The Molecular Genetical And Biochemical Study Of Transcription Factor TCP2 And CRY1 Interaction In Arabidopsis

Light regulates growth and development of plants via multiple photoreceptors, including phytochromes and cryptochromes. Arabidopsis CRY1(CRYPTOCHROME 1) as a blue light receptor has been reported to involve in several blue light signal pathways and regulate growth and development in plants, for example inhibition on the hypocotyls elongation; de-etiolation of seedlings; circadian clock; accumulation of anthocyanin and flowering time, ect. CRY1 protein initiates blue light signal transduction in a protein interaction manner, consequently causes the photomorphogenesis in plants. Until now, the mechanisms underlying the photoreceptor function have been extensively studied, but how cryptochrome 1 regulates photomorphogenesis remains elusive. Multiple proteins have been studied to interact with CRY1 and involve in CRY1 blue light signals transduction pathway. However, no any transcription factor protein is reported to directly interact with CRY1 and participate in the CRY1 blue light signals transduction pathway at present. To better understand and optimize the mechanism of CRY1 functions on photomorphogenesis, we employed Yeast Two Hybrid way to screen the Arabidopsis transcription factor yeast library and obtained the transcription factor proteins which would interact with CRY1 and involve in the photomorphogenesis of CRY1 signal pathway. The main results are as follows:CRY1 was proved to interact with the preliminary screened transcription factor protein-TCP2(TEOSINTE- LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR 2) via the verification of repeated yeast two hybrid experiments and further examination of Bi FC(Bimolecular Fluorescence Complementation) and co-IP(co-immunoprecipitation). The results of subcellular localization in Arabidopsis and tobacco cells show that TCP2 is a nucleus protein, and is speculated to interact with the CRY1 in nucleus. This study finds and demonstrates a novel transcription factor protein interacts with CRY1 directly for the first time. Their interaction shows obvious blue light specificity in yeast cells and tobacco leaf cells, but this character disappears in Arabidopsis. We presume that the reasons that yeast and tobacco systems are free of Arabidopsis proteins may result in this difference, and it was hypothesized that other Arabidopsis proteins involved in their interaction.Domains components of CRY1 and TCP2 were analyzed by bioinformatics analysis. Several of domain fragments of CRY1 and TCP2 were cloned and the results of Yeast two hybrid and Bi FC show that the N terminus of CRY1(including 1-515 amino acids, containing the PHR domain of CRY1) interacts with the feature domain TCP(including 1-174 amino acids) of TCP2. Moreover, The R domain which is included in the TCP domain would influence the blue light dependent of TCP domain and CRY1 interaction.Genetic studies suggest that TCP2 inhibits the hypocotyls elongation with blue light dependent, this kind of blue light specificity is not only performed on the light wavelength but also the light intensity. TCP2 functioned on the cotyledon morphology under red and blue light. Loss-function mutants showed the late flowering phenotype. Our results also verified TCP2 protein functions on leaf size and morphology regulation.In view of the significance of light as ambient factor on plant protein expression and CRY1 on blue light signal transduction, we studied TCP2 protein expression change in response to different light. Immunoblots were employed to test the protein levels in the transgenic lines of Myc-TCP2/WT and Myc-TCP2/cry1 and found that TCP2 was a light response protein. It sharply accumulated in the blue light, but degraded in darkness and far red. It also accumulated in the red light but both the quantity and speed of accumulation were far less and lower than in the blue light. Meanwhile, CRY1 was required to keep TCP2 protein stability and promote its accumulation speed. The results of protein inhibitor MG132 treatment demonstrate that the degradation of TCP2 is via 26 S proteasome ubiquitin pathway. Luciferase assay was used to examine LUC-TCP2 protein change in different backgrounds(e.g. WT, cry1, cry1cry2 and ztl3-1) of LUC-TCP2 over expression transgenic lines with or without the treatment of translation inhibitor CHX, when moved from dark to blue light. The results suggest that TCP2 accumulation in blue light is also regulated by other blue light receptors, and also influenced by its m RNA translation level. It also demonstrates indirectly that these blue light receptors regulate TCP2 protein accumulation by their regulations on the TCP2 m RNA.Studies on the photoreceptors effects on the TCP2 m RNA suggest that, TCP2 m RNA is light specifically regulated by several photoreceptors. TCP2 is a nucleus localization transcription factor, positively regulates CRY1 downstream genes HY5, HYH, CHS and CAB m RNA expression levels. RBSS(Radom binding site selection) showed the TCP2 specific binding site GGGGNCC. Meanwhile, the Ch IP-q PCR revealed that TCP2 protein blue light specific bound HY5 and HYH promoter regions which contained TCP2 binding site or similar binding sites. The above results support a hypothesis that TCP2 is a transcription activator that acts downstream from multiple photoreceptors, including CRY1.To investigate the interaction of CRY1 and TCP2 better in plant, we designed a dual vector system p DTs(including p DT1, p DT7 and p DT7G) to simultaneously express two proteins in plant with one time transformation. In this study, we constructed several paired genes to the dual vectors and transformed them into tobacco or Arabidopsis. The results of immunoblots, real time PCR analysis, fluorescence microscope assay, co-IP assay, transgenic lines phenotype analysis and co-expression efficiency analysis validated these vectors utility. The main results as follow:(1) several paired genes co-expressed successfully in plant;(2) c GR of p DT7 G vector achieved its nucleus-cytoplasm translocation function;(3) the biochemical and physiological functions of co-expressed genes were not affected;(4) these dual vectors realized two genes expressed in plant cells with high co-expression efficiency.