Screening And Identification Of Target Genes For GIS, GIS2and ZFP8in The Regulation Of Arabidopsis Trichome

Trichome development has provided a simple model to study mechanistic insight into the control of cell fate and morphogenesis in Arabidopsis. Significant progress has been made in understanding the genetic control of trichome development in Arabidopsis. A number of genes involved in different stages of trichome development have been isolated. Functional analyses of the corresponding genes have revealed a first glimpse into the underlying molecular mechanisms of trichome development and regulation. In our previous study, we demonstrated that GIS and its homologous genes ZFP8and GIS2postively regulated trichome development by integrating cytokinin and gibberellin signaling. We also showed that GIS, GIS2and ZFP8are positive activators for trichome production and they play specialized distinct roles in response to GA and cytokinin to control trichome development. In this study, we found that GIS regulates trichome branching by genetically interacting with SIM in Arabidopsis. More importantly, using dexamethasone-inducer overexpression and microarray data, the genes up and down regulated by GIS, GIS2and ZFP are identifed and all these genes expression were compared and analyzed in gis, gis2, zfp8single mutants, double mutants gisgis2, gisgis2ZFP8RNAi line and wild types, Dex-inducer overexpression of GIS, GIS2and ZFP8genes and no inducer. Through GO analyses and q-PCR confirmation, we determined a few direct target genes for GIS and GIS2by using chromatin immune-precipitation and DEX+CHX experimentsWe firstly screened out176downstream genes regulated by GIS. Then we determined the relative expression level of these genes and found out that the expressions of40genes are significantly differenent betweeen gis and overexpression GIS lines through q-PCR. Then, we selected9genes as the candidate target genes of GIS. Through dexamethasone and cycloheximide treatment and ChIP experiments, we demonstrated that At3g04720(PR-4), At2gO5160and At3g57920(SPLI5) are the direct target genes of GIS. Further GA induction experiments have shown that the three genes are also induced by GA.We screened out142downstream genes regulated by GIS2. Then we determined the relative expression level of these genes and found out that the expressions of40genes are significantly differenent betweeen gis2and overexpression GIS2lines through q-PCR. We also selected7genes as the candidate target genes of GIS2. Through dexamethasone and cycloheximide treatment and ChIP experiments, we confirmed that At5g24150(SQE5) and At4g35770(SENI) are the direct target genes of GIS2. Further GA induction experiments has shown that At5g24150(SQE5) genes is also induced by GA. We also screened out138downstream genes regulated by ZFP8. Through q-PCR, we identified a few important downstream genes regulated by ZFP8. Compared the downstream genes of GIS, GIS2and ZFP8, we only found one common gene, but GO analyses results showed that the functions of the downstream genes of GIS, GIS2and ZFP8were very similar. Gene expression analyses between the double mutants gisgis2and gisgis2ZFP8RNAi line, we found that GIS, GIS2and ZFP8could play common roles in the response to environmental stimuli.The findings from this study identifed the direct target gens of GIS and GIS2, which will provide more information and make us better understanding the molecualr mechanism of GIS and GIS family genes control trihcome development through plant hormone signaling. This study has an important academic significance in improving plant trichomes development regulation network. The clonings and function verifications of GIS, GIS2, ZFP8and their downstream genes provide important new genetic resources for improving crop resistance, crop yields and plant molecular breeding.