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MicroRNAs (miRNAs) are known to regulate expression of genes under stress. We report here the deep sequencing of small RNAs expressed during control, short and prolonged heat stress and recovery. Genome-wide identification of miRNAs in tolerant (Nagina 22) and susceptible (Vandana) rice cultivars was performed in 16 samples representing root and shoot of 13-day-old seedlings. The expression profile of miRNAs was analysed in 36 pairwise combinations to identify the genotype-, treatment- and tissue-dependent expression of miRNAs. Small-RNA sequencing of 16 libraries yielded ~271 million high-quality raw sequences; 162 miRNA families were identified. The highly expressed miRNAs in rice tissues were miR166, miR168, miR1425, miR529, mR162, miR1876, and miR1862. Expression of osa-miR1436, osa-miR5076, osa-miR5161, and osa-miR6253 was observed only in stressed tissue of both genotypes indicating their general role in heat stress response. Expression of osa-miR1439, osa-miR1848, osa-miR2096, osa-miR2106, osa-miR2875, osa-miR3981, osa-miR5079, osa-miR5151, osa-miR5484, osa-miR5792, and osa-miR5812 was observed only in Nagina 22 during high temperature, suggesting a specific role of these miRNAs in heat stress tolerance. This study provides details of the repertoire of miRNAs expressed in root and shoot of heat susceptible and tolerant rice genotypes under heat stress and recovery.

Transposons are generally kept silent by epigenetic mechanisms including DNA methylation. Here, we identified a pair of Harbinger transposon-derived Rroteins （HDPs）, HDP1 and HDP2, as anti-silencing factors in Arabidopsis. hdp1 and hdp2 mutants displayed an enhanced silencing of transgenes and some transposons. Phylogenetic analyses revealed that HDP1 and HDP2 were co-domesticated from the Harbinger transposon-encoded transposase and DNA-binding protein, respectively. HDP1 interacts with HDP2 in the nucleus, analogous to their transposon counterparts. Moreover, HDP1 and HDP2 are associated with IDMI, IDM2, IDM3 and MBD7 that constitute a histone acetyltransferase complex functioning in DNA demethylation. HDP2 and the methyl-DNA-binding protein MBD7 share a large set of common genomic binding sites, indicating that they jointly determine the target specificity of the histone acetyltransferase complex. Thus, our data revealed that HDP1 and HDP2 constitute a functional module that has been recruited to a histone acetyltransferase complex to prevent DNA hypermethylation and epigenetic silencing.

DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation.

DNA methylation-dependent heterochromatin formation is a conserved mechanism of epigenetic silencing of transposons and other repeat elements in many higher eukaryotes. Genes adjacent to repetitive elements are often also subjected to this epigenetic silencing. Consequently, plants have evolved antisilencing mechanisms such as active DNA demethylation mediated by the REPRESSOR OF SILENCING 1 (ROS1) family of 5-methylcytosine DNA glycosylases to protect these genes from silencing. Some transposons and other repeat elements have found residence in the introns of genes. It is unclear how these intronic repeat elements-containing genes are regulated. We report here the identification of ANTI-SILENCING 1 (ASI1), a bromo-adjacent homology domain and RNA recognition motif-containing protein, from a forward genetic screen for cellular antisilencing factors in Arabidopsis thaliana . ASI1 is required to prevent promoter DNA hypermethylation and transcriptional silencing of some transgenes. Genome-wide DNA methylation analysis reveals that ASI1 has a similar role to that of the histone H3K9 demethylase INCREASE IN BONSAI METHYLATION 1 (IBM1) in preventing CHG methylation in the bodies of thousands of genes. We found that ASI1 is an RNA-binding protein and ensures the proper expression of IBM1 full-length transcript by associating with an intronic heterochromatic repeat element of IBM1 . Through mRNA sequencing, we identified many genes containing intronic transposon elements that require ASI1 for proper expression. Our results suggest that ASI1 associates with intronic heterochromatin and binds the gene transcripts to promote their 3′ distal polyadenylation. The study thus reveals a unique mechanism by which higher eukaryotes deal with the collateral effect of silencing intronic repeat elements.

The genomes of an elite rice restorer line KMR3 (salinity-sensitive) and its salinity-tolerant introgression line IL50-13, a popular variety of coastal West Bengal, India, were sequenced. High-quality paired-end reads were obtained for KMR3 (147.6 million) and IL50-13 (131.4 million) with a sequencing coverage of 30X-39X. Scaffolds generated from the pre-assembled contigs of each sequenced genome were mapped separately onto the reference genome of Oryza sativa ssp. japonica cultivar Nipponbare to identify genomic variants in terms of SNPs and InDels. The SNPs and InDels identified for KMR3 and IL50-13 were then compared with each other to identify polymorphic SNPs and InDels unique and common to both the genomes. Functional enrichment analysis of the protein-coding genes with unique InDels identified GO terms involved in protein modification, ubiquitination, deubiquitination, peroxidase activity, and antioxidant activity in IL50-13. Linoleic acid metabolism, circadian rhythm, and alpha-linolenic acid metabolism pathways were enriched in IL50-13. These GO terms and pathways are involved in reducing oxidative damage, thus suggesting their role in stress responses. Sequence analysis of QTL markers or genes known to be associated with grain yield and salinity tolerance showed polymorphism in 20 genes, out of which nine were not previously reported. These candidate genes encoded Nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC) domain-containing protein, cyclase, receptor-like kinase, topoisomerase II-associated protein PAT1 domain-containing protein, ion channel regulatory protein, UNC-93 domain-containing protein, subunit A of the heteromeric ATP-citrate lyase, and three conserved hypothetical genes. Polymorphism was observed in the coding, intron, and untranslated regions of the genes on chromosomes 1, 2, 4, 7, 11, and 12. Genes showing polymorphism between the two genomes were considered as sequence-based new candidates derived from Oryza rufipogon for conferring high yield and salinity tolerance in IL50-13 for further functional studies.

Raffinose family oligosaccharides (RFOs) are known to have important physiological functions in plants. However, the presence of RFOs in legumes causes flatulence, hence are considered antinutrients. To reduce the RFOs content to a desirable limit without compromising normal plant development and functioning, the identification of important regulatory genes associated with the biosynthetic pathway is a prerequisite. In the present study, through comparative RNA sequencing in contrasting genotypes for seed RFOs content at different seed maturity stages, differentially expressed genes (DEGs) associated with the pathway were identified. The DEGs exhibited spatio-temporal expression patterns with high RFOs variety showing early induction of RFOs biosynthetic genes and low RFOs variety showing a late expression at seed maturity. Selective and seed-specific differential expression of raffinose synthase genes ( AhRS14 and AhRS6 ) suggested their regulatory role in RFOs accumulation in peanut seeds, thereby serving as promising targets in low RFOs peanut breeding programs. Despite stachyose being the major seed RFOs fraction, differential expression of raffinose synthase genes indicated the complex metabolic regulation of this pathway. The transcriptomic resource and the genes identified in this study could be studied further to develop low RFOs varieties, thus improving the overall nutritional quality of peanuts.

Globally, soil salinity has been on the rise owing to various factors that are both human and environmental. The abiotic stress caused by soil salinity has become one of the most damaging abiotic stresses faced by crop plants, resulting in significant yield losses. Salt stress induces physiological and morphological modifications in plants as a result of significant changes in gene expression patterns and signal transduction cascades. In this comprehensive review, with a major focus on recent advances in the field of plant molecular biology, we discuss several approaches to enhance salinity tolerance in plants comprising various classical and advanced genetic and genetic engineering approaches, genomics and genome editing technologies, and plant growth-promoting rhizobacteria (PGPR)-based approaches. Furthermore, based on recent advances in the field of epigenetics, we propose novel approaches to create and exploit heritable genome-wide epigenetic variation in crop plants to enhance salinity tolerance. Specifically, we describe the concepts and the underlying principles of epigenetic recombinant inbred lines (epiRILs) and other epigenetic variants and methods to generate them. The proposed epigenetic approaches also have the potential to create additional genetic variation by modulating meiotic crossover frequency.

Rice ( Oryza sativa L.), a major dietary source, is often cultivated in soils poor in available inorganic orthophosphate (Pi), which is a key nutrient for growth and development. Poor soils are amended by phosphorus (P) fertilizer, which is derived from the non-renewable rock phosphate reserves. Therefore, there is a need for developing rice varieties with high productivity under low P conditions. At the ICAR-IIRR, ethyl methanesulfonate (EMS) mutagenized rice genotype Nagina22 (N22) were screened for high grain yield in Pi-deprived soil, which led to the identification of ~ 10 gain-of-function mutants including NH787 . Here, detailed comparative morphophysiological, biochemical, and molecular analyses of N22 and NH787 were carried out in hydroponics and potting soil under different Pi regimes. Under Pi-deprived condition, compared with N22, NH787 exhibited higher root and vegetative biomass, the number of tillers, and grain yield. The augmented agronomic traits of NH787 were corroborated with significantly higher photosynthetic rate, pollen fertility, stigma receptivity, and the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). Further, several genes involved in the maintenance of Pi homeostasis (GPH) were differentially regulated. The study thus revealed a wide-spectrum influence of the mutation in NH787 that contributed towards its higher Pi use efficiency (PUE).

Sheath blight disease of rice caused by Rhizoctonia solani Kühn (teleomorph: Thanatephorus cucumeris) remains a global challenge due to the absence of reliable resistance genes and poor understanding of pathogen biology. Pectin, one of the most vital constituents of the plant cell wall, is targeted by pectin methylesterases, polygalacturonases, and few other enzymes of fungal pathogens. In this study, we catalogued the expressed genes of the fungal genome from RNAseq of R. solani infected four rice genotypes. Analysis of RNAseq revealed 3325 pathogen genes commonly expressed in all rice genotypes, in which 49, 490, and 83 genes were specific to BPT5204, Tetep, and Pankaj genotypes, respectively. To identify the early and late responding genes of R. solani during plant cell wall degradation, a real-time PCR analysis of 30 pectinolytic enzymes was done at six different time points after inoculation. The majority of these genes showed maximum induction at the 72 h time point, suggesting that it is the most crucial stage of infection. Pankaj showed lesser induction of these genes as compared to other genotypes. Leaf-blade tissue and 45 days old-growth stage are more favorable for the expression of pectin degradation genes of R. solani. Additionally, the expression analysis of these genes from four different strains of R. solani suggested differential regulation of genes but no distinct expression pattern between highly virulent and mild strains. The implications of the differential regulation of these genes in disease development have been discussed. This study provides the first such comprehensive analysis of R. solani genes encoding pectin degrading enzymes, which would help to decipher the pathogen biology and sheath blight disease development.

The harvested plant products, specifically, the grains of cereals are major drivers of soil phosphorus (P) depletion. However, the breeding or biotechnology efforts to develop low P seeds have not been attempted because of possible adverse effects on seedling vigour and crop establishment. Several studies have contradictory observations on influence of seed P on seedling vigour. Lack of appropriate genetic material has been the major bottleneck in reaching the consensus. In this study, we used 30 EMS induced mutants of rice cultivar Nagina22 to understand the role of seed P on seedling vigour and associated physiological processes. Seedling vigour, morpho-physiological characteristics, acid phosphatases, alpha-amylase, and expression of P transporter genes were analyzed in seedlings obtained from seeds of high and low grain P mutants. The study suggests that seed P has a significant role on seedling vigour, chlorophyll content and photosynthesis process of young seedlings, and P transport from roots. Notably, we identified few mutants such as NH4791 , NH4785 , NH4714 , NH4663 , NH4614 , and NH4618 which showed least influence of low seed P on seedling vigour and other metabolic processes. Therefore, these mutants can be used in breeding programs aiming for development of low P grains. Also, these and other identified mutants can be used to decipher the genetic and molecular mechanisms regulating the differential response of seed P on germination, seedling vigour and several other physiological processes influencing the crop growth and establishment.