Identification And Functional Characterization Of Small Heat Shock Protein Genes In Creeping Bentgrass (Agrostis Stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is a common perennial cool-season turfgrass, which has excellent characteristic of cold, salt and shade resistance. It can form a graceful landscape lawn and has been widely used for golf courses and grass court due to its quick turf-establishment and good texture. However, it experiences "summer-kill" in warm and transition area which reduces the landscape and quality of summer turf due to its poor heat resistance. Currently, the molecular mechanisms of creeping bentgrass heat resistance and heat responsiveness are still unclear and therefore, becoming a research hotspot for turfgrass investigators.In this study, we cloned three small heat shock protein (sHsp) genes, AsHsp17, AsHsp26.8 and AsHsp26.7 from creeping bentgrass and investigated their roles in plant response to high temperature and other environmental stresses. The experimental results are as follows:AsHsp17, the creeping bentgrass sHsp gene was isolated by RT-PCR, which encodes a protein of 151 amino acids. Sequence alignment showed that it contains a classic Hsp20/a-crystallin domain. Phylogenetic analysis and subcellular localization prediction suggested that it belongs to cytosolic class I sHsp subfamily. The expression of AsHsp17 gene was strongly induced by heat stress in both leaves and roots and was also triggered in roots by salt stress or ABA treatment. Sequence analysis of AsHsp17 promoter showed that it contains heat stress-related cis elements such as HSE. Heterologous expression of AsHsp17 gene led to reduced chlorophyll content and photosynthesis rate in transgenic Arabidopsis plants under normal conditions and increased sensitivity in plant response to heat, salt and ABA.AsHsp26.8, a small heat shock protein gene encoding a protein of 243 amino acids was isolated from creeping bentgrass by RT-PCR. In addition to a classic Hsp23/a-crystallin domain, it contains a chloroplast transit peptide, a Met-rich region and two consensus regions as well. Sequence comparison, phylogenetic analysis and subcellular localization prediction indicated that AsHsp 26.8 belongs to Chloroplastic sHsp subfamily. AsHsp26.8 gene expression was significantly induced by heat in both leaves and roots. Some heat stress-related cis elements such as HSE, AT-rich element and TATA were found in AsHsp26.8 promoter. GUS analysis showed that AsHsp26.8 promoter was activated by heat stress. Transgenic Arabidopsis plants overexpressing AsHsp26.8 exhibited hypersensitivity to heat, salt and ABA.Another small heat shock protein gene, AsHsp26.7 encoding a protein of 242 amino acids was isolated from creeping bentgrass. It contains a classic Hsp23/α-crystallin domain as well as a chloroplast transit peptide, a Met-rich region and two consensus regions. According to the results of sequence alignment, phylogenetic analysis and subcellular localization prediction, we found that AsHsp26.7 is a member of the chloroplastic sHsps subfamily. AsHsp26.7 gene expression was intensively stimulated by heat stress in both leaves and roots and slightly induced by drought treatment in roots. Promoter sequence analysis showed that it contained heat stress-related cis elements such as HSE, AT-rich element and TATA et al. GUS expression was preferentially found in the anther, pollen, and stigma after heat treatment. Heterologous expression of AsHsp26.7 increased the survival rate of transgenic Arabidopsis plants under heat stress condition.Taken together, these results shed new light on the roles of sHSPs in plant response to various abiotic stresses and provided evidence allowing further study on creeping bentgrass stress resistance.