Functional Analyses Of DWF4,a Key Enzyme In Brassinosteroid Biosynthesis Pathway In Arabidopsis

Brassinosteroids(BRs)are a group of plant steroidal hormones,originally identified and purified from rape pollens.They are critical for many processes during plant growth and development,such as stem elongation,leaf and root development,pollen tube growth,vascular differentiation,photomorphogenesis and skotomorphogenesis,seed germination and stress tolerance.Using combinational approaches including genetics,analytic chemistry,biochemistry and molecular biology,major enzymes catalyzing many critical steps of the BR biosynthesis pathway have been elucidated.Most of the enzymes identified belong to members of cytochrome P450 family.DWF4,a P450 enzyme,is believed to catalyze several rate-limiting steps in the BR biosynthesis pathway.Previous studies showed that overexpression of a transcription factor TCP1 can up-regulate DWF4,which suppresses the defective phenotypes of bri1-5,a weak mutant of a major BR receptor BRI1.The defective phenotypes of the bri1-5 can also be rescued by overexpression of DWF4,but not other enzymes involved in BR biosynthesis.These results are unexpected because bri1-5 is known to be insensitive to exogenously applied brassinolide(BL),the final product of the BR biosynthetic pathway and the most active BR.These results suggest that DWF4 may have additional functions besides its roles in regulating BR biosynthesis.Our analyses showed that overexpression of DWF4 can suppress the defective phenotypes of bri1-5 and bri1-301,suggesting that there is no allele specificity regarding the effect of DWF4 in regulating the growth of weak bri1 mutants.Overexpression of other BR biosynthetic genes cannot restore defective phenotypes.Endo H digestion experiment indicated that overexpression of DWF4 does not affect the retention of bri1-5 in the endoplasmic reticulum;meanwhile,phosphorylation and dephosphorylation status of BES1 in bri1-5 35S-DWF4-GFP was not altered before and after brassinolide treatment.These results indicated that the role of DWF4 in rescuing bri1-5 is unlikely via increasing endogenous concentration of BRs or via activating the BR signaling pathway.We therefore analyzed whether DWF4 regulates BR signaling pathway through activating the biosynthetic or signaling pathways of auxin and gibberellin(GA).The results of Real-time PCR showed that expression levels of auxin biosynthesis gene YUC3,and GA3OX1 and GA3OX2 required for GA biosynthesis were altered significantly in DWF4-overexpressed lines.Additionally,exogenous application of auxin and GA can slightly promote the growth of bri1-5 seedlings.Interestingly,overexpression of DWF4 can significantly restore the defective phenotypes of a GA insensitive mutant gai-1,indicating that DWF4 may be involved in promoting GA signaling transduction downstream DELLA protein.Further analysis is still needed to elucidate the detailed mechanisms of DWF4 in regulating GA signaling pathway.It is likely that overexpression of DWF4 increases the signaling pathways of both auxin and GA,which contribute significantly to the suppressed phenotype of bri1-5.