Phylogenetic and expression analysis of HSP20 gene family in Rhododendron species of different altitudes

Small heat shock proteins (sHSPs/HSP20s) play important roles in regulating plant growth, development and stress responses, especially heat stress. Rhododendron plants are major components in landscaping or potting, but poor high-temperature tolerance limits their wide application. To elucidate the adaptive differences between Rhododendron species inhabiting high- and low-altitude regions, this study identified a total of 265 HSP20 genes across seven Rhododendron species, with categorizing into 11 subfamilies. In the CI subfamily, low-altitude Rhododendron species had more HSP20 genes than high-altitude species. Ka/Ks analysis indicated that nearly all HSP20 genes in the seven Rhododendron species have undergone purifying selection, with only a few in the low-altitude species exhibiting positive selection. Analysis of cis-acting elements revealed that most HSP20 genes in R. ovatum and R. simsii could respond to a variety of plant hormones and stresses. Expression pattern analysis revealed that HSP20 members are implicated in flower development and response to heat stress, with the CI subfamily being the main branch responsible for the heat stress response in low-altitude Rhododendron species. Heat stress treatment of transgenic yeast further validated the crucial role of CI subfamily genes in heat stress tolerance. This study provides the first analysis of evolutionary differences in the HSP20 gene families between high- and low-altitude Rhododendron species. It offers insights into the evolutionary direction of HSP20 genes and identifies key genes related to heat tolerance. Additionally, it highlights the role of CI subfamily genes in heat stress tolerance, contributing to the development of heat-tolerant Rhododendron varieties and advancing flower development research.