Vikram Singh Gaur

Influence of nitrogen on the expression of TaDof1 transcription factor in wheat and its relationship with photo synthetic and ammonium assimilating efficiency

Nitrogen is a crucial macronutrient needed in the greatest amount of all mineral elements required by plants. Development of crop varieties with high nitrogen use efficiency (NUE) is imperative for sustainable agriculture. Understanding how plant genes respond to different nitrogen conditions is essential for formulating approaches, for manipulating genes, for improving NUE. In the present study we analyzed the activity of three different enzymes involved in nitrogen assimilation viz., GS, GOGAT and GDH along with physiological parameters like chlorophyll variable yield (Fv/Fmax), photosynthesis rate and total chlorophyll content at four different growth stages of wheat plant development under different nitrogen treatments. For this study two different wheat varieties UP-2644 and Raj-4097 having high and low protein content, respectively in the grains were chosen. Gene expression profile of a Dof transcription factor (TaDof1 of wheat) was also included in the study to assess its role in nitrogen metabolism. Densitometry analysis at S2 and S3 stage of wheat spikes of both the wheat varieties grown at different nitrogen treatments showed that TaDof1 expression was up-regulated in low nitrogen treatment. In S3 stage, in high protein content wheat variety UP-2644, TaDof1 expression was elevated in low and normal nitrogen treatment as compared to high nitrogen treatment. The gene expression profile of Dof 1 was found to coincide with the enzyme activities of GS, GOGAT at the S3 stage. The activities of these enzymes were prolonged in the high protein content variety. Since, Dof transcription factor(s) have been previously reported to control the expression of genes involved nitrogen assimilation i.e., GS and GOGAT and may be the elevated expression of Dof 1 at the grain filling stage over expresses the GS and GOGAT genes thereby prolonging their activities.

Transcriptional profiling and in silico analysis of Dof transcription factor gene family for understanding their regulation during seed development of rice Oryza sativa L.

Seed development is a complex process controlled by temporal and spatial expression of many transcription factors (TF) inside the developing seed. In the present study, transcript profiles of all the 30 members of rice DofTFs from flowering to seed development stages were analyzed. It was found that 16 Dof genes besides a previously characterized Dof gene ‘RPBF’ are differentially expressed during the seed development and unlike RPBF are not seed specific. Based on the expression patterns, these rice DofTFs were categorized into four groups-6 genes were constitutive while 4 genes were up-regulated and 3 genes were down regulated and four genes were maximally expressed at specific stages of seed development viz. one gene at flowering, two genes at watery ripe and one gene at milky stage. The involvement of more than one gene at different stages of seed development is suggestive of combinatorial regulation of their downstream genes involved in seed development. In silico expression analysis of wheat and Arabidopsis Dof Tfs also revealed that more than 50% of the Dof genes are expressed during the seed development process. Further in silico study of regulatory elements present in the promoters of these genes revealed the presence of some unique and common motifs in the promoters of rice and wheat Dof genes which indicate that Dof genes are possibly involved in ethylene and jasmonate signaling pathways affecting grain filling and grain quality. These Dof genes containing ethylene responsive motifs in their promoter region could possibly be the targets of recently identified Sub1 gene which codes for a ethylene responsive factor.

Systems Biology for Smart Crops and Agricultural Innovation: Filling the Gaps between Genotype and Phenotype for Complex Traits Linked with Robust Agricultural Productivity and Sustainability.

In recent years, rapid developments in several omics platforms and next generation sequencing technology have generated a huge amount of biological data about plants. Systems biology aims to develop and use well-organized and efficient algorithms, data structure, visualization, and communication tools for the integration of these biological data with the goal of computational modeling and simulation. It studies crop plant systems by systematically perturbing them, checking the gene, protein, and informational pathway responses; integrating these data; and finally, formulating mathematical models that describe the structure of system and its response to individual perturbations. Consequently, systems biology approaches, such as integrative and predictive ones, hold immense potential in understanding of molecular mechanism of agriculturally important complex traits linked to agricultural productivity. This has led to identification of some key genes and proteins involved in networks of pathways involved in input use efficiency, biotic and abiotic stress resistance, photosynthesis efficiency, root, stem and leaf architecture, and nutrient mobilization. The developments in the above fields have made it possible to design smart crops with superior agronomic traits through genetic manipulation of key candidate genes.

In Silico Analysis of Expression Data for Identification of Genes Involved in Spatial Accumulation of Calcium in Developing Seeds of Rice

The calcium (Ca(2+)) transporters, like Ca(2+) channels, Ca(2+) ATPases, and Ca(2+) exchangers, are instrumental for signaling and transport. However, the mechanism by which they orchestrate the accumulation of Ca(2+) in grain filling has not yet been investigated. Hence the present study was designed to identify the potential calcium transporter genes that may be responsible for the spatial accumulation of calcium during grain filling. In silico expression analyses were performed to identify Ca(2+) transporters that predominantly express during the different developmental stages of Oryza sativa. A total of 13 unique calcium transporters (7 from massively parallel signature sequencing [MPSS] data analysis, and 9 from microarray analysis) were identified. Analysis of variance (ANOVA) revealed differential expression of the transporters across tissues, and principal component analysis (PCA) exhibited their seed-specific distinctive expression profile. Interestingly, Ca(2+) exchanger genes are highly expressed in the initial stages, whereas some Ca(2+) ATPase genes are highly expressed throughout seed development. Furthermore, analysis of the cis-elements located in the promoter region of the subset of 13 genes suggested that D of proteins play essential roles in regulating the expression of Ca(2+) transporter genes during rice seed development. Based on these results, we developed a hypothetical model explaining the transport and tissue specific distribution of calcium in developing cereal seeds. The model may be extrapolated to understand the mechanism behind the exceptionally high level of calcium accumulation seen in grains like finger millet.

Relationship of Nitrogen Use Efficiency with the Activities of Enzymes Involved in Nitrogen Uptake and Assimilation of Finger Millet Genotypes Grown under Different Nitrogen Inputs

Nitrogen responsiveness of three-finger millet genotypes (differing in their seed coat colour) PRM-1 (brown), PRM-701 (golden), and PRM-801 (white) grown under different nitrogen doses was determined by analyzing the growth, yield parameters and activities of nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase; GOGAT, and glutamate dehydrogenase (GDH) at different developmental stages. High nitrogen use efficiency and nitrogen utilization efficiency were observed in PRM-1 genotype, whereas high nitrogen uptake efficiency was observed in PRM-801 genotype. At grain filling nitrogen uptake efficiency in PRM-1 negatively correlated with NR, GS, GOGAT activities whereas it was positively correlated in PRM-701 and PRM-801, however, GDH showed a negative correlation. Growth and yield parameters indicated that PRM-1 responds well at high nitrogen conditions while PRM-701 and PRM-801 respond well at normal and low nitrogen conditions respectively. The study indicates that PRM-1 is high nitrogen responsive and has high nitrogen use efficiency, whereas golden PRM-701 and white PRM-801 are low nitrogen responsive genotypes and have low nitrogen use efficiency. However, the crude grain protein content was higher in PRM-801 genotype followed by PRM-701 and PRM-1, indicating negative correlation of nitrogen use efficiency with source to sink relationship in terms of seed protein content.

Fluctuation of Dof1/Dof2 expression ratio under the influence of varying nitrogen and light conditions: involvement in differential regulation of nitrogen metabolism in two genotypes of finger millet (Eleusine coracana L.)

In order to gain insights into the mechanism of high nitrogen use efficiency (NUE) of finger millet (FM) the role of Dof2 transcription factor (TF), which is a repressor of genes involved in C/N metabolism was investigated. The partial cDNA fragment of EcDof2 (912-bp; GenBank acc. no. KF261117) was isolated and characterized from finger millet (FM) that showed 63% and 58% homology with Dof2 of Zea mays at nucleotide and protein level, respectively. Its expression studies were carried out along with the activator EcDof1 in two genotypes (GE3885, high protein genotype (HPG); GE1437, low protein genotype (LPG)) of FM differing in grain protein contents (13.8% and 6.2%) showed that EcDof2 is expressed in both shoot and root tissues with significantly (p≤0.05) higher expression in the roots. The diurnal expression of both EcDof1 and EcDof2 in shoots was differential having different time of peak expression indicating a differential response to diurnal condition. Under continuous dark conditions, expression of EcDof1 and EcDof2 oscillated in both the genotypes whereas on illumination, the fold expression of EcDof1 was higher as compared to EcDof2. Under increasing nitrate concentration, EcDof2 expression increases in roots and shoots of LPG while it remains unchanged in HPG. However, the EcDof1 expression was found to increase in both genotypes. Further, time kinetics studies under single nitrate concentration revealed that EcDof2 was repressed in the roots of both genotypes whereas EcDof1 oscillated with time. The EcDof1/EcDof2 ratio measured showed differential response under different light and nitrogen conditions. It was higher in the roots of HPG indicating higher activation of genes involved in N uptake and assimilation resulting in high grain protein accumulation. The results indicate that both light and nitrogen concentration influence Dof1 and Dof2 expression and suggests a complex pattern of regulation of genes influenced by these plant specific TFs. In nutshell, the Dof1/Dof2 ratio can serve as an index for measuring the N responsiveness and NUE of crops and can be further validated by Dof2 knock down approach.

Gene Discovery and Advances in Finger Millet [Eleusine coracana (L.) Gaertn.] Genomics-An Important Nutri-Cereal of Future.

The rapid strides in molecular marker technologies followed by genomics, and next generation sequencing advancements in three major crops (rice, maize and wheat) of the world have given opportunities for their use in the orphan, but highly valuable future crops, including finger millet [Eleusine coracana (L.) Gaertn.]. Finger millet has many special agronomic and nutritional characteristics, which make it an indispensable crop in arid, semi-arid, hilly and tribal areas of India and Africa. The crop has proven its adaptability in harsh conditions and has shown resilience to climate change. The adaptability traits of finger millet have shown the advantage over major cereal grains under stress conditions, revealing it as a storehouse of important genomic resources for crop improvement. Although new technologies for genomic studies are now available, progress in identifying and tapping these important alleles or genes is lacking. RAPDs were the default choice for genetic diversity studies in the crop until the last decade, but the subsequent development of SSRs and comparative genomics paved the way for the marker assisted selection in finger millet. Resistance gene homologs from NBS-LRR region of finger millet for blast and sequence variants for nutritional traits from other cereals have been developed and used invariably. Population structure analysis studies exhibit 2–4 sub-populations in the finger millet gene pool with separate grouping of Indian and exotic genotypes. Recently, the omics technologies have been efficiently applied to understand the nutritional variation, drought tolerance and gene mining. Progress has also occurred with respect to transgenics development. This review presents the current biotechnological advancements along with research gaps and future perspective of genomic research in finger millet.

Differential induction of two different cystatin genes during pathogenesis of Karnal bunt (Tilletia indica) in wheat under the influence of jasmonic acid

In the present study, expression patterns of two different wheat cystatins (WCs) were studied under the influence of jasmonate signaling in triggering resistance against Karnal bunt (KB). Cystatins are cysteine proteinase inhibitors (CPI) constituting a multigene family which regulate the activity of endo- and/or exogenous cysteine proteinases (CP). Two wheat varieties HD-29 (resistant, R) and WH-542 (susceptible, S) were pre-conditioned with jasmonate and then artificially inoculated with sporidial suspension of Tilletia indica to study its influence in inducing defense by regulating cystatin genes. On the transcriptional level, WC4 and WC5 gave different temporal expression patterns. Expression of WC4 was higher in boot emergence stage which is most susceptible to KB and then slowly declined in both varieties. Expression of WC5 showed an entirely reverse pattern of expression, which kept on rising as the grains matured. Cystatin activity determination by inhibitor assay gave higher activity in resistant variety and under JA treatment. Estimation of specific activity of total cystatin at different days after inoculation (DAI) showed that JA positively induced cystatin expression in both varieties but R variety always registered a greater cystatin expression than the susceptible one (P<0.05). In plants inoculated with pathogen, initially there was a rise in cystatin activity which gradually decreased 7 DAI when compared with the un-inoculated plants. Based on these findings it is clearly demonstrated that jasmonate acts as a potential activator of induced resistance by up-regulating cystatin expression and provides the conditioning effect prior to infection through the maintenance of critical balance of CP/CPI interaction. However, different cystatin genes show different temporal expression patterns and may play different roles at various developmental stages of the grain.

PREDICTION OF DOWNSTREAM INTERACTION OF TRANSCRIPTION FACTORS WITH MAPK3 IN ARABIDOPSIS THALIANA USING PROTEIN SEQUENCE INFORMATION

Protein*Protein interactions (PPIs) are vital to most biological processes thus the identification of PPIs is of primary importance. In the present work, we endeavor to identify the downstream interaction partners of Mitogen Activated Protein Kinase3 (MAPK3) in Arabidopsis Thaliana using the information of protein sequences through Support Vector Machine (SVM) approach. The approach here used is supervised learning based on physiochemical properties of protein sequences through which we predict whether the MAPK3 proteins interact with downstream transcription factor proteins viz., Myb, bZIP, WRKY, Myb*related proteins, AP2/EREBP, and NAC with which its interaction is almost unknown. The Myb*related transcription factor family is showing maximum interaction percentage i.e. 71.14% with MAPK3 while minimum interaction percentage is 21.15% which is shown by NAC transcription factor family. The interaction percentage shown by the gene loci of rest transcription factor family i.e . Myb, bZIP, AP2/EREBP, WRKY are 67.78%, 68.05%, 21.91% and 58.33% respectively. The results of our study clearly revealed the complexity of MAPK3 interaction with several variants of different transcription factors and the same can be verified by different methodology of wet lab experimentation for elucidating the role in various biological processes.

Identification of biomarker for determining genotypic potential of nitrogen-use-efficiency and optimization of the nitrogen inputs in crop plants

Worldwide, the nitrogen use efficiency (NUE) for crop plants is of great concern. The burgeoning world population needs crop genotypes that respond to higher nitrogen and show a direct relationship to yield with use of nitrogen inputs, i.e. high nitrogen-responsive genotypes. However, for fulfilling the high global demand of organic produce, it requires the low nitrogen responsive genotypes with greater NUE and grain yields. The lack of knowledge about precise regulatory mechanisms to explain NUE in crop plants hampers the goal of agricultural productivity. Understanding the molecular basis of NUE will enable to provide handle for crop improvement through biotechnological means. With the advent of modern genomics and proteomics approaches such as subtractive hybridization, differential display, and microarray techniques are revolutionizing to identify the candidate genes which play a pivotal role in the regulation of NUE. Beside it, quantitative real-time polymerase chain reaction technology is also being used to establish marker-trait association for NUE. The identification of potential candidate genes/proteins in the regulation of NUE will serve as biomarker(s) for screening genotypes for their nitrogen responsiveness for optimization of nitrogen input in agriculture. This paper describes the molecular basis of NUE with respect to nitrogen metabolism and its intimate relationship with carbon metabolism, use of molecular-physiological-genetics approaches for understanding the role of various genes/proteins, and their validation to use as biomarker(s) for determining genotypic potential for NUE. Since NUE in plants is a complex trait which not only involves the primary process of nitrogen uptake and assimilatory pathways but also a series of events, including metabolite partitioning, secondary remobilization, C-N interactions, as well as molecular signalling pathways and regulatory control outside the metabolic cascades. Therefore, identification of novel nitrogen responsive genes and their cis- and trans-acting gene elements is essential. Thus, fishing out a single gene, biomarker or a master regulator controlling complex trait of NUE could serve as an appropriate strategy for nitrogen management in agriculture.