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The cinnamic acid pathway and hispidin biosynthesis in cultures of Polyporus hispidus Fries Perrin, Peter William 1972

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THE UPTAKE OF SULPHUR, CALCIUM, AND MAGNESIUM AND THEIR DISTRIBUTION IN PHASEOLUS VULGARIS L. AS AFFECTED BY CYCLOHEXANECARBOXYLIC ACID by DAVID R. PEIRSON B.Sc. U n i v e r s i t y of Waterloo, 1 9 6 3 M.Sc. U n i v e r s i t y of Waterloo, 1 9 6 9 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of BOTANY We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re ference and s tudy . I f u r t h e r agree t h a t pe rmiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s understood that copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n p e r m i s s i o n . Department of BOTANY The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date August JO, 1972 i i ABSTRACT Cyclohexanecarboxylic acid (CHCA) has one of the lowest molecular weights of the naphthenic acids, a group of com-pounds which have been recognized recently as stimulators of plant growth and metabolism. Potassium cyclohexanecarboxy-l a t e ' s (KCHC) e f f e c t s on the mineral n u t r i t i o n of bush bean plants (Phaseolus vulgaris L., c u l t i v a r Top Crop) were ex-amined i n four separate studies. 1. The f o l i a g e of two-week-old plants, growing i n sulphur-free nutrient s o l u t i o n was treated with 1 X 10~ 2 M KCHC soluti o n and subsequently the roots were given a ^ hr 35 = . exposure to SO^ i n the nutrient solution. S l i g h t l y more sulphur - 3 5 was taken up by treated plants than untreated control plants, but the difference was not s t a t i s t i c a l l y s i g n i f i c a n t . However, s i g n i f i c a n t l y more sulphur - 3 5 was detected i n the leaves of treated plants. The differences i n d i s t r i b u t i o n were attributed to metabolic changes brought about by treatment. 2. Plants were handled as above and the leaves were harvested one, two, and f i v e days a f t e r feeding sulphur - 3 5 * The l e a f tissue was separated, chemically, into sulphate, l i p i d , free amino acid, p e r c h l o r i c acid soluble, and protein f r a c t i o n s . A portion of the protein f r a c t i o n was hydrolyzed to permit separation of the suiphur-amino acids by chromato-graphy. The bulk of the sulphur - 3 5 was incorporated into the acid soluble f r a c t i o n f i r s t and subsequently into the protein f r a c t i o n . KCHC treated leaves contained s i g n i f i c a n t l y more i i i a c i d soluble S i n i t i a l l y and more protein S f i n a l l y than con t r o l leaves. Control leaves contained more sulphate S than treated leaves two days a f t e r sulphur-35 feeding. These r e s u l t s indicated that KCHC treatment stimulated the incor-poration of sulphur into protein, and this was consistent with other reports of increased protein formation due to naphthenate treatment. 3. Bush bean plants growing i n complete nutrient s o l -ution were treated and exposed to calcium-45. Treated plants took up s l i g h t l y less calcium than controls, and they retained s i g n i f i c a n t l y more of i t i n t h e i r roots. These r e s u l t s are i n contrast to the e f f e c t of treatment on sulphur-35 uptake and d i s t r i b u t i o n . Bush bean plants growing i n complete nutrient s o l -ution were treated with KCHC sol u t i o n at two weeks of age. One week l a t e r the d i s t r i b u t i o n of sulphur, calcium, and magnesium was examined i n l e a f blades, stems plus p e t i o l e s , and roots. No s i g n i f i c a n t e f f e c t of treatment on uptake was found f o r any of the elements. D i s t r i b u t i o n of sulphur and calcium within the plant was not affected by treatment, but control plants contained s i g n i f i c a n t l y more magnesium i n t h e i r leaves than treated plants. The lack of s i g n i f i c a n t e f f e c t on ion uptake indicated that the increased growth found i n many studies, when plants were treated with naphthenic acids, apparently was not the r e s u l t of improved uptake of sulphur, calcium, or magnesium. i v TABLE OF CONTENTS ABSTRACT n LIST OF TABLES v i i LIST OF FIGURES ix ABBREVIATIONS x ACKNOWLEDGEMENTS x i INTRODUCTION AND LITERATURE REVIEW 1 Naphthenic acids 1 Cyclohexanecarboxylic acid 8 Sulphur compounds 11 Sulphur uptake 12 Sulphur translocation 14 Sulphur d i s t r i b u t i o n 16 Sulphate reduction. 17 Calcium 19 Magnesium 23 MATERIALS AND METHODS , 2 5 Uptake and D i s t r i b u t i o n of Sulphur - 3 5 25 A. Growth of plants. 25 B. Preparation of, and treatment with, cyclo-hexanecarboxylic acid solution 27 C. Exposure to 3 5 s 27 D. Harvest 28 E. Digestion.. 28 F. Radioactive counting 29 V Uptake and D i s t r i b u t i o n of Calcium - 4 5 . 3 4 Total Uptake and D i s t r i b u t i o n of Nonradioactive Sulphur, Calcium, and Magnesium _ 3 5 G. Growth of plants 3 5 H. Harvest 3 5 I. Digestion 3 6 J. Analysis of sulphur 3 ? K. Measurement of calcium 3 7 L. Magnesium measurement • 3 8 D i s t r i b u t i o n of Sulphur - 3 5 i n Leaf Fractions 3 9 M. Growth of plants 3 9 N. Harvest 3 9 0. Fractionation of the l e a f material 3 9 RESULTS ^ 5 A. Total uptake of sulphur - 3 5 by bean plants 4 5 B. The d i s t r i b u t i o n of sulphur - 3 5 within bean plants expressed as a c t i v i t y per gram of fresh weight......... 4 5 C. The d i s t r i b u t i o n of sulphur - 3 5 within bean plants expressed as a c t i v i t y per plant organ.. 48 D. The d i s t r i b u t i o n of sulphur - 3 5 among the plant organs as a percent of the t o t a l a c t i v i t y i n the plant. 5 3 E. Uptake and d i s t r i b u t i o n of t o t a l sulphur...... 5 3 F. The d i s t r i b u t i o n of sulphur - 3 5 among various f r a c t i o n s of bean leaves 5 7 G. Cysteine/cystine and methionine incorporation into bean l e a f protein 6 0 H. Total uptake of calcium - 4 5 by bean plants..... 6 6 v i I. The d i s t r i b u t i o n of calcium - 4 5 within bean plants expressed as a c t i v i t y per gram dry weight 66 J. The d i s t r i b u t i o n of calcium - 4 5 within bean plants expressed as a c t i v i t y per plant organ.. 69 K. The d i s t r i b u t i o n of calcium - 4 5 among the plant organs as a percent of the t o t a l a c t i v i t y i n the plant 71 L. The uptake and d i s t r i b u t i o n of t o t a l calcium.. 71 M. The uptake and d i s t r i b u t i o n of t o t a l magnesium 75 N. Comparison of fresh weight, dry weight and dry weight as a percent of fresh weight 75 DISCUSSION 79 Sulphur uptake. 79 Sulphur d i s t r i b u t i o n . 81 Sulphur - 3 5 d i s t r i b u t i o n within l e a f f r a c t i o n s 83 The e f f e c t of KCHC treatment on the incorporation of sulphur amino acids into protein 89 Calcium-45 uptake and d i s t r i b u t i o n 90 Uptake of nonradioactive sulphur, calcium and magnesium 92 SUMMARY 96 BIBLIOGRAPHY 98 v i i LIST OF TABLES TABLE PAGE I Volumes of molar stock solutions used for preparing one l i t e r of a IX nutrient solution. 26 II The e f f e c t of KCHC on t o t a l uptake of sulphur - 3 5 by bush bean plants at various harvest times on a per plant basis 46 III Fresh weights of plant parts in sulphur - 3 5 uptake and d i s t r i b u t i o n experiments 4 7 IV The e f f e c t of KCHC treatment on sulphur - 3 5 d i s t r i b u t i o n within bean plants at various harvest times on an a c t i v i t y per gram basis... 4 9 V The e f f e c t of KCHC treatment on sulphur - 3 5 d i s t r i b u t i o n within bean plants at various harvest times on an a c t i v i t y per plant part basis 5 2 VI The e f f e c t of KCHC treatment on sulphur - 3 5 d i s t r i b u t i o n within bean plants at various harvest times on a percent per plant part basis 5 4 VII The e f f e c t of KCHC treatment on t o t a l sulphur uptake and d i s t r i b u t i o n i n three week old bean plants 5 6 VIII The e f f e c t of KCHC treatment on sulphur - 3 5 d i s t r i b u t i o n among several f r a c t i o n s of bean le a f tissue at various harvest times 5 8 IX Leaf weights, t o t a l a c t i v i t y , and recovery of a c t i v i t y i n the le a f f r a c t i o n a t i o n experiment. 61 X The e f f e c t of KCHC treatment on cysteine/ cystine and methionine incorporation into the protein f r a c t i o n of bean l e a f tissue 64 XI The e f f e c t of KCHC treatment on t o t a l uptake of calcium - 4 5 by. bush bean plants at various harvest times on a per plant basis.. 6 7 v i i i TABLE PAGE XII The e f f e c t of KCHC treatment on the d i s t r i -bution of calcium - 4 5 within bean plants at various harvest times on a per gram basis..**. 68 XIII The e f f e c t of KCHC treatment on calcium - 4 5 d i s t r i b u t i o n within bean plants at various harvest times on an a c t i v i t y per plant part basis • .•  70 XIV The e f f e c t of KCHC treatment on calcium - 4 5 d i s t r i b u t i o n within bean plants at various harvest times on a percent basis •- 72 XV The e f f e c t of KCHC treatment on t o t a l calcium uptake and d i s t r i b u t i o n i n three week old bean plants 73 XVI The e f f e c t of KCHC treatment on t o t a l magnesium uptake and d i s t r i b u t i o n i n three week old bean plants 76 XVII A comparison of fresh weight, dry weight, and the dry weight as a percent of fresh weight of plants used i n t o t a l uptake and d i s t r i b u t i o n of sulphur, calcium, and magnesium, and the uptake and d i s t r i b u t i o n of calcium-4-5 experiments - 77 ix LIST OF FIGURES FIGURE PAGE 1. Flow sheet for separation of S - 3 5 containing compounds i n bean leaves 4 4 / 4 5 2 . The e f f e c t of KCHC treatment on sulphur - 3 5 d i s t r i b u t i o n i n bean plants at various harvest times 5 ° 3 . The e f f e c t of KCHC treatment on percentage d i s t r i b u t i o n of sulphur - 3 5 i n bean plant organs at various harvest times 5 5 4 . The e f f e c t of KCHC treatment on sulphur - 3 5 d i s t r i b u t i o n among some fr a c t i o n s of bean l e a f tissue at various harvest times 5 9 5 . The e f f e c t of KCHC treatment on sulphur - 3 5 incorporation into protein amino acids 6 3 X ABBREVIATIONS Cyclohexanecarboxylic acid CHCA Potassium cyclohexanecarboxylate KCHC Potassium naphthenates Knap Naphthenic growth substances NGS Indoleacetic acid IAA 2,4-Dichlorophenoxyacetic acid 2,4-D Deoxyribonucleic acids DNA Ribonucleic acids RNA Nicotinamide adenine dinucleotide phosphate NADPH2 Adenosine triphosphate ATP Adenosine-5'-phosphosulphate APS 3'phosphoadenosine-5*-phosphosulphate PAPS 3'phosphoadenosine phosphate 3'PAP Protein d i t h i o l Pr(SH) 2 Protein disulphide PrS 2 Ethylenediaminetetraacetic acid EDTA 2,5-diphenyloxazole PPO 1,4-bis(-2(5-phenyloxazole))-benzene POPOP Angstrom A Parts per m i l l i o n ppm Counts per minute cpm Disintegrations per minute dpm Hectare ha x i ACKNOWLEDGMENTS Dr. D.J. Wort's p a t i e n c e , counsel, and enthusiasm d u r i n g the course of t h i s work were much a p p r e c i a t e d . His comments d u r i n g the p r e p a r a t i o n of the manuscript were most v a l u a b l e . Dr. I.E.P. T a y l o r and Dr. J . Maze gave h e l p f u l advice and c r i t i c a l l y reviewed the manuscript. Dr. B. Bohm served on the authors committee and h i s encouragement was a p p r e c i a t e d . Dr. G. Eaton's a s s i s t a n c e w i t h the s t a t i s t i c a l a n a l y s i s was i n v a l u a b l e . The manuscript was f u r t h e r a p p r a i s e d by Dr. G.H.N. Towers, Dr. G.E. Rouse, and Dr. V.C. Runeckles, and I thank them f o r t h e i r comments. Thanks are due to Mr. G. Kowalenko f o r t e c h n i c a l i n f o r -mation, and Dr. L.E. Lowe, Department of S o i l Science, f o r the use of h i s l a b o r a t o r y f a c i l i t i e s . The understanding and p a t i e n c e of my f a m i l y have made i t p o s s i b l e to complete t h i s work. INTRODUCTION AND LITERATURE REVIEW During the p a s t few y e a r s , an i n c r e a s i n g number of r e -p o r t s have been p u b l i s h e d documenting the e f f e c t s of naphthenic a c i d s as s t i m u l a t o r s of p l a n t growth. Much of the e a r l y work has d e a l t w i t h the r e l a t i v e i n c r e a s e s i n y i e l d s t h a t can be expected from v a r i o u s crop p l a n t s under treatment (80). In most cases, the c o n c e n t r a t i o n and mode of a p p l i c a t i o n of the naphthenate, and the p l a n t ' s o n t o g e n e t i c c o n d i t i o n were d e s c r i b e d since, these are c r i t i c a l f a c t o r s a f f e c t i n g the response (110,112). A f t e r the i n i t i a l stages of d e t e r m i n i n g growth responses, work i n our l a b o r a t o r y has been aimed a t understanding the b a s i c p h y s i o l o g i c a l responses of the p l a n t s to naphthenate treatment. In t h i s p a r t i c u l a r study, the e f f e c t of one of the naphthenic a c i d s , c y c l o h e x a n e c a r b o x y l i c a c i d , on some aspects o f s u l p h u r , c a l c i u m , and magnesium n u t r i t i o n i n bush beans has been examined. The sulphur status, of the p l a n t s was looked at i n some d e t a i l , w h i le o n l y the uptake and d i s t r i b u t i o n of the other two elements were con s i d e r e d . Naphthenic a c i d s Naphthenic a c i d s are monocarboxylic a c i d s of the naphthene ( a l i c y c l i c ) s e r i e s of hydrocarbons (60). They occur i n and are r e c o v e r e d from petroleum. There i s a wide range of compounds which f a l l i n t o t h i s group, but they a l l share a g e n e r a l s t r u c t u r e which may be w r i t t e n as R(CH 2) nC00H, where R i s 2 a c y c l i c nucleus composed of one or more rings. These rings may contain f i v e , s i x , or seven carbon atoms ( 6 0 ). Many methods are available f o r separation of naphthenic acids from crude o i l , but the simplest involves extraction with aqueous sodium hydroxide. The amount and composition of the naphthenic acid f r a c t i o n vary with the source of the crude. As an ex-ample, gas chromatography, mass spectrometry, and d i s t i l l a t i o n procedures were used to determine that an Austrian crude oil^con-tained 120 d i f f e r e n t components ( 20 ). Eider ( 38 ) found as many as 60 components i n Venezuelan crude o i l . Large amounts of naphthenic acids are recovered annually, and they are used commercially as lubricants, d r i e r s , c a t a l y s t s , preservatives, corrosion i n h i b i t o r s , emulsifiers, and napalm explosives ( 60 ) . The l i t e r a t u r e on the use of naphthenic acids i n plant research has been extensively reviewed by Severson ( 8 3 ) . The f i r s t known report of naphthenic acid used as a metabolic stimulant was i n 1921 when Neuberg and Sandberg ( 75 ) found that potassium naphthenate stimulated the a l c o h o l i c sugar s p l i t -t i n g a c t i v i t y of yeast, but c r e d i t f o r i n i t i a t i o n of the pres-ent boom i n th i s area belongs to workers i n Bulgaria, Albania, and the USSR. Huseinov ( 57 ) reported the r e s u l t s of several experiments conducted i n the Soviet Union. He noted that t h i s work had begun about 1 9 5 °» but his report to the 7 t h Internat-io n a l Congress of S o i l Science, held at Madison, Wisconsin i n i 9 6 0 , i s one of the few references available that describe any of the early Russian work. Some of the r e s u l t s he reported then 3 w i l l be incorporated into the following discussion. Application of naphthenic acids to a wide range of crop plants has, i n many cases, caused economically useful changes i n t h e i r growth patterns. Reports of quite remarkable i n -creases i n the amount of potato tubers produced are noted. Krasnova et a l ( 6 5 ) observed increases of 48$ with some var-i e t i e s . Abolina and Ataullaev ( 1 ) found 3 0 to 40$ increases i n production, and starch accumulation was up 2 0 $ when the s o i l was treated with 5 0 0 rol of naphthenate growth substances per hectare. Wort and Hughes ( 1 1 1 ) have shown that one treat-ment with a 5 0 0 0 ppm f o l i a r spray of the potassium s a l t of naphthenic acids (Knap) 3 3 days before harvest resulted i n a s i g n i f i c a n t increase (42$) i n t o t a l fresh weight of tubers per plant, but there was no s i g n i f i c a n t e f f e c t on the number of tubers per plant, the percentage of dry matter, or starch produced. Plants which received the f i r s t treatment of Knap as well as a d d i t i o n a l treatments of 2 5 0 0 ppm of Knap 1 9 and f i v e days before harvest showed no s i g n i f i c a n t increase i n any of the parameters measured, although the fresh weights of tubers per plant were higher than controls. The increased starch y i e l d reported by the Russian workers was not found, and i t was suggested that v a r i e t a l differences may have been the reason. Cotton's response to naphthenates has been examined by a number of workers. Naghibin ( 74 ) found that a seed-soak with naphthenic growth substances (NGS) gave 5 to 1 0 $ i n -creases in y i e l d while sprays at the rate of 2 5 0 g/ha caused 4 a 20$ increase i n y i e l d . Other favourable influences such as accelerated ripening, e a r l i e r opening of b o l l s , and i n -creased weight of cotton per b o l l were also noted. In countries where regulations might require the approval of additives to food crop plants, the t o x i c i t y of naphthenates, or lack of i t , w i l l l i k e l y have to be determined before i t i s used commercially. However, even i n these countries, large scale t e s t i n g could reasonably be c a r r i e d out on crops such as cotton and f o r e s t trees. In the USSR cotton i n areas of over 2 0 0 , 0 0 0 hectares has been subjected to treatment with NGS ( 7 4 ) . In addition to the above, s i g n i f i c a n t increases i n the y i e l d of bush bean pods ( 1 1 0 , 1 1 2 ) , of carrots, cabbage, beet and onion (116), corn ( 1 1 3 ) , r i c e ( 57 ), sugar beets (113 ) , tomato ( 2 , 5 7 ), and wheat (86 ,113 )• have'been found. In-creases are noted for many other plants (see Severson, 83 ) but the examples above serve to i l l u s t r a t e the wide range of species which respond favourably to treatment. In general, the treated plants appear to grow more ra p i d l y than control plants. Frequently, the increased growth occurs i n parts of the plant which are economically important, thus making Knap of i n t e r e s t from an agronomic point of view. Since i t has been established that increased y i e l d s are quite a common occurrence, the questions that a r i s e are p h y s i o l o g i c a l i n nature. One wonders what changes occur i n the plants to cause the responses. Certainly i t appears that the plants are producing more b u i l d i n g materials and are:transporting them more rapidl y . Increased a c t i v i t y implies that higher l e v e l s 5 of r e s p i r a t i o n and photosynthesis occur. Enzyme a c t i v i t y would be expected to increase, and this would be manifested i n higher s p e c i f i c a c t i v i t i e s or i n increased amounts of enzyme. The treated plants would be expected to absorb and u t i l i z e more mineral elements than the less active control plants. More complete understanding of these processes i s being- obtain-ed slowly, since papers describing p h y s i o l o g i c a l effects are f a r outnumbered by reports considering the agronomic aspects. At t h i s point, i t might be well to summarize some of the relevant p h y s i o l o g i c a l information that i s available. Increased amounts of photosynthetic pigments have been reported by several authors (71,30,45 ). Although Chu ( 3 0 ) observed only minor increases of chlorophyll a and b, due to a 5°°° PP m Knap spray on tomato, she reported increases of 1.4 to 1 3 . 8 $ i n carotenoid pigments. Fattah (45 ) presented evidence of s i g n i f i c a n t increases in the chlorophyll a content of bean leaves two and three weeks a f t e r treatment with Knap but only nonsignificant increases of chlorophyll b and carot-enoids. He found a s i g n i f i c a n t increase i n t o t a l pigments three weeks af t e r treatment. Increased l e v e l s of protein have been found in several studies (77f78,62,114 ), and free amino acid l e v e l s were higher at the beginning and end of vegetative growth of maize treated with naphthenate growth substances (78 ). Severson also reported increased free amino acid levels ( 84), and Padmanabhan et a l ( 77 ) have shown increased l e v e l s of several free amino acids. 6 Bazanova determined the auxin and i n h i b i t o r content of cotton seedlings treated with 0 . 0 0 5 , 0 . 0 0 2 5 , and 0 . 0 0 1 2 5 $ solutions of Sh-8 (an organic compound belonging to the cyclohexenyl butanols, 8 ) ( 1 0 ) . He found evidence of r e d i s -t r i b u t i o n of auxins and i n h i b i t o r s i n various organs. There was an increased translocation of growth regulating substances from vegetative to reproductive organs and an increased a c t i v i t y of the natural growth regulating substances i n the reproductive organs. Huseinov ( 5 7 ) reported that petroleum growth helping substances (naphthenic acids) caused oat c o l e o p t i l e bends of 9 * 7 ° to 1 2 ° , whereas heteroauxin caused bends of 6.8° to 8.4°, and hybberellic acid ( g i b b e r e l l i c acid) 7 . 7 ° to 8 . 0 ° . These r e s u l t s , though i n t e r e s t i n g , are somewhat su r p r i s i n g and possibly suspect, because he reports an auxin e f f e c t f o r g i b b e r e l l i c acid which has not been shown to q u a l i f y as an auxin (82 ). Loh ( 7 2 ) has found several auxin-l i k e properties associated with Knap. He found that Knap treatment caused elongation of pea stem segments and promoted the i n i t i a t i o n of adventitious root formation by bean stem and azalea cuttings. Loh also found increased biosynthesis of IAA i n dark-grown bean plants treated with Knap. The a c t i v i t y of enzymes associated with IAA destruction was also stimulated. These r e s u l t s suggest that, rather than being auxins, the naphthenic acids may stimulate auxin a c t i v i t y . Several enzyme assays have been conducted to determine naphthenate e f f e c t s . Russian workers report increased per-oxidase a c t i v i t y ( 8 ) and increased catalase a c t i v i t y ( 6 3 ) . 7 The a s c o r b i c a c i d content o f bean pods was 2 6 . 5 $ g r e a t e r i n Knap t r e a t e d p l a n t s than c o n t r o l s ( 4 5 ) , but decreased l e v e l s were found i n tomato f r u i t s ( 3 ° ) . T h i s c o u l d , of course, r e f l e c t d i f f e r e n c e s i n response by the two s p e c i e s . S t u d i e s of enzymes i n our l a b o r a t o r y have shown some pos-i t i v e e f f e c t s due to 5 ° 0 0 P P m Knap sprays on bush bean l e a v e s . S e v e r a l n i t r o g e n m e t a b o l i z i n g enzymes were examined. F a t t a h and Wort ( 4 7 ) found v a r i a b l e r e s u l t s w i t h n i t r a t e r e d u c t a s e . In a l l cases the enzyme a c t i v i t y was i n c r e a s e d , but only under c e r t a i n c o n d i t i o n s were the i n c r e a s e s s i g n i f i c a n t . Wort e_t a l (114) determined t h a t Knap treatment i n c r e a s e d n i t r a t e r e ductase a c t i v i t y 1 5 2 $ compared to c o n t r o l , when measured seven days a f t e r treatment. Under s i m i l a r experimental con-d i t i o n s , Wort et a l (114) found g l u t a m i c - o x a l o a c e t i c t r a n s -aminase a c t i v i t y i n c r e a s e d 1 0 $ over c o n t r o l , glutamine synthe-tase 17$i and g l u t a m i c dehydrogenase a c t i v i t y was u n a f f e c t e d . F a t t a h ( 4 5 ) d i s c o v e r e d t h a t g l u t a m i c - p y r u v i c transaminase a c t i v i t y was i n c r e a s e d s i g n i f i c a n t l y under medium and high l i g h t c o n d i t i o n s . A 7 2 $ i n c r e a s e i n cytochrome oxidase a c t i v i t y was r e p o r t e d by Wort e t a l (114). The presence of h i g h e r c o n c e n t r a t i o n s of n u c l e i c a c i d s i n naphthenate t r e a t e d p l a n t s ( 4 6 , 7 7 ) and the g r e a t e r m i t o t i c a c t i v i t y t h a t was found i n some s t u d i e s ( 5 0 ) i n d i c a t e t h a t naphthenates may be f u n c t i o n i n g at the g e n e t i c l e v e l ( 7 7 ) . EJubov and Issaeva ( 3 9 ) r e p o r t e d i n c r e a s e d a v a i l a b i l i t y o f phosphorus and m i n e r a l n i t r o g e n compounds i n s o i l t r e a t e d w i t h naphthenates, as w e l l as i n c r e a s e d l e v e l s of n i t r o g e n and phosphorus content i n maize and l u c e r n e p l a n t s . Yur'eva ( 1 1 5 ) 8 found an increased phosphorus content i n the leaves of sugar beets. Babaev (8 ) determined that the amount of t o t a l P increased i n i n i t i a l growth phases i n cotton treated with eithe r a f o l i a r or s o i l a p p l i c a t i o n of naphthenates. Severson ( 83 ) found that the uptake of phosphorus by beans grown i n eithe r complete or phosphorus free nutrient solutions was un-affected by Knap treatment. He did f i n d that Knap treatment 32 enhanced the acropetal movement of J P from the roots of plants grown i n a phosphorus free nutrient solution. Severson ( 8 3 ) also determined that naphthenate treatment increased the rate of incorporation of into acid soluble (sugar phosphates, free nucleotides, phospholipids) and acid insoluble (nucleic acids, phosphoproteins) f r a c t i o n s of leaves. He did not f i n d that naphthenate treatment affected the amount of P. i n the two f r a c t i o n s when compared with controls. Cyclohexanecarboxylic acid One of the simplest of the naphthenic acids, cyclohexane-carboxylic acid (CHCA) has been used extensively i n our lab-oratory at the University of B r i t i s h Columbia. Many properties of t h i s compound have made i t a t t r a c t i v e as a test material for research purposes. CHCA i s a simple molecule f o r which the structure i s known, while the naphthenic acids used i n many of the studies are a complex mixture of chemical compounds. A C v 0 s \ - S - O H V c ' Cyclohexanecarboxylic acid 9 CHCA can be obtained i n a radioactive form, which i s useful for studies of the metabolism of the compound i t s e l f . Stimulation of vegetative and reproductive growth of bush beans i s very pronounced with t h i s compound. Wort and Patel (113)» reported increases of 23.5$ i n reproductive growth of bush beans with a 1 X 10 M f o l i a r a p p l i c a t i o n of potassium cyclohexanecarboxy-l a t e (KCHC) two weeks a f t e r planting. In many t r i a l s the stimulative e f f e c t s were c l e a r l y v i s i b l e when the treated plants were placed beside t h e i r controls. Wort and Patel (113) pointed out that gas chromatographic analysis of naphthenic acids ob-tained from Venezuelan crude oil.showed that low molecular weight compounds with carbon numbers close to that of CHCA were present i n t h i s f r a c t i o n . They suggested that i t was possible that the low molecular weight components of the naphthenate mixture could be the active agents. In studies of the metabolism of cyclohexanecarboxylic acid by bean plants, Severson et a l ( 85 ) found that CHCA formed conjugates with glucose and aspartic acid. Padmanabhan ( 76 ) has reported the appearance of a glucose ester of CHCA 1 4 0.125 hr a f t e r a p p l i c a t i o n of KCHC-7- C as droplets on the adaxial surface of the primary leaves, and the aspartate conjugate appeared a f t e r one hour. Free acid had disappeared within s i x hours of app l i c a t i o n . Considering these f a c t s , Severson ( 83 ) has suggested that the conjugated form of CHCA rather than the free acid may be the growth stimulating form. Severson ( 84 ) has also reported studies of the metabolism of ^C-glucose by bean root t i p s treated with naphthenic acids 10 and KCHC. Glucose incorporation by the roots was stimulated by both these substances, with KCHC having a somewhat stronger e f f e c t . The l e v e l s of r a d i o a c t i v i t y incorporated into 10 ethanol-soluble amino acids from (^C) glucose were determined, and i t was found that i n a l l cases a c t i v i t y was greater In the treated roots. Serine and valine l e v e l s were s i g n i f i c a n t l y increased by both Knap and KCHC, whereas isoleucine/leucine was s i g n i f i c a n t l y increased only by naphthenate treatment. In the case of hydrolysates of ethanol-insoluble material (the protein amino aci d s ) , KCHC s i g n i f i c a n t l y increased the l e v e l of radioactive aspartic acid, glutamic acid, and alanine, while Knap s i g n i f i c a n t l y stimulated incorporation of alanine. This e f f e c t of the naphthenates and KCHC on protein amino acids i s not su r p r i s i n g when It i s r e c a l l e d that protein l e v e l s are Increased under t h e i r influence ( 77 ). 11 Sulphur compounds A comprehensive l i s t of the sulphur-containing organic compounds present i n plants was given by Freney (49 ). The metabolically important ones included the amino acids cysteine, cystine, and methionine, and the vitamins thiamine and b i o t i n as well as S-adenosyl-methionine, l i p o i c acid, coenzyme A, and the t r i p e p t i d e glutathione. Quite a number of other compounds containing sulphur have been i d e n t i f i e d i n plants, but t h e i r metabolic r o l e has not been established. These include the mustard o i l glucosides of some Cruciferae (49 ) and the s u l p h o l i p i d of spinach ( 14 ). Sulphur-containing compounds are extremely important i n plants both s t r u c t u r a l l y and metabolically. Tertiary protein structure i s determined, i n a large part, by the cross linkage and f o l d i n g caused by the formation of the covalent disulphide bonds of cystine ( 4 ). Sulphydryl groups may be the s i t e f o r attachment of divalent cations which also a f f e c t the con-formation of proteins ( 4 ). The hydrophobic, thioether groups of methionine, by i n t e r a c t i n g with other hydrophobic groups, can a f f e c t the t e r t i a r y structure of proteins. Sulphydryl groups may also act as points of attachment fo r substrates or coenzymes associated with an enzyme ( 4. ). Thus, sulphur compounds as components of proteins, and therefore enzymes, are involved i n a wide v a r i e t y of plant a c t i v i t i e s . Allaway and Thompson (4) reviewed some of the other metabolic a c t i v i t i e s of nonprotein sulphur compounds. These include roles i n f a t t y acid biosynthesis, oxidation of keto acids, a c e t y l a t i o n reactions. 12 c a r b o x y l a t i o n s , a n d m e t h y l t r a n s f e r . S u l p h u r u p t a k e P l a n t s g e n e r a l l y o b t a i n s u l p h u r f r o m t h e s o i l a s s u l p h a t e ( 1 9 . 1 0 9 ) , b u t s u l p h u r - c o n t a i n i n g a m i n o a c i d s c a n a l s o _ b e a n i m p o r t -a n t s o u r c e f o r some p l a n t s ( 9 ) . S u l p h u r e x i s t s i n t h e s o i l a s s u l p h a t e , o r g a n i c s u l p h u r c o m p o u n d s , a n d s u l p h u r - c o n t a i n i n g m i n e r a l s s u c h a s p y r i t e ( F e S g ) , s p h a e r i t e ( Z n S ) , c h a l c o p y r i t e ( C u F e S g ) , a n d c o b a l t i t e ( C o A s S ) ( 2 1 ) , b u t i n h u m i d a n d s e m i -a r i d r e g i o n s m o s t o f t h e s u l p h u r i s I n o r g a n i c f o r m s (105 ) . O x i d a t i o n o f w e a t h e r e d m i n e r a l s a n d a i r b o r n e SOg. a n d m e t a b o l i c d e g r a d a t i o n o f t h e o r g a n i c f o r m s b y v a r i o u s s o i l b a c t e r i a a n d f u n g i / make s u l p h u r . a v a i l a b l e t o t h e p l a n t s ( ^9 ) . T h e m i c r o -o r g a n i s m s a r e s t r o n g c o m p e t i t o r s f o r s u l p h u r , a n d i f s u f f i c i e n t a m o u n t s o f c a r b o h y d r a t e a r e p r e s e n t i n t h e s o i l t o s u p p l y t h e m w i t h e n e r g y , t h e m i c r o b e s c a n t i e u p s o m u c h s u l p h u r t h a t p l a n t s w i l l e x p e r i e n c e d e f i c i e n c y c o n d i t i o n s ( 49 ) • S u l p h u r may e n t e r t h e s o i l w i t h r a i n w a t e r ( 19, 58 ) a n d b y t h e a d d i t i o n o f f e r t i l i z e r s . U n t i l r e c e n t l y , t h e u s e o f s u l p h u r - c o n t a i n i n g p e s t i c i d e s c o n t r i b u t e d s i g n i f i c a n t a m o u n t s o f s u l p h u r t o t h e s o i l , b u t o r g a n i c s u b s t i t u t e s f o r t h e s e p e s t -i c i d e s h a v e a l m o s t e l i m i n a t e d t h i s s o u r c e ( 19, 4 0 ) . N e a r i n d u s t r i a l c e n t e r s s u l p h u r may b e o b t a i n e d a s SO2 t h r o u g h t h e l e a v e s ( 4 3 , 1 0 2 ) . T h e s u l p h u r d i o x i d e d i s s o l v e s i n t h e m o i s t s u r f a c e o f m e s o p h y l l c e l l s p r o d u c i n g s u l p h u r o u s a c i d w h i c h i s n e u t r a l i z e d b y i n o r g a n i c a n d o r g a n i c b a s e s , s u c h a s a m i n o g r o u p s a n d o t h e r n i t r o g e n o u s c o m p o u n d s , o r i t may u n i t e w i t h a l d e h y d e s t o f o r m a d d i t i o n p r o d u c t s . T h e s e p r o d u c t s a r e s u b s e q u e n t l y 1 3 oxidized to form sulphate ( 9 5 )• Excess sulphur dioxide can be injurious to vegetation ( 43 ), and the recent eco l o g i c a l consciousness has caused industries to use low-sulphur con-tai n i n g f u e l s and to take steps to remove SO2 from smokestack emmissions. These changes i n practice, along with the increas-ing use of high analysis f e r t i l i z e r s which contain very l i t t l e sulphate ( 40 ), are expected to produce sulphur d e f i c i e n t con-d i t i o n s i n many areas within a few years. In one of the few studies done with higher plant tissue, Leggett and Epstein ( 6 9 ) found that uptake of sulphate by excised roots of barley exhibited t y p i c a l enzyme k i n e t i c s f or the range of external sulphate from 0 . 0 0 5 meq/1 to 0 . 0 5 meq/1. The uptake was apparently metabolically dependent since anaerobic conditions stopped i t . Wedding and Black ( 1 0 7 ) reported that low temperatures i n h i b i t e d the uptake of sulphate by C h l o r e l l a . Leggett and Epstein ( 6 9 ) found that the sulphate uptake was competitively i n h i b i t e d by selenate but not by n i t r a t e or phosphate, which indicated considerable s p e c i f i c i t y on the part of the c a r r i e r . They reported, i n addition, that double r e c i p r o c a l plots (Lineweaver-Burk plots) gave a curved l i n e as the concentration of substrate increased. This Indicated the presence of ad d i t i o n a l uptake s i t e s having d i f f e r e n t a f f i n i t i e s f o r sulphate. In plants exposed to conditions of sulphur s u f f i c i e n c y ranging from d e f i c i e n t to just adequate, most of the sulphur i s present i n the form of protein ( 4 0 ), When excess sulphur l s available i n the s o i l or nutrient solution, sulphate w i l l 14 be accumulated (81,88). Sulphur t r a n s l o c a t i o n Once sulphur, as s u l p h a t e , was absorbed by the r o o t s , i t was a p p a r e n t l y t r a n s l o c a t e d to other p a r t s of the p l a n t v i a the xylem ( 1 0 0 ) . The younger d e v e l o p i n g l e a v e s of Red Kidney bean were found to accumulate a r e l a t i v e l y g r e a t e r q u a n t i t y of l a b e l l e d s u l phur than o l d e r primary l e a v e s ( 1 5 ) . C r a f t s and C r i s p ( 3 2 ) noted t h a t r o o t a p p l i c a t i o n of ^ S to bean p l a n t s r e s u l t e d i n uniform l a b e l l i n g of the whole p l a n t w i t h s l i g h t l y h i g h e r c o n c e n t r a t i o n s i n stem a p i c e s and margins of l e a v e s . They s a i d t h a t t h i s i n d i c a t e d an a p o p l a s t i c type of t r a n s p o r t through the n o n l i v i n g continuum of c e l l w a l l s and xylem v e s s e l s . L e v i ( ? 0 ) found t h a t -^S moved d i r e c t l y out of bean l e a f m i d r i b s i n t o mesophyll areas w i t h no s p e c i a l accumulation i n the v e i n s . Sulphur t r a n s l o c a t e d i n t o the l e a v e s of a growing p l a n t was v e r y r a p i d l y trapped i n newly formed p r o t e i n l e a v i n g v e r y l i t t l e a v a i l a b l e f o r r e c i r c u l a t i o n ( 1 5 ) . T h i s phenomenon was p r o b a b l y r e s p o n s i b l e f o r the e a r l y concept t h a t sulphur was r e l a t i v e l y immobile i n the p l a n t ( 1 0 9 ) . K y l i n (66) found t h a t deseeded wheat p l a n t s f e d -^S u n t i l the f i r s t l e a f was h a l f formed and then t r a n s f e r r e d to S d e f i c i e n t n u t r i e n t s o l u t i o n had r e l o c a t e d only s m a l l amounts i n t o the d e f i c i e n t t h i r d l e a v e s . However, s e v e r a l s t u d i e s ( 1 6 , 2 3 , 9 8 ) have shown t h a t s u l p h u r was r e a d i l y r e l o c a t e d when i t was p r e s e n t i n excess. Biddulph et a l ( 1 6 ) u s i n g r a d i o a c t i v e t r a c e r , have shown t h a t a p o r t i o n of the t o t a l s u l phur w i t h i n the Red Kidney bean 15 remained mobile and moved f r e e l y from one organ to another, and they have suggested that the rate of downward movement of sulphur i n the phloem of the stem was s i m i l a r to that f o r phos-phorus, and exceeded 40 cm/hr. That i t does t r a v e l down v i a the phloem was shown by S.F. Biddulph ( 18 ), when autoradio-35 graphs of stem cross sections from plants fed S v i a the l e a f were superimposed on photomicrographs. The a c t i v i t y was c l e a r l y associated with the phloem connected to the t r e a t -ed l e a f . Thomas et a l ( 98 ) have also shown that 35§Q= i s r e c i r c u l a t e d within the plant. Using an a l f a l f a plant with several shoots, they dipped the leaves at the t i p of one of 35 = these shoots into a solution of J SO^ f o r one, two, or f i v e days. The sulphate was absorbed by the immersed leaves and translocated to other parts of the plant. At harvest, a higher concentration of sulphur - 3 5 was found in the leaves of undipped shoots than i n the roots of the same plant. Because of the structure of the a l f a l f a plant, i t was clear that the sulphur - 3 5 must have been transferred down the shoot which had been exposed to the radioactive material, through' the root or root crown, and up into the unexposed shoots. Bouma ( 23 ) determined that the retranslocation depended la r g e l y on the sulphur status of the plant. Mobility increased as sulphur d e f i c i e n t plants recovered from t h e i r deficiency and, i n addition, mobility was greater i n plants grown in nut-r i e n t solutions containing sulphur than i n plants that suffered sulphur deficiency. It i s l i k e l y that the n u t r i t i o n a l status of the p l a n t / i s the major factor c o n t r o l l i n g sulphur mobility. Because of the nature of the compounds containing sulphur, very 16 l i t t l e can be m o b i l i z e d f o r t r a n s f e r even when other p a r t s of the same p l a n t are e x p e r i e n c i n g severe d e f i c i e n c y . Only when there i s excess s u l p h u r p r e s e n t , as su l p h a t e or s m a l l m o l e c u l a r weight o r g a n i c compounds, can the p l a n t t r a n s f e r s u lphur from i one organ to another. Sulphur d i s t r i b u t i o n In a review, Thomas e t a l ( 9 7 ) co n s i d e r e d the range of sulphur l e v e l s n o r m a l l y found i n p l a n t s . In a d d i t i o n to the va l u e s obtained from others, they r e p o r t e d the r e s u l t s of n e a r l y 1000 o f t h e i r own measurements. They found, i n g e n e r a l , t h a t t o t a l s u l p h u r was q u i t e v a r i a b l e . The a v a i l a b i l i t y of su l p h u r from the source, e i t h e r s o i l or atmosphere, had a s i g n i f i c a n t e f f e c t on p l a n t sulphur content. Sulphate v a l u e s p a r a l l e l e d the t o t a l s while o r g a n i c s u l p h u r l e v e l s v a r i e d o n l y by a s m a l l amount, u s u a l l y 0.2 to 0.4$ on a dry weight b a s i s . T o t a l s u l p h u r f o r some p l a n t s was g i v e n as f o l l o w s : soybeans 0.20 to 0 . 3 0 $ of dry weight, c l o v e r 0.21 to 0 . 3 0 $ , a l f a l f a 0.18 to 0.24$, c o n i f e r needles n o r m a l l y had as l i t t l e as 0.1$, and some p l a n t s such as cabbage, rutabagas, rape and r a d i s h had 0.40 to 0.60 $ or more ( 9 7 ). Thomas e t a l ( 9 6 )found t h a t under adequate s u l p h u r c o n d i t i o n s a l f a l f a tops contained 0 . 3 0 $ s u l p h u r , leaves 0.60$ su l p h u r , and r o o t s 0.20$ sulphur. Ensminger and Freney, ( 40 ) claimed t h a t the c r i t i c a l p e r c e n t -age ( i . e . the minimum c o n c e n t r a t i o n o f a m i n e r a l n u t r i e n t i n a p l a n t a t which there was u n r e s t r i c t e d growth) of sulphur p r o b a b l y v a r i e d l i t t l e f o r a g i v e n s p e c i e s under w i d e l y v a r y i n g c o n d i t i o n s . They r e p o r t e d the c r i t i c a l percentage f o r a l f a l f a 17 as 0.20$ sul p h u r , and the probable range f o r most other s p e c i e s as 0.15$ to 0.30$. Thompson e t a l (99 ) claimed t h a t over 90$ of the o r g a n i c s u l p h u r i n p l a n t s was p r e s e n t as c y s t e i n e and methionine. How-ever, t h i s was pro b a b l y not the case f o r some o f the C r u c i f e r a e (and L i l i a c e a e ) mentioned e a r l i e r , i n which there were q u i t e l a r g e amounts of s u l p h u r - c o n t a i n i n g o r g a n i c compounds of no apparent f u n c t i o n other than to supply odour or t a s t e . Thomas e t a l (95 ) found 60 to 80$ of the r a d i o a c t i v e s u l p h u r i n the p l a n t was t r a n s l o c a t e d to the g r a i n of b a r l e y and wheat. Beaton (11 ), i n h i s review, s t a t e d t h a t s u l p h u r was l a r g e l y p r e s e n t i n the l e a v e s of p l a n t s b e f o r e f l o w e r i n g , but a t the time of f r u i t development, the o r g a n i c sulphur of le a v e s was converted to s u l p h a t e and t r a n s l o c a t e d to the seeds where i t was recom-bined i n o r g a n i c form. Sulphate r e d u c t i o n Allaway ( 3 ) suggested t h a t "the s y n t h e s i s o f s u l f u r amino a c i d s from s u l f a t e s i n p l a n t s i s one of the key r e a c t i o n s i n b i o l o g y , comparable i n importance to the r e d u c t i o n o f carbon i n p h o t o s y n t h e s i s . " Sulphate r e d u c t i o n must occur before c y s t e i n e and methionine can be s y n t h e s i z e d . The f o l l o w -i n g i s a proposed scheme f o r t h i s r e d u c t i o n (99 )• F ° r a b b r e v i a t i o n s see page x. 18 ATP + SO^ APS 1 APS + ATP PAPS 2 PAPS + ( P r ( S H ) 2 ) 3'PAP + SO" + ( P r S 2 ) ( y e a s t ) 3 SO r s u l p h i t e reductase 3 s= S + O - a c e t y l s e r i n e O - a c e t y l s e r i n e s ulphydrase c y s t e i n e + a c e t a t e 5 The f o r m a t i o n of s u l p h i t e as d e s c r i b e d f o r y e a s t i n r e a c t i o n 3 has never been demonstrated i n h i g h e r p l a n t s . I t was thought t h a t the s u l p h i t e may remain bound to an enzyme complex before undergoing f i n a l r e d u c t i o n to S =, but f r e e s u l p h i t e c o u l d a l s o a c t as a s u b s t r a t e f o r s u l p h i t e r e d u c t a s e . NADPH.2 p r o b a b l y f u n c t i o n e d as the e l e c t r o n donor f o r regener-a t i o n o f the p r o t e i n d i t h i o l and f o r s u l p h i t e r e d u c t a s e . S e r i n e was thought to be the ac c e p t o r f o r s u l p h i d e , but i t has been shown t h a t O - a c e t y l s e r i n e may be a more l i k e l y c a n d i date. I t was found to be more a c t i v e than s e r i n e , and p l a n t s contained a s e r i n e a c e t y l a s e ( 99 ). A l s o , i t was found t h a t c y s t e i n e i n h i b i t e d the s e r i n e a c e t y l a s e , and t h i s was taken as evidence f o r a feedback c o n t r o l w i t h i n the system. 19 Calcium Calcium i s f r e q u e n t l y the most abundant c a t i o n i n the s o i l . P l a n t s o b t a i n calcium e i t h e r from the s o i l s o l u t i o n or by d i r e c t c o n t a c t of the r o o t s with the s o i l p a r t i c l e s to v/hich the calcium i s bound ( 9 1 ) . Biddulph e t a l ( 1 5 ) found t h a t c a l c i u m moved through the r o o t system more s l o w l y than sul p h u r . They suggested t h a t the d e l a y c o u l d be due to the a c t i v i t y of the a b s o r p t i o n system, and the mechanism of t r a n s f e r a c r o s s the c o r t e x may be more complex than f o r s u l p h u r . A b s o r p t i o n of c a l c i u m i n t o the nonvacuolated. t i p s of corn r o o t s was found to be nonmetabolic, but uptake i n v a c u o l a t e d s e c t i o n s was s t r o n g l y temperature dependent and t h e r e f o r e l a r g e l y m e t a b o l i c ( 5 1 )• Higinbotham e t a l ( 5 5 ) showed t h a t the e l e c t r o c h e m i c a l g r a d i e n t f o r c a l -cium (and magnesium) was from the o u t s i d e to the i n s i d e of the c e l l i n pea and oat r o o t s and concluded t h a t t h i s i o n would move p a s s i v e l y i n t o the c e l l s . They suggested t h a t c a l -cium may e i t h e r be excluded from the c e l l s by some mechanism, or i f a c t i v e t r a n s p o r t was i n v o l v e d , i t would pump the ions out. A commonly h e l d concept of i o n t r a n s l o c a t i o n from the r o o t to the l e a v e s i s t h a t , a f t e r the m i n e r a l s are loaded i n t o the xylem, the flow of water w i l l c a r r y them al o n g u n t i l they are dumped a t the s i t e of t r a n s p i r a t i o n a l l o s s of the water ( 3 3 ). Biddulph e t a l ( 17 ) and B e l l and Biddulph ( 13 ) have conducted a number of experiments which c o n t r a d i c t e d t h i s concept and suggested another mode of t r a n s p o r t a t l e a s t f o r c a l c i u m . They found t h a t the c a l c i u m d i d not move d i r e c t l y 20 through the xylem v e s s e l s of Red Kidney bean, but r a t h e r moved as though the xylem and s u r r o u n d i n g t i s s u e s were a c t i n g as an exchange column. C a l c i u m - 4 5 was found i n the bark as w e l l as •.•in the xylem area, but o n l y a s m a l l amount of t h i s a c t i v i t y became f i x e d i n the bark ( 1 7 ). I t was shown t h a t when un-l a b e l l e d c a l c i u m f o l l o w e d the t r a c e r up the stem,exchange ac-t i v i t y r e s u l t e d i n movement of the t r a c e r on up the stem to be r e p l a c e d by c o l d c a l c i u m ( 1 7 ). One other s i g n i f i c a n t obser-v a t i o n which argued a g a i n s t the h y p o t h e s i s of mass flow was t h a t the c a l c i u m d i d not move a t the same r a t e as l a b e l l e d water ( 1 7 ). B e l l and Biddulph ( 1 3 ) found t h a t there was a r a p i d uptake phase of c a l c i u m - 4 5 i n t h e i r t e s t t i s s u e f o l l o w -ed by a slow accumulation phase. When r a d i o a c t i v e f e e d i n g s were f o l l o w e d by c o l d c a l c i u m , the bul k of the r a d i o a c t i v e mat-e r i a l absorbed d u r i n g the r a p i d uptake phase was d i s p l a c e d from t h i s t i s s u e . T h i s was c o n s i d e r e d to be good evidence f o r ex-change. The mode of t r a n s l o c a t i o n proposed by these workers was seen to p r o v i d e g r e a t e r c o n t r o l o f i o n movement, s i n c e char-a c t e r i s t i c s of the i o n and i t s exchange s i t e s , u t i l i z a t i o n of the i o n , and growth co u l d a l l i n f l u e n c e the d e s t i n a t i o n of the i o n s , whereas the mass flow h y p o t h e s i s o f f e r e d no means o f d i r -e c t i n g the flow. Biddulph e_t a l ( 1 7 ) found t h a t c a l c i u m - 4 5 f e d through the r o o t s of Red Kidney beans was t r a n s p o r t e d most r a p i d l y to the youngest t r i f o l i a t e l e a f s e t , w h i l e the second and o l d e s t t r i f o l i a t e s r e c e i v e d p r o g r e s s i v e l y l e s s and the p r i m a r i e s l e a s t . A f t e r s i x hours, there was l e s s spread i n a b s o l u t e amount of 21 tracer In each of the sets of leaves, but when expressed as a c t i v i t y on a fresh weight basis, the younger leaves had a d e f i n i t e advantage i n securing calcium-45. Bukovac and Wittwer ( 28 ) found that calcium-45 applied to l e a f tissue was r e a d i l y transported out into adjacent parts of the leaf but not to any other organ of the plant. Biddulph et a l ( 1 5 ) showed that calcium was almost completely Immobile in the phloem of kidney bean. Since calcium remained i n the plant part to which i t was transported i n i t i a l l y , i t meant that calcium would have to be supplied to plants continuously to en-sure that newly formed tissues received s u f f i c i e n t amounts ( 1 5 ) . Calcium i s an es s e n t i a l element f o r plant growth, and usually a concentration of about 0»S%* on a dry weight basis i s considered to be adequate f o r most plants ( 82 ). Concen-tr a t i o n s of several percent may be found i n the f o l i a g e of legumes ( 2 to J%) tomato, tobacco ( 3 to 4$), and other dicoty-ledonous plants, while cereals and grasses contain from 0 . 2 to 0.5 percent ( 6l ). In both groups, the roots have less calcium than the tops. Although i t i s present i n macro qua n t i t i e s , the established requirement for calcium i s so low that i t could be considered a micronutrient ( 2 9 ). Epstein ( 41 ) noted that algae required very low l e v e l s of calcium. Higher plants have been grown successfully i n nutrient solutions containing only 0.05 mil calcium (Hoagland solution may have as high as 5*0 mM), but other divalent cations also had to be kept at low concen-tr a t i o n s (106 ). Under normal circumstances, the higher l e v e l of calcium may be necessary to o f f s e t the toxic e f f e c t s of some 22 of the other divalent metals (106 ). Calcium has a very weak biochemical i d e n t i t y and has been i d e n t i f i e d as a component of only one metalloenzyme, o<amylase ( 4 l ). Even here i t apparently can be substituted f o r by magnesium or strontium ( 2 9 ), and the binding of calcium to the enzyme found i n barley was quite weak (41 ). Tagawa and Bonner ( 9 3 ) showed that calcium treatment of Avena c o l e o p t i l e tissue repressed the p l a s t i c i z i n g e f f e c t of indoleacetic acid (IAA) on the c e l l wall. In contrast, pot-assium treatment augmented IAA's softening e f f e c t . Calcium and potassium had the same e f f e c t on the p h y s i c a l properties of p e c t i c materials as they did on c e l l wall p l a s t i c i t y . There-fore, Tagawa and Bonner concluded that the p e c t i c materials of the c e l l wall, i n association with calcium, may be of import-ance i n determining the mechanical strength of the tissue, and may also have a role i n regulating growth (91 ). Formation of calcium oxalate c r y s t a l s may be a method of i n a c t i v a t i n g toxic organic acids (91 ). Epstein (41 ) considered i n some d e t a i l the e s s e n t i a l role of calcium i n maintenance of healthy membranes. Calcium deficiency r a p i d l y led to break down of the physical structure of membranes and to the loss of the membranes' a b i l i t y to exert control over the movement of other elements and compounds. Epstein thought that t h i s was probably one of the most s i g -n i f i c a n t a c t i v i t i e s of calcium i n the plant. Jones and Lunt ( 6 l ), Burstrom ( 2 9 )» and Epstein ( 4 l ) provide more extensive reviews of the present l e v e l of under-standing of calcium function i n plants. 23 Magnesium Magnesium i s present i n the s o i l as a component of the s o i l p a r t i c l e s (27 ), as the divalent cation adsorbed to s o i l p a r t i c l e s , or i n the s o i l solution. Magnesium i s apparently taken up a c t i v e l y but not at the same s i t e s as calcium, barium, or strontium (42 ). B e l l and Biddulph (13 ), i n a study of calcium transport i n the xylem (discussed e a r l i e r ) noted that magnesium was able to exchange for calcium i n the bean stem. This would suggest that magnesium might move up through the stem by a mechanism s i m i l a r to that proposed f o r calcium, but apparently no v/ork has been done i n t h i s area yet. 0 0 Bukovac and Wittwer ( 28 ) found that Mg would move into adjacent l e a f tissue when applied as a drop but was apparently not translocated to other parts of the plant. On the other hand, Tammes and Van Die ( 94 ) found measurable quantities of magnesium i n the phloem exudates of Yucca inflorescences, while calcium l e v e l s were predictably very low. Steucek and Koontz ( 8 9 ) reexamined th i s problem and concluded that magnesium was quite mobile i n the phloem and was exported from bean leaves at rates s i m i l a r to those f o r sulphur. They sug-gested that one of the reasons, among others, for the f a i l u r e of Bukovac and Wittwer to observe phloem mobility was that they did not apply enough 2^Mg to the leaves. Like sulphur, magnesium i s bound into immobile fr a c t i o n s from which i t can not be r e a d i l y retranslocated even i f a deficiency does occur elsewhere i n the plant. This factor was also considered tc be one of the reasons f o r confusion over magnesium mobility ( 8 9 ) . 24 An adequate supply of magnesium i n most plants i s consi-dered to be about 0.2$ of dry weight (82). The only major stable compound containing magnesium i s chlorophyll which i s a magnesium porphyrin ( 4 l ) . Only 10 percent of the l e a f magnesium i s t i e d up i n chlorophyll, but the chloroplasts contain 5 0 percent or more of the t o t a l magnesium i n the leaf. Magnesium i s a very important a c t i v a t o r of many enzymes including, i n p a r t i c u l a r , those which act on phosphorylated substrates, so i t s presence i n chloroplasts i n forms other than chlorophyll i s not s u r p r i s i n g (41). In many instances of enzyme a c t i v a t i o n , manganese and some other divalent cations can substitute f o r magnesium. 2 5 MATERIALS AND METHODS Uptake and D i s t r i b u t i o n of S u l p h u r - 3 5 A. Growth of p l a n t s Uniform seeds of the dwarf bush bean p l a n t , Phaseolus  v u l g a r i s L., c u l t i v a r Top Crop, were sown i n v e r m i c u l i t e con-t a i n e d i n wooden f l a t s , and the f l a t s were p l a c e d on benches i n a growth room. The s e e d l i n g s were watered w i t h tap water. Throughout the experiment, c o n d i t i o n s i n the growth room were maintained as f o l l o w s : 1 6 . 3 5 k i l o l u x a t the top of the p l a n t s , a 14 hour p h o t o p e r i o d , a day/night temperature regime of 2 2 ± 2 . 5 c / 2 0 ± 2 C, and a day/night r e l a t i v e humidity range o f 5 0 to 6 5 $ / 5 5 to 70%. The l i g h t i n the growth room was s u p p l i e d by co o l - w h i t e f l u o r e s c e n t tubes (Westinghouse) and 6 0 watt incandescent lamps. On the n i n t h day a f t e r sowing, the r o o t s of the seed-l i n g s were washed f r e e of v e r m i c u l i t e , and 1 2 uniform p l a n t s were p l a c e d i n each of f o u r , 2 0 cm X 2 5 cm X 1 5 cm deep p l a s t i c t r a y s . The t r a y s were p a i n t e d b l a c k on the o u t s i d e to reduce a l g a l growth, and the p l a n t s ' stems were wrapped w i t h c o t t o n and supported i n s l o t t e d , opaque p l a s t i c s toppers s i t t i n g i n h o l e s cut i n the l i d of the t r a y . Four l i t e r s of c o n t i n u o u s l y a e r a t e d s u l p h u r - f r e e Hoagland-Arnon's s o l u t i o n was put i n each t r a y (Table I ) . 26 TABLE I Volumes of molar s t o c k s o l u t i o n s of major ions used f o r p r e p a r i n g one l i t e r of a IX n u t r i e n t s o l u t i o n . M o d i f i e d a f t e r Hoagland and Arnon ( 5 6 ) . m l / l a Stock s o l u t i o n complete -S K H 2 P O 4 1 1 KNO3 5 5 C a ( N 0 3 ) 2 . 4 H 2 0 5 5 MgSO^HgO 2 — NaCl (o.lM) 1 1 MgCl 2 2 Fe EDTA ( 5 m g/ml) 1 1 A - 5 b 1 1 a d e i o n i z e d water ( 0 . 2 ppm, as NaCl) b. The A - 5 m i c r o n u t r i e n t s o l u t i o n was prepared by d i s s o l v i n g the f o l l o w i n g i n 1 l i t e r of d i s t i l l e d water: 2 . 8 6 g H3BO0 1.81 g M n C l 2 . 4 H 2 0 , 0 . 2 2 g ZnSO/j.. 5 H 2 0 , 0 . 0 8 g CuS04.5H|o, and 0 . 0 2 g Na^vloO^.h^O. 27 B. P r e p a r a t i o n o f , and treatment v/ith, c y c l o h e x a n e c a r b o x y l i c  a c i d s o l u t i o n -2 A 1 X 10 M potassium s a l t s o l u t i o n of cyclohexane-c a r b o x y l i c a c i d (KCHC) was prepared by d i s s o l v i n g O.56 g of KOH i n 750 ml of water f o l l o w e d by the a d d i t i o n of 1.28 g of CHCA. A f t e r s t i r r i n g f o r s e v e r a l hours, 3 g of the w e t t i n g agent, Tween 20 ( p o l y o x y e t h y l e n e s o r b i t a n monolaurate) was added, and the volume of s o l u t i o n was brought to one l i t e r . On the t h i r t e e n t h day a f t e r sowing, the l e a v e s of the bean p l a n t s i n two t r a y s were sprayed to d r i p w i t h the 1 X _2 10 M s o l u t i o n of KCHC. P l a n t s i n the other two t r a y s were sprayed w i t h 0.3$ w/v s o l u t i o n of Tween 20. C. Exposure to -^S Twenty-four hours a f t e r s p r a y i n g , the r o o t s of both t r e a t e d and c o n t r o l p l a n t s were immersed i n a c o n t i n u o u s l y . • 35 35 a e r a t e d , complete n u t r i e n t s o l u t i o n c o n t a i n i n g S as Na2 SO/j. (New England Nuclear, Boston, Mass.). For the three s u l p h u r uptake and d i s t r i b u t i o n experiments r e p o r t e d , the l e v e l of a c t i v i t y f e d was 34 to 37 ^ c / l . A f t e r exposing the r o o t s f o r f o u r hours, the r a d i o a c t i v e s o l u t i o n was siphoned o f f i n t o a l a r g e r e c e i v i n g f l a s k , and room temperature tap water was poured i n t o the t r a y s . The complete r i n s i n g sequence l a s t e d f o r one hour and c o n s i s t e d of f o u r changes of tap water, one change of 0.004 M magnesium su l p h a t e s o l u t i o n , and a f i n a l change of tap water. A f t e r r i n s i n g , the s u l p h u r - f r e e n u t r i e n t s o l u t i o n was added to the t r a y s . 28 D. Harvest At each of the four harvest times (4, 8, 12, and 24 hours a f t e r the conclusion of radioactive loading), two plants were taken from each tray. They were quickly separated into three p a r t s : roots, stems plus p e t i o l e s , and l e a f blades. Like parts of both plants from one tray were combined, weighed, and analyzed as a unit. The plant f r a c t i o n s were then placed i n separate 100 ml Kjeldahl f l a s k s f o r digestion. E. Digestion Fresh or dry plant tissue was placed i n a 100 ml Kjeldahl f l a s k and heated gently with 4 ml of HNO^ u n t i l the tissue disintegrated. One and one-half ml of HCIO^ was added to the mixture and heated ( 1 1 5 to 1 2 5°C) u n t i l a colourless or s l i g h t l y yellowish l i q u i d remained. The digest was transferred to a centrifuge tube and neutralized by the addition of KOH p e l l e t s to p r e c i p i t a t e the p e r c h l o r i c acid as KCIO^. When necessary, the s o l u t i o n was brought to pH 6 or less by the addition of small amounts of HCl. The s o l u t i o n was c e n t r i -fuged at 3 0 0 0 X g ( S o r v a l l RC2 centrifuge, 4 . 2 5 head) fo r 10 minutes, and the KClOij. p r e c i p i t a t e was washed three times with d i s t i l l e d water. The combined supernatants were usually brought to 1 0 ml by evaporation. To minimize the amount of KClOij, c a r r i e d over i n solution, a l l the wash steps were carried out at 4°C or l e s s . As an a l t e r n a t i v e to the tedious centrifugation procedure, i t was l a t e r found that f i l t r a t i o n through a 4 . 2 5 cm Buchner funnel using Whatman #42 f i l t e r paper was equally s a t i s f a c t o r y . 2 9 The p r e c i p i t a t e trapped on the f i l t e r paper was s t i r r e d and washed s e v e r a l times w i t h i c e c o l d water. Under circumstances where the volume of the f i n a l s o l u t i o n c o u l d be 2 0 ml or l a r g e r , the p r e c i p i t a t e c o u l d be l e f t i n the bottom of the s o l u t i o n . F. R a d i o a c t i v e Counting A r o u t i n e procedure was developed f o r c o u n t i n g r a d i o a c t i v e s u l p h u r which p r o v i d e d r e l i a b i l i t y , convenience, and the e f f i -c i e n c y n e c e s s a r y f o r h a n d l i n g hundreds of samples. A sheet of Whatman # 1 f i l t e r paper, ( 4 cm x 7 . 5 cm) was p l e a t e d a c r o s s i t s width to form f i v e , 4 cm x 1 . 5 cm pan e l s ( 1 0 1 ) . The paper was h e l d by f o r c e p s or a paper c l i p a t t a c h e d to one corner, and 0 . 3 ml of d i g e s t e d sample was p i p e t t e d evenly over the s u r f a c e . The paper was supported u p r i g h t on a bench by the paper c l i p and l e f t to dry completely. When dry, the paper was i n s e r t e d i n t o a s c i n t i l l a t i o n v i a l f i l l e d to the shoulder w i t h a s c i n -t i l l a t i o n f l u i d c o n s i s t i n g o f 4 g of PPO ( 2 , 5 - d i p h e n y l o x a z o l e ) p e r l i t e r of tol u e n e . The samples c o u l d be counted immediately. The expense of prepared standards and the r e l a t i v e l y s h o r t h a l f l i f e of prompted an a l t e r n a t i v e method of de t e r m i n i n g c o u n t i n g e f f i c i e n c y . F i v e l e a f samples were d i g e s t e d as des-c r i b e d and v a r i o u s amounts of KOH were added r a n g i n g from none to complete n e u t r a l i z a t i o n . The volumes were brought to 1 0 ml, and 5 nil o f each o f these s o l u t i o n s was mixed w i t h 5 roi °f a water s o l u t i o n c o n t a i n i n g a hig h l e v e l o f 3 5 S a c t i v i t y ( 6 x IO-* cpm/ml)« A s i x t h was prepared by combining 5 ml of water and 5 ml of the r a d i o a c t i v e s o l u t i o n . D u p l i c a t e 0 . 2 0 ml samples were p i p e t t e d onto f i l t e r papers, p l a c e d i n v i a l s as d e s c r i b e d , 3 0 and counted. It was found that a smooth curve was obtained when e f f i c i e n c y was plotted against the values obtained by channels r a t i o counting. The most e f f i c i e n t l y counted sample was considered to be 1 0 0 $ , and a l l the others were plotted i n r e l a t i o n to this one. A long term decay curve was prepared, and each time the "standards" were counted, the count rate was related back to the day they were prepared. Only very minor flu c t u a t i o n s from true decay values were noted over a period of ten months. A l l radioactive samples were counted i n the same Nuclear Chicago, 7 2 0 s e r i e s , s c i n t i l l a t i o n counter. Sulphur - 3 5 was counted at the same settings as used f o r carbon-14. When the a c t i v i t y i n a sample was adjusted to 1 0 0 $ with respect to the quench curve, t h i s value was designated dpm (disintegrations per minute) to di s t i n g u i s h i t from the cpm (counts per minute) obtained d i r e c t l y from the machine. A l -though these were not true dpm, the f a c t that a l l samples counted were related to one standard value put them on the same r e l a t i v e scale. True counting e f f i c i e n c y for 3 5 S was i n the 6 3 to 6 5 $ range. The use of f i l t e r paper, as a c a r r i e r of the radioactive sample, offered many advantages and no apparent disadvantages. A very serious problem associated with counting -^S in solu t i o n was i t s tendency to adsorb to the glass walls of the s c i n t i l -l a t i o n v i a l s (108). This changed i t s geometry with respect to the s c i n t i l l a t i o n f l u o r and resulted i n an uncontrolled and unpredictable decline i n measured a c t i v i t y ( 1 0 3,108). Calcium-4 5 and phosphorus -32 were also apparently susceptible to thi s 31 e r r o r (79). One s o l u t i o n f o r t h i s problem has been to use a t h i x o t r o p i c g e l , such as C a b o s i l , which prevented the mig-r a t i o n of the ions to the g l a s s (108). This seemed to be s a t i s f a c t o r y but was messy to handle. Another was to s i l i c o n -i z e the s c i n t i l l a t i o n v i a l s to prevent a d s o r p t i o n (79). The 3^S0^, immobilized on the f i l t e r paper, was surrounded by s c i n t i l l a t o r and a c t i v i t y d e c l i n e d a c c o r d i n g to normal decay p a t t e r n s . Calcium-45 a l s o behaved normally when counted i n t h i s way. Pure toluene based s c i n t i l l a t o r s can not be used f o r samples c o n t a i n i n g water (3^)« The a d d i t i o n of e t h a n o l to the mixture allowed i n c o r p o r a t i o n of l i m i t e d amounts of water, but e f f i c i e n c y dropped due to quenching from both the water and the e t h a n o l (34). Dioxane based mixtures c o u l d accommodate a l a r g e r p r o p o r t i o n of water (24), but t h i s was a r e l a t i v e l y expensive mixture. Use of the f i l t e r paper p e r m i t t e d evapora-t i o n of the water from a sample, and then the s i m p l e s t and most e f f i c i e n t s c i n t i l l a t o r c o u l d be used. I t was p o s s i b l e to l o a d 0.3 ml of sample on the paper a t one time, and l a r g e r amounts co u l d be put on i f the paper was allowed to dry between l o a d i n g s (101). The choice of a more absorbent paper, such as some of the f i b e r - g l a s s types, p e r m i t t e d the a d d i t i o n of l a r g e r amounts a t each time, but the e f f e c t s of these t h i c k e r papers on c o u n t i n g e f f i c i e n c y have not been examined th o r o u g h l y i n t h i s i n v e s t i g a t i o n . When the f i l t e r paper was removed from a v i a l , the r a d i o -a c t i v e sample was removed w i t h i t , and the v i a l u s u a l l y count-ed a t background l e v e l s . I t c o u l d then be used f o r a second 3 2 sample, r e s u l t i n g i n a g r e a t s a v i n g of c l e a n i n g time as w e l l as m a t e r i a l s . The f i l t e r paper procedure d e s c r i b e d was used f o r count-i n g 3 5 S o ^ i o n s , ^ 5 C a + + i o n s , l a b e l l e d l e u c i n e , 3 5 S l a b e l -l e d c y s t i n e and methionine, and % l a b e l l e d DNA. Of these, o n l y methionine was found i n the s c i n t i l l a t o r a f t e r the paper was removed, and t h i s a c t i v i t y was b a r e l y above background. I t appeared t h a t t h i s procedure c o u l d be used f o r a wide v a r i e t y of i s o t o p e s e i t h e r i n the form of i n o r g a n i c or o r g a n i c compounds (see a l s o T o n z e t i c h , 1 0 1 ) . P e r c h l o r i c a c i d was a s t r o n g quenching agent and e l i m i -n a t i o n of i t by p r e c i p i t a t i o n improved c o u n t i n g e f f i c i e n c y d r a m a t i c a l l y . NaOH n e u t r a l i z a t i o n was a l s o e f f e c t i v e , but i t l e f t a l a r g e amount of s a l t d i s s o l v e d i n the s o l u t i o n . POPOP l , 4 - B i s - ( 2 - ( 5 - p h e n y l o x a z o l e ) ) - b e n z e n e , commonly a c o n s t i t -uent of s c i n t i l l a t i o n mixtures, turned green i n the presence of s m a l l amounts of p e r c h l o r i c a c i d c a u s i n g s e r i o u s c o l o u r quenching problems. The beta p a r t i c l e s emitted by s u l p h u r - 3 5 have high enough energy t h a t POPOP*s c o n t r i b u t i o n to c o u n t i n g e f f i c i e n c y was minimal ( 1 0 3 ) . Therefore, i t was omitted from the mixture to a v o i d the d i f f i c u l t y t h a t c o u l d a r i s e i f some p e r c h l o r a t e remained i n the samples. P r e c i p i t a t i o n of sulphate as barium s u l p h a t e by the a d d i t i o n of barium c h l o r i d e , f o l l o w e d by suspension of the p r e c i p i t a t e i n a C a b o s i l , dioxane based s c i n t i l l a t o r (Bray's s o l u t i o n , 24), has been suggested as a means of c o u n t i n g 3 5 $ from l a r g e samples of v e g e t a t i o n ( 1 0 8 ) . V/ith s m a l l samples, 33 a c o n s i d e r a b l e l o s s of barium s u l p h a t e o c c u r r e d when u s i n g t h i s procedure. D u p l i c a t e samples were im p o s s i b l e to o b t a i n . T h i s was pr o b a b l y due to the f a c t t h a t v e r y s m a l l aggregates of BaSO^ p a r t i c l e s are c o l l o i d a l i n nature (108) and c o u l d not be c e n t r i f u g e d i n t o a p e l l e t i n the round bottom tubes of the S o r v a l RC-2 c e n t r i f u g e . Dr. J . Tonzetich, of the F a c u l t y o f D e n t i s t r y a t the U n i v e r s i t y of B r i t i s h Columbia, had no d i f f i c u l t y w i t h the p r e c i p i t a t i o n o f sulphate as BaSO^ from s a l i v a . In t h i s case, he used c o n i c a l c e n t r i f u g e tubes i n a swinging bucket, I n t e r n a t i o n a l c e n t r i f u g e . T h i s combination of equipment was a p p a r e n t l y s u i t a b l e f o r b r i n g i n g down the p r e c i p i t a t e . Obser-v a t i o n of h i s procedure l e d d i r e c t l y to the method developed f o r c o u n t i n g r a d i o a c t i v e samples i n t h i s study. T o n z e t i c h " d i s s o l v e d " the BaSO^ i n a s m a l l amount of e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) s o l u t i o n . The EDTA-BaSO^ complex was p l a c e d on the f o l d e d f i l t e r paper, d r i e d , and counted as d e s c r i b e d e a r l i e r . P r e c i p i t a t i o n of su l p h a t e as BaSO^ from p l a n t d i g e s t s , f o l l o w e d by complexing wi t h EDTA would be a way of s e p a r a t i n g the s u l p h a t e from other i n t e r f e r i n g i o n s , and the concen-t r a t i o n o f a c t i v i t y c o u l d e a s i l y be r e g u l a t e d by v a r y i n g the amount of EDTA s o l u t i o n used to complex the BaSO^. However, i t was unnecessary to take these a d d i t i o n a l s t e p s when the s i m p l e r procedure of p u t t i n g p l a n t d i g e s t s d i r e c t l y on the f i l t e r paper worked s a t i s f a c t o r i l y . 3 4 Uptake and D i s t r i b u t i o n of Calcium-45 Except f o r the changes noted below, the methods used f o r th i s experiment were the same as those f o r the sulphur-35 uptake and d i s t r i b u t i o n experiments. In t h i s case, complete Hoagland-Arnon's so l u t i o n (Table I) was supplied to the plants both before and a f t e r the feeding of radioactive calcium. Harvests were made at 12, 24, and 48 hours a f t e r radio-active calcium feeding ceased. At each harvest time, four plants were taken at random from each of the four trays and each plant was analyzed separately. Consequently, there were 8 treated and 8 control plants from each of the harvest times. The plants were separated into roots, stems plus p e t i o l e s , and l e a f blades and weighed immediately. The parts were then sandwiched between paper towels and dried i n a fan oven at 6 0 ° c f o r 24 hours. Dry weights of the parts were recorded. The plant parts were digested as described i n section E. After n e u t r a l i z a t i o n with KOH, about 0 . 5 ml concentrated HCl was added to the digest to strongly a c i d i f y i t , and thus prevent the formation of insoluble calcium hydroxide. The method used f o r counting calcium-45 was v i r t u a l l y the same as used f o r sulphur -35 . Calcium standards were pre-pared as before except that solutions of increasing concen-t r a t i o n of pe r c h l o r i c acid were used instead of the plant digests (section F). Both methods produced suitable quench curves. 35 Total Uptake and D i s t r i b u t i o n of Nonradioactive Sulphur, Calcium, and Magnesium G. Growth of plants Bush bean seeds were sown as described i n section A, and placed i n a greenhouse on August 9» 1971. Seventy-two l i t e r s of complete Hoagland-Arnon's solut i o n was prepared i n one large container, and four l i t e r s of this s o l u t i o n was placed i n each of 18, one-gallon crocks. Nine days a f t e r sowing, two of the bean plants were transferred from the vermiculite to each of the crocks. They were supported by cotton batting wrapped around t h e i r stems and held i n a s l o t i n a wooden l i d . The solutions were aerated continuously. When the plants were two weeks old, uniform specimens were selected and placed one per crock. The leaves of one h a l f of the plants were sprayed to drip with 1 X 10"2M KCHC i n 0.3$ Tween 20 sol u t i o n while the leaves of the other h a l f were sprayed with Tween 20 solution. The treated and control plants were placed a l t e r n a t e l y i n two p a r a l l e l rows along one bench i n the greenhouse. H. Harvest Three weeks a f t e r sowing, f i v e treated and f i v e control plants were harvested, separated into roots, stems plus pet-i o l e s , and l e a f blades. The roots were rinsed i n two changes of tap water, blotted to uniform dryness, and the fresh weights of a l l three organs recorded. The material was dried i n a fan oven f o r 2k hours at 60°c, weighed, and stored i n in d i v i d u a l p l a s t i c bags. 36 The t i s s u e was ground to a powder with a p e s t l e i n a mortar and p l a c e d i n s m a l l v i a l s . The powder was d r i e d and weighed. Approximately 0.4 g a l i q u o t s of t h i s powder were taken f o r d i g e s t i o n and a n a l y s i s . A second experiment with some m o d i f i c a t i o n s was begun August 271 1971. Nine-day-old p l a n t s were t r a n s f e r r e d from v e r m i c u l i t e to t r a y s ( s e c t i o n A) c o n t a i n i n g 0.5 X Hoagland-Arnon's s o l u t i o n , and kept i n the growth room u n t i l they were two weeks o l d . While s t i l l i n the t r a y s , the p l a n t s were t r e a t e d w i t h KCHC or Tween 20 as be f o r e . Two p l a n t s were then t r a n s f e r r e d to each of the o n e - g a l l o n crocks and grown i n a e r a t e d 0.5 X Hoagland-Arnon*s s o l u t i o n . Two p l a n t s were h a r v e s t e d from each of e i g h t t r e a t e d and c o n t r o l crocks one week a f t e r treatment. The two p l a n t s were combined to gi v e one sample and the same h a r v e s t i n g , d r y i n g , and g r i n d i n g procedure was used as befo r e . I. D i g e s t i o n A f t e r d i g e s t i o n w i t h HNO^ and HCIO^ was complete, 6.0 ml 6N HCl was added to the f l a s k and b o i l i n g was continued u n t i l a l l the HCl evaporated. N e u t r a l i z a t i o n w i t h KOH and s e p a r a t i o n o f the i n s o l u b l e p e r c h l o r a t e s was done as d e s c r i b e d ( s e c t i o n E ) . R e a c i d i f i c a t i o n w i t h 0.5 ml co n c e n t r a t e d HCl was c a r r i e d out immediately a f t e r n e u t r a l i z a t i o n to ensure t h a t no i n s o l -u b l e c a l c i u m or magnesium hydroxides were formed. The volume of each d i g e s t was brought to 25 ml wi t h water. Only hi g h n i t r a t e and high p e r c h l o r a t e c o n c e n t r a t i o n s i n the wet ashed samples were known to i n t e r f e r e w i t h the measurement of t o t a l s u l p h a t e ( 59 ). P e r c h l o r i c a c i d was 3 7 e f f e c t i v e l y removed by the p r e c i p i t a t i o n s t e p , and n i t r a t e was a p p a r e n t l y converted to v o l a t i l e n i t r o s y l c h l o r i d e by the 6N HC1 added a t the end of the d i g e s t i o n sequence ( 5 9 ) . J . A n a l y s i s of s u l p h u r The method used f o r su l p h u r a n a l y s i s was a m o d i f i c a t i o n of Johnson and N i s h i t a ' s technique ( 5 9 ) . Dean ( 3 5 ) developed -a method i n which bismuth n i t r a t e v/as used as the c o l o u r reagent r a t h e r than the methylene blue used by Johnson and N i s h i t a . Kowalenko and Lowe (64) have m o d i f i e d the equipment to p r o v i d e s i m p l e r h a n d l i n g and more r e l i a b l e r e s u l t s . One ml of p l a n t d i g e s t was heated i n a d i s t i l l a t i o n f l a s k w i t h an h y d r i o d i c a c i d r e d u c i n g mixture (12). H^S produced was swept up through a condenser by a stream o f n i t r o g e n and t r a p -ped i n 10 ml of IN NaOH i n a r e c e i v i n g tube. F i v e ml of b i s -muth n i t r a t e reagent (12) was added to the NaOH-H^S s o l u t i o n and bismuth s u l p h i d e was formed as a c l e a r brown c o l l o i d a l d i s -p e r s i o n . The absorbance of the bismuth s u l p h i d e c o l l o i d was measured a t 400 nm i n a S p e c t r o n i c 20 c o l o r i m e t r i c photometer. Amounts of s u l p h u r were determined from a standard curve prepared from sodium s u l p h a t e standards. K. Measurement of c a l c i u m Calcium was measured a t 4227 A wit h a P e r k i n Elmer, Model 3 0 3 atomic a b s o r p t i o n spectrophotometer. Except f o r d i g e s t i o n , the procedures f o l l o w e d were as d e s c r i b e d i n the machine manual (6) . One-half ml of p l a n t d i g e s t and 5 ml of a s o l u t i o n c o n t a i n i n g $% w/v lanthanum were combined and brought to a 38 t o t a l of 25 ml. Therefore, the f i n a l concentration of lanth-anum was one percent. The machine was zeroed with a one percent lanthanum solu t i o n . A 10 jag/ml calcium standard was also prepared i n one percent lanthanum. Measurements were taken d i r e c t l y from a d i g i t a l readout device attached to the spectrophotometer which, when adjusted to the standard, gave values expressed as ug/ml. L. Magnesium measurement Magnesium was measured with the atomic absorption spectrophotometer at a wave length of 2852 A. Magnesium solutions were prepared by d i l u t i n g one ml of plant digest to 50 ml with d i s t i l l e d water. The machine was zeroed with. d i s t i l l e d water. Stock magnesium standards were prepared from magnesium metal turnings as described i n the atomic absorption spectrophotometer manual ( 6 ). Standards of 2 jig/ml were used to c a l i b r a t e the measurements. 39 D i s t r i b u t i o n of Sulphur - 3 5 i n Leaf F r a c t i o n s M. Growth of p l a n t s Bean p l a n t s were grown, t r e a t e d w i t h KCHC, and exposed to r a d i o a c t i v e sulphur as d e s c r i b e d i n s e c t i o n s A, B, and C. However, there was o n l y one t r a y of t r e a t e d p l a n t s and one t r a y of c o n t r o l p l a n t s . N. Harvest One day a f t e r r a d i o t r a c e r l o a d i n g , three c o n t r o l and three t r e a t e d p l a n t s were har v e s t e d . The l e a f blades were removed and weighed. They were then p l a c e d i n an aluminum-f o i l - l i n e d mortar, f r o z e n with l i q u i d n i t r o g e n , and ground wi t h an aluminum-foil-covered p e s t l e . The f o i l prevented the ground p l a n t m a t e r i a l from s t i c k i n g to e i t h e r the mortar or p e s t l e . When g r i n d i n g was complete, the al u m i n u m - f o i l was f o l d e d s l i g h t l y and p l a c e d i n a 400 ml beaker. The beaker was covered w i t h Saran Wrap and s t o r e d a t -10°C u n t i l a l l p l a n t s had been har v e s t e d . From the c o l d s t o r a g e , the beakers were p l a c e d i n a f r e e z e - d r y i n g u n i t and l e f t f o r one day. A f t e r f r e e z e d r y i n g , the m a t e r i a l was c o l l e c t e d , weighed, p l a c e d i n a v i a l and s t o r e d i n a d e s i c c a t o r . A d d i t i o n a l h a r v e s t s were 35 made 2 days and 5 days a f t e r yyS l o a d i n g . A second batch o f p l a n t s was grown, t r e a t e d , l a b e l l e d and h a r v e s t e d . Harvest days were two and three days a f t e r 35s l a b e l l i n g . 0. F r a c t i o n a t i o n of the Leaf M a t e r i a l a. Approximately 50 m i l l i g r a m s of dry l e a f m a t e r i a l was weighed a c c u r a t e l y , p l a c e d i n a K j e l d a h l f l a s k , and d i g e s t e d 4o as d e s c r i b e d i n s e c t i o n E. The a c t i v i t y i n t h i s sample was used to determine t o t a l s u l p h u r ( T o t a l S ) . b. The remainder of the t i s s u e was weighed, p l a c e d i n a 2 0 ml V i r t i s " 4 5 " b l e n d e r cup w i t h 1 5 ml of water, blended f o r three minutes a t high medium speed, and poured i n t o 6 5 ml of b o i l i n g 9 5 $ e t h a n o l . A f t e r b o i l i n g 5 "to 1 0 minutes, the m a t e r i a l was t r a n s f e r r e d to a c e n t r i f u g e tube and c e n t r i f u g e d a t 1 2 , 0 0 0 X g f o r 1 0 minutes. The supernatant was decanted and saved, and the r e s i d u e was washed and c e n t r i f u g e d three times with 80$ e t h a n o l . This was a m o d i f i c a t i o n of the procedure used by F o r e s t and Wightman (48 ) f o r the e x t r a c t i o n of f r e e amino a c i d s from bush bean s e e d l i n g s . c. The supernatants from p a r t b were combined, and the e t h a n o l was evaporated on a steam bath. The m a t e r i a l remain-i n g was t r a n s f e r r e d to a s e p a r a t o r y f u n n e l by r i n s i n g w i t h water and chloroform. The water and c h l o r o f o r m - s o l u b l e f r a c t i o n s were separated. d. The c h l o r o f o r m - s o l u b l e f r a c t i o n from p a r t c was poured i n t o a K j e l d a h l f l a s k and the c h l o r o f o r m evaporated. The m a t e r i a l was then d i g e s t e d and c a l l e d the L i p i d S f r a c t i o n ( 6 7 ) . e. The water s o l u b l e p o r t i o n of p a r t c was poured i n t o a Dowex-50 ( 1 6 - 5 0 mesh) c a t i o n exchange column i n the H + con-d i t i o n . The column (a c y l i n d e r , 4 cm x 1 3 cm) c o n t a i n e d about 1 2 5 ml of r e s i n and was washed with 2 5 0 ml of water a f t e r the sample had been poured i n i t . The e l u a t e was c o l l e c t e d , reduced i n volume by h e a t i n g on a steam bath, and e i t h e r 41 digested or counted d i r e c t l y as the Sulphate S f r a c t i o n . The column was then eluted with 2 0 0 ml of 2 N NH4OH followed by 2 0 0 ml of water. The eluate was reduced to dryness i n a fan oven at 60°C, transferred to f i l t e r paper with a l i t t l e water, and counted as the Free Amino Acid S f r a c t i o n (48). f. About 25 ml of 0 . 3 M HCIO^ was added to the centrifuge tube containing the residue from part b. The tube was held i n a water bath at 75°C f o r 30 minutes, cooled, and c e n t r i -fuged. The residue was washed once with 1% HC1, centrifuged, and the two supernatants combined. The material was either digested and counted or counted d i r e c t l y . This was c a l l e d the Acid Soluble S f r a c t i o n (67). g. The residue of part f was considered to be the protein f r a c t i o n . It was freeze-dried, weighed, and stored i n v i a l s i n a desiccator u n t i l needed. Three, 10-mg aliquots of t h i s material were weighed, and placed i n separate small test tubes. Six normal H C 1 was added, and the tubes were evacu-ated and sealed. The material was hydrolyzed, i n vacuo, at 110°C f o r 24 hours. The tubes were opened, and the HC1 evaporated under vacuum i n a desiccator. The contents of two or three of the tubes were combined by r i n s i n g them out with water and f i l t e r i n g a l l the l i q u i d through a one-cm Buchner funnel. The f i l t r a t e was freeze-dried, taken up i n a small amount of water, and placed as a 6 cm-long, transverse band about 8 cm from one end of a ?.5 cm x 46 cm s t r i p of Whatman #1 chromatographic paper. The paper was hung i n a closed chromatocab, and the chromatogram was developed with a 42 phenol-water (80:20) mixture i n a descending d i r e c t i o n . Forest and Wightman (48) developed t h e i r chromatograms i n 80$ phenol i n the presence of ammonia vapours. However, the ammonia was omitted here because i t gave an undesirable, blue colour reaction with the phenol, and i t s omission did not i n t e r f e r e with the separation of the sulphur-containing amino acids. After developing for about 24 hours, the chromatogram was dried i n a fume hood f o r two days. It was then cut trans-versely into 2-cm sections which were pleated and counted i n the toluene-PPO s c i n t i l l a t o r . Authentic methionine, cysteine, and cystine were run concurrently i n the same system, and t h e i r positions were indicated by dipping the paper s t r i p i n a cadmium acetate-ninhydrin reagent (54). Peaks of radio-a c t i v i t y corresponded very well with the location of these amino acids. h. The remainder of the dried residue from part f was weighed, placed i n a Kjeldahl f l a s k , digested, and counted. The r e s u l t s of t h i s step were used to calculate t o t a l Protein S. ^3 F i g . 1 . Flow sheet f o r s e p a r a t i o n of S-35 c o n t a i n i n g  compounds i n bean leaves l e a f t i s s u e » weigh 40 mg * p l a c e i n a K j e l d a h l f l a s k weigh the remainder of the t i s s u e d i g e s t count ( T o t a l S) g r i n d w i t h water i n a s m a l l b l e n d e r cup, 3 min 1 . I — • c o l l e c t supernatants pour i n t o b o i l i n g 9 5 $ e t h a n o l I b o i l , 5 min c e n t r i f u g e , 1 2 , 0 0 0 x g, 10 mi wash w i t h 80$ e t h a n o l — c e n t r i f u g e (repeat 3X) 1 r e s i d u e CHCI3 i n t o K j e l d a h l f l a s k , evaporate a l l CHCl^ evaporate e t h a n o l 1 t r a n s f e r to a sep f u n n e l w i t h CHCl^ and H 20 separate CHCl^ and H 20 po_ur through Dowex-50 H exc lange column wash w i t h d i s t i l l e d H 20 d i g e s t count ( L i p i d S) reduce volume* d i g e s t I count (Sulphate S) - c o l l e c t e l u a t e pour 2 N NH4OH i n t o the column! wash w i t h water ! c o l l e c t the e l u a t e reduce volume, count a l l or chromatograph (Free amino a c i d S) Continued overleaJ 44 F i g . 1. cont. r e s i d u e heat r e s i d u e w i t h 0 . 0 3 M , — c o l l e c t the supernatants HCIO^, ? 0°C, 3 0 min I d i g e s t I c e n t r i f u g e , 12,000 x g, 20 min 1 count ( A c i d s o l u b l e S) r e s i d u e l add water and f r e e z e s o l i d rlO mg of dry r e s i d u e i n a s m a l l pyrex t e s t tube ^ j — j I add 6N HC1 r e s i d u e i n . a K j e l d a h l f l a s k - | j evacuate and s e a l the tube d i g e s t I count ( P r o t e i n S) heat a t 110°C f o r 24 hr open the tube and dry i n a d e s i c c a t o r f i l t e r h y d r o l y s a t e I f r e e z e dry the f i l t r a t e I t r a n s f e r f i l t r a t e to chromatogram develop w i t h 80$ phenol cu t the chromatogram i n t o s t r i p s t r a n s v e r s e l y I count the s t r i p s (Amino a c i d S i n p r o t e i n ) 45 RESULTS A. T o t a l uptake of s u l p h u r - 3 5 py bean p l a n t s The r o o t s of f o u r t e e n - d a y - o l d c o n t r o l and cyclohexane-c a r b o x y l i c a c i d - t r e a t e d bean p l a n t s which had been growing i n s u l p h u r - f r e e n u t r i e n t s o l u t i o n were t r a n s f e r r e d to a complete n u t r i e n t s o l u t i o n c o n t a i n i n g 35$o=, A f t e r f o u r hours i n the r a d i o a c t i v e s o l u t i o n , the r o o t s were r i n s e d s e v e r a l times with tap water over a p e r i o d of one hour and r e t u r n e d to s u l p h u r -f r e e n u t r i e n t s o l u t i o n . The r i n s i n g was n e c e s s a r y to remove adsorbed ^5so^ from the r o o t s u r f a c e s . In p r e l i m i n a r y experiments, the n u t r i e n t s o l u t i o n was checked on the h a r v e s t day and no a c t i v i t y was found above background, so i t can be assumed t h a t SO^ had been removed completely and t h a t there was no s i g n i f i c a n t e f f l u x of a c t i v i t y from the p l a n t s . Table I I i s a summary of the t o t a l a c t i v i t y data obtained from three runs of the experiment d e s c r i b e d i n the m a t e r i a l s and methods s e c t i o n s A, B, C, and D. CHCA treatment, had no s i g n i f i c a n t e f f e c t (p = O . 0 5 ) on t o t a l uptake of s u l p h u r - 3 5 from the n u t r i e n t s o l u t i o n , although t r e a t e d p l a n t s contained about 6 . 3 p e r c e n t more a c t i v i t y than c o n t r o l s . B. The d i s t r i b u t i o n of s u l p h u r - 3 5 w i t h i n bean p l a n t s  expressed as a c t i v i t y per gram of f r e s h weight In most cases there was a p r o g r e s s i v e i n c r e a s e i n the weight of the organs from one h a r v e s t to the next (Table I I I ) , but o n l y the weight of t r e a t e d leaves and stems had i n c r e a s e d s i g n i f i c a n t l y (p = O . 0 5 ) over the 2h hour p e r i o d . The i n c r e a s i n g weight of the p l a n t p a r t s due to growth w i l l have 46 TABLE II The e f f e c t of KCHC on t o t a l uptake of sulphur - 3 5 by bush bean plants at various harvest times on a per plant b a s i s a . Harvest Total sulphur - 3 5 a c t i v i t y 1 3 time i n hours C o n t r o l 0 Treated 4 8 6 8 , 4 5 3 d 977,577 8 9 1 7 , 8 3 8 904,244 12 8 9 0 , 0 9 9 1,013,829 24 9 5 3 , 8 1 3 964,818 Mean 9 0 7 , 8 1 3 9 6 5 , 1 1 7 Percent of control I O 6 . 3 Plants grown i n a minus sulphur nutrient so l u t i o n before and after they were exposed f o r four hours to a sulphur - 3 5 nutrient solution. Two plants were combined and treated as one. Disintegrations/minute/plant. Control: 0.3% Tween 20 spray; Treated: 1 X 10" 2M KCHC spray. Average of s i x values. TABLE I I I Fre s h weights of p l a n t p a r t s i n s u l p h u r - 3 5 uptake and d i s t r i b u t i o n e x p e r i m e n t s a . Weight (g) Leaf blades Steins Roots Harvest time i n hours C T C T C T 4 5.7^5 5 . 7 7 0 2 . 7 5 2 2 . 7 5 7 5 . 0 8 0 5 . 3 ^ 7 8 6 . 0 6 5 5 . 9 5 2 2 . 8 6 2 2.840 5 . 4 6 5 5 . 0 7 7 1 2 6 . 4 7 2 6 . 4 5 3 2.947 2 . 9 5 5 5 . 4 1 5 5.575 24 6 . 5 9 2 6 . 8 2 2 2 . 9 4 5 3 . 1 5 2 5 . 7 3 5 5 . 6 6 3 See Table I I Average of 6 v a l u e s . See Table I I A continuous v e r t i c a l l i n e to the r i g h t of a s e t of numbers i n d i c a t e s v a l u e s which do not d i f f e r s i g n i f i c a n t l y from each other a t the 0 . 0 5 l e v e l . 48 d i l u t e d the a c t i v i t y , and values calculated on a per gram basis were se n s i t i v e to these changes. The d i s t r i b u t i o n of sulphur-35 among the three organs, roots, stems plus p e t i o l e s , and l e a f blades, was measured four, eight, 12, and 24 hours from the time 35so^ loading concluded. Because loading lasted four hours, plants at the four hour harvest, f o r example, had been translocating radio-active sulphur f o r eight hours. Sulphur-35 a c t i v i t y i n l e a f blades, expressed as a c t i v i t y per gram of fresh weight, was greater i n treated plants than i n control plants at each of the harvest times and s i g n i f i c a n t l y greater (p = 0.05) at the 4 hr and 12 hr harvest (Table IV and Fig . 2). There was no s i g n i f i c a n t differences due to treatment i n any of the values associated with roots and stems, or i n l e a f blades at the 8 hr and 24 hr harvests. The l e v e l of a c t i v i t y at the 24 hr harvest was greater than the a c t i v i t y at the four hour harvest f o r control leaves, and control and treated stems. In addition, there was a s i g n i f i c a n t decline i n sulphur-35 a c t i v i t y i n control and treated roots between each of the four harvest times. These r e s u l t s indicated that sulphur was being translocated from the roots to the stem and leaves. They also indicated that some of the a c t i v i t y was becoming incorporated into stem tissue as time passes. C. The d i s t r i b u t i o n of sulphur-35 within bean plants  expressed as a c t i v i t y per plant organ The t o t a l values f o r a c t i v i t y on a per plant basis are shown i n Table II. When these were broken down to give the TABLE IV The e f f e c t o f KCHC treatment on su l p h u r - 3 5 d i s t r i b u t i o n w i t h i n bean p l a n t s a t v a r i o u s h a r v e s t times on an a c t i v i t y per gram f r e s h weight b a s i s 3 - . Sulphur - 3 5 a c t i v i t y b Harvest time i n hours Leaf blades Stems Roots 4 8 12 24 7 2 , 7 0 5 c 76,286 7^,791 88 ,073 8 3 , 6 2 7 * 8 5 , 0 5 8 9 1 , 7 1 1 * 9 0 , 9 1 8 13,928 15,243 15,801 22,724 | 14,733 14 ,205 16,571 20,468 81 , 8 8 9 7 4 , 9 0 6 6 7 , 0 5 9 5 2 , 4 0 7 85,427| 73,55^1 67 ,613| 51,2581 Mean 77,963 87,578* 1 6 , 9 2 4 1 6 , 4 9 4 6 9 , 0 6 4 6 9 , 4 6 3 P e r c e n t o f c o n t r o l 1 1 2 . 3 97.4 1 0 0 . 6 a c See Table I I . b D i s i n t e g r a t i o n s / m i n u t e / g r a m f r e s h weight. d Average o f s i x v a l u e s . * S i g n i f i c a n t l y d i f f e r e n t from the comparable c o n t r o l v a l u e a t the 0 . 0 5 l e v e l . V e r t i c a l l i n e , see Table I I I 50 o o o FIGURE 2 The e f f e c t o f KCHC treatment on s u l p h u r - 3 5 d i s t r i -b u t i o n i n bean p l a n t s a t v a r i o u s h a r v e s t times. Graphic i l l u s t r a t i o n of the data i n Table IV. 1 0 0 x 9 0 4 c •H >> -P •H > •H •P O f°\ I U a rH C o n t r o l — Treated --Leaf + Stem * Root o 3 0 I 2 0 1 1 0 1 8 Harvest Time i n Hours 51 amount i n each organ and analyzed s t a t i s t i c a l l y , i t was found that there was no s i g n i f i c a n t difference i n a c t i v i t y between comparable treated and control plants f o r any of the organs at any of the harvest times (Table V). However, when expressed i n t h i s way, there was a s i g n i f i c a n t e f f e c t due to harvest time. Leaves and stems s t e a d i l y accumulated sulphur-35 while roots l o s t i t . There was a s i g n i f i c a n t difference in the l e v e l of a c t i v i t y i n the 24 hr harvest i n every case when compared to the 4 hr harvest. The treated roots showed a s i g n i f i c a n t decline only between 12 and 24 hr. Expressing the data i n t h i s way gave an idea of the absolute a c t i v i t y present i n each organ. These data were not s e n s i t i v e to changes i n the weight of the organs. It was noted that the l e v e l of activity^ as indicated by the means, showed that the treated leaves contained more sulphur-35 than controls while roots and stems were almost i d e n t i c a l to the controls. It appeared that the extra sulphur-35 taken up by the treated plants (Table II) had been moved to the leaves. This suggested a s l i g h t l y more rapid movement of sulphur-35 within the treated plants. TABLE V The e f f e c t of KCHC treatment on sulphur - 3 5 d i s t r i b u t i o n within bean plants at various harvest times on an a c t i v i t y per plant part b a s i s a . Sulphur -35 a c t i v i t y Harvest time i n hours Leaf blades Stems T Roots c o n t r o l 4 4 1 7 , 0 8 0 4 8 1 , 2 3 6 3 8 , 6 7 1 41 , 4 0 6 412 ,701 4 5 4 , 9 3 5 | 8 4 6 3 , 6 8 9 4 9 4 , 8 1 4 4 3 , 5 9 3 39,880 410 ,555 3 6 9 , 5 5 0 12 482 ,718 5 8 9 , 3 6 3 46 ,877 4 9 , 1 7 9 3 6 0 , 5 0 3 3 7 5 , 2 8 5 24 5 8 4 , 9 2 3 6 1 4 , 3 2 2 6 7 , 1 2 9 6 3 , 7 5 1 3 0 1 , 7 6 0 286,744| Mean 4 8 7 , 1 0 2 5 4 4 , 9 3 4 4 9 , 0 6 7 48 , 5 5 4 3 7 1 , 3 8 0 371,628 Percent of 111.9 9 9 . 0 1 0 0 . 1 a c See Table II. ^ Disintegrations/minute/plant part. ^ Average of 6 values. V e r t i c a l l i n e , see Table III 53 D . The d i s t r i b u t i o n of sulphur - 3 5 among the plant organs  as a percent of the t o t a l a c t i v i t y in the plant . The percent of t o t a l plant a c t i v i t y present i n treated leaves at the 8 and 12 hr harvests, as well as the averages of these values, was s i g n i f i c a n t l y greater than i n the control leaves. Also, roots of treated plants, on the average, con-tained a s i g n i f i c a n t l y lower percentage of the t o t a l sulphur - 3 5 i n the plant. This means that the treatment had stimulated the rate of translocation of sulphur - 3 5 from the roots to the leaves (see also r e s u l t s , section B). The data are presented i n Table VI and Fig. 3 . E. Uptake and d i s t r i b u t i o n of t o t a l sulphur For an explanation of how the plants were grown, treated, and harvested, see materials and methods sections G, H, I, and J. Analysis f o r t o t a l sulphur according to the method of Kowalenko and Lowe (64) gave the r e s u l t s shown i n Table VII. Since there was a difference i n the size of the plants between the two experiments, the data from each experiment, expressed as mg of sulphate per plant (or plant p a r t ) , were presented separately. These data were not analyzed s t a t i s t i -c a l l y . When the data were expressed as mg of sulphate per g dry weight, the r e s u l t s of both experiments were combined and analyzed s t a t i s t i c a l l y . There was no apparent e f f e c t of treatment on t o t a l uptake of sulphur by these plants or on the d i s t r i b u t i o n of sulphur among the organs. TABLE VI The e f f e c t of KCHC treatment on s u l p h u r - 3 5 d i s t r i b u t i o n w i t h i n bean p l a n t s a t v a r i o u s h a r v e s t times on a p e r c e n t per p l a n t p a r t b a s i s a . Percent of t o t a l s u l p h u r - 3 5 a c t i v i t y -Leaf blades Stems Roots Harvest ; time i n hours c c T C T C T 4 48 .11° 4 9 . 3 7 | 4.42 4 . 1 6 47.46 46 .47 8 50.49 5 4 . 7 4 * | 4 . 8 7 4 . 4 5 4 4 . 64 40.8 l * | 12 54.06 5 8 . 2 3 * | 5.28 4 . 8 9 40 .67 3 6 . 8 8 * | 24 61.22 6 3 . 7 3 | 6 .96 6 .57 3 1 . 8 3 | 2 9 . 7 0 | Mean 53-^7 5 6 . 5 1 * 5 . 3 8 5 . 0 2 41 .15 3 8 . 4 7 * a See Table I I c See Table I I d Average of s i x values * See Table IV V e r t i c a l l i n e , see Table I I I 55 1 0 0 FIGURE 3 The e f f e c t of KCHC treatment on percentage d i s -t r i b u t i o n of sulphur - 3 5 i n bean p l a n t organs a t v a r i o u s h a r v e s t times. Data from Table VI. C o n t r o l — Treated — Leaf + 9 0 4 Stem * Root o 8 0 4 204 10-L — t ~~ «= 1 — Harvest Time i n Hours 5 6 TABLE V I I The e f f e c t of KCHC treatment on t o t a l s u l phur uptake and d i s t r i b u t i o n i n three week o l d bean p l a n t s . Treatment P l a n t Source of data p a r t C o n t r o l Treated Leaf 4.61 4 . 7 1 mg/plant p a r t Stem 1 . 1 2 1.16 Root 1 . 7 6 1 . 9 4 a Experiment T o t a l / p l a n t 7 . 5 3 7 . 81 One (mg) Leaf 6 1 . 7 6 0 . 5 $ / p l a n t p a r t Stem 14 . 9 14.8 Root 2 3.4 2 4 . 7 Leaf 2 . 7 3 2.82 mg/plant p a r t Stem • 7 2 . 6 1 Root I O ? . I . 2 3 Experiment T o t a l / p l a n t 4 . 7 9 4 . 6 5 Two (mg) Leaf 5 6 . 6 60.3 % / p l a n t p a r t Stem 1 5 . 0 1 3 . 1 Root 28.4 2 6 . 6 Leaf 2.82 2.82 Combined Expt. 1 + mg/g dry wt. Stem 2 .14 1 . 9 1 Expt. 2 Root 4 . 2 0 4. 3 2 a P l a n t s grown i n IX Hoagl and-Arnon's s o l u t i o n f o r one week a f t e r treatment. b P l a n t s grown i n 0.5X Hoa gland-Arnon* s s o l u t i o n f o r one week a f t e r treatment. See Table I I 5 7 F. The d i s t r i b u t i o n of s u l p h u r - 3 5 among v a r i o u s f r a c t i o n s  of bean l e a v e s These experiments were c a r r i e d out i n an attempt to f i n d whether CHCA treatment had any e f f e c t on the d i s t r i b u t i o n o f su l p h u r among v a r i o u s compounds i n bean l e a v e s . I t was de c i d e d to analyze o n l y leaves, because 24 hours a f t e r f e e d i n g s u l p h u r - 3 5 - over 60% of the t o t a l a c t i v i t y o f the p l a n t was found i n the l e a v e s (Table V I ) . The p l a n t s were grown i n s u l p h u r - f r e e n u t r i e n t s o l u t i o n so t h a t the o n l y s u l p h u r a v a i l -a ble to the p l a n t had come from the seed. There was no i n -d i c a t i o n of s u l p h u r d e f i c i e n c y i n two-week-old p l a n t s i n these experiments or i n any of the other experiments i n which p l a n t s were grown i n s u l p h u r - f r e e n u t r i e n t s o l u t i o n . However, i t was l i k e l y t h a t the p l a n t s were sulphur d e f i c i e n t to some extent. The p l a n t s were grown, f e d ^^S0^t h a r v e s t e d , and the le a v e s f r a c t i o n a t e d as d e s c r i b e d i n m a t e r i a l s and methods s e c t i o n s M, N, and 0. P l a n t s were grown on two separate o c c a s i o n s , and these were r e f e r r e d to as the f i r s t and second run o f the experiment. For the f i r s t run, h a r v e s t I, I I , and I I I were taken 1 , 2, and 5 days, r e s p e c t i v e l y , a f t e r the p l a n t s were f e d r a d i o a c t i v e s u l p h a t e . Only h a r v e s t I I was used from the second run of the experiment. Table V I I I shows the d i s t r i b u t i o n o f the a c t i v i t y i n each of the f r a c t i o n s a t the v a r i o u s h a r v e s t times. The a c t i v i t y i n each f r a c t i o n was expressed on the b a s i s of the dry weight of the l e a f m a t e r i a l . There was s i g n i f i c a n t l y (p = 0 . 0 5 ) more su l p h a t e S i n the c o n t r o l l e a v e s than t r e a t e d TABLE V I I I The e f f e c t of KCHC treatment on s u l p h u r - 3 5 d i s t r i b u t i o n among s e v e r a l f r a c t i o n s of bean l e a f t i s s u e a t v a r i o u s h a r v e s t times 3 -. F r a c t i o n s S u l p h u r - 3 5 a c t i v i t y 1 3 Harvest I d Harvest I I e Harvest I I I d c C T C T C T Sulphate 8 3 , 2 1 6 9 3 , 5 9 6 242 , 7 8 9 1 5 8 , 5 3 1 * 26 , 1 2 0 29,644 L i p i d 2 4 , 1 5 8 2 3 , 3 ^ 6 2 0 , 2 1 9 2 1 , 1 6 8 1 2 , 5 3 3 12,644 Free amino a c i d 2 3 , 3 3 3 21,644 7 , 9 8 2 9 , 2 3 0 6 , 2 5 0 7 , 7 2 4 A c i d s o l u b l e 6 9 5 , 1 5 9 8 2 1 , 9 4 3 * 4 4 9 , 0 2 8 5 4 7 , 2 8 3 * 5 3 , 0 3 5 4 0 , 4 8 2 P r o t e i n 2 3 6 , 6 9 5 2 5 9 , 1 5 5 3 7 8 , 0 5 7 4 4 6 , 8 2 5 * 4 6 3 , 7 2 3 5 2 1 , 4 7 8 * a P l a n t s grown i n -S n u t r i e n t s o l u t i o n before and a f t e r a 4 hr f e e d i n g o f S - 3 5 i n a complete n u t r i e n t s o l u t i o n . b D i s i n t e g r a t i o n s / m i n u t e / g r a m dry weight. c See Table I I . a Average of 3 v a l u e s . e Average of 6 v a l u e s . • I n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e between t r e a t e d and c o n t r o l v a l u e s a t the 0 . 0 5 l e v e l . H arvests I, I I , and I I I are 1 , 2 , and 5 days r e s p e c t i v e l y a f t e r s u l p h u r - 3 5 f e e d i n g . 59 FIGURE 4 The e f f e c t of KCHC treatment on sulphur-35 d i s t r i b u t i o n among some fractio n s of bean le a f tissue at various harvest times. Harvest Time in Days 60 leaves at harvest II. Treated leaves had s i g n i f i c a n t l y more acid-soluble sulphur than control leaves at harvests I and II. Treated leaves contained more protein S than control leaves at each harvest, with s i g n i f i c a n t l y greater amounts i n harvest II and I I I . L i p i d S and free amino acid S values were not s i g -n i f i c a n t l y d i f f e r e n t f o r control and treated leaves at any harvest. However, i t was i n t e r e s t i n g to note that there was a steady decline i n the amount of a c t i v i t y i n the l i p i d and free amino acid f r a c t i o n s as time passed. This could be p a r t l y due to the d i l u t i n g e f f e c t of growth (see Table IX). Figure 4 i l l u s t r a t e s the d i s t r i b u t i o n of the a c t i v i t y i n the three major f r a c t i o n s . It was apparent that protein S content increased at the expense of the acid soluble S.and sulphate S f r a c t i o n s . It was also clear from th i s graph that the treated plants had a higher concentration of sulphur - 3 5 than controls. This was i n agreement with, the information i n r e s u l t s , section B. G. Cysteine/cystine and methionine incorporation into bean  le a f protein Protein hydrolysates were placed as bands near the end of long s t r i p s of chromatographic paper (see materials and methods section 0, part g) and developed with 80% phenol sol u t i o n for a distance of 2 5 to 3 5 cm. A l l samples from one harvest were run i d e n t i c a l distances. When these s t r i p s were cut into 2 cm sections and the a c t i v i t y on these sections determined by counting i n a l i q u i d s c i n t i l l a t i o n counter, i t was found that TABLE IX Leaf weights, t o t a l a c t i v i t y , and recovery of a c t i v i t y i n the l e a f f r a c t i o n a t i o n experiment. Leaf dry Total sulphur - 3 5 Sum of f i v e f r a c t i o n s . Percent weight (g) a c t i v i t y / g b sulphur - 3 5 a c t i v i t y / g D recovery Harvest C ° T C T C T C T run one . 3 8 6 6 .4194 1,144,163 1,262,266 1,062,559 1,219,675 91.1 94.9 I I run one run two .4072 .4462 . 4 5 0 7 . 4 7 2 0 1 ?L48 , 4 3 6 1 , 2 9 0 , 3 7 6 1 , 2 4 3 , 1 9 4 1 , 2 0 8 , 0 3 1 1 , 0 8 5 , 9 7 3 1 , 2 5 2 , 0 8 2 1 , 1 1 8 , 9 3 2 1 , 1 2 0 , 7 4 4 9 4 . 5 9 7 . 1 8 8 . 6 9 1 . 6 III run one . 5 8 9 2 . 5 8 7 2 7 3 3 , 3 5 5 6 9 1 , 5 5 0 5 6 1 , 6 6 3 6 1 1 , 9 7 2 7 7 . 2 8 8 . 6 D Disintegrations/minute/gram. c See Table II d See Table VIII ON 62 two r e g i o n s of a c t i v i t y e x i s t e d . The curves i n f i g u r e 5 were from harvest- I I of the f i r s t run but were r e p r e s e n t a t i v e of the s i t u a t i o n a t o t h e r h a r v e s t s as w e l l . Each p o i n t Was the average of three d e t e r m i n a t i o n s and the f i g u r e i l l u s t r a t e d the d i s -t r i b u t i o n of the a c t i v i t y on the chromatogram and the r e l a t i o n -s h i p of t r e a t e d to c o n t r o l . A c c o r d i n g to the p o s i t i o n s of the a u t h e n t i c compounds, the two peaks corresponded to the c y s t e i n e / c y s t i n e group and the methionine group (methionine, methionine sulphone, and methionine sulphoxide can o c c u r ) . I t was a r b i t r a r i l y decided to separate these two groups a t the p o i n t between the two which showed the minimum a c t i v i t y . The methionine r e g i o n o c c a s i o n a l l y appeared as a sharp peak (methionine) or as a more d i f f u s e peak (a combination of the three p o s s i b l e forms). Attempts to convert a l l the c y s t e i n e / c y s t i n e to c y s t e i c a c i d and a l l the methionine to methionine sulphone by o x i d i z i n g w i t h ^2^2 a n ^ molybdate (36 ), or with p e r f o r m i c a c i d were g e n e r a l l y u n s u c c e s s f u l . Both treatments tended to smear the o r i g i n over a wide area and n e i t h e r was able to convert a l l of the m a t e r i a l i n t o j u s t two c l e a r l y s e parated compounds. The b e s t s e p a r a t i o n , with the l e a s t t r o u b l e , was o b t a i n e d when p r o t e i n h y d r o l y s a t e was chromato-graphed d i r e c t l y . From Table X i t was apparent t h a t KCHC treatment had some e f f e c t on the i n c o r p o r a t i o n of sulphur - 3 5 i n t o p r o t e i n amino a c i d s . The sulphur - 3 5 a c t i v i t y i n c y s t e i n e / c y s t i n e and methionine was g r e a t e r i n c o n t r o l l e a v e s than t r e a t e d l e a v e s FIGURE 5 The e f f e c t of KCHC treatment on s u l p h u r - 3 5 i n c o r p o r a t i o n i n t o p r o t e i n amino a c i d s . Data from experiment one, h a r v e s t II,. Distance from the Center of the O r i g i n (cm) 64 TABLE X The e f f e c t of KCHC treatment on c y s t e i n e / c y s t i n e and methionine 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 f r a c t i o n of bean l e a f t i s s u e a . Sulphur-•35 a c t i v i t y ^ Cys te i n e / c y s t i n e Methionine Harvest C C T C T I I I I I I 7 5 , 7 8 1 5 7 , 7 9 0 6 9 , 7 3 9 6 5 , 1 8 9 6 7 , 9 0 4 1 0 5 , 2 7 0 172,481 248,229 3 0 8 , 2 5 1 158,548 273,043 4 1 7 , 1 7 4 Mean 6 5 , 2 7 5 76,567 244,297 2 8 0 , 4 5 2 * P e r c e n t o f c o n t r o l 1 1 7 . 3 114.8 a b See Table V I I I c See Table I I I n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e between t r e a t e d and c o n t r o l v a l u e s a t the O .05 l e v e l . Harvests I, I I , and I I I are 1, 2 , and 5 days r e s p e c t i v e l y a f t e r s u l p h u r - 3 5 f e e d i n g . 6 5 at harvest I, but at harvest II and III , the treated leaves contained a greater amount of a c t i v i t y i n both amino acids. There was no s i g n i f i c a n t difference between comparable control and treated values at any harvest. The mean value f o r methionine a c t i v i t y was s i g n i f i c a n t l y greater (p = 0 . 0 5 ) f o r treated than control. Total sulphur - 3 5 a c t i v i t y i n the protein amino acids of treated leaves was 15*3% greater than i n control leaves. In summary, i t was found that KCHC treated bean plants took up s l i g h t l y more sulphur - 3 5 than control plants and accumulated s i g n i f i c a n t l y higher concentrations of sulphur - 3 5 i n t h e i r leaves than controls. When the a c t i v i t y i n each organ, at each harvest time, was expressed as a percent of t o t a l a c t i v i t y i n the plant, i t was clear that treated plants were moving sulphur from the roots to the shoots more vigor-ously than control plants. Total sulphur uptake over a period of one week a f t e r treatment was not s i g n i f i c a n t l y affected by the treatment. Treated leaves had higher concentrations of sulphur - 3 5 i n the acid soluble and protein f r a c t i o n s , while at the second harvest, control leaves had higher concentrations of sulphate. This indicated that treated plants were con-v e r t i n g sulphate into metabolically active compounds more quickly than control plants. Cysteine/cystine and methionine l e v e l s were higher i n treated leaves than control leaves suggesting the presence of more protein. 66 H. Total uptake of calcium-45 by bean plants Control plants accumulated more calcium-45 than treated plants, but the difference was not s i g n i f i c a n t (p «= 0 . 0 5 ) (Table X I ) . I. The d i s t r i b u t i o n of calcium-45 within bean plants  expressed as a c t i v i t y per gram dry v/eight Bean plants were grown, treated, fed calcium -45 , harvested, and analyzed as described i n materials and methods section t i t l e d "Uptake and d i s t r i b u t i o n of calcium -45". A treatment e f f e c t was found only i n the l e v e l of a c t i v i t y i n the roots (Table X I I ) . The treated roots at 48 hr contained a greater concentration (p = O . 0 5 ) of a c t i v i t y than control roots. No s i g n i f i c a n t difference was observed i n the l e v e l of a c t i v i t y i n leaves or stems at any of the harvest times. A s i g n i f i c a n t drop i n the l e v e l of a c t i v i t y i n the leaves at the 48 hr harvest can probably be explained i n terms of growth d i l u t i o n . R e l a t i v e l y small amounts of calcium-45 remained to be trans-located into the leaves from the roots and stems a f t e r twenty four hours. The increase i n the weight of the leaves over thi s period was s u f f i c i e n t to account for the decline i n a c t i v i t y expressed on a per gram basis. The dry weight of leaves at the 48 hr harvest was considerably greater than at the 12 or 24 hr harvests (Table XVII). For example, i n the case of the treated plants, there was a 3 7 $ increase i n weight from the 12 hr harvest to the 48 hr harvest and a 2 3 $ drop i n calcium-45 a c t i v i t y . The difference between these two numbers indicated that a d d i t i o n a l calcium - 4 5 was imported by the leaves, but the rate of accumulation between 24 and 48 hr was considerably below the e a r l i e r periods. 67 TABLE XI The e f f e c t of KCHC treatment on t o t a l uptake of calcium-45 by bush bean p l a n t s a t v a r i o u s h a r v e s t times on a per p l a n t b a s i s a . H a r vest T o t a l calcium-45 a c t i v i t y 1 3 time i n hours C o n t r o l 0 Treated 12 2 0 3 , 3 1 1 d 197,494 24 217,400 203,796 48 . ' 214,649 200,843 Mean 211,787 200,711 Pe r c e n t of c o n t r o l 94.8 P l a n t s grown i n a complete n u t r i e n t s o l u t i o n before and a f t e r they were exposed f o r f o u r hours to a calcium-45 n u t r i e n t s o l u t i o n . P l a n t s were handled i n d i v i d u a l l y . D i s i n t e g r a t i o n s / m i n u t e / p l a n t . C o n t r o l - 0 . 3 $ Tween 20 spray; Treated - 1 X 10" 2M KCHC spray. Average of e i g h t v a l u e s . f o r a l l but 12 hour h a r v e s t , C and T l e a v e s , s i x v a l u e s . TABLE X I I The e f f e c t of KCHC treatment on the d i s t r i b u t i o n o f calcium-45 w i t h i n bean p l a n t s a t v a r i o u s h a r v e s t times on a per gram b a s i s 3 - . Calcium-45 a c t i v i t y Harvest Leaf blades Stems Roots time i n hours c c T C T C T 12 24 48 5 1 5 , 1 8 8 d 541,9;+5 4 4 7 , 2 3 6 5 1 4 , 2 7 1 5 6 1 , 4 8 7 4 1 8 , 1 9 1 2 5 9 , 3 6 4 171 ,709 1 1 6 , 6 6 5 2 3 8 , 4 5 7 1 5 0 , 1 6 0 1 1 2 , 4 2 7 5 6 , 3 1 0 50,149 4 3 , 3 1 4 7 0 , 2 6 2 7 1 , 5 1 1 5 8 , 7 l l * | Mean 5 0 1 , 4 5 1 4 9 7 , 9 7 8 182,578 1 6 7 , 0 1 3 4 9 , 9 2 4 6 6 , 8 2 7 * P e r c e n t o f c o n t r o l 9 9 . 3 9 1 . 5 1 3 3 . 9 See Table XI Dis i n t e g r a t i o n s / m i n u t e / g r a m . See Table XI Average o f e i g h t v a l u e s f o r a l l but 12 hour h a r v e s t , C and T l e a v e s , s i x v a l u e s . V e r t i c a l l i n e , see Table I I I . 69 J. The d i s t r i b u t i o n of calcium - 4 5 w i t h i n bean p l a n t s  expressed as a c t i v i t y per p l a n t organ The a b s o l u t e l e v e l s of calcium - 4 5 i n the v a r i o u s organs of the p l a n t a r e . g i v e n i n Table X I I I . A s i g n i f i c a n t l y g r e a t e r amount of calcium - 4 5 was found i n the stems of c o n t r o l p l a n t s than t r e a t e d p l a n t s a t the 12 and 24 hr h a r v e s t and i n the mean va l u e f o r the stems. The leaves of c o n t r o l p l a n t s con-t a i n e d more calcium than l e a v e s of t r e a t e d p l a n t s . On the other hand, the mean value f o r the r o o t s a t a l l h a r v e s t s showed s i g n i f i c a n t l y g r e a t e r a c t i v i t y i n the t r e a t e d p l a n t s than i n the c o n t r o l s . The s i t u a t i o n here appeared to be j u s t o p p o s i t e to t h a t f o r sulphur uptake and d i s t r i b u t i o n . Leaves continued to accumulate calcium - 4 5 up to the 48 hr h a r v e s t (Table X I I I ) . These data expressed the. amount of a c t i v i t y i n the organ and were, t h e r e f o r e , i n s e n s i t i v e to changes i n the weight of the organ. However, i t was c l e a r t h a t the i n c r e a s e i n a c t i v i t y i n the leaves over the f i n a l 24 hours of the experiment was minimal. S i g n i f i c a n t decreases i n the l e v e l of a c t i v i t y i n the stems from the 12 hr h a r v e s t to the 48 hr h a r v e s t suggested t h a t the stems were c o n t r i b u t i n g the c a l c i u m being accumulated by the l e a v e s . Very l i t t l e d e c l i n e i n r o o t l e v e l s o c c u r r e d over the p e r i o d of the experiment. TABLE X I I I The e f f e c t of KCHC treatment on calc i u m - 4 5 d i s t r i b u t i o n w i t h i n bean p l a n t s a t v a r i o u s h a r v e s t times on an a c t i v i t y per p l a n t p a r t basis 3- . Calcium - 4 5 a c t i v i t y 0 Harvest Leaf blades Stems Roots time i n hours c c T C T C T 12 1 6 2 , 2 7 3 d 1 5 7 , 7 2 6 3 2 , 1 3 1 28 ,325* 8 , 2 7 6 1 0 , 4 6 5 24 186,076 172,048 24,112 2 0 , 8 6 3 * 7 , 9 9 6 1 0 , 0 8 9 48 188,250 1 7 3 , 7 8 5 18,920 1 7 , 3 0 2 7 , 4 7 9 9 , 7 5 6 Mean 1 7 8 , 8 6 6 1 6 7 , 8 5 3 2 5 , 0 5 4 2 2 , 1 6 3 * 7 ,917 1 0 , 1 0 3 * P e r c e n t of c o n t r o l 9 3 - 8 8 8 . 5 1 2 7 . 6 a See Table XI D i s i n t e g r a t i o n s / m i n u t e / p l a n t p a r t . c See Table XI d See Table XI V e r t i c a l l i n e , see Table I I I 71 K. The d i s t r i b u t i o n o f calcium-45 among the p l a n t organs  as a p e r c e n t o f the t o t a l a c t i v i t y i n the p l a n t The data from Table X I I I were r e c a l c u l a t e d to g i v e the percentage d i s t r i b u t i o n of calcium a c t i v i t y i n each organ. When viewed i n t h i s manner (Table XIV), the same p a t t e r n o f d i s t r i b u t i o n and t r a n s l o c a t i o n appeared as i n Tables XII and X I I I . The steady accumulation of calcium-45 by the le a v e s was l a r g e l y a t the expense of the stems, while the r o o t s a p p a r e n t l y c o n t r i b u t e d v e r y l i t t l e . The v e r y minor decrease i n a c t i v i t y o f the r o o t s from 12 to 48 hr suggested t h a t some of the calcium-45 became i n c o r p o r a t e d i n t o the r o o t t i s s u e . L. The uptake and d i s t r i b u t i o n of t o t a l c a l c i u m P l a n t s were grown, t r e a t e d and ha r v e s t e d as d e s c r i b e d i n m a t e r i a l s and methods s e c t i o n s G, H, I, and J. The e f f e c t of KCHC treatment on t o t a l c a l c i u m uptake and d i s t r i b u t i o n was d e t a i l e d i n Table XV. The data from Experiment One and Experiment Two, expressed as mg of ca l c i u m per p l a n t (or p l a n t p a r t ) , were pre s e n t e d s e p a r a t e l y i n the t a b l e . These data were n o t analyzed s t a t i s t i c a l l y . When the amount of c a l c i u m i n the p l a n t s was expressed as mg/g dry weight, the data from both experiments were combined and analyzed s t a t i s t i c a l l y . There were no apparent d i f f e r e n c e s i n the amount of c a l c i u m p r e s e n t i n the p l a n t t i s s u e due to treatment r e g a r d l e s s of how the data were expressed. The percentage d i s t r i b u t i o n of o r d i n a r y c a l c i u m w i t h i n the p l a n t was not u n l i k e t h a t f o r the r a d i o a c t i v e c a l c i u m a t the 12 hour h a r v e s t . As time passed, a g r e a t e r percentage of the r a d i o a c t i v e c a l c i u m was accumulated by the l e a v e s . The TABLE XIV The ef f e c t of KCHC treatment on calcium - 4 5 d i s t r i b u t i o n within bean plants at various harvest times on a percent b a s i s a . Percent of t o t a l calcium - 4 5 a c t i v i t y Leaf blades Stems Roots Harvest time i n hours c c T C T C T 12 79. 6 d 7 9 . 5 1 6 . 1 | 15.0 4 .2 5 . 5 24 8 5 . 1 84.0 1 1 . 3 1 0 . 6 3-6 5-5 48 8 7 . 6 8 6 . 3 9 . 0 8 . 7 3 . ^ 5 . 0 a See Table XI c See Table XI d See Table XI V e r t i c a l l i n e , see Table III 7 3 TABLE XV The e f f e c t of KCHC treatment on t o t a l c a l c i u m uptake and d i s t r i b u t i o n i n three week o l d bean p l a n t s . Treatment Source of data P l a n t p a r t C o n t r o l Treated Leaf 43.885 41.801 mg/plant p a r t Stem 6.693 7 . 1 5 0 Root 3.022 2 . 9 6 8 Experiment 3 , One T o t a l / p l a n t (mg) . Leaf 53.580 81.9 51.919 80.5 $ / p l a n t p a r t Stem 12.5 13.9 Root 5.6 5.7 Leaf 2 7 . 0 5 1 26.548 mg/plant p a r t Stem 4 . 6 3 4 4.468 Root 2.804 2.444 Experiment^ Two T o t a l / p l a n t (mg) Leaf 34.489 78.3 33.459 79.3 $ / p l a n t p a r t Stem 13.6 13.3 Root 8.2 7.2 Combined Expt. 1 + Expt. 2 mg/g dry wt. Leaf Stem Root 27.480 1 3 . 5 0 0 8.248 26 . 2 7 3 13.024 7.809 a D See Table V I I See Table I I 7 4 d i f f e r e n c e s may be e x p l a i n e d by the f a c t t h a t the r a d i o a c t i v e data r e p r e s e n t e d the d i s t r i b u t i o n o f ca l c i u m obtained over one s h o r t p e r i o d o f time, w h i l e the d i s t r i b u t i o n of c o l d c a l c i u m was the r e s u l t o f a l l c a l c i u m movements from the time o f germin a t i o n . The p l a n t s analyzed i n t h i s experiment accumulated c a l c i u m f o r two weeks before treatment with KCHC and f o r one week a f t e r treatment. I f there was going to be an e f f e c t due to treatment, i t had to occur d u r i n g the t h i r d week. When the dry weight data from the two-week-old p l a n t s used i n the c a l c i u m - 4 5 uptake and d i s t r i b u t i o n experiments ( 1 2 hr h a r v e s t ) were compared wi t h the dry weights f o r the three-week-old p l a n t s used i n t h i s experiment (Table XVII), a three to f i v e f o l d i n c r e a s e i n weight was noted f o r the whole p l a n t d u r i n g the t h i r d week. I t seems reasonable t h a t the uptake of m i n e r a l s d u r i n g the t h i r d week would a l s o amount to s e v e r a l times as much as was accumulated i n the f i r s t two weeks when the p l a n t s were q u i t e s m a l l . I f treatment had a d i r e c t e f f e c t on uptake, i t might be expected to show up d u r i n g the t h i r d week, but t h i s was not e v i d e n t . In summary, i t was found t h a t t r e a t e d p l a n t s took up somewhat l e s s c a l c i u m - 4 5 than c o n t r o l p l a n t s , but r e t a i n e d s i g n i f i c a n t l y more of i t i n t h e i r r o o t s . T o t a l c a l c i u m uptake by c o n t r o l p l a n t s over a p e r i o d o f one week a f t e r treatment was not s i g n i f i c a n t l y g r e a t e r than i n t r e a t e d p l a n t s . The d i s t r i b u t i o n of calcium w i t h i n these p l a n t s was a l s o v e r y s i m i l a r f o r t r e a t e d and c o n t r o l . 75 M . T h e u p t a k e a n d d i s t r i b u t i o n o f t o t a l m a g n e s i u m T h e s a m e p l a n t s u s e d f o r t o t a l s u l p h u r a n d c a l c i u m u p t a k e w e r e a n a l y z e d f o r m a g n e s i u m c o n t e n t . F o r d e t a i l s , s e e m a t -e r i a l s a n d m e t h o d s s e c t i o n s G , H , I , a n d J . T h e d a t a f r o m E x p e r i m e n t O n e a n d E x p e r i m e n t T w o , e x p r e s s e d a s m g o f m a g n e s i u m p e r p l a n t ( o r p l a n t p a r t ) , a r e p r e s e n t e d s e p a r a t e l y i n T a b l e X V I . T h e s e d a t a w e r e n o t a n a l y z e d s t a t -i s t i c a l l y . W h e n t h e a m o u n t o f m a g n e s i u m i n t h e p l a n t s w a s e x p r e s s e d a s m g / g d r y w e i g h t , t h e d a t a f r o m b o t h e x p e r i m e n t s w e r e c o m b i n e d a n d a n a l y z e d s t a t i s t i c a l l y . T h e r e w a s s l i g h t l y m o r e m a g n e s i u m i n c o n t r o l p l a n t s t h a n t r e a t e d p l a n t s . W h e n e x p r e s s e d a s m g o f m a g n e s i u m / g d r y w e i g h t , t h e r e w a s s i g n i f i c a n t l y m o r e i n c o n t r o l l e a v e s t h a n t r e a t e d l e a v e s . N . C o m p a r i s o n o f f r e s h w e i g h t , d r y w e i g h t a n d d r y w e i g h t  a s a p e r c e n t o f f r e s h w e i g h t T a b l e X V I I c o n t a i n s a v e r a g e v a l u e s f o r f r e s h w e i g h t , d r y w e i g h t , a n d d r y w e i g h t a s a p e r c e n t o f f r e s h w e i g h t f o r t h e e x p e r i m e n t s i n w h i c h d r y w e i g h t w a s d e t e r m i n e d . F o r t h e t o t a l s u l p h u r , m a g n e s i u m , a n d c a l c i u m e x p e r i m e n t s , i t w a s c l e a r t h a t t h e p l a n t s i n E x p e r i m e n t O n e w e i g h e d m o r e t h a n t h o s e i n E x p e r i m e n t T w o . T h e p l a n t s i n t h e s e c o n d e x p e r i m e n t w e r e g r o w n o n 0.5X H o a g l a n d - A r n o n ' s s o l u t i o n a s o p p o s e d t o t h e I X s t r e n g t h s o l u t i o n u s e d i n E x p e r i m e n t O n e . I n a d d i t i o n t o t h i s , t h e p l a n t s o f E x p e r i m e n t T w o w e r e g r o w n l a t e r i n t h e s e a s o n w h e n l i g h t l e v e l s w e r e n o t a s h i g h a s f o r E x p e r i m e n t O n e . 7 6 TABLE XVI The e f f e c t of KCHC treatment on t o t a l magnesium uptake and d i s t r i b u t i o n i n three week o l d bean p l a n t s . Treatment Source of data P l a n t p a r t C o n t r o l 0 Treated Leaf 9 . 5 1 7 9 . 1 1 0 mg/plant p a r t Stem l . 0 2 5 1 . 1 2 1 Root 1.619 I . 6 9 O Experiment 3 - T o t a l / p l a n t One (mg) Leaf 1 2 . 0 5 9 7 8 . 9 1 1 . 9 2 0 7 6 . 4 $ / p l a n t p a r t Stem 8 . 5 9 . 4 Root 12.6 14 .1 Leaf 6 . 0 3 1 5 . 9 1 2 mg/plant p a r t Stem . 7 5 8 . 7 4 7 Root 1 . 6 9 0 1.466 Experiment 1 3 T o t a l / p l a n t Two (mg) Leaf 8 . 4 7 9 7 1 . 2 8 . 1 2 5 7 3 . 1 $ / p l a n t p a r t Stem 9 . 4 9 . 1 Root 1 9 . 9 1 7 . 9 Combined Expt. 1 + mg/g dry wt. Expt. 2 Leaf Stem Root 6 . 0 6 9 2 . O 6 3 4 . 7 2 1 5 . 7 8 3 * 2 . 1 2 6 4 . 6 1 1 a b See Table V I I . c See Table I I . * S i g n i f i c a n t l y d i f f e r e n t from the comparable c o n t r o l v a l u e a t the 0 . 0 5 l e v e l . 77 TABLE XVII A comparison of fresh weight, dry weight, and the dry weight as a percent of fresh weight of plants used i n t o t a l uptake and d i s t r i b u t i o n of sulphur, calcium, and magnesium, and the uptake and d i s t r i b u t i o n of calcium - 4 5 experiments. Experiment Plant Fresh Weight Dry Weight Dry weight as description part (g) (g) a percent of fresh weight C T C T C T Total Ca, Leaf S, and Mg. Stem Expt. 1. Root Leaf Expt. 2 . Stem Root 16. 01 1 5 . 6 3 1 .74 , 7 . 2 3 7. 06 . 5 8 9 . 4 5 9 . 7 0 .46 9 . 9 0 1 0 . 1 1 . 9 5 4 . 4 7 4 . 6 6 • 33 6 . 3 3 6 . 0 1 . 3 0 1 .75 1 0 . 8 7 1 1 . 2 0 • 59 8 . 0 2 8 . 3 6 . 4 5 4 . 8 7 4.64 . 9 8 9 . 6 0 9 . 7 3 . 3 4 7.28 7 . 2 9 .28 4 . 7 4 4.66 Calcium-45 Leaf 3 . 0 1 2 . 8 3 . 3 1 0 9 . 3034 IO .33 1 0 . 7 2 uptake and d i s t r i b u t i o n Stem 1 .56 1 . 4 4 .1248 . 1174 8 . 0 0 8 . 1 5 12 hour harvest Root 2 . 3 9 2 . 2 5 .1477 . 1 4 8 1 6.18 6 . 5 8 Leaf 3 . 4 3 3 . 0 7 . 3 4 1 7 . 3 0 5 9 9 . 9 6 9 . 9 7 24 hour Stem 1 .76 1 .52 . 1 4 1 7 . 1 2 0 2 8 . 0 5 7 . 9 0 harvest Root 2.46 2 . 3 3 . 1574 . 1 5 3 7 6.41 6 . 6 1 Leaf 4 . 2 3 4.14 . 4 2 2 6 . 4 1 6 9 9 . 9 9 1 0 . 0 7 48 hour Stem 1 .99 1 .92 . 1 6 2 8 . 1 5 4 8 8.18 8 . 06 harvest Root 2 . 7 0 2 . 5 1 . 1 6 9 8 . 1677 6 . 2 9 6 . 6 8 78 The dry weight as a p e r c e n t of f r e s h weight f o r the r o o t s was q u i t e c o n s i s t e n t i n both runs of the t o t a l c a l c i u m , s u l p h u r , and magnesium uptake experiment, but the stem and l e a f v a l u e s were lower i n run two. The d i f f e r e n c e i n water content of the shoots may have been the r e s u l t of d i f f e r e n t c o n d i t i o n s which e x i s t e d a t the h a r v e s t times. T h i s n o t i o n was strengthened when examining the data f o r pe r c e n t dry weight from the three h a r v e s t s of the calcium-45 uptake and d i s t r i b u t i o n experiments. The v a l u e s f o r the lea v e s of 24 and 48 hr h a r v e s t s were v e r y s i m i l a r but were lower than the v a l u e f o r the 12 hr h a r v e s t . The 12 hour h a r v e s t was made j u s t a t the end of the d a i l y l i g h t c y c l e when temperatures were lower, w h i l e the other two were h a r v e s t e d about midv/ay through the c y c l e . The c o n d i t i o n s a t the d i f f e r e n t times of the d a i l y c y c l e c o u l d have an i n f l u e n c e on the water content of' the t i s s u e . Although these data were not analyzed s t a t i s t i c a l l y , . ' i t appeared t h a t treatment w i t h KCHC had no e f f e c t on the dry weight as a p e r c e n t of f r e s h weight. 79 DISCUSSION For th i s discussion, i t i s assumed that the e f f e c t of cyclohexanecarboxylic acid on plant metabolism i s represen-t a t i v e of the e f f e c t of naphthenic acid treatment. This i s a reasonable assumption since CHCA q u a l i f i e s as a naphthenic acid on the basis of structure ( 60 ), and i t has been found i n the naphthenic acid mixture ( 38 ). Moreover, studies of plant responses to CHCA treatment have revealed that d i f f e r -ences are i n degree rather than i n character (84,113 )• Sulphur uptake Active accumulation of ions by roots from s o i l or a solu t i o n culture i s accomplished by•expenditure of energy obtained from the respiratory a c t i v i t i e s of root c e l l s ( 91 ). Naphthenic acid treatment has been shown to stimulate processes which are associated with increased energy l e v e l s i n the plant. Increased rates of photosynthesis and dark res-p i r a t i o n were found i n the shoots of Knap treated beans at 7, 14, and 21 days a f t e r treatment ( 47 ), and increased photo-synthesis was reported f o r treated grape ( 63 ). Increased production of photosynthate can be implied from studies which showed s i g n i f i c a n t increases i n vegetative or reproductive growth due to naphthenate (47,112,113) or CHCA treatment (113). In addition to increased l e v e l s of metabolites, the a c t i v i t y of some enzymes associated with energy production has been stimulated by naphthenates. Fattah and Wort ( 47 ) reported stimulated phosphorylase and phosphoglycerate kinase a c t i v i t y due to Knap treatment and increases have also been found for cytochrome oxidase (114). It was suggested that increased 80 rates of photosynthesis would lead to a greater supply of reduced nucleotides, ATP, and photosynthate f o r use i n bio-chemical processes, and the hexose phosphate supply would be increased due to the stimulated phosphorylase a c t i v i t y ( 4 7 ) . Increased cytochrome oxidase a c t i v i t y implies more active electron transport. It i s l i k e l y that increased meta-b o l i c a c t i v i t y of the shoot causes stepped-up rates of trans-port of metabolites to the roots where they are u t i l i z e d i n the energy r e q u i r i n g process of ion uptake. It would not be s u r p r i s i n g to f i n d that CHCA treatment had stimulated uptake of sulphur - 3 5 from the nutrient solution. However, the increase i n uptake, when compared to the control, amounted to only 6 .3 percent and did not d i f f e r s i g n i f i c a n t l y from the amounts accumulated by control plants. There were several possible reasons f o r these r e s u l t s . The increased metabolic a c t i v i t i e s mentioned above have a l l been reported f o r plants at l e a s t seven days a f t e r treatment. It was e n t i r e l y possible that not enough time had passed to allow the treatment to become f u l l y e f f e c t i v e . There was the second p o s s i b i l i t y that the shoots were i n a better competitive p o s i t i o n with respect to increased metabolites for the f i r s t two or three days a f t e r treatment. Padmanabhan ( 76 ) has found that a f t e r 14 . . . one week 72% of the C a c t i v i t y remained i n the primary leaves of bean plants which had received a f o l i a r a p p l i c a t i o n 14 of KCHC-7- C. She also observed that only a very small amount 14 of the KCHC-7- C a c t i v i t y spotted on primary leaves of bean had been translocated to the roots. As yet i t i s not known 81 how much CHCA must be p r e s e n t i n a t i s s u e to be e f f e c t i v e as a s t i m u l a t o r of m e t a b o l i c a c t i v i t y . The f a c t t h a t there i s on l y a s m a l l amount of CHCA i n an organ i s not n e c e s s a r i l y a reason f o r low a c t i v i t y , but i t co u l d be. Sulphur d i s t r i b u t i o n Under the c o n d i t i o n s of the experiments, a s i g n i f i c a n t l y g r e a t e r amount of sulphur-35 was t r a n s l o c a t e d to the le a v e s of t r e a t e d p l a n t s than c o n t r o l p l a n t s . This was true when the data were expressed as a c t i v i t y per gram f r e s h weight or as a p e r c e n t of t o t a l p l a n t content. When the t o t a l a c t i v i t y i n c o n t r o l and t r e a t e d l e a v e s was compared, the t r e a t e d l e a v e s had more, but the d i f f e r e n c e was not s i g n i f i c a n t . The l a c k of s i g n i f i c a n c e i n the l a t t e r f i g u r e s may i n d i c a t e g r e a t e r v a r i a b i l i t y among l e a f f r e s h weights than among the v a l u e s f o r c o n c e n t r a t i o n o f a c t i v i t y i n each l e a f . These r e s u l t s suggested t h a t the su l p h u r absorbed by t r e a t e d p l a n t s over and above the amount obtained by the c o n t r o l p l a n t s was moved to the l e a v e s r a t h e r than b e i n g d i s t r i b u t e d evenly among the three organs. Si n c e s u l p h a t e moves by mass flow i n the xylem stream (33 )» t r a n s l o c a t i o n to the leaves would be a f u n c t i o n of the r a t e o f xylem l o a d i n g i n the r o o t s , i f t r a n s p i r a t i o n a l l o s s o f water were constant. The r a t e of xylem l o a d i n g would be a f u n c t i o n o f the a v a i l a b i l i t y o f sulphate and the me t a b o l i c a c t i v i t y o f the c e l l s r e s p o n s i b l e f o r l o a d i n g . Since t r e a t e d p l a n t s may have h i g h e r m e t a b o l i c l e v e l s and they d i d take up more (not s i g n i f i c a n t l y more) sulphate than c o n t r o l p l a n t s , 82 i t might be expected t h a t s u l p h a t e was more a v a i l a b l e to the xylem sap of t r e a t e d p l a n t s . When t h i s e x t r a s u l p h a t e reached the l e a v e s , the s m a l l d i f f e r e n c e i n uptake became a s i g n i f i -cant d i f f e r e n c e i n s u l p h u r l e v e l s i n the l e a v e s . Severson ( 8 3 ) r e p o r t e d an experiment i n which the r a t e of t r a n s p i r a t i o n was decreased i n Knap-treated p l a n t s . I t i s not known whether KCHC treatment has the same e f f e c t . I f i t does, i t would l i k e l y slow the r a t e of s u lphate movement to the l e a v e s i n the t r a n s p i r a t i o n stream r a t h e r than a c c e l e r a t i n g i t . However, r a t e of flow of the xylem sap i s o n l y one f a c t o r which determines t r a n s l o c a t i o n r a t e s . The c o n c e n t r a t i o n of the s u l p h a t e would a l s o a f f e c t d e l i v e r y r a t e s , and c o n c e i v a b l y , the s u l p h u r - 3 5 l e v e l s i n xylem sap might have been somewhat h i g h e r i n t r e a t e d than i n c o n t r o l p l a n t s . I t would be i n t e r -e s t i n g to compare the r e s p e c t i v e l e v e l s . o f a c t i v i t y - i n the xylem sap to t e s t t h i s i d e a . Naphthenate treatment had a s i g n i f i c a n t e f f e c t on the amount of t r a n s l o c a t e d from the r o o t to the shoot o f bean p l a n t s ( 8 3 ). Severson suggested t h a t phosphorus t r a n s l o c a t i o n c o u l d have occu r r e d i n the symplast where the i n c r e a s e d energy p r o d u c t i o n a s s o c i a t e d w i t h naphthenate treatment c o u l d con-t r i b u t e to more r a p i d phosphorus t r a n s l o c a t i o n . A s t r o n g s i n k e f f e c t due to phosphorus d e f i c i e n c y i n the l e a v e s was c o n s i d -ered to be an important f a c t o r i n t h i s mechanism ( 8 3 ) . C e r t a i n l y , the same e x p l a n a t i o n c o u l d be put forward f o r the i n c r e a s e d r a t e of movement of s ulphur to the l e a v e s , but i t does not seem necessary. 8 3 S u l p h u r - 3 5 d i s t r i b u t i o n w i t h i n l e a f f r a c t i o n s W i t h i n twenty-four hours of f e e d i n g s u l p h u r - 3 5 to two-week-old bean p l a n t s which had been growing i n s u l p h u r - f r e e n u t r i e n t s o l u t i o n , 60 to 6 5 $ of the s u l p h u r was p r e s e n t i n the l e a v e s (Table V I ) . This s u l p h u r a c t i v i t y c o u l d be separated i n t o f i v e f r a c t i o n s . E x t r a c t i o n w i t h 80$ v/v e t h a n o l was expected to remove f r e e amino a c i d s ( 9 0 ), s u l p h a t e ( 1 0 7)» and l i p i d a s s o c i a t e d s u l p h u r ( 1 0 7 ) . Subsequent e x t r a c t i o n of the r e s i d u e w i t h a mixture of hot e t h a n o l and toluene ( 3 * 1 ) ( 6 7 ) confirmed t h a t a l l l i p i d a s s o c i a t e d with s u l p h u r - 3 5 had been e x t r a c t e d by the 80$ e t h a n o l . A f t e r the e t h a n o l was evaporated, the l i p i d a s s o c i a t e d s u l p h u r .was.. separated from the other two f r a c t i o n s by e x t r a c t i o n w i t h c h l o r o f o r m . The water s o l u b l e p o r t i o n of the e t h a n o l e x t r a c t was passed through a c a t i o n exchange column to separate the f r e e amino a c i d s , which were trapped on the r e s i n , and the s u l p h a t e which had passed through ( 8 7 ) • The r e s i d u e from the e t h a n o l e x t r a c t i o n was heated w i t h 0 . 3 M H C I O 4 to 7 5°C f o r 3 0 minutes ( 6 7 ). The supernatant c o n t a i n e d something c a l l e d s u l phur bound to DNA, or DNA s u l p h u r , by Hase e t a l ( 5 2 , 5 3 ). They had e x t r a c t e d DNA with hot p e r c h l o r i c a c i d and had found s u l p h u r i n the e x t r a c t . In t h i s i n v e s t i g a t i o n l a r g e amounts of a c t i v i t y were found i n t h i s f r a c t i o n a t some h a r v e s t s , but s e p a r a t i o n of DNA by Marmur's method ( 7 3 ) r e v e a l e d t h a t very l i t t l e s u l p h u r - 3 5 was associate.', d i r e c t l y w i t h DNA. As the p r o t e i n was removed from the DNA i n p u r i f i c a t i o n s t e p s , the l e v e l of a c t i v i t y a l s o 84 declined. Consequently, sulphur i n t h i s f r a c t i o n was -simply referr e d to as acid-soluble sulphur. The residue remaining was considered to be protein sulphur ( 6 7 ). Hydrolysis of t h i s material yielded ninhydrin respon-sive substances and the only sulphur-containing compounds i n the hydrolysate were apparently cysteine/cystine and methionine. The bulk of the sulphur -35 a c t i v i t y was found i n the pro-t e i n , acid soluble, and sulphate f r a c t i o n s , and there were major changes i n the amounts present i n each of these f r a c t i o n s from day to day. From figure 3 i t can be seen that sulphur was moving from the acid soluble f r a c t i o n into the protein f r a c t i o n . Sulphate l e v e l s increased u n t i l day two and f e l l thereafter. It i s d i f f i c u l t to determine where sulphate sulphur f i t s into the sequence of tra n s f e r . Sulphur i s apparently delivered to the leaves as sulphate ( 1 0 0 ). It must then be reduced and incorporated into cysteine from which i t may be converted into methionine or some other forms ( 9 9 ). The cysteine and methionine formed w i l l presumably become part of the free amino acid pool u n t i l incorporated into protein or metabolized i n other ways. There are at l e a s t two possible sequences f o r the movement of sulphur through the various f r a c t i o n s . I. Sulphate S reduction Acid soluble Sulphate S-Free amino acid S »»protein S 85 ? ( - - - - - - ( reduction ) I I . Sulphate S » Acid soluble S - *• Free amino acid S ; • P r o t e i n S Scheme I gives the acid soluble portion the r o l e of storage sulphur. Remembering that the plants i n t h i s i n v e s t i -gation were i n i t i a l l y , and subsequently, low i n sulphur, i t was possible that during the sulphur feeding period, there was a very rapid i n f l u x of sulphur and the leaves may have needed to f i n d a way to store i t . Sulphate l e v e l s increased s l i g h t l y as acid soluble l e v e l s dropped d r a s t i c a l l y between day one and two, and then both declined together f o r the next three days. These observations would be consistent" with scheme I. Scheme II assigns a more s i g n i f i c a n t r o l e to the acid soluble f r a c t i o n . A r a t e - l i m i t i n g step i n the reduction of sulphate or formation of cysteine could have caused the p i l e up of an acid-soluble intermediate compound. Apparently there i s a known feedback mechanism In which cysteine i n h i b i t s the formation of O-acetylserine ( 99 ) thus regulating i t s own formation. This mechanism would keep cysteine l e v e l s low and slow up preceeding steps. If the acid soluble sulphur was one of the intermediates i n the reduction of sulphate, i t could have Increased in quantity u n t i l protein formation caught up with the supply. Chromatography of the free amino acid f r a c t i o n revealed that cysteine l e v e l s were quite low i n these leaves. There have been other reports of s i g n i f i c a n t amounts of acid-soluble sulphur, although the extraction procedures were not exactly as they were here (68,95 ). Moreover, i t should be emphasized that the amount of acid-soluble sulphur - 3 5 86 r e p o r t e d here (?0$ of the t o t a l a t h a r v e s t I) was much l a r g e r than i n the ot h e r s t u d i e s . The h i g h l e v e l o f a c i d - s o l u b l e s u l p h u r - 3 5 was l i k e l y a r e s u l t o f the p a r t i c u l a r experimental c o n d i t i o n s experienced by these p l a n t s . I d e n t i f i c a t i o n o f these compounds promises to be a very i n t e r e s t i n g a r e a f o r f u t u r e r e s e a r c h e s p e c i a l l y s i n c e CHCA treatment had such a pronounced e f f e c t on t h e i r f o r m a t i o n . At h a r v e s t I I , the le a v e s of c o n t r o l p l a n t s c o n t a i n e d s i g n i f i c a n t l y l e s s a c i d - s o l u b l e S and p r o t e i n S. This i n d i -c a t e d t h a t treatment had caused a more r a p i d c o n v e r s i o n from the m e t a b o l i c a l l y i n a c t i v e sulphate form to more u s e f u l o r g a n i c forms. Conversion of s u l p h a t e to p r o t e i n ( e i t h e r v i a the a c i d -s o l u b l e f r a c t i o n or d i r e c t l y ) would r e q u i r e c o n s i d e r a b l e expen-d i t u r e of energy. From p r e v i o u s d i s c u s s i o n , i t may be r e c a l l e d t h a t t r e a t e d l e a v e s were p r o b a b l y i n a b e t t e r p o s i t i o n to supply t h i s energy, and i t might be expected t h a t they would i n c o r p o r -ate s u l p h u r i n t o the amino a c i d s more r a p i d l y than c o n t r o l l e a v e s . In t r e a t e d p l a n t s the h i g h e r l e v e l o f sulphur i n p r o t e i n and lower l e v e l i n sulphate agreed v/ith t h i s concept. The i n c r e a s e d r a t e s of 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 mention-ed e a r l i e r (47 ) would c o n t r i b u t e NADPH2 and ATP which are needed f o r the r e d u c t i o n o f s u l p h a t e . At h a r v e s t I I I , f i v e days a f t e r s u l phate f e e d i n g , the su l p h a t e S and a c i d - s o l u b l e S f r a c t i o n s were n e a r l y d e p l e t e d . The p r o t e i n f r a c t i o n c o n t a i n e d more a c t i v i t y than i t d i d a t the second h a r v e s t and the t r e a t e d p l a n t s once agai n showed a g r e a t e r amount of a c t i v i t y than c o n t r o l s . However, the t o t a l amount of a c t i v i t y i n the leaves had dropped when measured on 87 a p e r gram b a s i s . I t was c l e a r t h a t some o f t h i s drop was due to the d i l u t i n g e f f e c t of growth, but a t the same time there was a l s o a decrease i n the a b s o l u t e amount of sulphur - 3 5 i n these l e a v e s . The appearance of the p l a n t s and the p r e s -ence of some su l p h a t e i n the l e a v e s showed t h a t they had not y e t used up a l l t h e i r s u l p h u r , but there was p r o b a b l y a sulphur s t r e s s b u i l d i n g again. Some of the d e c l i n e i n a c t i v i t y i n the l e a v e s c o u l d be a t t r i b u t e d to m o b i l i z a t i o n of s u l p h a t e and i t s r e t r a n s l o c a t i o n to other p a r t s of the p l a n t , but i t was not known i f t h i s was the case. P l a n t s grown f o r seven days under the c o n d i t i o n s of these experiments were b e g i n n i n g to show v i s i b l e s i g n s of s u l p h u r d e f i c i e n c y . I t appeared from these r e s u l t s t h a t KCHC treatment d i d have a p o s i t i v e e f f e c t on the r a t e of c o n v e r s i o n of imported s u l p h a t e i n t o p r o t e i n . I t was not l i k e l y though, t h a t i t was t h i s a c t i v i t y which accounted f o r the g e n e r a l growth e f f e c t s of KCHC found i n other s t u d i e s . Instead, they were pro b a b l y a r e f l e c t i o n of s t i m u l a t e d a c t i v i t y o c c u r r i n g elsewhere. A growing body of evidence obtained i n our l a b o r a t o r y and e l s e -where (46 ) p o i n t s to the l i k e l i h o o d t h a t naphthenates s t i m u l a t e m e t a b o l i c a c t i v i t y a t the g e n e t i c l e v e l . T h i s a c t i v i t y i n t u r n would promote the r a t e of f o r m a t i o n of enzymatic p r o t e i n and subsequently most ot h e r c e l l u l a r a c t i v i t i e s (77 ). The i n c r e a s e d i n c o r p o r a t i o n of s u l p h u r - 3 5 i n p r o t e i n observed here was an obvious m a n i f e s t a t i o n of t h i s a c t i v i t y . The f a c t t h a t p r o t e i n f o r m a t i o n i s so v i t a l l y dependent upon sulphur (37 ) makes i t a good t e s t parameter f o r s t u d i e s of naphthenate 88 e f f e c t s on p r o t e i n f o r m a t i o n . Recovery of sul p h u r - 3 5 a c t i v i t y from the f r a c t i o n s was not 100$ when compared to a va l u e obtained f o r the t o t a l a c t i v i t y i n the f r a c t i o n (Table IX). Recoveries were u s u a l l y o f the order o f 90$ or b e t t e r , but the f a t e of the oth e r 10$ was not known. I t was assumed t h a t a l l f r a c t i o n s were a f f e c t e d e q u a l l y . C e r t a i n l y , a l l were t r e a t e d with equal care. One source of d i f f i c u l t y c o u l d be t h a t the s m a l l sample taken f o r t o t a l s u l phur measurements amounted to l e s s than 10$ of the l e a f m a t e r i a l . A s m a l l e r r o r i n t h i s measurement c o u l d have been m a g n i f i e d when c a l c u l a t i o n s were made to convert the val u e to t o t a l a c t i v i t y . V a r i a t i o n s i n r e c o v e r y between t r e a t e d and c o n t r o l l e a v e s a f f e c t the numbers s l i g h t l y but not the g e n e r a l c o n c l u s i o n s . 89 The e f f e c t of KCHC treatment on the incorporation of  sulphur amino acids into protein KCHC-treated leaves incorporated more radioactive cysteine and methionine into protein than control leaves. The average incorporation of methionine for the three harvests was s i g n i f i c a n t l y greater i n treated than i n control. Several studies have found that protein l e v e l s were higher i n treated plants than controls (62,77,78,114), and the treated plants i n t h i s study were no exception. . The higher l e v e l of sulphur -35 i n the protein f r a c t i o n of treated leaves compared to control leaves implied that there was more protein i n the treated leaves on a per gram basis. Again, the e f f e c t of treatment could be i n d i r e c t . If naphthenate treatment caused increased production of DNA and RNA, i t almost surely would lead to increased protein production. Increased l e v e l s of enzymatic protein would cause increased metabolic a c t i v i t y . 9 0 Calcium-45 uptake and d i s t r i b u t i o n Calcium-45 uptake i s shown i n T a b l e XI. Since the f e e d -i n g time was the same f o r each p l a n t , i t was p o s s i b l e to make a comparison of t o t a l uptake. In t h i s case, c o n t r o l p l a n t s took up 5«2# more c a l c i u m than t r e a t e d p l a n t s , but the d i f f e r -ence was not s i g n i f i c a n t . D i s t r i b u t i o n of calcium-45 w i t h i n the p l a n t gave r e s u l t s which were i n c o n t r a s t to the sulphur-35 d a t a . When the con-c e n t r a t i o n of a c t i v i t y was c o n s i d e r e d ( i . e . calcium-45 a c t i v i t y per gram dry weight) the l e a v e s of t r e a t e d and c o n t r o l c o n t a i n e d equal amounts of calcium-45, on the average, a l t h o u g h there was some v a r i a t i o n from one h a r v e s t to the next. The stems of c o n t r o l p l a n t s c o n t a i n e d more calcium-45 than the stems of t r e a t e d p l a n t s , and the r o o t s of t r e a t e d p l a n t s have r e t a i n e d s i g n i f i c a n t l y more calcium-45 than the c o n t r o l s . E i t h e r the r o o t s of c o n t r o l p l a n t s used l e s s c a l c i u m than the r o o t s of t r e a t e d p l a n t s , or i t was t r a n s p o r t e d out of c o n t r o l r o o t s f a s t e r than t r e a t e d r o o t s . The decrease i n l e v e l o f calcium-45 In r o o t s from 12 to 48 hours was v e r y s l i g h t . A p p a r e n t l y the r e l o c a t i o n observed a t these h a r v e s t times and f o r these exper-imental c o n d i t i o n s was mainly from stem to l e a f . The c a l c i u m content of stems c o n s t i t u t e d a r e l a t i v e l y h i g h percentage of the t o t a l c a l c i u m a t the 12 hour h a r v e s t and d e c l i n e d as time passed, whereas sulphur-35 content of stems i n c r e a s e d w i t h time. The c a l c i u m i n stems was probably exchangeable c a l c i u m and was moved s l o w l y up the stem as non-r a d i o a c t i v e c a l c i u m came up from below to r e p l a c e i t . T h i s was i n agreement w i t h the concept of c a l c i u m t r a n s l o c a t i o n 91 described by B e l l and Biddulph ( 13 ) . The s i g n i f i c a n t l y greater amounts of calcium - 4 5 retained by the treated roots may be due to increased trapping or u t i l -i z a t i o n . If treatment caused increased formation of organic acids, or stepped-up secretion of hydrogen ions, cations such as calcium would become associated with these negatively charged acids and be retained i n the t i s s u e . 92 Uptake of n o n r a d i o a c t i v e sulphur, calcium and magnesium F o l i a r a p p l i c a t i o n of 1 X 1 0 " 2 M KCHC had no e f f e c t on the t o t a l amount of s u l p h u r , c a l c i u m , and magnesium taken up by bush beans from a complete n u t r i e n t s o l u t i o n over a p e r i o d of one week. These r e s u l t s were c o n s i s t e n t w i t h the e a r l i e r o b s e r v a t i o n t h a t n e i t h e r s u l p h u r - 3 5 nor calcium - 4 5 uptake over a f o u r hour p e r i o d was a f f e c t e d by a KCHC treatment a p p l i e d 24 hours before the r a d i o a c t i v e m a t e r i a l was a d m i n i s t e r e d to the r o o t s . S i m i l a r l y , Severson ( 83 ) found t h a t a 5000 ppm f o l i a r a p p l i c a t i o n of Knap had no e f f e c t on the uptake of ^2p when the p l a n t s were grown e i t h e r i n phosphate-free or complete n u t r i e n t c o n d i t i o n s . In c o n t r a s t , workers i n B u l g a r i a and the USSR have found t h a t v a r i o u s naphthenate treatments have i' s t i m u l a t e d the uptake of m i n e r a l elements from s o i l . Increased uptake of n i t r o g e n and phosphorus, due to naph-thenate treatment, has been r e p o r t e d f o r cabbage ( 7 ), c o t t o n ( 8 ), potato ( 1 ), and tomatoes ( 2 ). Peterburgsky and Karamete ( 78 ) found t h a t naphthenates' e f f e c t on N, P, and K uptake was g r e a t e s t f o r maize p l a n t s growing i n s o l u t i o n c u l -t u r e , somewhat l e s s i n sand c u l t u r e , and l e a s t i n s o i l . These r e s u l t s would appear to be a d i r e c t c o n t r a d i c t i o n of the r e s u l t s of Severson ( 83 ) and the others mentioned above. D i f f e r e n c e s i n s p e c i e s , treatment, growing p e r i o d s , and environmental f a c -t o r s c o u l d a l l be reasons f o r these d i f f e r e n c e s , but the uncer-t a i n n a ture of our knowledge i n t h i s area i s e v i d e n t . I f treatment w i t h KCHC s t i m u l a t e d m e t a b o l i c a c t i v i t y , i t may be asked why there was no evidence of t h i s i n c r e a s e d a c t i v i t y i n h i g h e r l e v e l s o f i n o r g a n i c i o n uptake. S e t t i n g a s i d e 93 Peterburgsky and Karamete's f i n d i n g s f o r the moment and con-s i d e r i n g the r e s u l t s of t h i s study and some o t h e r s , i t may be p o s s i b l e to o f f e r some e x p l a n a t i o n . Normally, p l a n t s w i l l accumulate more ions than they need. This i s q u i t e p o s s i b l e from a n u t r i e n t s o l u t i o n i n which ions are p r e s e n t i n r e a d i l y a v a i l a b l e forms. T h e r e f o r e , i n c r e a s e d m e t a b o l i c a c t i v i t y due to CHCA treatment would not n e c e s s a r i l y produce a s i n k e f f e c t which would l e a d to n o t i c e a b l y d i f f e r e n t r a t e s o f i o n uptake by p l a n t s growing i n s o l u t i o n c u l t u r e . Furthermore, measure-ment of the t o t a l amount of an i o n p r e s e n t i n a t i s s u e i s no i n d i c a t i o n of i t s m e t a b o l i c involvement. I t i s necessary to determine the chemical form i n which the element e x i s t s . For _ example, su l p h u r , as s u l p h a t e , p r o b a b l y would not c o n t r i b u t e to the m e t a b o l i c a c t i v i t y o f the p l a n t whereas sulphur i n p r o t e i n would. Two s i m i l a r p l a n t s c o u l d c o n t a i n the same amount of su l p h u r , but the more a c t i v e m e t a b o l i c a l l y would p r o b a b l y have more i n the form of p r o t e i n . Although there i s now much evidence t h a t naphthenate and CHCA e f f e c t s occur w i t h i n the p l a n t , s e v e r a l y ears ago Huseinov ( 57 ) expressed the i d e a t h a t naphthenate e f f e c t s were due to i t s i n f l u e n c e on s o i l a c t i v i t y . When p l a n t s growing i n s o i l are t r e a t e d w i t h f o l i a r sprays of naphthenic a c i d s or CHCA, some of these compounds ent e r the s o i l . Also, i t i s p o s s i b l e t h a t they c o u l d e n t e r the s o i l by being e x c r e t e d from the r o o t s . Voinova-Raikova (104) has r e p o r t e d t h a t naphthenic growth sub-stances improved the n i t r o g e n c o n d i t i o n s i n the s o i l by s t i m u l a -t i n g the a c t i v i t y of n i t r o g e n f i x i n g b a c t e r i a and d e p r e s s i n g the a c t i v i t y o f d e n i t r i f y i n g b a c t e r i a . He concluded t h a t t h i s 94 improved the nitrogen s i t u a t i o n i n the s o i l i n two ways: 1 ) by increasing the amount of nitrogen i n forms available to plants, and 2) by reducing the a c t i v i t y of organisms that convert nitrogen into r e a d i l y leached forms. In thi s way naphthenates may have an important e f f e c t on the mineral n u t r i -t i o n of plants. However, when plants grow in soluti o n culture where, presumably, no element i s l i m i t i n g , i t would be impos-s i b l e f o r naphthenate treatment to have an important influence on ion uptake i n the way mentioned above. In a review, Brian ( 25 ) noted that there was no clear understanding of how herbicides may a f f e c t ion uptake, but he offered three suggestions. Auxin herbicides could depress ion uptake by reducing t r a n s p i r a t i o n and c e l l permeability, but these e f f e c t s could be o f f s e t by a concurrent increase i n r e s p i r a t i o n which would contribute to ion uptake. Very low concentrations of 2,4-D were found to stimulate uptake of phosphate but the r e s u l t s were not always consistent ( 26 ). Cooke ( 3 1 ) found that 2,4-D ap p l i c a t i o n to bean leaves caused a stimulation of ion uptake shortly a f t e r treatment, but an i n h i b i t i o n a f t e r 24 hours. The difference was attributed to the f a c t that, i n the early stages, a low concentration of the 2,4-D had accumulated i n the roots and was acting as a stimulus to r e s p i r a t i o n and ion uptake, but as the concentration i n -creased i n time, the e f f e c t was reversed. This e f f e c t was not very l i k e l y f o r Knap or CHCA tre a t -ment at the concentrations being administered. It has not been shown that concentrations of Knap from 0 to 5U0° ppm °r CHCA 9 5 up to 1 X 1 0 " ^ M have a n y t h i n g other than s t i m u l a t i v e p r o p e r t i e s ( 1 1 0 ) . Swenson and Burstrom ( 9 2 ) b e l i e v e d t h a t auxins do not a f f e c t the uptake of ions e i t h e r d i r e c t l y or as a r e s u l t of t h e i r growth i n h i b i t i o n , but r a t h e r by t h e i r e f f e c t on some p r o p e r t y of the c e l l s conducive to both c a t i o n and water a b s o r p t i o n . The l a c k of an immediate e f f e c t o f CHCA or Knap t r e a t -ment on i o n uptake would i n d i c a t e t h a t they do not a f f e c t i o n uptake d i r e c t l y e i t h e r . Increased uptake of n i t r o g e n and phosphorus r e p o r t e d by the Russian workers was l i k e l y the r e s u l t of a l o n g term e f f e c t . The i n c r e a s e i n m e t a b o l i c a c t i v i t y and growth due to treatment e v e n t u a l l y b r i n g s about i n c r e a s e d uptake, but the e f f e c t may be i n d i r e c t . In a study of phosphorus metabolism, Fang and Butts ( 4 4 ) r e p o r t e d t h a t 2 , 4-D, i n d o l e a c e t i c a c i d , i n d o l e b u t y r i c a c i d , and naphthalene a c e t i c a c i d a l l caused changes i n the p a t t e r n 32 of J P d i s t r i b u t i o n w i t h i n bean p l a n t s . CHCA treatment was a l s o r e s p o n s i b l e f o r changes i n the d i s t r i b u t i o n p a t t e r n s of the elements examined i n t h i s study. S u l p h u r - 3 5 accumulated i n excess i n the l e a v e s and c a l c i u m - 4 5 i n the r o o t s of t r e a t e d p l a n t . Magnesium c o n c e n t r a t i o n was lower i n the l e a v e s of t r e a t e d p l a n t s than c o n t r o l s . No p a r t i c u l a r p a t t e r n emerges from these r e s u l t s , but perhaps t h a t i s not s u r p r i s i n g when B r i a n ( 2 5 ) comments, a f t e r 2 0 years of i n t e n s i v e study of 2 , 4-D, t h a t i t i s p o s s i b l e to do l i t t l e more than catalogue i t s e f f e c t s on the d i s t r i b u t i o n of elements w i t h i n p l a n t s . 96 SUMMARY Four separate s t u d i e s were c a r r i e d out with bean p l a n t s to determine the e f f e c t of c y c l o h e x a n e c a r b o x y l i c a c i d (CHCA) treatment on: 1. uptake and d i s t r i b u t i o n o f sulphur-35, 2. uptake and d i s t r i b u t i o n o f calcium-45, 3. uptake and d i s -t r i b u t i o n o f t o t a l s u l phur, calcium, and magnesium i n p l a n t s grown f o r one week a f t e r treatment, and 4. d i s t r i b u t i o n of sulphur-35 w i t h i n compounds of bean l e a v e s . S e v e r a l c o n c l u s i o n s may be drawn from the r e s u l t s of these experiments. 1. Uptake of sulphur-35 and calcium-45, measured one day a f t e r treatment, and su l p h u r , calcium, and magnesium uptake measured one week a f t e r treatment was not d i r e c t l y a f f e c t e d by CHCA treatment. 2. D i s t r i b u t i o n of the elements w i t h i n the p l a n t was a f f e c t e d by treatment. Sulphur-35 was moved more r a p i d l y to the l e a v e s o f t r e a t e d p l a n t s than c o n t r o l s , while calcium-45 was r e t a i n e d i n excess by the r o o t s of t r e a t e d p l a n t s . C o n t r o l p l a n t s accumulated more magnesium i n the le a v e s than t r e a t e d p l a n t s . 3. Treatment s i g n i f i c a n t l y a f f e c t e d the i n c o r p o r a t i o n o f sulphur-35 i n t o o r g a n i c compounds of bean l e a v e s . One and two days a f t e r s u p p l y i n g 35§Q=f ^he a c t i v i t y i n a c i d -s o l u b l e compounds was s i g n i f i c a n t l y g r e a t e r i n t r e a t e d than c o n t r o l l e a v e s , and the l e v e l of sulphate was lower i n t r e a t e d than c o n t r o l on the second day. Treated p l a n t s had s i g -n i f i c a n t l y more a c t i v i t y i n p r o t e i n than c o n t r o l s . 97 Furthermore, the l e v e l of a c t i v i t y i n cysteine/cystine and methionine also increased over the l e v e l i n controls as time passed. Evidence has accumulated i n favour of the concept that naphthenates activate nucleic acid metabolism, which i n turn would stimulate the formation of enzymatic protein. Increased metabolic a c t i v i t y may be expected to follow. The r e s u l t s of this study agree with t h i s concept. Increased l e v e l s of sulphur i n protein, and protein amino acids, suggested stepped-up metabolic a c t i v i t y . The more rapid conversion of sulphate into organic sulphur compounds by treated leaves compared to control leaves was consistent with the protein increases. D i s t r i b u t i o n e f f e c t s due to treatment, e s p e c i a l l y i n the sulphur - 35 d i s t r i b u t i o n experiment, sug-gested changed metabolic patterns. These plants were some-what d e f i c i e n t i n sulphur, and the more rapid transport to the leaves may have been the r e s u l t of an increased sink e f f e c t due to the stimulation of metabolic a c t i v i t y by CHCA. The retention of calcium i n treated roots could also be due to increased a c t i v i t y . A reasonable explanation f o r the reduced l e v e l s of magnesium i n treated leaves was not evident. The lack of a s i g n i f i c a n t e f f e c t of treatment on ion uptake suggested that CHCA does not stimulate plant growth by improving the uptake of sulphur, calcium, or magnesium. 98 BIBLIOGRAPHY 1. Abolina, G.I. and N. Ataullaev. 1969. 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