Significance Of BAK1 In Regulating BR Signaling Pathway And Molecular Mechanism Of ARF7 Protein Degradation

Both brassinosteroids(BRs)and auxin are important phytohormones.Individually or synergistically,they regulate multiple growth and development progresses in plants,including cell division and elongation,tissue and organ differentiation,flowering and fruiting,maturation and senescence,and responses to biotic or abiotic stresses.Phytohormone activities are controlled by their metabolism and distribution,and mainly depend on signal perception and transduction.The signaling pathways of these two hormones have attracted much attention,and great progresses have already been made by employing various approaches,such as genetics,biochemistry,pharmaceutical chemistry,molecular biology and omics.Although relatively complete BR and auxin signal transduction pathways have been built,little are known about the molecular mechanisms regulating the components in these two pathways.Previous studies indicated that BAK1 mediated the early events of BR signaling via interaction with BRI1.However,the significance of BAK1 was not clearly demonstrated in previous studies largely due to lacking of genetic data about the loss-of-function of BAK1 and its closely related redundant homologues.Whether BAK1 plays an essential role is one of the remaining scientific questions in this field.ARF transcription factors play key roles in the auxin signaling pathway.They regulate downstream gene transcription after receiving an auxin signal,to regulate plant growth and development from zygotic embryogenesis to senescence and death.However,mechanisms maintaining the homeostasis of this group of proteins are poorly understood.In this dissertation,I focused on the aforementioned two questions and obtained the following results:Phylogenetic and transgenic analyses indicated that in Col-0 only four members of the LRR? subfamily,including SERK1,SERK2,SERK3/BAK1 and SERK4/BKK1,play functionally redundant roles in regulating BR signaling.Two independent sets of triple T-DNA insertion mutants of serkl bak1 bkk1 show a de-etiolated phenotype similar to that of bri1-701,a null bril T-DNA insertional mutant,including shortened hypocotyls,opened cotyledons in the darkness.The phenotypes of the triple mutant can be complemented by the corresponding SERK genes,confirming that the observed triple mutant phenotypes are truly resulted from the null mutations of the corresponding SERKs.The serkl bakl bkkl triple mutant showed no response to exogenously applied BR in a classic root growth inhibition assay,and the expression levels of CPD and DWF4 did not change when treated with BR.Further more,the phosphorylation level of BES1 is not responsive to BR in the mutant.Finally,overexpression of BRI1 has no effect on the triple null mutant,and the phosphorylation of BRI1 remains at an extremely low level regardless of BR treatment in the triple null mutant.These results suggest that BR signaling is terminated at the very early stage in the triple mutant.The mutant aafl-D with short hypocotyl in the darkness was obtained via activation tagging to find BR signaling regulators.Molecular cloning and recapitulation analyses confirmed that the activated gene in this mutant is AAF1 which encodes an F-box protein with unknown functions.Overexpression of both AAF1 and its homologous gene AAF2 showed phenotypes similar to the the loss-of-function mutant arf7,with obvious auxin mutant phenotypes and reduced sensitivity to exogenous auxin treatment.On the contrary,the aafl aaf2 double loss-of-function mutant is more sensitive to auxin treatment.In addition,transgenic plants overexpressing AAF1 are insensitive to blue light,but the aafl aaf2 double mutant showed an exaggerated phototropism response to blue light treatment.Further in vivo and in vitro experiments proved that AAF1 directly interacts with ARF7,and AAF1 overexpression can accelerate ARF7 degradation through a 26S proteasome conjugated ubiquitination pathway.Furthermore,when exposed to unilateral blue light,the expression level of AAF1 is increased and the poly-ubiquitination level of ARF7 is enhanced with reduced ARF7 protein accumulation.In conclusion,our studies proved that BAK1 and its functionally redundant proteins play essential roles in the early events of BR signaling pathway by genetics and biochemistry approaches.Elegant genetics and biochemistry data showed that an F-box protein AAF1 mediates the polyubiquitination degradation of ARF7 to negatively regulate auxin and blue light signaling pathway.These results provided new molecular evidences to understand how plant cells maintain the balance between ARF protein biosynthesis and degradation.