The Regulatory Mechanisms Of Arabidopsis WRKY12 And WRKY13 Transcription Factors Involved In Age-mediated Flowering

For plants,the onset of flowering is a major developmental transition from vegetative phase to reproductive phase and that is critical for reproductive success.Flowering time is a critical determinant of regional adaptability of plants,and thus crop yields.However,flowering is a very complicated process,which is controled by both of internal genetic factors and external environmental conditions.Past two decades have seen fundamental advances in our knowledge of the flowering regulation pathway of plants,especially the model plant Arabidopsis thaliana,remarkable progresses have been achieved.At present,it has been indentified five flowering time pathways of arabidopsis thaliana,known as vernalization pathway,photoperiodic pathway,gibberellin pathway,age pathway and autonomic pathway.In short-day conditions,GA and age pathways play the predominant roles in controlling flowering time.In our previous study,we have identfied that WRKY12 and WRKY13 partipated in gibberellin pathway to regulate flowering through interacting with DELLA proteins.In addition,their expression patterns had a close related to the plant development,the WRKY12 expression level climbed steadily,while the WRKY13 expression level decreased.In the age-pathway,the expression level of miR156 is reduced with age,while the level of miR156-targeted SPLs increased.All of these results suggested that WRKY12 and WRKY13 may modulate age-mediated flowering.To investigate this hypothesis,we examined the expression patterns of WRKY12 and WRKY13 by quantitative real-time PCR(RT-qPCR)analysis in wild-type(WT),35S::MIR156 and 35S::MIM156 plants during development under SD conditions.The result showed that the expression levels WRKY12 and WRKY13 were regulated by miR156.Previous study had shown that miR156 regulates the expression of downstream genes via SPL transcription factors,the Y1H,EMSA and ChIP assay indicated that SPL10 and its homologous genes could directly bind to the promoters of WRKY12 and WRKY13,and the transient expression assays suggested that SPL10 activated the expression of WRKY12,and repressed the expression of WRKY13.The genetic assays indected that the early-flowering phenotype of pSPL10::rSPL10:GFP was significantly inhibited in the wrky12-2 mutant background.Moreover,overexpression of WRKY13 in SPL 10 misexpression background conferred plants flowering significantly later than pSPL10::rSPL10:GFP plants.At the protein level,we used yeast two-hybrid(Y2H)assays to test whether WRKY12 and WRKY13 could interact with SPL transcription factors.Our result suggested that WRKY12 and WRKY13 physically interacte with SPL10.Additionally,the widespread interactions between WRKY12/WRKY13 and SPLs(SPL2/6/11/12)were also confirmed by Y2H.Previous study showed that SPL10 regulated the transition from vegetative to reproductive by direct activation of miR72b.Y1H,EMSA and ChIP assays showed that WRKY12 and WRKY13 could directly bind to the promoter of miR172b.Transient expression assays showed that miR172b transcription was activated by WRKY12/SPL10 and repressed by WRKY13.The genetic assays indicted that the late-flowering phenotypes of the wrky12-2 mutant and WRKY13-OV plants were partially rescued by miR172b overexpression.In addition,WRKY12 and SPL10 form a complex on the miR172b promoter and increase the transcriptional activation activity of SPL10,while WRKY13 interact with SPL 10 and partly inhibit its transcriptional function to activate miR172b expression.In summary,our study found that not only on transcriptional level,but also on protein level WRKY12 and WRKY13 participated in age-pathway regulate flowering.In the juvenile phase,high levels of miR156 repressed the expression of SPL10,leading to the accumulation of WRKY13 while WRKY12 is maintained at a very low level.WRKY13 interacts with a small amount of SPL10 and inhibit its transcriptional function to activate miR172b expression,which maintains plants at the correct developmental stage.As plants growth,the age-dependent decline in miR156 results in increase SPL 10 expression,which promotes the expression of WRKY12 and suppresses the expression of WRKY13.The abundant WRKY12 protein then cooperates with SPL10 to promote flowering through activation of miR172b.