Substrates regulate the phosphorylation status of nitrate reductase

The effect of substrates on the phosphorylation status of nitrate reductase (NR; EC 1.6.6.1) was studied. The enzyme was obtained from the first leaf of 7-day-old oat (Avena sativa L. cv. Suregrain) plants, grown in the light. When desalted crude extracts were incubated with ATP, NR was strongly phosphorylated, as evidenced by the inhibition of the enzyme's activity in the presence of Mg²⁺. NR sensitivity to Mg²⁺ remained unchanged when 10 mM nitrate was added to crude extracts after ATP. Addition of nitrate before or simultaneously with ATP slightly decreased Mg²⁺ inhibition of NR, which was strongly diminished in the presence of 10 mM NO₃^?+ 100 µM NADH. Incubation with NADH alone did not affect the enzyme's susceptibility to Mg²⁺ inhibition. When ammonium sulfate was added to crude extracts, NR was recovered in a 0-40% saturation fraction (F1). After incubation of F1 with ATP, the sensitivity of the enzyme to Mg²⁺ inhibition remained low, but it strongly increased after mixing F1 with a 45-60% saturation fraction (F2) suggesting that also in oats an additional factor (inactivating protein, IP), which probably binds to phospho-NR, would be required to keep the phosphorylated enzyme inactive in a +Mg²⁺ medium. Addition of 10 mM NO₃^?+ 100 µM NADH together with desalted F2 did not prevent Mg²⁺ inhibition suggesting that NO₃^? did not interfere with IP binding to phospho-NR. Again, incubation of F1 with both substrates during in vitro phosphorylation kept the enzyme active after adding F2, even in the presence of Mg²⁺, After in vitro phosphorylation, NR in crude extract was hardly reactivated when incubated alone or in the presence of 10 mM NO₃^? at 30°C. On the other hand, a strong and very rapid reactivation was found when the extract was incubated with both nitrate and NADH. Microcystine, an inhibitor of types 1 and 2A phosphoprotein phosphatases, inhibited the reactivation of phospho-NR induced by the substrates. The results presented here show that the substrates could prevent NR phosphorylation and induce the enzyme's dephosphorylation, but they were effective only after their binding to the NR protein. Thereby, they seemed to affect the NR protein itself and not the phosphatase- or the kinase-proteins. It has been reported that nitrate binding to the enzyme's active site induces conformational changes in the NR protein. We propose that this conformational change would prevent NR phosphorylation, by converting the enzyme into a form in which the site recognized by the protein kinase is no longer accessible, and, simultaneously, stimulate NR dephophorylation by allowing the specific phosphatases to recognize NR.     

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.     

Metabolic activators of spinach leaf nitrate reductase: Effects on enzymatic activity and dephosphorylation by endogenous phosphatases

Nitrate reductase (NR; EC 1.6.6.1) in spinach (Spinacia oleracea L.) leaves was inactivated in the dark and reactivated by light in vivo. When extracted from dark leaves, NR activity was lower and more strongly inhibited by Mg²⁺ relative to the enzyme extracted from leaves harvested in the light. When dark extracts were desalted at pH 6.5 and preincubated at 25° C prior to assay, enzyme activity (assayed either in the presence or absence of Mg²⁺) remained essentially constant, i.e. there was no spontaneous reactivation in vitro. However, addition of certain metabolites resulted in a time- and concentration-dependent activation of NR in vitro. Effective activators included inorganic phosphate (Pi), 5-AMP, and certain of its derivatives such as FAD and pyridine nucleotides (both oxidized and reduced forms). All of the activators increased NR activity as assayed in the absence of Mg²⁺, whereas some activators (e.g. Pi, 5-AMP and FAD) also reduced Mg²⁺ inhibition. The reduction of Mg²⁺ inhibition was also time-dependent and was almost completely prevented by a combination of okadaic acid plus KF, suggesting the involvement of dephosphorylation catalyzed by endogenous phosphatase(s). In contrast, the activation of NR (assayed minus Mg²⁺) was relatively insensitive to phosphatase inhibitors, indicating a different mechanism was involved. Compounds that were not effective activators of NR included sulfate, ribose-5-phosphate, adenosine 5-monosulfate, coenzyme A, ADP and ATP. We postulate that NR can exist in at least two states that differ in enzymatic activity. The activators appear to interact with the NR molecule at a site distinct from the NADH active site, and induce a slow conformational change (hysteresis) that increases NR activity (assayed in the absence of Mg²⁺). Possibly as a result of the conformational change caused by certain activators, the regulatory phospho-seryl groups are more readily dephosphorylated by endogenous phosphatases, thereby reducing sensitivity to Mg²⁺ inhibition. Preliminary results suggest that light/dark transitions in vivo may alter the distribution of NR molecules between the low- and high-activity forms.Abbreviations AP₅A P¹, P⁵-di(adenosine-5)pentaphosphate - DTT dithiothreitol - Mops 3-(N-morpholino)propanesulfonic acid - NR NADH:nitrate reductase - NRA nitrate reductase activity Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643. This work was also supported in part by grants from the U.S. Department of Energy (Grant DE-AIO5-91 ER 20031) and USDA-NRI (Grant 93-373-5-9231). The authors thank Dr. W.M. Kaiser (Lehrstuhl Botanik I der Universität, Würzburg, Germany) for discussions and Dr. C. Lillo (Rogaland University Center, Stavanger, Norway) for sharing results prior to publication.     

Modifications of the activity of nitrate reductase from cucumber roots

The regulatory properties of NADH-dependent nitrate reductase (NR) in desalted root extracts from hydroponically grown cucumber (Cucumis sativus L.) seedlings were examined. The lowest activity of NR was detected in extracts incubated with Mg²⁺ and ATP. An inhibitory effect of Mg-ATP was cancelled in the presence of staurosporine (the protein kinase inhibitor) and completely reversed after addition of ethylenediaminetetraacetate (EDTA) as well as AMP into reaction mixture. Reactivation of enzyme due to AMP presence, contrary to the chelator-dependent NR activation, was sensitive to microcystin LR (the protein phosphatase inhibitor). Above results indicated that the nitrate reductase in cucumber roots was regulated through reversible phosphorylation of enzyme protein. A drop in the activity of NR was also observed after incubation of enzyme at low pH. At low pH, the presence of ATP alone in the incubation medium was sufficient to inactivate NR, indicating that H⁺ can substitute the Mg²⁺ in formation of an inactive complex of enzyme. ATP-dependent inactivation of NR at low pH was prevented by staurosporine and reversed by AMP. However, AMP action was not altered by microcystin LR suggesting that in low pH the nucleotide induced reactivation of NR is not limited to the protein phosphorylation.     

Effect of preincubation temperature on in vitro light saturated photosystem I activity in thylakoids isolated from cold hardened and nonhardened rye

Thylakoids isolated from winter rye (Secale cereale L. cv Muskateer) grown at 5°C or 20°C were compared with respect to their capacity to exhibit an increase in light saturated rates of photosystem I (PSI) electron transport (ascorbate/dichlorophenolindophenol → methylviologen) after dark preincubation at temperatures between 0 and 60°C. Thylakoids isolated in the presence or absence of Na⁺/Mg²⁺ from 20°C grown rye exhibited transient, 40 to 60% increases in light saturated rates of PSI activity at all preincubation temperatures between 5 and 60°C. This increase in PSI activity appeared to occur independently of the electron donor employed. The capacity to exhibit this in vitro induced increase in PSI activity was examined during biogenesis of rye thylakoids under intermittent light conditions at 20°C. Only after exposure to 48 cycles (1 cycle = 118 minutes dark + 2 min light) of intermittent light did rye thylakoids exhibit an increase in light saturated rates of PSI activity even though PSI activity could be detected after 24 cycles. In contrast to thylakoids from 20°C grown rye, thylakoids isolated from 5°C grown rye in the presence of Na⁺/Mg²⁺ exhibited no increase in light saturated PSI activity after preincubation at any temperature between 0 and 60°C. This was not due to damage to PSI electron transport in thylakoids isolated from 5°C grown plants since light saturated PSI activity was 60% higher in 5°C thylakoids than 20°C thylakoids prior to in vitro dark preincubation. However, a two-fold increase in light saturated PSI activity of 5°C thylakoids could be observed after dark preincubation only when 5°C thylakoids were initially isolated in the absence of Na⁺/Mg²⁺. We suggest that 5°C rye thylakoids, isolated in the presence of these cations, exhibit light saturated PSI electron transport which may be closer to the maximum rate attainable in vitro than 20°C thylakoids and hence cannot be increased further by dark preincubation.     

Rapid Modulation of Spinach Leaf Nitrate Reductase Activity by Photosynthesis : I. Modulation in Vivo by CO(2) Availability

It has been shown recently that in spinach leaves (Spinacia oleracea) net photosynthesis and nitrate reduction are closely linked: when net photosynthesis was low because of stomatal closure, rates of nitrate reduction decreased (WM Kaiser, J Förster [1989] Plant Physiol 91: 970-974). Here we present evidence that photosynthesis regulates nitrate reduction by modulating nitrate reductase activity (NRA, EC 1.6.6.1). When spinach leaves were exposed to low CO₂ in the light, extractable NRA declined rapidly with a half-time of 15 minutes. The inhibition was rapidly reversed when leaves were brought back to air. NRA was also inhibited when leaves were wilted in air; this inhibition was due to decreased CO₂ supply as a consequence of stomatal closure. The modulation of NRA was stable in vitro. It was not reversed by gel filtration. In contrast, the in vitro inhibition of nitrate reductase (NR) by classical inhibitors such as cyanide, hydroxylamin, or NADH disappeared after removal of free inhibitors by gel filtration. The negative modulation of NRA in —CO₂-treated leaves became manifest as a decrease in total enzyme activity only in the presence of free Mg²⁺ or Ca²⁺. Mg²⁺ concentrations required for observing half-maximal inhibition were about 1 millimolar. In the presence of EDTA, the enzyme activity was always high and rather independent of the activation status of the enzyme. NRA was also independent of the pH in the range from pH 7 to pH 8, at saturating substrate and Mg²⁺ concentrations. The apparent substrate affinities of NR were hardly affected by the in vivo modulation of NR. Only V_max changed.     

Rapid Modulation of Spinach Leaf Nitrate Reductase by Photosynthesis : II. In Vitro Modulation by ATP and AMP

Assimilatory nitrate reductase activity (NRA) in crude spinach leaf (Spinacia oleracea) extracts undergoes rapid changes following fluctuations in photosynthesis brought about by changes in external CO₂ or by water stress (WM Kaiser, E Brendle-Behnisch [1991] Plant Physiol 96:363-367). A modulation of NRA sharing several characteristics (stability, response to Mg²⁺ or Ca²⁺, kinetic constants) with the in vivo modulation was obtained in vitro by preincubating desalted leaf extracts with physiological concentrations of Mg²⁺ and ATP (deactivating) or AMP (activating). When nitrate reductase (NR) was inactivated in vivo by illuminating leaves at the CO₂ compensation point, it could be reactivated in vitro by incubating leaf extracts with AMP. For the in vitro inactivation, ATP could be replaced by GTP or UTP. Nonhydrolyzable ATP analogs (β, γ-imido ATP, β, γ-methyl-ATP) had no effect on NR, whereas γ-S-ATP caused an irreversible inactivation. This suggests that NR modulation involves ATP hydrolysis. In contrast to NR in crude leaf extracts, partially purified NR did not respond to ATP or AMP. ATP and AMP levels in whole leaf extracts changed in the way predicted by the modulation of NRA when leaves were transferred from photosynthesizing (low ATP/AMP) to photorespiratory (high ATP/AMP) conditions. Adenine nucleotide levels in leaves could be effectively manipulated by feeding mannose through the leaf petiole. NRA followed these changes as expected from the in vitro results. This suggests that cytosolic ATP/AMP levels are indeed the central link between NRA in the cytosol and photosynthesis in the chloroplast. Phosphorylation/dephosphorylation of NR or of NR-regulating protein factors is discussed as a mechanism for a reversible modulation of NR by ATP and AMP.     

Post-translational control of nitrate reductase activity responding to light and photosynthesis evolved already in the early vascular plants

Regulation of nitrate reductase (NR) by reversible phosphorylation at a conserved motif is well established in higher plants, and enables regulation of NR in response to rapid fluctuations in light intensity. This regulation is not conserved in algae NR, and we wished to test the evolutionary origin of the regulatory mechanism by physiological examination of ancient land plants. Especially a member of the lycophytes is of interest since their NR is candidate for regulation by reversible phosphorylation based on sequence analysis. We compared Selaginella kraussiana, a member of the lycophytes and earliest vascular plants, with the angiosperm Arabidopsis thaliana, and also tested the moss Physcomitrella patens. Interestingly, optimization of assay conditions revealed that S. kraussiana NR used NADH as an electron donor like A. thaliana, whereas P. patens NR activity depended on NADPH. Examination of light/darkness effects showed that S. kraussiana NR was rapidly regulated similar to A. thaliana NR when a differential (Mg²⁺ contra EDTA) assay was used to reveal activity state of NR. This implies that already existing NR enzyme was post-translationally activated by light in both species. Light had a positive effect also on de novo synthesis of NR in S. kraussiana, which could be shown after the plants had been exposed to a prolonged dark period (7 days). Daily variations in NR activity were mainly caused by post-translational modifications. As for angiosperms, the post-translational light activation of NR in S. kraussiana was inhibited by 3-(3,4-dichlorophenyl)-1*1-dimethylurea (DCMU), an inhibitor of photosynthesis and stomata opening. Evolutionary, a post-translational control mechanism for NR have occurred before or in parallel with development of vascular tissue in land plants, and appears to be part of a complex mechanisms for coordination of CO₂ and nitrogen metabolism in these plants.     

Effect of light/dark cycles on expression of nitrate assimilatory genes in maize shoots and roots

The level of nitrate reductase (NR) and nitrite reductase (NiR) varied in both shoot and root tissue from nitrate-fed Zea mays L. grown under a 16-hour light/8-hour dark regime over a 10-day period postgermination, with peak activity occurring in days 5 to 6. To study the effect of different light regimes on NR and NiR enzyme activity and mRNA levels, 6-day-old plants were grown in the presence of continuous KNO₃ (10 millimolar). Both shoot NRA and mRNA varied considerably, peaking 4 to 8 hours into the light period. Upon transferring plants to continuous light, the amplitude of the peaks increased, and the peaks moved closer together. In continuous darkness, no NR mRNA or NR enzyme activity could be detected by 8 hours and 12 hours, respectively. In either a light/dark or continuous light regime, root NRA and mRNA did not vary substantially. However, when plants were placed in continuous darkness, both declined steadily in the roots, although some remained after 48 hours. Although there was no obvious cycling of NiR enzyme activity in shoot tissue, changes in mRNA mimicked those seen for NR mRNA. The expression of NR and NiR genes is affected by the light regime adopted, but light does not have a direct effect on the expression of these genes.     

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.     

Inactivation of nitrate reductase involves NR-protein phosphorylation and subsequent ‘binding’ of an inhibitor protein

The function of two proteins (P67 and P100) required for the MgATP-dependent inactivation of nitrate reductase (NR) from spinach leaves (Spinacia oleracea L.) was studied. When NR was incubated with -[³²P]ATP and P67, NR-protein was phosphorylated, but without a change in NR activity. Protein P100 by itself was neither able to phosphorylate nor to inactivate NR, and when added together with P67 it did not change the extent of NR phosphorylation. However, when NR was first phosphorylated with MgATP and P67, subsequent addition of P100 after removal of unreacted ATP caused an immediate NR inactivation. In presence of both P67 and P100 the time-course of ATP-dependent NR phosphorylation paralleled the time course of inactivation. The extent of NR phosphorylation and of NR inactivation (in the presence of P67 plus P100) was similarly affected by metabolites or high salt concentrations. Magnesium (Mg²⁺) played a dual role in the inactivation process: the phosphorylation of NR by P67 was strictly Mg²⁺-dependent. Further, phospho-NR (+P100) was inactive only in the presence of Mg²⁺, but active in the presence of excess EDTA. Dephospho-NR appeared to be Mg²⁺-insensitive. The observations suggest that phosphorylation of NR by P67 is obligatory, but not sufficient for inactivation. In addition to protein phosphorylation, inactivation requires binding of an inhibitor protein (P100) to phospho-NR.Abbreviations G6P glucose-6-phosphate - NR NADH-nitrate reductase - NRA nitrate reductase activity The skilled technical assistance of Elke Brendle-Behnisch is gratefully acknowledged. We also wish to thank Dr. C. MacKintosh, University of Dundee, UK, who supplied us with an immuno-affinity column for NR purification. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 251).     

The protein kinase, protein phosphatase and inhibitor protein of nitrate reductase are ubiquitous in higher plants and independent of nitrate reductase expression and turnover

Nitrate reductase activity and NR protein levels in various leaf tissues were drastically decreased (<3.5% of normal activity) either by keeping detached leaves in continuous darkness for up to 6 d (spinach), or by growing plants (pea, squash) hydroponically on ammonium as the sole N-source, or by germinating and growing etiolated seedlings in complete darkness (squash). The presence of nitrate reductase protein kinase (NRPK), nitrate reductase protein phosphatase (NRPP) and inhibitor protein (IP) was examined by measuring the ability of NR-free desalted extracts to inactivate (ATP-dependent) and reactivate (5-AMP/EDTA-dependent) added purified spinach NR in vitro. Extracts from low-NR plants (ammonium-grown pea and squash) were also prepared from leaves harvested at the end of a normal light or dark phase, or after treating leaves with anaerobiosis, uncouplers or mannose, conditions which usually activate NR in nitrategrown normal plants. Without exception, extracts from NR-deficient plant tissues were able to inactivate and reactivate purified spinach NR with normal velocity, irrespective of pretreatment or time of harvest. Considerable NRPK, NRPP and IP activities were also found in extracts from almost NR-free ripe fruits (cucumber and tomato). Activities were totally absent, however, in extracts from isolated spinach chloroplasts. The NRPK and IP fractions were partially purified with normal yields from NR-deficient squash or spinach leaves, following the purification protocol worked out for nitrate-grown spinach. The Ca²⁺/Mg²⁺-dependent kinase fraction from NR-deficient squash or spinach phosphorylated added purified spinach NR with -[³²P]ATP and inactivated the enzyme after addition of IP. It is suggested (i) that the auxiliary proteins (NRPK, IP, NRPP) which modulate NR are rather species- or organ-unspecific, (ii) that they do not turn over as rapidly as does NR, (iii) that they are probably expressed independently of NR, and (iiii) that they are not covalently modulated, but under control of metabolic and/or physical signals which are removed by desalting.Abbreviations IP inhibitor protein - NR NADH-nitrate reductase - NRA nitrate reductase activity - NRPK nitrate reductase protein kinase - NRPP nitrate reductase protein phosphatase - PK protein kinase This work was supported by the Deutsche Forschungsgemeinschaft (SFB 251).     

Short-term modulation of nitrate reductase activity by exogenous nitrate in Nicotiana plumbaginifolia and Zea mays leaves

Maize (Zea mays L.) grown on low (0.8 mM) NO ₃ ^- , as well as untransformed and transformed Nicotiana plumbaginifolia constitutively expressing nitrate reductase (NR), was used to study the effects of NO ₃ ^- on the NR activation state. The NR activation state was determined from the relationship of total activity extracted in the presence of ethylenediaminetetracetic acid to that extracted in the presence of Mg²⁺. Light activation was observed in both maize and tobacco leaves. In the tobacco lines, NO ₃ ^- did not influence the NR activation state. In excised maize leaves, no correlation was found between the foliar NO ₃ ^- content and the NR activation state. Similarly, the NR activation state did not respond to NO ₃ ^- . Since the NR activation state determined from the degree of Mg²⁺-induced inhibition of NR activity is considered to reflect the phosphorylation state of the NR protein, the protein phosphatase inhibitor microcystin LR was used to test the importance of protein phosphorylation in the NO ₃ ^- -induced changes in NR activity. In-vivo inhibition of endogenous protein phosphatase activity by microcystin-LR decreased the level of NR activation in the light. This occurred to the same extent in the presence or absence of exogenous NO ₃ ^- . We conclude that NO ₃ ^- does not effect the NR activation state, as modulated by protein phosphorylation in either tobacco (a C₃ species) or maize (a C₄ species). The short-term regulation of NR therefore differs from the NO ₃ ^- -mediated responses observed for phosphoenolpyruvate carboxylase and sucrose phosphate synthase.Abbreviations Chl chlorophyll - MC microcystin-LR - PEP-Case phosphoenolpyruvate carboxylase - SPS sucrose-phosphate synthase We are indebted to Madeleine Provot and Nathalie Hayes for excellent technical assistance. This work was funded by EEC Biotechnology Contract No. BI02 CT93 0400, project of technical priority, Network D — Nitrogen Utilisation and Efficiency.     

Effect of one night with "low light'on uptake, reduction and storage of nitrate in spinach

During the night, shoot nitrate concentration in spinach (Spinacia oleracea L. cv. Vroeg Reuzenblad) increased due to increased uptake of nitrate by the roots. When the plants were subjected to a one night “low light’period at 35 μmol m^?2 s^?1, the shoot nitrate concentration did not increase and was reduced by 25% compared to control plants in the dark. The major contribution to this decrease was located in the leaf blades, where the nitrate concentration was decreased by 60%, while the petiole nitrate concentration decreased by only 9%. Nitrate accumulated in the leaf blade vacuoles during a dark night, but this was not the case during the “low light’period. This decrease in vacuolar nitrate concentration, compared to control plants in the dark, was not caused by increased amounts of leaf blade nitrate reductase (NR; EC 1.6.6.1). During a “low light’night period, the cytoplasmic soluble carbohydrate concentration was increased compared to the control plants in the dark. Calculations showed in situ NR activity to be higher than in the control plants in the dark. This increase in NR activity, however, was not large enough to account for the total difference found in the shoot nitrate concentration. Net uptake of nitrate by the roots was increased during the initial hours of the dark night, while vacuolar nitrate concentration in the leaf blades increased at the same time. During the “low light’night period, however, net uptake of nitrate by the roots did not increase, and vacuolar nitrate concentration did not change. We conclude that nitrate uptake by the roots and vacuolar nitrate concentration in the leaf blades are tightly coupled. The decreased shoot nitrate concentration is mainly caused by a reduction in net uptake of nitrate by the roots. During the “low light’night period, carbohydrates and malic acid partly replaced vacuolar nitrate. A “low light’period one night prior to harvest provides a valuable tool to reduce shoot nitrate concentrations in spinach grown in greenhouses in the winter months.     

The role of nitrate and ammonium ions and light on the induction of nitrate reductase in maize leaves

Corn seedlings (Zea mays cv W64A × W182E) were grown hydroponically, in the presence or absence of NO₃⁻, with or without light and with NH₄Cl as the only N source. In agreement with earlier results nitrate reductase (NR) activity was found only in plants treated with both light and NO₃⁻. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer of the proteins to nitrocellulose paper and reaction with antibodies prepared against a pure NR showed that crude extracts prepared from light-grown plants had a polypeptide of approximately 116 kilodaltons (the subunit size for NR) when NO₃⁻ was present in the growth medium. Crude extracts from plants grown in the dark did not have the 116 kilodalton polypeptide, although smaller polypeptides, which reacted with NR-immunoglobulin G, were sometimes found at the gel front. When seedlings were grown on Kimpack paper or well washed sand, NR activity was again found only when the seedlings were exposed to light and NO₃⁻. Under these conditions, however, a protein of about 116 kilodaltons, which reacted with the NR antibody was present in light-grown plants whether NO₃⁻ was added to the system or not. The NR antibody cross-reacting protein was also seen in hydroponically grown plants when NH₄Cl⁻ was the only added form of nitrogen. These results indicate that the induction of an inactive NR-protein precursor in corn is mediated either by extremely low levels of NO₃⁻ or by some other unidentified factor, and that higher levels of NO₃⁻ are necessary for converting the inactive NR cross-reacting protein to a form of the enzyme capable of reducing NO₃⁻ to NO₂⁻.     

Steryl glucoside and acyl glucoside biosynthesis in maturing pea seeds

Sterol glucosyltransferase activity was found in a particulate fraction of pea seeds. The activity was stimulated by Ca²⁺ and Mg²⁺ and inhibited by Zn²⁺, Cu²⁺, Hg²⁺, EDTA and EGTA. Iodoacetamide was without effect but p-chloromercuribenzoate completely inhibited the enzyme. N -Ethylmaleimide gave 60–70 % inhibition over a wide range of concentrations. The activity was stimulated by ATP in the presence of Mg²⁺. Under such conditions, steryl acyl glucoside was formed. The acyl derivative was barely detectable in the presence of Ca²⁺ either with or without ATP. Both oleyl CoA and palmityl CoA stimulated acyl glucoside synthesis. Of the four nucleoside triphosphates, ATP, GTP, UTP and CTP both ATP and CTP stimulated acylation in the presence of Mg²⁺. The observations suggest that acyl donors other than digalactosyl diglyceride and phospholipids may function in steryl acyl glucoside synthesis in plants.     

Nitrate reductase dephosphorylation is induced by sugars and sugar-phosphates in corn leaf segments

The in situ and in vitro regulation of nitrate reductase (NR; EC 1.6.6.1) activity by glucose (Glc) and glucose‐6‐phosphate (Glc‐6P) was studied in leaf segments of 7‐day‐old corn plants. In situ, Glc and Glc‐6P not only prevented NR inactivation, but also slightly activated the enzyme relative to that in fresh attached leaves in the light. Glc and Glc‐6P also reactivated NR that had previously been inactivated by incubating the segments for 30 min in the dark. Sugars were effective, even in the presence of cycloheximide, but not of cantharidin, an inhibitor of type 2A phosphoprotein phosphatase (PP2A). In segments kept in the dark, the inhibition of protein dephosphorylation by cantharidin showed that the phosphorylation of NR was not inhibited by either Glc or Glc‐6P, as the enzyme was inactivated to the same extent whether or not sugars (P) were present in the incubation medium. In vitro, as in situ, neither Glc nor Glc‐6P could prevent NR phosphorylation. In spite of some reports showing that sugar‐phosphates can act on kinases and prevent NR phosphorylation, the results presented here suggest that, in corn leaves, sugars and their phosphorylated derivatives probably activate NR in situ mainly by inducing protein dephosphorylation. The incubation of crude extract in a water bath at 27°C for 45 min resulted in the activation of NR that was blocked by cantharidin, but was not increased by either Glc or Glc‐6P. This result suggests that the presence of another metabolite(s) and the maintenance of cell functionality may be necessary for the sugar‐induced activation of NR. A sugar‐triggered signalling pathway independent of protein synthesis may be involved in the process. l ‐Glc and 6‐deoxyglucose were ineffective in reactivating NR in darkened segments, whilst 2‐deoxyglucose was as effective as Glc itself. The effect of sugar analogues shows that, although Glc has to enter the cell and be phosphorylated to activate NR, further metabolism is not necessary. As sugar‐phosphates, such as Glc‐6P and fructose‐6‐phosphate (Fru‐6P), also activate NR, it seems that hexokinases are not involved in the pathway that leads to the in situ dephosphorylation of NR. In vitro, Glc‐6P mildly but rapidly activated NR by a mechanism insensitive to cantharidin. The addition of an increasing concentration of Mg²⁺ to crude extract containing Glc‐6P increased the Mg²⁺ inhibition of NR. This result suggests that the hexose‐phosphate does not prevent Mg²⁺ association with NR. It is possible that Glc‐6P activates NR in vitro by inducing the dissociation of 14‐3‐3 from the phospho‐NR (pNR)/Mg/14‐3‐3 complex.