Background: High-temperature (HT) stress poses a significant threat to plant growth and productivity, necessitating a deeper understanding of thermotolerance mechanisms in economically important species like Oncidium orchids. This study investigates the physiological and molecular responses of heat-tolerant (GR) and heat-sensitive (HC) Oncidium cultivars under HT stress to identify key adaptive strategies. Results: Physiological analyses revealed that GR maintained superior chlorophyll retention, membrane stability, and metabolic flexibility under HT stress, while HC exhibited severe photosynthetic collapse and oxidative damage. Transcriptomic profiling identified 26,683 differentially expressed genes (DEGs) in GR, with pronounced upregulation of heat shock proteins (HSP20, HSP70, HSP90), antioxidant enzymes (glutathione peroxidase), and chloroplast-stabilizing genes. Functional enrichment analyses highlighted GR’s coordinated activation of protein homeostasis (GO:0044267), photosynthetic protection (GO:0009522), and metabolic reprogramming (ko01100), including glutathione metabolism (ko00480) and phenylpropanoid biosynthesis (ko00940). Weighted gene co-expression network analysis (WGCNA) further underscored GR’s robust transcriptional network, dominated by heat-shock proteins (HSPs) and heat stress transcription factors (HSFs), whereas HC displayed fragmented stress responses. Conclusions: Collectively, these results demonstrate that the thermotolerant GR cultivar employs a multi-layered defense strategy, including: (1) predominant upregulation of small heat shock proteins (HSP20) rather than canonical HSP70/90; (2) chloroplast protection via oxygen-evolving enhancer proteins; and (3) a well-coordinated gene regulatory network centered on HSFA2. Notably, thylakoid membrane stability emerged as an orchid-specific thermotolerance trait. Comparative analysis demonstrated that GR’s multi-layered defense strategy contrasts sharply with HC’s fragmented responses, characterized by protein homeostasis collapse and oxidative damage. Our findings provide both fundamental insights into orchid stress physiology and practical targets (HSP20, chloroplast HSP70, phenylpropanoid biosynthesis) for developing climate-resilient orchids through molecular breeding approaches.

Many species of the Dendrobium genus are traditional Chinese herbal medicine and ornamental plants. Flower fragrance is one of the most important horticultural ornamental characters and plays a crucial role in the ecology, economy, and aesthetics of plants. However, the volatile constituents and key regulatory genes related to floral biosynthesis are poorly understood. In this experiment, the flowers from two species of Dendrobium with high-scent smells, Dendrobium moniliforme (L.) Sw. (D. moniliforme), and light-scent smells, Dendrobium nobile “H1” (D. “H1”), were selected. The aim of this study was to explore the key gene expression profiles of floral biosynthesis by combining volatile constituent determination and transcriptome analysis in two different Dendrobium species. Physiological determination results showed that 60 volatile compounds were identified in D. moniliforme and 52 volatile compounds were identified in D. ‘H1’ flowers in four flowering stages, and the full bloom stage was the most complicated stage because there were 41 and 33 volatile compounds, respectively. These compounds belong to terpenes, aromatics, fatty acids, nitrogenous compounds, ketones, alcohols, and alkanes, respectively. The components identified in the gynandrium and petals revealed that the petals were probably the most important sites affecting the release of volatiles. The relative content of terpene compounds was the highest, with 77.25% (D. moniliforme) and 50.38% (D. “H1”), respectively. Transcriptome analysis showed that differentially expressed genes (DEGs) were highly enriched in terpenoid backbone biosynthesis and that four linalool synthetase (LIS) genes were up-regulated in high-scent smell species. This study is helpful to explore the key genes of flower fragrance and provides a theoretical basis for further understanding of the regulatory molecular functions of floral synthesis and release, as well as for the cultivation of new aromatic species.

The genetic improvement of Phalaenopsis-type Dendrobium, a valuable ornamental and medicinal orchid, is hindered by the lack of a complete reference genome. In this study, a transcriptome-based approach was employed to develop and validate insertion–deletion (InDel) markers for genetic analysis and variety identification. RNA-seq was performed on two distinct varieties, resulting in the de novo assembly of 156,108 unigenes. A bioinformatics pipeline was developed to identify 5083 high-quality InDel loci, from which 1029 potential markers were designed. Fifty primer pairs were selected and validated experimentally, with 84% successfully amplifying clear products, and 76% showing polymorphism. The polymorphism information content (PIC) of the markers ranged from 0.25 to 0.78, indicating their high potential for use in genetic diversity studies. These markers were used to classify 24 Phalaenopsis-type Dendrobium varieties into distinct genetic clusters. This work provides a scalable and robust platform for molecular breeding, DNA fingerprinting, and germplasm management in non-model species that lack a reference genome. By leveraging transcriptome data, these markers will contribute to the efficient genetic improvement of Dendrobium and other similar crops.

Denphal-group Dendrobium, a globally renowned orchid variety cultivated for both cut flowers and potted plants, is primarily distributed and grown in tropical regions but remains highly susceptible to low-temperature injury in many subtropical areas. To improve the cold tolerance of Denphal-group Dendrobium, in this study, a cold-responsive protein gene, DhCOR413PM1, was identified from the cultivated variety Denphal-group Dendrobium Sonia 'Hiasakul'. The DhCOR413PM1 ORF (612 bp) encodes a 203-amino-acid protein, containing five transmembrane domains and a conserved COR413 domain, showing the closest homology to DtCOR413PM1 (Den. thyrsiflorum). DhCOR413PM1 is ubiquitously expressed across all tissues of the plant, with predominant transcript accumulation in roots and reproductive organs. DhCOR413PM1 expression was significantly upregulated not only under low temperature (4 °C) but also in response to drought (15% PEG-6000) and salinity (50 mM NaCl) stresses. Transgenic Arabidopsis lines overexpressing DhCOR413PM1 exhibited markedly enhanced tolerance to multiple stresses, including higher survival under freezing stress, increased germination rates, and longer roots under osmotic and salt stress, indicating significantly enhanced tolerance to cold, drought, and salt stresses and demonstrating the unique role of the gene in conferring multi-stress resistance. These results suggest that DhCOR413PM1 is a stress-responsive gene potentially involved in multiple abiotic stress tolerance pathways in Dendrobium. Further functional characterization of DhCOR413PM1 may provide valuable insights into the molecular mechanisms underlying stress adaptation in orchids.

Background: Oncidium orchids are a core economic genus in the global tropical ornamental flower industry, while sustained high temperature has become the key bottleneck restricting its commercial production. The coordinated physiological mechanism of heat adaptation, unified screening temperature threshold, and practical physiological biomarkers for heat tolerance evaluation in Oncidium remain insufficiently characterized. Methods: This study systematically evaluated the heat tolerance of 36 Oncidium cultivars through natural field heatwave screening, controlled environment validation, and physiological mechanism dissection under graded temperature stress. Results: We screened 8 highly heat-tolerant cultivars from 36 tested materials, and identified 40.00°C as the optimal discriminative temperature for heat tolerance screening. Relative electrolyte conductivity (REC), proline content, and peroxidase (POD) activity were confirmed as the core physiological biomarkers (coefficient of variation > 50.00%). Principal component analysis revealed that membrane stability and osmotic adjustment were the primary determinants of Oncidium heat adaptation, explaining 80.95% of the total variance. Notably, our data suggested that excessive proline accumulation in heat-sensitive cultivars was a passive compensatory response after cell damage, rather than an active protective adaptation, which differs from the conventional view that higher proline accumulation indicates stronger stress tolerance. Conclusion: These findings characterize physiological responses associated with heat tolerance in Oncidium and provide practical reference markers and a physiological basis for future genetic and molecular studies in orchids.

Introduction: Phalaenopsis-type Dendrobium is a commercially important tropical orchid that is highly sensitive to chilling, which can cause premature leaf senescence and abscission. Methods: We evaluated the protective effect of exogenous 24-epibrassinolide (EBR) and integrated time-resolved RNA-seq with physiological traits previously characterized in the same experimental framework. A concentration of 2.0 mg/L was selected for transcriptomic analysis based on the refined dose-response experiment. WGCNA was performed using 18 EBR-treated libraries. Global GO and KEGG outputs were retained as descriptive overviews, whereas quantitative functional inference was based on module-specific analyses with complete multiple-testing-adjusted statistics. Results: EBR delayed leaf yellowing and abscission. Differential-expression analysis revealed time-dependent transcriptional responses during chilling and recovery. WGCNA identified trait-associated modules with contrasting temporal relationships to physiological traits: the pink module was positively associated with MDA and proline and negatively associated with chlorophyll, whereas the antiquewhite4 (AW4) module showed the opposite pattern. Pink-module enrichment emphasized central carbon and energy metabolism, lipid turnover, peroxisomal processes, autophagy, and cellular transport/homeostasis. AW4 GO enrichment was dominated by transmembrane and metabolite transport; no AW4 KEGG pathway remained significant after multiple-testing correction. Discussion: These results support an association between EBR treatment and coordinated metabolic, membrane-homeostasis, and stress-response adjustments during chilling and recovery in Phalaenopsis-type Dendrobium .