Research Scholar, Bose Institute
Dissecting the complex regulation of miRNA biogenesis during stresses in tomato plant
miR167 is a regulator of auxin response factors in different plants. We have observed that level of miR167 was increased during tomato plant development from plumule to formation of true leaves. miR167 level was upregulated during necrotrophic fungus as well as in biotrophic virus infections. Similarly, upregulation was also noted when the plants were exposed to cold stress. On the contrary miR167 level was down regulated during exposure to salt, drought and heat, indicating different stresses activated alternative mechanisms for miR167 expression regulation. The mechanism of mature miR167 biogenesis under different stress is not known. In NGS analysis of small RNA transcriptome of tomato (Pusa Ruby) two major isoforms miR167a and miR167b having 3 base pairs mismatch were identified. To investigate on the mechanism of regulation we have monitored pri and pre-miR167 levels of both the isoforms. Transcriptional regulation was predominant during biotic, drought and salt stresses and pre-miR167 processing regulation was important during thermal stress. As a prerequisite for promoter demarcation, 5’ RACE analysis was done to identify the transcription start site of miR167a gene. Promoter fragments of miR167a gene were cloned in a reporter vector. Reporter gene analysis indicated that NAC, CRT/DRE, W-box and MYB transcription factors are important regulators. Precursor-miR167a and precursor-miR167b (mostly expressed) levels has also been found to be differentially regulated under above conditions and mature-miR167 and Precursor-miR167 expression does not always follows similar pattern. This indicates the importance of processing in miR167 regulation. To explore the transcriptional regulation, transcription start site of miR167a gene (mostly processed) has been identified and promoter fragments of miR167 genes have been cloned and analyzed in planta. EMSA analysis confirms DREB as a regulator of miR167a biogenesis. Alternatively an invitro processing experiment has been carried out to understand the role of processing. Promoter-reporter transgenic plants have been developed to monitor the spatio-temporal pattern of regulation of miR167 gene during stress. Additionally, we have predicted some novel miR167 target genes that are involved in developmental and stress related pathways. Analyses of expressions of these genes and their nucleo-cytoplasmic distribution during stress indicated that miR167 is a key regulator of multiple stress related genes in tomato.
Abstract: Besides their definite role in plant developmental processes miR167 also serve as mediator of stress response. Although differential expression of miR167 occurs during stresses, the regulatory-mechanism of biogenesis remained elusive. Therefore, using tomato as the model plant we have explored the mechanism of regulation of miR167a expression during stresses. Fungus or virus infections and exposure to cold stress raised the level of miR167a expression. Whereas, salt, drought and heat treatments resulted in the downregulation, indicating different stresses activated alternative mechanisms for miR167a regulation. Interestingly, the relative expression level of precursors in control versus temperature stressed plants differed from the pattern observed in the mature miR167a expression, suggesting that both transcriptional and processing regulation were important for biogenesis. The promoter-regulatory sequence of the major isoform MIR167a harbours several development and stress-related regulatory sites. Accordingly, promoter assays using transient transformation and transgenic tobacco plants proved stress-dependent regulation of the promoter. Further analyses corroborated the role of tomato DREB2A protein in the transcriptional regulation during temperature stress. Finally, in vitro assays established the importance of processing factors in cold-stress dependent efficient processing of MIR167a precursors. These data confirm distinct role of transcriptional and processing machinery in stress-influenced regulation of tomato miR167a biogenesis.
Pub.: 13 Oct '17, Pinned: 19 Dec '17
Join Sparrho today to stay on top of science
Discover, organise and share research that matters to you