Study On The Roles Of Two BHLH Transcription Factors(AtLPl And AtLP2)in Cell Elongation Of Arabidopsis Thaliana

The bHLH/HLH transcription factor is a key transcription factor that regulates plant growth and development.In recent years,although there are many reports on bHLH/HLH transcr:iption factors,the molecular mechanism of this type of transcription factors regulating plant cell elongation has not been clearly analyzed,and further research is needed.In Arabidopsis,We isolated and identified two bHLH family transcription factors,AtLP1 and AtLP2.Studies have shown that two genes are involved in cell elongation and polar growth in Arabidopsis.Phylogenetic tree analysis showed that AtLP1 and AtLP2 are in the same evolutionary branch and belong to the bHLH/HLH family S25 subfamily.This paper studies the function of AtLPl and AtLP2 in the growth and development of Arabidopsis thaliana and the molecular mechanism regulating cell elongation.The main results obtained are summarized as follows:1.AtLPl and AtLP2 can promote cell polarity elongationLeaf morphogenesis is a complex biological problem that requires the coordination of many different genetic factors and environments.AtLPl and AtLP2 overexpressed transgenic Arabidopsis homozygotes were obtained by continuous planting for four generations.The phenotypic analysis revealed that leaves were curled and yellowed,and these phenotypes could be stably inherited.AtLPl and AtLP1 overexpressing transgenic Arabidopsis plants have long and narrow leaves,and the morphology of leaf mesophyll cell was observed under light microscope.Compared to the wild type,transgenic Arabidopsis leaf mesophyll cell are elongated in polarity,resulting in elongated leaves.In addition,the cotyledons length and cotyledons stalks length of AtLPl and AtLP2 overexpressing Arabidopsis were also increased compared to wild type.2.AtLPl and AtLP2 can promote epidermal hair developmentThe epidermal hairs of Arabidopsis thaliana are derived from the epidermal cells of the leaves.The epidermal hair cells are branched and have three branches,four branches and five branches.The wild-type and AtLP1 and AtLP2 overexpressing transgenic Arabidopsis plants were selected as experimental materials,and the sixth true leaves were selected for de-epidermal hair treatment.The morphology of epidermal hair was observed under the microscope.Compared with the wild type,AtLPl and AtLP2 overexpressed the transgenic Arabidopsis leaves,and the epidermal hair volume was significantly larger and the morphology was different.This suggests that AtLPl and AtLP2 can regulate epidermal hair cell development.3.AtLPl/AtLP2 can interact with AtIBH1/AtIBL1 proteinBioinformatics analysis and qRT-PCR analysis indicated that AtLPl and AtLP2 may form heterodimers with atypical bHLH transcription factors AtIBH1 and AtIBL1,which may affect their transcriptional activity and thus affect the transcriptional regulation of downstream genes.In order to verify the interaction between AtLP1,AtLP2 and AtIBH1 and AtIBL1 proteins,AtIBHl and AtIBL1 genes were first cloned and constructed into pGBKT7 vector,and yeast two-hybrid assay was performed with pGADT7-AtLPl and pGADT7-AtLP2.The results showed AtIBHl and AtIBL1 can interact with AtLPl and AtLP2.Further,the above interaction was verified by the bimolecular fluorescence complementation(BIFC)technique.The results showed that AtLPl,AtLP2 and AtIBHl and AtIBLI could interact in the cell.4.AtLPl and AtLP2 can activate AtLNG2 and AtLNGl gene expression,respectively.AtLNGl and AtLNG2 genes regulate leaf morphology by regulating longitudinal polar cell elongation.The dominant mutant lng1-1D phenotype is long and narrow,and the length of petiole and hypocotyl is longer.The expression levels of AtLNGl and AtLNG2 in AtLPl and AtLP2 overexpressing transgenic Arabidopsis were significantly up-regulated.The AtLNGl and AtLNG2 promoter sequences were analyzed and found to contain two G-box binding elements in their promoter sequences.Yeast one-hybrid experiments showed that AtLP1 and AtLP2 can bind to the AtLNG2 and AtLNG1 promoters,respectively,that is,AtLP1 can bind to the promoter of AtLNG2,and AtLP2 can bind to the promoter of AtLNGl.Dual luciferase transcriptional activation experiments indicated that AtLPl is able to activate the promoter activity of AtLNG2,AtLP2 activates the promoter activity of AtLNGl.These results indicate that AtLP1 and AtLP2 can directly bind to the promoters of the AtLNG2 and AtLNGl,respectively,and activate the expression of AtLNGl and AtLNG2 genes,thereby affecting the elongation of Arabidopsis cells.