Delineating the tissue-mediated drought stress governed tuning of conserved miR408 and its targets in rice.

Engineering drought tolerance in rice needs to focus on regulators that enhance tolerance while boosting plant growth and vigor. The present study delineated the concealed function and tissue-mediated interplay of the miR408/target module in imparting drought stress tolerance in rice. The plant miR408 family comprises three dominant mature forms (21 nt), including a distinct monocot variant (F-7 with 5' C) and is divided into six groups. miR408 majorly cleaves genes belonging to the blue copper protein in addition to several other species-specific targets in plants. Comparative sequence analysis in 4726 rice accessions identified 22 sequence variants (SNP and InDELs) in its promoter (15) and pre-miR408 region. Haplotype analysis of the sequence variants indicated eight haplotypes (three: Japonica-specific and five: Indica-specific) of the miR408 promoter. In drought-tolerant Nagina 22, miR408 follows flag leaf preferential expression. Under drought conditions, its levels are upregulated in flag leaf and roots which seems to be regulated by a differential fraction of methylated cytosines (mCs) in the precursor region. The active pool of miR408 regulated targets under control and drought conditions is impacted by the tissue type. Comparative expression analysis of the miR408/target module under different sets of conditions features 83 targets exhibiting antagonistic expression in rice, out of which 12 genes, including four PLANTACYANINS (OsUCL6, 7, 9 and 30), PIRIN, OsLPR1, OsCHUP1, OsDOF12, OsBGLU1, glycine-rich cell wall gene, OsDUT, and OsERF7, are among the high confidence targets. Further, overexpression of MIR408 in drought-sensitive rice cultivar (PB1) leads to the massive enhancement of vegetative growth in rice with improved ETR and Y(II) and enhanced dehydration stress tolerance. The above results suggest that miR408 is likely to act as a positive regulator of growth and vigor, as well as dehydration stress, making it a potential candidate for engineering drought tolerance in rice.

Understanding the evolution of miRNA biogenesis machinery in plants with special focus on rice.

miRNA biogenesis process is an intricate and complex event consisting of many proteins working in a highly coordinated fashion. Most of these proteins have been studied in Arabidopsis; however, their orthologs and functions have not been explored in other plant species. In the present study, we have manually curated all the experimentally verified information present in the literature regarding these proteins and found a total of 98 genes involved in miRNA biogenesis in Arabidopsis. The conservation pattern of these proteins was identified in other plant species ranging from dicots to lower organisms, and we found that a major proportion of proteins involved in the pri-miRNA processing are conserved. However, nearly 20% of the genes, mostly involved in either transcription or functioning of the miRNAs, were absent in the lower organisms. Further, we manually curated a regulatory network of the core components of the biogenesis process and found that nearly half (46%) of the proteins interact with them, indicating that the processing step is perhaps the most under surveillance/regulation. We have subsequently attempted to characterize the orthologs identified in Oryza sativa, on the basis of transcriptome and epigenetic modifications under field drought conditions in order to assess the impact of drought on the process. We found several participating genes to be differentially expressed and/or epigenetically methylated under drought, although the core components like DCL1, SE, and HYL1 remain unaffected by the stress itself. The study enhances our present understanding of the biogenesis process and its regulation.