| The miR396 family, one of the most conserved miRNA families, plays an important regulation role in plant growth and development. In Arabidopsis, miR396 targets seven members of GRF family and bHLH74. bHLH74, also referred to as Activator 2 for cell elongation, belongs to the basic Helix-Loop-Helix transcription factor family. Previous studies indicate that miR396/bHLH74 module takes part in the regulation of elongation zone in roots of Arabidopsis seedlings. However, other biological functions and relative mechanisms are largely unknown.In this study, we focused on how At-miR396 regulates hypocotyl elongation through bHLH74 repression and hormone signaling pathway using mutants of At-miR396 and its target. The results obtained are as follows.1 miR396-mediated bHLH74 repression negatively regulates hypocotyl elongation1.1 Phenotype observation of 6-d-old seedlings indicated that the hypocotyl length of bhlh74-1 is significantly shorter than that of wild-type. However, the hypocotyl length of over-expression lines,35S:mbHLH74, is significantly longer than that of wild-type. It indicated that the transcription factor bHLH74 is an activator for cell elongation.1.2 After growing for 6 days in darkness, At-miR396 over-expression lines (OX396a and OX396b) displayed shorter hypocotyl than wild-type, which was similar to that of bhlh74-1.In addition, the levels of bHLH74 transcript in these lines were also significantly down-regulated, compared with that of wild-type. It indicated that miR396-mediated bHLH74 repression negatively regulates hypocotyl elongation.1.3 The results of quantitative RT-PCR showed that the levels of downstream genes, including EXP1、EXPL2、EXP8 and XTH4, were significantly decreased in bhlh 74-1 and OX396a#7 transgenic lines. It suggested that miR396 negatively regulates downstream genes in BR signaling pathway to influence hypocotyl elongation.2 At-miR396 regulates hypocotyl elongation through BR signaling pathway2.1 At-miR396 negatively regulates hypocotyl elongation through enhancing BR sensitivity in darkness. Hypocotyl elongation in OX396a#7 and bhlh.74-1 were more sensitive to exogenous BR in darkness. Quantitative RT-PCR analysis of 4 cell elongation activating genes in BR signaling pathway showed that miR396 influenced the expression of these genes under control and BR-treatment conditions, which contributed to the alteration of BR sensitivity.2.2 Exogenous BR promotes cotyledon growth in miR396 over-expression lines and bhlh74-1mutant, but inhibits cotyledon growth of wild-type in light. It indicated that miR396 regulates BR sensitivity, which results in the alteration of cotyledon sizes in light.2.3 The growth of hypocotyl in wild-type is sensitive to temperature, with its length increasing when the temperature rises within 20℃ to 30℃. However, the hypocotyl growth in bhlh.74-1, OX396a#7 or OX396b#8 is insensitive to temperature. It suggested that miR396 regulates hypocotyl elongation through alteration of sensitivity to temperature.2.4 Exogenous hormones and environmental temperature influence the expression level of miR396.1 nM exogenous eBL induces the expression of miR396b in the aboveground parts of seedlings.2,4-D induces miR396a expression in the roots, while inhibits miR396a/b expression in the aboveground parts. In darkness,2,4-D or GA induced miR396b expression in the hypocotyls. In addition, temperature also affects miR396b expression in the hypocotyls. These data demonstrated that the accumulation of miR396 in different tissues is influenced by hormone and environmental signals.Based on the above results and previous studies, we propose a model for the regulation of hypocotyls cell elongation by miR396/bHLH74 through BR signaling pathway. At-miR396 mediates the post-transcriptional repression of target bHLH74, which might influence the interaction between bHLH74 and IBH or PIF4 transcription factor. At-miR396 also down-regulates the expression of downstream genes, leading to hypocotyl elongation inhibition and alters hypocotyl sensitivity to BR or environmental signals. Meanwhile, the expression of miR396 is regulated by Auxin, BR, GA and environmental temperature. In summary, this study identifies miR396/bHLH74 as a novel component involved in BR-regulated hypocotyl elongation. |
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