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Chemical control of growth in sugar beet (Beta saccharifera L.) Singh, Bharat 1968

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The University of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of BHARAT SINGH B.Sc, Banaras Hindu University, Varanasi, India, 1958 M„ Sc., Ranchi University, Ranchi, India, 1961 FRIDAY, MAY 31, 1968, AT 9:30 A.M. ROOM 3332, BIOLOGICAL SCIENCES BUILDING COMMITTEE IN CHARGE Chairman: Dr. I. McT. Cowan A. Gronlund G.H.N. Towers D.P. Ormrod D.J. Wort G.E. Rouse S.H. Zbarsky External Examiner: Dr. Albert U l r i c h Department of S o i l s and Plant N u t r i t i o n University of C a l i f o r n i a Berkeley, C a l i f o r n i a Research Supervisor: D.J„ Wort CHEMICAL CONTROL OF GROWTH IN SUGAR BEET (BETA SACCHARIFERA L.) ABSTRACT Metabolic inhibitors and growth regulators were used in an attempt to control the growth of sugar beet plants at the time of "ripening" of the roots. Maleic hydrazide (MH), pyrocatechol (PC), and vanadium sul-fate (VS) were found to be most effective in control" ling growth regardless of the age of the plants. The solutions containing MH, PC, or VS were applied to the foliage of 4.5-month-old plants and the effects on leaf expansion and content of sucrose, reducing sugars, n i t r i t e , nitrate, ammonia, amino acid, protein and total nitrogen were determined 7, 14, and 21 days after treatment. The rate of photosynthesis and respiration and the activity of nitrate reductase, transaminase, invertase, adenosine triphosphatase (ATP-ase), glucose-1-, glucose-6-, fructose-6-phospha-tase, uridine diphosphate glucose pyrophosphorylase (UDPG-pyrophosphorylase), sucrose synthetase and sucrose phosphate synthetase was measured. Compared with untreated plants, with few excep-tions, a l l treatments affected the growth; the chemical compositions the rate of photosynthesis and respira-tion, and the activities of enzymes measured, in a similar manner. Growth of the plants was determined by measuring the leaf area. MH, PC, and VS significantly inhibited growth of leaves under both "summer" and " f a l l " conditions. In the treated plants, the percentage reducing sugars, based on fresh weight of the root, decreased and percentage sucrose increased steadily. Application of MH, PC, and VS resulted in a significant decrease in n i t r i t e and an increase in nitrate content of roots. Ammonium nitrogen of the plants treated with MH was more than that of the untreated plants on the 7th, 14th, and 21st day after treatment. Plants reated with PC and VS had a lower ammonium content on the 7th and the 14th day but more on the 21st day. The soluble amino acid content of the roots of MH-treated plants was higher than that of the controls. PC-treated plants had a lower amino acid content on the 7th day but a higher content on the 14th and 21st day. VS caused a reduction in amino acid content of the roots on a l l dates of harvest. The rate of photosynthesis was measured by infra-red technique. MH and VS caused a stimulation in the rate of net C02 assimilation, however, PC inhibited the rate of net C02 assimilation on the 7th day after treatment. The rate of respiration of the storage roots, measured by the Warburg technique, was lower than that of the control plants in the case of MH-and VS-treatedpplants and i t was higher in the PC-treated plants. The results indicated that the application of MH, PC, and VS caused significant reduction in the activity of nitrate reductase, transaminase, inver-tase, ATP-ase, glucose-1-, glucose-6-, and fructose-6-phosphatase. These treatments also resulted in the stimulation of the activity of UDPG-pyrophos-phorylase, sucrose synthetase and sucrose phosphate synthetase. The inhibition of growth by MH, PC, and VS is discussed on the bases of the reductions in the ac-t i v i t i e s of invertase, nitrate reductase, and transaminase. The increase in sucrose content of the roots is explained on the bases of low invertase and high sucrose synthetase and sucrose phosphate synthetase activities in the treated plants. The possible participation of the phosphatases in the regulation of sucrose biosynthesis is indicated by the negative correlations between the activities of phosphatases and sucrose phosphate synthetase. GRADUATE STUDIES F i e l d of Study: Botany (Plant Physiology) Plant Physiology D.J. Wort Plant Biochemistry G.H.N. Towers Radioisotopes i n Botany E.B. Tregunna Other Studies: Plant Responses to Controlled D.P. Ormrod Environment A g r i c u l t u r a l Climatology V.C. Brink Biochemistry Biochemistry Faculty Advanced S t a t i s t i c a l Methods A. Kozak CHEMICAL CONTROL OF GROWTH IN SUGAR BEET (Beta saccharifera L) by BHARAT SINGH B . S c , Banaras Hindu University, Varanasl, India, 1958 M . S c , Ranch! University, Ranch!,- India, 1961 A THESIS SUBMITTED IN PARTIAL 'FULFILMENT. OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY . in the Department of Botany We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1968 In presenting this thesis in part ia l fulfilment of the requirements for an advanced degree at the University of Brit i sh Columbia, I agree that the Library shall make it freely available for reference and Study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by hits representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Botany The University of Brit i sh Columbia Vancouver 8, Canada Date May 3 1 , 1968 ABSTRACT Metabolic inhibitors and growth regulators were used In an attempt to control the growth of sugar beet plants at the time of "ripening" of the roots. Maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) were found to be most effective I n controll ing growth regardless of the age of the plants. The solutions containing MH, PC, or VS were applied to the foliage of ^.5-month-old plants and the effects on leaf expan-sion and content of sucrose, reducing sugars, n i t r i t e , ni trate , ammonia, amino acid, protein and total nitrogen were determined 7» 1^, and 21 days after treatment. The rate of photosynthesis and respiration and the act iv i ty of nitrate reductase, trans-aminase, invertase, adenosine triphosphatase (ATP-ase), glucose-1-phosphatase, glucose-6-phosphatase, fructose-6-phosphatase, uridine diphosphate glucose pyrophosphorylase (UDPG-pyrophos- . phorylase), sucrose synthetase and sucrose phosphate synthetase was measured. Compared with untreated plants, with few exceptions, a l l treatments affected the growth; the chemical composition; the rate of photosynthesis and respiration, and the ac t iv i t i e s of enzymes measured, in a similar manner. Growth of the plants was determined by measuring the leaf area. MH, PC, and VS s ignif icantly inhibited growth of leaves under both "summer" and "fa l l" conditions. In the treated plants, the percentage reducing sugars, based on fresh weight of the root, decreased and percentage sucrose increased steadily. i l Application of MH, PC, and VS resulted in a significant decrease in n i t r i t e and an increase in nitrate content of roots. Ammonium nitrogen of the plants treated with MH was more than that of the untreated plants on the 7th, l^th, and 21st day after treatment. Plants treated with PC and VS had a lower ammonium content on the 7th and the l^th day "but more on the 21st day. The soluble amino acid content of the roots of MH-treated plants was higher than that of the controls. PC-treated plants had a lower amino acid content on the 7th day but a higher content on the l^ J-th and 21st day. VS caused a reduction in amino acid con-tent of the roots on a l l dates of harvest. The rate of photosynthesis was measured by infrared tech-nique. MH and VS caused a stimulation in the rate of net CO2 assimilation, however, PC inhibited the rate of net CO2 assimi-la t ion on the 7th day after treatment. The rate of respiration of the storage roots, measured by the Warburg technique, was lower than that of the control plants in the case of MH- and VS-treated plants and i t was higher in the PC-treated plants. The results indicated that the application of MH, PC, and VS caused significant reduction in the act iv i ty of nitrate reduc-tase, transaminase, invertase, ATP-ase, glucose-l-phosphatase, glucose-6-phosphatase and fructose-6-phosphatase. These treat-ments also resulted in the stimulation of the act iv i ty of UDPG-pyrophosphorylase, sucrose synthetase and sucrose phosphate synthetase. The inhibit ion of growth by MH, PC, and VS is discussed on the bases of the reductions in the act iv i t i es of invertase,nit-rate reductase, and transaminase. The increase in sucrose I i i c o n t e n t o f the r o o t s i s e x p l a i n e d on the bases o f low i n v e r t a s e and h i g h s u c r o s e syn the tase and sucrose phosphate s y n t h e t a s e a c t i v i t i e s i n the t r e a t e d p l a n t s . The p o s s i b l e p a r t i c i p a t i o n o f the phosphatases i n the r e g u l a t i o n o f sucrose b i o s y n t h e s i s i s i n d i c a t e d by the n e g a t i v e c o r r e l a t i o n s between the a c t i v i -t i e s o f phosphatases and s u c r o s e phosphate s y n t h e t a s e . iv Acknowledgements I am deeply indebted to Professor D . J . VJort for his direct ion, advice, and encouragement during the period when this, research was conducted. In addition I wish to thank him for reviewing the manuscript c r i t i c a l l y . My thanks are due to Drs. S.H. Zbarsky, A.P. Gronlund and G.H.N. Towers for their continuing interest in the problem and c r i t i c a l review and helpful suggestions during the preparation of this manuscripto I would also l ike to acknowledge the help of Drs. E .B . Tregunna and P . V . SubbRao at various stages of this study; to Dr. G.W. Eaton and .Mr. S.VJ. Borden for their help in s ta t i s t i ca l analysis of the experimental resul ts . I am grateful to the Department of Botany for the use of the f a c i l i t i e s and equipment. Thanks are also due to the Beet Sugar Foundation of America whose f inancial support made this study possible. Special thanks are due to my wife for her help in many ways. V TABLE OP CONTENTS PAGE Abstract. i Acknowledgement iv Table of Contents v-L i s t of Figures v i i i L i s t of Tables x CHAPTER I . THE SUGAR BEET PLANT . . . . 1 1. Discovery. . . 1 2 . Production 1 3 . Gross Morphology 2 Jj>. Anatomy 2 II . NITROGEN METABOLISM OF THE SUGAR BEET AS RELATED TO LATE SEASON GROWTH WHICH RESULTS IN A LOW SUCROSE CONTENT OF THE ROOT 4 1. Introduction ^ 2 . Requirements of Growth and Di f ferent ia t ion . . . 6 3 . Carbon and Nitrogen Sources for Protein Syn- 8 thesis and growth of sugar beet leaves ^ . Interrelationship of Nitrogen Metabolism and Carbohydrate Metabolism.. 10 5» Nitrate Reduction in Relation to Respiration and Photosynthesis • • • • 12 III . CONTROL OF GROWTH IN SUGAR B E E T . . . . . 23 1. Cultural Practices. 23 2 . Selection and the Breeding of the new Varieties 26 3 . Disadvantages of the Prevalent Methods 26 ^ . Chemical Control of growth i n Sugar B e e t . . . . . 27 v i TABLE OF CONTENTS (cont'd) CHAPTER ?age I V . MATERIAL AND METHODS 32 1 . Growth of the Plants 32 2 . Mode of Application of Chemicals.. 33 3 . Determination of Leaf Area 33 k. Determination of the Chemical Composition of the Root 35 5 . Determination of the Rate of Respiration of Root 4-3 6. Determination of Photosynthesis and Respira-tion of Whole P l a n t s . . . . -1+3 7 . Determination of the Act iv i t ies of Nitrate Reductase and Transaminase.... ^5 8 . Determination of Invertase A c t i v i t y . . 9 . Determination of Act iv i t i es of Phosphatases.. ^8 1 0 . Determination of the Act iv i t ies of Sucrose Synthetase, Sucrose Phosphate Synthetase and UDPG-pyrophosphorylase 50 V . EXPERIMENTAL RESULTS 53 1 . Growth of the Leaves 53 2 . Sucrose Content of the Roots 56 3 . Reducing Sugars Content of the Roots . . . . 56 4 . Nitrogenous Constituents of the Roots 63 5 . Photosynthesis and Respiration 69 6. Nitrate Reductase and Transaminase A c t i v i t y . . 82 7 . Invertase Act iv i ty 89 8 . Phosphatases 89 9 . UDPG-pyrophosphorylase 100 1 0 . Enzymes of Sucrose Synthesis 101 1 1 . Simple Correlation Coefficients 10^ v i i TABLE OF CONTENTS (cont'd) CHAPTER PAGE VI. DISCUSSION 1 1 ° Conclusions 135 Literature c i t e d . . . . . 137 : v i i i LIST OF FIGURES FIGURE PAGE 1 . Interrelationships of nitrogen metabolism and carbohydrate metabolism... 20 2. Effect of maleic hydrazlde (MH), pyrocatechol (PC) and vanadium sulfate (VS) on leaf growth of ... sugar beet 55 3 . Effect of maleic hydrazide (MH), pyrocatechol (PC) and vanadium sulfate (VS) on leaf growth of sugar beet under summer growth c o n d i t i o n s . . . . . . . 57 4 . Effect of maleic hydrazide, pyrocatechol and vanadium.sulfate on leaf growth of sugar beet under f a l l conditions 58 5 . Effect of maleic hydrazide on sucrose and reducing sugar content of root of sugar beet grown under summer and f a l l conditions. , 60 6. Effect of pyrocatechol on sucrose and reducing sugar content of root of sugar beet grown under summer and f a l l conditions 6 l 7 . Effect of vanadium sulfate on sucrose and reducing sugar content of root of sugar beet grown under summer and f a l l condi t ions . . . . 62 . 8 . Effect of maleic hydrazide (MH),. pyrocatechol (PC) and vanadium sulfate (VS) on leaf growth and root reducing sugar and sucrose of sugar beet under summer growth conditions 65 9 . Effect of maleic hydrazide (MH), pyrocatechol (PC) and vanadium sulfate (VS) on leaf growth and root reducing sugar and sucrose of sugar beet under f a l l conditions 66 1 0 . Effect of maleic hydrazide on the nitrogen content of root of sugar beet.. 71 1 1 . Effect of pyrocatechol on the nitrogen content of root of sugar beet 72 1 2 . Effect of vanadium sulfate on the nitrogen content of root of sugar beet . . 73 1 3 . Effect of maleic hydrazide on photosynthesis and respiration of leaves and on respiration of root of sugar beet grown under summer c o n d i t i o n s . . . . . 76 -ix FIGURE . PAGE 14. Effect of maleic hydrazide on photosynthesis and respiration of leaves and on respiration of root of sugar beet under f a l l condi t ions . . . . 77 15« Effect of pyrocatechol on photosynthesis and respiration of leaves and on respiration of root of sugar beet grown under summer c o n d i t i o n s . . . . . 78 1 6 . . Effect of vanadium sulfate on photosynthesis and respiration of leaves and on respiration of root of sugar beet grown under summer conditions 79 17. Effect of vanadium sulfate on photosynthesis and respiration of leaves and on respiration of root of sugar beet under f a l l condi t ions . . . . 80 18. Effect of maleic hydrazide on nitrate reductase and transaminase ac t iv i ty in leaf and root of sugar beet 86 19. Effect of pyrocatechol on nitrate reductase and transaminase act iv i ty in leaf and root of sugar beet 87 2 0 . Effect of vanadium sulfate on nitrate reductase and transaminase ac t iv i ty in leaf and root of sugar beet 88 2 1 . Effect of maleic hydrazide on enzymes of sucrose synthesis and hydrolysis in the leaf of sugar beet 91 2 2 . Effect of pyrocatechol on enzymes of sucrose synthesis and hydrolysis in the leaf of sugar beet °2 2 3 . Effect of vanadium sulfate on enzymes of sucrose synthesis and hydrolysis in the leaf of sugar bee t . . . . 93 24. Effect of maleic hydrazide on enzymes of sucrose synthesis and hydrolysis in the root of sugar beet 9^ 2 5 . Effect of pyrocatechol on enzymes of sucrose synthesis and hydrolysis in the root of sugar beet. 95 2 6 . Effect of vanadium sulfate on enzymes of sucrose synthesis and hydrolysis in the root of sugar beet. . x :. LIST OF TABLES TABLE PAGE I . Effect of inhibitors on the growth of one-month-old sugar beet plants • • • • 54 II . Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on the growth of the leaves of 4.5-month-old sugar beet plants 5 4 a III . Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on the sucrose content of the roots of 4.5-month-old sugar beet plants 59 IV. Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on reducing sugar of the roots of 4.5-month-old sugar beet plants. 64 V. Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on the nitrog-enous constituents of the roots of 4.5-month-old sugar beet plants 67 VI. Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on the protein content of the roots of 4.5-month old sugar beet plants 70 VII . The summary of the effects of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) under summer conditions on the chemical composition of the roots of 4.5-month-old sugar beet plants 70 VIII. Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on photosyn-thesis, and respiration of 4.5-month old sugar beet plants 74 VIII(a) Summary of the effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on photosynthesis and respiration of sugar beet 83 IX. Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on nitrate reductase and transaminase ac t iv i ty of the foot and leaf of 4.5-month-old sugar beet plants 85 xi-TABLE PAGE X. Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on the inver-tase act iv i ty of the root and leaf of 4 . 5 -month-old sugar beet plants 90 XI . Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on the ac t iv i ty of phosphatases of leaf and root of 4.5-month-old sugar beet plants 97 XII Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on UDPG-pyro-phosphorylase ac t iv i ty of root and leaf of 4.5-month-old sugar beet plants 102 XIII. Effect of maleic hydrazide (MH), pyrocatechol (PC)., and vanadium sulfate (VS) on sucrose-P-synthetase and sucrose synthetase of root and leaf of 4.5-month-old sugar, beets 103 XIV. Summary of the effects of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on enzyme ac t iv i t i e s 105 XV. Relationship of growth to different variables . . 106 XVI. Relationship of sucrose to different variables. 109 1 I . THE SUGAR BEET PLANT 1. Discovery The sugar beet (Beta saccharifera) i s unique among crop plants because i t is a modern development with a well documented history. A chemist A.S . Marggraff (17^7) of the Royal Academy of Science i n Ber l in proved that the "white mangold", the so-cal led sugar root, and red mongold or red beet (called by Marggraff Beta rubra) a l l contained a sugar identical with that from sugar cane. About 1799 experimental and promotional work was done by Franz Karl Achard in Germany and by Delessert in France. Mean-while, the development of the white Si les ian beet root, from which a l l modern strains have come, was carried out by M o r i t z von Koppy. 2 . Production The sugar beet is the source of 40 percent of world's sugar. The principal sugar beet growing areas are the U.S.S.R. (European and As ia t i c ) , United States, France, Poland, Germany, Czechoslo-vakia, Canada and the United Kingdom. The main climatic requirements for sugar beet growth are ra in or other frequent application of water during the growing season, mean temperatures near 70°F , and cool dry weather at the end of the season. A succession of warm days and cool nights give the best growth. The plant w i l l grow as long as temperature and water conditions are favorable. A long growing season means higher tonnage« 2 Sugar beets grow in soi ls ranging from deep clay to s i l t loam or sometimes in fine sandy loam or muck soi l s which are alkaline to s l ight ly acid, well drained, and free from hard pan. The crop is very tolerant to sa l in i ty and responds to available phosphorus and to abundant nitrogen. 3 . Gross Morphology The sugar beet, a member of the family Chenopodiaceae, i s a herbaceous, biennial dicotyledon. The mature beet root is an elongate pear-shaped body composed morphologically of three regions - the crown, the neck, and the root. The crown bears a tuft of large succulent leaves and leaf bases. Adjoining the crown, is the neck, which constitutes ontogenetically, the thickened hypo-coty l . The root region, which forms by far the bulk of the beet tissues, is cone shaped and terminates in a slender tap root. The leaves are arranged on the crown i n close spira l with the divergence of 5/13« The lamina of the leaf i s elongate triangular with rounded t ip and undulate margin, the base is cordate and decurrent on the petiole. 4 . Anatomy The anatomy of sugar beet has been described in very great deta i l by Artschwager ( 1 9 2 6 ) . The young seedling root has a central strand of vascular tissue enclosed by a cortex and bounded at the periphery by an epidermis. Secondary growth of the root involves the act iv i ty of a primary cambium and of secondary cambium. The cambium arises apparently as a continuous band, but forms more or less separate bundles, bands of conjunctive parenchyma developing between the vascular s tr ips . The position of each new cambium, as i t arises in the perlcycle, i s such that i t encloses a few layers of per i -cyc l ic c e l l s . These rapidly multiply and build up a parenchym-atous layer as rapidly or even more rapidly than the cambium builds up the vascular layer. Alternate bands of proliferated pericycle and of vascular bundles are thus formed. The bundles are themselves largely parenchymatous. Growth continues through a l l layers - in the bundles apparently both by cambial ac t iv i ty and by prol i ferat ion of the. parenchyma of the xylem and phloem. Thus, the beet root increases in diameter by growth throughout i t s layers. The epidermis of the lamina of the leaf is similar on both surfaces. The stomates are of a simple type. The pores are sur-rounded by a pair of specialized guard c e l l s . There are no acces-sory c e l l s . Stomates are found on both upper and lower surfaces, but are more numerous on the lower. The mesophyll of the leaf is normally divided into palisade tissue and spongy parenchyma. Brovchenko (1965) found that the thin vascular bundles of sugar beet leaves contained 3-k times more sugar than the adjacent assimilating tissues. Thus, movement from the mesophyll to the thin bundles occurred against concentration gradient, which was an indication of the active character of the process. Brovchenko also observed that sucrose synthesis took place along the whole conducting system with the rate of synthesis of the disaccharide being part icularly high in the thin bundles of the leaf blade. He has suggested that part ia l or complete hydro-lys i s of sucrose precedes i t s entrance into the conducting bundles of the sugar beet leaves. 4 II . NITROGEN METABOLISM OF THE SUGAR BEET AS RELATED TO LATE  SEASON GROWTH WHICH RESULTS IN A LOW SUCROSE  CONTENT OF THE ROOT 1 . Introduction Nitrogen i s an essential element for the growth of a l l plants. Plants can u t i l i z e nitrogen sources such as ammonia, n i t r i t e , and amino acids hut in general nitrate w i l l support growth equal to, or better than other sources (Ghosh and Burris , 1 9 5 0 ) . There has been a great increase in the rate of application of nitrogen f e r t i l i z e r s on sugar beets during the last few decades. Associated with increased yields , which follow the use of nitrogen f e r t i l i z e r s , there has been a decline in qual i ty . Processing losses have increased. The depressing effect of high nitrogen nutrit ion on sugar beet quality was reported by Headden as early as 1 9 1 2 . Later studies by Gardner and Robertson (19^2) showed that excessive nitrogen reduced the sugar percentage and the purity . These authors estimated that the reduction in sugar percentage was an approximately l inear function of nitrate nitrogen in the beets at harvest and that each 0 . 0 2 5 percent nitrogen in the beet reduced the sugar percentage by approxi-mately one percent. In his experiments with nutrient solutions, Ulrich (19^2) found that whenever the nitrate content of the petioles was high, the sugar percentage of the beets was lower than in corresponding beets in which the nitrate content was low. When the nitrate 5 content of the beets was high, and the growing conditions favorable, rapid top growth took place, and continued unt i l the nitrogen supply was depleted. Thereafter the growth of the tops, as measured by their fresh weight, decreased gradually while root weights increased rapidly at f i r s t , and then more slowly unt i l the time of the last harvest. He maintained that at the harvest time, the sugar content of the beets would fee a function of growth, leaf area, l ight Intensity, temperatore, etc. Ogden et a l . (1958) found that an excessive application of nitrogen f e r t i l i z e r resulted in a decreased percentage of sucrose and an increased percentage of non-sugar carbohydrates in three different varieties of sugar beets. Several other investigators from different parts of the United States and Canada reported that the excessive nitrogen lowered the sucrose content of the beets (Ulrich, 1 9 5 0 ; Loomis and Ulr ich , 1 9 5 9 ; Tolman and Johnson, 1 9 5 8 ; Stout, 1961 ; Baldwin and Davies, 1 9 6 6 ; Hale and Mi l l er , 1966) Baver (1964) gave an account of the work done in Germany, The studies at the Institute of Sugar Beet Investigations at Gottingen established two rather s ignif icant points. In the f i r s t place, there was an increasing concentration of unasslmi-lated nitrogenous compounds in the beet juice as the amount of nitrogen applied as f e r t i l i z e r was increased. This nitrogen, given the name "harmful nitrogen", consists primarily of amino acids and related compounds. In the second place, increased nitrogen f e r t i l i z a t i o n raised the amount of ash in the juice. Both these factors had a deleterious effect rapon the thin juice purity and the extraction of the sugar. Adam ( i 9 6 0 ) and Tinker (19&5) from Great Br i ta in , -Okanenko (1959) from Russia, Anita et a l . ( 1963) from Romania and Noda Kenji et a l . (1963) from Japan, noted that increase in supply of N gave more vigorous top growth. The shift from root growth to root "ripening" was retarded by high N. Thus, the extensive studies in Europe, United States and Japan on the influence of nitrogenous f e r t i l i z e r s applied to sugar beets have resulted in the general conclusion that excessive quantities of nitrogen depress the sugar content of the beets, delay "ripening" at harvest time, cause excessive top growth, and increase the "harmful nitrogen" content of the beets. To explain the above mentioned facts the folloxtfing sections under-l ine the requirements for growth and the relationships of N-metab-olism to carbohydrate metabolism. 2. Requirements of growth and differentiat ion (a) Ce l l expansion as a process of growth:-(i) changes in water content — The changes in the volume of the c e l l i s due primarily to an increase in water content which may be twenty-fold. ( i i ) changes in the dry weight and the c e l l wall mass — The increase in dry weight of the expanding ce l l s is due primar-i l y to the growth of the wal l . The wall of the meristematic c e l l i s probably composed principal ly of polygalacturonides. Ce l lu -lose becomes incorporated into the primary wall and the propor-tion of this increases with time. The synthesis of polygalac-turonides and of cellulose involves u t i l i z a t i o n of sugars, either stored in the root, as in case of sugar beet, or formed i n the process of photosynthesis. ( i i i ) changes in the protein content. Heyes ( i 9 6 0 ) recorded that in the pea root apex, the protein content increased about five fold during the expansion phase of growth. Protein synthesis requires amino acids and energy (ATP). The production of both is at the expense of stored sugar or newly formed sugar diverted for this purpose. (iv) changes in the nucleic acid content. The RNA con-tent follows closely that of protein. Heyes ( i 9 6 0 ) reported a constant RNA-protein rat io in expanding ce l l s of pea root apex. During expansion, DNA content also increased. Jansen (1956) found that with progressing age of the ce l l s of the root t ip of V ic ia faba an i n i t i a l increase, an intermediate short phase of constant DNA content, and a f ina l stage of increasing content occurred. (v) expansion of the metabolic system of the c e l l . Growth also involves an active metabolic system. This might indeed be expected from the fact of protein increase, but is shown by a variety of determinations of which the most instructive possibly are some on the rate of oxygen uptake. An increase in the rate of respiration has been found in the expanding ce l l s of root and shoot apex (Sanderland and Brown, 1 9 5 6 ) . (b) Ce l l d i f ferent iat ion. In c e l l differentiat ion one can • note:-(i) changes in enzyme ac t iv i t i e s and respiration rates, ( i i ) changes in protein ,and nucleic acid composition. Robinson and Brown (1952) recorded the act iv i ty of invertase, phosphatase and dipeptidase and the protein content in broad bean (Vicia faba) roots for different distances from apex. They found the maximum c e l l volume' and protein content at about 8 mm behind 8 the t i p . After the peak values, protein decreased. The a c t i v i -t ies of invertase and phosphatases follow the protein curve very closely. They increased during the phase of expansion and decreased during that of early maturity. The enzyme ac t iv i t i e s showed that the growing c e l l i s a metabolieally expanding system and that this i s a consequence of greater enzyme ac t iv i ty , a t t r i -buted, at least in part, to an accumulation of protein. The base composition of RNA and DNA from the apex and 8.00 mm from i t , has been determined in pea roots (Robinson and Brown, 1 9 5 2 ) . In DNA there is no change in composition with increase in distance from the apex, the base composition of RNA differed only s l i ght ly . It is suggestive that the material from the older tissue had a higher purine to pyrimidine rat io than that from the younger. The changes in the composition of the RNA suggests a corresponding change in the composition of the protein. 3 . Carbon and nitrogen sources for protein synthesis and growth  of sugar beet leaves The above discussion brings out the fact that for satisfac-tory growth of young leaves, abundant supplies of carbon and nitrogen are required. Joy (1964) pointed out that the young leaves of sugar beet import carbon as photosynthate from older leaves, but become increasingly independent u n t i l at about two-thirds maximum size their own photosynthetic processes are able to supply a l l their requirements. In 1967 Joy further noted that carbon from autonomous photosynthesis and from translocated sucrose is u t i l i zed for synthesis of most amino acids, with a considerable preference for the former source. While much nitrogen arrives in young leaves in inorganic form, an important contribution is also made by glutamine/glutamic acid translocated in the phloem from roots. Using C-^C-2, Joy demonstrated that carbon fixed in autono-mous photosynthesis contributed a greater proportion to the protein increase than to the insoluble carbohydrate increase in younger leaves. When sucrose from older leaves is available there is a tendency for i t to contribute more carbon to Insoluble carbo-hydrate than to protein. Labelled sucrose was supplied direct ly to roots of beet seedlings, and after 18 hours various parts of the seedlings were analyzed (Joy, 1 9 6 7 ) . Radio carbon had been translocated and greatest ac t iv i ty was found in the youngest leaves. Glutamic acid was the predominantly labelled amino acid in both root and leaf . In young leaf protein glutamic acid was again the main labelled componentc Joy (1967) concluded that fibrous roots must therefore synthesize considerable amounts of glutamic acid (or glutamine) from the sucrose, normally translocated to them from mature leaves, and sucrose carbon is then re-exported ih this form to young leaves. Siskajan and co-workers in Russia (19^8, 1 9 5 1 , 1952) made a very different approach to the study of the metabolic changes in the sugar beet, especially with respect to sucrose storage. Their investigations have concentrated on change in the act iv i ty of leucoplasts extracted from roots at various stages of develop-ment. They regard the leucoplasts as constituting a depot of absorbed enzymes which can enter the metabolism of the c e l l when this is cal led for by the ontogenetic development of the plant. Leucoplasts in autolysing tissue liberated much invertase. 10 In storage roots of sugar beet, the leucoplasts become progres-sively less dense and less granular, but this is associated with decreasing invertase a c t i v i t y . During the vegetative period of the sugar beet plant, the invertase act iv i ty of leucoplasts in the root increased greatly, concurrently with a decrease in the invertase ac t iv i ty of the chloroplasts of leaves. This implies that when growth of new leaves or new fibrous roots i s going on, stored sucrose i s hydrolyzed to supply the carbon skeleton of the proteins and the insoluble carbohydrates. • r 4 . . Interrelationships of nitrogen, metabolism and carbohydrate  metabolism Nitrate is the principal source of nitrogen u t i l i z ed by higher plants. Nitrate uptake and metabolism require energy derived at the expense of sugar accumulation. (a) Nitrate uptake. Energy is required for the intake of nitrate into the roots, where Its concentration i s usually many times greater than in the s o i l solution (Lundergardh, 1 9 5 0 ) * The energy for this accumulation of ions against a concentration gradient is derived from the respiration of stored food, p r i n c i -pally sugar. (b) Nitrate reduction. 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 nitrate reductase (NRase). NRase was isolated from soya bean and Neurospora by Evans and Nason (1953) and later from many plants including bacteria, algae, fungi, and higher plants. Nitrate reductase from sugar beet was isolated 11 by Yang ( 1 9 6 4 ) . This enzyme system ut i l i ze s a reduced pyridine nucleotide as the H-donor and contains f lav in adenine dinucleo-tide (FAD) as the prosthetic group. Molybdenum has been shown as an essential,component of the nitrate reductase system (Nicholas and Nason, 195^; Nicholas et a l . 1 9 5 4 ) . The sulfhydryl nature of the enzyme was indicated by the reversal of p-chloromercuri-benzoate inhibi t ion by the sulfhydryl reagents glutathione and cysteine, and also by the strong inhibit ion by iodoacetate and cupric sulfate (Nicholas and Nason, 195^; Nicholas et a l . 195^? Yang, 1 9 6 4 ) . Enzyme systems capable of catalyzing the reduction of n i -t r i t e have been prepared from both Neurospora and soybean (Nason and Evans, 195^)• Medina and Nicholas (1957) demonstrated in Neurospora that hyponitrite is a product of n i t r i t e reduction. The n i t r i t e reductase of Neurospora is an NADPH2 - dependent flavoprotein containing FAD, Fe, and Cu. Copper may be involved in the transfer of electrons from reduced FAD to n i t r i t e ; the role of iron is unknown. An enzyme has been found in Neurospora that w i l l catalyze the conversion of hyponitrite to hydroxylamine (Medina and Nicholas, 1957 ) • Frear and Burel l (1955) have shown that plant preparations are capable of reducing labelled hyponitrite to ammonia. Studies with Neurospora extracts indicate that iron and copper are required for the act iv i ty of hyponitrite reductase (Medina and Nicholas, 1 9 5 7 ) . The f i n a l step in the sequence is the conversion of hydroxyl-amine to ammonia. The enzyme catalyzing this reaction hydroxyl-amine reductase, has been found in Neurospora by Zucker and Nason 12 (1955) and in higher plants by Frear and Burel l ( 1 9 5 5 ) . Mn + + has been found to be associated with hydroxylamine reductase. The overal l reaction for the reduction of nitrate may be represented: Mo „ Fe, Cu Fe, Cu Mn N0 3 - N0 2 - i -L—> HNO ! » NH2OH » NH3 +5 +3 +1 - 1 - 3 Nitrate Ni tr i te Hyponitrite Hydroxylamine Ammonia The above reaction sequence Involves 2-electron changes at each step. It means that before nitrate can be incorporated into usable compounds, i t requires eight hydrogen ions for each nitrate ion. The energy i s derived at the expense of sugar accumulation, whether i t i s from the direct metabolism of sugar or from the photolysis of water. 5 . Nitrate reduction in re lat ion to respiration and photosynthesis The main sources of reducing power in intact plants are the respiratory breakdown of carbohydrate and the photolysis of water in the chloroplasts. It i s known that the ultimate H-donors in the i n i t i a l step and in the subsequent steps of nitrate reduction are the reduced pyridine nucleotides. This would be true whether nitrate reduction were coupled to respiration or to photolysis of water. A number of enzymes involved in plant respiration such as glucose-6-phosphate dehydrogenase, malic dehydrogenase, glutamic dehydrogenase, glyceraldehyde phosphate dehydrogenase and i s o c i t r i c acid dehydrogenase are known to be linked to p y r i -dine nucleotides. The oxidative reactions catalyzed by these enzymes generate reduced pyridine nucleotides, and in this way 13 the energy for the reduction of nitrate is derived direct ly from the oxidative breakdown of carbohydrates. In this respect, many investigators have observed that under conditions of high nitrate reduction and assimilation in the dark, carbohydrate levels in the plant are s ignif icantly lowered. Lower sucrose percentage and high "harmful nitrogen" and continued growth are the common observation in the sugar beet under the high nitrate f e r t i l i z a t i o n in the f i e l d . The decrease of carbohydrate content of the ce l l s can be explained on the bases of nitrate reduction and incorpora-t ion into amino acids and protein. In photosynthetic tissues in the l ight , another potent source of reducing power is available, i . e . , the formation of reduced NADP, Thus, Evans and Nason (1953) showed that the combination of NRase and grana from soybean leaves could reduce nitrate to n i t r i t e in l ight i f catalytic amounts of NADP were present. Ramirex et a l . (1964) reported the findings of non-cyclic photosynthetic electron flow, in which f lav in nucleotides mediate the direct transfer of electrons from illuminated grana to nitrate with the aid of NRase. Yang (1964) found the coupling of photoreduction of FAD with the reduction of nitrate by NRase and suggested the probability of participation of NRase in a f lav in nucleotide - catalyzed enzymatic photoreduction of n i trate . When nitrate reduction is coupled to respiration, the n i t -rate replaces oxygen as the terminal oxidant or H-acceptor. Extra carbohydrate must be broken down to provide energy for the reduction and extra carbon dioxide is released without a corre-sponding increase in 02 consumption, i . e . , R. Q. increases. Thus, i t was observed by Hamner (1935) that nitrogen deficient plants had large carbohydrate reserves. When they were supplied 14 with nitrate they exhibited an increased rate of carbon dioxide production (measured in the dark), a depletion of carbohydrate reserves, and the temporary formation of n i t r i t e in the leaves. Cramer and Myers (1948) showed that nitrate reduction in the dark by chlorel la in a glucose medium is accompanied by an increase in R. Q. from 1.2 to 1.6 caused by an increased rate of carbon dioxide production. During photosynthesis at low l ight intensity, nitrate u t i l i z a t i o n in this organism is accompanied by a decrease in assimilation quotient from 0.9 to 0.7 owing to the decreased C0£ uptake (Van Niel et a l . 1953). Under these conditions presumably carbon dioxide and nitrate are alternative acceptors of the reduced H-carriers generated by the photolysis of viater. Evidence, then, suggests that photosynthesis and respira-t ion supply the necessary reducing power for nitrate reduction. Photosynthesis, however, plays i t s part in a l l probability through i t s photosynthates via respiration (Beevers et a l . 1964). In sugar beet, the formation of NADH2 for the reduction of nitrate to ammonia and the depletion of sugars for i t , can be summarized as follows: Sucrose C02 present in tracheids photosynthesis Sucrose stored in ^ - p , — i n v e r t a s e > Glucose Respiration NADH2 or NADPH2 root / * glycolysis Fructose Krebs cycle Sucrose formed during N O 3 -photosynthesis _ Ammonia -r 15 (c) The u t i l i z a t i o n of ammonia The ammonia formed "by the reduction of nitrate enters into organic combination by three main reactions: (i) as an oc-amino group ( i i ) as the amide group of asparagine and glutamine ( i l l ) as carbamyl phosphate, an intermediate In the synthesis of c i t r u l l i n e and pyrimld.lnes. Formation of amino acids Knoop and Osterl in ( 1925) postulated the formation of amino acids from ct-keto acids and ammonia by reductive amination. In 1937 Gregory and Sen discussed the interrelationship of carbohydrate and nitrogen metabolism. They regarded some carbon from carbohydrate as entering into the constitution of protein. Krebs and Johnson (1937) suggested that the c i t r i c acid cycle plays a major role in carbohydrate oxidation in animals. Chibnall (1939) realized i t s importance and suggested that the c i t r i c acid cycle occupies the central and key position, in the carbohydrate, protein, and fat metabolism of plant c e l l s . The importance of the Krebs cycle was further emphasized when Euler and Heyman (1933)showed that the dihydrocodehydrase reduces iminoglutaric acid to glutamic ac id . Thereafter the following reactions have been proposed by which ammonia, derived from nitrate can form the <-amino group of the amino ac id . (i) Fumarate + NH^ ^ aspartate This reaction is catalyzed by the enzyme aspartase which is present in bacteria, but i t s presence in higher plants is doubt-f u l . 16 ( i i ) Pyruvate + NH3 + NADH2 , ^ alanine + NAD +.H 20 This reaction has been demonstrated in bacteria and l iver mito-chondria. ( i i i ) c£ - K e t o g l u t a r a t e + NH3 + NADH2 r > glutamate + NAD + H 2 0 This reaction is generally regarded as quantitatively the most important reaction. Evidence to support this view has been provided in the case of Torulopsis u t l l i s (Folkes, 1 9 5 9 ) • The reaction is reversible and proceeds in two steps. oC-Keto ©C-imino NADH? glutarate + NH3 ^—* glutarate „ c glutamate + NAD glutamic \ dehydrogenase The f i r s t step probably proceeds spontaneously, but the second reaction i s catalyzed by the enzyme glutamic dehydrogenase and requires the presence of reduced NAD. Because of the central importance of glutamate in the synthesis of other amino acids and because of the higher proportion of glutamate formed in this manner by the plant, this reaction represents the "port of entry" into the metabolic system for inorganic nitrogen. However, i t has been suggested by Smith et a l . (1961) that the synthesis of alanine during photosynthesis does not involve transamination from glutamic ac id . It is suggested by the authors that alanine is formed by the reductive amination of phosphenol pyruvate. The discovery of transamination, i . e . , the transfer of the amino group from an amino acid to a keto acid, by Braunstein and Kritzman (1937) also placed the keto acids in a position such that they might receive amino groups from another source and produce both primary and secondary amino acids. The cofactor of transaminases is pyridoxal-5-phosphate or pyridoxamlne-5-17 phosphate, which is t ight ly bound to the enzyme but may be, at least partly, removed during precipitation of the protein with ammonium sulfate. A survey of transaminase act iv i ty in various plants has been made by Leonard and Burrls (19^7)• They found that the transamination rate per unit of tissue decreased with age in the growing plant. Wilson et a l . (195^)» have used C-^-dC-ketogluta-rate to study transamination in plants. Although transamination reactions involving glutamic acid are by far the most prevalent In the plant, other transamination reactions have been found. For example, transamination reactions involving aspartic acid and alanine has been found in higher plants (Wilson et a l . 1 9 5 4 ) . It is generally accepted now that reductive amination of ©t-keto acids followed by transamination is a major pathway of amino acid formation. These processes account for formation of the dicarboxyl amino acids, and of alanine from which a l l the other naturally occurring amino acids except proline and hydroxy-proline may be derived. The formation of amides:— These compounds are present in plants as part of protein and as soluble amides. Glutamine synthetase, requiring ATP for i t s operation, Is responsible for the amidation of glutamic acid and has been extracted and pur i -f ied from tissue of higher plants ( E l l i o t , 1 9 5 3 ; Varner and Webster, 1955)« The synthesis proceeds by the overall reaction: glutamate + ATP + NH3 glutamine + ADP + Pi The synthesis of asparagine probably occurs by reactions similar to those involved in glutamine synthesis, aspartate + ATP + NH3 ^ asparagine + ADP + Pi 18 An asparagine synthetase has been isolated and purified from lupine seedlings (Webster and Varner, 1 9 5 5 ) * Joy (1967) suggested that in sugar beets, glutamine is synthesized mainly in the roots and the carbon source is sucrose. Glutamine was also found to be present in the tracheids. Perhaps glutamine or glutamate is transported from the roots to the leaves. The lower sucrose content in the root along with increased leaf area and increased amino acids with added nitrogen, suggested that in the sugar beet plant the synthesis and metabolism of sucrose are influenced by available nitrogen. To test this poss-i b i l i t y , Snyder and Tolbert (1966) exposed mature sugar beet plants 14 grown on a complete nutrient solution, to C 0 2 in sunlight. After one hour of photosynthesis, the blades of the - N plants had 14 a s ignif icantly greater percentage of C in sucrose and smaller percentage in c i t r i c , malic, and amino acids than the blades of the + N plants. After 24 hours, the - N plants retained approx-imately 40$ less in the blades and proportionately more in roots than the + N plants. The foots of the - N plants contained 39$ of the C i / + while those of the + N plants contained only 1 9 $ . Of the retained in the blades of the - N plants, 36$ resided in sucrose and 21$ in c i t r i c plus malic plus amino acids, whereas 14 the blades of the + N plants contained 16$ of the C in sucrose and 4 4 $ in c i t r i c , malic, and the amino acids. The above data suggest that plants having a continuous and adequate supply of nitrogen may preferential ly snythesize the c i t r i c acid cycle products and their amino acid counterparts and thus produce less sucrose. Also with adequate nitrogen, 19 photosynthetic products may be channelled preferentially into new growth at the expense of sucrose being synthesized and trans-ported to the root (Fig. 1 ) . Protein synthesis - The current concept of protein biosyn-thesis has been summarized by Davies, Giovanelli and AP Rees (1964) as follows: (1) Activation of amino acid Enzyme + amino acid + ATP T * Enz - AMP - AA + PPi (2) Formation of aminoacyl - RNA • Enz' - AMP - AA + s-RNA <c > AA - s-RNA + AMP + Enz (3) Formation of peptide bonds n (AA - s-RNA) + ribosome ^ * polypeptide on ribosome + n (s-RNA) (4) Release of polypeptide Polypetide on ribosome > polypeptide + ribosome This scheme indicates that for protein synthesis amino acids and ATP are required. Since respiration furnishes the stores of usable energy (as ATP), i t is to be expected the resp i -rat ion w i l l be intimately connected to protein synthesis. Since the Krebs cycle can operate as a route.of synthesis, producing keto acids as "ports of entry" for nitrogen, respiration is envolved here also as a means of mobilizing carbon from carbohy-drate metabolism. Formation of nucleotides for nucleic acids - In an earl ier section, the work of Heyes (1960)was quoted in which he found that the RNA content of the growing c e l l closely followed that of protein. Steward and Durzan (1965) drex-r attention to the similar fact that agents l ike coconut milk which promote growth and protein synthesis do so, in part, because they.affect the (6) Sucrose- >Fructose + glucose Photosynthesis Fructose diphosphate Glycolysis Pyruvate Sucrose phosphate (5) Fructose-6-phosphate * (1) Fructose Glucose-6- Glucose-1-'phosphate ^—* phosphate (2) Glucose (3) Glucose -y Citrate -> Ketoglutarate ENZYMES 1 . Fructose-6-phosphatase 2 . Glucose-6-phosphatase 3 . Glucose-l-phosphatase h.UDPG-pyrophosphorylase 5 . Sucrose-P synthetase 6 . Invertase 7 . Nitrate reductase 8. Transaminase Uridine diphosphate glucose CO Uridine triphosphate -NH^ N 0 2 1 --» Glutamic acid • (8) Other amino acids I Protein F i g . 1. Interrelationships of Nitrogen Metabolism and Carbohydrate Metabolism (7) ' NO3-o ,21 effectiveness of the RNA in those ce l l s for protein synthesis. The formation of pyrimidine bases involves ATP, aspartic ac id , NH3»C02 and phosphoribosyl pyrophosphate. The synthesis of nucleotides containing adenine bases involves amino acids, for example, glycine, glutamine and aspartic acid, and also CO2, formate and phosphoribosyl pyrophosphate. The ultimate source of carbon for amino acids, ATP and phosphoribosyl pyrophosphate are the products of carbohydrate metabolism. Thus, increase in nucleic acid content of the growing ce l l s w i l l again involve the depletion of sucrose in sugar beet. v The above discussion outlines the main causes of the low sugar content in roots of sugar beets under continued supply of nitrate f e r t i l i z e r . The following tabulation w i l l summarize the events from nitrate uptake to i t s assimilation, and also the involvement of carbohydrate metabolism. 22 Physiological process Depletion of sucrose or diversion of sucrose synthe-slzlng system provides:  {1) Nitrate uptake (2) Nitrate reduction to ammonia (a) Nitrate to n i t r i t e (b) Nitrate to hyponitrite (c) Hyponitrite to hydroxyl-amine (d) Hydroxylamine to ammonia Usable energy as ATP Reduced NAD (or NADP) Reduced NAD Reduced NAD Reduced NAD (3) Formation of amino acids (a) Reductive amination (b) Transamination (4) Formation of amides (5) Protein synthesis (6) Formation of nucleotides (a) Pyrimidine containing nucleotides (b) Purine containing nucleotides Carbon skeleton as keto acids, and reduced NAD Carbon skeleton as keto acids Carbon skeleton as amino acids, and usable energy as ATP Carbon skeleton as amino acids Carbon skeleton as aspartate, phosphoribosyl pyrophos-phate, and usable energy as ATP Carbon skeleton as glycine, formate, phosphoribosyl pyrophosphate, and usable energy as ATP 23 III CONTROL OF GROWTH IN SUGAR BEET. The growth and development of sugar beet plants appear to be closely related to the sucrose economy of the beet plants, namely sucrose formation and u t i l i z a t i o n . An adequate supply of nitrogen stimulates growth of new leaves and fibrous roots. The conversion of large amounts of sugar to top and root growth would result in a storage root with a low sucrose concentration. The inverse relationship between the nitrogen status of the plant and the sucrose concentration of the beet root has been observed many times and has led to the suggestion that for a I. period before harvest, nitrate supply should be greatly reduced to prevent the reinvestment of sucrose in the production of surplus foliage and fibrous roots. The following discussion w i l l give some of the prevalent cultural practices to reduce the nitrogen supply at the time of "ripening" of sugar beet. It w i l l also present the outline of the more re l iable and definite control of growth by the use of metabolic inhibi tors . 1. Cultural practices (a) The use of optimum amount of n i trate : - This method was advocated by Ulrich ( 1 9 ^ 2 ) . The method generally used is to estimate the optimum amount of nitrogen to apply earl ier in the season so that nitrate w i l l be depleted to the proper extent in the root zone at the right time. In an average year i t is assumed that 80 to 100.pounds per acre of actual nitrogen w i l l be u t i l i zed by the crop and the beets w i l l run out of nitrogen by the end of growing season. 24 (b) The c r i t i c a l level of nitrate ferti l izer:—• Ulrich (1950) found that nitrate nitrogen of the petioles of the sugar beet leaves inversely correlated to the sucrose content and could be used for the estimation of c r i t i c a l level of nitrate f e r t i l -izer in the f i e l d . Thus, the method being used is to test the nitrate nitrogen content in the beet petiole and when i t drops below 1000 ppm, wait three weeks and then harvest. This method works f a i r l y well in areas where there are long harvest seasons. (c) Smaller beets:— Loomis and Ulrich (1959) investigated the response of sugar beets to nitrogen depletion in re lat ion to root s i z e . v Starting from a high nitrogen status, small beets increased faster in sucrose concentration with the onset of nitrogen deficiency than did large beets. The authors suggested that u n t i l sugar beet varieties are available that w i l l "ripen" naturally to a high sucrose concentration under high nitrogen conditions, i t may be possible for the grower to take advantage of the knowledge that small beets respond more readily than large beets to changes in nitrogen status. It would not be practicable for a grower to reduce the mean root size by delaying planting date. In that case two poss ib i l i t i e s remain: (1) reduce the average plant spacing, thus increasing the plant population, and ( 2 ) alter the length of the period of nitrogen deficiency prior to harvest (Loomis and Ulr ich , 1 9 5 9 ) * (d) N- fer t i l i za t ion and date of planting:—Schmehl et a l . (1963) determined that interactions of the rate of nitrogen f e r t i l i z a t i o n , date of planting, and plant spacing in the row, on yie ld and quality of the beet. They found that early planted beets produced higher yields and more sucrose than late planted 25 beets. They suggested the need to adjust the rate of nitrogen f e r t i l i z a t i o n with date of planting i n climatic areas where the harvest date cannot be extended. (e) Irrigat ion schedules:—Wolley and Bennet (1Q62) found that the use of moderate amounts of nitrogen f e r t i l i z e r with an i rr igat ion schedule that allowed the s o i l moisture to be main-tained near f i e l d capacity produced the highest y ie ld of roots and sucrose. The authors believed that for any given i rr igat ion regime there is a nitrogen level best calculated to give maximum sugar production. (f) Redistribution of nitrate in the soi l :— Stout (1961, 1964) drew attention to the fact that the nitrate ion moves very freely with moisture in so i l s . He suggested that according to the relat ive pattern of distr ibut ion of n i trate , one can develop cultural practices to help nature put the nitrate where the plants cannot get i t . For example, planting beets so close as to give enough fo l i ar protection to the s o i l near the root zone in case of ra in before the harvest, avoiding sprinkler i rr iga t ion , and the use of some product which might reduce the mobility of nitrogen nutrients either by reducing the rate of n i t r i f i c a t i o n of ammonium salts or by reducing so lubi l i ty of nitrate by incorporating i t into more slowly soluble forms. Stout (1964) further suggested that any supplemental nitrate added to row crops after the f i r s t i rr iga t ion should be placed below the bottom of i rr igat ion furrows in order to lengthen the period of a v a i l -a b i l i t y to plants before i t reaches the dry surface layer of s o i l . 26 2. Selection and the breeding of the new variet ies Payne et a l . (1961) suggested the poss ib i l i ty of improving the quality of sugar beet by plant breeding In association with f e r t i l i z e r practices. In their chemical genetic studies,the above authors found that genetic v a r i a b i l i t y was associated with total nitrogen, betaine and glutamic ac id . On the bases of their data, they claimed that increases in percentage sucrose can be obtained on high f e r t i l i t y soi ls by breeding populations of sugar beets adapted to growing under these conditions. They found two hybrids which were capable of producing high percentage sucrose at higher f e r t i l i t y levels . 3 • Disadvantages of the prevalent methods The above outline methods are common i n sugar beet f ie lds but they have some obvious disadvantages. The major disadvantage of the use of optimum amount of N- fer t i l i zer i s the dependency on 'average' weather condition to ensure complete use of nitrogen. Tolman ( i 9 6 0 ) very well points out another disadvantage of this method. The greatly increased number of micro-organisms competes with the beet for the available nitrate when the beets need i t most. Late in the summer, the micro-organisms begin to die and release nitrate at a time when the supply to the beets should be greatly reduced. The disadvantage of the method of estimation of c r i t i c a l level of nitrate in the petioles of the beet, is the gradual sup-plying of nitrate from the s o i l in the areas where there are short growing seasons and there is no cutting off this nitrate supply (Hussel, 1 9 6 5 ) . 27 Stout (1964) studied the nitrate content of i rr igat ion and drainage waters and found that, under normal conditions, i r r i g a -t ion practices for leaching of nitrate from soi ls have been over-estimated. The breeding of a new variety is the best answer to the problem, but the process is very slow. In the sugar beet, the breeding on the l ine of Vilmorin takes at least 8-10 years. And also the performance and s tab i l i ty of a new variety depend on the variables l ike climatic conditions etc. Thus, i t i s very d i f f i c u l t or Impossible to control the depletion of sucrose by the above mentioned methods under the conditions of high nitrogen f e r t i l i z a t i o n , because uncertainty i s always associated with them. 4 . Chemical control of growth in sugar beet Alternatives to the foregoing methods might involve the control of growth of the sugar beets at the time of "ripening" (two to three weeks before the harvest) of the roots by the use of metabolic inhibitors or growth regulators. In plants, four types of growth regulators have been recog-nized: auxins, gibberel l ins, kinins and inhibitors . The term auxin includes two types of materials: the growth hormones, which are natural plant constituents and which regulate c e l l enlarge-ment in the manner of indoleacetic acid, and synthetic materials, which can also stimulate c e l l enlargement in the manner of indoleacetic ac id . The gibberell ins also regulate growth, but through a type of action which is dist inct ive in the sense that they do not 28 stimulate growth of roots, and their translocation is not in a polar fashion. The kinins regulate growth, at least in part, by stimulating c e l l d iv i s ion . It seems l i k e l y , however, that gibberell ins and kinins require the presence of auxin for their growth effects. The inhibitors include a wide array of chemical entit ies which may inhibit growth or developmental functions or may inhib-i t some component reactions relat ing to the growth regulators. Examples of natural inhibitors are the various phenolic compounds, such as benzoic acid, cinnamlc acid, chlorogenic acid, caffeic acid, p-coumaric acids, etc. The synthetic inhibitors are compounds l ike maleic hydrazide, aminotriazole, cycocel and various metallic ions. A depression of growth and the control of the depletion of sucrose can result from a great variety of inhibitory mechanisms in sugar beets. Some of the l i k e l y mechanisms are as follows: (a) Inhibition of systems important for the synthesis of precursors necessary for a process. For example, (i) inhibit ion of nitrate reductase, the enzyme responsible for the reduction of nitrate to n i t r i t e which on further reduction gives r i se to ammonia required for amino acid synthesis (hence protein synthe-s i s ) , ( i i ) inhibit ion of transaminases, the systems necessary for the synthesis of most of the amino acids. ( i i i ) inhibit ion of 3-d- eo xy-D- arabinoheptulosonic acid 7-phosphate (DAHP) synthe-tase, the f i r s t enzyme of the shikimic acid pathway for the syn-thesis of aromatic amino acids. (b) Inhibition of the systems which supply the substrates for respiration; e .g. , inhibit ion of invertase act iv i ty which 29 hydrolyzes sucrose to glucose and frutose and makes available the hexoses for u t i l i z a t i o n in respirat ion. (c) Inhibition of respiratory pathways and energy genera-ting system. Intermediates of the Krebs cycle may be Involved in the synthesis of l i p i d s , proteins, nucleic acids etc. Respira-tion also provides usable energy as ATP for the synthetic reactions and also the reduced coenzymes for nitrate reduction and reductive amination. (d) Inhibition of synthesis of some growth regulators, e .g . , inhibi t ion of tryptothan synthetase which w i l l result in the inhibi t ion of the synthesis of indoleacetic ac id . (e) Inhibition of some steps in protein or nucleic acid synthesis. For example, use of chloramphenicol which binds with 50S ribosomal part ic le and inhibits the protein synthesis; Actinomycin D which binds to guanine in the minor groove of DNA and blocks RNA synthesis. (f) Incorporation of an abnormal analogue into a synthetic pathway resulting in a metabolic block i f one of the abnormal substances formed cannot undergo further metabolism or is unable to function as i t s normal analogue. For example, use of 5-bromo-u r a c i l , which replaces normal bases, causing H-bonding errors and mistakes in incorporation and repl icat ion of DNA. The result is the change in codon for amino acids. Similarly , the use of benzimidazole or 2-aminopurine which cause mistakes in incorpo-ration of adenine. (g) Direct disturbance of the mitotic sequence (such as inhibit ion of spindle formation, or of protoplasmic movements with cleavage or the breakdown of chromosomes e .g. , by maleic 30 hydrazide). (h) Inhibition of auxin ac t iv i ty , e .g. , the use of an ant i -auxin such as p-phenylbutyric acid, trans-cinnamic acid or maleic hydrazide. The present investigation is an attempt to control the growth of sugar beet at the time of "ripening" of roots by the use of metabolic inhibitors or growth regulators. It i s consis-tent with the discussion presented in the preceding sections, that the growth and development of plants are the products of the metabolic systems mediated by the enzymes. It is also consistent with the fact that the enzyme act iv i ty can be affected by inter-action with a small molecule •— inhibitor or act ivator. Since nitrate is considered to be the prime source of nitrogen available to sugar beet for continued growth, emphasis has been given the use of inhibitors of nitrate reductase. Nitrate reductase has a major role in regulating nitrogen meta-bolism in plants. Its regulatory nature is evident in the sense that i t i s (a) the f i r s t enzyme in the pathway of nitrate reduc-tion; (b) inducible by substrate ( N O 3 - ) (Beevers et a l . 1 9 6 5 ) ; l ab i l e in vivo under environmental stress (Mattas and Paul i , 1 9 6 5 ) » (c) variable in level diurnally (Yang, 1 9 6 4 ) ; (d) sensi-tive to the inhibitors of protein synthesis e.g. , cycloheximide (Shrader and Hageman, 1 9 6 7 ) ; (e) index of the total protein producing potential of the grain in wheat (Croy, 1 9 6 7 ) ; (f) and regulated by hormones as indicated by the d i f ferent ia l affects of 2 ,4 -D on mono- and dicotyledonous plants (Beevers et a l . 1 9 6 5 ) . The following other inhibitors have also been used: Benzimidazole - A synthetic analogue of adenine and found to 31 inhibi t the growth of the plants (Rebstock et. 1955)* Chloramphenicol - Inhibits protein synthesis and auxin-induced growth in plants (Nooden and Thimann, 1 9 6 5 ) . Caffeic acid - A phenolic acid and naturally occurring growth inhibitor in plants (Leopold, 1964) Cycocel (2-chloroethyl trlmethylammonium chloride) - is a growth retarding chemical which exerts i t s influence on plant growth processes through the enhancement of auxin destruction (Kuralshi and Muir, 1 9 6 3 ) . Maleic hydrazide - Inhibits growth of the plants by reacting with several plant constituents (Leopold and Kle in , 1 9 6 2 ; Nooden, 1 9 6 7 ) . The sequence of steps in the overall Investigation is as follows: (1) Various inhibitors were applied to the leaves of one-month-old plants. The inhibit ion of growth has been determined by measuring the leaf area, seven days after treatment. (2) The effect of the three most effective inhibitors of the growth of leaves of one-month-old plants were determined, 7 , 14, and 21 days after the treatment of 4.5-month-old plants on: (a) the growth of the leaves (b) the chemical composition of the roots (3) To explain the observed effects on the growth of leaves and the changes in the chemical composition of the roots due to the treatments, effects on metabolic processes have been determined. 32 IV MATERIAL AND METHODS 1. Growth of the plants Beta saccharifera seed, S .K.E. .R-11 , obtained from the Br i t i sh Columbia Sugar Refining Co. , Vancouver, B . C . , was sown in wooden f lats f i l l e d with vermiculite. They were watered once every day with nutrient solution containing 0 .005 M C a ( N 0 3 ) 2 , 0 . 0 0 0 5 M KH2P0^, 0.002 M MgSO ,^ 0 .05 M KNO3, 0 . 5 ppm Fe as FeEDTA, 0.04 ppm of' Cu as CuSO^, 0 .25 PPm of Mo as Na2Mo^, 2 . 0 ppm of boron as Na^BO .^, 0.02 ppm of Zn as ZnSOj^  and 0 . 5 ppm of Mn as MnCl 2 . When the seedlings were nine days old (early 2-leaf stage), they were transplanted to t in cans of 5 " diameter containing vermiculite or to one-gallon crocks containing sandy loam. The plants were grown in either a controlled environment room in a greenhouse at the University of Br i t i sh Columbia. The conditions in the controlled environment room were: photo-period, 16 hours; l ight intensity 1800 foot-candles at the top of the plants; temperature, day 21-26°C, night 18-22°C and relat ive humidity, day 62-70$, night 65-80$. These conditions are desig-nated as "summer" growth conditions. Three series of growth room experiments were conducted: the f i r s t , in which the effects of various chemicals on the expansion of leaves of one-month-old plants grown in vermiculite in 5 " diameter t in cans, were determined; the second, in which the effects of maleic hydrazide, pyrocatechol and vanadium sulfate (the three most effective inhibitors of leaf expansion of one-month-old plants) on the inhibi t ion of leaf expansion of 4 . 5 -month-old plants grown in one-gallon crocks, were determined; and 33 the third, in which the effects of maleic hydrazide, pyrocatechol and vanadium sulfate on the photosynthesis and respiration of 4.5-month-old plants were determined. Three series of greenhouse experiments were conducted with 4.5-month-old plants to f ind out the effects of maleic hydrazide, pyrocatechol and vanadium sulfate on (1) the nitrogenous constit-uents of the roots and on the respiration of the root discs; (2) the hydrolytic enzymes of roots and leaves; (3) the reducing sugar and sucrose percentage of the roots, and the enzymes of nitrogen metabolism and of sucrose synthesis. In three series of growth room experiments, 4.5-month-old plants were transferred to a controlled environment room with the following conditions; photoperiod 12 hours; l ight intensity at the top of the plants 1800 foot-candles; temperature day l 4 - l 6 ° C , night 5-7°C; and relat ive humidity, day 5 5 - 7 0 $ , night 75-80$. These conditions are designated as "fa l l" growth conditions. The effects of maleic hydrazide, pyrocatechol and vanadium sul-fate under the f a l l conditions on (1) leaf area; (2) sucrose and reducing sugar percentage and respiration of root discs and (3) photosynthesis and respiration of whole plants were determined. 2. Mode of application of chemicals The aqueous solutions of the inhibitors were sprayed to wet the leaf surfaces of the sugar beet plants. Vanadium compounds were applied, however, with 0.2$ Tween 20 (a wetting agent). 3 • Determination of leaf area Leaf area was determined by the use of the formula BL x BW/ 34 1.645. Here BL = blade length from the base of the blade to the t ip and BW = maximum blade width. The factor 1.645 was obtained by a preliminary experiment to account for the difference of the actual area of the blade from the value obtained by BL x BW. A grid-dot sheet was used to determine the actual area (BA) of the tracings of the blades of 7 leaves from each of 7 plants. The maximum width (BW) and length (BL) of each of the leaf blades were measured, and BW x BL calculated. The formula BW x BL/BA was used to obtain the correction factor which could be employed to calculate blade area from BW and BL measurement. Plant A B C D E F G Average true area (cm )^ 5 5 . 5 1 7 6 . 4 7 7 2 . 1 2 33.14 4 2 . 7 5 6 7 . 3 3 3 9 . 3 7 Average of BL x BW (cm )^ 85.14 1 2 6 . 6 9 1 2 3 . 6 8 5 2 . 0 6 7 4 . 5 9 1 1 3 . 4 5 6 2 . 9 0 BL X BW/BA 1 . 5 4 5 1 . 6 5 7 1 .715 1 .571 1 . 7 4 5 1 . 6 8 5 1 . 5 9 8 Average of BL x BW/BA 1.645 of 49 leaves from 7 plants  To test the accuracy of the method, the area of a leaf taken from each of 10 different plants was determined by the grid-dot sheet method and also by the formula. The following data show the correspondence of area values obtained by the two methods. 35 BA BL z B W / 1 > 6 ^ 5 Difference % difference (cm r^y (cm2) (9) (10) (l) (2) 43.24 6 1 . 9 2 4 7 . 2 0 20.48 I 8 . 5 6 38.36 69.23 24.96 48.12 62.24 41.91 6 3 . 0 1 4 5 . 1 2 I 8 . 8 5 19.95 37.20 71.80 26.37 45.48 59.^1 1 . 3 3 2 . 1 9 2.08 I . 6 3 1 . 3 9 1 .16 2 . 6 7 1.41 3.64 2 . 8 3 3.1 3.5 4 . 5 7 .8 6.9 2.9 3 .8 5.7 4.4 Avg. 5.63 2 f . Determination of the chemical composition of the root Preparation of the samples The beets were trimmed of leaves and small roots, washed, and the crowns removed. The entire remaining portion was finely-chopped and blended in a Waring blendor for 3-4 minutes. Three 50-gram aliquots of the blended material were used for sucrose and reducing sugars determinations, and another three 50-gram samples were heated at 110°C for five minutes to stop enzymatic changes, dried at 85°C to a constant weight, and then ground, redried and stored over calcium chloride in desiccators. (a) Determination of reducing sugars and sucrose Preparation of the extract Fi f ty grams of the freshly blended beet root was added to 250 ml boil ing 95$ ethanol in a 600 ml beaker, boiled for 10 minutes on a water bath, and then cooled. The l iqu id was f i l -tered through dry f i l t e r paper into a 500 ml volumetric f lask. 36 The residue was covered with 80$ ethanol, heated gently for 30 minutes on a water bath and then allowed to cool to room temper-ature. The l i q u i d was f i l t ered into the flask containing the f i r s t extract. This process was repeated eight times for two days. After the f i n a l addition of the l iqu id through the f i l t e r , the volume was made to 500 ml with 80$ ethanol. One hundred ml of the above extract was reduced to about 5 ml by heating on a hot water bath. Then 5 ° m l of water was added. The result ing solution was warmed to soften the gummy residues and bring the sugars into solution. A rubber-tipped rod was used vigorously to break up the gummy residues. The hot water extract was transferred quantitatively to a 250 ml volumetric f lask. The solution in the flask was cooled. Two ml of saturated lead acetate was added to remove tannins and other reducing impurities. The volume was made to 250 ml, mixed thoroughly and f i l t ered through dry f i l t e r paper into a 5 ° ° ml erlenmeyer flask containing about 0 . 3 g anhydrous potassium oxalate. By adding a drop of di lute lead acetate, the deleaded solution was tested for excess of oxalate. If a heavy precipitate did not appear, more oxalate was added. Toluene was added and mixed wel l . The flask was stoppered and allowed to stand overnight. The solution was then decanted through a dry f i l t e r into a dry f lask. This solu-tion was used for reducing sugars and sucrose determinations (Loomis and Shull , 1 9 3 7 ) . 37 Determination of reducing sugar Reducing sugar was determined by the arsenomolybdate reagent method of Nelson ( 1 9 ^ 4 ) . To 1 ml of the sugar solution was added 1 ml of the copper reagent. The solution was then heated for 20 minutes in a boil ing water bath. At the end of this time, the tubes were cooled, and 1 ml of arsenomolybdate reagent was added. The mixture was di luted, i f required, and the optical density was measured at 520 mu with a Beckman Model B spectrophotometer. The amount of reducing sugar was caluculated from a standard curve and expressed as percentage of the fresh weight. The copper reagent was a mixture of 25 parts of reagent A and one part of reagent B. Reagent A was prepared by dissolving 25 g of NagCO^ (anhydrous), 25 g Rochelle sal t , 20 g of NaHCO-j and 200 g of NagSO^ (anhydrous)in about 800 ml of water and d i l u t -ing to one l i t e r . Reagent B was 15$ copper sulfate containing one or two drops of concentrated sulfuric acid per 100 ml. The arsenomolybdate reagent was prepared by dissolving 25 g ammonium molybdate in 450 ml of d i s t i l l e d water. To this was added 21 ml of concentrated sulfuric acid and 3 g of sodium arsenate dissolved in 25 ml of H^O. The contents were mixed and placed in an incubator at 37°C for 48 hours. Determination of sucrose In order to determine sucrose, 25 ml of the cleared and de-leaded extract was pipetted into a 400 ml beaker. Then two drops of methyl red, 4 drops of invertase solution and 5 drops of 10$ 38 acetic acid were added. The contents were mixed and allowed to stand overnight at about 25°C. The total reducing sugar was determined by the arsenomolybdate reagent. The amount of sucrose present was calculated by subtracting the value of reducing sugars before hydrolysis of sucrose, from the total reducing sugars and multiplying the remainder by 0 .95* Sucrose was ex-pressed as percentage of fresh weight of the roots. (b) Determination of ammonium content Ammonium content of the root was determined by an adapta-t ion of the method of Vickery and Pucher ( 1 9 2 9 ) , on the alcohol extract made for reducing sugars and sucrose determinations. The volume of 100 ml of alcohol extract was reduced to about 20 ml on a boi l ing water bath. Then 50 ml of water was added to i t and the volume was reduced for the second time. The concentrated alcohol-free extract was quantitatively transferred to a d i s t i l -la t ion f lask. A few glass beads, a small piece of paraffin and 1 to 2 g of magnesium oxide were added. The flask was f i t ted for d i s t i l l a t i o n . The d i s t i l l a t i o n tube was dipped beneath the surface of 3 ml of 0.1 N HC1 containing a drop of methyl red solution. The contents of the d i s t i l l a t i o n flasks were mixed and heated to boi l ing with a micro-burner at such a rate that steam began to r ise in 3 minutes. D i s t i l l a t i o n was continued for 5 minutes. The end of the d i s t i l l a t i o n tube was washed with a few drops of water. The tube was then removed. The d i s t i l l a t e was allowed to cool and then transferred to a 100-ml flask containing 3 g permutit together with enough water 39 to make about 15 ml. The flask was shaken for 5 minutes and l a i d on i t s side for one minute. The f lu id was then decanted. The permutit was washed as above three more times. The permutit was then rinsed to the bottom of the flask with about 5 ml of water. Thereafter 1 ml of 10% NaOH was added and shaken for 3 minutes. About 65 ml water was added, the flask agitated, and then 5 ml of Nessler's reagent added. The contents were diluted to 100 ml and the optical density was read at 450 mu. Ammonium sulfate was used to make the standard curve. (c) Determination of n i t r i t e , nitrate and amino acid content Preparation of extract N i t r i t e , nitrate and amino acid contents were determined on an aqueous extract prepared from the dry powder. To 0 . 5 g of dried powder was added 35 ml of d i s t i l l e d water and boiled for 5 minutes. The extract was then cooled', made to 5 ° ml volume, centrifuged and f ina l l y f i l t ered through glass wool. Determination of n i t r i t e content Ni tr i te content of the aqueous extract was determined by the method of Wolley et a l . ( 1 9 6 0 ) o To 1 ml of the extract was added 9 ml of 20% acetic ac id . By the use of a measuring scoop, 0 . 8 g of an intimate mixture of 100 g of barium sulfate, 75 S of c i t r i c acid, 4 g of su l fani l i c acid and 2 g of 1-naphthylamine was added. The sample was shaken for about 15 seconds and was s imilarly shaken 3 minutes la ter . After 3 minutes more, the sample was shaken for the third time and centrifuged for 3 40 minutes at 1000 x g. The supernatant solution was poured through a small loose plug of borosil icate glass wool. The l ight absorbance was measured at a wavelength of 520 mu and the amount of n i t r i t e was calculated from a standard curve using potassium n i t r i t e . Nitrate content Nitrate content was measured by the method of Wolley et a l . ( i 9 6 0 ) . Nine ml of 20$ acetic acid containing 0 . 2 ppm of copper as copper sulfate was added to 1 ml of the aqueous extract. To this was added 0 .8 g of an intimate mixture containing 10 g of manganous sulfate dihydrate, 2 g of powdered zinc, 100 g of barium sulfate, 75 g of c i t r i c acid, 4 g of. su l fan i l i c acid and 2 g of 1-naphthylamine. The sample was shaken for about 15 seconds and was s imilarly shaken three minutes la ter . The sample was shaken for the third time after another three minutes and centrifuged for three minutes at 1000 x g. The supernatant solu-tion was poured through a small loose plug of borosil icate glass wool. The l ight absorbance was measured at a wavelength of 520 mu and the amount of nitrate was determined from a standard curve using potassium nitrate-, allowance being made for the n i t r i t e content. Determination of amino acid content The amino acid content of the aqueous extract was deter-mined by the method of Rosen ( 1 9 5 7 ) . One ml of the sample was heated for 15 minutes in a boi l ing water bath after the addition of 0 . 5 ml of cyanide - acetate buffer ( 0 . 0 0 0 2 M NaCN in sodium acetate - acetic acid buffer, pH 5 .4) and 0 . 5 ml of 3$ ninhydrin in methyl cellosolve (ethylene glycol monoethyl ether). 41 Immediately after removing from the water bath, 5 ml of isopropyl alcohol - water ( 1 : 1 ) diluent was added, shaken vigorously, and allowed to cool to room temperature. The intensity of color was read at 570 mu. The concentration of amino acids was calculated from a standard curve prepared from L - leucine. (d) Determination of total nitrogen Total nitrogen was determined by the standard Kjeldahl method described by Loomis and Shull ( 1 9 3 7 ) . Two 1.00-gram samples of. the dry powder and a 1 . 0 0 gram of sucrose were weighed on 7-cm f i l t e r papers. F i l t e r papers were folded and placed in 800 ml Kjeldahl f lasks. A selenized crystai was added to each f lask. Then 25 ml sulfuric - sa l i cy l i c acid mixture was poured in and allowed to react In the cold for 30 minutes. Thereafter 5 g of sodium thiosulfate was added. The flasks were warmed s l ight ly for 5 minutes and then cooled. By the use of a measure 8 g sodium sulfate - copper sulfate mixture was added to the f lasks. The flasks were allowed to heat gently on the digestion rack unt i l danger of frothing was over and then strongly unt i l c lear . The heating was continued for another 30 minutes. Then the flasks were allowed to cool in a fume closet. Several pieces of zinc, 250 ml of water and 100 ml of 33% NaOH were added and the flasks f i t ted for d i s t i l l a t i o n . The d i s t i l l a t i o n tube was dipped beneath the surface of 50 ml of 0 . 1 N HC1 containing a drop of methyl red in a wide mouth 500-ml Erlenmeyer f lask. After 150 ml was d i s t i l l e d , the receiver flasks were lowered, d i s t i l l a t i o n was continued for another 5 minutes, and the outside of the tube was rinsed with water. 42 Unused acid was t i t r a t e d with 0 . 1 N NaOH using methyl red-methylene blue i n d i c a t o r . Sucrose was used i n place of the plant material for the blank. "Net t i t r a t i o n " was determined (blank minus the nitrogen t i t r a t i o n ) . Total nitrogen for the "net t i t -r a t i o n " was calculated on the basis that 0 . 0 1 N NaOH equals 1.4 mg of nitrogen. (e) Determination of t o t a l protein Preparation of the TGA insoluble extract Samples for the determination of protein of root were pre-pared according to the method of 'West ( 1 9 6 2 ) , by grinding 25 g of fresh root material with 100 ml of cold demineralized water i n a Waring blendor for four minutes at 0°-4°C. The homogenate was centrifuged at 3800 x g for f i v e minutes to remove c e l l walls, and debris. Aliquots of the supernatant which had been cleared by centrifugation were added to equal volumes of 10$ TCA to pre-c i p i t a t e the TCA insoluble protein. The pr e c i p i t a t e was sedimen-ted at 5°0 x g for f i v e minutes and dissolved-in 0 . 1 N NaOH. The volume was made to 20 ml. Determination of protein Protein was determined by the method of Lowry et a l . ( 1951)* To 0 . 4 ml of the sample was added 2 ml of a l k a l i n e copper solution. (This solution was composed of 50 ml of 2$ Na2C02 i n 0 . 1 N NaOH, and 1 ml of 0 . 5 $ CuSO^ i n 1$ sodium potassium t a r t a r a t e ) . The contents were mixed. After 10 minutes 0 . 2 ml of 1 N F o l i n -Ciocalteau phenol reagent was added, the contents were mixed, and 43 the optical density was read at 500 mu. Protein content was obtained by comparison with a standard curve prepared by the use of crystal l ine bovine albumin and was expressed as mg per g of fresh weight. 5» Determination of the rate of respiration of root The rate of respiration of root was determined by the method of Wort and Shrimpton ( 1 9 5 9 ) . After the roots had been cleaned and trimmed, a trans-section 3*75 cm in height was cut from each root just below the region of greatest diameter. One-mm thick trans-sl ices were cut from these cylinders by a Spencer hand microtome. Discs one cm in diameter were cut by a steel cork borer from the trans-sl ices , avoiding the periphery and the core of the beet. The discs were rinsed with d i s t i l l e d water for 30 minutes. Twenty discs were blotted dry, quickly weighed and placed in a Warburg vessel containing a total of 4.0 ml of a solution whose composition was 0.4 M sucrose, 0 .05 M phosphate buffer, pH 6 . 8 , and 0.04 M K C 1 . After 15 minutes equil ibration, the oxygen consumption at 25°C was determined at 20 minute intervals for periods up to two hours. The rate of shaking was 120 strokes per minute. Respira-tion rates were expressed as microliters of oxygen consumed per hour per gram fresh weight of the discs . 6 . Determination of photosynthesis and respiration of whole plants The rate of CO2 uptake was measured in an open system with Beckman infrared analyzer IR 215 and a Heath Built Servo -44 R e c o r d e r , model EUW-20A. The a n a l y z e r was connec ted to a p l a n t chamber by tygon t u b i n g . The p l a n t chamber was a 2 0 - l b c a p a c i t y p o l y t h e n e bag o f 3 m i l t h i c k n e s s , the open end of which was s e a l e d by making p l e a t s and t i e d around a t h r e e - h o l e d r u b b e r s topper w i t h a s t r i n g . The s o i l s u r f a c e of the p o t t e d p l a n t was c o v e r e d w i t h s i l i c o n e r u b b e r f o r the d u r a t i o n o f the measurement. The a i r c o n t a i n i n g about 3Q0 ppm of CO2 was passed i n t o the chamber from a tank through a tygon t u b i n g w i t h a Matheson C o . f low meter o f tube s i z e R - 2 - 1 5 - B , w i t h a r e g u l a t e d r a t e o f 1 l i t e r per m i n u t e . Temperature i n the chamber w i t h the p l a n t r e -mained from 2 8 - 3 1 ° C d u r i n g the measurements, and was m o n i t o r e d by a t e l e thermometer from the Y e l l o w S p r i n g Instrument C o . The l i g h t i n t e n s i t y i n s i d e the chamber was 1600 f o o t - c a n d l e s . A l l the measurements were made i n the c o n t r o l l e d environment room where the p l a n t s were grown. At the s t a r t o f each measurement, CO2 gas i n the a n a l y z e r sample chamber was f l u s h e d out by n i t r o g e n gas and the i n s t r u m e n t was brought to z e r o . The CO2 c o n c e n t r a t i o n i n the tank was de termined b e f o r e c o n n e c t i n g the tank to the chamber. In the i l l u m i n a t e d system, a drop i n the CO2 c o n c e n t r a t i o n compared w i t h the t a n k , was c o n s i d e r e d as due to the CO2 f i x a t i o n (apparent p h o t o s y n t h e s i s ) . I n the dark system the i n c r e a s e i n CO2 concen-t r a t i o n was c o n s i d e r e d as due to the C0£ l i b e r a t i o n by the p l a n t (dark r e s p i r a t i o n ) . The p r o d u c t o f f low r a t e by the d i f f e r e n c e i n the C 0 2 c o n c e n t r a t i o n of the a i r b e f o r e and a f t e r p a s s i n g through the chanber gave the r a t e o f C 0 2 exchange ( f i x a t i o n i n an i l l u m i -n a t e d system and l i b e r a t i o n i n a d a r k system) of the e n c l o s e d 45 plant, which was expressed as microliters per hour per dm^ of the leaf area. 7 . Determination of the ac t iv i t i e s of nitrate reductase and  transaminase Preparation of the crude extracts Crude extract was prepared by grinding one weight of f inely chopped leaf blades or root material with 4 weights of cold 0 . 1 M phosphate buffer, pH 7 . 8 , containing 10~3 ^ reduced glutathione, i n a Waring blendor (at f u l l speed) for 1 to 2 minutes, at 0-4°C. The homogenate was strained through 4 layers of cheesecloth. For transaminase, the homogenate was diluted 5 times by the addition of cold d i s t i l l e d water and was used for assay. For nitrate reductase, i t was centrifuged in a Servall centrifuge at 2 0 , 0 0 0 x g for 20 minutes at 0 - 4°C and the resulting green, c e l l free supernatant solution was used for the assay of the ac t iv i ty . Assay of nitrate reductase (NRase) ac t iv i ty The ac t iv i ty of NRase was measured by a modification of the method of Evans and Nason ( 1 9 5 3 ) . A mixture containing 0 . 2 ml of crude enzyme, 0 . 1 ml 0 . 1 M KNO3, 0 . 0 5 ml 2 x 10"-5 M FAD, 0 . 0 5 ml 2 x 1 0 - 3 M DPNH, and 0 . 1 M phosphate buffer pH 7 . 0 in a total volume of 0 . 5 ml was incubated at 30°C for 30 minutes. The reac-tion was stopped by the addition of 1 ml of H£0 and 1 ml of 1% (w/v) sulfanilamide reagent. One ml of 0 . 2 2 $ (w/v) N-(l-naph-thyl)-ethylene diamine hydrochloride reagent was added and the contents mixed by inverting the tubes. The color was allowed to develop 15 minutes and the optical density was determined with a . 4 6 Beckman Model B spectrophotometer against a blank solution (complete but containing boiled enzyme) at 5^0 mu. Ni tr i te content.was obtained from a standard curve. The specific act iv-i ty was defined as mumoles of n i t r i t e formed per mg protein per 30 minutes. Protein in the enzyme preparation was determined by the method of Lowry et a l . (1951) Assay of alanine-glutamate transaminase ac t iv i ty Alanine-glutamate transaminase act iv i ty was assayed by an adaptation of the method of Reitman and Frankel ( 1 9 5 7 ) • One ml of <-ketoglutarate - alanine substrate (100 uM each) was pipetted into a test tube and placed in 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 30 minutes at 37°C. Immediately after removing the tubes from the water bath, 1 . 0 ml of 2,4-dinitrophenylhydrazine reagent (made by dissolving 1 9 . 8 mg of 2,4-dinitrophenylhydrazine in 100 ml of 1 N hydrochlo-r i c acid) , was added. This reagent stops further transaminase ac t iv i ty . After the tubes were allowed to stand at room temper-ature for 20 minutes, 10 ml of 0 . 4 N sodium hydroxide was added. A clean rubber stopper was inserted to each tube and the contents were mixed by inversion. At the end of exactly 30 minutes, the color intensity 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 for each homo-genate, was prepared. One ml of the substrate, 0 . 2 ml of the homogenate, and 1 ml of 2,4-dinitrophenylhydrazine reagent were mixed in a test tube. After 20 minutes 10 ml of 0 . 4 N sodium M h y d r o x i d e was added and a f t e r a f u r t h e r 30 minutes the c o l o r i n t e n s i t y was measured as a b o v e . The d i f f e r e n c e i n t r a n s m i t t a n c e between the i n c u b a t e d tubes and the a p p r o p r i a t e c o n t r o l was d e t e r m i n e d , and the c o n c e n t r a t i o n o f p y r u v a t e formed was c a l c u l a t e d from a s t a n d a r d c u r v e . S p e c i f i c a c t i v i t y was expres sed as urn p y r u v i c a c i d per mg of p r o t e i n per 30 minutes under the c o n d i t i o n s o f the a s s a y . 8. D e t e r m i n a t i o n o f i n v e r t a s e a c t i v i t y P r e p a r a t i o n o f crude e x t r a c t The crude e x t r a c t f o r the d e t e r m i n a t i o n o f i n v e r t a s e a c t i v -i t y was p r e p a r e d by a. m o d i f i c a t i o n o f the method of P r e s s y ( 1 9 6 6 ) , by g r i n d i n g 100 g f i n e l y chopped r o o t or l e a f w i t h 100 ml o f c o l d d i s t i l l e d water a t 0 - 4 ° C i n a Waring b l e n d o r f o r 1 to 2 m i n u t e s . The s l u r r y was squeezed through 4 l a y e r s o f c h e e s e c l o t h and the homogenate o b t a i n e d was c l a r i f i e d by c e n t r i f u g a t i o n a t 1 0 , 0 0 0 x g f o r 20 minutes a t 0 - 4 ° C i n a S e r v a l l c e n t r i f u g e . Two m i l l i l i t e r s o f sodium s u l f i t e , 0 . 0 1 M, was added to p r e v e n t d a r k e n i n g o f the s u p e r n a t a n t s o l u t i o n . The s o l u t i o n was then d i a l y z e d a g a i n s t 20 volumes o f 0 . 0 1 K NaCl a t 0 - 4 ° C w i t h 5 changes , f o r 24 h o u r s . The s m a l l amount of p r e c i p i t a t e formed d u r i n g d i a l y s i s was removed by c e n t r i f u g a t i o n . I n v e r t a s e assay The i n c u b a t i o n m i x t u r e f o r i n v e r t a s e a s say c o n t a i n e d 400 ;umoles sodium a c e t a t e b u f f e r , pH 4 . 7 . 730 ;umoles s u c r o s e , and a s u i t a b l e a l i q u o t o f i n v e r t a s e p r e p a r a t i o n , i n a t o t a l volume of 48 5 m l . The sample and a heated enzyme control, were incubated at 37°C for one.hour. The reaction was terminated by addition of 5 ml of 0 . 5 M dibasic sodium phosphate and heating in a boil ing bath for 5 minutes. One ml of the solution was then analyzed for reducing sugars by heating with 1 ml of copper reagent in a boi l ing water bath for 20 minutes. The solution was cooled and 1 ml of arsenomolybdate reagent was added. The sample was diluted and the optical density was measured at 520 mu. Enzyme act iv i ty was expressed as mumoles of reducing sugars formed per hour per mg of protein under the conditions of assay. 9 . Determination of ac t iv i t i e s of phosphatases Preparation of the extract Determinations of the ac t iv i t i e s of phosphatases were made by an adaptation of the method of Hinde and Finch ( 1 9 6 6 ) . The leaf or root material was cut into small pieces and ground in a small volume of medium A (sucrose, O.35 M; KHCO3, 0 . 0 3 5 M; KCl, 0 . 0 2 5 M; MgCl 2 , 0.004 M) for 1-2 minutes at 0 - 4 ° C . The homo-genate was squeezed through 4 layers of cheesecloth and then centrifuged in a Servall centrifuge at 1 5 , 0 0 0 x g for 15 minutes at 0°C. The supernatant was again centrifuged at 1 0 5 , 0 0 0 x g for 60 minutes in a Model L Preparative Spinco u l tra centrifuge. The supernatant of this second centrifugation was the extract used for enzyme assay. Assay of the act iv i ty of the enzyme The phenylphosphatase and adenosine triphosphatase (ATP-ase) 4 9 a c t i v i t i e s were a s s a y e d by i n c u b a t i o n o f 0 . 6 ml o f enzyme p r e p a -r a t i o n made up to 1 . 0 ml w i t h a s o l u t i o n which was 100 mM w i t h r e s p e c t to sodium a c e t a t e - a c e t i c a c i d b u f f e r , pH 5«1» and 3 mM w i t h r e s p e c t to pheny lphosphate or A T P . The t ime was 15 minutes and the t emperature 2 7 ° C . G l u c o s e - 1 - , g l u c o s e - 6 - , and f r u c t o s e - 6 - p h o s p h a t a s e a c t i v -i t i e s were de termined by the i n c u b a t i o n o f 0 . 6 ml o f enzyme p r e -p a r a t i o n w i t h a s o l u t i o n c o n t a i n i n g g l u c o s e - 1 - , g l u c o s e - 6 - , or f r u c t o s e - 6 - p h o s p h a t e , 3 mM, and 100 mM t r i s (hydroxymethylamino) methane- HC1 ( t r i s - H C l ) b u f f e r a t pH 8 . 2 i n a t o t a l volume of 1 . 0 m l , a t 27°C f o r 15 m i n u t e s . The, amount o f phosphate o r i g i n a t i n g from the enzyme p r e p a -r a t i o n was d e t e r m i n e d by the use o f the a p p r o p r i a t e amount o f b u f f e r , water and 0 . 6 ml o f enzyme p r e p a r a t i o n i n a t o t a l volume a 1 m l , i n c u b a t e d a t 2 7 °C f o r 15 m i n u t e s . The r e a c t i o n was s topped by the a d d i t i o n o f 3 ml o f c o l d 10$ t r i c h l o r a c e t i c a c i d to the i n c u b a t i o n m i x t u r e . I n o r g a n i c phosphate was de termined by the method o f F i s k e and SubbaRow ( 1 9 2 5 ) . P r i o r to a n a l y s i s , the TCA p r e c i p i t a t e d p r o t e i n from the i n c u b a t i o n m i x t u r e was removed by f i l t r a t i o n . One ml o f the f i l t e r a t e was d i l u t e d to 8 ml w i t h w a t e r . To t h i s was added 1 . 0 ml of 2 . 5 $ ammonium molybdate i n 5 N s u l f u r i c a c i d and 0 . 4 ml o f 0 . 2 5 $ a m i n o - n a p h t h o l s u l f o n i c a c i d r e a g e n t , ( p r e p a r e d by d i s s o l v i n g 0 . 5 g o f aminonaphtho l s u l f o n i c a c i d , 2 8 . 5 g NaHCO-j and 30 ml o f 10$ sodium s u l f i t e i n 90 ml o f d i s -t i l l e d water and making the volume 200 m l ) . Water was added to make the f i n a l volume 10 m l . C o l o r was a l l o w e d to deve lop f o r 5 minutes and r e a d i n a c o l o r i m e t e r (equipped w i t h a r e d f i l t e r ) 50 which had been adjusted to zero optical density on the blank solution prepared by di lut ing 5 ml of TCA to 8 ml and adding to i t 1 , 0 ml of ammonium molybdate reagent and 0 . 4 ml of amino-naphthol sulfonic reagent in a total volume of 10 ml. The concentration of inorganic phosphate was determined from a stan-dard curve prepared from KH^POj^ . Enzyme act iv i ty was expressed as mum Pi formed per mg of protein per 15 minutes 1 0 . Determination of the ac t iv i t i e s of sucrose synthetase, sucrose phosphate synthetase and UDPG-pyrophosphorylase Preparation of the extract Crude extracts were prepared by the method of Rorem et a l . ( I 9 6 0 ) . One hundred grams of fresh and thoroughly washed root or leaf materials was f inely chopped and blended in a Waring blendor with 100 ml of 0 . 0 5 M phosphate buffer, pH 7 . 2 , for 1-2 minutes, at 0 - 4 ° C . The homogenate was then squeezed through 4 layers of cheesecloth. The homogenate obtained was centrifuged at 1 3 , 0 0 0 x g for 15 minutes. The supernatant was gradually taken to pH 4 . 9 with acetic acid and immediately centrifuged at 1 3 , 0 0 0 x g for 15 minutes. The precipitate was discarded and the clear yellowish supernatant was l e f t overnight at 4 ° C . The flocculent precipitate which then formed was collected by centrif-ugation and washed several times with 0 . 0 2 M, pH 4 . 9 acetate buffer. The recentrifuged precipitate was then dissolved in sufficient 0 . 0 5 ' M phosphate buffer, pH 7 . 2 , to make a thick slurry and this f ina l fraction was then dialyzed against 0 . 0 2 M, pH 7 . 2 phosphate buffer. This enzyme fraction was used to deter-mine the ac t iv i ty of sucrose synthetase, sucrose phosphate. 51 synthetase, and UDPG-pyrophosphorylase. Assay of ac t iv i t i e s of sucrose synthetase and sucrose  phosphate synthetase The complete reaction mixture for sucrose synthetase con-tained 1.5 Ji'M UDPG, 4 uM fructose, 0.1 ml enzyme preparation, 0.002 ml of 0.1 M MgCl 2 and 0.01 ml of 1 M tr i s -HCl buffer in a total volume of 0.2 ml. The reaction mixture used for the determination of sucrose phosphate synthetase ac t iv i ty was identical to that used for sucrose synthetase except that 4 uM fructose-6-phosphate was substituted for the fructose, and in addition 0.01 ml of 1 M KF was present as a phosphatase inhib-i t o r . After 2 hours incubation at 37°C, the tubes containing the enzyme and substrate were placed in a boil ing water bath for 5 minutes and then cooled. In order to eliminate interference by reducing sugar, 0 .8 ml of a solution of 0.025 N NaOH containing 15 mg sodium borohydrate was added to each tube. This was followed by a few drops of ethanol as a foam retardant. These tubes were l e f t for 1 hour at room temperature and were then covered with glass stoppers and placed in a boi l ing water bath for 5 minutes to complete the reduction of the hexoses to their corresponding sugar alchols . The mixtures were cooled and acidi f ied with a few drops of acetic ac id . The sucrose was determined by the method of Roe (193^). To each tube, 2 ml of resorcinol solution ( 0 . 1 $ , w/v. in absolute ethanol) and 6 ml of 30$ HC1 were added. The tubes were heated for 30 minutes in a water bath adjusted to 80°C. 52 After cooling, the colors were measured at 490 mu and sucrose content was calculated from a standard curve. The enzyme act iv i ty was expressed as mumoles of sucrose or sucrose phos-phate formed per mg of protein per 2 hours under the conditions of assay. Assay of the ac t iv i ty of UDPG-pyrophosphorylase The enzyme assay was carried out by a modification of the method of Gander (1966) as follows: 5 umoles of UDPG, 1 pmole of inorganic pyrophosphate, 100 ;umoles of t r i s -HCl buffer, pH 7 . 5 , and 1 ml of enzyme preparation in a total volume of 2 ml were allowed to react for 15 minutes at room temperature. The reaction was stopped by heating the tubes for 5 minutes in b o i l -ing water, followed by rapid cooling in cold running water. One ml of the digest was analyzed for glucose-l-phosphate in a 3-ml quartz cuvette by addition of 0.1 jimole of 2 ,6 -d ich lo -rophenol indophenol, 0 . 3 umole of phenazine methosulfate, 0.5 umole of NADP, 100 umole of t r i s - H C l buffer, pH 7*5, one inter-national unit of phosphoglucomutase and glucose-6-phosphate dehydrogenase in a tota l volume of ^ ml. The absorbance at 600 mu was compared with that of a blank prepared from the inac t i -vated enzyme plus the other reagents. The concentration of glucose-l-phosphate was calculated from a standard curve. The enzyme act iv i ty was expressed as mumoles of glucose-l-phosphate formed per mg of protein per 15 minutes. 53 V EXPERIMENTAL RESULTS In the text, reference to an increase or decrease Is to be taken to mean an Increase or decrease compared with the appro-priate value found in untreated plants, unless spec i f i c ia l ly stated otherwise. 1. Growth of the leaves The effects of the various inhibitors on growth of one-month-old plants 7 days after the treatment are given in Table I . More than 30$ inhibi t ion was achieved by maleic hydrazide (MH), mercuric chloride, amino tr iazole , iodoacetate, vanadium sulfate (VS), pyrocatechol (PC) and copper sulfate. The effect of 8 x 10~3 M MH, 10~2 M PC and 1 0 - 2 M VS on the growth of 4.5-month-old sugar beet plants is given in Table II and F i g . 2. Under both summer and f a l l conditions MH, PC, and VS caused significant inhibi t ion of the leaf growth. The analysis of variance and comparison of the means by Student-Newman-Keuls1 test (Steel and Torrie , i 9 6 0 ) revealed that a l l the three treat-ments resulted in a highly significant decrease in the leaf area. Under summer conditions the maximum inhibi t ion of the leaf area by MH, 6 5 $ , was evident on the 7th day after treatment. PC and VS caused maximum inhibit ion of the leaf area on the 14th day after treatment. The inhibitions were 72$ and 60$ respec-t ive ly . Under f a l l conditions, the maximum inhibitions by MH, PC, and VS were 4 9 $ , 48$, and 46$ respectively. Evidently the treatments were more effective when applied under summer growth TABLE I Effect of inhibitors on the growth of leaves of one-month-old sugar beet plants Compound Type of action Concentration used Reduction in leaf expansion, 7 days after treatment A. Nitrate reductase (NRase) inhibitors 1 . Amlnotriazole 2 . Copper sulfate 3 . Iodoacetate 4 . Mercuric chloride 5 . Pyrocatechol (PC) 6 . Vanadium (a) Ammonium vanadate (b) Vanadium sulfate (VS) 7 . PC + Copper sulfate 8. PC + VS 9. VS + Copper sulfate B. Other Inhibitors 1 . Benzimidazole 2 . Caffeic acid 3 . Chloroamphenicol 4 . Cycocel 5 . Maleic hydrazide 6 . p-phenylenediamine (a) NRase inhibitor (b) Inhibitor of chlorophyll synthesis NRase inhibitor Inhibitor of NRase and dehydrogenases Inhibitor of NRase and invertase NRase inhibitor NRase inhibitor A synthetic analogue of adenine. Inhibits nucleic acid synthesis and growth A phenolic acid, action not known Binds with 5OS rlbosomal par t i c l e , interferes in protein synthesis "Growth retardant" Growth Inhibitor Transaminase inhibitor 1 x 10*"2M 1 x 10"?M 1 x 10"^M 1 x 1 0 ~ V 1 x 10~ 2 M 1 x 10" 2 M 1 x 10~ 2 M 1 x 10" 2 M 1 x 10" 2 M 1 x lO'^M 1 x 10"^M 5 x 1 0 " 3 M 1 x 1 0 " 2 M 2 x 10" 2 M 8 x 10-VM 1 x 10" 2 M 45 .1 3 0 . 9 $ 4 4 . 6 $ 5 1 . 9 $ 3 3 . 0 $ 1 2 . 5 $ 3 6 . 1 $ 2 7 . 9 $ 3 1 . 2 $ 3 7 . 1 $ 2 9 . 0 $ 24.0$ 21.6$ 24.0$ 6 1 . 0$ 20.7$ TABLE II Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on the growth of the leaves of 4 x /2 -month-old sugar beet plants ' Percentage Growth Days after Increase in leaf area conditions treatment . .:• •'. T/C {%) **CK MH PC VS £ 1 . 0 1 MH PC VS Summer conditions 7 46.32 1 5 . 7 9 1 9 . 6 2 29 .40 14 64.15 2 3 . 0 3 1 7 . 7 2 2 5 . 6 9 21 8 8 . 7 0 3 9 . 7 0 2 8 . 3 0 4 9 . 3 9 1.08 1.22 3 4 . 0 9 42.40 6 3 . 6 3 3 5 . 7 9 2 7 . 6 2 40 .05 4 4 . 7 5 3 1 . 9 0 5 5 . 6 9 F a l l conditions 11 7 62 .35 37-93 32.33 33.54 14 65.50 41.46 36.17 40.18 21 73.85 45.45 45.66 47.73 I . 6 3 1 .92 6 0 . 8 3 5 1 . 8 5 5 3 . 7 9 6 3 . 2 9 5 5 . 2 2 6 1 . 3 4 6 1 . 5 4 61.82 64.63 *Q - Significant difference according to the Student - Newman-Keuls• test . **CK - Control •8 days after treatment of one-month-old plants *21 days after treatment of 4.5-month-old plants 56 conditions. The inhibitory effects of each three compounds were found to decline by the 14th day after treatment under f a l l conditions (Fig. 4) . Under summer conditions, the effect of MH on growth of the leaves followed the same pattern as under f a l l conditions, while in case of PC and V S , the maximum inhibitions were on the 14 th day. By the 21st day the effects were found to decline (Fig. 3). 2 . Sucrose content of the roots The percentage of sucrose in the roots of .treated/ beets were s ignif icantly higher than in the roots of untreated beets. Of significance, too, was the interaction between treatments and the time of harvest. The data are given in Table III and Figs. 5 . 6,and 7 . The maximum sucrose percentage, 28$, more than control, under summer conditions was recorded 7 days after treatment when VS was used. Under f a l l conditions also, the maximum increase, 2 7 $ , in sucrose percentage was due to VS treatment. This was measured on the 14th day after treatment. The maximum in sucrose percentage under summer condition, induced by MH and PC was 22$ and 10$ respectively. The maximum increase, induced by MH under f a l l conditions was 11.5$ ° n the 21st day and by PC 16$ on the 14 th day after treatment. 3. Reducing sugars content of the roots A significant decrease in percentage reducing sugars was found due to each treatment under both, summer and f a l l conditions 57 PIG. . 3. EFFECT OF MALEIC -HYDRAZIDE (MH), PYROCATECHOL (PC)  AND VANADIUM 5ULFATE (V5) ON LEAF GROWTH OF . SUGAR BEET UNDER SUMMER GROWTH CONDITIONS 100 80 OF CONTROL 60 40 20 V \ \ Age when t r e a t e d : A\ months *<j V 5 10~2 M P C \ \ r \ \ M H \ 8 x 10"3 M \ 10~2 M -o •o— 14 21 DAYS AFTER TREATMENT 58 F I G , 4. EFFECT OF MALEIC HYDRAZIDE, PYROCATECHOL, AND  VANADIUM SULFATE ON LFAF GROWTH OF SUGAR RFFT UNDER FALL GROWTH CONDITIONS 100 80 OF 60 CONTROL 40 20 Age when treated: 4| months X V ^ -- 2 -— ^=-« '~~ — ^ o *-v p c _ l : — 7 14 DAYS AFTER TREATMENT 21 TABLE III Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on the sucrose content of the roots of 4.5-month -old sugar beet plants « Growth Days after Sucrose content as condition treatment percent of the fresh weight *Q T/C (%) MH PC VS .05 .01 MH PC VS Summer 0 14.3 • • • • • • • • • 0.15 0.17 it 7 14.7 17.2 16.2 18.8 117.46 110.90 1 2 8 . 4 0 it 14 17.3 18.1 18.4 1 9 . 8 104.43 105.97 114.35 it 21 1 3 . 9 1 7 . 0 15.1 1 7 . 3 122 .14 1 0 8 . 5 0 123.87 F a l l 0 14.6 • • • • • • • • • 0.55 0.62 n 7 14.3 1 5 . 9 16.2 1 7 . 9 110.75 112.88 124.72 it 14 13.6 14.5 1 5 . 9 17.3 106.57 116.80 127.29 ti 21 13.1 14.6 14.2 1 5 . 8 111.51 108.48 120.67 •"•Q - Significant difference according to the Student-Newman-Keuls test *»CK - Control * 6o FIG. 5. EFFECT' OF MALEIC HYDRAZIDE ON SUCROSE AND. REDUCING SUGAR  CONTENT OF ROOT OF, SUGAR BEET GROWN UNDER SUMMER  AND FALL CONDITIONS DAYS AFTER TREATMENT 61 FIG. . 6 . EFFECT OF PYROCATECHOL DIM SUCROSE AND REDUCING SUGAR 140 _ CONTENT OF ROOT OF 5UGAR BEET GROWN UNDER SUMMER AND FALL CONDITIONS 120 OF" ' „ 100 CONTROL 80 60 40 SUCROSE - F a l l SUCROSE - Summer " REDUCING - F a l l \ REDUCING - Summer V -7 14 DAYS AFTER TREATMENT 21 62 140. _ 120 100 OF CONTROL 80 60 40 FIG.. 7 . EFFECT OF VANADIUM SULFATE ON SUCROSE AND REDUCING  SUGAR CONTENT OF ROOT OF SUGAR BEET GROWN UNDER SUMMER AND FALL ' CONDITIONS • Cj"^ SUCROSE - F a l l C REDUCING - Summer . .-X \ REDUCING - F a l l - -o _L 7 14 DAY5 AFTER TREATMENT 21 63 (Table IV and Figs 5# 6 , 7 ) . Under summer conditions, the maximum reductions in reducing sugars were on the 14th day after treatment. These reductions were 22$, 3 4 $ , and 10$ for MH, and VS respectively. The relat ive effects of MH, PC, and VS on leaf growth, re-ducing sugars and sucrose content of the roots have been summa-rized for summer and f a l l conditions in Figs . 8 and 9 respec-t ive ly . 4 . Nitrogenous constituents of the roots Nitrate N - The treated plants had s ignif icantly higher (0.01 level) nitrate content in their roots than in the untreated plants. The data are given in Table V. In general, the maximum increase in nitrate was found to be on the 7 t h day after treat-ment. The content values on the 7 t h day after treatment were 8 9 , 121, 100, and 125 jig/gm of the dry weight of the root for control, MH-, PC-, and VS-treated beets respectively. Ni tr i te N - Nitr i te content of the roots is given in Table V. The analysis of variance showed that treatments caused significant reduction in n i t r i t e content of the roots. Compar-ison of the means with those of the control plants by Q test Indicated that except on the 7 t h day after treatment in PC-treated beets, a l l the treatment means were s ignif icantly lower (0.01 l eve l ) . The maximum reduction in the n i t r i t e content of the roots, up to 5 9 $ , was caused by MH on the 7 t h day after treatment. Ammonium N - In MH-treated plants ammonium N was s i g n i f i -cantly more than in the control on a l l dates of harvest. The TABLE IV Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on reducing sugar of the roots of 4.5-month-old sugar beet plants Growth condition Days after treatment Reducing sugars as percent of the fresh weight T/C {%) **CK MH PC VS .OS .01 MH PC VS Summer ti 11 it 0 7 1 4 21 0.17 0.21 0.19 0 o 2 4 • • • 0.17 0.15 0.22 • « • 0.16 0.13 0.21 • • • 0 o l 9 0.17 0.22 . 0 0 8 1 .0092 8 0 . 0 9 7 8 . 4 8 9 1 . 6 6 77.38 65.82 8 6 . 6 3 91.92 90.18 9 1 . 6 6 F a l l it it tt 0 7 1 4 21 0.63 0.71 0.77 0 . 8 3 • • • 0.36 0.61 0.65 • • • 0.34 0.52 0 . 6 9 * • • 0.35 O . 5 6 0.57 .051 .059 5 0 . 2 6 79.86 77.69 4 7 . 6 3 6 7 . 3 0 8 3 . 0 4 4 8 . 4 7 7 2 . 8 9 6 8 . 4 5 *Q - Significant difference according to the Student-Newman-Keuls test . **CK - Control +40 INCREASE +20 r OVER CONTROL •20 DECREASE UNDER CONTROL •40 • 60 FIG. 8 EFFECT OF MALEIC HYDRAZIDE (MH), PYROCATECHOL (PC) AND VANADIUM SULFATE (VS) ON LEAF GROWTH AND ROOT REDUCING SUGAR AND SUCROSE OF SUGAR BEET SUMMER GROWTH CONDITIONS :i ' . ' . ( - • • • II l i i f i 1 * • • f !-:• t SK3BS i i 11 i l l 1 i i i l GROWTH MH PC VS III i i I Ui MH rrf il i i l ii I i I ! -I_L PC VS 7 DAYS AFTER TREATMENT 14 DAYS AFTER TREATMENT 21 DAYS AFTER TREATMENT ON +4or INCREASE OVER CONTROL +20 F I G . 9 EFFECT OF MALEIC HYDRAZIDE (MH), PYROCATECHOL (PC) AND VANADIUM SULFATE (VS) ON LEAF GROWTH AND ROOT REDUCING SUGAR AND SUCROSE OF SUGAR BEET FALL GROWTH CONDITIONS - 2 0 r DECREASE UNDER CONTROL - 4 0 -7 DAYS AFTER TREATMENT 14 DAYS AFTER TREATMENT 21 DAYS AFTER TREATMENT ON OS TABLE V Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on the nitrogenous constituents of the roots of 4.5-month-old sugar beet plants Days after Kind of Nitrogen content as treatment nitrogen Jig/N gram of dry weight T/C {%) **CK MH PC VS .05 .01 MH PC VS 0 Nitrate 95 • • • • • • • • • 7.86 8.92 7 n 89 121 100 125 135.61 111.93 139.72 14 II 108 126 119 149 116.47 1 1 0 . 0 9 132.36 21 II 118 148 130 119 126.17 110.86 1 0 0 . 8 6 0 Ni tr i te 19 • • « • • • • • • « 1.31 1.48 7 II 31 13 30 19 1 40 .77 97.84 62.84 14 ti 32 20 19 14 63.49 61.51 44.40 21 it 45 29 25 28 • .1 65.73 55-57 6 2 . 0 6 0 Ammonia 143 • • • • • • • • • 7.94 9.01 7 it 163 183 127 145 112.28 77.86 89.16 14 it 125 182 117 112 146.06 94.22 8 9 . 8 9 21 it 98 199 125 105 130.36 1 2 7 . 8 5 107.15 0 Amino I850 • • • • • • • • • 7 0 . 9 0 80.44 7 it 2325 2175 2050 2053 93.55 8 8 . 1 7 8 8 . 3 0 14 it 1875 2800 2425 1604 149.33 129.33 85.56 21 1725 2700 2100 1492 156.52 121.74 8 6 . 4 7 0 Total N 11427 • • • • • • • • • 1458.1 1 6 5 4 . 4 7 it 12127 12000 9395 12750 98.95 76.77 105.10 14 it 14980 12250 12040 12850 81.77 8 0 . 3 7 85.70 21 ti 18190 14140 12340 13967 77.73 67.84 76.70 *Q - Significant difference according to the Student-Newman-Keuls test . **CK - Control TABLE V Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on the nitrogenous constituents of the roots of 4.5-month-old sugar beet plants Days after treatment Kind of nitrogen Nitrogen ug/N gram < content as of dry weight T/C •{%) **CK MH PC VS .05 .01 MH PC VS 0 7 14 21 Nitrate 11 11 11 95 89 108 118 • • • 121 126 148 • • • 100 119 130 • • • 125 149 119 7.86 8.92 135.61 116.47 126.17 111.93 1 1 0 . 0 9 110.86 139.72 132.36 1 0 0 . 8 6 0 N i tr i t e 19 1.31 1.4-8 7 " 31 13 30 19 40.77 97.84 62.84 14 " 32 20 19 14 6 3 . 4 9 61.51 44 .40 21 » 45 29 25 28 6 5 . 7 3 55.57 6 2 . 0 6 0 Ammonia 143 7.94 9 .01 7 » 163 I83 127 145 112.28 77.86 89.16 14 " 125 182 117 112 146.06 94.22 8 9 . 8 9 21 " 98 129 125 105 1 3 0 . 3 6 127.85 107.15 0 Amino I850 7 0 . 9 0 8 0 . 4 4 7 11 2325 2175 2 ° 5 0 2053 93.55 88.17 8 8 . 3 0 14 " I875 2800 2425 1604 149.33 129.33 8 5 . 5 6 21 » 1725 2700 2100 1492 156.52 121.74 8 6 . 4 7 0 Total N 11427 1458.1 1654.4 7 " 12127 12000 9395 12750 98.95 76.77 105 .10 14 » 14980 12250 12040 12850 81.77 8 0 . 3 7 8 5 . 7 0 21 it 18190 14140 12340 13967 77.73 67.84 76.70 *Q - Significant difference according to the Student-Newman-Keuls test . **CK - Control 68 ammonium content of the PC-treated plants was s ignif icantly low-er at the 0.01 level on the 7th day and at the 0.05 l evel on the 14th day after treatment. On the 21st day after treatment, the ammonium content of PC-treated beets was s ignif icantly higher than the control plants at the 0.01 l eve l . In VS-treated beets, the. ammonium content was lower by 11 and 10$ respectively on the 7th day and the 14th day. The increase of the ammonium content of the VS-treated beets on the 21st day after the treatment was not s t a t i s t i c a l l y significant (Table V) Amino N - Amino N was decreased in the roots of a l l the treated plants on the 7th day after treatment. These decreases, significant at the 0.01 l eve l , were 6 . 5 , 11.8 and 11.6$ for MH-, PC- , and VS-treated beets respectively. On the l4th and 21st day after treatment, there was a significant r i se in the amino acid content of the MH- and PC-treated beets. This r i se in amino acid content of the roots was 49 to 56$ in MH-treated beets and 21 to 29$ in the PC-treat-ed beets. VS-treated beets had always a s ignif icantly lower amino acid content in their roots (Table V) . 1 Total N - There was no significant difference between the total N content of the treated and of the untreated plants on the 7th day after treatment. However, the total N content of the treated beets was s ignif icantly lower on the 14th and 21st day after treatment. The maximum reduction i n the total N con-tent in the roots of the treated beets was on the 21st day after treatment. The values were 18.19, 14.14, 12.34, and 13.96 mg per gram of the dry weight of the roots, for the control, MH-, PC- and VS-treated plants respectively (Table V) . 69 Protein - There was a significant reduction in the protein content of the roots of the treated plants. The maximum reduc-tion in the protein content of the roots was on the 7 t h day-after treatment by PC, the 2 1 s t day by MH and the 14th day by VS. In general VS caused the maximum reduction. The protein content was 6 9 . 6 0 , 66.84 and 69.15$ of the control on the 7 t h , 14th and 21st day after treatment respectively in the roots of the VS-treated beets (Table VI) . 5 • Photosynthesis and respiration Photosynthesis and dark respiration of leaves The effect of MH, PC and VS on C 0 2 f ixat ion, C 0 2 l ibera-tion (dark respiration) , and the true photosynthesis ( C 0 2 f ixa-t ion + dark respiration) under summer and f a l l conditions are given in Table VIII and Figs . 13, 14, 15, 16, and 17. The rate of net C 0 2 f ixat ion, under summer conditions in the MH-treated plants was lower by 8$, on the 7 t h day after treatment. The difference was not significant: s t a t i s t i c a l l y . A significant decrease in the rate of net C 0 2 f ixation, in PC-treated plants was measured on the 7 t h day after treatment. VS enhanced the rate of net C 0 2 f ixation by 6 . 6 $ on the 7 t h day, 9.8$ on the l 4 t h day and J0,0% on the 21st day after treatment. The difference between the values in control and VS-treated plants was significant at the 0.01 level on a l l the three dates of observation. MH and PC also stimulated the rate of net C 0 2 f ixation signif icantly on the l 4 t h and the 2 1 s t day after treat-ment. The stimulation by MH were 4 l and 49$ and by PC 34 and 8$ 70 TABLE VI Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on the protein content of the roots of 4V2 -month-old sugar beet plants : . Days after treatment Protein content (mg/gm of the fresh weight Q T/C (%) • CK MH PC VS 0.05 0.01 MH • PC VS 0 7 14 21 9 • 17 • • • 9 . 0 3 7.33 6.23 6 . 2 7 8.23 7.12 5.84 5 . 5 0 8.57 6.65 6 . 3 9 ' 5 . 9 3 0.880 O.98O 81.45 69.14 86.64 70.98 77.60 74.63 6 9 . 6 0 66.84 69.15 Q - significant difference according to Student-Newman-Keuls' test The effects of each treatment on the various nitrogenous constituents of the roots have been summarized separately Figs. 10, 11 and 12. The effects of a l l the three treatments on the chemical composition of the roots of sugar beet are summarized in Table VII. TABLE VII The summary of the effects of maleic hydrazide (MH), pyrocatechol (PC), and vanadium'sulfate (VS) under summer conditions on the chemical composition of the roots of 41/2-month-old sugar beet plants  ^ # Effect of treatment with Item MH PC VS Sucrose + + + + + + + + + Reducing sugar - - - - - - - - -Nitrate N + + + + + + + + + Nitr i te N - - - n. s . - - - - -Ammonium N + + + - - — - n .s . Amino N - + + - + + — — — Total N n.s - n. s . - - n.s . — — Protein N - - - - - - — - _ # The symbols + and - refer to increase and decrease respectivel; compared to the control plants. Three symbols in each treatment correspond to harvests made. 7.14, and 21 days after treatment, n.s . - not significant at . 05 and .01 l eve l . * Significant only at .05 l eve l . 71 FIG. 10. EFFECT OF MALEIC HYDRAZIDE DiM THE NITROGEN CONTENT OF ROOT OF SUGAR BEET 160 140 120 OF 100 CONTROL 80 60 40 AMINO N NITRATE N \ TOTAL N • / NITRITE N . \ ••-a— JL 7 14 DAYS AFTER TREATMENT 140 FIG. 11. EFFECT OF PYROCATECHOL ON THE NITROGEN CONTENT OF ROOT OF 5UGAR BEET 120 OF 100 CONTROL 80 60 40 / AMINO N / < > / NITRATE N O -/- * AMMONIUM N NITRITE N 7 14 DAYS AFTER TREATMENT 73 FIG. 12. EFFECT OF VANADIUM SULFATE ON THE NITROGEN CONTENT OF ROOT OF SUGAR BEET 7 14 DAYS AFTER TREATMENT TABLE VIII Effect of maleic hydrazide (MH), pyrocatechol ( P C ) , and vanadium sulfate (VS) on photosynthesis and respiration of 4.5-month-old sugar "beet plants  Growth condition Days after treatment Rate of net CO2 fixation (yxl CO?/dm2/hr of the leaf area) *Q T/C (%) **CK MR PC VS .05 .01 MH PC VS Summer it ii ti 0 i l 21 1434 1386 1385 1426 1412 1373 670 1506 1012 1433 1360 1112 949 i 4 i 4 1033 1238 55.92 6 3 . 4 5 92.23 141.57 149.00 46.54 134.32 1 0 8 . 8 9 106.65 1 0 9 . 8 9 130.41 Rate of dark respiration (ul C0?/dm2/hr of the leaf area) 0 7 14 21 444 470 392 430 610 444 879 493 517 315 455 376 352 3^9 474 201 72.77 6 0 . 8 0 99.13 144.12 87.98 134.76 80.84 72.71 65.28 Rate of true photosynthesis Cul C02/dm2/hr of the leaf area) 0 7 14 21 1878 1857 1773 1856 2023 1817 1552 1999 1530 1748 1815 1446 1301 1763 1508 1141 8 9 . 8 5 114.25 135.51 76.74 118.64 115.86 97.21 9 8 . 8 5 87.70 Rate of net photosynthesis (ill C0?/dm2/hr of the leaf area) 74.24 P a l l 0 7 14 1510 1495 1452 1445 1562 1931 1514 2183 1652 1849 1748 2192 68.18 123.57 111.94 9 8 . 6 3 105.81 139 .70 132.65 Rate of dark respiration (ill C02/dm2/hr of the leaf area) 0 7 14 569 567 576 606 962 748 902 8?2 994 635 957 554 77.76 6 3 . 8 6 93.76 96.22 9 0 . 5 8 55.76 *Q - Significant difference according to the Student-Newman-Keuls test •**CK - Control TABLE VIII ( c o n t ' d ) E f f e c t o f m a l e i c h y d r a z i d e (MH), p y r o c a t e c h o l ( P C ) , and vanadium s u l f a t e (VS) on p h o t o s y n t h e s i s and r e s p i r a t i o n o f 4 . 5 - m o n t h - o l d sugar bee t p l a n t s  Growth c o n d i t i o n Days a f t e r t rea tment Rate o f ne t C02 f i x a t i o n ( u l C O ? / d m 2 / h r o f the l e a f a r e a ) T / C (%) * * C K MH PC VS . 0 5 . 0 1 MH PC VS F a l l Rate o f t r u e p h o t o s y n t h e s i s ( u l C O ? / d m 2 / h r o f the l e a f a r e a ) 0 7 14 20?9 2062 2525 2679 2868 2484 2028 2416 2705 2051 3054 2745 1 0 6 . 1 2 9 3 . 1 3 9 5 . 7 1 1 0 1 . 3 6 1 2 1 . 0 1 1 0 2 . 9 3 Summer Rate o f r e s p i r a t i o n ( u l Op/gm f r e s h w o f r o o t t / h r ) 0 7 14 21 82 82 49 75 47 66 46 84 75 68 • • 64 51 55 1 . 3 3 1 .51 5 9 . 2 4 6 2 . 9 7 6 9 . 4 8 1 0 1 . 5 8 1 0 0 . 1 2 1 0 1 . 5 9 7 8 . 2 5 6 7 . 7 7 8 3 . 9 8 F a l l 0 7 14 21 65 66 44 73 52 73 54 56 57 60 43 46 48 1 . 8 8 I . 9 6 6 6 . 0 7 7 1 . 1 5 7 3 . 9 8 8 4 . 8 8 7 8 . 7 6 8 I . 6 3 64.43 6 2 . 5 9 6 5 . 9 5 *Q - S i g n i f i c a n t d i f f e r e n c e a c c o r d i n g to S tudent -Newman-Keuls 1 t e s t * * C K - C o n t r o l FIG. 13. EFFECT OF MALSIC HYDRAZIDE ON PHOTOSYNTHESIS  AND RESPIRATION OF LEAVES AND ON  RESPIRATION 0?' ROOT OF SUGAR BEET GROWN UNDER SUMMER CONDITIONS 130 100 % o f control 70 40 10 LEAF CARBON ASSIMILATION (PSYN) LEAF RESPIRATION ROOT RESPIRATION _L 7 14 DAYS AFTER TREATMENT 77 FIG. 14. EFFECT OF MALEIC HYDRAZIDE ON PHOTOSYNTHESIS AND RESPIRATION OF LEAVES AND ON RESPIRATION OF ROOT OF SUGAR 3EET UNDER .FALL CONDITIONS OF 140 120 100 CONTROL 80 6o 40 PSYN + RESP o—...S^ CARBON ASSIMILATION LEAF RESPIRATION t J - ' ROOT RESPIRATION ~~T^« •••••••••• .o O 14 21 DAYS AFTER TREATMENT 78 OF FIG. 1 5 . EFFECT OF PYROCATECHOL ON PHOTOSYNTHESIS AND RESPIRATION OF LEAVES AND ON RESPIRATION . OF ROOT OF SUGAR BEET GROWN . UNDER SUMMER CONDITIONS 130 100 70 CONTROL 40 10 A S LEAF RESPIRATION / / O'* c ROOT RESPIRATION f's V %\ \ ( _o-\ PSYN + RSSP /I • * y 1 LEAF CARBON ASSIMILATION (PSYN) 14 21 DAYS AFTER TREATMENT 79 FIG. 16. EFFECT OF VANADIUM SULFATE ON PHOTOSYNTHESIS 130 100 OF 70 CONTROL 40 10 AND RESPIRATION OF LEAVES AND ON  RESPIRATION OF ROOT OF SUGAR BEET GROWN. UNDER SUMMER CONDITIONS. LEAF CARBON ASSIMILATION (PSYN) ^zS^L u • • * *• mnxs^»0 t * a t nun 4 PSYN + RESP A -o— —. ROOT RESPIRATION* '.....a» LEAF RESP IRAT ION^. V J - L 7 . 1 4 DAYS AFTER TREATMENT 21 80 F I G . 17. E F F E C T OF VANADIUM 5 U L F A T E DIM P H O T O S Y N T H E S I S  AND R E S P I R A T I O N OF L E A V E S AND ON R E 5 P I R A T I 0 N OF ROOT OF SUGAR B E E T UNDER F A L L C O N D I T I O N S 1 4 0 1 2 0 OF 1 0 0 CONTROL 8 0 6 0 _ 4 0 .O-. . C A R B O N A S S I M I L A T I O N •<c > / / . - " P S Y N + R E S P L E A F R E S P I R A T I O N V \ \ ROOT R E 5 P I R A T I 0 N *" V " ° v V O """)^ X 7 1 4 D A Y S A F T E R TREATMENT 2 1 81 , on the 14th and 21st day after treatment, respectively. The rate of dark respiration under summer conditions in MH-treated plants was slowed down. It was 7 2 . 7 , 6 0 . 8 , and 99.1% of the control values on the on the 7 t h , 14th and 21st day after treatment, respectively. There was a stimulation in the rate of dark respiration by PC on the 7 t h and the 21st day of 4 4 . 1 and 3 4 . 7 $ respectively. VS caused a consistent reduction in the rate of dark respiration on a l l the three dates of observations. The rate of net CO2 f ixation under f a l l conditions was stimulated by MH and PC on the 7 t h and the 21st day after treat-( • • • V -ment. PC also stimulated the rate of net CC>2 f ixation on the 14th day. The maximum stimulation, 32 to 39%, was by VS under f a l l conditions. The dark respiration was slowed down by a l l three treat-ments under f a l l conditions. Due to MH, the reduction in the rate of dark respiration on the 7 t h day was 22.2% and on the l4th day, 3 6 . 0 $ . PC caused the reduction in the rate of dark respiration under f a l l conditions on the 7 t h day by 6.2$ and on the 14th day by 3*7%. The reductions in the rate of dark respi -ration under f a l l conditions by VS were 9 .4 and 4 4 . 3 $ on the 7th and the 14th day respectively. The rate of true photosynthesis was calculated by addition of net CO2 f ixation and CO2 l iberat ion in the dark. The rate of true photosynthesis under summer conditions was lower than the control on a l l the three dates of observations in the VS-treated plants. In the MH- and PC-treated plants i t was lower than the control on the 7 t h day but higher on the 14th and 21st day of observations. 82 The rate of true photosynthesis under f a l l conditions was higher on the 7th day and lovrer on the l4th day after treatment in MH-treated plants. PC-treated plants had a lower rate of true photosynthesis on the 7th day but higher on the 14th day after treatment. VS-treated plants had a higher rate of true photosyn-thesis on both days of observations. Respiration of the roots The oxygen uptake by the beet tissue s l ices were determined by the Warburg method and is given i n Table VIII. MH and VS inhibited the rate of oxygen uptake under summer and f a l l condi-t ions. The inhibi t ion by MH was 38$ under summer and 3^$ under f a l l conditions on the 7th day after treatment. The inhibi t ion by VS was 33$ under summer and 38$ under f a l l conditions. The differences between the values for respiration on the MH- and PC-treated plants and the control plants were significant s t a t i s t i -c a l l y . PC stimulated the rate of oxygen uptake under summer condi-tions. However, the differences between control and the treated plants were not significant except on the 7th day after treatment. Under f a l l conditions, PC inhibited the rate of respiration up to 20$ and this inhibit ion was significant at the 0.01 l eve l . The effects of MH, PC, and VS on photosynthesis and respira-tion are summarized in Table VIII(a). 6. Nitrate reductase and transaminase ac t iv i ty MH-treated and the control plants showed a steady increase in nitrate reductase (NRase) ac t iv i ty from 7 days to 21 days after 83 TABLE VIII (a) .... Summary of the effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on photosynthesis and respiration of sugar beet Growth Item condition •Effect of treatment with MH PC VS Net Summer ns + + - + + + + + photosynthesis F a l l + + N ns + N + + N Dark Summer _ + - + respiration of leaf '• F a l l - - N - - N - N True Summer - + + - + + photosynthesis F a l l + - N - + N + + N Respiration Summer _ — x ns ns _ of roots F a l l *The symbols +, - and N refer to increase, decrease, and not determined, respectively. Three symbols for each treatment correspond to the harvests made 7 , 14, and 21 days after treatment. ns - not significant at 0 . 0 1 level x - Significant only at 0 . 0 5 l eve l . 84 treatment in the root and the leaf . In PC- and VS-treated plants the lowest specific act iv i ty recorded was on the l 4 t h day after treatment. A l l three treatments caused significant inhibit ion of specific NRase ac t iv i ty in the root and in the leaf on a l l the dates of harvest (Table IX and Figs . 18, 19 and 20). The specific ac t iv i ty values for the leaves of MH-treated plants were 7 8 . 8 , 6 9 . 5 and 59 .1$ of the control values, respec-t ively on the 7 t h , 14th and 21st day after treatment (Fig. 18). In PC-treated plants, the specific ac t iv i ty values for leaves were 72 .1$ on the 7 t h day, 62 .1$ on the 14 th day, and 4 4 . 4 $ on the 21st day, of the control values (Fig. 19). The maximum inhibit ion of the ac t iv i ty of NRase was due to VS and occurred in the leaves. These inhibitions were 42, $1, and 55$ respectively on the 7 t h , l 4 t h and 21st day after treatment (Fig. 20). VS also caused the maximum inhibi t ion of the root NRase ac t iv i ty . The ac t iv i ty of transaminase in the root and the leaf was inhibited s ignif icantly by a l l three treatments. The maximum inhibi t ion in the leaf was due to V S . The values for transaminase ac t iv i ty in the leaf of VS-treated plants were 40.5$ on the 7 t h day, 4 3 . 5 $ on the 14th day, and 52 .2$ on the 21st day, of the control values. The maximum reductions of transaminase act iv i ty in the leaf of MH- and PC-treated plants were on the 14th day after treatment. The maximum reductions in transaminase act iv i ty in the root of MH- and VS-treated plants were on the 7 t h day after treatment. These reductions were about 40 and 44$ by MH and VS respectively. The maximum reduction in the transaminase ac t iv i ty of the root of the PC-treated plants was about 25$ on the l 4 t h day after treatment. TABLE IX E f f e c t o f m a l e i c h y d r a z i d e (MH), p y r o c a t e c h o l ( P C ) , and vandium s u l f a t e (VS) on n i t r a t e r e d u c t a s e and transaminase a c t i v i t y o f the r o o t and the l e a f o f 4 l / 2 - m o n t h - o l d sugar bee t p l a n t s P l a n t Days a f t e r Enzyme p a r t t r ea tment S p e c i f i c a d b i v i t y v a l u e •Q T / C (%) CK MH PC VS 0 . 0 5 0 .01 MH PC VS N i t r a t e L e a f 0 33 .40 • • • • * • • • • 2 . 3 9 2 . 7 1 r e d u c t a s e it it 7 2 0 . 8 8 1 5 . 6 3 1 5 . 0 5 1 2 . 0 5 7 8 . 8 3 7 2 . 0 6 5 7 . 7 2 II ti 14 2 0 . 7 6 1 5 . 8 3 1 2 . 1 2 1 1 . 1 5 6 9 . 5 7 6 2 . 0 5 4 8 . 9 9 it tt 21 3 3 . 3 2 1 9 . 7 0 14.79 14 .99 59.14 44 .41. 4 5 . 0 1 it Root 0 24 .30 • • • • • • • • • 0.46 0 . 5 3 it tt 7 14.54 1 2 . 9 0 1 2 . 1 2 7 . 4 7 8 8 . 7 5 8 3 - 3 9 5 1 . 3 8 it .ti 14 1 7 . 3 3 14.37 7 . 8 8 6.46 8 2 . 9 0 . 46.46 3 7 . 3 1 it ti 21 1 9 . 8 0 1 6 . 2 6 1 3 . 0 6 1 0 . 1 5 82.13 6 6 . 0 3 5 1 . 2 2 T r a n s - L e a f 0 4 5 . 4 7 • • • • • • • • • 0 . 8 6 1 . 5 6 aminase it tt 7 6 9 . 2 3 5 4 . 0 1 4 0 . 1 9 28.03 7 8 . 0 2 5 8 . 0 5 40.48 it it 14 6 3 . 2 2 40 .27 3 6 . 0 3 2 7 . 5 3 6 3 . 6 9 5 6 . 9 9 4 3 . 5 4 ti it 21 7 8 . 2 8 5 7 . 6 3 3 9 . 7 9 40 .85 7 3 . 6 1 5 0 . 8 3 52 .18 „ Root 0 8 0 . 2 0 • • • • • • • • • . 0 3 1 . 0 7 8 60.42 9 3 . 6 8 5 6 . 7 6 it it 7 9 3 . 9 8 5 6 . 7 8 8 8 . 0 5 5 3 . 3 5 it it 14 82.22 7 6 . 0 9 6 2 . 2 2 48.13 9 2 . 5 4 7 5 . 6 8 5 8 . 5 4 ti it 21 9 6 . 3 4 6 9 . 6 6 8 7 . 3 2 7 6 . 3 3 7 2 . 3 0 9 0 . 6 3 7 9 . 2 3 S p e c i f i c a c t i v i t y o f NRase - mum N0 2 /mg o f p r o t e i n , s p e c i f i c a c t i v i t y f o r t ransaminase -$tm p y r u v i c / m g p r o t e i n s i g n i f i c a n t d i f f e r e n c e a c c o r d i n g t o Student-Newman—Keuls* t e s t 86 F I G . 18. EFFECT OF MALEIC HYDRAZIDE ON NITRATE REDUCTASE _AND TRANSAMINASE ACTIVITY IN LEAF AND ROOT OF SUGAR BEET 100 OF 80 CONTROL 60 40 TRAN5AMINA5E Root ( \ NRase Root TRANSAMINASE L e a f ^ — ~ o - — NRase Lea f 14 21 DAYS AFTER TREATMENT 8? FIG. 19 EFFECT OF PYROCATECHOL ON' NITRATE REDUCTASE AND TRANSAMINASE ACTIVITY IN LEAF AND ROOT OF SUGAR BEET 100 ^ T ; -\ W A % OF 80 CONTROL . 60 40! \ V . V NRase TRANSAMINASE Root " - v.. J> NRase L ea f \ — S P * 1 TRANSAMINASE Lea f "** • -\ - — — ~ ° * ^ ^ r - — s' 7 14 DAYS AFTER TREATMENT 21 88 F J G . 20. EFFECT OF VANADIUM SULFATE ON NITRATE REDUCTASE AND TRANSAMINASE ACTIVITY IN LEAF AND ROOT OF SUGAR BEET 100 * OF 80 CONTREL 60 40 \ v - . \ 20 \ TRANSAMINASE \ \ \ Leaf, TRANSAMINASE Root J*' NRase L ea f \ NRase. Root JL 7 14 DAYS AFTER TREATMENT-21 89 7. Invertase act iv i ty The invertase ac t iv i ty of the leaves of the treated plants was reduced s igni f icant ly . Maximum inhibit ion of the leaf inver-tase was achieved through PC treatment. These inhibitions were 55.7$ on the 7th day, 7 8 . 3 $ on the l4th day and 6 0 $ on the 21st day after treatment. Root invertase act iv i ty was also inhibited most by pyrocatechol. The root invertase act iv i ty of the PC-treated plants was 71$. 62$ and 75•7$ of the control act iv i ty on the 7th, l4th, and 21st day after treatment. The root invertase act iv i ty of the MH-treated plants was not s ignif icantly different from those of the untreated plants on the 7th day after treatment, but on subsequent days of harvest the differences were highly s ignif icant . The invertase act iv i ty is given in Table X . The inhibit ion by MH and VS increased progressively in the leaf (Fig. 21 and 2 3 ) . Figure 22 shows that maximum inhibi t ion by PC was on the l4th day after treatment. The maximum inhibit ion of invertase act iv i ty of the root by MH and PC was also on the l4th day (Figs. 24 and 25) and by VS on the 21st day after treatment (Fig. 2 6 ) . 8 . Phosphatases Table XI indicates that phosphatases in general were inhib-ited by the treatments. Phenylphosphatase act iv i ty of the roots and the leaves was s ignif icantly inhibited by a l l the three treat-ments. The maximum inhibit ion of phenylphosphatase act iv i ty of leaf by MH, PC, and VS was on the 14 th day after treatment. The values on this day were 35.7$ for MH, 75.4$ for PC and 57*7$ of TABLE X Effect of maleic hydrazide (MH), pyrocatechol (PC) and vanadium sulfate (VS) on the invertase a c t i v i t y of the root and the l e a f of /A/2 -month-old sugar beet plants Plant part Days after treatment (mum reducing sugar/mg protein) s" -•H •Q CK MH PC VS 0 . 0 5 U.Ul MH:. PC VS Leaf 0 7 14 21 9955 7373 7150 9283 • * • 6501 4045 4633 3268 1556 3716 5920* 4695 4185 4 6 3 . 8 6 5 2 6 . 2 1 8 8 . 1 7 5 6 . 5 7 4 9 . 9 0 4 4 . 3 2 80 .29 2 1 . 7 0 6 5 . 6 6 40 .03 4 5 . 0 8 Root 0 7 14 21 7843 6089 3936 5122 6047 3402 4907 4294 2441 3877 • • • 5559 3648 4 4 0 7 167 .19 189.70 99.96 8 6 . 4 3 95.80 70.98 91.29 6 2 . 0 1 92.68 7 5 . 6 8 86 .04 *Q - s ignif icant difference according to Student-Newman-Keuls1 test 91 F I G . 21. EFFECT OF MALEIC HYDRAZIDE ON ENZYMES OF SUCROSE 220 r SYNTHESIS AND HYDROLYSIS IN THE LEAF OF . SUGAR BEET 180 OF 140 CONTROL 100 60 20 SUCROSE PHOSPHATE SYNTHETASE ./' * V SUCROSE SYNTHETASE. / * UDPG-PYR0PH05PH0RYLA5F"" INVERTASE I 7 14 DAYS AFTER TREATMENT 21 F I G . 22. EFFSCT OF PYROCATECHOL ON ENZYMES OF SUCROSE SYNTHESIS AND HYDROLYSIS IN THE LEAF OF SUGAR BEETS OF 220 180 140 100 CONTROL 60 20 V SUCROSE PHOSPHATE .SYNTHETASE V : SUCROSE SYNTHETASE ^..y. —. v' '•^OJDPG-PYROPHOSPHORYLASS X J L c-INVSRTASE 14 I 21 DAYS AFTER TREATMENT :93; FIG. 23. EFFECT OF VANADIUM SULFATE ON ENZYMES OF SUCROSE SYNTHESIS 220 180 % OF 140 CONTROL 100 AND HYDROLYSIS IN THE LEAF OF SUGAR BEET UDPG -PYR0PH0SPH0RYLA5E / / / X / / ^ 5UCR0SE SUCROSE ••' PHOSPHATE .SYNTHETASE SYNTHETASE 6 0 INVERTASE 20 J — J 7 14 DAYS AFTER TREATMENT . . J 21 94 FIG. 24. EFFECT OF MALEIC' HYDRAZIDE ON ENZYMES OF SUCROSE SYNTHESIS AND HYDROLYSIS IN THE ROOT OF SUGAR-' . BEET OF 220 180 140 loq CONTROL 6d 20 UDPG-PYROPHOSPHORYLASE SUCROSE PHOSPHATE SYNTHETASE >R^ OS E / / X V / SUCR03 / SYNTHETASE / / // •o-< \ \ A INVERTASE 7 14 DAYS AFTER TREATMENT 21 FIG. 2<. EFFECT OF PYROCATECHOL ON EN2YME5 OF SUCROSE SYNTHESIS AND HYDROLYSIS IN THE ROOT OF SUGAR BEET 220 180 OF 140 CONTROL 100 // // / / // SUCROSE PHOSPHATE SYNTHETASE UDPG- \ PYROPHOSPHORYLASE \ \ \ \ r _ . \ 5UCR0SE SYNTHETASE \ V V V \ V 60 -o. INVERTASE \ 40 J - ! „ 7 14 DAYS AFTER TREATMENT 21 96 F I G . 26. EFFECT OF VANADIUM SULFATE ON ENZYMES OF SUCROSE SYNTHESIS - AND HYDROLYSIS IN THE ROOT OF SUGAR BEET '220 180 OF 140 CONTROL 10D 60 SUCROSE PHOSPHATE ..•*>" SYNTHETASE O^UDPG - V / / ' PYR0PH05PH0RYLASE / / \ \ / / . * \ / / • ^-•'""SUCROSE V —^, / / A SYNTHETASE "•. \ / / ' / / . 0 * • INVERTASE 7 14 DAYS AFTER TREATMENT 21 TABLE X I E f f e c t o f m a l e i c h y d r a z i d e (MH), p y r o c a t e c h o l ( P C ) , and vanadium s u l f a t e (VS) on the a c t i v i t y of phosphatases o f l e a f and r o o t o f 4 . g - m o n t h - o l d sugar beet p l a n t s P l a n t Days a f t e r S p e c i f i c a c t i v i t y Enzyme p a r t t reatment (mumoles P i / m g p r o t e i n ) *Q T / C (%) * * C K MH PC VS •05 . 0 1 MH PC VS P h e n y l - L e a f 0 312.24 • • • • • • • • • 1 3 . 5 8 15.41 phosphatase 148.69 it it 7 1 9 5 . 8 0 181.02 1 2 2 . 4 7 75.94 92.44 6 2 . 8 1 n it 14 293 .64 104.76 2 2 1 . 4 4 1 5 1 . 5 3 3 5 . 6 7 75.41 5 7 . 7 2 ii ti 21 2 7 5 - 7 5 245.10 248.00 1 6 5 . 6 8 8 8 . 7 8 9 2 . 0 2 6 0 . 2 5 it Root 0 61.53 « • • • * • • 4 • 1 . 9 5 2 . 2 1 n II 7 42.35 26 .17 29.99 3 5 - 3 0 61 .79 70.81 8 3 . 3 6 II II 14 36.84 34.13 34 .37 32.42 92.65 93.28 8 7 . 9 9 II II 21 40.31 29.32 13.37 33.29 7 2 . 7 4 33.16 8 2 . 6 0 Adenos ine L e a f 0 5 3 8 . 7 0 • • • • • • • • • 14.20 1 6 . 1 1 t r i -phosphatase 7 249.20 2 4 3 . 6 5 147.84 2 3 7 . 8 6 97.77 5 9 . 1 7 9 5 . 5 3 n it 14 5 2 5 . 2 2 2 0 6 . 3 4 117.73 427 .35 39.28 22 .41 81.37 a ti 21 570 .15 508 .24 427 .35 466.03 89 .14 7 4 . 9 4 8 I . 7 6 ti ROOt 0 46.74 • • • • • • • • • 1.94 2.19 tt ti 7 15.73 13.50 14.47 I I . 8 5 8 5 . 8 7 92.01 7 5 - 3 8 it it 14 1 1 . 6 1 1 0 . 8 5 8 . 2 7 8 . 8 0 93.43 71.23 75.81 . ti it 21 2 1 . 0 5 2 3 . 2 1 1 3 . 7 8 14.33 1 1 0 . 2 5 :65 .44 6 8 . 7 0 G l u c o s e - 1 - L e a f 0 3 6 1 . 5 0 • • • • • • • • • • 3 0 . 5 0 34.62 phosphatase ti 133 .48 41.82 it it 7 3 9 1 . 2 5 104.64 7 2 . 9 8 32 .77 2 2 . 8 5 it II 14 1 3 8 . 5 0 1 3 2 . 0 3 1 3 8 . 8 5 84.07 9 5 . 3 2 100 .24 6 0 . 7 0 it ti 21 1 0 9 . 6 8 7 6 . 9 5 108.92 8 5 . 0 7 7 0 . 1 6 99.38 *Q,. - S i g n i f i c a n t d i f f e r e n c e a c c o r d i n g to Student-Newman-Keuls* t e s t . * * C K - C o n t r o l TABLE X I ( c o n t ' d ) E f f e c t o f m a l e i c h y d r a z i d e (MH), p y r o c a t e c h o l ( P C ) , and vanadium s u l f a t e (VS) on the a c t i v i t y o f phosphatases o f l e a f and r o o t o f 4 . 5 - m o n t h - o l d sugar beet p l a n t s P l a n t Days a f t e r S p e c i f i c a c t i v i t y Enzyme p a r t t rea tment (mumoles P l / m g p r o t e i n ) T / C {%) •'••CK MH PC VS . 0 5 . 0 1 MH PC VS G l u c o s e - 1 - Root 0 64 .26 • • • • • • • • • 2 . 3 2 2 . 6 3 phosphatase ti it 7 49.90 2 6 . 5 1 48 .36 5 0 . 2 6 5 3 . 1 2 9 6 . 9 1 100.71 it it 14 48 .36 21.54 3 6 . 2 7 4 7 . 3 1 4 4 . 5 5 6 2 . 6 0 97.84 it tt 21 3 9 . 2 5 18 .44 1 9 . 0 6 3 8 . 5 2 46 .99 48 .56 9 8 . 2 9 G l u c o s e - 6 - L e a f 0 3 4 3 . 8 0 • • • • • • • • • 2 0 . 3 9 2 3 . 1 3 phosphatase it it 7 2 6 0 . 7 7 1 2 2 . 1 9 1 0 0 . 3 6 1 8 6 . 6 3 46 .85 42 .38 7 1 . 5 7 tt tt 14 145.25 l 6 2 . l l l 4 l . 0 1 9 0 . 4 3 111 . 16 9 7 . 0 7 6 2 . 2 6 tt tt 21 119.09 1 0 5 . 8 5 1 2 5 . 1 8 7 3 . 6 2 88.28 1 0 5 . 1 2 61.82 " Root 0 48 .02. • • • • • • • • • 1.54 1 .75 it tt 7 26.45 2 7 . 0 7 1 7 . 0 7 1 7 . 9 1 1 0 2 . 3 3 64.54 6 1 . 7 2 tt ti 14 29 .90 26.08 12.04 18 .73 8 7 . 2 2 40 .27 62.64 tt tt 21 45.53 I 8 . 6 3 28 .08 3 6 . 7 7 40 .93 63.80 7 8 . 1 0 F r u c t o s e - 6 - L e a f 0 1 8 6 . 1 9 • • • • • • • • • 2 5 . 9 1 2 9 . 4 0 phosphatase 9 6 . l l 5 7 . 6 1 tt tt 7 1 9 7 . 5 8 1 8 9 . 9 0 1 1 3 . 9 9 1 3 8 . 9 5 7 0 . 3 3 it it 14 1 5 5 . 4 9 1 3 3 - 4 2 1 3 7 . 6 0 118.35 85 .8O 8 8 . 4 9 76.14 , tt tt 21 3 6 0 . 0 0 3 2 0 . 0 1 388.11 3 0 0 . 6 3 8 8 , 8 9 1 0 7 . 8 0 8 3 . 5 1 " Root 0 5 0 . 0 9 • • • • • • • • • 2 . 5 9 ' 2 . 9 4 it it 7 . 1 2 . 4 7 1 2 . 5 6 10.82 ' 7 . o i - IOO .73 8 6 . 7 6 5 6 . 2 9 it tt 14 1 7 . 3 8 1 3 . 8 6 1 3 . 1 0 i o . 8 7 7 9 . 6 5 7 5 . 3 8 6 2 . 5 8 it ti 21 1 3 . 4 7 10.86 1 5 . 1 2 SO? 80.64 1 1 2 . 2 8 6 1 . 8 3 . 99 the control for VS. The maximum inhibit ion of the root phos-phatase was on the 7th day by MH and on the 21st day by VS and PC. The ac t iv i ty of ATP-ase was s ignif icantly lower in the leaves of the treated plants except 7 days after treatment in MH-treated plants where the act iv i ty was not s ignif icantly different from the control plants. The inhibit ion caused by MH was 6l% and hy PC, 7&% on the l4th day after treatment. The act iv i ty of root ATP-ase in the treated plants was also s ignif icantly lower than the control plants except on the 14th and 21st day in MH- and the 7th day In 'PC-treated plants. The act iv i ty of glucose-l-phosphatase in the leaf of MH-treated plants was decreased. However, the decreases were not significant s ta t i s t i ca l l y on the 14th and the 21st day. The difference between glucose-l-phosphatase act iv i ty in the leaves of PC-treated and the control plants was not significant on the 14th day. PC-treated plants had lower, glucose-l-phosphatase act iv i ty in their leaves on the 7th and the 21st day. VS-treat-ed plants had s ignif icantly lower act iv i ty of glucose-l-phos-phatase on the 7th and the l4th day. The maximum inhibit ion of glucose-l-phosphatase act iv i ty was about 77% "by MH on the 7th day after treatment. The root glucose-l-phosphatase act iv i ty was signif icantly lower in MH-treated plants on a l l the dates of harvest. In PC-treated beets, the root glucose-l-phosphatase was inhibited s ignif icantly on the l4th and the 21st day after treat-ment. VS-treated plants had no significant effects on glucose-l-phosphatase act iv i ty of the roots. There was a significant decrease in glucose-6-phosphatase act iv i ty in the leaves of the VS-treated plants. The values for . 100 glucose-6-phosphatase ac t iv i ty were 71•6%, 62.3% and 61.8$ of the control values in the leaves of VS-treated plants. There was considerable decrease in the glucose-6-phosphatase act iv i ty in the leaves of MH-, and PC-treated plants 7 days, after the treat-ment but on the subsequent days the values in the treated plants were not s ignif icantly different from the control plants. The glucose-6-phosphatase ac t iv i ty in the root of the MH-treated plants was s ignif icantly lower than the control plants on the l4th and the 21st day after treatment. The maximum inhibit ion;of glucose-6-phosphatase ac t iv i ty , 60$ was caused by PC on the 14th day after treatment. VS inhibited fructose-6-phosphatase act iv i ty in the root and the leaf on a l l the dates of harvest. The maximum decrease of the fructose-6-phosphatase act iv i ty in the leaf of VS-treated plants was 30$ on the 7 t h day. PC also caused the inhibit ion of the act iv i ty of fructose-6-phosphatase in the leaves and the roots. The maximum inh ib i -t ion in the leaf was 42 .4$ on the 7 t h day and in the root 25$ on the 14th day in PC-treated plants. MH signif icantly inhibited the act iv i ty of fructose-6-phos-phatase in the leaf on the l4th day only and in the root on the l4th and 21st day. •The maximum Inhibition of the leaf fructose-6-phosphatase by MH was 11.1$ on the 21st day after treatment. Inhibition in the root up to 20$ was achieved by MH on the l4th and 21st day after the treatment. 9. UDPG-pyrophosphorylase UDPG-pyrophosphorylase ac t iv i ty was stimulated signif icantly 101 by MH and VS on a l l days of harvest (Table XII) . PC-treated plants showed low UDPG-pyrophosphorylase act iv i ty in the leaf on the 7 t h day and in the root on the 21st day after treatment. Maximum stimulation, up to 48 .7$, of UDPG-pyrophosphorylase ac t iv i ty of the leaf by MH was on the 7 t h day. The maximum stimulation of the leaf UDPG—pyrophosphorylase in PC- and V S -treated plants were 5 ° . 3 and 95•7% respectively on the 14 th day after treatment. Compared to untreated, more than double act iv-i ty was recorded in the root on the l4th day in case of MH-treat-ed and VS-^reated plants, and on the 7 t h day in PC-treated plants. 10. Enzymes of sucrose synthesis The presence of the enzymes sucrose synthetase and sucrose phosphate synthetase in the sugar beet leaf and root has been shown by Rorem et a l . (I960) and Dutton et a l . ( 1 9 6 1 ) . Accord-ing to the results obtained in the present investigation the specific act iv i ty of the sucrose synthesizing enzymes, sucrose synthetase and sucrose phosphate synthetase, was. greater in the leaf than in the root of both control and treated sugar beet plants (Table XIII) A l l three treatments stimulated the act iv i ty of both sucrose synthetase and sucrose phosphate synthetase very s i g n i f i -cantly. The act iv i ty of sucrose phosphate synthetase in the leaf of a l l treated plants was approximately double that of the control plants on the l4th day after treatment, the actual stimulation percentages being 104, 116 and 79 for MH, PC and VS respectively. TABLE X I I E f f e c t o f m a l e i c h y d r a z i d e (MH), p y r o c a t e c h o l ( P C ) , and vanadium s u l f a t e (VS) on UDPG-pyrophosphory lase a c t i v i t y o f r o o t and l e a f o f 4 .5 -month-o ld sugar beet p l a n t s  Days a f t e r S p e c i f i c a c t i v i t y P l a n t p a r t t r ea tment (mumole G - l - P / m g o f p r o t e i n ) *Q T / C {%)  *»CK MH PC VS j_0J .01 MH PC VS L e a f 0 137 -72 . . . . . . . . . 12.78 13.08 7 110.54 164.25 8 5 . 9 9 199.72 148.68 77.79 1 8 0 . 6 7 14 74.17 98.93 115.94 145.16 133.38 1 5 6 . 2 9 195.69 21 98.59 120.95 116 .12 177.28 122.67 117.78 179.81 Root 0 1 6 8 . 8 9 . . . . . . . . . 16.97 19.26 7 79.28 9 8 . 7 8 168.43 151.52 124.59 212.45 191.12 14 60.72 142.50 103 .39 140.05 234 .66 170.27 230.62 21 98.17 142.28 79.40 119.69 145.13 80.88 121.92 *Q - S i g n i f i c a n t d i f f e r e n c e a c c o r d i n g to Student -Newman-Keuls 1 t e s t * * C K - C o n t r o l TABLE XIII Effect of maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulfate (VS) on sucrose-P-synthetase and sucrose synthetase of root and leaf of 4.5-month-old sugar beets Plant Days after Specific ac t iv i ty Enzyme part treatment (mumol es sucro se/mg protein) *Q T/C (%) **CK MH PC VS . 05 . 0 1 MH PC VS Sucrose-P- Leaf 0 2 0 7 . 7 8 • • • • • • • • • 4 . 5 9 5 .21 synthetase 2 5 6 . 9 8 2 1 3 . 8 6 it tt 7 202 .41 242.03 1 2 6 . 9 6 1 1 9 . 5 7 1 0 5 . 6 5 ti n 14 1 2 6 . 1 9 2 5 8 . 8 3 2 7 3 . 1 5 226 .46 204.95 216 .46 179 .46 it II 21 1 1 3 . 8 6 1 9 6 . 0 9 114.54 144.92 1 7 2 . 2 1 IOO.59 1 2 7 . 3 2 it Root 0 1 9 . 6 2 • • • • * • • • • 2 . 0 9 2 . 3 9 n II 7 1 1 . 3 1 24.45 2 3 . 3 1 24.24 216 .24 2 0 6 . 0 1 214.34 it II 14 1 3 . 4 0 2 5 . 9 0 2 6 . 9 9 24.35 193 .04 2 0 1 . 3 7 1 8 1 . 6 8 ti It 21 18.37 2 2 . 9 2 2 2 . 3 2 2 2 . 0 2 124.78 1 2 1 . 5 2 1 1 9 . 8 8 Sucrose Leaf 0 6 1 . 6 9 • • • • * • • • • 7 . 0 1 7 . 9 6 synthetase it II 7 6 1 . 6 2 6 7 . 2 7 8 2 . 9 5 9 7 . 8 7 1 0 9 . 1 7 1 3 4 . 6 1 1 5 8 . 7 8 it it 14 7 2 . 0 5 1 2 3 . 4 9 81.65 I O 4 . 5 O 1 7 1 . 3 9 113 .33 145.04 " II 21 57*6? 92.80 9 0 . 9 2 118.02 1 6 0 . 6 8 157.64 204.63 it Root 0 4 7 . 6 0 • • • • • * • • • 0 . 9 2 1.04 it it 7 2 7 . 4 3 33.89 33.65 38 .55 1 2 3 . 5 9 1 2 2 . 6 9 140.54 it it 14 55.49 1 0 9 . 7 4 8 5 . 7 7 8 9 . 7 3 1 9 7 . 7 8 154.82 1 6 1 . 7 1 it it 21 4 7 . 0 7 61.64 5 1 . 9 5 7 0 . 0 0 1 3 0 . 9 4 IIO .37 148.87 *Q - Significant difference according to Student-Newman-Keuls' test **CK - Control o to i:o4 The maximum stimulation of root sucrose phosphate synthetase ac t iv i ty , 116%, was induced by 'MH on the 7th day after treatment. The stimulated sucrose synthetase ac t iv i ty in the leaves and roots of MH-treated plants followed the same pattern as that of sucrose phosphate synthetase, reaching a maximum level on the 14th day (Fig. 21 and 2 4 ) . Although both PC and VS treatment s ignif icantly stimulated the ac t iv i ty of sucrose synthetase in the leaves, the maximum occurred on the 21st day, and the curves for the ac t iv i ty of the two enzymes dif fered. (Fig. 22 and 2 3 ) . The act iv i t i e s of sucrose synthetase in roots of VS-treated plants are shown in F i g . 26, and of PC-treated plants in F i g . 2 5 . The effects of MH, PC, and VS on the act iv i ty of various enzymes have been summarized in Table XIV. 11. Simple correlation coefficients The simple correlation coefficients have been determined to f ind out the relationship between the groifth of the leaf and the various parameters measured after the treatment. These.are given in Table XV. The data indicate that growth of the leaf in MH-treated plants was posit ively and signif icantly correlated with the reducing sugars, n i t r i t e , n i trate , amino acids, protein and the total N content of the roots. It was also posit ively corre-lated with photosynthesis, respiration of the root, nitrate reduc-tase (leaf and root) , transaminase (root) and invertase act iv i ty (root and leaf ) . In PC-treated plants the growth was correlated s ignif icant-ly and posit ively with reducing sugars, n i t r i t e , . n i t r a t e and protein content of the root and nitrate reductase, transaminase 105 TABLE X I V Summary of the e f f e c t s o f m a l e i c h y d r a z i d e '(MH), p y r o c a t e c h o l (PC) and vanadium s u l f a t e (VS) on enzyme a c t i v i t i e s Enzyme \ ^ E f f e c t s of t rea tment w i t h  MH PC - VS N i t r a t e r e d u c t a s e L e a f II Root Transaminase L e a f II Root ns I n v e r t a s e L e a f ti Root P h e n y l -phosphatase L e a f ti Root Adenos ine t r i -phosphatase L e a f ns - - - + - - -tt Root - -. + ns - - - ns -G ' l u c o s e - 1 -phosphatase L e a f - ns ns - ns - - - ns II Root - - - ns - - ns ns ns G l u c o s e - 6 -phosphatase L e a f - ns ns - ns +ns - - -II Root ns F r u c t o s e - 6 -Phosphatase L e a f ns - - ns - + - -II Root ns - - - ns - - + -UDGP-pyro-p h o s p h o r y l a s e L e a f + + + - + + + + + II Root + + + + + - + + + Sucrose s y n t h e t a s e L e a f ns + + + + + + + + it Root + + + + + + + + + S u c r o s e - P -phosphetase L e a f + + + + + + + + it Root + + + + + + + + + *The symbols +, - , r e f e r to s t i m u l a t i o n and i n h i b i t i o n . Three symbols f o r each t rea tment c o r r e s p o n d to the h a r v e s t made 5 . 14, and 21 days a f t e r t r e a t m e n t . ns - . means not s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l a t 0 .05 and 0 . 0 1 . TABLE XV Relationship of growth to different variables Correlation coefficients ( r ) Variables MH PC VS Growth 1.00 1 .00 1 .00 Sucrose n .s . n . s . n .s . Reducing sugar 0 .627 0 .577 0 .878 Nitr i te 0 .612 0 .583 0 . 5 7 9 Nitrate 0 .981 0 .872 • n.s . Ammonia - 0 . 6 9 1 - 0 . 7 5 6 - 0 . 6 5 9 Amino acids 0.828 n.s . n .s . Protein 0.946 0.844 0 .815 Total N 0 .874 n.s . 0 .990 Photosynthesis O .905 n.s . n.s . Respiration (root) O .851 n.s . 0 .791 Nitrate reductase (leaf) O.698 0 .907 0 .817 " " (root) 0.628 0 .585 0 .727 Transaminase (leaf) n.s . n.s . n.s . » " (root) 0.646 n.s . n .s . Phenyl phosphate (leaf) n .s . - 0 . 7 7 9 n.s . " " (root) - 0 . 8 9 0 -0.906 -O . 856 ATP-ase (leaf) n.s . n .s . n .s . •» (root) -0.618 - 0 . 9 1 7 - 0 . 7 9 1 Glucose-l-phosphatase (leaf) -0.849 -0.946 - 0 . 8 8 6 " » (root) -O . 893 -O . 898 - 0 . 9 2 5 Glucose-6-phosphatase (leaf) - 0 . 8 6 9 - 0 . 5 7 5 - 0 . 7 2 8 " » (root) -0.913 -O . 670 n.s . Fructose-6-phosphatase(leaf) - 0 . 8 6 8 -O .76O - 0 . 8 7 5 " »' (root) -0.841 -O . 887 - 0 . 8 7 5 Invertase (leaf) • 0 803 0 .866 0 .755 " (root) 0 . 8 0 0 0 .725 0 .765 n.s . - not significant at .05 level Significant correlation coefficient ( . 0 5 ) = O .576 3.0? and i n v e r t a s e a c t i v i t y of the r o o t and the l e a f . I n V S - t r e a t e d p l a n t s , the growth was p o s i t i v e l y and s i g n i f -i c a n t l y c o r r e l a t e d w i t h r e d u c i n g s u g a r s , n i t r i t e , p r o t e i n , the t o t a l N c o n t e n t and r e s p i r a t i o n o f the r o o t . The growth was a l s o p o s i t i v e l y c o r r e l a t e d w i t h the a c t i v i t y o f the enzymes n i t r a t e r e d u c t a s e and t ransaminase (Tab le X V ) . The s u c r o s e c o n c e n t r a t i o n i n the r o o t o f M H - t r e a t e d bee t s was s i g n i f i c a n t l y and p o s i t i v e l y c o r r e l a t e d w i t h the n i t r a t e and amino a c i d c o n t e n t s o f the r o o t , p h o t o s y n t h e s i s , and sucrose phosphate s y n t h e t a s e a c t i v i t y . I t was s i g n i f i c a n t l y and i n v e r s e -l y c o r r e l a t e d w i t h the p r o t e i n c o n t e n t and the r a t e o f r e s p i r a -t i o n , A T P - a s e , g l u c o s e - l - p h o s p h a t a s e , g l u c o s e - 6 - p h o s p h a t a s e , f r u c t o s e - 6 - p h o s p h a t a s e , i n v e r t a s e , and n i t r a t e r e d u c t a s e a c t i v -i t y o f the r o o t and the l e a f (Table X V I ) . The sucrose c o n c e n t r a t i o n o f the r o o t o f the P C - t r e a t e d p l a n t s showed s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n s w i t h the a c t i v -i t y o f sucrose phosphate s y n t h e t a s e and n e g a t i v e c o r r e l a t i o n s w i t h p r o t e i n , r e d u c i n g sugars and ammonium c o n t e n t o f the r o o t . There was a l s o a s i g n i f i c a n t c o r r e l a t i o n between the s u c r o s e c o n t e n t o f the r o o t and the a c t i v i t y o f the enzyme, A T P - a s e , g l u c o s e - 6 - p h o s p h a t a s e , f r u c t o s e - 6 - p h o s p h a t a s e , i n v e r t a s e , n i t r a t e r e d u c t a s e and t ransaminase (Tab le X V I ) . V S - t r e a t e d p l a n t s had s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n s between sucrose and the n i t r a t e c o n t e n t o f the r o o t and sucrose phosphate s y n t h e t a s e a c t i v i t y o f the r o o t and the l e a f . The sucrose c o n t e n t a l s o h a d . s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n s w i t h p r o t e i n c o n t e n t and the r e s p i r a t i o n o f the r o o t , a l l the p h o s -phatases measured (except f r u c t o s e - 6 - p h o s p h a t a s e ) , I n v e r t a s e , 108 n i t r a t e r e d u c t a s e and transaminase a c t i v i t y o f the r o o t and the l e a f (Tab le X V I ) . 109 TABLE XVI Relationship of sucrose to different variables (Storage root) Correlation coefficients (r) Variables MH. PC VS Sucrose 1.00 1 .00 1 .00 Reducing sugar n.s . rus. n. s. Ni tr i te n.s . n .s . n.s . Nitrate 0 .780 n.s. O.966 Ammonia n .s . - 0 . 8 3 9 n.s . Amino acids 0 .838 0 .913 n.s . Protein - 0 . 8 0 9 - 0 . 7 3 7 - 0 . 8 9 2 Total N n.s . n.s . 0 .587 Photosynthesis 0 .723 n.s . n.s . Respiration (root) - 0 . 9 6 0 n.s . - 0 . 8 5 5 Phenyl phosphatase (leaf) - 0 . 8 8 0 n.s . - 0 . 8 3 4 » " (root) -O . 836 n.s . - 0 . 9 0 5 ATP-ase (leaf) - 0 . 7 5 8 -0.845 -0.666 " (root) - 0 . 9 7 7 -O . 656 - 0 . 9 4 7 Glucose-l-phosphatase (leaf) - 0 . 9 1 9 n.s . - 0 . 9 0 9 11 " (root) -0.945 n.s . -O . 632 Glucose-6-phosphatase (leaf) -0.881 - 0 . 7 9 8 - 0 . 7 8 7 » " (root) - 0 . 8 5 9 -O . 883 - 0 . 9 7 2 Pructose-6-phosphatase(leaf) -O . 859 n.s . -n . s . » « (root) - 0 . 7 8 2 -0.682 - 0 . 9 0 7 Invertase (leaf) - 0 . 9 2 7 - 0 . 8 2 2 - 0 . 8 7 7 (root) - 0 . 9 1 5 - 0 . 8 2 6 - 0 . 8 5 6 Nitrate reductase (leaf) - 0 . 9 7 3 - 0 . 6 7 2 - 0 . 9 1 2 " " (root) -O . 903 n.s . -0.954 Transaminase (leaf) n.s . -O . 765 -0.940 »• " (root) - 0 . 9 0 2 - 0 . 8 1 5 - 0 . 9 3 8 Sucrose-P-synthetase (leaf) - 0 . 5 7 8 - 0 . 6 9 2 - 0 . 7 3 8 " » (root) - 0 . 9 3 0 - 0 . 9 1 0 - 0 . 6 8 9 Significant correlation coefficient ( . 05 ) = 0 .576 n.s . - not significant at . 05 level 110 VI D i s c u s s i o n MH i n h i b i t e d the growth of the l e a v e s and caused them to become narrower and c u r l e d . The e f f e c t s appeared to be perma-n e n t . A s i m i l a r r e s u l t has been no ted by P e t e r s o n and N a y l o r (1953) i n t o b a c c o . The a u t h o r s sugges ted t h a t p o s s i b l y the m a r g i n a l m e r i s t e m a t l c a c t i v i t y was more e a s i l y i n h i b i t e d - by MH trea tment than the e a r l i e r d e v e l o p i n g m i d - r i b m e r i s t e m a t i c c e l l s , the r e s u l t b e i n g development o f narrow l e a v e s . The i n h i b i t o r y e f f e c t on NRase and t ransaminase a c t i v i t y i n v i v o , c o u p l e d w i t h lower p r o t e i n and h i g h e r amino a c i d c o n t e n t , suggest t h a t e i t h e r p r o t e i n h y d r o l y s i s , or i n h i b i t i o n o f the p r o t e i n s y n t h e s i s , or b o t h , occur under the c o n d i t i o n s o f MH i n h i b i t i o n . T a b l e XV l i s t s the c o r r e l a t i o n c o e f f i c i e n t s o f s e v e r a l v a r i a b l e s measured w i t h g r o w t h . Root and l e a f n i t r a t e r e d u c t a s e shows a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n c o e f f i c i e n t w i t h growth . Root t ransaminase a l s o has a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n c o e f f i c i e n t w i t h g r o w t h . In view of the f a c t t h a t g l u t a m i c a c i d or g lu tamine i s s y n t h e s i z e d m a i n l y i n the r o o t o f sugar beet (Joy , 1 9 6 7 ) , i t i s p r o b a b l e t h a t t r a n s a m i n a t i o n r e a c -t i o n s a r e more i m p o r t a n t f o r amino a c i d s y n t h e s i s i n the r o o t than i n the l e a v e s . The NRase a c t i v i t y i s r e l a t e d to p r o t e i n p r o d u c i n g p o t e n -t i a l o f the p l a n t s and i s i m p o r t a n t f o r the maintenance o f the c o n t i n u e d growth , have been demonstrated by s e v e r a l w o r k e r s . F o r example, Croy (1967) found t h a t NRase a c t i v i t y was l i n e a r l y r e l a t e d to the t o t a l g r a i n p r o t e i n p r o d u c t i o n i n wheat, w i t h i n a g i v e n genotype . F i l n e r (1966) demonstrated t h a t the r e p r e s s o r s I l l o f NRase o f tobacco c e l l s grown i n s u s p e n s i o n c u l t u r e , i n h i b i t e d the growth of the t i s s u e and d e r e p r e s s o r s p r e v e n t e d t h i s i n h i b i -t i o n . The v a r i a t i o n i n the l e v e l of NRase w i t h the age o f the t i s s u e (Shrader and Hageman, 1 9 6 7 ; W a l l a c e and P a t e , 1 9 6 7 ) and w i t h the p l a n t m a t u r a t i o n ( Z i e s e r l e t a l . 1 9 6 3 ; Yang , 1 9 6 4 ; W a l l a c e and P a t e , 1 9 6 7 ) a l s o suggested the r o l e o f NRase i n the r e g u l a t i o n of g r o w t h . The r e d u c t i o n i n sugar beet NRase a c t i v i t y by MH d u r i n g the p r e s e n t I n v e s t i g a t i o n can be e x p l a i n e d on the b a s i s o f the p o s s i b l e i n t e r a c t i o n w i t h the — SH groups o f the p r o t e i n ( I s e n b e r g , 1 9 5 1 ) « On the b a s i s o f h i s s t u d i e s w i t h C - ^ - M H , Nooden ( 1 9 6 7 ) r e p o r t e d t h a t MH can b i n d w i t h p r o t e i n s v e r y t i g h t l y . Thus , i n h i b i t i o n o f NRase by MH i s a l s o p o s s i b l e by the b i n d i n g o f MH w i t h the enzyme p r o t e i n . N i t r a t e r e d u c t a s e i s an i n d u c i b l e enzyme (Beevers e t a l . 1 9 6 5 5 F i l n e r , 1 9 6 6 ; Shrader and Hageman, 1 9 6 7 ) . Beevers e t a l . ( 1 9 6 5 ) found t h a t the i n h i b i t i o n of DNA-dependent RNA s y n t h e s i s r e s u l t e d i n the i n h i b i t i o n o f the i n d u c t i o n o f NRase. T h e n , t h e r e d u c t i o n i n the a c t i v i t y o f NRase c o u l d a l s o be p o s s i b l e by the i n h i b i t i o n of i t s s y n t h e s i s by MH, because MR i s known to i n h i b i t DNA s y n t h e s i s i n whole i n t a c t c o r n r o o t s i n about 1 6 h o u r s , and RNA s y n t h e s i s a few hours l a t e r (Nooden, 1 9 6 7 ) R e p r e s s i o n o f NRase s y n t h e s i s i s p o s s i b l e by ammonium i o n s ( S y r e t t and M o r r i s , 1 9 6 3 ) and amino a c i d s ( F i l n e r , 1 9 6 6 ) . A l a r g e r c o n t e n t o f ammonium and amino a c i d s a f t e r MH treatment was r e c o r d e d d u r i n g the p r e s e n t i n v e s t i g a t i o n . The p r o g r e s s i v e i n c r e a s e i n the s o l u b l e amino a c i d c o n t e n t of the r o o t s and the c o r r e s p o n d i n g decrease i n the n i t r a t e r e d u c t a s e a c t i v i t y o f the •112 l e a v e s and the r o o t s suggest the p o s s i b i l i t y t h a t amino a c i d s might be t a k i n g p a r t i n the i n h i b i t i o n o f n i t r a t e r e d u c t a s e s y n -t h e s i s . However, i t i s premature to suggest t h a t i n h i b i t i o n o f growth i s o n l y due to the e f f e c t o f MH on n i t r a t e r e d u c t a s e and t ransaminase a c t i v i t y i n sugar b e e t . S e v e r a l o t h e r types o f ' s i g n i f i c a n t e f f e c t s have been no ted d u r i n g t h i s i n v e s t i g a t i o n , and a l s o by the o t h e r s . MH was d i s c o v e r e d to have s i g n i f i c a n t e f f e c t s on the r a t e o f p l a n t r e s p i r a t i o n . The r a t e o f r e s p i r a t i o n o f bee t s torage r o o t and of l e a v e s d u r i n g t h i s i n v e s t i g a t i o n was r e d u c e d by 30 to 40$ and 40$ r e s p e c t i v e l y . The r e s p i r a t i o n o f the s t o r a g e r o o t was c l o s e l y c o r r e l a t e d w i t h the g r o w t h . A s i g n i f i c a n t c o r r e l a t i o n c o e f f i c i e n t , O . 8 5 I , was found between the r a t e o f r e s p i r a t i o n o f the r o o t and the growth o f the l e a v e s o f sugar beet (Tab le X V ) . I n h i b i t i o n o f the r a t e o f r e s p i r a t i o n f o l l o w i n g MH t r e a t -ment has been no ted a l s o by s e v e r a l w o r k e r s . The r e s p i r a t i o n o f r o o t t i p s o f a number o f p l a n t s p e c i e s has been found to be r e d u c e d , f o l l o w i n g MH t r e a t m e n t , and B r i a n (1964) c o n c l u d e d t h a t MH competes f o r a c t i v e s i t e s o f an enzyme concerned w i t h r e s p i r a -t i o n . MH i n t e r a c t s w i t h the t h i o l s , t h u s , the i n h i b i t i o n o f s u c c i n i c dehydrogenase i s p o s s i b l e ( B r i a n , 1964) a n d , i n f a c t p a r t i a l i n a c t i v a t i o n o f one or more dehydrogenase has been s u g -g e s t e d by I senberg et a l . (1951) a s a r e s u l t o f s t u d i e s on o n i o n . I n v e r t a s e a c t i v i t y of l e a f and r o o t o f sugar beet was i n h i b i t e d by MH. S i n c e i n v e r t a s e a c t i v i t y was found to be p o s i -t i v e l y c o r r e l a t e d w i t h growth o f the l e a v e s (Tab le X V ) , the observed growth i n h i b i t i o n by MH d u r i n g t h i s i n v e s t i g a t i o n may a l s o 113 be a t t r i b u t e d I n p a r t to the i n h i b i t o r y e f f e c t o f MH on i n v e r t a s e . That i n v e r t a s e a c t i v i t y was a s s o c i a t e d w i t h growth was a l s o demon-s t r a t e d by Wort and White ( 1 9 5 6 ) . In the work of these a u t h o r s the tops of mature f i e l d grown sugar beets were f r o z e n or removed by k n i f e and the l e a v e s a l l o w e d to r e g r o w . I n the e x t e n s i v e s t u d i e s o f the m e t a b o l i c changes i n sugar bee t l e u c o p l a s t s , S i s a k j a n and c o - w o r k e r s (19^8, 1 9 5 1 . 1953) found t h a t d u r i n g the v e g e t a t i v e p e r i o d o f the sugar beet p l a n t i n v e r t a s e a c t i v i t y o f l e u c o p l a s t s i n the r o o t i n c r e a s e d g r e a t l y , w h i l e i n the s t o r a g e r o o t s the i n v e r t a s e a c t i v i t y i n l e u c o p l a s t s d e c r e a s e d . ^ Hatch and G l a s z i o u (1963) found t h a t r a t e o f e l o n g a t i o n o f i n t e r n o d e s i n sugar cane remained c o r r e l a t e d w i t h a c i d i n v e r t a s e a c t i v i t y i r r e s p e c t i v e o f whether the independent v a r i a b l e was age o f t i s s u e , t e m p e r a t u r e , or water r e g i m e . Under e n v i o r n m e n t a l c o n d i t i o n s which gave r a p i d growth of immature i n t e r n o d e s , t h e r e was no ne t sugar s t o r a g e i n the mature i n t e r n o d e s o f the same s t a l k . The r e v e r s e was a l s o t r u e , i n d i c a t i n g t h a t growth and s t o r a g e a r e r e c i p r o c a l l y r e l a t e d , presumably because of c o m p e t i -t i o n f o r a v a i l a b l e p h o t o s y n t h a t e s . I n immature s torage t i s s u e of sugar cane , a v a i l a b i l i t y o f c a r b o h y d r a t e i n the c e l l c y t o p l a s m appears to be r e g u l a t e d by the l e v e l o f two a c i d i n v e r t a s e s , one l o c a t e d i n the o u t e r space (which i n c l u d e s the c e l l w a l l ) and the second i n the s t o r a g e compartment (Sacher e t a l . 1 9 6 3 ) . C o n t r o l o f i n v e r t a s e l e v e l appeared to these a u t h o r s to be mediated through a u x i n , which may i n c r e a s e or decrease the l e v e l depending on c o n c e n t r a t i o n , and by a f e e d back system i n v o l v i n g the l e v e l o f sugar p r e s e n t i n the m e t a b o l i c compartment ( c e l l c y t o p l a s m ) . 114 Work w i t h t i s s u e s l i c e s from r a p i d l y expanding i n t e r n o d e s o f sugar cane i n d i c a t e s t h a t the l e v e l o f i n v e r t a s e i s a f u n c t i o n o f the b a l a n c e between s y n t h e s i s and d e s t r u c t i o n o f m-RNA ( G l a s z i o u e t a l . 1 9 6 6 ) . From s t u d i e s w i t h i n h i b i t o r s of p r o t e i n and RNA s y n t h e s i s , G l a s z i o u and h i s coworkers (1966) c o n c l u d e d t h a t a u x i n i n c r e a s e s the r a t e o f s y n t h e s i s and g l u c o s e i n c r e a s e s the r a t e o f d e s t r u c t i o n o f messenger RNA r e q u i r e d f o r the p r o d u c -t i o n o f i n v e r t a s e . M a l e i c h y d r a z i d e might t h e n , cause a decrease i n the l e v e l of i n v e r t a s e ( i . e . , i t s s y n t h e s i s ) a c t i n g as an a n t i -a u x i n . That MH has an a n t i - a u x i n p r o p e r t y , has been demonstrated by L e o p o l d and K l e i n ( 1951 , 1 9 5 2 ) . I t has a l s o been r e p o r t e d to a c c e l e r a t e the o x i d a t i o n o f i n d o l e a c e t i c a c i d (Kenten , 1 9 5 5 ) • Kaufman et a l . (1968) found t h a t the i n c r e a s e i n i n v e r t a s e a c t i v i t y c l o s e l y p a r a l l e d the growth p r o m o t i o n t h a t was caused by g i b b e r e l l i c a c i d (GA^) i n Avena stem segments i n c u b a t e d i n the d a r k a t 2 3 ° C . C y c l o h e x i m i d e , an i n h i b i t o r of p r o t e i n s y n t h e s i s a b o l i s h e d a l l GA-^-promoted growth and i n v e r t a s e a c t i v i t y i n Avena stem segments . T h i s a g a i n r e v e a l s the f a c t t h a t i n v e r t a s e a c t i v -i t y i s a s s o c i a t e d w i t h the growth and MH can i n h i b i t i t by i t s a n t i - a u x i n p r o p e r t y or through the i n h i b i t i o n o f DNA-dependent RNA s y n t h e s i s r e q u i r e d f o r p r o t e i n s y n t h e s i s . MH can i n h i b i t growth perhaps through s e v e r a l o t h e r s i t e s . F o r example, I t o and Y o s h i n a k a (1964) found t h a t RNA and p r o t e i n c o n t e n t i n M H - t r e a t e d o n i o n was r e d u c e d . Nooden (1967) found t h a t MH was bound s t r o n g l y to p r o t e i n w i t h i n the p l a n t . T h i s might e x p l a i n the i n h i b i t i o n o f s e v e r a l enzymes by m a l e i c h y d r a -z i d e . P o v o l o t k a y a ( 1 9 6 l ) o b s e r v e d t h a t u r a c i l r e s t o r e d normal growth and development of M H - t r e a t e d p l a n t s . He suggested t h a t 115 MH f u n c t i o n s as a u r a c i l a n t l m e t a b o l i c . M c l e i s h (1953) r e p o r t e d t h a t m i t o s i s i n a l l a c t i v e l y growing t i s s u e i n a v a r i e t y o f p l a n t s p e c i e s was suppres sed by MH.. T h i s o b s e r v a t i o n i s c o n s i s t e n t w i t h the f a c t t h a t DNA s y n t h e s i s i s i n h i b i t e d by MH (Nooden, 1 9 6 7 ) . Thus MH can i n h i b i t growth by i n h i b i t i n g c e l l d i v i s i o n and a l s o a u x i n - i n d u c e d c e l l e l o n g a t i o n . P h e n y l p h o s p h a t a s e , A T P - a s e , g l u c o s e - l - p h o s p h a t a s e . , g l u c o s e -6 - p h o s p h a t a s e , and f r u c t o s e - 6 - p h o s p h a t a s e were i n h i b i t e d by MH, Moreover , s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n s e x i s t between growth and these a c t i v i t i e s . Thus , the h y d r o l y s i s o f these s u b s t r a t e s (pheny lphosphate , ATP, g l u c o s e - l - p h o s p h a t e and g l u c o s e s - p h o s -phate ) does not f a v o r growth i n m a l e i c h y d r a z i d e - t r e a t e d p l a n t s . Maevskya and A l e k s e e v a (1964) observed i n c r e a s e d ATP-ase a c t i v i t y i n a p i c a l buds of boron d e f i c i e n t sunf lower p l a n t s b e f o r e s i g n s o f d e f i c i e n c y were v i s i b l e . Hinde and F i n c h (1966) noted i n c r e a s e d a c t i v i t y of pheny lphosphatase and ATP-ase i n boron d e f i c i e n t bean r o o t s . These works demonstrate t h a t the i n c r e a s e i n a c t i v i t y o f these enzymes i s a s s o c i a t e d w i t h the decrease i n the growth . T h i s i s u n d e r s t a n d a b l e , because hexose phosphates a r e a c t i v e i n t e r m e d i n a t e s o f the r e s p i r a t o r y pathways . T h e i r removal from the pathway might cause an i n h i b i t i o n o f the r e s p i r a t i o n through g l y c o l y s i s and the K r e b s . c y c l e . The i n h i b i t i o n o f r e s p i r a t i o n w i l l l e a d to c e s s a t i o n o f g r o w t h . ATP i s r e q u i r e d f o r the a c t i v a t i o n o f amino a c i d s f o r p r o t e i n s y n t h e s i s . H i g h r a t e o f the h y d r o l y s i s o f ATP by ATP-ase may cause an i n h i b i t i o n o f the p r o t e i n s y n t h e s i s and hence g r o w t h . The i n h i b i t i o n o f phosphatases by m a l e i c h y d r a z i d e i s p o s s i b l e e i t h e r by i t s b i n d i n g w i t h the p r o t e i n o f these enzymes 11.6 or by d i m i n u t i o n o f the DNA and RNA r e q u i r e d f o r the s y n t h e s i s o f the enzyme p r o t e i n s . PC was found to i n h i b i t growth of l e a v e s more e f f e c t i v e l y t h a n MH and V S . L i k e M H . i t a l s o i n h i b i t e d n i t r a t e r e d u c t a s e , t r a n s a m i n a s e , i n v e r t a s e and p h o s p h a t a s e s , but no t r e s p i r a t i o n , r a t h e r i t s t i m u l a t e d the r e s p i r a t i o n o f l e a v e s (dark r e s p i r a t i o n ) by 44$ on the 7 t h day a f t e r t r e a t m e n t . The r e s p i r a t i o n o f r o o t was a l s o s l i g h t l y s t i m u l a t e d . The growth i n P C - t r e a t e d p l a n t s was s i g n i f i c a n t l y c o r r e l a t e d w i t h r e d u c i n g s u g a r s , n i t r i t e , amino a c i d s and p r o t e i n c o n t e n t o f the r o o t and a c t i v i t y o f i n v e r t a s e , n i t r a t e r e d u c t a s e and t r a n s a m i n a s e . In these r e s p e c t s , r e s u l t s of PC trea tment c l o s e l y resemble those o b t a i n e d by MH. Yang (1964) d e m o n s t r a t e d , i n v i t r o , t h a t sugar beet n i t r a t e r e d u c t a s e was i n h i b i t e d c o m p l e t e l y by PC a t a c o n c e n t r a t i o n o f 1 x 1 0 " ^ M. N i t r a t e r e d u c t a s e i s a s u l f h y d r y l c o n t a i n i n g enzyme. I n a c t i v a t i o n o f t h i s , and o t h e r enzymes r e s p o n s i b l e f o r p r o t e i n s y n t h e s i s and growth r e g u l a t i o n i s p o s s i b l e , i f one c o n s i d e r s PC to be an o - d i p h e n o l , e a s i l y o x i d i z e d by o - d i p h e n o l : 0 2 o x i d o -r e d u c t a s e ( o - d i p h e n o l o x i d a s e ) to q u i n o n e . O - d i p h e n o l ox idase i s v e r y commonly found i n h i g h e r p l a n t s ( S h i r o y a et a l . 1 9 5 5 ; C l a y t o n , 1964 ; P i e r p o i n t , 1 9 6 6 ) . That t a n n i n s and qu inones i n h i b i t enzymes through the - S H group was shown by Mason, 1955 ; Young, 1965 ; and S l a c k , 1 9 6 6 . Anderson and.Rowan (1967) found t h a t t h i o l s or o t h e r r e d u c i n g agents p r o t e c t enzymes c o n t a i n i n g - S H group from i n a c t i v a t i o n by qu inones and t a n n i n s . T r a n s a m i n a s e , i n v e r t a s e and phosphatase may be i n h i b i t e d by PC e i t h e r through the o x i d a t i o n o f t h e i r s u l f h y d r y l groups or through the b i n d i n g of PC or i t s po lymers to the p r o t e i n . 117 The i n h i b i t i o n o f n i t r a t e r e d u c t a s e s y n t h e s i s i n v i v o , by PC i s perhaps p o s s i b l e on the b a s i s o f the f a c t s t h a t v a r i o u s p l a n t pheno l s i n h i b i t p r o t e i n s y n t h e s i s i n e x c i s e d p l a n t t i s s u e s , (Parupus , I 9 6 7 ) and some of the p h e n o l i c compounds have been found to i n h i b i t the i n d u c t i o n o f n i t r a t e r e d u c t a s e i n c o r n (Shrader and Hageman, 1 9 6 7 ) . The p o s s i b i l i t y o f r e p r e s s i o n o f n i t r a t e r e d u c t a s e by ammonium and amino a c i d a l s o e x i s t s on the b a s i s o f i n c r e a s e d ammonium and amino a c i d c o n t e n t o f the r o o t s , and a concomi tant decrease i n NRase a c t i v i t y f o l l o w i n g PC t r e a t -ment, r e c o r d e d d u r i n g the p r e s e n t i n v e s t i g a t i o n . The h i g h r a t e of r e s p i r a t i o n i n P C - t r e a t e d p l a n t s may be e x p l a i n e d by P C ' s p a r t i c i p a t i o n i n r e s p i r a t o r y metabo l i sm i n p l a n t s and m i c r o - o r g a n i s m s . Towers (1964) has r e c e n t l y rev i ewed the r o l e of PC as the key i n t e r m e d i a t e i n the breakdown o f benzene and s i m p l e p h e n o l s e s p e c i a l l y i n m i c r o - o r g a n i s m s In the f o l l o w i n g two ways: (1) OH °2 COOH ,COOH - H 2 0 > COOH ^ 0 + H 2 0 - A C a t e c h o l c i s - c i s - K u c o n i c a c i d CH^COSCoA , A c e t y l CoA + /B - K e t o a d i p i c a c i d CH 2 COSCoA A c e t o a c e t y l CoA 118 (2) an a l t e r n a t e pathway i s a l s o p o s s i b l e : t s . C a t e c h o l oC-Hydroxymuconic P y r u v i c Semialdehyde a c i d However, p a r t i c i p a t i o n o f PC as a r e s p i r a t o r y s u b s t r a t e . i n h i g h e r p l a n t s has no t been demonstrated i n the above manner. Wort and Shr impton (1959) i n t h e i r exper iment w i t h mature sugar beet r o o t d i s c s , observed t h a t the a d d i t i o n o f c a t e c h o l to the medium r e s u l t e d i n a v e r y c o n s i d e r a b l e i n c r e a s e i n r e s p i r a -t i o n . T h u s , the p r o p o s a l t h a t has r e c e i v e d c o n s i d e r a b l e a t t e n t i o n i s t h a t o - d i p h e n o l t o g e t h e r w i t h v a r i o u s pheno l o x i d a s e s , f u n c -t i o n s as a t e r m i n a l ox idase as i l l u s t r a t e d below w i t h c a t e c h o l : As l o n g as the o -quinone i s r a p i d l y r e d u c e d , b e f o r e i t can p o l y -m e r i z e or be degraded by o ther r e a c t i o n s , such a system w i l l r e m a i n c y c l i c . o - D i p h e n o l o x i d a s e mediates the a e r o b i c o x i d a -t i o n o f p y r o c a t e c h o l to o - q u i n o n e . Quinone r e d u c t a s e c a t a l i z e s the r e d u c t i o n o f quinones w i t h NADH. Whi le i t cannot be den ied t h a t the c a p a c i t y of t h i s type o f o x i d a t i o n e x i s t s i n many p l a n t s , the u t i l i z a t i o n of these enzymes i n t h i s f a s h i o n i n the i n t a c t p l a n t seems negated by a number of o b s e r v a t i o n s (Nakabayash i , 1 9 5 M . I t must a l s o be p o i n t e d out 119 t h a t i t has no t been p o s s i b l e to demonstrate c o u p l e d o x i d a t i v e p h o s p h o r y l a t i o n w i t h the above o x i d a s e sys tem, and u n t i l t h i s i s a c h i e v e d i t seems p r e f e r a b l e to a s s i g n the r e s p i r a t o r y r o l e o f p l a n t s to the c y t o c h r o m e - c o n t a i n i n g systems (Hanson and Z u c k e r , 1967). I t appears t h e n , t h a t a l t h o u g h r a t e o f CO2 e v o l u t i o n and oxygen consumpt ion was i n c r e a s e d by PC d u r i n g the p r e s e n t i n v e s -t i g a t i o n , i t was no t a t the expense o f sugar s t o r e d or sugar newly formed i n the l e a v e s . H i g h s u c r o s e p e r c e n t a g e o f the r o o t s s u p p o r t s t h i s c o n t e n t i o n . T h i s i n c r e a s e i n r a t e of r e s p i r a t i o n (C0£ e v o l u t i o n ) was, p e r h a p s , u n a b l e to g e n e r a t e ATP n e c e s s a r y f o r growth , and t h a t may be the r e a s o n t h a t r a t e o f r e s p i r a t i o n i n P C -t r e a t e d p l a n t s was not s i g n i f i c a n t l y c o r r e l a t e d w i t h the growth of the l e a v e s (Tab le X V ) . The r e s p i r a t i o n under f a l l c o n d i t i o n s was i n h i b i t e d by P C . I t would seem t h a t the a c t i o n on r e s p i r a t i o n o f t h i s compound i s t emperature dependent . Perhaps one o f the enzymes n e c e s s a r y f o r the a c t i v e p a r t i c i p a t i o n o f PC i n t e r m i n a l o x i d a t i o n was i n a c t i -v a t e d under low temperature c o n d i t i o n s . P o s s i b l y i t was quinone r e d u c t a s e , because i n the absence o f the a c t i v i t y o f t h i s enzyme quinone may a c t i v e l y p a r t i c i p a t e i n the i n a c t i v a t i o n o f the enzymes o f Krebs c y c l e c o n t a i n i n g - SH g r o u p s , f o r example, < - k e t o g l u t a r i c dehydrogenase and s u c c i n i c dehydrogenase . I n h i -b i t i o n o f r e s p i r a t i o n may t h e n be p o s s i b l e , under such c o n d i t i o n s . Vanadium b e l o n g s to the t r a n s i t i o n group meta l s a l o n g w i t h t i t a n i u m , chromium,manganese, and i r o n . L i t t l e i n f o r m a t i o n i s a v a i l a b l e on the e f f e c t s o f vanadium i n b i o l o g i c a l sys tems . P r e v i o u s i n v e s t i g a t i o n s have i n d i c a t e d a r o l e f o r t h i s m e t a l i n : (a) r e p l a c i n g molybdenum i n m i c r o b i a l n i t r o g e n f i x a t i o n pathway 120 (Burk , 1 9 3 ^ ) . (b) i n e f f e c t i n g a l t e r a t i o n im mammalian l i p i d o x i d a t i o n and s y n t h e s i s ( C u r r a n and Costella> : , 1 9 5 7 ) . (c) i n c o u n t e r a c t i n g the e f f e c t o f Mn, and p o s s i b l e I n t e r a c t i o n w i t h i t i n f l a x , soybean and oat ( W a r r i n g t o n , 1 9 5 7 ) » (d) i n i n h i b i t i n g the growth o f Mycobacter ium t u b e r c u l o s i s beiaag c o m p e t i t i v e w i t h Mn and chromium ( a n a t a g o n i s t i c ) ( C o s t e l l o ani l Hedgecock, 1 9 5 9 ) » (e) i n i n h i b i t i n g n i t r a t e r e d u c t a s e v e r y e f f e c t i v e l y i n wheat embyro (Spencer , 1959) and sugar bee t (Yang,, 1 9 6 4 ) , ( f ) i n the u n c o u p l i n g o f o x i d a t i v e p h o s p h o r y l a t i o n i n m i t o c h r o n d r i a i s o l a -t e d from l i v e r o f c h i c k s (Hathcock et a l . 1 9 & 1 ) . . . . I n the p r e s e n t i n v e s t i g a t i o n , VS was f o u n d to i n h i b i t the growth o f sugar beet l e a v e s under b o t h summer and f a l l c o n d i t i o n s . The i n h i b i t i o n o f growth was accompanied by Ithe i n h i b i t i o n o f a c t i v i t y o f the enzymes n i t r a t e r e d u c t a s e , t r a n s a m i n a s e , i n v e r t a s e and p h o s p h a t a s e s . R e s p i r a t i o n was a l s o i n h i T b i t e d . S i g n i f i c a n t c o r r e l a t i o n o f the growth w i t h n i t r a t e r e d u c t a s e , i n v e r t a s e and r e s p i r a t i o n might e x p l a i n the p o s s i b l e cause o f growth i n h i b i t i o n by vanadium. Vanadium b e i n g a heavy m e t a l may i n h i b i t n i t r a t e r e d u c t a s e through i t s a c t i o n on s u l f h y d r y l g r o u p s . I t c a n a l s o i n t e r a c t w i t h molybdenum, the p r o s t h e t i c m e t a l f o r nllfcrate r e d u c t a s e , or may b i n d w i t h the enzyme p r o t e i n as R o c k o l d a n d T a l v i t i e , (1956), d e m o n s t r a t e d . I n h i b i t i o n o f r e s p i r a t i o n r a t e by VS may be e x p l a i n e d on the b a s i s o f i t s p o s s i b l e i n t e r a c t i o n w i t h Mm ( C o s t e l l o and Hedgecock, 1959 ; W a r r i n g t o n , 1 9 5 1 ) ' Mn i s r e q u i r e d f o r the a c t i v i t y o f v a r i o u s enzymes o f the g l y c o l y t i c pathway and Krebs c y c l e , v i z . h e x o k i n a s e , i s o c i t r i c dehydrogenase , m a l i c d e h y d r o -i 2 r genase , m a l i c enzyme, o x a l o a c e t a t e d e c a r b o x y l a s e and condens ing enzyme. I t i s thus p o s s i b l e f o r vanadium to i n h i b i t the a c t i v i t y o f the above ment ioned enzymes, e s p e c i a l l y i s o c i t r i c d e h y d r o -genase and m a l i c dehydrogenase where Mn i s the a b s o l u t e r e q u i r e -ment, by c o u n t e r a c t i n g the e f f e c t o f Mn. Mn i s a l s o p a r t o f some o f the enzymes o f n i t r o g e n metabo-l i s m . A Mn f l a v o p r o t e i n was a s s o c i a t e d w i t h n i t r i t e r e d u c t a s e , the enzyme r e s p o n s i b l e f o r the r e d u c t i o n o f n i t r i t e to h y d r o x y l -amine (Nason et a l . 1 9 5 ^ ) . Thus , n i t r i t e r e d u c t a s e may a l s o be i n h i b i t e d by vanadium. o A l e x a n d e r (19^5) found i n sugar cane t h a t a c t i v e a c i d i n v e r t a s e s a r e p r o t e i n - sugar - Mn complexes , i n which the p r o t e i n c o n s t i t u e n t i s v i r t u a l l y i n a c t i v e i n the absence o f Mn o r s u g a r . I f t h a t i s t r u e f o r sugar beet i n v e r t a s e , vanadium i n h i b i t i o n o f i n v e r t a s e c o u l d be i n t e r p r e t e d as a c o u n t e r a c t i o n o f Mn i n the a c t i v e i n v e r t a s e sys t em. I n c r e a s e i n sucrose c o n t e n t of the r o o t I n c r e a s e i n s u c r o s e c o n t e n t o f the r o o t o f sugar beet was r e c o r d e d a f t e r the t reatment o f p l a n t s w i t h MH, PC and VS d u r i n g the p r e s e n t i n v e s t i g a t i o n . I n c r e a s e i n p e r c e n t sucrose f o l l o w i n g to MH a p p l i c a t i o n to sugar bee t has been noted by many o ther workers (Wittwer and Hansen, 1952 ; K a l i n i n e t a l . 1 9 6 5 ) . E x c e s -s i v e sucrose a c c u m u l a t i o n has been noted to r e s u l t from MH a p p l i -c a t i o n to o t h e r p l a n t s p e c i e s a l s o ( G r e u l a c h , 1953 ; Samborski and Shaw, 1957; P e t e r s o n and N a y l o r , 1953 ; A l e x a n d e r , 1965) The l a r g e r sucrose c o n t e n t observed i n the r o o t o f sugar beet may be the r e s u l t o f the f o l l o w i n g : (a) s t i m u l a t i o n of 122 p h o t o s y n t h e s i s , (b) i n h i b i t i o n o f i n v e r t a s e a c t i v i t y , (c) i n h i -b i t i o n o f r e s p i r a t i o n , (d) s t i m u l a t i o n o f s u c r o s e s y n t h e s i z i n g enzymes (e) i n h i b i t i o n of the enzymes which h y d r o l y z e the sub-s t r a t e s o f the s u c r o s e s y n t h e s i z i n g enzymes e . g . , phosphatase s . These mechanisms w i l l i n v o l v e the i n h i b i t i o n o f many p r o c e s s e s and s t i m u l a t i o n o f o t h e r s . T h i s was encountered i n the p r e s e n t i n v e s t i g a t i o n . S t i m u l a t i o n o f the r a t e o f p h o t o s y n t h e s i s was observed i n t r e a t e d p l a n t s . Perhaps t h i s was a s s o c i a t e d w i t h the i n h i b i t i o n o f n i t r a t e r e d u c t a s e . Cramer and Myers (1948) showed t h a t d u r -i n g p h o t o s y n t h e s i s by c h l o r e l l a a t low l i g h t i n t e n s i t i e s n i t r a t e u t i l i z a t i o n was accompanied by a decrease i n the a s s i m i l a t i o n q u o t i e n t from 0 . 9 to 0 . 7 , owing to a d e c r e a s e d c a r b o n d i o x i d e u p t a k e . Yang (1964) found t h a t n i t r a t e r e d u c t a s e a c t i v i t y was h i g h d u r i n g the day i n the l e a v e s o f sugar b e e t . Presumably , p h o t o s y n t h e s i s s u p p l i e s r e d u c e d coenzymes toward n i t r a t e r e d u c -t i o n . The c h a n n e l i n g o f r e d u c e d coenzymes toward n i t r a t e r e d u c -t i o n might cause a r e d u c t i o n i n the r a t e o f p h o t o s y n t h e s i s i n the p l a n t s when n i t r a t e r e d u c t a s e a c t i v i t y i s v e r y h i g h . I n h i b i t i o n o f n i t r a t e r e d u c t a s e t h e n , might r e s u l t i n a s t i m u l a t i o n o f c a r b o n r e d u c t i o n i n p h o t o s y n t h e s i s . T h i s may have happened i n the t r e a t e d p l a n t s d u r i n g t h i s e x p e r i m e n t . P h o t o s y n t h e s i s o f r e d k i d n e y beans has been found to be i n c r e a s e d by m a l e i c h y d r a z i d e (Sorensen , 1 9 5 6 ) . C a l l a g h a n and Norman (1956) a l s o observed an i n c r e a s e i n the r a t e o f p h o t o s y n -t h e s i s i n Swiss c h a r d and t o b a c c o . They e x p l a i n e d the i n c r e a s e d r a t e o f p h o t o s y n t h e s i s on the b a s i s o f h i g h e r c h l o r o p h y l l c o n t e n t per u n i t a r e a o f l e a v e s a f t e r t rea tment w i t h h i g h e r c o n c e n t r a t i o n s 123 . o f m a l e i c h y d r a z i d e . M a l e i c h y d r a z i d e and vanadium i n h i b i t e d the growth of the l e a v e s . Mutua l s h a d i n g o f the l e a v e s , i n t h a t s i t u a t i o n w i l l be m i n i m i z e d , which w i l l r e s u l t i n the h i g h e r e f f i c i e n c y o f the l e a v e s to p h o t o s y n t h e s i z e . T h i s c o u l d be another r e a s o n f o r the observed i n c r e a s e i n the r a t e o f p h o t o s y n t h e s i s per u n i t o f l e a f a r e a . Observed s t i m u l a t i o n o f the enzymes o f sucrose s y n t h e s i s may a l s o e x p l a i n the i n c r e a s e d p e r c e n t a g e o f s u c r o s e i n the t r e a t e d p l a n t s . I t might seem p a r a d o x i c a l f o r an enzyme i n h i b i t o r to i n -c r e a s e the r a t e o f some phase o f m e t a b o l i s m , but a c t u a l l y such s t i m u l a t i o n i s n o t uncommon. "It i s p r o b a b l y j u s t i f i a b l e to say t h a t the g r e a t e r the number of i n t e r r e l a t e d enzymes i n the t o t a l sys tem, and g r e a t e r the c o m p l e x i t y and o r g a n i z a t i o n o f the meta-b o l i c pathways, the more l i k e l y w i l l i t be t h a t s t i m u l a t i o n can o c c u r " (Webb, 1 9 6 3 ) . Examples o f such a phenomenon a r e many. A r s e n i t e , a l t h o u g h a g e n e r a l enzyme i n h i b i t o r has been found to s t i m u l a t e p a p a i n , m a l i c dehydrogenase (Green , 1 9 3 & ) , u r i c a s e (Mahler e t a l . 1 9 5 5 ) . and the P i - ATP exchange enzyme ( P l a n t , 1 9 5 7 ) « I n the p r e s e n t exper iment a l s o , we note s i m i l a r phenom-e n a . A l t h o u g h m a l e i c h y d r a z i d e , p y r o c a t e c h o l and vanadium s u l -f a t e i n h i b i t the a c t i v i t y o f n i t r a t e r e d u c t a s e , t r a n s a m i n a s e , i n v e r t a s e and phosphatase , they s t i m u l a t e the a c t i v i t y of sucrose phosphate s y n t h e t a s e , s u c r o s e s y n t h e t a s e and U D P G - p y r o p h o s p h o r y l -a s e . Webb (1963) suggests some of the ways by which s t i m u l a t i o n o f the enzyme c o u l d be a c h i e v e d by an enzyme i n h i b i t o r : 124 (a) removal or i n a c t i v a t i o n o f some i n h i b i t i n g s u b s t a n c e , (b) a l t e r a t i o n o f m e t a b o l i c f low i n mult ienzyme e . g . , i n a d i v e r -gent p o l y l l n e a r c h a i n when one pathway i s i n h i b i t e d , a n o t h e r i s s t i m u l a t e d . There the appearance o f s t i m u l a t i o n w i l l depend .on the a s p e c t o f the t o t a l system one i s e x a m i n i n g . T h i s mechanism i s a c t u a l l y a d i v e r s i o n o f metabo l i sm r a t h e r than a t r u e s t i m u -l a t i o n , (c) remova l o f a dynamic e q u i l i b r i u m (d) d e p r e s s i o n o f a r e a c t i o n - c o n t r o l l i n g mechanism. The a b i l i t y o f a l i v i n g c e l l to a d j u s t i t s metabo l i sm a c c o r d i n g to i t s f u n c t i o n a l a c t i v i t y makes i t n e c e s s a r y f o r the p r i n c i p a l m e t a b o l i c pathways to be under some s o r t o f c o n t r o l l i n g or r e g u l a t i n g mechanism. The r e -moval o f the r e s t r i c t i n g c o n t r o l w i l l m a n i f e s t i t s e l f as s t i m u -l a t i o n , (e) the i n h i b i t i o n o f an enzyme t h a t d e s t r o y s some i m p o r t a n t m e t a b o l i c subs tance may i n c r e a s e the s teady s t a t e l e v e l o f t h i s substance and a c c e l e r a t e r e a c t i o n s w i t h which i t i s c o n -c e r n e d . I n h i b i t i o n o f A T P - a s e , f o r example can s e c o n d a r i l y i n f l u e n c e many r e a c t i o n s dependent on A T P . The i n h i b i t o r may a l s o i n c r e a s e p e r m e a b i l i t y i n some manner and thus s t i m u l a t e r e a c t i o n s whose r a t e s a r e l i m i t e d by the a c c e s s o f the s u b s t r a t e , or i t may e i t h e r d i r e c t l y or i n d i r e c t l y damage the membrane so t h a t the s t r u c t u r a l d i s o r g a n i z a t i o n w i l l a l l o w r e a c t i o n s to be r e l e a s e d from t h e i r normal c o n t r o l , which may be s i m p l y a s p a t i a l s e p a r a t i o n of r e a c t a n t . The i n h i b i t o r s a p p l i e d i n t h i s exper iment w i t h sugar beet have e f f e c t s which suggest a l t e r a t i o n o f m e t a b o l i c f low i n m u l t i -enzyme sys tems . One pathway was i n h i b i t e d , the o ther was s t i m u -l a t e d . The pathways o f p r o t e i n s y n t h e s i s , sucrose h y d r o l y s i s and c a r b o h y d r a t e breakdown ( r e s p i r a t i o n ) were i n h i b i t e d . The pathway 125 f o r the s y n t h e s i s o f s u c r o s e was s t i m u l a t e d . The i n t e r r e l a -t i o n s h i p o f these systems i s g i v e n i n F i g . 1, page 2 0 . N i t r a t e r e d u c t a s e and s u c r o s e r e l a t i o n s h i p The i n v e r s e r e l a t i o n s h i p o f s u c r o s e c o n c e n t r a t i o n and the a c t i v i t y o f n i t r a t e r e d u c t a s e i s e v i d e n t from the r e s u l t s o b t a i n e d d u r i n g the p r e s e n t i n v e s t i g a t i o n . I t has been p o i n t e d out e a r l i e r t h a t the c o n v e r s i o n s o f n i t r a t e to n i t r i t e , h y d r o x y l -amine and ammonia a r e e n e r g y - r e q u i r i n g p r o c e s s e s which must be c o u p l e d to c a r b o h y d r a t e breakdown. T h i s was c l e a r l y demonstrated by Hamner (1935) who found t h a t the a p p l i c a t i o n o f n i t r a t e to n i t r o g e n s t a r v e d tomato p l a n t s r e s u l t e d i n the f o r m a t i o n of n i t -r i t e , the d e p l e t i o n o f c a r b o h y d r a t e r e s e r v e s , and a marked i n c r e a s e i n r e s p i r a t i o n . The d a t a from the exper iment w i t h sugar bee t by Snyder and T o l b e r t (1966) suggest t h a t i f the n i t r o g e n s u p p l y i s no t c u t down p l a n t s may p r e f e r e n t i a l l y s y n t h e s i z e the c i t r i c a c i d c y c l e p r o d u c t s and t h e i r amino a c i d c o u n t e r p a r t s , and thus produce l e s s s u c r o s e . I n v e r s e r e l a t i o n s h i p between n i t r a t e c o n t e n t o f the p e t i o l e and the sucrose percentage was shown by U l r i c h (195° ) i n sugar b e e t . I t seems l i k e l y t h a t t h i s r e l a t i o n -s h i p r e q u i r e s hexose d e g r a d a t i o n b o t h f o r the s u p p l y o f energy needed f o r n i t r a t e r e d u c t i o n and as a source of m a t e r i a l f o r o r g a n i c a c i d s y n t h e s i s . T h u s , the low n i t r a t e r e d u c t a s e a c t i v i t y c o u p l e d w i t h weak i n v e r t a s e and phosphatase (hexose monophospha-t a s e s and A T P - a s e ) a c t i v i t i e s would f a v o r s u c r o s e a c c u m u l a t i o n . T h i s agrees w e l l w i t h the e x p e r i m e n t a l r e s u l t s . The s i t u a t i o n which i s encountered i n the t r e a t e d p l a n t s i s t h a t perhaps the c a r b o h y d r a t e s or o ther energy y i e l d i n g 126 s u b s t r a t e s a r e abundant but amino n i t r o g e n i s absent due to i n h i b i t i o n o f n i t r a t e r e d u c t a s e and t ransaminase (perhaps g l u -tamic dehydrogenase a l s o ) . The r e s u l t a n t i n a b i l i t y o f the c e l l t o s y n t h e s i z e p r o t e i n and c a r r y out e n e r g y - r e q u i r i n g growth p r o c e s s e s would r e s u l t i n a c c u m u l a t i o n o f A T P . I n h i b i t i o n o f ATP-ase a c t i v i t y would f u r t h e r f a v o r the a c c u m u l a t i o n o f A T P . T h i s compound i s a n e g a t i v e e f f e c t o r o f c i t r a t e synthe tase which c a t a l y z e s the e n t r y o f a c e t y l CoA i n t o the Krebs c y c l e , thus under these c o n d i t i o n s the c y c l e w i l l compete weakly f o r a c e t y l CoA ( A t k i n s o n , 1 9 6 5 ) . When the c o n c e n t r a t i o n of ATP i s h i g h e r , the AMP c o n c e n -t r a t i o n i s n e c e s s a r i l y low and i s o c i t r i c dehydrogenase w i l l be i n h i b i t e d (Hathway and A t k i n s o n , 1 9 6 3 ) . AMP modulates the c a t a l y s i s by p h o s p h o f r u c t o k i n a s e o f f r u c t o s e - 6 - p h o s p h a t e to f r u c t o s e d i p h o s p h a t e . ATP i s the n e g a t i v e e f f e c t o r o f phospho-f r u c t o k i n a s e . Thus , i n a c e l l w i t h h i g h ATP and low AMP c o n c e n -t r a t i o n s the k i n e t i c b e h a v i o r o f p h o s p h o f r u c t o k i n a s e w i l l be such as to l e a d to h i g h c o n c e n t r a t i o n s of f r u c t o s e - 6 - p h o s p h a t e and i t s p r e c u r s o r g l u c o s e - 6 - p h o s p h a t e . The r e v e r s a l of Krebs c y c l e and g l y c o l y t i c pathway w i l l be f a v o r e d i n energy r i c h c e l l s . The r e s u l t w i l l be l e s s u t i l i z a t i o n and more s torage o f the c a r b o h y d r a t e s and l i p i d s ( sucrose i n case o f sugar b e e t , s t a r c h or f a t s i n o t h e r p l a n t s , g l y c o g e n or f a t s i n a n i m a l s ) . T h i s k i n d o f c o n t r o l mechanism by ATP and AMP (or ADP) i s d e s i g -n a t e d by A t k i n s o n as " r e g u l a t i o n by a d e n y l a t e s . " ATP, ADP or AMP are termed " r e g u l a t o r y e f f e c t o r s . " 127 Phosphatases and sucrose b i o s y n t h e s i s The s t u d i e s o f L e l o i r and c o - w o r k e r s ( 1 9 5 3 . 1955) w i t h wheat germ, Rorem e t a l . ( i 9 6 0 ) w i t h sugar bee t l e a f , D u t t o n e t a l . (1961) w i t h sugar beet r o o t , Hatch e t a l . (1963) w i t h sugar cane s t o r a g e t i s s u e , B i r d e t a l . (1965) w i t h tobacco l e a f c h l o r o -p l a s t s , and Haq and H a s s i d ( 1965 )w i th sugar cane l e a f c h l o r o p l a s t s have a l l p r o v i d e d c o n v i n c i n g ev idence which i s c o n s i s t e n t w i t h the s y n t h e s i s o f sucrose by f o l l o w i n g o v e r a l l r e a c t i o n s : (1) ATP + UDP r ^ UTP + ADP n u c l e o s i d e d i p h o s p h a t e k i n a s e (2) G l u c o s e - 6 - p h o s p h a t e ,. i G l u c o s e - l - p h o s p h a t e phosphoglucomutase (3) UTP + g l u c o s e - l - P r _ _ i UDP-g lucose + pyrophosphate UDP-g lucose p y r o p h o s p h o r y l a s e (4) F r u c t o s e - 6 - p h o s p h a t e + UDP-g lucose „. s. sucrose-P+UDP s u c r o s e - 6 - p h o s p h a t e s y n t h e t a s e (5) Sucrose - P + H 2 0 > sucrose + H^PO^ s u c r o s e - 6 - P phosphatase An a l t e r n a t e system would s u b s t i t u t e s u c r o s e s y n t h e t a s e f o r r e a c t i o n (4) and (5) (6) F r u c t o s e + UDP - g l u c o s e ^ sucrose + UDP s u c r o s e s y n t h e t a s e The r e s u l t of the p r e s e n t i n v e s t i g a t i o n i n d i c a t e s t h a t s u c r o s e - 6 - p h o s p h a t e s y n t h e t a s e i s more a c t i v e than the sucrose s y n t h e t a s e i n the l e a f o f the sugar b e e t . The r e s u l t s o b t a i n e d by Burma and Mort imer (1956) w i t h sugar beet l e a v e s a l s o i n d i -c a t e d t h a t s u c r o s e - 6 - p h o s p h a t e s y n t h e t a s e was the enzyme m a i n l y 128 concerned with sucrose production. Bird et a l . (1965) sug-gested that synthesis of sucrose as a result of photosynthesis in leaves proceeds mainly, i f not solely, via sucrose phosphate i n chloroplasts. Furthermore a consideration of the equilibrium constants for the two synthetases shows that sucrose phosphate rather than sucrose synthesis is favored (Mendicino, i 9 6 0 ) . A specific sucrose phosphatase from the stem tissue and leaves of sugar cane which catalyses the hydrolysis of sucrose-6-phosphate to sucrose and phosphate was isolated by Hawker ( 1 9 6 6 ) . Sucrose-6-phosphatase according to Hawker is situated near or at the tonoplast membrane of the vacuole. It i s possibly concerned with movement and accumulation of sucrose. Hexose phosphatases and ATP-ase were inhibited by MH, PC, and VS during the present investigation. The influence of these phosphatases on sucrose biosynthesis is evident from the involve-ment of hexose phosphates and ATP in the overall scheme of. the synthesis. ATP could enter the scheme outlined above via i t s a b i l i t y to phosphorylate UDP to UTP (Reaction 1 ) . Glucoses-phosphate and glucose-l-phosphates are involved in reactions, (2) and ( 3 ) . Fructose-6-phosphate supplies the fructosyl moiety of the sucrose molecule. The enzymes ATP-ase, glucose-l-phos-phatase, glucose-6-phosphatase and fructose-6-phosphatase would be able to alter the equilibrium of Reactions ( 1 ) , (2), (3) and (4) and may be able to inhibi t the sucrose synthesis as a whole. The inhibit ion of these during the present work, thus favors sucrose biosynthesis. The correlation coefficient values for different phos-phatases with sucrose concentrations of the root are given in 129 T a b l e X V I . A l l the phosphatases had a n e g a t i v e c o r r e l a t i o n w i t h the s u c r o s e . The f o l l o w i n g c o r r e l a t i o n c o e f f i c i e n t v a l u e s f o r A T P - a s e , g l u c o s e - l - p h o s p h a t a s e , g l u c o s e - 6 - p h o s p h a t a s e and f r u c t o s e -6-phosphatase w i t h sucrose phosphate s y n t h e t a s e p r o v i d e ev idence t h a t the phosphatases were n e g a t i v e l y c o r r e l a t e d to the enzyme sucrose phosphate s y n t h e t a s e . CORRELATION COEFFICIENTS ( r ) G l u c o s e - 1 - G l u c o s e - 6 - F r u c t o s e - 6 -V a r l a b l e s A T P - a s e phosphatase phosphatase phosphatase 1 .00 1 .00 0.288 - 0 . 6 3 6 0 .302 - O . 8 3 8 0 . 1 1 6 - 0 . 9 1 4 S i g n i f i c a n t r ( . 0 5 ) = O .57 A l e x a n d e r (1965) r e p o r t e d t h a t the i n h i b i t i o n s o f phos -phatase by molybdenum r e s u l t e d i n the h i g h e r s u c r o s e c o n t e n t i n the sugar c a n e . He a l s o no ted the i n v e r s e r e l a t i o n s h i p between ATP-ase a c t i v i t y and the sucrose c o n t e n t i n the l e a v e s o f sugar cane ( A l e x a n d e r , 1 9 6 5 a ) . N i t r o g e n o u s c o n s t i t u e n t s of the r o o t s H i g h n i t r a t e and low n i t r i t e c o n t e n t o f the t r e a t e d p l a n t s c o u l d be l o g i c a l l y i n t e r p r e t e d on the b a s i s o f low n i t r a t e r e d u c -t a s e a c t i v i t y due to i n h i b i t i o n o f MH, PC and V S . I n c r e a s e i n n i t r a t e n i t r o g e n i n the molybdenum d e f i c i e n t p l a n t s was no ted even b e f o r e the a c t u a l d i s c o v e r y o f n i t r a t e r e d u c t a s e (Leeper , 1 9 4 l ; H e w i t t and J o n e s , 1 9 4 7 ) . The vanadium, s u p p l i e d through the r o o t s to f l a x , oa t s and soybeans , caused a 1 .00 1 .00 S u c r o s e - P -synthetase(MH) - 0 . 8 7 1 - 0 . 3 4 0 » » (PC) - 0 . 8 8 6 - 0 . 5 3 1 » " (VS) - 0 . 8 1 0 - 0 . 3 0 2 130 r i s e i n n i t r a t e n i t r o g e n was n o t e d by W a r r i n g t o n ( 1951 ) • A g r e a t e r n i t r a t e c o n t e n t i n M H - t r e a t e d tobacco p l a n t s was r e -c o r d e d by P e t e r s o n and N a y l o r (1953)* A l l these r e s u l t s c o u l d be e x p l a i n e d on the b a s i s o f i n h i b i t i o n o f n i t r a t e r e d u c t a s e a c t i v i t y . MH t rea tment r e s u l t e d i n an i n c r e a s e i n the ammonium c o n -t e n t o f the r o o t s . H i g h ammonia c o n t e n t sugges ts t h a t e i t h e r ammonia was no t i n c o r p o r a t e d i n t o the amino a c i d s , p o s s i b l y by the i n h i b i t i o n o f g l u t a m i c a c i d dehydrogenase , or some of the amides a r e h y d r o l y z e d and g i v e r i s e to more f r e e ammonia. Amino a c i d c o n t e n t o f the r o o t o f M H - t r e a t e d p l a n t s was lower t h a n the c o n t r o l 7 days a f t e r the t rea tment but h i g h e r on the 14th and 2 1 s t d a y . That the i n t e r v a l from trea tment to h a r -v e s t i s one o f the i m p o r t a n t f a c t o r s i n d e t e r m i n i n g changes w i t h i n the p l a n t , i s w e l l i l l u s t r a t e d by the above r e s u l t s . A l s o F u l t s and Payne (1956) and L i v i n g s t o n (195*0 no ted a s i m i l a r t r e n d i n M H - t r e a t e d sugar beet p l a n t , where they found t h a t 5 days a f t e r t rea tment the c o n t e n t o f a l a n i n e , l y s i n e , t y r o s i n e and g l u t a m i c a c i d was lower i n the r o o t s o f t r e a t e d p l a n t s , but 60 days a f t e r s p r a y i n g a l l o f these amino a c i d s were p r e s e n t i n l a r g e r amounts i n the t r e a t e d r o o t s . The I n c r e a s e i n f r e e amino a c i d s a f t e r MH a p p l i c a t i o n was a l s o no ted by P e t e r s o n and N a y l o r (1953) who found t h a t t r e a t e d tobacco p l a n t s had more s o l u b l e n i t r o g e n i n c l u d i n g f r e e ammonia, g l u t a m i n e , a s p a r a g i n e , uncombined amino a c i d , and n i t r a t e n i t r o -gen but l e s s p r o t e i n . An i n c r e a s e i n a l c o h o l - s o l u b l e N was noted i n wheat by Samborski and Shaw (1957) a f t e r MH t r e a t m e n t . .131 In the Pc-treated plants ammonia was less than In the control plants up to 14 days after treatment, but i t was more on the 21st day. The poss ib i l i ty of increased formation of ammonia through nitrate reductase i s very small, because 21 days after treatment, PC-treated plants had the lowest rate of nitrate reduc-tase act iv i ty in the leaf . Then, the increase in free ammonia might be due to a greater breakdown of amides by 21 days after treatment. The amino acid content of the PC-treated beets was lower than the control on the 7th day after treatment but higher on the 14th and 21st day. The lower rate of nitrate reduction and of transamination which was measured, suggests a slowing of new amino acid synthesis. Any increase in amino acid content might be accounted for on the basis of protein degradation. Low total nitrogen and protein content of the roots support this sugges-t ion . In VS-treated plants amino acid, ammonium, and protein con-tent was lower than in the control plants. The lower protein content and low act iv i ty of nitrate reductase and transaminase provide the evidence that protein formation was inhibited by vanadium through nitrate reductase. It has been shown by Klotz (1954) that Mn stabil izes the peptidases and the amidases and by Warrington (1951) that vana-dium counteracts with Mn. If that be true, vanadium could cause an inhibi t ion of the enzymes responsible for amide and peptide hydrolysis, hence would exclude the poss ib i l i ty of an increase in ammonia and amino acids by degradation of protein or the amides. Perhaps this is why an accumulation of the ammonia and amino acids 132 i n the V S - t r e a t e d p l a n t s d u r i n g the p r e s e n t i n v e s t i g a t i o n was not r e c o r d e d . That amino a c i d s d e r i v e d from p r o t e i n breakdown may accumu-l a t e and may not be u t i l i z e d f o r p r o t e i n s y n t h e s i s was n o t e d l o n g ago by Gregory and Sen ( 1 9 3 7 ) • Steward and B i d w e l l (19^5) p o i n t out t h a t (a) the mere presence o f f r e e amino a c i d s i n the c e l l i s no t s u f f i c i e n t to ensure t h e i r d i r e c t i n c o r p o r a t i o n i n t o the p r o t e i n , i n f a c t they may w e l l be d e b a r r e d from t h i s . (b) o n l y i f the p r o t e i n p r e c u r s o r s a r e g e n e r a t e d a t the r i g h t s i t e , or a l t e r n a t i v e l y from the r i g h t source ( i . e . , one which reaches t h i s s i t e ) may they be i n c o r p o r a t e d d i r e c t l y i n t o the p r o t e i n , and they can do so w i t h o u t m i n g l i n g w i t h the f r e e amino a c i d p o o l , (c) These s i t u a t i o n s can p r e v a i l o n l y i n a h i g h l y o r g a n i z e d s y s -tem w i t h d i s c r e t e compartments , the i d e n t i t y and separa te f u n c -t i o n s o f which can be p h y s i c a l l y or c h e m i c a l l y m a i n t a i n e d and s u b j e c t e d to some form of r e g u l a t o r y c o n t r o l . H e l l e b r u s t and B i d w e l l (1963) found t h a t i n wheat l e a v e s , the p r o t e i n - b o u n d s e r i n e and g l y c i n e were d e r i v e d by a r o u t e which bypassed the b u l k o f s o l u b l e p o o l s o f amino a c i d s . B i d w e l l e t a l ' (1964) have p r e s e n t e d c o n s i d e r a b l e ev idence f o r the e x i s t e n c e of two types of amino a c i d p o o l s i n c a r r o t t i s s u e , one b e i n g a " p r o t e c t e d p o o l of amino a c i d s en r o u t e to p r o t e i n " i n t o which c a r b o n from sugars c o u l d p a s s , and the o ther b e i n g a s torage p o o l i n t o which exogenous ly s u p p l i e d amino a c i d s passed and which i s m a i n l y the p r o d u c t o f breakdown of p r o t e i n s . Dav ies and C o c k i n g (1967) found t h a t amino a c i d s l a b e l l e d a f t e r s u p p l y -i n g C - ^ b i c a r b o n a t e pass more r e a d i l y i n t o p r o t e i n s , than amino a c i d s exogenous ly s u p p l i e d . I t was suggested by these a u t h o r s 133 t h a t d i f f e r e n t types and p o o l s o f amino a c i d s e x i s t i n the l o c u l e c e l l s o f tomato . The above c o n s i d e r a t i o n s suggest t h a t i n MH- and P C - t r e a t e d p l a n t s the excess amino a c i d s ( s o l u b l e ) a r e the d e g r a d a t i o n p r o d -u c t s o f p r o t e i n s and they a r e not t a k i n g p a r t i n the s y n t h e s i s o f new p r o t e i n s . The f o l l o w i n g d iagram based on the d i s c u s s i o n p r e s e n t e d i n the p r e c e d i n g paragraphs w i l l i l l u s t r a t e the p o s i t i o n of the two amino a c i d p o o l s i n sugar b e e t . S u c r o s e , r e c e n t l y s y n t h e t l z e d or s t o r e d i n r o o t i n v e r t a s e s y n t h e t a s e Hexoses NR> C 0 2 P h o t o s y n t h e s i s 3 - F h o s p h o g l y c e r i c a c i d Phosphoenol p y r u v a t e •4/ Pyruvate -s' Krebs c y c l e A s p a r t l c a c i d G l u t a m i c a c i d -^Alanine P o o l o f amino a c i d s on d i r e c t Transaminase r o u t e to p r o t e i n s y n t h e s i s i P r o t e i n I P r o t e i n breakdown i S t o r a g e p o o l of amino a c i d s 135 CONCLUSIONS The r e s u l t s show t h a t the f o l i a r a p p l i c a t i o n o f MH, P C , and VS to sugar beet evoked a number of s i m i l a r re sponses by the p l a n t s : (1) l e a f growth i n h i b i t i o n (2) r e d u c t i o n i n the c o n t e n t o f r e d u c i n g s u g a r s , n i t r i t e , and p r o t e i n i n r o o t t i s s u e (3) i n c r e a s e i n the c o n t e n t o f sucrose and n i t r a t e i n r o o t t i s s u e (4) i n h i b i t i o n o f the a c t i v i t y o f n i t r a t e r e d u c t a s e , t r a n s a m i n a s e , i n v e r t a s e and phosphatases i n l e a f and r o o t (5) s t i m u l a t i o n of the enzymes o f s u c r o s e b i o s y n t h e s i s ' i n l e a f and r o o t . Some r e s p o n s e s were d i s s i m i l a r : (1) w h i l e MH and PC caused an i n c r e a s e i n the c o n t e n t of ammonia and amino a c i d s o f the s torage r o o t s , VS caused a decrease i n the c o n t e n t o f these c o n s t i t u e n t s (2) the r a t e o f r e s p i r a t i o n o f the s t o r a g e r o o t s and the f o l i a g e was reduced by MH and VS but not by PC (3) the r a t e o f net CO2 a s s i m i l a t i o n by i n t a c t p l a n t s was i n c r e a s e d by VS and MH. PC a l s o caused a s i g n i f i c a n t i n c r e a s e , except on the 7 t h day a f t e r t r e a t -ment. On the b a s i s o f the r e s u l t s o b t a i n e d , and t h e i r s t a t i s t i c a l a n a l y s i s , i t seems r e a s o n a b l e to conc lude t h a t : (1) The growth of the sugar beet l e a v e s was p o s i t i v e l y c o r r e l a t e d w i t h the r e d u c i n g s u g a r , n i t r i t e , and p r o t e i n c o n t e n t , and w i t h the a c t i v i t y o f n i t r a t e r e d u c t a s e , t ransaminase and i n v e r t a s e . The r e d u c t i o n i n each of these i t e m s , f o l l o v r i n g the a p p l i c a t i o n o f MH, PC, and VS to the p l a n t , may then be a c a u s a l f a c t o r i n the r e d u c t i o n o f growth . (2) the sucrose c o n t e n t o f the r o o t s was n e g a t i v e l y c o r r e -l a t e d - w i t h i n v e r t a s e and phosphatase a c t i v i t y and p o s i t i v e l y c o r r e l a t e d w i t h sucrose phosphate synthe tase and sucrose synthe tase a c t i v i t y . The i n c r e a s e i n s u c r o s e , r e s u l t i n g from use of the t h r e e r e g u l a t o r s may have been the r e s u l t of t h e i r i n h i b i t i o n of i n v e r t a s e and phosphatases and t h e i r s t i m u l a t i o n of the enzymes of sucrose s y n t h e s i s ( sucrose s y n t h e t a s e and sucrose phosphate s y n t h e t a s e ) 136 (3) a l t h o u g h MH, P C , and VS a p p a r e n t l y a c t e d a t the same s i t e s i n s e v e r a l i n s t a n c e s , the magnitude o f t h e i r e f f e c t s on the c h e m i c a l c o m p o s i t i o n or m e t a b o l i c p r o c e s s e s was d i f f e r e n t (4) f o r each c h e m i c a l , the i n t e r v a l from trea tment to the day o f o b s e r v a t i o n was one o f the i m p o r t a n t f a c t o r s i n d e t e r m i n i n g the n a t u r e and magnitude o f changes w i t h i n the p l a n t (growth, c h e m i c a l c o m p o s i t i o n or m e t a b o l i c p r o c e s s e s ) (5) the g e n e r a l enhancement o f sucrose percentage o f the r o o t s by MH, P C , and VS i n d i c a t e d t h e i r importance as agents f o r the c o n t r o l o f growth and the i n d u c t i o n o f " r i p e n i n g " o f the r o o t s o f sugar b e e t . However, VS e x h i b i t e d most s u i t a b l e p r o p e r t i e s from the s t a n d -p o i n t o f p r a c t i c a l s u c r o s e p r o d u c t i o n i n t h a t ' i t a l s o d e c r e a s e d the c o n t e n t o f ammonia and amino a c i d s of the r o o t s . 137 LITERATURE CITED Adams, S . N . i 9 6 0 . The v a l u e o f c a l c i u m n i t r a t e and u r e a f o r sugar b e e t , and the e f f e c t o f l a t e n i t r o g e n o u s top d r e s s i n g . J . A g r i c . S c i . 54: 395-398. A l e x a n d e r , A . G . 1964. Sucrose-enzyme r e l a t i o n s h i p s i n immature sugar cane a f f e c t e d by v a r y i n g l e v e l s o f n i t r a t e and po tas s ium s u p p l i e d i n sand c u l t u r e . J . A g r . U n i v . P . R . 48: 1 6 5 - 2 3 1 . A l e x a n d e r , A . G . 1965* Changes i n l e a f sugar c o n t e n t and enzyme a c t i v i t y o f immature sugar cane f o l l o w i n g f o l i a r a p p l i c a t i o n o f l n d o l e - 3 - a c e t i c a c i d , 2 , 4 - d i c h l o r o p h e n o x y -a c e t i c a c i d , under m a l e i c h y d r a z i d e . J . A g r . U n i v . P . R . 4 9 : 1-34. A l e x a n d e r , A . G . 1 9 6 5 ( a ) . H y d r o l y t i c p r o t e i n s of sugar cane: The a c i d i n v e r t a s e s . J . A g r . U n i v . P . R . 49: 2 8 7 - 3 0 7 . A n d e r s o n , J . W . , and K . . S . Rowan. 1 9 6 7 . E x t r a c t i o n o f s o l u b l e l e a f enzymes w i t h t h i o l s and o t h e r r e d u c i n g a g e n t s . Phytochem. 6 : 1047-1056. A n i t a , N . , C . I l l e , and M. V o i c u l e s c u . 1963« The i n f l u e n c e o f v a r i a b l e doses o f p o t a s s i u m and n i t r o g e n on the y i e l d o f sugar beet r o o t s . A c d . Repub. P o p . Rom. S t u d . C e r c e t . B i o l . V e g . 1 5 : 479-498 A r t s c h w a g e r , E . F . 1926. On the anatomy o f the v e g e t a t i v e organs of the sugar b e e t . J . A g r i c . R e s . 33= 143-176. A t k i n s o n , D . E . 1965« B i o l o g i c a l f e e d back c o n t r o l a t the m o l e c u l a r l e v e l . S c i e n c e . 1 5 0 : 851-857. B a l d w i n , C . S . , and J . F . D a v i s . 1 9 6 6 . E f f e c t of t ime and r a t e of a p p l i c a t i o n of n i t r o g e n and date of h a r v e s t on the y i e l d and sucrose c o n t e n t of sugar b e e t s . A g r o n . J . 58: 3 7 3 - 3 7 6 . B a v e r , L . D . 1964. N i t r o g e n e f f e c t s on sugar c r o p s . J . Am. S o c . S u g . Beet T e c h n o l . 13: 21-26. B e e v e r s , L . D . , D . M . P e t e r s o n , J . C . Shannon, and R . E . Hageman. 1 9 6 3 . Comparat ive e f f e c t s o f • 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d on n i t r a t e metabol i sm i n c o r n and cucumber. P l a n t P h y s i o l . 3 8 : 675-679* B e e v e r s , L . D . , D. F l e s h e r , and R . H . B u r r i s . 1964. The p y r -i d i n e n u c l e o t i d e s p e c i f i c i t y o f n i t r a t e r e d u c t a s e i n h i g h e r p l a n t s and i t s r e l a t i o n to s u l f h y d r y l l e v e l . B i o c h . B i o p h y s . . A c t a . 8 9 : 453-464. 138 Beevers, L . D . , L . E . Shrader, D. Flesher, and R.H. Hageman. 1965* The role of l ight in the induction of nitrate reductase in radish cotyledons and maize seedlings. Plant Physiol . 40: 6 9 1 - 6 9 8 . Bidwell, R . G . S . , A. Barr, and F . C . Steward. 1 9 6 4 . Protein synthesis and turnover in cultured plant tissue. Source of carbon for synthesis and the fate of the protein breakdown products. Nature (Lond.), 2 0 3 : 3 6 7 - 3 7 3 . Bird , I . F . , H.K. Porter, and C.R. Stocking. 1 9 6 5 . Intercellular local izat ion of enzymes associated with sucrose synthesis in leaves. Biochem. Biophys. Acta. 1 0 0 : 3 6 6 - 3 7 5 . Braunstein, A . E . , and M.G. Kritzman. 1937* Uber den Ab- und Aufbau von AminosMuren durch Umaminierung. Enzymologia. 2: 1 2 9 - 1 3 7 . Brian, R.C. 1 9 6 4 . The c lass i f i cat ion of herbicides and types . of tox ic i ty . In; The Physiology and Biochemistry of Herbicides, ed. L . J . Audus. Academic Press, Lond. 1 -37• Brovchenko, M.I. 1 9 6 5 . Movement of sugar from mesophyll to the vascular bundles of sugar beet leaves. F i z i o l . Rast. 1 2 : 2 7 0 - 2 7 9 . Burk, A . E . 1 9 3 4 . Azotase and nitrogenase in Azotobacter. Ergeb. Enzymforsch. 3 : 2 3 - 5 6 . Burma, D .P . , and D.C. Mortimer. 1 9 5 6 . The biosynthesis of uridine diphosphate glucose and sucrose in sugar beet leaf . Arch. Biochem. Biophys. 62: 16-28. Callaghan, J . J . , and R.W. van Norman. 1 9 5 6 . Effect of fo l iar sprays of maleic hydrazide on photosynthesis. Science. 1 2 3 : 8 9 4 - 8 9 5 . Chibnall , A . C . 1 9 3 9 . Protein metabolism in the plants. Yale Univ. Press. New Haven. Costello, R . L . , and L.W. Hedgecock. 1 9 5 9 . Effect of metavana-date ion on the growth in v i tro of Mycobacteriurn tubercu-l o s i s . J . Bact. 7 7 : 7 9 4 - 7 9 9 . Cramer, M. , and J . Myers. 1 9 4 8 . Nitrate reductase and assimi-lat ion in ch lore l la . J . Gen. Physiol . 3 2 : 9 3 - 1 0 2 , Croy, L . I . I 9 6 7 . Nitrate reductase in wheat (Triticum aestlcum L . ) and i t s relationship to grain protein and y i e ld . PhD. thesis. Univ. of I l l i n o i s . Curran, G . L . , and R . L . Costel lo. 1957 • Reduction of excess cholesterol in the rabbit aorta by inhibi t ion of endogenous cholesterol synthesis. J . exp. Med. 1 0 3 : 4 9 - 5 6 . 139 D a v i e s , D . D . , J . G i o v a n e l l i , and T . AP Rees , 1 9 6 4 . P l a n t B i o c h e m i s t r y . B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s , O x f o r d . D a v i e s , J . W . , and E . C . C o c k i n g . 1 9 6 7 . P r o t e i n s y n t h e s i s i n tomato f r u i t l o c u l e t i s s u e : The s i t e o f s y n t h e s i s and pathway of c a r b o n i n t o p r o t e i n . P l a n t a . 7 6 : 285-305* D u t t o n , J . V . , A . C a r r u n t h e r s , and J . F . T . O l d f i e l d . 1 9 6 l . The s y n t h e s i s o f s u c r o s e by e x t r a c t s o f the r o o t o f the sugar b e e t . B iochem. J . 81: 2 6 6 - 2 7 2 . E c k e r s o n , S . H . 1 9 2 4 . P r o t e i n s y n t h e s i s by p l a n t s . I . N i t r a t e r e d u c t i o n . B o t . G a z . 7 7 : 3 7 7 - 3 9 0 . E l l i o t , W . H . 1953* I s o l a t i o n o f g l u t a m i n e s y n t h e t a s e and g l u t a m o t r a n s f e r a s e from g r e e n p e a s . J . B i o l . Chem. 2 0 1 : 6 6 1 - 6 7 2 . E v a n s , H . J . , and A . Nason . 1953* P y r i d i n e n u c l e o t i d e n i t r a t e r e d u c t a s e from e x t r a c t s o f h i g h e r p l a n t s . P l a n t P h y s i o l . 28: 2 3 3 - 2 5 4 . F i l n e r , P . 1 9 6 6 . R e g u l a t i o n o f n i t r a t e r e d u c t a s e i n c u l t u r e d tobacco c e l l s . B iochem. B i o p h y s . A c t a . 118: 2 9 9 - 3 1 0 . F i s k e , C . H . , and Y . SubbaRow. 1 9 2 5 . The c o l o r l m e t r i c d e t e r -m i n a t i o n o f p h o s p h o r u s . J . B i o l . Chem. 6 6 : 3 7 5 - 4 0 7 . F o l k e s , B . F . 1 9 5 9 . The p o s i t i o n o f amino a c i d s i n the a s s i m i -l a t i o n o f n i t r o g e n and the s y n t h e s i s o f p r o t e i n s i n p l a n t s . I n : Symp. S o c . exp . B i o l . 1 3 : 126-147. F r a n s , R . E . , E . E . L i n d , and W . E . L o o m i s . 1 9 5 7 . K i n e t i c s o f growth i n h i b i t i o n by h e r b i c i d e s . P l a n t P h y s i o l . 3 2 : 3 0 1 - 3 0 7 . F r e a r , D . S . , and R . C . B u r e l l . 1955« S p e c t r o p h o t o m e t r i c method f o r d e t e r m i n i n g h y d r o x y l a m i n e r e d u c t a s e a c t i v i t y i n h i g h e r p l a n t s . A n a l . Chem. 2 7 : 1 6 6 4 - 1 6 6 5 . F u l t s , J . L . , and M . G . Payne . 1 9 5 6 . E f f e c t s o f 2 , 4 - d i c h l o r o -p h e n o x y a c e t i c a c i d and m a l e i c h y d r a z i d e on f r e e amino a c i d s and p r o t e i n s i n p o t a t o , sugar b e e t , and bean t o p s . B o t . G a z . 118: 1 3 0 - 1 3 3 . -Gander , J . E . 1 9 6 6 . A c t i v a t i o n o f sorghum u r i d i n e - 5 ' - d i p h o s -phate g l u c o s e p y r o p h o s p h o r y l a s e by o t h e r r i b o n u c l e o s i d e - 5 1 -d i p h o s p h a t e compounds. Phytochem. 5 : 405-410. G a r d n e r , R . , and D.W. R o b e r t s o n . 1 9 4 2 . The n i t r o g e n r e q u i r e -ment o f sugar b e e t . C o l o r a d o A g r i c . E x p t . S t . T e c h . B u l l . 28. •• 140 Ghosh, B . P . , and R . H . B u r r i s . 1 9 5 0 . U t i l i z a t i o n o f n i t r o g e n o u s compounds by p l a n t s . S o i l S c i . 7 0 : I 8 7 - 2 0 3 . G l a s z i o u , K . T . i 9 6 0 . A c c u m u l a t i o n and t r a n s f o r m a t i o n o f sugars i n sugar cane s t a l k s . P l a n t P h y s i o l . 3 5 : 8 9 5 - 9 0 1 . G l a s z i o u , K . T . , and J . C . W a l d r o n . 1 9 6 4 . R e g u l a t i o n o f a c i d i n v e r t a s e l e v e l s i n sugar cane s t a l k s by a u x i n and m e t a b o l i t e - med ia ted c o n t r o l sys tems . Nature ( L o n d . ) . 2 0 3 : 541-542. G l a s z i o u , K . T . , J . C . W a l d r o n , and T . A . B u l l . 1 9 6 6 . C o n t r o l o f i n v e r t a s e s y n t h e s i s i n sugar c a n e . L o c i o f a u x i n and g l u c o s e e f f e c t s . P l a n t P h y s i o l . 41: 282-288. G r e g o r y , F . G . , and P . K . S e n . 1 9 3 7 . P h y s i o l o g i c a l s t u d i e s i n p l a n t n u t r i t i o n . V I . The r e l a t i o n o f r e s p i r a t i o n r a t e to the c a r b o h y d r a t e and n i t r o g e n metabo l i sm o f b a r l e y l e a f as de termined by n i t r o g e n and p o t a s s i u m d e f i c i e n c y . A n n . B o t . ( L o n d . ) . 1 : 5 2 1 - 5 6 1 . G r e u l a c h , V . A . 1955 • P y r i m i d i n e s as a n t a g o n i s t s o f m a l e i c h y d r a z i d e . J . E l i s h a M i t c h e l l S c l e n t . S o c . 7 1 : 2 . G r e e n , D . E . 1 9 3 6 . The m a l i c dehydrogenase o f a n i m a l t i s s u e s . B iochem. J . 3 0 : 2 0 9 5 - 2 1 1 0 . Haddock, J . L . 1 9 5 2 . The n i t r o g e n r e q u i r e m e n t o f sugar b e e t s . P r o c . Am. S o c . Sug . Beet T e c h n o l . 7 : 1 5 9 - I 6 5 . H a l e , V . Q . , and R . J . M i l l e r . 1 9 6 6 . R e l a t i o n s h i p between NO3"" N i n p e t i o l e s d u r i n g the growing season and y i e l d components o f sugar bee t s (Beta v u l g a r i s ) . A g r o n . J . 5 8 : 567-569. Hamner, K . C . 1935* E f f e c t s o f n i t r o g e n s u p p l y on r a t e s o f p h o t o s y n t h e s i s and r e s p i r a t i o n i n p l a n t s . B o t . G a z . 9 7 : 7 4 4 - 7 6 4 . Hanson, R . , M. Z u c k e r , and E . Sandheimer . 1 9 6 7 . The r e g u l a t i o n o f p h e n o l i c b i o s y n t h e s i s and the m e t a b o l i c r o l e of p h e n o l i c compounds i n p l a n t s . I n : P h e n o l i c compounds and m e t a b o l i c r e g u l a t i o n , e d . B . J . F i n k l e , and V . C . R u n e c k l e s . Academic P r e s s , New Y o r k . Haq, S . , and W . Z . H a s s i d . 1 9 6 5 . B i o s y n t h e s i s o f sucrose p h o s -phate w i t h sugar cane l e a f c h l o r o p l a s t s . P l a n t P h y s i o l . 40: 5 9 1 - 5 9 4 . H a t c h , M . D . , J . A . S a c h e r , and K . T . G l a s z i o u . 1 9 6 3 . The sugar a c c u m u l a t i o n c y c l e i n sugar c a n e . I . S t u d i e s on the enzymes o f the c y c l e . P l a n t P h y s i o l . 3 8 : 3 3 8 - 3 4 3 . 141 H a t c h , M . D . , and K . T . G l a s z i o u . 1963* Sugar a c c u m u l a t i o n c y c l e i n sugar c a n e . I I . R e l a t i o n s h i p o f i n v e r t a s e a c t i v i t y to sugar c o n t e n t and growth r a t e i n s t o r a g e t i s s u e o f p l a n t s grown i n c o n t r o l l e d e n v i r o n m e n t s . P l a n t P h y s i o l . 3 8 : 3 4 4 - 3 4 8 . H a t h c o c k , J . N . , C . H . H i l l , and S . B . T o v e . 1 9 6 6 . U n c o u p l i n g o f o x i d a t i v e p h o s p h o r y l a t i o n by v a n a d a t e . C a n . J . Biochem. 4 4 : 9 8 3 - 9 8 8 . Hathway, J . A . , and D . E . A t k i n s o n . 1 9 6 5 . K i n e t i c s o f r e g u l a t o r y enzymes: E f f e c t o f adenos ine t r i p h o s p h a t e on y e a s t c i t r a t e s y n t h e t a s e . B iochem. B i o p h y s . R e s . Comm. 2 0 : 6 6 1 - 6 6 5 . Hawker, J . S . 1 9 6 6 . S t u d i e s on the l o c a t i o n o f s u c r o s e phos -phatase i n p l a n t t i s s u e s . Phytochem. 1191-1199. Headden, J . 1 9 1 2 . D e t e r i o r a t i o n o f the q u a l i t y o f sugar bee t s due to n i t r a t e s formed i n the s o i l . C o l o r a d o A g r . E x p t . S t . B u l l . I 8 3 . H e l l e b r u s t , J . A . , and R . G . S . B i d w e l l . 1 9 6 3 . Sources o f c a r b o n f o r the s y n t h e s i s o f p r o t e i n amino a c i d s i n a t t a c h e d p h o t o s y n t h e s l z i n g wheat l e a v e s . C a n . J . B o t . 4 l : 985-994. H e w i t t , E . J . , and E . W . J o n e s . 1 9 4 7 . The p r o d u c t i o n o f molyb-denum d e f i c i e n c y i n p l a n t s i n sand c u l t u r e w i t h s p e c i a l r e f e r e n c e to tomato and b r a s s i c a c r o p s . J . Pomol . H o r t . S c i . 2 3 : 2 5 4 - 2 6 2 . Heyes , J . K . i 9 6 0 . N u c l e i c a c i d changes d u r i n g c e l l e x p a n s i o n i n the r o o t . P r o c . Roy. S o c . B . 1 5 2 : 2 1 8 - 2 3 0 . Heyes , J . K . , and R. Brown. 1 9 6 5 . C y t o c h e m i c a l changes i n c e l l growth and d i f f e r e n t i a t i o n i n p l a n t s . I n : E n c y c l o p a e d i a o f P l a n t P h y s i o l o g y , e d . W. R u h l a n d . S p r i n g e r . B e r l i n . 14: 189-212. H i n d e , R . W . , and L . R . F i n c h . 1 9 6 6 . The a c t i v i t i e s o f phos -p h a t a s e s , pyrophosphatases and a d e n o s i n e t r i p h o s p h a t a s e s from normal and b o r o n d e f i c i e n t bean r o o t s . Phytochem. 5 : 6 1 9 - 6 2 3 . I s e n b e r g , F . M . R . , M . L . O d l a n d , H .W. Popp, a n d C O . J e n s e n . 1 9 5 1 . The e f f e c t o f m a l e i c h y d r a z i d e on c e r t a i n dehydrogenases i n t i s s u e s o f o n i o n p l a n t s . S c i e n c e . 113: 5 8 - 6 0 . I s e n b e r g , F . M . R . , C O . J e n s e n , and M . L . Odlamd. 1 9 5 4 . E f f e c t o f m a l e i c h y d r a z i d e on the r e s p i r a t i o n o f mature o n i o n b u l b s . S c i e n c e . 2 0 : 464-465. I t o , H . , and Y . Y o s h l n a k a . 1 9 6 4 . The s t o r a g e o f o n i o n b u l b s . w i t h m a l e i c h y d r a z i d e . X I V . E f f e c t o f m a l e i c h y d r a z i d e f o l -i a g e sprays on the RNA and p r o t e i n c o n t e n t s o f the o n i o n b u l b s . Kobe Daigaku Kyoikugakubu Kenkyu S h u r o k u , S h i z e n Kagakenhen. 3 2 : 6 9 - 9 9 . 142 J a n s e n , W . A . 1 9 5 6 . On the d i s t r i b u t i o n o f n u c l e i c a c i d s i n the r o o t t i p o f V i c l a f a b a . E x p t . C e l l . R e s . 1 0 : 222-224. J o y , K . W . 1 9 6 2 . T r a n s p o r t o f o r g a n i c n i t r o g e n through the phloem i n sugar b e e t . Nature ( L o n d . ) 195* 6 1 8 - 6 1 9 . J o y , K . W . 1 9 6 4 . T r a n s l o c a t i o n i n sugar b e e t . I . J . E x p . B o t . 15: 4 8 5 - 4 9 5 . . J o y , K . W . 1 9 6 7 . Carbon and n i t r o g e n sources f o r p r o t e i n s y n -t h e s i s and growth o f sugar b e e t . J . E x p . B o t . 18: 140-150. K a l i n i n , F . L . , A . D . K u l a k a l i , V . S . D o l y a , A . G . M i s h u r e m i a , and G . I . Gogotov . 1 9 6 4 . H y d r a z i d e o f m a l e i c a c i d ; an e f f e c t i v e means o f i n c r e a s i n g the p r o d u c t i v i t y o f p l a n t s . K h i m . V . S e l ' s k . K h o z . 5:40-45. K e n t e n , R . H . 1 9 5 5 . The o x i d a t i o n o f i n d o l y l - 3 - a c e t i c a c i d by waxpod bean r o o t sap and p e r o x i d a s e sys tem. B iochem. J . 59: 1 1 0 - 1 2 1 . K l o t z , I . M . 1 9 5 4 . Thermodynamic and m o l e c u l a r p r o p e r t i e s o f some m e t a l - p r o t e i n complexes . I n ; The mechanism o f enzyme a c t i o n . Symp. M c C o l l u m - P r a t t I n s t i t u t e , e d . W.D. M c E l r o y and B . G l a s s . B a l t i m o r e , John Hopkins U n i v . P r e s s . 2 5 7 - 2 8 5 . Knoop, F . , and H . O s t e r l i n . 1925 • ttber d i e n a t u r l i c h e Synthese der Amino Sauren und i h r e e x p e r i m e n t e l l e R e p r o d u k t i o n . Hoppe - S e y l . Z . 148: 2 9 4 - 3 0 5 . K r e b s , H . A . , and W . A . J o h n s o n . 1 9 3 7 . C i t r i c a c i d i n i n t e r -mediate metabo l i sm i n a n i m a l t i s s u e s . E n z y m o l o g i a . . 4 : 148-156. K u r a i s h i , S . , and R . M . M u i r . I 9 6 3 . Mode of a c t i o n o f growth r e t a r d i n g c h e m i c a l s . P l a n t P h y s i o l . 30: 1 9 - 2 4 . L e e p e r , G.W. 1 9 4 1 . Manganese d e f i c i e n c y and a c c u m u l a t i o n of n i t r a t e s i n p l a n t s . J . A u s t . I n s t . A g r i c . S c i . 7- l 6 l - l 6 2 . L e l o i r , L . F . , and C . E . C a r d i n i . 1 9 5 3 . The b i o s y n t h e s i s o f s u c r o s e . J . Am. Chem. S o c . 75: 6 0 8 4 . L e l o i r , L . F . , and C . E . C a r d i n i . 1955* The b i o s y n t h e s i s o f sucrose p h o s p h a t e . J . B i o l . Chem. 214: 1 5 7 - 1 6 5 . L e o n a r d , M . J . K . , and R . H . B u r r i s . 1 9 4 7 . A survey o f t r a n s -aminases i n p l a n t s . J . B i o l . Chem. 1 7 0 : 7 0 1 - 7 0 9 . L e o p o l d , A . C . , and W . H . K l e i n . 1 9 5 1 . M a l e i c h y d r a z i d e as an a n t i - a u x i n . S c i e n c e . 114: 9 - 1 0 . L e o p o l d , A . C . , and W . H . K l e i n . 1 9 5 2 . M a l e i c h y d r a z i d e as an a n t i - a u x i n . P h y s i o l . P l a n t . 5: 9 1 - 9 9 . 143 Leopold, A . C . 1964. Plant Growth and Development. McGraw-Hill Book Co. New York. -Livingstone, C , M.G. Payne, and J . L . Ful t s . 1 9 5 4 . Effects of maleic hydrazide and 2,4-dichlorophenoxyacetic acid on the free amino acids in sugar beets. Bot. Gaz. 1 1 6 : 148-156. Loomis, W.E. , and C.A. Shul l . 1 9 3 7 . Methods in Plant Physiol-ogy. McGraw-Hill Book Co. New York. Loomis, R . S . , and A. U l r i c h . 1 9 5 9 . Response of sugar beets to nitrogen depletion in re lat ion to root s ize . J . Am. Soc. Sug. Beet Technol. 1 0 : 499-512. Lowry, O .H. , N . J . Rosenberg, A . L . Farr, and R . J . Randall. 1 9 5 1 . Protein measurement with the Fol in phenol reagent. J . B i o l . Chem. 1 9 3 : 2 6 5 - 2 7 5 . Lund, H . A . , A . E . Vatter, and J . B . Hanson. 1 9 5 8 . Biochemical and cytological changes accompanying growth and d i f ferent i -ation i n the roots of Zea mays. J . Biophys. Biochem. Cytol . 4 : 8 7 - 9 8 . Lundergardh, H. 1 9 5 0 . The translocation of salts and water through wheat roots. Physiol . Plant. 3: 1 0 3 . 1 5 1 . Maevskaya, A . N . , and K.A. Alekseeva. 1 9 6 4 . The effect of boron deficiency on adenosinetriphosphatase (ATP-ase) ac t iv i ty in the sunflower. Dokl. Akad. Nauk. S.S.S.R. 1 5 6 : 212-213. Mahler, H.R. , G. Hubscher, and H. Baum. 1955 • Studies on uricase. 1. Preparation, puri f icat ion, and properties of a cuproprotein. J . B i o l . Chem. 2 1 6 : 625-641. .Mason, H.S. 1955* Comparative biochemistry of the phenolase complex. Adv. Enzymol. 1 6 : 105-184. Mattas, R . E . , and A.W. Paul i . I 9 6 5 . Trends in nitrate reduction and nitrogen fractions in young corn (Zea mays L . ) plants during heat and mositure stress. Crop S c i . 5: 181-184. Mcleish, J . 1955* Radiation sensi t iv i ty and the mitotic cycle in V ic ia faba. Nature (Lond.) 1 7 5 : 8 9 O - 8 9 1 . Medina, A . , and D.J .D. Nicholas. 1 9 5 7 . Hyponitrite reductase in Neurospora. Nature (Lond.) 1 7 9 : 533-534. Medicino, J . i 9 6 0 . Sucrose phosphate synthesis in wheat germ and green leaves. J . B i o l . Chem. 2 3 5 : 3 3 4 7 - 3 3 6 2 . Nakabayashi, T. 1 9 5 4 . Browning of apple f r u i t . Nippon "Nogeikagaku Kaishi . 28: 212-217-144 Nason, A . , and H . J . E v a n s . 1 9 5 4 . T r i p h o s p h o p y r i d i n e n u c l e o -t i d e - n l t r a t e r e d u c t a s e i n N e u r o s p o r a . J . B i o l . Chem. 2 0 2 : 655-673. Nason, A . , R . G . Abraham, and B . C . A v e r b a c h . 1 9 5 4 . The enzymat ic r e d u c t i o n o f n i t r i t e to ammonia by r e d u c e d p y r i d i n e n u c l e o t i d e s . B iochem. B i o p h y s . A c t a . 1 5 : l 6 0 - l 6 l . N e l s o n , M. 1944. A p h o t o m e t r i c a d a p t a t i o n o f the somogyi method f o r d e t e r m i n a t i o n o f g l u c o s e . J . B i o l . Chem. 1 5 3 : 375-330. N i c h o l a s , D . J . D . , and A . Nason . 1 9 5 4 . Molybdenum and n i t r a t e r e d u c t a s e . I I . Molybdenum as a c o n s t i t u e n t o f n i t r a t e r e d u c t a s e from soybean l e a v e s . P l a n t P h y s i o l . 3 0 : 1 3 5 - 1 3 8 . N i c h o l a s , D . J . D . , A . Nason, and W.D. M c E l r o y . 1 9 5 4 . Molybdenum and n i t r a t e r e d u c t a s e . I . E f f e c t o f molybdenum d e f i c i e n c y on the Neurospora enzyme. J . B i o l . Chem. 2 0 7 : 341-351* Noda, K . , S . E g u c h i , K . I b a r a g i , and K . Ozawa. 1963* S t u d i e s on growing b e h a v i o r o f sugar bee t p l a n t i n the warmer d i s t r i c t s o f J a p a n . I I . E f f e c t o f n i t r o g e n f e r t i l i z e r on growing b e h a v i o r . P r o c . Crop S c . S o c . J a p a n . 3 2 : 2 6 - 3 0 . Nooden, L . D . 1 9 6 7 . S t u d i e s on the mechanism o f a c t i o n o f m a l e i c h y d r a z i d e . P l a n t P h y s i o l . 42: S - 5 0 . Nooden, L . D . , and K . V . Thimann. 1965* I n h i b i t i o n o f p r o t e i n s y n t h e s i s and o f a u x i n - i n d u c e d growth by c h l o r a m p h e n i c o l . P l a n t P h y s i o l . 40: 1 9 3 - 2 0 1 . Ogden, D . B . , R . F . F i n k e r , R . F . O l s o n , and P . C . H a n z a s . 1 9 5 8 . The e f f e c t o f f e r t i l i z e r t rea tment upon t h r e e d i f f e r e n t v a r i e t i e s i n the Red R i v e r V a l l e y o f Minneso ta f o r : I . -s t a n d , y i e l d , s u g a r , p u r i t y , and n o n - s u g a r s . J . Am. S o c . S u g . Beet T e c h n o l . 1 0 : 2 6 5 - 2 7 1 . Okanenko, A . S . 1 9 5 9 . The a c c u m u l a t i o n o f sugar In v a r i o u s forms and s t r a i n s o f bee t and p r o s p e c t s f o r f u r t h e r i n c r e a s e i n the sugar c o n t e n t o f sugar b e e t . R e f e r a t . Z h u r . B i o l . No. 3 9 2 7 7 . Owens, H . S . , C . L . Ramussen, and W.D. M a c l a y . 1 9 5 1 . P r o d u c t i o n and u t i l i z a t i o n o f sugar b e e t s . E c o n . B o t . 5- 348-366. P a r u p u s , E . V . 1 9 6 7 . E f f e c t o f v a r i o u s p l a n t pheno l s on p r o t e i n s y n t h e s i s i n e x c i s e d p l a n t t i s s u e . C a n . J . B iochem. 4 5 : 4 2 7 - 4 3 3 . Payne , M . G . , LeRoy Powers , and E . E . Remmenga. 1 9 6 l . Some - c h e m i c a l and g e n e t i c s t u d i e s p e r t a i n i n g to q u a l i t y i n sugar bee t s (Beta v u l g a r i s L . ) . J . Am. S o c . S u g . Beet T e c h n o l . 1 1 ; 6l0^6"28". 145 P e t e r s o n , E . L . , and A . W . N a y l o r . 1953* Some m e t a b o l i c changes i n tobacco stem t i p s accompanying m a l e i c h y d r a z i d e t r e a t -ment and the appearance o f f r e n c h i n g symptoms." P h y s i o l . P l a n t . 6: 816-828. P i e r p o i n t , W . S . 1966. The enzymat ic o x i d a t i o n o f c h l o r o g e n i c a c i d and some r e a c t i o n s o f the qu inone p r o d u c e d . B iochem. J . 98: 567-580. P l a u t , G . W . E . 1957* A s o l u b l e enzyme from m i t o c h o n d r i a c a t a l -y s i n g an exchange between i n o r g a n i c phosphate and adenos ine t r i p h o s p h a t e . A r c h . B iochem. B i o p h y s . 69: 320-333. P o v o l o t k a y a , K . L . I 9 6 I . Mechanism o f the a c t i o n o f m a l e i c h y d r a z i d e i n p l a n t . I z v e s t . A k a d . Nauk. S . S . S . R . 2 6 ; S e r . B i o l . No . 2 : 2 5 0 - 2 5 5 . P r e s s e y , R. 1 9 6 6 . S e p a r a t i o n and p r o p e r t i e s o f p o t a t o i n v e r -ta se and i n v e r t a s e i n h i b i t o r . A r c h . B iochem. B i o p h y s . 1 1 3 : 667-67k. Ramirex , J . M . , P . P . d e l Campo, A . Paneque, and M. L o s a d a . 1 9 6 4 . Mechanism o f n i t r a t e r e d u c t i o n s i n c h l o r o p l a s t s . B iochem. B i o p h y s . R e s . Comm. 1 5 : 2 9 7 - 3 0 2 . R e b s t o c k , T . L . , C D . B a l l , and C . L . Hamner. 1 9 5 5 . I n h i b i t i o n of p l a n t growth by 2 -mercaptobenz imidazo le a n a l o g s . P l a n t P h y s i o l . 3 0 : 382-384. Rei tman , S, and S . F r a n k e l . 1957* A c o l o r i m e t r i c method f o r the d e t e r m i n a t i o n o f serum g l u t a m i c o x a l o a c e t i c and g l u t a m i c p y r u v i c t r a n s a m i n a s e s . Am. J . C l i n . P a t h . 28: 56-63. R o b i n s o n , E . , and R . Brown. 1 9 5 2 . The development o f the enzyme i n growing r o o t c e l l s . J . E x p . B o t . 3: 356-374. R o c k h o l d , W . T . , and N . A . T o l v i t i e . 1956. Vanadium c o n c e n t r a t i o n o f u r i n e . R a p i d c o l o r i m e t r i c method f o r i t s e s t i m a t i o n . C l i n . Chem. 2: 188-194. Roe, J . 1934. A c o l o r i m e t r i c method f o r the d e t e r m i n a t i o n o f f r u c t o s e i n b l o o d and u r i n e . J . B i o l . Chem. 1 0 7 : 15-22. Rorem, E . S . , H . G . Walker j r . , and R . M . McCready . i 9 6 0 . B i o s y n t h e s i s o f s u c r o s e and s u c r o s e phosphate by sugar beet l e a f e x t r a c t s . P l a n t P h y s i o l . 35: 269-272. Rosen, H . 1957* A m o d i f i e d n i n h y d r i n c o l o r i m e t r i c a n a l y s i s f o r amino a c i d s . A r c h . B iochem. B i o p h y s . 6 7 : 1 0 - 1 5 . Rounds, H . G . , E . R u s h , D . L . Oldmeyer, C . P a r r i s h , and F . N . R a w l i n g s . 1958. A s tudy and economic a p p r a i s a l o f the e f f e c t of n i t r o g e n f e r t i l i z a t i o n and s e l e c t e d v a r i e t i e s on the p r o -d u c t i o n and p r o c e s s i n g o f sugar b e e t s . J . Am. S o c . Sug . Beet T e c h n o l . 1 0 : 97-116. 146 R u s s e l , F . 1 9 6 5 . The e f f e c t o f v a r y i n g r a t e s o f n i t r o g e n on beet y i e l d s and sugar p r o d u c t i o n . P r o c . 1 3 t h R e g i o n a l M e e t i n g . Am. S o c . Sug . Beet T e c h n o l . E a s t e r n U . S . , and E a s t e r n Canada. , 4 4 - 4 9 . S a c h e r , J . A . , M . D . H a t c h , and K . T . G l a s z i o u . I 9 6 3 . Sugar a c c u m u l a t i o n c y c l e i n sugar c a n e . I I I . P h y s i c a l and m e t a b o l i c a s p e c t s o f c y c l e i n immature s t o r a g e t i s s u e . P l a n t P h y s i o l . 3 8 : 3 4 8 - 3 5 4 . S a c h e r , J . A . , M . D . H a t c h , and K . T . G l a s z i o u . 1 9 6 3 ( a ) . The r e g u l a t i o n o f i n v e r t a s e s y n t h e s i s i n sugar cane by an a u x i n - and s u g a r - m e d i a t e d c o n t r o l sys tem. P h y s i o l . P l a n t . 1 6 : 836-842. S a m b o r s k i , D . J . , and M. Shaw. 1 9 5 7 . The p h y s i o l o g y of h o s t -p a r a s i t e r e l a t i o n s . T V . A . The e f f e c t o f m a l e i c h y d r a z i d e on the c a r b o h y d r a t e , n i t r o g e n and f r e e amino a c i d c o n t e n t o f the f i r s t l e a f o f k h a p l i wheat . C a n . J . B o t . 35: 4 5 7 - 4 6 1 . S c h r a d e r , L . E . , and R . H . Hageman. 1967* R e g u l a t i o n o f n i t r a t e r e d u c t a s e a c t i v i t y i n c o r n (Zea mays L . ) s e e d l i n g by endogenous m e t a b o l i t e s . P l a n t P h y s i o l . 42: 1 7 5 0 - 1 7 5 6 . Schmehl , W . R . , R. F i n k e r , and J . Swink . 1 9 6 3 . E f f e c t o f n i t r o -gen f e r t i l i z a t i o n on y i e l d and q u a l i t y o f sugar b e e t s . J . Am. S o c . S u g . Beet T e c h n o l . 1 2 : 5 3 8 - 5 4 4 . S h i r o y a , M . , T . S h i r o y a , and S . H a t t o r i . 1 9 5 5 . S t u d i e s on the browning and b l a c k e n i n g o f p l a n t t i s s u e s . I V . C h l o r o g e n i c a c i d i n the l e a v e s o f N i c o t i a n a tabacurn. P h y s i o l . P l a n t . 8 : 5 9 4 - 6 0 4 . S i s a k j a n , N . M . , and A . M . Kobyakova . 1 9 4 8 . A c t i v i t y and c o n d i t i o n o f enzymes i n p l a s t i d s . B i o k h i m . 1 3 : 8 8 - 9 4 . S i s a k j a n , N . M . , A . M . Z o l k o v e r , and V . I . B i r y u z o v a . 1 9 4 8 . S t r u c t u r e o f p l a s t i d s and a c t i v i t y o f enzymes. Doklady A k a d . Nauk. S . S . S . R . 6 0 : 1 2 1 3 - 1 2 1 5 . S i s a k j a n , N . M . , and A . M . Kobyakova . 1951* F o r m a t i o n and move-ment o f enzymes i n l i v i n g o r g a n i s m . B i o k h i m . 1 6 : 2 9 2 - 2 9 7 . S i s a k j a n , N . M . , V . I . B i r y u z o v a , and A . M . Kobyakova . 1951* Changes i n the s t r u c t u r e and enzymat ic a c t i v i t y o f p l a s t i d s i n the o n t o g e n e t i c c y c l e o f p l a n t deve lopment . B i o k h i m . 1 6 : 4 4 9 - 4 5 3 . S l a c k , C . R . 1 9 6 6 . I n h i b i t i o n o f UDPG-g lucose : D - f r u c t o s e - 2 -g l u c o s y l t r a n s f e r a s e from sugar cane stem t i s s u e by pheno l o x i d a t i o n p r o d u c t s . Phytochem. 5 : 3 9 7 - 4 0 3 . S m i t h , D . C . , J . A . Bassahm, and M. K i r k . 1 9 6 1 . Dynamics of the p h o t o s y n t h e s i s o f c a r b o n compounds. I I . Amino a c i d s y n t h e s i s . B iochem. B i o p h y s . A c t a . 48: 2 9 9 - 3 1 3 * 147 S n y d e r , F . W . , and N . E . T o l b e r t . 1 9 6 6 . I n f l u e n c e o f n i t r o g e n n u t r i t i o n and season on p h o t o s y n t h e t i c i n c o r p o r a t i o n o f C ^ 0 2 i n t o s u c r o s e and o t h e r compounds o f sugar b e e t . B o t . G a z . 1 2 ? : 1 6 4 - 1 7 0 . S o r e n s e n , L . O . 1 9 5 6 . E f f e c t o f m a l e i c h y d r a z i d e on p h o t o -s y n t h e s i s and r e s p i r a t i o n o f r e d k i d n e y b e a n . U n i v . m i c r o f i l m (Ann A r b o r , M i c h . ) Pub . No. 1 7 3 6 7 , 4 l p p . D i s s e r t a t i o n A b s t r . 1 6 : 1 5 6 9 . S t e e l , R . G . D . , and J . H . T o r r i e . i 9 6 0 . P r i n c i p l e s and p r o c e -dures o f s t a t i s t i c s . M c G r a w - H i l l Book C o . , New Y o r k . S teward , F . C , and D . J . D u r j a n . 1 9 6 5 . M e t a b o l i s m o f n i t r o -genous compounds. I n : P l a n t P h y s i o l , e d . F . C . S t e w a r d . Academic P r e s s . New Y o r k . 4 ( A ) : 379-684. S t e w a r d , F . C , and R . G . S . B i d w e l l . 1 9 6 6 . S torage p o o l s and t u r n o v e r systems i n . growing and non-growing c e l l s . Exper iments w i t h C ^ - s u c r o s e , C-1- - g l u t a m i c a c i d , C -^ - a s p -a r a g i n e . J . E x p . B o t . 1 7 : 726-741. S t o u t , M. 1 9 6 l . A new l o o k a t some n i t r o g e n r e l a t i o n s h i p s a f f e c t i n g the q u a l i t y o f sugar b e e t s . J . Am. S o c . S u g . Beet T e c h n o l . 1 1 : 3 8 8 - 3 9 8 . S t o u t , M. 1 9 6 4 . R e d i s t r i b u t i o n o f n i t r a t e i n s o i l and i t s e f f e c t s on sugar bee t n u t r i t i o n . J . Am. S o c . S u g . Beet T e c h n o l . 1 3 : 6 8 - 8 0 . S t r e e t , H . E . , and D . E . S h e a t . 1 9 5 8 . The a b s o r p t i o n and a v a i l a b i l i t y o f n i t r a t e and ammonia. I n : E n c y c l o p a e d i a o f P l a n t P h y s i o l o g y , e d . W. R u h l a n d . S p r i n g e r . B e r l i n . 8 : 1 5 0 - 1 6 5 . S u n d e r l a n d , N . , and R . Brown. 1 9 5 6 . D i s t r i b u t i o n o f growth i n the a p i c a l r e g i o n o f the shoot o f L u p i n u s a l b u s . J . E x p . B o t . 7 : 127-145. S y r e t t , P . J . , and I . M o r r i s . 1 9 6 3 . The i n h i b i t i o n o f n i t r a t e a s s i m i l a t i o n by ammonia i n c h l o r e l l a . B iochem. B i o p h y s . A c t a . 6 7 : 5 6 6 - 5 7 5 . T a k e t a , K . , and B . M . P o g e l l . 1 9 6 3 . R e v e r s i b l e i n a c t i v a t i o n and i n h i b i t i o n o f l i v e r f r u c t o s e - l , 6 - d i p h o s p h a t a s e by adenos ine n u c l e o t i d e s . B i o c h e m . B i o p h y s . R e s . Comm. 1 2 : 2 2 9 - 2 3 5 . T i n k e r , P « B . H . 1 9 6 5 . The e f f e c t o f n i t r o g e n , p o t a s s i u m , and sodium f e r t i l i z e r s on sugar b e e t . J . A g r i c . S c . 6 5 : 2 0 7 - 2 1 2 . Tolman, B . , and R . C . J o h n s o n . 1 9 5 8 . E f f e c t o f n i t r o g e n on the y i e l d and sucrose c o n t e n t o f sugar b e e t s . J . Am. S o c . Sug . Beet T e c h n o l . 10: 2 5 4 - 2 5 7 . 148 Towers , G . H . N . 1 9 6 4 . M e t a b o l i s m of p h e n o l i c s i n h i g h e r p l a n t s and m i c r o - o r g a n i s m s . I n : B i o c h e m i s t r y o f P h e n o l i c Compounds, e d . J . B . H a r b o r n e . Academic P r e s s . New Y o r k . 249-294. U l r i c h , A . 1 9 4 2 . The r e l a t i o n s h i p o f n i t r o g e n to the f o r m a t i o n o f sugar i n sugar b e e t s . P r o c . Am. S o c . S u g . Beet T e c h n o l . 66-80. U l r i c h , A . 1 9 5 0 . C r i t i c a l n i t r a t e l e v e l s o f sugar bee t s e s t i m a t e d from a n a l y s i s o f p e t i o l e s and b l a d e s w i t h s p e c i a l r e f e r e n c e to y i e l d and s u c r o s e c o n c e n t r a t i o n . S o i l S c i . 6 9 : 2 9 1 - 3 0 9 . Van N i e l , C . B . , M . B . A l l e n , and B . E . W r i g h t . 1 9 5 3 . On the p h o t o c h e m i c a l r e d u c t i o n o f n i t r a t e by a l g a e . B iochem. B i o p h y s . A c t a . 1 2 : 6 7 - 7 4 . V a r n e r , J . E . , and G . C . W e b s t e r . 1955 • S t u d i e s on the enzymat ic s y n t h e s i s o f g l u t a m i n e . P l a n t P h y s i o l . 3 0 : 393-402. V i c k e r y , H . B . , and G.W. P u c h e r . 1 9 2 9 . The d e t e r m i n a t i o n o f ammonia and amide n i t r o g e n i n tobacco by the use o f p e r m u t i t . J . B i o l . Chem. 8 3 : 1 - 1 0 . W a d l e i g h , C . H . 1 9 5 2 . F a c t o r s a f f e c t i n g h e a l t h y r o o t s . P r o c . Am. S o c . S u g . Beet T e c h n o l . 7 : 1 5 - 2 1 . W a l l a c e , W . , and J . S . P a t e . 1 9 6 7 . N i t r a t e a s s i m i l a t i o n i n h i g h e r p l a n t s w i t h s p e c i a l r e f e r e n c e to the c o c k l e b u r (Xanthium p e n n s y l v a n l c u m W a l l r . ) . A n n . B o t . ' N . S 3 1 : 213-228. W a r r i n g t o n , K . 1951* Some i n t e r r e l a t i o n s h i p s between manga-nese , molybdenum and vanadium i n the n u t r i t i o n o f soybeans , f l a x , and o a t s . A n n . A p p l . B i o l . 3 8 : 624-641. Webb, J . L . I 9 6 3 . Enzyme and M e t a b o l i c I n h i b i t o r s . V o l . I . Academic P r e s s . New Y o r k . Webs ter , G . C , and J . E . V a r n e r . 1955* A s p a r t a t e metabo l i sm and a s p a r a g i n e s y n t h e s i s i n p l a n t sys tems . J . B i o l . Chem. 2 1 5 : 9 1 - 9 9 . West, S . H . 1 9 6 2 . P r o t e i n , n u c l e o t i d e , and r i b o n u c l e i c a c i d metabo l i sm i n c o r n d u r i n g g e r m i n a t i o n under water s t r e s s . P l a n t P h y s i o l . 3 7 : 5 6 5 - 5 7 1 . W i l s o n , D . G . , K . W . K i n g , and R . H . B u r r i s . 1 9 5 4 . T r a n s a m i n a t i o n i n p l a n t s . J . B i o l . Chem. 208: 8 6 3 - 8 7 4 . W i t t w e r , S . H . , and C M . Hansen . 1 9 5 2 . Some e f f e c t s o f p r e -' h a r v e s t f o l i a r s p r a y s o f m a l e i c h y d r a z i d e on the sugar c o n t e n t and s t o r a g e l o s s e s o f sugar b e e t s . P r o c . Am. S o c . Sug . Beet T e c h n o l . 9 0 - 9 4 . 149 W o l l e y , D . G . , and W . H . B e n n e t . 1 9 6 2 . E f f e c t o f s o i l m o i s t u r e , n i t r o g e n f e r t i l i z a t i o n , v a r i e t y and h a r v e s t da te on r o o t y i e l d s and s u c r o s e c o n t e n t o f sugar b e e t s . J . Am. S o c . Sug . Beet T e c h n o l . 1 2 : 2 3 3 - 2 3 7 . W o l l e y , J . T . , G . P . H i c k s , and R . H . Hageman. i 9 6 0 . R a p i d d e t e r m i n a t i o n o f n i t r a t e and n i t r i t e In p l a n t m a t e r i a l . J . A g r . F d . Chem. 8 : 2 6 0 - 2 6 1 . Wort , D . J . , and D . M . S h r i m p t o n . 1 9 5 9 . T e r m i n a l o x i d a s e s o f the mature sugar bee t r o o t . J . Am. S o c . Sug . Beet T e c h n o l . 1 0 : 5 9 4 - 6 0 2 . Wort , D . J . , and G . A . W h i t e . 1 9 5 6 . C h e m i c a l and b i o c h e m i c a l r e s p o n s e s o f the mature sugar beet r o o t to d e f o l i a t i o n by f r o s t and k n i f e . C a n . J . B o t . 3 4 : 7 7 7 - 7 9 2 . W o s i l a i t , W . D . , A . Nason, and A . J . T u r r e l . 1955* P y r i d i n e n u c l e o t i d e - q u i n o n e r e d u c t a s e . I I . R o l e i n e l e c t r o n t r a n s p o r t . J . B i o l . Chem. 2 0 6 : 2 7 1 - 2 8 3 . Yang , K . J . 1 9 6 4 . D i p h o s p h o p y r i d i n e n u c l e o t i d e - n i t r a t e r e d u c -tase i n Be ta v u l g a r i s L . PhD. T h e s i s , U n i v . o f B r i t i s h C o l u m b i a . Yemm, E . W . 1 9 6 5 . The r e s p i r a t i o n o f p l a n t s and t h e i r o r g a n s . I n : P l a n t P h y s i o l , e d . F . C . • S t e w a r d . Academic P r e s s . New Y o r k . 4 ( A ) : 2 3 1 - 3 1 0 . Young, R . E . 1 9 6 5 . E x t r a c t i o n o f enzymes from t a n n i n - b e a r i n g t i s s u e . A r c h . B iochem. B i o p h y s . I l l : 1 7 4 - 1 8 0 . Z i e s e r l , J . F . , W . L . R i v e n b a r k , and R . H . Hageman. 1963* N i t r a t e r e d u c t a s e a c t i v i t y , p r o t e i n c o n t e n t and y i e l d o f f o u r maize h y b r i d s a t v a r y i n g p l a n t p o p u l a t i o n s . Crop S c i . 3 : 2 7 - 3 2 . Z u c k e r , M . , and A . Nason. 1955« A p y r i d i n e n u c l e o t i d e - h y d r o -xy lamine r e d u c t a s e from N e u r o s p o r a . J . B i o l . Chem. 2 1 3 : 4 6 3 - 4 7 8 . 

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