Molecular Mechanism Of Arabidopsis FRS6 And FRS8 In Regulation Of Hypocotyl Elongation

The Arabidopsis FRS(FAR1-related sequences)protein family has 14 family members including FHY3 and FAR1,together with FRS1 to FRS12.The sequence of the FRS protein family has high homology and conserved secondary structure,FRS proteins contain nuclear localization signals and coiled-coil domains.FRS genes are expressed in rosette leaves,cauline leaves,inflorescence stems,flowers,and is involved in regulating plant growth and development.Previous studies have shown that FRS6 and FRS8 may negatively regulate flowering time,but the downstream target genes and potential molecular mechanisms are unclear.In this study,we observed the phenotype of frs6,frs8,frs6 frs8 mutants and FRS6 overexpression lines(FRS6-OE-L1,FRS6-OE-L3),no obvious phenotype of flowering were found.However,it was found that frs6,frs8,and frs6 frs8 have a short hypocotyl phenotype,and FRS6-OE-L1,FRS6-OE-L3 have a long hypocotyl phenotype.We used molecular biology,biochemistry and other methods to explore the mechanism of FRS6 and FRS8 regulated hypocotyl elongation.The main results are as follows:FRS6 and FRS8 were localized in the nucleus through subcellular localization analysis.GUS staining analysis suggested that FRS6 and FRS8 were mainly expressed in the roots,veins and tips of cotyledons,hypocotyls in the seedling stage and expressed in stems,leaf tips,flowers and petioles in the adult seedling of Arabidopsis.The result suggested that frs6,frs8,frs6 frs8 have a short hypocotyl phenotype and FRS6-OE-L1,FRS6-OE-L3 have long hypocotyls compared to wild-type Col-0 under long-day or short-day conditions.After treatment with different light quality,the phenotype under red light is more obvious than white light.There is no significant difference in hypocotyl length under far red and blue light,indicating that FRS6 and FRS8 may positively regulate hypocotyl elongation by responding to red light signal transduction pathway.Our research showed that FRS6 and FRS8 can form homo and heterodimers through yeast two-hybrid experiments,but cannot interact with red light receptor phyB.HISTONEDEACETYLASE 15(HDA15)was interact with FRS6 and FRS8 through yeast screens and yeast two-hybrid experiments.Previous studies have shown that HDA15 is a negative regulator of hypocotyl elongation under red light.Therefore,we speculate that HDA15,FRS6,and FRS8 may antagonize each other to regulate hypocotyl in Arabidopsis thaliana in response to the red light signal transduction pathway.The result showed that there is no significant difference in the changes in the length of hypocotyl of frs6,frs8,frs6 frs8 and FRS6-OE-L1,FRS6-OE-L3 hypocotyls compared with wild-type Col-0 after applying IAA and NPA.The hypocotyl of FRS6-OE-L1,FRS6-OE-L3 were significantly elongated after applying GA,and the hypocotyl of FRS6-OE-L1,FRS6-OE-L3 were significantly inhibited after applying PAC.The results of RT-qPCR showed that Gibberellin synthetic genes are up-regulated in FRS6-OE-L1 and FRS6-OE-L3,and down-regulated in frs6.Yeast one-hybrid experiments confirmed that FRS6 and FRS8 can bind to the promoter of GA20OX4,indicated that FRS6 and FRS8 may regulate hypocotyl elongation by regulating the expression of gibberellin synthesis genes.In summary,these results suggested that FRS6 and FRS8 play a more important role in plant growth and development.FRS6 and FRS8 are co-localized in the nucleus,and they interact with each other and bind to HDA15,which may antagonize each other under red light to regulate hypocotyl growth.At the same time,FRS6 and FRS8 can bind to the promoter of GA20OX4 and participate in gibberellin synthesis to regulate the elongation of hypocotyls.The study of FRS6 and FRS8 has enriched the regulation mechanism of hypocotyl and provided new ideas for the functional exploration of other members of the FRS protein family.