Nitrate-responsive transcriptome analysis reveals additional genes/processes and associated traits viz. height, tillering, heading date, stomatal density and yield in japonica rice.

MAIN CONCLUSION: Our transcriptomic analysis expanded the repertoire of nitrate-responsive genes/processes in rice and revealed their phenotypic association with root/shoot, stomata, tiller, panicle/flowering and yield, with agronomic implications for nitrogen use efficiency. Nitrogen use efficiency (NUE) is a multigenic quantitative trait, involving many N-responsive genes/processes that are yet to be fully characterized. Microarray analysis of early nitrate response in excised leaves of japonica rice revealed 6688 differentially expressed genes (DEGs), including 2640 hitherto unreported across multiple functional categories. They include transporters, enzymes involved in primary/secondary metabolism, transcription factors (TFs), EF-hand containing calcium binding proteins, hormone metabolism/signaling and methytransferases. Some DEGs belonged to hitherto unreported processes viz. alcohol, lipid and trehalose metabolism, mitochondrial membrane organization, protein targeting and stomatal opening. 1158 DEGs were associated with growth physiology and grain yield or phenotypic traits for NUE. We identified seven DEGs for shoot apical meristem, 66 for leaf/culm/root, 31 for tiller, 70 for heading date/inflorescence/spikelet/panicle, 144 for seed and 78 for yield. RT-qPCR validated nitrate regulation of 31 DEGs belonging to various important functional categories/traits. Physiological validation of N-dose responsive changes in plant development revealed that relative to 1.5 mM, 15 mM nitrate significantly increased stomatal density, stomatal conductance and transpiration rate. Further, root/shoot growth, number of tillers and grain yield declined and panicle emergence/heading date delayed, despite increased photosynthetic rate. We report the binding sites of diverse classes of TFs such as WRKY, MYB, HMG etc., in the 1 kb up-stream regions of 6676 nitrate-responsive DEGs indicating their role in regulating nitrate response/NUE. Together, these findings expand the repertoire of genes and processes involved in genomewide nitrate response in rice and reveal their physiological, phenotypic and agronomic implications for NUE.

Heterotrimeric G-protein α subunit (RGA1) regulates tiller development, yield, cell wall, nitrogen response and biotic stress in rice.

G-proteins are implicated in plant productivity, but their genome-wide roles in regulating agronomically important traits remain uncharacterized. Transcriptomic analyses of rice G-protein alpha subunit mutant (rga1) revealed 2270 differentially expressed genes (DEGs) including those involved in C/N and lipid metabolism, cell wall, hormones and stress. Many DEGs were associated with root, leaf, culm, inflorescence, panicle, grain yield and heading date. The mutant performed better in total weight of filled grains, ratio of filled to unfilled grains and tillers per plant. Protein-protein interaction (PPI) network analysis using experimentally validated interactors revealed many RGA1-responsive genes involved in tiller development. qPCR validated the differential expression of genes involved in strigolactone-mediated tiller formation and grain development. Further, the mutant growth and biomass were unaffected by submergence indicating its role in submergence response. Transcription factor network analysis revealed the importance of RGA1 in nitrogen signaling with DEGs such as Nin-like, WRKY, NAC, bHLH families, nitrite reductase, glutamine synthetase, OsCIPK23 and urea transporter. Sub-clustering of DEGs-associated PPI network revealed that RGA1 regulates metabolism, stress and gene regulation among others. Predicted rice G-protein networks mapped DEGs and revealed potential effectors. Thus, this study expands the roles of RGA1 to agronomically important traits and reveals their underlying processes.

Transcriptomic and network analyses reveal distinct nitrate responses in light and dark in rice leaves (Oryza sativa Indica var. Panvel1).

Nitrate (N) response is modulated by light, but not understood from a genome-wide perspective. Comparative transcriptomic analyses of nitrate response in light-grown and etiolated rice leaves revealed 303 and 249 differentially expressed genes (DEGs) respectively. A majority of them were exclusive to light (270) or dark (216) condition, whereas 33 DEGs were common. The latter may constitute response to N signaling regardless of light. Functional annotation and pathway enrichment analyses of the DEGs showed that nitrate primarily modulates conserved N signaling and metabolism in light, whereas oxidation-reduction processes, pentose-phosphate shunt, starch-, sucrose- and glycerolipid-metabolisms in the dark. Differential N-regulation of these pathways by light could be attributed to the involvement of distinctive sets of transporters, transcription factors, enriched cis-acting motifs in the promoters of DEGs as well as differential modulation of N-responsive transcriptional regulatory networks in light and dark. Sub-clustering of DEGs-associated protein-protein interaction network constructed using experimentally validated interactors revealed that nitrate regulates a molecular complex consisting of nitrite reductase, ferredoxin-NADP reductase and ferredoxin. This complex is associated with flowering time, revealing a meeting point for N-regulation of N-response and N-use efficiency. Together, our results provide novel insights into distinct pathways of N-signaling in light and dark conditions.