Comparative miRNome and transcriptome analyses reveal the expression of novel miRNAs in the panicle of rice implicated in sustained agronomic performance under terminal drought stress.

MAIN CONCLUSION: Differential expression of 128 known and 111 novel miRNAs in the panicle of Nagina 22 under terminal drought stress targeting transcription factors, stress-associated genes, etc., enhances drought tolerance and helps sustain agronomic performance under terminal drought stress. Drought tolerance is a complex multigenic trait, wherein the genes are fine-tuned by coding and non-coding components in mitigating deleterious effects. MicroRNA (miRNA) controls gene expression at post-transcriptional level either by cleaving mRNA (transcript) or by suppressing its translation. miRNAs are known to control developmental processes and abiotic stress tolerance in plants. To identify terminal drought-responsive novel miRNA in contrasting rice cultivars, we constructed small RNA (sRNA) libraries from immature panicles of drought-tolerant rice [Nagina 22 (N 22)] and drought-sensitive (IR 64) cultivars grown under control and terminal drought stress. Our analysis of sRNA-seq data resulted in the identification of 169 known and 148 novel miRNAs in the rice cultivars. Among the novel miRNAs, 68 were up-regulated while 43 were down-regulated in the panicle of N 22 under stress. Interestingly, 31 novel miRNAs up-regulated in N 22 were down-regulated in IR 64, whereas 4 miRNAs down-regulated in N 22 were up-regulated in IR 64 under stress. To detect the effects of miRNA on mRNA expression level, transcriptome analysis was performed, while differential expression of miRNAs and their target genes was validated by RT-qPCR. Targets of the differentially expressed miRNAs include transcription factors and stress-associated genes involved in cellular/metabolic/developmental processes, response to abiotic stress, programmed cell death, photosynthesis, panicle/seed development, and grain yield. Differential expression of the miRNAs could be validated in an independent set of the samples. The findings might be useful in genetic improvement of drought-tolerant rice.

Epigenome editing for targeted DNA (de)methylation: a new perspective in modulating gene expression.

Traditionally, it has been believed that inheritance is driven as phenotypic variations resulting from changes in DNA sequence. However, this paradigm has been challenged and redefined in the contemporary era of epigenetics. The changes in DNA methylation, histone modification, non-coding RNA biogenesis, and chromatin remodeling play crucial roles in genomic functions and regulation of gene expression. More importantly, some of these changes are inherited to the next generations as a part of epigenetic memory and play significant roles in gene expression. The sum total of all changes in DNA bases, histone proteins, and ncRNA biogenesis constitutes the epigenome. Continuous progress in deciphering epigenetic regulations and the existence of heritable epigenetic/epiallelic variations associated with trait of interest enables to deploy epigenome editing tools to modulate gene expression. DNA methylation marks can be utilized in epigenome editing for the manipulation of gene expression. Initially, genome/epigenome editing technologies relied on zinc-finger protein or transcriptional activator-like effector protein. However, the discovery of clustered regulatory interspaced short palindromic repeats CRISPR)/deadCRISPR-associated protein 9 (dCas9) enabled epigenome editing to be more specific/efficient for targeted DNA (de)methylation. One of the major concerns has been the off-target effects, wherein epigenome editing may unintentionally modify gene/regulatory element which may cause unintended change/harmful effects. Moreover, epigenome editing of germline cell raises several ethical/safety issues. This review focuses on the recent developments in epigenome editing tools/techniques, technological limitations, and future perspectives of this emerging technology in therapeutics for human diseases as well as plant improvement to achieve sustainable developmental goals.

Epigenetic Modifications in Genome Help Remembering the Stress Tolerance Strategy Adopted by the Plant.

Genetic information in eukaryotic organisms is stored, replicated, transcribed, and inherited through the nucleus of a cell. Epigenetic modifications in the genetic material, including DNA methylation, histone modification, changes in non-coding RNA (ncRNA) biogenesis, and chromatin architecture play important roles in determining the genomic landscape and regulating gene expression. Genome architecture (structural features of chromatin, affected by epigenetic modifications) is a major driver of genomic functions/activities. Segregation of euchromatin (transcriptionally active) from heterochromatin (transcriptionally repressed chromosome) and positioning of genes in specific nuclear space in eukaryotic cells emphasise non-randomness in the organization of the genetic information. Not only does the base sequence of a gene carry the genetic information but the covalent modifications of bases, three-dimensional positioning of the genome, and chromatin loops are vital for switching on/off the gene and regulating its expression during growth/environmental stress. The epigenetic dynamics depend on the activities of writers and erasers under changing environmental conditions. The discovery of non-coding RNAs (one of the players in de novo methylation of DNA), increased DNA methylation protein (guide for the DNA demethylase), and methylation monitoring sequence (that helps keep a balance between DNA demethylation and methylation) have been some of the new developments in the era of epigenomics. To respond to environmental stimuli, plants depend on modulating gene expression through different mechanisms including biochemical, molecular, genetic, and epigenetic alterations. Studies on plants might provide better insights into epigenetic stress memory and molecular bases of adaptability to enable (epi)genome editing of crops for climate resilience and sustainable agriculture in the present era of multifaceted climate change.

DNA methylome analysis provides insights into gene regulatory mechanism for better performance of rice under fluctuating environmental conditions: epigenomics of adaptive plasticity.

MAIN CONCLUSION: Roots play an important role in adaptive plasticity of rice under dry/direct-sown conditions. However, hypomethylation of genes in leaves (resulting in up-regulated expression) complements the adaptive plasticity of Nagina-22 under DSR conditions. Rice is generally cultivated by transplanting which requires plenty of water for irrigation. Such a practice makes rice cultivation a challenging task under global climate change and reducing water availability. However, dry-seeded/direct-sown rice (DSR) has emerged as a resource-saving alternative to transplanted rice (TPR). Though some of the well-adapted local cultivars are used for DSR, only limited success has been achieved in developing DSR varieties mainly because of a limited knowledge of adaptability of rice under fluctuating environmental conditions. Based on better morpho-physiological and agronomic performance of Nagina-22 (N-22) under DSR conditions, N-22 and IR-64 were grown by transplanting and direct-sowing and used for whole genome methylome analysis to unravel the epigenetic basis of adaptive plasticity of rice. Comparative methylome and transcriptome analyses indicated a large number (4078) of genes regulated through DNA methylation/demethylation in N-22 under DSR conditions. Gene × environment interactions play important roles in adaptive plasticity of rice under direct-sown conditions. While genes for pectinesterase, LRK10, C2H2 zinc-finger protein, splicing factor, transposable elements, and some of the unannotated proteins were hypermethylated, the genes for regulation of transcription, protein phosphorylation, etc. were hypomethylated in CG context in the root of N-22, which played important roles in providing adaptive plasticity to N-22 under DSR conditions. Hypomethylation leading to up-regulation of gene expression in the leaf complements the adaptive plasticity of N-22 under DSR conditions. Moreover, differential post-translational modification of proteins and chromatin assembly/disassembly through DNA methylation in CHG context modulate adaptive plasticity of N-22. These findings would help developing DSR cultivars for increased water-productivity and ecological efficiency.

Comparative Genome-Wide Analysis of MicroRNAs and Their Target Genes in Roots of Contrasting Indica Rice Cultivars under Reproductive-Stage Drought.

Recurrent occurrence of drought stress in varying intensity has become a common phenomenon in the present era of global climate change, which not only causes severe yield losses but also challenges the cultivation of rice. This raises serious concerns for sustainable food production and global food security. The root of a plant is primarily responsible to perceive drought stress and acquire sufficient water for the survival/optimal growth of the plant under extreme climatic conditions. Earlier studies reported the involvement/important roles of microRNAs (miRNAs) in plants' responses to environmental/abiotic stresses. A number (738) of miRNAs is known to be expressed in different tissues under varying environmental conditions in rice, but our understanding of the role, mode of action, and target genes of the miRNAs are still elusive. Using contrasting rice [IR-64 (reproductive-stage drought sensitive) and N-22 (drought-tolerant)] cultivars, imposed with terminal (reproductive-stage) drought stress, we demonstrate differential expression of 270 known and 91 novel miRNAs in roots of the contrasting rice cultivars in response to the stress. Among the known miRNAs, osamiR812, osamiR166, osamiR156, osamiR167, and osamiR396 were the most differentially expressed miRNAs between the rice cultivars. In the root of N-22, 18 known and 12 novel miRNAs were observed to be exclusively expressed, while only two known (zero novels) miRNAs were exclusively expressed in the roots of IR-64. The majority of the target gene(s) of the miRNAs were drought-responsive transcription factors playing important roles in flower, grain development, auxin signaling, root development, and phytohormone-crosstalk. The novel miRNAs identified in this study may serve as good candidates for the genetic improvement of rice for terminal drought stress towards developing climate-smart rice for sustainable food production.

Biochemical and Epigenetic Modulations under Drought: Remembering the Stress Tolerance Mechanism in Rice.

A plant, being a sessile organism, needs to modulate biochemical, physiological, and molecular responses to the environment in a quick and efficient manner to be protected. Drought stress is a frequently occurring abiotic stress that severely affects plant growth, development, and productivity. Short- and long-term memories are well-known phenomena in animals; however, the existence of such remembrance in plants is still being discovered. In this investigation, different rice genotypes were imposed with drought stress just before flowering and the plants were re-watered for recovery from the stress. Seeds collected from the stress-treated (stress-primed) plants were used to raise plants for the subsequent two generations under a similar experimental setup. Modulations in physio-biochemical (chlorophyll, total phenolics and proline contents, antioxidant potential, lipid peroxidation) and epigenetic [5-methylcytosine (5-mC)] parameters were analyzed in the leaves of the plants grown under stress as well as after recovery. There was an increase in proline (>25%) and total phenolic (>19%) contents, antioxidant activity (>7%), and genome-wide 5-mC level (>56%), while a decrease (>9%) in chlorophyll content was recorded to be significant under the stress. Interestingly, a part of the increased proline content, total phenolics content, antioxidant activity, and 5-mC level was retained even after the withdrawal of the stress. Moreover, the increased levels of biochemical and epigenetic parameters were observed to be transmitted/inherited to the subsequent generations. These might help in developing stress-tolerant crops and improving crop productivity under the changing global climate for sustainable food production and global food security.

Meta-Analysis of Microarray Data and Their Utility in Dissecting the Mapped QTLs for Heat Acclimation in Rice.

In the current global warming scenario, it is imperative to develop crops with improved heat tolerance or acclimation, for which knowledge of major heat stress-tolerant genes or genomic regions is a prerequisite. Though several quantitative trait loci (QTLs) for heat tolerance have been mapped in rice, candidate genes from these QTLs have not been reported yet. The meta-analysis of microarray datasets for heat stress in rice can give us a better genomic resource for the dissection of QTLs and the identification of major candidate genes for heat stress tolerance. In the present study, a database, RiceMetaSys-H, comprising 4227 heat stress-responsive genes (HRGs), was created using seven publicly available microarray datasets. This included in-house-generated microarray datasets of Nagina 22 (N22) and IR64 subjected to 8 days of heat stress. The database has provisions for searching the HRGs through genotypes, growth stages, tissues, and physical intervals in the genome, as well as Locus IDs, which provide complete information on the HRGs with their annotations and fold changes, along with the experimental material used for the analysis. The up-regulation of genes involved in hormone biosynthesis and signalling, sugar metabolism, carbon fixation, and the ROS pathway were found to be the key mechanisms of enhanced heat tolerance. Integrating variant and expression analysis, the database was used for the dissection of the major effect of QTLs on chromosomes 4, 5, and 9 from the IR64/N22 mapping population. Out of the 18, 54, and 62 genes in these three QTLs, 5, 15, and 12 genes harboured non-synonymous substitutions. Fifty-seven interacting genes of the selected QTLs were identified by a network analysis of the HRGs in the QTL regions. Variant analysis revealed that the proportion of unique amino acid substitutions (between N22/IR64) in the QTL-specific genes was much higher than the common substitutions, i.e., 2.58:0.88 (2.93-fold), compared to the network genes at a 0.88:0.67 (1.313-fold) ratio. An expression analysis of these 89 genes showed 43 DEGs between IR64/N22. By integrating the expression profiles, allelic variations, and the database, four robust candidates (LOC_Os05g43870, LOC_Os09g27830, LOC_Os09g27650, andLOC_Os09g28000) for enhanced heat stress tolerance were identified. The database thus developed in rice can be used in breeding to combat high-temperature stress.

Mitochondrial markers differentiate two distinct phylogenetic groups in indigenous rice landraces of northeast India: an evolutionary insight.

The inheritance of the mitochondria genome and its diversity is unique for genetic and evolutionary studies relative to nuclear genomes. Northeast India and Himalayan regions are considered as one of the centres of indica rice origin. Also, rice diversity in northeast India is very distinct and highly suited for evolutionary studies. Although reports are available on the genetic diversity of indigenous northeast rice landraces, its relationship with the wild relatives is not yet properly explored and understood. In an attempt, mitochondrial markers were used to study the evolutionary relationship between the 68 landraces of northeast India and wild relatives (O. rufipogon and O. nivara) along with IR64 (indica) and Nipponbare (japonica) were taken as reference cultivars. Phylogenetically, the findings include two distinct clusters in the indigenous northeast India landraces representing indica and japonica groups. Further, the wild relatives and ~60% of northeast India landraces were identified to be closely related to the Nipponbare cluster. Besides, landraces of northeast India grouping with the indica group (IR64) are characterized by the absence of wild relatives. This indicates that there are two distinct evolutionary paths in the origin of northeast Indian rice landraces based on mitochondrial markers diversity and it is proposed that the inheritance of mitochondria, mitonuclear genome interactions, and bottleneck events could have genetically separated these two phylogenetically unique groups of northeast rice landraces.

Transcriptome and Physio-Biochemical Profiling Reveals Differential Responses of Rice Cultivars at Reproductive-Stage Drought Stress.

Drought stress severely affects the growth and development of rice, especially at the reproductive stage, which results in disturbed metabolic processes, reduced seed-set/grain filling, deteriorated grain quality, declined productivity, and lower yield. Despite the recent advances in understanding the responses of rice to drought stress, there is a need to comprehensively integrate the morpho-physio-biochemical studies with the molecular responses/differential expression of genes and decipher the underlying pathways that regulate the adaptability of rice at various drought-sensitive growth stages. Our comparative analysis of immature panicle from a drought-tolerant (Nagina 22) and a drought-sensitive (IR 64) rice cultivar grown under control (well-watered) and water-deficit/drought stress (treatment, imposed at the reproductive stage) conditions unraveled some novel stress-responsive genes/pathways responsible for reproductive-stage drought stress tolerance. The results revealed a more important role of upregulated (6706) genes in the panicle of N 22 at reproductive-stage drought stress compared to that (5590) in IR 64. Functional enrichment and MapMan analyses revealed that majority of the DEGs were associated with the phytohormone, redox signalling/homeostasis, secondary metabolite, and transcription factor-mediated mitigation of the adverse effects of drought stress in N 22. The upregulated expression of the genes associated with starch/sucrose metabolism, secondary metabolites synthesis, transcription factors, glutathione, linoleic acid, and phenylalanine metabolism in N 22 was significantly more than that in the panicle of IR 64. Compared to IR 64, 2743 genes were upregulated in N 22 under control conditions, which further increased (4666) under drought stress in panicle of the tolerant cultivar. Interestingly, we observed 6706 genes to be upregulated in the panicle of N 22 over IR 64 under drought and 5814 genes get downregulated in the panicle of N 22 over IR 64 under the stress. In addition, RT-qPCR analysis confirmed differential expression patterns of the DEGs. These genes/pathways associated with the reproductive-stage drought tolerance might provide an important source of molecular markers for genetic manipulation of rice for enhanced drought tolerance.

Bacteriophages diversity in India's major river Ganga: a repository to regulate pathogenic bacteria in the aquatic environment.

Bacteriophages are key viruses that can kill thousands of harmful microbes generally present at polluted sites. Such bacteriophages are abundantly present in the river Ganga, where millions of people in India and abroad drink its water and take baths every day for spiritual reasons. Besides bacteriophages, several pathogenic and zoonotic microbes are present in the river Ganga. It is interesting to study the diversity and abundance of bacteria and their respective phages present in polluted or non-polluted sites. Thus, the metagenomics study was carried out at the most polluted sites of river Ganga near Kanpur and non-polluted sites at Farakka, which harbors several harmful bacteria and their phages. The results revealed a significantly higher percentage of Microviridae phage family, ssDNA viruses, and Mimiviridae virus family near Kanpur than Farakka. In addition, compared to Kanpur, Farakka has a more significant percentage of Myoviridae, an unidentified phage family, and Retroviridae viral families. Despite heavy drainage of untreated and contaminated effluents from the leather industry, pesticide industry, paper mills, metropolitan cities, and other sources, the vast number of said phages kills several harmful pathogenic microbes in polluted sites to maintain the Ganga water's healing power or natural sterility. In a polluted aquatic environment, the varieties of bacteriophages were identified in the Ganga and their interaction with the microbial host. The taxonomic diversity of several bacteriophages found in pathogenic host systems was investigated to get exceptional knowledge of these small viruses in the aquatic environment.

Physiological Analysis of Source-Sink Relationship in Rice Genotypes with Contrasting Grain Yields.

Rice is a major staple food, and, hence, doubling its productivity is critical to sustain future food security. Improving photosynthesis, source-sink relationships and grain-filling mechanisms are promising traits for improvement in grain yield. To understand the source-sink relationship and grain yield, a set of contrasting rice genotypes differing in yield and biomass were studied for physiological, biochemical and gene-expression differences. The physiological and yield component traits of selected rice genotypes were analyzed in 2016 and 2017 under field conditions. This led to the categorization of genotypes as high yielding (HY) and high biomass, viz., Dular, Gontra Bidhan 3, Way Rarem, Patchai Perumal, Sahbhagi Dhan, Indira Barani Dhan-1, MTU1010, and Maudamani; while, low yielding (LY) and low biomass, viz. Anjali, Ghanteswari, Parijat, Khao Daw Tai, RKVY-104, Ghati Kamma Nangarhar, BAM4510 and BAM5850. The HY genotypes in general had relatively better values of yield component traits, higher photosynthetic rate (Pn) and chlorophyll (Chl) content. The study revealed that leaf area per plant and whole plant photosynthesis are the key traits contributing to high biomass production. We selected two good-performing (Sahbhagi Dhan and Maudamani) and two poor-performing (Ghanteswari and Parijat) rice genotypes for a detailed expression analysis of selected genes involved in photosynthesis, sucrose synthesis, transport, and starch synthesis in the leaf and starch metabolism in grain. Some of the HY genotypes had a relatively high level of expression of key photosynthesis genes, such as RbcS, RCA, FBPase, and ZEP over LY genotypes. This study suggests that traits, such as leaf area, photosynthesis and grain number, contribute to high grain yield in rice. These good-performing genotypes can be used as a donor in a breeding program aimed at high yields in rice.

MicroRNA: noncoding but still coding, another example of self-catalysis.

MicroRNAs (miRNAs) are known to interact with specific mRNAs to regulate gene expression at the post-transcriptional level by cleaving/repressing the translation process. MiRNA-mediated regulation of gene expression has become an interesting area of research on biological processes like growth, development, and stress responses. Studies suggest that some of the noncoding RNAs possess short open reading frames (ORFs) that code for micropeptides (miPEPs) having a regulatory function. Dual functions of some MIR genes are being deciphered, wherein the gene is transcribed into a longer transcript having a stem-loop structure and a shorter alternatively spliced transcript with no stem-loop. While the longer transcript is processed into miRNA, the shorter one is translated into miPEP. The miPEP enhances the transcription/production of the pri-miRNA from which it originates. Regulatory action of miPEP being species-specific, synthetic miPEP being is tested for exogenous application on crop plant to improve stress tolerance/agronomic performance. Deployment of the miPEP-mediated regulatory function might be a promising strategy to modulated miRNA-facilitated regulation of gene/trait of interest towards developing climate-resilient crops. In this review, we describe the newly identified and verified function of the MIR gene in the coding of miPEPs along with the comparison of the features of miRNA and miPEP in plant. We also discuss about their potential role in crop improvement and some of the yet unanswered question about miPEP.

Molecular basis of genetic plasticity to varying environmental conditions on growing rice by dry/direct-sowing and exposure to drought stress: Insights for DSR varietal development.

Rice requires plenty of water for its cultivation by transplanting. This poses several challenges to its cultivation due to erratic rainfall resulting in drought, flood, and other abiotic stresses of varying intensity. Dry/direct-sown rice (DSR) has emerged as a water-saving/climate-smart alternative to transplanted rice (TPR). The performance of a rice cultivar on growing by different methods of planting under varying environmental conditions varies considerably. However, the molecular basis of the observed phenotypic plasticity of rice to varying environmental conditions is still elusive. Resilience to various environmental fluctuations is important to ensure sustainable rice production in the present era of global climate change. Our observations on exclusively up-regulated genes in leaf of Nagina 22 (N 22) grown by dry/direct-sowing and subjected to drought stress at panicle initiation stage (compared to that in leaf of IR 64), and another set of genes exclusively down-regulated in leaf of N 22 (compared to that in leaf of IR 64) indicate important roles of leaf in stress resilience. A large number of genes down-regulated exclusively in root of N 22 on dry/direct-sowing subjected to drought stress indicates a major contribution of roots in stress tolerance. The genes for redox-homeostasis, transcription factors, stress signaling, carbohydrate metabolism, and epigenetic modifications play important roles in making N 22 better adapted to DSR conditions. More importantly, the involvement of genes in rendering genetic plasticity to N 22 under changing environmental conditions was confirmed by reversal of the method of planting. To the best of our knowledge, this is the first report on decoding the molecular basis of genetic plasticity of rice grown by two different methods of planting subjected to drought stress at the reproductive stage of plant growth. This might help in DSR varietal development program to enhance water-productivity, conserve natural resources, and minimize the emission of greenhouse gases, thus achieving the objectives of negative-emission agriculture.

Identification of microRNAs That Provide a Low Light Stress Tolerance-Mediated Signaling Pathway during Vegetative Growth in Rice.

Low light intensity affects several physiological parameters during the different growth stages in rice. Plants have various regulatory mechanisms to cope with stresses. One of them is the differential and temporal expression of genes, which is governed by post-transcriptional gene expression regulation through endogenous miRNAs. To decipher low light stress-responsive miRNAs in rice, miRNA expression profiling was carried out using next-generation sequencing of low-light-tolerant (Swarnaprabha) and -sensitive (IR8) rice genotypes through Illumina sequencing. Swarnaprabha and IR8 were subjected to 25% low light treatment for one day, three days, and five days at the active tillering stage. More than 43 million raw reads and 9 million clean reads were identified in Swarnaprabha, while more than 41 million raw reads and 8.5 million clean reads were identified in IR8 after NGS. Importantly, 513 new miRNAs in rice were identified, whose targets were mostly regulated by the genes involved in photosynthesis and metabolic pathways. Additionally, 114 known miRNAs were also identified. Five novel (osa-novmiR1, osa-novmiR2, osa-novmiR3, osa-novmiR4, and osa-novmiR5) and three known (osa-miR166c-3p, osa-miR2102-3p, and osa-miR530-3p) miRNAs were selected for their expression validation through miRNA-specific qRT-PCR. The expression analyses of most of the predicted targets of corresponding miRNAs show negative regulation. Hence, miRNAs modulated the expression of genes providing tolerance/susceptibility to low light stress. This information might be useful in the improvement of crop productivity under low light stress.

Pup1 QTL Regulates Gene Expression Through Epigenetic Modification of DNA Under Phosphate Starvation Stress in Rice.

Cytosine methylation, epigenetic DNA modification, is well known to regulate gene expression. Among the epigenetic modifications, 5-methylcytosine (5-mC) has been one of the extensively studied epigenetic changes responsible for regulating gene expression in animals and plants. Though a dramatic change in 5-mC content is observed at the genome level, the variation in gene expression is generally less than that it is expected. Only less is understood about the significance of 5-mC in gene regulation under P-starvation stress in plants. Using whole-genome bisulfite sequencing of a pair of rice [Pusa-44 and its near-isogenic line (NIL)-23 harboring Pup1 QTL] genotypes, we could decipher the role of Pup1 on DNA (de)methylation-mediated regulation of gene expression under P-starvation stress. We observed 13-15% of total cytosines to be methylated in the rice genome, which increased significantly under the stress. The number of differentially methylated regions (DMRs) for hypomethylation (6,068) was higher than those (5,279) for hypermethylated DMRs under the stress, particularly in root of NIL-23. Hypomethylation in CHH context caused upregulated expression of 489 genes in shoot and 382 genes in root of NIL-23 under the stress, wherein 387 genes in shoot and 240 genes in root were upregulated exclusively in NIL-23. Many of the genes for DNA methylation, a few for DNA demethylation, and RNA-directed DNA methylation were upregulated in root of NIL-23 under the stress. Methylation or demethylation of DNA in genic regions differentially affected gene expression. Correlation analysis for the distribution of DMRs and gene expression indicated the regulation of gene mainly through (de)methylation of promoter. Many of the P-responsive genes were hypomethylated or upregulated in roots of NIL-23 under the stress. Hypermethylation of gene body in CG, CHG, and CHH contexts caused up- or downregulated expression of transcription factors (TFs), P transporters, phosphoesterases, retrotransposon proteins, and other proteins. Our integrated transcriptome and methylome analyses revealed an important role of the Pup1 QTL in epigenetic regulation of the genes for transporters, TFs, phosphatases, carbohydrate metabolism, hormone-signaling, and chromatin architecture or epigenetic modifications in P-starvation tolerance. This provides insights into the molecular function of Pup1 in modulating gene expression through DNA (de)methylation, which might be useful in improving P-use efficiency or productivity of rice in P-deficient soil.

Community structure and function of microbiomes in polluted stretches of river Yamuna in New Delhi, India, using shotgun metagenomics.

The large population residing in the northern region of India surrounding Delhi mostly depends on water of River Yamuna, a tributary of mighty Ganga for agriculture, drinking and various religious activities. However, continuous anthropogenic activities mostly due to pollution mediated by rapid urbanization and industrialization have profoundly affected river microflora and their function thus its health. In this study, potential of whole-genome metagenomics was exploited to unravel the novel consortia of microbiome and their functional potential in the polluted sediments of the river at Delhi. Analysis of high-quality metagenome data from Illumina NextSeq500 revealed substantial differences in composition of microbiota at different sites dominated by Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria and Chloroflexi phyla. The presence of highly dominant anaerobic bacteria like Dechloromonas aromatica (benzene reducing and denitrifying), Rhodopseudomonas palustris (organic matter reducing), Syntrophus aciditrophicus (fatty acid reducing) and Syntrophobacter fumaroxidans (sulphate reducing) in the polluted river Yamuna signifies the impact of unchecked pollution in declining health of the river ecosystem. A decline in abundance of phages was also noticed along the downstream river Yamuna. Mining of mycobiome reads uncovered plethora of fungal communities (i.e. Nakaseomyces, Aspergillus, Schizosaccharomyces and Lodderomyces) in the polluted stretches due to the availability of higher organic carbon and total nitrogen (%) could be decoded as promising bioindicators of river trophic status. Pathway analysis through KEGG revealed higher abundance of genes involved in energy metabolism (nitrogen and sulphur), methane metabolism, degradation of xenobiotics (Nitrotoluene, Benzoate and Atrazine), two-component system (atoB, cusA and silA) and membrane transport (ABC transporters). Catalase-peroxidase and 4-hydroxybenzoate 3-monooxygenase were the most enriched pollution degrading enzymes in the polluted study sites of river Yamuna. Overall, our results provide crucial insights into microbial dynamics and their function in response to high pollution and could be insightful to the ongoing remediation strategies to clean river Yamuna.

Transcriptome analysis of a near-isogenic line and its recurrent parent reveals the role of Pup1 QTL in phosphorus deficiency tolerance of rice at tillering stage.

Phosphorus (P) is essential for cellular processes like respiration, photosynthesis, biosynthesis of membrane phospholipids, etc. To cope with P deficiency stress, plants adopt reprograming of the expression of genes involved in different metabolic/signaling pathways for survival, growth, and development. Plants use transcriptional, post-transcriptional, and/or post-translational machinery to achieve P homeostasis. Several transcription factors (TFs), miRNAs, and P transporters play important roles in P deficiency tolerance; however, the underlying mechanisms responsible for P deficiency tolerance remain poorly understood. Studies on P starvation/deficiency responses in plants at early (seedling) stage of growth have been reported but only a few of them focused on molecular responses of the plant at advanced (tillering or reproductive) stage of growth. To decipher the strategies adopted by rice at tillering stage under P deficiency stress, a pair of contrasting genotypes [Pusa-44 (a high-yielding, P deficiency sensitive cultivar) and its near-isogenic line (NIL-23, P deficiency tolerant) for Pup1 QTL] was used for morphophysiological, biochemical, and molecular analyses. Comparative analyses of shoot and root tissues from 45-day-old plants grown hydroponically under P sufficient (16 ppm) or P deficient (4 ppm) medium confirmed some of the known morphophysiological responses. Moreover, RNA-seq analysis revealed the important roles of phosphate transporters, TFs, auxin-responsive proteins, modulation in the cell wall, fatty acid metabolism, and chromatin architecture/epigenetic modifications in providing P deficiency tolerance to NIL-23, which were brought in due to the introgression of the Pup1 QTL in Pusa-44. This study provides insights into the molecular functions of Pup1 for P deficiency tolerance, which might be utilized to improve P-use efficiency of rice for better productivity in P deficient soils. KEY MESSAGE: Introgression of Pup1 QTL in high-yielding rice cultivar modulates mainly phosphate transporters, TFs, auxin-responsive proteins, cell wall structure, fatty acid metabolism, and chromatin architecture/epigenetic modifications at tillering stage of growth under phosphorus deficiency stress.

RNA-seq analysis reveals the genes/pathways responsible for genetic plasticity of rice to varying environmental conditions on direct-sowing and transplanting.

Rice cultivation by transplanting requires plenty of water. It might become a challenging task in future to grow rice by transplanting due to the climatic change, water and labor scarcities. Direct-sown rice (DSR) is emerging as a resource-conserving and climate-smart alternative to transplanted rice (TPR). However, no specific variety has been bred for dry/direct-sown conditions. The present study was undertaken to decipher the molecular basis of genetic plasticity of rice under different planting methods. Comparative RNA-seq analysis revealed a number (6133) of genes exclusively up-regulated in Nagina-22 (N-22) leaf under DSR conditions, compared to that (3538) in IR64 leaf. Several genes up-regulated in N-22 were down-regulated in IR64. Genes for growth-regulation and nutrient-reservoir activities, transcription factors, translational machinery, carbohydrate metabolism, cell cycle/division, and chromatin organization/epigenetic modifications were considerably up-regulated in the leaf of N-22 under DSR conditions. Complementary effects of these factors in rendering genetic plasticity were confirmed by the agronomic/physiological performance of rice cultivar. Thus, growth-regulation/nutrient-reservoir activities, transcription factors, and translational machinery are important molecular factors responsible for the observed genetic plasticity/adaptability of Nagina-22 to different planting methods. This might help to develop molecular markers for DSR breeding, replacing TPR with DSR for better water-productivity, and minimizing greenhouse-gas emission necessary for negative emission agriculture.

Overexpression of a DUF740 family gene (LOC_Os04g59420) imparts enhanced climate resilience through multiple stress tolerance in rice.

Functional characterization of stress-responsive genes through the analysis of transgenic plants is a standard approach to comprehend their role in climate resilience and subsequently exploit them for sustainable crop improvement. In this study, we investigated the function of LOC_Os04g59420, a gene of DUF740 family (OsSRDP-Oryza sativa Stress Responsive DUF740 Protein) from rice, which showed upregulation in response to abiotic stress in the available global expression data, but is yet to be functionally characterized. Transgenic plants of the rice OsSRDP gene, driven by a stress-inducible promoter AtRd29A, were developed in the background of cv. Pusa Sugandh 2 (PS2) and their transgene integration and copy number were confirmed by molecular analysis. The three independent homozygous transgenic plants (AtRd29A::OsSRDP rice transformants) showed better resilience to drought, salinity, and cold stresses, but not heat stress, as compared to the non-transformed PS2, which corresponded with their respective relative transcript abundance for OsSRDP. Transgenic plants maintained higher RWC, photosynthetic pigments, and proline accumulation under drought and salinity stresses. Furthermore, they exhibited less accumulation of reactive oxygen species (ROS) than PS2 under drought stress, as seen from the transcript abundance studies of the ROS genes. Under cold stress, OsSRDP transgenic lines illustrated minimal cell membrane injury compared to PS2. Additionally, the transgenic plants showed resistance to a virulent strain of rice blast fungus, Magnaporthe oryzae (M. oryzae). The promoter analysis of the gene in N22 and PS2 revealed the presence of multiple abiotic and biotic stress-specific motif elements supporting our observation on multiple stress tolerance. Based on bioinformatics studies, we identified four potential candidate interaction partners for LOC_Os04g59420, of which two genes (LOC_Os05g09640 and LOC_Os06g50370) showed co-expression under biotic and drought stress along with OsSRDP. Altogether, our findings established that stress-inducible expression of OsSRDP can significantly enhance tolerance to multiple abiotic stresses and a biotic stress.