Comparative studies of the light modulation of nitrate reductase and sucrose-phosphate synthase activities in spinach leaves

We recently obtained evidence that the activity of spinach (Spinacia oleracea L.) leaf nitrate reductase (NR) responds rapidly and reversibly to light/dark transitions by a mechanism that is strongly correlated with protein phosphorylation. Phosphorylation of the NR protein appears to increase sensitivity to Mg²⁺ inhibition, without affecting activity in the absence of Mg²⁺. In the present study, we have compared the light/dark modulation of sucrose-phosphate synthase (SPS), also known to be regulated by protein phosphorylation, and NR activities (assayed with and without Mg²⁺) in spinach leaves. There appears to be a physiological role for both enzymes in mature source leaves (production of sucrose and amino acids for export), whereas NR is also present and activated by light in immature sink leaves. In mature leaves, there are significant diurnal changes in SPS and NR activities (assayed under selective conditions where phosphorylation status affects enzyme activity) during a normal day/night cycle. With both enzymes, activities are highest in the morning and decline as the photoperiod progresses. For SPS, diurnal changes are largely the result of phosphorylation/dephosphorylation, whereas with NR, the covalent modification is super-imposed on changes in the level of NR protein. Accumulation of end products of photosynthesis in excised illuminated leaves increased maximum NR activity, reduced its sensitivity of Mg²⁺ inhibition, and prevented the decline in activity with time in the light seen with attached leaves. In contrast, SPS was rapidly inactivated in excised leaves. Overall, NR and SPS share many common features of control but are not identical in terms of regulation in situ.     

Reversible light/dark modulation of spinach leaf nitrate reductase activity involves protein phosphorylation.

Spinach (Spinacia oleracea L.) leaf nitrate reductase (NADH:NR;NADH:nitrate oxidoreductase, EC 1.6.6.1) activity was found to rapidly change during light/dark transitions. The most rapid and dramatic changes were found in a form of NR which was sensitive to inhibition by millimolar concentrations of magnesium. This form of NR predominated in leaves in the dark, but was almost completely absent from leaves incubated in the light for only 30 min. When the leaves were returned to darkness, the NR rapidly became sensitive to Mg2+ inhibition. Modulation of the overall reaction involving NADH as electron donor was also found when reduced methyl viologen was the donor (MV:NR), indicating that electron transfer had been blocked, at least in part, at or near the terminal molybdenum cofactor site. Changes in activity appear to be the result of a covalent modification that affects sensitivity of NR to inhibition by magnesium, and our results suggest that protein phosphorylation may be involved. NR was phosphorylated in vivo after feeding excised leaves [32P]Pi. The NR subunit was labeled exclusively on seryl residues in both light and dark. Tryptic peptide mapping indicated three major 32P-labeled phosphopeptide (Pp) fragments. Labeling of two of the P-peptides (designated Pp1 and 3) was generally correlated with NR activity assayed in the presence of Mg2+. In vivo, partial dephosphorylation of these sites (and activation of NR assayed with Mg2+) occurred in response to light or feeding mannose in darkness. The light effect was blocked completely by feeding okadaic acid via the transpiration stream, indicating the involvement of type 1 and/or type 2A protein phosphatases in vivo. While more detailed analysis is required to establish a causal link between the phosphorylation status of NR and sensitivity to Mg2+ inhibition, the current results are highly suggestive of one. Thus, in addition to the molecular genetic mechanisms regulating this key enzyme of nitrate assimilation, NR activity may be controlled in leaves by phosphorylation/dephosphorylation of the enzyme protein resulting from metabolic changes taking place during light/dark transitions.     

Studies on the dark/light regulation of maize leaf pyruvate, orthophosphate dikinase by reversible phosphorylation

In maize leaves, pyruvate, orthophosphate dikinase (PPDK) is deactivated in the dark and reactivated in the light. Studies in vitro using purified PPDK and a partially purified regulatory protein from maize confirmed previous reports correlating deactivation/reactivation with the reversible phosphorylation/dephosphorylation of a threonyl residue. By monitoring the stability of the exogenous 32P-labeled adenylate substrates during deactivation, we have firmly established ADP as the specific phosphate donor. In isolated maize leaf mesophyll protoplasts preilluminated with 32Pi, we observed a three- to fivefold higher PPDK activity in situ in the light, and a corresponding three- to fivefold higher level of phosphorylation of the 94-kDa PPDK protomer in the dark. HPLC-based phosphoamino acid analysis of PPDK purified from maize leaves of both light- and dark-adapted plants revealed the presence of P-serine. The inactive enzyme from dark-adapted plants (inactivated in vivo) also contained P-threonine. Total phosphate content of PPDK purified from leaves of light-adapted plants was approximately 0.5 mol/mol protomer, and 1.5 mol/mol protomer from leaves of dark-adapted plants. Since the difference between enzyme purified from light-adapted (active PPDK) and dark-adapted (inactive PPDK) plants is the presence of P-threonine in the latter, this suggests an inactivation stoichiometry in vivo of 1 mol P-threonine/mol 94-kDa protomer. These complementary studies with maize leaf PPDK in vitro, in situ, and in vivo provide convincing evidence for the dark/light regulation of this key C4-photosynthesis enzyme by reversible phosphorylation.     

Nitrate-reductase expression is under the control of a circadian rhythm and is light inducible in Nicotiana tabacum leaves

Over a 24-h light-dark cycle, the level of mRNA coding for nitrate reductase (NR; EC 1.6.6.1) in the leaves of nitrate-fed Nicotiana tabacum L. plants increased throughout the night and then decreased until it was undetectable during the day. The amount of NR protein and NR activity were two-fold higher during the day than at night. When plants were transferred to continuous light conditions for 32 h, similar variations in NR gene expression, as judged by the above three parameters, still took place in leaf tissues. On the other hand, when plants were transferred to continuous dark conditions for 32 h, the NR-mRNA level continued to display the rhythmic fluctuations, while the amount of NR protein and NR activity decreased constantly, becoming very low, and showed no rhythmic variations. After 56 h of continuous darkness, the levels of NR mRNA, protein and activity in leaves all became negligible, and light reinduced them rapidly. These results indicate the circadian rhythmicity and light dependence of NR expression.     

Regulatory seryl-phosphorylation of C4 phosphoenolpyruvate carboxylase by a soluble protein kinase from maize leaves

A reconstituted system composed of purified phosphoenolpyruvate carboxylase (PEP-Case) and a soluble protein kinase (PK) from green maize leaves was developed to critically assess the effects of in vitro protein phosphorylation on the catalytic and regulatory (malate sensitivity) properties of the target enzyme. The PK was partially purified from light-adapted leaf tissue by ammonium sulfate fractionation (0-60% saturation fraction) of a crude extract and blue dextran-agarose affinity chromatography. The resulting preparation was free of PEPCase. This partially purified protein kinase activated PEPCase from dark-adapted green maize leaves in an ATP-, Mg2+-, time-, and temperature-dependent fashion. Concomitant with these changes in PEPCase activity was a marked decrease in the target enzyme's sensitivity to feedback inhibition by L-malate. The PK-mediated incorporation of 32P from [gamma-32P]ATP into the protein substrate was directly correlated with these changes in PEPCase activity and malate sensitivity. The maximal molar 32P-incorporation value was about 0.25 per 100-kDa PEPCase subunit (i.e., 1 per holoenzyme). Phosphoamino acid analysis of the 32P-labeled target enzyme by two-dimensional thin-layer electrophoresis revealed the exclusive presence of phosphoserine. These in vitro results, together with our recent studies on the light-induced changes in phosphorylation status of green maize leaf PEPCase in vivo (J. A. Jiao and R. Chollet (1988) Arch. Biochem. Biophys. 261, 409-417), collectively provide the first unequivocal evidence that the seryl-phosphorylation of the dark-form enzyme by a soluble protein kinase is responsible for the changes in catalytic activity and malate sensitivity of C4 PEPCase observed in vivo during dark/light transitions of the parent leaf tissue.     

Regulation by light and metabolites of ferredoxin-dependent glutamate synthase in maize

The regulation of Fd-glutamate synthase (Fd-GOGAT, EC 1.4.1.7) and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) was investigated in maize ( Zea mays L. cv. DEA) (1) during development starting from 7- to 11-day-old seedlings, (2) by treatment of 7-day-old etiolated leaves with intermittent light pulses to activate (red) and inactivate (far-red) phytochromes and (3) in 7-day-old green leaves grown under 16-h light/8-h dark cycles. Fd-GOGAT mRNA accumulated 4-fold, and the enzyme polypeptide (3-fold) and activity (3-fold) also increased in leaf cells, while NADH-GOGAT activity remained constantly low. Leaf-specific induction of Fd-GOGAT mRNA (3-fold) occurred in etiolated leaves by low fluence red light, and far-red light reversibly repressed the mRNA accumulation. Red/far-red reversible induction also occurred for Fd-GOGAT polypeptide (2-fold) and activity (2-fold), implicating the phytochrome-dependent induction of Fd-GOGAT. In contrast, NADH-GOGAT activity remained constant, irrespective of red/far-red light treatments. Fd-GOGAT showed diurnal changes under light/dark cycles with the maximum early in the morning and the minimum in the afternoon at the levels of mRNA, enzyme polypeptide and activity. Gln diurnally changed in parallel with Fd-GOGAT mRNA. The induction of Fd-GOGAT provides evidence that light and metabolites are the major signal for the Gln and Glu formation in maize leaf cells.     

Phosphorylation/dephosphorylation of Komatsuna (Brassica campestris) leaf nitrate reductase in vivo and in vitro in response to environmental light conditions: Effects of protein kinase and protein phosphatase inhibitors

Activity of nitrate reductase (NR; EC 1.6.6.1) in leaves of Komatsuna (Brassica campestris L. ssp. rapifera cv. Osome) was decreased by sudden darkness, and rapidly recovered upon reillumination. However, the amount of NR protein, estimated by western blots, did not fluctuate during short-term light/dark/light transitions. This suggests that rapid changes of NR activity in response to light/dark regimes are due to reversible modulation of the protein and not to de novo synthesis/degradation. In mannose-fed leaves, such light/dark changes in NR activity were not observed. When extracts from illuminated leaves were incubated with MgATP, NR activity decreased in a time-dependent manner. K-252a, a specific inhibitor of protein kinases, prevented the in vitro inactivation of NR. The radiolabel of [γ-³²P] ATP was incorporated into NR protein in vitro and the labelling of NR was blocked by K-252a. On the other hand, extractable NR from darkened leaves was activated by incubation at 30°C without further additions. The in vitro activation of NR was prevented by calyculin A, a potent and specific inhibitor of protein phosphatase. Moreover calyculin A abolished the in vivo activation of NR by illumination. Our results confirm a regulatory system by phosphorylation/dephosphorylation of NR. The data also suggest that the activity of NR depends on the relative phosphorylation/dephosphorylation activities subtly controlled in response to photon flux density.     

Rapid modulation of nitrate reductase in leaves and roots: Indirect evidence for the involvement of protein phosphorylation/dephosphorylation

Nitrate reductase activity (NRA; NADH-nitrate reductase, E. C. 1.6.6.1) has been measured in extracts from leaves of spinach ( Spinacia oleracea L.) in response to rapid changes in illumination, or supply of CO₂ or oxygen. Measured in buffers containing magnesium, NRA from leaves decreased in the dark and increased again upon illumination. It decreased also, when CO₂ was removed in continuous light, and was reactivated when CO₂ was added. Nitrate reductase (NR) from roots of pea ( Pisum sativum L.) was also rapidly modulated in vivo. It increased under anaerobiosis and decreased in air or pure oxygen. The half time for inactivation or reactivation in roots and leaves was 5 to 30 min.
When spinach leaves were harvested during a normal day/night cycle, extractable NRA was low during the night, and high during daytime. However, at any point of the diurnal cycle, NR could be brought to a similar maximum activity by preincubation of the desalted leaf extract with AMP and/or EDTA. Thus, the observed diurnal changes appeared to be mainly a consequence of enzyme modulation, not of protein turnover. In vivo, the reactivation of the inactivated enzyme from both leaves and roots was prevented by okadaic acid, and inhibitor of certain protein phosphatases. Artificial lowering of the ATP-levels in leaf or root tissues by anaerobiosis (dark), mannose or the uncoupler carbonyl cyanide m -chlorophenyl hydrazon (CCCP), always brought about full activation of NR.
By preincubating crude leaf or root extracts with MgATP, NR was inactivated in vitro. Partial purification from spinach leaves of two enzymes with molecular masses in the 67 kD and 100 kD range, respectively, is reported. Both participate in the ATP-dependent inactivation of NR.
Alltogether these data indicate that NR can be rapidly modulated by reversible protein phosphorylation/dephosphorylation, both in shoots and in roots.     

In Vivo Regulation of Wheat-Leaf Phosphoenolpyruvate Carboxylase by Reversible Phosphorylation

Regulation of C3 phosphoenolpyruvate carboxylase (PEPC) and its protein-serine/threonine kinase (PEPC-PK) was studied in wheat (Triticum aestivum) leaves that were excised from low-N-grown seedlings and subsequently illuminated and/or supplied with 40 mM KNO3. The apparent phosphorylation status of PEPC was assessed by its sensitivity to L-malate inhibition at suboptimal assay conditions, and the activity state of PEPC-PK was determined by the in vitro 32P labeling of purified maize dephospho-PEPC by [[gamma]-32P]ATP/Mg. Illumination ([plus or minus]NO3-) for 1 h led to about a 4.5-fold increase in the 50% inhibition constant for L-malate, which was reversed by placing the illuminated detached leaves in darkness (minus NO3-). A 1 -h exposure of excised leaves to light, KNO3, or both resulted in relative PEPC-PK activities of 205, 119, and 659%, respectively, of the dark/0 mM KNO3 control tissue. In contrast, almost no activity was observed when a recombinant sorghum phosphorylation-site mutant (S8D) form of PEPC was used as protein substrate in PEPC-PK assays of the light plus KNO3 leaf extracts. In vivo labeling of wheat-leaf PEPC by feeding 32P-labeled orthophosphate showed that PEPC from light plus KNO3 tissue was substantially more phosphorylated than the enzyme in the dark minus-nitrate immunoprecipitates. Immunoblot analysis indicated that no changes in relative PEPC-protein amount occurred within 1 h for any of the treatments. Thus, C3 PEPC activity in these detached wheat leaves appears to be regulated by phosphorylation of a serine residue near the protein's N terminus by a Ca2+ -independent protein kinase in response to a complex interaction in vivo between light and N.     

Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro.

NO (nitric oxide) production from sunflower plants (Helianthus annuus L.), detached spinach leaves (Spinacia oleracea L.), desalted spinach leaf extracts or commercial maize (Zea mays L.) leaf nitrate reductase (NR, EC 1.6.6.1) was continuously followed as NO emission into the gas phase by chemiluminescence detection, and its response to post-translational NR modulation was examined in vitro and in vivo. NR (purified or in crude extracts) in vitro produced NO at saturating NADH and nitrite concentrations at about 1% of its nitrate reduction capacity. The K(m) for nitrite was relatively high (100 microM) compared to nitrite concentrations in illuminated leaves (10 microM). NO production was competitively inhibited by physiological nitrate concentrations (K(i)=50 microM). Importantly, inactivation of NR in crude extracts by protein phosphorylation with MgATP in the presence of a protein phosphatase inhibitor also inhibited NO production. Nitrate-fertilized plants or leaves emitted NO into purified air. The NO emission was lower in the dark than in the light, but was generally only a small fraction of the total NR activity in the tissue (about 0.01-0.1%). In order to check for a modulation of NO production in vivo, NR was artificially activated by treatments such as anoxia, feeding uncouplers or AICAR (a cell permeant 5'-AMP analogue). Under all these conditions, leaves were accumulating nitrite to concentrations exceeding those in normal illuminated leaves up to 100-fold, and NO production was drastically increased especially in the dark. NO production by leaf extracts or intact leaves was unaffected by nitric oxide synthase inhibitors. It is concluded that in non-elicited leaves NO is produced in variable quantities by NR depending on the total NR activity, the NR activation state and the cytosolic nitrite and nitrate concentration.     

Adenine nucleotides are apparently involved in the light-dark modulation of spinach-leaf nitrate reductase

Nitrate reductase (NR; EC 1.6.6.1) in spinach (Spinacia oleracea L. cv. Polka F1) leaves showed reversible modulation, being activated in the light and inactivated in the dark (t/2 = 20–30 min). The large changes in enzyme activity during light-dark transients were observed only when assayed in buffers containing free Mg²⁺. In the presence of EDTA (5 mM), the enzyme activity was high and the light modulation was barely evident.The inactivation of NR in the dark could be totally prevented by anaerobiosis, or by feeding mannose or 2,4-dinitrophenol through the leaf petiole. All these treatments drastically decreased ATP levels and increased AMP levels in leaf extracts, thus pointing to a close correlation between adenine-nucleotide levels and NR activity. Treatment of leaves in the dark with 2,4-dinitrophenol or with anaerobiosis brought about an accumulation of nitrite, thus confirming that under these conditions NR remained active also in vivo. The in-vivo dark-inactivated enzyme was reactivated in vitro by preincubating a leaf extract with AMP in the presence of the myokinase inhibitor p¹,p⁵-di(adenosine 5)pentaphosphate. It is suggested that NR responds to artificially induced drastic changes in cytosolic adeninenucleotide levels, being active when ATP is low and AMP is high. Adenine nucleotides also appear to participate in the light-dark modulation of NR, but additional regulatory factors have to be postulated.     

Induction and Turnover of Nitrate Reductase in Zea mays (Influence of Light)

Both light and NO3- are necessary for the appearance of nitrate reductase (NR) activity (NRA) in photosynthetic tissues. To define the light effect more precisely, we examined the response to light/dark transitions on NRA, NR protein (NRP), and NR mRNA in 6-d-old maize (Zea mays cv W64A x W182E) seedlings that had been grown in a light/dark regime for 5 d and then induced with 5 mM KNO3 for 24 h. The decay of NRA and NR mRNA in the shoot was immediate, but there were only minor changes in NRP during the initial 4 h in the dark. In root tissues, in contrast, there was a 4-h delay in the loss of NRA, NRP, and NR mRNA after transfer to the dark. When the seedlings were returned to light after a 2-h interval in the dark, shoot NRA reached 92% of the initial levels within 30 min of illumination. These results indicate that in the shoots (a) NR message production requires light and (b) the NRP that appears with light treatment and that is active is inactivated in the dark. The NRP can be reactivated when the light is turned on after short periods of darkness (2 h). Root tissues, on the other hand, probably respond to the supply of photosynthetically produced metabolites rather than to immediate products of the light reactions of photosynthesis.     

Light-responsive subtilisin-related protease in soybean seedling leaves.

Protease C1 (E.C. 3.4.21.25), the soybean (Glycine max L. Merrill) proteolytic enzyme responsible for initiating the degradation of soybean storage proteins in seedling cotyledons appears at even higher levels in seedling leaves. This was manifested at the mRNA level through northern blot analysis, at the protein level through western blot analysis, through determination of enzyme activity, and also through isolation and partial sequencing of active leaf enzyme. Comparison of cDNA and amino acid sequences, as well as characterization of enzyme activity, is consistent with the leaf enzyme being identical to or highly similar to the cotyledon enzyme. Protease C1 mRNA and protein are also present in stems of soybean seedlings, but is very low to absent in the roots. This presence in the aerial tissues is consistent with the higher steady state level of gene expression at both the mRNA and protein levels when the seedlings are grown in a 12-h light: 12-h dark photoperiod as compared to seedlings grown in continuous darkness. Transfer of dark-grown seedlings to light is followed by marked elevation in protease C1 protein as seen in western blots.     

Antibodies to assess phosphorylation of spinach leaf nitrate reductase on serine 543 and its binding to 14-3-3 proteins.

To monitor site-specific phosphorylation of spinach leaf nitrate reductase (NR) and binding of the enzyme to 14-3-3 proteins, serum antibodies were raised that select for either serine 543 phospho- or dephospho-NR. The dephospho-specific antibodies blocked NR phosphorylation on serine 543. The phospho-specific antibodies prevented NR binding to 14-3-3s, NR inhibition by 14-3-3s, NR dephosphorylation on serine 543, and did not precipitate 14-3-3s together with NR. Together, this confirms that 14-3-3s bind to NR at hinge 1 after it has been phosphorylated on serine 543. The amounts of individual NR forms were determined in leaf extracts by immunoblotting and immunoprecipitation. The phosphorylation state of NR on serine 543 increased 2-3-fold in leaves upon a light/ dark transition. Before the transition, one-third of NR was already phosphorylated on serine 543 but was not bound to 14-3-3s. Phosphorylation of serine 543 seems not to be enough to bind to 14-3-3s in leaves.     

Light modulation and in vitro effects of adenine nucleotides on leaf nitrate reductase activity in cucumber (Cucumis sativus)

Nitrate reductase (NR, EC 1.6.6.1) activity in attached cucumber ( Cucumis sativus L. cv. Ashley) leaves changed rapidly and reversibly during light/dark transitions, especially when assayed in the presence of free Mg²⁺. Light decreased and darkness increased the sensitivity of the enzyme to inhibition by Mg²⁺. The NR activation state, i.e. activity in the presence of Mg²⁺ relative to activity in the absence of Mg²⁺, increased with light intensity up to 400 μmol m⁻² s⁻¹ PAR (photosynthetically active radiation). When a desalted crude extract from illuminated leaves was preincubated with ATP, NR was gradually inactivated. Inactivation was only observed when activity was assayed in the presence of Mg²⁺. The ATP-inactivated NR remained inactive after removing the excess of ATP by gel filtration and it did not occur in partially purified NR preparations. NR extracted from darkened attached leaves was markedly activated when preincubated with 5'-AMP. These results support the view that inactivation/activation of cucumber-leaf NR in response to light/dark signals most likely involves phosphorylation/dephosphorylation of the enzyme catalysed by endogenous proteins. A substantial activation of NR by preincubation with 5'-AMP was also observed when activity was assayed in the absence of Mg²⁺, thus indicating that 5'-AMP can directly activate NR. Irradiation of an extract from darkened leaves containing FAD promoted a partial activation of NR. This effect was observed both in the +Mg²⁺ and in the −Mg²⁺ assay, indicating that activation was caused by photoexcited flavin and did not involve dephosphorylation of the enzyme.