Journal of the Xndiar :t of Sciemce,...

Journal of the Xndiar :t ..-s of Sciemce, Baagalore 5 - -- -- - . - -- -- --

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V*RI&TiOYS i'r ilRGLN.4SE AND OTCASE LE\ELS DURIXi GROWTH I N A.rperxiYkrs nidi:lun,~

Nr.erujn Raarnkrisirnal~ rmd E. R. B. , Shrn~n~~~~aranda~a t~~ 63

Srrwi~s. cu s o h f t HMP PATHWAY E N Z Y . ~ ~ IN gal XUTANTS OF asp erg ill^.^ nidillans S. Mdathi and E. R. B. Skanmzqcn.~~mrlnram 67

GIBBLRELLIX METAHOLISM .4XD RECGLATiOY OF PAMYLASE ISOENZYMES IN HIGHER Pill YTS

.r. P. Maclwinli and U. K. Ydkii 73

BOOR REVIEW3 97

CVDEX 101

Joor, h d . hat. Sc. 61 LC), Dcc. 1970, Pp. 5!-62 d Prinlcd in Mia.

Tryptophan-phenylpyrwate aminolransferase of Agrobaetcria~t~ trrmefaeiens: Purification and general properties of the enzyme

N K SUKANYA A N D C . S. VAIDYANATNAN Department of B~ochemstry, Indian lnsl~tuie of Sclencc, Bangalore 560 012, Indla.

Received 011 M m h 8, 1979; Revfsed on September 12, 1979.

Evidence has been presented to show that the first step in the convers~on nf tr?olop.han to indole acetic acid by .4~~nbucteriunr tiirmfaciem involves Lransamination. The properties of a 58-fold purified t ~ y p i o - phm-p1:cnylpyruvatr. amino tramfcrase preparation with respect to specikcitj, optimbm pH, rempc- ralnre and grotectioii against tiiermal denaiuraLio;~ by subsimtes aud cofactoral avc bcen studied.

Key words : Amiuotrmslerase, trailsanination and plant turnours.

Z. Introduction

The production of plant tunlours by the genus Agr.obucteriu~ri is a well knoun pheno- menon. Tumorigenesis in plants by Agvobacierium tumejaciens has received the attention of several research gronps hecause of the great virnlence and wide host ranre of the organism. In recent ycars several excellent reviews have appeared which deal in detaii with the biochen~ical and molecular aspects of tumour induction by Agi.obu~teriicnl izime-

f ~ c i e n s l - ~ .

1.t has been shown by w e r a l authors that hornlone-like substances are synthesized by Ag-robacteriuin furnefucieus and ~hese in turn may be respmsible for tumour production in plants. The role of I k 4 in tumour formation has been reviewed by Braun'. The kyper-awric condition of tomato crown-gall tissues has been demonstrated by Link and Eggersz and Dye r l ai6. Lippincotts7 have clearly established that bacteria of Bgrobacteiiuin genus actuallv produce the hormones in plant cells.

That IAA is clezrly involved in tumour production by Rgrobacteriwn rumefu~icns is shown by the fact that some strains of rhis bacterium fail to induce tumour on ronmo stems w-ithout addzd auxina-< Klein and Link' have suggested thai bacterial auain is required either ro promote transformed cells into continued multiplication or as cocarcinugen in the transformation process.

51

Production of other hormones such as cytoliinin and cytokinin active substanceslu-11 and cytokinesd"ave also bee11 shoi%n.

The role of tryptophan in the bioge-enesis of auxin and also the probable intermediate involved have been discussed by Fawcett13. The conversion of tryptophan to the plant hormone may involve either the initial deaminstion of the aminoacid fo l lo~ed by &. carbo~ylation'~-'~ or initial decarboxylation followed by deamination1"ls. The available evidence'+-" seems to support the view that the first step involves the formation of the keto acid which in turn forms indole acetic acid via indole acetaldehyde.

Kaper and Veldstra'3 have reported the accumulation of indole pyruvic acid in the culture filtrates of A. tzcmefacier~s grown in media containing tryptophan, which further suggests that the conversion of tryptophan to indole acetic acid involves the formation of indole pyruvic acid as an intermediate, probably by transamination.

Preliminary studies in our laboratory showed that crude extracts of A. tumefacien, contained transarninase activity with various aminoacids as amino donors and a number of keto acids as amino group acceptors. It was observed that transatninase activity with tryptophan and either a-0x0-glutarate or phenylpyruvate was significant. Further, it was found that the two activities could be readily separated.

The present comlnunication deals with the partial purification of the tryptophan. phenylpyruvate transaminase.

2. Materials and methods

2.1 . Organism Agvobacterium fzimefaciens, strain Be, was used throughout this work. The virulence of the organism was shown by the development of galls subsequent to inoculation of a suspension of the bacterium on the tomato (Lycopersicum esculentum) and sunflower (Hehnt/rus annuus) plants.

48-hour cultures of bacteria grown on potato-sucrose-agar slants were transferred to liquid media containing nitrate as the sole nitrogen source" and incubated for 48 hours at 30' C. The bacterial cells were collected by centrif~igation at 5,000 g for 15min. Cells were washed with 0 .1 M phosphate buffer pH 7.0. Extracts were prepared by suspending the cells in the same buffer and exposing them for 20 min to sonic vibration in a Baytheon 10 kc oscillator. After removal of the cell debris by centrifugation in the cold (0-4") at 13,000 g for 15 rnin the supernatant was used for further purification.

2.2 . Assay of the tryptophan-phenylpy~wate transaminase

The enzyme assay was performed in 2 ml reaction mixture containiilg 1 pmole of L-tryptophan, 2 ,moles of phenylpyruvate, 0~05pmoIes of pyridoxal phosphaie, 25 pmoles of veronal : HCl buffer pH 9.6, 0.5 ml of enzyme preparation and water up to 2.0 ml Incubation was carried out at 1.5' C for 60 pin. The reaction was stopped by the addi-

tion of 0 . 5 ml of 15% trichloroacetic acid. 4 ml of peroxide-free ether was added to each tube. The tubes were shakeu and the layers separated by centrifugation. , ~ ~ i ~ ~ ~ t ~ from the aqueous layer were taken for the colorimetric estimation o l tryptopl~an according to ihe method of Horn and Jones"". In the case of controls, the enzyme was added after the addit~on of trichloroacetic acid.

2.3. Estimation of phenylpyvuvatc

phenylpyruvate was estimated spectrophotonletrically by the method of Lin r t &c,

The deprotcinized reaction mixture was suitably diluted and treated with hog kidney tau.tomerase prepared by the inelhod 01 Knox and P~ t t ' ~ . Bol-ate was added at a Enal ~oncentraiion of 0-57 M, pH adjusted to 6.3 and the absorption measured at 299

2.4. Estimation of phenylulanine

~ h c amino acid was identified and estimated by circular paper chromatographic tech- nique" using Wllatrnan No. 1 paper and butanol : acetic acid : water (4 : 1 : 5) as the solvent system.

2.5 . Estimation of protein

Thc protein content of the enzyme preparations was determined by the method of Lowry et nl",using crystalline bovine serum albumin as standard.

2. 6. Substrates and cofactov

L-tryptophan, a-.oxoglutara?e and pyridoxal phosphate %ere obtained from Hoffman -La- Roche Co. Ltd. Phenylpyruvaie was prepared by the method of Herbst and Stemin30. The p~ri-ty OF the phenylpyruvatc was checked spectrophotometrically according to tke method of Liu et alLt.

3. Results

3.1. PuriJication procedure fop trypfopllan-phenylpyruraic transaminase

All the steps were carried oiit in the cold (0-5" C).

s tep I : I t was observed that treatnient of the cmde extract with tricalcium gel at pH above 6 . 5 resulted in rctcntion of some of the inactive proteins by

the gel but lirtle adsorption of the transaminase. The tolai activity of the supernatant, conlpared to the crude, increased, presumably duc lo the removal of an inhibitor.

The crnde extract (pH 7.0) was treated with half its volume or tricalcium phosphate eel (15 1ng/m1)~'. After stirring for 15 min, the suspension mas centrihrged and the precipitate discarded. This negative adsorption of the extract resul.ted in a 6 .6 fold purification.

Step 2 : The pH of the supernatant of step 1 was adjusted to 5 by the dropwise addition of 6N acetic acid. The precipitated inactive protein was removed by centrifugation a t 13,000 g for 15 min.

Step 3 : The supernatant from the previous step was treated with twice its volume of tricalciurn phosphate gel. The suspension was stirred for 20 min and centrifuged. The supernatant was discarded. The enzyme uras eluted by stirring the gel with 0.1 M phosphate buffer pH 8.0 for 15 min and centrifuging.

Step 4 : The eluate was subjected to a second negative adsorption by adjusting the pH of the enzyme to 7.0 with dilute acetic acid and treating with an equal volume of tricalcium phosphate gel for 15 min. The supernatant obtained after centrifugation of the suspension was used for further purification.

Step 5 : The supernatant from step 4 was adjusted to pH 5.0 with 6N acetic acid and treated with alumina C, ge13"0.Z ml geljml enzyme). The mixture was centrifuged after 15 min and the supernatant discarded.

The enzyme was eluted by treatment with 0.1 M phosphate buffer pH 8.0 for 15 min followed by centrifugation.

A summary of the steps involved, the degree of pur&ation achieved and the penen- tage recovery obtained is given in Table I.

Though the crude extract was stable for over three months at - 20" C, the partially purified preparation was found to lose activity either on freezing or storing at 0-4' C for more than 72 hr.

Table I

Progress of purification of tryptophau-phenylpyruvate transaminase from A. tumefaciens.

Step Sp. activity Fold puri- Total % recovew a. tNp. fication activity dissapp./mg protein

Crude 20 - 2700 - Calcium phosphate negative adsorption 133 6 6 3000 111 pH 5.0 supernatant 150 7.5 3000 111 Calcium phosphate eluate 300 15.0 3000 111 Second negative adsorption on calcium phosphate gel 370 18.5 3000 111 Alumina C, eluate 1350 67.5 2300 90

3.2. Specificity The partially purified preparation was conipletely inactive with tryptophan, histidine, methioniae, leucine. valine or isoleucine as the amino donor and a-0x0-glutarate as acceptor. However, phenylpymvate, dimethyl-pymvate and hydroxypbenylpyn!vate were very similar in their ability to act as amino group acceptors, with the above- mentioned amino acids as amino group donors.

Of the variaus amino acids tested with phenylpyruvate, tryptophan, leucine, valine. isoleucine and tyrosine showed higher activity while niethionine and bistidine were less active. DL-aspartic acid, DL-alanine, DL-threonine, L-arginine, L-gh~xamine and DL- serine were nd. effective as an~ino donors (Table IT).

Under the conditiom of incubation up to 90 min, within reasonable limits, there was a correlation in the disappearance of tryptophan and phenylpymvatb. Because of its very unstable nature and progressive decomposition with time indolepyruvate was not estimated.

3.4. pH-aetivity curve

The activity of the enzyme at various pH values was stndied by usingphospllate (5.8 to 8.0), tris : HC1 (8.0 to 9 . l), veronal : HCI (8.0 to 9.6) and glycine : NaOH (8.6 to 10.6). A steady increase in activity was observed up tO pH 9.6 and there u.as a drop in activity at higher pH values. The activity was quite s i d c a n t even at pH 10.6 (Fig. I).

Table U

Phenylalanine formation with various amino donors

Reaction mixtures : 2 pmoles of pl-,c.nylpyruvate, 2 pmoles of the amino donor, 0.1 pmoles of pyridoxal phosphate, 25pmoles of veronal: HCI buffer pH9.6 and enzyme ; incubation was at 45'C for 60 min.

Amino donor pmoles Amino donor moles phenylalanine phenylalanine formed formed

L-Tryptophan J 0- 50 LGlutamic acid 0.02 DL-Histidine 0.09 DL-Aspartic acid 0.00 DL-Mkthionine 0.20 DGAlanine 0.00 DL-Leucine 0.51 DGThreoniue 0.00 DLValine 0.41 GArgininc 0.00 DGIsoleucine 0.51 JAiluia~nine 0.00 L-lhmsine 0.45 D L k i n e 0.00

N. K. SUKANYA AND C. S. VAIDYANATHAN

FIG. 1. pH activity curve. Reaction mixtures were the same as mentioned under Materials and Methods except that glycine: NaOH buffer was used at the pHs indicated (i.e., pH 8.6 to 9.6).

Temperatme, "C

Fro. 2. Effect of temperature. Reaction mixtures were same as in Materials and Methods, incubated at different temperatures for 60 min.

3. 5. Effect of temperature

The activity of the enzyme was found to increase with temperature from 25 to 50' C. At higher temperatures the activity levelled off (Fig. 2).

TRYPTOPHAN-PHBNYLPYRUVATE AMINOTRAWSFERASE OF A. tumefaciens 57

Table m[

Heat inactivation in the presence and in the absence of substrates and cofactors.

Reaction mixtures : Same as mentioned under Materials and Meti-ods (Section 2). The eozyme was heated at the indicated temperatures with or wjtIiout the substrates or cofactor for 5 rnin and chilled immediately. Incubation was continued folr 60 miu at 45" C aftt-r the addiiion ol other constituents.

Temperatwe moles tryptophan disappeared when tEe enzyme was "C. heated with

No addition

Pyridoxal phosphate

3.6. Heat stability of the enzyme

The enzyme was found to be markedly heal. stable in the presence of substrates or pyri, doxal phosphate. Heating the enzyme in the absence of substrates or cofactor for even 5 min at 50" C resulted in a drop in activity, and complete inactivation was observed at 70" C (Table TIT).

Though all the three components of the reaction mixture, namely, tryptophan, phenyl, pyruvate and pyridoxal phosphate afforded protection against heat denaturation, pyridoxal phosphate was found to be the most effectme, phenylpyruvate and tryptophan being less effective in that order.

Incubation of the enzyme with pyridoxal phosphate at 45" C for 30 min, under the onditions involved, did not result in any reduction in activity. Incubation was therefore routincly performed at this temperature.

3.7. Effect of enzyme cnneentration

The activity of the enzyme increased with increaae in protein concentration from 10 to c100 pg (Fig. 3).

ug enzyme protein

FIG. 3. Relationship of enzyme concentration to activity. Reaction mixtures were the same as mentioned in the text except for the vnriation in eJlZyMe concen- rrations.

4. Discussion

Accumulation of indolepyruvic acid in culture filtrates of A. tumefaciens grown on tryptophan con~aining media has been demonstrated by Kaper and VeldstraZ3. Their conclusion was based an the comparison of the chromatographic pattern of the decomposition products of indolepyruvic acid with those of the bacterial metabolites. More recently intermediates in this biosynthetic sequence havc been studied by paper chromato- graphyZ3. But the enzymic conversion of tryptophan to indolepymvic acid has not been demonstrated. However, recent studies on the formalion of JAA by A . tumefaciens suggest that more than one pathway may operate. Rodriguez et ala4 have reported a sequence

Tryptophan -, Indole-3-pyruvate -+ Indole-3-glycolic acid. .1

IAA

Tt is of interest to note that the sequence of steps in the formation of IAA is different in A. tum$uciens from its fixed L, forma5. The authors36 have put forward a novel scheme for the synthesis of JAA in the L. forms of A . tumefaciens, the sequence being

Tryptophan -. Indole-3-acetonitrile -t lndole-3-acetic acid.

In addition, indole 3 acetonitrile has been identified as intermediate in the biosynthetic sequence tryptophan to IAA in several other tumour producing bacteria a1s0~~.

The formation of indolepyruvic acid has been demonstrated in the tryptophan a-oxoglutarate system (unpublished results). The method was not found suitable in the present system due to the interference of colour development by phenylpymvate. However, since the stoichiometry was q ~ i t e convincing, the rate of tryptophan disappearance was routinely followed in the estimation of enzyme activity.

Results of the present investigation support the view that the ffrst step in the metabolism of tryptophan to indole acetic acid by A. tumefaciens involves transamination and the formation of indokpymvic acid. Indole acetic acid is formed from indolepymvic acid presumably by oxidative decarboxylation. Extracts of A. tumefacieizs seem to contain more than one transaminase which converts tryptophan to indolepymvic acid. Though an absolute specificity for tryptophan as amino donor and phenylpymvate as acceptor was tacking for the enzyme purified by the present method, a complete separation fram a tryptophan-a-oxoglutarate amino transferase could be achieved.

The optimum pH for the activity of the enzyme was found to be 9.6. Though decrease in activity was observed at higher pH values, the activity was quite significant even at pH 10-6. Lack of sharp drop in activity at high pH values could be compared to that of the tyrosine-a-oxogtutarate enzyme reported by Sentheshanmugaoathana7.

In its heat stability the enzyme resembles the glutamic-aspartic transaminase reported by Jenkins et aIse and the tyrosine-a-oxoglutarate t r ansamina~e~~ .~~ . This property has been advantageously employed by these authors for the removal of inactive proteins fram their enzyme preparations.

Protection against heat denaturation afforded by substrates is similar tothat observed in the case of gtutamic-aspattic transaminase of pig hearts8 and the tyrosine-a-oxogtuta rate transaminase isolated from rat livers'.

The activity of the enzyme at elevated temperatures and prevention of denaturation even at such a high temperatureas 70' C in the presence of pyridoxal phosphate, phenyl.. pyruvate or tryptophan could be explained as due to the protection afforded by the substrates or cofactors against thermal denat~rat ion~~.

1. Evidence has been presented to show that the first step involved in the conversion of tryptophan to iudole acetic acid by A. tumefaciens involves transamination.

2. A 58-fold purification of the enzyme has been achieved.

3. The optimum pH for the enzyme activity has been shown to be 9.6. The activity at higher pHs, though less than at optimum, was quite significant.

4. Substrates and pyridaxal phosphate were found to stabilize the enzyme against heat denaturation.

The authors wish to tiianlc Dr. H. R. fama for his mierest in this woi-k and Dr. R. M KIein Tor the cii1tu1-e of A. t i ; inrfclciu~s red in th is investigation.

References

5. L m . G . K. K. AM)

Ewm, V.

6. DYE, M. 11. CLARKP, C. AM) WAIN. R L.

8. B u r n , A. C. AND LAsKARls, T.

9. KLEIN, R. M ANV LIED(. G. K . K .

10. MnLFR, C . 0.

11. KAIS CKATMAV, R. W. AND MORRIS, R. 0.

13. Woon, H. N., R ~ N N ~ : KahlP, M. E., Bowrhi, D. B., FLELD, F. H. AM)

BR~uL~, A. C.

15. WJWMA~W, S. G.. FERRI, M. G. AND

BONNm, J.

16. CLARK, A. J. AND

MANS, P. J. G.

i 7. Carny, E E. AND

Worn, i'. J.

IS. DAhmXBrnCI, W. N. AND

L I W , 3. L.

Am. Rev. Phyiopnlh., 1976, 14, 265.

B~onkhaveii. Syinp. Biol., 1977.

Ann. Rev. Plant Pltysiol., 1962, 13, 533

Bot. Gnz , 1941, 103, 87.

Proc. Roy. Soc. (R), 1962, 155, 478

Proe. Natl. Acod. Sci. (USA), 1942, 28, 468.

Proc. Hall. Acnd. Sci. (USA), 1952, 38, 1066.

Pmc. Nall. Acail. Sci. (USA), 1974, 71, 334.

Bioc1,rm Biophj3. Res. Coam%., 1977, 76. 463

Pwc . Nail. Acnd. Sci. (LISAJ, 1974, 71, 4140

Ann. Rev. Plant. Physiol., 1961, 12, 345

J. Biol. Ciiem., 19.15, 109, 279.

Arch. Biochern. Biophys., 1947. 13, 131.

TRYPTOPHAN-PHENYLPYRUVATE AENNOTRANSFERASE OF A . tamefaciens 61

19. STOWE, B. B. Biochem. J., 1955, 61, ix.

DANNENBZIRO, W. N. AND LNERMAN, J. L.

LIYERMAN. J. L. AND

DANNENBURG. W. N.

KAPER, J. hl. AND VELDSTRA

HORN, M. J. AND

JONES, D . B.

LINN, E. C. C., Prrr, B. M., CNEN, M. AND KNOX, E.

KNOX, W. E. AND PITT, B. M.

GEU, K. V. AND Rao, N. A. N.

HERBST, R. M. AND

Suam, D.

K E I L ~ , D. AND H A R ~ E , E. F.

WILLSTATIER, R., KRANT, H. AND ERBASHER, 0 .

OHARA, H., AND KOYIM~, M.

BELEU, R., MARCILLA, P., RODIUGUEZ D6 LECFA, J. AND DBLA ROSA, C.

Pmgress in the chemisrrv of organic nuturn1 pro&ts, ~ d . zech- nleister, Springer Verlag, Vienna, 1959, 17, 248.

Plant. Physiol., 1957, 33, 263.

Plant. Physiol., 1957, 32, Suppl. xviii.

Biochim. Biophys. Acfa, 1958, 30, 401.

Fundnme~~fal prinapies of bacteriology, ~McGraw-Hill Book Co., Inc., 1954, 135.

J . Biol. Chem., 1945, 157, 153.

J. Biol. Chem., 1958, 233, 668.

J . Biol. Chem., 1957, 225, 675.

J. Bdian Insf. Sci., 1952, 34, 95.

J. Biol. Chem., 1951, 193, 265.

Organic synrbesis roll. VoI. 2, John Wiley and Sons, 1943, 519.

Proc. Roj,. S<JC. (B), 1937, 124, 397.

Ber., 1925, 58, 24.18.

Doshisho Jmhi Dnigohr~ Gnklcjirlree K P I I ~ Y I I Nenrpo, 1970, 21, 38 -53.

Mimhiol. Exp., 1970, 21. 1.

62 V. K. SZIKANYA A T D C . S. VAIDYANATHAN

38. JENKrhs, W. T., YPHAWIS, J . Bid. Chem., 1959, 234, 51. D. A. 4PID S ~ R , 1. W.

39. ~ E Y , F. T. J. Bid. Chenr., 1962, 237, 1605.

40. BURTON, K. Biocheni. J., 1951, 48, 458.

Sour. Ind. Inst. Sc. 61 (C), bec. 1979, Pp. 63-66 0 Printed in India.

Short Communication

Variations in arginase and OTCase levels during growth in AspergiUas nidulans

NEERAJA R.AMAKRISHNAN AND E. R. B. SHANMUGASUNDAW University Biochemical Laboratory, University of Madras, Madras 600 025.

Received on December 3, 1970.

Abstract The variations in the enzyme levels of arginase and ornithine tmscarbarnylase ( O ~ ~ a s e ) during the progressive days of growth was assayed in the wild strain of A. nidulnns. A signi6cant increase in both the enzyme activities was seen with the onset of growth.

Key words : Acginase, OTCase levels, nitrogen sources.

The importance of the Kreb's-Henseleit cycle amino acids and urea as nitrogen sources in plants and microorganisms is now well doc~mented~-~. Arginine, being particularly rich in nitrogen, is recycled to form other nitrogenous compounds.

It has been established that in Aspergillus nidulms, a homothaIlic, mononucleate fungus, the pathway for arginine synthesis is similar to that observed in E. COP. Since the enzyme arginase (L-arginine ureohydrolase, E.C. 3.5.3.1) is responsible for the catabolism of arginine, we examined (i) the induction of the enzyme by varying the concentration of arginine in the medium, (ii) the variations in the levels of enzyme activity brought about during the period of growth of the organism. The enzyme ornithine transcarbamylase (Carbamoyl phosphate : L-ornithine carbamoyl transferase E.C. 2.1.3.3), involved in the biosynthesis of arginine was simultaneously studied.


2.1. Strain and medium The wild strain of Aspsrgillus niduians, bearing green conidia, was cultured on liquid minimal medium prepared according to the method described by Pontecorvo er a16 and

64 NEmAJA RAMAKRISrnAV AND E. R. B. SHAh'ML3GAFUNDARA.M

incubated at 37" C. For our experimental studies, arginine was added in three different concentrations, (i) 50 mg L-argininellitre, (ii) 100 mg L-arginine/lifre, (iii) 210 mg L-arginine/liire. The niycelia used for the experiments were 24 hours to five days old.

2.2 . Preparation of extract

The mycelial pad was carefully removed and washed with distilled water to free it of the notrient medium. It was then blotted on a fllter paper and homogenised in 0.1 M phosphate buffer, pH 6.7 , in a mortar and pestle. The homogenate was centrifuged at 8,000 g for 10 min and the supernatant used in the assay of arginase and omithine iraussarbamylase. The above operations were carried out at 4" C.

2.3. Enzyme assay

For the assay of arginase activity, 0.1 ml of the enzymatic extract was activated at first with 2Ojm1olcr of CoCI? in 0 .1 M Tris-HC1 buffer pH 7.0, at 42" C for 10 mio. The addition of 20 pmoles of arginine started 1 hour of incubation a t 30" C. The reaction was s top~ed with the addition of 0 .5 ml of 10% TCA and the samples were assayed for ornithine by the niethod of Chinard'. One unit of enzyme activity is defined as the amount of enzyme required to form 1 pmole of ornithine per hour a t 30" C. The speciffc activity is expressed as units/mg protein.

Ornithine transcarhamylase activity was estimated by measuring the citmlline produced by the procedure of Archibalds as modified by Ohshita et UP. One unit of the enzyme activity is deiined as that amount of enzyme required to form 1 pmole of citrulline a t 37" C in half an hour.

protein was estimated by the method of Lowry et a P .

3. Results and discussion

~t is obvious ihat the onset of growth of the organism begins with a dramatic increase in the activity of arginase, catabolising arginine to ornithine and urea. The ornithine so produced may be channelled into the formation of glulamate, proline or med in poly- amine synthesis. As seen from Fig. 1, even in the case of the wild strain grown on mini. ma1 medium without supplemented arginine, there is an elevation in the arginase activity initially, which then gradually falls down with progressive days of growth, showing that the catabolism of arginine is a preliminary necessity for growth. However, the enzyme being induzible by nature, there is a significant enhancement in arginase activity as the amount of exogenous arginine is increased. I t is interesting to note that, as evident in the figure, the rate of the catabolism of arginine is highest during the first two days of growth in all the cases and then there is a rapid decrease.

The pattern of variation of oruithine transcarbamylase activity with the age of the mycelium provides interesting material. In the case of the wild strain grown on minimal

VARIATIONS IN ~R@INAS& AND OTCASE LEVELS DURING GROWTH IN A. nidulans 65 i

ilmo Idoys:

Fro. 1. Wild grown on *-+ Mlnimal Medium (MM)

a-a MM + SO mg atgininellitre

A-A MM I- 100 mg arginimlitre A-A MM + 210 mg arginine/Lire

medium alone, without added argiaine, there appears an almost uniform level of activity being maintained in the first four days of growth. While, the wildculturesonmedium containing 210 mg ar@nine/litre show a significantly higher level orornitbine transcarbamylase activity on the first and second day, the increase in activity is not so ot\.ions in the wild strain grown on minimal medium, containing (i) 50 mg argininellitre, (ii) 100 mg arginine/litre.

Our cantention is that in the case of the wild strain grown in arginine supple~nented medium, the catabolisin of arginine proceeds at a rapid rate initially, providing the organism an environment with a high amount of ornithine. This causes a conconlitant increase in ornithine transcarbamylase activity. This phenomena is n:ost evident in the wild strain grown on 210 mg arginine/litre. That there is no obvious repression of ornithine transcarbamylase in the presence of arginine is significant. Data indicating that some ornithine production during the onset of arginine catabolism is reused for arginine synthesis has been reported1'.

4. Acknowledgement

The financial aid given to one of us (N. R.) by the University Grants Conm~ission is gratefully acknowledged.

66 N E E R i J R4MAKRfSHNAh A S D B. R. B. S H A N M U G A S $ ~ ~ ~ ~ ~

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Clin. Chim. A m , 1976, 67, 145.

four. h d . Inst. SC. 61 (0, Dec. 1979, Pp. 67-12 B Prioted in India

Short Communication

Studies on some HMP pathway enzymes in gal mutants of Aspergillus nidulans

S. MALATHI AND E. R. B. SHANMUGASUNDARAM Department of Biochemistry, University of Madras, Madras 600 025.

Received on December 11, 1979.

Abstract

Biochemical studies carried out with galactose non-utilising mutants of microorganisms demonstrated that the enwmic defect in these mutants is similar to the enzymic defect seen in galactosemics. In the present study, we carried out investigations on three enzymes of t b HMP pathway in some gal mutants of A9pergillus nidufuns. The study indicates that this pathway is disturbed in these mutants when they are cultured in the presence of galactose, which may suggest that galactose and some of its metabolites which are said to accumulate in the galactosemic condition may impair the HMP pathway in Cis state.

Key words : Microorganisms, galactose, gal mutants, genetic studies.

1. Iutroduction

Mutants of microorganisms unable to utilise galactose as the sole sanrce of carbon have been isolated and some biochemical studies have been carried out nith Galac- tose non-fermenting mutants of Aspergillus nidulans have been prodmed and extensive genetic studies have been carried out with thenl%8. The enzymic defect in these mv.tanls parallels the enzymic lesion in the galactosemic individuals. In the present investiza- tion we report the changes in some of the HMP pathway enzymes in some galactose non- fermenting mutants of Aspergillus nidulans. -

It has been established that one of the important pathways through which gl~lrose is metabolised in the lens is through glx~.cose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Hence, the HMP pathway has a pivotal role to play with respect to the metabolic activities of the lens. It has also been shown that the lens is one of the main targets affected in the galactosemic condition.

Carter and Bull' had reported that an increase in the growth rate of tke aild wain of A. nidulans in a medium containing glucose in both continuous and batch c ~ t l n ~ s

67

68 S. MALATHI AND E. R. B. SHANMUGASUNDAAAM

led to an increase in the proportion of glucose cycled through tb-e HMP pathway concomitantly bringing about an increase in the levels of G6PD. This implicates tht important role theHMP pathway plays with respect to the growthand nutrition of thif fungus.


Six galaqtose non-utilising mutants of A. nidulatzs, namely,

bi, ; 1 ; gal, - constitutive mutant

bi, ; 1 ; gal, - total nmtant; kinase deffcient

bil ; ""' & - } r~oir growing mutants bi, ; w, : gul, - bi,; wI; p l , - total mutant; transferase deBcient

pyre, ; gall, - partial mutant

and the wild strain were nsed in the present study. These strains were maintained on minimal medium slants suitably sv.pplemented by fortnightly subculiure. Minirra.1 medium was prepared according to the method of Pontecorvo et als.

Three conditions of growth were tested. Minimal medium containing

I - glucose and galactose in 1 : 1 proportion

I1 - galactose and glucose in 3 : 1 proportion; and

111 - only galactose.

50 mi of the medium was dispensed in 250 ml Erlenmeyer flasks and these uere steri- lised at 15 lb per sqinch pressure for 15 min. The flasks were inoculated with a spore suspension of the various strains, containing roughly about lo8 spores per ml of the medium. The flasks were incubated for 3 days a t 37' C. At the end of the period of growth, the mycelia were harvested, washed and ground with buffer. The mycelial extract served as the enzyme source.

Three key enzymes of the HMP pathway, G6PDH (assayed by the method of Ells and Kirkman'), 6-phosphogluconate dehydrogenase and ribose-5-phosphate isomerase (assayed a:cording to the methods of Kinglo and Borenfreund'l respectively) uere studied.

3. Results

The changes in the activities of G6PDH, 6-phosphog1u.conate dehydrogenase and rlbose- 5-phosphate isomerase in the various strains, when grown in the different media, can be seen in Figs. 1, 2 and 3 respectively.

Strain Strain I Strain

Strain Strotn

11 1x1

HMP PATHWAY ENZYMES IN gal MUTANTS OB A. nidulaws 69

L.0 L.0 Il --q--y---

-

_ I

FIG. 1 . Activity of CTBPDH in the mycolia of various strains grown for three days a t 37'C. Activity is expressed as change in OD per min per mg protein.

Fro. 2. Activity of 6-ph~~phogluconate dehydrogenase in the mycelia of various strains grown for three days a t 3 7 O C . Activity is expressed as change in OD per min per mg protein.

FIG. 3 . Activity of ribose-5-phosphate isomerase in the mycelia of various strains grown for three days a t 37'C. Activity is expressed as mc moles of ribulose-5-phosphate formed per rng protein.

Common legends for Figs. 1, 2 and 3 : Key: 1-wild; 2-gal,; 3-gal,; 4--gal,; 5-gal,; 6- gal,,,; 7-E&.

I - Medium containing glucose and galactose in 1 : 1 proportion. 11 - Medium containing galactose and glucose in the ratio of 3 : 1.

1J.I - Medium containing Ody galactose.

70 S. MALATHI km E. R. B. SHANMUGP.S~NDARAM .

From Fig. 1 it could be seen that in the medium where glucose and galactose are present in eqaal propxtions, the activity of the enzyme is reduced in the mutant strains, maxi- mal changes being seen in the case of gal, and gal,. A further study of the results presented in the A s r e shows that the activity of the enzyme is further reduced in all the mutants in the medium u-here the proportion of galactose is still greater. Marked reduc- tions in enzyme activity are seen in the case of gal, and gal,. In a medium uhere only galactose is present as the carbon source, the activity of the enzyme is significantly redtxced in all :he mutants, the chanees in gal, and gal, being particularly pronox~nced, tke level of activity in these strains being about 45 per cent of the activity present in the wild strain.

It is observed from Fig. 2 that the activity of t te enzyme 6-phorphoglucona'e dell! dro- genase is also reduced in the gal mntants when grown in the variovs media indicated.. The extent of decrease depends on the nature of th.e medium, the decrease becoming pro- nounced as the proportion of galactose in the medium increases. In a medium containing only galactose as the sole source of carbon, the enzyme activity is redued by 40 per cent in the case of the strains gal, and gal,. About 80 per cent of the conirol activity is expressed by strains gal,, gal, and gal,. About 84 per cent of the activity in the wild strain is present in the mu ant gal,,. In media containing a lesser proportion of galactose, the activity of the enzyme in the mutants is higher tF.an in a ~ e d i c m containing on!y galactose as the carbon source, but the activity in the total mutants js lower than that of other strains.

The activity of the enzyme ribose-5-phosphate isomerase in the various strains grown in the media described are presented in Fig. 3. In a medium containing equal propor- tions of glucose and galactose, it is seen that the enzyme activity is markedly reduced in the nmtants gal, and gal, and less so in the other mutants. In a medium where the proportion of galactose is greater, the activity of the enzyme in gal, and gal, is significantly reduced. Strains gal,, gal, and gal, record almost similar levels of activity. The activity of the enzymeingal,, isinbetween that of theother mutants tested. In amedium containing only galactose, the ribose-5-phosphate isomerase activity decreased in the order, gal,, gal, < gals < gal, < gal, < gal,,.

L. Discussion

in important metabolic psthway of glucose in the lens is through glucose-6-phosphate 3 6-p:~osphogluconate, catalysed by G6PDH. The activity of this enzyme-is lowin ataractous lenses from galactose fed rats. Lermml%as claimed that in normal lenses 1 vitro, a significant reducion in the activity of this enzyme is caused by adding galac- xe-I-phosphate to the culture medium. Rawal and Raola studied the effect of galac- x e on the metabolism of rat lenses in vitro. They observed that lensescultured in 20 mM mcentration of galactose and harvested at variom time intervals showed lower levels f G6PDH activity than the lenses maintained in a medium devoid of galactose. A milar observation was made when the lenses were treated with different concentrations

HMP PATHWAY ENZYMES IN gal MUTANTS OF A. nidulans 71

of gala-tose but harvested after 24 hours. Thus, their results shoued that galactose has an inhibitory effect on the activity of G6PD.

Rats fed on a diet rich in galactose and thus maintained in the ' galactosemic state ' have shown to have dist~~rbances in the fmctioning of the HMP pathway14. Since the metabolism of glucose through the HMP pathway depends on the activity of G6PD, it is suggestive that the inhibitory effrst of gah.ctose on G6PD, may disturb the HMP shunt1'. It way against this background that we undertook a study of three enzymes concerned with the HMP pathway.

From our stu-dies we have observed a decrease in the activity of the ihree enzymes studied, in the galactose non-fermenting mutants, when cultured in media conaining galactose, as compared to the activity in the normal wild strain. It is seen that the activity of the enzymes depends hoth on the nature of the growth medium and the nature of the mutation in the strains. From our results it may be conclnded that galactose toxicity affects the HMP pathway. The depression in the activity of G6PD, the key enzyme in the pathway may bring about a conlornitant decrease in the other e n q z e s involved in the sequence of reactions.

In the galactosemic individuals and rats, as well as in the galactose negative microorganisms, the accnmulation of galactose-l-phosphate has been establishedW7. In addition, it has been shown that the accumulation of galactose itself covld cause changes in the enzyme activityxs. In our study, we have used both kinaseless and transferase- less motants of A. nidulans, wherein the accumulation of galactose and galactose-1-ph-0s- phate should take place. Onr s:udies with these mutants indicate that both these toxicity factors may interfere with the HMP pathway in these mutants.

5. Acknowledgements

The authors wish to thank Dr. Ogata of the Fougal Genetic Stock Centre, USA, for having kindly sent some of the strains. One of US (S.M.) is grateful to the University Grants Commission for the award of a Junior Fellowship. References

1 . NIK~IW, H. Bioeirim. Biophys. Acta, 1961, 48, 460.

2. BLUM, W. AND Engymologia, 1968, 35, 40. SERZDELL~, A.

3. DOUGUS, H. C . Biochim. Biophys. Acta, 1961, 52, 209.

.4. ROBERTS, C. F. J. Gen. Microbial., 1959, 20, 540.

5 . ROBERTS, C. F. J. Gea. Microbial., 1963, 31, 285.

6. ROBERTS, C. F. Biochim. Biophys. Acta, 1970, 201, 267.

7. CARTER, B. L. A. AND Biotechnol. Bioeng., 1969, 11, 785. BULL, A. T.

72 S. MALATHI AVD E. R. B. SH9h'KUGASUNDARAM

8. POE~TECOR'.'O, G., ROPER J. A,, HEMMONS, L. M., MCDOWALO, K. D. AID BUTTON, A. W. J.

9. ELLS, H. A. AND

KTRKMAN, H. N.

15. KURAH~SHI, E. AND

WAHBA, A. 3.

17. QUAK-MA, R. AND

WELLS, W.

Adc. Gemies, 1953, 5, 141.

Pmc. Soc. Exp. Biol. Med, 1961, 106, 607.

In Practical Clinical Enzymology, D. Van Nostrand Co., London 1965, p. 127.

3. Bid . Chem., 1951, 192, 583

Science, 1959, 130, 1473.

h d . 3. Exp. Biol., 1978, 16, 499

Biochim. Biophys. A m , 1958, 30, 298

Arch. Dis. Child., 1960, 35, 428.

Biochem. Biophys. Res. Commw., 1965, 20, 486.

Arch. Biochem. Biophys., 1967, 120, 239.

Jour. Ind. Inst. S:. 61 (C), Dec. 1979, pp, 73-95 O Prinred in India

Gibberellin metabolism and regulation of a-amylase isoenzymes in higher plants

J. P. M A C H A I A H AND U. K. V A K I L Biochemistry and Food Technology Division, Bhabha Atomic Research Centre, Trombay, Bombay MO 085.

Received on October 22, 1979.

Abstract

So far 52 gibberellins (GA) have been isolated from fungi and higher plants. Structural elucidation, biogenetic sequence of the intermediates and the nature of regulating enzymes involved in t t e biosm- thesis of GA from their precursors, are well establisked. TLC patf.;aps fn'm mcvalonic aud (LWA) ro G.4, ,-aldehyde are common in fungi and higher ~ l a n f s and [hen d~ffcr. denendine uoon r1.e order of hyd~~xylation. The structural ~e.equi&ment fo;spe&c bioIogi~al functiod O ~ G A is-well establi .?,ed. Numerous conventional methods and newer approaches are used for tentative identification and quanti- tation of GA metabolites. 2,&h~droxyIation and glycosylation of GAS are conelated with seed develop. ment, maturation and storage of GAS in inactive forms. The subsequent release of active GAS during germination follows the enzymatic hydrolysis of the endogenous ones. However, environmental stresses have deleterious effects on GA metabolism: in y-irradiated seeds reduced GA formation during germination adversely affects GA-controlled metabolic processes such as seedling growth and devekpment of alpha-amylase isoenzymes. Physiological aspects of the role of GA in breaking dormancy, germination and in protein, carbotydrate and lipid metabolism have been dircusaed. The tonnonal role of GA in biogenejis of alpha-amylase and its isoenzymes have been olrtlined in detail. Recently, media- tion of GAS in organelle biogenesis and in the formation of subcellular bi~membranes, has been envisaged.

Key words : GiShellins biogenesis, environmental stress, physiological role, a1pf.a-amylase, isoenzymes.

1. Introduction

Gibberellins (GAS), comprising a large family o f naturally occurring diterpenoid acids, have a hormonal. function in higher plants and a r e essential for normal growth a n d development. They were originally isolated a s secondary metabolitesfrom Gibberella fujikuroi, t he causative fungus of t h e ' Bakanne ' disease o f rice. Tkongh intensive studies

on various aspects o f G A metabolism including their turnover, biosyntkesis a n d re@!- l a t ion have been made in recent years, their mode o f action at molecular level is n o t well understood.

73

GAS display a wide speciri[m of structural array in higher plants and 52 derivatives, isolated to date, are danozed by the trivial names GA, to GA,,. Systematic nomen- clature of naturally occurring GAS is based on their gibbane or gibberellane skeleton with steric conffgwation of cyclic diterpene (-) kaurene (Fig. 1) and on their biological

GIBBANE

FIG. 1. The smctures of gibbane, gibberellane, ent-gibberellane and ent-kaurene. Heavy lines or wedgm indicate bonds lying above the plane of tke ring system; broken lines indicate bonds lying below this plane'.

properties. GAS are broadly classified into two distinct groups, depending upon the number and position of hydroxyl groups. The C,, GAS have one COOH group in position 7 (gibberel!ane numbering) and a lactone conRgurationin the A ring with one C atom less; whereas C,, GAS have full complement of diterpenoid C atoms. If GA has a single functional hydroxyl group, it is always in 3 and 13 posi.tions in firngi and iigher plants, respectively1. For physiologicai experiments with plants, "C-GAS with ligh speciffc activity are prepared by growing Gibberella fujikuroi in a medium containing 4Gacetate2. Large amount of SH-GA, can be produced during cata!ytic reduction hrough the exchange of tritium of 3H-GA,S. Several chemical processes, involved in :onversion of relatively inactive CXo GAS into biologically active Cl, GAS, Have been uggested using cell free fungal sys?emsa. Recently, Bearder et al5 have envisaged a bio- ogical sequential mechanism for GA syntliesis, involving Baeyer-Villiger type oxidation, ~ydrolysis and lactonisation. However, the reaction, responsible for loss of the angular '. inthe conversion of C,, into C,, GA, remains unresolved. So far, the absolute sterea-

GIBBERELLIN METABOLISM IN ITTGFlER PLANTS 7 5

chemisrry of only few chemically related GAS (like GA,, GA,,, GA,,, etc.) has been established, rest are assigned by analogy.

2. Occurrence, transport and storage of GA

2.1. Occurrence

In higher plants, GAS are synthesized at diverse sites including endosperm and cotyledons of immature seeds, scutellum, embryonic axis, shoot apex, root tip, etc.' The comp!ete GA-biosynrhetic pa;ihway may be operating in plastids, since GA-like substances can be extracted from this organelle. Since ch1orop:ast membrane is imper- meable to MVA" this precarsor may be synthesized in chloroplasts. This statement is supported by demonstrating the presence of MVA-activating enzyme and biosynthesis of ent-kaurene in chloroplasts, isolated from non-aqueous mediav. It is suggested that the etio?lasts have access to cytopl.asmic MVA, but lose this property when they Ax C0,8. Phytozhromes present in the membrane of barley etioplasts are shown to regolate GA levels. This is attributed to red-light stimulated increase in the membrane permeability * or t o the release of ' bound ' GAs from the membraneLo.

2.2. Transport

GAI are transporred from roots t o longer distances both via xylem and phloem, speci- 6cally by sieve tubes. They are .transported passively with the flow of water and are assimilated. However, the short distance transport of GA from the sites of synthesis to the site of action i s shown to be non-polar and slow by donor-receptor techniq~el. Very little is known about the occirrrence and naiure of GA-receptors. Mwgrave ef all1 have shown by determining the distribution pattern of applied 3H-GA, in peas that gcoaing p ~ r l i o n of the axis is a GA-target tissue. A positive correlation between GA upiake in b~r l ey alenrone layers and its physiological potency are reported, though binding sitea in cellular compartments are not localizedE. A specific GA receptor and two non-covalent GA protein complexes have been isolated from pea seedlings treated with "-GA,, though their physiological significance is not clear15.

2.3. Storage

Active terminal GAS like CAI, GA, and GA,, present during maturation, are conjugated mainly wlrh sogar through a hydroxyl group and are stored as stable but biologically inactive conp!exes in dormant seeds. 2B-liydrox>lation of GAS, e.g., GAT + GA, in P h a s e o h vulguris and G.42, -t G.k2, in Pirum sufiixm, results in marked reduction in their biological activityl4 and thns plays an important role in metabolic control and seed developnent. An ampho:eric conjugate of 3H-GA,, metabolica1i.y bound to a pep- tide chain is isdated from barley a!eurone layers1< Several such inert acelyl, glueosyl as well as g1.ncosidic conjugates, in which sxgar is linked to the 7-carboxyl g o ~ l p of GA: with different relative biological ac~ivities are isolated or s~nihesized chemically and ~haraclerised'~. These are cleaved by endogenous p-glucosidases and re1 ased as free functionally active GAS during eal-ly seedling growth'?. Th~ts, plucosyl ester formation

76 I. P. MACHATAH .4.'4D U. K. VhKIL

and hydrolysis of GAS regulate their levels daring seed maturation and early germin&. tion. Recently, a s~il.phur containing deactivation product of GA,, gibberethione, is isolated from immature seeds of Pliarbitis d l s .

3. Biogenesis of GA

GAS are oxurring in diverse forms in nature and no single biosynthetic paihnay exists in fungi and higher plants. The steps up to the synthesis of GA,,-aldehyde, the first intermediate with entgibberelline skeleton, are common in both fungi and higher plants, as established by Pztsarimn ntoniliformr and by cell free systems from immatrre seeds of Cucwbitaceae, Rieimis conz~izrwis and Pislmi sativunzl" ; ihereafter the pathways diverge. The current understanding of intermediates and interconversions involved in GA biosynthesis has been extensively reviewed by Hedden et aP2. Initially (Fig. 2 )

, -

CH~-SCOA------ do--C-CH~ ----i, M - C - C W j I CH2CH2- OH ~ H ~ C H ~ O @

ACETYL COA MEVALONATE MEVALONATE-5 -a A/

D

W

F d-ISOPENTENYL-PP trans-GERANY L GERANYL-PP

!?

, i

H3C CH3 - ent (-) KAURENE COPALYL P P

re. 2. Outline of the gibberellin-biogenetic pathway from acetyl-CoA and mevalonale to (-) kaureitj le conversions are mediated by soluble enzymes having a requirement for Mg++ and ATPI.

~osphoryl.ation of MVA to MVA-5-phosphate (MVAP) is catalysed by cytop!asmic VA-kinase, a rate-limiting enzyme for the formation of various polyprenoid com- unds in plants and microorganisms. The svbsequent phosphoryla,ion of MYAP d the sequen:ial. formaiion of pyrophosphate (PP) intermediates, are cti.alysed by a .ies of kinases, including phosphomevalonate kinase. Further, prenyl rransferases a group catalyse the seqcential condensations of isopentenyl-PP (IPP) and longer :nyl-PP to produce pools of farnesyl-PP (FPP, C,,) and geranyl geranyl-PP (GGPP,

GiBBERnLLlN METABOLISM IN HIGHER PLANTS 77

C,,) as thc major products of chain elongation. Two forms of geraoyl transferase (I and I[), which catn!yse ihc specific formation of FPP and. GGPP from IPP, have been isolated together from castor bean They undergo protein-protein interaction, which modulates their catalytic properties. However, Shinika et aP4 have purified GGPP synthetase from pumpkin fruit, which is free of FPP qnthetase and caralyses the conden- sation of 1PP wit11 either FPP or GPP lo give GGPP as the final product. ALI these soluble enzymes have been isolated, p,!riiled and characierised. T31cy have pH optimum between 7.8 and 8.0, u'tilise only phosphate or pyrophosphate as donors and require M,n+l- for activation'? The final cyclization of GGPP to tetrzyclic diterpene ent (-) kaurene proceeds via a two stage mechanism. Th.e reaction is catalysed by a high molecrlar weight enzyme complex, kaurene synthetase (KS)'" The enzyme can be resolved into two catalytic activities (A and B) with mbstrate specacity and activated by divalmt metal ions". Activity A catalyses the conversion of GGPP to copalyl PP (CPP), a bicyclic intermediate, probably through a proton initiated cyclization? Acti- vity B mediates f~lrther cyclization, where pyrophosphate is lo?t from CPP, fa,lo%ed by rearrangement of the resulting carbonium ion to produce ent i-) kaurenc. This reaction is a potenlial site of ~ e g ~ l a t i o n of GA biosynthetk pathway in higher plants, since GGPP is a branch point melal'olite in the production of dite~penoids and caroieno:ds20.

In a second part, ent (-) kaurene is metabolized via a series of oxidative steps: the biosynlhetic sequence, ent kaurene + kaurenA + kanren~l + kaurenoic scid, has Leen established by r~feeding the ;@termediatcs and isolation of ihe end products. Xaurenoi.. acid on fcrther hydrcxylaiicn in 78posirion f d m s lhc last product in the serirs, namely, 7-1 (OH) kaurenoic a c i d . All these steps are catalysed by the mlcrosomal prepa~ation in 6. macrocarpa seeds and require oxygen, ATP and NADPH as cofaciow, suggesting that they are mixed function oxidasesl", having eleitron transport systen~ similar to that found in liver. The presence or cytochron~c P,,,, and a high mclecular weight ent- kaurene carrier protein 111 high speed supernatant from the co-iyledons of P. sativrrm2s, is analogous to the sterol carrier proteinfol!nd in rat liver Bcmogenatcs2~ These com- poilnds exhibit GA aciivities and sf mulate a-amylase formation in embryoless barley seedsl. Further, by contraction of B ring from a 6 to 5 carbon ring with C, being extruded, 7-a (OFI) kaurenoic acid is convened to a relatively inactive GA confiyraticn with aldehyde group in C7 positionz'. The rate lilniiing step appears to be the removal of a hydrogen from the ent 6-positioc. The resulting high energy intermediate. e g . , carbonirm ion could undergo rearrangement to give GA12-aldehyde!z.

Lastly (Fig. 3), GAl,-aldehyde is sequentially oxidized in vifro in higher plants at C , to farm GA,,, followed by 38 hydroxylaiion to give GA,,. Further oxidation at C,, t o the carboxylic acid yields GA,,, which is then hydroxyiated at the 2fl position to the final produ.ct. The conversion is catalysed by the soluble enzymes which are distinct from the membrane-bound mic osomal oxidase responsible for GA,,-aldehyde forn~ation from ent-kawene3'. Conversion oi GAT,-aldehyde th ro~gh GA,, to GA, is the first demon%tra(ion of in vitro transformation of C2" to CIS GA by 3 fi-hydroxyla-

I. P. MACHAIAH AND U. K. VAKIL

PARTICULATE ENZYMES. NADPH. Q 2 a

pw. 3. Sequence of convanion pathway from ent(-) kaurene to g~bberellins, in F. moniliforme and in h i i ~ e r plants.

tion pathway in higher plants. Interconversion of GA, to GA, and t o GA,, are mediated by a single hydroxylation reactiona? A biogenetic sequence for GAl,, GA,, GA,, GA1 and GA,, in which GAsare both 38 and 131-hydroxylated, is well established using cell free systemss3. In dormant seeds GA, and GA,, more non-polar and inactive GAS, are predominant. These are oxygenated to more polar and biologically active GA, and GA, during germinations4. In the fmgi, both 3P-hydroxylation and non- 3-hydroxylation pathways forthe synthesis of GAS from GA,,aldehyde are established using a number of GA-prodwing strains of C. f ir j iku~oi and their mutants. In mntant R-9, 13-hydroxylation reaction is blocked and as a result, GA1 and GA, are not formed. Similarly, in mutant El-4ia, GA1, and GA,, do not act as precursors of GA, and since 3fl-hydroxylation pathway does not operate, GA,, is not metabolised". All these hydroxylation processes are mediated by hydroxylases, having specific require-

GIBBERELLIN METBOLISM IN HIGHER PLANTS 79

ment for reduced co-substrates and are inhibited by EDTA. Some of them are puri. fied and characterised3c.

4. Structure-function relationships of GA

Two hypotheses have been postulated to explain tke structure-fi'ncto relationship of GA, which depends upon several fac:ors like transpori, biosyntketic pattern and cata- b ~ l i s r n ~ ~ . The biological activity of GA directly depends 1.pon the degree to which it 8;s t o hypothe~ical receptor molecule or site in the cell. It is, mainly, associated with 38-OH, 13-a-OH and y-lactone structure; GAS, having a 19, 10 or 19,20 lactonic bridge a t the receptor site, usually show svbstantial. b i o p o t e n ~ y ~ ~ . Tt-e modiftcation in the usual hydroxyl conffguration by substitution with 20-carboxbl or ~xethyl group completely destroys the activity, whereas moleciilar rearrangement in lactone ring partz- ally reduces GA activity. Four major decomposition products of GAS (iso-GAS, dlo-GA,, epiab-GA, and i:9 (1 1)-dehydro GA,), exhibit partial or total loss of acti- vityS8. This furiher ascertains the structl~.ral reqvirement for biological activity Secondly, efficiency of GA interconversion of inactive to active metabolites (e.g., G A , p GA,) also controls the biopotency of GA derivatives. However, the assessment of the relationship depends upon the bioassay methods employed. Dwarf rice bioassay method responds to all G.4s excepi GA,,, GA,, and GA,, because of their rapid irterconversion in rice seedlings, whereas barley aleurone responds to only limited number of G A P .

5. Identification and estimation of GAS

GAS are generally extracted from fungal or p!ant systems with appropriate organic sol- vents and buffers. They are firrther pfriffed by partition colcmn chromatography3* and by agar diffusion t e c h n i q ~ e ~ ~ . Treatment with polyvinyl-pyrrolidine is highly effective in puriffcation of GA from the extracts of vegetative tiss~tes, presumably by selective removal of inhibitory impurities such as phenolic compounds, ahscicic acid and glucosides of active GAs41.

Separation, identification and characterisation of minute quantities of GA metabolites i n plants have been achieved by employing sensitive techniques based on TLC, GLC, fluorimetry, paper chromatography, silicagel adsorption, e!c4? Isotope d,ilution method is shown to correlate well with bioassay methodsP3. Deuterium-labeled substrates are used as internal standard for the determination of native GAS. In metabolic studies, these can be distingvished from the 1,abeled metabolites by usingdoubly labeled substrates with tritium, and quantified by GLC and mass-spe~troscopy"~. More recently, using GLC with 3 liquid phases, all GAS and their glucosides have been separated and charac- terised45. Similariy, a conclusive identifica'ion and sequence of interconversions of GA derivatives present in sub-n~icrogram quantities have been achieved by combined gas liquid radio chromatography-mass spectrometr~"~ and radioimm~inoassay techniqv.e7. X-ray analysis and NMR spectroscopy are useful in determining the stereochernistry of natural G A P .

A namber of bioassay meihods for qi!anlitatlve analysis of GA activity are developed The barley aleurone a-amylase bioassa), though laborionsand time consuming, is one of the most sensitive one". fn addition to widely cmp?oyed dwarf mutant lesis, rapid bioassay methods wing nileat, rice or bark)- endosperms. are routinely nsed; GA %tivity is measnred in terms of a!pha-amylase production and reduc~ng sugars re:eased in thc n~edi~mni. Sin~ilarly, total proteins released from barley aleurolles are measured to determine GA-!ike activity over a wide range of concentrationdu. at temp:^ have been made to use sirnp!.~ systems snch as pale ma1t"i and sol.uble starcl-" as substrates for rapid assay of GA activitj. The methods baced on stimulation of peashoot gowth, lithia or cucumber hypocotyl growth o r I-etardation of senescence in leaf discsoT Tauu. xacunz afftcirzaie and Rumex ~ h t i l ~ i f o l i z ~ ~ , have been reported for detection of GAS"".

6 . Inhibitors of GA biogenesis

A number of synthetic coxpounds including, 2-chloroethyl trin~ethyl amn-oniom cbloride (CCC), 2'-isopropyl-4'-(tri111eth~~laii11noni~n chloride)-5'-methyl phenyl piperidme-I. carboxy!a?e (AMO-16i8), tribu?yl-2, 4-dichlorobenzyl phosphonium chloride (Phos- phon D), etc., are known growth retardants1. Some hydrazonium analopes of CCC also have the same potency for growth retardation in wheat". CCC and phosphon D interact with GAS in a specitlc, non-competitive way for the sites of action1. The available evidon-e indicates that AMO-I618 inhibits in vitvo incorporation of GGPP into (-) kanrene in castor beans55. Quarternary ammonium compounds derived from a-ionone and isophorones are more potent inhibitors of GA biosynthesis than AMO-1618, with a similar site of actions6. CCC is shown to inhibit cyclization of GGPP t o (-) kanrene, causing the accumulation of the Latter in F. nzonilfoime culture"'. HON ever, Paleg et ~158 have indicated that CCC inhibits acetate to mevalonate conversion, thereby blocking the metabolism of barley embryos grown in soluble slalcb. Dennis et al" have sugsested that the sites of action of these two potent growth retardants on GA, may be different and AMO-1618 may have a multiple action as shown schematically in Fig. 4. Fnrther, thesc inhibirors uncouple oxidative phosphorylation in pea seedlings, t hm reducingthe availahil~ty of ATP for cell division and GA synthesisG0. A bitter principle cncurbitacins

4 ACETATE- MEVALGNATE- ISOPENTENYL PP-

t I

Iccc 1 FARNESYL PP

GIBBERELLINS (GA3) +- 1-) KAURENE c--- GERANYLGERANVL PP

1 -4 SQUALENE AMO-1618 1

STEROLS

FIG. 4. Scilenlatic representation of sites of action of growth retardants on GA biosynthetic pathww

isolated from cucurbitaceae has also a pronou.nced anri GA activity and rcdvces GA,- induced plant growth"'. Thus, these retardants, having high specificity of action limited to one single slcp, can serve as very u s e f ~ ~ l tools in studies of GA physiology. Trcatnent with CCC resalts rn increased cold-hardiness of winter wheat, cabbage, and peas'?. This may be atiributed to CCC-ind~iced rcdr!ction in GA levels, which controls leaf size and sten1 growth, both of which are retarded in cold temperatnres.

7. Effects of environmental stress on GA metabolism

Environmental factors s x h as light, photoperiod, vernalization, wratiffcation and radiation bring aboxt quantitative and q11al:tative changes in GA levels in higher p!ants. fnvolvement of endogenom GAS in photoinorphogenic events have been proved con- vincingly. Inhibitory effects of red light on stem growrh in peas is attribl*.ted to the depression of endogenous GA levels in tissnes and such physio!ogical duarfism can be reversed by exogenous GASF3. On the other hand, expon8re to red ligtl stin~ulates ;he synthesis of extracLable bnt transient GA, in etiolated barleq'l and wheat" leaves. Elimi- nation of thik GA by incubafion of leaf sections with AMO-1618 or CCC, suggests that new synthesis of hormone is occurring. The appearance of diffi.sible GA IS increased in unilaterally i!hminated or geotropically stimulated sonilouer sf.ooi tips. dl!e to increased GA biosyntt-esis and transportR? GA-induced inhibition and pronlotion of sprooting in aerial tnbers is directly correlated wilh photoperiodic treatment and wber growth stages7. The phytochrome-mediated dormancy of cekry seeds responds lo GA,, and treated secds may germinate in dark at high temperatrresH8. GA synthesis is inhibited and biologically active acidic GAS areconverted into relalively inactive polar GAS o r GA--g!y.;osi.des in cold-hardened wheat". However, cold treatment triggers GA synthesis in tulip and plmn seeds? This suggests, that possible in'.eracting effect of vernalization on GA metabolism may be a complex phenon~enon. It is shown that mechanical stress given by moderate shaking also retards seedling growth and GA pro- dnction in P. ~w1gari.s".

ionizing radiations indxcc nnmerous histological and cytological changes in plants. Thongh DNA is shown as the primary site of damage'" the lesion and/or depletion of thc merkiem polulaiion, the siteof auxin and GA synthesis during gemination of seeds. is strikingly radio-sensitiveT3. Stiml!lation of mitotic activity in snboptical region of meristern and spindle orientation, preceding the cell elongation, are the earliest effects of GA treatment7$. Treatment with exogenous GA, reverses the inhibition of seedling growth in sub-lethally irradiated wheatG by stiml~lating tf e synthesisof mRNA7'or of pclydisperse RNA fraction and thereby lriggeringa-amylase synthesis". Hi& doses of gamma irradiation induces physiological dormancy. Loss oi'viability in il-radlated see& is attributed to the metabolic disturbances in the biosyntlretic events lead;% to protein synthesis7%wh1ch controls the in'racell1~.lar GA concentration. G h is degraded by gamma-irradiation in v i r w and in vi fr07~. Jncorporation cf radioa~tive precursors in GA,, the most bioactive GA derivative in wheat, is reduced in gamma-irradiated wheat d ~ r i n g germniation and the responseis dose-dependenPo. Initiations of RSA, DNA and protejn syuihcses are delayed during ea~ly stages of germination in irrad'ated wheat".

82 J. P. MALHAIAH A ~ D c. K. VAKIL

Dunham and Cherrys' have shownthat gamma-:rradiation inhibits de n o w synthesis of RNA polymerase, subsequently leading t o reducedRNA formation in sugar beer tissues. Gamma izradiation also affects the transcription and translation processes leading to altered protein conformarions3. Recenily, ii has been reporteds4 that gamma- irradiation stimulates repair mechanisn~ in barley aleurones and this is accelerated by GA treatment. The underisin$ mechanism which adversely affects tke slrncture and fmction of GAS, is elucidated in irradiated wheat dnringgermination. The activities of MVA kinase, ATPase and kaurene synthetase, involved in various intermediary steps leading to GA synthesis from precursorp. areimpaired due to radiation treatment o f wheat (Fig. 5):8. Further. efficiency c f interconversion of a less active G A to a highly aciive one is adversely affected. This resum in acccmu1a;ion of GA, and GA,, wh:ch are biologically less active intermed;ate precnrsor products on GA bjosynthetic p a t h ~ a y leading to GA, format i~n '~ . Differential requirements fcr GA, to stimulate the synthesis of total and of three majot a-amylase isoenzymes (ax , a2, a3) are noted in germ!. na,ing control and irradiated ~eecis. a,-;soenzyme 's the most rad;olabi!e and does not appear in germinating wheat, irradiated a t 200 krad. However, pre-treatizent ot seeds wirh GA triggers its formation (Fig. F). Th's si!ggests that two aggregate systems

FIG. 5. Individual peak activities of tF.e enzymes, reaching meximum during ~ermination were measured for MVA kinase (at 4 hr), ATPase (at 7 hr) and kaurene synthetase (at 56 hr). The results are ex- ~essed as % of tF.e activity in irradiated seeds taking control (in unirradiated seeds) as 100% activity.

GIBBERELLIN METABOLISM IN HIGHER PLANTS 83 differing in their radiosens;tivity and response to GA, application, are operating for the synthesis of functional alpha-amylase molecllle in germinating wheat8'.

FIG. 6. Effect of GA trextment on the formation of alpha-amylase isoenzymes in irradiated wheat. Control and irradiated (20 or 200 had) szeds were imbibed for 16 hr either with water or GA tion and for 4 days. Zymograms depict the polyacrylamide gel electrophoretic pattern of the isoenzymes stained with starch +I, solution.

8. Physiological role of GA

In recent years, GAS have been implicated in several biochemical reactions and shown to play an important role in physiological processes of diverse natnre. These include : breaking of dormancy, regulation of germination processes, control of synthesis and release of various hydrolysing enzymes, etc. The processes controlled and stimnlated by GA in germinating grain, are summarised in Fig 7.

8.1 . Dormancy and germination

Dormant seed embryos can be activated by the removal of seed coat, a physical barrier for cell expansion, hy increasing 0, supply and leaching out the endogenons inhibitors. Various environmental conditions like light, photoperiod, vernalization, etc., bring about changes in endogenous metabolism^. GAS are implicated in reversal of thermo- and phota-dormancy as well as osmotic dormancy imposed by mannitol". Jarvis et ala7 have suggested that GA, breaks seed dormancy by derepression of dormant, repressed gene in embryonic cells. Increase in mitotic activity, DNA template availability for transcription and ANA p3lymerase activity in embryonic axis, are the earliest visible

AMYLASES

Fro. 7. Schematic representation of biochemical reactions responding to GA action in germinating grains.

and measurable effects of GA treatmenta8; although the exact inter-relationship, is still obscure. Although GA, has no effect onendomitotic DNA synthesis, i t increases epi- cotyl DNA content and p~lyploidy of a celP" which is an important factor in the control of cell lengthq'. GA increases total RNA content and enhances DNA-depenhnt nuclear RNA synthesis in isolated pea and sugar beet nucleig'. Application of GA stimulates protein synthesir in shoot and root meristems and in the developing vasculsr tissues of charlock seedss2. Increase in the synthesis of cell wall, with concurrent elongation of kternodes in Averza stem, is propo~tional to GA, concentratlons3. This is attributed to stimulation of cell wall polysaccharide synthetase activ~ty by GA,, nhich in turn causes increased cell wall depositiona4. GA, stimulates de novo synthesis and release of hydrolyzing enzymes including a-amylase, ribonnclease and protease in aleu- mne layers" and other enzymes involved in intermediary metabolism in cotyledons (Fig. 7). Importance of GA, has been recognised in regulating the mobilisation and absorption of the solutes produced by the action of these enzymes for growth and development of embryos. However, GA,-induced inhibition in germination of various species has also been repxted. Existence of two counteractive reactions (inhibition and

GIBBWELLIN ME.T.ABOLISM IN HIGHER PLANTS 85

promotion of sprouting) activated by GA,, are shown in the penus Diosc~reo'~. Thesc reactions are related to light quality. In germinating D. tokoro, application of exogenous GA, inhibits the seedling growth in dark and red but promotes in blue and far red lights'.

8.2. Balance oJ hournones jbr germination

Though GAS are effective endogenous regulator?, it is now fairly v.e!l established tl-at appropriate balance between GA, growth promoters icytokiains and mdolacelic acid) and inhibito~s (e.g., abscisic acid (ABA)) is necessary for the regu1at;on ot grcwth and development of plant in nor~nal and stress conc';t ion~~~. Both ABA and GA are bic- synthesised from MVA by common isoprenoid pathway" and during the later stages of growth, ABA accumulates in the seed1". A11 GAS-stimulated germinative and enzymatic processes are strongly inhibited by ABA. ABA also blocks DNA and RNA syntheses and delays the onset of germination. Similarly, alpha-amylase and protease production in barley grains are inhibited by ABA and reversed by cytokinins but not hy GAlo1. These observations on competitive interactions between cytokinins and GA and non.competitive interactions of ABA and GAlo5 SUppori the prevalent inhibitor promoter hypothesisXoa. Recently, KhansB has suggested several passible hormonal situations, which can control dormancy and germination, depending upon physiologi- cally effeztive or non-effective levels of GA, cytokinins and inhi5itors. Thus, GA plays a primary role in germination, whereas inhibitors and cytokinins are secondary and essentially preventive m d permissive, respectively.

8.3. Carbohydpute metuBoEism

Besides the wel! defined primary action of GA in alpha-amylase formation for initiation of germination, the detailed characterisation of other carbohydrases is not achieved. However, GA is shown to increase de novo synthesis and release of laminarinase (,&(I -+ 3) glncanase), alpha-glucosidase and diastase activities in germinating bar!ejJ04

8.4 . Lipid metabolism

Role of GA in lipid meiaholism is elucidated by events occurring during lag phase of 2 to 8 hr picceding nearly germinuion, and therefore, camlot be considered as its primary action. GA, enhances the rate of W!-cho5nelo5 and 3'P"Bin~0rp0ration into phospho- lipid moieties of semi-purified endoplasmic reticulum of aleurone layers of germinating barley1". This is attributed to the activation of two key transferases, namely, phos- phoryl choline-cytidyl and pP.osphory1 choline glyceride, involved in lecithin b ios~nae - sislos. Similarly, GA stimulates the activities of the enzymes of gluconeogenic Pathnay in castor bean seedlingsus.

8.5. GA-induced changes in membrune properties

GA is implicated in the biogenesis of endoplasmic retic~lum'~!', nuclearmunbraner5, mitochondria1 cristae"1, and binding of polysoines to the endoplasmic membranes"'., GA, also stimulates extensive proiiferation of cellular membranes during early gsrmi

86 J. P. MhCHALAH AND U. K. VAXIL

nationn3. GA, is sho;+n to enhance the synthesis of marker enzymes of glyoxysomal and mitochondria1 (inner and outer) inernhi-anes in germinating almond seeds114. How- ever, GA, is shoun io inhibi~ cell-wall pentosail synthesis and membrane-bound arabi- nosy1 :ransferase ac~iricy. present in barley aleurone layerP3. A possible interaction between GA and phospholipids is shoun to govern the permeability and trans- meinbraze potential of jiposomal membranes'lG. GA, also affects the phase transition temperatwe ofliposomes as a function of concentration117. Recently, studies with dwarf maize mutant by eleclron microprobe anat)sis, have indicated that GA, selectively changes the permeability of aleurone plasmalemma and thereby regulates the inflvx of manova!eni (Nx, K-, C i ) and divalent (Mg'-+) ions'ls. Thus GA3 alters the micro- enviroilinent in difiermt cell compartments. The synergistic effects beineen GA and low pH compoands (i..g., EDTA), may be ria removal of inhibitors and/or alteration of membrane permeability through the chelaiing activity of EDTA'la. However, acid growth hlpothesis, which exp!ains auxin-induced elongation growth by cell-wall acid~ffcationl" is not applicable to GA3-induced elongation in lettuce hypocotyl ~ e c l i o n s l ~ ~ .

8.6. Role of G.P in alpha-amylase synihesis

GA-induced alpha-amylase synthesis is one of the best understood hormone-controlled me;hanis:ns in planis. In germinating barley and wheat, the enzyme is synthesised by the microsomal fraction12z of the aleurone layers in response to GAlZ3. GA-stimulated for:na!ion ofpalyribosomes is shown as a pre-requisite for the enzyme synthesis in aleurone layers. It has been observed that GA stimulates the synthesis of specific mRNA for a-amyiase indnciiou and accumulation of helerodispcrse minor RNA fractions*24. G.4 also activates some preformed RNA and inhibits the breakdoun of mRNA. Carl- son125 has postulated that GA regulates a t the post-transcriptional level. Houever, Chandral?"las suggested that GA can bring abont speciffc stmctnral modiffcations of the transcribed RNAs, and consequently, a translational modulation of prolein(s), with mo1e:ular heterogeneity f ~ l l o w s ~ ~ ' . Thus the effects of GA on synthesis, activation and stability of mRNA are not clearly distinguished.

B-sides GA, many naturally occurring compounds including abscisic acid (ABA), helminthosporal, (-) kaurene, cyclic 3'-5' AMP, and ATP as well as osmotic stress and concentration of hydrolytic prod~cts, are shown to reg~late alpha-amylase synthesis:$. ABA-inhibited alpha-amylase synthesis is conntera~ted by GA in barley128 and musiardl'%eedlings. Photoinactivation of the enzjme in barley on exposure to light, is corrdated with the chlorophyll content and a change in the endogenous GA- inhibitor balance13@.

The subcellular pathway of the release of alpha-amylase is not yet uell defined. Evi- dence for enzyme packaging in lysosome-like bodies in GA-treated wheat ale~!rones is reported131 but not confirmed in barleyls2. The hypothesis, that the enqme is trans- portedfrom the cytoplasm both inside and outside the cell by distinct secretory vesicles133,

GIBBERELLIV METETRBOLTSM IN HIGHER PLNTS 87

is not supported by cytological evidences. I t is snggestedl" that alpha-amylase beilip a soluble enzyme, is directly released from the cytoplasm across ibe alenronc cellplasra- lemma, without the involvement of discrete secretory organelles.

8.7. Ho~rnunul regulation of @ha-amylase bocnzymcs

Highly purified alpha-.amylase is selectively oblained by coiuplexing with gljcogen, thou& two apparently independent bivding sites on thc eiizynie are demons~rated for the formation of rnultimolccular complex135. Heterogeneity of a-amylases ?.ale been reported and 5 isoenzymes ar-c separated from barley13% 11socnz)mcs are sepal-a!cd by starch or polyacrylamide gel electrophorcsis, isoelectric Evcussing and by iimirnoc'iffu- tion inethodsL3~. Quantitative separation can be obtained by ion-exchange column chromatography using a linear salt gradient.

I t has been shown that in barley aleurones, GA-induced alpha-amjlase isoenzymes, synlhesised in vitro and in ~'ivo, are not similar'". Two sets of isoenzynes haviug different electrophoretic mobilities have been isolaled from germinaling bal-iey13H ; GA-(reat- ment accelerates the ronnation of all the isoen~yrnes'~\ Faster moving set of isoenzymes is comparable to green alpha-amylase of imrnatnre wheat kernel and their quaniiiy varier a t diWrent stages or seed development. I t is s h o ~ n to be responsible for louered amylograph viscosity and thus governs the ultimate quality of wheat""'.

Studies on physico-chemical properties of four alpha-an~ylase isoemyaes of malred wheat revealed that they have similar Km values, p H optima, activation energy and molecular weights, but different electrophoretic and irnrnunological properties141. In barley, seven alpha-amylare isoenzymes are present differing only in their net electrical charges'b3. High acidic aniino acid content of the engmc explains their stability in acidic pH. However, their individual contribution in governing lhe rheological and bread making properties of wheat dough are no1 known. The failures to interconvert alpha-amylase isoenzyines or lo ilnplicaie them as preparation artifacts, si!@x!s that they have differen1 pnetic origin. The isoenzy~nes from barley, \!heat and maize reflect saine differences in their tuolecular characteristics, which result from base pair a1:era- tions in their structural genes'43. Multiple loci controlling alpha- and beta-amylase isoenzymes have been reported in wheat'4i. Furlria ct all4; hale conchded that no appreciable changes in the nuclear DNA content of A and B genornes of co~nlnon u,heat have o3c irred during the entire course of evolution. The genetic characteristics of alpha. amylase isoenzymes from genuine and reconstitnted strains of tetra and hexaploid uheat are compared and used to analyse the genetic regulatory ~nechanism in p l a n t ~ ' ~ t The illdirect fluorescent antibody techniq~~.e is used lo analyse the celiular and inrracelliriar localization of the enzyme more precisely alld this helps to screcn mutation in p I a n l ~ ~ * ~ .

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77. ZWAR, 3. A. AND Plan1 i%ysi01., 1972, 49, 1000. JACOBSEN, 3. V.

78. MACHAW, J. P., VAKIL, E ~ V ~ I . O I I . Exptl. Bot., 1976, 16, 131. U. K. AND SREEXVASAN, A.

79. SWERI~, E. G., NAWAR, Rarliaf. Bof., 1971, 11, 309. M. M. AND NILAN, R. A.

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94. MowAGUB, M. J. m Plant Physiol., 1978, 62, 391. '

IINMA, H.

GIRBERELLIN METABOLISM IN H I G I m PLANTS

97. OKAGAMI, N. AND KAWAI, M.

100. KING, R. w. 101. W N , A. A.

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103. WEBB, D. P., VANSTADEN, I. AND WARBING, P. F.

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KENDE, H.

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LORD, I. M.

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BOOK REVIEWS

The Study of Behaviour by J. D. Carthy, revised by Philip E. Howse. The lnstitnte of Biobgy's Srudies in Biology, No. 3. Publislied by Edward Arnold, London. Second edition, 1979, pp 68; price f 1,913.

This is one of over a hundred short books on specific topics in biology sponsored by the Institute of Biology. It is meant to provide an authoritative and an up-to-date view of the current status of the study of behaviour in simple 1a"nguage and with examples designed to encourage the reader to begin his or her own investigations. 1 found it a useful account for a beginner, covering a whole variety of topics: orientation and navi- gation, sensory factors, courtship, rhythm, hormones andpheromones, learning, social behaviour and behaviour and survival. Many of the interesting facts recently discovered about these topics have been incorporated in the account and are well summarized.

Unfortunately, the book lacks a unifying framework. It treats the various facets of animal behaviour in isolation, and makes only the most cursory attempts to acquaint the reader with the exciting new conceptual developments that have taken piace in the study of behaviour over the last fifteen years. These developments have flown from a clearer understanding of the process of adaptation through natural selection, following the work of W. D. Hamilton, J. Maynard Smith, G. C. Williams and IE. L. Trivers. In particular, it is now accepted that natural selection acls primarily at the level of the individual. Howse seems quite unaware of this crucial point, regardless of his reference to the ideas on evolution of altruism. For on page 57 he says ' on the whole, a hierar- chial system decreases the amount of inter-individual aggression. . . in a group . . . This is advantageous because Bghting.. . .takes up time which is wasted from the

of view of the group.' This major failure means that the book is not an up-to- date and atrthoritative view of the study of behaviour at all; rather i~ is a useful, but conceptually outdated review of a variety of topics related to behaviour.

MAnmv G m

Introdnction to Experimental Ecology by T. Lewis and L. R. Taylor. Published by ELBS and Academic Press, London. ELBS edn., 1979, pp xi 4- 401 ; price £2.80.

This is the low-priced edition of one of the most popular books on experimental ecology. Meant for both students and teachers, the book shows in an elegant manner, how simple observations on various living forms in the surroundings can be made and how to inter- pret them. Many of these experiments can easiiy be conducted by high school students

97

98 BOOK REVIEWS

which will make them rake a livelier interest in their surroundings. Since this is supposed to be an introduc!or! book in experimental ecoiogy excessive theory has been avoided hut has been foriifled with a Large number of numerical examples. The authors have also done well to caution the student aginst the common pitfalls he should guard against.

After a brief introduction on the scope and principles of ecology the book deals sub- stantially wiih the basir ana!)tical methods in ecology. Then follows a fairly long list of experiments both sb.orr term and long term which can be trdy enlightening. The authors !labe provided i~ small key to these experiments classifying them based on sub- ject and illore interestingly on spec& requiremenrs like apparatus needed, season, site* labour, duration, etc. A resourcefiii teacher aith motivated students can proiltably use rhe concepts developed in the hook for exploring the region. Most of the colleges in lndia lack the saphii:isated equipment needed for the modern biochemical students. These colleges c:m take up intensive studies on the local flora and fauna, which in the long and the short rlin can benei?t the community a great deal.

Unfort~mately the hook has a defeci in the sense that all the exercises listed are centered around animals and ~nostly insects. Concedmg the fact that these experiments are more easily conxived and condgcted to convey an ecological concept, several neat experiments involving plants covld have been included. I would think experiments on dele- pathic interaction of plants, soil characters affecting seed germination and growth, light and shade on the response of plants to mention a few must have found a place in a book of this type.

The book also contains a number of appendices useful to an experimental ecology student and a subjecr index. Bnt I should also say even the low-priced edition is beyond the reach of an Indian student.

ANNOUNCEMENTS

World Congress on brain in health and disease

The first World Congress of the International Brain Resear& Organisation (IBRO) will be held a1 Lai~ssnne, Switzerland, from April I to 6, 1982. The Congress will bring lo,7:the; n:xoi:ientists and slinical neurologists from all over the world to discuss the latest developmenfsin braln research and their clinical application.. The Congress will d i m s s developments in the following fields : ( I ) Nenromatomy, (2) Neoro- chemistry, (3) Neiiroendosrii~olog~~, (4) Nei!ropharmacoiogy, ( 5 ) Nenrophysiology, (6) Behavioural sciences, (7 ) Neurocornmmicatior.~ and biophysics, (8) Brain pathology, (9) Clinical and health-related brain science.

Those desirous of participating and or presenting papers should contact Prof. L. J. Garey a t the Institute of Anatomy, University of Lausanne, Rue de Bugnon 9, 10 1 1, Lausanne, Switzerland. The organisers are hopeful of extending limited financial support for travel and accommodation.

International coIIoquium on lipid metabolism and its pathology

An in'ernational colloquiun~ on lipid ineraholism and its pathology will be held a t Lisbon, Portugal, kom December 8 lo 11, 1980. The colloquinn~ which will act as an international forum will focus attention on lipoprotein structure and metabolism, hiperlipemies, lipids and coagulation, phospolips, lipidosis and techniques of lipid study. On December 12 and 13, 1980, a practical course on lipid determining techniques on hiperlipemies screening, high resolution thin layer chromatography, gas chromatography, ultracentrif~tgation, electrophoresis and methods for study of apopro- toins and HDL cholesterol will be conducted, For the benefit of the participants, an exhibition of eqg!ipment and reagen!s ~ised in lipid biochemislry will also be hold. Further details regarding the colloquium ran be had from Professor Manuel JOdice Halpern, Departmento de Bioquimica, FacuXade de Ciencias Medicas da UNL, Canlpo dos Mirtires da Pitria, !LOO, Lisboa, Portugal.

Journal of the

Indian Institute of Science Volume 61 (C), 1979

I N D E X

Indian Institute of Science Bangalore 560 012

JOURNAL OF THE INDIAN INSTITUTE OF SCIENCE

Volume 6 1, January-December 1979

SECTION C

Title Index

Adaptive significance ol' the relation between soot and shoot growth MADHAV GADGIL AND SULOCHANA GADG~L 25

Gibberellin metabolism and regulation of a-amylase isoenzymes in higher plants J . P. MACFIAIAH AND U. K. VARIL 73

Regulation of glutamine synlhetase activity S. SEETH.ALAKSHMI, C. S. VAIDYA~ATHAN AND N. APPAJI RAO 1

Studies on some HMP pathway enzymes in gul mutants of Aspergillus itidiilmzs S. MALAWI AND E. R . B. SHAN~~UCA- SUNDARAM 67

Toxicity and persistence of effectiveness of some organophosphorns insecticides

against green aphid Aphis gossypij glover on apple l e n e s S. F. HAMEBD 4 N D C. L. DIVABANDHOO

41

Tryptophan-pl~cnylpyruvnie aminotransferase of Agrobnclerhtni tu??lifaciens : Puri- fication and general aronerties of the . a

enzymes N. K. SUKANYA AND C. S. VADYA- NATHAN 51

Variations in arginase and OTCase levels dnsing growth in A s p e ~ g i h s nidzi lm NECRAJA RAMAKRISHNAN AND E. R. B. SHANML~GASUNDARAM 63

X-anti-iz bodies H. SESHI

Author Index

APPAJI RAO, N. Adaptive significance of the relatioil S'M SEETHALARSHMI. S.. VAIDYANATIIAV. between KlOt and shoot growth 25 ... - -

C . S. AND APPAJI RAO, N. 1 GADCIL, SULOCHANA

DINABANDHO~, C. L. See Gadgil. Madhav and Gadsil. Spe Hainecd, S. F. and Dinabandhoo, Sulochsna 25 c1 T dl C . Li.

HAMEED, S. F. AND DINMANDI~OO, C. L. GADCIL, MADRAV AW GATIGIL, Su1.0- Toxicity and persistence of effectiveness CIXAN A of some organophosphorus insecticides

104 INDEX

against green aphid Aphis gossypii glover on apple leaves 41

MACHAIAH, J. P. AXD VAKIL, U. K. Gibberellin metabolism and regulation of a-anylase isoenz>mes in higher plants

73

E. R. B. Studies on some HMP pathway enzymes in gal mutants of Aspergillvs izidzrlaw

67

RAXIAKRISHNAN, NEERAJA AND SHANMGASUNDARA~U, E. R. B. Variations in arginase and OTCase levels during growth in AsprrgiNus nidzilars

63

SERTHALAKSHMI, S., VAWYANATHAN, C. S. AND APPAJI RAO, N. Regulation of glutamine synthetase activity 1

Alpha-amylase Aminotransferase Antibodies Antigens Anti-n bodies Arginase

Biogenesis

Environmental stress

Gal mutants Galactose Genetic studies Gibberellins Crlutamine synthetw

X-anti-n bcdies 21

SHANMUGASUNDARAM, E. R. B. See Malathi, S. and Shanmugasandaram, E. R. B. 67

See Ramakrishnan, Neeraja, and Shanmngasundaram, E. R. B. 63

SUKANYA, N. K. AND VAIDYANATHAN, C. S. Tryptophan-phenplpyruvate aminotransferase of Agrobacterltmn tumifaciens : Purification and general properties of the enzyme 51

VAIDYANATHAN, C. S. See Seethalakshmi, S., Vaidyanathan, C. S. and Appaji Rao, N. Z

See Sukanya, N. K . and Vaidyanathan, C. S. 51

VAKIL, U. K. See Machaiah, J. P. and Vakil, U. K. 73

Key Word Index

73 Isoenzymes 5 1 ?! Microorganisms 41 21 Nitrogen sources 63

OTCase levels 73

Phenotype strategies 73 Physiological role

Plant tumonrs 67 67 Regulation 67 Resource allocation 73 Root-to-shoot ratio

Transamination

INDEX

Book Review Index

Introduction to exverimeutal ecology by bv T. N. Pattabiraman 47 T. Lewis and L. R. Taylor, reviewed by V. N. Vasantharajan 97 The study of behaviour by J. D. Carthy,

revised hy Philip E. Howse, revie~ed by Microbial ecology of the g ~ l t edited by Madhav Gadgil 97 R. T. J. Clarke and T. Bauchop, reviewed

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