| Perennial ryegrass(Lolium perenne L.)is the most extensively cultivated perennial cool season grass species in the temperate regions of the world.It is widely utilized as turf and forage.Perennial ryegrass is sensitive to high temperature,and turf quality is frequently challenged by heat stress with decreased landscape effect.bZIP is a large family of transcription factors in plants that participate in stress responses and hormone signal transduction.bZIP plays a crucial role in the regulation of growth and development,as well as biotic and abiotic stress.In this study,LpbZIP gene family was identified through a genome-wide analysis and transcriptomic expression profile analysis under high temperature stress.In addition,mechanisms of two LpbZIP genes under high temperature stress in perennial ryegrass were characterized,providing the theoretical basis for breeding of heat tolerant perennial ryegrass and other cold-season grass species.The results are as follows.1.Genome-wide identification of the LpbZIP gene family of perennial ryegrass and expression profile analysis of LpbZIP genes under abiotic stress.Totally 75 LpbZIP genes were identified in perennial ryegrass genome after functional annotation of the amino acid sequences of all protein-coding genes.Phylogenetic tree was constructed using the complete amino acid sequences of the bZIP family in both perennial ryegrass and rice.The 75 perennial ryegrass bZIP genes were divided into 10 subfamilies.Seventy-one out of the 75 LpbZIP genes are unevenly distributed across seven chromosomes,and four are located at the scalfold level.The analysis of transcriptome data revealed that several LpbZIP genes in groups A,B,C,and S were primarily up-regulated by four abiotic stresses in relative to the control group,whereas groups D,H,and I exhibited no significant change in gene expression levels under stress conditions.In contrast,the majority of genes in groups E and F were downregulated following stress treatments.2.LpbZIP40 and regulated heat stress response through incorporating ABA signaling pathway: existing studies have shown that the bZIP family genes are involved in the ABA signal channel and are regulated by ABA.ABA has significantly enhanced the expression of the LpbZIP40 gene.The expression of LpbZIP40 under high temperature coercion was upregulated.Heterologous overexpression of this gene Arabidopsis revealed that LpbZIP40 constitutively increased the heat tolerance of Arabidopsis.Expressions of Lp HSFA2 A,Lp HSFA2 B,Lp DREB2 A,and Lp HSP70.4 were upregulated in LpbZIP40 overexpressed protoplasts.High temperature induced the expression of ABA pathway key rate limiting enzyme NCED genes,with the Lp NCED4 gene being upregulated 60-fold in relative to the control.Lp NCED4 gene promoter was cloned and cis-regulatory motifs were analyzed.The results revealed the presence of G-box element which is one of the binding sites for members of the bZIP family.Yeast one hybrid and EMSA assay revealed that LpbZIP40 binds to the promoter of Lp NCED4 gene to stimulate its expression.The LpbZIP40 can be verified by the yeast single miscellaneous and EMSA experiments that can combine the Lp NCED4 gene promoter to promote its expression.These data indicated that LpbZIP40 was up-regulated by heat stress,respectively,to regulate expression of Lp NCED4 to fine-tune ABA biosynthesis and improve high temperature stress tolerance.3.Identification and functional characterization of LpbZIP40 interacting proteins.In order to identify potential interacting proteins of LpbZIP40,c DNA library screening was performed using yeast two-hybrid approach.One of the interacting proteins of LpbZIP40 is LpbZIP65,a member of the C subfamily.Yeast two hybrid and tobacco Bi FC assays showed that S subfamily LpbZIP40 interact with LpbZIP65 to form heterodimers.Lp RHC1 A was identified as another interacting protein of LpbZIP40,which encodes E3 ubiquitination ligase.Interactions between LpbZIP40 and Lp RHC1 A proteins were further demonstrated by yeast two hybrid,Bi FC,and pull-down assays.Heterologous overexpression of LpbZIP65 and Lp RHC1 A in Arabidopsis and perennial ryegrass protoplast showed a decrease in heat tolerance.4.Characterization of upstream and downstream transcriptional regulatory network for LpbZIP40.A DREB transcription factor binding element(DRE)was identified through cis-acting element analysis in the promoter region of LpbZIP40.The yeast one hybrid and Dual Luciferase assay confirmed that Lp DREB2 A binds to and stimulates the expression of the LpbZIP40 gene.Epression analysis revealed that Lp HSFA2 a was significantly induced by high temperature.LpbZIP40 can bind to G-box region of Lp HSFA2 a promoter and activated the expression of Lp HSFA2 a.These results showed that Lp DREB2 A functions as upstream regulator of LpbZIP40,which further regulates expression of Lp HSFA2 a.These data provided further evidence to enrich the bZIP gene regulatory network during plant heat stress response.In summary,bZIP gene family in perennial ryegrass was identified at the whole genome level.The results showed that LpbZIP40 positively regulated Lp NCED4 expression.The mechanisms of Lp DREB2A-LpbZIP40-Lp HSFA2 a cascade regulated heat stress response were also characterized in perennial ryegrass.Protein interaction analysis showed that LpbZIP65 interacts with LpbZIP40 to form heterodimersa,and an ubiquitination related protein Lp RHC1 A interacts with LpbZIP40,to negatively regulated LpbZIP40.These results partially revealed the molecular mechanisms of heat stress response of perennial ryegrass. |
