Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa.

14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1-5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses.

Nitrogen stress-induced alterations in the leaf proteome of two wheat varieties grown at different nitrogen levels.

Inorganic nitrogen (N) is a key limiting factor of the agricultural productivity. Nitrogen utilization efficiency has significant impact on crop growth and yield as well as on the reduction in production cost. The excessive nitrogen application is accompanied with severe negative impact on environment. Thus to reduce the environmental contamination, improving NUE is need of an hour. In our study we have deployed comparative proteome analysis using 2-DE to investigate the effect of the nitrogen nutrition on differential expression pattern of leaf proteins in low-N sensitive and low-N tolerant wheat (Triticum aestivum L.) varieties. Results showed a comprehensive picture of the post-transcriptional response to different nitrogen regimes administered which would be expected to serve as a basic platform for further characterization of gene function and regulation. We detected proteins related to photosynthesis, glycolysis, nitrogen metabolism, sulphur metabolism and defence. Our results provide new insights towards the altered protein pattern in response to N stress. Through this study we suggest that genes functioning in many physiological events coordinate the response to availability of nitrogen and also for the improvement of NUE of crops.

Relevance of proteomic investigations in plant abiotic stress physiology.

Plant growth and productivity are influenced by various abiotic stresses. Stressful conditions may lead to delays in seed germination, reduced seedling growth, and decreased crop yields. Plants respond to environmental stresses via differential expression of a subset of genes, which results in changes in omic compositions, such as transcriptome, proteome, and metabolome. Since the development of modern biotechnology, various research projects have been carried out to understand the approaches that plants have adopted to overcome environmental stresses. Advancements in omics have made functional genomics easy to understand. Since the fundamentals of classical genomics were unable to clear up confusion related to the functional aspects of the metabolic processes taking place during stress conditions, new fields have been designed and are known as omics. Proteomics, the analysis of genomic complements of proteins, has caused a flurry of activity in the past few years. It defines protein functions in cells and explains how those protein functions respond to changing environmental conditions. The ability of crop plants to cope up with the variety of environmental stresses depends on a number of changes in their proteins, which may be up- and downregulated as a result of altered gene expression. Most of these molecules display an essential function, either in the regulation of the response (e.g., components of the signal transduction pathway), or in the adaptation process (e.g., enzymes involved in stress repair and degradation of damaged cellular contents), allowing plants to recover and survive the stress. Many of these proteins are constitutively expressed under normal conditions, but when under stress, they undergo a modification of their expression levels. This review will explain how proteomics can help in elucidating important plant processes in response to various abiotic stresses.

Genotypic variability among soybean genotypes under NaCl stress and proteome analysis of salt-tolerant genotype.

The present investigation was conducted to evaluate salt tolerance in ten genotypes of soybean (Glycine max L.). Twelve-day-old seedlings, grown hydroponically, were treated with 0, 25, 50, 75, 100, 125 and 150 mM NaCl for 10 days. Growth, lipid peroxidation and antioxidant enzyme activities were evaluated. Growth, measured in terms of length, fresh weight and dry weight of plants, was drastically reduced in Pusa-24 while there was little effect of NaCl treatment on Pusa-37 genotype of soybean. High level of lipid peroxidation was observed in Pusa-24 as indicated by increased level of malondialdehyde. Activities of superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase were maximum in Pusa-37 where 9-, 1-, 5- and 6-fold increase over control were observed, respectively. The results suggested that Pusa-24 and Pusa-37 are salt-sensitive and salt-tolerant genotype of soybean, respectively, and antioxidant defence system is involved in conferring the sensitiveness and tolerance in these genotypes. Salt-tolerant genotype Pusa-37, was further analysed by 2-dimensional gel electrophoresis to analyse the differential expression of proteins at high salt stress. In the present study, 173 protein spots were identified. Of these, 40 proteins were responsive to salinity in that they were either up- or downregulated. This study could help us in identifying the possible regulatory switches (gene/s) controlling novel proteins of the salt-tolerant genotype of the crop plants and their possible role in defence mechanism.

Proteomic analysis for low and high nitrogen-responsive proteins in the leaves of rice genotypes grown at three nitrogen levels.

Nitrogen (N) is an essential nutrient for plants. Increase in crop production is associated with increase in N fertilizers. Excessive use of N fertilizers and the low nitrogen utilization efficiency by crop plants is a major cause for environmental damage. Therefore, to reduce the N-fertilizer pollution, there is an urgent need to improve nitrogen use efficiency. Identification and/or development of genotypes which can grow and yield well at low nitrogen levels may provide a solution. Understanding the molecular mechanism of differential nitrogen use efficiency of the genotypes may provide some clues. Keeping the above facts in mind, in this study we have identified the high N-responsive and low N-responsive contrasting rice genotypes, out of 20 genotypes that were grown at low (1 mM), moderate (10 mM), and high (25 mM) levels of N (KNO(3)). Proteome analysis of leaves revealed that the proteins involved in the energy production/regulation and metabolism in plant leaf tissues are differentially expressed under N treatments. Moreover, some disease-resistant and stress-induced proteins were found to be overexpressed at high levels of N. The present study could be useful in identifying proteins responding to different levels of nitrogen fertilization, which may open new avenues for a better understanding of N use efficiency, and for developing new strategies to enhance N efficiency in cereal crops.

Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR.

The real time quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes), ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes), in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.

Sulfur starvation and restoration affect nitrate uptake and assimilation in rapeseed.

We analyzed the effect of omission of sulfur (S) from the nutrient solution and then restoration of S-source on the uptake and assimilation of nitrate in rapeseed. Incubation in nutrient solution without S for 1-6 days led to decline in uptake of nitrate, activities, and expression levels of nitrate reductase (NR) and glutamine synthetase (GS). The nitrite reductase (NiR) and glutamate synthase (GOGAT) activities were not considerably affected. There was significant enhancement in nitrate content and decline in sulfate content. Evaluation of amino acid profile under S-starvation conditions showed two- to fourfold enhancement in the contents of arginine, asparagine and O-acetyl-L-serine (OAS), whereas the contents of cysteine and methionine were reduced heavily. When the S-starved plants were subjected to restoration of S for 1, 3, 5, and 7 days, activities and expression levels of NR and GS recovered within the fifth and seventh days of restoration, respectively. Exogenous supply of metabolites (arginine, asparagine, cysteine, glutamine, OAS, and methionine) also affected the uptake and assimilation of nitrate, with a maximum for OAS. These results corroborate the tight interconnection of S-nutrition with nitrate assimilation and that OAS plays a major role in this regulation. The study must be helpful in developing a nutrient-management technology for optimization of crop productivity.

Variability in Indian bread wheat (Triticum aestivum L.) varieties differing in nitrogen efficiency as assessed by microsatellite markers.

Wheat (Triticum aestivum L.) is a staple food for half of the world. Its productivity and agronomical practices, especially for nitrogen supplementation, is governed by the nitrogen efficiency (NE) of the genotypes. We analyzed 16 popular cultivated Indian varieties of wheat for their NE and variability estimates using a set of 21 simple sequence repeat (SSR) markers, derived from each wheat chromosome. These genotypes were categorized into three groups, viz., low, moderate, and high nitrogen efficient. Of these 16 genotypes, we have reported six, eight, and two genotypes in high, moderate, and low NE categories, respectively. The differential NE in these genotypes was supported by nitrogen uptake and assimilation parameters. The values of average polymorphic information content and marker index for these SSR markers were estimated to be 0.32 and 0.59, respectively. The genetic similarity coefficient for all possible pairs of varieties ranged from 0.41 to 0.76, indicating the presence of considerable range of genetic diversity at molecular level. The dendrogram prepared on the basis of unweighted pair-group method of arithmetic average algorithm grouped the 16 wheat varieties into three major clusters. The clustering was strongly supported by high bootstrap values. The distribution of the varieties in different clusters and subclusters appeared to be related to their variability in NE parameter that was scored. Genetically diverse parents were identified that could potentially be used for their desirable characteristics in breeding programs for improvement of NE in wheat.