Biochimica et Biobqo'sicaActa, 1137(1992) 121-126

121

g: 1992ElsevierScience PublishersB.V. All rightsreserved0167-4889/92/$05.00

BBAMCR 10293

Rapid Report

Regulation of spinach-leaf nitrate reductase by reversible phosphorylation Carol MacKintosh MRC Prote~nPhosphoo'lation Unit. Departmentof Biochemistry. Unil'ersityof Dundee, Dundee (UK)

(Received 10June 1992)

Keywords: Nitrate reduclase; Proteinphosphowlation:(Spinachleaf) Okadaic acid and micro~s~n (but trot the inactive methyl esters of these toxins) prevented the rapid light-induced activation of nitrate rednctase (NR) in intact spinach leaves, in vitro, nitrate reductase was inactivated by a protein kinase and activated by PP2A. The role of reversible protein phosphorylatiou in regulation of light-coupled cytoplasmic metabolism is discussed.

Protein phnsphata~e 2A (PPZA) dephosphorylates and regulates several enzymes of cytnsolic metabolism in higher plants. Physiological substrates for PP2A in plants include sucrose-phoxphate synthase (SPS) [1], PEP carboxylase [2] and quinate dehydrogenase [3]. For example, upon illumination of darkened spinach leaves SPS is converted from a ~o~-activhy form to a high-activity form [4,5]. The high-activity form is less sensitive .to inh~ition by Pi and has a lower K~ for Fru6~. The effect of light on SPS acdvi:y ;s an ind[re~ cop~eq*!epce of increased rates of photosynthesis [4,5]. Experiments performed with purified enzymes and in spinach-leaf extracts have demonstrated that the low activity form of SPS is phosphorylated, and can be activated (dephosphorylated) by PP2A but not by protein phosphatases I and 2C (PPI or PF2C) [I]. Furthermore, in vivo okadaic acid and microeystin-LR (two potent and specific inh~itors of PPI and PP2A) both prevent the light-induced activation of SPS [1,4] and decrease the rate "~.fsu~z-.)se biosynthesis [1]. How the relevant SPS kinase(s) a n d / o r PP2A are controlled in response to changing rates of photosynthesis is not yet understood. One of the questions raised by these observations is whether other light-coupled cytoplasmic enzymes (i.e., those whose activities are dependent on metabolites generated by light-dependent reactions in the chloro-

Correspondence to: C. MacKintosh.MRC Protein Phosphmylation Unit, Department of Biochemist~,Universityof Dundee, Dundee. DD! 4FIN,UIC

plast) might also be regulated by reversible protein phnsphorylation during the normal day/night cycle. Nitrate reductase (NR) in leaves is one such enzyme. NR in spinach leaves is a cytoplasmic NADH-dependent enzyme which reduces nitrate to nitrite. Nitrite is then converted to ammonia by reduced-ferredoxin-dependent nitrite reductase (NiR) in the chloroplast. The expression of leaf NR in various plant species is highly regulated by light (e.g. see Ref. 6), external nitrate [7] and a circadian rhythm [8] at the level of gene transcription [6--8] and possibly by changes in rate of protein turnover. However, in addition there are reasons for suspecting that NR might be a candidate for short-term post-translational regulation during the normal dark/light cycle. Firstly, some mechanism to ensure tight co-ordination of the activities of NR and NiR might be expected so that nitrite (which is very toxic) is never allowed to build up [9] under conditions (e.g., a s u ' d e n drop in illumination) where reduced ferredoxin supply and hence NiR activity falls. Secondly, there are several reports that the activity of NR in leaf extracts does not always match the amount of NR protein measured by immunological methods [10]. In particular, short-term changes in NR activity (within minutes) are sometimes observed with changing light conditions or CO2 levels, i.e., conditions which alter the rate of pLotosynthesis [11,12]. Recently, Kaiser et a!. [12-14] have found that NR activity in extracts of spinach leaves harvested after ! h of illumination could be inactivated in a tlme-dependent manner in ~.he presence of MgATP. Upon darkening of leaves or exposure to CO2-free air, NR activity was reduced by

122 4 - 1 0 - f o l d w i t h i n 1 h b u t c o u l d b e a c t i v a t e d i n v i t r o in t h e p r e s e n c e o f A M P [13,14]. T o g e t h e r w':.h m e a s u r e m e n t s of .cytusolic metabolite concentrations, these results l e d t h e a u t h o r s t o s u g g e s t t h e p o s s i b i l i t y t h a t N R a c t i v i t y is r e g u l a t e d b y r e v e r s ~ l e p h u s p h o r y l a t o n i n a s y s t e m t h a t r e s p o n d s t o a l t e r a t i o n s in cytosolic m e f a b o rite c o n c e n t r a t i o n s ( s u c h a s c h a n g e s in A T P a n d A M P

levels) g e n e r a t e d by c h a n g i n g r a t e s o f p h o t o s y n t h e s i s

Herein, experiments in intact spinach [eaves, in leaf extracts and using purified enzymes and specific protein p h o s p h a t a s e i n h i b i t o r s (okaclaic a c i d [15], m i c ~ r , : i n [16] aral i n h ~ i t o r 2, a specific i n h i b i t o r o f P P I [17,11, a r e r e p o r t e d , t h e r e s u l t s o f w h i c h s t r o n g l y s u g g e s t

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Fi~ !. Effect of iHuraioation and protein phosphatase inhibitors on NR and protein phnsphatase activity in intact ~¢,~inaeh . leaves. (AI Leaves were treated with micrnc~n-LR and then either illumi,aiL;d (o) or kept in the dark (zx) or were treated with raethyl-raicrncystin-LR and illuminated (n). Spinach (Spinmea oferacea L ~'dr M, d:nia) was grown in a greenhouse or growth cabinet in soil-based compost in an II-h fight/13-h dark cycle for 4-6 weeks. Small leaves (app~ ~x.2 g) were detached at the end of the light period and petioles were cut under degassed water and placed individually into 2 ml of 10 mM KNO 3 and incubated in the dark in a growth cabinet for 2 h at 2{)~C. Leaves were then transferred in the dark to fresh l-nd solutions of 10 ram KNO3 containing the iod.;cated concentrations of microc~tin-LR, raet byl-raicrocystio-LR or 0.04% DMSO (control leaves) and kept in the dark for a further 2 h. Toxins were diluted from stock solutions (5 raM in DMSO). Leaves were then Hlurainated to 600 p.reot photons/m e per s (o, ra) at 2WC or left in the dark (~). After 30 mio leaves were frozen in liquid N:, petioles and midribs removed and leaf lamina was ground to a fine powder and stored in liquid N2. For each data point, powder from st× individually-treated leaves was pooled and homogenates were prepared by mixing approx. 0.5 g of powder in 3 ,mrs. of ice-cold buffer consisting of 50 mM Hepes-KOH (pH 7.4), 0.1 raM EGTA, 0.2% Triton X-1000 ! raM dithiothreitol, 10% (v/v) glycerol. I mM henzarai0ine. 10 ttM leupeptio. I mM phetrflrnethylsulphonyl fluoride and I /zM raicrocystin-LR. Homogenatns were filtered through fine nylon mesh and 50 p.I of the supernatant assayed for NR activity immediately. Protein was mea;ored by the Bradford method using BSA as standard [23]. NR ussa)~ were carried out for 4 rain at 3ffC in a total volume of 0.5 ml in extraction buffer contaioir,/; 2 raM KNO 3, 0.3 mM NADH plus 5 raM MgCI:. Reactions were started by addition of extract and stopped by addition of 0.1 ml of 0.5 M zinc acetate plus 0.1 ral of 150 ItM phenazine raethnsulphate. After addition of 0.3 ml 1% (w/v) sulphanilamide in 3 N HCI plus 0.3 ml N-(I-naphthylLetbylenediamioe. tubes were incubated at room temperature for 20 mio. centrifuged at 151~)× g and absorbance measured at 540 nra [24]. Nitrite concentrations were determined by comparison wi~ a ~tandard cur~e prepared under identical conditions. Controls were performed in the absence of NADH and v,ith addition of zinc acetate before addition of enzyme. Results are shown for duplicate assays which varied by less than 5 ~ from leaf powder taken from the same experiment. Similar results were seen in three individual experiments performed over a 6-month period. (B) Leaf powder from the same experiment shown in A was assayed as before, except that the NR assays contained 5 mM EDTA instead of 5 raM MgCI2. Leaves were treated with microcystin-LR and then illuminated (o) or kept in the dark ( • ) or were treated with metbyl.raicrocystin-LR and illuminated ( • ) us for A. ((2) Phospho~lase phosphatase activity ext.racted from leaves treated with raicrocystin-LR (o,o) or metbyl-microc,.~stio-LR(E],•) and illuminated. Leaf powder from the same experiment shown in A was homogenized and centrifuged as before in extraction buffer minus raicrne~stin. Supernatant (i ral) was passed through a column (15 cmx I cm) of Sephadex G-50 Superfine. Fractions containing protein were pooled tapprox. 2.3 ral) and a 5*p.I sample retained for protein determination [23]. Bovine serum album;, ~? mg/ml final) and buffer were added to give a final volume of 2.5 ral for each and 50-p.I aliqouts were frozen in liquid N2. Protein phusphatase activity was assayed at a 10-fold dilution using 32P-lahelled g~ycogen phcr~pho~lasa as substrate [25]. Results are expressed as total activities tclosed symbols) or specific activities (open symbols) relative to control. Control activity 1100%) was 3.25 raU total or i.75 raU/rag of protein representing a 105% rectwery of activity from the extract. One unit of protein phosphatase was that amount which eatalysed the dephnsphoulation of I/tmol of glycogen phosphor,jlaso in I rain.

!23 that NR is activated upon illumination due to dephosphorylation by PP2A. In vitro, NR from illuminated plants can be inactivated by a protein kinase which can be separated from NR by chromatography on BlueSepharose. When spinach leaves incubated in the dark were illuminated for 30 rain, subsequent assay in the presence of 5 mM MgCI 2 revealed that NR activity was increased 4 to 8-fold compared with leaves that remained in the dark (Fig. IA). In agreement with Kaiser et al. [12-14], observed ch~.~ges in NR activity were less marked when NR a~says were performed in the presence of 5 mM EDTA (Fig. IB). In leaves that had been incubated with microcystin-LR or okadaic acid before illumination, activation of NR was prevented with 1 p.M microcystin-LR causing a decrease of 47 + 11% (6 determinations) (also Fig. IA) and 1 p.M okadaic acid causing a decrease of 53 +_8% (6 determinations), similar to the concentrations of these toxins that prevented light-stimulated activation of SPS in spinach-leaf discs [1]. Furthermore, tautomycin, which is also a potent inhibitor of PPl and PP2A [18] reduced the light-stimulated activation of NR by 70% when used at 1 p,M. Therefore, three structurally different compounds which are potent ~nh~itors of PPI and PP2A all had very similar effects. None of the toxins had any effect on NR activity in leaves that had been kept in the dark throughout the experiment (Fig. IA and B). Methylated microcystin which is not an inhibitor of PPI or PP2A (C.M., unpublished data, Fig. IC) showed only slight inh~ition of NR activation at !0 p.M (Fig. IA). This slight effect may be due to hydrolysis of the methyi linkage by plant erzymes, leading to regeneration of active microcystin toxin (Fig. 1C). Methylated okadaic acid, which likewise does not inh~it of PP1 or PP2A [19], had no effect on NR activity when supplied to leaves at concentrations between 50 nM and 5 p.M (data not shown). During these experiments it was noticed that toxintreated leaves took up less solution than untreated leaves or leaves treated with methylated compounds (approximately 350 p.I removed from each vial of 10 # M microcystin compared with 550 p.I from control vials). Therefore, the amount of protein phosphate inhibitor reaching the leaf lamina was determined by assaying phosphorylase phosphatase (PPI plus PP2A) activity in desalted extracts of leaves that had been pretreated with microcystin. Microcystin does not dissociate from PPI and PP2A during gel filtration ([20]; C.M., unpublished). Fig. IC shows that decreased recovery of active phospho~lase phosphatase (comprising PP1 plus PP2A) from treated leaves occurs over the same range of microcystin concentrations as does prevention of NR activation (Fig. IA). Phusphorylase phosphatase activity in desalted extracts from control plants was not inhibited by addition of desalted extract

from leaves treated with 10 p.M microcystin-LR. This confirms that there was no free toxin in the pool of protein after gel filtration. Similar experiments were not possible with okadaic acid or tautomycin which dissociate from protein phosphatases during gel filtration. These experiments (Fig. 1) suggested that NR is phosphorylated in the dark and is activated upon illumination due to dephosphorylation by either PP1 or PP2A. in order to find out which of the protein phosphatases in spinach leaves are responsible for dephusphor~lating NR, extracts were prepared from the spinach leaves that had been harvested after 10 h of light followed by 2 h of darkness in the absence of protein phosphatase inhibitors. Extracts were rapidly fractionated from 0 to 55% ammonium sulphate and desalted as in [1,3]. As was found for SPS [1], NR activity remained in a relatively low-activity state provided that the procedure was performed rapidly and at 0°C. Subsequent incubation at 3ff'C led to a 3 to 5-fold increase in NR activity, measured in the presence of 5 mM MgCi 2, within 10 min (Fig. 2) in 7 experiments, performed with 4 separate hatches of plants. Activation of NR could be prevented by addition of 5 p.M okadaic acid or microcystin-LR (Fig. 2). However, inclusion of 0.5 p.M inhibitor 2 to the incubation had no effect (data not shown). This concentration of inhibitor 2 is sufficient to inhibit all of the PP1 in the spinach-leaf extract with no effect on PP2A activity [3]. These experiments suggested that PP2A is the major phosphatase in spinach-leaf extracts which dephosphorylares and activates NR. This result is in contrast to Kaiser et al. [13,14] as activation of NR in the present study had no absolute dependence upon the presence of AMP (Fig. 2 and data not shown). Longer incubations led to a sharp drop in NR activity measured in the presence of either 5 mM MgCI 2 or 5 mM EDTA (Fig. 2) and the enzyme could not then be reactivated. This decline is likely to be due to proteolysis. NR is well known to be extremely susceptible to proteolysis in leaf extracts. However, inclusion of trypsin inhibitor, e-amino-n-caproic acid and benzamidine had no effect. The properties of the kinase(s) responsible for the inactivation of NR were examined in a concentrated and desalted spinach-leaf extract prepared rapidly from spinach leaves that had been incubated in the dark for 4 h and then illuminated for 30 rain as described in Ref. 1 and in the legend of Fig. 1. NR activity decreased in a time-dependent manner at 30°C in the presence of 5 mM MgCI 2 plus 2 mM ATP (Fig. 3). Several previously identified plant protein kinases are Ca2+-dependent [21]. However, addition of Ca 2+ (0.3 mM; buffer contained 0.1 mM EGTA) in the presence or absence of bovine brain calmodulin or addition of

124 W h e n partially-purified N R , inactivated in the prese n c e o f 1 /zM microcystin (Fig. 4) was desalted by gel filtration a n d t h e n i n c u b a t e d with 60 m U / m l o f P P 2 A catalytic subonit purified from rabbit skeletal muscle [18] t h e r e w a s a 3-fold increase in N R activity m e a s u r e d in the p r e s e n c e o f 5 m M MgCi 2 from 0.15 _+ 0.03 (n=3) n m o l / m i n p e r m g to 0.49_+0.059 ( n = 3 ) n m o l / m i a p e r mg. T h e increase in N R activity d u e to a d d e d P P 2 A w a s completely abolished by 1 /~M microcystin-LR. A d d i t i o n o f 60 m U / m l o f P P I did not affect the activity o f N R . This result is in a g r e e m e n t with the finding t h a t P P 2 A w a s the m a j o r N R - a c t i v a t i n g activity present in s p i n a c h - l e a f extracts (Fig. 2). A l t h o u g h the c o n c e n t r a t i o n o f P P 2 A catalytic subunit r e q u i r e d to activate N R w a s r a t h e r high (60 m U / m i ) it should be n o t e d t h a t as in m a m m a l i a n tissues, the native f o r m o f P P 2 A in s p i n a c h - l e a f extracts is not the free catalytic subonit but is a high m o l e c u l a r m a s s complex [3] which

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Time (rain) Fig. 2. Activation of nitrate reductase by endogenous protein phosphatase in an ammonium sulphate fraction prepared from spinach leaves har,,'ested in the dark. Spinach plants were preillumioated for 10 h, darkened for ! h and a desalted ammonium sulphate fraction was prepared rapidly [l.3] containing NI¢, in the low-activity form Aliquots of the fraction were frozen and stored in liquid N2 before use. The extract was incubated at 3WC with 0.02% DMSO (control. o,o) or I ,aM micrtg~tin-LR ([3.11), and pliquots of 5/zl removed at the times indicated and assayed for NR in the presence of 5 mM MgCI2 (open ~ b o l s ) or 5 mM EDTA (closed symbols). Results are shown mean values from 3 experiments performed with extracts from the same batch of leaves. Similar results were seen in 7 experiments performed with 4 batches of leaves over a 6-month period.

m E G T A at 2 m M h a d n o effect o n the r a t e N R inactivation ( d a t a not shown). Extract f r o m illuminated leaves w a s passed t h r o u g h a B l u e - S e p h a r o s e c o l u m n a n d N R activity w a s e l u t e d with 100 g M N A D H . In c o n t r a s t to the c r u d e extract (Fig. 3) N R activity in this fraction could not be inactiv a t e d rapidly unless a s a m p l e o f the 0.6 M NaCI e l u a t e f r o m the s a m e c o l u m n w a s a d d e d to the incubation (Fig. 4). T h u s the inactivating N R kinase (or possibly a n accessory factor) was s e p a r a t e d f r o m N R by chrom a t o g r a p h y o n Biue-Sepharose. F u r t h e r m o r e , p a r tially-purified N R c o n t a i n e d n o d e t e c t a b l e c o n t a m i n a t ing PP1 o r P P 2 A activity, b e c a u s e these enzymes both b i n d to B l u e - S e p h a r o s e a n d a r e e l u t e d a l o n g with the kinase activity in the 0.6 M NaC! e l u a t e (PP1, 10.9 m U / m g protein; P P 2 A , 3.8 m U / m g ) .

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Time (mln) Fig. 3. Inactivation of NR by endogenous kinase in an ammonium sulphate fraction prepared from leaves han~ested after 30 mio illumination. Spinach plants were darkened for 4 h. illuminated by 600 ,amol photons/m-" per s for 30 rain and a desalted ammonium sulphate fraction was prepared rapidly at 0°C containing NR in the high-activity form. Immediately after preparation the extract was incubated at 3WC in extraction buffer (Fig. I). in the presence of 5 mM MgCiz plus 50 /zM I-5-di(adenosine-5'-pentaphosphate) (to inhibit myokinase activity) and in the presence (o,e) or absence ( [3,11) of 2 mM ATP. NR assays were performed in the presence of 5 rnM MgCI: (open symbols) or 5 mM EDTA (closed symbols).

125 (e.g. see Ref. 22). In o r d e r to answer the question of how reversible phosphorylation of NR and SPS contributes to these different patterns of regulation, it will be crucial to determine how the activities of N R and SPS kinases (or kinase) a n d / o r PP2A are controlled in vivo. In principle, activation of N R upon illumination

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of leaves could be due to activation of the phosphatase or inhibition of the kinase or both and experiments are underway to determine the nature o f the 'signals" that operate to regulate the phosphorylation state o f NR.

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Acknowledgements

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! would like to thank Professor Philip Cohen (in whose laboratory this study was carried out), Dr. John Wray, University of St. A n d r e w s and Dr. Roger Fido, Long Ashton Research Station for helpful discuz~ions. Thanks also to Mr. Barry Robertson, Scottish Crop Research Institue, lnvergowrie, for growing spinach plants and Dr. Charles F.B. Holmes for the gift of okadaic acid methyl ester. T h e work was supported by a Royal Society of Edinburgh Personal Fellowship and an S E R C / A F R C Research Grant.

Time (min) Fig. 4. Inactk'afon of NR partially purified by Blue-Sepharose chromatographyfrom spinach plants harvested after illumination at 600 #tool photons/m 2 per s for 30 rain. 50 g of spinach leaves was harvested by freezing in liquid N2 after 30 rain of illumination. Lcavaswere ground in 125 ml of extra,ion buffer (as Fig. I. except minus micrncystin) and extract was filtered through 2 layers of cheesecloth and centrifuged at 200flOxg for 20 rain. Supernatant was mixed with 15 ml of Blne-Sepharose and poured into a column. drained and washed with extraction buffer (minus micrncysfin)until the A ~ of the eluate was zero. NR was elnled b~ IllO/zM NADH in extraction buffer (minus micrncystin)concentrated to 2 ml using a Centricon-30 concentrator and stored in aliqouts in liquid N2. Par* tinily purified NR was incubated at 30~Cin extraction buffer (including microcyslin), in the presence of 5 mM MgCI,., 50 /zM I-5d/{adcnosine-5'-penlaphospbatel plus 2 mM ATP with no further additions ( [] . I }or in the presence of a IO-folddilution of a desalted 0.h M NaCI eluate from the Blue-Sepharose column (o,o). NR assays were peffoJmed in the prcsCtlt.~ of 5 mM MgCI2 (open s3'mbols)or 5 mM EDTA (closed s~ymbols).

may have different activity towards N R c o m p a r e d with the catalytic subunit. T h e results presented in this p a p e r d e m o n s t r a t e that N R is regulated by p h o s p h o r y l a t i o n / d e p h o s p h o r y l a tion in a similar m a n n e r to SPS. Both enzymes are dephosphorylated and activated by PP2A upon illumination of leaves and it is tempting to speculate that inactivation of both N R and SPS may be catalysed by the same protein kinas¢. However, interrelationships between sucrose biosynthesis, nitrate assimilation and photosynthesis in leaves are very complex and vary enormously u n d e r different physiological situations

References I SiegL G.. MacKintosh, C. and Stilt. M. (1990) FEBS Len. 27. 198-202. 2 Carter, PJ.. Nimmo. H.G.. Few,on. C.A. and Wilkins. M.B. (1991) EMBO J. 10. 2063-2068. 3 MacKintosh. C.. Coggins, J. and Cohen, P. (1991)Biochem. J. 273. 733-738. 4 Stitt. M.. Wilke, F., [Ceil, R. and Heldt. H.W. (1988) Planta 174, 217-230. 5 Huher, J.L.A. and Huher, S.C. (1992) Bidchem. J. 283. 877-882, 6 Faure. J.D.. Vincentz. M., Kroncnherger. J. and Caboche M. (1991) The Plant Journal L 107-113. 7 Rajasekhar. V.IL and Oelmuller, R. (1987) Physiologia Plantarn,'q. 71. 517-521. 8 Deog, M.-D.. Moureaux. T., Leydecker. M.-T. and Caboche. M. (1990) Planta 180.257-261. 9 Riens, B. and Heldt. H.W. (1992) Plant Physiol. 98, 573-577. 10 LUlo,C. (1991) Plant Sci. 73, 149-154. II Remmler. J.L. and Campbell, W.H. (1986) Plant Physiol. 80. 442-447. 12 Kaiser. "~'.M.and Brendle-Behnisch, E. (1991) Plant Ph)siol. 96, 363-3647. 13 Kaiser, W.M. and Spill, D. (1991) Plant Physiol. 96. 368-375. 14 Kaiser. W.M.. Spill, D. and Brendle-Behniseb, E. (19921 Planta 186, 236-240. 15 Bialojan, C. and Takai, A. (1988) Biochem.J. ~6. 283-290. 16 MacKintosh.C., Beanie, K.A., Klumpp. S., Cohen, P. and Codd, G.A. (1990) F/~BS Len. 264, 187-192. 17 Cohen. P., Foulkes. J.G.. Holmes, C.F.B., Nimrod. G.A. and Tonks, N.K. (1988) Methods Eozymol. 159, 427--437. 18 MacKintosh.C. and Klumpp. S. (1990) FEBS Len. 277.137-140. 19 Holmes. C.F.B., Luu, H.A. and SchmiULFJ. (1{~0) FEBS Left. 270. 216-218. 20 Robinson, M.A.. Pace. J.G., Matson. C.F., Miura. G.A. and Lawrence, W.B. (199;)J. Pharmacol. Exp. Ther. ~6. 176-182.

126 21 Poovaiah, B.W. and Reddy, A.S.M. (1987) CRC Cr/L Re,.,. Plant Sci. 6. 4-103. 22 OOY, L - V . Foyer. C. and Champigny. M.-L (1991) Plant Physiol. 97,1476-148"Y_ 23 Bradford, M.M. (1976) Anal. B/ochem. 72 248-254.

24 Wra): .LL and Fido. R.L (It~O) Methods Plant Biochem. 3. 241-256. 2.5 Cohen. P_ Klumpp. S. and Schelling. D.L (1989) FEBS I~lt. ~_50. 59f~600.

Regulation of spinach-leaf nitrate reductase by reversible phosphorylation.

Okadaic acid and microcystin (but not the inactive methyl esters of these toxins) prevented the rapid light-induced activation of nitrate reductase (N...
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