Functional characterization of CiHY5 in salt tolerance of Chrysanthemum indicum and conserved role of HY5 under stress in chrysanthemum

Abstract

Among various abiotic stresses, secondary soil salinization poses a significant threat to plant productivity and survival. Cultivated chrysanthemums (Chrysanthemum morifolium), widely grown as ornamental crops, are highly susceptible to salt stress, and their complex polyploid genome complicates the identification of stress resistance genes. In contrast, C. indicum, a native diploid species with robust stress tolerance, serves as a valuable genetic resource for uncovering stress-responsive genes and improving the resilience of ornamental chrysanthemum cultivars. In this study, we cloned, overexpressed (OE-CiHY5), and silenced (RNAi-CiHY5) the CiHY5 gene in C. indicum. OE-CiHY5 plants exhibited larger leaves, sturdier stalks, and higher chlorophyll content compared to wild-type plants, while RNAi-CiHY5 plants displayed weaker growth. Under salt stress, OE-CiHY5 plants demonstrated significantly improved growth, enhanced osmotic adjustment, and effective ROS scavenging. In contrast, RNAi-CiHY5 plants were more sensitive to salinity, showing higher electrolyte leakage and impaired osmotic regulation. Transcriptomic analyses revealed that CiHY5 regulates key hormonal pathways such as zeatin (one of cytokinins), abscisic acid and jasmonic acid, as well as metabolic pathways, including photosynthesis, carbohydrate metabolism, which collectively contribute to the enhanced stress resilience of OE-CiHY5 plants. Promoter-binding assays further confirmed that CiHY5 directly interacts with the CiABF3 promoter, highlighting its critical role in ABA signaling. Evolutionary analyses showed that HY5 is conserved across plant lineages, from early algae to advanced angiosperms, with consistent responsiveness to salt and other abiotic stresses in multiple Chrysanthemum species. These findings establish CiHY5 as a key regulator of salt tolerance in C. indicum, orchestrating a complex network of hormonal and metabolic pathways to mitigate salinity-induced damage. Given the conserved nature of HY5 and its responsiveness to various stresses, HY5 gene provides valuable insights into the molecular mechanisms underlying stress adaptation and represents a promising genetic target for enhancing salt stress resilience in chrysanthemums.