Calcium regulates primary nitrate response associated gene transcription in a time- and dose-dependent manner.

Nitrate (NO3-) is the primary source of nitrogen preferred by most arable crops, including wheat. The pioneering experiment on primary nitrate response (PNR) was carried out three decades ago. Since then, much research has been carried out to understand the NO3- signaling. Nitrate is sensed by the dual affinity NO3- transceptor NPF6.3, which further relays the information to a master regulator NIN-like protein 7 (NLP7) through calcium-dependent protein kinases (CPK10, CPK30, CPK32), highlighting the importance of calcium ion (Ca2+) as one of the important secondary messengers in relaying the NO3- signaling in Arabidopsis. In a previous study, we found that Ca2+ regulates nitrogen starvation response in wheat. In this study, 10 days old NO3--starved wheat seedlings were exposed to various treatments. Our study on time course changes in expression of PNR sentinel genes; NPF6.1, NPF6.2, NRT2.1, NRT2.3, NR, and NIR in wheat manifest the highest level of expression at 30 min after NO3- exposure. The use of Ca2+ chelator EGTA confirmed the involvement of Ca2+ in the regulation of transcription of NPFs and NRTs as well the NO3- uptake. We also observed the NO3- dose-dependent and tissue-specific regulation of nitrate reductase activity involving Ca2+ as a mediator. The participation of Ca2+ in the PNR and NO3- signaling in wheat is confirmed by pharmacological analysis, physiological evidences, and protoplast-based Ca2+ localization.

miRNA mediated regulation of nitrogen response and nitrogen use efficiency of plants: the case of wheat.

Nitrogen (N) is needed for plant growth and development and is the major limiting nutrient due to its higher demand in agricultural production globally. The use of N fertilizers has increased considerably in recent years to achieve higher cereal yields. High N inputs coupled with declining N use efficiency (NUE) result in the degradation of the environment. Plants have developed multidimensional strategies in response to changes in N availability in soil. These strategies include N stress-induced responses such as changes in gene expression patterns. Several N stress-induced genes and other regulatory factors, such as microRNAs (miRNAs), have been identified in different plant species, opening a new avenue of research in plant biology. This review presents a general overview of miRNA-mediated regulation of N response and NUE. Further, the in-silico target predictions and the predicted miRNA-gene network for nutrient metabolism/homeostasis in wheat provide novel insights. The information on N-regulated miRNAs and the differentially expressed target transcripts are necessary resources for genetic improvement of NUE by genome editing.

Phenomics based prediction of plant biomass and leaf area in wheat using machine learning approaches.

Introduction: Phenomics has emerged as important tool to bridge the genotype-phenotype gap. To dissect complex traits such as highly dynamic plant growth, and quantification of its component traits over a different growth phase of plant will immensely help dissect genetic basis of biomass production. Based on RGB images, models have been developed to predict biomass recently. However, it is very challenging to find a model performing stable across experiments. In this study, we recorded RGB and NIR images of wheat germplasm and Recombinant Inbred Lines (RILs) of Raj3765xHD2329, and examined the use of multimodal images from RGB, NIR sensors and machine learning models to predict biomass and leaf area non-invasively. Results: The image-based traits (i-Traits) containing geometric features, RGB based indices, RGB colour classes and NIR features were categorized into architectural traits and physiological traits. Total 77 i-Traits were selected for prediction of biomass and leaf area consisting of 35 architectural and 42 physiological traits. We have shown that different biomass related traits such as fresh weight, dry weight and shoot area can be predicted accurately from RGB and NIR images using 16 machine learning models. We applied the models on two consecutive years of experiments and found that measurement accuracies were similar suggesting the generalized nature of models. Results showed that all biomass-related traits could be estimated with about 90% accuracy but the performance of model BLASSO was relatively stable and high in all the traits and experiments. The R2 of BLASSO for fresh weight prediction was 0.96 (both year experiments), for dry weight prediction was 0.90 (Experiment 1) and 0.93 (Experiment 2) and for shoot area prediction 0.96 (Experiment 1) and 0.93 (Experiment 2). Also, the RMSRE of BLASSO for fresh weight prediction was 0.53 (Experiment 1) and 0.24 (Experiment 2), for dry weight prediction was 0.85 (Experiment 1) and 0.25 (Experiment 2) and for shoot area prediction 0.59 (Experiment 1) and 0.53 (Experiment 2). Discussion: Based on the quantification power analysis of i-Traits, the determinants of biomass accumulation were found which contains both architectural and physiological traits. The best predictor i-Trait for fresh weight and dry weight prediction was Area_SV and for shoot area prediction was projected shoot area. These results will be helpful for identification and genetic basis dissection of major determinants of biomass accumulation and also non-invasive high throughput estimation of plant growth during different phenological stages can identify hitherto uncovered genes for biomass production and its deployment in crop improvement for breaking the yield plateau.

Nitrate supply regulates tissue calcium abundance and transcript level of Calcineurin B-like (CBL) gene family in wheat.

Calcium ion (Ca2+) is the most ubiquitous signalling molecule and is sensed by different classes of Ca2+ sensor proteins. Recent evidences underscore the role of calcium signalling in plant response to nitrogen/nitrate supply. Recently we found that under nitrate deficiency, a short-term supply of calcium could improve the plant biomass, nitrate assimilation, anthocyanin accumulation and expression of nitrate uptake and signalling genes. Long-term calcium supply, on the other hand, was not beneficial. Calcineurin B-like (CBL) proteins are one of the vital plant Ca2+ sensory protein family which is essential for stress perception and signaling. To understand the dynamics of CBL-mediated stress signalling in bread wheat, we identified CBL genes in bread wheat (Triticum aestivum) and its progenitors, namely Triticum dicoccoides, Triticum urartu and Aegilops tauschii with the aid of newly available whole-genome sequence. The expression of different CBLs and the changes in root Ca2+ localization in response to nitrate provision or deficiency were analysed. Expression of the CBLs were studied in two bread wheat genotypes with comparatively higher (B.T. Schomburgk, BTS) and lower (Gluyas early, GE) nitrate responsiveness and nitrogen use efficiency. High N promoted the expression of CBLs in seedling leaves while in roots the expression was promoted by N deficiency. At the 5 days after anthesis stage, nitrate starvation downregulated the expression of CBLs while nitrate supply enhanced the expression. At anthesis stage, expression of CBL6 was significantly promoted by HN in panicles of both the genotypes, the highest expression was recorded in BTS. Expression of CBL6 was significantly upregulated by short term nitrate treatment also suggesting its role in Primary nitrate response (PNR) in wheat. There was a significant down regulation of CBL6 expression post nitrate starvation, making it a probable regulator of nitrogen starvation response (NSR) as well. In seedling roots, the tissue localization of Ca2+ was increased both by high and low nitrate treatments, albeit at different magnitudes. Our results suggest that calcium signalling might be a major signalling pathway governing nitrogen responsiveness and CBL6 might be playing pivotal role in NSR and PNR in wheat.

Long term nitrogen deficiency alters expression of miRNAs and alters nitrogen metabolism and root architecture in Indian dwarf wheat (Triticum sphaerococcum Perc.) genotypes.

The important roles of plant microRNAs (miRNAs) in adaptation to nitrogen (N) deficiency in different crop species especially cereals (rice, wheat, maize) have been under discussion since last decade with little focus on potential wild relatives and landraces. Indian dwarf wheat (Triticum sphaerococcum Percival) is an important landrace native to the Indian subcontinent. Several unique features, especially high protein content and resistance to drought and yellow rust, make it a very potent landrace for breeding. Our aim in this study is to identify the contrasting Indian dwarf wheat genotypes based on nitrogen use efficiency (NUE) and nitrogen deficiency tolerance (NDT) traits and the associated miRNAs differentially expressed under N deficiency in selected genotypes. Eleven Indian dwarf wheat genotypes and a high NUE bread wheat genotype (for comparison) were evaluated for NUE under control and N deficit field conditions. Based on NUE, selected genotypes were further evaluated under hydroponics and miRNome was compared by miRNAseq under control and N deficit conditions. Among the identified, differentially expressed miRNAs in control and N starved seedlings, the target gene functions were associated with N metabolism, root development, secondary metabolism and cell-cycle associated pathways. The key findings on miRNA expression, changes in root architecture, root auxin abundance and changes in N metabolism reveal new information on the N deficiency response of Indian dwarf wheat and targets for genetic improvement of NUE.

Variation in nitrogen partitioning and reproductive stage nitrogen remobilization determines nitrogen grain production efficiency (NUEg) in diverse rice genotypes under varying nitrogen supply.

Nitrogen (N) is an important macronutrient needed for grain yield, grain N and grain protein content in rice. Grain yield and quality are significantly determined by N availability. In this study, to understand the mechanisms associated with reproductive stage N remobilization and N partitioning to grain 2 years of field experiments were conducted with 30 diverse rice genotypes during 2019-Kharif and 2020-Kharif seasons. The experiments were conducted with two different N treatments; N deficient (N0-no external N application, available soil N; 2019-234.15 kgha-1, 2020-225.79 kgha-1) and N sufficient (N120-120 kgha-1 external N application, available soil N; 2019-363.77 kgha-1, 2020-367.95 kgha-1). N application increased the NDVI value, biomass accumulation, grain yield, harvest index and grain N accumulation. Post-anthesis N uptake and N remobilization from vegetative tissues to grain are critical for grain yield and N harvest index. Rice genotypes, Kalinga-1, BAM-4234, IR-8384-B-B102-3, Sahbhagi Dhan, BVD-109 and Nerica-L-42 showed a higher rate of N remobilization under N sufficient conditions. But, under N deficiency, rice genotypes-83929-B-B-291-3-1-1, BVD-109, IR-8384-B-B102-3 and BAM-4234 performed well showing higher N remobilization efficiency. The total amount of N remobilization was recorded to be high in the N120 treatment. The harvest index was higher in N120 during both the cropping seasons. RANBIR BASMATI, BAM-832, APO, BAM-247, IR-64, Vandana, and Nerica-L-44 were more efficient in N grain production efficiency under N deficient conditions. From this study, it is evident that higher grain N accumulation is not always associated with higher yield. IR-83929-B-B-291-3-1-1, Kalinga-1, APO, Pusa Basmati-1, and Nerica-L-44 performed well for different N use efficiency component traits under both N deficient (N0) and N sufficient (N120) conditions. Identifying genotypes/donors for N use efficiency-component traits is crucial in improving the fertilizer N recovery rate and site specific N management.

Upregulation of genes encoding plastidic isoforms of antioxidant enzymes and osmolyte synthesis impart tissue tolerance to salinity stress in bread wheat.

Wheat genotype Kharchia is a donor for salt tolerance in wheat breeding programs worldwide; however, the tolerance mechanism in Kharchia is yet to be deciphered completely. To avoid spending energy on accumulating organic osmolytes and to conserve resources for maintaining growth, plants deploy sodium (Na+) ions to maintain turgor. The enhanced ability to tolerate excess ion accumulation and ion toxicity is designated as tissue tolerance. In this study, salt-tolerant wheat genotype (Kharchia 65) and sensitive cultivars (HD2687, HD2009, WL711) were exposed to vegetative stage salinity stress (for four weeks). Kharchia 65 showed better tissue tolerance to salinity than the other genotypes based on different physiological parameters. Gene expression and abundance of chloroplast localized antioxidant enzymes and compatible osmolyte synthesis were upregulated by salinity in Kharchia 65. In Kharchia 65, the higher abundance of NADPH Oxidase (RBOH) transcripts and localization of reactive oxygen species (ROS) suggested an apoplastic ROS burst. Expression of calcium signaling genes of SOS pathway, MAPK6, bZIP6 and NAC4 were also upregulated by salinity in Kharchia 65. Considering that Kharchia local is the donor of salt tolerance trait in Kharchia 65, the publically available Kharchia local transcriptome data were analyzed. Our results and the in-silico transcriptome analysis also confirmed that higher basal levels and the stress-induced rise in the expression of plastidic isoforms of antioxidant enzymes and osmolyte biosynthesis genes provide tissue tolerance in Kharchia 65. Thus, in salinity tolerant genotype Kharchia 65, ROS burst mediated triggering of calcium signaling improves Na+ exclusion and tissue tolerance to Na+. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01237-w.

Genome-wide characterization and identification of cyclophilin genes associated with leaf rust resistance in bread wheat (Triticum aestivum L.).

Cyclophilins (CYPs) are a group of highly conserved proteins involved in host-pathogen interactions in diverse plant species. However, the role of CYPs during disease resistance in wheat remains largely elusive. In the present study, the systematic genome-wide survey revealed a set of 81 TaCYP genes from three subfamilies (GI, GII, and GIII) distributed on all 21 wheat chromosomes. The gene structures of TaCYP members were found to be highly variable, with 1-14 exons/introns and 15 conserved motifs. A network of miRNA targets with TaCYPs demonstrated that TaCYPs were targeted by multiple miRNAs and vice versa. Expression profiling was done in leaf rust susceptible Chinese spring (CS) and the CS-Ae. Umbellulata derived resistant IL "Transfer (TR). Three homoeologous TaCYP genes (TaCYP24, TaCYP31, and TaCYP36) showed high expression and three homoeologous TaCYP genes (TaCYP44, TaCYP49, and TaCYP54) showed low expression in TR relative to Chinese Spring. Most of the other TaCYPs showed comparable expression changes (down- or upregulation) in both contrasting TR and CS. Expression of 16 TaCYPs showed significant association (p < 0.05) with superoxide radical and hydrogen peroxide abundance, suggesting the role of TaCYPs in downstream signaling processes during wheat-leaf rust interaction. The differentially expressing TaCYPs may be potential targets for future validation using transgenic (overexpression, RNAi or CRISPR-CAS) approaches and for the development of leaf rust-resistant wheat genotypes.

Integrated breeding approaches to enhance the nutritional quality of food legumes.

Global food security, both in terms of quantity and quality remains as a challenge with the increasing population. In parallel, micronutrient deficiency in the human diet leads to malnutrition and several health-related problems collectively known as "hidden hunger" more prominent in developing countries around the globe. Biofortification is a potential tool to fortify grain legumes with micronutrients to mitigate the food and nutritional security of the ever-increasing population. Anti-nutritional factors like phytates, raffinose (RFO's), oxalates, tannin, etc. have adverse effects on human health upon consumption. Reduction of the anti-nutritional factors or preventing their accumulation offers opportunity for enhancing the intake of legumes in diet besides increasing the bioavailability of micronutrients. Integrated breeding methods are routinely being used to exploit the available genetic variability for micronutrients through modern "omic" technologies such as genomics, transcriptomics, ionomics, and metabolomics for developing biofortified grain legumes. Molecular mechanism of Fe/Zn uptake, phytate, and raffinose family oligosaccharides (RFOs) biosynthesis pathways have been elucidated. Transgenic, microRNAs and genome editing tools hold great promise for designing nutrient-dense and anti-nutrient-free grain legumes. In this review, we present the recent efforts toward manipulation of genes/QTLs regulating biofortification and Anti-nutrient accumulation in legumes using genetics-, genomics-, microRNA-, and genome editing-based approaches. We also discuss the success stories in legumes enrichment and recent advances in development of low Anti-nutrient lines. We hope that these emerging tools and techniques will expedite the efforts to develop micronutrient dense legume crop varieties devoid of Anti-nutritional factors that will serve to address the challenges like malnutrition and hidden hunger.

Genome Editing Targets for Improving Nutrient Use Efficiency and Nutrient Stress Adaptation.

In recent years, the development of RNA-guided genome editing (CRISPR-Cas9 technology) has revolutionized plant genome editing. Under nutrient deficiency conditions, different transcription factors and regulatory gene networks work together to maintain nutrient homeostasis. Improvement in the use efficiency of nitrogen (N), phosphorus (P) and potassium (K) is essential to ensure sustainable yield with enhanced quality and tolerance to stresses. This review outlines potential targets suitable for genome editing for understanding and improving nutrient use (NtUE) efficiency and nutrient stress tolerance. The different genome editing strategies for employing crucial negative and positive regulators are also described. Negative regulators of nutrient signalling are the potential targets for genome editing, that may improve nutrient uptake and stress signalling under resource-poor conditions. The promoter engineering by CRISPR/dead (d) Cas9 (dCas9) cytosine and adenine base editing and prime editing is a successful strategy to generate precise changes. CRISPR/dCas9 system also offers the added advantage of exploiting transcriptional activators/repressors for overexpression of genes of interest in a targeted manner. CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) are variants of CRISPR in which a dCas9 dependent transcription activation or interference is achieved. dCas9-SunTag system can be employed to engineer targeted gene activation and DNA methylation in plants. The development of nutrient use efficient plants through CRISPR-Cas technology will enhance the pace of genetic improvement for nutrient stress tolerance of crops and improve the sustainability of agriculture.

Genome-Wide Identification and Expression Analysis of the Thioredoxin (Trx) Gene Family Reveals Its Role in Leaf Rust Resistance in Wheat (Triticum aestivum L.).

Bread wheat (Triticum aestivum L.; Ta) is the staple cereal crop for the majority of the world's population. Leaf rust disease caused by the obligate fungal pathogen, Puccinia triticina L., is a biotrophic pathogen causing significant economic yield damage. The alteration in the redox homeostasis of the cell caused by various kinds of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in response to pathogenic infections is controlled by redox regulators. Thioredoxin (Trx) is one of the redox regulators with low molecular weight and is thermostable. Through a genome-wide approach, forty-two (42) wheat Trx genes (TaTrx) were identified across the wheat chromosome groups A, B, and D genomes containing 12, 16, and 14 Trx genes, respectively. Based on in silico expression analysis, 15 TaTrx genes were selected and utilized for further experimentation. These 15 genes were clustered into six groups by phylogenetic analysis. MicroRNA (miRNA) target analysis revealed eight different miRNA-targeted TaTrx genes. Protein-protein interaction (PPI) analysis showed TaTrx proteins interact with thioredoxin reductase, peroxiredoxin, and uncharacterized proteins. Expression profiles resulting from quantitative real-time PCR (qRT-PCR) revealed four TaTrx genes (TaTrx11-5A, TaTrx13-5B, TaTrx14-5D, and TaTrx15-3B) were significantly induced in response to leaf rust infection. Localization of ROS and its content estimation and an assay of antioxidant enzymes and expression analysis suggested that Trx have been involved in ROS homeostasis at span 24HAI-72HAI during the leaf rust resistance.

Interactive effect of elevated CO2 and nitrogen dose reprograms grain ionome and associated gene expression in bread wheat.

Wheat crop grown under elevated CO2 (EC) often have a lowered grain nitrogen (N) and protein concentration along with an altered grain ionome. The mechanistic understanding on the impact of CO2 x N interactions on the grain ionome and the expression of genes regulating grain ionome is scarce in wheat. In the present study, the interactive effect of EC and N dosage on grain yield, grain protein, grain ionome, tissue nitrate, and the expression of genes contributing to grain ionome (TaNAM-B1 and TaYSL6) are described. Three bread wheat genotypes were evaluated under two CO2 levels (Ambient CO2 (AC) of 400 ± 10 ppm and elevated CO2 (EC) of 700 ± 10 ppm) and two N levels (Low (LN) and Optimum N (ON). In EC, wheat genotypes HD2967 and HI 1500 recorded a significant decrease in grain nitrate content, while leaf and stem nitrate showed a significant increase. BT. Schomburgk (BTS), showed a significant increase in unassimilated nitrate and a decline in grain N and grain protein under EC. There was a general decline of grain ionome (N, P, K, Ca, Fe) in EC, except for grain Na content. The expression of genes TaNAM-B1 and TaYSL6 associated with protein and micronutrient remobilization to grains during senescence were affected by both EC and N treatments. For instance, in flag leaves of BTS, the expression of TaNAM-B1 and TaYSL6 were lower in EC-LN compared to AC-LN. In maturing spikes, transcript abundance of TaNAM-B1 and TaYSL6 were lower in EC in BTS. The altered transcript abundance of TaYSL6 and TaNAM-B1 in source and sink supports the change in grain ionome and suggests an N dependent transcriptional reprogramming in EC.

Analysis of NIA and GSNOR family genes and nitric oxide homeostasis in response to wheat-leaf rust interaction.

Nitric oxide (NO) modulates plant response to biotic and abiotic stresses by S-nitrosylation-mediated protein post-translational modification. Nitrate reductase (NR) and S-nitrosoglutathione reductase (GSNOR) enzymes are essential for NO synthesis and the maintenance of Nitric oxide/S-nitroso glutathione (NO/GSNO) homeostasis, respectively. S-nitrosoglutathione, formed by the S-nitrosylation reaction of NO with glutathione, plays a significant physiological role as the mobile reservoir of NO. The genome-wide analysis identified nine NR (NIA) and three GSNOR genes in the wheat genome. Phylogenic analysis revealed that the nine NIA genes +were clustered into four groups and the 3 GSNORs into two groups. qRT-PCR expression profiling of NIAs and GSNORs was done in Chinese spring (CS), a leaf rust susceptible wheat line showing compatible interaction, and Transfer (TR), leaf rust-resistant wheat line showing incompatible interaction, post-inoculation with leaf rust pathotype 77-5 (121-R-63). All the NIA genes showed upregulation during incompatible interaction in comparison with the compatible reaction. The GSNOR genes showed a variable pattern of expression: the TaGSNOR1 showed little change, whereas TaGSNOR2 showed higher expression during the incompatible response. TaGSNOR3 showed a rise of expression both in compatible and incompatible reactions. Before inoculation and after 72 h of pathogen inoculation, NO localization was studied in both compatible and incompatible reactions. The S-nitrosothiol accumulation, NR, and glutathione reductase activity showed a consistent increase in the incompatible interactions. The results demonstrate that both NR and GSNOR plays significant role in defence against the leaf rust pathogen in wheat by modulating NO homeostasis or signalling.

Interaction of elevated CO2 and form of nitrogen nutrition alters leaf abaxial and adaxial epidermal and stomatal anatomy of wheat seedlings.

Plant's stomatal physiology and anatomical features are highly plastic and are influenced by diverse environmental signals including the concentration of atmospheric CO2 and nutrient availability. Recent reports suggest that the form of nitrogen (N) is a determinant of plant growth and nutrient nitrogen use efficiency (NUE) under elevated CO2 (EC). Previously, we found that high nitrate availability resulted in early senescence, enhanced reactive oxygen species (ROS), and reactive nitrogen species (RNS) production and also that mixed nutrition of nitrate and ammonium ions were beneficial than sole nitrate nutrition in wheat. In this study, the interactive effects of different N forms (nitrate, ammonium, mixed nutrition of nitrate, and ammonium) and EC on epidermal and stomatal morphology were analyzed. Wheat seedlings were grown at two different CO2 levels and supplied with media devoid of N (N0) or with nitrate-N (NN), mixed nutrition of ammonium and nitrate (MN), or only ammonium-N (AN). The stoma length increased significantly in nitrate nutrition with a consistent reduction in stoma width. Guard cell length was higher in EC treatment as compared to AC. The guard cell width was maximum in AN-grown plants at EC. Epidermal cell density and stomatal density were lower at EC. Nitrate nutrition increased the stomatal area at EC while the reverse was true for MN and AN. Wheat plants fertilized with AN showed a higher accumulation of superoxide radical (SOR) at EC, while in NN treatment, the accumulation of hydrogen peroxide (H2O2) was higher at EC. Reactive oxygen species, particularly H2O2, can trigger mitogen-activated protein kinase (MAPK) mediated signaling and its crosstalk with abscisic acid (ABA) signaling to regulate stomatal anatomy in nitrate-fed plants. The SOR accumulation in ammonium- and ammonium nitrate-fed plants and H2O2 in NN-fed plants might finely regulate the sensitivity of stomata to alter water/nutrient use efficiency and productivity under EC. The data reveals that the variation in anatomical attributes viz. cell length, number of cells, etc. affected the leaf growth responses to EC and forms of N nutrition. These attributes are fine targets for effective manipulation of growth responses to EC.

Expression dynamics of genes encoding nitrate and ammonium assimilation enzymes in rice genotypes exposed to reproductive stage salinity stress.

Understanding the reproductive stage salinity stress tolerance is a key target for breeding stress tolerant rice genotypes. Nitrate and ammonium are equally important nitrogen forms utilized by rice. We evaluated nitrate and ammonium assimilation during reproductive stage in control and salinity (10dSm-1 using NaCl) stressed rice plants. Osmotic stress tolerant rice genotype Shabhagidhan (SD) and high yielding yet osmotic and salinity stress sensitive genotype Pusa sugandh-5 (PS5) were evaluated. Salinity stress was given to plants during panicle emergence and flag leaves was collected after 1d, 3d 5d, 7d, 9d,12d and 15d after anthesis. Reproductive stage salinity stress resulted in decrease of membrane stability, relative water content and osmotic potential of rice plants. Reproductive stage salinity stress decreased the expression of nitrate reductase (OsNIA), nitrite reductase (OsNiR), Glutamine synthetase (OsGLN1.1, OsGLN1.2, OsGLN2) and glutamate synthase/GOGAT (OsFd-GOGAT, OsNADH-GOGAT) in flag leaves. In response to stress, SD showed better stress tolerance than PS5 in terms of higher yield stability. Variety SD showed higher leaf nitrate and ammonium content and maintained comparatively higher nitrate and ammonia metabolism enzyme activity than PS5. Salinity stress upregulated the activity of glutamate dehydrogenase enzyme and indirectly contributed to the higher proline content and maintenance of favourable osmotic potential in SD. Expression of GS2 which has role in photo respiratory ammonia assimilation was upregulated by salinity stress in PS5 in comparison to SD. Rice genotype showing better induction of nitrogen assimilatory genes will be more tolerant to reproductive stage salinity stress.

Raffinose accumulation and preferential allocation of carbon (14 C) to developing leaves impart salinity tolerance in sugar beet.

Sugar beet is a salt-tolerant crop that can be explored for crop production in degraded saline soils. Seeds of multigerm genotypes LKC-2006 (susceptible) and LKC-HB (tolerant) were grown in 150 mM NaCl, from germination to 60 days after sowing, to decipher the mechanism of salinity tolerance at the vegetative stage. The biomass of the root and leaf were maintained in the tolerant genotype, LKC-HB, under saline conditions. Na+ /K+ ratios were similar in roots and leaves of LKC-HB, with lower values under salinity compared to LKC 2006. Infrared temperatures were 0.96°C lower in LKC-HB than in LKC-2006, which helped regulate the leaf water status under stressed conditions. Pulse-chase experiment showed that 14 C photosynthate was preferentially allocated towards the development of new leaves in the tolerant genotype. The sugar profile of leaves and roots showed accumulation of raffinose in leaves of LKC-HB, indicating a plausible role in imparting salinity tolerance by serving as an osmolyte or scavenger. The molecular analysis of the genes responsible for raffinose synthesis revealed an 18-fold increase in the expression of BvRS2 in the tolerant genotype, suggesting its involvement in raffinose synthesis. Our study accentuated that raffinose accumulation in leaves is vital for inducing salinity tolerance and maintenance of shoot dry weight in sugar beet.

Elevated CO2 differentially regulates root nitrate transporter kinetics in a genotype and nitrate dose-dependent manner.

The nitrogen (N) and protein concentration of wheat crop and grain often decline as a result of exposure of the crop to elevated CO2 (EC). In our earlier studies, it was found that the exacerbated production of nitric oxide (NO) represses the transcription of nitrate reductase (NR) and high affinity nitrate transporters (HATS) in EC grown wheat seedlings receiving high N. High N supply under EC also resulted in accumulation of reactive oxygen species (ROS), and reactive nitrogen species (RNS; NO and S- nitrosothiols) ensuing faster senescence and reduced N metabolite concentration in wheat. In this study, the effect of short-term exposure to EC on nitrate uptake kinetics was studied. The impact of EC on constitutive and inducible components of high affinity and low affinity nitrate uptake systems (HATS and LATS) were delineated in two wheat genotypes diverse in terms of nitrate uptake and assimilation capacities. Nitrate dose-response of NR was suppressed by EC in both leaf and root tissues. Plants grown under EC displayed a marked reduction in nitrate uptake kinetic components of LATS. Wheat genotype with high leaf nitrate assimilation capacity was able to maintain considerably higher nitrate uptake rate under EC albeit at a lower rate in comparison to ambient CO2. Wheat leaves exposed to EC displayed a comparatively higher abundance of NO and showed incremental abundance depending on increase in nitrate supply. Exogenous NO supply significantly suppressed the nitrate uptake rate of EC grown plants. Hence, EC-induced production of NO downregulates LATS kinetics in a genotype and nitrate dose-dependent manner.

Role of protein phosphatases in the regulation of nitrogen nutrition in plants.

The reversible protein phosphorylation and dephosphorylation mediated by protein kinases and phosphatases regulate different biological processes and their response to environmental cues, including nitrogen (N) availability. Nitrate assimilation is under the strict control of phosphorylation-dephosphorylation mediated post-translational regulation. The protein phosphatase family with approximately 150 members in Arabidopsis and around 130 members in rice is a promising player in N uptake and assimilation pathways. Protein phosphatase 2A (PP2A) enhances the activation of nitrate reductase (NR) by deactivating SnRK1 and reduces the binding of inhibitory 14-3-3 proteins on NR. The functioning of nitrate transporter NPF6.3 is regulated by phosphorylation of CBL9 (Calcineurin B like protein 9) and CIPK23 (CBL interacting protein kinase 23) module. Phosphorylation by CIPK23 inhibits the activity of NPF6.3, whereas protein phosphatases (PP2C) enhance the NPF6.3-dependent nitrate sensing. PP2Cs and CIPK23 also regulate ammonium transporters (AMTs). Under either moderate ammonium supply or high N demand, CIPK23 is bound and inactivated by PP2Cs. Ammonium uptake is mediated by nonphosphorylated and active AMT1s. Whereas, under high ammonium availability, CIPK23 gets activated and phosphorylate AMT1;1 and AMT1;2 rendering them inactive. Recent reports suggest the critical role of protein phosphatases in regulating N use efficiency (NUE). In rice, PP2C9 regulates NUE by improving N uptake and assimilation. Comparative leaf proteome of wild type and PP2C9 over-expressing transgenic rice lines showed 30 differentially expressed proteins under low N level. These proteins are involved in photosynthesis, N metabolism, signalling, and defence.

Elevated CO2 alters tissue balance of nitrogen metabolism and downregulates nitrogen assimilation and signalling gene expression in wheat seedlings receiving high nitrate supply.

Tissue and canopy-level evidence suggests that elevated carbon dioxide (EC) inhibits shoot nitrate assimilation in plants and thereby affects nitrogen (N) and protein content of the economic produce. It is speculated that species or genotypes relying more on root nitrate assimilation can adapt better under EC due to the improved/steady supply of reductants required for nitrate assimilation. A study was conducted to examine the effect of EC on N assimilation and associated gene expression in wheat seedlings. Wheat genotypes, BT-Schomburgk (BTS) with comparatively high leaf nitrate reductase (NR) activity and Gluyas Early (GE) with high root NR activity were grown in hydroponic culture for 30 days with two different nitrate levels (0.05 mM and 5 mM) in the climate controlled growth chambers maintained at either ambient (400 ± 10 µmol mol-1) or EC (700 ± 10 µmol mol-1) conditions. Exposure to EC downregulated the activity of enzyme NR and glutamate synthase (GOGAT) in leaf tissues, whereas in roots, activities of both the enzymes were upregulated by exposure to EC. In addition, EC downregulated N assimilation and signalling gene expression under high N availability. Root N assimilation was less affected in comparison with shoot N assimilation; thereby, the proportion of root contribution towards total assimilation was higher. The results suggest that EC could alter and re-programme N assimilation and signalling in wheat seedlings. The genotype and tissue-specific effects of EC on N assimilation also warrants the need for identification of suitable genotypes and revision of fertiliser regime for tapping the beneficial effects of EC conditions.