then washed with approximately 100 ml of distilled water. The concentrated eluate from the Dowex
50 X 8 column was placed on the Dowex 1 X 8 column and followed by 100 ml or more of distilled water. The rate of flow as adjusted to
give 10 drops/minute. In this case, the eluate was discarded. The




15
organic acids remained on the column. The organic acids were eluated from the column with 25 ml of 90.5 per cent formic acid (J. T. Baker Chemical Company, Phillipsburg, N.J.) followed by 100 ml of distilled water. The eluate containing the various organic acids was taken to dryness under reduced pressure and the residue was taken up in an appropriate volume of 10 per cent isopropanol to give 1 ml of solution/g original fresh weight of tissue.
Paper Chromatography. Qualitative determination and quantitative measurements of the organic acids of the Krebs' cycle were made by paper chromatography of the concentrated tissue extracts. Aliquots of 50 pl of the concentrated extracts were chromatographed ascendingly on strips of Whatman No. 1 filter paper (50 X 4 cm) in N-butanol formic acid : water (10 : 2 : 15v/v/vupper phase). The strips were equilibrated for 5 hours with a portion of the solvent before being developed for a period of 36 hours. Equilibration and development were conducted at 240 20. The developed paper strips were initially dried at room temperature for a period of 5 hours after which they were subjected to a series of alternate 10-minute periods of forced hot (700) and cool air to remove all traces of solvent. A color developer for observing and determining the amount of the organic acid (Smith, 1958) was made by placing 1 g of xylose in 3 ml of distilled water and then adding 1 ml of aniline; the final volume was made up to 100 ml with methanol. The paper strips were dipped in the developer and then were
0
air dried for 30 minutes. They were then heated at 70-80 for 20-30




16
minutes. The organic acids appeared as reddish-brown spots on a white background. Although it was difficult to obtain a white background, by carefully following the above procedure it was accomplished. Quantitative measurements were made by eluting the spots in 50 per cent ethanol and determining the absorbance of the solutions at 395 mp in a Spectronic 20 (Bausch and Lomb) spectrophotometer. Standard curves were prepared by spotting known amounts of organic acids directly on the paper.
Effect of Storage on Organic Acids. In preparing standard curves from solutions of known amounts of organic acids which were stored at -15, a reduction in the slope of the curves with duration of storage was noticed. This suggested some loss in the amounts of organic acids. In order to determine the extent of loss due to storage (probably via esterification), mixtures of authenic acids were prepared in 10 per cent isopropanol and also in 80 per cent ethanol. Standard curves were obtained for both mixtures at the time of preparation and following storage for periods of 1, 2, 3 and 4 weeks.
A typical loss in acid content of stored solutions, as shown for citric acid, is given in Figure 1. As can be seen, the amount of loss is higher when acids are stored in 80 per cent ethanol. Accordingly, storage of standards and extracts in alcohol was eliminated and all the analyses were done immediately after harvesting the plant.
Per Cent Recovery of Organic Acids. The per cent recovery of organic acids was determined by addition of known amounts of authentic




Figure 1. Loss of organic acids on storage.




18
IN 10% ISOPROPANOL, 0,--**--*
NO STORAGE PERIOD .1 N 80% ETHANOL, NO STORAGE PERIOD IN 10% ISOPROPANOL, STORED FOR 4WEEKS IN 80% ETHANOL,
STORED FOR 4WEEKS
0.5
0.4
9
Lo -0.2
, 0.1
0 -o I I I I
0 20 40 60 80
/G OF' CITRIC ACID ADDED TO THE PAPER
FIG. I




19
organic acids to the plant extracts. The difference between the values obtained from the plant extract alone after going through the entire preparative procedures and those obtained from plant extract plus the known amounts of acids -was compared to a standard curve. This standard curve was prepared from a known mixture of authentic organic acids spotted directly on the paper. The per cent recovery of the different acids is given in Table 2. Subsequent determinations of organic acids in experimental material were corrected for recovery, using the average recoveries given in Table 2 as the recovery factors. D. Chemical Treatments
Various chemicals for treatment of supraoptimal temperature I"lesions" were added to the agar medium under aseptic conditions. These additives included: sucrose, biotin, phosfon-S (2,4 dichlorobenzyl tributyl ammonium chloride, Virginia-Carolina Chemical Corporation) and malic acid. A series of concentrations of sucrose (1-5%) in agar was prepared in the test tubes and the standard procedures for handling seeds and seedlings were followed. Since phosfon-S, malic acid and biotin are unstable to heat, they were not mixed with the agar solution in the test tubes before autoclaving the agar. In the case of phosfon-S the seeds were soaked in a solution containing 20 p.p.m. of the material before they were transferred to the agar medium. Stock solutions of biotin and malic acid were sterilized by passing the solutions through membrane filters (pore diameter 0.22 p). Aliquots containing either 6 pg of biotin or 3.35 mg of malic acid




TABLE 2
PER CENT RECOVERY OF ORGANIC ACIDS
Recovery of Acids
Amount of Acid
Added o Ant Citric Malic Succinic Fumaric
Added to Plant
Extract Amount* Per Amount Per Amount Per Amount Per
(mg) (mg) Cent (mg) Cent (mg) Cent (mg) Cent
20 17 85 15 75 17 85 14 70
40 42 lO5 36 90 32 80 34 85
60 63 105 60 100 51 85 56 93.3
Average Recovery
Per Cent 98 88 83 83
The difference between plant extract alone and that recovered from plant extract plus the given amount of added organic acid.
0




21
were added to the sterile test tubes just before solidification of the autoclaved and still warm agar. Since the pH of the agar medium would change on addition of the malic acid, the pH of the 1 M malic acid was adjusted to 6.0 by addition of concentrated KOH before sterilization. Transfer of seed to the agar and procedures for handling seeds and seedlings were the same as given above.
Problems in Identification of Organic Acids. Citric, malic, succinic and fumaric acid were identified in extracts of Arabidopsis thaliana by paper chromatography. However, there were three unknown
compounds which gave positive organic acid color reactions., The unknown compounds had Rf values less than the known acids. These unknowns could not be successfully identified by co-chromatography with known organic acids.
Since the pKa's of the carboxyl groups of acidic amino acids were lower than the pH of the plant extract, it was assumed that these acids would not be held tightly by the Dowex-50 column. Accordingly the effluent from this column was checked to find out whether any amino acids had leaked off with the organic acids. Paper chromatograms of the effluent were sprayed with nirhydrin and it was found that there were two spots giving a positive ninhydrin test. These ninhydrinpositive spots corresponded in Rf values to the unknown spots having the two lowest Rf values in the chromatograms of the organic acids. Further testing by chromatography in other solvent mixtures identified these two amino acids as glutamic and aspartic acids. To obtain additional




22
information about the behavior of glutamic acid and aspartic acids under the conditions employed in our experiments, a sample of plant extract containing C14 labeled glutamic and aspartic acids was placed on the Dowex-50 column. It was found that 25 per cent of the glutamic acid and 20 per cent of the aspartic acid passed through the column. Acidification of the extract to pH 2.5 prior to passing through the column reduced the leakage of glutamic and aspartic acids to 19 and 14 per cent, respectively. However, the use of a finer resin (200-400 mesh) and increasing the column length (1.5 times) reduced the leakage of the mixture of radioactive glutamic and aspartic acids to 2.5 per cent. Therefore, the larger column and finer resin were adopted as standard for all data reported here. The third unknown (found at a greater Rf value than those for glutamic and aspartic acids) could not be unequivocally identified although the Rf values for the unknown when chromatographed in two different solvents were close to those for tartaric acid.




RESULTS AND DISCUSSION
Effect of Supraoptimal Temperature on Growth Characteristics
In order to determine the proper optimal and supraoptimal temperatures, plants were grown in different temperature regimes as shown in Figure 2. Fresh weights of the plants were determined at 1, 2, 3 and 4 weeks after planting. A regime of 25-day, 18-night temperatures was chosen for the optimal condition and 320-day, 25night temperatures was selected for the supraoptimal temperature treatments. Growth characteristics were measured under these two, temperature regimes with the following results.
A. Length of the Main Root
At 8 days after planting, supraoptimal temperatures had caused 45 per cent reduction in the length of the main root as compared to plants grown at optimal temperature (Table 3, data for basal medium only). The maximum increase in the length of the main root in plants grown under the optimal condition occurred at 7 days after planting, while under the supraoptimal temperature condition the maximum daily growth rate occurred at 4 days after planting. This could be due to acceleration in breakdown of seed reserves under the supraoptimal temperature regime.
23




Figure 2. Effect of varying temperatures on fresh weight of
the plant.




140
250 DAY, 180 NIGHT
2 230 DAY, 170 NIGHT
0----o 270 DAY, 210 NIGHT 210 DAY, 150 NIGHT
100 290 DAY, 230 NIGHT
0 O----O 320 DAY, 250 NIGHT
80 /
/////
K/ /
60
iz / //,
,:~~ //,",
40 3 4
WEEKS AFTER PLANTING
FIG.. 2




TABLE 3
EFFECT OF TEMPERATURE ON LENGTH OF THE MAIN ROOT OF ARABIDOPSIS THALIANA
Length of Main Root (mm)
Addition Time after Planting
to MW
Bs 3 days 4 days 5 days 6 days 7 days 8 days
Basal
Medium Ot St 0 S 0 S 0 S 0 S 0 S
None 2.7 1.7 6.5 5.1 9.9 7.6 13.8 9.5 18.7 10.8 21.9 12.0
Biotin 2.1 2.0 4.9 4.3 9.2 7.9 14.2 11.3 24.8 19.1 27.6 26.7
Malic Acid 2.5 2.4 5.9 4.8 9.3 7.3 10.5 9.1 11.4 10.6 12.1 11.8
Phosfon-S 2.4 2.7 6.3 5.6 10.7 8.4 14.6 10.6 18.7 13.0 21.3 14.4
1% Sucrose 2.7 2.5 7.5 4.8 11.3 7.1 14.8 9.1 21.4 12.0 24.1 14.6
*Average of 2 trials, 5 plants per trial.
*-No measurable root appeared prior to 3 days after planting. t 0 = Optimal temperature.
S = Supraoptimal temperature.




27
B. Number of Secondary Roots
The number of secondary roots of plants grown at supraoptimal temperature was reduced by 27 per cent at 12 days after planting (Table 4, data for basal medium only). Daily increases in the number of secondary roots were constant with time within each treatment.
Supraoptimal temperature slightly decreased the time required for the appearance of the secondary roots (Approximately 1 day); again, perhaps because of enhancement of the breakdown of the seed reserves.
C. Length of the Main Stem
During a 19-day period following planting, the length of the main stem and also the rate of stem elongation, were greater in plants
grown under supraoptimal temperature (Table 5, data for basal medium only). The maximum daily rate of elongation occurred at 21 days after planting at supraoptimal temperature and 23 days after planting at the optimal temperature.
D. Number of Seed Pods and Seeds
The numbers of seed pods and seeds were reduced under the
supraoptimal temperature regime (Table 6, data for basal medium only). Actually, the effect was on the number of the seed pods since the average number of seeds per pod remained unchanged under both temperature regimes (approximately 12 seeds per seed pod). However, the sizes of the seed pods and seeds were much smaller under the supraoptimal temperature condition.




TABLE 4
EFFECT OF TEMPERATURE ON NUMBER OF SECONDARY ROOTS OF ARABIDOPSIS THALIANA
Number of Secondary Roots
Addition Time after Planting
to
Basal 6 days- 7 days 8 days 9 days 10 days 11 days 12 days
Basal
Medium 0O St 0 S 0 S 0 S 0 S 0 S 0 S
None 0.0 0.3 0.9 1.1 2.8 2.3 4.3 3.1 6.2 4.3 7.3 5.8 9.2 6.7
Biotin 0.9 0.0 2.4 2.8 4.8 4.6 7.8 7.0 11.3 9.0 15.2 11.6 20.5 14.8
Malic Acid 1.2 0.0 4.4 2.4 5.8 3.9 7.3 5.8 9.0 6.7 10.7 7.8 12.8 10.8
Phosfon-S 0.0 0.0 0.0 1.0 1.4 2.3 3.3 3.4 4.1 4.1 5.1 5.1 7.2 6.3
1% Sucrose 0.0 0.4 0.0 1.6 1.2 2.7 1.9 4.1 3.6 5.4 4.0 7.3 4.9 8.8
Average of 2 trials, 5 plants per trial.
*No secondary roots appeared prior to 6 days-after planting.
0 = Optimal temperature.
SS = Supraoptimal temperature.
NC




-TABLE 5
EFFECT OF TEMPERATURE ON LENGTH OF THE STEM OF ARABIDOPSIS THALIANA
Length of Stem (mm)
Addition to Basal Medium
Days None Biotin Malic Acid Phosfon-S 1% Sucrose
after
Planting Ot St 0 S 0 S 0 S 0 S
1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
2 2.3 2.1 0.0 2.7 2.1 0.0 2.0 0.0 2.0 2.2
3 3.0 3.0 2.0 3.6 2.2 2.1 2.5 2.5 3.0 3.0
4 3.0 3.5 2.5 3.9 3.0 2.7 3.0 3.4 3.1 3.2
5 3.0 4.5 3.2 4.7 3.2 3.0 3.7 4.2 3.5 3.7
6 3.1 5.0 3.5 5.0 3.8 4.0 3.9 4.2 3.7 4.0
7 3.5 5.5 4.2 5.8 4.1 4.4 4.4 4.5 4.3 4.1
8 3.8 6.5 4.3 5.8 4.2 4.9 4.4 5.2 4.5 5.1
9 3.9 7.0 4.4 5.8 4.2 5.5 5.0 5.5 4.7 5.4
10 4.1 8.0 4.5 6.2 4.5 5.9 5.3 5.6 5.2 5.4
11 4.3 8.3 4.8 6.3 5.3 6.2 5.4 6.1 5.3 5.4
12 4.6 8.4 4.9 6.4 5.3 6.4 6.0 6.5 5.5 5.9




TABLE 5 (Continued)
Length of Stem (mm)
Days Addition to Basal Medium
after None Biotin Malic Acid Phosfon-S 1% Sucrose
Planting OT S4 0 S 0 S 0 S 0 S
13 5.0 9.0 5.3 7.2 5.4 7.4 6.1 6.5 5.7 6.5
14 5.5 9.6 5.6 8.0 5.7 8.5 6.5 7.1 6.4 7.2
15 5.8 10.5 6.0 10.0 6.2 10.3 6.8 7.5 6.8 8.7
16 6.6 11.9 6.7 13.9 6.4 13.0 7.3 8.3 7.4 11.0
17 7.9 15.1 7.7 18.3 7.4 16.8 8.0 10.3 8.7 15.8
18 10.8 22.0 9.4 26.3 9.2 23.7 9.2 15.8 12.0 28.1
19 17.1 32.7 14.1 47.9 12.6 33.3 12.3 21.9 17.6 46.4
20 29.4 44h.7 24.1 68.2 19.4 44.9 17.8 33.4 26.0 71.0
21 46.2 64.8 37.3 93.7 29.6 61.0 29.6 47.9 36.7 96.0
22 64.3 80.0 -- -- -- -- -- -- -- -23 87.1 96.3 -- -- -- -- -- -- -- -24 98.9 104.1 -- -- -- -- -- -- -- -Average of 2 trials, 5 plants per trial. S0 = Optimal temperature.
S = Supraoptimal temperature.
0




31
TABLE 6
EFFECT OF TEMPERATURE AND ADDED METABOLITES ON NUMBER OF SEED PODS AND SEEDS OF ARABIDOPSIS THALIANA
Addition to Number of Seed Pods* Number of Seeds*
Basal
Medium Ot S* 0 S
None 10.0 3.5 127.9 44h.h
Biotin 11.5 10.5 197.7 127.2
Malic Acid 10.2 6.0 248.9 59.8
Phosfon-S 10.9 7.0 166.4 81.2
1% Sucrose 16.0 7.6 235.1 '1105.2
Average of 2 trials, 5 plants per trial. o0 = Optimal temperature. S = Supraoptimal temperature.




32
E. Fresh and Dry Weights
The most striking effect of supraoptimal temperature on growth was reflected in the fresh and dry weights of the plant (Figures 3 and 4, data for basal medium only). The fresh and dry weights of the plants grown under supraoptimal temperature were reduced by 48 and 38 per cent, respectively, at 4 weeks after planting as compared to those plants grown under optimal temperature. The maximum rates of weekly increase in fresh weight were obtained at 4 weeks after planting at both optimal and supraoptimal temperatures. The effects of the different temperatures on dry weight were similar to those obtained for the fresh weight.
Chemical Cure of the "Lesions" Caused by Supraoptimal Temperatures
The effects of different metabolites on plant growth characteristics at different temperatures are given in Tables 3-8 and Figures 3 and 4. Dry and fresh weights were used as the main criteria for the comparison of the effectiveness of the added metabolites.
Biotin, sucrose, and phosfon-S exhibited positive effects in
alleviating detrimental effects of supraoptimal temperature. The addition of sucrose or biotin alone gave the most effective results, and hence a combination of both was tried under both temperature regimes.
Measurements of growth (which had to be terminated after 8 days in this case because of a malfunction of growth chambers) showed an even better
response to the combination as compared to the addition of sucrose or biotin alone to the plant medium.




Figure 3A. Effect of added metabolites on fresh weight of plants
grown at optimal temperature.
Figure 3B. Effect of added metabolites on fresh weight of plants
grown at supraoptimal temperature.




34
o BASAL MEDIUM ONLY A
160- Q BASAL MEDIUM + BIOTIN
A BASAL MEDIUM + PHOSFON-S
0 BASAL MEDIUM + 1% SUCROSE
120- BASAL MEDIUM + MALIC ACID
. 80-.
S40
0
B
- L160 o BASAL MEDIUM ONLY
0 BASAL MEDIUM + BIOTIN
A BASAL MEDIUM + PHOSFON-S
(6 120120 0 BASAL MEDIUM + 1% SUCROSE
a BASAL MEDIUM + MALIC ACID
80
00
40 -s_" sS
0-1
0 I 2 3 4
WEEKS AFTER PLANTING
FIG. 3




Figure 4A. Effect of added metabolites on dry weight of plants
grown at optimal temperature.
Figure 4B. Effect of added metabolites on dry weight of plants grown
at supraoptimal temperature.




36
* BASAL MEDIUM ONLY A
16- e BASAL MEDIUM + BIOTIN
A BASAL MEDIUM + PHOSFON-S
0 BASAL MEDIUM + 1% SUCROSE
12- BAA
2 BASAL MEDIUM + MALIC ACID
S8
*""6
- 1-J
.
0:4
0
B
0 BASAL MEDIUM ONLY
W 16 0 BASAL MEDIUM + BIOTIN
A BASAL MEDIUM + PHOSFON-S S12- 0 BASAL MEDIUM + 1% SUCROSE
a BASAL MEDIUM + MALIC ACID
8 -OIL
cri
4
0
ai? i I i
0 I 2 3 4
WEEKS AFTER PLANTING
FIG. 4




Biotin alone improved all the growth characteristics studied and prevented the detrimental effects of supraoptimal temperature. This promotion of growth as a result of biotin treatment was observed in both temperature regimes but the degree of the enhancement of growth was much higher at supraoptimal temperature. The increases in fresh and dry weights per plants grown under optimal temperature was 38 and 15 per cent, respectively, while under supraoptimal temperature the increases were 54 and 66 per cent, respectively.
These results are in agreement with those obtained by Langridge and Griffing (1959) for the same plant growing under constant 250 optimal and 31.50 supraoptimal temperatures.
Sucrose was even more effective than biotin in alleviating the injuries caused by supraoptimal temperature. The addition of 1 per cent sucrose to the medium increased the dry weight only 2 per cent under optimal temperature while the increase under supraoptimal was 70 per cent. Surprisingly, sucrose decreased the fresh weight under optimal temperature (12%) at the 4-week sampling period, while it increased the fresh weight by 58 per cent at the same time under supraoptimal temperature (Figures 3 and 4). In addition to increasing the fresh and dry weights, sucrose improved most of the other growth characteristics which were adversely affected by supraoptimal temperature (Tables 3-5).
Prior to application of 1 per cent sucrose, an agar medium containing 10 per cent sucrose as was suggested by Ketellapper and Bonner (1961) was used. However, seeds did not germinate at this high




38
concentration of sucrose perhaps because of unfavorable osmotic conditions. To determine the most suitable concentration of sucrose, an experiment with agar mediums containing 1-5 per cent sucrose was conducted under both temperature regimes. The fresh and dry weights of the plants grown under different sucrose concentrations were measured (Tables 7 and 8). The results obtained showed 1 per cent sucrose to be the most. suitable concentration for further experimentation.
A possible mechanism of the protective action of sugars at
supraoptimal temperatures has been suggested by Molotkovsky and Zhestkova (1964). They showed that application of several sugars, especially sucrose, prevented the drop in the rate of respiration at supraoptimal temperatures. Stabilization of respiration under the influence of sucrose was sufficient to render the plants insensitive to the action of respiratory inhibitors. They further showed that other sugars and even metabolically inert mannitol had a similar effect but to a lesser degree. Sucrose also prevented the uncoupling of oxidative phosphorylation and respiration as measured by accumulation of inorganic phosphate.
Referring to the work of Moshanskii (discussed by Molotkovsky and Zhestkova, 1964), which showed that mitochondria became swollen at
supraoptimal temperature with eventual dissociation of oxidative phosphorylation and respiration and that of Lehninger (also discussed by Molotkovsky and Zhestkova, 1964), which showed that sugars blocked the swelling of the mitochondria, they hypothesized that sugars have a ",conserving" effect on mitochondria and that this explains the "protective action" of sugars.




TABLE 7
EFFECT OF TEMPERATURE AND VARYING CONCENTRATIONS OF SUCROSE ON FRESH WEIGHT OF ARABIDOPSIS THALIANA
Fresh Weight per Plant (mg)
Per Cent. Time after Planting
Sucrose Added
Sucrose Added 1 week 2 weeks 3 weeks 4 weeks
to
Basal Medium Ot St 0 S 0 S 0 S
0 1.0 0.75 15.1 5.9 61.3 24.70 118.7 44.1
1 0.99 0.93 14.3 9.20 44.2 45.97 106.1 94.5
2 0.99 0.90 14.7 8.7 50.1 31.30 115.3 66.9
3 1.0 0.80 15.3 4.5 51.1 11.ho40 103.4 25.7
4 0.8 0.4o0 5.8 1.8 22.7 10.5 46.1 20.9
5 0.7 0.30 5.1 1.5 16.4 9.8 40.7 18.8
Average of 2 trials, 5 plants per trial. S0 = Optimal temperature.
SS = Supraoptimal temperature.




TABLE 8
EFFECT OF TEMPERATURE AND VARYING CONCENTRATIONS OF SUCROSE ON DRY WEIGHT OF ARABIDOPSIS THALIANA
Dry Weight per Plant (mg)
Per -Cent Time after Planting
Sucrose Added
o 1 week 2 weeks 3 weeks 4 weeks
to
Basal Medium O1 St 0 S 0 S 0 S
0 0.08 0.06 1.15 0.46 4.91 2.61 10.8 5.3
1 0.09 0.07 1.36 0.75 4.32 4.95 11.1 10.20
2 0.09 0.07 1.48 0.80 4.90 4.60 12.0 8.2
3 0.08 0.06 1.40 0.66 6.0 1.50 11.9 3.9
4 0.06 O.05 0.65 0.26 2.9 2.1 5.6 3.0
5 0.06 0.05 0.65 0.33 2.2 1.9 5.4 2.9
Average of 2 trials, 5 plants per trial. t 0 = Optimal temperature.
SS = Supraoptimal temperature. 0




41
Phosfon-S also gave positive results in alleviating some of the injuries caused by supraoptimal temperature (Tables 3-6) as well as increasing the fresh and dry weights of the plant (Figures 3 and 4). However, phosfon-S was not as effective as sucrose or biotin in increasing the fresh and dry weights of the plants grown at supraoptimal temperature. Phosfon-S caused a 27 per cent increase in the fresh weight under optimal temperature and a 30 per cent increase under
supraoptimal temperature. The increase in dry weight under optimal' and supraoptimal temperatures was 9 and 16 per cent, respectively.
The addition of malate to the medium reduced the fresh and dry weight of plants under both temperature regimes (Figures 3 and 4). It was also interesting to note a striking effect of malate on the seed pods and seeds. Addition of this metabolite resulted in the production of large seed pods and also in increases in both size and the number of seeds from plants grown at both temperatures (Table 6). The data fail to show any effect of malate in reducing or alleviating any of the detrimental effects of supraoptimal temperature on the
growth of these plants (Tables 3-6). In this regard, our results are largely in disagreement with those of Petinov and Molotkovsky (1957). They demonstrated the reduction of the injuries caused by supraoptimal temperature in sunflower, pumpkin, oats and corn by spraying the leaves of plants with organic acids, mainly citric and malic acids. However, the absence of ameliorative effects in our experiments could be because of two reasons: (1) Although malic acid was effective on plants used by Petinov and Molotkovsky (1957), it is possible that it is not




42
effective on Arabidopsis thaliana. In this regard it has been shown that even two species of the same genus may have two different requirements (Langridge and Griffing, 1959). (2) The supraoptimal temperature used by Petinov and Molotkovsky was extremely high (460) which could cause the accululation of ammonia and eventual self-toxification of the plant. In this case, the applied organic acids probably formed the organic salts of ammonia or amides thus removing the NH3 and eliminating the detrimental effects of the supraoptimal temperature. Under the temperature regime selected for our experiment, the accumulation of ammonia in concentrations which could be toxic to the plant seems improbable since Anastasia (1966) could not find any detectable amounts of ammonia in pea plants grown under the same temperature regimes.
Although the results obtained with sucrose and biotin were obviously significant, the data were statistically analyzed according to the t test. This test showed the results for dry and fresh weights to be highly significant at the .01 level.
Effect of Supraoptimal Temperature on the Contents of the Krebs' Cycle Acids
Organic acid (citric, malic, succinic and fumaric) contents of the shoots were measured at 2, 3 and 4 weeks after planting and the results are given in Tables 9-11 and Figures 5-9.
A. Two Weeks after Planting
The organic acid contents of the plants grown under supraoptimal temperature were usually slightly higher than those of the plants grown




43
at optimal temperature (Table 9). The ratios of various acids in the plants grown under supraoptimal temperature to those grown at optimal temperature ranged from 1 to 1.54 while the ratio of total acids was 1.21. The increase at supraoptimal temperature was greater for malic
acid than any other acid in this sampling period. Since the quantity of the other acids did not differ greatly under the two temperature regimes
it is possible that supraoptimal temperature caused either enhancement in formation of malic acid, probably from other pools (Lips and Beevers, 1966), or possibly affected the activity of the enzyme, malic dehydrogenase resulting in the accumulation of malic acid.
B. Three Weeks after Planting
In contrast to the results obtained at 2 weeks (and again at
4 weeks) the contents of the organic acids, with the exception of citric, were higher in plants grown under optimal temperature (Table 10). The total acid content (the sum of citric, malic, succinic and fumaric)
of plants grown under supraoptimal temperature was 42 per cent of the total found in plants grown at optimal temperature. The difference between fumaric acid contents in plants grown in these two temperature regimes was the most pronounced of all. One possible explanation for the reversal of the acid contents under these two different regimes between 2-week and 3-week periods is that flowering and maximum increase in the rate of stem elongation in plants grown under optimal temperature occurred at this time. The increase in stem length during the 21st day after planting was approximately equal to the total stem elongation obtained during the first 19 days of growth!




TABLE 9
EFFECT OF TEMPERATURE ON ORGANIC ACID CONTENT
OF THE SHOOT OF ARABIDOPSIS THALIANA
2 WEEKS AFTER PLANTING
p Moles per Gram Fresh Weight Organic Temperature
Acid Optimal Supraoptimal Ratio
Citric 4.8 5.1 1.06
Malic 9.3 14.4 1.54
Succinic 5.1 5.1 1.00
Fumaric 8.3 8.7 1.05
Total 27.5 33.3 1.21
Supraoptimal over optimal.




TABLE 10
EFFECT OF TEMPERATURE ON ORGANIC ACID CONTENT
OF THE SHOOT OF ARABIDOPSIS THALIANA
3 WEEKS AFTER PLANTING
p Moles per Gram Fresh Weight Temperature
Organic
Acid Optimal Supraoptimal Ratio
Citric 3.8 5.4 1.42
Malic 12.5 8.3 0.66
Succinic 5.8 3.7 0.63
Fumaric 19.1 6.6 O.35
Total l41.2 24.O 0.58
Supraoptimal over optimal.




46
C. Four Weeks after Planting
The amounts of organic acids, with the exception of fumaric acid, were higher in plants grown at supraoptimal temperature (Table 11). The most pronounced change caused by supraoptimal temperature occurred in the citric acid content (8.36 times higher in high temperature).
In general the amounts of organic acids increased at supraoptimal temperature at 2 and 4 weeks after planting. Citric acid was the only acid which remained higher in plants grown under supraoptimal temperature at all three sampling times.
In retrospect, it might have been better to check the effect of supraoptimal temperature on organic acids by sampling plants at the end of dark period, the end of light period, as well as at the intermediate time chosen for this study.
It should be -mentidned' at this point that the physiological
and chronological ages of the plants grown at optimal and supraoptimal temperatures were approximately the same at all sampling periods as shown by plastochrome indices.




47
TABLE 11
EFFECT OF TEMPERATURE ON ORGANIC ACID CONTENT
OF THE SHOOT OF ARABIDOPSIS THALIANA
4 WEEKS AFTER PLANTING
p Moles per Gram Fresh Weight Organic Temperature
Acid Optimal Supraoptimal Ratio
Citric 1.1 9.2 8.36
Malic 6.8 13.4 1.97
Succinic 4.3 5.3 1.23
Fumaric 12.5 8.9 0.71
Total 24.7 36.8 1.49
Supraoptimal over optimal.




Figure Effect of temperature on citric acid content
at different ages.




20- CITRIC ACID
16- OPTIMAL TEMPERATURE
SSUPRAOPTIMAL TEMPERATURE S12La:
Q) 8
(I)
S4
0
o
2 3 4
WEEKS AFTER PLANTING FIG. 5




Figure 6. Effect of temperature on malic acid content
at different ages.




MALIC ACID
20
SOPTIMAL TEMPERATURE
fl SUPRAOPTIMAL TEMPERATURE L16
L 12
S04
2 3 4
WEEKS AFTER PLANTING FIG. 6




Figure 7. Effect of temperature on succinic acid content
at different ages.




20
2SUCCINIC ACID S16:I I OPTIMAL TEMPERATURE
12 SUPRAOPTIMAL TEMPERATURE
8
O4
(I)
0
2 3 4
WEEKS AFTER PLANTING FIG. 7




Figure 8. Effect of temperature on fumaric acid content
at different ages.




FULKV\RIC ACID
OPTIMAL IEWPERATURE 20 SUPRAOPTIIAL TEMPERATURE
16
LU
FIG 8




Figure 9A. Effect of optimal temperature on organic acid content
at different ages. (C citric acid, M malic acid,
S succinic acid, F fumaric acid.)
Figure 9B. Effect of supraoptimal temperature on organic acid
content at different ages. (C citric acid,
M malic acid, S succinic acid, F fumaric acid.)




A7
F
18
L F
ZU M F
S0
B
18
m M
12
6A c SC. F S
2 3 4
WEEKS AFTER PLANTING FIG. 9




SUMMARY
The effects of supraoptimal temperature on growth characteristics and Krebs' cycle acid content of Arabidopsis thaliana (L.) Heynh were investigated. The small, rapidly-growing crucifers were grown under aseptic conditions at two temperature regimes (optimal was 250 during day and 180 during night; supraoptimal was 320 during day and 250 during night; light intensity was 1100 200 f.c. for both conditions) on agar in large test tubes. Various metabolites were added to the agar medium in attempts to prevent by chemical means the detrimental effects of supraoptimal temperature. The characteristics studied included root length, number of the secondary roots, stem length, number of seed pods and seeds, fresh weight, and dry weight. Supraoptimal temperatures depressed all of the measures of growth and reproductive performance investigated except for the length of the main stem. It was found that supraoptimal temperature caused a reduction of 45 per cent in the root length, 27 per cent in number of secondary roots, .65 per cent in number of seed pods, 65 per cent in the number of seeds, 48 per cent in fresh weight and 38 per cent in the dry weight of the plant. The main sbems of the plants grown under
58




supraoptimal temperature were longer than those of plants grown under optimal temperature up to 2h days after planting. However, the stems of the plants grown under optimal temperature caught up with those grown under supraoptimal temperature.
In attempts to cure or prevent the detrimental effects of supraoptimal temperature, biotin, sucrose, malic acid and phosfon-S
were individually added to the agar medium under both temperature regimes. The fresh and dry weights of the plants were used as measures
for comparison of the effects of these metabolites on the growth. An ideal curative added metabolite is considered to be one which would increase the fresh and dry weights of the plant under supraoptimal temperature but not, or very little under optimal temperature. Biotin and 1 per cent sucrose approached this ideal, completely preventing any deleterious effect of the supraoptimal temperatures employed.
Phosfon-S partially removed the injuries caused by supraoptimal temperature. Malic acid was ineffective, or actually injurious. Quantitatively, the effect of the additives was as follows: Biotin induced increases of 54 per cent in fresh weight and 66 per cent in dry weight
of the plant under the supraoptimal temperature, while these increases in fresh and dry weights for the plant grown under the optimal temperature were 38 and 15 per cent, respectively. Addition of 1 per cent sucrose induced 58 per cent increase in fresh and 70 per cent in dry weights of the plants grown under supraoptimal temperatures, while under
optimal temperature conditions, sucrose actually reduced the fresh weight by 12 per cent and had only slight (2%) promotive effect on




60
dry weights. Phosfon-S increased the fresh and dry weight 30 per cent and 16 per cent, respectively, under the supraoptimal temperature, and 27 per cent and 9 per cent under the optimal temperature. Malic acid caused a reduction in both fresh and dry weights under both temperature
regimes, although it was observed to increase the size and the number of seed pods and seeds.
The amount of several of the organic acids of the Krebs' cycle in the shoots of the plants were determined by paper chromatography. Citric, malic, succinic and fumaric acids were identified and measured at 2, 3 and 4 weeks after planting. These measurements revealed that the total amount of the identified acids of the shoot increased by 21 per cent under supraoptimal temperature 2 weeks after planting. This increase was maximum for malic acid (55%). At three weeks after planting, the amount of organic acids of the shoots decreased by 42 per cent under the supraoptimal temperature. This decrease was most pronounced for fumaric acid (65%). The amounts of all acids but
fumaric were higher in the shoot of the plant grown under the supraoptimal temperature 4 weeks after planting. Citric acid was 8.3 times (830%) greater under the supraoptimal than under the optimal regime. Increases in the other acids were very much less. Since the amounts of organic acids in general were higher at 2 weeks and again at 4.weeks after planting at the supraoptimal temperature, the contrast observed at 3 weeks was attributed to the flowering and also maximum (extremely rapid) rate of stem elongation, both of which occurred at this time.




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Geromino, J. and Harry Beevers. 1964. Effects of aging and temperature on respiratory metabolism of green leaves. Plant Physiol.
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Hoagland, D. R. and D. I. Arnon. 1938. The water-culture method for
growing plants without soil. Univ. of Cal.Ag. Exp. Sta. Circ.
347.
Houlahan, M. B. and H. K. Mitchell. 1947. A suppressor in Neurospora
and its use as evidence for allelism. Proc. Nat. Acad. Sci.
33:223-228.
Ketellapper, H. J. 1963. Temperature-induced chemical defects in higher
plants. Plant Physiol. 38:175-179.
Ketellapper, H. J. and J. Bonner. 1961. The chemical basis of temperature responses in plants. Plant Physiol. 36 suppl. XXI.
Kliewer, W. M. 1964. Influence of environment on metabolism of organic
acids and carbohydrates in Vitis vinifera. I. Temperature.
Plant Physiol. 39:869-880.
Knox, R. 1951. The formation of bacterial urease. Gen. Microbiol.
5:xx.
Knox, R. 1953. The effect of temperature on enzymic adaptation, growth
and drug resistance. Symp. Soc. Gen. Microbiol. 3:184-195.
Langridge, J. 1957. The aseptic culture of Arabidopsis thaliana
(L.) Heynh. Aust. J. Biol. Sci. 12:2h3-252.
Langridge, J. 1963. Biochemical aspects of temperature response.
Annual Rev. of Plant Physiol. 14:441-462.
Langridge, J. and B. Griffing. 1959. A study of high temperature
lesions in Arabidopsis thaliana. Aust. J. Biol. Sci. 12:
117-135.
Levitt, J. 1951. Frost, drought, and heat resistance. Annual Rev.
of Plant Physiol. 2:245-268.
Levitt, J. 1962. A Sulfhydryl-disulfide hypothesis of frost injury
and resistance in plants. J. Theoret. Biol. 3:355-391.




63
Lips, S. H and Harry Beevers. 1966. Compartmentation of organic
acids in corn roots. II. The cytoplasmic pool of malic acid.
Plant Physiol. 4:713-717.
Lockhart, J A. 1958. The role of gibberellin in the control of pea
growth by temperature. Planta, Bd. 52:250-258.
Luke, H. H and T. E. Freeman. 1965. Effects of victorin on Krebs'
cycle intermediates of a susceptible oat variety. Phytopathology 55:967-969.
Maas, W. K. 1950. A temperature sensitive pantothenicless mutant
of Escherichia coli. Bact. Proc. 128-129.
Maas, W. K. and B. D. Davis. 1952. Production of an altered pantothenate-synthesizing enzyme by a temperature-sensitive mutant of Escherichia coli. Proc. Nat. Acad. Sci. Wash. 38:785-797.
McCune, D. 1956. Biology 1956. Cal. Inst. Tech. (Reference obtained
from Davern, 1959.)
McElroy, W. D. and H. K. Mitchell. 1946. Enzyme studies on a temperature-sensitive mutant of Neurospora. Federation Proc. 5:
376-379.
Mitchell, H K. and M. B. Houlahan. 1945. Neurospora. IV. A temperature-sensitive riboflavinless mutant. Am. J. Botany 33:
31-35.
Molotkovsky, Yu. G. 1961. Changes in adnosine triphosphate activity
of subcellular units in heat-treated plants. Fiziologiya
Rastenii 8:669-672.
Molotkovsky, Yu. G. and I. M. Zhestkova. 1964. Mechanism of the
protective action of sugars at high temperatures. Translated
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Petinov, N. S. and Yu. G. Molotkovsky. 1957. Protective reactions
in heat-resistant plants induced by high temperatures.
Fiz:iologiya Rastenii 4:225-233.
Petinov, N. S. and Yu. G. Molotkovsky. 1960. The effect of respiratory inhibitors on heat resistance in plants. Translated from
Fiziologiya Rastenii 7:665-672.
Pollock, M. R. 1945. The influence of temperature on the adaptation
of "Tetrathionasel in washed suspensions of Bact. paratyphosum
B. Brit. J, Exptl. Pathol. 26:410-416.




64
Pollock, M. 1950. Penicillinase adaptation in B. cereus: Adaptive
enzyme formation in the absence of free substrate. Brit. J.
Exptl. Pathol. 31:739-753.
Rabson, R. and F. C. Steward. 1959. (From Steward, F. C., F. Cane,
K. Millar, R. M. Zacharius, R. Rabson and D. Margolis. 1959.) Nutritional and environmental effects on the nitrogen metabolism of plants. Symposia on the society for experimental
biology. Printed in Great Britain.
Rahn, 0. and W. R. Schroeder. 1941. Inactivation of enzymes as the
cause of death. Biodynamica 3:199-208.
Shokraii, E. H. 1965. Ph.D. Dissertation, The effect of high temperature on the free amino acids on common pea (Pisum sativum L.).
University of Florida, Gainesville, Florida.
Smith, I. 1958. Chromatographic techniques. William Reinemann.
Medical Books, Ltd., London, Interscience Publishers, Inc.,
New York.
Went, F. W. 1953. The effect of temperature on plant growth. Annual
Rev. of Plant Physiol. 4:347-362.
Went, F. W. 1957. Some aspects of effects of temperature on plants.
In: Influence of Temperature on Biological System.
Johnson, ed. John Wiley and Sons Company, New York.
Ying, Huii-Kuen. 1965. Ph.D. Dissertation, The effect of temperature
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to cell division and cell elongation of Pisum sativum "'Alaska."
University of Florida, Gainesville, Florida.




BIOGRAPHICAL SKETCH
The author, Azizollah Shiralipour Soltanabadi, was born on
May 5, 1935, in Ahwaz, Iran. He received his elementary and secondary
education at Danesh Pahlavi Elementary School. and Dr. Hessabi High School in Ahwaz.
In 1955 he entered the University of Ahwaz, Iran. Three yea2s later he received the degree of Bachelor of General Agriculture as the top student of his college and hence was awarded a four-year scholarship from the Government of Iran to further his studies in the United States. He was employed by Khuzestan Development Service in the fertilizer program at his home town for the period of ten months prior to coming to the United States.
In September, 1959, he entered the University of Florida and received the degree of Bachelor of Science in Agriculture in August, 1960. In September, 1960, he enrolled in the Graduate School of the University of Florida, majoring in Soils and received the degree of Master of Agriculture in June, 1962.
He was employed by the Agricultural Experiment Station, Department of Botany, in June, 1962. He also continued his studies toward the degree of Doctor of Philosophy in the Department of Botany.
He is a member of Alpha Zeta, Phi Sigma, agricultural and
biological honor fraternities, respectively, and American Society of Plant Physiologists.
65




66
The author is married to the former Miss Joan Strother and they have one child, Laleh.




This dissertation was prepared under the direction of the chairman of' the candidate's supervisory committee and has been approved by all members of that committee. It was submitted to the Dean of the College of Agriculture and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December, 1966
a, a' College of Agriculture
Dean, Graduate School Supervisory Committee:
Chira




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THE EFFECT OF SUPRAOPTIMAL TEMPERA.TURES ON THE GROWTH AND KREBS' CYCLE ACID CONTENTS OF ARABJDOPSIS THAL/ANA By AZIZOLLAH SHIRALIPOUR SOLTANABADI A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILO S OPHY UNIVERSITY OF FLORIDA December, 1966

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ACKNOWLEDGMENTS The author wishes to express his deep gratitude to Professor D.S. Anthony, chairman of the supervisory committee, for his close and continuous supervision during the course of this research and the preparation of this dissertation. He also would like to extend his appreciation to Dr. T. E. Humphreys for his valuable suggestions and for the use of his equip ment. The writer is also grateful to Dr. H. H. Luke and Dr. W. L. Pritchett, members of his committee, for their advice and suggestions during the course of his research. Special appreciation is extended to Dr. L.A. Garrard for his suggestions made during the course of this research and his guidance in preparing this manuscript. The author acknowledges with much gratitude the patience, encouragement and help of his wife, Joan, without whom this degree could not have been completed. ii

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ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION TABLE OF CONTENTS . . Effect of Supraoptimal Temperature on Growth Characteristics .... Chemical Cure of the "Lesions" Caused by Supraoptimal Temperatures Page ii iv V 1 11 23 23 32 Effect of Supraoptimal Temperature on the Contents of the Krebs' Cycle Acids 42 SUMMARY LITERATURE CITED BIOGRAPHICAL SKETCH . . . . iii 58 61 65

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LIST OF TABLES Table 1. GROWING CONDITIONS IN GROWTH CHAMBER 2. PER CENT RECOVERY OF ORGANIC ACIDS . 3. EFFECT OF TEMPERATURE ON LENGTH OF THE MAIN ROOT OF Page 12 20 ARABIDOPSIS THALIANA 26 4, EFFECT OF TEMPERATURE ON NUMBER OF SECONDARY ROOTS OF ARABIDOPSIS THALIANA 28 5. EFFECT OF TEMPERATURE ON LENGTH OF THE STEM OF ARABIDOPSIS THALIANA 29 6. EFFECT OF TEMPERATURE AND ADDED METABOLITES ON NUMBER OF SEED PODS AND SEEDS OF ARABIDOPSIS THALIANA 31 7. EFFECT OF TEMPERATURE AND VARYING CONCENTRATIONS OF SUCROSE ON FRESH WEIGHT OF ARABIDOPSIS THALIANA 39 8. EFFECT OF TEMPERATURE AND VARYING CONCENTRATIONS OF SUCROSE ON DRY WEIGHT OF ARABIDOPSIS THALIANA 40 9. EFFECT OF TEMPERATURE ON ORGANIC ACID CONTENT OF THE SHOOT OF ARABIDOPSIS THALIANA 2 WEEKS AFTER PLANTING 44 10 EFFECT OF TEMPERATURE ON ORGANIC ACID CONTENT OF THE SHOOT OF ARABIDOPSIS THALIANA 3 WEEKS AFTER PLANTING 45 11. EFFECT OF TEMPERATURE ON ORGANIC ACID CONTENT OF THE SHOOT OF ARABIDOPSIS THALIANA 4 WEEKS AFTER PLANTING 47 iv

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LIST OF FIGURES Figure 1 Loss of organic acids on storage 2 Effect of varying temperatures on fresh weight of the plant . . .... 3A. Effect of added metabolites on fresh weight of plants grown at optimal temperature 3 B. Effect of added metabolites on fresh weight of plants grown at supraoptimal temperature 4A. Effect of added metabolites on dry weight of plants grown at optimal temperature . . .. 4B. Effect of added metabolites on dry weight of plants grown at supraoptimal temperature .... 5. Effect of temperature on citric acid content at different ages .... 6. Effect of temperature on malic acid content at different ages . . . 7. Effect of temperature on succinic acid content different ages . . . at 8. Effect o f t e mperature on fumaric acid content at differen t a ge s . . . . 9A. Effec t of opt i m al temperature on orga n ic acid content at dif fere nt ages . . . 9B. Effect of supraoptimal temperature on organic acid content at different ages . . . V Page 18 25 34 34 36 36 49 51 53 55 57 57

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INTRODUCTION Many of the environmental variables affecting the growth of plants have been brought under control, but temperature is still a major limiting factor (Davern, 1959). Although a large amount of information concerning the effects of supraoptimal temperatures on plant growth and development is available, lmowledge of the biochem ical effects of such temperatures is limited. Since much of the data concerning the effects of temperature on the chemistry of growth proc esses of plants were derived from experiments with bacteria and algae, further biochemical studies on higher plants are necessary. It was suggested that, under some extreme temperatures, plant growth is depressed by inactivation of one or more reactions which are sensitive to such temperatures (Langridge and Griffing, 1959). This depression in growth caused by extreme temperatures was termed 11 climatic lesions" by Bonner (1957). The causes of lesions produced in plants by supraoptimal temperatures are not yet known, although several theories have been proposed by diff e ren t workers. Some of t h ese theories have been revi ewed b y Langridge (1963). Levitt (1951; 196 2 ) believes that frost, heat and drought injuries are related and are all caused by denaturation of the protoplasmic proteins. The thiol or sulfhydryl groups in adjacent protein mole cules in the cells of the plants grown in optimal temperatures are 1

PAGE 7

sufficiently far apart to preclude formation of disulfide bridges between adjacent molecules. Adverse climatic conditions supposedly result in intracellular rearrangement in which free thiol groups of protein constituents closely approach each other with the subsequent formation of disulfide bonds. When optimum conditions again prevail, protein denaturation results from the tensions created by this inter molecular bonding. 2 It has been shown that the elevation of temperature from opti mal to supraoptimal has a pronounced effect on the respiration of higher plants (Went, 1957; Petinov and Molotkovsky, 1957; Geromino and Beevers, 19 6 4). The decrease in respiratory activity as a result of supra optimal temperatures is believed to result from protein denaturation (Went, 1957). Self-toxifica t ion by ammoni a resulting from intensified proteolysis is one of the earliest views explaining the cause of "plant destruction" ("lesions'' in Bonner's terminology) induced by supraoptimal temperatures. The toxic action of ammonia, formed endogenously in the plant was reflected by impaired respiration wilting and eventual death of the plant (Petinov and Molotkovsky, 1 957 ) Decrease in availability of gases is another hypothesis for the cause of heat injury. Borek and Waelsch (1951) have shown that, when the bacterium Lactobacillus arabinosus was transferred from optimal to supraoptimal temperatures, addition of phenylalanine, tyrosine and aspartic acid became necessary for growth unless CO 2 tension was in creased. These amino acids were also required at optimal temperatures,

PAGE 8

if the bacteria was grown under reduced CO 2 tension, These observa tions suggest that "lesions" may result from decreased co 2 avail ability. 3 Pollock (1945) and Knox (1953) were among the first who showed that the formation of a particular enzyme was inhibited by supraoptimal temperatures, although the enzyme itself had maximum stability at such temperatures. This particular effect of supraoptimal temperatures, in inhibiting enzyme formation has been observed also by other workers (Pollock, 195a'; Knox, 1951; Bernheim, 1955; Halpern, 1961). According to Bernheim (1955) inhibition of enzyme formation results from the destruction of RNA during exposure to supraoptimal temperatures. Inactivation of enzymes at supraoptimal temperatures is another hypothesis under consideration. In this regard,Rahn and Sch~oeder (1941) found that treating Bacillus cereus at 46 f ::, ::.o minutes reduced the activity of peroxidase by 14 per cent and catalase activity by 20 percent. Supraoptimal temperatures can accelerate the breakdown of enzymes and other metabolites. Organisms vmich can function at such temperatures must synthesize protein at ~ates sufficie~tly high to replace the enzyme protein broken down by heat (Allen, 1950). The 'uncoupling of respiration and oxidative phosphorylation caused by supraoptimal temperatures is another theory for the explana tion of heat injury. It has been shown (Molotkovsky, 1961) that the activity of AT?. ase decreased with increasing temperatures, but the amount of inorganic phosphate increased continuously in excised leaves.

PAGE 9

4 Since exposure of plant tissues growing at optimal temperatures to 2,4-dinitrophenol also resulted in the accumulation of inorganic phos phate, it was concluded that the observed accumulation of inorganic phosphate in plants grown at supraoptimal temperaturesresulted from the formation of uncoupling agents at such temperatures. It has been shown that the growth of many organisms ceases at temperatures only slightly above optimal, but that normal growth can be restored by the addition of only a single organic substance. However, by raising the temperature a little higher an additional substance becomes necessary to counteract the temperature inhibition of growth. Thus, as the temperature is increased, these additive requirements to restore growth become progressively more numerous. Apparently, higher temperatures do not affect the genes because the growth rate can be fully restored by transferring the plants to optimal temperatures (Langridge, 1963). Bonner's proposal of the possibility of "chemical cure of cli matic lesions" is of particular interest because of its considerable potential economic importance. Although the higher plant is conaidered an autotrophic organism, it has been possible to cure temperature induced lesions in a few varieties of a few species by external supplies of organic compounds. The first so-called temperature-sensitive mutants Neurospora crassa and Escherich:iacoli have been extensively studied (Atwood and Mukai, 1953; McElroy and Mitchell, 1946; Maas, 1950; Maas and Davis, 1952; Mitchell and Houlahan, 1945). It has been shown that a mutant

PAGE 10

5 of Neurospora could grow at or below 25, but that growth decreased at temperatures above 25 and was completely arrested at 28. The addi tion of adenine to the medium partially restored growth; however, normal growth was obtained only by the addition of adenine, histidine and methionine. Histidine and methionine were ineffective when supplied singularly or together in the absence of adenine. Other mutants with different requirements for protection against heat injury have been found. In this regard a riboflavin-requiring, temperature-sensitive mutant (Mitchell and Houlahan, 1945), a uracil-requiring mutant of Neurospora(Houlahan and Mitchell, 1947) and a pantothenate-requiring, temperature-sensitive mutant in E. coli (Maas, 1950; Maas and Davis, 1952) should be mentioned. The removal of supraoptimal temperatur e inhibition of growth in excised pea stem sections by adenine has been studied by Galston and Hand (1949). They showed that high temperature lesions in Pisum sativum can be greatly reduced by the application of adenine as a foliar spray. A temperature-adenine growth response interaction was obtained. The amount of growth obtained by application of adenine at 30 was half the increment obtained at 35. These results indicate that high temperature growth inhibition in pea plants could be in part due to an adenine deficiency. However, additional studies (Galston, 1957) in the temperature-controlled facilities of Earhart Laboratory g ave a little if any indication that adenine was effective in reduc. ing supraoptimal temperature lesions. In this experiment, the day temperature was kept constant and the night temperature was varied from

PAGE 11

low to high levels. This could indicate that the adenine limitation was the result of high temperatures during the day period. 6 In another experiment (Galston, 1959), with a different temper ature regime but with the same variety of pea (night temperature was 6 lower than the day temperature), the investigator was unable to show any growth response to adenine at any temperature tested. Despite these negative results from studies with whole plants, the adenine effect may have some significance in that the t otal adenine level of a high temper ature-tolerant pea strain increases with increasing temperature, while the total adenine level of a supraoptimal temperature-sensitive strain decreases with increasing temperatures (Highkin, 1958). These changes in adenine level may reflect changes in RNA status associated with meri stematic activity, which are the results and not the cause of primary lesions. In the whole plant other supraoptimal temperature-induced lesions could be occurring which would preclude the detection of an adenine effect unless these other lesions were also removed. The results of Galston and Hand (1949) were confirmed by Highkin (1958). On the other hand, other workers did not find the application of adenine to be effective for the removal of the "lesions" caused by supraoptimal tem peratures in pea plant (Lockhart, 1958; Ketellapper, 19 63 ). However, it was found that a vitamin B mixture or ribosides increased the dry weight up to 40 per cent at supraoptimal temperatures (Ketellapper and Bonner, 1961). Sucrose was found to be effective at temperatures only slightly above optimal but not at still higher temperatures (Ketellapper, 1963). This suggests that the effect of sucrose was temperature specific.

PAGE 12

7 Adenosine and guanine were added to Lemna minor cultured at supraoptimal temperatures by McCune (1956) who observed that they pre vented the death of the plants at such temperatures. A genotype-dependent auxin limitation in two varieties of corn was investigated and was shown to be affected by temperature (Galston, 1957). The 11 silkiness 11 variety gave a 50 per cent elongation response to addition of indoleacetic acid at 20 day and 15 night temperatures where the 11 normal 11 variety gave a 16 per cent elongation response to the application of auxin while the 11 silkless 11 variety showed no response. The application of gibberellin to Alaska peas which were grow ing under a supraoptimal temperature regime gave a striking effect in delaying the supraoptimal temperature-accelerated senescence which accompanies the onset of maturity (Lockhart, 1958). Spraying casein hydrolysate on clover plants Trifolium subter raneum grown under supraoptimal temperatures, caused a significant alleviation of supraoptimal temperature lesions whereas the plants did not respond to hydrolysa te under optimal conditions (Davern, 1959). Petinov and Molotkovsky (1957 ) reduced the supraoptimal tem perature lesions in sunflower, corn, pumpkin and oats by spraying the leaves with orga ni c acids. Citric and malic acids were particularly effective in this regard. In wor ki ng with Arabidopsis thaliana a very cl ea r-cut case of effective ch emo therapy of high temperature growth and developmental inhibition has been obtained by Langridge and Griffing (1959). Plants were grown with aseptic culture techniques and under controlled

PAGE 13

8 environmental conditions. Forty-three different races were grown under three different temperature regimes, 25, 30 and 31.5. Five races (PI, BLA, m, HI, and LS) showed particular sensitivity (depressed in growth and morphologically abnormal) to th~ 31.5 temperature regime; three of these (PI, LA, and BLA) responded to chemical supplements. Further study showed that in two races (PI and BLA) addition of 6 or 3 g/plant of biotin to the culture medium completely prevented the supraoptimal temperature lesions. The third race (LS) partially responded to cytidine applied at the rate of 0.25 mg/plant. Two other races (DI and HI) did not respond to any applied supplements. It is possible that the metabolites required by nonresponding races were not present in the supplements which were tested, although the yeast extract used might be expected to contain most of the diffusible substances needed by plants. Of course, it is also possible that certain applied supplements were not taken up by the plants. The "lesions" caused by supraoptimal temperatures may be com pletely eliminated by providing the plant with one or more metabolites which have been destroyed by such temperatures. Since the replacement of temperature-sensitive cultivated plants by theit temperature resistant alternate varieties is a slow and limited process and because of the great ~conomic importance of this problem, it seems reasonable to direct more attention to the 11 chemical cure of climatic lesion. 11 The ideal compounds are those, which do not affect the plant growth under optimal temperatures but improve its growth under supraoptimal temper atures (Langridge and Griffing, 1959).

PAGE 14

9 In general, very little is known about specific biochemical compounds or kinds of metabolism which are particularly sensitive to supraoptimal temperatures in higher plants. There are relatively few published reports showing the quantitative effects of supraoptimal tem peratures on specific biochemical constituents of higher plants. In this respect, the effect of day length, mineral nutrition, and night temperature on nitrogenous compounds (Rabson and Steward, 1959), the effect of supraoptimal temperatures on free amino acids (Petinov and Molotkovsky, 1960; Shokraii, 1965), some fragmentary data on organic acids (Geromino and Beevers, 1964; Kliewer, 1964) and a recent study on RNA content (Ying, 1965; Brown, 1965) should be mentioned. In the biochemical work reported in this paper, attention will be confined to the effect of supraoptimal temperatures on the organic acids of the Krebs' cycle. In order to understand the biochemical effects of temperature on plants the effect of such temperatures on the individual life and growth processes must also be known. Growth, especially elongation of cells, has a high Q 10 indicating a chemically rather than physically controlled phenomenon (Chao and Loomis, 1947). Due to this fact, a variety of growth measurements as well as chemical analyses were made. There are numerous reports concerning the effect of temper ature on growth and development of plants. Some of these have been reviewed by Went (1953) and Langridge (1963). For the kind of work presented here it is imperative to use aseptic methods, since this eliminates at least one variable, the

PAGE 15

10 microorganisms. It is also important to select a small plant which has a short life cycle and which is sensitive to supraoptimal temperatures. The ideal test plant should be able to be grown aseptically on an agar medium in test tubes. This permits easy application of additives for the "chemical cure of climatic lesions" to the agar medium, and allows many replications in a small space. A plant belonging to the tribe Arabideae of the Cruciferae family, Arabidopsis thaliana (L.) Heynh (Variety PI, mouse-ear kress), was selected as meeting all of the requirements as a desirable test plant for these studies. It is a very small plant with a short life cycle (24 1 days), is sensitive to supraoptimal temperatures, and can be grown aseptically on agar medium in test tubes.

PAGE 16

MATERIALS AND METHODS A. Plant Materials and Growing Conditions Seeds of Arabidopsis thaliana were soaked in sterilized, distilled water between two filter papers in a petri dish. The petri dish was placed in a refrigerator for 24 hours, at 4, This cold treatment insured more uniform gennination of the seeds (Langridge, 1957). Following their cold treatment, the seeds were washed for 3 minutes in 10 per cent Chlorox solution after which they were washed several times with distilled water. The seeds were then transferred to sterile agar in 150 X 25 mm (pyrex) test tubes. The agar medium was prepared by a method modified from that of Langridge (1957), A 0,8 per cent agar solution was prepared by adding 8.0 g of agar (Bacto-Agar, Difeo Laboratories, Detroit, Mich igan) to one liter of Hoagland solution (Hoagland and Arnon, 1938), The pH of the resulting agar suspension was adjusted to pH 6.0 with concentrated KOH. The agar suspension was liquified by placing in an autoclave at 125 and 15 psi for 20 minutes, Aliquots of approx imately 10 ml were placed in the test tubes, and the tubes were plugged with nonabsorbent cotton. The culture tubes were then ster ilized in an autoclave at 125 and 15 psi for 15 minutes After re moval from the autoclave and solidification of the agar, the seeds were transferred to the surface of the medium under aseptic conditions 11

PAGE 17

12 in a transfer box. The culture tubes were then placed in growth cham bers set to provide either a supraopttmal temperature or an optimum temperature regime. The temperatures were those actually observed for the agar medium (Table 1). TABLE 1 GROWING CONDITIONS IN GROWTH CHAMBER Temperature Phototemperature Nyctotemperature Light Condition (16 hr) (8 hr) Intensity Optimum 1 1 1100 200 f.c. Supraoptimal 1 1 1100 200 f.c. The source of light was a combination of fluorescent (cool white) and incandescent bulbs. B. Growth Measurements The seedlings were observed daily for a 5-week growth period during which a number of growth measurements were made. These measure ments included: length of the primary root and number of secondary roots, length of main stem, number of leaves, number of seed pods and seeds, and the fresh and dry weights of the shoots at 2, 3 and 4 weeks of age. In order to determine the effects of supraoptimal temperatures on the g rowth of the plants and the effect of various chemicals in cur ing "climatic lesions" resulting from supraoptimal temperatures the

PAGE 18

13 following procedure was followed: groups of 40 seedlings each were grown under both supraoptimal and optimal temperature regimes with or without chemical additives in the agar medium. On the first day following germination five more uniform plants were selected from each treatment to be used for daily growth measurements. Additional five seedling samples were selected at random at 1, 2, 3 and 4 weeks after planting for the determination of fresh and dry weights. Each experiment was replicated 2 or 3 times. C. Chemical Analysis Preparation of Tissue Extracts. Alcoholic extracts were pre pared from the shoots of plants at 2, 3 and 4 weeks of age for the determination of the organic acids of the Krebs' cycle. Whole shoots (1-2 g) were cut in small pieces and extracted for 5 minutes in 50-100 ml of boiling 80 per cent ethanol. The extract was cooled to room temperature and filtered through Whatman No. 1 filter paper. The plant residue was homogenized in a glass homogenizer with an addi tional 40 ml of hot 80 per cent ethanol. This extract was again fil tered through Whatman No. 1 filter paper and was combined with the first filtrate. The volume of the combined filtrate was reduced under vacuum in a rotary evaporator at room temperatur e T h e concentrated filtrate was centrifuged at 10300 X g for 20 minu t es at 0 ~ The super natant fraction was collected by decanting and the pellet was taken up in a s~ all amount of distilled water. The suspended pellet was again centrifuged and the supernatant fraction combined with that from

PAGE 19

the first centrifugation. The volume of the supernatant was reduced to 10 ml. 14 The concentrated supernatant was passed through a Dowex 50 X 8 (Hydrogen form) cation-exchange column (150 X 12.7 mm) and the column was rinsed by subsequent passage of 100 ml of distilled water. The rate of flow of the column was controlled to give 10 drops/minute or less. The amino acids of the extract remained on this column while the eluate contained the organic acids of the Krebs' cycle. The vol ume of the eluate was reduced to 10 ml before further separation. Separation of Organic Acids. The organic acids of the eluate from the Dowex 50 column were then separated on a Dowex 1 X 8 anion exchange column in the formate form (Luke and Freeman, 1965). Dowex 1 X 8 resin in the chloride form was washed successively with portions of 2 N formic acid until ch loride could no longer be detected, The resin was then freed of excess acid by washing with distilled water. The resin (now in the formate form) was oven-dried at 50-60. A 150 X 6.5 mm column was prepared by placing 3 g of the dried resin in a 500 X 6.5 mm glass tube. The tip of the tube was tapered and plugged with a small piece of glass wool. The resin bed was capped with another glass wool plug. The column was then washed with approximately 100 ml of distilled water. The concentrated eluate from the Dowex 50 X 8 column was placed on the Dowex 1 X 8 column and followed by 100 ml or more of distilled water. The rate of flow as adjusted to give 10 drops/minute. In this case, the eluate was discarded. The

PAGE 20

15 organic acids remained on the column. The organic acids were eluated from the column with 25 ml of 90.5 per cent formic acid (J. T. Baker Chemical Company, Phillipsburg, N.J.) followed by 100 ml of distilled water. The eluate containing the various organic acids was taken to dryness under reduced pressure and the residue was taken up in an appropriate volume of 10 per cent isopropanol to give 1 ml of solu tion/g origincf!fresh weight of tissue. Paper Chromatography. Qualitative determination and quantita tive measurements of the organic acids of the Krebs' cycle were made by paper chromatography of the concentrated tissue extracts. Aliquots of 50 l of the concentrated extracts were chromatographed ascendingly on strips of Whatman No. 1 filter paper (50 X 4 cm) in N-butanol: formic acid: water (10: 2: 15v/v/v,upper phase). The strips were equilibrated for 5 hours with a portion of the solvent before being developed for a period of 3 6 hours. Equilibration and development were conducted at 24 2. The developed paper strips were initially dried at room temperature for a period of 5 hours after which they were sub jected to a series of alternate 10-minute periods of forced hot (70) and cool air to remove all traces of solvent. A color developer for observing and determining the amount of the organic acid (Smith, 1958) was made by placing 1 g of xylose in 3 ml of distilled water and then adding 1 ml of aniline; the final volume w a 3 made up to 100 ml with methanol. The paper strips were dipped in t h e developer and then were air dried for JO minutes. They were then heated at 70-80 for 20-30

PAGE 21

16 minutes. The organic acids appeared as reddish-brown spots on a white background. Although it was difficult to obtain a white background, by carefully following the above procedure it was accomplished, Quan titative measurements were made by eluting the spots in 50 per cent ethanol and determining the absorbance of the solutions at 395 m in a Spectronic 20 (Bausch and Lomb) spectrophotometer. Standard curves were prepared by spotting known amounts of organic acids directly on the paper. Effect of Storage on Organic Acids. In preparing standard curves from solutions of known amounts of organic acids which were stored at -15, a reduction in the slope of the curves with duration of storage was noticed. This suggested some loss in the amounts of organic acids. In order to de~ermine the extent of loss due to stor age (probably via esterification), mixtures of authenic acids were prepared in 10 per cent isopropanol and also in 80 per cent ethanol. Standard curves were obtained for both mixtures at the time of prepa ration and following storage for periods of 1, 2, 3 and 4 weeks. A typical loss in acid content of stored solutions, as shown for citric acid, is given in Figure 1. As can be seen, the amount of loss is higher when acids are stored in 80 per cent ethanol. Accordingly, storag e of standards and extracts in alcohol was eliminated and all the analyses were done immediately after harvesting t he plant. Per Cent Recovery of Organic Acids. The per cent recovery of organic acids was determined by addition of known amounts of authentic

PAGE 22

Figure 1. Loss of organic acids on storage.

PAGE 23

....... lO 0) I\') '< "{ 0:) 0c: C) (/) Cl) 0.5 0.4 0.3 0.2 0.1 IN 10% ISOPROPANOL, NO STORAGE PER I OD 0 .1 N 80% ETHANOL, NO STORAGE PER I OD ~s'NTORlEOo/oD ISOPROPANOL, FOR 4WEEKS IN 80% ETHANOL, o,---o STORED FOR 4WEK S 18 0--+-----.----.---------------__, 0 20 40 60 80 -G OF CITRIC ACID ADDED TO THE PAPER FIG. I

PAGE 24

19 organic acids to the plant extracts. The difference between the values obtained from the plant extract alone after going through the entire preparative procedures and those obtained from plant extract plus the known amounts of acids was compared to a standard curve. This stand ard curve was prepared from a known mixture of authentic organic acids spotted directly on the paper. The per cent recovery of the di ff erent acids is given in Table 2. Subsequent determinations of organic acids in experimental material were corrected for recovery, using the average recoveries given in Table 2 as the recovery factors. D. Chemical Treatments Various chemicals for treatment of supraoptimal temperature 11 lesions 11 were added to the agar medium under aseptic conditions. These additives included: sucrose, biotin, phosfon-S (2,4 dichloro benzyl tributyl ammonium chloride, Virginia-Carolina Chemical Corpora tion) and malic acid. A series of concentrations of sucrose (1-5 % ) in agar was prepared in the test tubes and the standard procedures for handling seeds and seedlings were followed. Since phosfon-S, malic acid and biotin are unstable to heat, they were not mixed with the agar solution in the test tubes before autoclaving the agar. In the case of phosfon-S the seeds were soaked in a solu t ion containing 20 p.p.m. of the ma t erial before they were t ransferred to the agar medium. Stock solutions of biotin and malic acid were sterilized by passing the solutions through membrane filters (pore diameter 0.22 ). Aliquots containing either 6 g of biotin or 3.35 mg of malic acid

PAGE 25

TABLE 2 PER CENT RECOVER Y OF ORGANIC ACIDS Recovery of Acids Amount of Acid Citric Malic Succinic Fumaric Added to Plan t -,( Extract Amount P er Amount Per Amount Per Amount Per (mg) (mg) Cent (mg) Cent (mg) Cent (mg) Cent 20 17 85 15 75 17 85 14 70 40 42 105 3 6 9 0 32 80 34 85 60 63 10 5 60 1 00 51 85 56 93.3 Average Reco ve r y Per Cent 98 88 83 83 The difference bet wee n pl a nt ext ract alone and that recovered from plant e x tract plus the given amount of added organic acid. I\) 0

PAGE 26

21 were added to the sterile test tubes just before solidification of the autoclaved and still warm agar. Since the pH of the agar medium would change on addition of the malic acid, the pH of the 1 M malic acid was adjusted to 6.0 by addition of concentrated KOH before sterilization. Transfer of seed to the agar and procedures for handling seeds and seedlings were the same as given above. Problems in Identification of Organic Acids. Citric, malic, succinic and fumaric acid were identified in extracts of Arabidopsis thaliana by paper chromatography. However, there were three unknown compounds which gave positive organic acid color reactions The un known compounds had Rf values less than the known acids. These unknowns could not be successfully identified by co-chromatography with known organic acids. Since the pKa's of the carboxyl groups of acidic amino acids were lower than the pH of the plant e xtract, it was assumed that these acids would not be held tightly by the Dowex-50 column. Accordingly the effluent from this column was checked to find out whether any amino acids had leaked off with the org a nic acids. Paper chromatograms of the efflu en~ were sprayed with nim y drin and it was found that there were two spo t s giving a positive ninhydrin test. These ninhydrin positive spots corresponded in Rf values to the unknown spots having the two lowest Rf values in the chromatograms of the organic acids. Further testing by chromatography in other solvent mixtures identified these two amino acids as glutamic and aspartic acids. To obtainadditional

PAGE 27

22 information about the behavior of glutamic acid and aspartic acids under the conditions employed in our experiments, a sample of plant extract containing c 14 labeled glutamic and aspartic acids was placed on the Dowex-50 column. It was found that 25 per cent of the glutamic acid and 20 per cent of the aspartic acid passed through the column. Acid ification of the extract to pH 2.5 prior to passing through the column reduced the leakage of glutamic and aspartic acids to 19 and 14 per cent, respectively. However, the use of a finer resin (200-400 mesh) and increasing the column length (1.5 times) reduced the leakage of the mixture of radioactive glutamic and aspartic acids to 2.5 per cent. Therefore, the larger column and finer resin were adopted as standard for all data reported here. The third unknown (found at a greater Rf value than those for glutamic and aspartic acids) could not be unequiv ocally identified although the Rf values for the unknown when chroma tographed in two different solvents were close to those for tartaric acid.

PAGE 28

RESULTS AND DISCUSSION Effect of Supraoptimal Temperature on Growth Characteristics In order to determine the proper optimal and supraoptimal temperatures, plants were grown in different temperature regimes as shown in Figure 2. Fresh weights of the plants were determined at 1, 2, 3 and 4 weeks after planting. A regime of 25-da~ 18-night 0 0 temperatures was chosen for the optimal condition and 32 -da~ 25 night temperatures was selected for the supraoptimal temperature treatments. Growth characteristics were measured under these two temperature regimes with the following results. A. Length of the Main Root At 8 days after planting, supraoptimal temperatures had caused 45 per cent reduction in the length of the main root as compared to plants grown at optimal temperature (Table 3, data for basal medium only). The maximum increase in the length of the main root in plants grown under the optimal condition occurred at 7 days after planting, while under the supraoptimal temperature condition the maximum daily growth rate occurred at 4 days after planting. This could be due to acceleration in breakdown of seed reserves under the supraoptimal temperature regime. 23

PAGE 29

Figure 2. Effect of varying temperatures on fresh weight of the plant.

PAGE 30

25 1 4 0 0 0 25 DAY, 18 NI GH'r A A 230 DAY, 170 NIGHT 120 0 21 o--o 27 DAY, NIGHT 0 21 DAY, 15 NIGHT 0 100 0 DAY, 230 NIGHT --d. 29 Q: 0 DAY, 25 NIGHT o---o 32 (!) 8 0 f..... / ::c: 60 // p (!) 4 0 ::c: e: 20 0 2 3 4 W EEKS AF T ER PLANTING FIG 2

PAGE 31

TABLE 3 EFFECT OF TEMPERATURE ON LENGTH OF THE MAIN ROOT OF ARABIDOPSIS THALIANA Length of Main Root (mm)* Addition Time after Planting to 3 days H~ 4 days 5 days 6 days 7 days 8 days Basal Medium ot st 0 s 0 s 0 s 0 s 0 s None 2.7 1.7 6.5 5.1 9.9 7.6 13.8 9.5 18.7 10.8 21.9 12.0 Biotin 2.1 2.0 4.9 4.3 9.2 7,9 14.2 11.3 24.8 19.1 27 .6 26.7 Malic Acid 2.5 2.4 5.9 4.8 9.3 7.3 10.5 9 1 11.4 10.6 12.1 11.8 Phosfon-S 2.4 2.7 6.3 5.6 10.7 8.4 14.6 10.6 18.7 13.0 21.3 14.4 1% Sucrose 2.7 2.5 7. 5 4.8 11.3 7 .1 14.8 9.1 21.4 12.0 24.1 14.6 Average of 2 trials, 5 plants per trial. -".-No measurable root appeared prior to 3 days after planting. t O = Optimal temperature. S = Supraoptimal temperature.

PAGE 32

27 B. Number of Secondary Roots The number of secondary roots of plants grown at supraoptimal temperature was reduced by 27 per cent at 12 days after planting (Table 4, data for basal medium only). Daily increases in the number of secondary roots were constant with time within each trea tm ent. Supraoptimal temperature slightly decreased the time required for the appearance of the secondary roots (Approximately 1 day); again, perhaps because of enhancement of the breakdown of the seed reserves. C. Length of the Main Stem During a 19-day period following planting, the length of the main stem and also the rate of stem elongation, were greater in plants grown under supraoptimal temperature (Table 5, data for basal medium only). The maximum daily rate of elongation occurred at 21 days after planting at supraoptimal temperature and 23 days after planting at the optimal temperature. D Number of Seed Pods and Seeds The numbers of seed pods and seeds were reduced under the supraoptimal temperature regime (Table 6, data for basal medium only). Actually, the effect was on the number of the seed pods since the aver age number of seeds per pod remained unchanged under both temperature regimes (approx;i.mately 12 seeds per seed pod). However, the sizes of the seed pods and seeds were much smaller under the supraoptimal tem perature condition.

PAGE 33

TABLE 4 EFFECT OF TEMPERATURE ON NUMBER OF SECONDARY ROOTS OF ARABIDOPSIS THALIANA ~Number of Secondary Roots Addition Time after Planting to 6 days H} 7 days 8 days 9 days 10 days 11 days Basal ot s* Medium 0 s 0 s 0 s 0 s 0 s None 0.0 0.3 0.9 1.1 2.8 2 3 4.3 3.1 6.2 4.3 7.3 5.8 Biotin 0.9 o.o 2.4 2.8 4.8 4.6 7.8 7.0 11.3 9.0 15.2 11.6 Malic Acid 1.2 o.o 4.4 2.4 5.8 3.9 7.3 5.8 9.0 6.7 10.7 7.8 Phosfon-S 0.0 0.0 0.0 1.0 1.4 2.3 3.3 3.4 4.1 4.1 5.1 5.1 1% Sucrose o.o 0.4 0.0 1.6 1. 2 2.7 1.9 4.1 3.6 5.4 4.0 7.3 Average of 2 trials, 5 plants per trial. ~~No secondary roots appeared prior to 6 d ay s after planting. t 0 = Optimal temperature. =I= s = Supraoptimal temperature. 12 days 0 s 9.2 6.7 20.5 14.8 12.8 10.8 7.2 6.3 4.9 8.8 I\) O:>

PAGE 34

TABLE 5 EFFECT OF TEMPERATURE ON LENGTH OF THE STEM OF ARABIDOPSIS THALIANA .,,_ Length of Stem (mm)~ Addition to Basal Medium Days None Biotin Malic Acid Phosfon-S 1% Sucrose after Planting ot s:J: 0 s 0 s 0 s 0 s 1 0.0 o.o 0.0 0.0 o.o 0.0 0.0 0.0 o.o 0.0 2 2.3 2.1 0.0 2.7 2.1 0.0 2.0 0.0 2.0 2.2 3 3.0 3.0 2.0 3.6 2.2 2.1 2.5 2.5 3.0 3.0 4 3.0 3.5 2.5 3.9 3.0 2.7 3.0 3.4 3.1 3.2 5 3.0 4.5 3.2 4.7 3.2 3.0 3.7 4.2 3.5 3,7 6 3.1 5.0 3.5 5.0 3.8 4.o 3,9 4.2 3.7 4.o 7 3.5 5.5 4.2 5.8 4.1 4.4 4.4 4.5 4.3 4.1 8 3.8 6.5 4.3 5.8 4.2 4.9 4.4 5.2 4.5 5.1 9 3.9 7.0 4.4 5.8 4. 2 5.5 5.0 5.5 4,7 5.4 10 4.1 8.0 4.5 6.2 4.5 5.9 5.3 5.6 5.2 5.4 11 4.3 8.3 4.8 6.3 5.3 6.2 5.4 6.1 5.3 5.4 12 4.6 8.4 4,9 6.4 5.3 6.4 6.0 6.5 5.5 5.9

PAGE 35

TABLE 5 (Co nti nu e d) Leng t h of St em ( mm ) Day s Addition to B asa l M edium aft e r Non e Biotin Malic Acid Phosfon-S 1 % Sucrose Planting ot s* 0 s 0 s 0 s 0 s 1 3 5.0 9. 0 5 3 7.2 5 4 7.4 6.1 6.5 5.7 6.5 14 5.5 9. 6 5 6 8.0 5.7 8.5 6.5 7.1 6.4 7.2 1 5 5. 8 10. 5 6 0 1 0 0 6. 2 10. 3 6.8 7 .5 6 8 8 7 16 6 6 11. 9 6 .7 1 3 .9 6.4 1 3 .0 7. 3 8. 3 7. 4 11.0 17 7.9 15 1 7.7 18 3 7 .4 16 8 8.0 10 .3 8 7 15.8 18 1 0 .8 2 2 0 9 4 2 6.3 9 .2 2 3 .7 9.2 15.8 12 0 28 1 19 1 7. 1 3 2.7 14 1 47. 9 1 2.6 33 3 12 3 21.9 17. 6 4 6. 4 20 29 4 44 .7 24.1 6 8 2 1 9. 4 44 9 17.8 33 4 26.0 71.0 21 4 6 .2 6 4. 8 3 7. 3 93 .7 2 9 6 61.0 29 6 47.9 3 6.7 96.0 22 64 .3 80 0 23 87.1 9 6 3 24 98 9 1 0 4.1 *Average of 2 trials, 5 plants per trial f O = Optimal temperat u re. + s = Supraoptimal te m perature. \.,J 0

PAGE 36

TABLE 6 EFFECT OF TEMPERATURE AND ADDED METABOLITES ON NUMBER OF SEED PODS AND SEEDS OF ARABIDOPSIS THALIANA Addition to ~ Number of Seed Pods Basal Medium ot s=1= None 10~0 3.5 Biot:i,.n 11.5 10.5 I M alic Acid 10.2 6 0 Phosfon-S 10.9 7. 0 1 % Sucrose 1 6 .0 7. 6 ~ Average of 2 trials, 5 plants per trial. r O = Optimal temperature. S = Supraoptimal temperature. Number 0 127.9 197. 7 248.9 166.4 235.1 31 of Seeds s 44.4 127.2 59.8 81. 2 1 105.2

PAGE 37

32 E. Fresh and Dry Weights The most striking effect of supraoptimal temperature on growth was reflected in the fresh and dry weights of the plant (Figures 3 and 4, data for basal medium only). The fresh and dry weights of the plants grown under supraoptimal temperature were reduced by 48 and 38 per cent, respectively, at 4 weeks after planting as compared to those plants grown under optimal temperature. The maximum rates of weekly increase in fresh weight were obtained at 4 weeks after planting at both optimal and supraoptimal temperatures. The effects of the different temperatures on dry weight were similar to those obtained for the fresh weight. Chemical Cure of t he 11 Lesions 11 Caused by Supraoptimal Temperatures The effects of different metabolites on plant growth character istics at different temperatures are given in Tables J-8 and Figures J and 4. Dry and fresh weights were used as the main criteria for the comparison of the effectiveness of the added metabolites. Biotin, sucrose, and phosfon-S exhibited positive effects in alleviating detrimental effects of supraoptimal temperature. The addi tion of sucrose or biotin alone gave the most effective results, and hence a combination of both was tried under both temperature regimes Measurements of growth (which had to be terminated after 8 days in this case because of a malfunction bf growth chambers) showed an even better response to the combination as compared to the addition of sucrose or biotin alone to the plant medium.

PAGE 38

Figure JA. Effect of added metabolites on fresh weight of plants grown at optimal temperature. Figure JB. Effect of added metabolites on fresh weight of plants grown at supraoptimal temperature.

PAGE 39

3 4 0 BASAL M ED I UM ONLY A 160 G, BASAL MED I UM + BIOTIN A BASAL M ED I UM + P HOSFON-S 0 BASAL MED I UM + 1 % SUCROSE 120 BASAL MED I UM + MALIC ACID ..... ..... ..... '} ..... ..... ':% .......... ..... ..... 4 0 ..... ..... ..... _,_ ,..,.. V ,,,, ......... .... 4 ..... ~ .... .... .... --.1 ~ ---:<::;.----a--0 0 2 3 4 W EEKS AFTER PLANTING FIG. 3

PAGE 40

Figure 4A. Effect of added metabolites on dry weight of plants grown at optimal temperature. Figure 413. Effect of added metabolites on dry weight of plants grown at supraoptimal temperature.

PAGE 41

36 o BASAL MED I UM ONLY A 16 0 BASAL MED I UM + BIOTIN A BASAL MED I UM + PHOSFON-S o BASAL MED I UM + llJD SUCROSE 12 BASAL MED I UM + MALIC ACID -... I<: 8 q: -.J Cl. Q:: 4 Lu Cl. (.!:) 0 'IB :x: (.!:) e BASAL MED I UM ONLY 16 Lu 0 BASAL MED I UM + BIOTIN >... BASAL MED I UM + PHOSFON-S Q:: 12 o BASAL MED I UM + 1% SUCROSE I Q BASAL MED I UM + MALI C ACID 8 L ~, 4 ~;'~,~ /}<:.f ~-,. .;:. -~--a" 0 0 2 3 4 WEEKS AFTER PLANTING FIG. 4

PAGE 42

Biotin alone improved all the growth characteristics studied and prevented the detrimental effects of supraoptimal temperature. )7 This promotion of growth as a result of biotin treatment was observed in both temperature regimes but the degree of the enhancement of growth was much higher at supraoptimal temperature. The increases in fresh and dry weights per plants grown under optimal temperature was JS and 15 per cent, respectively, while under supraoptimal temperature the increases were 54 and 66 per cent, respectively. These results are in agreement with those obtained by Langridge and Griffing (1959) for the same plant growing under constant 25 opti mal and 31.5 supraoptimal temperatures. Sucrose was even more effective than biotin in alleviating the injuries caused by supraoptimal temperature. The addition of 1 per cent sucrose to the medium increased the dry weight only 2 per cent under optimal temperature while the increase under supraoptimal was 70 per cent. Surprisingly, sucrose decreased the fresh weight under optimal temperature (12 % ) at the 4-week sampling period, while it increased the fresh weight by 58 per cent at the same time under supraoptimal temperature (Figures 3 and 4). In addition to increasing the fresh and dry weights, sucrose improved most of the other growth characteristics which were adversely affected by supraoptimal temper ature (Tables 3-5). Prior to application of 1 per cent sucrose, an agar medium con taining 10 per cent sucrose as was suggested by Ketellapper and Bonner (1961) was used. However, seeds did not germinate at this high

PAGE 43

38 concentration of sucrose perhaps because of unfavorable osmotic condi tions. To determine the most suitable concentration of sucrose, an experiment with agar mediums containing 1-5 per cent sucrose was con ducted under both temperature regimes. The fresh and dry weights of the plants grown under different sucrose concentrations were meas ured (Tables 7 and 8). The results obtained showed 1 per cent sucrose to be the most suitable concentration for further experimentation. A possible mechanism of the protective action of sugars at supraoptimal temperatures has been suggested byMolotkovsky and Zhest kova (1964). They showed that application of several sugars, especi ally sucrose, prevented the drop in the rate of respiration at supra optimal temperatures. Stabilization of respiration under the influ ence of sucrose was sufficient to render the plants insensitive to the action of respiratory inhibitors. They further showed that other sugars and even metabolically inert mannitol had a similar effect but to a lesser degree. Sucrose also prevented the uncoupling of oxidative phosphorylation and respiration as measured by accumulation of inorganic phosphate. Referring to the work of Moshanskii (discussed by Molotkovsky and Zhestkova, 1964), which showed that mitochondria became swollen at supraoptimal temperature with eventual dissociation of oxidative phos phorylation and respiration and that of Lehninger (also discussed by Molotkovsky and Zhestkova, 1964), which showe~ that sugars blocked the swelling of the mitochondria, they hypothesized that sugars have a "conserving" effect on mitochondria and that this explains the "pro tective action" of sugars.

PAGE 44

TABLE 7 EFFECT OF TEMPERATURE AND VARYING CONCENTRATIONS OF SUCROSE ON FRESH WEIGHT OF ARABIDOPSIS THALIANA Fresh Weight per Plant .,,_ (mg) n Per Cent Time after Planting Sucrose Added 1 week 2 weeks 3 weeks to Basal Medium ot st 0 s 0 s 0 1.0 o. 75 15.1 5.9 61.3 24. 70 1 0.99 0.93 14.3 9.20 44.2 45.97 2 0.99 0.90 14.7 8.7 50.1 31.30 3 1.0 0.80 15.3 4.5 51.1 11.40 4 o.8 o.4o 5.8 1.8 22.7 10.5 5 0.7 0.30 5.1 1.5 16.4 9,8 *Average of 2 trials, 5 plants per trial. t O = Optimal temperature. S = Supraoptimal temperature. 4 weeks 0 s 118.7 44.1 106.1 94.5 115.3 66.9 103.4 25.7 46.1 20.9 40.7 18.8

PAGE 45

TABIE 8 EFFECT OF TEMPERAWRE AND VARITNG CONCENTRATIONS OF SUCROSE ON DRY WEIGHT OF ARABIDOPSIS THALIANA Dry Weight per Plant (mg) Per Cent Time after Planting Sucrose Added 1 week 2 weeks 3 weeks 4 weeks to Basal Medium ot s=1= 0 s 0 s 0 s 0 0.08 0.06 1.15 o.46 4.91 2.61 10.8 5.3 1 0.09 0.07 1.36 o. 75 4.32 4.95 11.1 10.20 2 0.09 0.07 1.48 0.80 4,90 4.60 12.0 8.2 3 0.08 0.06 1.40 0.66 6.0 1.50 11.9 3.9 4 0.06 0.05 0.65 0.26 2.9 2.1 5.6 3.0 5 0.06 0.05 0.65 0.33 2.2 1.9 5.4 2.9 Average of 2 trials, 5 plants per trial. t O = Optimal temperature. S = Supraoptimal temperature. .i:-0

PAGE 46

41 Phosfon-S also gave positive results in alleviating some of the injuries caused by supraoptimal temperature (Tables 3-6) as well as increasing the fresh and dry weights of the plant (Figures 3 and 4). However, phosfon-S was not as effective as sucrose or biotin in increas ing the fresh and dry weights of the plants grown at supraoptimal temperature. Phosfon-S caused a 27 per cent increase in the fresh weight under optimal temperature and a 30 per cent increase under supraoptimal temperature. The increase in dry weight under optimal and supraoptimal temperatures was 9 and 16 per cent, respectively. The addition of malate to the medium reduced the fresh and dry weight of plants under both temperature regimes (Figures 3 and 4). It was also interesting to note a striking effect of malate on the seed pods and seeds. Addition of this metabolite resulted in the produc tion of large seed pods and also in increases in both size and the number of seeds from plants grown at both temperatures (Table 6). The data fail to show any effect o~ malate in reducing or alleviating any of the detrimental effects of supraoptimal temperature on the growth of these plants (Tables 3-6). In this regard, our results are largely in disagreement with those of Petinov and Molotkovsky (1957). They demonstrated the reduction of the injuries caused by supraoptimal temperature in sunflower, pumpkin, oats and corn by spraying the leaves of plants with organic acids, mainly citric and malic acids. However, the absence of ameliorative effects in our experiments could be because of two reasons: (1) Although malic acid was effective on plants used by Petinov and Molotkovsky (1957), it is possible that it is not

PAGE 47

42 effective on Arabidopsis thaliana. In this regard it has been shown that even two species of the same genus may have two different require ments (Langridge and Griffing, 1959). (2) The supraoptimal temperature used by Petinov and Molotkovsky was extremely high (46) which could cause the accululation of ammonia and eventual self-toxification of the plant. In this case, the applied organic acids probably formed the organic salts of ammonia or amides thus removing the NH 3 and elimin ating the detrimental effects of the supraoptimal temperature, Under the temperature regime selected for our experiment, the accumulation of ammonia in concentrations which could be toxic to the plant seems improbable since Anastasia (1966) could not find any detectable amounts of ammonia in pea plants grown under the same temperature regimes. Although the results obtained with sucrose and biotin were obviously significant, the data were statistically analyzed according to the t test. This test showed the results for dry and fresh weights to be highly significant at the .01 level. Effect of Supraoptimal Temperature on the Contents of the Krebs' Cycle Acids Organic acid (citric, malic, succinic and fumaric) contents of the shoots were measured at 2, 3 and 4 weeks after planting and the results are given in Tables 9-11 and Figures 5-9. A. Two Weeks after Planting The organic acid contents of the plants grown under supraoptimal temperature were usually slightly higher than those of the plants grown

PAGE 48

43 at optimal temperature (Table 9). The ratios of various acids in the plants grown under supraoptimal temperature to those grown at optimal temperature ranged from 1 to 1.54 while the ratio of total acids was 1.21. The increase at supraoptimal temperature was greater for malic acid than any other acid in this sampling period. Since the quantity of the other acids did not differ greatly under the two temperature regimes it is possible that supraoptimal temperature caused either enhancement in formation of malic acid, probably from other pools (Lips and Beevers, 1966), or possibly affected the activity of the enzyme, malic dehy drogenase resulting in the accumulation of malic acid. B. Three Weeks after Planting In contrast to the results obtained at 2 weeks (and again at 4 weeks) the contents of the organic acids, with the exception of citric, were higher in plants grown under optimal temperature (Table 10). The total acid content (the sum of citric, malic, succinic and fumaric) of plants grown under supraoptimal temperature was12 per cent of the total found in plants grown at optimal temperature. The difference between fumaric acid contents in plants grown in these two temperature regimes was the most pronounced of all. One possible explanation for the reversal of the acid contents under these two different regimes between 2-week and 3-week periods is that flowering and maximum increase in the rate of stem elongation in plants grown under optimal temperature occurred at this time. The increase in stem length during the 21st day after planting was approximately equal to the total stem elonga tion obtained during the first 19 days of growthl

PAGE 49

Organic Acid Citric Malic Succinic Furnaric Total TABLE 9 EFFECT OF TEMPERATURE ON ORGANIC AQID CONTENT OF THE SHOOT OF ARABIDOPSIS THALIANA 2 WEEKS AFTER PLANTING Moles per Gram Fresh Weight TemEerature Optimal Supraoptimal 4.8 5. 1 9.3 14.4 5.1 5.1 8 3 8 .7 27 .5 33.3 *supraoptimal over optimal. 44 Ratio* 1.06 1.54 1.00 1.05 1.21

PAGE 50

Organic Acid Citric Malic Succinic Fumaric Total TABLE 10 EFFECT OF TEMPERATURE ON ORGANIC ACID CONTENT OF THE SHOOT OF ARABIDOPSIS THALIANA 3 WEEKS AFTER PLANTING Moles per Gram Fresh Weight Temperature Optimal Supraoptimal J 8 5.4 12.5 8 .3 5 8 3.7 19.1 6.6 41.2 24.0 ~ Supraoptimal over optimal. 45 R t ~ a J.O 1.42 0.66 0.63 0.35 0.58

PAGE 51

46 C. Four Weeks after Planting The amounts of organic acids, with the exception of fumaric acid, were higher in plants grown at supraoptimal temperature (Table 11). The most pronounced change caused by supraoptimal temperature occurred in / the citric acid content (8.36 times higher in high temperature). In general the amounts of organic acids increased at supra optimal temperature at 2 and 4 weeks after planting. Citric acid was the only acid which remained higher in plants grown under supraoptimal temperature at all three sampling times. In retrospect, it might have been better to check the effect of supraoptimal temperature on organic acids by sampling plants at the end of dark period, the end of light period, as well as at the inter mediate time chosen for this study. It should be mentioned at this point that the physiological and chronological ages of the plants grown at optimal and supraoptimal temperatures were approximately the same at all sampling periods as shown by plastochrome indices.

PAGE 52

Organic Acid Citri c Malic Succinic Fumaric Total TABLE 1 1 EFFECT OF TEMPERATURE ON ORGANIC ACI D C ONTENT O F THE SHOOT OF ARABIDOPSIS THAL I AN A 4 WEEKS AFTER PLANTING Moles pe r Gram Fr esh Weight Temperature Optimal Supraoptima l 1.1 9 2 6 8 13 4 4 3 5 3 12 5 8 9 24 7 36 8 ~Supraoptimal over opt i mal. 47 Rat io 8 36 1. 97 1.23 0 71 1. 49

PAGE 53

Figure 5. Effect of t e mperature on citric acid content at differ e nt ages.

PAGE 54

2 0 CIT R IC ACID 1 6 OPTIMAL TEMPERATURE SUPRAOPTIMAL TEMPERATURE 1 2 8 4 Q---L--~--'-------Ji.J:~L-----~--'-------1 2 3 4 WEEKS AFTER PLANT I NG F I G. 5

PAGE 55

Figure 6 Effect of temperature on malic acid content at different ages

PAGE 56

MALIC ACID 20 OPTIMAL TE MPERATURE 0 SUPRAOPTIMAL TEMPERATURE 2 3 4 AFTER PLANTING FIG. 6

PAGE 57

Figure 7. Effect of temperature on succinic acid content at different ages.

PAGE 58

1-:t (!) ..._ :t Cl) Lu 20 SUCCINIC ACID 1 6OPTIMAL TEMPERATURE 12 SUPRAOPTIMAL TEMPERATURE 0 __L ___ ~_.._ ___ ___.,b;~.L----~l.-..L.----' 2 3 4 WEEKS AFTER PLANTING FIG. 7 Vl. \.,J

PAGE 59

Figure 8. Effect of temperature on fumaric acid content at different ages.

PAGE 60

20 ..... lu FU L~!\RIC ACID 0 PT lf, ~A L HU.PER A TUR E SUPRAOPTI MA L TEMPERATURE WEEKS AFTER PLANTING FIG. 8 \.Tl. \.Tl.

PAGE 61

Figure 9A. Effect of optimal temperature on organic acid content at different ages. (C ci t ric acid, M m alic acid, S succinic acid, F fumaric acid .) Figure 9B. Effect of supraoptimal temperature on organic acid content at different ages. (C citric acid, M malic acid, S succinic acid, F fumaric acid.)

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57 A F 18 ..... :t: F (.!) 12 ::t:: 6 t3 Pc 0 l( (!) B -.I C) ::t_ 18 M M 12 F F M C s --i C s C F s s 0 2 3 4 W EE K S A F TER PLANTING FIG. 9

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SUMMARY The effects of supraoptimal temperature on growth character istics and Krebs' cycle acid content of Arabidopsis thalian a (1.) Heynh were investigated. The small, rapidly-growing crucifers were grown under aseptic conditions at two temperature regimes (optimal was 25 during day and 18 during night; supraoptimal was 3 2 during day and 25 during night; light intensity was 1100 200 f.c. for both conditions) on agar in large test tubes. Various metabolites were added to the agar medium in attempts to prevent by chemical means t h e detrimental effects of supraoptimal te m perature. The characteristics studied included root length, number of the secondary root~ stem length, number of seed pods and seeds, fresh weight, and dry weight. Supra optimal temperatures depressed all of the measures of growth and reproductive performance investigated except for the len g th of the main stem. It was found that supraoptimal te m perature caused a reduc tion of 45 per cent in the root length, 27 per cent in nu m ber of secondary roots, 65 per cent in number of seed pods, 65 per cent in t h e number of seeds, 48 per cent in fresh weight and 38 per cent in the dry weight of the plant. The main s~ems of the plants grown under 58

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59 supraoptimal temperature were long e r than those of plants grown under optimal temperature up to 24 days after planting. However, the stems of the plants grown under optimal temperature caught up with those grown under supraoptimal temperature. In attempts to cure or prevent the detrimental effects of supraoptimal temperature, biotin, sucrose, malic acid and phosfon-S were individually added to the agar medium under both temperature re gimes. The fresh and dry weights of the plants were used as measures for comparison of the effects of these metabolites on the growth. An ideal curative added metabolite is considered to be one which would increase the fresh and dry weights of the plant under supraoptimal temperature but not, or very little under optimal temperature. Biotin and 1 per cent sucrose approached this ideal, completely preventing any deleterious effect of the supraop t imal temperatures employed. Phosfon-S partially removed the injuries caused by supraoptimal tem perature. Malic acid was ineffective, or a c t ua l ly injurious. Quanti tatively, the effect of the additives wa s a s f o l lo w s: Biotin induced increases of 54 per cent in fresh wei ght a n d 66 per cen t in dry weight of the plant under the supraoptimal tempe ra t ure, while these increases in fresh and d r y w e igh t s for the plan t grown under the optimal temper ature were 38 an d 1 5 per cent, respec t ively. Addition of 1 per cent sucrose induced 58 per cent increase in fresh and 70 per cent in dry weights of the plants grown under supraoptimal temperatures, while under optimal temperature conditions, sucrose actually reduced the fresh weight by 12 per cent and had only slight (2 % ) promotive effect on

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60 dry weights. Phosfon-S increased the fresh and dry weight 30 per cent and 16 per cent, respectively, under the supraoptimal temperature, and 27 per cent and 9 per cent under the optimal temperature. Malic acid caused a reduction in both fresh and dry weights under both temperature regimes, although it was observed to increase the size and the number of seed pods and seeds. The amount of several of the organic acids of the Krebs' cycle in the shoots of the plants were determined by paper chromato graphy Citric, malic, succinic and fumaric acids were identified and measured at 2, 3 and 4 weeks after planting. These measurements revealed that the total amount of the identified acids of the shoot increased by 21 per cent under supraoptimal temperature 2 weeks after planting. This increase was maximum for malic acid (55%). At three weeks after planting, the amount of organic acids of the shoots decreased by 42 per cent under the supraoptimal temperature. This decrease was most pronounced for fumaric acid (65%). The amounts of all acids but fumaric were higher in the shoot of the plant grown under the supra optimal temperature 4 weeks after planting. Citric acid was 8.3 times ('830%) greater under the supraoptimal than under the optimal regime. Increases in the other acids were very much less. Since the amounts of organic acids in general were higher at 2 weeks and again at 4 weeks after planting at the supraoptimal-temperature, the contrast ob served at 3 weeks was attributed to the flowering and also maximum (extremely rapid) rate of stem elongation, both of which occurred at this time.

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LITERATURE CITED Allen, M. B. 1950. The dynamic nature of thermophily. J. Gen. Physiol. 33:205-214. Anastasia, J. V. 1966. M.S. Thesis, The effect of supraoptimal tem perature on protein synthesis and turn over in Pisum sativum L. University of Florida, Gainesville, Florida. Atwood, K. C. and F. Mukai. 1953. Indispensable gene function in Neurospora. Proc. Nat. Acad. Sci. Wash. 39:1027-1035. Bernheim, F. 1955. The effect of temperature on adaptive enzyme. Arch. Biochem. Biophys. 59:252-259. Bonner, J. 1957. The chemical cure of climatic lesions. Eng. and Sci. 20:28-30. Borek, E. and H. Waelsch. 1951. The effect of temperature on the nutritional requirement of microorganism. J. Biol. Chem. 190:191-196. Brown,G N. 1965. Temperature controlled growth rates and ribonucleic a cid characteristics in Mimosa epicotyl tissue. Plant Physiol. 40:557-561. Chao, M. D. and W. E. Loomis. 1947. Bot. Gaz. 109:225-231.(~eference obtained from Went, 1953.) Davern, C. I. 1959. Ph.D. Dissertation, Chemotherapy of high temper ature inhibition of plant growth. Cal. Inst. of Tech., Pasa de na, California. Galston, A. W. 1957. In: The Experimen t al Control of Plant Growth. F. W. Went, ed. Chronica Botanica Co., Waltham, Mass. Pp. 313-317. Galston, A. W. 1959, Adenine and plant growth. Science 129:357, Galston, A. W. and M. E. Hand. 1949. Adenine as a growth factor for etiolated peas and its relation to the thermal inactivation of growth. Arch. Biochem. 22:434-443. 61

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62 Geromino, J. and Harry Beevers. 1964. Effects of aging and temper ature on respiratory metabolism of green leaves. Plant Physiol. 39:786-793, Halpern, Y. S. 1961. Temperature-dependent inducer requirement for the synthesis of glutamic acid decarboxylase by Escherichia coli. Biochem. Biophys. Res. Commun. 6:33-37. Highkin, H. R. 1 0 1958. Temperature-induced variation in peas. Am. J. Botany Li5 : 631-632. Hoagland, D.R. and D. I. Arnon. 1938. The water-culture method for growing plants without soil. Univ. of Cal.Ag. Exp. Sta. Circ. 347. Houlahan, M. B. and H.K. Mitchell. 1947. A suppressor in Neurospora and its use as evidence for allelism. Proc. Nat. Acad. Sci. 33:223-228. Ketellapper, H.J. 1963. Temperature-induced chemical defects in higher plants. Plant Physiol. 38:175-179. Ketellapper, H.J. and J. Bonner. ature responses in plants. 1 96 1. The chemical basis of temper Plant Physiol. 36 suppl. XXI. Kliewer, W. M. 1964. Influence of environment on metabolism of organic acids and carbohydrates in Vitis vinifera. I. Temperature. Plant Physiol. 39:869-880. -Knox, R. 1951. The formation of bacterial urease. Gen. Microbial. 5:xx. Knox, R. 1953. The effect of temperature on enzymic adaptation, growth and drug resistance. Syrop. Soc. Gen. Microbial. 3:184-195, Langridge, J. 1957. The aseptic culture of Arabidopsis thaliana (1.) Heynh. Aust. J. Biol. Sci. 12:243-252. Langridge, J. 1963. Biochemical aspects of temperature response, Annual Rev. of Plant Physiol. 14:441-4 6 2. Langridge, J. and B. Griffing. 1959. A study of high temperature lesions in Arabidopsis thaliana. Aust. J. Biol. Sci. 12: 117-135. Levitt, J. 1951. Frost, drought, and heat resistance. Annual Rev. of Plant Physiol. 2:245-268. Levitt, J. 1962. A Sulfhydryl-disulfide hypothesis of frost injury and resistance in plants. J. Theoret. Biol. 3:355-391,

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63 Lips, S. H ~nd Harry Beevers. 1966. Compartmentation of organic acids in corn roots. II. The cytoplasmic pool of malic acid Plant Physiol. 4:713-717. Lockhart, J A. 1958. The role of gibberellin in the control of pea growth by temperature Planta, Bd. 52:250-258. Luke, H. H and T. E. Freeman. 1965. Effects of victorin on Krebs' cycle intermediates of a susceptible oat variety. Phytopath ology 55:967-969. Maas, W. K. 1950. A temperature sensitive pantothenicless mutant of Escherichia coli. Bact. Proc. 128-129. Maas, W. K. and B. D. Davis. 1952. Production of an altered panto thenate-synthesizing enzyme by a temperature-sensitive mutant of Escherichia coli. Proc. Nat. Acad. Sci. Wash. 38:785-797. McCune, D. 1956. Biology 1956. Cal. Inst. Tech. (Reference obtained from Davern, 1959.) McElroy, W. D. and H.K. Mitchell. 1946. Enzyme studies on a temper ature-sensitive mutant of Neurospora. Federation Proc. 5: 376-379. Mitchell, H K. and M. B. Houlahan. 194 5 Neurospora. IV. A temper ature-sensitive riboflavinless mutant. Arn. J. Botany 33: 31-35. Molotkovsky, Yu. G. 1961. Changes in adnosine triphosphate activity of subcellular units in heat-treated plants. Fiziologiya Rastenii 8:669-672. Molotkovsky, Yu. G. and I. M. Zhestkova. 1964. Mechanism of the protective action of sugars at high temperatures. Translated from Fiziologiya Rastenii 11:301-307. Petinov, N. S. and Yu. G. Molotkovsky. 1957. Protective reactions in heat-resistant plants induced by high temperatures. Fiz : iologiya Rastenii 4: 225-233. Petinov, N. S. and Yu. G. Molotkovsky. 1960. The effect of respira tory inhibitors on heat resistance in plants. Translated from Fiziologiya Rastenii 7:665-672, Pollock, M. R. 1945. The influence of temperature on the adaptation of "Tetrathionase" in washed suspensions of Bact. paratyphosum B. Brit. J Exptl. Pathol. 26:410-416.

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Pollock, M. 1950. Penicillinase adaptation in B. cereus: enzyme formation in the absence of free substrate. Exptl. Pathol. 31:739-753. Adaptive Brit. J. 64 Rabson, R. and F. C. Steward. 1959. (From Steward, F. C., F. Cane, K. Millar, R. M. Zacharius, R. Rabson and D. Margolis. 1959.) Nutritional and environmental effects on the nitrogen metab olism of plants. Symposia on the society for experimental biology. Printed in Great Britain. Rahn, 0. and W.R. Schroeder. 1941. Inactivation of enzymes as the cause of death. Biodynamica 3:199-208. Shokraii, E. H. 1965. Ph.D. Dissertation, The effect of high temper ature on the free amino acids on common pea (Pisum sativum L.): University of Florida, Gainesville, Florida. Smith, I. 1958. Chromatographic techniques. William Reinemann. Medical Books, Ltd., London, Interscience Publishers, Inc., New York. Went, F. W. 1953. The effect of temperature on plant growth. Annual Rev. of Plant Physiol. 4:347-362. Went, F. W. 1957. Some aspects of effects of temperature on plants. Ying, In: Influence of Temperature on Biological System. Johnson, ed. John Wiley and Sons Company, New York. Huii-Kuen. 1965. Ph.D. Dissertation, The effect of temperature on growth and the metabolism of ribonucleic acid in relation to cell division and cell elongation of Pisum sativum 11 Alaska. 11 University of Florida, Gainesville, Florida.

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BIOGRAPHICAL SKETCH The author, Azizollah Shiralipour Soltanaba di, was born on May 5, 1935, in Ahwaz, Iran. He received his elementary and secondary education at Danesh Pahlavi Elementary School and Dr. Hessabi High School in Ahwaz. In 1955 he entered the University of Ahwaz, Iran. Three yea~s later he received the degree of Bac helo r of General Agriculture as the top student of his college and hence wa s awarded a four-year scholar ship from the Government of Iran to further his studies in the United States. He was employed by Khuzestan Development Service in the fertilizer program at his home town for the period of ten months prior to coming to the United States. In September, 1959, he entered the University of Florida and received the degree of Bachelor of Science in Agriculture in August, 1960. In September, 1960, he enrolled in the Graduate School of the University of Florida, majoring in Soils and received the degree of Master of Agriculture in June, 1962. He was employed by the Agricultural Experiment Station, Depart ment of Botany, in June, 1962. He also co ntinue d his studies toward the degree of Doctor of Philosophy in the Department of Botany. He is a member of Alpha Zeta, Phi Sigma, agricultural and biological honor fraternities, respectively, and American Society of Plant Physiologists. 65

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The author is married to the former Miss Joan Strother and they have one child, Laleh. 66

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This dissertation was prepared under the direction of the chairman of the candidate's supervisory committee and has been approved by all members of that committee. It was submitted to the Dean of the College of Agriculture and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December, 1966 Supervisory Committee: ~ -:!7--4 ~ College of Agriculture Dean, Graduate School