Studies On The Mechanism Of BIC1 Involved In Brassinosteroid Signaling Pathway In Regulating The Growth Of Arabidopsis And Wheat

Brassinosteroid(BR)is a kind of very important plant hormone,which is involved in regulating many important processes of plant growth and development and the response of plants to biotic and abiotic stresses.The study of BR signal transduction is of great scientific significance and is the frontier and hot field of international life science.As a green plant growth regulator,the potential application value of BR in improving plant architecture has also attracted people’s extensive interest.Through the research of scientists,BR signal transduction pathway has been established.BR signal is sensed by the co-receptor BRINSENSITIVE1(BRI1)and BRI1ASSOCIATEDPROTEINKINASE1(BAK1)on the plant cell surface,and then the negative regulator GSK3 kinase BRASSINOSTEROID INSENSITIVE 2(BIN2)is inactivated through a series of signal transduction events,which leads to the de-phosphorylation of(BRASSINAZOLE RESISTANT1)BZR1,a core transcription factor of BR signal pathway,which then transfers from the cytoplasm to the nucleus,and binds to the promoters of downstream target genes,so as to activate or inhibit the expression of target genes.Although the BZR1 transcription factor plays an important role in BR signal transduction,the molecular mechanism of how BRZ1 activates target genes is not clear.In this study,using the firefly luciferase complementation imaging(LCI)assay-based screening for BZR1 interacting proteins,we identified a BZR1-interacting protein Blue-light Inhibitor of Cryptochrome 1(BIC1).It has been reported that BIC1 acts as an inhibitor of cryptochrome 2(CRY2)function through suppressing the blue light-dependent dimerization,phosphorylation and degradation of CRY2.We further demonstrated that BIC1 interacts with BZR1 in vitro and in vivo via Pull down assay,BiFC assay and Co-IP assay.Then,phenotypic analyses showed that the bic1bic2 double mutant exhibited less sensitivity to BR treatment in hypocotyl elongation and BR-induced downstream gene expression compared to wild type,indicating that BIC1 positively regulates BR response.Genetic evidence showed that BIC1 and BZR1 synergistically promote hypocotyl elongation and BR response.Further,we demonstrated that the BZR1-induced hypocotyl elongation and hypersensitivity to BR treatment were largely dependent on the function of BIC1 and BIC2.Next,we found that BIC1 has the transcriptional activation activity via transactivation activity assays in yeast cells and transient dual-luciferase reporter system(DLR).Then,we performed DLR assays and the results showed that BIC1 and BZR1 synergistically activate PRES.Previous studies have shown that PHYTOCHROME INTERACTING FACTOR(PIF4)interacts with BZR1 and they synergistically activate common target genes.We performed LCI assay,Pull down assay and Co-IP assay to demonstrate that BIC1 interacts with PIF4 in vitro and in vivo.Furthermore,genetic evidence illustrated that BIC1 and PIF4 synergistically and interdependently promote hypocotyl elongation and downstream gene expression.To further investigate the biological function of BIC1 homologs in bread wheat,we cloned the BIC1 and BZR1 homologous genes from bread wheat.We demonstrated the interaction between TaBIC1 and TaBZR1 also occurs in bread wheat via LCI assay,Pull down assay and BiFC assay.We also generated TaBIC1-overexpression transgenic wheat plants which exhibited appropriately increased plant height compared with the control KN199(the transformation background)at mature stage.