Luciferase-assisted Proteome Detection And The Functional Study Of COL7in The Regulation Of Branch

Protein degradation is one of the most conspicuous processes involved in the regulations ofdevelopment in response to environmental variations.26s proteasome pathway plays a vital role in theprotein degradation. Luciferase is a powerful reporter for the detection of protein dynamic changesbecause of its high sensitivity, stability and authenticity. In this study, we obtained nearly5000constructs containing different genes fused with luciferase, and obtain a large population of transgenicplants representing1000independent constructs. Based on this transgenic populations, we screenedcandidate proteins, the levels of which is changed in response to light, auxin, cytokinin andstrigolactones. We totally identified262candidate proteins and chose one of them, named COL7for thefunctional study. We found that COL7is unstable in dark but stabilized by phyB in white light.Over-expression of COL7promotes branching in white light with a high red/far red light ratio (R/FR)but not in shade with a low F/FR. The major results of this study were described as following:1.We obtained a large transgenic populations representing1000independent genes fused withluciferase;2.We screen the transgenic population and obtained262candidate genes, the protein level ofwhich response to the light, auxin, cytokinin and strigolactones;3.We study the half-life of plant proteins, the results indicate that the overall half-life distributenormally with the peak at the90min;4.COL7is unstable in dark but accumulates continually in far-red and blue light condition;Interestingly, COL7protein accumulates first but then degrade in red light condition;5.Over-expression of COL7results a more branch phenotype, which could be suppressed whenthe plants were grown with a higher density; Moreover, the function of COL7in branchpromotion is dependent on the phyB;6.Root-excited assay, grafting assay and DR5rec::GFP genetic assay showed that COL7possiblyregulates branch development through the auxin synthesis pathway;7.In vivo dual-luc assay indicated that COL7could activate the recombinant promoter containingthe CCAAT-box, and COL7has the transcriptional activity in yeast. But in vitro DNA-bindingassay indicated that the COL7could not bind the CCAAT box directly, implying that COL7may form protein complex with unknown partners to target the promoter containing theCCAAT-box.8.Real-time PCR results indicated that the COL7could suppress the expression of auxin relatedgenes such as TAA, YUC2. Moreover, it could increase the expression of SUR2, a suppressor ofauxin biosynthesis.In vivo dual-LUC assay indicated that the COL7can activate the expressionof LUC reporter genes under the driven of SUR2promoter.Taking together that the protein stability of COL7is regulated by phyB in response to the lightradiations and COL7enhances branching via activating the expression of SUR2and suppressing the synthesis of auxin, we argue that C0L7isa key factor linking rfom the light perception to branchdevelopment.