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.

Deciphering the melatonin-mediated response and signalling in the regulation of heavy metal stress in plants.

MAIN CONCLUSION: Melatonin has a protective effect against heavy metal stress in plants by immobilizing HM in cell walls and sequestering them in root cell vacuoles, reducing HM's translocation from roots to shoots. It enhances osmolyte production, increases antioxidant enzyme activity, and improves photosynthesis, thereby improving cellular functions. Understanding the melatonin-mediated response and signalling can sustain crop production in heavy metal-stressed soils. Melatonin is a pleiotropic signal molecule that plays a critical role in plant growth and stress tolerance, particularly against heavy metals in soil. Heavy metals (HMs) are ubiquitously found in the soil-water environment and readily taken up by plants, thereby disrupting mineral nutrient homeostasis, osmotic balance, oxidative stress, and altered primary and secondary metabolism. Plants combat HM stress through inbuilt defensive mechanisms, such as metal exclusion, restricted foliar translocation, metal sequestration and compartmentalization, chelation, and scavenging of free radicals by antioxidant enzymes. Melatonin has a protective effect against the damaging effects of HM stress in plants. It achieves this by immobilizing HM in cell walls and sequestering them in root cell vacuoles, reducing HM's translocation from roots to shoots. This mechanism improves the uptake of macronutrients and micronutrients in plants. Additionally, melatonin enhances osmolyte production, improving the plant's water relations, and increasing the activity of antioxidant enzymes to limit lipid peroxidation and reactive oxygen species (ROS) levels. Melatonin also decreases chlorophyll degradation while increasing its synthesis, and enhances RuBisCO activity for better photosynthesis. All these functions contribute to improving the cellular functions of plants exposed to HM stress. This review aims to gain better insight into the melatonin-mediated response and signalling under HM stress in plants, which may be useful in sustaining crop production in heavy metal-stressed soils.

From source to sink: mechanistic insight of photoassimilates synthesis and partitioning under high temperature and elevated [CO2].

Photosynthesis is the vital metabolism of the plant affected by abiotic stress such as high temperature and elevated [CO2] levels, which ultimately affect the source-sink relationship. Triose phosphate, the primary precursor of carbohydrate (starch and sucrose) synthesis in the plant, depends on environmental cues. The synthesis of starch in the chloroplasts of leaves (during the day), the transport of photoassimilates (sucrose) from source to sink, the loading and unloading of photoassimilates, and the accumulation of starch in the sink tissue all require a highly regulated network and communication system within the plant. These processes might be affected by high-temperature stress and elevated [CO2] conditions. Generally, elevated [CO2] levels enhance plant growth, photosynthetic rate, starch synthesis, and accumulation, ultimately diluting the nutrient of sink tissues. On the contrary, high-temperature stress is detrimental to plant development affecting photosynthesis, starch synthesis, sucrose synthesis and transport, and photoassimilate accumulation in sink tissues. Moreover, these environmental conditions also negatively impact the quality attributes such as grain/tuber quality, cooking quality, nutritional status in the edible parts and organoleptic traits. In this review, we have attempted to provide an insight into the source-sink relationship and the sugar metabolites synthesized and utilized by the plant under elevated [CO2] and high-temperature stress. This review will help future researchers comprehend the source-sink process for crop growth under changing climate scenarios.

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.

CO2 Elevation Accelerates Phenology and Alters Carbon/Nitrogen Metabolism vis-à-vis ROS Abundance in Bread Wheat.

Wheat is an important staple food crop of the world and it accounts for 18-20% of human dietary protein. Recent reports suggest that CO2 elevation (CE) reduces grain protein and micronutrient content. In our earlier study, it was found that the enhanced production of nitric oxide (NO) and the concomitant decrease in transcript abundance as well as activity of nitrate reductase (NR) and high affinity nitrate transporters (HATS) resulted in CE-mediated decrease in N metabolites in wheat seedlings. In the current study, two bread wheat genotypes Gluyas Early and B.T. Schomburgk differing in nitrate uptake and assimilation properties were evaluated for their response to CE. To understand the impact of low (LN), optimal (ON) and high (HN) nitrogen supply on plant growth, phenology, N and C metabolism, ROS and RNS signaling and yield, plants were evaluated under short term (hydroponics experiment) and long term (pot experiment) CE. CE improved growth, altered N assimilation, C/N ratio, N use efficiency (NUE) in B.T. Schomburgk. In general, CE decreased shoot N concentration and grain protein concentration in wheat irrespective of N supply. CE accelerated phenology and resulted in early flowering of both the wheat genotypes. Plants grown under CE showed higher levels of nitrosothiol and ROS, mainly under optimal and high nitrogen supply. Photorespiratory ammonia assimilating genes were down regulated by CE, whereas, expression of nitrate transporter/NPF genes were differentially regulated between genotypes by CE under different N availability. The response to CE was dependent on N supply as well as genotype. Hence, N fertilizer recommendation needs to be revised based on these variables for improving plant responses to N fertilization under a future CE scenario.

Elevated CO2-induced production of nitric oxide differentially modulates nitrate assimilation and root growth of wheat seedlings in a nitrate dose-dependent manner.

Wheat is a major staple food crop worldwide contributing approximately 20% of total protein consumed by mankind. The nitrogen and protein concentration of wheat crop and grain often decline as a result of exposure of the crop to elevated CO2 (EC). The changes in nitrogen (N) assimilation, root system architecture, and nitric oxide (NO)-mediated N signaling and expression of genes involved in N assimilation and high affinity nitrate uptake were examined in response to different nitrate levels and EC in wheat. Activity of enzyme nitrate reductase (NRA) was downregulated under EC both in leaf and root tissues. Plants grown under EC displayed enhanced production of NO and more so when nitrate supply was high. Based on exogenous supply of NO, inhibitors of NO production, and NO scavenger, regulatory role of NO on EC mediated changes in root morphology and NRA was revealed. The enhanced NO production under EC and high N levels negatively regulated the transcript abundance of NR and high affinity nitrate transporters (HATS).

Visualization of Nitric Oxide, Measurement of Nitrosothiols Content, Activity of NOS and NR in Wheat Seedlings.

Nitric oxide (NO), is a redox-active, endogenous signalling molecule involved in the regulation of numerous processes. It plays a crucial role in adaptation and tolerance to various abiotic and biotic stresses. In higher plants, NO is produced either by enzymatic or non-enzymatic reduction of nitrite and an oxidative pathway requiring a putative nitric oxide synthase (NOS)-like enzyme. There are several methods to measure NO production: mass spectrometry, tissue localization by DAF-FM dye. Electron paramagnetic resonance (EPR) also known as electron spin resonance (ESR) and spectrophotometric assays. The activity of NOS can be measured by L-citrulline based assay and spectroscopic method (NADPH utilization method). A major route for the transfer of NO bioactivity is S-nitrosylation, the addition of a NO moiety to a protein cysteine thiol forming an S-nitrosothiol (SNO). This experimental method describes visualization of NO using DAF-FM dye by fluorescence microscopy (Zeiss AXIOSKOP 2). The whole procedure is simplified, so it is easy to perform but has a high sensitivity for NO detection. In addition, spectrophotometry based protocols for assay of NOS, Nitrate Reductase (NR) and the content of S-nitrosothiols are also described. These spectrophotometric protocols are easy to perform, less expensive and sufficiently sensitive assays which provide adequate information on NO based regulation of physiological processes depending on the treatments of interest.