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UBC Theses and Dissertations

Growth and metabolic responses of the bush bean to potassium naphthenates Fattah, Quazi Abdul 1969

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5oe# GROWTH AND METABOLIC RESPONSES OF THE BUSH BEAN TO POTASSIUM NAPHTHENATES by QUAZI ABDUL FATTAH M.Sc. Dacca University, East Pakistan B.Sc. (Hons.), Bristol University, England A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILCSOPHY in the Department of BOTANY We accept this thesis as ccnforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department of Botany The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada September 26, 1969 i ABSTRACT Recent investigations have shown that application of appropriate concentrations of naphthenate induces greater growth and yield of several crop plants. However, reports are lacking on the effect of naphthenate on plants grown under various temperature and light conditions and also on physiological and biochemical changes induced in bush bean (Phaseolus vulgaris L.Var. Top Crop) plants following naphthenate treatment. In the course of the present work the following aspects were investigated: 1) juvenile growth, as measured by fresh and dry weight of roots, stem and leaves, number and area of leaflets and plant height; 2) reproductive growth, as measured by flower number, number and fresh weight of pods, and number and weight of dry seeds; 3) chemical composition, such as moisture content of roots, stem, leaves, and pods, chlorophyll and carotenoid content of leaves, ascorbic acid content of green pods and loss of ascorbic acid by pods during storage for five days, and 4) such physiological and metabolic changes as rates of apparent photosynthesis and dark respiration, activities of the enzymes nitrate reductase, glutamic-pyruvic transaminase, i i phosphorylase and phosphoglyceryl kinase. Subsequent to KNap treatment, plants i n some experi-ments were grown i n growth rooms provided with 26°/26°, 26°/21° and 15°/15°C, day/night temperature. At 26°/26° and 15°/15° plants were grown under three d i f f e r e n t l i g h t i n t e n s i t i e s , 1500, 1000 and 500 f t - c . The results revealed that: (1) treatment with KNap resulted in increases i n plant height, number and area of l e a f -l e t s , fresh and dry weight of roots, stem and leaves, and t o t a l chlorophyll content i n leaves; (2) measurements made with intact plants using an infraredd C0£ analyzer revealed increases i n rates of apparent photosynthesis and dark r e s p i r a t i o n i n treated plants; (3) the a c t i v i t y of the four enzymes mentioned was stimulated i n plants treated with KNap; (4) increases i n number and fresh weight of green pods, number and weight of seed were observed i n treated plants; (5) treatment resulted i n higher ascorbic acid content i n green pods at harvest and the treatment had a protective action on ascorbic acid loss during storage. Different plant organs were found to respond d i f f e r e n t l y to treatment depending on temperature and l i g h t i n t e n s i t y i n which the plants were grown. The maximum r e l a t i v e stimulatory ef f e c t of KNap treatment was found mostly at 26°/21° and i t was i i i followed by 26°/26° and 15°/15°, i n plants grown under a l i g h t i n t e n s i t y of 1500 f t - c . Plants grown at 26°/26° showed maximum r e l a t i v e stimulation i n most instances i n high l i g h t . The maximum r e l a t i v e stimulation for plants grown at 15°/15° was i n medium l i g h t generally speaking. In proposing a physiological and biochemical basis for the stimulation of growth and y i e l d following KNap treatment, the following points may be emphasized: (a) the stimulated rate of photosynthesis produced a larger amount of photosynthate which could be u t i l i z e d i n the biosynthesis of a l l c e l l constituents and serve as substrate for r e s p i r a t i o n and other chemical processes; (b) the stimulated rate of re s p i r a t i o n and a c t i v i t y of phosphoglyceryl kinase resulted in an increased supply of available energy, as ATP and reduced nucleotides, for biosynthesis; (c) the augmented supply of amino acids r e s u l t i n g from the greater a c t i v i t y of n i t r a t e reductase and transaminase would be favorable for enhanced synthesis of protein, evident i n stimulated growth. iv ACKNOWLEDGEMENTS The author, in deepest gratitude, wishes to acknowledge the guidance, advice and encouragement extended to him by Professor D.J. Wort throughout the progress of this investigation. In addition, the author wishes to thank him for his constructive review of the entire manuscript. Sincere gratitude and thanks go to Dr. J.J.R. Campbell, Professor of Microbiology, Dr. B.A. Bohm and Dr. E.B. Tregunna, Department of Botany and Dr. D.P. Ormrod, Department of Plant Science, for their continued interest and helpful suggestions during this study. The author acknowledges with thanks the help of Mr. S.W. Borden in s t a t i s t i c a l analysis of the experimental results. Thanks are also due to the Ministry of Education, Government of Pakistan, for awarding the author a Central Overseas Scholarship, and to the University of Dacca, East Pakistan, for granting him study leave. The study was carried out in the Department of Botany, U.B.C., and the author wishes to express his appreciation of the excellent f a c i l i t i e s made available to him. V TABLE OF CONTENTS PAGE Abstract i Acknowledgement iv Table of Contents v List of Tables ' x List of Figures x i i INTRODUCTION 1 NAPHTHENIC ACIDS 5 REVIEW OF EFFECTS OF NAPHTHENATES 8 A. Effect of naphthenates on growth and yield 8 1. Growth responses 8 2. Yield 10 B. Effects on plant composition 17 1. Moisture content 17 2. Pigments 17 3. Ascorbic acid 18 C. Effect on metabolism 19 1. Photosynthesis 19 2. Respiration 20 3. Enzymes 20 4. Other metabolic changes 22 v i PAGE MATERIALS AND METHODS 25 A. Plant material 25 B. Preparation of the potassium naphthenate 26 C. Spray treatment 26 D. Measurement times 27 E. Juvenile growth measurements 27 F. Yield measurement 28 G. Analytical procedures 28 1. Pigment analysis 28 2. Ascorbic acid 29 H. Determination of photosynthesis and 30 respiration rates I. Determination of enzyme activities 33 1. Nitrate reductase 33 2. Glutamic-pyruvic transaminase 34 3. Phosphorylase 36 4. Phosphoglyceryl kinase . 38 5. Determination of protein 39 RESULTS 41 I. Plants grown at 26°/21°C temperature 41 A. Juvenile growth 41 1. Fresh weights 41 2. Dry weights 44 v i i PAGE 3. Shoot elongation 44 4. Leaflet number and area 47 B. Effects on plant compostion 47 1. Moisture content 47 2. Chlorophyll and carotenoid content 47 3. Ascorbic acid content of pods 50 C. Yiel d 50 1. Pod number 50 2. Pod fresh weight 50 D. Moisture content of pod 50 II. Plants grown at 15°/15°C temperature 53 A. Juvenile growth 53 1. Fresh weights 53 2. Dry weights 56 3. Shoot elongation 56 4. Leaflet number and area 56 B. Chemical composition 59 1. Moisture content 59 2. Chlorophyll and carotenoid content 61 3. Ascorbic acid content of fresh pods 61 C. Yield 64 1. Flower number 64 2. Pod number 64 3. Pod fresh weight 64 v i i i PAGE D. Moisture content of pods 64 III. Plants grown at 26°/26°C temperature 67 A. Juvenile growth 67 1. Fresh weights 67 2. Dry weights 70 3. Shoot elongation 70 4. Leaflet number and area 73 B. Chemical composition 73 1. Moisture content 73 2. Chlorophyll and carotenoid content 75 3. Ascorbic acid content of pods 75 C. Yield 78 1. Flower number 78 2. Pod number 78 3. Pod fresh weight 81 4. Seed yield 81 D. Moisture content of pods 81 E. Photosynthesis and respiration rates 84 1. Photosynthesis 84 2. Respiration 84 F. Enzyme activities 84 1. Phosphorylase 87 2. Phosphoglyceryl kinase 87 3. Nitrate reductase 4. Glutamic-pyruvic transaminase DISCUSSION CONCLUSION BIBLIOGRAPHY X LIST OF TABLES TABLE PAGE I II Enzyme nomenclature 32 Effect of KNap spray on fresh weights, dry weights and moisture content (% moisture) 42 of roots, stem and leaves of bush bean plants. I l l IV V VI VII VIII IX X Effect of KNap spray on plant height, leaf-let number and leaf area of bush bean plants. Effect of KNap spray on chlorophyll and carotenoid content of bush bean leaves. Effect of KNap spray on yield, moisture content and ascorbic acid content of pods, of bush bean plants. Effect of KNap spray on fresh and dry weights and percentage moisture contents of roots, stem and leaves of bush bean plants. Effect of KNap spray on plant height, and number and area of leaflets in bush bean plants. Comparative effects of treatment on juvenile growth of bush bean plants with KNap spray in three light intensities at 15°/15°C. Effect of KNap spray on chlorophyll and carotenoid content of bush bean leaves. Effect of KNap spray on flower number, number and weight of pod, moisture content and ascorbic acid content of pods of bush bean plants. 45 48 51 54 57 60 62 65 XI Effect of KNap spray on fresh and dry weights and moisture contents of roots, stem and 68 leaves of bush bean plants. xi TABLE XII XIII XIV XV XVI XVII XVIII XIX XX XXI Effect of KNap spray on plant height, and numbers and area of leaflets in bush bean plants. Comparative effects of treatment on juvenile growth of bush bean plants with KNap spray in three light intensities at 26°/26°. Effect of KNap spray on chlorophyll and carotenoid content of bush bean leaves. Effect of KNap spray on ascorbic acid content, moisture content, and their loss during 5 days storage, in pods of bush bean plants. Effect of KNap spray on yield of bush bean plants. Effect of KNap on photosynthetlc and respiration rates of the aerial portions of intact bush bean plants. Effect of KNap spray on the activity of phosphorylase and phosphoglyceryl kinase in leaves of bush bean plants. Effect of KNap spray on the activity of nitrate reductase in bush bean plants. Effect of KNap spray on the activity of glutamic-pyruvic transaminase in bush bean leaves. Relative stimulation of juvenile growth, number, weight and ascorbic acid content of pod in plants grown in 1500 ft-c and at three temperatures. PAGE 71 74 76 79 82 85 88 91 95 99 x i i LIST OF FIGURES FIGURE PAGE 1. Ef f e c t of KNap spray on fresh and dry weights of roots, stem and leaves of 43 bush bean plants grown at 26°/21°. 2. Eff e c t of KNap spray on plant height, and number and area of l e a f l e t s of bush 46 bean plants grown at 26°/21°. 3. Eff e c t of KNap spray on chlorophyll a, b and carotenoid, and t o t a l pigment content 49 of leaves of bush bean plants grown at 26°/21°. 4. Eff e c t of KNap spray on number, weight, moisture and ascorbic acid content of ^ green pods of bush bean plants grown at 26°/21°. 5. E f f e c t of KNap spray on fresh and dry weights of roots, stem and leaves of bush bean plants 55 grown at 15°/15°. 6. E f f e c t of KNap spray on plant height, and number and area of l e a f l e t s of bush bean 58 plants grown at 15°/15°. 7. E f f e c t of KNap spray on chlorophyll a, b and carotenoid, and t o t a l pigment content 63 of leaves of bush bean plants grown at 15°/15°. 8. Eff e c t of KNap spray on flower number, and number, weight and ascorbic acid content of 66 green pods of bush bean plants grown at 15°/15°. 9. E f f e c t of KNap spray on fresh and dry weights of roots, stem and leaves of bush bean plants 69 grown at 26°/26°. 10. Eff e c t of KNap spray on plant height, and number and area of l e a f l e t s of bush bean 72 plants grown at 26°/26°. x i i i FIGURES PAGE 11. E f f e c t of KNap spray on chlorophyll a, b and carotenoid, and t o t a l pigment 77 content of leaves of bush bean plants grown at 26 ° /26 ° . 12. Eff e c t of KNap spray on ascorbic acid content, and i t s loss during 5 days storage, i n green pods of bush bean plants grown at 26°/26°. 18. Rate of apparent photosynthesis i n bush bean plants. 80 13. E f f e c t of KNap spray on flower number, and number and weight of pod and seed 83 of bush bean plants grown at 26°/26°. 14. E f f e c t of KNap spray on photosynthetlc and r e s p i r a t i o n rates of the a e r i a l g£ portions of intact bush bean plants grown at 26°/26°. 15. E f f e c t of KNap spray on phosphorylase and phosphoglyceryl kinase a c t i v i t i e s i n 89 leaves of bush bean plants grown at 26°/26°. 16. E f f e c t of KNap spray on n i t r a t e reductase a c t i v i t y i n roots and leaves of bush bean 92 plants grown at 26°/26°. 17. Eff e c t of KNap spray on glutamic-pyruvic transaminase a c t i v i t y i n leaves of bush 96 bean plants grown at 26°/26°-110 19. Rate of dark re s p i r a t i o n i n bush bean plants. 112 20. A diagram to show the points where stimula-tion by KNap was observed i n the investiga-tion . 116 1 INTRODUCTION The use of naphthenic acid and its salts as plant growth stimulators is rather recent and as such they have been utilized primarily by Russian and Bulgarian workers. In low concentrations naphthenic compounds have been found to promote vegetative growth and yield, and to affect physiological and biochemical activities of a number of plants. In higher concentrations the compounds act as herbicides. Naphthenic acids (HNap) have also been found to stimulate growth of a number of species of animals. The mechanism of the action of HNap on plants is relatively unknown. Apparently there are stages in the l i f e of a plant at which HNap application is most effective, but there is a lack of specific information in this regard. As is the case with many chemical growth regulators the fin a l results of HNap application are determined not only by the general nature of the acids used but also by the concentration used, the pH, the carrier, the method of application and the size of droplet or dust particle applied. The species of plant, the part of the plant to which the chemical is applied, the plant"s age, vigor and growth, a l l may play a part in determining the response. Moreover, weather conditions such as temperature, light intensity 2 and humidity; s o i l conditions such as the nature of the s o i l and the level and availability of its nutrients, are of importance in determining the outcome. Information concerning the effect of many of the factors mentioned above is very sparse, particularly that having to do with the relationship of temperature and light intensity to the response of plants to the application of naphthenic compounds. There are no previous reports on the effects of different condi-tions of light and temperature on the growth, development, yield, and physiological and biochemical processes of bush bean plants to potassium naphthenates (KNap) application. In view of the considerations outlined above, i t was f e l t very desirable to investigate changes that occur during the different stages of growth of the bush bean plant following KNap treatment. In the course of the present work the following aspects were investigated at different stages of growth subsequent to the application of an aqueous spray of 5000 parts per million (ppm) KNap to 2-week-old bush bean (Phaseolus vulgaris L. Var. Top Crop) plants: 1. Juvenile growth: Plant height, leaf number and area, fresh and dry 3 weights of roots, stems and leaves. 2. Chemical composition: Moisture content of leaf, stem, rootsand pods; chlorophyll a and b and carotenoid content of leaves, ascorbic acid content of green pods and loss of ascorbic acid by pods during storage for five days. 3. Yield response: Number and weight of pods, seed number and seed weight per plant. 4. Metabolic changes: Rates of photosynthesis and respiration, activities of four enzymes of nitrogen and carbohydrate metabolism, viz., nitrate reductase, glutamic-pyruvic transaminase, phosphor-ylase, phosphoglyceryl kinase. A l l the preceding aspects were investigated in bush bean plants grown under a constant day and night temperature of 26 1°C, and aspects concerning juvenile growth, chemical composition and yield in plants grown under a constant day and night temperature of 15 * 1°C. These two sets of investigations could indicate the response of bush bean to KNap under a high and a comparatively low temperature. In both temperatures bush beans were grown under three different light intensities, 1500, 1000, and 500 foot candles 4 (ft-c). In addition, juvenile growth, chemical composition and yield were investigated in bush bean plants grown ; under a temperature of 26 * 1°C during the light period and 21 * 1°C during the dark period. An attempt has been made to interpret the results obtained. 5 NAPHTHENIC ACIDS The name, naphthenic acids, was f i r s t suggested in 1 8 8 3 by Markovnikoff and Ogloblin for the C ^ - ^ H ^ Q O ^ acids of unknown structure which Hell and Medinger ( 1 8 7 4 ) had recovered from Rumanian o i l . Currently the term is used to denote the carboxylic acids occurring in and recovered from petroleum. They are also known as "petroleum acids", because work has shown that aliphatic acids and phenols are also present in some crude o i l s . Commercial naphthenic acid (HNap) is a product which contains a l l the acidic components of * the crude, and varying amounts, usually less than 1 0 % , of ' o i l ' , that is to say nonacidic compounds, most-ly hydrocarbons. While the structure of the higher molecular weight HNap has yet to be determined, i t is known that the acids may be carboxylic derivatives of cyclopentane, cyclohexane and cyclo-heptane. The average molecular weight of the mixtures usually lies in the range 2 0 5 to 3 3 0 . Generally the carboxylic group is not attached directly to the ring, but through a methylene group of a chain containing five or more methylene groups. The general formula may be written as RCC^^COOH, where R is a cyclic nucleus composed of one or more rings. The simplest acid conforming to this definition 6 when n = 1 is cyclopentane acetic acid (Mol. wt. 128), ;HoCC00H CH 2 •CH. 2 Naphthenic acids have a characteristic odor which varies with the acid source, degree of refinement, and content of phenol and sulfur compounds. Naphthenic acids are soluble in a l l proportions in hydrocarbons, and the lower members have slight solubility in water. driers, catalysts, preservatives, emulsifiers, corrosive inhibitors and fungicides (Jolly, 1967) . Recently HNaps and their salts of sodium, potassium, copper, cobalt, manganeese and nickel have been used as growth stimulators of plants and animals and also as herbicides. have been named differently by different workers, viz., naphthenic growth stimulators or naphthenic growth substances (NGS), petroleum growth substances or stimulators (PGS), petroleum Naphthenic acids have been used variously, as lubricants, The plant growth stimulators obtained from petroleum 7 nutrient, petroleum growth promoters, petroleum growth helping substances, R.V. ( i n i t i a l letters of Russian words meaning 'growth helping substance'), and naphthenic acid (HNap). Detailed information about occurrence, composition, properties, recovery and refining, purification and economic aspects and uses of HNap has been given by Jolly (1967). Potassium naphthenate (KNap) used in this investigation was made by adding KOH solution to HNap. HNap was obtained from Eastman Organic Chemicals, Rochester, N.Y., as "Naphthenic acids (practical)". The average molecular weight of this HNap was stated to be 230. o 8 REVIEW OF EFFECTS OF NAPHTHENATES The l i t e r a t u r e cited i n this review is related to the effects of HNap and i t s salts on growth, y i e l d , chemical composi-tion , photosynthesis, r e s p i r a t i o n and enxymic a c t i v i t i e s of plants. Emphasis has been put on the l i t e r a t u r e pertaining to the ef f e c t of naphthenates i n the areas investigated by the author. Reports of other growth regulators have been included for comparative purposes. I n a c c e s s i b i l i t y of o r i g i n a l Russian a r t i c l e s has necessitated reference to Chemical and B i o l o g i c a l Abstracts i n a number of cases. In the Naphthenic Acids i t has been indicated that a variety of abbreviations have been used for the extractives of petroleum which apparently are more or less i d e n t i c a l with the mixture described i n this thesis as "naphthenates". The l a t t e r term w i l l be used for convenience. A. Effect of naphthenates on growth and y i e l d . The p r a c t i c a l application of naphthenates to plants i n Russia dates back to 1943. Various species were stimulated. In this section the responses of a number of plants are summarized. 1. Growth responses Weak solutions (0.0001%) of naphthenates stimulated root growth of cotton by 36%, cucumber 96%, onion 166% and winter 9 wheat 1 9 3 7 o as compared with the control (Huseinov, 1 9 6 0 ) . Increase in the leaf surface of potato plants due to naphthenate treatment was obtained by Ladygina ( 1 9 6 5 ) . Naphthenate treatment increased the plant growth and the leaf surface of tobacco plant (Zamanov, 1 9 6 6 ) . Kosobokov ( 1 9 6 5 ) applied 0 . 0 0 5 to 0 . 0 1 % naphthenates on peas, feedbeans and corn and the treatment gave a favorable effect on germination and the subsequent growth and development of the plants. The use of 0 . 0 1 % naphthenate during root formation in green cuttings of cherries, gooseberries and black currants favorably affected the formation of roots and subsequent develop-ment of cuttings. (Polikarpova, 1 9 6 5 ) . Naphthenate treatment was also found by the same investigator to increase the growth intensity of shoots. Spraying of eggplant with 0 . 0 5 7 o naphthenates by A l i Zade and Guseinov ( 1 9 6 5 ) resulted in a 2 1 % increase in plant height, 1 2 % increase in the aerial mass of the plants, 1 3 % decrease in the root system, and 6 % increase in the total weight of the plant. There was an appreciable increase in the number of branches and also a 1 4 3 7 o increase in the assimilation area of leaves. Gurvich ( 1 9 6 8 ) reported that seeds of Allium 1 3 7 fistulosum irradiated by "tf -rays from Cs at the dose of 1 kr, in the state of physiological rest, showed a delay in the onset of mitosis after the beginning of seed germination and an inhibition of the total mitotic activity for 5 4 to 5 7 hours. There was a temporary delay of mitosis and i t was compensated 1 0 d u r i n g t h e f o l l o w i n g 6 0 t o 6 3 h o u r s . W h e n t h e i r r a d i a t e d s e e d s w e r e g e r m i n a t e d i n 0 . 0 0 1 t o 0 . 0 0 0 1 % s o d i u m n a p h t h e n a t e s o l u t i o n s , h o w e v e r , t h e r e w a s a n i n t e n s i f i c a t i o n o f t h e m i t o t i c a c t i v i t y . H e f u r t h e r m e n t i o n e d t h a t i n c o m p a r i s o n t o o t h e r f r a d i o - p r o t e c t i v e c o m p o u n d s r e p o r t e d i n t h e l i t e r a t u r e , s o d i u m n a p h t h e n a t e g a v e t h e 1 3 7 m a x i m u m r a d x o p r o t e c t i o n f r o m C s . W e a k c o n c e n t r a t i o n s o f n a p h t h e n a t e s , 0 . 0 0 0 1 t o 0 . 0 0 0 7 % , s h o w e d a f a v o r a b l e e f f e c t o n p o l l e n g e r m i n a t i o n a n d t h e s u b s e q u e n t g r o w t h o f p o l l e n t u b e s ( K r a w c h a n k a , 1 9 6 6 ) . A t h i g h e r c o n c e n t r a t i o n s n a p h t h e n a t e s o l u -t i o n s w e r e f o u n d t o d e p r e s s t h e p r o c e s s o f f e r t i l i z a t i o n . H i g h c o n c e n t r a t i o n s o f n a p h t h e n a t i s w e r e s h o w n t o b e h e r b i c i d a l b y Z h u k o v a ( 1 9 6 5 ) «, A 9 5 % k i l l w a s r e p o r t e d . A c c o r d i n g t o K a z a k o v a ( 1 9 6 5 ) 0 . 0 0 0 1 t o 0 . 0 1 % n a p h t h e n a t e s s t i m u l a t e d b o t h g r o w t h a n d g e r m i n a t i o n o f w e e d s . 2 . Y i e l d T o b a c c o ; H u s e i n o v ( 1 9 6 0 ) a n d z a m a n o v ( 1 9 6 6 ) r e p o r t e d t h a t w e a k s o l u t i o n s o f n a p h t h e n a t e s i n c r e a s e d t h e g r o w t h a n d l e a f a r e a o f t h e t o b a c c o p l a n t . T h e r e s u l t s h o w e d t h a t w h e n u s e d a s s p r a y t h e b e s t r e s u l t s w e r e o b t a i n e d w i t h 0 . 0 0 5 % n a p h t h e n a t e s o l u t i o n . R e p e a t e d s p r a y i n g w i t h 0 . 0 0 5 % n a p h t h e n a t e s d u r i n g t h e v e g e t a t i v e p e r i o d g a v e 1 2 t o 3 5 % i n c r e a s e i n y i e l d i n c o m p a r i s o n w i t h w a t e r 11 spraying. The application of naphthenates promoted accumulation of dry substance and improved the quality of the tobacco. By applying naphthenates at the " f i r s t pair of collateral leaves" stage of tobacco plants Popoff and Hristzov ( 1 9 6 6 ) obtained a 5 . 8 to 9 . 6 7 0 increase in the yield. However, there was no clearly expressed effect on the quality of the leaves. Potato The application of 0 . 0 2 and 0 . 0 5 7 o naphthenates to two species of potato plants (Ladygina, 1 9 6 5 ) resulted in an increase in tuber yield of 9 to 9 . 6 7 o . Increases in yield of tubers were reported by Krasonova et al ( 1 9 6 5 ) 4 8 7 o , Abolina and Ataullaev ( 1 9 6 6 ) 3 5 to 4 0 7 o , and Popoff and Boikov ( 1 9 6 6 ) 2 7 7 0 . Corn: Yureva ( 1 9 6 5 ) found that the application of naphthenates favorably affected the growth of maize plants, resulting in more corn silage and a 1 3 to 4 3 7 = increase in cobs. The yield of corn increased due to naphthenate treatment (Szekely and Gleria, 1 9 6 6 ) . According to Eyubov and Issaeva ( 1 9 6 6 ) the best results with respect to growth and yield were obtained by spraying maize plants with 0 . 0 0 5 7 o naphthenates during the flowering period. They also found that soaking the seeds in naphthenate solution for 1 2 hours before sowing considerably increased the plant growth, and 12 stimulated the development of the vegetative and the generative organs of maize and lucerne. Seed-soaking augmented the maize grain yield by 18 to 35%. Cabbage: In 1956, Guseinov e_t al reported that naphthenates increased cabbage yield by 206% when the chemical was used along with normal mineral f e r t i l i z e r material. Cabbage plants treated with weak solutions of naphthenates gave an increase in yjLeJLd by 15 to 207c. The most effective concentration was found to be 0.005% (Huseinov, I960). Asadov (1965) found that naphthenates added to f e r t i l i z e r or sprayed on cabbage plants increased the cabbage crop by 15 to 26% and 12 to 28% respectively. In an earlier work Asadov (1943) applied sodium naphthenate at the time of planting of cabbage seedlings, 50 or 100 g/ha, and during growth as spray solution, 25 to 100 g/ha in combination,, with trace mineral f e r t i l i z e r s containing Fe, Al, Zn, Cu, Co, Mn, Mo, and Ag. Ripening of the cabbage heads was accelerated and the yield was increased by 12 to 27% with naphthenate treatment and when used with trace mineral f e r t i l i z e r s the yield was increased by 13 to 32%. Presowing treatment of carrot, cabbage, beet and onion seeds with naphthenates together with organomineral f e r t i l i z e r s increased the yield by 5 to 20% (Zhukova, 1965). Muskmelon, cabbage, tomato and carrots^treated with 0.0005, 0.005 13 and 0.01% naphthenates showed activated metabolism, greater growth and development and a larger y i e l d . The increase i n y i e l d i n cabbage was 307o (Ataullaev, 1965). Tomato: Guseinov e_t al (1956) used naphthenates along with normal mineral f e r t i l i z e r and as a r e s u l t tomato f r u i t y i e l d was increased up to 138%. The f r u i t y i e l d to tomato plants treated with 0.005 and 0.05% naphthenate solution was increased by 40 to 50% (Huseinov, 1960). An increase of 30 to 37% i n y i e l d of tomato f r u i t followed the application of naphthenate solutions by A l i e v (1965). The most e f f e c t i v e modes of application of naphthenates were i n s o i l and as spray. Seed treatment with naphthenates and naphthenates i n combination with trace elements increased the tomato f r u i t y i e l d and the average weight of berries (Szekely and G l e r i a , 1966). Popoff and Boikov (1966) obtained early ripeness by spraying tomato plants with naphthe-nates. By using 2500 and 5000 ppm KNap as f o l i a r spray on 3-week-old tomato plants Chu (1969) observed that the tomato f r u i t y i e l d , based on number and fresh weight, was decreased by 2500 ppm treatment but was increased by 5000 ppm treatment. Wheat and Rye: After soaking winter wheat seeds with 0.4% naphthenates 14 p r i o r to sowing the grain y i e l d increased by 30% (Shaki-Zade, 1963). Popoff and Boikov (1966) obtained increases up to 10% i n the y i e l d of grain when naphthenates were applied to winter wheat and winter rye. Under green house conditions naphthenates increased the number of fruit-bearing stems and also raised the lodging resistance. Grape: Addition of naphthenates and th e i r similar Bulgarian preparation HTI, to Bordeaux mixture used to spray d i f f e r e n t vine v a r i e t i e s before the blossoming season and 10 to 14 days a f t e r i t , produced an increase of the weight of the clusters and there was an increase i n y i e l d up to 28% (Popoff and Boikov, 1966). Kolesnik (1965) found that 0.0057o naphthenate spray increased grape y i e l d by 10%. The stimulative e f f e c t was also evident i n the following years. Beet: Yureva (1965) reported that wetting sugarbeet seeds for 16 hours i n 0.005% aqueous naphthenate solutions and/or spray-ing of plants resulted i n an increase of beet root crop. Cotton: Soaking cotton seeds with naphthenate solution at 15 sowing time increased the amount of cotton boll s up to 3.1% (Guseinov and Guseinov, 1958). Moistening cotton seeds with 0.01% naphthenates at sowing time increased the crop from 480 to 590 kg/ha (Shaki-Zade, 1963). Naghibin (1966) reported that ::naphthenates exerted a favorable influence upon;: the growth of the cotton plant and accelerated the ripening of the plant. The opening of the b o l l s was 2 or more days e a r l i e r than i n the control plants. The weight of the cotton wool i n a b o l l i n -creased. The increase i n y i e l d was more than 5 to 10% i f the seeds were soaked for 3 to 4 hours i n a 0.01% solution of the chemical. He also found that i f sprinkled with naphthenate solutions at the flower stage y i e l d was increased by 207o. By treating cotton plants with 0.01% naphthenate solution, Bazanova and Akopova (1966) obtained an increase in the crude f i b e r y i e l d . Other plants: Huseinov (1960) reported the use of 0.005, 0.01 and 0.05% naphthenates on aubergines (eggplant), tea, r i c e and cucumbers. In aubergines the 0.005% naphthenate treatment gave an increase i n crop y i e l d of 32%. For tea, r i c e and cucumber the febest naphthenate concentration was found to be 0.05% and i t gave increases of 39, 28 and 40% respectively. In carnation naphthe-nate treatment increased the size of flower. The number of flowers and seeds was found to be increased by 27 and 54% 16 respectively (Popoff and Boikov, 1966). In the case of apple, Sardarova et a l (1958), observed that naphthenate treatment had stimulatory effects on the growth and development of seedlings. Spraying of tangerine plants with 0.05% naphthenates before and during flowering increased the y i e l d by 18.6 to 21.2% and also increased the average weight of the f r u i t (Marshaniya et a l , 1965). Guseinov and Masiev (1965) sprayed 0.005 and 0.0005% naphthenate solutions on mulberry and o l i v e trees and the treat-ment accelerated the growth and development of above ground parts of the plants. Beans: There are only a few reports on the ef f e c t of naphthe-nates on bean plants. The stimulatory effect of naphthenates upon the pod y i e l d of china bean has been observed by Bachramov (1957). Experiments made by Popoff e_t a l (1966) indicated that naphthenates obtained from Bulgarian naphtha markedly stimulated the growth of bean plants. Popoff and Boikov (1966) obtained an increase i n y i e l d of f i e l d beans by 4 to 14%. In the papers mentioned the genus and the species of the beans were not stated. Investigators at the University of B r i t i s h Columbia obtained enhanced growth i n various plants treated with KNap. Maize, radish, sugarbeet, tobacco, spinach, sunflower and bush bean (Phaseolus vulgaris L. Var. Top Crop) 17 were found to be stimulated by KNap treatment (Wort, In Press). B. Effects on plant composition: 1. Moisture content Moisture content change is one of the early symptoms of hormone action on plants (Freiberg and Clark, 1952; Wort, 1951). There i s , however, a dearth of reports available on moisture content and i t s loss or gain due to the action of naphthenates. An increase in water absorption by potato plants due to naphthe-nate treatment was obtained by Krasonova et al_ (1965) ; but he did not mention the moisture content of different parts of the potato plant.' The corn silage was more moist due to naphthenate treat-ment of the plants (Yureva, 1965), but in this case also no detailed report was available. 2. Pigments: Chlorophylls: There is a small number of reports available on the effect of naphthenates on chlorophyll pigments of plants. Soak-ing tubers of two species of potatoes in 0.00057o naphthenate for one hour before planting increased the assimilative surface per plant and also the chlorophyll content of the leaves (Ladygina, 1965). Yureva (1965) obtained an increase in 18 chlorophyll content in the leaves of corn and sugarbeets due to the application of naphthenates. In the case of sugarbeets he observed that more chlorophyll was formed i f the seeds were soaked for sixteen hours in 0.01 and 0.005% naphthenates and subsequently the plants were double sprayed with 0.005% aqueous solution of the chemical. In their experiments with cotton, Bazanova and Akopova (1966) found that 0.01% naphthenate increased the chlorophyll concentration in the leaves of non-fertilized plants or in the case of a single N-P application. Higher doses of f e r t i l i z e r decreased the chlorophyll content. Abolina and Ataullaev (1966) reported that application of naphthenates gave more chlorophyll in potato leaves. They also obtained an increase in chlorophyll content in melon leaves when the melon seeds were soaked with 0.005 to 0.0005% naphthenate solutions prior to sowing. Chu (1969) noticed that chlorophyll a and b contents increased to a small extent but the carotenoid contents increased more in tomato leaves when the plants were sprayed with 5000 ppm KNap solutions. 3. Ascorbic acid Agakishev and Bazanova (1965) measured increased ascorbic acid (vitamin C)content in cotton leaves following naphthenate treatment. Similarly, Babaev (1966) reported in-creased biosynthesis of vitamin C in cotton roots in a l l growth 19 and developmental periods of the plant when the seeds were treated withO.001% naphthenate solution before sowing. Bazanova and Akopova (1966) also found that a 0.01% naphthenate solution used as f o l i a r spray on the cotton plant had a p o s i t i v e e f f e c t on the vitamin C content of leaves. An increase i n the amount of vitamin C i n the tomato f r u i t , when the plants were sprayed with 0.005% : NaNap was obtained by A l i e v (1965). However, Chu (1969) found that the vitamin C content was decreased i n tomato f r u i t s when the plants were treated with 5000 ppm KNap solution. Naphthenate added to f e r t i l i z e r s or sprayed on cabbage plants increased the vitamin C content i n the plants (Asadov, 1965). Abolina and Ataullaev (1966) found that naphthenates augmented vitamin C content i n melon f r u i t s . C. E f f e c t on Metabolism 1. Photosynthesis: Cotton plants treated with naphthenates showed an increase i n the i n t e n s i t y of the photosynthetlc rate (Agakishev and Bazanova, 1965). Bazanova and Akopova (1966) also observed stimulation of photosynthesis i n cotton plants treated with 0.01% naphthenates. An increased rate of photosynthesis was obtained by Kolesnik (1965) by the application of 0.005% naph-thenates to the grape plant. A higher concentration of naph-thenates (0.05%) and double spraying with 0.005% i n i t i a l l y 20 decreased photosynthesis, but at the end of the vegetative period there was considerable increase i n photosynthesis. Ladygina (1965) found only a s l i g h t change i n the photosynthetic a c t i v i t y of two species of potatoes. Abolina and Ataullaev (1966) observed that photosynthesis proceeded more energetically i n potato plants treated with naphthenates as f o l i a r spray. The apparent photosynthesis of three-week-old tomato plants treated with 5000 ppm KNap as f o l i a r spray was diminished two weeks afte r treatment (Chu, 1969), however, the treatment resulted i n 4.2% higher rates i n apparent photosynthesis four weeks afte r the treatment. 2. Respiration: Bazanova and Akopova (1966) found that 0.01% naphthenate stimulated r e s p i r a t i o n of cotton plants when the chemical was used as a spray. An increase i n r e s p i r a t i o n i n potato leaves due to naphthenate treatment of the plants was obtained by Abolina and Ataullaev (1966). Chu (1969) showed that application of 5000 ppm KNap produced a decrease i n rate of r e s p i r a t i o n i n the above-ground parts of tomato plants a f t e r two weeks, followed by a 9.7% increase i n r e s p i r a t i o n a f t e r four weeks. 3. Enzymes Catalase Kolesnik (1965) obtained an increase i n catalase 21 a c t i v i t y i n grape plants due to the application of naphthenates. A higher concentration (0.05%) of naphthenates and double-spray-ing with a lower concentration (0.005%) i n i t i a l l y decreased catalase a c t i v i t y but increased i t l a t e r on. Only a s l i g h t e f f e c t on catalase a c t i v i t y i n cotton plants was obtained by Bazanova and Akopova (1966) when the plants were treated with 0.01% naphthenates. Peroxidase Agakishev and Bazanova (1965) found that cotton plants treated with naphthenates and grown on sulfate s a l i n i z e d s o i l s showed a higher rate of peroxidase a c t i v i t y i n the leaves. Naphthenates activated peroxidase a c t i v i t y i n the root system of the cotton plant (Babaev, 1966). The peroxidase a c t i v i t y increased greatly when the seeds were soaked i n naphthenate solutions and increased less when the chemical was applied to the s o i l . Ascorbic acid oxidase: The a c t i v i t y of ascorbic acid oxidase of the cotton plant increased due to naphthenate treatment when the plants were grown i n chloride s a l i n i z e d s o i l but did not increase i n sulfu r s a l i n i z e d s o i l (Agakishev and Bazanova, 1965). Babaev (1966) also found that naphthenate treatment activated ascorbic acid oxidase i n cotton plant roots. 22 Amylase: The a c t i v i t y and the formation of amylase was found to be promoted i n Aspergillus usamii, when the mold was treated with 0.0005% naphthenate solution (Burachevskii, 1965). Other enzymes: Chu (1969) found that i n KNap treated tomato plants the a c t i v i t y of phosphorylase increased i n plant leaves. The glutamic-pyruvic transaminase a c t i v i t y increased only s i x weeks af t e r treatment. .However, n i t r a t e reductase, phosphoglyceryl kinase and succinic dehydrogenase a c t i v i t i e s decreased at a l l times of her observations,; v i z . 2 and 4 weeks after treatment. 4. Other metabolic changes Zhukova (1965) found that 100 g/ha naphthenate applied to s o i l increased the available phosphorus i n s o i l and also increased the nitrogen content of plants. Naphthenates added to f e r t i l i z e r s or sprayed on cabbage plants increased the nitrogen, phosphorus and sugar contents i n the heads (Asadov, 1965). Spraying tomato plants with 0.005% aqueous naphthenate solution caused changes i n proteins and nucleic acids in leaves and vegetative apices. The content of c^-nucleoprotein complex increased and that of f$-nucleoprotein complex decreased. The 23 protein N i n vegetative apices increased, but i t decreased i n old leaves (Pakhomova, 1965). By soaking seeds or spraying during the vegetative period with 0.005% naphthenates, Yureva (1965) obtained more protein and starch i n corn cobs and more protein and phosphorus i n sugar beet leaves. By spraying tomato plants with 0.0057o naphthenates A l i e v (1965) found that there was an increase i n the uptake of s a l t s of nitrogen and phosphorus by the treated plants. Asadov (1965) also reported that naphthe-nate treatment at the rate of 50 or 100 g/ha at the time of planting of cabbage seedlings and during growth as a spray solution, increased the amount of nitrogen and phosphorus i n the cabbage heads. When potato tubers were soaked i n 0.005% aqueous solution of naphthenates before sowing, the plants showed increased content of nitrogenous substances i n leaves, roots and stems (Gruodiene and Buciene, 1967). Increases i n nitrogen-phosphorus metabolism and t o t a l and protein nitrogen content i n the roots of cotton plants were obtained by Babaev (1966) when the cotton seeds were treated with 0.00017o naphthenates before sowing. Guseinov- (.1958) found that naphthenates i n combination with f e r t i l i z e r s applied to the s o i l increased the uptake of NPK f e r t i l i z e r s by plants by 78% and that of NP f e r t i l i z e r s by 43%, when the chemical was added at the rate of 56 to 280 g/ha. The addition of naphthenates alone or i n combination with mineral f e r t i l i z e r s (N-P) to s o i l increased the available phosphorus 24 content i n tomatoes and cabbage (Guseinov et al_, 1956). More-over, the sum.of ammonical and n i t r a t e N i n the plant tissue also increased. Cotton plants treated with 0.01% naphthenates increased the concentration of free carbohydrates i n the leaves (Bazanova and Akopova, 1966). Guyot (1964) found that sugarcane treated with naphthenates before harvest yielded more sugar than the control. By applying 0.05% naphthenate on tangerine leaves during the flowering period, Marshaniya e_t a l , 1965) obtained increased sugar content i n the tangerine f r u i t . The application of 0.005% naphthenates to the grape plant by Kolesnik (1965) increased the sugar content of f r u i t s . However, a higher concentration (0.05%) and double spraying with 0.005% decreased the concentration of disaccharides. At the end of vegetative period sucrose i n leaves disappeared. The sugar content of grapes was higher by 1.6% than i n controls. Chu (1969) found that 5000 ppm KNap treatment increased reducing sugar, sucrose and t o t a l sugars i n the mature tomato f r u i t s . The loss of sugar content during the course of storage was also less i n tomato f r u i t s from KNap treated plants. T i t r a t a b l e acid of the mature tomato f r u i t under the influence of KNap was higher at the end of 4 days storage but lower as the duration of storage was longer. MATERIALS AND METHODS A. Plant Material Bush bean (Phaseolus vulgaris L. var. Top Crop) was used i n a l l the experiments. Seeds were obtained from Buckerfield 1s Limited, New Westminster, B r i t i s h Columbia. Seeds v i s u a l l y selected for uniformity were sown i n p l a s t i c pots (16 ?5 x 11.4 cm) containing steam-treated garden s o i l , at the rate of seven seeds per pot. After 7 days, seed-lings were thinned to 1 or 2 uniform plants i n each pot, as required. The seeds, and l a t e r the plants, were watered d a i l y with tapwater. In a l l the experiments, for the f i r s t 14 days plants were grown i n a growth room provided with a 14-hour photoperiod, 26 ± 1°C i n the l i g h t and 21 - 1°C i n the dark period. The l i g h t i n the growth room was supplied by cool-white fluorescent tubes (Westinghouse, USA) and 60 Watt incandescent lamps. The l i g h t intensity^as measured by a Weston illumination meter (Weston Model 756 without cosine filter),was 1500 f t - c at the tops of 14-day old plants. The r e l a t i v e humidity was 60 to 707o i n the l i g h t and 70 to 807o i n the dark period. At the end of every four days the pot locations were changed i n order to average l o c a l environmental v a r i a b i l i t y . 26 In the f i r s t experiment, subsequent to the f o l i a r - s p r a y treatment, plants were grown i n the conditions mentioned above. But i n other experiments, a f t e r the spray treatment, plants were grown : i n the growth room provided with constant day and night temperature, either at 15 - 1°C or 26 ± 1°C. In the constant day-night temperature experiments, plants were divided into three groups on the 14th day. One group received l i g h t of 1500, the second 1000, and the t h i r d group 500 f t - c . B. Preparation of the potassium naphthenate (KNap) aqueous  solution from naphthenic acid (HNap). KOH solution (2.1 g KOH dissolved i n 17 ml d i s t i l l e d water) was added to a f l a s k containing 5 g naphthenic acid (HNap) and the f l a s k was shaken for 10 to 15 minutes. The solution was allowed to stand for 5 minutes and then was made to a volume of 25 ml with d i s t i l l e d water. The solution thus prepared was the stock solution containing 250 mg of KNap/ml. By d i l u t i n g 1 ml of the stock solution to 50 ml with d i s t i l l e d water 5000 ppm KNap solution was obtained. The pH value of the diluted solution was adjusted to about 10 by the addition of d i l u t e HC1. C. Spray treatment Plants i n a l l the experiments were sprayed with 5000 ppm aqueous KNap solution at the age of 14 days, at which time the primary leaves were f u l l y expanded, and the f i r s t 27 t r i f o l i a t e leaf was s t i l l in bud. The aerial parts of the plants were sprayed to drip and were allowed to dry before transferring the plants back into the growth room. plant age is after planting. D. Measurement times: Measurements of juvenile growth, photosynthesis, respiration and enzymic activities were made 7, 14 and 21 days after treatment. Pods were picked five weeks after the treat-ment and the seeds were harvested 12 weeks after the spraying. A l l harvests were made in morning hours. E. Juvenile growth measurements: Plants were measured for their stem height from s o i l surface to the stem apex. The leaflet number, excluding the primary pair of leaves, was recorded. The primary leaf pair was not ; considered in any further measurements of juvenile growth; and stem and petioles were considered together. The leaf area measurements were made by employing light-sensitive Ozalid papers and a planimeter (G. Coradi, Zurich). Roots were carefully washed with cold running water to free a l l entangled s o i l and humus particles, and then dried by pressing gently between paper towels, and then weighed. Dry weights of roots, stems and leaves were obtained after they had 28 been dried i n an oven at 75°C for 24 hours. F. Yield measurement: Green pods, 4 cm and larger, were harvested ; 5 . : weeks af t e r the treatment. The number of pods per plant and the moisture content of pods were determined. To ascertain seed production pods were harvested 12 weeks af t e r treatment. The number, and weight of seeds per plant were determined. G. A n a l y t i c a l procedures: 1. Pigment analysis: Chlorophyll a and b and t o t a l carotenoids were determined spectrophotometrically (Beckman Model B). Leaf material was cut into small pieces, mixed thoroughly, and 1 g of the leaf material was blended i n 60 ml of cold 80% acetone i n a c h i l l e d Waring blendor for two minutes. The homogenate was f i l t e r e d through Whatman Number 1 f i l t e r paper and was made up to 100 ml volume with cold 80% acetone. The o p t i c a l density (O.D.) of each solution was measured at 663, 645 and 440.5 m/u against 80% acetone as blank i n a 1-cm c e l l . The amounts of chlorophyll a and b were determined by using s p e c i f i c absorption c o e f f i c i e n t s of McKinney (1940) and the formula of MacLachalan and Zal i k (1963). The 29 formulas used were as follows: . (12.3 D - 0.86 D 6 4 5) V °a • d x 1000 x W (19.3 D 6 4 5 - 3.6 D 6 6 3 ) V C = d x 1000 x W where, C = concentration i n mg/g fresh weight, a = chlorophyll a, b = chlorophyll b, D = O.D. at wave length indicated, V = f i n a l volume of extract, W = fresh weight of leaf material used, d = length of l i g h t path i n cm. The amounts of carotenoids were determined by the equation of von Wettstein (1957): C c = 4.695 D 4 4 0 > 5 - 0.268 C (a+b) where, c = concentration of carotenoids i n mg/1. The carotenoid content was then converted to mg/g fresh leaf weight. 2. Ascorbic acid: The extraction procedure used was es s e n t i a l l y the same as described by L o e f f l e r and Ponting (1942) 30 except that 0.5% oxalic acid was used rather than metaphosphoric acid, and the r a t i o of pods to acid was modified to 1:7. The method for determination of ascorbic acid i n the pod extract was the indophenol reduction technique as modified by Schuster (1950) for use with a Klett-Summerson colorimeter. A standard regression l i n e was drawn from readings obtained with graded solutions of pure ascorbic acid (N u t r i t i o n a l Biochemical Corporation, Cleveland). Quantitative determinations of ascorbic acid contents of fresh pods were made within 2 hours of harvest and aft e r 5 days of storage at room temperature. Results were expressed as mg of ascorbic acid per 100 g fresh weight. H. Determination of photosynthesis and r e s p i r a t i o n rates The rate of CO2 exchange was measured i n the growth room i n an open system with a Beckman infrared analyzer IR-215. The pot of the plant was enclosed i n a 2-mil polythene bag sealed around the plant stem to prevent CO2 escape from the s o i l . The plant chamber was a 20-lb (9-kg) capacity polythene bag of 3 mil thickness. A 3-hole rubber stopper provided i n l e t and exit for the gas and for the i n s e r t i o n of the thermistor probe. The analyzer was connected to the plant chamber by tygon tubing. The a i r , containing about 300 ppm C^, was passed into the chamber at a constant rate of 2000 ml per minute. The temperature i n the chamber was monitored by a Tele-thermometer (Yellow Spring Instrument Company Inc., Ohio). For photosynthesis measurement the chamber was exposed to l i g h t of 1500, 1000 or 500 f t - c depending on the l i g h t regime i n which the plant had been growing. During the determination of the rate of r e s p i r a t i o n , the plant chamber was covered with two layers of thick black c l o t h to exclude l i g h t . The C0£ concentration of the a i r i n the tank was determined before connecting the tank to the chamber. In the illuminated system, a drop i n the C0£ concentration compared with that i n the tank was considered to be due to the CO2 f i x a t i o n i n apparent photo-synthesis. While i n the dark system the increase i n C0£ concentration was considered to be due to the l i b e r a t i o n of CO2 by the plant i n dark r e s p i r a t i o n . The product of the flow rate by the difference i n the CO2 concentration of the a i r before and af t e r passing through the chamber gave the rate of CO^ exchange i n the chamber. Ozalid paper was used to determine the leaf area. The results were expressed as m i c r o l i t r e s of CO^ used or formed per 2 hour per dm of l e a f blade area. TABLE I Enzyme nomenclature Number 1 Systematic name''" T r i v i a l name''" Reaction''" Other comments 1. 6.6. 1 NADH^ : nitrate oxido-reductase NADH2 nitrate reductase NADH?+nitrate= NAD+nitrite+H20 Also known as nitrate reductase 2. 6.1. 2 L-alanine: 2-oxoglutarate aminotransferase Alanine aminotransferase L-alanine+2-oxo-glutarate=pyruvate+ glutamate Also known as glut-amic-pyruvic trans-aminase 2. 7.2. 3 ATP: D-3 phosphoglycerate 1-phosphotransferase Phosphoglycerate Kinase ATP+D-3 phospho-glycerate=ADP+D-1,3-phosphoglyceric acid Also known as phosphoglyceryl Kinase 3. 1.3. 17 Phosphorylase phosphohydrolase Phosphorylase phosphatase Phosphorylase a+^ Also known as 4H20=2 phosphorylase phosphorylase b+4H3P04 1. According to the 'Report of the Commission on Enzymes1961. 33 I. Determination of enzyme a c t i v i t i e s The enzymes investigated were n i t r a t e reductase, glutamic-pyruvic transaminase, phosphorylase and phosphoglyceryl kinase (Table I ). Enzyme a c t i v i t i e s of leaves were assayed 7, 14 and 21 days a f t e r the treatment. Nitrate reductase a c t i v i t y was measured i n leaves and roots. In a l l cases, the plants for enzyme assays were harvested between 9 and 10 a.m. except for n i t r a t e reductase, i n which case harvests were made i n the a f t e r -noon between 1 and 2 p.m. Roots were thoroughly washed i n cold running water, root nodules were removed and the roots were dried gently between paper towels before use. Enzyme a c t i v i t i e s were determined i n homogenates prepared from freshly harvested tissues. 1. Nitrate Reductase Preparation of the crude extract: Crude enzyme extract was prepared by grinding 10 g of f i n e l y chopped leaf blade or root material with 50 ml of cold 0.1 M phosphate buffer, pH 7.8, containing 10 M reduced glutathione, i n a Waring blendor, at f u l l speed, for 2 to 3 minutes, at 0 to 4°C. The homogenate was ra p i d l y strained through four layers of cheesecloth. The homogenate was centrifuged i n a Se r v a l l centrifuge at 20,000 x g for 20 minutes at 0 to 4°C and the r e s u l t i n g c e l l - f r e e supernatant solution was used for the assay of the a c t i v i t y . 34 Assay procedure: The activity of nitrate reductase was measured by the procedure of Evans and Nason (1953) as modified by Yang (1964). At zero time 0.2 ml of the enzyme preparation was added to a reaction mixture containing 0.1 ml of 0.1 M KN03, 0.05 ml of 2 x 10" 5 M FAD, 0.05 ml of 2 x 10" 3 M NADH£ and 0.1 M phosphate buffer, pH 7.0, to give a total volume of 0.5 ml. The resulting mixture was incubated for 30 minutes in a constant temperature water bath at 30°C. The reaction was stopped by the addition of 1 ml of d i s t i l l e d water and 1 ml of 170 (W/V) sulfanilamide reagent. One ml of 0.227o (W/V) N - (1-napthyl) - ethylene diamine hydrochloride reagent was added and the contents were mixed by inverting the test tube. The color was allowed to develop for 15 minutes. By using a Spectronic•20 spectrophotometer the O.D. of each experimental solution and i t s blank (complete except for NADR^) was measured at 540 m/u . The amount of the n i t r i t e formed was determined from a standard curve prepared from O.D. measurements of known quanti-ties of n i t r i t e . The specific activity was defined as m/u moles of n i t r i t e formed per mg protein in the enzyme extract per hour. 2. Glutamic-pyruvic transaminase. Preparation of the crude extract: Same as for nitrate reductase. 35 Assay procedure: Glutamic-pyruvic transaminase a c t i v i t y was assayed by an adaptation of the ; method of Reitman and Frankel (1957). One ml of ©O-Ketoglutarate-alanine substrate was pipetted into a test tube and this was placed i n a water bath at 37°C for 10 minutes. Upon the addition of 0.2 ml of the crude extract, the contents were mixed and incubated for exactly 30 minutes i n a constant water bath at 37°C. Immediately a f t e r removing the tubes from the water bath, 1.0 ml of 2,4-dinitrophenyl hydrazine reagent was added to the reaction mixture i n the tubes. This reagent stops further transaminase a c t i v i t y . After the tubes were allowed to stand at room temperature for 20 minutes, 10 ml of 0.4 N sodium hydroxide was added. A clean rubber stopper was inserted i n each tube and the contents were mixed by inversion. At the end of exactly 30 minutes, the color i n t e n s i t y of the solution was measured by a Klett-Summerson colorimeter equipped with a green f i l t e r . While the samples were incubating, a control of each homogenate was prepared. One ml of the substrate, 0.2 ml of the homogenate and 1 ml of 2,4-dinitrophenyl-hydrazine reagent were mixed i n a test tube. After 20 minutes 10 ml of 0.4 N NaOH was added and a f t e r a further 30 minutes the color i n t e n s i t y was measured as above. Thus the only difference i n control tubes was that the 2,4-dinitrophenyl-hydrazine reagent was added to the 36 reaction mixture before incubation. The difference i n transmittance between the incubated tubes and the appropriate control was determined. The amount of pyruvic acid formed was determined by the use of a standard curve prepared from O.D. measurements of °.known quantities of pyruvic acid. The s p e c i f i c a c t i v i t y was expressed as •urn pyruvic acid formed per mg of protein i n the enzyme extract per 30 minutes under the conditions of the assay. 3. Phosphorylase Preparation of the crude extracts Crude enzyme extract was prepared by blending 10 g of coarsely chopped leaf tissue with 50 ml of cold d i s t i l l e d water i n a p r e - c h i l l e d Waring blendo.r run at f u l l speed for 2 to 3 minutes. The r e s u l t i n g homogenate was rapid l y strained through four layers of cheesecloth, and was used for the enzyme assay. Assay procedure Sumner's (1950) method, which was a modification of Fiske and Subbarow's method (1925), was employed to assay the enzyme phosphorylase by running the reaction i n the d i r e c t i o n of starch synthesis and measuring the amount of the inorganic phosphate li b e r a t e d . The reaction mixture consisted of 1 ml of 37 enzyme preparation and 2 ml of buffered substrate. The buffered substrate was prepared by dissolving 1 g glucose-l-phosphate in 50 ml d i s t i l l e d water. The solution was shaken with dry Ca(0H)2 to remove inorganic phosphate and then was filtere d . The f i l t r a t e was neutralized with drops of HC1. Equal volumes of f i l t r a t e and citrate buffer, pH 6.0, were mixed. This buffered substrate was kept in a refrigerator. Before,use, the buffered substrate was mixed with an equal volume of 17c potato starch solution, and a small crystal of thymol was added. The reaction was continued for one hour at 30°C in a constant temperature water bath. At the end of the incubation period the reaction was terminated with 5 ml of 6.6670 ammonium molybdate. Addition of 5 ml 7.5 N H 2S0 4 followed by 5 ml of 470 acidic ferrous sulfate developed a deep blue color. The solution was diluted with 10 ml of d i s t i l l e d water and the optical density of each solution and i t s corresponding blank (ammonium molybdate was added before the addition of the substrate) was read in a Klett-Summerson colorimeter equipped with a red f i l t e r . The amount of phosphate formed was read from a standard curve, prepared with known amounts of phosphate using the same reagents as above. Specific activity was defined as Aig of inorganic phosphate liberated per mg protein, in the enzyme extract, per hour. 38 4. Phosphoglyceryl Kinase Preparation of the crude extracts. Ten g of the leaf tissue was blended with 30 ml of cold succinate buffer, pH 6.2 Pin a Waring blendor at f u l l speed for 2 to 3 minutes. The homogenate was r a p i d l y strained through four layers of cheesecloth and the solution was centrifuged at 2000 x g for 10 minutes i n a Sorvall centrifuge at 0 to 4°C. The clear supernatant was used for the enzyme a c t i v i t y determination. Assay procedure The method described by Axelrod and Bandurski (1953) was employed. Notwithstanding the d i f f i c u l t y i n preparing 1,3-diphosphoglycerate, a procedure for measuring the enzyme through the u t i l i z a t i o n of 3-phosphoglyceric acid as substrate i n the presence of ATP was followed. The diphosphoglycerate was trapped with hydroxylamine, and the anhydride thus formed was measured c o l o r i m e t r i c a l l y by the hydroxamic test of Lippman and Tuttle (1945). To each reaction tube were added 1 ml of 0.1 M succinate buffer, pH 6.2, 1 ml of 2 M hydroxylamine hydrochloride, pH 6.2, 0.5 ml of 0.01 M ATP, 0.25 ml of the crude enzyme prepara-tion , 1 drop of 0.1 M NaF and 1 drop of 0.01 M MgCl 2, i n the above order. After the addition of each reagent the tube was 39 shaken. The control tube at this time received 2 ml of FeCl^-TCA-HCl reagent to stop the enzyme activity. The reaction in the experimental tube was then initiated by the addition of 1 ml of 0.01 M 3-phosphoglyeric acid (barium salt). The reaction was allowed to continue for 1 hour at 30°C in a constant tempera-ture water bath and was terminated by the addition of 2 ml of FeCl3-TCA-HCl reagent to the experimental tubes. Twenty minutes were given for the color development. The optical density of the solution from each experimental tube, and i t s blank, was determined by a Spectronic 20 spectrophotometer at 430 m/u . The specific activity was defined as the increase of 0.05 in optical density per mg protein in the enzyme extract per hour. 5'.. Determination of Protein The method of Lowry et_ al_ (1951) was employed to determine the protein content of a l l the enzyme preparations mentioned above. To 0.2 ml of the enzyme preparation 2 ml of alkaline copper solution (prepared by mixing 50 ml of 2% Na2C03 in 0.1 N NaOH, and 1 ml of 0.5% CuS04 . 5H20 in 1% sodium potassium tartrate) was added and the contents were mixed. After 10 minutes 0.2 ml of 1 N Folin Ciocalteau phenol reagent (Fisher Scientific Company, Fairlawn, N.J.) was added, and the 40 contents were mixed. The optical density of each experimental solution and its blank (Folin-Ciocalteau phenol reagent was replaced by an equal volume of d i s t i l l e d water) was determined at 500 m/u with a Spectronic 20 spectrophotometer. Protein content was determined by comparison with a standard curve prepared from O.D. measurements of known amounts of Bovine albumin. The values were expressed as mg protein per ml of the enzyme extract. in the tables "8 measurements," etc. means that a different plant was used for each of 8 measurements etc. 41 RESULTS I. Plants grown at 26°/21°C temperature Results given from page 41 to 52 concern plants grown at day/night temperature of 26°/21°C and in 1500 ft - c . The values obtained as a result of KNap treatment are expressed as percent-ages of corresponding control plant values in the following text and in figures. Tukey's CO test has been used to test the significance of means at 0.05 level.. A. Juvenile growth 1. Fresh weights (Table II , Fig. l) Roots: The spray containing KNap decreased fresh weights of roots nonsignificantly at 7 days and increased significantly at 14 and 21 days after the treatment. The increases were 28.2 and 21.72%. Stem: Following KNap treatment fresh weights of stem increased at a l l times of observation. The increases following the treatment were 1.2, 21.1 and 32.07% at 7, 14 and 21 days. The increases were significant at 14 and 21 days after the treat-ment . Leaves: A temporary depressive effect of KNap, 4.470, at 7 days was overcome by the 14th day. At the latter age KNap TABLE II Effec t of KNap spray on fresh weights, dry weights and moisture ^ content (% moisture) of roots, stem and leaves of bush bean plants ' * Days after treatment 14 21 Treat- 3 ment Roots Stem Leaves Roots Stem Leaves Root Stem Leaves C 4 3.72 2.46 0.89 4.57 4.25 1.79 7.24 6.19 7.53 Fresh weight g T 3.27 2.49 0.85 5.87*5 5.14* 2.44 8.82* 8.17* 11.18* C 272 250 140 390 488 238 594 918 1034 Dry weight mg T 255 269 137 502* 619* 332 734* 1224* 1467* C 92.59 89.88 83.75 91.44 88.57 86.80 91.72 85.01 86.03 Moisture 7o T 92.18 89.09 83.62 91.49 88.0 86.59 91.67 85.05 86.85 1. Plants grown at day/night temperature of 26°/21° 2. Average value per plant of 16 measurements each. 3. Exclusive of the pair of primary leaves. 4. C = control (no spray); T = 5000 ppm KNap spray. 5. In Tables II to V , Tukey's oo test has been used to test the significance of means. Means d i f f e r i n g s i g n i f i c a n t l y at 0.05 l e v e l from the respective control mean are indicated by *. fS3 o u •u c o CJ o CD CO n o c 50 40 30 20 10 0 •10 •20 Fresh weight IMlt 14 I I Root llllllllllllllll Stem M///////M Leaf Dry weight 21 7 Days after treatment 14 Fig. 1 Effect of KNap spray on fresh and dry weights of roots stem and leaves of bush bean plants grown at 26°/21°. 44 resulted i n nonsignificantly higher leaf weights. This stimula-tory e f f e c t was continued further u n t i l the observation at 21 days. At this l a s t time of measurement leaf weight increased s i g n i f i c a n t l y by 48.45%. 2. Dry weights. (Table II , F i g . l ) Roots: Root dry weight followed c l o s e l y the pattern of i n i t i a l depression and subsequent stimulation as observed i n fresh weight of roots. There was an i n i t i a l nonsignificant decrease of 2.3% at 7 days and 28.85 and 23.567o s i g n i f i c a n t increase at 14 and 21 days. Stem: Dry weights of stem increased at a l l times of observation, nonsignificantly at 7 days (7.46%) and s i g n i f i c a n t l y at 14 and 21 days by 26.78 and 33.3%. Leaves: Dry weight of leaves followed the pattern of i n i t i a l depression and subsequent stimulation as observed i n the fresh weight. A temporary depressive e f f e c t of KNap, 2.5%, at 7 days was overcome by the 14th day. At the l a t t e r age KNap resulted i n nonsignificantly higher leaf weights. The leaf dry weight increased s i g n i f i c a n t l y (41.97o) 21 days a f t e r treatment. 3. Shoot elongation: (Table I I I , F i g . 2 ) Heights of treated plants increased following KNap TABLE III Effect of KNap spray on plant height, leaflet number and leaf area of bush bean plants. > 7 Days after 14 treatment 21 Treat-ment Plant Height cm Leaflet Number Leaf Area^ -•dm Plant Height cm Leaflet Number Leaf Area -"dm Plant Height cm Leaflet Number Leaf Area •;dm c 4 15.01 6.5 1.94 30.31 10.0 3.50 35.29 18.0 4.59 T 15.71 8.5* 2.04* 33.22* 13.5* 3.97* 40.36* 22.0* 5.74* 1. Plants grown at day/night temperature of 26 / 2 1 ° . 2. Average value per plant of 16 measurements each. 3. Exclusive of the pair of primary leaves. 4. C = control (no spray); T = 5000 ppm KNap spray. Increase as % of control to o o •j>-o **! OQ K> M -f> Co W P Hi N> CL Hi " 1—1 CD CD O H. rt fD CD O Hi O a Hi CD h-> CD CO fD "O CD CD Hi CO Hi 1—1 T 3 rt fD l-{ fD rt Cu l-{ CO rt h-1 O O rj -P> Hi P fD CD CT T 3 rt N 3 P t-* 3 t-1 CO CD fD 3 - P P rt rt cr fD . P" CD fD P H-OQ TD P" 1—1 rt J13 %# P rt Co co p n P £ 3 h-1 P a 4 -f> fD CD l-{ rt N 3 K 3 p- •x) fD h-1 H* CD OQ P P" rt rt P f-1 P fD 3 CD a 4 Hi fD h-> H. fD rt CD r 4 H. fD fD CO CD Hi h-1 fD rt to O 9^ 7 47 treatment. There was a nonsignificant increase of 4.667o at 7 days and a significant increase of 9.59 and 14.38% at 14 and 21 days. 4. Leaflet number and area: (Table I I I , Fig. 2) The spray containing KNap increased leaflet number significantly at a l l the three times of observation. The increases caused by KNap at 7, 14 and 21 days were 30.7, 35.0 and 22.270. Leaflet area, exclusive of the pair of primary leaves, increased significantly by 5.1, 13.6 and 25% at 7, 14 and 21 days. B. Effects on plant composition 1. Moisture content: (Table II) The moisture content of the plant parts mentioned, roots, stem and leaves, was not affected significantly by KNap treatment. 2. Chlorophyll and carotenoid content: (Table IV, Fig._3) Following KNap treatment chlorophyll a increased at a l l times of observation. The increase was significant only 14 and 21 days after treatment. A nonsignificant increase in chlorophyll b content was found only 14 and 21 days after treatment. A non-TABLE IV Effect of KNap spray on chlorophyll and carotenoid content of bush bean leaves. » Treat- Chloro- Chloro- Carote- Total ments phyll a 3 . phyll b 3' noids • pigment c 4 0.52 0.34 0.26 1.12 7 days after treatment T 0.55 0.34 0.27 1.16 C 0.64 0.36 0.26 1.26 14 days after treatment T 0.75* 0.43 0.31 1.49 C 0.78 0.58 0.30 1.66 21 days after treatment T 0.89* 0.63 0.35 1.87* 3. 1. Plants grown at day/night temperature of 2 6 ° / 2 1 ° . 2. Average value of 2 samples (4 determinations). 3. mg/g fresh leaf weight. 4. C = control (no spray); T = 5000 ppm KNap spray. chlorophyll a chlorophyll b carotenoid total pigment «-• 30 r o U 4J d o o 4-1 O 6-8 CO CO CD CO CO <D U O C 20 10 -W/ 0 w. 14 21 Days after treatment Fig. 3 Effect of KNap spray on chlorophyll a, b and carotenoid, and total pigment content of leaves of bush bean plants grown at 26°/21°. . 50 s i g n i f i c a n t increase i n the carotenoid content was found at a l l times of observation. Total pigment (chlorophyll a, b and carotenoid) increased nonsignificantly at 7 and 14 days a f t e r treatment. There was a s i g n i f i c a n t (12.2%) increase of t o t a l pigment at 21 days. 3. Ascorbic acid content of pods The ascorbic acid content of green pods, measured at harvest, (mg/100 g fresh weight) was increased s i g n i f i c a n t l y . The increase by KNap treatment was 26.5% (Table V , F i g . 4 ). C. Yi e l d 1. Pod number: (Table V , F i g . 4 ). The number of pods per plant, 4 cm and above, harvested (5 weeks af t e r the treatment, was increased s i g n i f i c a n t l y by 18.88%. 2. Pod fresh weight: (Table V , F i g . 4 ). The weight of green pods per plant harvested 5 weeks aft e r the treatment was increased s i g n i f i c a n t l y by 16.5%. D. Moisture content of pods (Table V , F i g . 4 ). The e f f e c t of KNap on pod moisture content was ne g l i g i b l e . TABLE . V E f f e c t of KNap spray on y i e l d , moisture content and ascorbic acid content of pods, of bush bean plants. Number of ^ Pod weight/ Moisture Ascorbic acid Treatment pods/plant plant content,'pod 7o content of^pod g at harvest c 4 5.62 29.75 89.51 11.68 T 6.69* 34.67* 88.02r 14.77* 1. Plants grown at day/night temperature of 26°/21 . 2. 4 cm and longer as harvested 5 weeks afte r treatment. Average value of 16 determinations each. 3. mg/100 g fresh weight. Average value of 2 samples each. 4. C = control (no spray); T = 5000 ppm KNap spray. 30 20 -10 0 number weight moisture ascorbic acid -10 Fig. 4 Effect of KNap spray on number, weight, moisture and ascorbic acid content of green pods of bush bean plants grown at 26°/21°. 5 3 II. Plants grown at 1 5 ° / 1 5 ° C temperature Results given from page 5 3 to 6 6 concern plants grown at 1 5 ° / 1 5 ° C temperature and in three different light intensities, 1 5 0 0 , 1 0 0 0 and 5 0 0 f t - c . For convenience, 1 5 0 0 , 1 0 0 0 and 5 0 0 ft-c w i l l be mentioned as high, medium and low light. The values obtained as a result of KNap treatment are expressed as percentages of corresponding control plant values in the following text and in figures. Tukey's CO test has been used to test the significance of means at 0 . 0 5 level. A. Juvenile growth 1 . Fresh weights: (Table VI , Fig. 5 ) . Roots: The spray containing KNap increased fresh weights of roots nonsignificantly in a l l the three light inten-sities measured 2 1 days after the treatment. The increases were 9 . 8 5 , 6 7 . 2 1 and 4 9 . 2 1 7 o in high, medium and low light. Stem: Due to KNap treatment fresh weights of stem, 2 1 days after the treatment, increased in a l l the three light intensities, but only significantly in medium light. The increases were 1 . 2 8 , 2 7 . 3 7 and 2 5 . 5 8 7 o in high, medium and low light. Leaves: Fresh weights of leaves decreased ( 3 . 9 1 7 c ) nonsignificantly in high light, but increased significantly in both medium ( 4 1 . 3 5 7 0 ) and low light ( 4 5 . 7 8 7 o ) following KNap treat-ment . TABLE VI Effect of KNap spray on fresh and dry weights and percentage moisture contents of roots, stem and leaves of bush bean plants. 21 days after treatment Fresh weight g Dry weight mg Moisture content % Treatment i Treatment^ Letter Roots Stem Leaves^ Roots Stem Leaves Roots Stem Leaves c 1500 a 3. 69 cef 3. 68 cef 4. 61 cef 558 cef 626 cef 736 cdef 83.8 c 84. 7 be 84. 1 cdef T it b 4. 05 cef 3. 73 cef 4. 43 ce 561 cef 667 cdef 687 cdef 85.7 ce 81. 9 aef 84. 5 cef C 1000 c 1. 62 ab 2. 69 abd 2. 98 abdf 322 abd 430 abde 373 abd 79.5 abf 84. 0 ef 87. 4 abe T II d 2. 72 3 . 42 ce 4. 22 ce 470 ce 576 beef 596 abcef 82.3 83. 1 ef 86. 0 aef C 500 e 1. 52 ab 2. 37 abd 2. 71 abdf 263 abd 297 abed 293 abdf 81.0 b 87. 4 abed 89. 2 abed T II f 2. 27 ab 2. 97 ab 3. 96 ace 333 ab 376 .abd 437 abde 84.8 c 87. 1 bed 89. 0 abd 1. 2. 3. 4. 5. Plants grown at day/night temperatures of 1 5 ° / 1 5 ° . Average value per plant of 8 measurements each. Exclusive of the pair of primary leaves. In Tables VI to XXI Tukey's&J test has been used to test the significance of means. For s t a t i s t i c a l comparison of values letters have been assigned to treatments as follows: a, b, c, d, e, and f for C 1500, T 1500, C 1000, T 1000, C 500 and T 500 respectively. Each treatment value differs significantly at the 0.05 level from the values of those treatments whose letters appear after i t . C T C T C T 1500 1500 1000 1000 500 500 = Control at 1500 ft-c Treated Control Treated Control Treated 1000 it 500 ti II ti II at 1500 ft-c o u i-y c o u M-l O ctl CD CO «0 CD !^  O C M 70 60 50 40 30 20 10 0 •10 Fresh weight T J MM- at 1000 ft-c at 500 ft-c Dry weight Root Stem Leaf Root Stem Leaf Fig. 5 Effect of KNap spray on fresh and dry weights of roots, stem and leaves of bush bean plants grown at 15°/15°. 5 6 2 . Dry weights: (Table VI , Fig. 5 J Roots: The dry weights of roots in treated plant increased in a l l the three light intensities, significantly only in medium light. The increases were 0 . 4 4 , 4 5 . 7 5 and 2 6 . 5 4 7 o in high, medium and low light. Stem: Treated plants showed increases in dry weight of stems in a l l the three light intensities, significantly only in medium light, 2 1 days after the treatment. The increases were 6 . 5 7 , 3 4 . 0 3 and 2 6 . 4 7 , in high, medium and low light. Leaves: There was a nonsignificant decrease ( 6 . 6 3 7 0 ) in dry weights of leaves in high light and significant increases in both medium ( 5 9 . 5 3 7 0 ) and low light ( 4 8 . 9 2 7 , ) at 2 1 days after treatment. 3 . Shoot elongation: -(Table VII , Fig. 6 ) . Heights of treated plants increased significantly at a l l times of observation and in a l l the three light intensities. In high light the increases were 1 9 . 8 2 , 2 1 . 4 2 and 1 9 . 8 2 7 0 ; in medium light 1 5 . 3 8 , 2 1 . 9 and 3 0 . 8 8 7 o and in low light 8 . 9 6 , 1 2 . 6 2 and 1 7 . 4 8 7 o at 7, 1 4 and 2 1 days. 4 . Leaflet number and area: (Table VII , Fig. 6 ) . Following KNap treatment leaflet number increased in TABLE VII Eff e c t of KNap spray on plant height, and number and area of leaves i n bush bean plants. ' Days afte r treatment 7 14 21 21 Leaflet Plant height cm numberJ Treatment Treatment Letter c 1500 a 14.31 bdef 4 15.57 bdef 17. 61 bdf 11.25 be 1.34 cef T t i b 17.15 ac 18.91 ace 21. 10 ace 14.25 acdef 1.37 cdef C 1000 c 15.11 bdef 16.10 bdef 16. 87 bdef 9.00 b 1.19 abdef T II d 17.44 ac 19.51 ace 22. 08 ace 9.75 be 1.31 beef C 500 e 16.45 acf 17.72 abcdf 18. 66 bcdf 6.75 abd 1.03 abcdf T i t f 17.92 ace 19.96 ace 21. 92 ace 9.00 b 1.26 abede 21 Leaf area "dm 1. Plants grown at day/night temperature of 1 5 ° / 1 5 ° . 2. Average value per plant of 8 measurements each. 3. Exclusive of the pair of primary leaves. 4. Treatment value d i f f e r s s i g n i f i c a n t l y from these treatments. F i g . 6 E f f e c t o f KNap spray on p l an t he igh t , and number and area of l e a f l e t s of bush bean p l an t s grown at 1 5 ° / 1 5 ° . . 5 9 a l l the three light intensities, but significantly only in the. high light. The increases were 2 6 . 6 6 , 8 . 3 3 and 3 3 . 3 3 % in high, medium and low light. Leaflet area, exclusive of the pair of primary leaves, increased nonsignificantly in high light ( 2 . 7 3 % ) , and significant-ly in medium ( 9 . 7 5 % ) and low light ( 2 2 . 6 6 % ) following KNap treat-ment . A summary of the comparative effects of treatment on juvenile growth of bush bean plants with KNap spray in three light intensities at 1 5 ° / 1 5 ° C is given (TableVIII). B. Chemical composition 1 . Moisture content: Table VI presents data on the moisture content of roots, stem and leaves 2 1 days after the treatment. There were nonsignificant increases in the percentage moisture content of roots in a l l the three light intensities and the increases were 2 . 2 8 , 3 . 5 1 and 4 . 7 5 7 o in high, medium and low light. The percent-r age moisture content of stem decreased in a l l the light intensi-ties, significantly in the high light ( 3 . 2 9 % ) and nonsignificantly in the medium ( 1 . 0 3 % ) and in the low light ( 0 . 4 6 % ) . The moisture content of leaves increased only in high light and decreased in other light conditions. The differences were nonsignificant. TABLE VIII Comparative effects of treatment on juvenile growth of bush bean plants with KNap spray in three light intensities at 15°/15°C. High light Medium light Low light Fresh weight + + + Root Dry weight + +* + Fresh weight + +* + Stem Dry weight + +* + Fresh weight _ +* +* Leaf Dry weight - +* +* Leaflet number +* + + Leaf area + +* Plant height +* +* +* + increase in comparison to control value. decrease in comparison to control value. * significant at 0.05 level. 61 q 2. Chlorophyll and carotenoid content: (Table IX-, _ • F i g . 7 ) Due to the treatment, chlorophyll a increased s i g n i f i c -antly i n a l l the l i g h t i n t e n s i t i e s . The increases were 39.34, 12.96 and 14.28% i n high, medium and low l i g h t respectively. Chlorophyll b increased s i g n i f i c a n t l y (13.33%) i n treated plants grown i n high l i g h t but decreased nonsignificantly i n both medium (4.17%) and low l i g h t (4.53%). Carotenoid content remained the same i n both control and treated plants i n high l i g h t and i t decreased nonsignificantly i n medium l i g h t (1.83%) and s i g n i f i c -antly i n low l i g h t (6.677e) i n treated plants. As a r e s u l t of the above changes, the t o t a l pigment (chlorophyll a, b and carotenoid) i n leaves of treated plants increased i n a l l the three l i g h t i n t e n s i t i e s due to the large increases i n chlorophyll a, s i g n i f i c a n t l y only i n high l i g h t . The increases were 20.27, 2.5 and 0.65% i n high, medium and low l i g h t . 3. Ascorbic acid content of fresh pods: (Table Xj Fi g . 8 ) The ascorbic acid content of green pods, measured at harvest (mg/100 g fresh weight), increased s i g n i f i c a n t l y i n treated plants i n a l l the three l i g h t i n t e n s i t i e s . The highest increase was found i n medium l i g h t (31.25%) followed by low l i g h t (16.93%) and high l i g h t (13.75%). TABLE IX Effect of KNap spray on chlorophyll and carotenoid content of bush bean leaves.^"* Treatment „ q q Treatment Letter Chlorophyll a Chlorophyll b Carotenoids Total pigment C 1500 a 0.61 beef 4 0.45 b 0.42 cdef 1.48 bedef T n b 0.85 acdef 0.51 adef 0.42 cdef 1.78 acdef C 1000 c 0.54 abde 0.48 ef 0.58 abef 1.60 abef T II d 0.61 beef 0.46 bf 0.57 abe 1.64 abef C 500 e 0.49 abedf 0.44 be 0.60 abedf 1.53 abed T it f 0.56 abde 0.42 bed 0.56 abce 1.54 abed 1. Plants grown at day/night temperatures of 1 5 ° / 1 5 ° . 2. Average value of 2 samples (4 determinations), measurements taken 21 days after treatment. 3. mg/g fresh leaf weight. 4. Treatment value differs significantly from these treatments. at 1500 ft-c at 1000 ft-c 40 r Chlorophyll 2 at 500 ft-c Chlorophyll Carotenoids Total pigments 0 - _£zzza Fig. 7 Effect of KNap spray on chlorophyll a, b and carotenoid, and total pigment content of leaves of bush bean plants grown at 15°/15°. O N 64 C. Yield (Table X , Fig. 8 ) 1. Flower number: There was a significant increase in flower number in high light (23.45%), a nonsignificant i n -crease (6.5%) in medium light and a nonsignificant decrease ( 7 . 7 7 o ) in low light due to KNap treatment. 2. Pod number: The number of pods, 2 cm and longer, harvested ; 5-weeks after the treatment was increased nonsig-nif icantly in a l l the three light intensities. The increases were 14.28, 7 7 . 7 7 and 38.46% in high, medium and low light respectively. There was no significant difference between pod numbers from different light intensities. 3. Pod fresh weight: The weights of green pods per plant, harvested g5Lx weeks after the treatment, were increased significantly in the high (21.24%) and the medium light (54.53%) and increased nonsignificantly in the low light (34.95%), due to KNap treatment. D. Moisture content of pods: (Table X ,) The effect of KNap on moisture content of pods from a l l the light intensities was negligible. TABLE X Effect of KNap spray on flower number, number and weight of pod, moisture content and ascorbic acid content of pods of bush bean plants. Treat-ment Treat-ment Letter Flower Number/ plant Number of pod/ 2' 3 plant Pod weight/ 2' 3 plant g Moisture content of pod % Ascorbic acid content of fresh pod 4 C 1500 a 8.00 bcdef 5 1.75 8.64 bcdef 83.87 10.76 beef r£ 11 b 9.87 acdef 2.00 10.48 acdef 83.18 12.24 acdef C 1000 c 5.75 ab 1.12 4.13 abd 85.82 8.16 abdf IJ1 II d 6.12 abf 2.00 6.39 abcef 85.48 10.71 beef C 500 e 4.87 ab 1.62 2.61 abd 87.75 8.15 abdf rj-l II f 4.50 abd 2.25 3.53 abd 87.26 9.53 abede 1. Plants grown at day/night temperatures of 15°/15°. 2. 2 cm and longer as harvested '5 weeks after treatment. 3. Average value of 8 measurements each. 4. mg/100 g fresh weight. Average value of 2 determinations each. 5. Treatment value differs significantly from these treatments. Flower Pod I at 1500 ftHo \ mmm at 1000 f t - c Pod Ascorbic weight acid Fig. 8 Effect of KNap spray on flower number, and number, weight and ascorbic acid content of green pods of bush bean plants grown at 15°/15°> 67 I I I . Plants grown at 26°/26°C temperature Results given from page 67 to 96 concern plants grown at 26°/26°C temperature and i n three d i f f e r e n t l i g h t i n t e n s i t i e s , 1500, 1000, and 500 f t - c . For convenience, 1500, 1000, and 500 f t - c w i l l be mentioned as high, medium and low l i g h t respect-i v e l y . The values obtained as a r e s u l t of treatment are expressed i n the following text and i n figures as percentages of correspond-ing control plant values. Tukey's 60 test has been used to test the s i g n i f i c a n c e of means at 0.05 l e v e l . A. Juvenile growth 1. Fresh weights. (Table XI , F i g . <9 ) Roots: The spray containing KNap increased fresh weights of roots nonsignificantly i n the high and the low l i g h t , and s i g n i f i c a n t l y i n the medium l i g h t measured 21 days a f t e r the treatment. The increases were 15.95, 32.16 and 7.017o i n high, medium and low l i g h t . Stem: Due to KNap treatment fresh weights of stem, measured 21 days a f t e r the treatment, increased i n a l l three l i g h t i n t e n s i t i e s , but s i g n i f i c a n t l y only i n high l i g h t . The increases were 18.57, 13.46 and 11.9270 i n high, medium and low l i g h t . TABLE XI Effect of KNap spray on fresh and dry weights and moisture contents of roots, stem and leaves of bush bean plants.-"-J^ 21 days after treatment Fresh weight g . Dry weight mg Moisture content % Treatment Treatment Letter Roots Stem Leaves Roots Stem Leaves Roots Stem Leaves c 1500 a 10. 50 cef ^ 13. 90 beef 20. 63 beef 1068 cdef 1955 cef 2320 beef 89. 6 ed 85. 8 cef 88. 7 ef T it b 12. 18 cdef 16. 48 acdef 24. 20 acdef 1196 cdef 2237 cdef 2720 acdef 90. 1 ed 86. 4 ce 88. 7 ef C 1000 c 6. 87 abdef 11. 79 abef 17. 93 abdef 515 abdef 1463 abdef 1765 abdef 92. 6 abef 87. 6 abd 89. 9 T tt d 9. 08 beef 13. 38 bef 19. 69 beef 721 abcef 1835 beef 2161 beef 92. 1 abef 86. 3 ce 88. 8 e C 500 e 2. 60 abed 7. 44 abed 10. 98 abed 269 abed 901 abed 1040 abed 89. 5 ed 88. 1 abd 90. 6 abd T ti f 2. 78 abed 8. 33 abed 12. 15 abed 281 abed 1072 abed 1216 abed 89. 5 ed 87. 1 a 90. 0 ab 1. Plants grown at day/night temperature of 2 6 ° / 2 6 ° . 2. Average value per plant of 8 measurements each. 3. Exclusive of the pair of primary leaves. 4. Treatment value differs significantly from these treatments. 00 at 1500 ft-c o u •u c o o «w o to CO <u CO CO CD U O a 40 30 20 -10 -0 L 1 Fresh weight 1 at 1000 ft-c at 500 ft-c Dry weight m 7/A Root Stem Leaf Root Stem Fig. 9 Effect of KNap spray on fresh and dry weights of roots, stem and leaves of bush bean plants grown at 26 /26°. 7 0 Leaves: Fresh weights of leaves increased s i g n i f i c -antly in high ( 1 7 . 2 9 7 Q ) and medium ( 9 . 7 7 7 o ) light, and nonsig-nif icantly in low light ( 1 0 . 6 3 % ) . 2 . Dry weights (Table X I , Fig. 9 ) Roots: Root dry weight followed closely the pattern observed in fresh weight of roots. There were nonsignificant increases in high ( 1 1 . 9 3 7 o ) and low light ( 4 . 6 5 % ) and significant increases in medium light ( 4 0 . 0 6 % ) . Stem: Dry weights of stem increased in a l l the three light intensities, significantly only in the medium light. The increases were 1 4 . 4 5 , 2 5 . 3 6 and 1 8 . 9 9 % in high, medium and low light. Leaves: Dry weights of leaves followed the pattern observed in fresh weight of leaves. There were significant increases in high ( 1 7 . 2 4 % ) and medium light ( 2 2 . 4 5 7 o ) and a non-significant increase in low light ( 1 6 . 9 5 7 0 ) . 3 . Shoot elongation: (Table X I I , Fig. 1 0 ) Height of treated plants was increased at a l l times of observation and in a l l the three light intensities. Measured 7 days after treatment, the increases were nonsignificant in a l l the three light intensities and measured 1 4 days after treatment TABLE XII E f f e c t of KNap spray on plant height and numbers and area of l e a f l e t s i n bush bean plants.*-2 Leaflet Leaf area Plant height cm number3 ddm2 Days afte r treatment Treatment 7 14 21 21 21 Treatment Letter G 1500 a 19. 48 d e f 4 37.60 cdef 40.43 bcdef 23.25 beef 8.02 bcdef T u b 20. 85 ef 40.16 cdf 44.51 adf 28.25 acdef 9.28 acdef C 1000 c 21. 21 f 43.97 ab 46.62 ae 18.00 abd 6.80 abdef T II d 23. 16 a 44.94 ab 48.46 abe 24.00 beef 7.57 abcef C 500 e 23. 67 ab 43.02 afq 43.57 acdf 16.50 abd 4.07 abedf T it f 25. 69 abc 46.76 abe 48.12 abe 18.75 abd 4.56 abede 1. Plants grown at day/night temperatures of 26°/26°. 2. Average value of 8 determinations each. 3. Exclusive of the pair of primary leaves and average value of 4 determinations each. 4. Treatment value d i f f e r s s i g n i f i c a n t l y from these treatments. at 1500 ft-c at 1000 ft-c ^ at 500 ft-c o u c o o O to CO CO co CO CD U O C M 40 30 20 10 0 L Plant height 777 in i Leaflet number 1 Leaflet area 14 21 21 21 Days after treatment Fig. 10 Effect of KNap spray on plant height, and number and area of leaflets of bush bean plants grown at 26°/26°> there was s i g n i f i c a n t increase only in low l i g h t . There were s i g n i f i c a n t increases i n plant height i n high and low l i g h t measured 21 days a f t e r treatment. In high l i g h t the increases were 6.99, 6.81 and 10.07%; i n medium l i g h t 9.19, 2.18 and 3.94% and i n low l i g h t 8.50, 8.68 and 10.44% measured at 7, 14 and 21 days. 4. Leaflet number and area: (Table XII } Fig.10) Due to KNap treatment l e a f l e t number increased i n a l l the three l i g h t i n t e n s i t i e s , but s i g n i f i c a n t l y i n high and medium l i g h t i n t e n s i t i e s only. The increases were 21.50, 33.33 and 13.63% i n high, medium, and low l i g h t respectively. Leaflet area, exclusive of the pair of primary leaves, increased s i g n i f i c a n t l y i n a l l the three l i g h t i n t e n s i t i e s due to KNap treatment. The increases were 15.66, 11.21 and 11.85% in high, medium and low l i g h t respectively. A summary of the comparative effects of treatment on juvenile growth of bush, bean plants with KNap spray i n three l i g h t i n t e n s i t i e s at 26°/26° i s given (Table XIII). B. Chemical composition: 1. Moisture content: (Table XI) There were nonsignificant increases i n the moisture TABLE XIII Comparative effects of treatment on juvenile growth of fresh bean plants with KNap spray in three light intensities at 26/26 C. High light Medium light Low light Fresh weight + +* + Root Dry weight + +* + Fresh weight + + Stem Dry weight + +* + Fresh weight +* +* + Leaf Dry weight +* +* + Leaflet Number +* +* + Leaf Area +* +* +* Plant Height +* + +* + increase in comparison to control value. * significant at 0.05 level. content of roots i n high l i g h t and nonsignificant decreases i n medium and low l i g h t . The moisture content of stem decreased i n medium and low l i g h t and increased i n high l i g h t . The d i f f e r -ence was s i g n i f i c a n t only i n medium l i g h t (1.56%). Changes i n the moisture content of leaves were nonsignificant. 2. Chlorophyll and carotenoid content: (Table XIV, F i g . 11). Due to the treatment, chlorophyll a increased non-s i g n i f i c a n t l y i n high l i g h t and s i g n i f i c a n t l y i n medium and low l i g h t . The increases were 3.13, 37.07 and 15.54%. Chlorophyll b increased s i g n i f i c a n t l y i n a l l the l i g h t i n t e n s i t i e s . The increases were 9.58, 30.06 and 13.35% i n high, medium and low l i g h t respectively. Carotenoid content decreased s i g n i f i c a n t l y i n high (12.77%) and medium (5.61%) l i g h t , and increased s i g n i f i c -antly (3.7%) i n low l i g h t . As a r e s u l t of the above changes, the t o t a l pigment (chlorophyll a, b and carotenoid) i n treated plants increased i n a l l the three l i g h t i n t e n s i t i e s , n onsignificantly i n high l i g h t (1.37%) and s i g n i f i c a n t l y i n medium (23.37%) and low l i g h t (12.43%). 3. Ascorbic acid content of pods: (Table XV , F i g . 12) Fresh pods: The ascorbic acid content of green pods, measured at harvest (mg/100 g fresh weight), increased s i g n i f i c a n t l y i n treated plants i n a l l the three l i g h t i n t e n s i t i e s . The TABLE XIV Effect of KNap spray on chlorophyll and carotenoid content of bush bean leaves. ' ' Treatment Treatment Letter Chlorophyll a chlorophyll b carotenoids Total pigment C 1500 a 0.84 c d e f 4 0.55 bcdef 0.43 bede 1.82 cef rj, 11 b 0.87 cef 0.60 acef 0.37 acdef 1.84 cef C 1000 c 0.68 abdef 0.46 abdef 0.41 abdef 1.55 abdef IJI II d 0.93 aef 0.60 acef 0.39 abcef 1.91 ef C 500 e 0.95 abef 0.64 abedf 0.40 abedf 2.00 abef II f 1.10 abede 0.73 abede 0.42 bede 2.25 abede 1. Plants grown at day/night temperatures of 26°/26°. 2. Average value of 4 determinations each. Measurements made 21 days a f t e r treatment. 3. mg/g fresh leaf weight. 4. Treatment value d i f f e r s s i g n i f i c a n t l y from these treatments. o u J-) a o o m o to <u co CO <D ! - l O c 40 r 30 20 10 0 -10 1 -20 L chlorophyll chlorophyll a b at 1500 ft-c at 1000 ft-c at 500 ft-c i carotenoids total pigmen Fig. 11 Effect of KNap spray on chlorophyll a, b and carotenoid, and total pigment content of leaves of bush bean plants grown at 26°/26°. 78 highest increase was found in medium light ( 3 9 . 2 7 7 o ) followed by low light ( 2 9 . 3 9 7 o ) and high light ( 2 0 . 5 3 7 o ) . Ascorbic acid loss during storage! At the end of 5 days storage, control pods from a l l the three light intensities lost more ascorbic acid than did pods from treated plants. Control pods from plants grown in high, medium and low light lost 4 4 . 8 6 , 5 5 . 5 3 and 5 5 . 7 9 7 Q of the original ascorbic acid, whereas the pods of treated plants grown in high, medium and low light lost 2 3 . 8 , 5 2 . 1 7 and 4 8 . 8 3 7 o . As a result of the different rates of ascorbic acid loss from pods of treated and control plants grown in different light intensities, at the end of 5 days storage, treated pods contained 6 6 . 5 7 , 4 9 . 8 0 and 4 9 . 7 5 7 0 more ascorbic acid than their corresponding controls grown in high, medium and low light. C. Yield (Table X V I , Fig. 1 3 ) 1 . Flower number: There were nonsignificant increases in flower number in a l l the three light intensities. The in-creases were 1 0 . 7 5 , 2 1 . 4 9 and 5 . 6 1 7 o in high, medium and low light. 2 . Pod number: The number of pods 4 cm and longer, harvested f5> weeks after the treatment was increased significantly in high and medium light and nonsignificantly in low light. The TABLE XV Effec t of KNap spray on ascorbic acid content, moisture content, and th e i r loss during 5 days storage, i n pods of bush bean plants. Ascorbic acid Moisture Treatment Treatment Letter at harvest mg/100 g 2 a f t e r 5 days storage mg/100 g loss during storage % J at harvest % a f t e r 5 days storage % loss during storage 7o J C 1500 a 13.73 bcdef 4 7.57 44.86 84.71 66.50 21.49 T 11 b 16.55 acdef 12.61 23.80 84.02 68.80 18.11 C 1000 c 11.38 abdef 5.06 55.53 88.47 68.20 22.91 d 15.85 abcef 7.58 52.17 87.46 69.00 21.10 C 500 e 9.32 abedf 4.12 55.79 90.38 71.20 21.22 f 12.06 abede 6.17 48.83 89.21 71.40 19.96 1. Plants grown at day/night temperatures of 26°/26°. 2. Fresh weight. 3. Of o r i g i n a l content. 4. Treatment value d i f f e r s s i g n i f i c a n t l y from these treatments. 70 o u 4-1 c o o •4-1 o CO cfl cu CO CO CU u CJ 60 50 40 30 20 10 0 -10 -20 -30 -40 -50 at 1500 ft-c at 1000 ft-c 7/ I Fresh After storage 7o loss at storage Fig. 12 Effect of KNap spray oh ascorbic acid content, and its loss during 5 days storage, in green pods of bush bean plants grown at 26/26°. GO o 81 increases were 28.57, 33.33 and 6.06%. 3. Pod fresh weight: The weights of green pods harvested 5 weeks afte r the treatment were increased s i g n i f i c a n t -l y i n the high (14.73%) and the medium l i g h t (12.87%), and increased nonsignif i c a n t l y i n the low l i g h t (9.557.), due to KNap treatment. 4. Seed y i e l d : Seed y i e l d was measured i n terms of the number of f u l l y mature seeds and weight of air-d r y seeds per plant. (a) Number of seeds: S i g n i f i c a n t increases i n the number of seeds per plant were produced i n a l l the l i g h t i n t e n s i -t i e s . The increases were 38.26, 27.18 and 30.64% i n high, medium and low l i g h t . (b) Weight of seeds: Seed weight per plant was also s i g n i f i c a n t l y increased i n a l l the l i g h t i n t e n s i t i e s . The increases were 36.40% i n high l i g h t , 35.98% in medium l i g h t and 31.34% i n low l i g h t . D. Moisture content of pods There were nonsignificantldecreases i n the pod moisture content due to KNap treatment i n a l l the three l i g h t regimes. Treatment did not afford protection from moisture loss during 5 days storage. TABLE XVI Effect of KNap spray on yield of bush bean plants. Treat- Treatment Flower no./ Number Pod weight/ Number Weight dry ment Letter plant pods/plant » plant (g)^j^ seeds/plant^ seeds/plant^ (g) c 1500 a 19.75 cef 7. 87 beef 40.07 beef 19.16 bdef 5. 53 bdef T it b 21.87 cdef 10. 12 acef 45.97 acdef 26.50 acdef 7. 55 acef C 1000 c 13.37 ab 6. 37 abdef 35.21 abdef 17.16 bdef 5. 00 bdef T tl d 16.25 bef 8. 50 beef 39.75 beef 21.83 abcef 6. 80 acef C 500 e 11.12 abd 4. 12 abed 16.59 abed 10.33 abcdf 2. 74 abcdf T it f 11.75 abd 4. 37 abed 18.17 abed 13.50 abede 3. 60 abede 1. Plants grown at day/night temperature of 2 6 ° / 2 6 ° . 2. Average value of 8 determinations each. 3. 4 cm and longer as harvested (5 weeks after treatment. 4. Treatment value differs significantly from these treatments. CO at 1500 ft-c o u •u c o o 4H o 50 r 40 30 % 20 CD U O c 10 0 at 1000 ft-c Flower number Pod number I Pod weight Seed number Seed weight Fig. 13 Effect of KNap spray on flower number, and number and weight of pod and seed of bush bean plants grown at 26°/26°. 00 84 E. Photosynthesis and Respiration Rates (Table XVII, F i g . 14)'. 1. Photosynthesis: Rate of apparent photosynthesis of treated plants i n comparison to thei r respective controls i n -creased s i g n i f i c a n t l y at a l l times of observation and i n a l l the three l i g h t i n t e n s i t i e s . Measured 7 days afte r treatment the increases were 15.73, 8.39 and 9.647, i n high, medium and low l i g h t . In high l i g h t the increases were 13.9 and 16.157,, and in medium l i g h t 13.01 and 8.377o measured at 14 and 21 days af t e r treatment. Thus, the rate of apparent photosynthesis showed maximum increase i n high l i g h t on the 21st day a f t e r treatment, and i n medium and low l i g h t on 14th and 7th day respectively. 2. Respiration: Due to KNap treatment the rate of dark r e s p i r a t i o n , measured 7 days afte r treatment increased s i g n i f i c a n t l y i n high (11.477,) and medium (29.497,) and nonsignif i c a n t l y i n low (11.96%) l i g h t . S i g n i f i c a n t increases i n the rate of re s p i r a -t i o n were found i n a l l the l i g h t i n t e n s i t i e s measured at 14 and 21 days af t e r treatment. The increases were 36.14 and 43.447o i n high l i g h t , 35.66 and 35.21% i n medium l i g h t and 17.83 and 20.24% i n low l i g h t , measured 14 and 21 days af t e r treatment. F. Enzyme a c t i v i t y A c t i v i t y of some enzymes involved i n carbohydrate and nitrogen metabolism i n c e l l - f r e e homogenates of leaves from plants TABLE XVII' Effect of KNap on photosynthetlc and respiration rates of the aerial portions of intact bush bean plants . •*-> ^ » Days after treatment 7 14 21 Treat-Treat- ment Apparent Dark Apparent Dark Apparent Dark ment Letter psyn. Respiration psyn. Respiration psyn. Respiration c 1500 a 1646 4 bcdef 762 bcdef 2995 bcdef 1335 bcdef 2191 bcdef 1251 beef T it b 1905 acdef 850 acdef 3412 acdef 1817 acdef 2545 acdef 1794 acdef C 1000 c 1406 abdef 525 abdef 2481 abdef 901 abde 1844 abdef 893 abdef T II d 1524 abcef 680 abcef 2804 abcef 1223 abcef 1998 abcef 1207 beef C 500 e 1130 abcdf 255 abed 1493 abcdf 740 abcdf 1290 abcdf 600 abcdf T II f 1239 abede 285 abed 1628 abede 872 abde 1405 abede 721 abede 1. Plants grown at day/night temperatures of 2 6 ° / 2 6 ° . 2. Average value of two determinations each. 3. Ail C02/dm2 leaf surface/hour. 4. Treatment value differs significantly from these treatments. CO at 1500 ft-c at 1000 ft-c 50 r 2 40 a o o CO 30 3 20 o c M 10 o L ^ at 500 ft-c Photosynthesis Respiration Photosynthesis Respiration Photosynthesis Respiration I 1 1 14 Days after treatment i 21 J Fig. 14 Effect of KNap spray on photosynthetic and respiration rates of the aerial portions of intact bush bean plants grown at 26°/26° 00 O N 87 grown at 26°/26° temperature, and in three light intensities, 1500, 1000, and 500 ft-c, were determined 7, 14 and 21 days after KNap treatment. Activity of nitrate reductase was also determined in cell-free homogenates of roots at the days mention-ed above. Results, expressed as specific activities under the specified conditions, are presented in Tables XVIII to XX. Figures 15 to 17 depict the percentage values of enzyme activities with respect to control values. 1. Phosphorylase (Table XVIII, Fig. 15) Phosphorylase- activities in the leaves of treated plants grown at 26°/26° temperature increased significantly at a l l times of observation and in a l l the three light intensities. Measured 7 days after treatment the increases were 42.78, 29.16 and 28.47% in high, medium and low light respectively. On subsequent observa-tions, the stimulatory effect of treatment decreased in high and medium light. But in low light the maximum stimulatory effect of KNap on phosphorylase activity was found 21 days after treat-ment . .2. Phosphoglyceryl Kinase (Table XVIII, Fig. 15) Measured 7 days after treatment the phosphoglyceryl Kinase activity in the leaves of treated plants grown at 26°/26° temperature showed a significant increase only in high light TABLE XVIII Eff e c t of KNap spray on the a c t i v i t y of phosphorylase and phosphoglyceryl Kinase i n leaves of bush bean plants. *• Days afte r treatment 7 14 21 Specific a c t i v i t y of Treat-ment Treat-ment Letter Phos . 2 » 4 P .G.l Kinase 3'^ Phos • P. G.: Kinase Phos • P. G . l Kinase C 1500 a 20.1 bcdef 6 3.10 bcdef 21.5 beef 7. 20 bcdef 20.6 bcdef 7. 22 bcdef i p i t b 28.7 acdef 3.40 acdef 29.6 acdef 8. 45 acdef 26.5 acdef 8. 25 acdef C 1000 c 16.8 abdef 2.42 abef 17.0 abdef 5. 45 abdef 16.7 abdef 5. 57 abdef I J I II d 21.7 abcef 2.55 abef 21.9 beef 6. 20 abcef 18.0 abcef 6. 10 abcef C 500 e 14.4 abedf 1.54 abed 14.8 abedf 3. 60 abed 12.1 abedf 3. 45 abedf r£ II f 18.5 abede 1.65 abed 19.0 abede 3. 95 abed 17.4 abede 3. 82 abede 1. Plants grown at day/night temperature of 26°/26°. 2. Phosphorylase. 3. Phosphoglyceryl Kinase. 4. /ug of inorganic phosphate formed/mg protein/hour. 5. An increase of 0.05 O.D./mg protein/hour. 6. Treatment value d i f f e r s s i g n i f i c a n t l y from these treatments. Fig. 15 Effect of KNap spray on phosphorylase and phosphoglyceryl kinase activities in leaves of bush bean plants grown at 26°/26°. 9 0 ( 9 . 5 % ) and nonsignificant increases in medium ( 5 . 1 5 % ) and low light ( 7 . 1 4 % ) . Higher activities were observed in a l l the light intensities measured 1 4 days after treatment. The increases were 1 7 . 5 2 , 1 3 . 7 6 and 9 . 7 2 % in high, medium and low light. The increases were significant in high and medium lights only. Significant increases in the enzyme activity were observed in a l l the light intensities ^measured 2 1 days after treatment. In comparison to the second observation the stimulatory effect of treatment decreased in high and medium light and increased in low light. ,3,. Nitrate reductase: (Table XIX , Fig. 16 ) The activity of nitrate reductase in the roots of treated plants grown at 2 6 ° / 2 6 ° temperature showed a nonsignif-icant increase in a l l the light intensities measured 7 days after treatment. The increases were 4 . 0 , 7 . 1 4 , and 6 . 2 5 7 o in high, medium and low light respectively. At the 14-day observation, the activity of this enzyme showed a significant increase in high light ( 3 0 % ) and nonsignificant increases in medium ( 1 3 . 3 3 % ) and low light ( 1 5 . 3 8 % ) . Measured 2 1 days after treatment, there was a significant increase in enzyme activity only in high light ( 2 5 . 0 % ) . In medium ( 1 4 . 2 8 % ) and low light ( 1 2 . 5 0 % ) , however, the increases were nonsignificant. TABLE XIX Effec t of KNap spray on the a c t i v i t y of n i t r a t e reductase i n bush bean plants. Days a f t e r treatment 7 14 21 Specific a c t i v i t y of Nitrate reductase 2' 3 Treat-Treat- ment ment Letter Root Leaves Root Leaves Root Leaves C 1500 a 25.0 c e f 4 35.0 bcdef 35.0 bcdef 60.0 bcdef 45.0 bcdef 75.0 bcdef T 11 b 26.0 cef 31.1 acdef 45.5 acdef 76.2 acdef 56.2 acdef 97.5 acdef C 1000 c 17.5 ab 23.8 abef 26.2 abef 42.0 abdef 31.5 abe 54.0 abde T II d 18.7 b 22.1 abef 29.7 abef 49.2 abcef 36.0 abef 65.2 abcef C 500 e 12.0 ab 15.4 abed 18.2 abed 28.8 abed 21.6 abed 41.2 abed T I I f 12.7 ab 14.0 abed 21.0 abed 33.0 abed 24.3 abd 48.0 abd 1. Plants grown at day/night temperature of 26°/26°. 2. mvuml n i t r i t e formed per mg protein per hour. 3. Each figure represents mean of 2 r e p l i c a t e s . 4. Treatment value d i f f e r s s i g n i f i c a n t l y from these treatments. at 1500 ft-c 40 r 30 20 O U 4J C o o •4-1 o £ 10 CO cu w CO o o 0 c M -10 • 20 1 Root 14 at 1000 ft-c '/£ at 500 ft-c Nitrate Reductase 1 21 7 Days after treatment Leaf 1 I 14 Fig. 16 Effect of KNap spray on nitrate reductase activity in roots and leaves of bush bean plants grown at 26 /26 , 21 V O 9 3 Nitrate reductase activity in leaves of plants grown at 2 6 ° / 2 6 ° temperature was found to decrease, due to treatment, measured 7 days after treatment. There was a significant decrease ( 1 1 . 0 7 o ) in high light, and nonsignificant decreases in medium ( 7 . 3 6 7 . ) and low light ( 9 . 1 7 o ) . At the 14-day observation, the activity of this enzyme in leaves showed significant increase in both high ( 2 7 . 0 7 o ) and medium light ( 1 7 . 1 4 7 o ) and nonsignificant increase in low light ( 1 4 . 5 8 % ) . The same pattern, as observed in the second week, was found 2 1 days after treatment. The increases were significant and 3 0 . 0 , 2 0 . 8 3 , and 1 6 . 3 6 % in high, medium and low light. 4. Glutamic-pyruvic transaminase: (Table XX , Fig. 17)'-The activity of glutamic-pyruvic transaminase in the leaves of plants grown at 2 6 ° / 2 6 ° temperature showed nonsignificant decrease in a l l the light intensities measured 7 days after treat-ment. The decreases were 1 2 . 7 7 , 6 . 4 6 and 1 0 . 0 % in high, medium and low light. At the 14-day observation, the activity of this enzyme showed significant increases in high ( 2 4 . 8 1 % ) and medium ( 1 6 . 7 5 % ) light, and nonsignificant increase in low light ( 1 3 . 8 5 % ) . Measured 2 1 days after treatment, there were significant increases in high ( 2 7 . 9 % ) and medium ( 1 8 . 0 % ) light and nonsignificant increases ( 1 2 . 1 5 % ) in low light. Thus there was an i n i t i a l depression and subsequent increase in activity in high and medium light. In low light, however, subsequent increases following the i n i t i a l depression never attained significance. TABLE XX. Effect of KNap spray on the activity of glutamic-pyruvic transaminase in bush bean leaves.  Days after treatment Treatment — 1 2 1 Treatment Letter Specific activity of glutamic-pyruvic transaminase » c 1500 a 1.41 cdef 4 2.82 bcdef 3.62 bcdef T II b 1.23 cdef 3.52 acdef 4.63 acdef C 1000 c 0.93 abef 1.91 abdef 2.50 abdef T 11 d 0.87 abef 2.23 abcef 2.95 abcef C 500 e 0.60 abed 1.30 abed 1.81 abed T II f 0.54 abed 1.48 abed 2.03 abed 1. Plants grown at day/night temperature of 2 6 ° / 2 6 ° . 2 . Aim pyruvic acid formed/mg protein/hour. 3. Each figure represents mean of two replicates. 4. Treatment value differs significantly from these treatments. o u 4-1 a o CJ MH O CO CO CD co CO CD U O c 40 30 20 10 0 -10 Transaminase at 1500 ft-c at 1000 ft-c at 500 ft-c 1 14 21 -20 L Days after treatment Fig. 17 Effect of KNap spray on glutamic-pyruvic trans• aminase activity in leaves of bush bean plants grown at 26°/26°. ON 97 DISCUSSION The results presented herein are consistent with the view that when applied in appropriate doses to plants at the correct ontogenetic stage and under appropriate climatic condi-tions, naphthenate can bring about significant stimulation in juvenile growth, and that this may fi n a l l y lead to improved yield and composition. It is noticed from the data that following KNap treatment some metabolic activities, eg. apparent photosynthesis, dark respiration and enzyme activity, are stimulated. These aspects w i l l be discussed and evaluated in the light of available literature. An attempt has been made to interpret the results, obtained. 1. Juvenile growth Juvenile growth of various plants has been found to be stimulated following naphthenate treatment by various workers which have been mentioned earlier (see Review). In this experiment the juvenile growth of bush bean plants has been found to be stimulated following KNap spray. However, different plant organs were found to respond differently to treatment depending on temperature and light intensity in which the plants were grown. The maximum relative stimulatory effect of KNap treatment on vegetative growth was found at 98 26 /21 , except i n case of leaf area, and i t was followed by 26°/26° and 15°/15°, i n plants grown under a l i g h t i n t e n s i t y of 1500 f t - c (Table XXI). plants grown at 26°/26° showed maximum r e l a t i v e stimulation i n some cases i n high l i g h t eg. fresh weight of stem and leaf, and leaf area, and i n some other cases i n medium l i g h t eg. fresh and dry weight of root, dry weight of stem and leaf and l e a f l e t number. Only i n the case of shoot length the maximum r e l a t i v e stimulation was i n low l i g h t . The maximum r e l a t i v e stimulation of juvenile growth for plants grown at 15°/15° was found i n medium l i g h t followed by low and high l i g h t . I t may be mentioned that i n plants grown at 15°/15° there were large variations among the r e p l i c a t e s and thus i n many cases a r e l a t i v e l y large stimulation lacked s t a t i s -t i c a l s i g n i f i c a n c e . 2. y i e l d Several investigators have reported increases obtained i n commercial y i e l d following application of naphthe'nates (see Review). Data presented i n t h i s thesis indicate that following KNap treatment the pod weight of bush bean i n a l l the three temperature and l i g h t conditions increased. Thus the present work shows r e s u l t s similar to those found by other workers. TABLE XXI Relative stimulation of juvenile growth, number, weight and ascorbic acid content of pod i n plants grown i n 1500 f t - c and at three temperatures. Root wt. Stem wt. Leaf wt. Fresh g Dry mg Fresh g Dry mg Fresh g Dry mg Plant Leaf height number cm Leaf area dm2 Ascorbic Pod acid numb mg/lOOg e" r Pod 7 wt. g C 26°/26° 10.50 1068 13.90 1955 20.63 2320 40.43 23.25 8.02 13.73 7.87 40.07 C:.26°/21° 7.24 594 6.19 918 7.53 1034 30.31 18.0 4.59 11.68 5.62 29.75 C 15°/15° 3.69 558 3.68 626 4.61 738 17.61 11.25 1.34 10.76 1.75 8.64 T 26°/26° 12.18 1196 16.48 2237 24.20 2720 44.51 28.25 9.28 16.55 10.12 45.97 % c o n t r o l * 16.0 11.98 18.56 14.42 17.3 17.24 10.07 21.5 15.66 20.53 28.57 14.73 T 26°/21° 8.82 734 8.17 1224 11.18 1467 33.22 22.0 5.74 14.77 6.69 34.67 % c o n t r o l * 21.72 23.56 32.07 33.3 48.45 41.0 10.38 22.2 25.0 26.5 18.88 16.5 T 15°/15° 4.05 561 3.73 667 4.43 687 21.0 14.25 1.37 12.24 2.0 10.48 7o c o n t r o l * 9.85 0.44 1.28 6.57 -3.91 -6.63 19.82 26.6 2.73 13.75 14.28 21.24 * Increase as % of control V O 100 3. Stimulation of growth Stimulation of growth and development of plants follow-ing naphthenate treatment as observed by the author and others can result from a variety of stimulatory mechanism in plants. Some of the likely mechanisms are as follows: (i) stimulation of nitrate reductase, the enzyme responsible for the reduction of nitrate, which on further reduction gives rise to ammonia required for amino acid synthesis (hence protein synthesis). NO — > N0 2 »» HNO a-NHoOH N^Ho reductase J ( i i ) stimulation of transaminases, the systems necessary for the synthesis of most of the amino acids; ( i i i ) stimulation of the systems which supply the substrates for respiration, eg. stimula-tion of phosphorylase activity which converts starch to glucose which may be Cifcilized in respiration; (iv) stimulation of respiratory pathway enzymes. Intermediates in the respiratory pathway are involved in the synthesis of lipids, proteins and nucleic acids. Respiration also provides usuable energy as ATP and also the reduced coenzymes for various synthetic reactions; (v) stimulation of synthesis of some growth regulators eg. stimula-tion of the enzymes responsible for the synthesis of indole acetic acid, gibberellin etc.; (vi) stimulation of some steps in protein 101 and nucleic acid synthesis; (vii) stimulation of the activity of plant growth regulators. Out of the above-listed stimulatory mechanisms a few which have been investigated by the author and by others are discussed below. Among the investigated enzymes, phosphorylase activities in the leaves of treated plants increased significantly. The increased phosphorylase activity in treated plants would solubilize starch to hexose at a faster rate. Starch ^ Glucose-l-phosphate The hexose thus formed can act as substrate for the respira-tory pathway and could be utilized for other purposes eg. synthesis of c e l l wall material, synthesis of ascorbic acid etc. Activity of phosphoglyceryl kinase, a key enzyme of respiratory system, showed significant increase following KNap treatment. Due to increased phosphoglyceryl kinase activity there would be greater production of utilizable energy as ATP Phosphoglyceryl kinase 1,3-diphosphoglyceric acidf+ ADP ^ ,v 3-phosphoglyceric acid + ATP Activity of nitrate reductase, a key enzyme of nitrogen metabolism, indicated that different organs, i.e. roots and leaves, 102 responded differently to the KNap treatments and i t fluctuated with time. The variation in the level of nitrate reductase activity has been noted by other workers too, eg. with the age of the tissue (Shrader and Hageman, 1967; Wallace and Pate, 1967) and with the plant age (Zieserl et_ a l 1963; Yang, 1964; Wallace and Pate, 1967). Nitrate is the principal source of nitrogen utilized by higher plants and nitrate must be reduced before i t can be incorporated into usable compounds. The f i r s t step in nitrate reduction is conversion of nitrate to n i t r i t e which is catalyzed by the enzyme nitrate reductase, NADH2 -\,^~ FAD-^^2Mo 5 + + 2 H + ^ ^ - NO3" NAD *S X»FADH 2 \ r 2Mo!6+ J/ V * N0 2 + H20 The nitrate reductase activity is thus related to protein producing potential of the plants and is important for growth. The increase in activity of nitrate reductase by KNap treatment observed by the author can be explained as follows: (a) as nitrate reductase utilizes a reduced pyridine nucleotide (NADH^) as the hydrogen-donor and contains flavin adenine dinucleotide (FAD) as the prosthetic group, the increased photo-103 synthesis and re s p i r a t i o n i n KNap-treated plants could enhance the supply of the necessary reducing power and as a re s u l t the ni t r a t e reductase a c t i v i t y increased; or (b) the treatment caused enhanced a c t i v i t y of n i t r a t e reductase i n a dif f e r e n t way, the proposed mechanism of which w i l l be discussed l a t e r . A c t i v i t y of glutamic-pyruvic transaminase, another key enzyme of nitrogen metabolism, showed a r e l a t i v e l y s i m i l a r pattern of a c t i v i t y to that of n i t r a t e reductase i n roots of treated plants. Although transamination reactions involving glutamic acid are by far the most prevalent i n plants, other transamination reactions have been found. It i s generally accepted now that reductive amination of oC-Keto acids followed by transamination i s a major pathway of amino acid formation. Amino acids thus formed may be used for protein synthesis. H2N.CH2.CH2.COOH ^Glutamic- CH3.CO.COOH pyruvic + transaminase + ^ y H00C.CH2.CH2.CO.COOH HOOC.CH2.CH2.CH.NH2.COOH Following naphthenate treatment there was increased catalase a c t i v i t y i n grape plants (Kolesnik, 1965) and i n cotton plants (Bazanova and Akopova, 1966). It i s known that a co r r e l a -t i o n usually exists between the catalase a c t i v i t y of a plant and i t s metabolic status; and measurements of the catalase a c t i v i t y 104 of a plant are, therefore, often accepted as an index of the intensity of metabolic activity in that plant. So, the results mentioned would indicate that naphthenate treatment increased the intensity of metabolic activity in treated plants, as now dis-covered in the present investigation. It is known that the metabolic reactions in a living organism are made possible by individual enzymes or enzyme systems, and thus i t seems reasonable to expect that the fundamental action of a growth stimulator such as KNap would be upon enzymes. Experiment by the author showed that KNap treatment causesan increase in activity of several enzymes and thus KNap can be regarded as a general stimulator rather than a specific one. The stimulation of enzyme activity varied in bush bean grown under different environmental conditions eg. light intensity (TablesXVIII-XX,Figs.15-17 ). The variation in enzyme activity with time following chemical treatment of plants as observed in this experiment is also evident in the results of other workers (Wort and Cowie, 1953; Chu, 1969). It should be mentioned, however, that the determination of enzyme activity in tissue of a plant previously treated with naphthenate certainly does not reveal the mode of action of the chemical nor does i t identify the substances or conditions which 105 are d i r e c t l y responsible for the change i n enzyme a c t i v i t y , but i t does present a summation eff e c t i n the l i v i n g plant, measured at a given time. 4. Ascorbic acid Ascorbic acid occurs i n plants mainly i n the reduced form, but a small and not e a s i l y determined portion may be i n the oxidized form, dehydro-ascorbic acid. Data presented i n this thesis show that the ascorbic acid content of green pods from KNap-treated plants showed s i g n i f i c a n t increases i n comparison to that of respective control plants i n a l l the temperature and l i g h t conditions i n which the plants were grown. However, the r e l a t i v e stimulatory e f f e c t of treatment varied with temperature and l i g h t conditions i n which the plants were grown. Isherwood and Mapson (1962) also found that external factors had considerable e f f e c t on the metabolism of ascorbic acid. Following naphthenate treatment both increase and de-crease of ascorbic acid content of tomato f r u i t s (Aliev, 1965; Chu, 1969); increases i n cotton leaves (Agakishev and Bazanova, 1965; Bazanova and Akopova, 1966), cotton roots (Babaev, 1966), cabbage leaves (Asadov, 1965) and i n melon f r u i t s (Abolina and Ataullaev, (1966) have been reported. Thus, the results of the present work are s i m i l a r to those found by most of the workers, i n that naphthenate 106 treatment increased ascorbic acid content in plant parts. The actual mechanism, or mechanisms, by which temper-ature and light intensity influenced the metabolism of ascorbic acid in pods was not ascertained. A consideration of the bio-chemistry of ascorbic acid would appear useful at this point of discussion as a means of identifying some of the potential sites of KNap interaction. Ascorbic acid in plants is derived from D-glucose by a sequence of enzymic conversions. D-glucose is converted to Drglucuronolactone, which is reduced to the lactone of an aldonic acid (L-gulonic acid). Oxidation of this lactone gives L-ascorbic acid, which can also arise by oxidation of L-galactano-X-lactone, derived from D-galactose via D-galacturonic acid. The enzymes responsible for these conversions have not yet been characterized, but i t appears likely that the reduction of hexuronic acid to gulonolactone or galactonolactone is affected by a pyridine nucleotide dependent hydrogenase (Fruton and Simmonds, 1960). It may be assumed that KNap could interact with some of the enzymes involved in the biosynthesis of ascorbic acid. Moreover, a synthesis from glucose may go in darkness, but a light dependent synthesis which in some way is coupled to photosynthesis does also occur (Aberg, 1961) and this could explain the variation in ascorbic acid content of green pods in various light intensities observed in this 107 experiment. Ascorbic acid and dehydroascorbic acid constitute a redox system in biological materials, which participates in various oxidation-reduction mechanisms in living cells, including the phenol oxidase systems of plants, tyrosinase, and mechanisms involving the interconversion of disulfide and sulfhydryl groups. Ascorbic acid thus activates sulfhydryl enzymes and coenzymes. Moreover, an enhanced production of NADPR^  could lead to a more reduced state of the glutathione and ascorbic acid systems (Key, 1962). There are enzyme systems in plants which oxidize ascorbic acid and others which reduce dehydro-ascorbic acid. Ascorbic acid may, therefore, function as a respiratory carrier. Due to oxidation, the amount of ascorbic acid f a l l s during storage. The oxidation of ascorbate is accelerated by traces of iron and copper and also by oxidizing enzymes. Protein and certain hydroxyacids such as c i t r i c acid, which form complexes with copper, protect ascorbic acid from such oxidation. In the present experiment ascorbic acid loss during storage was less in pods from treated plants in comparison to that of the control. It can be explained by assuming that KNap treatment either decreased the ascorbic acid oxidizing enzymes of accelerated the formation of complexes of protein and c i t r i c 108 acid with copper thereby protecting ascorbic acid from oxidation. The protective action of KNap treatment was found to be highest in pods from treated plants grown in high light, and thus light in some way, s t i l l uncertain, played its role. A catalytic function of ascorbic acid in photosynthetlc phosphorylation (cyclic photophosphorylation) has been suggested (Arnon et a l , 1957; Avron, 1960; Jogendorf and Neumann, 1965; Avron and Neumann, 1969). A participation of ascorbic acid in photosynthesis as a hydrogen-donor in clorophyll-sensitized NADP ^ reduction has also been mentioned (Rabinowitch, 1956). Ascorbic acid has also been found to reduce toxic products generated by the action of phenoloxidase (Wetmore and Morrel, 1949). Ascorbic acid may also be an important regulator in iron metabolism in plants (Aberg, 1961). In summary, there may be several explanations of the effect of KNap on ascorbic acid metabolism and its subsequent influence on plant growth and development. One possible explana-tion may be that KNap treatment of plants causes a shift towards a more reduced state of ascorbic acid, soluble sulfhydryl compounds and pyridine nucleotides, accompanied by a net increase in the concentration of the total ascorbic acid and pyridine nucleotides. Moreover, ascorbic acid could participate in the activation of various enzyme systems and stimulates the production of ATP by 109 acting as an electon-donor in photosynthetic and oxidative phosphorylation. The action of ascorbic acid thus could create a favorable balance for synthesis of nucleic acids and so enabling the process of growth and development to proceed at a fast rate. 5. Photosynthesis Increase i n the i n t e n s i t y of photosynthetic rate follow-ing naphthenate treatment was observed i n cotton plants (Agakishev and Bazanova, 1965; Bazanova and Akopova, 1966), i n grape plant (Kolesnik, 1965) and i n potato plants (Ladygina, 1965; Abolina and Ataullaev, 1966). Chu (1969) found that i n tomato plants KNap treatment resulted i n higher rates i n apparent photosynthesis four weeks af t e r treatment. The data presented i n this thesis indicate that KNap can r e s u l t i n higher rates of apparent photosynthesis, as was evident i n works mentioned above. The rates d i f f e r e d i n plants of d i f f e r e n t ages and also i n plants grown under d i f f e r e n t l i g h t i n t e n s i t i e s . The rates of apparent photosynthesis increased more or less l i n e a r l y with increasing l i g h t i n t e n s i t y i r r e s p e c t i v e of age or treatment of plants. The rates of photosynthesis of treated and control plants, measured 21 days after treatment, are much lower than those measured at 14 days (Fig.18 ). It should be noted that by 21 days the f r u i t s are quite advanced i n develop-ment . 14T 14C 2 IT 21C 7T 7C 500 1000 Light intensity (ft-c) 1500 7C - Control on 7th day after treatment 7T - Treated on 7th day after treatment 14C - Control on 14th day after treatment 14T - Treated on 14th day after treatment 21C - Control on 21st day after treatment 21T - Treated on 21st day after treatment Fig. 18 Rate of apparent photosynthesis in bush bean plants I l l The observed increase in apparent photosynthesis could be important in providing more carbohydrate and organic acids, ATP and NADPl^ and thereby resulting in increased metabolic activities and growth of treated plants. No reports are yet available on the effect of naphthenates on enzymes of photosynthetic process. How this chemical enhanced photosynthesis remains unexplained. 6. Respiration KNap was found to have significant effects on the rate of dark respiration of aerial parts of bush bean plants. Stimula-tion of respiration following naphthenate treatment has also been noted by several workers (Bazanova and Akopova, 1966; Abolina and Ataullaev, 1966; and Chu, 1969). In this experiment the relative stimulatory effect of KNap on rates of dark respiration differed in plants of different ages and also in plants grown under different light intensities. Respiration rate increased with increased light intensity. There seemed to be no effect of plant age on the respiratory rates of treated and control plants measured 14 and 21 days after treatment and grown at 1000 and 1500 ftre (Fig. 19 ). The rate of respiration of some plants is normally limited by the capacity of the phosphorylating system. It may 500 1000 Light intensity (ft-c) 14T 21T 14C 21C 7T 7C 1500 7C - Control on 7th day after treatment 7T - Treated on 7th day after treatment 14C - Control on 14th day after treatment 14T - Treated on 14th day after treatment 21C - Control on 21st day after treatment 21T - Treated 'on 21st day after treatment Fig. 19 Rate of dark respiration in bush bean plants 113 well be that the effect of KNap is on the phosphorylative portion of the respiratory process since the activity of phosphoglyceryl kinase was found to be stimulated following KNap treatment of plants. 7. Mechanism of KNap action Either or both of the two subsequent statements could suggest a possible role of KNap in the stimulation of enzyme activity: 1) KNap could increase the synthesis of one or more enzymes. For example, the synthesis of amylase was found to be promoted in naphthenate-treated Aspergillus usamii (Burachevskii, 1965). An answer to the action of KNap may l i e in the control of the mechanism by which enzymes are made in the c e l l eg. at the transcription or translation level. Pakhomova (1965) reported an increase in the content of exl-nucleoprotein complex in leaves and vegetative apices of naphthenate treated plants. 2) The direct stimulation of available enzyme may arise through incorporation of this chemical into a substrate-regulator-enzyme complex with a lower energy of activation than that of a substrate-enzyme complex. A similar proposal as the second one was made by Wort (1962) regarding 2,4-D (2,4-dichlorophenoxyacetic acid). KNap 114 saturation of enzyme and substrate separately could explain the slowing of reaction with high KNap concentration, as found in tomato by Chu (1969), This hypothesis proposes the formation of a complex in which both regulator and substrate participate, and the regulator can move out of the complex once the substrate has been converted to the products. Recently, Severson e_t al_ (In Press) found that when cyclo-hexane carboxylic acid-7-C-'-^, a low molecular weight naphthenic acid, was fed to bush bean leaf discs conjugates with glucose and aspartic acid were formed. In a similar study Seaforth ejt al (Personal communication) were able to show that bush bean plants fed with KNap would also form glucose and aspartic acid conjugates with KNap. Without further extensive investigations i t is d i f f i c u l t to say whether these conjugates are involved in the mechanism by which KNap operates in the plant. The formation of photosynthate, which serves as the ultimate source of sugars catabolized during respiration, as well as a source of carbon supply for a l l biosynthetic processes, was found to be stimulated by KNap treatment. This increased photo-synthesis may well supply necessary substrates for increased respiration. KNap treated plants also maintained a higher respiratory activity by virtue of enzyme stimulation and available substrate. The net result of this should be greater production 115 of utilizable energy, and an increase in supply of different carbon compounds from the respiratory process, for other bio-synthetic reactions. Increased nitrate reductase and trans-aminase activity could lead to increased synthesis of amino acids and hence protein synthesis. The augmented carbon and energy supply may lead to the formation of larger amounts of new protoplasm and c e l l wall material, essential for new growth. Fig. 20 indicates the positions where stimulations by KNap were measured in the present investigation. 116 2H Ascorbic acid «- — 7 1. Photosynthesis measured as CO^ uptake „ ^  „ Phosphorylase Sugars £. v J Starch I I I 1,3-diphosphoglyceric acid A DP Phosphoglyceryl kinase ATP 3-Phosphoglyceric acid Glutamic ^ -pyruvic transaminase Alanine e»<-Ketoglutarate CO-cG-Ketoglutaric acid CO, 4. Respiration measured as CO2 evolution. r Glutamic acid NH-NO, Nitrate reductase NO-Fig. 20 A diagram to show the points where stimulation by KNap was observed in the investigation. 117 CONCLUSION On the basis of the results obtained i t seems reason-able to conclude that: 1) when applied in appropriate dose, 5000 ppm, to 2-week-old bush bean plants, f o l i a r sprays of KNap lead to stimulation of juvenile growth, as measured by fresh and dry weight of roots, stem and leaves, number and area of leaflet and plant height; 2) the stimulation of vegetative growth led to improved yield as measured by number and fresh weight of pods and number and weight of dry seeds; 3) treatment resulted in higher ascorbic acid content in green pods at harvest and the treatment had a protective action to reduce ascorbic acid loss during storage; 4) following KNap treatment, the physiological processes,apparent photosynthesis and dark respiration, and the biochemical processes, activities of phosphorylase, phosphoglyceryl kinase, nitrate reductase and transaminase, were stimulated; 5) the time elapsed between the application of KNap and time of observation, and the temperature and light intensity in which the plants were grown were found to be important in determining response to KNap stimulation. 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