Glutamate Dehydrogenase Activity and Assimilation of Inorganic Nitrogen in Maize Seedlings

Bioehom. PhysioI. Pflanzen 177, 633-642 (1982)

Glutamate Dehydrogenase Activity and Assimilation of Inorganic Nitrogen in Maize Seedlings RAN"A P. SINGH and H. S. SRIVASTAVAl ) Department of Life Sciences, University of Ind ore, India K ey T erm Ind ex : enzyme activity, gluta mat e dehydro genase, nitr ogen assimilation ; Zea mays

Summary Supply of KNOa, NH 4Cl or NH 4N Oa during seedling growth in creased assimilatory glutamate dehydro genase (NADH-GDH) activit y as well as organic nitrogen in th e roots, shoots and leaves of maize (Zea mays L. cv. Ganga safed-2) seedlings. An increas e in enzyme activity and organic nitrogen was also observed duri ng a 3 h in cubation of excised tissues but only in the presence of amm onium when either excised tissues from ammonium grown seedlings or enzyme extracted from such tissues were incubated for 24 h in th e absence of a nitro gen sour ce, the activity declined, both in vivo and in vitro . Supply of NH 4CI protect ed in vivo decline in enzyme activity in secondary leaves. Cycloheximide abolish ed this prot ection of enzyme activ it y by ammonium. In most cases, th e supply of inorganic nitro gen result ed in decreased levels of dissimilatory glut amat e dehydrogenase (NAD+-GDH). Under non-indu cing conditions, t his enzyme declined at faster rate than t hat of NADH-GDH and supply of ammonium did not prote ct th is decline. Althou gh the experiments demonstr ate a variati on in th e response of NADH-GDH and NADt-GDH to nitrogen in different organs, it appears th at th e assimilat ory enzyme is operativ e in th e assimilat ion of ammonium as well as nitrate in th e maize tissues. The experiments also demonstrate th at ammonium prot ects t he enzyme activity in leav es and possibly thi s prote ction involves prot ein synt hesis.

Introduction

Synthesis of glutamic acid from 2-oxoglutarate and ammonium by the enzyme glutamate dehydrogenase (GDH, EC 1.4.1.2) has been considered to be the key reaction in the assimilation of inorganic nitrogen (JOY 1973). However, in the last few years, the demonstration of glutamate synthase (GOGAT, EC 1.4.7.1) in several plant systems (DOU GALL 1974; BOLAND and BENNY; OAKS et al. 1979), has lead to the belief that glutamate is synthesized from glutamine rather than from ammonium (LEA and MIFLIN 1974). In the light of the information available about GDH and GOGAT, it may be postulated that the relative importance of these alternate pathways of glutamate synthesis depends upon the tissues , form of inorganic nitrogen and possibly some other factors. In order to assess the role of GDH in the assimilation of inorganic nitrogen in a tissue, it is necessary to find out the correlation between the enzyme activity and the 1) To whom all correspond ences should be addressed. Abbr evations: GDH, glutamate dehydrogenase; GOGAT, gluta mine oxoglutarate amino transferase or glutama te synthase; NAD+, nicotinamide adenine dinucleotide; OIl, cycloheximide. 42 Biochem. Physiol. Pfl anzen , Bd. 177

634

R. P. SING H and H. S. SHIVASTAV A

formation of organic nitrogen during nitrogen supply. Although th e increase in asssimilatory GDH activity (Ja y 1969; TSENOVA 1975 ; EHMKE and HART)IANN 1976; BUCZEK 1979) and in some components of organic nitrogen (OJI and IZAwA 1972; NAQUIB and IBRAHIlII 1973; BOSE and SRIVASTAVA 1979) upon the supply of either ammonium or nitrate has been demonstrated in various tissues, studies correlating GDH activity and organic nitrogen are rare. In one such study with detached oat leaves, BARASH et al. (1974) demonstrated an increase in GDH activity together with some amino acids and amides during ammonium supply. The changes in other components of organic nitrogen and with nitrate supply were not examined. In almost all the cases examined, supply of ammonium resulted in increased GDH activity (SHEl'ARD and THURMAN 1973 ; BARASH et al. 1974; 1975; 1976; BIELAWSKI and RAFALSKI 1979). However, in most of these cases, the effect of ammonium has been examined after a long period of incubation with ammonium, and it is difficult to decide, whether ammonium induces enzyme synth esis or protects its turnover. The present investigation was undertaken with this perspective and with the twin objectives to study (1) possible correlation between GDH activity and organicnitrogen during nitrogen supply and (2) possible effects of ammonium on th e stability of th e enzyme. The act ivity of dissimilatory (NAD+) GDH was also determined in order to evaluate the overall role of GDH in primary nitrogen assimilation in maize seedlings. Materials and Methods Seeds of Zea mays L. cv, Ganga safe d-z were purchased fro m Natio na l Seed Cor poratio n, New Delhi. Seeds were sur face steri lised with 0.1 % HgCl2 for a bout 5 min and then washed t horo ughly with dist illed wate r before plan t ing th em eit her on moist filt er pap er in pe tri dishes or in washed sa nd contained in sma ll pla st ic pot s. Seedlings were rai sed in continuous light of about 65 W m- 2 ra dia nt flux density supplied by inca ndescent bulbs and fluoresce nt tu bes at 25 ± 2 °C, either for 4 d (for roots a nd shoots) or for 9 d (for leaves). Th ey were watered dail y wit h modi fied 1/2stre ngth Hoag lands solution withou t a ny n itrogen or eonta ining nitrogenou s salts as desir ed. In cubati on wit h vario us salts was also carri ed on in sa me envi ronme nt. Th e p H of all media was 6.0. When in cubation was to be carrie d for a lon ger durati on (24 h), chlora mp henicol (1 mf1,/ 50 ml) W,IS added in the in cubation mixture t o prevent an y bact erial contamination. For nit rogen determination, an aliq uout of 100 mg tissue fro m th e oven dried (at 60°C for 24 h) sa mp le was extracted thoroughly with 80 % ethanol. The nitrogen in t he supern at ant (soluble) an d pellets (insoluble) was determin ed separate ly by a modified mikro kjeldahl met hod (LANG 1958). Th e enzy me was extracted and assaye d following the measur ement of oxidat ion/reduction of NAD(H) (B ULE N 195G). Th e extractio n medium consisted of sodium phospha t e buffer 150 p,moles (p H 7.4), sucrose 1,200p,mol es, E DTA (as sodiu m salt) Gp,moles, cyste in 3 /lmo les in a total volume of 3.0 ml. The ratio of plan t ma t erial to extraction medium was 1: 4 (w/v) . The samples were extracted in t he cold at 0 to 4 °C and centrifuged at a bout G,OOO g for 45 min at sa me te mperat ure. Th e supernat an t was used as enzy me pr epar ation. The en zy me activity in t he pellets was ve ry low, less th au 10 % of that in t he snperna tant . The assay medium for NADH-GD H consiste d of sodium ph osph at e bu ffer 300 /lm oles (p H 8.1), (X-keto glutarate 40 !(moles, (NH4)2S0 4 45/l moles, NA D H O.G Ilmo les a nd enzy me extract 0.1 ml in a tota l volume of 3.0 ml. Th e ox idatio n of NAD H was re cord ed at 340 nm on a do nble beam spectroph ot om et er (Varian model 634). The assay medium for NAD+-GD H consisted of pho sphat e buff er 300 /Imoles (p H 8.1), gluta mic add GO Ilmoles, NA D+ 0.6 p,moles and enzyme extract 0.1 ml in a

635

Glutamate Dehydro gen ase Activity and Assimilation of Ni t roge n

fin al volume of 3.0 ml, In crease in ab sorbance was recorded at 340 mn. In ea ch case the amount of NA D(H) oxidi sed or redu ced was calc ulate d from It standard curve. In it preliminary investigation , it was observed that the enz yme was ac t iv e with NAD(P )H a lso, alt houg h with slightly low er efficiency. The response of NAD(P) H enz yme howev er , to the sup ply of inorganic nitro gen was almost the sa me as of NAD( H) a nd therefore the data for onl y N AD( H) are given . Th e r esul t s of ot her preliminary invest igation s r elated to the st abilit y of en zy me ar e publish ed earlier (J AIN a n d SHIL\STA VA 1( 81).

Re sults

Glutamate dehydrogenase act 1'vity in seedlings raised with differentnitrogens sources A significant level of assimilatory glutamate dehydrog enase (NADH-GDH) activity was detected in roots, shoot s and leaves of ma ize seedlings raised in t he absence of any nitrogen source (Table 1). Supply of 10 mM RNO a to the seedlings increased enzym e activity ver y lit tl e in roots and shoots but it caused considerable increase (upto 46 %) in the leaves. Supply of 10 mm NH 4CI, increased enzyme act ivity significantly in all tissues examined. The maximum increase was recorded in t he secondar y leaves. The increase in the pr esence of NH 4NO a (5 mM) was almost similar to that with NH 4Cl. The control level of dissimilatory glutamate dehydrogenase (NAD+-GDH) activity was relati vely lower in all the tissu es examined (Table 1). In th e tissu es of RNO a and NH 4N Oa grown seedlings, the enzyme activity was always lower than the control a ctivi ty. No enzyme a ctivi ty could be detected in the shoots of the seedlings raised with an y nitrogen sour ce. In the roots and in secondary leaves from NH 4CI grown seedlings, the enzyme activit y was almost the same as in control. The enzyme activity was considerably higher in th e pr imary leaves of NH 4C l grown seedlings compared with control. Tab le 1. Glutamate dehydrogel/flse a.ctivity i ll maize seedlings raised with differellt nitrogen sources. See dlings were ra ised with 1/2 st re ng t h Hoa glands solution either for 4 d (for roots and sh oots) or for U £I (for lea v es). Nitrogenous sa lts were included in the nutri ent solut ion as follows : Control .-nOIlO, KN0 3 - 10 111M, :'IHI CI - 10 IllJI, NH~N03 - 5 mJ!. Th e valu es relative to control are given under bracket Nit ro gen sources

Enzym e a ctivity, n moles NA D H min"! g- l fr. wt. Ro ot

~ADH -GDH

Con tro l K N0 3 N H 1Cl N H~ N 03

697 797 1,09 2 1,022

Shoot

± ~ 8 (100) ± 25 (114) ± 52 (161) ± 22 (14 5)

1,040 1,124 1,345 1,:m

± 17 (100) ± 12 (108) ± 28 (129) ± 35(117)

± S.E.

Pri. L eaf

Sec. Leaf

± 25 (100) ± 22 (130) ± 38( 156) 1,43.t ± 27 (155)

640 934 1,114 1,113

9 ~5

1,2 02 1,443

N AD+-GD H Cont rol

K:'W s NHtCI N Ht ~ 03 42*

541 364 509 389

± 07 (100) ± 67 (67)

± 11 ± 72

(94) (72)

128 0 0 0

± 05 (100)

b22 ± 406 1)93 ± 472

18 (100) (78) 29 (133) 17 (90)

± 21 ±

846 629 846 551

± 13 (100) ± 36 (146) ± 30(174) ± 35 (174) ± 29 (100) ± 13

(74)

± 23

(65)

± 34 (100)

636

R. P.

S INGH

and H.

S . SHlYAST AYA

Table 2. Organic nitrogen of the waize seed/il/gs raised 'with different nitrogen sources. Growth conditi ons and other det ails as in Table 1 Nitrogen Orga nic nitrogen mg, g- l fr owt. sourc es Root Shoot Etha nol-soluble nitro gen Cont rol KN0 3 NHtCI NH tN03

± 0.05 (100) ± 0.02 (112)

1.07 1.20 1.45 ± 1.13 ±

0.01 (136) 0.01 (122)

± 0.01 (100) ± 0.01 (106)

1.12 1.19 1.45 ± 1.28 ±

In solubl e nitro gen Control K NO:1 NH 4 Cl NH,\N 0 3

1.59 1.68 1.83 1.80

± 0.04 (100) ± 0.01 (105) ± 0.01 (115) ± 0.02 (113)

± S.E.

2.84 2.56 3.00 2.81

0.04 (120) 0.0 (114)

± 0.01 (100) ± 0.05 (90) ± 0.01 (106) ± 0.04 (99)

Pri . Leaf

± 0.08 (100)

0.74 1.02 ± 1.15 ± 1.11

± 0.0

0.0 (138) 0.0 (155) 0.01 (150)

0.71 0.79 ± 1.06 ± 0.82 ±

(100) 0.01 (111) 0.0 (149) 0.01 (115)

± 0.02 (100) ± 0.05 (107) ± 0.0 (116) ± 0.02 (109)

2.16 ± 2.23 2.52 ± 2.34

0.04 (100)

±

2.03 2.18 2.35 2.22

Sec. Leaf

± 0.06 (103) 0.05 (117)

± 0.05 (108)

Organic nitrogen in the maize seedlings raised with differentnitrogen sources In order to investigate the possible correlation between glutamate dehydrogenase activity and organi c nitrog en, both 80 % ethanol soluble and insoluble nitrogen were determined in root s, shoots and leaves of seedlings rai sed with different nitro genous salts (Table 2). Supply of KNOa caused increase of soluble nitrogen by 38 % in primary leaves (relat ive to control), to 11 %in secondary leaves and 6- 12 %in root s and shoots . Supply of NH 4NOa also increased soluble nitro gen to almost th e same magnitude. Ammonium chloride caused a higher increase in soluble nitrogen especially in th e roots and leaves. Supply of KNOa had little effect on insoluble nitrogen and in shoots th e salt caused a decline. With NH4CI, the insoluble nitrogen increased slightly in all th e t issues, the maximum increase in nitro gen being 16-17 % in the leaves. Ammonium nitrate increased insoluble nitro gen in th e leaves by 8- 13 %. There was a good correlation between increase in assimilatory GDH and organic nitro gen durin g nitro gen supply, which was particularly app arent with the soluble nitrogen and in the root s (Fig. 1).

Effect of short term supply of nitrogenous salts on glutamate dehydrogenase activity Nitrogenous salts supplied for 3 h to the tissues from th e seedlings raised without any nitrogen caused variable increase in the level of NADH-GDH activity (Table 3). Potassium nitrate increased relativ e to control , enzyme level; very little in roots and shoots bu t to a considerable exte nt in t he leaves. The increase in enzyme activity with NH4NOa was almost similar to NH4CI in th e leaves, although it caused only a slight increase in th e root s and shoots. Durin g short term supply of nitrogenous salts, NAD+-GDH activity was inhibited in th e roots and shoots, th e inhibi tion being more pronoun ced in the first mentioned t issue (Table 3). Nitro genous salts causcd a significant increase in enzyme activity in the leaves exccpt for KNOa, which inhibited enzyme activity by 8 % in th e secondary

leaves.

Glutam at e Dehydrogenase Activity and Assimilation of Nitrogen

637

% INCREASE OVER CONTROL 1 0 0 , - - - - - - - - - - - - - - ----,

o

ENZYI1E ACTIVITY

ILZI SOLUBLE NITROGEN IIIIIl INSOLUBLE NITROGEN 80 I - - - - - - - - - - - - - - - - - - - - i

601-----------

Y.:;.: .... . ......

201-------

NH'f CI

Fig. 1. Correlation between N .-IDH glutamate dehydrogenase activity and organic nitrogen in the roots of maize seedlings supplied with different nitrogen sources. Seedlings were raised for 4 d wit h modified 1/2 strengt h Hoagland s soluti on conta ining either no nitro gen or nitrogenous salt as desired. Dat a tak en from Tabl e 1 and 2.

Effect of short term supply of nitrogenous salts on organic nitrogen Supply of KNOa incr eased ethanol soluble nitro gen slightly in the primary leaves while in oth er tissues it caused a decline (Tab. 4). Ammonium chloride increased soluble nitro gen in both primary and secondary leaves. Potassium nitrate had either no effect on insoluble nitrogen or stimulated it slightly as in roots. A slight increase was observed also with NH4CI except in the shoots. A considerable increase in insoluble nitrogen with NH 4N Oa was recorded in roots only.

I n vivo and in vitro stability of glutamate dehydrogena.se a.ctivity as affected by ammonium and cycloheximide Root and secondary leaf tissue s from the seedlings raised with NH 4 CI were incubated with NH 4 CI, ClI or NH 4CI + en for 24 h and enzyme activity measured to

examine wheth er amm onium affect ed th e stability of th e enzyme (Table 5). In roots,

638

R. P.

SINGH

and H. S.

Table 3. Effect of short term supply of uitrocenoun salts

SIUVASTAYA

011

glutaillate dehydrogenase activity in maize

seedlings.

Seedlings were raised with modified 1/2 strength Hoaglands solution containing no nitrogen either for 4 d (for roots and shoots) or for 9 d (for lea Yes). Detached plant tissues were floated on desired nitrogenous salt for 3 h in light. Other details as in Table 1 Enzyme activity, n moles NADII mirr !

Nitrogen sources

Hoot

NADH-GDH Control K:.\"03 NH~CI

NH~:\03

Shoot

(;21 ± 642 ± vss ± G83 ±

22 (100) 14 (1m) 12 (119) 08 (110)

1,(HiG 1,12il 1,2''>1 1,123

g~1

Ir. wt.

± S.E.

Pri. Leaf

± 21 (100) ± 13 (105) ± 14 (117) ± ;\4 (105)

See. Leaf

± 24 (100) ± 41 (112) ± GO (155) ± 70 (149)

939 1,080 1,451 1,338

± G4(100) ± 19 (115) ± GO (155) ± 44 (142)

(;52 730 1,011 972

478 894 8GG 929

± 07 (100) ± 41 (187) ± 23 (181) ± 23 (194)

857 ± 788 ± 1,431 ± 1,lGG ±

NAD+-GDH Control KT\"()3

517 364 343 393

NH~CI

NH J:\03

± 04 (100) ± 04 (70) ± 21 (GG) ± 18 (7G)

104 ± 94 ± G8 ± 83 ±

10 (100) Oil (90) 08 (65) 11 (80)

41 (100) 04 (92) 15 (lG7) 21 (13G)

Table 4. Effect of short term supply of nitroqenoue salts on organic nitrogen of maize tissues. Growth conditions and other details as in Table 3 Nitrogen Organic nitrogen mg g-1 Ir. wt, sources Root Shoot

± S.E. Pri. Leaf

Sec. Leaf

Ethanol-soluble nitrogen Control K:\03 NH~CI

NH~N03

± 0.04 (100) ± 0.04 (96) ± 0.0 (136) i.os ± 0.01 (99)

1.04 1.00 1.41

1.13 1.09 1.35 1.08

± 0.0 (100) ± 0.02 (9G) ± 0.02 (119) ± 0.0 (9G)

0.55 ± 0.59 ± 0.G9 ± 0.63 ±

2.91 2.84 2.90 2.98

± 0.03 (100) ± 0.01 (98) ± 0.01 (100) ± 0.01 (102)

1.63 1.70 1. 77 1.75

0.0 0.0 0.01 0.02

(100) (107) (125) (114)

O.GO ± 0,48 ± 0.[)8 ± 0.G4 ±

0.01 (100) (1.04 (80) 0.01 (97) 0.04 (106)

Insolu ble nitrogen l.51 ± 1.G7 ± 1.80 ± NH~Cl NH~N03 1.76 ± Control KN0 3

0.05 0.01 0.03 0.04

(100) (111) (119) (117)

± 0.04 (100) ± 0.0 (104) ± 0.01 (108) ± 0.02 (107)

1.81 1.85 2.01 1.79

± 0.01 (100) ± 0.01 (102) ± 0.01 (111) ± 0.0 (99)

the activity of NADH-GDH declined by about 13% in control. While NH4CI had no effect on this decline, CH either alone or with NH4CI caused a further decrease in enzyme activity. In secondary leaves, on the other hand, NH4CI caused further increase in enzyme activity while CH accelerated the decline. This effect of CH was partially abolished when NH4CI was also included with CH The NAD+-GDH activity also declined when the tissues wcre incubated for 24 h without any nitrogen (Table 5). In roots, either NH4CI or CH accelerated this decline to some extent. In the leaves, the decline in enzyme activity in the presence of NH4CI, CH or NH 4CI + CH was not as pronounced as in control.

Glutamate Dehydrogenase Activit y and Assimil ation of Nitro gen

639

Table 5. I II vivo stability of glutamate dehydrogenase as affected by ammonium and eycloheximide supply in maize tissues. Seedlings wer e rai sed with modified 1/ 2 strengt h Hoaglands solutio n containing 10 m~I N H 4C l as sole nitrogen source either for 4 d (for roots) or for 9 d (for leav es). Th e ex cised tissues from these seedlings wer e in cub ated in th e desir ed solutio n for 24 h in light. Th e concent rations wer e N H 4 Cl 10 mM ; cycloheximide - 5 mg/ml In cub ation media

Enzym e a ctivity, nm oles NADH mirr-' g- l Ir. wt, ± S.E. Roo t

Secon dary Leaf

NADH-G DH

± 29 (100)

1125

±

875 855 563 634

± 30 ± 24 ± 05 ± 15

(87) (88) (56) (63)

866 1,485 563 866

± 17 (77) ± 60 (132) ± 31 (50) ± 19 (77)

At 0 h

502

± 09 (100)

835

± 10 (100)

Aft er 24 h in No ne (control) N R 4C l CR N H 4C l + CR

374 326 252 347

± 09 ± 14 ± 02 ± 12

618 701 751 776

± 52 ± 22 ± 28 ± 05

At 0 h

1006

After 24 h None (control) N H 4Cl CR NR4Cl + CR NAD +-GDR

(75) (65) (50) (69)

20 (100)

(74) (84) (90) (93)

Tabl e 6. I n vitro stability of glutamate dehydrogenase as affected by allllnollium and cycloheximide. Qrowth conditi ons and other det ails as in Ta ble 5. The enz ym e extracte d from maiz e tis sues was in cub ated with NH 4C I, CR or both together as in Table 5 for 24 h In cubation media

En zyme activity, nmol es NA DH min- ' g-l frowt. ± S.E . Root

NA DH-GDR At Oh

1,006

Aft er 24 h in None (control) NR4Cl CR NH 4Cl CH

±

After 24 h in None (cont rol) N R 4 Cl

OR

N R 4 CI

+ CR

± 29 (100)

1,125

± 20 (100)

583 795 785 976

± 07 ± 05 ± 10 ± 07

(58) (79) (78) (97)

472 427 495 506

± 09 ± 09 ± 09 ± 16

502

± 09 (100)

835

± 10 (100)

397 190 247 343

± 15 ± 08 ± 06 ± 09

559 584 276 518

NAD+-GDH At 0 h

Secon da ry leaf

(63) (38) (49) (68)

± 09

± 13

± 23 ± 08

(42) (38) (44) (45)

(67) (70)

(33) (62)

640

R. P.

SINGH

and H.

S. SRIVASTAVA

An in vitro incubation of enzyme extract at 25°C for 24 h demonstrated a significant decline in enzyme activity (Table 6). Supply of NH 4CI, CH or NH 4CI + CH protected this decline to some extent in roots but not in leaves. The activity of NAD+-GDH appeared to be more stable than that of NADH-GDH under in vitro conditions. In roots, NH 4CI or CH accelerated the decline, but when both were supplied together the enzyme level was almost the same as in control. In the leaves, the enzyme level in NH 4CI was almost the same as in control but a further decline occured in CH. Discussion

The experiments demonstrate that assimilatory GDH activity in maize tissues is increased by nitrate or ammonium either supplied individually or together. In most of the tissues examined, the maximum enzyme level is recorded with NH 4CI followed by that obtained with NH 4N03 and KN0 3 • This response of GDH activity to nitroge nous salts is in general agreement with the results obtained in other tissues (TSENOVA 1975; BUCZEK 1979). There is some variation in enzyme activity and its response to nitrogenous salts among the tissues also. The age of the tissue also appears to matter as in some cases there was a difference between primary and secondary leaves also. While ammonium may increase enzyme activity by inducing the synthesis of the enzyme (BIELAWSKI and RAFALSKI 1979), nitrate can do so only after its reduction and conversion to ammonium ion. Therefore, increase in GDH activity with nitrate supply during a 3 h treatment is not as pronounced as when the seedlings are raised with it. A correlation between NADH-GDH and organic nitrogen (Fig. 1), demonstrates that operation of GDH pathway for the assimilation of inorganic nitrogen is effective in maize tissues. SKOKUT et al. (1978) have suggested that while primary amination reaction during nitrate supply was due to glutamine synthetase and GOGAT, the GDH pathway is operative mainly during ammonium supply. Although in our experiments also increase in GDH activity and organic nitrogen was maximum in the presence of NH 4Cl, the increase in these two components in the presence of KN0 3 , especially in the primary leaves was significant enough for deriving the conclusion that GDH pathway operates also in the assimilation of nitrate nitrogen, in the maize leaves. Increase in GDH activity in the presence of ammonium during a 3 h incubation is quite low as compared to when the seedlings are raised with it. When the samples from ammonium grown seedlings are transferred to ammonium free medium, the activity declines. Supply of NH 4Cl protects this decline in the leaves. It may be concluded that this decline in the leaves was due to enzyme inactivation which is protected by the ammonium. Also in Lemna gibba (SHEPARD and THURMAN 1973), increase in GDH activity due to ammonium was possibly the consequence of enzyme activation rather than synthesis, as inhibitors such as chloramphenicol, lincomycin and puromycin inhibited ammonium induced GDH activity only partially and that too after 2 d. Further, in our experiments this protection or positive modulation on enzyme activity appears to be dependent upon protein synthesis as it is not observed in the presence of

Glutamate Dehydrogenase Activity and Assimilation of Nitrogen

641

cycloheximide and in in vitro experiments. Cacloheximide alone in general causes more decrease in enzyme activity than control and therefore, it appears that the stability of the enzyme depends upon continuous synthesis of protein even in the absence of ammonium. Another indirect evidence for possibility of enzyme modulation by ammonium comes from the observation that it had different effect on NADHGDH and NAD+-GDH. These two enzyme activities are functions of the same protein aggregate. Differential effect of ammonium on these two activities indicates that it changes the physico-chemical nature of the enzyme protein rather than its total amount. The dissimilatory GDH in most cases is inhibited by the supply of inorganic nitrogen in the roots and shoots. It is not surprising because the enzyme is involved in the deamination of glutamate and ammonium thus mobilised is used for the synthesis of other amino acids. In cultured soybean cells also the activity of NAD+-GDH is low and the fluctuation is not marked in response to the change in the level of nitrate, ammonium or glutamate (CHIU and SHARGOOL 1979). Further, in vivo NAD+-GDH appears to be much more stable than NADH-GDH. Excision of plant tissues from the seedlings causes a drastic decline in enzyme level, even if the tissue is incubated in NH 4CI. Thus the dissimilatory enzyme appears to be more sensitive to the changes in the nutrient level than the assimilatory one. Acknowledgement This research was financially supported by a research grant (no. 37(377)80 EMRII) from C.S.I.R. New Delhi to HSS.

References BARASH, I., SADON, T., and MOR, H.: Relationship of glutamate dehydrogenase level to free amino acids, amides and ammonia in excised oat leaves. Plant Cell Physiol. 15, 563-566 (1974). BARASH, J., MOR, H., and SADON, T.: Evidence for ammonium dependent de novo synthesis of glutamate dehydrogenase in detached oat leaves. Plant. Physiol. 56, 856-858 (1974). BARASH, I., MOR, H., and SAD OW, T.: Isozymes of glutamate dehydrogenase from oat leaves: Properties and light effect on synthesis. Plant Cell Physiol. 17, 493-500 (1976). BIELAWSKI, W., and RAFALSKI, A.: Glutamate dehydrogenase and glutamate synthase in rye seedlings supplied with ammonium and nitrate. Acta Biochem. Pol. 26, 383-396 (1979). BOLAND, M. J., and BENNY, A. G.: Enzymes of nitrogen metabolism in legume nodules: Purification and properties of NADH dependent glutamate synthase from lupine nodules. Eur. J. Biochern. 79, 355-362 (1977). BOSE, B., and SRIVASTAVA, H. S.: Role of nitrate in delaying senescence of detached leaves. Indian J. Expt. BioI. 17, 932-934 (1979). BUCZEK, J.: Ammonium and potassium effect on nitrate assimilation in cucumber seedlings (Cueumis sativus cv. Monaskyrsti). Acta SOl'. Bot. Pol. 48, 157-170 (1979). BULEN, W. A.: The isolation and characterisation of glutamic dehydrogenase from corn leaves. Arch. Biochem. Biophys. 62, 173-183 (1956). CHIU, J. Y., and SHARGOOL, P. D.: Importance of glutamate synthase in glutamate synthesis by soybean cell suspension culture. Plant Physiol. 63, 409-415 (1979). DOUGALL, D. K.: Evidence for the presence of glutamic synthase in extract of carrot cell cultures. Biochem. Biophys. Res. Commn, 58, 639-646 (1974).

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R. P. SINGH and H. S SRIVASTAVA, Glutamate Dehydrogenase Activity and Assimilation etc.

EHMKE, A., and HARTMANN, T.: Properties of glutamate dehydrogenase from Lemna minor. Phytochemistry 15, 1611-1718 (1976). JAIN, A., and SRIVASTAVA, H. S.: Effect of salicylic acid on nitrite reductase and glutamate dehydrogenase activities in maize roots. Physiol. Plant. 53, 285-288 (1981). JOY, K. W.: Nitrogen metabolism of Lemna minor. II. Enzymes of nitrate assimilation and some aspects of their regulation. Plant Physiol. 44, 849-853 (1969). JOY, K. W.: Control of glutamate dehydrogenase from Pisum sativum roots. Phytochemistry 12,

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Receiced March 16, 1982 Author's address: H. S. SRIVASTAVA, Department of Life Sciences, Vigyan Bhawan, Khandwa Road, University of Indore, Indore - 452001, India.