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Heat shock factors (HSFs) are the key regulators of heat stress responses and play pivotal roles in tissue development and the temperature-induced regulation of secondary metabolites. In order to elucidate the roles of HSFs in Cymbidium ensifolium, we conducted a genome-wide identification of CeHSF genes and predicted their functions based on their structural features and splicing patterns. Our results revealed 22 HSF family members, with each gene containing more than one intron. According to phylogenetic analysis, 59.1% of HSFs were grouped into the A subfamily, while subfamily HSFC contained only two HSFs. And the HSF gene families were differentiated evolutionarily between plant species. Two tandem repeats were found on Chr02, and two segmental duplication pairs were observed on Chr12, Chr17, and Chr19; this provided evidence for whole-genome duplication (WGD) events in C. ensifolium. The core region of the promoter in most CeHSF genes contained cis-acting elements such as AP2/ERF and bHLH, which were associated with plant growth, development, and stress responses. Except for CeHSF11, 14, and 19, each of the remaining CeHSFs contained at least one miRNA binding site. This included binding sites for miR156, miR393, and miR319, which were responsive to temperature and other stresses. The HSF gene family exhibited significant tissue specificity in both vegetative and floral organs of C. ensifolium. CeHSF13 and CeHSF15 showed relatively significant expression in flowers compared to other genes. During flower development, CeHSF15 exhibited markedly elevated expression in the early stages of flower opening, implicating critical regulatory functions in organ development and floral scent-related regulations. During the poikilothermic treatment, CeHSF14 was upregulated over 200-fold after 6 h of heat treatment. CeHSF13 and CeHSF14 showed the highest expression at 6 h of low temperature, while the expression of CeHSF15 and CeHSF21 continuously decreased at a low temperature. The expression patterns of CeHSFs further confirmed their role in responding to temperature stress. Our study may help reveal the important roles of HSFs in plant development and metabolic regulation and show insight for the further molecular design breeding of C. ensifolium. [ABSTRACT FROM AUTHOR]

• Cymbidium orchids are the most valuable flowers with a long vegetative phase (2–3 years). • We obtained leafless flowers for three Cymbidium species through a special media combination in tissue culture. These can be excellent resources to study abiotic stress tolerance. • Comparative transcriptome between leafless flowers and healthy leaves provides important clues about melatonin-mediated flowering, which has not been documented before in orchids. • A number of DEGs were found for Tryptophan pathway with contrasting expression patterns between leafless flowers and healthy leaves of three Cymbidium species. Abiotic stress is a major constraint in the successful growth and development of ornamental plants under severe conditions. Since antiquity, melatonin homeostasis has been an effective contrivance in the strategies of abiotic stress tolerance in plants, although it is completely neglected in orchids, the champagne of flowers. Orchids with exceptionally long vegetative phase face a number of stresses and, therefore, stress alleviation has been a hot topic in orchid cultivation. The in-vitro flowering is a useful method for early identification of hybrids in crop improvement programs. We successfully achieved in-vitro leafless flowering in three Cymbidium orchids (C. sinense, C. goeringii and C. ensifolium), which are elite flowers in terms of versatile flower shapes, esthetic preference and price. Transcriptomic analysis was performed to compare the leafless flowers with leaves to identify genes related to tryptophan pathway and melatonin biosynthesis. More than 50 differentially expressed genes (DEGs) were identified in each species with contrasting expressions in the leafless flowers and leaves. The important melatonin pathway genes included serotonin N-acetyltransferase (SNAT), caffeic acid 3-O-methyltransferase (CAMT) and tryptophan decarboxylase (TDC). Moreover, clustering analysis pointed out the significant abiotic stress tolerance-related genes. This opening report, thus, provides a robust cradle to idealize the melatonin as an effective abiotic stress regulator to aid the long journey of vegetative growth and then flowering in precious orchids. [ABSTRACT FROM AUTHOR]