Identification and Analysis of Aluminum-Activated Malate Transporter Gene Family Reveals Functional Diversification in Orchidaceae and the Expression Patterns of Dendrobium catenatum Aluminum-Activated Malate Transporters.

Aluminum-activated malate transporter (ALMT) genes play an important role in aluminum ion (Al3+) tolerance, fruit acidity, and stomatal movement. Although decades of research have been carried out in many plants, there is little knowledge about the roles of ALMT in Orchidaceae. In this study, 34 ALMT genes were identified in the genomes of four orchid species. Specifically, ten ALMT genes were found in Dendrobium chrysotoxum and D. catenatum, and seven were found in Apostasia shenzhenica and Phalaenopsis equestris. These ALMT genes were further categorized into four clades (clades 1-4) based on phylogenetic relationships. Sequence alignment and conserved motif analysis revealed that most orchid ALMT proteins contain conserved regions (TM1, GABA binding motif, and WEP motif). We also discovered a unique motif (19) belonging to clade 1, which can serve as a specifically identified characteristic. Comparison with the gene structure of AtALMT genes (Arabidopsis thaliana) showed that the gene structure of ALMT was conserved across species, but the introns were longer in orchids. The promoters of orchid ALMT genes contain many light-responsive and hormone-responsive elements, suggesting that their expression may be regulated by light and phytohormones. Chromosomal localization and collinear analysis of D. chrysotoxum indicated that tandem duplication (TD) is the main reason for the difference in the number of ALMT genes in these orchids. D. catenatum was chosen for the RT-qPCR experiment, and the results showed that the DcaALMT gene expression pattern varied in different tissues. The expression of DcaALMT1-9 was significantly changed after ABA treatment. Combining the circadian CO2 uptake rate, titratable total acid, and RT-qPCR data analysis, most DcaALMT genes were highly expressed at night and around dawn. The result revealed that DcaALMT genes might be involved in photosynthate accumulation. The above study provides more comprehensive information for the ALMT gene family in Orchidaceae and a basis for subsequent functional analysis.

Transcription factor DcbZIPs regulate secondary metabolism in Dendrobium catenatum during cold stress.

Cold stress seriously affects plant development and secondary metabolism. The basic region/leucine zipper (bZIP) is one of the largest transcription factor (TFs) family and widely involved in plant cold stress response. However, the function of bZIP in Dendrobium catenatum has not been well-documented. Cold inhibited the growth of D. catenatum and increased total polysaccharide and alkaloid contents in stems. Here, 62 DcbZIP genes were identified in D. catenatum, which were divided into 13 subfamilies. Among them, 58 DcbZIPs responded to cold stress, which were selected based on the transcriptome database produced from cold-treated D. catenatum seedlings. Specifically, the expression of DcbZIP3/6/28 was highly induced by cold treatment in leaves or stems. Gene sequence analysis indicated that DcbZIP3/6/28 contains the bZIP conserved domain and is localized to the cell nucleus. Co-expression networks showed that DcbZIP6 was significantly negatively correlated with PAL2 (palmitoyl-CoA), which is involved in flavonoid metabolism. Moreover, DcbZIP28 has significant negative correlations with various metabolism-related genes in the polysaccharide metabolic pathway, including PFKA1 (6-phosphofructokinase), ALDO2 (aldose-6-phosphate reductase) and SCRK5 (fructokinase). These results implied that DcbZIP6 or DcbZIP28 are mainly involved in flavonoid or polysaccharide metabolism. Overall, these findings provide new insights into the roles of the DcbZIP gene family in secondary metabolism in D. catenatum under cold stress.

Unveiling the Catalytic Roles of DsBBS1 and DsBBS2 in the Bibenzyl Biosynthesis of Dendrobium sinense.

Dendrobium sinense, an endemic medicinal herb in Hainan Island, is rich in bibenzyl compounds. However, few studies have explored the molecular mechanisms of bibenzyl biosynthesis. This study presents a comprehensive analysis of DsBBS1 and DsBBS2 function in D. sinense. A molecular docking simulation revealed high-resolution three-dimensional structural models with minor domain orientation differences. Expression analyses of DsBBS1 and DsBBS2 across various tissues indicated a consistent pattern, with the highest expression being found in the roots, implying that they play a pivotal role in bibenzyl biosynthesis. Protein expression studies identified optimal conditions for DsBBS2-HisTag expression and purification, resulting in a soluble protein with a molecular weight of approximately 45 kDa. Enzyme activity assays confirmed DsBBS2's capacity to synthesize resveratrol, exhibiting higher Vmax and lower Km values than DsBBS1. Functional analyses in transgenic Arabidopsis demonstrated that both DsBBS1 and DsBBS2 could complement the Atchs mutant phenotype. The total flavonoid content in the DsBBS1 and DsBBS2 transgenic lines was restored to wild-type levels, while the total bibenzyl content increased. DsBBS1 and DsBBS2 are capable of catalyzing both bibenzyl and flavonoid biosynthesis in Arabidopsis. This study provides valuable insights into the molecular mechanisms underlying the biosynthesis of bibenzyl compounds in D. sinense.

Genome-wide identification of Ankyrin (ANK) repeat gene families in three Dendrobium species and the expression of ANK genes in D. officinale under gibberellin and abscisic acid treatments.

BACKGROUND: Dendrobium Sw. represents one of the most expansive genera within the Orchidaceae family, renowned for its species' high medicinal and ornamental value. In higher plants, the ankyrin (ANK) repeat protein family is characterized by a unique ANK repeat domain, integral to a plethora of biological functions and biochemical activities. The ANK gene family plays a pivotal role in various plant physiological processes, including stress responses, hormone signaling, and growth. Hence, investigating the ANK gene family and identifying disease-resistance genes in Dendrobium is of paramount importance. RESULTS: This research identified 78 ANK genes in Dendrobium officinale Kimura et Migo, 77 in Dendrobium nobile Lindl., and 58 in Dendrobium chrysotoxum Lindl. Subsequently, we conducted comprehensive bioinformatics analyses on these ANK gene families, encompassing gene classification, chromosomal localization, phylogenetic relationships, gene structure and motif characterization, cis-acting regulatory element identification, collinearity assessment, protein-protein interaction network construction, and gene expression profiling. Concurrently, three DoANK genes (DoANK14, DoANK19, and DoANK47) in D. officinale were discerned to indirectly activate the NPR1 transcription factor in the ETI system via SA, thereby modulating the expression of the antibacterial PR gene. Hormonal treatments with GA3 and ABA revealed that 17 and 8 genes were significantly up-regulated, while 4 and 8 genes were significantly down-regulated, respectively. DoANK32 was found to localize to the ArfGAP gene in the endocytosis pathway, impacting vesicle transport and the polar movement of auxin. CONCLUSION: Our findings provide a robust framework for the taxonomic classification, evolutionary analysis, and functional prediction of Dendrobium ANK genes. The three highlighted ANK genes (DoANK14, DoANK19, and DoANK47) from D. officinale may prove valuable in disease resistance and stress response research. DoANK32 is implicated in the morphogenesis and development of D. officinale through its role in vesicular transport and auxin polarity, with subcellular localization studies confirming its presence in the nucleus and cell membrane. ANK genes displaying significant expression changes in response to hormonal treatments could play a crucial role in the hormonal response of D. officinale, potentially inhibiting its growth and development through the modulation of plant hormones such as GA3 and ABA.

DoSPX1 and DoMYB37 regulate the expression of DoCSLA6 in Dendrobium officinale during phosphorus starvation.

BACKGROUND: Dendrobium officinale Kimura et Migo (D. officinale) is parasitic on rocks or plants with very few mineral elements that can be absorbed directly, so its growth and development are affected by nutritional deficiencies. Previous studies found that phosphorus deficiency promotes polysaccharides accumulation in D. officinale, the expression of DoCSLA6 (glucomannan synthase gene) was positively correlated with polysaccharide synthesis. However, the molecular mechanism by which the low phosphorus environment affects polysaccharide accumulation remains unclear. RESULTS: We found that DoSPX1 can reduce phosphate accumulation in plants and promote the expression of PSIs genes, thereby enhancing plant tolerance to low phosphorus environments.Y1H and EMSA experimental show that DoMYB37 can bind the promoter of DoCSLA6. DoSPX1 interact with DoMYB37 transiently overexpressed DoSPX1 and DoMYB37 in D. officinale protocorm-like bodies, decreased the Pi content, while increased the expression of DoCSLA6. CONCLUSIONS: The signaling pathway of DoSPX1-DoMYB37-DoCSLA6 was revealed. This provides a theoretical basis for the accumulation of polysaccharide content in D. officinale under phosphorus starvation.

Genome-Wide Identification of Glutathione S-Transferase Family from Dendrobium officinale and the Functional Characterization of DoGST5 in Cadmium Tolerance.

Glutathione S-transferases (GSTs) are members of a protein superfamily with diverse physiological functions, including cellular detoxification and protection against oxidative damage. However, there is limited research on GSTs responding to cadmium (Cd) stress. This study classified 46 GST genes in Dendrobium officinale (D. officinale) into nine groups using model construction and domain annotation. Evolutionary analysis revealed nine subfamilies with diverse physical and chemical properties. Prediction of subcellular localization revealed that half of the GST members were located in the cytoplasm. According to the expression analysis of GST family genes responding to Cd stress, DoGST5 responded significantly to Cd stress. Transient expression of DoGST5-GFP in tobacco leaves revealed that DoGST5 was localized in the cytoplasm. DoGST5 overexpression in Arabidopsis enhanced Cd tolerance by reducing Cd-induced H2O2 and O2- levels. These findings demonstrate that DoGST5 plays a critical role in enhancing Cd tolerance by balancing reactive oxygen species (ROS) levels, offering potential applications for improving plant adaptability to heavy metal stress.

Underlying mechanism of Dendrobium huoshanense resistance to lead stress using the quantitative proteomics method.

Lead affects photosynthesis and growth and has serious toxic effects on plants. Here, the differential expressed proteins (DEPs) in D. huoshanense were investigated under different applications of lead acetate solutions. Using label-free quantitative proteomics methods, more than 12,000 peptides and 2,449 proteins were identified. GO and KEGG functional annotations show that these differential proteins mainly participate in carbohydrate metabolism, energy metabolism, amino acid metabolism, translation, protein folding, sorting, and degradation, as well as oxidation and reduction processes. A total of 636 DEPs were identified, and lead could induce the expression of most proteins. KEGG enrichment analysis suggested that proteins involved in processes such as homologous recombination, vitamin B6 metabolism, flavonoid biosynthesis, cellular component organisation or biogenesis, and biological regulation were significantly enriched. Nearly 40 proteins are involved in DNA replication and repair, RNA synthesis, transport, and splicing. The effect of lead stress on D. huoshanense may be achieved through photosynthesis, oxidative phosphorylation, and the production of excess antioxidant substances. The expression of 9 photosynthesis-related proteins and 12 oxidative phosphorylation-related proteins was up-regulated after lead stress. Furthermore, a total of 3 SOD, 12 POD, 3 CAT, and 7 ascorbate-related metabolic enzymes were identified. Under lead stress, almost all key enzymes involved in the synthesis of antioxidant substances are up-regulated, which may facilitate the scavenging of oxygen-free radical scavenging. The expression levels of some key enzymes involved in sugar and glycoside synthesis, the phenylpropanoid synthesis pathway, and the terpene synthesis pathway also increased. More than 30 proteins involved in heavy metal transport were also identified. Expression profiling revealed a significant rise in the expression of the ABC-type multidrug resistance transporter, copper chaperone, and P-type ATPase with exposure to lead stress. Our findings lay the basis for research on the response and resistance of D. huoshanense to heavy metal stress.

Recent Advances and New Insights in Genome Analysis and Transcriptomic Approaches to Reveal Enzymes Associated with the Biosynthesis of Dendrobine-Type Sesquiterpenoid Alkaloids (DTSAs) from the Last Decade.

Dendrobium species, which are perennial herbs widely distributed in tropical and subtropical regions, are notable for their therapeutic properties attributed to various bioactive compounds, including dendrobine-type sesquiterpenoid alkaloids (DTSAs). The objective of this review article is to provide a comprehensive overview of recent advances in the biosynthesis of DTSAs, including their extraction from Dendrobium species and endophytes, elucidation of associated genes through genomic and transcriptomic sequencing in both Dendrobium spp. and endophytes, exploration of the biosynthetic pathways of DTSAs, and drawing conclusions and outlining future perspectives in this field. Alkaloids, predominantly nitrogen-containing compounds found in medicinal orchids, include over 140 types discovered across more than 50 species. DTSAs, identified in 37 picrotoxane alkaloids, have a distinctive five-membered nitrogen heterocyclic ring. This review highlights endophytic fungi as alternative sources of DTSAs, emphasizing their potential in pharmaceutical applications when plant-derived compounds are scarce or complex. Genomic and transcriptomic sequencing of Dendrobium spp. and their endophytes has identified key genes involved in DTSAs biosynthesis, elucidating pathways such as the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways. Genes encoding enzymes, such as acetyl-CoA C-acetyltransferase and diphosphomevalonate decarboxylase, are positively associated with dendrobine production. Despite significant advancements, the complexity of terpenoid biosynthesis in different subcellular compartments remains a challenge. Future research should focus on leveraging high-quality genomic data and omics technologies to further understand and manipulate the biosynthetic pathways of DTSAs and enhance their medicinal use.

Multi-omics analyses and botanical perfumer hypothesis provide insights into the formation and maintenance of aromatic characteristics of Dendrobium loddigesii flowers.

Dendrobium loddigesii, a member of the Orchidaceae family, is a valuable horticultural crop known for its aromatic qualities. However, the mechanisms responsible for the development of its aromatic characteristics remain poorly understood. To elucidate these underlying mechanisms, we assembled the first chromosome-level reference genome of D. loddigesii using PacBio HiFi-reads, Illumina short-reads, and Hi-C data. The assembly comprises 19 pseudochromosomes with N50 contig and N50 scaffold sizes of 55.15 and 89.94 Mb, respectively, estimating the genome size to be 1.68 Gb, larger than that of other sequenced Dendrobium species. During the flowering stages, we conducted a comprehensive analysis combining volatilomics and transcriptomics to understand the characteristics and biosynthetic mechanisms pathways of the floral scent. Our findings emphasize the significant contribution of aromatic terpenoids, especially monoterpenoids, in defining the floral aroma. Furthermore, we identified two crucial terpene synthase (TPS) genes that play a key role in maintaining the aroma during flowering. Through the integration volatilomics data with catalytic assays of DlTPSbs proteins, we identified specific compounds responsible for the aromatic characteristics of D. loddigesii. This integrated analysis of the genome, transcriptome, and volatilome, offers valuable insights into the development and preservation of D. loddigesii's aromatic characteristics, setting the stage for further exploration of the botanical perfumer hypothesis.

The Whole Mitochondrial Genome Sequence of Dendrobium loddigesii Rolfe, an Endangered Orchid Species in China, Reveals a Complex Multi-Chromosome Structure.

Dendrobium loddigesii is a precious traditional Chinese medicine with high medicinal and ornamental value. However, the characterization of its mitochondrial genome is still pending. Here, we assembled the complete mitochondrial genome of D. loddigesii and discovered that its genome possessed a complex multi-chromosome structure. The mitogenome of D. loddigesii consisted of 17 circular subgenomes, ranging in size from 16,323 bp to 56,781 bp. The total length of the mitogenome was 513,356 bp, with a GC content of 43.41%. The mitogenome contained 70 genes, comprising 36 protein-coding genes (PCGs), 31 tRNA genes, and 3 rRNA genes. Furthermore, we detected 403 repeat sequences as well as identified 482 RNA-editing sites and 8154 codons across all PCGs. Following the sequence similarity analysis, 27 fragments exhibiting homology to both the mitogenome and chloroplast genome were discovered, accounting for 9.86% mitogenome of D. loddigesii. Synteny analysis revealed numerous sequence rearrangements in D. loddigesii and the mitogenomes of related species. Phylogenetic analysis strongly supported that D. loddigesii and D. Amplum formed a single clade with 100% bootstrap support. The outcomes will significantly augment the orchid mitochondrial genome database, offering profound insights into Dendrobium's intricate mitochondrial genome architecture.

Visualization of the Infection and Colonization Process of Dendrobium officinale Using a Green Fluorescent Protein-Tagged Isolate of Fusarium oxysporum.

Dendrobium officinale soft rot is a widespread and destructive disease caused by Fusarium oxysporum that can seriously affect yield and quality. To better understand the fungal infection and colonization, we successfully created an F. oxysporum labeled with green fluorescent protein using the Agrobacterium tumefaciens-mediated transformation method. Transformants had varying fluorescence intensities, but their pathogenicity did not differ from that of the wild type. Fluorescence microscopy revealed that F. oxysporum primarily entered the aboveground portion of D. officinale through the leaf margin, stomata, or by direct penetration of the leaf surface. It then colonized the mesophyll and spread along its vascular bundles. D. officinale exhibited typical symptoms of decay and wilting at 14 days postinoculation, accompanied by a pronounced fluorescence signal in the affected area. The initial colonization of F. oxysporum in the subterranean region primarily involved attachment to the root hair and epidermis, which progressed to the medullary vascular bundle. At 14 days postinoculation, the root vascular bundles of D. officinale exhibited significant colonization by F. oxysporum. Macroconidia were also observed in black rot D. officinale tissue. In particular, the entire root was surrounded by a significant number of chlamydospore-producing F. oxysporum mycelia at 28 days postinoculation. This approach allowed for the visualization of the complete infection process of F. oxysporum and provided a theoretical foundation for the development of field control strategies.

The Evolution of the WUSCHEL-Related Homeobox Gene Family in Dendrobium Species and Its Role in Sex Organ Development in D. chrysotoxum.

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.

Metabolic and Transcriptomic Profile Revealing the Differential Accumulating Mechanism in Different Parts of Dendrobium nobile.

Dendrobium nobile is an important orchid plant that has been used as a traditional herb for many years. For the further pharmaceutical development of this resource, a combined transcriptome and metabolome analysis was performed in different parts of D. nobile. First, saccharides, organic acids, amino acids and their derivatives, and alkaloids were the main substances identified in D. nobile. Amino acids and their derivatives and flavonoids accumulated strongly in flowers; saccharides and phenols accumulated strongly in flowers and fruits; alkaloids accumulated strongly in leaves and flowers; and a nucleotide and its derivatives and organic acids accumulated strongly in leaves, flowers, and fruits. Simultaneously, genes for lipid metabolism, terpenoid biosynthesis, and alkaloid biosynthesis were highly expressed in the flowers; genes for phenylpropanoids biosynthesis and flavonoid biosynthesis were highly expressed in the roots; and genes for other metabolisms were highly expressed in the leaves. Furthermore, different members of metabolic enzyme families like cytochrome P450 and 4-coumarate-coA ligase showed differential effects on tissue-specific metabolic accumulation. Members of transcription factor families like AP2-EREBP, bHLH, NAC, MADS, and MYB participated widely in differential accumulation. ATP-binding cassette transporters and some other transporters also showed positive effects on tissue-specific metabolic accumulation. These results systematically elucidated the molecular mechanism of differential accumulation in different parts of D. nobile and enriched the library of specialized metabolic products and promising candidate genes.

Genomic identification and expression analysis of acid invertase (AINV) gene family in Dendrobium officinale Kimura et Migo.

BACKGROUND: Dendrobium officinale Kimura et Migo, a renowned traditional Chinese orchid herb esteemed for its significant horticultural and medicinal value, thrives in adverse habitats and contends with various abiotic or biotic stresses. Acid invertases (AINV) are widely considered enzymes involved in regulating sucrose metabolism and have been revealed to participate in plant responses to environmental stress. Although members of AINV gene family have been identified and characterized in multiple plant genomes, detailed information regarding this gene family and its expression patterns remains unknown in D. officinale, despite their significance in polysaccharide biosynthesis. RESULTS: This study systematically analyzed the D. officinale genome and identified four DoAINV genes, which were classified into two subfamilies based on subcellular prediction and phylogenetic analysis. Comparison of gene structures and conserved motifs in DoAINV genes indicated a high-level conservation during their evolution history. The conserved amino acids and domains of DoAINV proteins were identified as pivotal for their functional roles. Additionally, cis-elements associated with responses to abiotic and biotic stress were found to be the most prevalent motif in all DoAINV genes, indicating their responsiveness to stress. Furthermore, bioinformatics analysis of transcriptome data, validated by quantitative real-time reverse transcription PCR (qRT-PCR), revealed distinct organ-specific expression patterns of DoAINV genes across various tissues and in response to abiotic stress. Examination of soluble sugar content and interaction networks provided insights into stress release and sucrose metabolism. CONCLUSIONS: DoAINV genes are implicated in various activities including growth and development, stress response, and polysaccharide biosynthesis. These findings provide valuable insights into the AINV gene amily of D. officinale and will aid in further elucidating the functions of DoAINV genes.

MIR396-GRF/GIF enhances in planta shoot regeneration of Dendrobium catenatum.

Recent studies on co-transformation of the growth regulator, TaGRF4-GIF1 chimera (Growth Regulating Factor 4-GRF Interacting Factor 1), in cultivated wheat varieties (Triticum aestivum), showed improved regeneration efficiency, marking a significant breakthrough. Here, a simple and reproducible protocol using the GRF4-GIF1 chimera was established and tested in the medicinal orchid Dendrobium catenatum, a monocot orchid species. TaGRF4-GIF1 from T. aestivum and DcGRF4-GIF1 from D. catenatum were reconstructed, with the chimeras significantly enhancing the regeneration efficiency of D. catenatum through in planta transformation. Further, mutating the microRNA396 (miR396) target sites in TaGRF4 and DcGRF4 improved regeneration efficiency. The target mimicry version of miR396 (MIM396) not only boosted shoot regeneration but also enhanced plant growth. Our methods revealed a powerful tool for the enhanced regeneration and genetic transformation of D. catenatum.

DhuFAP: a platform for gene functional analysis in Dendrobium huoshanense.

BACKGROUND: Dendrobium huoshanense, a traditional medicinal and food plant, has a rich history of use. Recently, its genome was decoded, offering valuable insights into gene function. However, there is no comprehensive gene functional analysis platform for D. huoshanense. RESULT: To address this, we created a platform for gene function analysis and comparison in D. huoshanense (DhuFAP). Using 69 RNA-seq samples, we constructed a gene co-expression network and annotated D. huoshanense genes by aligning sequences with public protein databases. Our platform contained tools like Blast, gene set enrichment analysis, heatmap analysis, sequence extraction, and JBrowse. Analysis revealed co-expression of transcription factors (C2H2, GRAS, NAC) with genes encoding key enzymes in alkaloid biosynthesis. We also showcased the reliability and applicability of our platform using Chalcone synthases (CHS). CONCLUSION: DhuFAP ( www.gzybioinformatics.cn/DhuFAP ) and its suite of tools represent an accessible and invaluable resource for researchers, enabling the exploration of functional information pertaining to D. huoshanense genes. This platform stands poised to facilitate significant biological discoveries in this domain.

Physiological and transcriptomic comparisons shed light on the cold stress response mechanisms of Dendrobium spp.

BACKGROUND: Dendrobium spp. comprise a group of tropical orchids with ornamental and medicinal value. Dendrobium spp. are sensitive to low temperature, and the underlying cold response regulatory mechanisms in this group are unclear. To understand how these plants respond to cold stress, we compared the transcriptomic responses of the cold-tolerant cultivar 'Hongxing' (HX) and the cold-sensitive cultivar 'Sonia Hiasakul' (SH) to cold stress. RESULTS: Chemometric results showed that the physiological response of SH in the later stages of cold stress is similar to that of HX throughout the cold treatment. Orthogonal partial least squares discriminant analysis (OPLS-DA) revealed that soluble protein content and peroxidase activity are key physiological parameters for assessing the cold tolerance of these two Dendrobium spp. cultivars. Additionally, weighted gene co-expression network analysis (WGCNA) results showed that many cold response genes and metabolic pathways significantly associated with the physiological indices were enriched in the 12 detected modules. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) enrichment analyses of the 105 hub genes showed that Dendrobium spp. adapt to cold stress by regulating signal transduction, phytohormones, transcription factors, protein translation and modification, functional proteins, biosynthesis and metabolism, cell structure, light, and the circadian clock. Hub genes of the cold stress response network included the remorin gene pp34, the abscisic acid signaling pathway-related genes PROTEIN PHOSPATASE 2 C (PP2C), SNF1-RELATED PROTEIN KINASE 2 (SnRK2), ABRE-BINDING FACTOR 1 (ABF1) and SKI-INTERACTING PROTEIN 17 (SKIP17), the Ca2+ signaling-related GTP diphosphokinase gene CRSH1, the carbohydrate-related gene STARCH SYNTHASE 2 (SS2), the cell wall biosynthesis gene CINNAMYL ALCOHOL DEHYDROGENASE (CAD7), and the endocytosis-related gene VACUOLAR PROTEIN SORTING-ASSOCIATED PROTEIN 52 A (VPS52A). CONCLUSIONS: The cold-responsive genes and metabolic pathways of Dendrobium spp. revealed in this study provide important insight to enable the genetic enhancement of cold tolerance in Dendrobium spp., and to facilitate cold tolerance breeding in related plants.

DcERF109 regulates shoot branching by participating in strigolactone signal transduction in Dendrobium catenatum.

Shoot branching fundamentally influences plant architecture and agricultural yield. However, research on shoot branching in Dendrobium catenatum, an endangered medicinal plant in China, remains limited. In this study, we identified a transcription factor DcERF109 as a key player in shoot branching by regulating the expression of strigolactone (SL) receptors DWARF 14 (D14)/ DECREASED APICAL DOMINANCE 2 (DAD2). The treatment of D. catenatum seedlings with GR24rac/TIS108 revealed that SL can significantly repress the shoot branching in D. catenatum. The expression of DcERF109 in multi-branched seedlings is significantly higher than that of single-branched seedlings. Ectopic expression in Arabidopsis thaliana demonstrated that overexpression of DcERF109 resulted in significant shoot branches increasing and dwarfing. Molecular and biochemical assays demonstrated that DcERF109 can directly bind to the promoters of AtD14 and DcDAD2.2 to inhibit their expression, thereby positively regulating shoot branching. Inhibition of DcERF109 by virus-induced gene silencing (VIGS) resulted in decreased shoot branching and improved DcDAD2.2 expression. Moreover, overexpression of DpERF109 in A. thaliana, the homologous gene of DcERF109 in Dendrobium primulinum, showed similar phenotypes to DcERF109 in shoot branch and plant height. Collectively, these findings shed new insights into the regulation of plant shoot branching and provide a theoretical basis for improving the yield of D. catenatum.

[Identification and expression profiling of SAPK gene family members in Dendrobium officinale].

To investigate the potential roles of stress-activated protein kinase (SAPK) gene family members in Dendrobium officinale, we employed multiple bioinformatics methods to identify the members of this family. The physicochemical properties, chromosomal localization, phylogenetic relationship, gene structure, and cis-acting elements of each D. officinale SAPK (DoSAPK) member were analyzed. In addition, their expression profiles in different tissues and under the low-temperature or salt stress treatment were determined by real-time fluorescence quantitative PCR. The results showed that D. officinale carried eight DoSAPK family members, which belonged to three groups (groups Ⅰ, Ⅱ, and Ⅲ). These genes were located on seven chromosomes, and there were two pairs of genes with replication. The DoSAPK members within the same group had similar gene structures, conserved motifs, and secondary structures. The cis-acting elements in the promoter regions of DoSAPK genes included abundant hormone and stress response elements. DoSAPK family members presented tissue-specific expression in D. officinale. Furthermore, they were differentially expressed under the low-temperature or salt stress treatment, which suggested that they might be involved in the responses to low-temperature and salt stress. Intriguingly, DoSAPK1 might play a role in the abiotic stress responses. The results laid a foundation for in-depth study of the members and roles of the DoSAPK gene family.

Drought Stress Induced Different Response Mechanisms in Three Dendrobium Species under Different Photosynthetic Pathways.

Many Dendrobium species, which hold a high status and value in traditional Chinese medicine, grow on barks and rocks in the wild, often encountering harsh environments and facing droughts. However, the molecular mechanisms underlying the shift in the photosynthetic pathway induced by drought remain unclear. To address this issue, three Dendrobium species with different photosynthetic pathways were selected for sequencing and transcriptome data analysis after drought treatment. The findings included 134.43 GB of sequencing data, with numerous Differentially Expressed Genes (DEGs) exhibiting different response mechanisms under drought stress. Gene Ontology (GO)-KEGG-based enrichment analysis of DEGs revealed that metabolic pathways contributed to drought tolerance and alterations in photosynthetic pathways. Phosphoenolpyruvate Carboxylase (PEPC) was subjected to phylogenetic tree construction, sequence alignment, and domain analysis. Under drought stress, variations were observed in the PEPC gene structure and expression among different Dendrobium species; the upregulation of Dc_gene2609 expression may be caused by dof-miR-384, which resulted in the shift from C3 photosynthesis to CAM, thereby improving drought tolerance in Dendrobium. This study revealed the expression patterns and roles of PEPC genes in enhancing plant drought tolerance and will provide an important basis for in-depth research on Dendrobium's adaptation mechanisms in arid environments.