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The effect of 3-amino-1,2,4-triazole on the uptake, retention, distribution, and utilization of labelled.. 1961

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THE EFFECT OF 3-AMIN0-l ,2,4-TRIAZ0LE ON THE UPTAKE, RETENTION, DISTRIBUTION, AND UTILIZATION OF LABELLED PHOSPHORUS BY YOUNG- BEAN PLANTS. by Donald E . LaBerge B . S c . , . Univers i ty of B r i t i s h Columbia, 1959 A Thesis Submitted i n P a r t i a l Ful f i lment of the Requirements f o r the Degree of Master of Science In the Department of Botany We accept th i s thesis as conforming to the required standard The Univers i ty of B r i t i s h Columbia June,. 1961 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of 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 reference and study. I f u r t h e r agree that permission f o r extensive copying of 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 of my Department or by h i s representatives. It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department Date *&k\ The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8 , Canada. ABSTRACT Bean plants were grown in a phosphate-free nutrient solution to the early t r i f o l i a t e stage. At this time, they were transferred to a minus phosphate nutrient solution containing 100 p.p.m. 3~amino-l,2,4-triazole for 48 hours, and then placed into a labelled phosphate nutrient solution for another hour. The plants were then returned to a phosphate-free nutrient solution and harvested one, 24, 48, and 96 hours after the period of i n i t i a l phosphate uptake. AT-treatment did not affect uptake of P52 but did decrease loss of p32 to the phosphate-free nutrient solutions after i t had been absorbed by the plants. The proportion of absorbed phosphate found in the stems and leaves of AT-treated plants was higher than in these organs in the control plants. This phosphate represented an increase in both acid-30luble activity and acid-insoluble activity. The accumulation of acid- soluble activity in the shoots of AT-treated plants was an accumulation of inorganic phosphates, sugar phosphates, and nucleotides. AT appeared to inhibit downward trans- location of acid-soluble and acid-insoluble activity. The incorporation of p32 into esterified compounds (i.e., nucleotides and sugar phosphates) was unaffected by AT indicating that AT does not interfere with oxidative phosphorylation nor with glycolysis. However, AT did inhibit transfer of P ^ 2 from the acid-soluble fraction - i i - to the a c i d - i n s o l u b l e f r a c t i o n . Therefore, the p r i n c i p a l e f fec t of AT i s to i n h i b i t the incorporat ion of phosphate into one or more of the nuc le ic a c i d , phosphol ipid , or phosphoprotein f r a c t i o n s . - i i i - TABLE OF CONTENTS INTRODUCTION 1 EXPERIMENTAL PROCEDURES 14 The M6 l i q u i d counter 19 Analys i s of r e s u l t s 20 RESULTS 22 Intake, re tent ion and d i s t r i b u t i o n of P ^ 2 23 F i g . 1: The e f fect of AT-treatment on the uptake and re tent ion of phosphate by whole bean plants 24 Table I : Up:take and re tent ion of P - 5 2 . . ; 24 Table I I : D i s t r i b u t i o n of P?2 i n leaves, stems, and roots 25 E f f e c t of AT-treatment on the d i s t r i b u t i o n of a c i d - s o l u b l e and a c i d - i n s o l u b l e P^2 26 F i g . 2: D i s t r i b u t i o n of P ^ 2 i n roots , stems and leaves 27 Table I I I : D i s t r i b u t i o n of ac id - so lub le P32 i n bean plants 29 Table IV: D i s t r i b u t i o n of a c i d - i n s o l u b l e P32 i n bean plants 30 F i g . 3s D i s t r i b u t i o n of ac id - so lub le P ^ 2 i n leaves, stems, and roots of young bean plants 31 F i g . 4 : D i s t r i b u t i o n of a c i d - i n s o l u b l e P ^ 2 i n the leaves, stems, and roots of young bean plants 32 Incorporation of P32 into a c i d - i n s o l u b l e f r a c t i o n s • 33 Table V: D i s t r i b u t i o n of P ^ 2 between a c i d - soluble and ac id-Inso luble f r a c t i o n s i n each organ 34 F i g . 5: Incorporation of p32 in to a c i d - inso luble f rac t ions 35 - i v - Effect of AT-treatment on the e s t e r i f i c a t i o n of phosphate and on the d i s t r i b u t i o n of the various acid-soluble components 36 Table VI: Effect of AT on the e s t e r i f i c a t i o n of phosphate 37 Fig. 6: D i s t r i b u t i o n of inorganic P32 within the plant 38 Fig. 7i D i s t r i b u t i o n of sugar phosphate i n young bean plants 40 Fig. 8: D i s t r i b u t i o n of nucleotide P32 i n young bean plants 42 DISCUSSION 43 BIBLIOGRAPHY; 50 BIOGRAPHY 54 - V - ACKNOWLEDGMENTS. The author wishes to express s incere thanks to Dr. D . J . Wort, Dept. of Botany, Univers i ty of B r i t i s h Columbia, for h i s valuable advice and encouragement during the course of th i s work and for h i s kind help during the preparat ion of th i s thes i s . The author i s a l so indebted to Dr. P. Townsley of the Research Laboratory, C D .A., and to ind iv idua l s w i th in the Dept. of Botany who gave the i r ass i s tance . These studies were aided through funds supplied by the Nat ional Research Counci l of Canada. - 1 - INTRODUCTION. Formerly used only i n small quant i t ies i n photographic work (6), 3-amino-l,2,4—triazole underwent f i e l d tests i n 1952 as a new absciss ion-promoting and growth- inhib i t ing chemical (5,21). In 1954, W.W. A l l e n , chief formulat ing chemist of the American Paint Co. (now known as Amchem Products I n c . ) , was granted a patent for the use of 3 -amino- l ,2,4 - tr iazole as a herb ic ide (6). Known a lso as amino t r i a z o l e , a m i t r o l , AT, or ATA, th i s herbic ide i s formulated and sold by Amchem under the trade name of "Amizol ." The herbic ide i s a l so sold under the trade names of "Weedazol" and "Amino T r i a z o l e Weed-k i l l e r ." AT i s a he terocyc l i c compound possessing the fo l low- ing s t r u c t u r a l formula: HN N I I HC C —NH- 2 AT has a molecular weight of 84.5 and a melt ing point between 153 - 159°C (21). I t i s water soluble and w i l l react with acids and bases to form acetate, l a c t a t e , hydrochlor ide , n i t r a t e , phosphate and sodium sa l t s (29). AT can a lso be d iazot l zed i n the presence of n i t r i t e and phenol to form a yellow azo dye (3). Because th i s reac t ion appears to be s p e c i f i c for AT, i t affords a use fu l technique for i d e n t i f y i n g amino t r i a z o l e on paper chrom- atograms. Other chromatographic techniques have a lso been developed for detect ing AT (31,33,40). Amino t r i a z o l e - 2 - forms stable complexes with several metals, inc lud ing i r o n , n i c k e l , cobal t , copper and magnesium (40). The importance of amino t r iazo l e -meta l complex formation w i l l be discussed l a t e r . Amino t r i a z o l e came to a p o s i t i o n of n a t i o n a l prominence i n the United States during the Thanksgiving hol iday of 1959 i n what has become known as the "Cranberry C r i s i s . " Amchem recommended amino t r i a z o l e as a post- harvest spray for the c o n t r o l of redroot (Lachnanthss t l n c t o r i a ) i n cranberr ies , and In A p r i l of 1958 appl ied to the Federal Department of A g r i c u l t u r e (FDA) for a one p .p .m. tolerance for AT on cranberries (17). A f t e r i n v e s t i g a t i n g t o x i c i t y data, FDA n o t i f i e d Amchem i n May of 1959 that a tolerance for amino t r i a z o l e could not be es tabl i shed. The compound caused thyroid adenomas i n ra t s at a l e v e l a;s low as 10 p .p .m. i n the d i e t . Amchem therefore withdrew i t s p e t i t i o n . On Nov. 9, 1959, Health , Education and Welfare Secretary Arthur S. Fleming urged that there be no fur ther sales of cranberries or cranberry products produced i n Washington and Oregon because of poss ib le contamination with amino t r i a z o l e . He said that th i s herbic ide caused cancer i n the thyro id of ra t s when i t was contained i n t h e i r d i e t s . He was ac t ing i n con- junct ion with the Delaney clause of the Food Addi t i ve Amendment (24) which p r o h i b i t s the addi t ion to foods of any substance that has been shown to produce cancer i n laboratory animals when fed at any dosage. Because a l l - 3 - cranberries became suspect i n the eyes of the p u b l i c , cranberries from Wisconsin, New Jersey, and Massachusetts were impl icated and the bottom f e l l from the cranberry market. In order to c l ear s u f f i c i e n t cranberries f or the Thanksgiving trade, a crash program invo lv ing 100 FDA inspectors and 60 chemists was establ ished to analyze the cranberry crop. The cranberry c r i s i s cost the U . S . Federal Government $>10 m i l l i o n i n Indemnity to cranberry growers (25) and i n a d d i t i o n , the government bought the unsold cranberries for government use. The wave of controversy which resu l ted from th i s c r i s i s has done much to stimulate in teres t i n the metabolic e f fects of AT-treatment both i n animal and i n plant t i s sues . Numerous a r t i c l e s have appeared recent ly on several aspects of plant metabolism as af fected by amino t r i a z o l e and these w i l l be discussed b r i e f l y i n the fo l lowing text . Extensive experiments have indicated that many plants r e a d i l y absorb amino t r i a z o l e through the roots or through the a e r i a l parts ( 7,26 , 3 0 ) . Surface agents of the a l k y l - a r y l sulfonate type ( e .g . , X -77) and the a l k y l a r y l po ly - oxyethelene g l y c o l type ( e .g . , M u l t i f i l m C and M u l t i f i l m L) were both e f fec t ive i n increas ing the amount of AT absorbed through the leaves (19). G i r d l i n g experiments were used to determine how AT i s translocated (21). When AT i s appl ied to the s o i l or i s taken up from a nutr ient so lu t ion , i t i s translocated upward i n the xylem. This i s ind icated by the ch loros i s which occurs i n the growing regions above the bark- - 4 - g i r d l e d regions of cotton p lants . However, f o l i a r app l i ca t ions of amino t r i a z o l e ind icate that the phloem i s involved i n t rans loca t ion when amino t r i a z o l e i s absorbed through the leaves. When cotton plants are b a r k - g i r d l e d half-way up the main stem, the amino t r i a z o l e i s not translocated past the g i r d l e whether the herbic ide i s appl ied to the leaves above the g i r d l e d bark or to the leaves below the g i r d l e d bark. Thus, plants appear to trans locate AT i n a manner s i m i l a r to that used to trans locate other herb ic ides . Further d e t a i l s regarding the t rans loca t ion and accumulation of AT by plants have resu l ted from the use of amino t r i a z o l e-5 - C 1 ^ and from radioautography ( 7 , 1 6 , 3 3 , 3 5 ) . When l a b e l l e d AT was appl ied to the cotyledons of cotton p lants , i t was absorbed and translocated to the roots w i th in two hours ( 1 6 ) . Ana lys i s of the cu l ture so lu t ion showed no i n d i c a t i o n of leakage from the roots . Amino t r i a z o l e moved In the d i r e c t i o n of food transport and at the same r a t e . The e f f i c i ency of AT as a herbic ide depends upon i t s a b i l i t y to penetrate and to translocate r e a d i l y to a l l parts of a plant while r e t a i n i n g s u f f i c i e n t t o x i c i t y to k i l l the v i t a l ' o r g a n s . Radioautograms ind icate that AT (or some e f f ec t ive metabolite) d e f i n i t e l y tends to accumulate i n the meristems, such as ac t ive buds, root t i p s , etc. I t i s scarce or l ack ing i n dormant buds, storage parenchyma and mature t i ssues i n general ( 7 1 ) . This accumulation of AT i n young growing t issue coincides - 5 - with the s i t e s where AT i s known t o a t t a i n i t s maximum t o x i c e f f e c t . F u r t h e r e x p e r i m e n t s have i n d i c a t e d t h a t movement of AT t h r o u g h the phloem i s dependent upon p r o d u c t i o n of p h o t o s y n t h a t e . S t a r c h - d e p l e t e d n u t g r a s s p l a n t s were g i v e n one d r o p of r a d i o a c t i v e AT on one l e a f (7). When t h i s l e a f was exposed t o s u n l i g h t , AT c o u l d move r e a d i l y from t h i s l e a f t o a l l p a r t s of the p l a n t s . Hov/ever, i f the l e a f was k e p t i n darkness w h i l e t h e remainder of the p l a n t was exposed t o l i g h t , t he AT remained i n t h e darkened l e a f . T h i s I n d i c a t e d t h a t p h o t o s y n t h e s i s was e s s e n t i a l t o t h e t r a n s l o c a t i o n of AT by n u t g r a s s . B e s i d e s a f f e c t i n g growth, AT i s known t o cause c h l o r o s i s i n many p l a n t s and i n many ca s e s , c h l o r o s i s i s f o l l o w e d by the. d e a t h of the p l a n t . Measurements of c h l o r o p h y l l c o n t e n t i n c o t t o n r e g r o w t h - l e a v e s have shown t h a t AT reduces the c o n c e n t r a t i o n s of b o t h c h l o r o p h y l l a and b (2 8 ) . S i m i l a r d e p r e s s i o n s i n c h l o r o p h y l l c o n t e n t have been measured i n beans ( 3 0 ) , tomatoes ( 4 1 ) , b a r l e y and p o t a t o e s ( 3 2 ) . AT-treatment a l s o d e c r e a s e s c a r o t e n e and x a n t h o p h y l l c o n t e n t of v a r i o u s p l a n t s (1,28). The o v e r a l l e f f e c t of AT i s a t w o - f o l d i n c r e a s e i n the ant h o - c y a n i n c o n t e n t of c o t t o n l e a v e s ( 2 8 ) . S p e c t r a l a n a l y s i s of the c o m p o s i t i o n o f the a n t h o c y a n i n s i s u n a l t e r e d by AT (28) . I n t h e i r e a r l y work, M i l l e r e t a l (21) found t h a t t i s s u e s formed a t the time o f , or subsequent t o the a b s o r p t i o n o f amino t r i a z o l e were c h a r a c t e r i z e d by c h l o r o s i s . - 6 - This suggested that ch lorophy l l synthesis was affected- Further work indicated that AT did not e n t i r e l y i n h i b i t the change from protoch lorophyl l to ch lorophy l l but that AT had i t s p r i n c i p l e effect during the biogenesis of c h l o r o p h y l l . Laboratory studies have shown that AT possesses the a b i l i t y to form stable complexes with i ron- copper, and magnesium (40). Though M i l l e r et a l (21) could not demonstrate that the r e s t r i c t i o n of c h l o r o p h y l l synthesis was due to an Immobilization of Mg, Fe, Mn, N, P or K, i t has been shown recent ly that ferrous ion can p a r t i a l l y reverse the i n h i b i t i o n of ch lorophy l l synthesis as w e l l as completely reverse the i n h i b i t i o n of carotenoid synthesis and m u l t i p l i c a t i o n of several microorganisms (1). This suggests that AT acts as a che lat ing agent. There i s further evidence which suggests that amino t r i a z o l e chelates metals. AT i n h i b i t e d the a c t i v i t y of a phosphorylase preparat ion from the blue-green a lga , O s c i l l a t o r i a princeps (20). This i n h i b i t i o n appeared to be the r e s u l t of the che lat ion by AT of the e s sent ia l metal required by th i s enzyme since i n h i b i t i o n could be e f f e c t i v e l y reversed by the add i t ion of manganese or f e r r i c ions . It has been suggested (1,21,40) that the s i m i l a r i t y of s tructure of the t r i a z o l e r i n g to the pyrro le r ings of c h l o r o p h y l l together with the a b i l i t y of AT to form stable complexes with metals was such that amino t r i a z o l e might subst i tute for at l eas t one pyrro le r i n g thus b locking c h l o r o p h y l l synthesis p r i o r to the protoch lorophyl l stage. HN N HG PH I I C C H amino t r i a z o l e pyrro le Hov/ever, M i l l e r and H a l l (28) have almost completely excluded the l i k e l i h o o d that AT enters into the synthesis of pseudochlorophyllous porphyrins . Using rad ioac t ive AT, they recovered only extremely low C ^ a c t i v i t y from t h e i r pigment f r a c t i o n s . Gyto log ica l data has indicated that ch loros i s i s due to a lack of ch lorop las t s , rather than some ef fect on ' c h l o r o p h y l l per se (34). Microscopic examination of the c h l o r o t i c t i ssues of several plants has indicated that the p l a s t i d s are few i n number, shrunken and misshapen. Wort and Loughman (44) have shown that an a l t ered phosphol ipid metabolism may be responsible for the i n h i b i t i o n by AT of p l a s t i d development. Sund (41) found that AT blocked the synthesis of r i b o f l a v i n , and the need for c e r t a i n f l a v i n coenzymes might account for a decrease i n p l a s t i d development. Both groups of inves t igators a l so presented evidence which indicated that AT i n h i b i t e d prote in synthesis . I t i s Interes t ing to note the ef fect of AT on various iron-*porphyrin enzymes since these enzymes are also pyrro le containing compounds. Pyfrom et a l (32) were among the f i r s t inves t igators to study the e f fects of AT on catalase a c t i v i t y . Using barley and potato plants with low con- - 8 - centrat ions of AT, they showed that catalase a c t i v i t y i s depressed, whenever AT i s present i n the t i s sues . I t has been shown recent ly that AT i n h i b i t s the synthesis of tryptophane peroxidase-oxidase, an i ron-porphyr in enzyme found i n the l i v e r of rats ( 8 ) . Whereas the cytochrome oxidase enzyme i n chlorot ic . corn t i s sue was not af fected by AT-treatment ( 2 7 ) , the cytochrome oxidase a c t i v i t y of e t i o l a t e d wheat seedlings i s increased by AT-treatment ( 4 3 ) . There i s no information ava i lab le to indicate whether or not AT in ter feres with other cytochrome•enzymes which commonly occur i n p lants . I t has been suggested ( l) that the main ac t ion of AT i n v ivo i s to i n t e r f e r e with the synthesis of porphyr in- containing enzymes, and only when AT i s present i n high concentrations does i t i n h i b i t e x i s t i n g enzymes. I f AT acts p r i m a r i l y as an i r o n chelator , i t i s more l i k e l y to prevent synthesis by making i r o n unavailable rather than by making i t i n e f f e c t i v e when i t i s already part of the enzyme molecule. I n h i b i t i o n of catalase a c t i v i t y r e s u l t s i n an increase of H2O2 i n plant t i s sues . Racusen ( 3 3 ) has proposed that an increase i n HgOg would lower the amount of IAA by ac t ion of indoleacet ic ac id oxidase. This would r e s u l t i n an i n d i r e c t AT-induced growth i n h i b i t i o n . Russe l l ( 3 6 ) has found that AT does increase the peroxide l e v e l of i n d i v i d u a l root c e l l s . He recognized the s ign i f i cance of an increased peroxide l e v e l with respect to the IAA reduct ion sequence. He therefore attempted to e s t a b l i s h whether or not IAA would reverse AT-growth i n h i b i t i o n . His experiments indicated that there v/as no such i n t e r a c t i o n . However, other workers (21) have shown that AT does antagonize the growth ef fects of IAA thus lending support to Racusen's hypothesis . Reduction of growth i n h i b i t i o n has also been obtained by adding purines and pyrimidines to plants simultaneously with AT (4,41,42). Because these compounds are necessary for the production of nuc le i c ac ids , i t - h a s been proposed that AT in terrupts pro te in synthesis since nuc le i c ac id i s necessary for pro te in production (41,44). Many workers have noted that AT stimulates r e s p i r a t i o n (21,27,29,36). Wort and Shrimpton (43) found that f o l i a r app l i ca t ions of 4000 p .p .m. AT resu l ted i n an immediate and continued increase i n r e s p i r a t i o n of both wheat and bean p lants . A l l concentrations of AT upJ to 840 mg/l increased the re sp ira tory rate of cotton l ea f d i scs (21,29). A p p l i c a t i o n of AT to homogenates of wheat seedlings grown i n the dark for 6 days a lso increased the oxidat ion of reduced cytochrome c (43) i n d i c a t i n g that cytochrome oxidase a c t i v i t y v/as increased by AT-treatment. Thus, i t appears that there may be several l o c i at which AT induces growth i n h i b i t i o n . B r i e f l y , these would seem to include such things as i n a c t i v a t i o n of c e r t a i n enzyme systems through che lat ion of e s sent ia l metal , oxidat ion of IAA by H 2 02, decreased photosynthesis (43) and increased r e s p i r a t i o n which lower the amount of a v a i l - able sugars, and f i n a l l y , a decreased phosphol ipid and - 10 - nuc l e i c a c i d production i n AT-treated p lants . Many plants can metabolize AT. The use of paper chromatography for the detect ion of AT i n plant extracts has given an ins ight into this problem of AT metabolism. A l d r i c h (4) removed AT from plant t issue by gr inding the t i ssues i n Q0% ethanol . The eluate was chromatographically separated on Whatman No. 1 paper us ing ethanol:water: 1-butanol (1:1:4) as the solvent. The paper was sprayed f i r s t with 5% KN0 2 and then with phenol i n 20% H C l . In the experiments using th is technique, two yellow spots appeared on the chromatograms. The assumption was made that the spot with the lower Rf value was "bound" AT, that Is , AT bound to p r o t e i n . The other spot was designated as "free" AT and appeared at a higher Rf value. While studying the t rans locat ion and fate of AT i n Canada t h i s t l e , Johnson grass and soybeans, Rogers ( 3 5 ) was able to demonstrate that even though soybeans were very suscept ible to AT, the plants were able to metabolize a l l of the AT suppl ied . In the case of soybeans and Canada t h i s t l e , he demonstrated the presence of an unknown ent i ty having a small Rf value . Chromatograms prepared from Johnson grass, which was l eas t susceptible to_ AT- treatment, d i d not have th is en t i ty , a fact which he f e l t might be of importance for expla in ing tolerance of plants to AT. Herrett (22) has found that the rate of AT-metabolism wi th in bindweed i s more rapid than i n t h i s t l e . He Isolated two metabolites which he has designated as Unknown I and - 1 1 - Unknown I I . In Canada t h i s t l e , he noted that there was a lag i n the transport of AT from the treated l ea f to the stem. The formation of a transportable form of AT v/as postulated as the l i m i t i n g reac t ion for i t s d i s t r i b u t i o n through the phloem from the l ea f of a p p l i c a t i o n to the remainder of the p lant . Herrett showed that movement of AT through the xylem d i d not require synthesis of a transportable form of AT and he supported the claim that the xylem i s not the major pathway of AT movement out .of leaves. The d i f ference i n s e n s i t i v i t y to AT by bindweed and t h i s t l e was concluded to be a r e s u l t of major d i f f e r - ences i n absorption and metabolism of the herb ic ide . In 1 9 5 7 j Racusen ( 3 3 ) undertook some experiments to determine whether AT i t s e l f or some product of AT-metabolism was the a c t u a l mater ia l which was toxic to c e r t a i n plant a c t i v i t i e s . He demonstrated that amino t r i a z o l e was t rans - formed into two new products i n the leaves of young bean p lant s . He designated these AT-metabolites as Compound X .and Compound Y. Because i t v/as not c e r t a i n that X and Y were a c t u a l l y transformation products of AT, proof of t h e i r o r i g i n v/as establ ished using C 1 ̂ "-labelled AT. Compound X v/as the major product of AT metabolism. I t v/as formed at a uniform rate i n young bean leaves for at l eas t 4 days. A f t e r 5 days, 9 3 $ of the AT had been i n - corporated into Compound X. Racusen eluted Compound X from his chromatograms and determined i t s t o x i c i t y using Lemna minor. Like AT, Compound X produced ch loros i s and stunting but i t s t o x i c i t y v/as lov/er than that of AT alone. Racusen - 12 - a l so i so la t ed Compound Y which he found to be non-toxic to Lemna cu l tures . He could not e s tab l i sh whether or not X and Y were merely d e t o x i f i c a t i o n by-products of AT metabolism as suggested by t h e i r lower t o x i c i t y , or whether they were formed as a d i r e c t consequence of some toxic react ion of AT. However, both compounds were characterized by the same r i n g system and free amino group of AT. Because healthy, turgid bean leaves were required for the ir formation, Racusen has suggested that the transformation of AT in to X and Y i s probably c a r r i e d out by some enzyme system i n the l ea f . Another approach to the problem of AT metabolism has been invest igated (12 ,15). Guided by the hypothesis that AT i n t e r f e r e s with the production of porphyrins which are e s sen t ia l for c h l o r o p h y l l production, Carter and Naylor (15) studied the metabolism of c e r t a i n porphyrin precursors i n AT-treated p lants . When excised t ips of bean plants were exposed to g l y c i n e - 1 , 2 - C 1 ^ i n a so lut ion of AT, large quant i t i e s of r a d i o a c t i v i t y appeared i n an unknown, n i n - hydrin sens i t ive compound which was not present i n the contro l s . The unknown was designated compound "1". Glyc ine - l -C 1 2 * ' , g lyc ine-2-C l i { ", s e r i n e - 1 , 2 , 3 - 0 1 ^ reacted s i m i l a r i l y whereas glucose, succinate and bicarbonate l a b e l l e d with G1^ were i n e f f e c t i v e . When A T - 5 - C 1 2 f was used, compound "1_" became heavi ly l a b e l l e d i n d i c a t i n g that a complex between AT and serine or g lyc ine was formed by bean p lant s . Mass in l (26) has recent ly i so la ted and p u r i f i e d an AT metabolite which he c a l l s ATX. ATX i s a white powder - 13 - composed of fi n e needle-like c r y s t a l s . I t has a melting point of 230-232°C. as compared to AT at approximately 155°C This compound has been i d e n t i f i e d as 3-amino- 1 , 2 , 4 - t r i a z o l y l alanine. Indications at present are that t h i s compound i s not the same as compound "1" described by Carter and Naylor nor i s l t s i m i l a r to Compound X described by Racusen. Rather, these l a t t e r compounds are thought to be i d e n t i c a l (13,14). Recent studies by Carter and Naylor (13) have shown that compound "1" ( i . e . Compound X) i s the p r i n c i p a l metabolic product formed i n beans, a l f a l f a , s i l v e r maple, and honeysuckle when treated v/ith AT. However, they also 14 demonstrated that nine other compounds derived C from AT thus demonstrating the complexity of AT metabolism i n plants. The present study i s an attempt to discover the effect of AT on several aspects of phosphate metabolism i n young bean plants, the roots of which were f i r s t exposed to 100 p.p.m. AT f o r 48 hours followed by a one hour exposure to radioactive phosphorus. The influence of AT on the uptake and retention of r a d i o a c t i v i t y and on the d i s t r i b u t i o n of a c t i v i t y within the plant was determined. Attempts were also made to study the manner i n which AT affected the incorporation of inorganic phosphate into organic compounds. A comprehensive study of the effect of AT on the phosphate metabolism of barley has already been made (44) and t h i s work w i l l be discussed l a t e r . - 14 - EXPERIMENTAL. Bean seeds (Phaseolus v u l g a r i s , Top Crop, i 9 6 0 ) obtained from Buckerf ie ld Seed Co. were sown i n vermicul i te saturated with phosphate-free nutr i en t s o l u t i o n . The plants were grown i n a constant environment chamber at a temperature of 7 2 ° F . , a r e l a t i v e humidity of 62%, and a l i g h t in tens i ty of approximately 2500 f . c . The plants were given 14 hours of l i g h t d a i l y . A f t e r s ix days, uniform seedlings were transplanted to bo t t l e s containing f resh phosphate-free nutr i en t so lu t ion . These so lut ions were given continuous aerat ion . Eleven days a f t e r germination, when the plants had grown to the ear ly three - l ea f stage, plants were again se lected for uniformity so that only eight bo t t l e s , each containing four p lants , remained. At midday (corresponding to seven hours of l i g h t ) , the plants i n four bot t l e s were qu ick ly transferred to bot t l e s containing 100 p .p .m. AT i n f re sh phosphate-free nutr i en t s o l u t i o n . The contro l p lants were' transferred to fresh phosphate-free nutr i en t so lut ions . These so lut ions were continuously aerated. At midday, 48 hours l a t e r , the roots of a l l the plants were quickly r insed with d i s t i l l e d water and then immersed i n a f re sh cu l ture so lu t ion to which had been added NaHgP^O^.. This so lut ion had an a c t i v i t y of 80 uC P 5 2 per 3000 mis. of cu l ture so lu t ion . A f t e r a one hour exposure to P ^ 2 , the roots of a l l the plants were quick ly r insed i n d i s t i l l e d water and the plants were returned to bot t l e s - 15 - containing f r e s h phosphate-free nutr ient so lu t ion . Four AT-treated and 4 contro l plants were removed one, 24, 48, and 96 hours a f t er the s tar t of P ^ 2 absorption for the ex trac t ion of phosphorus compounds. EXTRACTION OF LABELLED COMPOUNDS. Label led compounds were extracted from plant t i ssues by the procedures of Wort and Loughman (44). The two groups of four plants from the AT-treated and contro l groups were quick ly d iv ided into roots , stems and leaves . Each plant part was cut into smaller pieces and immersed i n 12 mis. of i c e - c o l d 16% t r i c h l o r a c e t i c ac id (TCA) contained i n c h i l l e d mortars. The cold TCA caused an immediate cessat ion of metabolic a c t i v i t y . A f t e r gr inding for approximately f i v e minutes, the mater ia l was transferred q u a n t i t a t i v e l y to 25 ml. screw cap v i a l s us ing a minimum amount of i c e - c o l d wash water. The v i a l s were then cent- r i fuged f o r 10 minutes at 3000 r . p . m . and the supernate was decanted into another v i a l . The residue was r e - extracted with f i v e mis. of i c e - c o l d 8% TCA and centrifuged i n a r e f r i g e r a t e d centrifuge for 10 minutes at 3750 r . p . m . This supernate was poured into the f i r s t supernate so lu t ion and the combined supernate was made to 25 mis. with i c e - c o l d water. This so lu t ion comprised the ac id - so lub le phosphate compounds ( i . e . , inorganic phosphates, nuc leot ides , and sugar phosphates). To determine the t o t a l ac id - so lub le count, a two ml. a l i q u o t was removed and made to 10 mis. i n a volumetric . This so lu t ion was counted i n a 20th Century - 16 - E l e c t r o n i c s M6 l i q u i d counter (see below). To reduce changes, the remainder of the ac id - so lub le phosphate so lu t ion was frozen s o l i d l y . The screw cap v i a l containing the plant residue was inverted on a paper towel to dry . This res idue, comprised of a c i d - i n s o l u b l e phosphate compounds ( i . e . , phosphol ipids , nuc l e i c ac ids and phosphoproteins) was transferred to I50 ml. K j e l d a h l f lasks and digested us ing f i ve mis. of con- centrated n i t r i c ac id and f i v e mis. of 60% _perchloric a c i d . The l i q u i d was reduced to f i v e mis. with heating. This required from two to three hours. The cooled solut ions were then made to 10 mis. with water and counted i n the M6 l i q u i d counter. This count represented the t o t a l a c i d - inso luble phosphate compounds. SEPARATION Q | ACID-SOLUBLE PHOSPHATE COMPOUNDS. The ac id - so lub le so lut ions , containing inorganic phosphates, sugar phosphates and nucleot ides , were allowed to thaw. The solut ions were centrifuged at 3750 r . p . m . i n order to remove polysaccharide mater ia l formed during f r e e z i n g . A 10-ml. a l iquot was taken from each so lu t ion and p l a c e d . i n a continuous l i q u i d - l i q u i d extract ion system. A three to four hour extract ion with e thy l ether was needed to remove the TCA. A f t e r ex tract ion , the solut ions were poured in to c h i l l e d v i a l s and i f any ether layer remained on these so lut ions , i t was removed with a c a p i l l a r y p ipe t te . I f stored overnight, these solut ions were frozen to reduce any chemical changes. When thawed, the solut ions were - 17 - again centrifuged at 3750 r . p . m . to remove any polysaccharide m a t e r i a l . T v / o - m i l l i l i t e r samples of these solut ions were reduced to approximately one-half ml . using an i n f r a red lamp and a current of warm a i r from a h a i r d r i e r . The reduced so lut ions were spotted on acid-washed 3 MM Whatman papers. (Sheets of 3 MM Whatman paper were placed i n a t ray , covered with 2N ace t i c a c i d , and allowed to stand overnight. The ac id was removed next morning and th i s wash was repeated several times. When the ac id so lut ion no longer gave a p r e c i p i t a t e when ammonium hydroxide and ammonium oxalate were added, the papers were thoroughly r insed with d i s t i l l e d water. This washing removed metal impur i t i e s , p a r t i c u l a r l y calcium, which i n t e r f e r e with the chromatography of c e r t a i n phosphate compounds.) The chromatograms were equ i l ibra ted i n the chromatography chambers i n an atmosphere of HgS. This converted any metals which remained on the papers to su l f ides and these su l f ides remained at the o r i g i n . This gas was generated by dropping crys ta l s of sodium su l f ide into 6N H C l . A f t e r one hour e q u i l i b r a t i o n , the solvent v/as added to the trays . The solvent used v/as t er t -butano l : v /a ter :p icr i c a c i d (80 mls:20 mls:2.0 gms). The solvent v/as allowed to run for 20 hours. Ih order to f i n d the areas of a c t i v i t y on each chrom- atogram, autoradiograms were prepared. Each chromatogram v/as placed on a piece of plywood covered with f i l t e r paper. The chromatogram was covered v/ith a sheet of Saran Wrap and i n the darkroom, X -ray f i l m , Du Pont Medical Type 508, v/as - 18 - placed over the chromatogram. A piece of b lack paper and a second piece of plywood were placed over the X-ray f i l m . The two pieces of plywood, together with t h e i r chromato- grams, X-ray f i l m , e t c . , were wrapped with l i g h t - p r o o f b lack paper. These packages were stored i n a press f or 48 to 72 hours depending on the r e l a t i v e a c t i v i t y of the spots. Before the f i lms were removed for development,' a p i n was thrust through each f i lm and chromatogram i n several spots to insure the correct replacement of the f i l m . The chromatograms were cut into segments containing inorganic phosphates, nucleot ides or sugar phosphates. Each segment was wet ashed i n a 150 ml. Kje ldah l f l a s k using two mis. of n i t r i c ac id and 2.2 mis. of p e r c h l o r i c a c i d . The digests were made to 10 mis. and counted i n the M6 l i q u i d counter. This count represented the r e l a t i v e a c t i v i t y of the a c i d - s o l u b l e f r a c t i o n s . These f r a c t i o n s were not further character ized . RETENTION OF P ^ 2 . To determine the amount of P-^2 which had been l o s t from the plants to the nutr i en t so lut ions , the so lut ions which remained a f t er the 24, 48, and 96 hour harvests were each made to one l i t e r i n a volumeteric f l a s k . A 10-ml. a l iquot of each of these so lut ions was counted i n the M6 l i q u i d counter. - 19 - THE M6 LIQUID COUNTER. The rad ioac t ive so lut ions were counted i n a 20th Century E l e c t r o n i c s M6 l i q u i d counter. The construct ion and uses of th i s counter are f u l l y described by Russe l l and Mart in (37,38). The counter tube was supported i n a lead cast le i n order to sh i e ld the tube from a l l but the: most penetrat ing cosmic rays thereby reducing the back- ground count to below 16 c .p .m. In order to record the number of emissions passing from the rad ioac t ive so lu t ion and through the counter, the tube was connected to a Nuclear-Chicago sca ler (Model 151A) equipped with, a Model T l t imer. The 20th Century E l e c t r o n i c s M6 l i q u i d counter i s a th in-wal led Geiger-Mul ler counter surrounded by a glass jacket into which a sample of l i q u i d 10 mis. i n volume can be placed. The counter i s designed to record up to 10% "52 of the emissions from a l i q u i d sample of Br . This type of counter has two d i s t i n c t advantages over conventional end window counters designed to assay dry samples of p lant m a t e r i a l . F i r s t l y , the p o s i t i o n of the rad ioac t ive sample r e l a t i v e to the p o s i t i o n of the counter i s always the same when a 10-ml. a l iquot i s added to the jacket surrounding the l i q u i d counter. Small v a r i a t i o n s i n the p o s i t i o n of dry samples r e l a t i v e to the end window counter often r e s u l t s i n large v a r i a t i o n s with regard to the number of p a r t i c l e s which w i l l pass from the dry sample through the counter. The M6 tube i s a lso designed so that samples of P ^ 2 s l i g h t l y - 20 i n excess of 10 mis. g i v e no a p p r e c i a b l e a l t e r a t i o n i n the c o u n t i n g r a t e . Secondly, l i q u i d samples are much e a s i e r to prepare than are d r y samples and l o s s e s which occur d u r i n g the p r e p a r a t i o n of l i q u i d samples are u s u a l l y n e g l i g i b l e compared to l o s s e s encountered when p r e p a r i n g s m a l l samples of dry m a t e r i a l . L i q u i d c o u n t i n g may have one disadvantage when u s i n g l i q u i d samples of P-^2. T h i s i s o t o p e tends t o adsorb to g l a s s s u r f a c e s . This l o s s may r e s u l t i n a p p r e c i a b l e experimental e r r o r s . A d s o r p t i o n l o s s e s are prevented by a d j u s t i n g the sample to pH 3 or by adding c a r r i e r phosphate to s o l u t i o n s which c o n t a i n l e s s than one p.p.m. P ? 2 . i f these p r e c a u t i o n s are not taken, then s o l u t i o n s which c o n t a i n low c o n c e n t r a t i o n s of P-^2 may l o s e as much, as 83$ of t h e i r a c t i v i t y a f t e r s i x days due to a d s o r p t i o n of P^ 2 on the w a l l s of g l a s s c o n t a i n e r s when the s o l u t i o n s are s t o r e d . COUNTING- PROCEDURE AND CORRECTION AND ANALYSIS OF RESULTS. Because the time i n t e r v a l between c o n s e c u t i v e d i s - i n t e g r a t i o n s i n a sample of P- 5 2 i s s u b j e c t to random v a r i a t i o n , g r e a t e r accuracy was obtained by counting a l a r g e r t o t a l number of counts. T h i s was done by c o u n t i n g a l l samples f o r 20 minutes or f o r 10,000 counts depending upon which came f i r s t . F u r t h e r , any s o l u t i o n s which, were more a c t i v e than 6800 counts per minute (c.p.m.) were d i l u t e d u n t i l they counted below t h i s r a t e . This was done i n order to prevent l o s s e s of counts which occured when the s c a l i n g u n i t was r e c o r d i n g above t h i s maximum co u n t i n g - 21 - rate. A l l samples were corrected f o r background and radio- active decay. The a c t i v i t y of each sample was corrected to a chosen -reference time, namely, to the a c t i v i t y of the sample one hour a f t e r i n i t i a l phosphate uptake by the plant. In order to determine whether or not the difference between sample means was due to treatment or to chance, the means of the small samples were compared using the 'Student's' t - t e s t ( 9 ) . X i - x 2 S.D. / 1 , 1 y n i *2 To determine the' significance of the t-value given by the above equation, the one-sided table f o r 'Student's' t - d i s t r i b u t i o n was used,.. - 22 - RESULTS. 1 The response of bean plants to AT-treatment i s quite s t r i k i n g . For the f i r s t 24 hours a f t e r treatment with 100 p.p .m. AT, treated plants look very s imi lar to contro l p lants with the exception that some chloros i s of the pe t io l e s of the f i r s t t r i f o l i a t e leaves can usual ly be found. However, a f t er 48 hours, the basa l port ions of the expanding f i r s t t r i f o l i a t e s become p a r t i a l l y c h l o r o t i c . A concentration of 100 p .p .m. AT does not re su l t i n the immediate cessat ion of growth so that any secondary t r i - f o l i a t e s which appear at th i s time are c h a r a c t e r i s t i c a l l y white, i n d i c a t i n g a complete lack of c h l o r o p h y l l and carotenoids. Areas wi th in the laminae of primary leaves a l so begin to lose t h e i r normal green color 48 hours a f t e r treatment. These leaves are usual ly quite f l a c c i d as compared to the primary leaves of contro l p lants , with the r e s u l t that the margins of treated leaves tend to c u r l upward. Some of the primary leaves may abscize during th i s per iod . A f t e r 96 hours, many of the treated plants have abscized both of the i r primary leaves or i f not , these leaves are very nearly ready for absc i s s ion . I f the p lant i s j a r r e d , these leaves w i l l usua l ly f a l l . The older t r i f o l i a t e leaves are c h a r a c t e r i s t i c a l l y yellow while the young t r i f o l i a t e s are white. Growth appears to have terminated 96 hours a f t er AT-treatment. - 23 - INTAKE, RETENTION AND DISTRIBUTION OF YJ . The 48 hour exposure of the roots of young bean plants to 100 p .p .m. AT had very l i t t l e ef fect on the t o t a l uptake of P - 5 2 . This i s c l e a r l y i l l u s t r a t e d i n F i g . 1. One hour a f t er the beginning of phosphate uptake, the t -value f o r "52 the P^ content of AT-treated plants did not d i f f e r s i g n i f i c a n t l y from that of the contro l p l a n t s . Thereafter , however, the roots of contro l plants l o s t ~z>2 •more P-' to the f i n a l phosphate-free nutr i ent solut ions than d id the roots of AT-treated plants (Table I ) . This e f fec t was most not iceable 24 hours a f t e r the end of A T - treatment by which time the roots of contro l plants had l o s t 31.8$ of t h e i r o r i g i n a l a c t i v i t y to the nutr i en t so lu t ion whereas the AT-treated plants l o s t only 1.7$. A l l plants continued l o s i n g a c t i v i t y to the nutr ient so lut ions for the durat ion of the experiment. This lo s s appeared to reach a steady state between the 48 hour and 96 hour harvests . N ine ty - s ix hours a f t e r the beginning of phosphate uptake, AT-treated plants had retained 6.9$ more of t h e i r o r i g i n a l a c t i v i t y than had the contro l s . 32 There v/as a marked increase i n the amount of P trans located to the leaves of AT-treated bean plants (Table I I ) . T-values d i f f e r e d s i g n i f i c a n t l y one hour a f t er the beginning of phosphate uptake. Ninety-s ix hours a f t er the beginning of phosphate uptake, the leaves of AT-treated plants had approximately 40,000 c .p.m. more a c t i v i t y than the leaves of contro l p lants . 24 - 300,000 250,000 Average c.p.m. per p l a n t 200,000 150,000 100,000 A T - t r e a t e d 0 24 48 96 Hours a f t e r the b e g i n n i n g of p32 uptake F i g . 1. The e f f e c t of AT-treatment on the uptake and r e t e n t i o n of phosphate by whole bean p l a n t s . Hours a f t e r i n i t i a l c o n t a c t w i t h P32 P l a n t TABLE I UPTAKE AND RETENTION OF p32 T o t a l a c t i v i t y p e r p l a n t (cpm) A c t i v i t y l o s t to the n u t r i e n t s o l u t i o n (cpm) Percentage of t o t a l i n i t i a l a c t i v i t y l o s t 1 24 48 96 AT C o n t r o l 268,880 266,675 AT 264 ,325 C o n t r o l 182,255 AT C o n t r o l AT C o n t r o l 158,418 125,154 141,006 122,886 4,555 84,420 110,462 141,521 127,874 143,789 1.7 31.8 41.0 53.7 47.5 53.9 - 25 - TABLE I I DISTRIBUTION OF P 5 2 IN LEAVES, STEMS AND ROOTS. Hours a f t e r i n i t i a l c o n t a c t w i t h P 3 2 P l a n t Organ T o t a l a c t i v i t y (cpm/organ) Percentage of t o t a l P 3 2 found i n each organ t - v a l u e 24 48 96 Treated Leave s 1,932 0.71 Stem 13,637 5 .06 Roots 253,311 94.22 32 .96 15 .38 C o n t r o l Leaves 281 0.10 16 .87 Stem 4,564 1.67 Roots 261,830 98 .22 Treated Leaves 7,265 2 - l 5 Stem 41 ,905 20.10 Roots 2 1 5 , 1 5 4 77.74 0.49 4 . 2 1 C o n t r o l Leaves 2 , 9 5 0 1 .64 3.27 « Stem 13,549 7 .49 Roots 165,756 90 .87 Treated Leaves 33,102 20 . 85 Stem 35,827 22.57 Roots 89,489 56.57 37 .61 41 .73 C o n t r o l Leaves 5 ,518 4 . 11 93 .28 Stem 12,718 10.34 Roots- 106,918 85 .54 Treated Leaves 44,721 31.96 Stem 22,966 16.27 Roots 73,319 51.76 4 . 41 it 7.70 # C o n t r o l Leaves 3 ,940 2 . 7 7 5 .48 •i'c Stem 1 5 , 2 2 5 12.12 Roots 103,721 85 .10 * s i g n i f i c a n t a t 0 . 0 5 l e v e l s i g n i f i c a n t a t 0 .01 l e v e l *** s i g n i f i c a n t a t 0 .001 l e v e l - 26 - When the a c t i v i t y i n the leaves, stems and roots i s expressed as a percentage of the t o t a l a c t i v i t y i n the p lant , a much c l e a r e r p ic ture of the d i s t r i b u t i o n of P ^ 2 with in the plant i s given (Fig . 2) . The roots of A T - treated plants l o s t a c t i v i t y to the shoots s teadi ly for 96 hours, at which, time only 51-8$ of the a c t i v i t y remained i n the roots . For the f i r s t 24 hours, the larges t amount of the a c t i v i t y translocated from the roots appeared p r i m a r i l y i n the stems. Thereafter, the leaves of A T - treated plants r a p i d l y accumulated while the a c t i v i t y i n the stems decreased s l i g h t l y . Thus, the o v e r - a l l p i c t u r e i s a steady loss of a c t i v i t y from the roots mirrored by a steady accumulation of P32 i n the leaves. The roots of c o n t r o l plants l o s t only 14.4$ of t h e i r i n i t i a l a c t i v i t y to the shoots during the f i r s t 48 hours a f t e r the beginning of phosphate .uptake. Of th i s 14.4$, 10.3$ was located i n the stems and only 4.1$ i n the leaves. However, a f t e r 48 hours, the roots d id not change i n t h e i r P ^ 2 content whereas the leaves l o s t some of t h e i r a c t i v i t y which reappeared i n the stems. . This f l u c t u a t i o n indicates that the leaves of contro l plants accumulate P 32 for the f i r s t 48 hours a f t er which time there i s a downward trans - l o c a t i o n of a c t i v i t y . EFFECT OF AT-TREATMENT ON THE DISTRIBUTION OF ACID-SOLUBLE AND ACID-INSOLUBLE ACTIVITY. Grinding plant mater ia l with cold 16$ TCA separates the a c i d - s o l u b l e phosphate compounds from the a c i d - i n s o l u b l e - 27 - 4 o . o r Percentage of a c t i v i t y i n the leaves Hours a f t er the beginning of phosphate uptake 25.0 Percentage of a c t i v i t y i n the stems o L i i 0 24 4 8 96 Hours a f t e r the beginning of phosphate uptake Percentage of a c t i v i t y i n the roots Hours a f ter the beginning of phosphate uptake F i g . 2: D i s t r i b u t i o n of P 3 2 i n roots , stems, and leaves. - 28 - phosphates. The ac id - so lub le phosphates include inorganic phosphates, sugar phosphates and nucleot ides while the nuc le i c ac ids , phosphol ipids , and phosphoproteins are included i n the a c i d - i n s o l u b l e f r a c t i o n . The r e l a t i v e a c t i v i t y and the d i s t r i b u t i o n of the ac id - so lub le and a c i d - inso luble f rac t ions wi th in bean plants i s presented i n Tables III and IV. It was noted i n the previous sect ion that treatment of bean plants with 100 p.p.m. AT for 48 hours increased the amount of P-^2 that moved from the roots into the stems and leaves. F ig s . 3 and 4 indicate that th i s increase represents an increase i n both the amount of ac id - so lub le P 3 2 and the amount of a c i d - i n s o l u b l e P^ 2 that moves from the roots in to the upper port ions of the treated p lants . The roots of AT-treated plants l o s t ac id - so lub le P ^ 2 s teadi ly for 96 hours u n t i l only 35.4% of the t o t a l a c t i v i t y i n the plant remained i n the roots . The leaves of treated plants accumulated 43.9% of the t o t a l ac id - so lub le a c t i v i t y present i n the p lant . The leaves of contro l plants d i d not accumulate ac id - so lub le P^2 ±n a s i m i l a r manner. Though 29% of the t o t a l ac id - so lub le a c t i v i t y of the plant had been translocated to the shoots a f t e r 96 hours, only k% of th i s a c t i v i t y appeared i n the leaves. The f l u c t u a t i o n of ac id - so lub le P-^2 wi thin the leaves between the 24 hour and 96 hour harvests together with the steady increase of a c t i v i t y i n the stems of contro l plants suggests that a c i d - soluble P32 i s being free ly translocated from the leaves to the roots as we l l as i n the reverse d i r e c t i o n . - 29 - TABLE III DISTRIBUTION OF AGID-SOLUBLE P 3 2 IN BEAN PLANTS. Hours a f t e r i n i t i a l contact with p 3 2 Plant Organ A c i d - s o l u b l e Percentage a c t i v i t y of t o t a l t - t e s t (cpm/organ) a c i d - s o l . a c t i v i t y i n organ 24 48 96 Treated Leaves Stem Roots Control Leaves Stem Roots Treated Leaves Stem Roots Contro l Leaves Stem Roots Treated Leaves Stem Roots Control Leaves Stem Roots Treated Leaves Stem Roots Contro l Leaves Stem Roots 1,630 0.9 11,765 6.5 4.02 167,281 92.6 180,676 8.90 12.74 155 0.1 3,631 2.2 153 ,525 97.6 157,311 3,375 2.1 27,840 26.0 103,500 71.9 1.11 134,715 2.68 1,043 2.2 2.07 6,547 13.7 44,784 84.1 52,374 16,865 23.8 18,890 26.7 34,684 49.5 70,439 5 .71 13.52 1,884 5.3- 16.85 6,298 18.9 24,785 75.8 32,967 19,434 43.9 9,250 20.7 ' 15,859 35.4 9 .50 44,543 1.99 1,003 3.9 17.54 6,259 24.5 18.256 71.5 25,518 tt tt * s i g n i f i c a n t at 0.05 l e v e l * * s i g n i f i c a n t at 0.01 l e v e l - 30 - TABLE IV DISTRIBUTION OF ACID-INSOLUBLE P 3 2 IN BEAN PLANTS. Hours a f t e r i n i t i a l contact with P32 Acid-insoluble Plant Organ a c t i v i t y (cpm/organ) Percentage of t o t a l a c i d - i n s o l . a c t i v i t y i n organ t - t e s t 1 Treated Leaves Stem Roots 301 . 1,871 86,030 88,202 0.2 2.1 97.5 9.18 12.16 4.10 Control Leaves Stem Roots 126 933 108,305 109,364 0.1 0.8 99.0 24 Treated Leaves Stem Roots 3,890 14,065 111,655 129,610 2.2 13.7 84.0 0.96 3.61 Control Leaves Stem Roots 1,906 7,002 120,972 1 2 9 , 8 8 0 1.5 5.4 91.1 2.85 48 Treated Leaves Stem Roots 16,236 16,936 54,805 87,977 18.5 19.3 62.2 39.83 35.22 Contro l Leaves Stem Roots 3,634 6,421 8 2 , 1 3 3 92,188 3.7 7.2 89.1 60.49 96 Treated Leaves Stem Roots 2 5 , 2 8 7 13,716 57,460 98,963 26.2 14.2 59.6 6 . 5 0 1.89 Contro l Leaves Stem Roots 2,937 8,966 85,465 97,368 2.4 8.7 88.9 1 5 . 9 0 * s i g n i f i c a n t at 0.05 l e v e l ** s i g n i f i c a n t at 0.01 l e v e l s i g n i f i c a n t at 0.001 l e v e l - 3 1 - 50.0 Percentage of a c i d - soluble a c t i v i t y i n the leaves Hours a f t er the beginning of phosphate uptake 30.0 p , • Percentage of a c i d - soluble a c t i v i t y i n the stems n l • i 1 0 24 48 96 Hours a f t e r the beginning of phosphate uptake 1 0 0 . 0 r ' 1 Percentage of a c i d - soluble 62.5 - a c t i v i t y i n the roots 2 5 . 0 I 1 — ' 1 0 24 48 96 Hours a f t er the beginning of phosphate uptake F i g . 3: D i s t r i b u t i o n of ac id - so lub le P 32 i n leaves, stems, and roots of young bean p lants . - 32 - 50.0 Percentage of a c i d - inso luble a c t i v i t y i n the leaves Hours a f t er the beginning of phosphate uptake 30.0 r 1 " Percentage of a c i d - inso luble a c t i v i t y i n the stems Hours a f t e r the beginning of phosphate uptake Hours a f t er the beginning of phosphate uptake F i g . 4: D i s t r i b u t i o n of a c i d - i n s o l u b l e P 3 2 i n the leaves, stems, and roots of young bean p lants . - 3 3 - The d i s t r i b u t i o n of a c i d - i n s o l u b l e P 3 2 with in treated and untreated bean plants a l so d i f f e r e d . In a l l cases, the percentage of P 3 2 i n the leaves of treated p lants , r e l a t i v e to the a c t i v i t y i n the ent ire p lant , increased over that of contro l p lants . A f t e r 9 6 hours, only 59.5% of the a c i d - inso luble a c t i v i t y remained i n the roots of AT-treated p lants . During th i s per iod , 2 7 $ of the a c t i v i t y i n the plant had accumulated i n the leaves. A f t e r 96 hours, the roots of contro l plants had 88.9% of the t o t a l a c i d - insoluble a c t i v i t y whereas the leaves had only 2 . 4 $ of the a c i d - i n s o l u b l e P-3 . The re su l t s indicate that rather than accumulating P 3 2 i n the a c i d - i n s o l u b l e form within the leaves, contro l plants tend to translocate a c t i v i t y i n e i ther d i r e c t i o n . INCORPORATION OF P3£ INTO ACID-INSOLUBLE FRACTIONS. When the P32 content of ac id - so lub le and a c i d - i n s o l u b l e f rac t ions i n the roots , stems and leaves was expressed as a percentage of the t o t a l P-^2 content i n these organs (Table V ) , the resu l t s ind icated that AT-treatment a f fec t s the d i s t r i - but ion of P32 b etween these two f r a c t i o n s . Though a l l p lants continued to incorporate P-^2 into a c i d - i n s o l u b l e f r a c t i o n s r e s u l t i n g i n a corresponding decrease i n a c i d - soluble a c t i v i t y , AT-treated plants incorporated less of the t o t a l P ^ 2 into th i s f r a c t i o n . A f t e r 96 hours, 5 6 . 6 $ of the 32 P i n the leaves was present i n the a c i d - i n s o l u b l e f r a c t i o n whereas 7 3 . 9 $ of the a c t i v i t y appeared n th is f r a c t i o n i n the leaves of the contro l plants (F ig . 5 ) . Though the - 3 4 - TABLE V DISTRIBUTION OF P ^ 2 BETWEEN ACID-SOLUBLE AND ACID-INSOLUBLE FRACTIONS IN EACH PLANT ORGAN Hours a f t e r i n i t i a l contact with P 3 2 Plant Organ A c i d - s o l u b l e as percent t o t a l a c t i v i t y i n organ A c i d - i n s o l u b l e as percent t o t a l a c t i v i t y i n organ t - t e s t 1 T Leaves 8 4 . 7 8 1 5 . 2 2 Stem 8 6 . 4 9 13.51 Roots 6 6 . 3 7 3 3 . 6 2 7 . 7 9 6 . 1 8 * C Leaves 5 2 - 2 7 4 7 . 7 3 3 . 5 1 * Stem 7 9 . 0 7 2 1 . 3 8 Roots 5 8 . 6 2 4 1 . 3 7 2 4 T Leaves 4 7 . 1 0 5 2 . 9 0 Stem 6 6 . 0 5 3 3 . 9 4 Roots 4 8 . 1 0 5 1 . 8 9 6 . 2 8 * 1 8 . 5 1 C Leaves 3 4 . 8 4 6 5 . 1 6 1 0 . 7 4 * * Stem 4 8 . 6 4 5 1 . 3 6 Roots ' 2 6 . 9 4 7 3 . 0 5 4 8 T Leaves 49.91 5 0 . 0 9 Stem 5 1 . 7 7 4 8 . 2 2 Roots 3 8 . 2 4 6 1 . 7 5 5 . 6 3 * 0 . 7 3 C Leaves 3 3 . 9 1 6 6 . 0 9 5 . 2 0 * Stem 4 9 . 9 2 5 0 . 0 8 Roots 2 4 . 6 1 7 5 . 3 9 9 6 T Leaves 4 3 . 4 2 5 6 . 5 8 Stem 4 0 . 1 9 5 9 . 8 1 Roots 2 2 . 5 2 7 7 . 4 7 7 . 1 6 * * C Leaves 2 6 . 1 3 7 3 . 8 7 3 . 2 0 * Stem 4 1 . 1 7 5 8 . 8 2 Roots 1 7 . 6 0 8 2 . 4 0 T " Treated C = Control * s i g n i f i c a n t at 0.05 l e v e l * * s i g n i f i c a n t at 0.01 l e v e l - 35 - 80.0 r Percentage of a c i d - inso luble 3 i n leaves based on t o t a l a c t i v i t y i n leaves ,32 40.0 - 0 24 48 96 Hours a f t er the beginning of phosphate uptake 60.0 Percentage of a c i d - - , 0 inso lub le P-^ i n stems 30.0 based on t o t a l a c t i v i t y i n stems 0 0 24 48 96 Hours a f t e r the beginning of phosphate uptake 100. O r 1 r Percentage of a c i d - inso luble P-^ i n roots based on t o t a l a c t i v i t y i n roots 62 .5" 25.0 AT-treated F i g . 5: 0 24 48 96 Hours a f t er the beginning of phosphate uptake Incorporation of P32 in to Ac id - Inso lub le F r a c t i o n s . - 36 - p e r c e n t a g e of P 3 2 i n t h e a c i d - i n s o l u b l e f r a c t i o n i n the stems of A T - t r e a t e d p l a n t s d i f f e r e d s i g n i f i c a n t l y from t h a t i n the c o n t r o l s f o r the f i r s t 24- h o u r s , t h i s d i f f e r - ence was n o t s i g n i f i c a n t af-ter-. 4-8 hou r s . A T - t r e a t m e n t a l s o i n h i b i t e d the i n c o r p o r a t i o n of P 3 2 i n t o a c i d - i n s o l u b l e f r a c t i o n s i n the r o o t . EFFECT OF AT-TREATMENT ON THE ESTERIFICATION OF PHOSPHATE AND ON THE DISTRIBUTION OF THE VARIOUS ACID-SOLUBLE COMPONENTS. When the P 3 2 c o n t e n t of i n o r g a n i c p hosphates, sugar phosphates and n u c l e o t i d e s i n the r o o t s , stems, and l e a v e s was e x p r e s s e d as a p e r c e n t a g e of the t o t a l a c i d - s o l u b l e P^2 c o n t e n t of t h e s e organs, the r e s u l t s I n d i c a t e d t h a t t r e a t - ment r e s u l t e d i n no change i n e s t e r i f i c a t i o n of P^2 f o r 96 hours (Table V I ) . Thus, A T - t r e a t e d p l a n t s and c o n t r o l p l a n t s e s t e r l f i e d a p p r o x i m a t e l y t h e same pe r c e n t a g e of the i n o r g a n i c phosphates p r e s e n t i n each organ f o r the f o r m a t i o n of n u c l e o t i d e s and sugar phosphates. When t h e i n o r g a n i c P-^2 c o n t e n t o f each organ was e x p r e s s e d as a p e r c e n t a g e of the t o t a l i n o r g a n i c P-^2 c o n t e n t of the p l a n t , the r e s u l t s i n d i c a t e d t h a t A T - t r e a t m e n t does a f f e c t the d i s t r i b u t i o n of i n o r g a n i c P^ 2 w i t h i n t h e p l a n t ( F i g . 6 ) . The l e a v e s of A T - t r e a t e d and c o n t r o l p l a n t s have about the same p e r c e n t a g e of the i n o r g a n i c a c t i v i t y p r e s e n t i n each p l a n t f o r the f i r s t 24- hours a f t e r A T - t r e a t m e n t . T h e r e a f t e r , however, the l e a v e s of t r e a t e d p l a n t s r a p i d l y a c cumulate i n o r g a n i c P-̂  f o r the d u r a t i o n of t h e experiment whereas the l e a v e s of c o n t r o l p l a n t s - 37 - TABLE V I EFFECT OF AT ON THE ESTERIFICATION OF PHOSPHATE. Hours a f t e r i n i t i a l P l a n t Organ c o n t a c t w i t h P32 P e r c e n t a c i d - s o l u b l e P e r c e n t a c t i v i t y i n the form o f : e s t e r i f i - N u c l e o - Sugar I n o r g a n i c c a t i o n t i d e s phos- phos- p h a t e s p h a t e s Leaves Stem Roots 27.4 18.6 22.4 13.2 25.0 21.2 59.4 56.4 56.4 40.6 43.6 43.6 Leaves Stem Roots 26.1 31.4 29.6 16.0 16.6 24.9 57.8 51.9 45.4 42.1 48.0 54.5 24 T Leaves Stem Roots 26.6 21.0 28.6 19.5 22.2 18.7 53.9 56.8 52.6 46.1 43.2 47.3 Leaves Stem Roots 20.3 27.4 30.8 22.5 18.2 25.5 57.2 54.4 43.7 42.8 45.6 56.3 48 Leaves Stem Roots 25.5 17.3 24.8 24.0 23.0 34.6 50.4 59.7 40.5 49.5 40.3 59.4 Leaves Stem Roots 32.3 27.9 25.3 17.1 23.9 54.9 50.8 45.0 49.2 96 Leaves Stem Roots 26.9 24.1 32.4 18.9 20.8 14.6 54.2 55.0 53.0 45.8 44.9 47.0 Leaves Stem Roots 19.8 18.9 20.7 22.9 25.5 22.6 57.3 55.6 56.7 42.7 44.4 43.3 T = A T - t r e a t e d C » C o n t r o l s - 38 - 50.0 r r Percentage of the t o t a l p lant inorganic a c t i v i t y i n the leaves Hours a f t er the beginning of phosphate uptake 40.0 r 1 1 Percentage of the t o t a l p lant inorganic 20.0 • a c t i v i t y i n the stems 0 24 48 96 Hours a f t er the beginning of phosphate uptake 100.0 r-r Percentage of the t o t a l p lant inorganic a c t i v i t y i n the roots Hours a f ter the beginning of phosphate uptake F i g . 6: D i s t r i b u t i o n of Inorganic P 32 wi th in the P lant . - 39 - a c t u a l l y d e c r e a s e s l i g h t l y i n t h e i r i n o r g a n i c a c t i v i t y . N i n e t y - s i x h o u r s a f t e r A T - t r e a t m e n t , the l e a v e s of AT- t r e a t e d p l a n t s c o n t a i n 4-7.3$ of the t o t a l i n o r g a n i c P-52 w h i l e the l e a v e s of the c o n t r o l p l a n t s c o n t a i n o n l y 1.5$ "32 of the t o t a l i n o r g a n i c P^ p r e s e n t i n the p l a n t . The steady d e c r e a s e of i n o r g a n i c P32 i n the r o o t s of A T - t r e a t e d p l a n t s r e f l e c t s the a c c u m u l a t i o n of a c t i v i t y i n the l e a v e s . N i n e t y - s i x hours a f t e r A T - t r e a t m e n t , the A T - t r e a t e d r o o t s had 75.6$ o f the i n o r g a n i c a c t i v i t y . The d i s t r i b u t i o n o f i n o r g a n i c a c t i v i t y i n the stems i s a l s o worthy of n o t e . The stems of A T - t r e a t e d p l a n t s have much more of the t o t a l i n o r g a n i c P^ 2 t h a n do t h e stems of c o n t r o l p l a n t s d u r i n g the f i r s t 48 hours a f t e r i n i t i a l phosphate u p t a k e . However, 96 hours a f t e r i n i t i a l phosphate u p t a k e , the a c t i v i t y i n the stems of each s e t of p l a n t s i s the same. I n s p i t e of t h i s , t h e l e a v e s of c o n t r o l p l a n t s do not' accumulate as much i n o r g a n i c P-52 as do the l e a v e s of A T - t r e a t e d p l a n t s . T h i s s u g g e s t s t h a t c o n t r o l p l a n t s r e d i s t r i b u t e i n o r g a n i c P--2 i n b o t h d i r e c t i o n s whereas the i n o r g a n i c P^ 2 i n AT- t r e a t e d p l a n t s i s accumulated i n the l e a v e s . Sugar phosphates a l s o a c c u mulated i n the l e a v e s of AT- t r e a t e d p l a n t s ( F i g . 7) whereas the l e a v e s of c o n t r o l p l a n t s f l u c t u a t e d markedly i n t h e i r sugar phosphate a c t i v i t y . A g a i n , c o n t r o l p l a n t s seem t o f r e e l y t r a n s p o r t sugar phos- phate from the l e a v e s t o the r o o t s as w e l l as i n t h e r e v e r s e d i r e c t i o n whereas A T - t r e a t m e n t r e s u l t s i n o n l y upward movement of a c t i v i t y . - 40 - 6o.o r Percentage of the t o t a l p lant sugar phosphate i n the leaves Hours a f t er the beginning of phosphate uptake 40. o r i • — — Percentage of the t o t a l p lant sugar phosphate i n the stems Hours a f t er the beginning of phosphate uptake Percentage of the t o t a l p lant sugar phosphate i n the roots Hours a f ter the beginning of phosphate uptake F i g . 7: D i s t r i b u t i o n of sugar phosphate i n young bean plants - 41 - The d i s t r i b u t i o n of nucleot ide a c t i v i t y wi th in each set of p lants i s given i n F i g . 8. As i n the case of inorganic phosphate a c t i v i t y and sugar phosphate a c t i v i t y , the nucleot ide a c t i v i t y i s a lso accumulated i n the leaves of AT-treated p lants . The l e v e l of nucleot ide a c t i v i t y reaches a steady state i n these leaves 48 hours a f t e r A T - treatment (38.7$). On the other hand, the nucleot ide a c t i v i t y of the leaves of contro l plants f luctuates with only 22.4$ of th i s f r a c t i o n oc'curing i n the leaves 96 hours a f t e r AT-treatment. The roots of AT-treated plants l o s t nucleot ide a c t i v i t y s t ead i ly for 48 hours and thereafter increased s l i g h t l y i n t h e i r nucleot ide a c t i v i t y . Though th i s suggests that some of the nucleot ides may have been transported from the stem to the roots during th i s per iod , nucleot ides may have been manufactured wi th in the roots during th i s period since there was a corresponding decrease i n inorganic and sugar P^ 2 during th i s same per iod . Thus, the accumulation of ac id - so lub le a c t i v i t y i n the leaves of AT-treated plants does not appear to be confined to any of the f rac t ions which comprise the ac id - so lub le a c t i v i t y , but rather , the accumulation represents an accumula- t ion of inorganic phosphates, sugar phosphates and nucleo- t ides . The leaves of contro l plants do not accumulate any of these f r a c t i o n s . Furthermore, the incorporat ion of p32 into e s t e r l f i e d compounds ( i . e . , nucleot ides and sugar phosphates), as revealed by the percentage e s t e r i f i c a t i o n i n each plant organ, i s unaffected by AT-treatment. - 42 - 40.0 Percentage of the t o t a l p lant nucleot ide a c t i v i t y i n the leaves Hours a f t e r the beginning of phosphate uptake 40.0|-i , , Percentage of the t o t a l p lant nucleot ide a c t i v i t y i n the stems oL i . 0 24 48 96 Hours a f t er the beginning of phosphate uptake 100.0 Percentage of the t o t a l p lant nucleot ide a c t i v i t y i n the roots Hours a f ter the beginning of phosphate uptake F i g . 8 : D i s t r i b u t i o n of nucleot ide P 3 2 i n young bean p lant s . - 43 - DISCUSSION A 48 hour exposure of the r o o t s of young bean p l a n t s t o 100 p.p.m. amino t r i a z o l e d i d n o t a f f e c t t h e i r a b i l i t y t o t a k e up phosphate from the n u t r i e n t s o l u t i o n . However, Wort and Loughman (44) found t h a t AT reduced the a b i l i t y of young b a r l e y p l a n t s t o absorb P32 and f u r t h e r m o r e , t h i s r e d u c t i o n i n a b s o r p t i o n i n c r e a s e d w i t h an i n c r e a s e of the l e n g t h of exposure t o AT. The d e c r e a s e i n a b s o r p t i o n of P32 was g r e a t e s t i n b a r l e y p l a n t s t r e a t e d w i t h 100 p.p.m. AT f o r 96 h o u r s p r i o r t o the a b s o r p t i o n p e r i o d . H e r r e t t and L i n c k (23) found t h a t t h e t r e a t m e n t o f P - d e f i c i e n t Canada t h i s t l e p l a n t s w i t h AT r e s u l t e d i n a marked r e d u c t i o n i n the upta k e of f o l i a r a p p l i e d These workers found t h a t the a c t i v i t y of AT was g r e a t l y reduced i n phosphorus- d e f i c i e n t p l a n t s as compared t o p l a n t s s u p p l i e d w i t h an adequate amount of phosphate. I f t h i s were a l s o the case i n bean p l a n t s , a phosphate d e f i c i e n c y might tend t o a n t a g o n i z e the A T - i n l i i b i t i o n of P-^2 u p t a k e by the r o o t s of t h e s e p l a n t s . However, the bean p l a n t s had no v i s i b l e s i g n s of phosphorus d e f i c i e n c y p r i o r t o phosphate u p t a k e , t h u s , t h i s l a t t e r p o s s i b i l i t y seems u n l i k e l y . A T - t r e a t e d bean p l a n t s r e t a i n more P^2 when r e t u r n e d t o a p h o s p h a t e - f r e e environment t h a n do c o n t r o l p l a n t s . T h i s i s a n a l o g o u s t o the e f f e c t of AT- t r e a t m e n t on the r e t e n t i o n of P^ 2 by b a r l e y p l a n t s (44) i n w h i c h i t v/as found t h a t p l a n t s l o s t up t o o n e - t h i r d of the a c t i v i t y d u r i n g 3 hours a f t e r the b e g i n n i n g of phosphate u p t a k e . Bean p l a n t s l o s t - 44 - a c t i v i t y s tead i ly for 48 hours and reached a r e l a t i v e l y stable state thereafter . At th is time, c o n t r o l plants had l o s t 53.7% of the i r i n i t i a l a c t i v i t y whereas treated plants l o s t only 41%. As reported by Wort and Loughman, the rapid i n i t i a l loss of P^2 m a y be a d i f f u s i o n process whereas the slower loss occuring a f t er 48 hours i s probably associated with metabolic processes occuring with in the p lant . AT-treated plants transport much more P 32 from the roots to the shoots than do contro l p lants . The steady r i s e of P 32 i n the leaves mirrored by the steady loss of a c t i v i t y from the roots indicates that the leaves of AT-treated plants accumulate P-^ 2. On the other hand, the P^2 content of the' leaves of c o n t r o l plants increases but l i t t l e and f luc tuat ions of a c t i v i t y appear to occur during the 96 hour per iod . This f l u c t u a t i o n together with the steady loss of a c t i v i t y from the roots and the steady increase i n P ^ 2 a c t i v i t y i n the stems suggests that contro l plants do not accumulate P ^ 2 i n the leaves. These re su l t s ind icate that AT i n h i b i t s downward t rans loca t ion of phosphate compounds. Wort and Loughman (44) have found that barley plants react i n a s i m i l a r manner to AT-treatment. They found that barley plants required an exposure to 100 p .p .m. AT of over 4 hours before increased transport of phosphate compounds from'the roots to the leaves occured. When the phosphate compounds were separated into a c i d - soluble P-*2 ( inorganic phosphates, sugar phosphates, and nucleot ides) and a c i d - i n s o l u b l e P^2 (phosphoproteins, nuc le i c ac ids and phosphol ip ids) , the re su l t s indicated that - 45 - b o t h o f t h e s e f r a c t i o n s a r e accumulated i n the l e a v e s of A T - t r e a t e d p l a n t s . The a c i d - s o l u b l e and a c i d - i n s o l u b l e c o n t e n t of the r o o t s of these p l a n t s d e c r e a s e d s t e a d i l y . Thus, the pronounced i n c r e a s e i n t r a n s l o c a t i o n of P^ 2 due t o A T - t r e a t m e n t I s an i n c r e a s e i n t h e amount of a c i d - s o l u b l e P^ 2 and a c i d - i n s o l u b l e P^ 2 w h i c h i s t r a n s l o c a t e d r a t h e r t h a n an i n c r e a s e d t r a n s p o r t of j u s t one of t h e s e f r a c t i o n s . F u r t h e r c h a r a c t e r i z a t i o n of the a c i d - s o l u b l e f r a c t i o n r e v e a l s the i n t e r e s t i n g f a c t t h a t the a c c u m u l a t i o n of a c i d - s o l u b l e a c t i v i t y i n the l e a v e s of A T - t r e a t e d p l a n t s i s n o t c o n f i n e d t o any of the f r a c t i o n s w h i c h comprise the a c i d - s o l u b l e a c t i v i t y . I n s t e a d , the a c c u m u l a t i o n r e p r e s e n t s an a c c u m u l a t i o n of I n o r g a n i c phosphates, n u c l e o t i d e s , and s ugar phosphates. S y n t h e s i s of c e r t a i n o r g a n i c phosphate compounds (e.g., p h o s p h o l i p i d s , ATP, r i b o n u c l e i c a c i d , d e o x y r i b o n u c l e i c a c i d ) i s e s s e n t i a l f o r normal growth i n p l a n t s (2). E s t e r i f i c a t i o n of i n o r g a n i c phosphate v i a reduced p y r i d i n e n u c l e o t i d e s and the cytochrome system ( o x i d a t i v e p h o s p h o r y l a t i o n ) o r v i a t h e p h o t o s y n t h e t i c p h o s p h o r y l a t i v e mechanism r e s u l t s i n the g e n e r a t i o n of ATP, a h i g h - e n e r g y phosphate, w h i c h i s t h e n u t i l i z e d f o r m e d i a t i n g v i t a l r e a c t i o n s r e q u i r i n g a s u p p l y of energy. I t has been shown t h a t growth i s dependent on the c o n t i n u o u s s y n t h e s i s of ATP (2). C e r t a i n growth i n h i b i t o r s d i r e c t l y o r i n d i r e c t l y e f f e c t t h e maintenance of the ATP p o o l , I o d o a c e t a t e , f o r example, b l o c k s sugar u t i l i z a t i o n by i n h i b i t i o n of g l y c o l y s i s . T h i s r e s u l t s i n a r e d u c t i o n of ATP. A r s e n a t e s u b s t i t u t e s f o r i n o r g a n i c phosphate i n the - 46 - o x i d a t i o n of t r i o s e phosphate t h e r e b y u n c o u p l i n g o x i d a t i o n from ATP s y n t h e s i s (10). A r s e n a t e i n h i b i t s the u p t a k e , i n c o r p o r a t i o n and t u r n o v e r of r a d i o p h o s p h o r u s i n mung bean s e e d l i n g s ( 2 ) . DNP ( 2 , 4 - d i n i t o p h e n o l ) a l s o u n c o u p l e s p h o s p h o r y l a t i o n from o x i d a t i v e m e t abolism and t h e r e b y reduces the i n c o r p o r a t i o n of phosphate i n t o ATP and o t h e r n u c l e o t i d e s ( 4 4 ) . However, DNP has no e f f e c t on t h e p r o - p o r t i o n of phosphate e n t e r i n g the n u c l e i c a c i d f r a c t i o n . U n l i k e t h e s e growth i n h i b i t o r s , AT does n o t a f f e c t the i n c o r p o r a t i o n of i n o r g a n i c phosphate i n t o sugar phosphates and n u c l e o t i d e s . T h i s s u g g e s t s t h a t A T - t r e a t m e n t does n o t a f f e c t o x i d a t i v e or p h o t o s y n t h e t i c p h o s p h o r y l a t i o n w h i c h a r e r e s p o n s i b l e f o r the p r o d u c t i o n of ATP. A T - t r e a t m e n t does n o t seem t o a f f e c t those g l y c o l y t i c r e a c t i o n s whereby sugars a r e p h o s p h o r y l a t e d . However, A T - t r e a t m e n t does i n - h i b i t t r a n s f e r of phosphate from the a c i d - s o l u b l e t o the a c i d - i n s o l u b l e f r a c t i o n . Thus, t h e p r i n c i p l e e f f e c t of AT i s on the i n c o r p o r a t i o n of phosphate i n t o one or more of the n u c l e i c a c i d , p h o s p h o l i p i d , or p h o s p h o p r o t e i n f r a c t i o n s . These f i n d i n g s a r e i n agreement w i t h those of Wort and Loughman (44) and t h e i r work w i t h b a r l e y p l a n t s . They have suggested t h a t i n h i b i t i o n of phosphate i n c o r p o r a t i o n i n t o the a c i d - i n s o l u b l e f r a c t i o n by AT-treatment may a c c o u n t f o r t h e d i v e r s i o n of phosphate t o the system r e s p o n s i b l e f o r upward t r a n s l o c a t i o n of a c t i v i t y . Though the a c t u a l mechanism of p r o t e i n s y n t h e s i s i s n o t known, t h e r e i s much e v i d e n c e t o suggest t h a t b o t h a s o l u b l e r i b o n u c l e i c a c i d (RNA) and a r i b o n u c l e o p r o t e i n a r e - 47 - I n v o l v e d I n t h e sequence o f r e a c t i o n s whereby amino a c i d s a r e i n c o r p o r a t e d i n t o p r o t e i n s (18,39). Because AT may i n h i b i t t he i n c o r p o r a t i o n o f phosphate i n t o e i t h e r one o r b o t h of the s e compounds, then the I n h i b i t i o n of growth due t o A T - t r e a t m e n t may a c t u a l l y be an i n h i b i t i o n of p r o t e i n s y n t h e s i s . Sund (41) and Wort and Loughman (44) have a l s o p o s t u l a t e d t h a t AT i n t e r f e r e s w i t h p r o t e i n s y n t h e s i s . Sund has found t h a t c e r t a i n p u r i n e p r e c u r s o r s , p u r i n e s o r p u r i n e r i b o s i d e s when added t o tomato p l a n t s s i m u l t a n e o u s l y w i t h low c o n c e n t r a t i o n s o f AT w i l l p a r t i a l l y a l l e v i a t e growth I n h i b i t i o n due t o AT - t r e a t m e n t . Of the p u r i n e s , a d e n i n e , guanine and h y p o x a n t h i n e were e f f e c t i v e . Growth i n h i b i t i o n of G h l o r e l l a p y r e n o i d o s a due t o AT-trea t m e n t i s a l s o r e v e r s e d by the a d d i t i o n of p u r i n e s (4.2). A l d r i c h (4) has found t h a t one of the p y r i m i d i n e s , u r a c i l , i s a l s o e f f e c t i v e f o r r e v e r s i n g AT-grpwth i n h i b i t i o n . These r e s u l t s i m p l y t h a t AT i n t e r f e r e s w i t h n o r m al p u r i n e and p y r i m i d i n e m e t a b o l i s m . C a r t e r and N a y l o r (15) have shown t h a t t h e r e i s a sharp r e d u c t i o n i n the f r e e g l y c i n e and s e r i n e p o o l s i n A T - t r e a t e d bean p l a n t s . B o t h these amino a c i d s can p a r t i c i p a t e i n the s y n t h e s i s of the p u r i n e r i n g (11). H0CH 2CHC00H *H oNCH oC00H NH 2 S e r i n e - 48 - A T - i n h i b i t i o n of i n c o r p o r a t i o n of i n t o a c i d - i n s . o l u b l e f r a c t i o n s c o u l d a l s o be due t o an a l t e r e d p h o s p h o l i p i d m e t a b o l i s m . Sund (41) has a l s o i n v e s t i g a t e d t h i s problem. Knowing t h a t p u r i n e s n o t o n l y s t i m u l a t e d ' r i b o f l a v i n p r o d u c t i o n b u t a l s o became i n c o r p o r a t e d i n t o t h e r i b o f l a v i n m o l e c u l e , Sund f e l t t h a t an e f f e c t comparable t o t h a t of p u r i n e s on AT-growth i n h i b i t i o n might be produced by r i b o f l a v i n . T h e r e f o r e , he added r i b o f l a v i n t o tomato p l a n t s s i m u l t a n e o u s l y w i t h AT. Because f l a v i n mononucleo- t i d e (FMN) and f l a v i n a d e n i n e d i n u c l e o t i d e (FAD) a c t as the p r o s t h e t i c group (coenzyme) of s e v e r a l f l a v o p r o t e i n enzymes and a r e r i b o f l a v i n d e r i v a t i v e s , these compounds were a l s o i n v e s t i g a t e d . Whereas the p u r i n e s and r e l a t e d compounds had been e f f e c t i v e i n p a r t i a l l y r e d u c i n g the growth i n h i b i t i o n due t o A T - t r e a t m e n t , r i b o f l a v i n , FMN, and FAD b r o u g h t about a marked r e d u c t i o n o f the i n h i b i t i o n of p l a s t i d f o r m a t i o n . These a l s o reduced AT-growth i n h i b i t i o n . Sund c o n c l u d e d t h a t the i n h i b i t i o n of p l a s t i d f o r m a t i o n and c o n s e q u e n t l y of c h l o r o p h y l l s y n t h e s i s by AT r e s u l t e d from the e f f e c t i v e ^ n e s s w i t h w h i c h AT b l o c k e d the s y n t h e s i s of c e r t a i n m e t a b o l i t e s , p a r t i c u l a r i l y r i b o f l a v i n , which, a r e n e c e s s a r y f o r n o r m a l c h i o r o p l a s t development. R i b o f l a v i n and i t s d e r i v a t i v e s a r e n e c e s s a r y f o r normal growth. Sund has a l s o found t h a t a l b i n i s t i c c o r n and peas t r e a t e d w i t h AT have much l e s s r i b o f l a v i n t han u n t r e a t e d t i s s u e s . - 49 - Aaronson (1) has a l s o p r o v i d e d some e v i d e n c e w h i c h i n d i c a t e s t h a t AT i n t e r f e r e s " w i t h p h o s p h o l i p i d m e t a b o l i s m . He found t h a t crude soybean l e c i t h i n c o u l d r e v e r s e AT- i n h i b i t i o n of c h l o r o p h y l l s y n t h e s i s i n Ochromonas d a n i c a , a p h y t o f l a g e l l a t e . I n the presence of i r o n , . l e c i t h i n was even more e f f e c t i v e i n r e v e r s i n g A T - i n h i b i t i o n . I n summary, t h e r e i s - m o u n t i n g e v i d e n c e w h i c h s u g g e s t s t h a t the i n h i b i t i o n by AT of p l a s t i d f o r m a t i o n and growth i s a c t u a l l y due t o an i n t e r f e r e n c e by AT of normal p r o t e i n and p h o s p h o l i p i d s y n t h e s i s . 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