REVIEW ARTICLE NUMBER 23

ROLE AND REGULATION OF L-GLUTAMATE DEHYDROGENASE ACTIVITY IN HIGHER PLANTS

H S. SRIV*~TAVA and RANA P. SINGH

Dcpnmenr of &o-W M D Unwcwty. Rohtak IZWI. lndu

(KKdlon!i 7 July 19%)

Key Wad Ida -Ammo ocxd rynthuaammonu asumdatlon. cnzymc n~nty. enzyme rqulatcon. higher plants. L-~luurmlc &hydro#abuc

-- -- - AUrut -L-Glutamate dehydrogenase aulysa the rrnniblc convcruon of 2-oxoglutaratc and ~-glutamate for the enrty of l mmoruum Into he orpuc cyck and for IU relax as well. Various ~sozymes of GDH are present ukqultously IO higher plant tusuu The enzyme. wllh a mokcular weight of 208ooO IO 27OooO. IS compovd of four IO SIX subunltr contams a free -SH group at the act~yc cent% and u assocmtd wth me~sl ION. Some ~lozymu of GDH are mdmbk and vary l ocordmg (0 the outritiorvl and ennroomental status of the tissues. The kvcl and actlnty of enzyme IS c~ther dirac~too II regulated by age. light/dark regime, morganic and or+ mtrogco. carboo and energy IUUIS growrth regukton rod some other frtors. The enzyme seems 10 be important m asurmlatlon of ammonba under strtu conditions such u dark starvation. htgh temperature. sabm~y. waler stress, ennronmental pollution. senextl~t and other l bnormabt~~.

- --_- --- ..- .-

I~ODlJC-lION

TIK morganc mtrosn acquired by plao~s 1s ult~ma~cly cooverted lo ammonium before bemg incorporated into orgamc moleculu. Although several hochemical rac- tlonr mvolvmg ammomum as a reactant are koowo [I]. the reductive umnal~on of 2~xoglutantc IO gluumic aad has long been considered u a mqor route of ammonu uurmlatlon. This revcruble ractlon IS a~- lysed by the enzyme L-gJutunrte debydropnw [L- gh~urnate NAD’/NAD(P)’ or NADP’ oxldoraluaw (daminatmg) GDH. EC I.4.1.24]. In the sevenlia. however. the discovery of a oew enzyme. glutamate synlhue. from bacteru [ 21 and hves of higher plaots [ 3 ] chaogaI this conapl and II IS now recognlLcd that ammomum 1s first mcorpora~cd Into glutamme by the actton of glutammc syotheuu (L-glutamate~ammonu hga.se. GS. EC 6.3. I 2)and rubsequco~ly Into glutamc rcrd

by glulamale synthase [ L-gluI.amale.

NAD(P)‘/fenedoxm oxldoreductase. GOGAT. EC I4.1.1>14 rod 1.4.7 I] (41

The path of ammoma uurmlatlon. mcludmg studla on GDH. has ban reviewed (l.ClO] However, the phyuolo@ role of large amounts of GDH present In the IIUUCS of higher plants u s1111 obxure Although the GS GOGAT prthwry 11 considered IO be the maJor route for ammoma uumllatloo m plants under oonrul growth CO~~IIIOIU the role of GDH under some envlroomental and nuinl~o~l conditloos cannot be excluded sod. there- fore. the posslbk factors under whKh GDH may plry a slgruficanl ammatIng or dcmxu~oo~ role IO ai! met&e lism are yet 10 be discussed. Moreover, oumerous new obscnrtio~ have appeared follownag the rtV*w on plant GDH by Stewart TV al [9] Our aim In thts artck IS lo

revlcw the bteraturc on htgher plant GDH and IO evslua~e the possJble phyuolo~cal rok of the enzyme III the nitrogen metabohrm of higher plaotr under dllTcrcnt envlronmental and nutrltlonal condltlons.

DISTRIDUTION A%D LOCALIZATION

L-Glutrma~c dehydrogeruse has been found IO be present umvcrsally in almost all types of organisms from mKrobes IO higher plants and ammals In higher plants, the enzyme activity has been detected m almost all spccla E3lCd

Organ splcJIc 5pecrra

Although the enzyme IS frequently dlstrlbuted In va- nous plant pans. IIS level and bhavlour appear IO k orgrn specific The rmmarmg (NADHdependcnt) enzyme act~nry IS hlghcr In roots than In leaves of Zca (Il. 121. SK& [I)] and Hordtum [l4] In PISIM and Pamwco. high GDH 1s found In the area of rapld growth and high phytochromc content [ 151 and In Amchts secdlmgs. the enzyme actlnty IS higher m the co~ykdons than In root or shoot portions [ 16) Further. the develop tng pods and seeds of PhPuolus and CaJanw possess higher GDH UIIVII~ than the leaves [ I?]. It may be noted that the conanlralion of ammomum In the seeds of CUJMW IS also grater than that In the kava The enzyme actrnty also vanes accordmg IO the age and tissue compoatlon of the orgao. For eumplc. In Zeo roots the enzyme ia much more LCIIVC In mature repon, [ 18. 191 rod In mcnsrems thrn In rpmI rwonr [ 18). In develop mg Hordrw grams. GDH actlvlty IS largely confined IO

Yam H S SRIVAS~AVA and R P SINW

the cndosperm tn contrast IO GS whrh 1s mostly m the testa pencarp [ 201.

The enzyme present m dtffercot nuues may be dtffercnt In IIS Isozymtc pattern and consequeotly In the regulatory nature In Trrruwn. the predommant form of the enzyme tn senescent leaves 1s a different tsozymc than the one present m young leaves (21) In Rrcuxw. the leaf chloro- plast and root enzymes scgrepte from the endosperm enrymc on polyacrytamtdc gel clatrophorcsts [22]. Organ spcoftc ~sorymes are also reported tn Mtduqo.

although they show stmtlar general and kmctlc propentcs [23] In <;~JWW roots and nodules, the enzyme 1s re- gulateddttfercnttally by urea (241. Further. the pnttcrnsof GDH tsozymcs arc different m nodules and roots of Lup~nu~ [ 25) Loyola-Varglr and de Jtmena [26] have reported the presence of dlfTercnt tsoorymes;conformen of GDH In root. callus and lava of Zeo which may vary depending on the nutrttlonal requtrement and state of dltfercnttatton II has ban suggcstcd that the character- ~sttc rpcc~ra of GDH tsoryma m the cndospcrm. embryo and pertcarp of Zto may be due to dtfferentul activity of gcnacontrollmg the enzyme syntheses m each ttuuc [27]

In Zea. C;omphrtna and Sorghum. NADH-GDH II found IO be loalized IO bundle sheath alb [26]. Further. m vartous C.-spa~cs 69-87 “/a of NADH-GDH IS equally dlstrtbuted tutwan mcsophyll and bondk sheath cells [ 2Y] This finding 1s further confirmed by Hare1 l f al. [ 301 In greening Zeo leaves and by Moore and Black [3l] tn Dryrrarra luves In nodulatcd p&nts the rekttvc dutrt- button of GDH shows three possible types of patterns (I) the enzyme 1s dtstrtbuted equally tn cytosol and bactenod fracttons in Luprnw [ 321. (II) tt 1s prtmartly loated IO the cytosol fraction tn Glycrnr [ 331 and tn Mtduago [ 341 and

(III) tt 1s higher m bactcrtods than tn cytosol m PIuueolw 1351 A htstologtcal study shows that a higher kvcl of GDH 1s present In the cells surroundmg the nod&s m Lupmus rools [ 36)

Inrrrbctllular Iocaitrarion

Vartous studies. using dtffertntral ccotnfupttoo and density gradunt tahntques. have demonstrated a mtto- chondrtal locatton of the cnxymc [22. 36471. Sma the cntymc IS eastly dissolved. It IS thought to k loalued IO the dtssolved matrix of the mttochoodna [48-501. However. the method of dtsrupttng mnochondria m- volvcd a frau/thaw treatment followed by sontatroo tn most cues. whwh could curly dtsaolvc the membranes. Yamaya tr d [46] usmg a gcotle durupttoo method (osmottc shock) demonstrated that the enzyme II loosely bound wtth the mttochondnal membrane. On the baats of Anhcmus plots of GDH acttvtty from Mayold Glycrrv axes and 11s analogy to that of Ytt~raa suocmatc oxrdaac (both consrst of thra phases), Duke tr al. [5I] deduced

that GDH 1s assocutcd wnh tncmbranc I~prds. A separate GDH species dtffcrmg m ammo propcnwr from tbc mttochondrul enzyme has ban charrtcrued from the chloroplasts of many plants [ 22.29.39.42.52-U). This enzyme 1s shown to be tightly bouod to the cbloroplut lamcllac. and cannot be relaaal by osmotrc shock. It can, however. be dtssolvai by a dctergcot tratmcot [52]. There IS some cvdena for assocxatton of GDH acttvrty with the plasttds of roots ax well (55.561. Many workers

have reported the occurrena of GDH acrlwty In the supcnutant fraction m addttton to mttochondnal fractton [ 18.54,57,58]. It has ban fouod that enzymes from both frrtrons poucaa Uuular physrcal and kmctrc propcrttcs. and hence It 1s suggested that the cytoplasmw enzyme 1s due to leakage from mrtochondna [57] However. CVI- detkz for a separate cytoplasmrc enzyme dtflermg In wrtun ktoetrc propcrt~a and tsofymc pattern has been dctnotutratal by some workers [ II. 4l.%]

Slruclwt and imzjmt palftrn

The structure of GDH from ammal l od fungal systems has ban cxtctutvcly studied and the htcrature has ban adoqustely rcvtcwod [5. IO]. A general account of the plant enzyme was also presented by Stewart tr ol. m 1980 [O]. The present dtscuasJon. therefore. will k ltmttal IO more rocort~ dcvclopmcotr IO the area. The plant enzyme IS thought to be a rnetalloprotcm hvmg an M, tn the range 208 -27OooO (41. The ctuymc 1s composed of tdenttcal subunlts hanog M, IO the range of 46OtW58 %O. There IS some ducrepoocy in the lttcnture regarding the number of subumu. As IIX elatrophorcttc bands appear after uosa-ltoktag of the enzyme wtth dnmtdatcs from Ptrum seeds. the enzyme 1s suggested to be a hcxamer [59] However, Schetd tf al. (601 using sodium dodayl sul- phate gel and by EM obscrvattons have demonstrated that GDH from &mna and PISUW sads 1s a tctramcr (M, 230000) wtth four tdcnttal subumts (M, 58 500) Although the hcxamcrac and tetramenc conhguratrons of the enzyme have ban reported from various orynisms [O]. further tovattyttonr uung modern tahmqucs arc required to cluadate whctbcr c~yma of two different configuntrons exist in vartous plant ~usucs. Schctd tr al [60] fuvc reported that the am100 actd composttton of the tetratncnc enzyme from LM~M and Puw 1s stmtlar to those of varrous hexamcrtc GDHs obtamcd from other organums and the /V-tcrtntnal ammo actd of the Ptrw enzyme II alatune. Further. the enzyme 1s constdercd IO contam fra -SH groups at act~vc ccntrcs [6l] which arc l pprcotly more abundant m the enzyme from hght growo Phastolus lava than In those from dark grown lavu [62]. The prescoa of Zn’ ’ as an assoctatcd metal ton u reported for Zta root [63] l od Lycop~s~ctun chloroplut [64] cnzymcs. Other dtvalcnt uttons such as Ca”. Mn”. Co”. Fe” also acttvatc the cnryotc acttvity [9].

Isozymtc number of the enzyme varies wtth plant spccla as well u other nutnttonal and cnvtronmcntal conditions. Scvcn isozymic forms have ban reported in Puum (60.65.66). Phpvolw [ 381. YICU, [ 671 and RICI~US [68]. The Dumber of ~soryara. however. 1s Increased by the l ddrtton of ammonium [25.58.69-711 and ammo act6 (25.721. and docrasod by the addttton of sucrose [25]. Founan ~sozyma have ban reported m Puwn [73]. Me&* (74) and Lupinw [ 251 and the number mcraacs to I7 IO young Pi.sum shoots prcmcubtcd wtth tap water [49]. The number l od banding pattern of isozymesarc also mgueoczd by growth stages [ 25.65. 741. pkot organs [23. 531. ottrogcn nutrttton [25. 58. 76721 Itghtdrk rzymt [70,7577J, carbohydrate status r25.66. 78) l od effazt~vc symbtosts wtth Rhlrobww

[25. 791.

Gluumr~c dehydrgnuc In higher plants 599

In legumes. 11 has ban su~tcd that the GDH lsozymc patIern 1s the result of an adaputlon of the cdl to mtrogcn meubohsm [25.72]. lndlv1dual plant organs may develop a typrcal GDH lsozyme pattern lor each organ dunng the growth stages whch may be correlated wvlth the known metabolr •CIIVI~ICS of those orpns as well as with external nuInt1onal and cnv1ronmental condltlons avallabk to the plant. This reveals a physrotop- 14 rok of GDH lsozymes 1n the rcsulatlon of nitrogen mttabohsm A hypothesis has ban proposed for the anabol~c and catabolr role of GDH Isozymcs rn plants (25.68.73.741 and Masurowa er al (25) have dcm- onstratcd m Lup~nus that l nabohc and utabolw forms of GDH rsozymcs may be present m IWO types of mrtochon. drrr. heavy muochondna scdlmcn1mg at w)(w)@ with bactermd fraction of the aoduks contain anabolrc ISO- zymes. and light mltochoodru sajrmcnttng II 2OOOOy contain both anabolrc and a~abohc Isozymcs DIflcrcnccs tn cnrymattc content bctwan heavy and light mltochon- dru [80] and the prcscna of rapnatory and synthcstnng mltcuhondru [8l) from higher plant t~ssucs have also ban shown. l-urthcr. cvldencc for glutamate synthesis m IsOlrlCd mltochondna has also ban prexntcd [46.49.82] H0wcvcr.m Mtdvoqoumllar kmcuc proper. IICI of the IWO forms of GDH Isoorymcs, prcvlously proposed as forms rcflcctmp dlfTerent physlologlal func- tlons [23.74] suggest thaw this hypothews may not hold unrvcrsally. or even m kgumcs Hence the proposal of Masuruwa t1 ol [25] should be tested carefully m other plants also PIsum GDH Isozymcs arc reported to be the same molaulc with dlflerent types of subunits [83] On the basis of gent analysis. II has also been suggcstcd thaI IWO non-allehc genes are raponslbk for the synthcsls of two types of subunus whrh could arbltrarlly assoclatc ma hexamcnc wmplcx glvm6 rise to several Isozymcs [&I] However. the cxlstcna of dehmtc Isozymcs rn Rtc~nus whrch do no1 change after reprcclpnarlon and rcckctro- phorcrls of the enzyme [68] and the prevna of Idental subunits In the Lmnu and Puum enzymes [60] do not suppon thls Idea

Coen:ymr sprcrficrr r. wartcon mrchancsm crnd ktnrrw brhob~our

Mult~plc forms of steady state and mduablc GDH enzyme from various organs of hiuhcr plants un use NADH and NADPH m ammattng and NAD ’ as well u NADP’ In dammatmg. as cocnzyma Most of the studms reveal that the cnzyn~ UIIIKU NADH and NAD’ prcfertnlully over NADPH and NADP’ to varymg dcyas [8.22.68.8>89]. In developing sads of Clpww three forms of GDH hav1ng d1flcrcnI nuckotldc spcs~- bclty have ban 1dcntihai[ 531. GDH-I. a md enzyme, IS active with both NADH and NADPH 1n ammatmg reaction and rcad1ly uses NAD’ and NADP’ In dam,- nation. GDH-2. a chloroplastw enzyme prefers NADPH in ammaboo buI NAD ’ m dcunttutton whllc GDH-3. a mltochondrul enzyme. 1s spaclfic for NAD’.

The rcutlon axocharusm of plant GDH u proposed u the compulsory ordered binding of NADH. 2-0x@ glutante and ammoma fdlowal by the ordered rckuc d glutamate and NAD’ and VKX versa for ntbcr brcctroa [90 921. However. a partsally random [23.93] and even a fully raodom [94] mechaIurm for the GDH atalyscd rcactlon have also been sugga~ai. The kmctyJ of GDH atalyscd ratloos have ban examined in several plantx

(91. II has ban 10Lcatcd that the aminatroS enzyme hu an unusually high K, for ammomum (nogcs from 5.2-70.0 mM). th fact often wed to qucatioo tbc ammat- 106 rok of the enzyme IO plant t~asua [I. 61. However. morcrcanIsIudksrevalth~~hc K,valuaforNH.‘and even for other substrata may be iollucocod by various external and mternal facton a~xd th value at~matcd may not be a true retition of wtut anfit be hpputing in sifu It has ban shown that IRK atalytr eff~~ncy of GDH ~nc~uses at low ammoruum aod low NADH cooanfra- I1ons. I c. K,,; dccra.ses by many fold [22.46.49] The

afyparcni K hAOH values are also reduad at low NADH [ 23.461. and oonudcrably at bw ammomum conantra- tlons [ 221 II may be noted Ihereforc that various kvcls of substrates for am1~1106 activity reveal a compkx k1nctK bchavlour of the enzyme wb~cb u highly adapttve for the substrate kvel The k1nctKs of the ammat1oo reaction of Luputw nodule enzyme arc also mllucnad by aaom-

pPnylng anions of NH; salts. as normal M~chacl~s Menton kloebca were obtomal with (NH,l,SO, whllc NH,Cl and ammoo~um aatate showed substraIc mhlblt1oa [OJ. 961. It baa ban suogcrtcd that anions bmd IO an allostcnc YIC on the enzyme aod ausc a change tn some of the rate constants of the rcutlon. I c. decrease the rates of wt1on of NADH and 2- oxoglutaratc wrth the enzyme and ~nc~casc the raIe of dssocutlon of NAD’

It has also ban suggested thaw SO: added as (NH,l,SO, acts as an activator of lhc enzyme (Y7] Further. K ,,u; 1s deaascd about seven-fold when the pH IS mcrcad from 7 3 to 9.0 tn green Nrcotrwm allus [98) In Phawolw kava GDH hu lower KICK value for the enzyme from dark grown lavcs than that from I&II grown and II has ban suggested that a more acuve GDH species IS generated In the dark [99] Ncsselhut and Harnlrhfcger [ 1001 and Furuhuhl and Takahash1 [98] have mdlcltal that the actual substrate for GDH may be NH, rather than NH;and replottlogof thedata for NH, conanIrat)ons reduas the KNn, value. However. II IS d1fliculI IO conclude tht a real subrtntc for plant GDH 1s NH, The spad with whrh l mmoItIa reacts with water makes II ddlicult to examme thu hypothests However. the assocutron ot GDH with mttochondrta may provdc a Itptd-rrch ‘mtuoclrmrte’ m which NH, IS more readily avallabk to the dehydropDue [82]

hturatlon curves for thra substrates IO NADH-GDH showed normal M~chael~r Menton kmctlcs In the pre- scnccof I mMCi” .andmthcabacnaofCa”anurkcd substn~c InhIbItIon was obscrvad [46.98) Sma. plant

mltochondru conum sumnt Ca’ * for this ruvatmn [46] II ~sev~dent that plant GDH ~salradyact~vatod with regard to Ca’ ‘. Moore l od Akcrman [IOI] have sug- gcslcd that cp” u a modulator of the m1tochondrol NADH dehydropcnutr UI UIW. as c’o” increases NADHox1datlon m ffdtan~hus Thae obscrvatrons. however. reveal a compkx kmetrc bchvlour ol the enzyme rn plant mltochondru and IIS mtegratcd lunclion naxls t’urthcr clarlfK.atlon

lltCULATlOh

Pbu agt and rhyOunuuy

Lcds 01 plant GDH vary roEordtnS to plant age A general mcrcasc rn enzyme actrv~ty hc ban obacrvcd by several workers In roou (25. 102. 1031. hypouttyl

600 H 5 SRIVUT~V~ and R P SINW

t 251. cotyladonr [67]. shoots [103] and Lava 99.102. IoClO6]. during thctr mttml growth phase

However, the per-rod taken for enzyme mductroo appears to vary wrth plant rpcc~~ and ts possrbly afTacted by other factors as well. On the other hand. m plant allcultura behnour of the enzyme seems to be ddTcrent For caampk. II rcrnaror unchanged dunng the mrtral growth perrod m Nicoruana [ 1071 and Cknopodrum [ 1081 all cultures and mcrcua during later phasea. I.C after 33 days Further, m Zeo [26] and m Soccharum [ 1091 allur GDH acbvlty doxascs consdtrably dunng the mrtvl perrod of growth l od dlffcrcnUuoo on MS medrum. but for non-shoot-formtogallur of s4tch4nun GDH actlvtty 111craacs conudmbly between 4-9 days of mcubatron and replaces the GS-GOGAT route for ammoout LUIIIII- latron [IW] In Lup~nw scullmgs. mdrvrdual organs dcvelopa GDH rsorymc pattern ~ypuzal for the metabolic requrremcnt of each organ during growth [25]

The enzyme IS last studied In mature plants; In C’IWUS trees. GDH kvcls Increase at the time of flowtrmg and frurtmg when the plants are suppl~cd with nitrate as an N- soura [ 1 IO] Further. m YIQM gcnotypesassoctatai with ddlercnt rhlrobul strams the enzyme actlvlty mcrcas~s consrdcrablyat arly flowering and early pod filling stages This mcrasc IS most pronounad rn genotype Ks,, assoctatai utth stram SI, [I I I] However. rn Addopsu roots and luvcs both NADH- and NAD’-GDH act- t~r~t~cs remamcd relatrvelyconstant untrl thedevelopment of Aowcr buds and then mcrcascd. accompanral by a gradual daxasc rn GS activity [ IO61

GDHactrv~tyaIsochangesdurmgsaddevclopment In 7iutcw. enzyme act~r~ty starts mcrasing I5 days after anthcsrs and reaches maximum at 40 days with three consprcuous praks a~ 25. 40 and 55 days (201 During development of P~turn embryos. the enzyme activity reaches Its hrughest point m the seed coat at 27 days and In the cotykdons at 40 days (112) A higher enzyme conantratron In the sad coat IS possrbly ~s.soc~tcd wrth a higher ammomum content. as hs ban reported in PhaKolw [I 131. l~p~nw ( II41 and Arum [I 15. II61 In drllcrent mutants of Zca. GDH acttrrty increased during kernel development bctwan I5 and 35 drys after polll- natron. the pcrlod of mtensrvc synthesis of stonge protein ( I 171 The mcrax IS. however. more pronounced m an opaque-2 mutant than in normal rnauc. It has ban suggested that the opaque-2 gene mcrascs GDH activity along wrth GOT and GPT and daruses lysinc at~bo- hsm rn the devclopmg cndosperm [ 1171

An mcraac In GDH l ctrvtty during lal scnesana appears to be a general faturc (77. 106. I I& 1221 The Increase IS reportal to b due to dr ruwo synthesis of the cruyme m Hordrwn during the tirst hour ot scncsana [ 1211 It has ban suggested that ammonu produad by protcolyrrs durmp scnesam could be raponsrble for dr now syntheus of the enzyme (71.76. 118. 123) It has been demonstrated recently that an mcrcw m GDH actrvrty follows a srmdar control mahnrsm whrch gov- erns the course 01 scncscena. and kmctm. whrh retards senescence. also retards the mcrusc In GDH activity (I??] Sma GS-GOGAT enlymer Jarusc raprdly durmg scncscencc (I??]. the activity 01 GDH may possibly serve as an rdaptatroo of leaves to detor~ty the ammomum generated durmg scncsana The consrda- able mcrcasc IO hAD’-GDH (dammatmg) during lat scncsana (ItM] demonrtnta that the enzyme also operates m the drratlon of energy gcncntron Sma the

prolmc cooteot also ituxaaa gratly tn detached Oryro lava dunnp sctma~ (1241. a part of the incrase In GDH octrnty In nthcr drrcctron. I.C artunat.tng or de- ammat~og. mry be caused by prolme accumulatron. as II has baeo shown In cxcucd Zw lava that toul NADH- and f%AD’-GDH actrnty ~ncraacs constdcnbly when prohtx u added to the madlum (I251 s+uOnal vanatrons have also been rcuxdcd for GDH actrvrty In Pynu. the hlghcst enzyme ac~nty 1s found m kam and wood in Gctobcr. and tn stem bark tn May ( 1261. lo Iksc~psiu and ZWM spoxs growmg on acdr or alcnrcour soils. maarmum levels of GDH xtrvrty arc observed tn February/March followal by a dabnc to a lower level whrh rerrums furly constant throughout the yar [ 1271

In hght grown Puum all cultures a 48hr rhythm IS observed for GDH actrvrty which IS. however. Irregular in the dark grown alls [ 1281 A cuadun rhythm. In Puum roots (I291 and In Tr~tuum leaves (IO0 and ultradan rhythms In Chopohm scxdlrngs [Ix)] hve also ban reported In C/ycuu and Zta scedlmgs Duke tr ol (511 demonstrated sorry correlatron of peaks m GDH acttvrty wrth soluble protan oontcnt of the scodhng. rcflcctmg that the penod~c~ty m enzyme actrvrty 1s of phystolo@ rtgnrfiana They also behere that dark strmulated ac- IIVII~ ot GDH may be part of a crrcadrrn pcrrodrcrty (511 Further. II has ban suggested that phytochromc kvel osctllatrons modulate clrcadun rhythm by couphng with the high frequency osctllatron of vartous btochemxal and physrologtcal functrons ( 131. 132) and the rhythm IS presumably due to altcrnatrons rn the anabolr and utabohc balana of orpnrc compounds (I331

Lyht. raduwon and phoropcrtod

Light has shown barable effats on GDH actlvlty tn dr(Tcrcnt plant specter In PLntm roots [ 1344). Cucw cotyledons [ 1351 and the leaves of Tmcum (77. I(#]. Cucurbrro 76). Syzygwm (I 36). Arabrdopsu [ 1061 and Phastolw I 62. 137). enzyme UIIVIIY IS ~nacascd In dark- ness The mhlbltory cllbtt of I@ on GDH IS often explained In terms of mcrcascd carbohydrate kvcl m the IIUUC. as exogenously suppi~cd sucrose rod somt 0th rugus mlublt enzyme actlv~ty (49. 106. 134. 13s 1421 POSIIUS and Jacob1 (761 observed a parallel ~ncrase m GDH actrvrty and endopcnousammontum amaotntlon dunng dark stress In Cucurblro kavcs and concludal that ammomum u the mduang factor dunog darkncaa. It has also ban shown that dark alters the ~sorynx pattern 01 GDH (75. 1431 and Induces dr MU)W) synthesis of one Isoryme. responslblc tor this altcratlon (771. wtih IS ldentlfied as the same ~sozymc whrh IS syntharzed when cxogenously supphcd ammomum (711 However, In Phastolw lava the Itxrasc In enzyme utwty In dark- ncss IS observed In both the abscna and prcscnoc of NH;- Salk. and the dark mduad enzyme IS apparently drffercnt In 11s kmctc constants lor vprlous substrates. In clutron profile through Scphadex G-XI (Yo] and In 1t.s reactton to thlol modulatmg compounds such as OTT and DTNB (621 It has ban proposal that darkous mduas the synthevs of a more active GDH spcc= whrch tus lower A’hH; than the enzyme synthestzul m light (62.991

On the other hand. m ALYM kava. NH;&pcndent InductIon of a new ~sozyme of GDH IS reported to be proportlonal to the hght mtennty and the Inductron IS mhrbrtcd by DC’MU (851 Kar and Facrabcnd (I 221 have also shown that the scnercna-Induced mcrcax m kaf

601

GDH rctrv~ty tn Tr~ruum 1s htgher tn I@ thn tn the duk A ltght dependent NAD’-GDH 1s rlso dcm- onrtrttal m ~rab&ps~ [lo61 Further. tn Puum md Pastt~~~o root.& GDH rttvtty mcr~ rfta tllumtnrtton with fu-rrd [ 151. The duntton of the pbotopcrtod W sams to rfTcct the enzyme wtwty. In Urruo lava [ I331 rnd tn Ltnvm roots [ 1021. longer pbotopermdc tncrusc the enzyme actwty but tn Lnur! lava the rltvtty IS not affatai by ~bccluogc tn photopcrtod [ 102). Although the n#hrntsm of thts mcrasc hu not ban followed It rppan tht the rcguhtton of GDH by Itghhl;‘duk 1s l compkx proccu rnd different tsozymcs of GDH mry bc synthcsixd tn *thcr axtdttmn dcpcndtng on the meu- boltc sutus of the cell If rmmonu raxmuhta tn higher

rmouolc II mduasdetoufiatton by the GDH ~sozymc In both 6rk rnd tn Itght

Inargonu nrfrogenow sah

The supply of rmmotuum. nttrrte rnd ura as nttrogcn sources tnflucnas GDH rcttv~ty tn plants Ammontum geoenlly ~ncxasa enzyme ac~ty. rlthough tn a Icw cases it has etthcr no cffat or even tnhtbts the enzyme rttvtty (Tabk I) Apprently the nrtabk cfTatr 01 rmmontum arc dctermtncd by several other frctorr such as dtuatton of rmmontum supply. IIUUC studtal. age of scalltngs as well IS pmcna of other tilts rnd mcuboltta

Tab& I EtTat of rutrga ults oo &utnatc dchydropnu rctwtty .- -

NItlOp soufu (=M)

Duntloo of 1twJbn1l00

whr~ Phot tusuI

Fuarc~oo of

th+=Y= rctinty EKat Rdmncu

----

-OIlJar

up to I43 I50 150

up to 21 5 100 70

100 90 I50 100 60

200 150 I50 100 100 150 200 ZOO 100 200 100 100 80

ZOO 90

Nttratc. 30 60

100 100 100 100 400 a00 100 400

2d 3d 3d 3d 5d 3d Jd

IJd 3d 9d

I5d 3d 3d 3d

i: 3d 3d 3d 4d IO hr 5 hr Jbr

2Od 3d

lU2d l6d l5d 9d 3 hr 3d 5 br

3d Jd 3 hr

3d

up to up to Um

100 4d 100 3d 100 3br I4 3 4d 80 2Od

100 2Od 80 3d 80 3d t30 3d

100 206

oly:o roots hmea pLuakt, AWM kaus Rosa CSIIU Lrrw p&aktr Lvruw anbwon~ ua z&l root1 ti shoot1 Clrnu moIl aad kaes TruuLA lavu zta Lava zta root8 Md sbootr Zta roots

A?obldopwl bus Araindopui roots Zto roots uxl shoots Zta lara ArobrbplLc roots Zea laws Z80 &Jur Glycuu orll whura PLmm sbootr Zta roots Zta roots l nd shoots Hodrum moo Zta roots Cunu roots lbd Lanr Pbuqp roots unI kwa Zta root8 and lava Zta lcwcs I Zta roots sod leaves Zta leave4 1 Zta roots Zta roots uxl kwu Phawolw lava 1 Zta roots sod lava Zta allus

Zta roots ArddopLs roou d bmva Zta rhoou md lava OIyra roots Hdtmu roou Clycw roou Zta roots rod lavu Zta roo4s rod Lava .

AouMtm)

Amuutq alId dc!unlMtlo(

Amurrtq

lDuuae

No ctkt

No clkt

louaa

No eJTscr

[581 (l4t31 [ I491 [I451

It:;

[l21 (1101 [‘II (121 r 1031 [261 [to61 [Ial

II:; [IWl [261 1261 [ I*61 [491 (191 [I21 It41 (261 (tlO1 [I511 [ttOl 1121 [I21 t261

;:I;

;l:;’ iI61

602 H S SRIV*~*W aod R P SIWH

CIC In L.+~tnw embryonr l au. hH; caused no eff~i block mcreua UI enzym ut~v~ty only 10 a IrmrIal cx1cnl. Jurmg a 24 hr mcubatron but mcra& enzyme acuity durmg 72 hr treatment [14l] Furrher. In Zeu rooIs.

atlcr a prolonged treatment of 2 days (I481 The ammat- mg and dammalmg wlrvltres 01 Ihc enzyme also respond

shoots and lubes a conlmuour prolongad supply of ammomum caused Increase supply Ihn a 3 hr tratmcnt

in drflercnt r~oncrs In Ztcr (12.261 However. In

[I?] Houcbcr. In the rooIs of malure C’rrrus plants. Arabdop~. cycbheumde compktcly suppresses Ihc

ammomum mcr& Ihc enzyme level only dunng rhc ammomum-iodua?d mcruse In the enzyme (106)

hrsl2 waks IruIrncr&and Ihereafter. up IO 6 weeks, there Althou& the CLUI mcchusum of enzyme acCllvuIron by

~1s no sr@ianI efiec~ [I IO] DIlTerenlIal cffeclr of l mmooIum 1s 001 known. the porrrkl~ty of an allostcnc

ammomum on drlTerenI IISUCS such as callus. nod&s. rcgulatron annot be ruled OUI ( 12). At least .%e~~sporo NADP’-GDH shows a complex rotcractron with NH;

rooIs. shoots and kava have also been demonslratad by barrous workers [ 12.24.26. 106). For exampk. In Glyc~n.

sod II Is suopatcd thrt ammonrum II actmg both a.s a substralc and as an acuvalor of the enzyme (I 531 A

ammomum. nilrate or urea mcrca& root enzyme whrk the same souras decrcasod noduk GDH activity (241

um~lar WI00 of ammonium on hi&r plant enzyme ma)

kurther \arrour forms 01 GDH activity. I c hADH-. also be propoJod baause II has ban shown that !~‘~“;l’or rhe enzyme IS modlfiad by the concenlrallon of NH; 11scl1

NADPH- rnd NAD’dcpcndenI ~CII\IMS respond drf- krcntly cbcn In primary and secondary ka\cs of the same

lsa krm khanour). Howc\cr. further crperlmcnts

age [I?] uung modero tahruqua arc requrral lo cluc~datc the propoaod asechamsm.

The cllatr 01 ammomum vary In magmtude wrth Jilkrcnl conantralions 01 ammonium In most cases [SO. W. 1451 Howcrcr. In CJyclruall cuhure [ l46]aod In some grassland species [ 1471 the concentratmn response IS not so marhcd and 11 appears. therefore. that some planIs,t~ssucs arc not adapted lo changes m abarlabk ammonia II IS also c\Jent from the above-menlroncd ,Iudrcs that not only the nature but also the magmtudc of the cllect of ammomum tons on GDH IS afTacted by many other Iactors as well 1 he clTcsI of ammomum upon the spa~fic IunctIons 01 GDH acct~w~y also vanes. as II

Increases NADH-GDH acmlty In Ztu roots. wbk dc- crearmg NAD’-GDH actlrtty [ 121 Loyola-Vargas and de Jrmcncz [26] have also shown dlflerent raponscs of IW ~CIIWIICI. In \arIous Zto IIUUCS. IO a supply of NH; and suggested that the NAD’:NADH raI1o tends IO darase ulth Ihc supply ol’ ammomum and urea Thus cnrymc bchrblour r&cIs a compkx mahnrsm tar ammonrum rcgulalion

Ammomum may mcreasc cnzymeacur~ty by mcrasmg the amounI of cnrymlc prorem oriand by modulatm8 IRK aclivily of cxislrng enzyme mokcuks Ik now) synihcsts of GDH In rnponsc 10 ammomum has ban dcmonstratal In LxmM [ 1481 and in Arobdopsr~ plantlets (1061. Ofyzcr rooIs (5x1. AWW (701 and Tr~rvum laves (7 I] E*tdena tor de noto synthesis ot the cruymc protern hns been dcrrvcd tram trmc course SW&~. the supply of protern synthews mhrbltors. dcnslty and radIoactIve labellmg Iazhruqua. gel clatrophorcsls (58.70. 106. 1481 and more rmntly uscng lmmunochcm~l approaches (7 I ] The synIhcsls 01 ncy Iwzymc IS bchcbcd IO take place at XUS nbosomcs and an Increase In cruymc IS obvrved only In the soluble lractron and not In the mrtochondrul tractIon (5X 1 Further. the mhlbltors of 70s rrbosomcs. such as t~thrwchloramphcnrcol Irncomyan and cry- thromyc>n.do not prevent the nse In GDH of Lrmrw [ 148) *her-s puromycm [ 1481 andcyclohcumuk (12].mhrbl- Iors of protccn synthcsls aI L(OS rlbosomcs. InhlbrI am- monrum induced mucases In L.mno and Zto. rcspcctrbcly

The etTat of nrlrate on GDH actl\~ty al- banes wllh spes~~. IIWC. time of mcubatron and some other factors (Tabk I). Gencnlly nitrate IS crthcr mhlbltory (12.I9.154] or has no cflai (14.43.58] on enzyme acliv~Cy dunq short term supply IO cxc~sed IISSUCS However. prolonged tratrncnl of muci seedlmgs with IulnlC I_ enzyme act~nty m various plants (12. 103. 151. 155) It IS Illrely that the strmuhIory cltat of mtnte durlog prolonged Iratment 1s mdrrat. possibly through ammoorum produad from the reduction OI nrlrate It IS also errdent from barrous siudlcs that Ihc &al of n~lrate is not as pronounad as that of ammomum (12. 103. I%, IS?] However. In one study. the presence 01 flowers and fru~tr caused Ihc GDH le~l In mlurc (‘trru, plants racnbmg NO; 10 rise 10 a kvcl srmllar IO those raelving ammomum (I IO]. In P/anfago lolrc-toloro. a spcs from a relatrvcly nutrent-poor habitat. NADH- GDH mcrcucd dunng 4 days after a rwvltch lrom 2”. IO 100’:. NO; -N nulntron (1511. uhcras In Pbnroyo nmlor. a grassknd spoxs from rcktnely nuIrrcnI rch habItaI. II remamal relatl*ely constant (I%] Urw. l no0ser nrtrogenous nutrrent. IS 1cs.s sIudrad. probably because 11 Is a.s~ly hydrolyscd IO prods ammomum and thus 11s cflau are erpatal 10 be the sarnc as Ihosc 01 ammomum Th~scxpecut~on. has. In IBcI. been rcalrral In Glycmr and Zta [ 24.261

Ammo 4th. amdts and amino wdi adoyut5

II 1s bcl~cved that a new tsozyme appears IO deiorrfy the excess ammomum absorbed by the IIUIK because IIS

accumuhtron IS harmlul IO all mcubolum (I231 Moreover. the pos~t~rc ellat of ammontum on enzyme act~r~ty may noI Involve only dt N)NJ syntheus 01 the cnzymc. as the Increase In activity 1s also observed durmg in I trro mcubatron ol the enzyme with ammomum ( 121 lurthcr. the cnhrbltors OI protein synthesis arc abk IO

Armno rds hvc bocn reported lo barubly allat boIh amtrutmg and darmnatmg actrbrIIcs of GDH In various I~ssues of plants. Glutamate Increases the ammatmg cruyrnc acI~n[y In hmna (1501 and Ihc dammatmg enzyme acttvlty in Zta rools ( 1251 and laba ( 125. I 5Y] However. II hardly aflau ammatmg actrvliy of the enzyme In Lrnvu, (148) and hum shoot (401 or even mhlblts the same In LXIWUI plantlets (160. 1611. Plum roots (1621. Camrllw rooIkIr I63 . Lto roots [26]. Ltu lam (125). hnusa pkntkts I50 . Luptnw cmbryonlc axes (I411 aod dammatm

I 1 enzyme activity In Ltu roots

(26.1251 and kaves (I25 II has ban suggested that f glutamIne would exert a pout~vc control on GDH but a negaI~+c ooe on GS, and lhereforc II may dctcrmmc the cnIry of ammorua vu either pathway (141. I SO. 1561 On the olhcr tmnd. gluummc mhlbm NADH-GDH In L!tcr roots (1251. and both actrv~t~es lNADH ad NAD’ I In Zto lavaand callus IIUUC (261 posubly baausc 01 some

bM H S Sawar4rr rd R P SIW;W

Adcnosmc InpbosphRtc RI corxcntrat~ons of I mM rod above IS an mhlktor of CDH actlnty In kth diwons (90) However. when 0 2 mM AMP. ADP and ATP were s~ppkd to Asparagus all cultm for ? days. AMP reduced the enzyme wttvlty whllc the other two had no cllat [ 1421 It has ban suRRcstcd th1 when btosynthctlc potcntul IS low. tk energy char~c rcdard and kvcb of NH; h@ In the tLuuc. GDH mry piry a more nnporunt role In the ustmllatlon of arnmonu than GS [ 19. lob. 161)

Ftmptrarurr Lower tcmpcrrturc rdvcrxly rffats GDH l ctlvny tn the roots of Trtruum [ 1671. Clycuu [l&t. 169. 170].Puvm[171]rndZto(172] Adacrcllccn enzyme rctinty in Glyccnt roots Rrown at lower tcmpcr8. turc IS possibly caused by a rrlrtlrc darax IO enzyme protcm content u well as by cho~cs m the tatto ktwan NADH.‘NADPH form, (168. 173) It rlso rpparr tkr tk rmlmtmR utlvlty of GDH may not be slyrlbcant at lower temperature as the ctxru of utlntlon [E, ] and

k..; UC hlRhcr at 12’ than at 25’ In FtIruum roots ( 173) The enzyme act~vny IL howcvcr. hardly aRated when planIs arc yown at hlRh culIlvrt,on tcmpcmtura such I) In CIyctnt (170) and Frutiunr ( 1731 roots and In Zta lava (174) The enzyme from Aqau Iuvcs has ban reported IO k hat sublc at %&TO’ (1751 and Frrlrtcur, root GDH ks ban found to k more thermostable than GS (I?)] Baclux of very hmltcd studlo dalmR with charactcrlstlcs of tk enzyme a1 dlfTcrtn1 IcmpcnIum and tack of Informatton rcyrdmR dumirxlinR GDH l w~t). tt IS dlfBcul1 IO dnw any dckntc conclurrons rcprdinR the physloloyal r~gnhcancz of the tkrmo. sIabllrty of the enzyme Howcrer. a possible role of Ihls cnrymc at hlRhcr Icmpcraturc may k propoxd &s an rdaptatcon of plant IIUUC for rmmomr dcto~lfiatlon when GS 1s macII*ated II hiRhcr temperature

W OIV r~rtt< A consistent darux In GDH •CIIVII~ 1s reported In the xcdlmRs of 8rass1cu juncta and B cam~~~ro ( 176). In rooI< and nodules of CO/MU ( 177) and m rooIs of Porrr~um ( 1271 In rcsponx IO IncrusmR water stress Water sIrc)s has also ban repor to mhlbn Ihc rbsorptlon of ammomum rnd nnrrtc (1781 On the other hand. In root nodult~ 01 .Hedrtoqo both l mlnatmR and damlnrIinR GDH l ctlrnla mcrax a~ hl@cr lc*cls of water strcu l~c luf waler porcntirl - I IO - 2 HRJ lnrokcd by wlthholdmR water (I 791 This discrepancy In the Incraturc 1s possibly baaux the mponu IO water sIrc\sdepcnds upon Ihc sIaRc a1 whtch the sIrcu IscraIcd and may *ary with the plant spates For cumplc. no rlRmhcan1 CharIRe In GDH actlvlty IS reported In the I-ICI 01 B pncto when wllIlnR IS crard at the sIllqua ,I+ but wllImR at tk RowcrmR str~c darrua GDH actn~~y (IRO] In Contras1 to thlr. stress at the RowcnnR strRc In B camplsrru ~mzm GDH l ct1r11y rhlk that RI Ihc xllqur suRc daraxs utlvlty (1801 Further. in shoots of Portrum ( 1271 and In Mtduogo root nodules [ 1791 water strw created by PEG b only a w~rnal elTat on enzyme actlvlty while strus cruxd by wlIhhold. mR waler Incrusts II The Increax In enzyme actlrlty IS kk\cd IO k a measure of dctorlkaIlon of rmmonir rclas& u a brakdown prodocc of protcms and ammo acids durmR water stress ( 1791 In rddttlon to this. prohn aaumulrIed JurmR water stress (181. 1821 may also

maax GDH rctlrlty u an mcrax In NAD’ and NADH.GDH acIIvIt_ auxd by prolmc hu ban rc. poned for clctxd tta lava ( 125)

Salus+ In tk txlophytc Sucuda. the enzyme actlnty IS rctlvrted by 25 mM N&l and mh&nted by h@cr lover 100 mMl ConanIrntlons of the ult ( 1811 An IO- In enzyme l rtlvny with a low krcl of uhalty ks rL0 bon obxwod~oOry:acmbryo( IM]aod~o hmna[ 1851 Thrs incrcIy In enzyme UtiviIy under vlinc condiuoru mry k Ihc conxqurna of mcrQsbd rrnmonu and unldc levels whtch raumulatc under thcx con&loos (I%) pou&Jy beaux of the ~nrbhty of the GSGOGAT route of ammona uxmIlation to function uodcr uhnc condition, (185. 187. 1881 However. h& vlmrty mhlbns GDH rctlnty In the roots of klophyta (187) rod Puum (189. 1%) posxbly by modIfymR the rfiolty of the enzyme for substrates and the aulytlc potency of the enzyme (I831 It rpparr tkrcforc. that GDH may cktoufy NH; only under low xhmty kvcls

Pollur~on The rmmatmR utlvlty of GDH IS quite xnuttvc IO ur pollutants and u such It ks ban rdvoated IO k the best cnzymr mcbator of pollution strcu (IPI. 1921 The enzyme atlrlty mcrasu dunng crposurc to SOI (19% I%]. NO, rod NH, (197.198) and H,S ( 1991 In Plurvolw Iarcs low kvtb of NO, do no1 rllat NADH.GDH actlvlty (I521 Funkr. the pollutant tNO,l ~srrunulrted vu tkGSGOGAT route. rlIhouRh rulmllrIlon l ~ a h@hcr level of tk polluunr may mvolvc the GDH prthwry (I 52) AlthouRh IkcucI makmsm of mcruunR GDH by ur pollutants IS not known. some postulra hate ban rdvrnad Sma the cfTa1 of SO, un be stmulated by acid tltltmcnt of 1so0Ltcd GDH. II IS kkvcd thrc SO, trnd pourbly NO, as wclll YIS by cratmR rtidny In the cnzymK cnvlron- men1 (2al] The polluUou may also IrKraM CnryaK

rctlnty through altered mcmbrrn pnnablhty It h, also ban shown that SO, chanta Ihc tsoorymc pottcm and clatrophorctr mobIlIty of GDH (831 The clTat of other pollutrnrs on the enzyme acuvny. however. warr. anIs furtkr InvccIlRatIon to ascertain the posslblc rok of IhIs enzyme during pollution stress

Parhuytnrr mjtcfron PaIho~nK infalions also rffat GDH actlwt) In Llmuncotyledons Infated with Rar rus1 funRus. cruymc l ctlrity shows 4 blphaslc mcrax. dunn( Ihc fIrsI 24 hr of mfauon and l ym l fIcr 7 days (2011 The enzyme durlnR each sIa(c. however. dlfTm IO its propcrtlc) and also possibly In IIS rcLlIlvc cmmncy in dummarmg rnd amlnrtmR racIIons II ts suRRcstad. however. Ihat In okby~mfataf cotyledons. GDH rp purs IO b an enzyme of RlutamrIc dcyrdatlon whllc in 7&y-tnfatal cotyledons it pLys a minor role 10 RluU. maIc synthtus Brrrllk tI al (2021 hvc ckmoostra1c.d tht a Iosm produad by Htlrrunrlrosporrurw mhlblts NAD’ Innsport In Zta mltahondru whwh ttuy con. xqucntly InRucna the dchydrornaxs More detaIled studlcs may RIVC some ms&It on GDH rcRutatIoa in plants dunnR dlxases However. In thcx sIudm arc should k taken IO usurc that tk enzymes of host and pmhogcn arc meuurad mdcpcndcntly

Grorrh rtplarors ad ofhtrs

A few studla hvc baa performed cooamrnR tbc cflal of yowth rcgulaIon on GDH utlvlty In Puum roots IAA and other l ulms mcrcue GDH level IO some

Glutamr~c dchydrogcnu In htgtur plants 605

extent but ktncttn has etthcr no mflucncc [ IJJ.203.204] or tnhtbns II [ 122. 2051. The enzyme IS also sttmulatod slightly but conststenlly when 001 mM sahcyl~c actd IS mcluded tn the mcubatton medtum although htficr concentnttons of the aad mhtbtt the enzyme b~twty

(2061 Chlorocthylphosphontc acrd (CEPA). a well known growth rc&ator. greatly stimulates enzyme ac- IIVII~ when supplied tn a co#mtratton range of 60-480 ppm IO Prrvrr.xrw sadlmp (2071. It 1s proposed. however. that CEPA dacomposcs tn plant nssucs IO release ethylene whtch may retard growth and cnhana enzyme acltvtly The exIcl mechanism IS yet lo be eluc~datcd. Appbcattons of 2.4-drhlorophcnoxyoatrc acrd (2.4-D) also mcruse GDH acuv~ty In Puum and Zro roots PI low conantrattons (2 x IO-* IO 2 x IO’ ’ Ml but tnhtbtt the same a~ h&r conantrattonr (2081 The shoot enzyme from PIWW IS also sttmulatcd strongly a~ lower conantrattons of the hcrbtctde. although that from Zca IS unaffatbd (2081. It has been suggu~od that under the mfluc~ of 2.4D.ammonium accumulates In the root and shoots of both plan& sttmulatmg enzyme acttvtty (2081 Aytn foltar applications of 2thloro-4.6 btstcthylamtno~s-trrne. 2-mcthylmcrupto-4cthyl- amino-blsobutyLmln~s-tr~ne and 2-mcthoxy-4- isopropylamino-s-tri?ne and 2-methoxy-4- Isopropylammo-6butytammo-s-trlwnc (2 me;11 mcrux NADH-GDH levels along with nttrate reductasc and protctn tn Ptsum and ZIP leaves (209) They postulate that sublethal conantrattons of the s-trtazmcs rttmulate general nttrogcn asstmtlatton and protein synthats DCMU. an uncoupler of photophosphorylatton. mhtbtts NADH-GDH In exc~scd Zro leaves. possibly by mtcrfcr- mg wtth NADH productton (89)

The enzyme activity IS stimulated by P- mcrcaptocthanol tn the leaves of Urrua and Sp~n~~to (2101 5.5-Dtthtobts(2-nttrobcnzoate) (DTNB) com- pletely tnhtbtts cnrymc acttvtty at I mM tn AQOUI lures (21 I]. However. the •CIIVII~ 1s parttally restored by cyrtetnc. dtthtothrettol. reduad glutathtonc and /I- mcraptocthanol when suppl~cd IO either exc~scd Phacoh leaves or to the enzyme prcparatton (62.991. lnhtbttton by DTNB 1s more pronounced for the cnryme from II& grown IIS.SUCS than hat from dark grown IISIUCS (621 It has tuen reported thaw the tnhtbttton 1s due IO mteraclton of DTNB wtth sylphydryl groups present at the acttve antre of the enzyme (611. whtch arc apparently more abundant tn the enzyme from II@ grown leaves than In that from dark grown (621. S~mc the UI LWO mhtbttton of enzyme by DTNB 1s dependent upon NADH conantratton tn the mcubatton mtxturc. II has been postulated that the enzyme first forms a complex wtth NADH and then this complex II acted upon by DTNB (621 The dummattng enzyme acttvtty in Pawn uzdltngs 1s mhtbttal by pthloromcrcurtphcnylsulphontc actd (PCMPSA) and phcnylmercurtc acetate (PMAL although the ICII~II~ IS restored by the l ddttton of glutathtonc (2 I2 ]

Melill bmdmg agents. 0-phcnanthrolmc. LL - dtpyrtdyl. EDTA. zmcon. ferron. nuroso-R salts and 8- hydroxyqumolme arc also reported IO tnhtbtt both the acttvtttts of GDH and these l CIIVIIICS arc restored by the addrtton of dtvaknt metal tons (212). Pyrtdoul 5- phosphate mhtbtts the enzyme act~vny tn Luprnus noduks [M6] and P&sum mttochondru (2131 This bchavtour reflects the auoctatton of metal tons wtth the l CIIVC form of GDH

Root nodultr

Ammonu IS the hrst stable product of dtnttrogcn fixation tn nodule bactcrtods. A large portton of II IS excreted from the bactertod IO the nodule cy~osol where II 1s asstmtlatcd Into organtc compounds (4. 123.214) Although the observed kmcttc studta have revealed tha ammonta produad from symbtotrcally fixed dmttrogcn IS l ~mtlatcd prtmartly via the GS-GGGAT pathway. the stgntfiancz of a large amount of NADH-GDH present In nodule cy~osol (214.215] 1s not understood.

Some cvtdcna. basal on pulse labcllmg. for the tn. volvcmcnt of GDH In the asstmtlatton of ammontum has been presented for nodule l~uucs of several legumes (2 16.2 181 In Luprnw root nodules. I4 tsotymes of GDH are considered to be phystol~lly ctgnthant and also change with the nttrogcn and sugar content of the II~SUC (251 The mvolvement of GDH cn ammonta asstmtlatton. especully at the urly flowertng and ac’11vc pod hllmg stages. has also ban shown tn YI~M genotypes symbtott- ally assocuttcd with vartous rhtzobnl strains (I I I ] On the basis of pulse lablltng and InhIbItor studies with nodules of Alnur. a non-legummous angrospcrm. and In (il~rmr. Schubert and Cooker (219) ruggcstcd that GDH may play a major role In the asstmtlatton of cxogcnourly suppIled ammontum Further. NH.’ supply tncrused GDH ~CIIVII~ constdcrably ktwan 4 and 24 days of apphcatton with a maxtmum at I3 days In an mcffattvc Mrdrcugo clone. MnPL-480 (220)

Dcfohatton and other typer of stress mdua nodule scnesccncc In a wtdc range of legumes (221-2231 Root nodules of Mrdrrfxgo have an adapttve upac~ly to un- dergo temporary locahud scncsansc m response IO harvcstmg and rpplmd N-ferttltur (223.2241 II has ban supOcsted that. sIthot@ noduk NADH-GDH IS not clovly assoc~a~cd wtth N, tixatton. II may be ruoc~atcd wtth ammonta a*llmtlatton durmg mduad nodule scncs- ana (2241 Further. when hfcdrccrgo nodules arc sub- )actcd IO water strew. the GS/NADH-GOGAT cycle IS opcrattonal tn normal or even mtldly strd plants When drought progrcsIcr NADH-GOGAT IS mhtbtted. and NAD’-and NADH-GDH tncrusc (&w = I 7 M Pak tn the absence ofa N-supply. The acttvtttcs arc matntamul al higher level when plants arc supplmd wtth 20 mM nttrate [ 1791 II has ban reported that NAD’-GDH IS relatively higher in root nodules in comparison wtth other IIUUCS. and the ratto of NADHINAD’ IS lower [ 179.220.2241 II has ban suMtested. therefore. that ammonrum released as result of protein hydrolyses. ammo rcld oxtdatton or mcrud NR l cttvtty durmg nodule scneyxnce may bcass~mtlated by the co-actton of NADH- GDH and GS and their relattve contrtbuttons may possibly be dependent on the nttropcn. carbon and energy II~IUS of the IISSUC ( 179.225). Further. when the coupltng of NADH-GGGAT wvlth GS 1s interrupted durtng htgher water stress. GDH IS sct~vr~cd IO provide glutamate for GS PCIIVII~ ( I791

II l wrs. therefore. I~JI although GS-GOGAT IS the mam route for the entry of symbtotcully fixed NH; Into the oryntc cycle. GDH. present In large amounts In the nodule cytosol. may also play some role under some nutritional and cnvtronmcntal condtttons. depending upon plant rpocus and type of symbtotrc l ssocutton Moreover. there are a number of N,-fixma symbtontr

606 H S SIIVAST*V~ and R P SINCH

mcludtag non-kgumc angsospcrmr. where httk IS known about the btahemutry of ammonu a.sumtlatton and nnrogcn lrrnsfcr. In A:olia. for example. a large amount of GDH 1s present compared IO GS However. GS- GOGAT act~tm mcreasc when l ssocsatton wtth AMborna OCTUN [226]

Roots and rusue culrurrs

Ammomum tn the root IS generally denvcd from absorptton or ustmtlatton of tnoryntc 41s avatlable In the sod. It does not accumulate m the plant IISSUCS because of tts IOIIC nature [ 123). Roots have been demonstrated IO bc the major SIIC for ammonu assimrlation m Hordtum. where 93 to of externally supphcd ’ ‘NH; was transported IO the shoot m the fotm of orgntc nttrogcn [i27) In PISW 1001s both NR and GDH are h&r m root lips [ 15, 138. 2281. whereas m Zea 1001s GDH WIIVIIY.U well as the kvels of NO; and NH;, are higher tn mature root portions [ IP] Generally. the supply of NH; and IO some extent NO; mcrcases ammatmg GDH activity con- stderably. which potenttata further wtth treatment trmc (see Tabk 11 A good conelatton between amtnattng GDH ac1tvtty l d soluble and protetn nttrogcn fracttons [ 121 and mcrcased fra ammo aads [229] dunnp tn. orpntc nrtrogcn supply have also Men shown

One of the malor obpcttons reprdtng the operatton of GDH tn the amtnattng dtroctton IS a htgher K.,; However thts nads raonstderatton as various factors have been reported to mtlucoa the KWH; for GDH (see kmettc bchav~our) PUIK labcllmg and tnhtbttor studtcs support the opcnllon of the GS-GOGAT route tn normal growth condtttons. However. tn most studtcs ghrtama~c ts lablkd alon with &naminc and it IS dtfficult IO exclude the contnbutton of GDH tn uumrlatton of lab&d ammontum [ 191. II may be raltzed that when mcthtontnc sulphoxtde (MSO) IS supphed wtth nttrate/ammonntm. a very low kvcl of ammontum accumulatton oaurs tn the roots of several C, and C, plants [230]. Further, MS0 Increases GS and NADH/NAD’-GDH when suppIled with IS mM NO;, and NADH/NAD’-GDH UIIVIIICS when suppl~cd wxth IS mM NH; [ 1061 These studm tndwr1e that GDH IS mvolvcd tn ammonu uumtlatton al

hi&r levels of nttrogcn and a1 the same time II gcncrrlcJ energy na the dammatton reaction. for the acllvll) of GS to rurmilate physiologsal levels of nitrogen in the roots [ 106). Further. II also appears that GDH IS a more s1abk enzyme than GS tn stress condtttons (see above) and II may play a st@ant role In ctther dtratton IO mamtam ptant metabolism during these condtttons For example. GDH has ban shown IO be more thermostable than GS [ 173. 1751 and II tncrascs dunng water stress [ 179) and sahntty (183 185. 1881. Further. GS and GOGAT cn- rymcs arc more labtk and they are macttvatcd or then couplinp dtsturbed during stress condtttonr [ 173. 179.183. 185. 187. 1881 In all culture of Saccharum. GDH has ban shown IO operate when normal growth and dtfferentutton do not occur and GS 1s very low [ 1091 II would tx tntcrattng IO study the primary l mmatton reactton tn acrophytcs wtth thus pcrspcslivc

Green shoots and lratvs

The mapr soura of ammonu tn the shoots and leaves IS the rcductton of nttratc in s~ru [ 2311 In l ddttton a large

amount of ammonu can be general IR SUM durto8 the photorcspnatory conventon of glyctnc IO scnnc tn mtto- chondru of yan 11ssuc [232.233] and the brcakdowo of asparrync nthcr vu transamtnasc [ 2341 or upuagma.~ [235.236] Many studies show that ammoou or/and nttrrtc supphcd dunng sadlinl growth increase GDH acttvtry constdcrably tn shoot and laf tissues (see Tabk I I Althoufi several studm have demonstrated the operatton of the GS-GOGAT pathway for ammonta asstmtlatton in shoot and kaf tissues [I. 4.91. they do not exclude the role of GDH compktcly. II has ban shown that the cqulhbrlum of mltochondrial GDH IS tn the dtrcctton of glutamate fonuatton. and ~solatcd Puum shoot mttochondna are able IO mcorpora~c “N from either 2 mM of “NH, or “N ~lyanc to glutamate [46.237.238] Necman er ol (2391 have shown tn their “N nuclur magnct~c resow- studm thal GS and GDH are both active for rasstmtlatton of ammonu rcluscd dunng photorcsptratton tn Ntcor~ana proto- plassts As mcnttoncd carher. GDH mcrascs dunnO vartous envtronmental stresses such u darkness. htgh temperature. water stress and ur pollutton (sbc cnvtron- mental stresses) tn leaves also In Phastolw lava. the enzyme from dark grown samples IS more act~vc than that from II&I grown [62] II may be proposed thaw durtng s~rcss condttlons. when the GS-GOGAT pathway 1s mcfhcrcnt. GDH IS acttvatcd. possibly IO detoxtfy ac- cumulated ammonu

S& dtwlopmrnr and grrmrnarron

Dunn8 the later stages of seed development. ammo acids from scncscmg ~~s.sucs arc an Important portion of the total nutncnt supply IO the pods l od dcvtloptn~ sads. The (;lyctru sa~4 GDH hu ban shown IO be aprbk of opcrrttng tn both ammattng and damnuttng dirattons and could posstbly provdc a ruttable rcverstblc hnk be1wan carbon and ammo actd metabolism [53.240]. The enzyme has also been shown 10 be Important dunng kernel dcvclopmcnr tn Zla genotypes and IS more abun- dant tn a high lystnc variety.. I e opoquc-2 [ Il7,241] A general mcrasc In the enzyme activity dunnp sad gcrmma1ton (sa plan1 age and rhythmnty section above) also suftgests some role of the enzyme tn ammoma mctaboltsm during thts proass A htgh concznlratton of ammonu In sad coats [ I IS I 161 and an increase tn GDH acttvtty tn Ktd coat and cotyledons [ I 121 and endospetm [ 201 may mdlatc a poutblc rok of GDH tn ~mtlattnp seal ammonta dunng gcrmtnatton

ADDEhDL M

Mutants of hahdops~ d&rent tn laf GOGAT ac~~vny [ 2421 and Hordtum lacktngchloroplut GS [243] have ban shown IO survive only under Ihe condtttona of nonphotoraplra1ton and htgh atmosphere. rapcctivclY.

rrthcr than the normal urigrowth condtttons. Further m scncsant Trrrrctun lava l mmonu rclascd dunng photoroptntton IS urtmtlated through laf GS rather than GDH [244] These obacwatroos Imply that GDH has no upthant role tn rasstmttatton of photorapt- ratory l mmonu even tn mutants lacking enzymes for the GS;GDGAT route and during leaf scMSCII)(X

Acknmvlrdgrmmrs WC UC crmmcly Ihmkrul 10 RoruJor

D P &mrod for thotoufi CdlIONl chak up and rcvc~ WC also thank MI Knsluu Hladun for ‘pcoczura~’ the mu~uscnpt

I MlRm. B J and La. P J t1980) IO 7’hr Blakmrsrry o/ P/am IMdm. B J cd I Vol 5. p 169 Aadcmr Rcss. New York

2 Tern-r. D W . Mccrr J L and Brown. C M (19701 Brothem J 117.405

3 La. P J rnd Mltlln B J 11974) Narwr 251. 614 4 Mrtlm. B J and La P J (1902) In Nw/rrc AC& Md

ProrrlN te flrurrs I IBoulrcr. D and Panha. B cdr I Vol 140. 9 5 Sprmgcr~Vcrl4~ Bcrlm

5 Smcth. E L . Awrcn. B M . Blumenthal. K M and Nyc J F (19751 In 77~ Lnz)mcs IBoycr. P D cd I Vol I I. p 293 AadcmK Press. New York

6 M~lbn B J rnd Lea P J 11976) PLyrochcmurr) 15. 873 7 MllIrn. B J rnd La. P J 11977) Annu Rm P/m Phyuol

zu 299 (I Tyler. B (1978) Annu Ret. Bwwkm 47. II27 9 Stcw~n. G R . Mann. A and Fcnlcai. P A tIPsO) II) The

Btochrmurr~ O/ P/am (Mdlch !, J cd ) Vol 5. p 271 Aadcmrc Preu. New York

IO Gort. M G (19811 J Btorkm II). 879

I I Punakova T. 011lrr G and Ntzrunskr A (1976) Blol P/am 18. 2U3

I2 SmgJt, R P and Srtvtiv& H S (1982) Bu~Arm Phystol P@n:m 177.633

I3 &lawckl. W rod RJfJbskl. A (1979) Am Bmckm PO/ 2.6. 383

I4 LCWIL 0 A M . JJW D M JIKI HC~III. t J I 19821 AM flor 49.39

I5 Duke. S H. Koukkarl. W L and Soulcn. T K 119751 PL)Jco/ P/anr 34.8

I6 Snvasrrba. G C Sirohr. <j S rnd Scngupu U K 119751 /ndt~ J P/am Physrol I& 26

I7 SamI% J K l bd SJIX. P V (197~1 J P/an1 Ph)sro/ 86. I07 18 Ouhr G , Pscnakorr T JIKI NUNM~. A (1978) ScOrq7lo

33. 35 I9 Chkr A S~ulcn. I. Jones. K . Wrrupcar. M I. Mun. S and

t)oocl. I L lI98OI Planro 148.477 20 Dullus. C M rnd Row. R (1978) Pfar Phpd 61. 570 21 buncrc. C . WeIrsnun. N and DawnI. J II98I) Ph)m/

P/ml 52 146 22 L~CX b M rnd Dennis. D T tI9UIb P/MI Phwd 6R827 23 N~gd. M Jnd HJrlnunn T 1I98bI Norwjowh 3S.a 24 Duke. S H rnd Ilrm. G E (1976) Plonr Cc// Phpd I?.

1937 25 Muurowr. H . RalaJczak. W and RJrJpJk. L 119801 Atro

Yh)d P/-r 2 I67 26 Loyolr-Vup* V M and dc J~mctxz. E S (1984) Piam

Physd 7& 534 27 SukborzbcnkJys. T B (1978) Oarogcn 9. 390 28 Mcllor. G E and Trcgulv E B (1971) CM J Boraay 49.

I37 29 Rathrurn.C K M and Edwards.G E (1976) Pbr Phynol.

s7. nu1 30 Hucl. E. La P J sod Mlllla B J (1977) Planra 1% I95 31 Moore. R and Bbck. C C. Jr (1979) P/anr Ph)sd. b& 3(@ 32 Brown,C M ti hlwortb. M J (1975) J Gcn Mu&/d

8& 39 33 Dunn S D rnd Kluu R V (1973) CM J Murob/ 19.

1493 34 Henson. C A. COIIIIU M and Duke. S H (1982) P&w Cd/

Phyd U. 227.

35 AwoDukcK 0,La.P J radMtbn,B J (198l)P/,wuSc1 &rrrn 23. I19

36 Ratqaak. L , R~u~aak. W. Muurowr H and Wuny. A ( 1979) Bwhcm fhyad f’jbuta 174 2.89

37 Ri1coour.G L.. Joy. K. W, Bu~ln& J god w II H II9671 P/an, Physd 42, 233

38 VIK. S c’ (1969) P/ant t’hyd 44.453. 39 LcrP J l ndThurmraD A (1972)J Exp BomnyU440 40 Pahlrh. E rnd Joy. K. W (197l)Can J Bwchm 49. I27 41 Thou. K H and Sphrr~csscr. W E (1972) P/w Phpwof

49. 550 42 Sahulka. J (1975) B& Lurr a. I5 43 Ehmkc. A and Hanmann T (1976) Phyrahmwry IS,

I61 I 44 Ema. M J sod Fowlcr. M W 11979) Pbuo 144.249 45 Surukl. A. 6dd. P rod Oaka, A (1911) Plnnra 151.457

46 Y~mayr T . OIkr A and Malwmolo. H (1984) P&uu Phpd 76. IOOP

47 Shclp. B J and Alkro& C A (1984) P/m Set Lmrrrs 3& 225

48 Bownan. E I. lkuma. H dad Stan. H J (1976) P/m Myd u, 426

49 NUJCU. W dad HJ~IIUCB~ T (1980) P&NO l4& 7 50 Pnatky. D A Jod Brunsmr J (1982) Phynd P/mu %.

303 51 Duke. S H. Fncdm. J W. ScJurdcr. L E. and Koukkan.

W L (1978) Phyd Planr 42 269 52 bah. R M god Kirk. P R lI968) Biockm Brofiys Res

cmulw 31685 53 Mckcruu. E A and lxca E M (1981) Arch BwArm

Bboph), 212 290 54 Vcnkrurunaor S rnd Du V S R t 1982) J P/aw Physd

105. 289 55 Mdlra B J (1974: Phr Phyd 54, 550 56 WLL)Itlm. I Jd SJIO. S (19771 P/-r Cc// Physw/ I& 505 57 Bone. D H (1959) Narwc IW990 58 KJIUUIO~~. T . Koonhk S end TJ~A~~II. E (1972) Physd

Planr u, I 59 Ktndt. R , PJhhch. E. and As&d. I (1980) Lw J Bnxhrm

II2 333 60 Scheld. H W . Ehmkc. A Jnd H~rm~on. T (1980) 2

tiurwJorsch 35Cc. 213 61 Anduroo. L E, Nchrlwh S C d C?umppy. M L

(197111 P/M~ Phyd 61.601 62 Punolk. R M god Snvutava. H S (1986) Phym&m.my

25. 803 63 Pol~krfpochklr&% R T (1975) L’SSR Phru Physlol 22.971 64 I@m& T I and Kow~~a. A V (1975) L’SSR Plan1

Phjsd 22 426 65 Thurm~DA.PaJm.Crodlqcuck.M V (1965)Narur

207. I93 66 HJ~ H . Pncss. I dad Rhbch. E (1976) Phyrrol Plant

x403 67 Fawok. M 0 (1977) Cm J ~&MM) 5% lU50 68 Laz D W (1973) Phyrochmurry I2 2631 69 Kretowch. W L. KM&M. T I.. Yykon. V sod

)‘lOfClUkJy& V (1974) CSSR Planr Plysol 21. 247 70 Buuh I. Moe. H and Sadoa T (1975) P/aru Phyrrol %.

71 i.i’tnm. C d Dnunrot. J (1983) f’hyd P/mu 5& 89 72 Rau~cmk. L. bqmk. W d Muurowa H (1977) Acre

sot lb PO/ 4& 347 73 HM~ T (1973) Planra Ill. I29 74 Hartmann. T.. N~pl M JD~ Ikrt. H 119731 Ploruo Ill.

II9

75 Nskluch. A (1979) BuwAtm Phyuol P@zrn 174, 80 76 Poat~ur. C and Jwok. G (1976) J P/OJU Phy,w/ 78, I33 77 l~urmc. C , Warsnun. N rnd busun~. J (1981) Phyd

P/anr sz 151 78 shultr. J 11974) Bco/ P/r~r II. m 79 zhclyuk. v M. M~aoclt. A v and Lobov* M A 11974)

tt:a/ BKX~WI Kul’r R-f ti 232 ll0 Wilson. S B rnd Bonacr. N D 11971) P/MI Phyuol U

MO

81 Fktchcr. J S 11972) Nafuw 230,466 ll2 kwa D D rnd Tclrcfn. A N I 1975) Phyrakrusrry I4

647

II3 PahlKh. t. 11972) Plonru 104. 78 84 Carnmrrrw D rnd Jrok. M (1983) Pl4r)f SCI hrm 31.

65

85 Buash. I. Mor. H wtd Sdon. T I 1976) P/-f Cd/ Physwl

17.493

86 stow S R . CopcLod. L rnd Ken&y. I R 11979) Pb,~ahrnu.w, I& 1273

87 Hwwnwtn. T and Ehmkc. A (1980) P/afo 149. 207 Jl8 TaL&whI. Y rnd Furuhsht. K lIYU0) P/MI Cd/ Ph)sw/

21. 1067 (I9 Smgh. R P (1984) Ph D ti. Dew Ahflyr

VtrhwrwdyaLya Indorc. ladu 90 Kln& J l bd Wu W Y (1971) PLJW&IWI~~ IO. 915 91 Stone. S R . Copeland. L rnd Hcydc. t: lI960) Arch

Bwchtm Bwphys In.550 92 Stone. S R . Hcyde. E rnd Cop&ad. L 11960) Arch

Bwckn Bwphys IW. 5M 93 Crlrnd. W J rnd Dcnnts. D T (1977) Arch Btakn

Btophys IBL 614 94 Grort. R <i and Soukn. J K (1977) Plum Phyuol 39.70 95 RhlKh. E rnd Gcrh~ C H R (IPSO) fhyfaknwry 19.

II 96 stone. S R utd Copclwl L 11982) Arch Bwkm

B@oph!s 214. 550 97 Pahhch. E 11971) P/wrfo I@. 222 98 Fur&&~. K and Tlkrhuht. Y 11982) P/rznf Cell Ph)sw/

23. I79 99 PunnIt. R M (19851 Ph D Therrr Dew A)ulyr

Vlshrrvldyaky& Indon. lndu IO0 Ncuclhuf. 1 rnd Hwnfuhfc#w. G 11980) Physwl Phf

w. I I01 Moore. A L end Akcrmrn. K E 0 llPR2l Burkm

fflOph)% Rrs C-WI 109. 513 I02 Sllnm. R K . Slroht. G S and Srwrsrrvr <i C’ 11975)

Ph, rrol Plcvlr 35. I26 I03 Quctr P C . Twhncr. R rnd Lotcfwn. H 119821 Buwhrm

Ph),to/ P+WI 177. 567 lo4 NKkltsh. A. Gcskc. W and Kohl. J G 11976) Blakm

Phjtrol f’jbtrrn 170. 85 IO5 Street. L. Fclkr. U rnd Erormn. K Ii llY79) Phf

Ph) W/ 63. 49 IO6 C’~mmrcnx D and Jw>b. M lIY851 Planto 163. 517 107 Bcrgnunn. L Groru. W and Koch. P II9761 J P/wr

Ph)tw/ r). 60

IOtl Camp&II. W H. Z~c~lcr. P and Bak. E 11984) flaw

PI) swl 74. 947 IO9 Dwval~. U N . Khn. B M , Rarrl. S K l nd MuarchnL

A F 11984) J Plunf Ph),ro/ 117. 7 I Ill Ranumur1hy.S l nd Ludden. P ll982)~qcn &I 5& 371 I I I Slnfi. 0. Rae. A S . Nwuwrtec. H S rnd Sun&h. R I 19841

Inf J lrop Aqrtr 2. 221 I I2 Murray. D R end KcnnaJy. I R 11980) P/w Ph)sro/ b6.

782

I I3 South. J G 11973) Plorv Physol 51.454 I I4 Arkms. C A. RIG. J S rnd Shrrkcy. P J 0975) P&N

Ph)sw/ W 807 I I5 Murny. D R ll97Y) P/unf Physd 64. 763 I I6 Murray. D R (1980) AM &fan) IS. 273 II7 %JkdOVK. V H T 11984) FLBS Letters 111. 59 II8 Thomu H 1197%) P/~No I42 I61 I I9 bng. S M rnd Tftur. J S 11980) Phyvd P/au SO. 2991 I20 Slmpsoo. R J rnd [)rllm&. M J (1981) Planfo ISI. 447 I21 Gucllo. J md .Q~LcT. B (1982) P&w Cell Physwl 2.3. %I 122 KJr. M ti Fcwaknd. J 11984) Phpwl P/ow 62 39 I23 Gwm C V (1979) Phyfahtausrry la 375 I24 Wan& Y C. Cknb S H rnd Kao. C H (1982) Ploru

Phpwl 69. I348 I25 Sm&. R P and Srtnsuvr. H S (1983) Physwl Planf 9.

549 I26 Coopt. D R and Htll-COIIIO+I~. D G 11974) Ph)w/

Ylonf 31. I93 I27 Taylor. A A, [)r-Fcln. J and Ha*111 D C 11982) New

Yh)ro/ 92. I41 l2U Plhlrh. t . M(rywJld. H Jnd Humr H 11976) Phpswl

P/hull 36 310 I29 Dukc.S H rnd Koukkwt. W L ll977)m XI/ /nfrrwww/

CUII~trrur o( fk lnrrrn sor for Ckonobwl 1975 Wuhm#ton D C. lhc PublIshIn House II. Ponlc. Mllrno. Irllu. p 705

130 bclrrr. <i F KemptO. 1 twha. S Jnd wJ),WX. k (1974) Phro II?. 29

131 WJ&M. Et Frosch. S rnd Kcmpf. 0 11974) Pfanf kf

hctrr, 3. 43 I12 Wqncr.E.Dcwrr.G F.FIwhcr.S.Frmh.S.Kempf.0

JIMI Srrocbck. L (1975) Bwrysfrau 7. 68 I33 Wcbndcr. M (1974) Ph)rw/ Pfonf W. 192 I )J Duke. S H rnd Koukkrn. W L I 1977) Ph)sw/ P/H 39.

67 I35 Kubtk.Dobow.G JndSorok&K 11979)ArfuSoc Bof PO/

4a.U)

I36 C?undkr. (3 ll9llll Nr* Phyfo/ 43. 1587 I37 ttlncr. B rnd Klein. A 0 (1968) Pfonf Phyd 43. I587 I38 SIhulk& I. Gaudfnovr. A rnd Ha&on. V I 1975) J P/a11

Ph)sw/ 75. 392 I39 S&hulk& J rnd Ltu. L I 1978) Bwl Phf 20. 444 I40 tihulkr. J Jd LIU. I_ 11980) Ph,sw/ P&n4 w). 32 I41 Rluynk. L. ~~qczak.. W Jnd Muurowa H 119811

Ph)sto/ P/MI 51. 277 I42 Trul. F . Rat~vo. R M . Pudeu. P P rnd Ciao. G I I9841

Ph,sd Plwtr 60. 61 I43 P~IIUS. C. Kkmmc. B rnd Jxobt. G 11976) J Planf

Ph)d 7& 122 IU Tunow E N 11975) L’SSR Plonr Phyuol I. 20 I45 <‘al& R A md Caldu. L S (1976) Physwl P/atf 37. I I I I46 Chlu. J Y Jnd Shqool. P D I 1979) P&xnf Physwl 63.409

147 Taylor. A A rnd Hwtll. D C 11981) h’rr Phyiol 87. 53 I4R Skpwd. D V rnd Thurman. D A 11973) Phyrakmurr)

I2 1937 I49 &r&h. I. Sldon. T god Mor. H I 1974) Phf Cdl Physwl

IS. 563 I50 Rhodcx 0. Rcndon. (3 A end Stewut. G R I I9761 Plcvlfo

129. .x3 151 Stukn. I. bntq. L . bmkrr H . Posthumur F . Von de

bJk. S J end Hofsfrr. R (1981) Physd P/an1 51. 93 I52 SWJUJVJ. H S rnd Olmrod. D P I 1984) Planf Physcol 76.

4IU I53 Wooron. J C’ 11983) Bwrkm J 2W. 527 I54 ln3c.J.Joy.K W J~~HJ~PR.H ll966)BmhJ

(iluonu~c dehydtgnrv to rUgha plants 609

loo. 517 I55 Imews. 0 A M and Ptobyn r A (1978) Nrr Phptal 01.

519 I56 We-n. G S (1972) Pht PhpmJ 49. I38 I57 Matwha. R S and J&MO. B 0 (1976) P/ant Phywol 57.

923 1% Stukn. I. bntrn& L . hbct& H . Posthumur ) . Von de

l)1Jk. S J and Holstta R 119811 Ph)sro/ P/am 51 I08 I59 Mladcnova Y I I 197U) J AQ~Y Food Ckm 26. 1274 I60 Joy. K W 11969, P/ant Ph)w/ 44. 849 161 S~cwrn. 6 R, and Rhodes. D (1977) New Ph~tol 97.257 162 Joy. K W 1lY731 Phytockmwry IL 1031

163 Takco. T (IY79) Agw Bw/ Ckm 43. 2257 164 Mnhta. s h and SrlwurJ. H s 11983) Lxprrtrntlo 39.

IIIR I65 C’unltfTc. D. lason. M. Patkm. I) and La P J 119831

Ph,tuchrnuttr) 22. 1357 I66 Sahulta. J rod &u&now A 11976) J Ploru Ph)sd 70.

IJ I67 Sn*astrw <; (’ and bowden. L (1972) J Exp fbtrm) 23.

921 I6U Duke. 5 II. Schndct. L E and Mtlkt. M G (1977) Plan,

Ph)sro/ 60. 716 169 Duke. S H Schtadct. L E, M&T. M G and NIOX. R L

I 1978) P/MI Ph,w/ 62 642 170 Duke. S H . Scbtrdcr. L E . Henston. C A, Scwut~ J E.

Vogelmng. R D and Rcndklon. J W (1979) Plant Physcol 63.956

I71 Sahulka. J and Llu. L 1197’4) Blo/ f%mr 21. I49 172 Alckhma. N D and Sokolva. S A 11975) L’SSR Plant

Phwol 22.97 I72 Alekhma. N D. KIytko\a.A I rndGtas~mova.S I 11984)

C’SSR P/ant Ph)w/ 31. 276 I74 Amos. J A and SchooL R L 119771 Crop Scl 17. 445 I75 Ramtta H. Dclapdo. J M and Pctcytn E G (1977) J

Plum Ph),w/ 67. UY I76 Gupta. P and Shawan. I S I 19791 Phytwknutry II. 1981 177 Shcotrn. I S. Luthtr. Y P. Khwd. M S and San&. R

(1981) Ph,torkrmstr)- 20. 2675 178 trota. J N t and Tucker. 1 C (197M) .h/ Scr Sor Am J

4x 75\ 179 Baana. M. Apttao.Tcp. P M and Sanchc~.Dw. M

11984) PhbJto/ Plant 61. 651 I80 Gupr P rnd Shconn. I S 11983) Plant Ph)llo/ Burhm

IO. 5

I%1 Hsuo. T C (19731 Anw Rm P/au Phywol 24. 519 IA2 Waklten. R P and Tutc. I P (1974) P/ant Sod 49. 689 IUI Bouaud. J and &Ibd. J P (1978) Physml P/m 44. 31 I84 I ut. N S and Skuhcntk. M A I IPRO) CSSR P/MI Phywol

27. 885 Ill5 Hgtut. W (1982) Buxkm Phrwol P)(awn 177. 259 I86 Hauu. M 11982) Acta Bar Pal 51. 263 187 Stcrat~. G R and Rhoda D 11978) %I* Ph,to/ W. 307 I81 8~llatd.J P and Bouud. J (IP1W)1Ph~l0ckrmll~~l9.1939 189 Klystw. L K. Kozhrnob. T K and Gtltnanor. M K

(1976) Fcrol BloAhur Kdt RPV & 284 I90 Kuynlbkov. B K . Klyshcr. L K and Rakovr N M

I I9801 F~:ro/ flrdrhm Kurt Rat I2 Iti9

I91 Wcllbum. A R . Capron. T M. C%an. H S and Hontnan. D C 11976) cn J&o of Au PO//YIMII m P/mu (Man&Id. T A cd) p I05 Cambttdgc Unwcntty Ptar. London

192 Ja~ct. H J and Kkln. H I 19771 J AIM PO//W (‘onrrd ~uor 21. 464

I93 Jqct. )I J Jnd PahItch. E (1972) tiobglo 9. I35

I94 Pabhch. E. Jrgcr. H J and Swubq. L (1972) Apm &t Y. I83

195 Jaw. H J (1975) Pjkuurn 2 Pjftanm KrMU P)fmrnxhut 2 81 I39

I96 Klan H and Jrpr. H J 11976) Z P@w:enkmn Kkrrrr, P.lkwmxhu: 83. 555

197 Wdlbutn. A R . Wtlson J and Aklnd~. P H 119801 tn1von PO//W 22. 219

198 Wcllbutn. A R . Hqpson C . Robmson. D and WaJmsky. C’ tl9Ul1 Nrw Phytol 8& 223

199 Stcubmb L and Jrpt. H J (1978) Agarw f/or 52. I37 2a) Jrgct. H J I 19821 ID Mowtowq/ o/Atr Pa//v~ts by P/WJ

lStcubm& L and JJ~c~. H J als 1 p 99 Junk. The HJ~UC

201 Saldct. R rnd Shaur. M (I9791 J P/am Phgstol 93. IO5 202 Bct*tlk. A. Ghu. A. Chatbonna. M and Botuvcnt. J F

IIYRII P/am Ph,d 76. 508 203 Sahulkr. J (IY72) Bw/ Pbnr 14. 330

204 Sahulkr J and Cudrnoa A (1975) Blo/ P/w If. 228 205 Watts. I) and Gopltn D (1970) P/am 91. I55

2M Jan. A and Stwutra. H S (1981) Physd P/am 53.2UJ 207 SankhL N aad Hukt. W (1974)Phytockrruury 13. 1319

2CUl Tkcmaladzc. G S (IPRI) CSSR P&at Phyd u. 1013 m Wu. M T . Smgh. B and Sllunkhc. D K (1971) P/am

Phhlco/ U 517 210 Wclandct. M (1978) Phyd Plant 43. 242 ?I I Rwn~te~. H end Pctcgnn. E G 11978) J Plaw Phyuo( 87.

89 212 YwnawLI. K and Suzuki. Y I 1969) Phjaxhwurr~ 8.963 213 Tcw~tr A R N and Dana. D D 11974) PhyruckruWr~

13.2071 214 Rawthrone. S. Mm&n F R. Summer&Id. R I,

Cookson. C and Coombr J I IPSO) Phytmkmutry 19.3441 215 Rata)ark. L. Muutowa. H , Raupak. W lad Womy. A

(1982) Acta Phjsd P/am 4. 73 216 Kennedy. I R 11966) Blothrm Bloph)~ Acto Iw). 285 217 Kcnnady. I R (1966) Btoc)um BlopAp Acre IX. 295 21% Kcnncdy. I R (1979) Pra Auf Blorhrm. Sot I2 Q I5 219 Schukn. K R l ndCookcr.Ci T. III (19811 P/orrr Physd

67.662 220 Groat. R G and Van0r.C P (1982) P/ant Physw/ 69.614 221 Puhu. A S and Cowla J R (1978) J Cm Mwo6~ I I I.

IO1 222 Pattason. R P. Rapa. C D. Jr JIKI Gtou H D (1979)

P/au Ph)d U. 551 223 VanaC P.Hachcl.G H.-D K.&yan.J Wand

Johnson. L E (1979) P/am Ph)sro/ 64. I 224 Grow R G end Vancr.C P (1981) Ploru Physd 67. II98 225 Apanno-Tcp. P M and Swchc~-Duz_ M (19112) P/m

Phgd 69.479 226 Ray.T B.Pc~ctxG A.TorR E.Jt rndMayne.B C

tl97R) P/ant Phyd 62 463 227 ,X.9% 0 A M mod chdwck. S 11983) Hr*- Phytd 9S.

635 228 Satktssun. G S and Fowkr. M W 11974) P/~IUO 119. 335 229 Cbuhan J S and Stwasw~ H S (1979) P/m Bwkm J

6. 7 230 Manm. F . Winspat. M 1, MacFuboc. J D and Daks. A

(1983) Pfanr Phyd 71. 177 231 Lwuuv~ H S (1980) Phytochrmwr, 19. 725

232 Keys A 1. Bwl. I F. CorncLur M 1. k P I.

Wdlsfron. R M and M&a B J (19781 Natwe 275.741 233 Kcya A J II98OO)m Thr &orkmutryo/P/amts (Mdkn B J

cd 1 Vd 5. p 359 Aadcmu Rcrr New York iW Lby4 N D H sod Joy. K. W (1978) Buwhrm &op+

RIJ C- .I. I86

610 H S SJIV~ST~V~ rnd R P SI*GM

235 La. P I. Hugha J rod Mlllm. B J (1979) J Exp BO(amy

30.65)

234 La P J rnd Mlflm. B J (1980) m ?lv Bwrknuswy oj flaws (Mdlm. B J al I Vol J. p 369 Aadcnvc Pras. Ncr York

238 YJ,“JyJ. T Jnd MJrsumolo. H (1987JSo1l Set P/-f Nufr

lm prasl 239 Nanun M AVIV. D. Utynl. H end Grlun. F tlY851

Plaw Phyd 77. 374

240 Mckcnnc. E A. Copcbnd. L rnd Lees. F M I 1981 I Arch

Bnxhrm Bmfi,s 21). 2% 241 bm.P C.Lodh.M L.Mchu.S L rndSmgh.J (1986)

Cvr scl 35. 307 242 Somcrwlk. C R Jnd Or&fen. W L 11980) Nutvr Z& 257 243 wJllSy0b-C. R M . Ken&II. A C. Turner. J C Jnd HJIt.

N P (1986) P14111 Physrol (0. 54

2U Bcr~cr.M (3.Woo.K C’.WoneS c’rndFak.H P (IIJ) Pfonr Phvd 78. 779