Function And Transcriptional Regulatory Mechanism Of Cold Stress Responsive Transcription Factors In Lily Stress Response

Abiotic stresses adversely affect the growth,development,and productivity of lily plants which are the most important constraints for lily outdoor planting.As a wild stress?resistant plant,Lilium lancifolium has been shown to have capacity for resisting multiple abiotic stresses,which could be an ideal model to study stress tolerance mechanisms and signaling regulation of Lilium spp.The transcriptional regulatory network is the key to initiating the complex abiotic stress defense system of plants,in which the transcription factors(TFs)act as the terminal of signal transduction and the beginning of transcriptional regulatory at the same time.Therefore,in this study,we aimed exploring the key stress responsive TF genes from L.lancifolium by gene co-expression network method;identifying and characterizing the function of explored TF genes,and revealing the involving transcriptional regulatory pathways of these TF genes.The main results are as follows:1.The gene co-expression network was constructed by re-analyzing the differentially expressed genes in the L.lancifolium 4? cold treated(CT)transcriptome(CK 0 h,CT 2 h,16 h).In this gene co-expression network,we identified 4 gene modules that were significantly enriched in various abiotic stress responses,as well as 3 TF genes from the MYB,NAC,and ZFHD families within these gene modules that might be key stress responsive TF genes.2.The full length of 3 TF genes were cloned and named as Ll MYB3,Ll NAC2,and Ll ZFHD4.Subcellular localization and transcriptional activation activity analysis revealed that 3 TFs are located in nucleus and have transcriptional activation activity.Gene tissue specificity and gene expression patterns analysis showed that the expression level of 3 genes are relatively higher in bulbs and flower petals of L.lancifolium,and all of them were induced by low temperature,drought,salt stress and ABA treatment.3.Overexpressing Ll MYB3 and Ll NAC2 in Arabidopsis thaliana showed ABA hypersensitivity and enhanced tolerance to cold,drought,and salt stresses;overexpression of Ll ZFHD4 could improve cold and drought resistance of transgenic plants.Furthermore,higher transcript levels of some stress-responsive genes from Arabidopsis thaliana accumulated in Ll MYB3,Ll NAC2,and Ll ZFHD4 transgenic plants.4.The promoter regions of 3 TF genes were isolated and cloned.Sequences of various putative stress-related and hormone-responsive cis-acting regulatory elements were identified,includingCRT/DRE,MYBRS,E-box,W-box,LTRE,ABRE,GARE-motif and so on.5.Gene tissue specificity and gene expression patterns analysis of the GUS reporter gene in promoter transgenic Arabidopsis thaliana showed that GUS gene driven by Ll MYB3 promoter specifically expressed in the leaf vein of transgenic Arabidopsis;Ll NAC2 promoter could efficiently initiate the expression of GUS gene after cold,salt and ABA treatments,resulted in significant enhanced GUS enzyme activity in transgenic Arabidopsis.6.The cold-responsive Ll DREB1 gene was cloned from L.lancifolium and proved to be able to bind to the CRT/DRE elements in the promoter regions of 3 TF genes by yeast one-hybrid(Y1H)assay.Ll NAC2 was shown to physically interact with Ll DREB1 and Ll ZFHD4 by bimolecular fluorescence complementation(Bi FC)assay.7.Ll CHS gene was confirmed significantly co-expressed with Ll MYB3 under cold treatment by q RT-PCR.Thus,we isolated and cloned the promoter regions of Ll CHS gene.Y1 H assays further demonstrated that Ll MYB3 could interact with Ll CHS promoter,suggesting Ll MYB3 might be able to bind to the MYBRS elements in Ll CHS promoter.