S92F Amino Acid Substitution In The R3 Domain Of GLABRA1 Leads To Inhibition Of Trichome Formation In Arabidopsis

Arabidopsis thaliana is the first plant with genome completely sequenced.It has been widely used in researches in plant genetics,molecular biology and developmental biology,and it is the typical model plant in scientific research.Trichomes of Arabidopsis thaliana are unicellular structures specialized from epidermal cells that cover the surface of plant.Trichomes are mainly distributed on leaves,stems and calyxes.Trichomes play a role in plant responses to environmental stresses,however,at least under the laboratory growth conditions,absence of trichomes does not affect the growth of Arabidopsis.Scientists investigated the regulation of trichome formation in Arabidopsis,and they found that several factors including transcription factors,phytomormones,and microRNAs are involved in the regulation of trichom formation.Based on the results obtained,they proposed a model to explain how trichome formation is regulated in Arabidopsis.In this model,R2R3 MYB transcription factors GLABRA1(GL1),WD40 repeat proteins TRANSPARENT TESTA GLABRA1(TTG1)and bHLH transcription factors GLABRA3/ENHANCER OF GLABRA3(GL3/EGL3)form a MBW complex to induce the experssion of GLABRA2(GL2)and some of the single-repeat R3 MYB transcription factor genes.GL2 positively regulates trichome formation,whereas R3 MYB transcription factors negatively regulate trichome formation by competing with GL1 for binding GL3/EGL3,thus inhibiting the formation of the MBW complex.GL1 is one of the first cloned trichome regulator genes in Arabidopsis.It encodes an R2R3 MYB transcription factor protein consistent of 228 amino acids.GL1 contain a conserved amino acid signature,[D/E]Lx2[R/K]x3Lx6Lx3R,that has been proven to be crucial for the interaction of MYB transcription factors with bHLH transcription factors,but it remains unclear whether the amino acids in the conserved signature may have other functions.In this study,We characterized the GL1-S92 F protein and the gl1-S92 F mutant.We found that no phenotypic differences were observed for gl1 and gl1-S92 F mutants through out their life cycle,suggesting that S92 F amino acid substitution in GL1 lead to lost of function in regulating trichome formation.RT-PCR results showed that the expression of GL2 in both gl1 and gl1-S92 F mutants was decreased at the same degree,suggesting that GL1-S92 F cannot activate the expression of GL2.We also generated a VPGL1-S92FGL3 construct,and used protoplast transfection assays to examine its ability to activate the report gene PGL2: GUS.The transfection results showed that the fusion protein VPGL1-S92FGL3 failed to activate the expression of the reptor gene,We also constructed transgenic Arabidopsis of over expressing VPGL1-S92FGL3,compared the phenotype of the selected homozygotic plant with the existing VPGL1GL3 over expression homozygotic plant,we found that over expression VPGL1-S92FGL3 transgenic homozygotic plant assumed that the trichome formation of all parts is decreased,and the RT-PCR results showed that the expression of GL2 is also decreased in VPGL1GL3 over expression homozygotic plant.Because S92 F amino acid substitution was located in the conservatived amino acid sequence [D/E]Lx2[R/K]x3Lx6Lx3R of GL1,and previously results have demonstrated that GL1-S92 F is still able to interact with GL3,we conclude that S92 is not required for the interaction of GL1 and GL3,but may play a role in binding of GL1 to the promoters of its target genes.