Nitrate signals determine the sensing of nitrogen through differential expression of genes involved in nitrogen uptake and assimilation in finger millet.

In order to understand the molecular basis of high nitrogen use efficiency of finger millet, five genes (EcHNRT2, EcLNRT1, EcNADH-NR, EcGS, and EcFd-GOGAT) involved in nitrate uptake and assimilation were isolated using conserved primer approaches. Expression profiles of these five genes along with the previously isolated EcDof1 was studied under increased KNO3 concentrations (0.15 to 1,500 µM) for 2 h as well as at 1.5 µM for 24 h in the roots and shoots of 25 days old nitrogen deprived two contrasting finger millet genotypes (GE-3885 and GE-1437) differing in grain protein content (13.76 and 6.15 %, respectively). Time kinetics experiment revealed that, all the five genes except EcHNRT2 in the leaves of GE-3885 were induced within 30 min of nitrate exposure indicating that there might be a greater nitrogen deficit in leaves and therefore quick transportation of nitrate signals to the leaves. Exposing the plants to increasing nitrate concentrations for 2 h showed that in roots of GE-3885, NR was strongly induced while GS was repressed; however, the pattern was found to be reversed in leaves of GE-1437 indicating that in GE-3885, most of the nitrate might be reduced in the roots but assimilated in leaves and vice-versa. Furthermore, compared with the low-protein genotype, expression of HNRT2 was strongly induced in both roots and shoots of high-protein genotype at the least nitrate concentration supplied. This further indicates that GE-3885 is a quick sensor of nitrogen compared with the low-protein genotype. Furthermore, expression of EcDof1 was also found to overlap the expression of NR, GS, and GOGAT indicating that Dof1 probably regulates the expression of these genes under different conditions by sensing the nitrogen fluctuations around the root zone.

Understanding the differential nitrogen sensing mechanism in rice genotypes through expression analysis of high and low affinity ammonium transporter genes.

Two rice genotypes, Kalanamak 3119 (KN3119) and Pusa Basmati 1(PB1) differing in their optimum nitrogen requirements (30 and 120 kg/ha, respectively) were undertaken to study the expression of both high and low affinity ammonium transporter genes responsible for ammonium uptake. Exposing the roots of the seedlings of both the genotypes to increasing (NH(4))(2)SO(4) concentrations revealed that all the three families of rice AMT genes are expressed, some of which get altered in a genotype and concentration specific manner. This indicates that individual ammonium transporter genes have defined contributions for ammonium uptake and plant growth. Interestingly, in response to increasing nitrogen concentrations, a root specific high affinity gene, AMT1;3, was repressed in the roots of KN3119 but not in PB1 indicating the existence of a differential ammonium sensing mechanism. This also indicates that not only AMT1;3 is involved not only in ammonium uptake but may also in ammonium sensing. Further, if it can differentiate and could be used as a biomarker for nitrogen responsiveness. Expression analysis of low affinity AMT genes showed that, both AMT2;1 and AMT2;2 have high levels of expression in both roots and shoots and in KN3119 are induced at low ammonium concentrations. Expressions of AMT3 family genes were higher shoots than in the roots indicating that these genes are probably involved in the translocation and distribution of ammonium ions in leaves. The expression of the only high affinity AMT gene, AMT1;1, along with six low affinity AMT genes in the shoots suggests that low affinity AMTs in the shoots leaves are involved in supporting AMT1;1 to carry out its activities/function efficiently.

Influence of different nitrogen inputs on the members of ammonium transporter and glutamine synthetase genes in two rice genotypes having differential responsiveness to nitrogen.

Two aromatic rice genotypes, Pusa Basmati 1 (PB1) and Kalanamak 3119 (KN3119) having 120 and 30 kg/ha optimum nitrogen requirement respectively, to produce optimal yield, were chosen to understand their differential nitrogen responsiveness. Both the genotypes grown under increasing nitrogen inputs showed differences in seed/panicle, 1,000 seed weight, %nitrogen in the biomass and protein content in the seeds. All these parameters in PB1 were found to be in the increasing order in contrast to KN3119 which showed declined response on increasing nitrogen dose exceeding the normal dose indicating that both the genotypes respond differentially to the nitrogen inputs. Gene expression analysis of members of ammonium transporter gene family in flag leaves during active grain filling stage revealed that all the three members of OsAMT3 family genes (OsAMT1;1-3), only one member of OsAMT2 family i.e., OsAMT2;3 and the high affinity OsAMT1;1 were differentially expressed and were affected by different doses of nitrogen. In both the genotypes, both increase and decline in seed protein contents matched with the expressions levels of OsAMT1;1, OsGS1;1 and OsGS1;2 in the flag leaves during grain filling stage indicating that high nitrogen nutrition in KN3119 probably causes the repression of these genes which might be important during grain filling.

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.

Genome-wide identification and characterization of seed storage proteins (SSPs) of foxtail millet (Setaria italica (L.) P. Beauv.).

We report the identification of 47 foxtail millet (Setaria italica (L.) P. Beauv.) seed storage proteins (SSPs) consisting of 14 albumins, 12 prolamins, 18 globulins and 3 glutelins using computational approaches and compared their essential amino acid composition with 225 SSPs of rice, barley, sorghum and maize. Comparative analysis revealed several unique foxtail millet SSPs containing high amounts of essential amino acids. These include three 2s-albumin proteins containing 11.9%, 10.9%, 9.82% lysine, one 10-kDa prolamin containing 20% methionine residues and one each 7S-globulin, 10-kDa prolamin, alpha-zein proteins containing 9.2% threonine, 9.35% phenylalanine and 2.5% tryptophan, respectively. High lysine containing albumins and high methionine containing prolamins were also detected in other cereals indicating that these SSPs are widespread in cereals. Phylogenetic studies revealed that the foxtail millet SSPs are closer to sorghum and maize. The lysine-rich albumins and the methionine-rich prolamins formed a separate cluster. Motif analysis of lysine-rich albumins displayed several lysine containing conserved motifs across cereals including foxtail millet. The 10-kDa prolamin protein containing 20% methionine was unique as it lacked the characteristic repeat motifs of methionine found in the high methionine containing zeins and kafirins. The motif "NPAAFWQQQQLL" was uniquely repeated in the foxtail millet high tryptophan prolamin protein. The findings of the present study provide new insights in foxtail millet seed storage protein characterization and their nutritional importance in terms of essential amino acid composition.

Identification and molecular characterization of Dof transcription factor gene family preferentially expressed in developing spikes of Eleusine coracana L.

We report 48 putative DNA binding with one finger (Dof) TF genes from genome and transcriptome data of finger millet (Eleusine coracana L.; FM), involved in plant developmental process. To characterize seed-specific Dof genes, transcript profiles of 32 EcDof identified from transcriptome data of developing spikes of FM genotypes were further analyzed in different tissues (root, stem, and leaf) and developmental stages of spikes (S1, S2, S3, and S4) in two FM genotypes [GE1437 (low protein genotype; LPG) and GE3885 (high protein genotype; HPG)]. More than 50% of identified EcDof genes showed expression during seed development processes. Among these, seven genes (EcDof 3, EcDof 5, EcDof 15, EcDof 18, EcDof 22, EcDof 23, and EcDof 31) expressed maximally at specific stages of seed development. Fourteen EcDof genes showed that differential transcript accumulation in vegetative tissue as well as in developing spikes suggests involvement during seed filling and also throughout the plant development. In addition, three EcDof genes (EcDof 9, EcDof 25, and EcDof 28) expressed preferentially at root and stem tissue. The 3D structural prediction of EcDof proteins showed variability in structural attributes. Molecular docking results showed strong binding affinity for seed-specific EcDof-EcO2 with α-prolamine promoters. The identified and characterized EcDof genes will help to dissect the roles of FM seed-specific Dof genes.

Identification and characterization of finger millet OPAQUE2 transcription factor gene under different nitrogen inputs for understanding their role during accumulation of prolamin seed storage protein.

In this study, we report the isolation and characterization of the mRNA encoding OPAQUE2 (O2) like TF of finger millet (FM) (Eleusine coracana) (EcO2). Full-length EcO2 mRNA was isolated using conserved primers designed by aligning O2 mRNAs of different cereals followed by 3' and 5' RACE (Rapid Amplification of cDNA Ends). The assembled full-length EcO2 mRNA was found to contain an ORF of 1248-nt coding the 416 amino acids O2 protein. Domain analysis revealed the presence of the BLZ and bZIP-C domains which is a characteristic feature of O2 proteins. Phylogenetic analysis of EcO2 protein with other bZIP proteins identified using finger millet transcriptome data and O2 proteins of other cereals showed that EcO2 shared high sequence similarity with barley BLZ1 protein. Transcripts of EcO2 were detected in root, stem, leaves, and seed development stages. Furthermore, to investigate nitrogen responsiveness and the role of EcO2 in regulating seed storage protein gene expression, the expression profiles of EcO2 along with an α-prolamin gene were studied during the seed development stages of two FM genotypes (GE-3885 and GE-1437) differing in grain protein content (13.8 and 6.2%, respectively) grown under increasing nitrogen inputs. Compared to GE-1437, the EcO2 was relatively highly expressed during the S2 stage of seed development which further increased as nitrogen input was increased. The Ecα-prolamin gene was strongly induced in the high protein genotype (GE-3885) at all nitrogen inputs. These results indicate the presence of nitrogen responsiveness regulatory elements which might play an important role in accumulating protein in FM genotypes through modulating EcO2 expression by sensing plant nitrogen status.

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.

Isolation, characterization and immunolocalization of a seed dominant CaM from finger millet (Eleusine coracana L. Gartn.) for studying its functional role in differential accumulation of calcium in developing grains.

To understand the exceptional high grain calcium accumulation in finger millet grains, a calmodulin (CaM) gene that is strongly expressed during developing spikes of high grain calcium genotype was further characterized. Using 5'-3' RACE, the full-length CaM open reading frame (ORF) was isolated and the deduced protein sequence showed the presence of four characteristic EF motifs. Phylogenetic analysis showed that the finger millet CaM (Eleusine coracana calmodulin [EcCaM]) was identical to the rice CaM 1-1. Southern hybridization showed the presence of at least four copies of CaM gene that might be located on different regions of the finger millet "AABB" genome. Immunodetection using monospecific polyclonal anti-EcCaM antibodies revealed that EcCaM is localized in the embryo and aleurone layer and accumulates in higher amounts in high grain calcium genotype compared to the low grain calcium genotype. Furthermore, in silico analysis showed that EcCaM interacts with aquaporin which indicates that calcium is probably delivered to developing spike via mass flow of water. These results indicate that higher expression of CaM might cause greater stimulation of the downstream calcium transport machinery operative in the aleurone layer leading to the higher calcium accumulation in the grains of high grain calcium genotype.

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.

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.