| miR393 is a conserved miRNA family in plants, involved in plant immune response, growth and development, and stress responses. So far, there is no report about the regulating mechanism and biological function of miR393/target gene in seed germination. As an important cereal crop, rice has unique growth habit of germinating and growing under submergence. When submerged, the hollow coleoptile elongates persistently to contact with water surface for sufficient oxygen, which is necessary for seed germination. In our prior reseach, we have identified two targets of miR393 in rice are OsTIR1 and OsAFB2, which are auxin receptor genes. Interestingly, miR393a strongly expressed in coleoptile tip. On this basis, we established a series of miR393 and its target genes associated transgenic rice, this study aims to explore the rote of miR393/target genes homo log module in rice growth and development via analysis of their transcriptional activity site, phenotype and transcription regulation mechanism. The main results are as follows:1. Features of transcriptional activity(1) Active transcription sites of OsTIRl and OsAFB2 are different Transcription activity of OsTIR1 is mainly observed at the root tip of primary root, adventitious root and crown root, also in young palea and lemma, auricle and node, while transcription activity of OsAFB2 was only found in young palea and lemma, lamina joint and node.(2) At the early stage of seed germination, miR393a and OsTIR1 present distinct spatial expression patterns in scute llar epitheliums, coleoptiles, radical roots and lateral roots.(3) In rice, coleoptile stoma consists of a pair of kidney-shaped guard cells, different from the typical dumbbell stomatal guard cells of leaves. miR393a and DR5 have obvious transcriptional activity in coleoptile stomatal guard cells. Especially, miR393a strongly expressed in the meristemoid, suggesting that miR393 may participate in the regulation of stomatal development2. Impacts of miR393 on rice growth and expression of related genes(1) miR393 promoted the primary root growth and suppressed lateral root growth. By microscopic observation we find that changes had taken place in the elongation zone length and cell number in the zone. Lateral roots of MIR393 overexpression lines were significantly shorter than the wild type, while that of MIM393 lines was longer than the wild type.(2) miR393 suppressed the growth of coleoptiles and stomata on its tips and the expression level of EXPA4, EXPA 7 and EXPB12.(3) miR393 induced free auxin accumulation in coleoptiles and increased transcrptional levels of OsYUCCA1 and OsYUCCA4.3. The regulatory mechanism of miR393(1) Under submergence, transcriptional activity of miR393a in coleoptile tips decreased, while DR5 reaction increased.(2) ABA treatment caused the cease of coleoptile growth, enhanced transcriptional activity of miR393a on coleoptile tips and the stomata, and weakened DR5 reaction. The growth inhibition of coleoptile by ABA is likely to be related to EXPA2 and EXPA4.(3) Ac cord ing to northern blot results, OsTIR1 promoted the expression of miR393 by feedback bop.Together, this study found that miR393a had strong transcriptional activity in radical root base, lateral root primordium, coleoptile tips and meristemoids, influenced growth and development of radical roots, lateral roots, coleoptiles and stomata through regulating auxin signaling pathways, and increased the accumulation of the endogenous auxin levels in coleoptiles. Moreover, the transcription ofmiR393a was suppressed by submergence while induced by ABA treatment, and adjusted by target gene OsTIR1 through positive feedback. Our study reveals the regulation of miR393a/ OsTIRl in the early stage of rice seed germination. |
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