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Background: RT-qPCR is a powerful strategy for recognizing the most appropriate reference genes, which can successfully minimize experimental mistakes through accurate normalization. Ludisia discolor, recognized for its ornamental value, features little, distinctive blossoms with twisted lips and gynostemium showing chiral asymmetry, together with striking blood-red fallen leaves periodically marked with golden blood vessels. Methods and Results: To ensure the accuracy of qRT-PCR, selecting appropriate reference genes for quantifying target gene expression levels is essential. This study aims to identify stable reference genes during the development of L. discolor. In this study, the entire floral buds, including the lips and gynostemium from different development stages, were taken as materials. Based upon the transcriptome information of L. discolor, nine housekeeping genes, ACT, HIS, EF1-α1, EF1-α2, PP2A, UBQ1, UBQ2, UBQ3, and TUB, were selected in this research study as prospect interior referral genes. The expression of these nine genes were found by RT-qPCR and afterwards comprehensively examined by four software options: geNorm, NormFinder, BestKeeper, and ΔCt. The outcomes of the analysis showed that ACT was the most steady gene, which could be the most effective inner referral gene for the expression evaluation of flower advancement in L. discolor. Conclusions: The results of this study will contribute to the molecular biology research of flower development in L. discolor and closely related species. [ABSTRACT FROM AUTHOR]

The TIFY family is a plant-specific gene family that is involved in regulating a variety of plant processes, including developmental and defense responses. The Cymbidium species have certain ornamental and ecological value. However, the characteristics and functions of TIFY genes in Cymbidium remain poorly understood. This study conducted a genome analysis of the TIFY gene family in Cymbidium goeringii, C. ensifolium, and C. sinense and investigated their physicochemical properties, phylogenetic relationships, gene structures, and expression patterns under heat stress in C. goeringii. C. goeringii (26), C. ensifolium (19), and C. sinense (21). A total of 66 TIFY genes were identified, and they were classified into four subfamilies (JAZ, ZML, PPD, and TIFY) based on their systematic evolutionary relationships. Sequence analysis showed that TIFYs contained a conserved TIFY domain and that genes within the same subfamily had structural similarity. Analysis of cis-regulatory elements revealed that these genes contain numerous light-responsive elements and stress-responsive elements. We subjected C. goeringii (16 h light/8 h dark) to 24 h of 38 °C high-temperature stress in a climate chamber. Additionally, results from RT-qPCR experiments showed that under heat stress, the expression levels of eight genes in C. goeringii show significant differences. Among them, the JAZ subfamily exhibited the strongest response to heat stress, initially showing upregulation followed by a downregulation trend. In conclusion, this study investigated the role of TIFY genes in three Cymbidium species, providing insights into C. goeringii under heat stress. [ABSTRACT FROM AUTHOR]

As an important genus in Orchidaceae, Cymbidium has rich ecological diversity and significant economic value. DNA binding with one zinc finger (Dof) proteins are pivotal plant-specific transcription factors that play crucial roles in the growth, development, and stress response of plants. Although the Dof genes have been identified and functionally analyzed in numerous plants, exploration in Orchidaceae remains limited. We conducted a thorough analysis of the Dof gene family in Cymbidium goeringii, C. ensifolium, and C. sinensis. In total, 91 Dof genes (27 CgDofs, 34 CeDofs, 30 CsDofs) were identified, and Dof genes were divided into five groups (I–V) based on phylogenetic analysis. All Dof proteins have motif 1 and motif 2 conserved domains and over half of the genes contained introns. Chromosomal localization and collinearity analysis of Dof genes revealed their evolutionary relationships and potential gene duplication events. Analysis of cis-elements in CgDofs, CeDofs, and CsDofs promoters showed that light-responsive cis-elements were the most common, followed by hormone-responsive elements, plant growth-related elements, and abiotic stress response elements. Dof proteins in three Cymbidium species primarily exhibit a random coil structure, while homology modeling exhibited significant similarity. In addition, RT-qPCR analysis showed that the expression levels of nine CgDofs changed greatly under heat stress. CgDof03, CgDof22, CgDof27, CgDof08, and CgDof23 showed varying degrees of upregulation. Most upregulated genes under heat stress belong to group I, indicating that the Dof genes in group I have great potential for high-temperature resistance. In conclusion, our study systematically demonstrated the molecular characteristics of Dof genes in different Cymbidium species, preliminarily revealed the patterns of heat stress, and provided a reference for further exploration of stress breeding in orchids. [ABSTRACT FROM AUTHOR]

The GRAS gene family, responsible for encoding transcription factors, serves pivotal functions in plant development, growth, and responses to stress. The exploration of the GRAS gene family within the Orchidaceae has been comparatively limited, despite its identification and functional description in various plant species. This study aimed to conduct a thorough examination of the GRAS gene family in Cymbidum goeringii, focusing on its physicochemical attributes, phylogenetic associations, gene structure, cis-acting elements, and expression profiles under heat stress. The results show that a total of 54 CgGRASs were pinpointed from the genome repository and categorized into ten subfamilies via phylogenetic associations. Assessment of gene sequence and structure disclosed the prevalent existence of the VHIID domain in most CgGRASs, with around 57.41% (31/54) CgGRASs lacking introns. The Ka/Ks ratios of all CgGRASs were below one, indicating purifying selection across all CgGRASs. Examination of cis-acting elements unveiled the presence of numerous elements linked to light response, plant hormone signaling, and stress responsiveness. Furthermore, CgGRAS5 contained the highest quantity of cis-acting elements linked to stress response. Experimental results from RT-qPCR demonstrated notable variations in the expression levels of eight CgGRASs after heat stress conditions, particularly within the LAS, HAM, and SCL4/7 subfamilies. In conclusion, this study revealed the expression pattern of CgGRASs under heat stress, providing reference for further exploration into the roles of CgGRAS transcription factors in stress adaptation. [ABSTRACT FROM AUTHOR]

ZF-HD transcription factors, which are unique to land plants, are involved in the regulation of abiotic stress response and related signaling pathways, and play a crucial role in plant growth and development. Dendrobium is one of the largest genera of orchids, with a high ornamental and ecological value. However, the specific functions of the ZF-HDs in Dendrobium remain unknown. In this study, we identified a total of 53 ZF-HDs from D. chrysotoxum (17), D. catenatum (23), and D. huoshanense (13), and analyzed their physicochemical properties, phylogenetic relationships, chromosomal locations, protein structures, conserved motifs, and expression patterns. The phylogenetic relationships revealed that 53 ZF-HDs were classified into six subfamilies (ZHDI–V and MIF), and all ZF-HD proteins contained motif 1 and motif 4 conserved domains, while a minority of these proteins had exons. The analysis of cis-elements in the promoters of ZF-HDs from three Dendrobium species showed that growth- and development-related elements were the most prevalent, followed by hormone response and abiotic stress response elements. Through collinearity analysis, 14 DchZF-HDs were found to be collinear with DhuZF-HDs, and 12 DchZF-HDs were found to be collinear with DcaZF-HDs. Furthermore, RT-qPCR analysis revealed that DchZF-HDs play a regulatory role in the development of lateral organs during the flowering process. The results indicated that DchZHD2 plays a role in the unpigmented bud stage, while DchMIF8 and DchZHD16 play significant roles during the pigmented bud and initial bloom stages. Hence, this study provides a crucial basis for further exploring ZF-HDs functions in regulating the floral organs of orchids. [ABSTRACT FROM AUTHOR]

The WUSCHEL-related homeobox (WOX) transcription factor plays a vital role in stem cell maintenance and organ morphogenesis, which are essential processes for plant growth and development. Dendrobium chrysotoxum, D. huoshanense, and D. nobile are valued for their ornamental and medicinal properties. However, the specific functions of the WOX gene family in Dendrobium species are not well understood. In our study, a total of 30 WOX genes were present in the genomes of the three Dendrobium species (nine DchWOXs, 11 DhuWOXs, and ten DnoWOXs). These 30 WOXs were clustered into ancient clades, intermediate clades, and WUS/modern clades. All 30 WOXs contained a conserved homeodomain, and the conserved motifs and gene structures were similar among WOXs belonging to the same branch. D. chrysotoxum and D. huoshanense had one pair of fragment duplication genes and one pair of tandem duplication genes, respectively; D. nobile had two pairs of fragment duplication genes. The cis-acting regulatory elements (CREs) in the WOX promoter region were mainly enriched in the light response, stress response, and plant growth and development regulation. The expression pattern and RT-qPCR analysis revealed that the WOXs were involved in regulating the floral organ development of D. chrysotoxum. Among them, the high expression of DchWOX3 suggests that it might be involved in controlling lip development, whereas DchWOX5 might be involved in controlling ovary development. In conclusion, this work lays the groundwork for an in-depth investigation into the functions of WOX genes and their regulatory role in Dendrobium species' floral organ development. [ABSTRACT FROM AUTHOR]

Cymbidium sinense, a type of orchid plant, is more drought-resistant and ornamental than other terrestrial orchids. Research has shown that many members of the NUCLEAR FACTOR Y (NF-Y) transcription factor family are responsive to plant growth, development, and abiotic stress. However, the mechanism of the NF-Y gene family's response to abiotic stress in orchids has not yet been reported. In this study, phylogenetic analysis allowed for 27 CsNF-Y genes to be identified (5 CsNF-YAs, 9 CsNF-YBs, and 13 CsNF-YC subunits), and the CsNF-Ys were homologous to those in Arabidopsis and Oryza. Protein structure analysis revealed that different subfamilies contained different motifs, but all of them contained Motif 2. Secondary and tertiary protein structure analysis indicated that the CsNF-YB and CsNF-YC subfamilies had a high content of alpha helix structures. Cis-element analysis showed that elements related to drought stress were mainly concentrated in the CsNF-YB and CsNF-YC subfamilies, with CsNF-YB3 and CsNF-YC12 having the highest content. The results of a transcriptome analysis showed that there was a trend of downregulation of almost all CsNF-Ys in leaves under drought stress, while in roots, most members of the CsNF-YB subfamily showed a trend of upregulation. Additionally, seven genes were selected for real-time reverse transcription quantitative PCR (qRT-PCR) experiments. The results were generally consistent with those of the transcriptome analysis. The regulatory roles of CsNF-YB 1, 2, and 4 were particularly evident in the roots. The findings of our study may make a great contribution to the understanding of the role of CsNF-Ys in stress-related metabolic processes. [ABSTRACT FROM AUTHOR]