Effects Of Cutin Alteration On Plant Skotomorphogenesis And Its Mechanism

An Arabidopsis thaliana mutant gfc1-1(grow fast on cytokinin)was obtained previously in our laboratory through forward genetic screening.When induced by cytokinin,the aerial part of this mutant grow larger than the wild type.GFC1 is identical to gene DCR(DEFECTIVE IN CUTICULAR RIDGES)encoding an acyltransferase,which plays a role in cutin synthesis(its catalytic activity in vivo has not been determined).In addition to the GFC phenotype,gfc1-1 also exhibits skotomorphogenetic deficient phenotype,displaying impairment in apical hook formation and hypocotyl elongation suppressing.Here,we study the function of GFC1/DCR in skotomorphogenesis and try to determine the catalytic activity.Double homozygous lines of gfc1-1 and DR5::GUS/GFP were constructed and observed by chemical staining and fluorescence,the asymmetric distribution of auxin in the apical hook is not different between gfc1-1 and Col-0.Through toluidine blue staining of cutin mutants,we found that all mutants that exhibit skotomorphogenetic deficient phenotype have deficient cuticle.In the subsequent study,we constructed transgenic lines of pGFC1::CDEF1(CDEF1 is a cutinase,located outside the cell,which can hydrolyze the cuticle),which showed abnormal apical hook development in darkness.Therefore,deficient cuticle structure rather than some cutin metabolites cause skotomorphogenetic deficient phenotype.In order to study its molecular mechanism,the expression levels of genes related to skotomorphogenesis in gfc1-1 were detected.as a result,the expression levels of genes PIF4 and HLS1 were reduced in dark-grown gfc1-1.In our study,when grown in darkness,the hypocotyl shortening of cuticle-deficient seedlings were depended on hypocotyl contact with medium,but the angle of the apical hook was not.So the effect of cuticle on the length of the hypocotyl was independent of the effect on the apical hook,and the length of the hypocotyl was affected by sucrose in the medium.Finally,the protein GFC1/DCR was expressed in the prokaryotic system.We tested enzymatic activity of the protein with speculated substrate in vitro,and found that a new product was detected by thin layer chromatography after the reaction.But,due to the limitations of the conditions,the product was unidentified.