Molecular Mechanisms Characterization Of Light Signal Protein FHY3 Interact With DET1 In Arabidopsis Thaliana

Light plays a vitol role in plant growth and development.Light not only act as a source of energy for photosynthesis,but also a signal to regulate plant growth and development.Plants had formed a light perception network consist of a series photoreceptors during the long history of evolution.Photoreceptors are used to preceive external light signal,and made plants adjust the growth and development through sensing the variation of light signal.When recieving the light changes of external environment,the protein structure and location of photoreceptors were changed,and transmit the light signal to the downstream pathways through interaction with other signal proteins to regulate plant growth and development and acclimatize the environment exchanges.FHY3(FAR-RED ELONGATED HYPOCOTYL 3)and its homologue protein FAR1(FAR-RED IMPAIRED RESPONSE 1)are two important transcription factors in light signal pathway.They directly bind to the FBS(FHY3/FAR1 Binding Sites)cis-element on the promoter and regulate the expression of downstream target genes.Previous studies have proved that FHY3/FAR1 involved in many biological process,such as ABA signal response,circadian clock regulation,chloroplast division,oxidative stress response and starch synthesis.DET1(DE-ETIOLATED 1)is a negative regulator of photomotphogenesis to be identified through genetic screening in early research of photomorphogenesis.DET1 loss-of-function mutant det1-1 shows constitutive de-etiolated phenotypes in dark.Previous studies indicate that DET1 interact with COP10 and DDB1 to form the CDD complex.CDD complex associated with the scaffolding protein CUL4 to form the CUL4-CDD complex,and regulate plant photomorphogenesis.In order to explore the regulation mechanisms of light signal protein FHY3,we used BD-FHY3 as the bait for yeast two hybrid based library screening,found FHY3 interact with DET1.Using choromatin immunoprecipitation,protoplast transient transformation and other experiment methods,we confirmed DET1 involved in regulating the actication of FHY3 downstream genes.We confirmed DET1 repress the activation of FHY3 downstream genes through analyzing the ABA sensitivity phenotypes of FHY3 and DET1 mutant.Main results are as follows:(1)We found FHY3 interact with DET1 through Yeast two hybrid based library screening,we separate DET1 and FHY3 into different part based on their protein structure and built the constructs for further yeast assay,the yeast two hybrid assay demonstrated FHY3 interact with DET1 in vitro.(2)Co-immunoprecipitation assay using DET1 and FHY3 transgenic crossing line confirmed FHY3 interact with DET1 in vivo.(3)Co-expression of DET1 and FHY3 in protoplast and decte the protein level of FHY3,we found FHY3 protein did not affect by DET1.(4)Chromatin immunoprecipitation analysis suggests that DET1 bind the FBS on FHY3 downstream target genes(CCA1,ELF4 and ABI5)promoter.(5)Yeast transcriptional activation and protoplast transformation assay indicated that FHY3 activated downstream genes(CCA1,ELF4 and ABI5)expression,while adding exongeous DET1 repressed this activation process.(6)FHY3 loss-of-function mutant fhy3-4 under No-0 ecotype background was insititive to exongeous ABA,we analyzed FHY3 mutant fhy3-11 under Col-0 ecotype background and DET1 mutant det1-1 ABA sensitivity,we found det1-1 was sensitive to ABA while fhy3-11 was insensitive.Through RT-qPCR test of the ABA signal response genes expression,we presumed ABI5 was overexpressed in det1-1,made det1-1 sensitive to ABA.(7)We analyzed the ABA sensitivity of fhy3det1 double mutants,absence of FHY3 in det1-1 partialy rescued the ABA sensitivity of det1-1 mutant,indicate that the ABA sensitive phenotype of det1-1 was depend on the ABI5 activation by FHY3.In conclusion,our data indicate DET1 interact with FHY3,and FHY3 transcriptional activation to CCA1,ELF4 and ABI5 was repressed by DET1,regulated the stability of plant circadian clock and sensitivity to ABA signal.