Functional Study Of Arabidopsis AT-hook Proteins

The plant cryptochromes CRY1 and CRY2 are the two receptors of blue light,and are involved in the control of numerous developmental events,including the regulation of flowering time and hypocotyl elongation.We screened out two late-flowering mutants through activation tagging method in background of the Arabidopsis cry1 cry2 double mutant.After genotyping,we found that the late-flowering phenotypes of the two mutants resulted from the overexpression of the two AT-hook genes AHL22 and AHL27,respectively.This suggests that Arabidopsis AT-hook genes may take part in the regulation of plant flowering.We cloned the genes AHL22 and AHL27,and another two genes AHL18 and AHL29 that shared highly identical sequence with AHL22 and AHL27,respectively.Bioinformatic analysis shows these AT-hook proteins contain an AT-hook motif and a PPC domain,Overexpression of these AT-hook genes in Arabidopsis wild type resulted in late flowering in both long days and short days and shorter hypocotyls in different lights with different fluence rates.Moreover,AHL22 overexpression can inhibit the growth of the primary roots.Furthermore,the quadruple mutant among the genes of AHL18,AHL22, AHL27 and AHL29 has phenotypes of early flowering and longer hypocotyls compared with wild type, even though the single and double mutants have no observable phenotype in flowering,suggesting that the four genes maybe involve redundantly in flowering regulation and photomorphogensis.Through the analysis of gene expression related to flowering time and hypocotyl elongation in AHL22- and AHL27-overexpressed plants and AHL18-AHL22-AHL27-AHL29 quadruple-RNAi mutant, we found that AHL22 delayed flowering resulting from primarily repression of photoperiodic genes CO and FT expression,that the late-flowering phenotype of AHL27 overexpressed plants resulted from the mRNA expression level change of FT and FLC,and that AHL18,AHL22,AHL27 and AHL29 inhibited the hypocotyl elongation through the repression of PIF4 gene expression.The analysis of mRNA expression of four AT-hook genes in different organs and promoter activity showed that they primarily expressed in roots and flowers.The result of Electrophoretic Mobility Shift Assay(EMSA) indicated that AHL22 and AHL27 proteins can directly bind to the AT-rich DNA sequence,which provided a line of evidence of the character of AT-hook proteins binding DNA.Meanwhile,the results of subcellular localization of GFP fused to AT-hook proteins showed the four AT-hook proteins are nuclear-localized proteins.The study proved experimentally that the four nuclear-localized AT-hook proteins AHL18,AHL22, AHL27 and AHL29 redundantly participate in regulating Arabidopsis flowering and hypocotyl elongation.