No abstract

Nitrogen-use efficiency (NUE) is a function of N-response and yield that is controlled by many genes and phenotypic parameters that are poorly characterized. This study compiled all known yield-related genes in rice and mined them from the N-responsive microarray data to find 1,064 NUE-related genes. Many of them are novel genes hitherto unreported as related to NUE, including 80 transporters, 235 transcription factors (TFs), 44 MicroRNAs (miRNAs), 91 kinases, and 8 phosphatases. They were further shortlisted to 62 NUE-candidate genes following hierarchical methods, including quantitative trait locus (QTL) co-localization, functional evaluation in the literature, and protein–protein interactions (PPIs). They were localized to chromosomes 1, 3, 5, and 9, of which chromosome 1 with 26 genes emerged as a hotspot for NUE spanning 81% of the chromosomes. Further, co-localization of the NUE genes on NUE-QTLs resolved differences in the earlier studies that relied mainly on N-responsive genes regardless of their role in yield. Functional annotations and PPIs for all the 1,064 NUE-related genes and also the shortlisted 62 candidates revealed transcription, redox, phosphorylation, transport, development, metabolism, photosynthesis, water deprivation, and hormonal and stomatal function among the prominent processes. In silico expression analysis confirmed differential expression of the 62 NUE-candidate genes in a tissue/stage-specific manner. Experimental validation in two contrasting genotypes revealed that high NUE rice shows better photosynthetic performance, transpiration efficiency and internal water-use efficiency in comparison to low NUE rice. Feature Selection Analysis independently identified one-third of the common genes at every stage of hierarchical shortlisting, offering 6 priority targets to validate for improving the crop NUE.

The biological improvement of fertilizer nitrogen use efficiency (NUE) is hampered by the poor characterization of the phenotype and genotype for crop N response and NUE. In an attempt to identify phenotypic traits for N-response and NUE in the earliest stages of plant growth, we analyzed the N-responsive germination, respiration, urease activities, and root/shoot growth of 21 Indica genotypes of rice (Oryza sativa var. indica). We found that N delays germination from 0 to 12 h in a genotype-dependent and source-dependent manner, especially with urea and nitrate. We identified contrasting groups of fast germinating genotypes such as Aditya, Nidhi, and Swarnadhan, which were also least delayed by N and slow germinating genotypes such as Panvel 1, Triguna, and Vikramarya, which were also most delayed by N. Oxygen uptake measurements in the seeds of contrasting genotypes revealed that they were affected by N source in accordance with germination rates, especially with urea. Germinating seeds were found to have endogenous urease activity, indicating the need to explore genotypic differences in the effective urea uptake and metabolism, which remain unexplored so far. Urea was found to significantly inhibit early root growth in all genotypes but not shoot growth. Field evaluation of 15 of the above genotypes clearly showed that germination rates, crop duration, and yield are linked to NUE. Slow germinating genotypes had longer crop duration and higher yield even at lower N, indicating their higher NUE, relative to fast germinating or short duration genotypes. Moreover, longer duration genotypes suffered lesser yield losses at reduced N levels as compared to short duration genotypes, which is also a measure of their NUE. Together, these results indicate the potential of germination rates, crop duration, urea utilization and its effect on root growth in the development of novel phenotypic traits for screening genotypes and crop improvement for NUE, at least in rice.

The genome-wide role of heterotrimeric G-proteins in abiotic stress response in rice has not been examined from a functional genomics perspective, despite the availability of mutants and evidences involving individual genes/processes/stresses. Our rice whole transcriptome microarray analysis (GSE 20925 at NCBI GEO) using the G-alpha subunit (RGA1) null mutant (Daikoku 1 or d1) and its corresponding wild type (Oryza sativa Japonica Nipponbare) identified 2270 unique differentially expressed genes (DEGs). Out of them, we mined for all the potentially abiotic stress-responsive genes using Gene Ontology terms, STIFDB2.0 and Rice DB. The first two approaches produced smaller subsets of the 1886 genes found at Rice DB. The GO approach revealed similar regulation of several families of stress-responsive genes in RGA1 mutant. The Genevestigator analysis of the stress-responsive subset of the RGA1-regulated genes from STIFDB revealed cold and drought-responsive clusters. Meta data analysis at Rice DB revealed large stress-response categories such as cold (878 up/810 down), drought (882 up/837 down), heat (913 up/777 down), and salt stress (889 up/841 down). One thousand four hundred ninety-eight of them are common to all the four abiotic stresses, followed by fewer genes common to smaller groups of stresses. The RGA1-regulated genes that uniquely respond to individual stresses include 111 in heat stress, eight each in cold only and drought only stresses, and two genes in salt stress only. The common DEGs (1498) belong to pathways such as the synthesis of polyamine, glycine-betaine, proline, and trehalose. Some of the common DEGs belong to abiotic stress signaling pathways such as calcium-dependent pathway, ABA independent and dependent pathway, and MAP kinase pathway in the RGA1 mutant. Gene ontology of the common stress responsive DEGs revealed 62 unique molecular functions such as transporters, enzyme regulators, transferases, hydrolases, carbon and protein metabolism, binding to nucleotides, carbohydrates, receptors and lipids, morphogenesis, flower development, and cell homeostasis. We also mined 63 miRNAs that bind to the stress responsive transcripts identified in this study, indicating their post-transcriptional regulation. Overall, these results indicate the potentially extensive role of RGA1 in the regulation of multiple abiotic stresses in rice for further validation.

Heterotrimeric G-proteins are implicated in several plant processes, but the mechanisms of signal-response coupling and the roles of G-protein coupled receptors in general and GCR1 in particular, remain poorly understood. We isolated a knock-out mutant of the Arabidopsis G-protein α subunit (gpa1-5) and analysed its transcriptome to understand the genomewide role of GPA1 and compared it with that of our similar analysis of a GCR1 mutant (Chakraborty et al. 2015, PLoS ONE 10(2):e0117819). We found 394 GPA1-regulated genes spanning 79 biological processes, including biotic and abiotic stresses, development, flavonoid biosynthesis, transcription factors, transporters and nitrate/phosphate responses. Many of them are either unknown or unclaimed explicitly in other published gpa1 mutant transcriptome analyses. A comparison of all known GPA1-regulated genes (including the above 394) with 350 GCR1-regulated genes revealed 114 common genes. This can be best explained by GCR1-GPA1 coupling, or by convergence of their independent signaling pathways. Though the common genes in our GPA1 and GCR1 mutant datasets constitute only 26% of the GPA1-regulated and 30% of the GCR1-responsive genes, they belong to nearly half of all the processes affected in both the mutants. Thus, GCR1 and GPA1 regulate not only some common genes, but also different genes belonging to the same processes to achieve similar outcomes. Overall, we validate some known and report many hitherto unknown roles of GPA1 in plants, including agronomically important ones such as biotic stress and nutrient response, and also provide compelling genetic evidence to revisit the role of GCR1 in G-protein signalling.