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Increase in in vivo Nitrate Reductase Activity in Bean Leaf Segments in the Presence of Ethanol
Biochem. Physiol. Pflanzen 178, 131- 138 (1983)
Increase in in vivo Nitrate Reductase Activity in Bean Leaf Segments in the Presence of Ethanol REKHA PURANIK and H. S. SRIVASTAVA') Department of Life Sciences, Vigyan Bhawan, University of Indore, India
Ke y Term Index: nitrate reductase activity, bean leaves, ethanol effects, enzyme activity
Summary The substrate inducibility of in vivo nitrate redu ctase (NR, EC 1.6.6.1) activity in bean leaf segments increased with the supply of 10 to 100 mM ethanol. Ethanol also in creased soluble protein, but to a lesser extent while it inhibited the peroxidase activity. The increase in enzyme activity in the presence of ethanol was more pronounced at lower concentrations of nitrate than at higher. While supply of cycloheximide inhibited the substrate induction of nitrate reductase, it had little effect on increase in enzyme activity due to ethanol. Chloramphenicol inhibited the ethanol·induced in crease slightly, while tungstate com pletely blocked it. While glucose had little effect, sucrose increased enzyme activity substantially in the presence of nitrate. Supply of ethanol had no effect on enzyme activity in the presence of sucrose, althou gh it increased enzyme activity in the presence of glucose. l''lalonate, in a concentration of 2 to 10 mM inhibited enzyme activity both in the absence as well as in the presence of ethanol. The experiments demonstrate that ethanol increased nitrate reductase activity, by increasing nitrate uptake and transport, and possibly also by accelerating the conversion of inactive apoenzyme to active enzyme molecules.
Introduction
The activity of nitrate reductase i8 known to be controlled by various nutritional and environmental factors (SRIVASTAVA 1980). There are reports of increase in enzyme activity by >everal endogenous factors, which are neither metabolites of the nitrogen pathway nor one of the cla8sical growth regulators (KNYl'L 1979; SHEN 1972; JAIN and SRIVASTAVA 1981). In most of the cases, however, either the mechanism or the physiological significance of such an increase is not known. Ethanol has been shown to increase nitrate reductase activity in Agrostemma gitkago embryos (BORRIS 1967). During a preliminary investigation on effects of methanol, ethanol, propanol , and butanol on in vivo nitrate reductase activity in bean leaves, only ethanol was found to increase enzyme activity at low concentrations. Further experime)lts were performed with an aim to find a possible mechanism(s) of increase in nitrate reductase activity in the presence of ethanol. The activity of peroxidase was also determined to examine whether the effects of ethanol were also extended to otheI enzymes. 1) To whom all correspondence should be addressed. 9'
132
R.
PUR ,\NIK
and H. S. SIUYASTAYA
Material and Method. Seeds of Phaseolus vulgaris cv. RAHtAH (red bean) werc purchased from a local dealer. The seeds were surface sterilised with 0.1 % HgCIJ! for about 1 min and then washed thoroughly with distilled water. Then they were planted in small pots containing washed sand and the seedlings were raised for 8 d in continuous light of about 65 w m- 2 radiant flux density (400- 700 nrn) supplied by inca.ndescent bulbs and fluorescent tubes, at 25 2 °c. The seedlings were watered daily with half strength Hoagla,nd's ~olution, which was modified to exclude nitrogen. In each case, 8 d old uniformly grown seedlings, which did not develop any nodules by that time, were used for sample collection. For various treatments, segments (about 0.25 cm 2 ) of primary leaves were floated on the desired solution 2°C in light. The pH of the solution was 6.0 in ea.ch case . for 24 h at 25 Nitrate reductase activity was assayed in vivo by the method of SRIVASTAVA (1975). Protein was estimated by the method of LOWRY et at. (1951). Peroxidase was extracted and its activity assayed by the method of MAEliLY (1954). The endogenous metabolic nitrate pool in the lea-ves was estimated by mea.suring the production of nitrite under anaerobic conditions (ASLAi'ol 1981). Each determination waS carried out at least 4 times . The data presented are the average ± S.E. of such determinations.
±
±
Results Effect of Ethanol on Nitrate Reductase and Peroxidase Activities and on Protein content of Bean leaf Segments Supply of 10 to 100 mM ethanol with nitrate increased nitrate reductase activity in leaf segments; the maximum increase being achieved with 20 to 60 mM ethanol (Table 1). It also increased protein content but not to the same extent. For example, 40 mM ethanol increased nitrate reductase activity by 70% while it caused only 30% increa,e in the protein. Unlike nitrate reductase activity, peroxidase was inhibited slightly by the supply of 10 to 100 mM ethanol. Effect of Ethanol and Nitrate on the Endogenous Nitrate Pool The amount of nitrite secreted in the medium (which contained no nitrate) which is taken to be an estimate of the metabolic nitrate pool, was low when leaf segments Table 1. Effect of ethanol on nitrate reductase act-ivity, protein content, and peroxidase activity i n b ean leaf segments. Leaf segments were floated on 10 mM KN0 3 containing the desired concentration of ethanol, for
24 h in light. The values relative to the control are given in brackets Concentration of ethanol,
ruM
Nitrate reductase activity, ,umol N0 2 h - 1 g_l
Protein content, mg g- l fr.wt.
Perox idase activity, O.D. min- 1 g_l
j
fro wt.
Ir. wt.
8 10 20 40 60 100
2.56 3.28 4.08 4.37 4.13 3.38
± 0.02 (100) ± 0.05 (128) ± 0.14 (159) ± 0.08 (170) ± 0.06 (161) ± 0.05 (132)
18.21 21.63 22.71 23.75 22.31 20.96
± 0.21 (100) ± 0.21 (118) ± 0.29 (124) ± 0.35 (130) ± 0.79 (122) ± 0.75 (115)
33.3 29.0 29.8 28.5 28.5 29.3
± 0,07 (100) ± 0.29 (87) ± 0.33 (89) ± 0.09 (86) ± 0.09 (86) ± 0.26 (88)
Nitrate Reductase Stimulatio n by Ethanol
J33
Table 2. Effect of ethanol and nitrate on the endogenous nitra,te pool. Leaf segments were floated on t he desired solution for 24 h in light. Secretion of nitrite fro m t h.~se lea.! segments was measured a.fter in cubating them anae robi call y (ASLAM 1981) Treatment
Nitrite produced, ,umoi h- 1 g - l fe. wt.
Control
0.05 0.43 1.88
±
1.93
± 0.Q4
Ethanol (40 mM) KNO, (10 mM) Ethanol (40 mM)
+
0.0
± 0.0 ± 0.03
KNO, (10 mM) Table 3. Effect of nitrate level on increase in nitrate reductase activity in the presence of ethanol. Leaf segments were floated on different concentration s of KN0 3 • containing either 0 or 40 ml\I ethanol , for 24 h in light Concentmtion of nitrate, m~f
N itrite red uctase a,c tivity, "mol N0 2 h- 1 g - l fro wt. - ethanol
0 0.5 1.0 2.0 5.0 10.0 50.0
0.17 0.22 0.43 0.72 1.60 2.56 7.98
± 0.Q2 ± 0.01 ± 0.03 ± 0.01 ± 0.0 ± 0.02 ± 0.15
+ ethanol 0.53 0.73 1.Z3 1.56 3.26 4.37 10.88
± 0.01 ± 0.0 ± 0.01 ± 0.03 ± 0.03 ± 0.08 ± 0.15
Increase in the presence of ethanol , percent
211 233 183 115 104 70 36
were pretreated with ethanol only (Table 2). Nitrite secreted from leaf segments pretreated with nitrate was high ; 1.88 ,umol h _1 g_1 IT. wt. Supply of ethanol with nitrate had no effect on this level, indicating thereby that ethanol caused actual increase in enzyme activity and did not only accelemte the diffusion of nitrite into the medium.
Effect of Nitrate Concentration on Nitrate Reductase Activity in the Presence or Absence of Ethanol Supply of 0.5 to 50 mM KN0 3 caused an almost linear increase in enz yme activity in bean leaves (Table 3). When 40 mM ethanol was also included in the medium , the activity increased with increasing concentration of nitrate, but it was always higher than the respective control (minus ethanol). Further, the in crease due to ethanol supply was higher at a lower level of nitrate and it decreased progressively as t he concentration of ni trate in creased .
Effect of Inhibitors on Nitrate Reductase Activity in the Absence or Presence of Ethanol Increase in nitrate reductase activity by nitrate was substantially inhibited by cycloheximide, chloramphenicol, or sod ium tungstate (Table 4). The inhibition was
134
R.
PURANIK
and H. S.
SR IVAS TAVA
Table 4. Effect of inh1:bitors on increase in nitrate reductase activity in the presence of ethanol. Leaf segments welC floated on 40 mM etha.nol or on 50 mM KN0 3 or on both together in the presence or absence of the corresponding inhibitor, for 24 in light
Treatment
Nitrate reducta se activity, ,amol N0 2 h- 1 g - l fr. wt. Inhibitor used
Cycloheximide (5 mg/I)
None (control)
Ethanol KNOa Ethanol K NO,
+
0.58 7.98 10.88
± 0.10 ± 0.15 ± 0.15
0.24 1.33 3.14
Chlora mphenicol (1 g/I)
± 0.05 ± 0.13 ± 0.18
0.33 0.72 1.33
± ± ±
Sodium tungstate (60 I'M)
0.01 0.03 0.02
0.29 0.67 0.65
± ± ±
0.0 0.01 0.02
Table 5. Effect of carbon compouuds on nitrate reductase activity in tile presence or absence of ethanol. Lea,f segments were incubated in a solution of the desited carbon compound with 10 mM KNO a either in the prese nce (40 mM) or absence of etha.nol, for 24 h in light Carbon source
Nitrate reductase a.ctivity J&mol N02 h- 1 g - 1 fro wt. -etha.nol
None (control)
Glucose (10 mM) Sucrose (5 mM) Oxaloacetate (2.5 mM)
2.56 2.98 7.21 0.92
±
0.02 (100)
± 0.10 (116) ± 0.12 (284) ± 0.04 (27)
+ ethanol 4.37 4.92 7.74 0.94
± 0.08 (100) ± 0.13 (112) ± 0.11 (117) ± 0.06 (22)
Increase in the presence of ethanol, percent
70 65 1 2
also observed in the absence of nitrate. Although the enzyme activity in the presence of ethanol was also inhibited by these compounds, the increase by ethanol, was little affected by cycloheximide; and slightly by chloramphenicol. Supply of tungstate on the other hand, completely inhibited the increase in enzyme activity due to ethanol.
Effect of Carbon Sources on Nitrate Reductase Activity in the Presence or Absence of Ethanol Although, 10 mM: glucose caused only a slight stimulation of enzyme activity, sucrose (5 mM:) stimulated it 3ignificantly (Table 5). Increase in enzyme activity due to ethanol wa~ observed only in the presence of glucose, but not in the presence of sucrose. Oxa.loacetate (2.5 mM:) inhibited enzyme activity Significantly, and again ethanol did not increase enzyme activity when this acid was included in the medium.
Effect of Malonate or, Nitrate Reductase Activity in the Absence or Presence of Ethanol Supply of malolHlte inhibited enzyme activity; the inhibition increasing gradually with the increase in concentration of malonate from 2 to 10 mM: (Fig. 1). The inhibition
135
Nitrate Reductase Sti mulation by Ethanol
5
"
"",t
" "
-'-'---' ... ,------------ -------t
o
o
2
5
10
[MALONATE] ,mM Fig. 1. Effect of different concell trations of malollate 011 nitrate reductase activity 1:'1/ tlte presence or absence of ethanol. Leaf segments were in cubated in the desired conce ntrations of malonate either in the presence (40 m~'f) or absence of ethanol. In ea.ch case ]0 mM "NO, was also included in the medium. Dashed lin es ~ min us etha nol ; solid lines ~ plus ethanol
was observed both in the absence as well as in the presence of ethanol. Further, the increase in enzyme activity due to ethanol was ob!erved at each concentration of malonate; although it was less. Discussion An increase in n'i tratc
re ducta~c
activity in the presence of ethanol and nitrate
is demonstrated. This increase appears to be specific for nitmte reductase activity beCiluse increase in total soluble protein was not as high as in enzyme activity. In
136
R.
PUHANIK
and H. S.
SRIVASTAVA
fact, an increase in protein may be the consequence of an increase in llitrate reducta:lC activity, as the enzyme is believed to be the rate limiting cnzyme in overall assimilation of nitrate (BEEVERS and HAGEMAN 1969). PEIVE et al. (1975) have suggested that peroxidase, under certain conditions, has also nitrate reducing ability in bean plants. Under the present conditions, the activity of nitrate reductase was increased by ethanol while that of peroxidase was decreased. This again demonstrates that increase in nitrate reductase activity by ethanol is very specific and other proteins which could possibly perform similar functions, are not induced. The supply of tungstate, a specific inhibitor of synthesis of nitrate reductase molecules (HEIMER et al. 1969), inhibits the increase in enzyme activity due to ethanol (Table 4). The increase in enzyme activity appears to be real and not an artifact of the methodology used, as the secretion of nitrite in the absence of nitrate in the assay medium was little affected by ethanol treatment (Table 2). Anaerobiosis causes increase in nitrate reductase activity in barley aleurone layers (FERRARI and VARNER 1970). Further, in vivo supply of oxygen inhibits nitrate reduction in cotton leaf discs (RADIN 1973). Anaerobiosis increases the activities of pyruvate decarboxylase and alcohol dehydrogenase also in rice roots (JOHN and GREENWAY 1976). It is likely that the increase in enzyme activity under anaerobic conditions is due to ethanol produced endogenously. Lipid components of the membrane, because of their solubility in ethanol, may be disrupted during ethanol treatment. This may lead to an increase in nitrate uptake and also in its intra-cellular mobilisation for enzyme induction. Increa!.e in nitrate reductase activity with ethanol was higher at lower concentrations of nitrate (Table 3). This may suggest that ethanol increased enzyme activity by increasing nitrate uptake. However, ethanol also increased enzyme activity at a high level (50 mM) of nitrate. Further, in another experiment (data not shown), when leaf segments were floated on 100 mM KNO. prior to treatment with 10 mM nitrate + ethanol, the increase in enzyme activity was observed as usual. This may suggest that increase in enzyme activity in the presence of ethanol was only partially mediated via increased uptake of nitrate. Supply of cycloheximide, an inhibitor of protein synthesis on 80 S ribosomes had little effect on increase in enzyme activity due to ethanol; while chloramphenicol, an inhibitor of protein synthesis on 70 S ribosomes, inhibited the process slightly. This may indicate that increase in nitra.!e reductase activity in the presence of ethanol was not dependent upon cytoplasmic protein synthesis, but mitochondrial and chi oroplastic activities contributed to the process to some extent. Since tungstate, which inhibits nitrate reductase activity by replacing molybdenum from the enzyme molecule (NOTTON and HEWITT 1971), inhibited the increase in enzyme activity completely, it appears that ethanol was somehow involved in the formation of an active enzyme molecule from apoenzyme. This activation may include the insertion of molybdenum in the presynthesized protein. Alternatively, it may be postulated that ethanol increased enzyme activity possibly by acting as a source of energy and/or carbon for the synthesis of enzyme molecules.
Nitra te Reductase Sti mul ation by Ethanol
137
However, glucose, a bet ter source of carbon/energy docs no t increase enzyme activity in t he bean leaves to t he extent ethanol does and increase due to etha nol ia observed even in the presence of glucose. Oxaloacetate, a n in tcrmediate of tricarbo xylic acid cycle, which could a lso provide require d carbon/energy, do cs not in crease enzyme activity in the absence or presence of ethanol ; instead it inhibi ted t he same. This inhibition by oxaloace tate is not understood at present . Sucrose, on the other hand, increases enzyme activity significantly a nd increase due to ethanol is no t obser ved in the presence of sucrose. In contrast to our this observation wi th t he leaf enzyme, the enz yme in the roots is stimulated both by glucose a nd sucrose (HANISCH TEN CATE and BRETELER 1981). Sucrose is believed to be a specific co-indu cer or derepressor in the induction of nitrate reductase activity in pea root~ (SAHULKA a nd LISA 1978). Involvement of R NA a nd protein sy nthesis during stimulation of nitrate reductase activity by sugars, also supports this hypothes is (ASLAM a nd OAKS 1975 ; S.IIHAG et al. 1979). It is likely that ethanol , like sucrose, acts as a specific inducer of nitrate reductase molecules. Inhibition of nitrate reductase activity by ma lonate has been reported also in wheat (RH IARAo et al. 1980). This indicates that reactions of the TCA cycle were involved in in vivo reduction of ni trate. Although, some inhi bit ion of ethanolinduced enha ncement of enz yme activity is observed in the presence of malonate, it is never complete ; indica ting thereby that involve ment of ethanol in stimulation of in vivo ni trate reductase activity through TCA cycle metabolism is onl y marginal. References ASLAM, M.: Re-evaluation of anaerobic ni tri te production as a.n index for th e measurement of metabolic pool of nitrate. Plant Phy, iol. 68, 305- 308 (1981). ASL,\ M, M., .tnd OAKS, A.: Effect of glucose on the ind uction of nitrate redu ctase in corn roots. Plant Phy, iol. 56, 634- 639 (1975). BEEVERS, L., a.nd HAGEM ,\ N, R. 1-1. : Nitrate redu ction in higher pla nts. Ann. Rev. Plant Physiol. 20 , 495- 522 (\969). BORHI S, H.: Untersuchu ngen fib er di e Steuern ng der En1.ymaktivitat in pflanzlichen Emb ryonen du rch Cytok ini ne. Wiss. Z. Univ. Rostock Math. Naturwiss. Reih e 18, 629 - 639 (1967). FERR ,\ IU, T. E., a.nd V,\RNE R, J. E.: Control of nitrate redu ctase activity in barley aleurone la.yers. Proc. Natl. Acad. Sci. U.S. A. 65, 729- 736 (1970). HA NISH TEN C.\ n:, C. H., a.nd RRETE LER, H.: Role of suga.rs in nitrate utilisation by roots of dwa.rf beall. Ph y, iol. Plan t. ,,2, 129- 135 (1981). HEDfE R, Y. M., WRA Y, J. L., and FILNE II, P.: The effec t of tungstate on nitra te assimilation in higher plants . Plant P hysiol. 44 , 1197- 1199 (1969). JAIN, A., an d SR IVAST ,\\'A, H. S.: Effect of salicyl ic ac id on nitrate redu ctase activity in maize seedlin gs. Phy'iol. Pla nt . • 1, 339- 342 (1981). JOli N, C. D., and GREENW ,"', H.: Al coholi c fer mentat ion an d activity of some enz ymes in rice roots und er anaerobiosis. Aust. J. Plant Ph ys iol. 3, 325-336 (1976). KNY PL, J. S.: Horm onal control of ni tmte assi milati on: Do phytoh orm ones and phytochrome control the activity of nitrate redu ctase. In : "Nitroge n Assimil atio n in Plants" . E. J. HEW ITT an d C. V. Cl:TT ING eds., pp. 541- 550, Academic Press, Lo ndon 1979. LOWRY, O. H., ROSE BOROUGH , N. J., FAIIH , A. L., an d R,\ NOAJ.L, R. J.: Protein measurement with Foli n- phenol reagent. J. BioI. Chem. 193, 265- 275 (1951). M.\ EII LY, A. C.: Perox ida se. In : "Method s of Bi ochem ica l Analysis" pp. 385- 386 (C. D. GL ICK ed) Tnterscience Pub!. New York 1954.
138
R. PURANIK and H. S. SRIVASTAVA, Nitrate Reductase Stimulation by Ethanol
NOTTON, B. A., and HEWITT, E. J.: Reversible cyanide inhibition of spinach (Spinacea oleracea L.) nitrate reductase and nonexchangibility in vitro of protein bound molybdenum and tungsten.
FEBS Letters 18, 19- 21 (1971). PEIVE, Y. U., I VANovA, N. N., OUCHARENKO, G. A., and SHIRI NSKANGA, M. C.: Possible participation of peroxidase in reduction of nitrates. Fiziol. Rast. (Moscow) 22 , 527- 536 (1975). RADIN, J. W.: In vivo assay of nitrate reductase in cotton leaf discs. Effects of oxygen and am-
monium. Plant Physiol. 51, 332-336 (1973). RAMARAO, C. S., SRINIVASAN, S., and NAIK, 1\'1. S.: Inhibition of in vivo dark ana.erobic nitrate reduction by succinate, malonate and D-malate. Plant Sci. Letters. 20, 219- 224 (1980). SA IIULKA, J., and LI S.\, L.: The influence of sugars on nitrate reductase induction by exogenous nitrate or nitrite in excised P£sum sativu'Il1 roots. BioI. Plant. 20, 359- 367 (1978). SHEN, T. C.: Nitrate Reductase of rice seed lings and its induction by organic nitro compounds.
Plant Physiol. 49, 546- 549 (1972). SU III AG, R. K., MUKHERJEE, S. G., and SOPORY, S. K.: Effect of ammonium, sucrose and light in the regulation of nitrate redu ctase level in Pisum sativlUn. Physiol. Plant. 4&, 281- 287 (1979). SRIVASTAVA, H. S.: Distribution of nitrate reductase in ageing bean seed lin gs. Plant Cell Physiol.
16, 995- 999 (1975). SRIVASTAVA, H. S.: Reglilution of nitrate reducta.se activity in higher plants. Phytochemistry 19,
725- 733 (1980).
Received May 26, 1982; accepted August 5,1982 Author's address: H. S. SR IVASTAVA, Department of Life Sciences, Vigyan Bha,wan, Khandwa Road, Uni versity of Indore, INDIA - 452001 Indore.
Increase in in vivo Nitrate Reductase Activity in Bean Leaf Segments in the Presence of Ethanol REKHA PURANIK and H. S. SRIVASTAVA') Department of Life Sciences, Vigyan Bhawan, University of Indore, India
Ke y Term Index: nitrate reductase activity, bean leaves, ethanol effects, enzyme activity
Summary The substrate inducibility of in vivo nitrate redu ctase (NR, EC 1.6.6.1) activity in bean leaf segments increased with the supply of 10 to 100 mM ethanol. Ethanol also in creased soluble protein, but to a lesser extent while it inhibited the peroxidase activity. The increase in enzyme activity in the presence of ethanol was more pronounced at lower concentrations of nitrate than at higher. While supply of cycloheximide inhibited the substrate induction of nitrate reductase, it had little effect on increase in enzyme activity due to ethanol. Chloramphenicol inhibited the ethanol·induced in crease slightly, while tungstate com pletely blocked it. While glucose had little effect, sucrose increased enzyme activity substantially in the presence of nitrate. Supply of ethanol had no effect on enzyme activity in the presence of sucrose, althou gh it increased enzyme activity in the presence of glucose. l''lalonate, in a concentration of 2 to 10 mM inhibited enzyme activity both in the absence as well as in the presence of ethanol. The experiments demonstrate that ethanol increased nitrate reductase activity, by increasing nitrate uptake and transport, and possibly also by accelerating the conversion of inactive apoenzyme to active enzyme molecules.
Introduction
The activity of nitrate reductase i8 known to be controlled by various nutritional and environmental factors (SRIVASTAVA 1980). There are reports of increase in enzyme activity by >everal endogenous factors, which are neither metabolites of the nitrogen pathway nor one of the cla8sical growth regulators (KNYl'L 1979; SHEN 1972; JAIN and SRIVASTAVA 1981). In most of the cases, however, either the mechanism or the physiological significance of such an increase is not known. Ethanol has been shown to increase nitrate reductase activity in Agrostemma gitkago embryos (BORRIS 1967). During a preliminary investigation on effects of methanol, ethanol, propanol , and butanol on in vivo nitrate reductase activity in bean leaves, only ethanol was found to increase enzyme activity at low concentrations. Further experime)lts were performed with an aim to find a possible mechanism(s) of increase in nitrate reductase activity in the presence of ethanol. The activity of peroxidase was also determined to examine whether the effects of ethanol were also extended to otheI enzymes. 1) To whom all correspondence should be addressed. 9'
132
R.
PUR ,\NIK
and H. S. SIUYASTAYA
Material and Method. Seeds of Phaseolus vulgaris cv. RAHtAH (red bean) werc purchased from a local dealer. The seeds were surface sterilised with 0.1 % HgCIJ! for about 1 min and then washed thoroughly with distilled water. Then they were planted in small pots containing washed sand and the seedlings were raised for 8 d in continuous light of about 65 w m- 2 radiant flux density (400- 700 nrn) supplied by inca.ndescent bulbs and fluorescent tubes, at 25 2 °c. The seedlings were watered daily with half strength Hoagla,nd's ~olution, which was modified to exclude nitrogen. In each case, 8 d old uniformly grown seedlings, which did not develop any nodules by that time, were used for sample collection. For various treatments, segments (about 0.25 cm 2 ) of primary leaves were floated on the desired solution 2°C in light. The pH of the solution was 6.0 in ea.ch case . for 24 h at 25 Nitrate reductase activity was assayed in vivo by the method of SRIVASTAVA (1975). Protein was estimated by the method of LOWRY et at. (1951). Peroxidase was extracted and its activity assayed by the method of MAEliLY (1954). The endogenous metabolic nitrate pool in the lea-ves was estimated by mea.suring the production of nitrite under anaerobic conditions (ASLAi'ol 1981). Each determination waS carried out at least 4 times . The data presented are the average ± S.E. of such determinations.
±
±
Results Effect of Ethanol on Nitrate Reductase and Peroxidase Activities and on Protein content of Bean leaf Segments Supply of 10 to 100 mM ethanol with nitrate increased nitrate reductase activity in leaf segments; the maximum increase being achieved with 20 to 60 mM ethanol (Table 1). It also increased protein content but not to the same extent. For example, 40 mM ethanol increased nitrate reductase activity by 70% while it caused only 30% increa,e in the protein. Unlike nitrate reductase activity, peroxidase was inhibited slightly by the supply of 10 to 100 mM ethanol. Effect of Ethanol and Nitrate on the Endogenous Nitrate Pool The amount of nitrite secreted in the medium (which contained no nitrate) which is taken to be an estimate of the metabolic nitrate pool, was low when leaf segments Table 1. Effect of ethanol on nitrate reductase act-ivity, protein content, and peroxidase activity i n b ean leaf segments. Leaf segments were floated on 10 mM KN0 3 containing the desired concentration of ethanol, for
24 h in light. The values relative to the control are given in brackets Concentration of ethanol,
ruM
Nitrate reductase activity, ,umol N0 2 h - 1 g_l
Protein content, mg g- l fr.wt.
Perox idase activity, O.D. min- 1 g_l
j
fro wt.
Ir. wt.
8 10 20 40 60 100
2.56 3.28 4.08 4.37 4.13 3.38
± 0.02 (100) ± 0.05 (128) ± 0.14 (159) ± 0.08 (170) ± 0.06 (161) ± 0.05 (132)
18.21 21.63 22.71 23.75 22.31 20.96
± 0.21 (100) ± 0.21 (118) ± 0.29 (124) ± 0.35 (130) ± 0.79 (122) ± 0.75 (115)
33.3 29.0 29.8 28.5 28.5 29.3
± 0,07 (100) ± 0.29 (87) ± 0.33 (89) ± 0.09 (86) ± 0.09 (86) ± 0.26 (88)
Nitrate Reductase Stimulatio n by Ethanol
J33
Table 2. Effect of ethanol and nitrate on the endogenous nitra,te pool. Leaf segments were floated on t he desired solution for 24 h in light. Secretion of nitrite fro m t h.~se lea.! segments was measured a.fter in cubating them anae robi call y (ASLAM 1981) Treatment
Nitrite produced, ,umoi h- 1 g - l fe. wt.
Control
0.05 0.43 1.88
±
1.93
± 0.Q4
Ethanol (40 mM) KNO, (10 mM) Ethanol (40 mM)
+
0.0
± 0.0 ± 0.03
KNO, (10 mM) Table 3. Effect of nitrate level on increase in nitrate reductase activity in the presence of ethanol. Leaf segments were floated on different concentration s of KN0 3 • containing either 0 or 40 ml\I ethanol , for 24 h in light Concentmtion of nitrate, m~f
N itrite red uctase a,c tivity, "mol N0 2 h- 1 g - l fro wt. - ethanol
0 0.5 1.0 2.0 5.0 10.0 50.0
0.17 0.22 0.43 0.72 1.60 2.56 7.98
± 0.Q2 ± 0.01 ± 0.03 ± 0.01 ± 0.0 ± 0.02 ± 0.15
+ ethanol 0.53 0.73 1.Z3 1.56 3.26 4.37 10.88
± 0.01 ± 0.0 ± 0.01 ± 0.03 ± 0.03 ± 0.08 ± 0.15
Increase in the presence of ethanol , percent
211 233 183 115 104 70 36
were pretreated with ethanol only (Table 2). Nitrite secreted from leaf segments pretreated with nitrate was high ; 1.88 ,umol h _1 g_1 IT. wt. Supply of ethanol with nitrate had no effect on this level, indicating thereby that ethanol caused actual increase in enzyme activity and did not only accelemte the diffusion of nitrite into the medium.
Effect of Nitrate Concentration on Nitrate Reductase Activity in the Presence or Absence of Ethanol Supply of 0.5 to 50 mM KN0 3 caused an almost linear increase in enz yme activity in bean leaves (Table 3). When 40 mM ethanol was also included in the medium , the activity increased with increasing concentration of nitrate, but it was always higher than the respective control (minus ethanol). Further, the in crease due to ethanol supply was higher at a lower level of nitrate and it decreased progressively as t he concentration of ni trate in creased .
Effect of Inhibitors on Nitrate Reductase Activity in the Absence or Presence of Ethanol Increase in nitrate reductase activity by nitrate was substantially inhibited by cycloheximide, chloramphenicol, or sod ium tungstate (Table 4). The inhibition was
134
R.
PURANIK
and H. S.
SR IVAS TAVA
Table 4. Effect of inh1:bitors on increase in nitrate reductase activity in the presence of ethanol. Leaf segments welC floated on 40 mM etha.nol or on 50 mM KN0 3 or on both together in the presence or absence of the corresponding inhibitor, for 24 in light
Treatment
Nitrate reducta se activity, ,amol N0 2 h- 1 g - l fr. wt. Inhibitor used
Cycloheximide (5 mg/I)
None (control)
Ethanol KNOa Ethanol K NO,
+
0.58 7.98 10.88
± 0.10 ± 0.15 ± 0.15
0.24 1.33 3.14
Chlora mphenicol (1 g/I)
± 0.05 ± 0.13 ± 0.18
0.33 0.72 1.33
± ± ±
Sodium tungstate (60 I'M)
0.01 0.03 0.02
0.29 0.67 0.65
± ± ±
0.0 0.01 0.02
Table 5. Effect of carbon compouuds on nitrate reductase activity in tile presence or absence of ethanol. Lea,f segments were incubated in a solution of the desited carbon compound with 10 mM KNO a either in the prese nce (40 mM) or absence of etha.nol, for 24 h in light Carbon source
Nitrate reductase a.ctivity J&mol N02 h- 1 g - 1 fro wt. -etha.nol
None (control)
Glucose (10 mM) Sucrose (5 mM) Oxaloacetate (2.5 mM)
2.56 2.98 7.21 0.92
±
0.02 (100)
± 0.10 (116) ± 0.12 (284) ± 0.04 (27)
+ ethanol 4.37 4.92 7.74 0.94
± 0.08 (100) ± 0.13 (112) ± 0.11 (117) ± 0.06 (22)
Increase in the presence of ethanol, percent
70 65 1 2
also observed in the absence of nitrate. Although the enzyme activity in the presence of ethanol was also inhibited by these compounds, the increase by ethanol, was little affected by cycloheximide; and slightly by chloramphenicol. Supply of tungstate on the other hand, completely inhibited the increase in enzyme activity due to ethanol.
Effect of Carbon Sources on Nitrate Reductase Activity in the Presence or Absence of Ethanol Although, 10 mM: glucose caused only a slight stimulation of enzyme activity, sucrose (5 mM:) stimulated it 3ignificantly (Table 5). Increase in enzyme activity due to ethanol wa~ observed only in the presence of glucose, but not in the presence of sucrose. Oxa.loacetate (2.5 mM:) inhibited enzyme activity Significantly, and again ethanol did not increase enzyme activity when this acid was included in the medium.
Effect of Malonate or, Nitrate Reductase Activity in the Absence or Presence of Ethanol Supply of malolHlte inhibited enzyme activity; the inhibition increasing gradually with the increase in concentration of malonate from 2 to 10 mM: (Fig. 1). The inhibition
135
Nitrate Reductase Sti mulation by Ethanol
5
"
"",t
" "
-'-'---' ... ,------------ -------t
o
o
2
5
10
[MALONATE] ,mM Fig. 1. Effect of different concell trations of malollate 011 nitrate reductase activity 1:'1/ tlte presence or absence of ethanol. Leaf segments were in cubated in the desired conce ntrations of malonate either in the presence (40 m~'f) or absence of ethanol. In ea.ch case ]0 mM "NO, was also included in the medium. Dashed lin es ~ min us etha nol ; solid lines ~ plus ethanol
was observed both in the absence as well as in the presence of ethanol. Further, the increase in enzyme activity due to ethanol was ob!erved at each concentration of malonate; although it was less. Discussion An increase in n'i tratc
re ducta~c
activity in the presence of ethanol and nitrate
is demonstrated. This increase appears to be specific for nitmte reductase activity beCiluse increase in total soluble protein was not as high as in enzyme activity. In
136
R.
PUHANIK
and H. S.
SRIVASTAVA
fact, an increase in protein may be the consequence of an increase in llitrate reducta:lC activity, as the enzyme is believed to be the rate limiting cnzyme in overall assimilation of nitrate (BEEVERS and HAGEMAN 1969). PEIVE et al. (1975) have suggested that peroxidase, under certain conditions, has also nitrate reducing ability in bean plants. Under the present conditions, the activity of nitrate reductase was increased by ethanol while that of peroxidase was decreased. This again demonstrates that increase in nitrate reductase activity by ethanol is very specific and other proteins which could possibly perform similar functions, are not induced. The supply of tungstate, a specific inhibitor of synthesis of nitrate reductase molecules (HEIMER et al. 1969), inhibits the increase in enzyme activity due to ethanol (Table 4). The increase in enzyme activity appears to be real and not an artifact of the methodology used, as the secretion of nitrite in the absence of nitrate in the assay medium was little affected by ethanol treatment (Table 2). Anaerobiosis causes increase in nitrate reductase activity in barley aleurone layers (FERRARI and VARNER 1970). Further, in vivo supply of oxygen inhibits nitrate reduction in cotton leaf discs (RADIN 1973). Anaerobiosis increases the activities of pyruvate decarboxylase and alcohol dehydrogenase also in rice roots (JOHN and GREENWAY 1976). It is likely that the increase in enzyme activity under anaerobic conditions is due to ethanol produced endogenously. Lipid components of the membrane, because of their solubility in ethanol, may be disrupted during ethanol treatment. This may lead to an increase in nitrate uptake and also in its intra-cellular mobilisation for enzyme induction. Increa!.e in nitrate reductase activity with ethanol was higher at lower concentrations of nitrate (Table 3). This may suggest that ethanol increased enzyme activity by increasing nitrate uptake. However, ethanol also increased enzyme activity at a high level (50 mM) of nitrate. Further, in another experiment (data not shown), when leaf segments were floated on 100 mM KNO. prior to treatment with 10 mM nitrate + ethanol, the increase in enzyme activity was observed as usual. This may suggest that increase in enzyme activity in the presence of ethanol was only partially mediated via increased uptake of nitrate. Supply of cycloheximide, an inhibitor of protein synthesis on 80 S ribosomes had little effect on increase in enzyme activity due to ethanol; while chloramphenicol, an inhibitor of protein synthesis on 70 S ribosomes, inhibited the process slightly. This may indicate that increase in nitra.!e reductase activity in the presence of ethanol was not dependent upon cytoplasmic protein synthesis, but mitochondrial and chi oroplastic activities contributed to the process to some extent. Since tungstate, which inhibits nitrate reductase activity by replacing molybdenum from the enzyme molecule (NOTTON and HEWITT 1971), inhibited the increase in enzyme activity completely, it appears that ethanol was somehow involved in the formation of an active enzyme molecule from apoenzyme. This activation may include the insertion of molybdenum in the presynthesized protein. Alternatively, it may be postulated that ethanol increased enzyme activity possibly by acting as a source of energy and/or carbon for the synthesis of enzyme molecules.
Nitra te Reductase Sti mul ation by Ethanol
137
However, glucose, a bet ter source of carbon/energy docs no t increase enzyme activity in t he bean leaves to t he extent ethanol does and increase due to etha nol ia observed even in the presence of glucose. Oxaloacetate, a n in tcrmediate of tricarbo xylic acid cycle, which could a lso provide require d carbon/energy, do cs not in crease enzyme activity in the absence or presence of ethanol ; instead it inhibi ted t he same. This inhibition by oxaloace tate is not understood at present . Sucrose, on the other hand, increases enzyme activity significantly a nd increase due to ethanol is no t obser ved in the presence of sucrose. In contrast to our this observation wi th t he leaf enzyme, the enz yme in the roots is stimulated both by glucose a nd sucrose (HANISCH TEN CATE and BRETELER 1981). Sucrose is believed to be a specific co-indu cer or derepressor in the induction of nitrate reductase activity in pea root~ (SAHULKA a nd LISA 1978). Involvement of R NA a nd protein sy nthesis during stimulation of nitrate reductase activity by sugars, also supports this hypothes is (ASLAM a nd OAKS 1975 ; S.IIHAG et al. 1979). It is likely that ethanol , like sucrose, acts as a specific inducer of nitrate reductase molecules. Inhibition of nitrate reductase activity by ma lonate has been reported also in wheat (RH IARAo et al. 1980). This indicates that reactions of the TCA cycle were involved in in vivo reduction of ni trate. Although, some inhi bit ion of ethanolinduced enha ncement of enz yme activity is observed in the presence of malonate, it is never complete ; indica ting thereby that involve ment of ethanol in stimulation of in vivo ni trate reductase activity through TCA cycle metabolism is onl y marginal. References ASLAM, M.: Re-evaluation of anaerobic ni tri te production as a.n index for th e measurement of metabolic pool of nitrate. Plant Phy, iol. 68, 305- 308 (1981). ASL,\ M, M., .tnd OAKS, A.: Effect of glucose on the ind uction of nitrate redu ctase in corn roots. Plant Phy, iol. 56, 634- 639 (1975). BEEVERS, L., a.nd HAGEM ,\ N, R. 1-1. : Nitrate redu ction in higher pla nts. Ann. Rev. Plant Physiol. 20 , 495- 522 (\969). BORHI S, H.: Untersuchu ngen fib er di e Steuern ng der En1.ymaktivitat in pflanzlichen Emb ryonen du rch Cytok ini ne. Wiss. Z. Univ. Rostock Math. Naturwiss. Reih e 18, 629 - 639 (1967). FERR ,\ IU, T. E., a.nd V,\RNE R, J. E.: Control of nitrate redu ctase activity in barley aleurone la.yers. Proc. Natl. Acad. Sci. U.S. A. 65, 729- 736 (1970). HA NISH TEN C.\ n:, C. H., a.nd RRETE LER, H.: Role of suga.rs in nitrate utilisation by roots of dwa.rf beall. Ph y, iol. Plan t. ,,2, 129- 135 (1981). HEDfE R, Y. M., WRA Y, J. L., and FILNE II, P.: The effec t of tungstate on nitra te assimilation in higher plants . Plant P hysiol. 44 , 1197- 1199 (1969). JAIN, A., an d SR IVAST ,\\'A, H. S.: Effect of salicyl ic ac id on nitrate redu ctase activity in maize seedlin gs. Phy'iol. Pla nt . • 1, 339- 342 (1981). JOli N, C. D., and GREENW ,"', H.: Al coholi c fer mentat ion an d activity of some enz ymes in rice roots und er anaerobiosis. Aust. J. Plant Ph ys iol. 3, 325-336 (1976). KNY PL, J. S.: Horm onal control of ni tmte assi milati on: Do phytoh orm ones and phytochrome control the activity of nitrate redu ctase. In : "Nitroge n Assimil atio n in Plants" . E. J. HEW ITT an d C. V. Cl:TT ING eds., pp. 541- 550, Academic Press, Lo ndon 1979. LOWRY, O. H., ROSE BOROUGH , N. J., FAIIH , A. L., an d R,\ NOAJ.L, R. J.: Protein measurement with Foli n- phenol reagent. J. BioI. Chem. 193, 265- 275 (1951). M.\ EII LY, A. C.: Perox ida se. In : "Method s of Bi ochem ica l Analysis" pp. 385- 386 (C. D. GL ICK ed) Tnterscience Pub!. New York 1954.
138
R. PURANIK and H. S. SRIVASTAVA, Nitrate Reductase Stimulation by Ethanol
NOTTON, B. A., and HEWITT, E. J.: Reversible cyanide inhibition of spinach (Spinacea oleracea L.) nitrate reductase and nonexchangibility in vitro of protein bound molybdenum and tungsten.
FEBS Letters 18, 19- 21 (1971). PEIVE, Y. U., I VANovA, N. N., OUCHARENKO, G. A., and SHIRI NSKANGA, M. C.: Possible participation of peroxidase in reduction of nitrates. Fiziol. Rast. (Moscow) 22 , 527- 536 (1975). RADIN, J. W.: In vivo assay of nitrate reductase in cotton leaf discs. Effects of oxygen and am-
monium. Plant Physiol. 51, 332-336 (1973). RAMARAO, C. S., SRINIVASAN, S., and NAIK, 1\'1. S.: Inhibition of in vivo dark ana.erobic nitrate reduction by succinate, malonate and D-malate. Plant Sci. Letters. 20, 219- 224 (1980). SA IIULKA, J., and LI S.\, L.: The influence of sugars on nitrate reductase induction by exogenous nitrate or nitrite in excised P£sum sativu'Il1 roots. BioI. Plant. 20, 359- 367 (1978). SHEN, T. C.: Nitrate Reductase of rice seed lings and its induction by organic nitro compounds.
Plant Physiol. 49, 546- 549 (1972). SU III AG, R. K., MUKHERJEE, S. G., and SOPORY, S. K.: Effect of ammonium, sucrose and light in the regulation of nitrate redu ctase level in Pisum sativlUn. Physiol. Plant. 4&, 281- 287 (1979). SRIVASTAVA, H. S.: Distribution of nitrate reductase in ageing bean seed lin gs. Plant Cell Physiol.
16, 995- 999 (1975). SRIVASTAVA, H. S.: Reglilution of nitrate reducta.se activity in higher plants. Phytochemistry 19,
725- 733 (1980).
Received May 26, 1982; accepted August 5,1982 Author's address: H. S. SR IVASTAVA, Department of Life Sciences, Vigyan Bha,wan, Khandwa Road, Uni versity of Indore, INDIA - 452001 Indore.