Genome-wide identification and functional analyses of heat shock transcription factors involved in heat and drought stresses in ryegrass

Italian ryegrass (Lolium multiflorum) is one of the most important and widely cultivated cool-season forage and turfgrass around the world. Heat and drought are two key factors limiting its growth and distribution. Heat shock transcription factors (Hsfs) are important regulators involved in various abiotic stresses; however, how the Hsf family responds to heat and drought stresses and the roles of LmHsfs in heat and drought tolerance remain to be determined in Italian ryegrass. In the present study, 16 putative Hsf genes were identified by whole-genome bioinformatics analysis, including eight HsfAs, five HsfBs, and three HsfCs. The subcellular localization, physiological and biochemical characteristics, conserved domains, gene structure, and phylogenetic relationship of the Hsf family were systematically analyzed in silico. Gene expression profiling analyses revealed that all Hsfs were responsive to heat and drought stresses. Most Hsfs, including LmHsfA2, LmHsfA3, LmHsfA4, LmHsfA5, LmHsfA6, LmHsfA7, LmHsfA8, LmHsfB1, and LmHsfB2, were induced to the highest expression levels at 1 h of heat stress treatment, while the majority of LmHsfs were up-regulated with a peak at 10 d of drought stress treatment. Subsequently, the biological function of LmHsfA5 which was induced by both heat and drought stresses was analyzed. Heterologous overexpression of LmHsfA5 improved heat and drought tolerance in transgenic Arabidopsis. The qRT-PCR analysis showed that the expression of APX2 and HSP18.2 were significantly induced in LmHsfA5-overexpressing lines under both normal and heat stress conditions. What's more, yeast one-hybrid, dual-luciferase assay, and electrophoretic mobility shift assay exhibited that LmHsfA5 could bind to the promoters of LmHSP18.2 and LmAPX2 and induce their expression, indicating that LmHsfA5 may positively regulate heat and drought tolerance via directly activating the expression of LmHSP18.2 and LmAPX2 in ryegrass. This study provides a basis for further research on LmHsfs functions in abiotic stress.