TCP1 Modulates The Exptessions Of DWF4 And Other Genes Via Directly Interagting With The GGNCCC Motifs In Their Promoter Regions In Arabidopsis Thaliana

Brassinosteroids (BRs) are endogenous plant growth-promoting hormones besides classic plant growth substances such as auxins, cytokinins, gibberellins, abscisic acid and ethylene. BRs were identified in almost all tested plants. Previous studies showed that BRs cannot be transported in a long distance manner, which is unique in phytohormones. Therefore, BR homeostasis must be maintained at tissue or even at cellular levels to ensure normal growth and development. Studies on various mutants helped to identify a number BR inactivation enzymes such as BSA1, SOB7 and BEN1. In the meantime, a number of transcription factors regulating BR biosynthesis were also identified such as BZR1/BES1 and CESTA. Using a gain-of-function genetic approach, our group identified a positive regulator of BR biosynthesis, TCP1. Subsequent analyses indicated that TCP1 is a positive regulator mediating BR biosynthesis via regulating the transcription of DWF4, one of the key BR biosynthetic genes. Overexpression of TCPI resulted in an increased level of DWF4 transcription, whereas overexpression of TCPI-SRDX resulted in a dramatically decreased level of DWF4 transcription. Detailed BR profile analyses also indicated that TCPI regulates BR biosynthesis via mediating the function of DWF4. Previous studies, however, did not experimentally demonstrate that TCPI can directly associate with the promoter region of DWF4. The objectives of this dissertation therefore include:1) investigating whether TCPI can directly interact with the promoter region of DWF4; 2) Using identified cis-elements and microarray technology to identify all possible genes directly regulated by TCP1 in Arabidopsis thaliana.TCP1 belongs to TCP family, a unique plant transcription factor family, TCP genes encoding proteins containing the so-called TCP domain, a conserved 59-amino acid basic helix-loop-helix (bHLH) motif that allows DNA binding and protein-protein interactions. TCPI is a member of class Ⅱ. GTGGNCCC motif was considered as a putative binding motif for TCPI. There is no such a sequence in the promoter region of DWF4. Instead, there are two GGNCCC motifs. ChIP analyses indiated that these two GGNCCC motifs can asssociate with TCP1. Analyses using EMSA further comfirmed that TCP1 can directly interact with the two GGNCCC motifs in the promoter region of DWF4. Site-directed mutagenesis suggested the flanking sequences of GGNCCC are less important for TCP1 binding. At the same time, we analyzed the importance of these two motifs in TCP1 controlled DWF4 expression by using a promoter-GUS transformation technique. Comparing to the same length of native DWF4 promoter, the modified promoter with two TCP1 motifs being replaced showed dramatically decreased transcription efficience. Because overexpression of TCP1 or TCP1-SRDX also showed phenotypes unrelated to BR biosynthesis or signaling, we carried out microarray analyses to compare the transcriptions of genes from wild-type, TCP1-SRDX and tcp1-1D plants, respectively in order to idenfy all potential TCP1-regulated genes in the Arabidopsis genome. We identified 520 genes in which there is at least one GGNCCC motif per gene and their expression levels are up- or down-regulated by at least two folds. These studies suggested that TCP1 not only regulate the expression of the key BR biosynthesis gene DWF4, but also other genes critical to plant growth and development.