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Effects of water deficit on the activity of nitrate reductase and contents of sugars, nitrate and free amino acids in the leaves and roots of sunflower and with lupin plants growing under two nutrient supply regimes
Abstract
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.
... NR activity (NRA) in situ is highly regulated by transcription factors and reversible phosphorylation, which determine its level of activation (Solomonson and Barber 1990;Lillo et al. 2004;Lea et al. 2006), which also depends on environmental factors such as light, CO 2 , oxygen, ATP, pH, and respiration inhibitors (Kaiser et al. 1999;Lea at al. 2006;Sánchez et al. 2009). The availability of NO 3 and the intracellular ammonium (NH 4 ? ) concentration regulate the synthesis and activation of NR (Solomonson and Barber 1990;Chen et al. 2004;Correia et al. 2005;Miller et al. 2007;Nicodemus et al. 2008;Dias et al. 2011). Moreover, photosynthetic activity is also able to activate 60-80 % of the enzyme (Kaiser and Brendle-Behnisch 1991). ...
... The NR is found in all plant organs (Bar-Akiva and Sagiv 1967; Carelli et al. 1990), with NO 3 reduction preferentially occurring in leaves. Nevertheless, higher NRA can be found in roots, which depends on specie, tissue age and type, environmental conditions, NO 3 availability, and plant ontogenetic state (Bar-Akiva and Sternbaum 1965;Bar-Akiva and Sagiv 1967;Chen et al. 2004;Correia et al. 2005;Oliveira et al. 2005). Therefore, it is necessary to clearly define the plant ontogenetic state and the sampled tissue to avoid bias when making comparisons. ...
... Plants differ in tissue localization of nitrate reduction and assimilation. Generally, substantial proportion of nitrate assimilation occurs in shoots of tropical and subtropical species (Andrews 1986), as found in peach palm (Oliveira et al. 2005) and sunflower (Correia et al. 2005). Nevertheless, the relative contribution of roots and shoots to NO 3 reduction is changed with environmental conditions, nutritional status, and plant age (Andrews et al. 1986;DaMatta et al. 1999;Dias et al. 2011). ...
The aim of this study was to assess factors that can affect the in vivo and in vitro assays of the nitrate reductase activity (NRA) in sweet orange trees and then standardize conditions for tissue sampling and analysis. Seven-month-old plants grown in pots were used, of which, mature and healthy leaves between the third and the fifth position from the branch apex, stems, and roots were assessed. One half of each leaf was used for in vivo tests, and the other half was used for in vitro tests. In addition to varied incubation time and temperature, in vivo KNO3 and n-propanol concentrations and in vitro KNO3 and NADH concentrations were evaluated. The optimum conditions for the in vivo NRA assay in leaves were: 200 mmol L−1 KNO3 and 1 % n-propanol at 40 °C for 20 min. The highest leaf NRA occurred at 11:00 h for the in vivo assay and at 13:00 h for the in vitro assay, with both analysis showing similar results. Overestimation of the in vitro NRA occurred as compared to the in vivo analysis when accessed early morning and late afternoon. Branches bearing fruits show reduced NRA in young mature leaves, whereas sprouting significantly increases NRA in correspondent leaves. For the root assays, the optimized conditions for the NRA estimation were the same as for leaves. Although roots and stems (bark) have shown some NRA, it was six times lower than leaf NRA. Our data indicate that NO3
− reduction occurs mostly in leaves and there is a significant effect of daytime and leaf position in relation to fruit or sprouts on NRA of citrus trees.
... During water deficit, NRA decreases more rapidly than most enzymes (Huffaker et al.,1970) and often presents more sensitive physiological indicators of water stress than either stomatal closure or photosynthesis (Bardzik et al., 1971;Hsiao et al., 1976). The negative effect of water deficit on the activity of this enzyme may result from decreased nitrate reductase protein or decreased activation of the existing protein (Correia et al., 2005). In our experiment, compared to the control, NRA appears to vary significantly in the leaves of family 2 in response to drought treatment. ...
... Decreases of stomatal conductance recorded in family 2 can be attributed to the inability of stomata to completely re-open (Kozlowski, 1982) or to the structural changes in stomata (Gallé and Feller, 2007). The increase of NRA after the 7-day period indicated an ongoing recovery process in the stressed plants, which is consistent with previous studies (Bardzik et al., 1971;Ferrario-Méry et al., 1998;Correia et al., 2005). ...
Pedunculate oak (Quercus robur L.) is economically and ecologically one of the most important tree species in lowland forests of Southeastern Europe, and it is endangered by numerous biotic and abiotic factors. In this study, we investigated the effect of drought and herbivore attack of gypsy moth (Lymantria dispar L.) on two families of young oak seedlings subjected to the following treatments: drought (D); gypsy moth (GM); both drought and gypsy moth (D+GM) and control (Ø) for a period of 15 days followed by a 7-day recovery period. During both treatment and recovery, physiological parameters-net photosynthesis (A), transpiration (E), stomatal conduct-ance (gs), sub-stomatal CO 2 concentration (Ci), water use efficiency (WUE), nitrate reductase activity (NRA) and chlorophyll content (Chl) were measured. Our results showed significant effects of stress factors on physiological processes in oak seedlings which could have potential impact on forest regeneration. Also, differences in the reaction between investigated families indicated the need for breeding and selection of more resistant progenies and provenances of pedunculate oak.
... In several prairie grasses, a close correlation existed between foliar N concentration and photosynthesis in response to water stress and recovery once water was restored (Heckathorn et al., 1997). Study of sunfl ower (Helianthus annuus L.) revealed that root NO 3 -concentration decreased during soil drying (Correia et al., 2005). Usually, nitrate reductase [NR (EC 1.6.6.1)] ...
... The foliar NO 3 -concentration was extraordinarily low when compared with that in roots of both drought-stressed and control apple plants in this study (Fig. 2). This differs from herbaceous plants such as maize (Zea mays L.), wheat (Triticum aestivum L.), and lettuce (Lactuca sativa L.), which have shown higher foliar NO 3 -concentrations (Correia et al., 2005; Ferrario-Méry et al., Foyer et al., 1998). In these species, NO 3 -can accumulate to a high concentration in the vacuoles of leaf cells as a result of the shortage of carbon skeletons for synthesis of nitrogen-containing organic compounds (Demšar et al., 2004). ...
Half or whole root systems of micropropagated 'Gala' apple (Malus xdomestica Borkh.) plants were subjected to drought stress by regulating the osmotic potential of the nutrient solution using polyethylene glycol (20% w/v) to investigate the effect of root drying on NO3(-) content and metabolism in roots and leaves and on leaf photosynthesis. No significant difference in predawn leaf water potential was found between half root stress (HRS) and control (CK), while predawn leaf water potential from both was significantly higher than for the whole root stress (WRS) treatment. However, diurnal leaf water potential of HRS was lower than CK and higher than WRS during most of the daytime. Neither HRS nor WRS influenced foliar NO3(-) concentration, but both significantly reduced NO3(-) concentration in drought-stressed roots as early as 4 hours after stress treatment started. This reduced NO3(-) concentration was maintained in HRS and WRS roots to the end of the experiment. However, there were no significant differences in NO3(-) concentration between CK roots and unstressed roots of HRS. Similar to the effect on root NO3(-) concentration, both HRS and WRS reduced nitrate reductase activity in drought-stressed roots. Moreover, leaf net photosynthesis, stomatal conductance and transpiration rate of HRS plants were reduced significantly throughout the experiment when compared with CK plants, but the values were higher than those of WRS plants in the first 7 days of stress treatment though not at later times. Net photosynthesis, stomatal conductance and transpiration rate were correlated to root NO3(-) concentration. This correlation may simply reflect the fact that water stress affected both NO3(-) concentration in roots and leaf gas exchange in the same direction.
... This may be attributed to the negative effects of deficit irrigation on the activity of nitrate reductase (NR), the enzyme governing the reduction of nitrate in plant organs. It may also be attributed to reduced absorption of nitrogen by roots under water stress (Correia et al., 2005). Under water deficit stress generally increased oxalate content of plants (Gouveia et al., 2020). ...
... Since the last two decades, agronomists have focused on choosing nitrogen-efficient cultivars, which means that these plants efficiently use the nitrate reductase (NR) enzyme to convert more nitrate ions from the soil into nitrite. NR is a key regulatory enzyme in this process and directly affected by salt stress (Chandrasekaran et al. 2017;Correia et al. 2005;Sepehr et al. 2012). Under salinity stress activity of NR enzyme decrease rapidly (also show decreased NO 3 − accumulation), but salt stress-tolerant CVS even exhibits enhanced NR activity (Katiyar and Dubey 1992;Rohilla and Yadav 2019). ...
Soil salinity is a global agricultural issue that decreases both plant production and survival. Plants synthesize secondary nitrogen metabolites, which use nitrogen fixed by plants, to withstand salinity stress. Soil salinity is a global agricultural problem that reduces the yield as well as survivability of plants. Plants fix nitrogen by nitrate reductase (NR) which converts nitrate (NO3−) to nitrite (NO2−). Salinity affects NR control differentially and shows both increased and decreased activity. This increase or decrease in activity was due to its control of transcriptional and post-translation (activation). We characterized the NR gene promoter from salt-sensitive and resistant CVS with an in vivo reporter gene (gusA) by raising transgenic rice plants. The major difference in their actions at different salt levels (100 mM, 200 mM, and 300 mM) is verified by qPCR and quantitative expression of gusA. We found a collinear association with previously reported NR activity outcomes between relative transcript number or reporter gene. In addition, sequence analysis of NR promoters reveals that the tolerant CVS promoter (pCNR3) contains a higher number of elements of GATA (cis-regulatory elements, CREs) that are directly responsible for the response to salinity stress. The hypothesis that the promoter of the NR gene has a significant function in the regulation of NR activity under salinity stress is confirmed by the existence of a higher number of salt-sensitive CREs. This study was designed by considering the contrasting salt-sensitive (Jaya and CSR36) cultivars to understand the function of the promoter (transcriptional regulation) (CVS). In addition, this information leads to a stress-responsive promoter of endogenous salinity that can be further used in programs for research and crop improvement.
... Aside from cellular growth roles, N has been found to play a role in protein metabolism, particularly under moisture stress [64,65]. Increased N uptake and the enzyme nitrate reductase, which is involved in nitrogen absorption, have both been linked to increased N availability in plants under water stress [66,67]. Furthermore, foliar application of N fertilisers at the reproductive stage, particularly in leguminous crops under drought conditions, significantly slows abscisic acid synthesis while accelerating cytokinin production, which promotes nodulation, cell elongation, apical dominance, shoot development, photosynthetic activity, and assimilates translocation to the sink organs [68,69]. ...
... Aside from cellular growth roles, N has been found to play a role in protein metabolism, particularly under moisture stress [64,65]. Increased N uptake and the enzyme nitrate reductase, which is involved in nitrogen absorption, have both been linked to increased N availability in plants under water stress [66,67]. Furthermore, foliar application of N fertilisers at the reproductive stage, particularly in leguminous crops under drought conditions, significantly slows abscisic acid synthesis while accelerating cytokinin production, which promotes nodulation, cell elongation, apical dominance, shoot development, photosynthetic activity, and assimilates translocation to the sink organs [68,69]. ...
Citation: Kumari, V.V.; Banerjee, P.; Verma, V.C.; Sukumaran, S.; Chandran, M.A.S.; Gopinath, K.A.; Venkatesh, G.; Yadav, S.K.; Singh, V.K.; Awasthi, N.K. Plant Nutrition: An Effective Way to Alleviate Abiotic Stress in Agricultural Crops. Int. J. Mol. Sci. 2022, 23, 8519.
... Aside from cellular growth roles, N has been found to play a role in protein metabolism, particularly under moisture stress [64,65]. Increased N uptake and the enzyme nitrate reductase, which is involved in nitrogen absorption, have both been linked to increased N availability in plants under water stress [66,67]. Furthermore, foliar application of N fertilisers at the reproductive stage, particularly in leguminous crops under drought conditions, significantly slows abscisic acid synthesis while accelerating cytokinin production, which promotes nodulation, cell elongation, apical dominance, shoot development, photosynthetic activity, and assimilates translocation to the sink organs [68,69]. ...
By the year 2050, the world’s population is predicted to have grown to around 9–10 billion people. The food demand in many countries continues to increase with population growth. Various abiotic stresses such as temperature, soil salinity and moisture all have an impact on plant growth and development at all levels of plant growth, including the overall plant, tissue cell, and even sub-cellular level. These abiotic stresses directly harm plants by causing protein denaturation and aggregation as well as increased fluidity of membrane lipids. In addition to direct effects, indirect damage also includes protein synthesis inhibition, protein breakdown, and membranous loss in chloroplasts and mitochondria. Abiotic stress during the reproductive stage results in flower drop, pollen sterility, pollen tube deformation, ovule abortion, and reduced yield. Plant nutrition is one of the most effective ways of reducing abiotic stress in agricultural crops. In this paper, we have discussed the effectiveness of different nutrients for alleviating abiotic stress. The roles of primary nutrients (nitrogen, phosphorous and potassium), secondary nutrients (calcium, magnesium and sulphur), micronutrients (zinc, boron, iron and copper), and beneficial nutrients (cobalt, selenium and silicon) in alleviating abiotic stress in crop plants are discussed.
... The nitrate assimilation process in eelgrass has been retained through a long evolutionary process, which means that its key enzymes may play a role in eelgrass adaption to the marine environment. Nitrate assimilation is affected by salt stress [8] with the effects of this stress on the expression of NR and NiR in many terrestrial plants being well documented [9,10]. During salt stress, the enzyme activities of NR and NiR in salt-sensitive plants decreased significantly, while the activities increased in salt-tolerant plants. ...
Nitrate reductase (NR) and nitrite reductase (NiR) play important roles in nitrate assimilation in plants. Previous studies have indicated that NR and NiR in eelgrass may contribute to its NaCl tolerance. This study investigated the expression characteristics and the biological functions of NR and NiR in eelgrass (Zostera marina), named as ZmNR and ZmNiR, were cloned, characterised and overexpressed in both bacteria and tobacco. The open reading frames of ZmNR and ZmNiR contain 2628 and 1773 nucleotides that encode 875 and 590 amino acids respectively. Amino acid sequence alignment indicated that the purported ZmNR and ZmNiR proteins presented low homology with other plant NR and NiR sequences. Real-time quantitative PCR revealed that the expression of ZmNR and ZmNiR was supressed when exposed to low salinity and induced by high salinity. Further physiological analyses demonstrated that blocking nitrate assimilation by adding Na2WO4 in eelgrass reduced its tolerance to NaCl stress. The heterologous expression of the ZmNR and ZmNiR genes in Escherichia coli and Nicotiana benthamiana could confer tolerance to NaCl stress. Physiological and growth analyses suggested that ZmNR and ZmNiR in plants could resist NaCl stress by regulating various physiological pathways and biochemical processes triggered by nitric oxide (NO). Taken together, these results suggested that NR-dependent NO synthesis may play an important role in NaCl tolerance in eelgrass.
... Nitrate concentration decreased approximately two times compared to the control plants (Table 2). Decrease in nitrate content under water stress was also observed in other plants [30,31]. Alternatively, applying silicon noticeably improved nitrate supply in wheat shoots, and the increase was 50% comparing to plants without silicic acid added. ...
The study aimed to examine the effect of silicon on spring wheat subjected to drought stress.
The experiment was conducted in hydroponic conditions on 10-day old wheat seedlings. Drought
stress was induced by polyethylene glycol (PEG 6000) at the concentration of 10% and 15% added to
nutrient medium. Silicon was used in the form of silicic acid (H4
SiO4
) at the doses of 1.0 and 1.5 mM.
Silicon evidently improved growth of all plants. Under stress conditions silicon significantly increased
concentration of photosynthetic pigments and lowered malondialdehyde content in leaves. Silicon also
had a positive effect on nitrate supply in leaves. Applying silicon, to some extent influenced proline
concentration in leaves and soluble carbohydrates content in roots.
Concluding, the application of silicic acid improves growth of wheat seedlings and effectively
alleviates the negative effects caused by drought stress.
... Nitrate is the main nitrogen source in agricultural soils and the mineral nutrient that most frequently limits plant growth. This nitrate accumulation has been previously observed in Spartina alterniflora plants under drought stress [80] and could be due to a reduction in the nitrate reductase activity, the first and rate-limiting step enzyme in the nitrate assimilation pathway, which has been observed to decline in many species even under mild water-stress [81]. The decline in root AAT activity (Table 5), another nitrogen metabolism-related enzyme, also agrees with this decrease in root nitrogen metabolism under water-deficit conditions. ...
Water-deficit stresses such as drought and salinity are the most important factors limiting crop productivity. Hence, understanding the plant responses to these stresses is key for the improvement of their tolerance and yield. In this study M. truncatula plants were subjected to 250 mM NaCl as well as reduced irrigation (No-W) and 250 g/L polyethylene glycol (PEG)-6000 to induce salinity and drought stress, respectively, provoking a drop to −1.7 MPa in leaf water potential. The whole plant physiology and metabolism was explored by characterizing the stress responses at root, phloem sap and leaf organ level. PEG treatment led to some typical responses of plants to drought stress, but in addition to PEG uptake, an important impairment of nutrient uptake and a different regulation of carbon metabolism could be observed compared to No-W plants. No-W plants showed an important redistribution of antioxidants and assimilates to the root tissue, with a distinctive increase in root proline degradation and alkaline invertase activity. On the contrary, salinity provoked an increase in leaf starch and isocitrate dehydrogenase activity, suggesting key roles in the plant response to this stress. Overall, results suggest higher protection of salt-stressed shoots and non-irrigated roots through different mechanisms, including the regulation of proline and carbon metabolism, while discarding PEG as safe mimicker of drought. This raises the need to understand the effect at the whole plant level of the different strategies employed to apply water-deficit stress.
... In addition, accumulation of free amino acids and proline are marked response of moisture deficiency in soil as well as in plants. In K0 the increment of free amino acids might be due to the denaturation and hydrolysis of protein by stress induces reactive oxygen species (Zhu 2016), which involved in osmotic adjustment of cellular contents and work as a sink for energy, nitrogen as well as a signal of senescence (Ashraf and Harris 2004;Correia et al. 2005). In fact, protein hydrolysis has been positively correlated with proline synthesis induced by moisture deficit (Hayat et al. 2012). ...
Uncertainty of rainfall intensity and distribution in association with fast depletion of soil moisture content affects sowing time, seed germination, and establishment of lentil in rice-fallows. Mid-season and terminal droughts often retard growth and development of rainfed lentil by affecting its critical growth stages. Delayed sowing further accelerates the moisture stress. A two-year field experiment was conducted in split-plot design with tillage (ZT—zero tillage and CT—conventional tillage) in main plots and strategic K application methods (K0—no potassium, Kb—basal application, Kf—foliar application, and K(b+f)—basal-foliar application) in sub-plots using rainfed lentil (var. B-77) as test crop in rice-fallow system. We assessed the yield, physiological, and biochemical changes of lentil and soil biological properties owing to the different treatments and treatment combination imposed. ZT appeared to have provided ~ 15 and 23.5% higher yield than CT during 2015 and 2016, respectively. Under both tillage practices, Kf and K(b+f) were able to boost lentil yield effectively. Also, soil microbial biomass carbon (MBC) and dehydrogenase activity (DHA) were seen to improve satisfactorily under Kf and K(b+f). Kf under ZT accumulated greatest amount water in leaves (RLWC) and provided the highest leaf area index (LAI), whereas K(b+f) showed similar results in CT. Irrespective of tillage and growth stages, there happened to be a corresponding hike in chlorophyll as well as carbohydrate content by ~ 24.3 and 41.5%, respectively, under K(b+f). In addition, the enhanced concentration of phenol, free amino acids, and proline in lentil apparently suggested suffering of moisture stress in K0 under both the tillage practices over the years. We can conclude that practicing zero tillage along with foliar application of potassium could be an efficient way to escape soil moisture stress sustaining lentil productivity by regulating the biochemical properties and improving physiological aspects of plants at initial stages in rice-fallow system.
... Drought is known to increase apoplastic pH, as shown for field bean (Karuppanapandian et al., 2017), sunflower (Gollan et al., 1992), asiatic dayflower (Commelina communis; Wilkinson and Davies, 1997), or maize (Goodger et al., 2005). Drought also has a significant effect on NRA in some species (Larsson et al., 1989;Azedo-Silva et al., 2004;Correia et al., 2005). Limited availability of NO 3 − as substrate for NRA due to the reduced uptake and transport under drought is one possible explanation for the downregulation of NRA (Azedo-Silva et al., 2004). ...
Soil drying combined with nitrogen (N) deficiency poses a grave threat to agricultural crop production. The rate at which nitrate (NO 3 ⁻ ) is taken up depends partly on the uptake and transpiration of water. Rapid changes in nitrate assimilation, in contrast to other N forms, may serve as a component of the plant stress response to drought because nitrate assimilation may lead to changes in xylem pH. The modulation of xylem sap pH may be relevant for stomata regulation via the delivery of abscisic acid (ABA) to guard cells. In several factorial experiments, we investigated the interactions between nitrate and water availability on nitrate fate in the plant, as well as their possible implications for the early drought-stress response. We monitored the short-term response (2–6 days) of nitrate in biomass, transport to shoot and reduction in Pisum sativum , Hordeum vulgare , Vicia faba , and Nicotiana tabacum and correlated this with sap pH and transpiration rates (TRs). Cultivation on inorganic substrate ensured control over nutrient and water supply and prevented nodulation in legume species. NO 3 ⁻ content in biomass decreased in most of the species under drought indicating significant decline in NO 3 ⁻ uptake. Hordeum vulgare had the highest NO 3 ⁻ concentrations in all organs even under drought and low NO 3 ⁻ treatment. This species can likely respond much better to the combined adverse effects of low NO 3 ⁻ and water scarcity. Nitrate reductase activity (NRA) was reduced in both roots and leaves of water deficient (WD) plants in all species except H. vulgare , presumably due to its high NO 3 ⁻ contents. Further, transient reduction in NO 3 ⁻ availability had no effect on sap pH. Therefore, it seems unlikely that NRA shifts from shoot root leading to the supposed alkalization of sap. We also did not observe any interactive effects of NO 3 ⁻ and water deficiency on transpiration. Hence, as long as leaf NO 3 ⁻ content remains stable, NO 3 ⁻ availability in soil is not linked to short-term modulation of transpiration.
... Two technical replicates were carried out for each sample. To measure soluble sugar content, 1 g of freeze-dried sample was ground into a fine powder in liquid nitrogen and approximately 500 mg of each sample was homogenized with 2 ml of 80% ethanol and incubated for 20 min at 80 °C according to the method described by Correia et al. (2005). The homogenate was then centrifugated at 10,000 rpm for 15 min, the pellet was resuspended in 4 ml ddH 2 O, and filtered through 0.2 mm Whatman filter paper. ...
In this study, the effect of varying nitrogen and sucrose concentrations in culture media was evaluated with respect to biomass production, accumulation of flavonoids, anthocyanin, and associated gene expression in rice callus. The callus was induced on control MS (M1), sugar-deficient (M2), sugar-excessive (M3), nitrogen-deficient (M4), and nitrogen-excessive (M5) media. The results indicated that the callus induction percentage (CIP) as well as the size and fresh weight of the callus were inhibited by all types of media compared with control media. Varying the sucrose and nitrogen concentration significantly affected callus morphology and caused a browning effect. Genes related to flavonoid biosynthesis (CHS, CHI, F3H, FLS and DFR) were upregulated in the callus cultured in all four media types compared with control media. Likewise, flavonoid and anthocyanin accumulation were higher in callus grown in excessive sugar-and nitrogen-containing media compared with control media. Unlike flavonoids, salicylic acid (SA) regulation was significantly higher in callus grown in sugar-and nitrogen-deficient media compared with control media. Sugar content was significantly higher in callus cultured in sugar-excessive media, whereas it was reduced in the callus cultured in the other media types compared with control media. Finally, chlorophyll was reduced in all callus media compared with the control media. Key message The current study concluded that, varying concentration of sugar and nitrogen inhibit callus development and morphology via alteration of secondary metabolites and their related genes.
... ance to WBPH and WBPH rearing") were collected in three replicates, after 10 days of WBPH infestation, at three time points within one week intervals. Approximately 0.5 g lyophilized tissues were ground into fine powder in liquid nitrogen and homogenized with 80% ethanol (2 ml) at 80 °C for 20 min, following previously described methods 47 . The homogenates were pelleted by centrifugation at 10,000 rpm for 15 min. ...
The whitebacked planthopper (WBPH), has become a devastating pest for rice crops, causes serious yield losses each year, and urgently needs biological control. Here, we developed a WBPH-resistant rice cultivar by overexpressing the OsF3H gene. A genetic functional analysis of the OsF3H gene confirmed its role in facilitating flavonoid contents and have indicated that the expression of the OsF3H gene is involved in regulation of the downstream genes (OsDFR and OsFLS) of the flavonoid pathway and genes (OsSLR1 and OsWRKY13) involved in other physiological pathways. OxF3H (OsF3H transgenic) plants accumulated significant amounts of the flavonols kaempferol (Kr) and quercetin (Qu) and the anthocyanins delphinidin and cyanidin, compared to the wild type, in response to the stress induced by WBPH. Similarly, OsF3H-related proteins were significantly expressed in OxF3H lines after WBPH infestation. The present study, indicated that the regulation of JA in OxF3H plants was suppressed due the overexpression of the OsF3H gene, which induced the expression of downstream genes related to anthocyanin. Similarly, the OsWRKY13 transcriptional factor was significantly suppressed in OxF3H plants during WBPH infestation. Exogenous application of Kr and Qu increased the survival rates of susceptible TN1 lines in response to WBPH, while decreased the survival rate of first instar WBPHs, indicating that both flavonols exhibit pesticide activity. Phenotypic demonstration also affirms that OxF3H plants show strong resistance to WBPH compared with wild type. Collectively, our result suggested that OsF3H overexpression led to the up-regulation of defense related genes and enhanced rice resistance to WBPH infestation.
... Generally, the activity of NR is an indicator of a plant's nitrogen status which is very important for its proper growth and development (Srivastava 1980;Loussaert et al. 2010;Foyer and Zhang 2011). NR activity also determines the stress-tolerant capacity of a plant (Ferrario-Méry et al. 1998;Cazetta and Villela 2004;Correia et al. 2005;Sepehr et al. 2012;Chandrasekaran et al. 2017). ...
Salt stress response includes alteration in the activity of various important enzymes in plants. Nitrate reductase (NR) is one of the known enzyme affected by salt stress. In this study, contrasting salt responsive cultivars (CVS) (IR64-sensitive and CSR 36-tolerant) were considered to study the regulation of NR genes under salt stress conditions. Using Arabidopsis genes Nia1 and Nia2, three different NR genes were identified in rice and their expression study was conducted. Under stress condition, salt-sensitive CVS (IR64) showed a decrease in NR activity under in vitro and in vivo conditions, whereas tolerant CVS showed an increase in NR activity. Different trends for NR activity in contrasting genotype are explained by the variable number of GATA element in the upstream region of the NR gene. This variation of NR activity in contrasting CVS further co-relates with the transcript level of NR genes. The transcript level of three different NR genes also evidenced the effect of CREs in gene regulation. Promoter (1-kb upstream region) of different NR genes contained different abiotic stress-responsive CREs, which explain the differential behavior of these genes towards the abiotic stress. Overall, this study concludes the role of CREs in the regulation of NR gene and indicates the importance of transcriptional control of NR activity under stress condition. This is the first type of report that highlights the role of the regulatory mechanism of NR genes under salt stress condition.
... This feature has also been detected in other crops such as barley (Lanzinger et al. 2015) and wheat (Bowne et al. 2012). Conversely, Moschen et al. (2017) found that the concentrations of several leaf amino acids were decreased under DS in sunflower (Correia et al. 2005). These contradictory results regarding amino acid responses might be due to water-stress intensity, sampling stage or differences in nitrogen nutrition. ...
Introduction
Plant and crop metabolomic analyses may be used to study metabolism across genetic and environmental diversity. Complementary analytical strategies are useful for investigating metabolic changes and searching for biomarkers of response or performance.
Methods and objectives
The experimental material consisted in eight sunflower lines with two line status, four restorers (R, used as males) and four maintainers (B, corresponding to females) routinely used for sunflower hybrid varietal production, respectively to complement or maintain the cytoplasmic male sterility PET1. These lines were either irrigated at full soil capacity (WW) or submitted to drought stress (DS). Our aim was to combine targeted and non-targeted metabolomics to characterize sunflower leaf composition in order to investigate the effect of line status genotypes and environmental conditions and to find the best and smallest set of biomarkers for line status and stress response using a custom-made process of variables selection.
Results
Five hundred and eighty-eight metabolic variables were measured by using complementary analytical methods such as ¹H-NMR, MS-based profiles and targeted analyses of major metabolites. Based on statistical analyses, a limited number of markers were able to separate WW and DS samples in a more discriminant manner than previously published physiological data. Another metabolic marker set was able to discriminate line status.
Conclusion
This study underlines the potential of metabolic markers for discriminating genotype groups and environmental conditions. Their potential use for prediction is discussed.
... At the end of the experiment, the A plants, followed by A/A, did not show any NR activity and g s dropped, while A/A25 maintained 64% enzyme activity and sustained g s compared to the control. Some nonstomatal effects, such as reduced nitrate availability in plants, could inhibit NR gene transcription and decrease the stability of NR-mRNAs or post-translational factors, including inactivation through protein phosphorylation, and the induction of proteases could rapidly occur by decreasing NR activity as a result of drought stress (Ferrario et al., 1995;Lillo et al., 2004;Correia et al., 2005). In most herbaceous plants, NO 3 − assimilation takes place predominantly in leaves (Scheurwater et al., 2002). ...
In vegetables, tolerance to drought can be improved by grafting commercial varieties onto drought tolerant rootstocks. Grafting has emerged as a tool that copes with drought stress. In previous results, the A25 pepper rootstock accession showed good tolerance to drought in fruit production terms compared with non-grafted plants and other rootstocks. The aim of this work was to study if short-term exposure to drought in grafted plants using A25 as a rootstock would show tolerance to drought now. To fulfill this objective, some physiological processes involved in roots (rootstock) and leaves (scion) of grafted pepper plants were analyzed. Pepper plants not grafted (A), self-grafted (A/A), and grafted onto a tolerant pepper rootstock A25 (A/A25) were grown under severe water stress induced by PEG addition (-0.55 MPa) or under control conditions for 7 days in hydroponic pure solution. According to our results, water stress severity was alleviated by using the A25 rootstock in grafted plants (A/A25), which indicated that mechanisms stimulated by roots are essential to withstand stress. A/A25 had a bigger root biomass compared with plants A and A/A that resulted in better water absorption, water retention capacity and a sustained CO2 assimilation rate. Consequently, plants A/A25 had a better carbon balance, supported by greater nitrate reductase activity located mainly in leaves. In the non-grafted and self-grafted plants, the photosynthesis rate lowered due to stomatal closure, which limited transpiration. Consequently, part of NO3- uptake was reduced in roots. This condition limited water uptake and CO2 fixation in plants A and A/A under drought stress, and accelerated oxidative damage by producing reactive oxygen species (ROS) and H2O2, which were highest in their leaves, indicating great sensitivity to drought stress and induced membrane lipid peroxidation. However, drought deleterious effects were slightly marked in plants A compared to A/A. To conclude, the A25 rootstock protects the scion against oxidative stress, which is provoked by drought, and shows better C and N balances that enabled the biomass to be maintained under water stress for short-term exposure, with higher yields in the field.
... The stomatal conductance and the assimilation of CO 2 in sunflower leaves reduces with an enhancement in the drought intensity (Correia et al., 2005;Iqbal et al., 2009;Ghobadi et al., 2013); nonetheless a genotypic variability exists for reduction in stomatal conductance and CO 2 assimilation in response to drought stress (Andrianasolo et al., 2016;Iqbal et al., 2009). In a study, Tezara et al. (2002) found an increase in net CO 2 assimilation rate in sunflower seedlings due to elevated CO 2 level with a simultaneous reduction in RuBisCO contents. ...
... The stomatal conductance and the assimilation of CO 2 in sunflower leaves reduces with an enhancement in the drought intensity (Correia et al., 2005;Iqbal et al., 2009;Ghobadi et al., 2013); nonetheless a genotypic variability exists for reduction in stomatal conductance and CO 2 assimilation in response to drought stress (Andrianasolo et al., 2016;Iqbal et al., 2009). In a study, Tezara et al. (2002) found an increase in net CO 2 assimilation rate in sunflower seedlings due to elevated CO 2 level with a simultaneous reduction in RuBisCO contents. ...
... The increase in NI content in the I 75 treatment at the 3 rd crop cycle is consistent with the literature data that show the negative effects of water shortage on NR activity and thus the increase in NI ( Santamaria et al., 1999;Weightman et al., 2012). On other hand, the opposite effect found in the last crop cycle can be attributed to a lower N absorption from the soil by the roots for the effect of increased and prolonged water limitation, which possibly caused reduction in N transport to the leaves through transpiration stream (xylem) ( Correia et al., 2005). However, the NI increase in the I 125 and I 150 treatments, can be at- tributed to possible conditions of root hypoxia due to the excess water, who may have had an adverse effect on N metabolism ( Tamme et al., 2010). ...
In Italy, the cultivation of wild rocket is still rising due to the increase of the market of minimally processed vegetables that requires innovative and high quality products. The present study focused on the interactive effect of irrigation regimes (IR) and nitrogen (N) supply on yield, water use efficiency (WUE), nitrogen use efficiency (NUE), morphological and quality parameters of wild rocket. The research was carried out on four crop cycles during autumn-spring season in Basilicata region, Southern Italy, on wild rocket grown in a plastic greenhouse. Four IR (corresponding to 75, 100, 125 and 150% of crop evapotranspiration, and labelled respectively as I75, I100, I125 and I150) and two N levels (60 and 120 kg ha⁻¹), were compared. The irrigation and N levels affected production traits of wild rocket. The highest yield was obtained by I100, while 8% and 6% decrease in yield was observed with I75 and I150, respectively. The greater yield was obtained with the higher N rate, to which contributed firstly the leaf number and secondly the leaf size. However, the higher N dose provided higher leaf nitrate content. Moreover, in conditions of greater water stress occurring in the last two crop cycles of I75, higher N rate adversely affected yield. Lower water and N supply improved phenols, carotenoids and antioxidant activity in rocket leaves. Both yield and biomass WUE increased in water shortage conditions (I75) at 1st crop cycle. Thereafter, both parameters tended to decrease because of the increase of water shortage, indicating that biomass and marketable yield losses were proportionally greater than the amount of water used by crops. Higher N rate improved WUE, but reduced NUE. The latter parameter was higher in water shortage conditions. Thus, adequate water and N supply are critical factors to ensure economically sustainable production levels and high quality features of wild rocket.
... Drought responses involve physiological and biochemical processes such as changes in gene expression and enzyme activities. Nitrate reductase transcription and enzymatic activity were reported to be reduced in different plants under drought conditions (Ferrario-Méry et al., 1998;Foyer et al., 1998;Correia et al., 2005) and these decreases have been correlated to stomatal closure and consequent lower internal CO 2 concentration (Kaiser and Fòrster, 1989). Therefore, while NO seems to play a crucial role in the control of guard cells movement in well-hydrated tissues, most likely in the stomatal closure occurring during the light/dark transition; it seems not to be required for the closure of stomata during dehydration. ...
The PII protein in plants has been associated to many different tissue specialized roles concerning the Nitrogen assimilation pathways. We report here the further characterization of L. japonicus transgenic lines overexpressing the PII protein encoded by the LjGLB1 gene that is strongly expressed in the guard cells of Lotus plants. Consistently with a putative role played by PII in that specific cellular context we have observed an alteration of the patterns of stomatal movement in the overexpressing plants. An increased stomatal closure is measured in epidermal peels from detached leaves of normally watered overexpressing plants when compared to wild type plants and this effect was by-passed by Abscisic Acid application. The biochemical characterization of the transgenic lines indicates an increased rate of the Nitric Oxide biosynthetic route, associated to an induced Nitrate Reductase activity. The phenotypic characterization is completed by measures of the photosynthetic potential in plants grown under greenhouse conditions, which reveal a higher stress index of the PII overexpressing plants.
... The cultivar N 22 showed higher NR activity of 13.10 with 4.4% reduction at PI and a value of 15.84 with 5.2% decrease over control at anthesis stage followed by TKM 9 with a content of 12.85 with 8.1% decrease at PI and 8.8 % increase over control at anthesis stage ( Figure 2). Correia et al. (2005) stated that, maintenance of NR activity has an imperative role by the tolerant genotypes for and 11.1 respectively. Whereas, N 22 recorded the lowest value of soluble protein content of 13.9 and 12.6 in control and value of 12.9 and 11.5 at PI and anthesis respectively. ...
... Inhibition of NO 3 − assimilation is more significant in leaves which have more difficulties in accessing NO 3 − than that in roots when NO 3 − supply is limited (Hawkesford et al., 2012). As eCO 2 inhibits plant transpiration and further limits N uptake and NO 3 − transportation to leaves (Correia et al., 2005;Jauregui et al., 2016;McDonald et al., 2002), it is possible that the inhibition of NO 3 − assimilation in leaves is greater than that in roots under eCO 2 . However, it lacks evidence indicating the tissue variation of NO 3 − assimilation under eCO 2 . ...
With respect to vegetables with high nitrate accumulation, very little information is available on how elevated CO2 affects their nitrate assimilation. In this study, cucumber (Cucumis sativus L.) plants were hydroponically grown for two stages (the seedling stage and the initial fruit stage) under three levels of CO2 [400 (aCO2), 625 (subeCO2) and 1200 (eCO2), μmol mol⁻¹] with three NO3⁻ concentrations [2 (low NO3⁻), 7 (moderate NO3⁻) and 14 (high NO3⁻), mmol L⁻¹] in open top chambers. Our results showed that subeCO2 had no significant effects on plant growth at either stage, whereas eCO2 increased both plant photosynthesis and biomass, with the increase being greater at the seedling stage. The alleviation of photosynthetic acclimation was not only at the seedling stage, but also in high NO3⁻ treatment indicated by higher net photosynthesis rates and plant biomass, higher C, fructose and glucose concentration in leaves, and lower starch concentration in leaves. The yield of cucumber increased by 73% under eCO2 in high NO3⁻ treatment, with no promotion in moderate NO3⁻ treatment. Our results also found that the alleviation of photosynthetic acclimation accompanied with higher N assimilation. Specifically, eCO2 increased N content of entire seedlings in high NO3⁻ supply and maintained N concentration in leaves. Elevated CO2 increased the ratio of NH4⁺-N to total N in roots more than that in leaves and decreased the ratio of NO3⁻-N to total N in roots greater than that in leaves, which implied that eCO2 probably promoted NO3⁻ assimilation in roots more than that in leaves. Elevated CO2 is more likely to inhibit N assimilation, and then decreases the sink strength, thus limits photosynthate transportation from leaves. Therefore, we suggest more NO3⁻ fertilizer is needed to match CO2 fertilization to enhance cucumber yield.
... Lawlor and Cornic (2002) stated that a water deficit could have a more marked effect on N assimilation than on the uptake process, as nitrate reductase activity diminished sharply, albeit in a reversible fashion, in line with low relative water content values. Other authors have also pointed out the particular sensitivity of nitrate reductase activity (Triboi-Blondel, 1978;Larsson et al., 1989;Larsson, 1992;Azedo-Silva et al., 2004;Correia et al., 2005). ...
Among the environmental factors that can be modified by farmers, water and nitrogen are the main ones controlling plant growth. Irrigation and fertilizer application overcome this effect, if adequately used. Agriculture thus consumes about 85% of the total fresh water used worldwide. While only 18% of the world's cultivated areas are devoted to irrigated agriculture, this total surface represents more than 45% of total agricultural production. These data highlight the importance of irrigated agriculture in a framework where the growing population demands greater food production. In addition, tighter water restrictions and competition with other sectors of society is increasing pressure to diminish the share of fresh water for irrigation, thus resulting in the decrease in water diverted for agriculture. The effect of water and nutrient application on yield has led to the overuse of these practices in the last decades. This misuse of irrigation and fertilizers is no longer sustainable, given the economic and environmental costs. Sustainable agriculture requires a correct balance between the agronomic, economic and environmental aspects of nutrient management. The major advances shown in this review are the following: (1) the measurement of the intensity of drought and N deficiency is a prerequisite for quantitative assessment of crop needs and management of both irrigation and fertilizer application. The N concentration of leaves exposed to direct irradiance allows both a reliable and high-resolution measurement of the status and the assessment of N nutrition at the plant level. (2) Two experiments on sunflower and on tall fescue are used to relate the changes in time and irrigation intensity to the crop N status, and to introduce the complex relationships between N demand and supply in crops. (3) Effects of water deficits on N demand are reviewed, pointing out the high sensitivity of N-rich organs versus the relative lesser sensitivity of organs that are poorer in N compounds. (4) The generally equal sensitivities of nitrifying and denitrifying microbes are likely to explain many conflicting results on the impact of water deficits on soil mineral N availability for crops. (5) The transpiration stream largely determines the availability of mineral N in the rhizosphere. This makes our poor estimate of root densities a major obstacle to any precise assessment of N availability in fertilized crops. (6) The mineral N fluxes in the xylem are generally reduced under water deficit and assimilation is generally known to be more sensitive to water scarcity. (7) High osmotic pressures are maintained during grain filling, which enables the plant to recycle large amounts of previously assimilated N. Its part in the total grain N yield is therefore generally higher under water deficits. (8) Most crop models currently used in agronomy use N and water efficiently but exhibit different views on their interaction.
... Reduced mobility due to drought is particularly important for nitrate ions, which are transported from the bulk soil to the root surface mainly through mass flow (Marschner, 1995). Besides limiting the acquisition of nitrate by roots (BassiriRad and Caldwell, 1992), drought may also restrict the ability of plants to reduce and assimilate nitrogen as a result of inhibition of the activities of enzymes involved in nitrogen metabolism (Correia et al., 2005). Thus, the lower crude protein content (i.e., low nitrogen content) could be the result of one or more effects of drought stress on nitrogen mobility, uptake and assimilation. ...
Enset [Ensete ventricosum (Welw.) Cheesman] is an important root crop serving as a
carbohydrate rich food source in Ethiopia. Perennial crops, like enset, are often exposed to recurrent dry
periods which could greatly affect their growth, physiology and yield. The effect of induced
drought/irrigation on the proximate composition and carbohydrate content of harvestable plant parts
(pseudostem and corm) of two field grown enset clones (Ameratye and Yesherakinkye) was investigated.
Proximate analysis showed that extended drought significantly (P £0.05) reduced crude protein, ash
content, potassium and phosphorus contents of enset pseudostem and corm of the two clones. On the
other hand, crude fibber content and calcium levels were significantly higher in droughted groups than
irrigated ones. There were no significant treatment effects on crude fat content and /or magnesium
levels. Moreover, drought resulted in a significant increase in the level of soluble sugars and a decline in
starch content of plant parts in both clones. Droughted plants accumulated 2–4 fold more soluble
sugars than irrigated groups while the latter group gained 14–23% more starch. There was no
significant difference between the two clones for the parameters considered. Low nutrient content of
droughted plants could be the result of reduced nutrient uptake and assimilation caused by low soil
water content and limited energy source (carbon skeleton). The observed increase in soluble sugars
could be an indication of osmotic adjustment mechanisms in droughted enset plants. On the other
hand, the decline in starch content could be due to reduced carbon assimilation and/or starch
degradation.
Key words/phrases: Carbohydrates, clones, drought, enset, proximate composition
... In relation to this, NR and GS activities also decreased under water deficit, salinity and in combination, but overall, tolerant genotypes exhibited higher GS and NR activities. A number of studies have reported decreased NR activity in response to water stress (Foyer et al., 1998;Correia et al., 2005) and salinity (Wang et al., 2007;Carillo et al., 2008). Salt was observed to inhibit nitrate transport to the leaf mainly because of nitrate ⁄ chloride competition, consequently affecting NR activity (Rao & Gnanam, 1990;Abd-El Baki et al., 2000). ...
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... Nitrogen uptake of the root system is reduced by water scarcity (Azedo-Silva et al., 2004;Correia et al., 2005;Gonzalez-Dugo et al., 2010;Larsson, 1992;Pandey et al., 2000;Pirmoradian et al., 2004). This could be due to a lower enzyme activity in nitrogen assimilation by the root system. ...
Cover cropping is developing in vineyards as it may induce numerous ecosystem services. However, soil cover also competes with grapevine for soil resources. This work aimed at evaluating both the period and intensity of the resulting water and nitrogen stress experienced by grapevine by comparing treatments with bare soil, permanent and non-permanent soil cover. The adopted stress indices were the Fraction of Transpirable Soil Water (FTSW) and the Nitrogen Nutrition Index (NNI).Cover cropping improved winter soil water refilling, but the grass transpiration during spring later led to similar water stress of grapevine among treatments. In dry years, summer water stress was not higher with non-permanent cover than with bare soil. The building of a critical nitrogen content curve for grapevine enabled to diagnose an early nitrogen stress, from budbreak to flowering, in cover cropped treatments. NNI dropped then in dry years, particularly in cover cropped treatments.The early growth limitation of grapevine observed in cover cropped treatments was the consequence of mild early nitrogen stress, which suggests that perennial nitrogen reserves were reduced because of an earlier competition with cover crop. After grapevine flowering, water appeared to be the most limiting factor for both grapevine growth and nitrogen availability.In water limited environment, nitrogen stress is highly dependant on water constraints. However, this work reveals the partial uncoupling of the dynamics of water and nitrogen stress during the grapevine cycle in water-limited cropping systems, which highlights the relevance of a co-ordinated management of water and nitrogen.
... Drought stress caused a marked decrease in NRA and the leaves' nitrate content. Similar results under water deficit were reported earlier in Helianthus annuus L., Lupinus albus L. (Correia et al., 2005), and Phaseolus vulgaris and Sesbania aculeata (Ashraf and Iram, 2005). Lobato et al. (2008) noted that NRA had an accumulated reduction of 70% after 6 days of water deficit and that rehydration promoted an increase of 96% in this parameter. ...
To assess the effect of NaCl on seed germination and early seedling growth of four Moroccan alfalfa (Medicago sativa L.) populations, five salt treatments (0, 50, 100, 150 and 200 mM) were applied during germination in Petri dishes at 25°C±1 for 8 days. Germination
(%) and germination velocity index were progressively inhibited as NaCl concentration increased with significant differences between populations. Thus, Tafilalet 1 population was less tolerant to NaCl but Tata population was more tolerant. The use of isotonic mannitol solutions indicated that
salinity effect on seed germination occurred by both an osmotic and an additional ion toxicity specific effect which caused an inhibition of seed reserve mobilization. Agro-physiological parameters were also assessed in the seedling. A salt treatment (0, 100 and 200 mM NaCl) was applied 14
days after seeds were sown in pots in a peat/sand mix and assessments of seedlings were taken 15 days later. Seedlings showed a reduction in growth parameters, and accumulation of proline, soluble sugars and inorganic ions with a clear difference between the populations particularly at the
highest NaCl concentration. This higher accumulation of solutes during salt stress was related to salt tolerance.
... Drought stress caused a marked decrease in NRA and the leaves' nitrate content. Similar results under water deficit were reported earlier in Helianthus annuus, Lupinus albus L. (Correia et al., 2005), and Phaseolus vulgaris and Sesbania aculeata (Ashraf and Iram, 2005). Lobato et al. (2008) noted that NRA had an accumulated reduction of 70% after 6 days of water deficit and that rehydration promoted an increase of 96% in this parameter. ...
The effect of water deficit on growth and some physiological and biochemical parameters related to water deficit tolerance was studied in 4 Moroccan alfalfa Medicago sativa L. populations that originated from the mountains and oases of Morocco. The experiment was conducted in greenhouse conditions. Seeds were allowed to germinate in pots filled with sand and peat at a 2:1 ratio, respectively. After 1 month of sowing, the plants were subjected to 3 water regimes: optimal irrigation (75% of field capacity, FC), moderate water deficit (50% FC), and severe water deficit (25% FC). At 45 days of stress, the plants were harvested and subjected to some agrophysiological and biochemical analyses related to tolerance. Results showed that the water deficit negatively affected dry biomass, membrane permeability, leaves' relative water contents, and nitrate and phosphorus contents. The behaviors of tested populations were significantly different for most of the considered parameters. The tolerance was positively correlated to the ability of plants to overcome oxidative stress by the induction of antioxidant enzyme activity, accumulation of inorganic ions (Na+ and K+), and maintenance of an adequate level of nitrate reductase and acid phosphatase activities.
... Lawlor and Cornic (2002) stated that a water deficit could have a more marked effect on N assimilation than on the uptake process, as nitrate reductase activity diminished sharply, albeit in a reversible fashion, in line with low relative water content values. Other authors have also pointed out the particular sensitivity of nitrate reductase activity (Triboi-Blondel, 1978;Larsson et al., 1989;Larsson, 1992;Azedo-Silva et al., 2004;Correia et al., 2005). ...
Among the environmental factors that can be modified by farmers, water and nitrogen are the main ones controlling plant growth.
Irrigation and fertilizer application overcome this effect, if adequately used. Agriculture thus consumes about 85% of the
total fresh water used worldwide. While only 18% of the world’s cultivated areas are devoted to irrigated agriculture, this
total surface represents more than 45% of total agricultural production. These data highlight the importance of irrigated
agriculture in a framework where the growing population demands greater food production. In addition, tighter water restrictions
and competition with other sectors of society is increasing pressure to diminish the share of fresh water for irrigation,
thus resulting in the decrease in water diverted for agriculture.The effect of water and nutrient application on yield has
led to the overuse of these practices in the last decades. This misuse of irrigation and fertilizers is no longer sustainable,
given the economic and environmental costs. Sustainable agriculture requires a correct balance between the agronomic, economic
and environmental aspects of nutrient management. The major advances shown in this review are the following: (1) the measurement
of the intensity of drought and N deficiency is a prerequisite for quantitative assessment of crop needs and management of
both irrigation and fertilizer application. The N concentration of leaves exposed to direct irradiance allows both a reliable
and high-resolution measurement of the status and the assessment of N nutrition at the plant level. (2) Two experiments on
sunflower and on tall fescue are used to relate the changes in time and irrigation intensity to the crop N status, and to
introduce the complex relationships between N demand and supply in crops. (3) Effects of water deficits on N demand are reviewed,
pointing out the high sensitivity of N-rich organs versus the relative lesser sensitivity of organs that are poorer in N compounds.
(4) The generally equal sensitivities of nitrifying and denitrifying microbes are likely to explain many conflicting results
on the impact of water deficits on soil mineral N availability for crops. (5) The transpiration stream largely determines
the availability of mineral N in the rhizosphere. This makes our poor estimate of root densities a major obstacle to any precise
assessment of N availability in fertilized crops. (6) The mineral N fluxes in the xylem are generally reduced under water
deficit and assimilation is generally known to be more sensitive to water scarcity. (7) High osmotic pressures are maintained
during grain filling, which enables the plant to recycle large amounts of previously assimilated N. Its part in the total
grain N yield is therefore generally higher under water deficits. (8) Most crop models currently used in agronomy use N and
water efficiently but exhibit different views on their interaction.
... The literature also presents controversy on the contribution of roots to nitrate reduction. For instance, in Lotus japonicum nitrate assimilation takes place predominantly in the roots (Pajuelo et al. 2002), whereas in Dactylis glomerata and Festuca ovina the main site for nitrate reduction in vivo was shown to be the leaves with very little in the roots (Scheurwater et al. 2002). in non-nodulated Lupinus albus plants, nitrate accumulation was reduced in response to a low nutrient supply regime whereas under drought conditions nitrate accumulation was not affected (Correia et al. 2005). Nr activity was also shown to be equivalent between leaves and roots of L. albus, contrary to what was reported by Pate et al. (1979). ...
In Jack bean (Canavalia ensiformis), nitrogen metabolism was shown to be drastically altered when different development stages were compared. During development, a change in the location of nitrate reduction and alterations in the relative concentrations of amino acids were observed. Nitrate reductase activity in the roots of C. ensiformis increased during the growth of the plants, being highest at the stage of seed production, whereas leaf nitrate reductase activity showed the reverse pattern, being highest at the vegetative stage and lowest at seed production.
... Dehydration adversely affected the activity of nitrate reductase in roots of sunflower (Azedo Silva et al. 2004) and in wheat ( Larsson et al., 1989), whereas contrasting results were observed in roots of maize and no effect of dehydration on nitrate reductase activity was recorded (Abd-El Baki et al., 2000). Correia et al. (2005) reported that the activity of nitrate reductase (NR; EC 1.6.6.6) in Helianthus annuus L. and non-nodulated Lupinus albus L. was negatively affected by soil drying and a decreased supply of nutrients and the observed changes in NR activity being linearly correlated with the depletion of nitrate. Possible mechanisms to minimize the detrimental effects of drought by improving water use efficiency with N nutrition were described by Waraich et al (2011) .Inorganic fertilization has been reported to mitigate the adverse effects of water stress on crop growth and development (Marschner, 1995;Payne et al., 1995;Raun and Johnson 1999). ...
Water, the most important component of life, is rapidly becoming a critically short commodity for humans and crop production. Limited water supply is one of the major abiotic factors that adversely affect agricultural crop production worldwide. Drought stress influences the normal physiology and growth of plants in many ways. It results in an increase of solute concentration outside the roots compared to the internal environment of the root and causes reverse osmosis. As a result, the cell membrane shrinks from the cell wall and may eventually lead to death of the cell. Water stress tends to shrink away from the interface with water-absorbing roots, creating a gap in the soil-plant-air continuum. As the plant continues to lose water via transpiration, water is drawn from root cells resulting in shrinkage of cell membranes and results in decreased integrity of the cell membrane and the living cell may be destroyed. Drought stress inhibits photosynthesis in plants by closing stomata and damaging the chlorophyll contents and photosynthetic apparatus. It disturbs the balance between the production of reactive oxygen species (ROS) and the antioxidant defence, causing accumulation of ROS which induces oxidative stress to proteins, membrane lipids and other cellular component. Mineral elements have numerous functions in plants including maintaining charge balance, electron carriers, structural components, enzyme activation, and providing osmoticum for turgor and growth .In this paper, an overview of some macronutrients (nitrogen, phosphorus, potassium, calcium and magnesium), micronutrients (Zinc, Boron, Copper) and silicon has been discussed in detail as how these nutrients play their role in decreasing the adverse effects of drought in crop plant.
... Water deficit has an important indirect effect on nutrient uptake, which may be as important as its effect on growth (Pugnaire et al., 1999). Maybe because of that, N content in the plant is reduced under water deficit (Dejong and Phillips, 1982;Mahieu et al., 2009) and the activity of NR is also depressed (Correia et al., 2005;Fresneau et al., 2007;Pugnaire et al., 1999). ...
Background and Objective: Drought is one of the main limitations of rice (Oryza sativa L.) productivity and is a severe problem in many
regions of the world. In Bangladesh, drought affects approximately 11.54 million ha of the area under rice cultivation and usually occurs
during the reproductive stage. Responses of six rice genotypes subjected to different drought levels (40% FC) and control (100% FC) were
investigated in the laboratory to evaluate drought tolerance mechanism(s) based on osmolytes at the reproductive stage in rice
genotypes. Materials and Methods: Twelve treatments (6 genotypes×2 irrigations) were arranged in CRD and the experiment was
carried out at Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh, Bangladesh, in two seasons. Drought was imposed at the
reproductive stage where important biochemical traits of flag leaf at 75 days after transplanting were estimated. Results: During drought
stress, proline and sugar accumulation increased and nitrate reductase activity reduced intolerant genotypes compared to sensitive ones.
Proline and total sugar significantly increased under drought and nitrate reductase activity reduced during the drought which
discriminated two genotypes consistently as drought tolerant. Conclusion: Due to increase osmolytes accumulation (proline and total
soluble sugar) and decrease osmolyte like nitrate reductase activity of flag leaf at the reproductive stage, it may be concluded that the
solute accumulation is one of the mechanisms for drought tolerance in rice genotypes.
Key words: Rice, drought stress, nitrate reductive activity, proline, total soluble sugar, reproductive stage
Drought causes not only the decrease of tobacco yield and quality, but also the lowering of net photosynthetic rate, leading to reactive oxygen species accumulation and even the death of plants. Salicylic Acid is involved in regulating many plant physiological processes and has increasingly been applied to improve tolerance in plants exposed to drought stress. To explore the regulating mechanism of SA, flue-cured tobacco K326 was used in the hydroponic experiments to design PEG drought stress. The photosynthetic characteristics, antioxidant enzymes activities and osmotic regulatory substances contents of tobacco seedlings under drought stress were investigated after 0.3 mmol L ⁻¹ SA treatment. Transcriptome sequencing and GO/KEGG analysis were also performed. The main results showed that SA-applied greatly increased the activities of SOD, POD, CAT activity, Pn, proline and soluble protein by 44.27%, 50.18%, 26.23%, 45.74%, 34.67% and 24.91% while reduced the MDA content by 23.89%. GO and KEGG analysis showed that SA treatment was able to up-regulate the genes involved in photosynthesis, carbon metabolism, porphyrin and chlorophyll metabolism, photosynthesis-antenna proteins. The conclusion is that SA application would effectively improve the ability of pigment biosynthesis and photosystem repair of tobacco under drought conditions, thus enhance the photosynthesis, reduce the accumulation of ROS and increase drought resistance, which would provide a measure for alleviating the damage of tobacco caused by drought stress.
The effect of water deficit on nitrogen nutrition is complex and requires a multiscale approach, from the membrane to the crop, and different media and their interfaces. The first and most important need is to separate growth-induced changes from the direct effects of water deficits. The main effect of water restriction is certainly a reduction in nitrogen demand due to the marked sensitivity of leaf area expansion. Nitrogen nutrition indices can reveal the processes that alter plant nutrition independently of its actual demand. Process-based crop growth simulation models must currently incorporate these concepts, either implicitly or explicitly. It is noteworthy, however, that they still do not take similar account of the interaction between nitrogen and water water deficit, thus reflecting the ongoing discussion concerning co-limitation analysis in crops. Indeed, some aspects remain poorly understood, especially concerning the establishment of the supply of nitrogen to meet plant needs. The main effect of water restriction is certainly a reduction in nitrogen demand due to the marked sensitivity of leaf area expansion. Nitrogen nutrition indices can reveal the processes that alter plant nutrition independently of its actual demand. Process-based crop growth simulation models must currently incorporate these concepts, either implicitly or explicitly. It is noteworthy, however, that they still do not take similar account of the interaction between nitrogen and soil water deficit, thus reflecting the ongoing discussion concerning co-limitation analysis in crops. Indeed, some aspects remain poorly understood, concerning the establishment of the supply of nitrogen to meet plant needs.
The tolerance of plant growth-promoting endophytes (PGPEs) against various concentrations of cadmium (Cd) and nickel (Ni) was investigated. Two glutathione-producing bacterial strains (Enterobacter ludwigii SAK5 and Exiguobacterium indicum SA22) were screened for Cd and Ni accumulation and tolerance in contaminated media, which showed resistance up to 1.0 mM. Both strains were further evaluated by inoculating specific plants with the bacteria for five days prior to heavy metal treatment (0.5 and 1.0 mM). The enhancement of biomass and growth attributes such as the root length, shoot length, root fresh weight, shoot fresh weight, and chlorophyll content were compared between treated inoculated plants and treated non-inoculated plants. Both strains significantly increased the accumulation of Cd and Ni in inoculated plants. The accumulation of both heavy metals was higher in the roots than in the shoots, however; Ni accumulation was greater than Cd. Heavy metal stress-responsive genes such as OsGST, OsMTP1, and OsPCS1 were significantly upregulated in treated non-inoculated plants compared with treated inoculated plants, suggesting that both strains reduced heavy metal stress. Similarly, abscisic acid (ABA) was increased with increased heavy metal concentration; however, it was reduced in inoculated plants compared with non-inoculated plants. Salicylic acid (SA) was found to exert synergistic effects with ABA. The application of suitable endophytic bacteria can protect against heavy metal hyperaccumulation by enhancing detoxification mechanisms.
Seed germination and seedling growth establishment are the most critical growth stages, and drought stress imposed at these stages highly limits crop productivity. In this regard, a hydroponic water culture experiment was conducted with the aim to assess the potential of 20 wheat genotypes against drought stress at the seedling stage. Water deficit was induced through polyethylene glycol (PEG-6000), by maintaining two osmotic potentials in water culture medium, i.e. −0.7 MPa (medium water stress) and −1.0 MPa (high water stress). After seed germination, drought stress was applied for 8 days. Seedlings shoot and root length and biomasses were restricted with an increase in osmotic deficit. Photosynthetic pigments and nitrate reductase activity (NRA) of wheat seedlings were reduced, while proline, total soluble sugars, total phenolics, and mineral ions (K⁺ and Ca²⁺) were augmented with the rise in water deficiency in most of the genotypes. On the basis of growth and biochemical attributes, six genotypes (NIA-AA-01, NIA-AA-08, NIA-AA-09, NIA-AA-13, NIA-AA-12, and NIA-AA-14) were categorized as drought tolerant, and three as medium tolerant. These genotypes exhibited better growth by showing the least reduction in root and shoot length, and fresh and dry biomasses, as well as modulation in biochemical processes to survive under water deficit. All studied traits indicated tolerance potential of these genotypes against moderate and extreme drought stress, which could also give better growth in arid and semi-arid regions of the country that facing water scarcity.
During a growing season, agricultural crops are frequently exposed to abiotic stresses, which hinder plant growth and reduce crop productivity. Abiotic stresses include extreme temperatures, drought, submergence, soil salinization, and nutrient imbalances, and many of these stresses are predicted to increase in frequency or severity in the coming century. Maintaining high levels of crop productivity under increasingly suboptimal growth environments will require the development of new agronomic tools. One new and promising strategy involves the utilization of microorganisms, which have been shown to be capable of mitigating abiotic stresses, and the plant microbiome has the potential of promoting growth and protecting the host through a variety of molecular mechanisms. While studies have begun to explore how specific members of the root microbiome act to enhance plant growth, we still lack a full understanding of the role of the broader root microbiome in shaping plant stress tolerance. Emphasizing three of the most agriculturally significant abiotic challenges, namely, drought stress, salinity stress, and phosphate stress, this chapter describes our current understanding of the specific mechanisms the root microbiome can employ to help boost plant fitness and summarizes new methods through which the plant root microbiome has been leveraged in agricultural settings to reduce damage from abiotic stresses.
The experiment entitled “Effect of Date of Sowing and Leaf Mulching on Mungbean under Custard Apple based Agri-Horti System” was carried out during kharif season of 2013-14. The experiment was carried out at the agroforestry block of Agronomy farm of Rajiv Gandhi South Campus, Brakachha Mirzapur (BHU). The field experiment was laid out in factorial RBD having eight treatments which were replicated thrice. The date of sowing of 4 August was found optimum as compared to other date of sowing (24 July, 14 August and 24 August) in mungbean under custard apple based agri-horti system of Vindhyan region. Leaf mulching 5 t ha-1 is more beneficial for higher yield and net return over no mulching in mungbean under custard apple based agri-horti system.
Cape Verde, off the coast of Senegal in western Africa, is a volcanic archipelago where soil and water conservation techniques play an important role in the overall subsistence of half a million inhabitants. The steep slopes in the more agricultural islands are a result of their volcanic origin. The country is located in the Sahel, and is characterized by a very irregular wet season, with high intensity rainfall events. The hard conditions led during the first half of the twentieth century to frequent cycles of drought with severe implications for the local populations. Large numbers of deaths occurred due to famine, and there was a decrease in the number of inhabitants by more than halve on some islands. To retain the soil and soil moisture was therefore a matter of survival, and the Cape Verdeans implemented over the last half century a number of soil and water conservation techniques that affect the whole landscape. In this chapter, we report the monitoring of a number of slope soil and water conservation techniques, such as terraces, "half moons", live barriers, etc., together with two agricultural management strategies, involving the planting of corn and beans on the one hand, and peanuts on the other, with a semiquantitative methodology, to evaluate their effectiveness. A discussion is given on the costs and effectiveness of the techniques to reduce overland flow production and therefore erosion, and to promote rainfall infiltration. Several variables were measured along 25 metres transects: soil resistance to penetration, percentage or rock outcrops, percentage of organic matter cover, percentage of vegetation cover, and two indexes derived from the observation of erosion and accumulation features. Results show the efficiency of the various soil conservation measures, despite the intensive use and the harsh soil mobilization techniques used to plant beans, maize and peanuts at steep slopes. Soil and water conservation techniques play an important role in the sustainability of local communities' livelihoods in fragile environments such as those in rural Cape Verde.
Effect of drought stress on gas exchange, physiological parameters and yield of tomato (Solanum lycopersicum) genotypes was investigated under pot culture conditions in rainout shelter. The drought condition was created at 15 days after transplanting based on field capacity of soil. Experiment was laid out with 18 genotypes by adopting CRD with three replications and two treatments viz, 100 and 50 % field capacity. As the stress increased from 100 % field capacity to 50 % field capacity, reductions in photosynthetic rate, leaf water potential, chlorophyll stability index (CSI), relative water content (RWC) and nitrate reductase activity (NRA) were noticed at all the growth stages. The genotypes LE 114, LE 57, LE 118 and LE 27 which showed significantly less reduction in the above parameters during drought were considered as drought tolerant. Genotypes LE 1, LE 3 and LE 20 which recorded the lowest photosynthetic rate, CSI, RWC, proline and NRA and ultimately poor yield were considered as drought susceptible.
Potassium is accumulated in substantial amount in water stressed plants, contributing to osmotic adjustment. Eight varieties of chickpea were field grown according to randomized block design with three replications during two consecutive crop seasons to explore the role of K uptake in drought tolerance of chickpea under rain-fed conditions. Plant water relation and other physiological parameters were measured at 70 days after planting, while potassium uptake and yield parameters were recorded at harvest. The varieties were divided into high (HKU), medium (MKU) and low (LKU) K uptake groups, using average K uptake across the seasons. All the data were statistically analyzed group-wise. The HKU, followed by MKU and LKU (i.e. HKU>MKU>LKU) gave the highest values for most of the parameters studied including seed yield and crop biomass. The HKU also exhibited the lowest osmotic potential together with maintenance of elevated water content and turgor in plants compared to MKU and LKU. There was significant season-group interaction regarding most parameters including seed yield and crop biomass. However, such an interaction was not significant regarding K uptake, though varietal group pattern of K uptake was similar to that of seed yield and crop biomass (i.e. HKU>MKU>LKU), indicating that promising chickpea varieties might be selected under rain-fed conditions using K uptake.
In order to set up large-scale acclimatization protocols of micropropagated plants, an in-depth knowledge of their physiological responses during in vitro to ex vitro transfer is required. This work describes the photosynthetic performance of Ulmus minor micropropagated plants during acclimatization at high irradiance (HI; 200 ± 20 μmol m-2 s-1 or low irradiance (LI; 100 ± 20 μmol m-2 s-1). During this experiment, leaf pigment content, chlorophyll a fluorescence, gas exchange, stomata morphology, the activity of the Calvin cycle enzymes and saccharides were measured in persistent and new leaves. The results indicated that HI induces a higher photosynthetic performance compared to LI. Therefore, plants acclimatized under HI are likely to survive better after field transfer.
(http://link.springer.com/content/pdf/10.1007%2Fs10535-012-0234-8.pdf#page-1)
In this article we discuss and update some of the effects of Cd toxicity on the photosynthetic apparatus in a model crop Lactuca sativa. Seeds of L. sativa were germinated in solutions with 0, 1, 10 and 50 μM of Cd(NO3)2 and then transferred to a hydroponic culture medium. After 28 days, the effects of Cd on the photosynthetic apparatus of lettuce were analysed. Exposure of lettuce to 1 μM Cd(NO3)2 affected already plant growth (dry biomass), but, did not induce serious damages in the photosynthetic apparatus. However, increasing concentrations of this metal to 10 and 50 μM promoted a strong reduction of the maximum photochemical efficiency of PSII and an impairment of net CO2 assimilation rate, putatively due to Rubisco activity decrease. This ultimately results in a strong inhibition of plant growth. Nutrient uptake and carbohydrate assimilation were also severely affected by Cd.
Maize (Zea mays L.) plants were grown to the nine-leaf stage. Despite a saturating N supply, the youngest mature leaves (seventh position on the stem) contained little NO3⁻ reserve. Droughted plants (deprived of nutrient solution) showed changes in foliar enzyme activities, mRNA accumulation, photosynthesis, and carbohydrate and amino acid contents. Total leaf water potential and CO2assimilation rates, measured 3 h into the photoperiod, decreased 3 d after the onset of drought. Starch, glucose, fructose, and amino acids, but not sucrose (Suc), accumulated in the leaves of droughted plants. Maximal extractable phosphoenolpyruvate carboxylase activities increased slightly during water deficit, whereas the sensitivity of this enzyme to the inhibitor malate decreased. Maximal extractable Suc phosphate synthase activities decreased as a result of water stress, and there was an increase in the sensitivity to the inhibitor orthophosphate. A correlation between maximal extractable foliar nitrate reductase (NR) activity and the rate of CO2 assimilation was observed. The NR activation state and maximal extractable NR activity declined rapidly in response to drought. Photosynthesis and NR activity recovered rapidly when nutrient solution was restored at this point. The decrease in maximal extractable NR activity was accompanied by a decrease in NR transcripts, whereas Suc phosphate synthase and phosphoenolpyruvate carboxylase mRNAs were much less affected. The coordination of N and C metabolism is retained during drought conditions via modulation of the activities of Suc phosphate synthase and NR commensurate with the prevailing rate of photosynthesis.
Nitrate uptake in pot-grown, well watered and water-stressed Artemisia tridentata seedlings was determined both during drought and during recovery from drought using >15NO3-. Water deficit caused a 40% decrease in NO3- uptake compared to well watered plants and the restricted NO3- uptake persisted 4 days after rewatering. Between 4 and 14 days after rewatering, NO3- uptake in previously stressed plants was the same as that of the controls. Root relative growth rate (RGR) during the drying cycle was about one-fourth that of the control, but recovered to the control level within 4 days after rewatering. Between 4 and 14 days after rewatering, the previously droughted plant roots exhibited nearly three times greater RGR than the control plant roots. Estimates of root solute content indicated that at no time during the stress and recovery periods did the droughted roots exhibit osmotic adjustment. Changes in root growth properties were uncoupled from turgor. During the recovery period, drought-induced adjustments in cell wall yielding properties are thought to have increased root growth in previously stressed seedlings. Nevertheless, the greater root growth of previously droughted plants did not result in more NO3- acquisition than in control plants. The pattern of NO3- uptake upon rewatering was apparently more closely associated with root uptake capacity.
The effects of drought on the activity of nitrate reductase (NR) were studied in Helianthus annuus L. plants subjected to soil drying and subsequent re-watering. Drought did not negatively affect the activation state of NR, but resulted in linearly-correlated decreases in the activity of the unphosphorylated active form and the total activity of NR, in both roots and leaves. The concentration of nitrate in roots, xylem and leaves also decreased in water-stressed plants, whereas the concentration of total amino acids was only transiently depressed at the leaf level. In contrast, soluble sugars accumulated both in roots and leaves of water-stressed plants. Drought-induced decreases in root NR activity were correlated with the observed changes in root nitrate concentration. A higher percentage of the decrease in foliar NR activity could be explained by the decline in nitrate flux to the leaves than by leaf nitrate content. Following re-watering, the extent of recovery of NR activity was higher in roots than in leaves. The delay in the recovery of foliar NR activity did not result from the persistence of reduced flux of nitrate through the xylem. Several hypotheses to explain the after-effect of soil drying on foliar NR activity are discussed.
In roots of Lotus japonicus (Regel) Larsen cv. Gifu, the
level of NADH-nitrate reductase (NR) activity and protein, as well as
ferredoxin-nitrite reductase activity and nitrate accumulation, were higher in
roots of young plants, and decreased in mature plants grown in seed trays.
When plants were grown in larger pots, the decrease in NR activity and nitrate
accumulation took place at a later stage of growth, suggesting that the
cessation of nitrate assimilation in mature plants could be related to both
ageing and a lower availability of space for roots. Low NR activity was
detected in leaves, whereas nitrate accumulation in this tissue could reach
relatively high levels. NR activity and protein, and nitrate accumulation,
also decreased in leaves of mature plants, indicating that the diminution in
nitrate accumulation and NR in roots from mature plants is not related to a
shift of the nitrate assimilation process from roots to leaves. Measurement of
the extent of total and active NR according to the
phosphorylation/dephosphorylation inactivating mechanism described for
spinach leaf NR, indicates that this mechanism is operative on NR from
L. japonicus roots, being responsible for a great
proportion of inactive NR protein. The amount of inactive NR protein in roots
due to the aforementioned phosphorylation mechanism also increased in mature
plants.
Double-barrelled nitrate-selective microelectrodes have been used to measure the time course of the remobilisation of vacuolar
stored nitrate in barley (Hordeum vulgare L. cv. Klaxon) root cells during 24 h of nitrate deprivation. These measurements showed that there are different time courses
for this process in epidermal and cortical cells of the same root. The remobilisation was much slower from cortical cell vacuoles
and had a time course which was similar to that obtained for tissue digests of the roots. The microelectrodes were also used
to measure the nitrate concentration in sap exuding from detopped seedlings. These measurements showed that there was a gradual
decrease in the delivery of nitrate to the shoot during this time. Root nitrate reductase activity of neither shoots nor roots
changed significantly during the first 24 h. Direct measurement of the cytosolic nitrate in a root epidermal cell showed that
during short-term changes, such as a 20-min exposure to zero external nitrate supply, cytosolic nitrate was maintained relatively
unchanged. Net nitrate efflux from the roots was measurable during the initial 5 h of the zero-nitrate incubation period;
after this time no further nitrate efflux was detectable. These measurements are discussed in relation to the nitrate budget
of a root cell and we conclude that during the first 24 h of nitrate withdrawal vacuolar nitrate can be readily mobilised
to supply the nitrogen demands of the seedling and to maintain the cytosolic nitrate concentration.
Three leafy vegetables, rape (Brassica campestris L.), Chinese cabbage (Brassica chinensis var. Oleifera Makino et Nenoto) and spinach (Spinacia oleracea L.), were grown in plastic pots with 5 kg soil per pot at five nitrate supply rates, 0.00 (N1), 0.15 (N2), 0.30 (N3), 0.45 (N4), and 0.60 (N5) g N kg−1 soil to investigate the effects of nitrate supply on plant growth, nitrate accumulation and nitrate reductase activity (NRA) 9 weeks after sowing. The optimum yield appeared at N3, while above N4, a strong decrease in plant growth occurred. The nitrate concentration increased with nitrate supply in the whole plant and the different organs except in roots where nitrate concentration at N5 decreased compared with N4. The nitrate concentration in both the metabolic pool (MP) and the storage pool (SP) of the leaf blades increased with nitrate supply. From N1 to N2, NRA increased most rapidly. The highest NRA occurred at N4. However, nitrate reductase (NR) activities were not significantly different between N3, N4 and N5, which imply that there is a threshold of nitrate concentration in MP (NMP) to induce NRA. The parameters of NR for nitrate were measured by the in vivo method. The Km values we obtained were similar to the reported values by the in vitro method, which confirms the feasibility of the anaerobic method for determining NRA and NMP. Finally, the effects of the posttranslational regulation of NR were discussed.
The impact of low humidity in ambient air on water relations, nitrate uptake, and translocation of recently absorbed nitrogen,
was investigated in 5-week-old tomato (Lycopersicon esculentum Mill cv. Ailsa Craig) plants grown hydroponically in a complete nutrient solution. Plants were subjected to dry air (relative
humidity 2–4% for 6 h. The transpiration rate increased several-fold and the shoot water content decreased by almost 20%,
whereas root water content was unaffected. No effect on in vitro nitrate reductase (NR) activity was detected when using an EDTA-contraining assay buffer. Replacement of EDTA with Mg2+ revealed a significant decline in shoot NR activity, which suggests phosphorylation of the enzyme during the stress treatment.
Plants were grown in a split-root system, in which one root half was fed 15N-nitrate during the treatment, in order to determine nitrate uptake and translocation of recently absorbed nitrogen in the
plants. Uptake of nitrate was substantially inhibited, but the proportion of absorbed 15N that was translocated to the shoots was only slightly affected. In untreated plants, 71% of the 15N recovered in roots had been retranslocated from the shoots, whereas in plants subjected to stress the delivery of 15N from shoots to roots appeared to be completely inhibited. The data show that lowered humidity in air has significant effects
on both uptake of nitrate as well as translocation of nitrogen within the plants. Some of these effects appear to be common
with those observed in plants subjected to reduced water potentials in the root environment and point to the possibility of
the shoot water relations being highly influential on nitrogen uptake and translocation.
Although nitrate reductase (NR. EC 1.6.6.1) is thought to control the rate of nitrate assimilation, mutants with 40-45% of wildtype (WT) NR activity (NRA) grow as fast as the WT. We have investigated how tobacco (Nicotiana tabacum L. cv. Gatersleben) mutants with one or two instead of four functional nia genes compensate. (i) The nia transcript was higher in the leaves of the mutants. However, the diurnal rhythm was retained in the mutants, with a maximum at the end of the night and a strong decline during the photoperiod. (ii) Nitrate reductase protein and NRA rose to a maximum after 3-4 h light in WT leaves, and then decreased by 50-60% during the second part of the photoperiod and the first part of the night. Leaves of mutants contained 40-60% less NR protein and NRA after 3-4 h illumination, but NR did not decrease during the photoperiod. At the end of the photoperiod the WT and the mutants contained similar levels of NR protein and NRA. (iii) Darkening led to a rapid inactivation of NR in the WT and the mutants. However, in the mutants, this inactivation was reversed after 1-3 h darkness. Calyculin A prevented this reversal. When magnesium was included in the assay to distinguish between the active and inactive forms of NR, mutants contained 50% more activity than the WT during the night. Conversion of [15N]-nitrate to organic compounds in leaves in the first 6 h of the night was 60% faster in the mutants than in the WT. (iv) Growth of WT plants in enhanced carbon dioxide prevented the decline of NRA during the second part of the photoperiod, and led to reactivation of NR in the dark. (v) Increased stability of NR in the light and reversal of dark-inactivation correlated with decreased levels of glutamine in the leaves. When glutamine was supplied to detached leaves it accelerated the breakdown of NR, and led to inactivation of NR, even in the light. (vi) Diurnal changes were also investigated in roots. In the WT, the amount of nia transcript rose to a maximum after 4 h illumination and then gradually decreased. The amplitude of the changes in transcript amount was smaller in roots than in leaves, and there were no diurnal changes in NRA. In mutants, nia transcript levels were high through the photoperiod and the first part of the night. The NRA was 50% lower during the day but rose during the night to an activity almost as high as in the WT. The rate of [15N]-nitrate assimilation in the roots of the mutants resembled that in the WT during the first 6 h of the night. (vii) Diurnal changes were also compared in Nia30(145) transformants with very low NRA, and in nitrate-deficient WT plants. Both sets of plants had similar low growth rates. Nitrate reductase did not show a diurnal rhythm in leaves or roots of Nia30(145), the leaves contained very low glutamine, and NR did not inactivate in the dark. Nitrate-deficient WT plants were watered each day with 0.2 mM nitrate. After watering, there was a small peak of nia transcript NR protein and NRA and, slightly later, a transient increase of glutamine and other amino acids in the leaves. During the night glutamine was low, and NR did not inactivate. In the roots, there was a very marked increase of nitrate, nia transcript and NRA 2-3 h after the daily watering with 0.2 mM nitrate. (viii) It is concluded that WT plants have excess capacity for nitrate assimilation. They only utilise this potential capacity for a short time each day, and then down-regulate nitrate assimilation in response, depending on the conditions, to accumulation of the products of nitrate assimilation or exhaustion of external nitrate. Genotypes with a lower capacity for nitrate assimilation compensate by increasing expression of NR and weakening the feedback regulation, to allow assimilation to continue for a longer period each day.
Transformed (cauliflower mosaic virus 35S promoter [35S]) tobacco (Nicotiana plumbaginifolia L.) plants constitutively expressing nitrate reductase (NR) and untransformed controls were subjected to drought for 5 d. Drought-induced changes in biomass accumulation and photosynthesis were comparable in both lines of plants. After 4 d of water deprivation, a large increase in the ratio of shoot dry weight to fresh weight was observed, together with a decrease in the rate of photosynthetic CO2 assimilation. Foliar sucrose increased in both lines during water stress, but hexoses increased only in leaves from untransformed controls. Foliar NO3- decreased rapidly in both lines and was halved within 2 d of the onset of water deprivation. Total foliar amino acids decreased in leaves of both lines following water deprivation. After 4 d of water deprivation no NR activity could be detected in leaves of untransformed plants, whereas about 50% of the original activity remained in the leaves of the 35S-NR transformants. NR mRNA was much more stable than NR activity. NR mRNA abundance increased in the leaves of the 35S-NR plants and remained constant in controls for the first 3 d of drought. On the 4th d, however, NR mRNA suddenly decreased in both lines. Rehydration at d 3 caused rapid recovery (within 24 h) of 35S-NR transcripts, but no recovery was observed in the controls. The phosphorylation state of the protein was unchanged by long-term drought. There was a strong correlation between maximal extractable NR activity and ambient photosynthesis in both lines. We conclude that drought first causes increased NR protein turnover and then accelerates NR mRNA turnover. Constitutive NR expression temporarily delayed drought-induced losses in NR activity. 35S-NR expression may therefore allow more rapid recovery of N assimilation following short-term water deficit.
Maize (Zea mays L.) plants were grown to the nine-leaf stage. Despite a saturating N supply, the youngest mature leaves (seventh position on the stem) contained little NO3- reserve. Droughted plants (deprived of nutrient solution) showed changes in foliar enzyme activities, mRNA accumulation, photosynthesis, and carbohydrate and amino acid contents. Total leaf water potential and CO2 assimilation rates, measured 3 h into the photoperiod, decreased 3 d after the onset of drought. Starch, glucose, fructose, and amino acids, but not sucrose (Suc), accumulated in the leaves of droughted plants. Maximal extractable phosphoenolpyruvate carboxylase activities increased slightly during water deficit, whereas the sensitivity of this enzyme to the inhibitor malate decreased. Maximal extractable Suc phosphate synthase activities decreased as a result of water stress, and there was an increase in the sensitivity to the inhibitor orthophosphate. A correlation between maximal extractable foliar nitrate reductase (NR) activity and the rate of CO2 assimilation was observed. The NR activation state and maximal extractable NR activity declined rapidly in response to drought. Photosynthesis and NR activity recovered rapidly when nutrient solution was restored at this point. The decrease in maximal extractable NR activity was accompanied by a decrease in NR transcripts, whereas Suc phosphate synthase and phosphoenolpyruvate carboxylase mRNAs were much less affected. The coordination of N and C metabolism is retained during drought conditions via modulation of the activities of Suc phosphate synthase and NR commensurate with the prevailing rate of photosynthesis.
To gain an insight into the diurnal changes of nitrogen assimilation in roots the in vitro activities of cytosolic and plasma membrane‐bound nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.7.7.1) and cytosolic
and plastidic glutamine synthetase (EC 6.3.1.2) were studied. Simultaneously, changes in the contents of total protein, nitrate,
nitrite, and ammonium were followed. Roots of intact tobacco plants (Nicotiana tabacum cv. Samsun) were extracted every 3 h during a diurnal cycle. Nitrate reductase, nitrite reductase and glutamine synthetase
were active throughout the day–night cycle. Two temporarily distinct peaks of nitrate reductase were detected: during the
day a peak of soluble nitrate reductase in the cytosol, in the dark phase a peak of plasma membrane‐bound nitrate reductase
in the apoplast. The total activities of nitrate reduction were similar by day and night. High activities of nitrite reductase
prevented the accumulation of toxic amounts of nitrite throughout the entire diurnal cycle. The resulting ammonium was assimilated
by cytosolic glutamine synthetase whose two activity peaks, one in the light period and one in the dark, closely followed
those of nitrate reductase. The contribution of plastidic glutamine synthetase was negligible. These results strongly indicate
that nitrate assimilation in roots takes place at similar rates day and night and is thus differently regulated from that
in leaves.
Simultaneous measurements of CO(2) (CER) and O(2) (OER) exchange in roots and shoots of vegetative white lupin (Lupinus albus) were used to calculate the flow of reducing power to the synthesis of biomass that was more reduced per unit of carbon than carbohydrate. On a whole-plant basis, the diverted reductant utilization rate (DRUR which is: 4 x [CER + OER]) of shoot tissue was consistently higher than that of roots, and values obtained in the light were greater than those in the dark. An analysis of the biomass being synthesized over a 24-h period provided an estimate of whole-plant DRUR (3.5 mmol e(-) plant(-1) d(-1)), which was similar to that measured by gas exchange (3.2 mmol e(-) plant(-1) d(-1)). Given that nitrate reduction to ammonia makes up about 74% of whole-plant DRUR, root nitrate reduction in white lupin was estimated to account for less than 43% of whole-plant nitrate reduction. The approach developed here should offer a powerful tool for the noninvasive study of metabolic regulation in intact plants or plant organs.
Assimilatory nitrate reductase (NR) of higher plants is a most interesting enzyme, both from its central function in plant primary metabolism and from the complex regulation of its expression and control of catalytic activity and degradation. Here, present knowledge about the mechanism of post-translational regulation of NR is summarized and the properties of the regulatory enzymes involved (protein kinases, protein phosphatases and 14-3-3-binding proteins) are described. It is shown that light and oxygen availability are the major external triggers for the rapid and reversible modulation of NR activity, and that sugars and/or sugar phosphates are the internal signals which regulate the protein kinase(s) and phosphatase. It is also demonstrated that stress factors like nitrate deficiency and salinity have remarkably little direct influence on the NR activation state. Further, changes in NR activity measured in vitro are not always associated with changes in nitrate reduction rates in vivo, suggesting that NR can be under strong substrate limitation. The degradation and half-life of the NR protein also appear to be affected by NR phosphorylation and 14-3-3 binding, as NR activation always correlates positively with its stability. However, it is not known whether the molecular form of NR in vivo affects its susceptibility to proteolytic degradation, or whether factors that affect the NR activation state also independently affect the activity or induction of the NR protease(s). A second and potentially important function of NR, the production of nitric oxide (NO) from nitrite is briefly described, but it remains to be determined whether NR produces NO for pathogen/stress signalling in vivo.
The mechanism of the post‐translational modulation of nitrate reductase activity (NR, EC 1.6.6.1) is briefly summarized, and
it is shown that by this mechanism nitric oxide production through NR is also rapidly modulated. New and partly unexpected
details on the modulation mechanism have been obtained by using immunological techniques. The phosphorylation state of NR
has been assessed with peptide antibodies raised against the serine phosphorylation motive of spinach NR. By co‐immunoprecipitation
experiments, 14‐3‐3 binding to phospho‐NR and the function of Mg2+ in that process has been elucidated. Conflicting data on the role of NR phosphorylation and 14‐3‐3 binding in controlling
NR proteolysis are discussed. A possible role of other NR inactivating proteins is also briefly considered and the regulation
of NR of Ricinus communis is described as an interesting special case that differs from the ‘normal’ mechanism in several important aspects.
The hypothesis was tested that slow‐growing grass species perform a greater proportion of total plant NO3– reduction in their roots than do fast‐growing grasses. Eight grass species were selected that differed in maximum relative
growth rate (RGR) and net NO3– uptake rate (NNUR). Plants were grown with free access to nutrients in hydroponics under controlled‐environment conditions. The site of in vivo NO3– reduction was assessed by combining in vivo NO3– reductase activity (NRA) assays with biomass allocation data, and by analysing the NO3– to amino acid ratio of xylem sap. In vivo NRA of roots and shoots increased significantly with increasing NNUR and RGR. The proportion of total plant NO3– reduction that occurs in roots was found to be independent of RGR and NNUR, with the shoot being the predominant site of NO3– reduction in all species. The theoretical maximum proportion of whole plant nitrogen assimilation that could take place in
the roots was calculated using information on root respiration rates, RGR, NNUR, and specific respiratory costs associated with growth, maintenance and ion uptake. The calculated maximum proportion that
the roots can contribute to total plant NO3– reduction was 0.37 and 0.23 for the fast‐growing Dactylis glomerata L. and the slow‐growing Festuca ovina L., respectively. These results indicate that slow‐growing grass species perform a similar proportion of total plant NO3– reduction in their roots to that exhibited by fast‐growing grasses. Shoots appear to be the predominant site of whole plant
NO3– reduction in both fast‐ and slow‐growing grasses when plants are grown with free access to nutrients.
The low-activity, phosphorylated form of nitrate reductase (NR) became activated during purification from spinach (Spinacia oleracea) leaves harvested in the dark. This activation resulted from its separation from an approximately 110-kd nitrate reductase inhibitor protein (NIP). Readdition of NIP inactivated the purified phosphorylated NR, but not the active dephosphorylated form of NR, indicating that the inactivation of NR requires its interaction with NIP as well as phosphorylation. Consistent with this hypothesis, NR that had been inactivated in vitro in the presence of NR kinase, ATP-Mg, and NIP could be reactivated either by dephosphorylation with protein phosphatase 2A or by dissociation of NIP from NR.
Nitrate uptake, nitrate reductase activity (NRA) and net proton release were compared in five grain legumes grown at 0.2 and 2 mM nitrate in nutrient solution. Nitrate treatments, imposed on 22-d-old, fully nodulated plants, lasted for 21 d. Increasing nitrate supply did not significantly influence the growth of any of the species during the treatment, but yellow lupin (Lupinus luteus) had a higher growth rate than the other species examined. At 0.2 mM nitrate supply, nitrate uptake rates ranged from 0.6 to 1.5 mg N g(-1) d(-1) in the order: yellow lupin > field pea (Pisum sativum) > chickpea (Cicer arietinum) > narrow-leafed lupin (L angustifolius) > white lupin (L albus). At 2 mM nitrate supply, nitrate uptake ranged from 1.7 to 8.2 mg N g(-1) d(-1) in the order: field pea > chickpea > white lupin > yellow lupin > narrow-leafed lupin. Nitrate reductase activity increased with increased nitrate supply, with the majority of NRA being present in shoots. Field pea and chickpea had much higher shoot NRA than the three lupin species. When 0.2 mM nitrate was supplied, narrow-leafed lupinreleased the most H+ per unit root biomass per day, followed by yellow lupin, white lupin, field pea and chickpea. At 2 mM nitrate, narrow-leafed lupin and yellow lupin showed net proton release, whereas the other species, especially field pea, showed net OH- release. Irrespective of legume species and nitrate supply, proton release was negatively correlated with nitrate uptake and NRA in shoots, but not with NRA in roots.
In higher plants, nitrate reductase (NR) is inactivated by the phosphorylation of a conserved Ser residue and binding of 14‐3‐3
proteins in the presence of divalent cations or polyamines. A transgenic Nicotiana plumbaginifolia line (S521) has been constructed where the regulatory, conserved Ser 521 of tobacco NR (corresponding to Ser 534 in Arabidopsis) was mutated into Asp. This mutation resulted in the complete abolition of activation/inactivation in response to light/dark
transitions or other treatments known to regulate the activation state of NR. Analysis of the transgenic plants showed that,
under certain conditions, when whole plants or cut tissues are exposed to high nitrate supply, post‐translational regulation
is necessary to avoid nitrite accumulation. Abolition of the post‐translational regulation of NR also results in an increased
flux of nitric oxide from the leaves and roots. In view of the results obtained from examining the different transgenic N. plumbaginifolia lines, compartmentation of nitrate into an active metabolic pool and a large storage pool appears to be an important factor
for regulating nitrate reduction. The complex regulation of nitrate reduction is likely to have evolved not only to optimize
nitrogen assimilation, but also to prevent and control the formation of toxic, and possibly regulatory, products of NR activities.
Phos phorylation of NR has previously been found to influence the degradation of NR in spinach leaves and Arabidopsis cell cultures. However, experiments with whole plants of N. plumbaginifolia, Arabidopsis, or squash are in favour of NR degradation being the same in light and darkness and independent of phosphorylation at the
regulatory Ser.
Partitioning and utilization of assimilated C and N were compared in nonnodulated, NO(3)-fed and nodulated, N(2)-fed plants of white lupin (Lupinus albus L.). The NO(3) regime used (5 millimolar NO(3)) promoted closely similar rates of growth and N assimilation as in the symbiotic plants. Over 90% of the N absorbed by the NO(3)-fed plants was judged to be reduced in roots. Empirically based models of C and N flow demonstrated that patterns of incorporation of C and N into dry matter and exchange of C and N among plant parts were essentially similar in the two forms of nutrition. NO(3)-fed and N(2)-fed plants transported similar types and proportions of organic solutes in xylem and phloem. Withdrawal of NO(3) supply from NO(3)-fed plants led to substantial changes in assimilate partitioning, particularly in increased translocation of N from shoot to root. Nodulated plants showed a lower (57%) conversion of C or net photosynthate to dry matter than did NO(3)-fed plants (69%), and their stems were only half as effective as those of NO(3)-fed plants in xylem to phloem transfer of N supplied from the root. Below-ground parts of symbiotic plants consumed a larger share (58%) of the plants' net photosynthate than did NO(3)-fed roots (50%), thus reflecting a higher CO(2) loss per unit of N assimilated (10.2 milligrams C/milligram N) by the nodulated root than by the root of the NO(3)-fed plant (8.1 milligrams C/milligram N). Theoretical considerations indicated that the greater CO(2) output of the nodulated root involved a slightly greater expenditure for N(2) than for NO(3) assimilation, a small extra cost due to growth and maintenance of nodule tissue, and a considerably greater nonassimilatory component of respiration in root tissue of the symbiotic plant than in the root of the NO(3)-fed plant.
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 CO(2) 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 CO(2) 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 -CO(2)-treated leaves became manifest as a decrease in total enzyme activity only in the presence of free Mg(2+) or Ca(2+). Mg(2+) 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 Mg(2+) concentrations. The apparent substrate affinities of NR were hardly affected by the in vivo modulation of NR. Only V(max) changed.
Young maize seedlings (Zea mays L. cv.Giza 2) were exposed to moderate salinity in hydroponic culture. NADH-nitrate reductase (NR) activity (E.C. 1.6.6.1), NR activation state, NR-mRNA-steady state levels and major solute contents in leaves and roots were investigated. With increasing external salt concentration, Na+, Cl-, sugars, amino acids and quarternary ammonium compounds accumulated in leaves and roots, with concentrations in leaves exceeding those in roots. The nitrate content of leaves decreased, but increased in roots. The diurnal pattern of NR activity and of NR-mRNA was also changed under salinity, but the NR activation state was not affected, In the first light phase, maximum NR activity increased rapidly in leaves of control plants, but was much slower in leaves from salinized plants. Thus, integrated over the whole day, the NR activity of salt-stressed plants was lower than in control plants. NR transcript levels of control plants were low in the early night phase, started to increase in the second night phase, followed by a distinct peak at 2 h into the light period, This large 'early morning peak' of NR-mRNA was hardly affected by salinity, whereas the initial slow increase of m-RNA levels in the early night phase was almost absent in salinized plants. This is considered as one reason for the low NR activity of salinized plants in the first half of the day. It is also suggested that nitrate is a major signal affecting NR expression and activity under salinity. Sugars and amino acids appeared less important.
While stomatal closure usually limits photosynthesis during drought, our previous results suggest that drought-related decreases in foliar nitrogen concentration (N-L) limit photosynthesis during recovery from drought in prairie grasses. Here we estimate the importance of decreases in N-L to decreased photosynthetic capacity (PScap) during drought and a subsequent recovery period in three perennial C-4 prairie grasses. PScap (O-2 evolution at light and CO2 saturation) decreased 69 to 78% during drought these grasses, and full recovery of PScap required 8 to 12 days, until younger leaves were expanded or older leaves were repaired, depending on species. Decreases in N-L explained 38 to 51% of the loss of PScap during drought and accounted for 51 to 69% of the total loss of PScap integrated over the post-drought recovery period. N-related loss of PScap appeared to result more from decreases in ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), phosphoenolpyruvate carboxylase (4.1.1.31), and other soluble photosynthetic enzymes, than from decreases in thylakoid N-containing compounds. Decreases in quantum yield of O-2 evolution and F-v/F-m (variable-to-maximum fluorescence of dark-adapted leaves) during drought were small, so we assumed that little damage to photsystem II (PSII) and thylakoid membrane function occurred. Further, F-o (minimum F) decreased or remained unchanged, dark F-o was greater than light F-o, and decreases in photochemical quenching (the fraction of oxidized PSII) were reversed within 1-3 days after drought. Therefore, prolonged increases in nonphotochemical quenching (q(n); thermal dissipation of excess light energy) during and after drought were indicative of protective downregulation and were likely associated with disproportionate loss of soluble photosynthetic proteins during drought. In support of this, post-drought recovery of q(n) paralleled recovery of N-L and PScap. Thus, in C-4 prairie grasses, loss of PScap during drought is largely the result of decreases in shoot N-L and of associated protective downregulation, decreasing carbon assimilation for 1-2 weeks after drought.
Transformed (cauliflower mosaic virus 35S promoter [35S]) tobacco (Nicotiana plumbaginifoliaL.) plants constitutively expressing nitrate reductase (NR) and untransformed controls were subjected to drought for 5 d. Drought-induced changes in biomass accumulation and photosynthesis were comparable in both lines of plants. After 4 d of water deprivation, a large increase in the ratio of shoot dry weight to fresh weight was observed, together with a decrease in the rate of photosynthetic CO2 assimilation. Foliar sucrose increased in both lines during water stress, but hexoses increased only in leaves from untransformed controls. Foliar NO3⁻decreased rapidly in both lines and was halved within 2 d of the onset of water deprivation. Total foliar amino acids decreased in leaves of both lines following water deprivation. After 4 d of water deprivation no NR activity could be detected in leaves of untransformed plants, whereas about 50% of the original activity remained in the leaves of the 35S-NR transformants. NR mRNA was much more stable than NR activity. NR mRNA abundance increased in the leaves of the 35S-NR plants and remained constant in controls for the first 3 d of drought. On the 4th d, however, NR mRNA suddenly decreased in both lines. Rehydration at d 3 caused rapid recovery (within 24 h) of 35S-NR transcripts, but no recovery was observed in the controls. The phosphorylation state of the protein was unchanged by long-term drought. There was a strong correlation between maximal extractable NR activity and ambient photosynthesis in both lines. We conclude that drought first causes increased NR protein turnover and then accelerates NR mRNA turnover. Constitutive NR expression temporarily delayed drought-induced losses in NR activity. 35S-NR expression may therefore allow more rapid recovery of N assimilation following short-term water deficit.
The effect of water stress on nitrate reductase (NR) activity was studied in detached oat leaves exposed to various levels of oxygen. Drought inhibited NADH:NR only in the presence of oxygen, the higher the concentration of oxygen in the atmosphere, the stronger the effect of water stress. The decrease in NADH:NR was always accompanied by an increase in FMNH:NR and NADH:cytochrome c reductase (CR) partial activities. Infiltration of segments with free-radical scavengers prior to drought and oxygen treatment completely impeded the decrease in NADH:NR and the increase in FMNH:NR and CR activities. A trypsin-like inhibitor only partially reversed the loss of NADH:NR. Phenylmethylsulfonyl fluoride (PMSF), a protease inhibitor, and EDTA-Na had no effect on NADH:NR.
The evidence presented here strongly suggests that drought and oxygen stress inhibit NR mainly via oxygen free radicals, which may stimulate the hydrolysis of the protein.
Wheat seedlings (Triticum aestivum L. cv. Timmo) were treated with up to 20% (w/v) polyethylene glycol (PEG, mol. wt. 3350) in the nutrient medium for 6 d.
Shoot growth and nitrate transport and metabolism were substantially affected by PEG treatment. At 20% PEG (corresponding
to a water potential of approximately −1.6 MPa), which caused plants to wilt within 1–2 h, activity of nitrate reductase (NR)
declined with a half-life of approximately 5 h in both roots and shoots. The decline was considerably slower at lower PEG
concentrations. Significant increases in levels of abscisic acid (ABA) only occurred in shoots. Application of ABA to intact
plants or excised shoots did not induce or accelerate decline in shoot NR activity. The rapid decline in NR activity during
wilting appears unrelated to both nitrate flux and ABA. At lower PEG concentrations and in the long-term, however, NR activity
corroborates rates of both transport and growth-related utilization of nitrate. The role of ABA in this context appears to
be indirect through its action on stomatal function which reduces water flux and gas exchange.
While stomatal closure usually limits photosynthesis during drought, our previous results suggest that drought‐related decreases in foliar nitrogen concentration (NL) limit photosynthesis during recovery from drought in prairie grassses. Here we estimate the importance of decreases in NL to decreased photosynthetic capacity (PScap) during drought and a subsequent recovery period in three perennial C4 prairie grasses. PScap (O2 evolution at light and CO2 saturation) decreased 69 to 78% during drought in these grasses, and full recovery of PScap required 8 to 12 days, until younger leaves were expanded or older leaves were repaired, depending on species. Decreases in NL explained 38 to 51% of the loss of PScap during drought and accounted for 51 to 69% of the total loss of PScap integrated over the post‐drought recovery period. N‐related loss of PScap appeared to result more from decreases in ribulose‐1,5‐bisphosphate carboxylase/oxygenase (EC 4.1.1.39), phosphoenolpyruvate carboxylase (4.1.1.31), and other soluble photosynthetic enzymes, than from decreases in thylakoid N‐containing compounds. Decreases in quantum yield of O2 evolution and Fv/Fm (variable‐to‐maximum fluorescence of dark‐adapted leaves) during drought were small, so we assumed that little damage to photsystem II (PSII) and thylakoid membrane function occurred. Further, Fo (minimum F) decreased or remained unchanged, dark Fo was greater than light Fo, and decreases in photochemical quenching (the fraction of oxidized PSII) were reversed within 1–3 days after drought. Therefore, prolonged increases in nonphotochemical quenching (qn; thermal dissipation of excess light energy) during and after drought were indicative of protective downregulation and were likely associated with disproportionate loss of soluble photosynthetic proteins during drought. In support of this, post‐drought recovery of qn paralleled recovery of NL and PScap. Thus, in C4 prairie grasses, loss of PScap during drought is largely the result of decreases in shoot NL
Publisher Summary This chapter focuses on the metabolite levels in specific cells and the subcellular compartments of plant leaves. Leaf material is difficult to fractionate because a typical plant cell contains several different, mechanically fragile subcellular organelles and is surrounded by a mechanically strong plant cell wall. Rather than attempting to isolate whole organelles or cells, leaves are frozen in liquid N 2 and then broken to small fragments that are enriched in material from a given compartment. These fragments are physically separated under conditions when the metabolic activity or redistribution of metabolites is prevented and subsequently their metabolism is quenched. The exclusion of water, or the use of extremely low temperatures, prevents metabolic activity during the fractionation procedures. The chapter describes the silicone oil centrifugation that allows chloroplasts to be separated from the remainder of the protoplast and to be quenched within 2–3 sec of disrupting the protoplast.
In higher plants, cytosolic NAD(P)H-nitrate reductase (NR) is rapidly modulated by environmental conditions such as light, CO2, or oxygen availability. In leaves, NR is activated by photosynthesis, reaching an activation state of 60–80%. In the dark, or after stomatal closure, leaf NR is inactivated down to 20 or 40% of its maximum activity. In roots, hypoxia or anoxia activate NR, whereas high oxygen supply inactivates NR. Spinach leaf NR is inactivated by phosphorylation of serine 543 and subsequent Mg2+-dependent binding of 14-3-3 proteins at, or close to, this phosphorylation site. At least three different protein kinases (NR-PK) have been identified in spinach leaves that are able to phosphorylate NR on serine 543. Two of them show up as calmodulin-like domain protein kinases (CDPKs), and one as a SNF1-like protein kinase. Dephosphorylation of serine 543 is catalyzed by a Mg2+-dependent protein phosphatase and by a type 2A protein phosphatase (NR-PP), which is regulated by a trimer/dimer interconversion. The NR-PKs, NR-PPs, and 14-3-3s are present even in NR-depleted plant tissues. Artificial activation of NR in vivo is achieved by cellular acidification, by respiratory inhibitors, or by mannose feeding. As for anoxia, these treatments seem to act, at least in part, via cytosolic acidification, mediated by low cytosolic ATP levels. Activation is also achieved by ionophore-induced release of divalent cations from the cytosol. In addition, cytosolic AMP and phosphate esters seem to regulate NR-PK and NR-PP activities, thereby adapting NR activity within minutes to the changing environment.
Young maize seedlings (Zea mays L. cv.Giza 2) were exposed to moderate salinity in hydroponic culture. NADH-nitrate reductase (NR) activity (E.C. 1·6.6·1), NR activation state, NR-mRNA-steady state levels and major solute contents in leaves and roots were investigated. With increasing external salt concentration, Na+, Cl−, sugars, amino acids and quarternary ammonium compounds accumulated in leaves and roots, with concentrations in leaves exceeding those in roots. The nitrate content of leaves decreased, but increased in roots. The diurnal pattern of NR activity and of NR-mRNA was also changed under salinity, but the NR activation state was not affected. In the first light phase, maximum NR activity increased rapidly in leaves of control plants, but was much slower in leaves from salinized plants. Thus, integrated over the whole day, the NR activity of salt-stressed plants was lower than in control plants. NR transcript levels of control plants were low in the early night phase, started to increase in the second night phase, followed by a distinct peak at 2 h into the light period. This large ‘early morning peak’ of NR-mRNA was hardly affected by salinity, whereas the initial slow increase of m-RNA levels in the early night phase was almost absent in salinized plants. This is considered as one reason for the low NR activity of salinized plants in the first half of the day. It is also suggested that nitrate is a major signal affecting NR expression and activity under salinity. Sugars and amino acids appeared less important.
Most of the nitrate reductase activity (80%;) in carob ( Ceratonia siliqua L. cv. Mulata) is localised in the roots. The nitrate concentration in the leaves is relatively low compared to that in the roots, suggesting that nitrate influx into the leaf may be a major factor limiting the levels of nitrate reductase in the shoot. Transport of nitrate from root to shoot appears limited by the entrance of nitrate into the xylem. In order to study this problem, we determined the nitrate concentrations and nitrate reductase activities along the roots of nitrate‐grown plants, as well as the composition of the xylem sap and the nitrate levels in the leaves. Some of the the bypocotyl, in order to bypass the loading of nitrate into the xylem of the roots. The results show that the loading of nitrate into the xylem is a limiting step.
The cation and anion concentrations of nitrate‐ and ammonium‐fed plants were similar, showing almost no production of organic anions. In both nitrate‐ and ammonium‐fed plants, the transport of nitrogen from root to shoot was in the form of organic nitrogen compounds. The nitrate reductase activity in the roots was more than sufficient to explain all the efflux of OH ⁻ into the root medium of nitrate‐fed plants. In carob plants the K‐shuttle may thus be operative to a limited extent only, corresponding to between 11 and 27%; of the nitrate taken up. Potassium seems to be the cation accompanying stored nitrate in the roots of carob seedlings, since they accumulate nearly stoichiometric amounts of K ⁺ and NO ⁻ 3 .
These experiments use Nia30(145), a tobacco nia1nia2 double null mutant transformed with a NIA2 construct, to define when sugar supply plays the dominating role in the regulation of nitrate reductase (NIA) expression. The null alleles of Nia30(145) are transcribed and translated to produce non-functional NIA transcript and NIA protein, providing an endogenous reporter system to track NIA expression at the transcript and protein level. The re-introduced NIA2 construct is expressed at low efficiency, providing a background in which the response to changes in sugar status is not complicated by simultaneous changes in the rate of nitrate assimilation and the levels of nitrate and glutamine. In an alternating light–dark regime, Nia30(145) contained high levels of nitrate and low levels of glutamine and other amino acids. This drives constitutive overexpression of NIA. After transfer of Nia30(145) to continuous darkness, nitrate remains high and glutamine low, but the NIA transcript level and NIA protein decreased significantly within 24 h and were undetectable from 48 h onwards. The decrease of the NIA transcript level was fully reversed and the decrease of NIA protein was partly reversed when leaves were detached from the pre-darkened plants and supplied with sucrose in the dark. The decrease was not reversed by nitrate or cytokinin. The NIA transcript disappeared when the leaf sugar content fell below 4 μmol hexose equivalents g−1 FW, and recovered when sugars rose above 8 μmol hexose equivalents g−1 FW. It is concluded that low sugar represses NIA, completely overriding signals derived from nitrate and nitrogen metabolism.
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 Mg ²⁺ to crude extract containing Glc‐6P increased the Mg ²⁺ inhibition of NR. This result suggests that the hexose‐phosphate does not prevent Mg ²⁺ 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.
Nitrate reductase (NR) activity in spinach leaf extracts prepared in the presence of a protein phosphatase inhibitor (50 μM
cantharidine) was measured in the presence of Mg2+ (NRact) or EDTA (NRmax), under substrate saturation. These in-vitro activities were compared with nitrate reduction rates
in leaves from nitrate-sufficient plants. Spinach leaves containing up to 60 μmol nitrate per g fresh weight were illuminated
in air with their petiole in water. Their nitrate content decreased with time, permitting an estimation of nitrate reduction
in situ. The initial rates (1–2 h) of nitrate consumption were usually lower than NRact, and with longer illumination time
(4 h) the discrepancy grew even larger. When leaves were fed through their petiole with 30 mM nitrate, initial in-situ reduction
rates calculated from nitrate uptake and consumption were still lower than NRact. However, nitrate feeding through the petiole
maintained the in situ-nitrate reduction rate for a longer time. Initial rates of nitrate reduction in situ only matched NRact
when leaves were illuminated in 5% CO2. In CO2-free air or in the dark, both NRact and in-situ nitrate reduction decreased, but NRact still exceeded in-situ reduction.
More extremely, under anoxia or after feeding 5-amino-4-imidazole carboxyamide ribonucleoside in the dark, NR was activated
to the high light level; yet in spite of that, nitrate reduction in the leaf remained very low. It was examined whether the
standard assay for NRact would overestimate the in-situ rates due to a dissociation of the inactive phospho-NR-14-3-3 complex
after extraction and dilution, but no evidence for that was found. In-situ NR obviously operates below substrate saturation,
except in the light at high ambient CO2. It is suggested that in the short term (2 h), nitrate reduction in situ is mainly limited by cytosolic NADH, and cytosolic
nitrate becomes limiting only after the vacuolar nitrate pool has been partially emptied.
To investigate the importance of the sugar supply for the regulation of nitrogen and organic acid metabolism, various sugars
and nitrogenous compounds were supplied for 8 h to detached tobacco leaves in low light. (i) In control leaves supplied with
water, there was a large decrease of the Nia transcript level, a 50% decline of nitrate reductase (NR) activity, starch increased and sugars remained low, nitrate decreased
by 50%, and amino acids increased only slightly during the 8 h incubation. About half of the nitrogen accumulating in amino
acids was present in glutamine (Gln). (ii) When 25 mM sucrose was supplied, the in-vivo rate of nitrate assimilation (estimated
from the accumulation of ammonium and amino acids) increased 2-fold. The Nia transcript level still decreased, but the decline of NR activity was less pronounced and NR activation was increased. The
in-vivo net rate of ammonium assimilation (estimated from the accumulation of amino acids) also doubled after feeding sucrose.
Ammonium and glutamate (Glu) decreased and Gln rose markedly, showing that in-vivo activity of glutamine synthetase had been
stimulated. Glutamine still accounted for about half of the nitrogen, indicating that sucrose does not selectively stimulate
glutamine synthase. Glutamate and aspartate decreased and all the minor amino acids increased, showing that the amino acid
biosynthesis pathways are activated by sucrose. There was a decrease of 3-phosphoglycerate (3PGA) and phosphoenolpyruvate
(PEP) and a large increase of α-oxoglutarate, showing that the flow of carbon from glycolysis into organic acids has been
stimulated by sucrose. (iii) The changes of 3PGA, PEP, α-oxoglutarate, Glu, aspartate and the minor amino acids were smaller
when 50 mM glucose was supplied, even though the internal levels of sugars at the end of the incubation resembled those found
after feeding 25 mM sucrose. This indicates that the signals that regulate nitrogen and respiratory metabolism are derived
from the uptake or metabolism of sucrose, rather than glucose. (iv) A different spectrum of changes was found when 20 mM nitrate
was supplied. The estimated rate of nitrate assimilation increased 2-fold, and this was accompanied by an increase of NR activity
but not of NR activation. Nitrate-feeding did not lead to a decrease of Glu, and the increase of minor amino acids was slightly
smaller than with sucrose. There was a decrease of sugars, starch, and hexose phosphates, but 3PGA and PEP were not significantly
decreased and isocitrate increased instead of α-oxoglutarate. (v) A different spectrum of changes was also found when 10 mM
Gln was supplied. The estimated rate of nitrate assimilation decreased, and this was accompanied by a decrease of NR activity
and NR activation. Glutamate did not decrease, and the increase of minor amino acids was smaller than with sucrose. Starch
and sugars remained high and, although hexose phosphates decreased, 3PGA and PEP were not significantly decreased. Isocitrate
remained unaltered and the increase of α-oxoglutarate was smaller than after supplying sucrose. (vi) When 25 mM sucrose was
added together with 20 mM nitrate or 10 mM Gln, the effect on NR activity, NR activation and the estimated rate of nitrate
assimilation was additive to the effect of nitrate, and antagonistic to the effect of Gln. Sucrose still led to a decrease
of Glu, an increase of the minor amino acids, a decrease of 3PGA and PEP, and an increase of α-oxoglutarate when it was supplied
together with nitrate or Gln. (vii) It is concluded that sucrose initiates a co-ordinate activation of nitrate assimilation,
ammonium assimilation, amino acid biosynthesis, and α-oxoglutarate synthesis. Sucrose acts in concert with nitrate and antagonistically
to Gln to increase NR activity and nitrate assimilation, and complements the action of nitrate and Gln to increase the flow
of nitrogen from ammonium into amino acids, and to increase α-oxoglutarate formation.
Sugars were found to interfere with the determinations of alpha-amino nitrogen and free proline with ninhydrin. Suitable modifications are proposed for the quantitative analysis of these compounds in the presence of large amounts of carbohydrates. In this new method of proline assay, the improvement consists of using a citrate buffer adjusted to pH 4.6. Free proline is assayed by ninhydrin reagent prepared without phosphoric acid. This latter procedure uses less ninhydrin and appears to be simplified in comparison with the commonly used method. Both procedures permit a simple, sensitive, and specific determination of nitrogenous compounds in crude extracts. Elimination of carbohydrate interference makes our procedure applicable to crude extracts from various food products such as fruits and plants that accumulate soluble carbohydrates.
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.
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).
The activity of nitrate reductase (+Mg2+, NRact) in illuminated leaves from spinach, barley and pea was 50–80% of the maximum activity (+EDTA, NRmax). However, NR from leaves of Ricinus communis L. had a 10‐fold lower NRact, while NRmax was similar to that in spinach leaves. The low NRact of Ricinus was independent of day‐time and nitrate nutrition, and varied only slightly with leaf age. Possible factors in Ricinusextracts inhibiting NR were not found. NRact from Ricinus, unlike the spinach enzyme, was very low at pH 7.6, but much higher at more acidic pH with a distinct maximum at pH 6.5.
NRmax had a broad pH response profile that was siilar for the spinach and the Ricinus enzyme. Accordingly, the Mg2+‐sensitivity of NR from Ricinus was strongly pH‐dependent (increasing sensitivity with increasing pH), and as a result, the apparent activation state of
NR from a Ricinus extract varied dramatically with pH and Mg2+concentration. Following a light–dark transition, NRact from Ricinus decreased within 1 h by 40%, but this decrease was paralleled by NRmax. In contrast to the spinach enzyme, Ricinus‐NR was hardly inactivated by incubating leaf extracts with ATP plus okadaic acid. A competition analysis with antibodies
against the potential 14‐3‐3 binding site around ser 543 of the spinach enzyme revealed that Ricinus‐NR containes the same site. Removal of 14‐3‐3 proteins from Ricinus‐NR by anion exchange chromatography, activated spinach‐NR but caused little if any activation of Ricinus‐NR. It is suggested that Mg2+‐inhibition of Ricinus‐NR does not require 14‐3‐3 proteins. The rather slow changes in Ricinus‐NR activity upon a light/dark transient may be mainly due to NR synthesis or degradation.
Nitrate reductase (NR; EC 1.6.6.1) is the first enzyme of the nitrate-assimilatory pathway and is regulated transcriptionally and post-translationally by several metabolic and environmental signals. To investigate whether NR is controlled by the rate of photosynthetic CO2 assimilation in cucumber (Cucumis sastivus L.), intact plants were exposed, after the dark period, to light under different atmospheric CO2 concentrations (100, 400 and 2,000 microL x L(-1)) for 2 h. The in-vivo rates of net CO2 assimilation correlated with atmospheric CO2 concentrations. The CO2-fixation rate under 2,000 microL x L(-1) CO2 was 2.4- and 5.4-fold higher than under 400 and 100 microL x L(-1), respectively. Stomatal conductances and transpiration rates were almost identical after the 2-h light period under the various CO2 concentrations tested. Increasing atmospheric CO2 concentrations caused concomitant increases in the contents of starch and soluble sugars in the leaves and a decrease in the nitrate content. The activity and activation state of NR were both higher under elevated CO2 than under low CO2. High CO2 also enhanced NR-gene expression in the leaves. Sugars were supplied via roots to intact carbohydrate-starved plants and NR mRNA levels were analysed after 7 h. Fructose markedly stimulated NR-gene transcription in both leaves and roots. It is concluded that, in cucumber plants, the rate of CO2 assimilation controls the rate of nitrate assimilation by modulation of NR expression and activity, and that sugars are presumably involved as regulatory metabolites.
The accumulation and reduction of nitrate in the presence of the nitrogen metabolites asparagine (Asn) and glutamine (Gln) and the carbon metabolite sucrose (Suc) were examined in maize (Zea mays L.) seedlings in an attempt to separate their effects on the nitrate uptake system and the nitrate reduction system. After 8 h of exposure to nitrate in the presence of 1 mM Asn, tissue nitrate accumulation was reduced at 250 [mu]M external nitrate, but not at 5 mM Asn. The induction of nitrate reductase (NR) activity was reduced at both external nitrate concentrations. In the presence of 1 mM Gln or 1% Suc, tissue nitrate concentration was not significantly altered, but the induction of root NR activity was reduced or enhanced, respectively. The induction of root nitrite reductase (NiR) activity was also reduced in the presence of Asn or Gln and enhanced in the presence of Suc. Transcript levels of NR and NiR in roots were reduced in the presence of the amides and enhanced in the presence of Suc. When Suc was present in combination with either amide, there was complete relief from the inhibition of NiR transcription observed in the presence of amide alone. In the case of NR, however, this relief from inhibition was negligible. The inhibition of the induction of NR and NiR activities in the presence of Gln and Asn is a direct effect and is not the result of altered nitrate uptake in the presence of these metabolites.
Zea mays (cv W64A x W182E) was used to investigate the induction and turnover of nitrate reductase (NR). In our system, 5 or 10 mM KNO3 gave the best growth over a 6-d growing period. With these NO3- levels, NR reached steady-state levels after 24 h. For the turnover experiments, the seedlings were transferred to a NO3--free medium after a 24-h induction. Shoot NR was less sensitive to the removal of NO3- than root NR, which declined almost as soon as NO3- was removed when the seedlings were induced with 5 or 10 mM NO3-. With 1 mM NO3-, however, removal of NO3- from medium resulted in declines in both NR activity and NO3- in shoot and root. Although there was a delay in the degradation of NR protein relative to the loss of NR activity, this protein was not reactivated when NO3- was resupplied. These results indicate that NO3- regulates NR by influencing the de novo synthesis of the NR protein and not by a reversible activation-inactivation of that protein.
Nitrate reductase (NR) in maize (Zea mays cv W64A x W182E) roots has been stabilized in vitro by the addition of chymostatin to extraction buffer. Contrary to previous observations, levels of NR were higher in the mature root than in root tip sections when chymostatin was included in the extraction buffer. Two forms of NR were identified, an NADH monospecific NR found mainly in the 1cm root tip and an NAD(P)H bispecific NR found predominantly in mature regions of the root. During the first 10 days of seedling growth, NR activity in the root ranged from 50 to 80% of the activities found in the leaf (a maximum of 2.4 micromoles NO(-) (2) produced per hour per gram fresh weight was measured at 4 days).
Nitrate reductase in Zea mays L. under salinity Effects of soil drying and subsequent re-watering on the activity A short-term exposure of cucumber plants to rising atmospheric CO 2 increases leaf carbo-hydrate content and enhances nitrate reductase expression and activity
- Abd
- Baki Gk
- F Siefritz
- H-M Man
- H Weiner
- R Kaldenhoff
- J Kaiser Wm Azedo-Silva
- J Osó
- F Fonseca
- B Correia Mj Larios
- E Agü
- P R De La Haba
- Maldonado
Abd-El Baki GK, Siefritz F, Man H-M, Weiner H, Kaldenhoff R, Kaiser WM (2000) Nitrate reductase in Zea mays L. under salinity. Plant Cell Environ 23: 515–521 Azedo-Silva J, Osó rio J, Fonseca F, Correia MJ (2004) Effects of soil drying and subsequent re-watering on the activity Larios B, Agü era E, de la Haba P, Pé-Vicente R, Maldonado JM (2001) A short-term exposure of cucumber plants to rising atmospheric CO 2 increases leaf carbo-hydrate content and enhances nitrate reductase expression and activity. Planta 212: 305–312