A comprehensive review on nutritional, nutraceutical, and industrial perspectives of perilla (Perilla frutscens L.) seeds - An orphan oilseed crop.

There is a growing need to mainstream orphan or underutilized crops to enhance nutritional security and sustainable agriculture. Among these, Perilla frutescens L. is an important crop due to its rich nutritional and phytochemical content which makes it significant in nutrition, medicine, and industrial sector. Perilla seeds are mainly rich in ω-3 fatty acids, dietary fiber, amino acids, vitamins, and minerals, high α-linolenic acid, which contributes to their health benefits. This review explores the nutritional profile of perilla seeds and highlights its unique composition compared to other oilseed crops. It also analyzes the phytochemical components of perilla seeds and their various biological activities, including antioxidant, antidiabetic, antiobesity, cardioprotective, anticancer, antimicrobial, neuroprotective, and anti-inflammatory effects. These activities demonstrate the potential of perilla seeds in both pharmaceutical and food sectors. The review also covers recent advancements in genomics and transgenic research discussing potential areas for crop improvement. Additionally, it explores the use of perilla seeds in functional foods, blending perilla oil with other oils, and their applications in enhancing product formulations. This review offers valuable insights for researchers, students, policymakers, environmentalists, and industry professionals by detailing the potential of perilla seeds across various sectors. The findings support sustainable agriculture, crop diversification, and innovative product development, thus contributing to the integration of perilla into mainstream agriculture.

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.

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.

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.

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.

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.

Understanding 3D Genome Organization and Its Effect on Transcriptional Gene Regulation Under Environmental Stress in Plant: A Chromatin Perspective.

The genome of a eukaryotic organism is comprised of a supra-molecular complex of chromatin fibers and intricately folded three-dimensional (3D) structures. Chromosomal interactions and topological changes in response to the developmental and/or environmental stimuli affect gene expression. Chromatin architecture plays important roles in DNA replication, gene expression, and genome integrity. Higher-order chromatin organizations like chromosome territories (CTs), A/B compartments, topologically associating domains (TADs), and chromatin loops vary among cells, tissues, and species depending on the developmental stage and/or environmental conditions (4D genomics). Every chromosome occupies a separate territory in the interphase nucleus and forms the top layer of hierarchical structure (CTs) in most of the eukaryotes. While the A and B compartments are associated with active (euchromatic) and inactive (heterochromatic) chromatin, respectively, having well-defined genomic/epigenomic features, TADs are the structural units of chromatin. Chromatin architecture like TADs as well as the local interactions between promoter and regulatory elements correlates with the chromatin activity, which alters during environmental stresses due to relocalization of the architectural proteins. Moreover, chromatin looping brings the gene and regulatory elements in close proximity for interactions. The intricate relationship between nucleotide sequence and chromatin architecture requires a more comprehensive understanding to unravel the genome organization and genetic plasticity. During the last decade, advances in chromatin conformation capture techniques for unravelling 3D genome organizations have improved our understanding of genome biology. However, the recent advances, such as Hi-C and ChIA-PET, have substantially increased the resolution, throughput as well our interest in analysing genome organizations. The present review provides an overview of the historical and contemporary perspectives of chromosome conformation capture technologies, their applications in functional genomics, and the constraints in predicting 3D genome organization. We also discuss the future perspectives of understanding high-order chromatin organizations in deciphering transcriptional regulation of gene expression under environmental stress (4D genomics). These might help design the climate-smart crop to meet the ever-growing demands of food, feed, and fodder.

Characterization of contrasting rice (Oryza sativa L.) genotypes reveals the Pi-efficient schema for phosphate starvation tolerance.

BACKGROUND: Phosphorus (P), being one of the essential components of nucleic acids, cell membranes and enzymes, indispensable for diverse cellular processes like photosynthesis/carbohydrate metabolism, energy production, redox homeostasis and signaling. Crop yield is severely affected due to Phosphate (Pi) deficiency; and to cope with Pi-deficiency, plants have evolved several strategies. Some rice genotypes are compatible with low Pi availability, whereas others are sensitive to Pi deficiency. However, the underlying molecular mechanism for low Pi tolerance remains largely unexplored. RESULT: Several studies were carried out to understand Pi-deficiency responses in rice at seedling stage, but few of them targeted molecular aspects/responses of Pi-starvation at the advanced stage of growth. To delineate the molecular mechanisms for low Pi tolerance, a pair of contrasting rice (Oryza sativa L.) genotypes [viz. Pusa-44 (Pi-deficiency sensitive) and its near isogenic line (NIL-23, Pi-deficiency tolerant) harboring Phosphorus uptake 1 (Pup1) QTL from an aus landrace Kasalath] were used. Comparative morphological, physiological, and biochemical analyses confirmed some of the well-known findings. Transcriptome analysis of shoot and root tissues from 45-day-old rice plants grown hydroponically under P-sufficient (16 ppm Pi) or P-starved (0 ppm Pi) medium revealed that Pi-starvation stress causes global transcriptional reprogramming affecting several transcription factors, signaling pathways and other regulatory genes. We could identify several significantly up-regulated genes in roots of NIL-23 under Pi-starvation which might be responsible for the Pi starvation tolerance. Pathway enrichment analysis indicated significant role of certain phosphatases, transporters, transcription factors, carbohydrate metabolism, hormone-signaling, and epigenetic processes in improving P-starvation stress tolerance in NIL-23. CONCLUSION: We report the important candidate mechanisms for Pi acquisition/solubilization, recycling, remobilization/transport, sensing/signalling, genetic/epigenetic regulation, and cell wall structural changes to be responsible for P-starvation tolerance in NIL-23. The study provides some of the novel information useful for improving phosphorus-use efficiency in rice cultivars.

Silicon-Solubilizing Media and Its Implication for Characterization of Bacteria to Mitigate Biotic Stress.

Silicon (Si), the second most abundant element on earth, remains unavailable for plants' uptake due to its poor solubility. Microbial interventions to convert it in soluble forms are well documented. However, studies on discrimination of Si and P solubilizing microbes due to common estimation method and sharing of solubilization mechanism are still obscure. A defined differential media, i.e. silicon-solubilizing media (NBRISSM) is developed to screen Si solubilizers. NBRISN13 (Bacillus amyloliquefaciens), a Si solubilizer, exhibiting antagonistic property against Rhizoctonia solani, was further validated for disease resistance. The key finding of the work is that NBRISSM is a novel differential media for screening Si solubilizers, distinct from P solubilizers. Dominance of Pseudomonas and Bacillus spp. for the function of Si solubilization was observed during diversity analysis of Si solubilizers isolated from different rhizospheres. Sphingobacterium sp., a different strain has been identified for silicon solubilization other than Pseudomonas and Bacillus sp. Role of acidic phosphatase during Si solubilization has been firstly reported in our study in addition to other pH dependent phenomenon. Study also showed the combinatorial effect of feldspar and NBRISN13 on elicited immune response through (i) increased Si uptake, (ii) reduced disease severity, (iii) modulation of cell wall degrading and antioxidative enzyme activities, and (iv) induced defense responsive gene expression.

Plant growth-promoting bacteria Bacillus amyloliquefaciens NBRISN13 modulates gene expression profile of leaf and rhizosphere community in rice during salt stress.

Growth and productivity of rice and soil inhabiting microbial population is negatively affected by soil salinity. However, some salt resistant, rhizosphere competent bacteria improve plant health in saline stress. Present study evaluated the effect of salt tolerant Bacillus amyloliquefaciens NBRISN13 (SN13) inoculation on rice plants in hydroponic and soil conditions exposed to salinity. SN13 increased plant growth and salt tolerance (NaCl 200 mM) and expression of at least 14 genes under hydroponic and soil conditions in rice. Among these 14 genes 4 (NADP-Me2, EREBP, SOSI, BADH and SERK1) were up-regulated and 2 (GIG and SAPK4) repressed under salt stress in hydroponic condition. In greenhouse experiment, salt stress resulted in accumulation of MAPK5 and down-regulation of the remaining 13 transcripts was observed. SN13 treatment, with or without salt gave similar expression for all tested genes as compared to control. Salt stress caused changes in the microbial diversity of the rice rhizosphere and stimulated population of betaine-, sucrose-, trehalose-, and glutamine-utilizing bacteria in salt-treated rice rhizosphere (SN13 + salt). The observations imply that SN13 confers salt tolerance in rice by modulating differential transcription in a set of at least 14 genes. Stimulation of osmoprotectant utilizing microbial population as a mechanism of inducing salt tolerance in rice is reported for the first time in this study to the best of our knowledge.