Screening And Function Analysis Of BZR1Target Genes

(Brassinosteroids)have been recognized as the sixth class of plant hormones aftercytokinin, auxin, gibberellins, ethylene and abscisic acid. Brassinosteroids regulate diverseplant growth and developmental processes such as promotion of stem elongation and celldivision, increase of tolerance to various stresses. Recently, more progresses were made infunctions analysis of Brassinosteroids, special in elucidating the brassinosteroid signalingpathway. BZR1(BRASSINAZOLE-RESISTANT1) is a key transcription factor in the BRsignaling pathway to regulate gene expression. How BZR1regulates the expression ofdownstream genes is still unclear. Analysis of the target genes of BZR1is a key forunderstanding the downstream of BR pathways and elucidating the transcriptional networkwith other signaling pathway, and also improving the bioactive substances of medicinal plant.In this study, we screened some BR regulated BZR1targets genes. We analyzed thegrowth phenotype of the over-expressing transgenic plants and their response to light andvarious hormones. Finally, we identified two genes, over-expression of which resulted inobvious phenotypes in the transgenic plants, PAR1(PHYTOCHROME RAPIDLYREGULATED1)and QQ1with unknown function. PAR1and its closest homolog PAR2arenegative regulators of shade-avoidance syndrome (SAS), they belong to the HLHtranscription factor family that lacks a typical basic domain required for DNA binding, andare believed to regulate gene expressions through DNA binding transcription factors that areyet to be identified. We used yeast-two-hybrid system to screen the possible genes which caninteract with the target genes. Further analysis of the mechanism by sorts of moleculartechniques such as DNA pull-down and chromatin immunoprecipitation assay identified thefunctions in vitro and vivo. The results show as follows:1. We generated overexpressing transgenic plants fused with MYC tag for candidategenes. Over expressing PAR1(PAR1OX) and QQ1(QQ1OX) transgenic plants displayeddwarfism with reduced petiole length and small leaves compared with wild-type Col-0.2. Here, we show that PAR1interacts with PIF4(phytochrome interaction factor4) inyeast-two-hybrid assay, bimolecular fluorescence complementation, in vitro pull down assayand Co-immunoprecipitation assay. Transgenic plants overexpressing PAR1arehypersensitive to light; the hypocotyl length of PAROX is shorter than wildtype Col-0understrong light density. PAR1protein level increased in etiolated seedlings under white light, and also under in red, far-red and blue light, indicating that PAR1protein stability is regulated bymultiple photoreceptors.3. PAR1inhibits PIF4direct target genes. The expression level of HFR1, PIL1andIAA29in PAR1OX are lower than wild type Col-0in qRT-PCR result. DNA pull-down andchromatin immunoprecipitation (ChIP) assays showed that PAR1inhibits PIF4DNA bindingin vitro and in vivo. We generated PAR1OX/PIF4OX double transgenic plants and thencompared hypocotyl length with each single transgenic plant. While PIF4OX dramaticallypromoted hypocotyl elongation, PAR1OX partially suppressed the long-hypocotyl phenotypeof PIF4OX, consistent with PIF4being less active in the PAR1OX background. In addition,PAR1is involved in the PIF4-mediated response, as transgenic plants overexpressing PAR1are insensitive to gibberellin (GA) or high temperature in hypocotyl elongation, similar to thepifq (PIF1, PIF3, PIF4and PIF5mutant).4. In addition to PIF4, PAR1also interacts with PRE1in yeast-two-hybrid andbimolecular fluorescence complementation assay. PRE1, belonging to HLH family, isactivated by brassinosteroids (BRs) and GA. We generated PAR1OX/PRE1OX doubletransgenic plants. The F1-generation plants showed long petioles and expended leaf blades,similar to PRE1OX plants. It indicated that overexpression of PRE1largely suppressed thedwarf phenotype of PAR1OX. These results indicate that PAR1–PRE1and PAR1–PIF4heterodimers form a complex HLH/bHLH network regulating cell elongation and plantdevelopment in response to light and hormones.5. In addition to PAR1, we also identified a BZR1directly target gene which hasobviously phenotype in transgenic plants, named QQ1. QQ1belongs to HLH family lacks atypical basic domain required for DNA binding. It is close to AIF1and PAR1after alignment.We found that QQ1interacts with PRE1in the yeast-two-hybrid assay. QQ1protein leveldramatically increased when exposed under short time of white light. BL treatment reducedits protein level which is consistent with the phenotype of the PAROX transgenic plant. Theresults indicated that QQ1might be another link for integrating the light and BR signalingpathway.