COG1 Regulates Brassinosteroids Biosynthesis Via PIF4 And PIF5 In Arabidopsis Thaliana

Brassinosteroids(BRs)are a group of naturally occurring plant hormones regulating many cellular and physiological processes during normal growth and development,such as cell division,cell elongation,photomorphogenesis,flowering,senescence and responses to biotic and abiotic stresses.An activation-tagging screen in a weak BR receptor mutant,bri1-5,was performed to isolate more components involved in BR biosynthesis,catabolism,and signal transduction.bri1-5 cog1-2D mutant was identified as a genetic modifier that suppresses the short hypocotyl of bri1-5.The cog1-2D single mutant showed a long-hypocotyl phenotype compared to that of wild-type seedlings.COG1 promotes vegetative growth and vascular development of Arabidopsis.The adult cog1-2D single mutant shows delayed flowering and senescence in comparison with that of wild type.TAIL-PCR was used to clone the flanking sequence of the T-DNA from the p BIB-BASTA-AT2 vector.RT-PCR analysis found that a Dof-type transcription factor gene,COG1,was activation-tagged in cog1-2D.Genetic,biochemical and molecular strategies were used to uncover the detailed mechanisms through which COG1 is involved in mediating hypocotyl growth.Inducible expression of a dominant-negative form of COG1,COG1-SRDX,with a 12-amino acid repressor sequence fused to COG1 at its C terminus,results in plants with shortened hypocotyls.The hypocotyl growth of cog1-2D showed decreased insensitivity to exogenously applied 24-epibrassinolide,and the short hypocotyl phenotypes of COG1-SRDX transgenic plants can be rescued by exogenously applied 24-epibrassinolide,indicating that COG1 is involved in modulating BR biosynthesis or inactivation.RNA-seq and q RT-PCR data showed that several BR biosynthetic genes were up-regulated in cog1-2D seedlings.However,transcript levels for genes known to be involved in BR inactivation displayed subtle expression changes in cog1-2D mutants compared with wild type.BR profile assay suggested that BR levels were indeed elevated in cog1-2D seedlings.RNA-Seq was carried out to identify potential target genes of COG1.Two basic helix-loop-helix transcription factors,PIF4 and PIF5,were found to be transcriptionally regulated by COG1.Genetic analysis and q RT-PCR data indicated that PIF4 and PIF5 are required for COG1 to regulate BR biosynthesis and hypocotyl elongation.Ch IP and EMSA analyses demonstrated that COG1 directly binds to the promoter regions of PIF4 and PIF5.PIF4 and PIF5 also directly bind to the promoter regions of at least two BR biosynthetic genes,DWF4 and/or BR6ox2.These studies demonstrate another transcriptional regulation mechanism through which plant maintains BR homeostasis that is critical for plant growth and development.