NR Activation Research Articles

Histone H3K4 trimethylation by MLL3 as part of ASCOM complex is critical for NR activation of bile acid transporter genes and is downregulated in cholestasis

The nuclear receptor Farnesoid x receptor (FXR) is a critical regulator of multiple genes involved in bile acid homeostasis. The coactivators attracted to promoters of FXR target genes and epigenetic modifications that occur after ligand binding to FXR have not been completely defined, and it is unknown whether these processes are disrupted during cholestasis. Using a microarray, we identified decreased expression of mixed lineage leukemia 3 (MLL3), a histone H3 lysine 4 (H3K4) lysine methyl transferase at 1 and 3 days of post-common bile duct ligation (CBDL) in mice. Chromatin immunoprecipitation analysis (ChIP) analysis revealed that H3K4me3 of transporter promoters by MLL3 as part of activating signal cointegrator-2 -containing complex (ASCOM) is essential for activation of bile salt export pump (BSEP), multidrug resistance associated protein 2 (MRP2), and sodium taurocholate cotransporting polypeptide (NTCP) genes by FXR and glucocorticoid receptor (GR). Knockdown of nuclear receptor coactivator 6 (NCOA6) or MLL3/MLL4 mRNAs by small interfering RNA treatment led to a decrease in BSEP and NTCP mRNA levels in hepatoma cells. Human BSEP promoter transactivation by FXR/RXR was enhanced in a dose-dependent fashion by NCOA6 cDNA coexpression and decreased by AdsiNCOA6 infection in HepG2 cells. GST-pull down assays showed that domain 3 and 5 of NCOA6 (LXXLL motifs) interacted with FXR and that the interaction with domain 5 was enhanced by chenodeoxycholic acid. In vivo ChIP assays in HepG2 cells revealed ligand-dependent recruitment of ASCOM complex to FXR element in BSEP and GR element in NTCP promoters, respectively. ChIP analysis demonstrated significantly diminished recruitment of ASCOM complex components and H3K4me3 to Bsep and Mrp2 promoter FXR elements in mouse livers after CBDL. Taken together, these data show that the "H3K4me3" epigenetic mark is essential to activation of BSEP, NTCP, and MRP2 genes by nuclear receptors and is downregulated in cholestasis.

Effects of water deficit on the activity of nitrate reductase and content of sugars, nitrate and free amino acids in the leaves and roots of sunflower and white lupin plants growing under two nutrient supply regimes

The effects of soil drying on the activity of nitrate reductase (NR; EC 1.6.6.6) were studied in Helianthus annuus L. and non‐nodulated Lupinus albus L. plants growing under two nutrient supply regimes. NR activity was assessed in leaf and root extracts by measuring the activity of the unphosphorylated active form (NRact), the maximal extractable activity (NRmax) and the activation state. To obtain an insight into potential signalling compounds, nitrate, free amino acids and soluble sugars were also quantified. In both species, foliar NRact and NRmax were negatively affected by soil drying and a decreased supply of nutrients, the observed changes in NR activity being linearly correlated with the depletion of nitrate. Similar results were obtained in the roots of sunflower. Conversely, in white lupin roots, NRmax was found to be independent of tissue nitrate concentration. Regardless of the species and organ, the activation state of the enzyme was unaffected by the nutrient supply regime. In well‐watered sunflower roots, only about 50% of the existing NR was unphosphorylated, but the activation state increased significantly in response to drought. In contrast, lupin roots always exhibited NR activation state values close to 80%, or even higher. At the leaf level, the NR activation state was hardly changed in response to soil drying. The observed changes in the concentrations of soluble sugars and free amino acids are discussed in terms of their possible contribution to the variations in NR activity.

Unusual regulatory nitrate reductase activity in cotyledons of Brassica napus seedlings: enhancement of nitrate reductase activity by ammonium supply.

The effect of supplying either nitrate or ammonium on nitrate reductase activity (NRA) was investigated in Brassica napus seedlings. In roots, nitrate reductase activity (NRA) increased as a function of nitrate content in tissues and decreased when ammonium was the sole nitrogen source. Conversely, in the shoots (comprising the cotyledons and hypocotyl), NRA was shown to be independent of nitrate content. Moreover, when ammonium was supplied as the sole nitrogen source, NRA in the shoots was surprisingly higher than under nitrate supply and increased as a function of the tissue ammonium content. Under 15 mM of exogenous ammonium, the NRA was up to 2.5-fold higher than under nitrate supply after 6 d of culture. The NR mRNA accumulation under ammonium nutrition was 2-fold higher than under nitrate supply. The activation state of NR in shoots was especially high compared with roots: from nearly 80% under nitrate supply it reached 94% under ammonium. This high NR activation state under ammonium supply could be the consequence of the slight acidification observed in the shoot tissue. The effect of ammonium on NRA was only observed in cotyledons and when more than 3 mM ammonium was supplied. No such NRA increase was evident in the roots or in foliar discs. Addition of 1 mM nitrate under ammonium nutrition halved NRA and decreased the ammonium content in shoots. Thus, this unusual NRA was restricted to seedling cotyledons when nitrate was lacking in the nitrogen source.

Effect of NO3‐ supply on N metabolism of potato plants (Solanum tuberosum L.) with special focus on the tubers

AbstractThe response of the tubers to NO3– was studied in comparison to the other organs of Solanum tuberosum var. Sava, with special focus on: (a) whether tubers are capable of primary N assimilation; (b) whether N assimilation is stimulated by NO3–; and (c) whether primary N assimilation in tubers is important for tuber growth. NO3– reduction via nitrate reductase (NR; EC 1.6.6.1) and NH4+ assimilation via glutamine synthetase (GS; EC 6.3.1.2) occurred predominantly in the shoots, but up to 20% was contributed by the tubers under low‐NO3– conditions. NR activation was highest in tubers (up to 90%) and declined in all organs with increasing NO3– supply. NR and GS activity responded with a decline in tubers and roots as opposed to an increase in the shoots. This corresponded to relative organ growth: growth of tubers and roots was stimulated relative to that of shoots at a limiting NO3– supply. Absolute growth of all organs was stimulated by NO3–, whereas tuber number declined. The concentration of N compounds increased with NO3– supply in all organs: NO3– increased most dramatically in the shoots (81‐fold), free amino acids most markedly in the tubers (three‐fold). The amount of patatin and of the 22 kDa protein complex in the tuber reached a minimum when the amount of Rubisco in the shoot reached maximum as a response to NO3– supply. Tuber sucrose and starch increased by 40%, whereas glucose and fructose declined two‐fold as plant N status increased. It is concluded that tubers are potentially N autotroph organs with capacity for de novo synthesis of amino acids. Primary N assimilation in tubers, however, declines with increasing NO3– supply and is not of major importance for tuber growth.

Nitrate reductase from winter wheat leaves is activated at low temperature via protein dephosphorylation.

The effect of short-term low temperature treatment on nitrate reductase (NR, EC 1.6.6.1) activity, NR protein and NR transcript levels in excised leaves of winter wheat (Triticum aestivum L. cv. Sadovo-1) was investigated. NR activity, measured in the presence of Mg2+ (NRact), doubled within 2 h at 4 degrees C, whereas NR activity, measured in the presence of EDTA (NRmax), did not respond to the cold treatment. Such an activation of NR occurred only if leaves were exposed to low temperature in the light but not in the dark. It was not affected by feeding cytoplasmic protein synthesis inhibitor, cycloheximide, or protein kinase inhibitor, staurosporin, but was completely prevented by okadaic acid, an inhibitor of protein phosphatases of the type 1 and 2 A. This inhibitory effect decreased gradually when okadaic acid-concentration in the nutrient solution was lowered below 1 &mgr;M and tended to disappear when leaves were fed with 10 nM okadaic acid. It was demonstrated that the cold-induced NR activation was dependent neither on cold-triggered calcium influx nor on high endogenous abscisic acid levels. The increased NRact in cold-exposed leaves was found to correlate with a higher level of NR transcript but not with an increased NR protein level. Feeding okadaic acid to these leaves prevented the cold-induced accumulation of NR mRNA. These data point to protein phosphatases of the type 2 A being involved in NR protein dephosphorylation and NR transcript accumulation as targets of activation by low temperature treatment.

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.

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 Km for nitrite was relatively high (100 μM) compared to nitrite concentrations in illuminated leaves (10 μM). NO production was competitively inhibited by physiological nitrate concentrations ( Ki =50 μM). 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.

Nitrate reductase activation state in leaves of molybdenum-deficient winter wheat

Summary Nitrate reductase (NR, EC 1.6.6.1) activity, NR activation state, and in vivo nitrate reduction were measured in leaves of winter wheat ( Triticum aestivum L. cv. Sadovo 1) in order to study the effect of molybdenum (Mo) deficiency on light-induced changes in NR activation. It was established that dark to light transition of the plants resulted in a fast increase in actual and maximum NR activities (NRA act and NRA max ) and vice versa; the deactivation of NR occurred soon after light to dark transition. The diurnal rhythm of NRA act and NRA max was similar in phase in Mo-deficient and Mo-supplied plants. However, the absolute values of NRA act and NRA max were about two to three times higher in Mosupplied compared to Mo-deficient plants, i.e. only the amplitude of the rhythm was affected by Mo deficiency. The activation state of NR, given as percentage ratio of NRA act to NRA max , was higher in Mo-deficient plants, reaching a maximum of 60-66% in the light, independent of the light intensity over 210 μmol m -2 s -1 . In both Mo-deficient and Mo-supplied plants, the level of NRA act — thought to reflect the in situ NR activity — exceeded by several times the rate of in vivo nitrate reduction. The results indicate that Mo deficiency itself does not alter the timing of light/dark changes in NR activity, but it reduces significantly the levels of NRA max and NRA act and results in a higher NR activation state in Mo-deficient plants. A possible mechanism of this effect is discussed.

Effects of cadmium on activity of nitrate reductase and on other enzymes of the nitrate assimilation pathway in bean

We examined the influence of cadmium (Cd) exposure on nitrate assimilation in bean ( Phaseolus vulgaris L. cv Morgane). Bean plants were submitted to either a short- (24 h) or long-term (7 d) supply of Cd in the nutrient solution. Addition of Cd decreases very significantly both the water and nitrate uptake of the treated plants when compared to untreated plants. Cadmium also induces a decrease in nitrate reductase (NR, EC 1.6.6.1) activation state after 24 h of exposure whereas, after 7 d, NR activation state was similar to that of the control bean plants. On the other hand, the level of NR protein was decreased by about 80 % after 7 d of Cd exposure and by only 15 % 1 d after Cd addition. We then investigated the in vitro effect of Cd on NR catalytic activities and inactivation by phosphorylation. The molybdenum cofactor-binding domain of NR seemed to be the most affected by Cd which did not interfere with the in vitro inactivation process of NR by MgATP. Glutamate synthase and NR activities were more inhibited by supply of Cd in the long-term experiment than the activities of nitrite reductase and glutamine synthetase. Conversely, an increase in glutamate dehydrogenase activity was observed in parallel with an increase in ammonium concentration. It thus appears that Cd treatment induces both rapid and long-term changes in the activity of the enzymes involved in nitrate assimilation, partly in response to reduced water and nutrient uptake. Moreover we have also shown that Cd itself can have a direct effect on the activities of these enzymes.

The activation state of nitrate reductase is not always correlated with total nitrate reductase activity in leaves

The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRA(max)) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRA(max) and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRA(max) and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRA(max) and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRA(max) was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRA(max) in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NR(max) by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRA(max).

Photosynthetic Post-Translational Activation of Nitrate Reductase

Experiments with barley ( Hordeum vulgare ) leaf segments were undertaken to elucidate the signal transduction chain linking photosynthesis in the chloroplasts and the NR activating system in the cytosol where NR (EC 1.6.6.1) is located. Fructose-1,6-bisphosphate phosphatase (FBAse) was used as a marker enzyme to reveal the reduction state of the ferredoxin pool, which possibly could be a link in the signal transduction chain. Photosynthesis in the presence of NaHCO 3 or inhibition of the Calvin cycle by DL-glyceraldehyde resulted in high activity of FBAse as well as NR. However, other treatments, i.e. illumination in the absence of NaHCO 3 , activated FBAse only; therefore, a reduced ferredoxin pool was not sufficient for triggering the activation of NR. Inhibitors known to induce an oxidized or reduced plastoquinone pool, respectively, both induced a low NR activity state, showing that different redox states of the plastoquinone pool did not alter the NR activity state. Infiltration of sucrose or glucose into the leaf slices resulted in activation of NR in the dark, indicating that sugars or sugar derivatives could be a link between photosynthesis and NR activation. This was also supported by testing a maize ( Zea mays ) mutant lacking Calvin cycle activity. In this mutant, light activation of NR was absent. On the other hand high Calvin cycle activity was not always necessary for light activation, because in the presence of DL-glyceraldhyde CO 2 -fixation was reduced by 97%; however, NR was still activated. Side effects of DL-glyceraldehyde may mask the effect of Calvin cycle inhibition. The ambivalent evidence for involvement of the Calvin cycle in NR activation points to the complexity of the activating system and likely existence of multiple signal transduction pathways influencing the NR activity state.

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.

Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status.

The NADH-dependent nitrate reductase (NR, EC 1.6.6.1) in roots of hydroponically grown barley seedlings was extracted, desalted and the activity measured in buffer containing either Mg2+ (10 mM) or EDTA (5 mM). The former gives the actual NR activity (NRact) equivalent to dephospho-NR, whereas the latter gives the maximum NR capacity of the dephospho-form (NRmax). Both values together permit an estimation of the NR-phosphorylation state. Changes in NRact and NRmax were followed in response to root aeration or to shoot illumination or shoot removal, and were correlated with sugar contents and adenylate levels. Ethanol formation was also measured in roots differing in NR activity in order to obtain information on the relation between anaerobic alcoholic fermentation and nitrate reduction. In aerated roots, NR was highly phosphorylated (about 80%) and largely inactive. It was partly dephosphorylated (activated) by anoxia or by cellular acidification (pH 4.8 plus propionic acid). Anaerobic activation (dephosphorylation) of NR was stronger at acidic external pH (5) than at slightly alkaline pH (8), although ATP levels decreased and AMP levels increased at pH 5 and at pH 8 to the same extent. Thus, rapid changes in the NR-phosphorylation state in response to anaerobiosis were not directly triggered by the adenylate pool, but rather by cytosolic pH. Under prolonged darkness (24 h) or after shoot removal. NRmax decreased slowly without a large change in the phosphorylation state. This decrease of NRmax was correlated with a large decrease in the sugar content, and was prevented by glucose feeding, which had only minor effects on the phosphorylation state. Cycloheximide also prevented the decrease in NRmax without affecting the phosphorylation state. In contrast, anaerobiosis or cellular acidification prevented the decrease of NRmax and at the same time decreased the NR-phosphorylation state. It is suggested that NR turnover in roots is controlled by several factors: NR synthesis appears to depend on sugar availability, which has little effect on the phosphorylation state; in addition, NR degradation appears to be strongly affected by the phosphorylation state in such a way that the inactive phospho-NR is a better substrate for NR degradation than the dephospho-form. The rate of anaerobic ethanol formation was not affected by NR activity, indicating that the purpose of NR activation under hypoxia or anoxia is not to decrease or prevent alcoholic fermentation.

Differential response of nitrate reductase and sucrose‐phosphate synthase‐activation to inorganic and organic salts, in vitro and in situ

Studies were conducted to compare the modulation of β‐nicotinamide adenine dinucleotide (NADH): nitrate reductase (NR; EC 1.6.6.1) and sucrose‐phosphate synthase (SPS: EC 2.4.1.14) with respect to regulation by the inorganic anions, phosphate (P1), sulfate and tungstate. Following inactivation of both enzymes in vivo by transferring spinach plants (Spinacia oleracea L. cv. Bloomsdale) to a darkened growth chamber, spontaneous reactivation occurred in vitro when desalted leaf extracts were preincubated at 25°C prior to assay. All three inorganic anions inhibited SPS activation in vitro and also reduced the light activation of SPS in situ when they were fed to excised leaves via the transpiration stream. As expected, feeding tungstate to excised leaves prevented the light‐dependent increase in extractable NR activity. However, in contrast to SPS, the light activation of NR in situ was relatively unaffected by Pi, and sulfate, and in vitro, both anions stimulated (rather than inhibited) the reactivation of NR. Part of the stimulation by Pi and sulfate was the result of increased ionic strength, and stimulation could also be demonstrated with other inorganic and organic salts. In the presence of high ionic strength (0.1 to 0.2 M KCl) the rate of NR activation in vitro was relatively constant when the pH of the preincubation medium was varied from pH 6.5 to 8.0, whereas in the absence of added salt the rate of activation was nearly zero at pH 6.5 but increased progressively as pH was raised. The stimulation by salts could be reversed, in part, by glycerol and ethylene glycol suggesting that hydrophobic interactions might play some role in the activation of NR.

Involvement of CO2 fixation products in the light‐dark modulation of nitrate reductase activity in barley leaves

Nitrate reductase (NR, NADH:nitrate oxidoreductase, EC 1.6.6.1) activity from leaves of barley (Hordeum vulgare L. cv. Hassan) is rapidly and reversibly inactivated during a light‐dark transition. A hyperbolic correlation exists between in vivo rates of CO2 fixation and extractable NR activity from the leaves, and feeding hexose and hexosephosphate protects against the dark‐inactivation; indicating that carbon‐assimilation products are regulatory factors of NR activity mediating both the light‐dark modulation and its dependence upon CO2 fixation. To corroborate this point, the effect of inhibiting CO2 fixation on NR activity in barley leaves has been analyzed. Glycolaldehyde (50 mM), an inhibitor of the regeneration phase of the Calvin cycle, was fed through the transpiration stream and inhibited CO2 fixation by more than 80% at the same time as it produced a parallel inhibition of NR light‐activation. Feeding mannose (10 mM), inhibited CO2 fixation by 35% but did not affect NR activity in illuminated leaves and completely protected against dark‐inactivation. Interestingly, feeding inorganic phosphate, Pi, (10 mM) alone or together with mannose also protected NR activity against dark‐inactivation. The mannose effect could be interpreted in terms of accumulation of mannose 6‐phosphate, an analog of glucose 6‐phosphate. After feeding either 10 mM glucose or dihydroxyacetone phosphate, NR activity from darkened leaves was significantly higher than that of darkened control leaves fed with water (P< 0.03). These treatments, as well as Pi feeding, also produce some increase in extractable NR activity from illuminated leaves. The results indicate that factors increasing the levels of hexose‐ and triose‐phosphate have positive effects on NR activation, supporting the contention that the NR activation system is sensitive to carbon‐assimilation products.

Role of light and CO2 fixation in the control of nitrate-reductase activity in barley leaves

Nitrate reductase (NR, NADH:nitrate oxidoreductase, EC 1.6.6.1) from barley (Hordeum vulgare L. cv. Hassan) leaves was inactivated during a light-dark transition, losing approx. 50% of activity after 30 min of darkness. The dark inactivation was reversed by illumination of the seedlings, the kinetics of reactivation being similar to those of inactivation. High extractable NR activity and significant differences between illuminated and darkened leaves were observed in media containing EDTA and inorganic phosphate (Pi). Addition of Ca2+ ions during extraction and assay decreased NR activity from illuminated and darkened leaves, enhancing the light-dark difference. While no clear correlation could be found between irradiance and NR activity, a hyperbolic correlation appeared between extractable NR activity and in-vivo rates of CO2 fixation, indicating that NR activation follows saturation kinetics with respect to CO2 fixation. Furthermore, hexoses and hexose-phosphates fed to the leaves via the transpiration stream protected against the dark-inactivation of NR. The results indicate that carbon-assimilation products are regulatory factors of NR activity in barley leaves, mediating both the light-dark modulation of NR and its dependence upon CO2 fixation.