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.

Comparative Transcriptome Profiling Reveals Two WRKY Transcription Factors Positively Regulating Polysaccharide Biosynthesis in Polygonatum cyrtonema.

Polygonatum cyrtonema (P. cyrtonema) is a valuable rhizome-propagating traditional Chinese medical herb. Polysaccharides (PCPs) are the major bioactive constituents in P. cyrtonema. However, the molecular basis of PCP biosynthesis in P. cyrtonema remains unknown. In this study, we measured the PCP contents of 11 wild P. cyrtonema germplasms. The results showed that PCP content was the highest in Lishui Qingyuan (LSQY, 11.84%) and the lowest in Hangzhou Lin'an (HZLA, 7.18%). We next analyzed the transcriptome profiles of LSQY and HZLA. Through a qRT-PCR analysis of five differential expression genes from the PCP biosynthesis pathway, phosphomannomutase, UDP-glucose 4-epimerase (galE), and GDP-mannose 4,6-dehydratase were determined as the key enzymes. A protein of a key gene, galE1, was localized in the chloroplast. The PCP content in the transiently overexpressed galE1 tobacco leaves was higher than in the wild type. Moreover, luciferase and Y1H assays indicated that PcWRKY31 and PcWRKY34 could activate galE1 by binding to its promoter. Our research uncovers the novel regulatory mechanism of PCP biosynthesis in P. cyrtonema and is critical to molecular-assisted breeding.

Integrating Transcriptomics and Hormones Dynamics Reveal Seed Germination and Emergence Process in Polygonatum cyrtonema Hua.

Polygonatum cyrtonema Hua is a traditional Chinese herb propagated using rhizomes, and excessive demand for seedlings and quality deterioration caused by rhizome propagation has highlighted that seed propagation may be an ideal solution to address these issues. However, the molecular mechanisms involved in P. cyrtonema Hua seed germination and emergence stages are not well understood. Therefore, in the present study, we performed transcriptomics combined with hormone dynamics during different seed germination stages, and 54,178 unigenes with an average length of 1390.38 bp (N50 = 1847 bp) were generated. Significant transcriptomic changes were related to plant hormone signal transduction and the starch and carbohydrate pathways. Genes related to ABA(abscisic acid), IAA(Indole acetic acid), and JA(Jasmonic acid) signaling, were downregulated, whereas genes related to ethylene, BR(brassinolide), CTK(Cytokinin), and SA(salicylic acid) biosynthesis and signaling were activated during the germination process. Interestingly, GA biosynthesis- and signaling-related genes were induced during the germination stage but decreased in the emergence stage. In addition, seed germination significantly upregulated the expression of genes associated with starch and sucrose metabolism. Notably, raffinose biosynthesis-related genes were induced, especially during the emergence stage. In total, 1171 transcription factor (TF) genes were found to be differentially expressed. Our results provide new insights into the mechanisms underlying P. cyrtonema Hua seed germination and emergence processes and further research for molecular breeding.

[Identification and expression of uridine diphosphate glycosyltransferase(UGT) gene family from Dendrobium officinale].

Uridine diphosphate glycosyltransferase(UGT) is a highly conserved protein in plants, which usually functions in secondary metabolic pathways. This study used the Hidden Markov Model(HMM) to screen out members of UGT gene family in the whole genome of Dendrobium officinale, and 44 UGT genes were identified. Bioinformatics was used to analyze the structure, phylogeny, and promoter region components of D. officinale genes. The results showed that UGT gene family could be divided into four subfamilies, and UGT gene structure was relatively conserved in each subfamily, with nine conserved domains. The upstream promoter region of UGT gene contained a variety of cis-acting elements related to plant hormones and environmental factors, indicating that UGT gene expression may be induced by plant hormones and external environmental factors. UGT gene expression in different tissues of D. officinale was compared, and UGT gene expression was found in all parts of D. officinale. It was speculated that UGT gene played an important role in many tissues of D. officinale. Through transcriptome analysis of D. officinale mycorrhizal symbiosis environment, low temperature stress, and phosphorus deficiency stress, this study found that only one gene was up-regulated in all three conditions. The results of this study can help understand the functions of UGT gene family in Orchidaceae plants and provide a basis for further study on the molecular regulation mechanism of polysaccharide metabolism pathway in D. officinale.

Morphological and stage-specific transcriptome analyses reveal distinct regulatory programs underlying yam (Dioscorea alata L.) bulbil growth.

In yam (Dioscorea spp) species, bulbils at leaf axils are the most striking species-specific axillary structure and exhibit important ecological niches. Genetic regulation underlying bulbil growth remains largely unclear so far. Here, we characterize yam (Dioscorea alata L.) bulbil development using histological analysis, and perform full transcriptional profiling on key developmental stages together with phytohormone analyses. Using the stage-specific scoring algorithm, we have identified 3451 stage-specifically expressed genes that exhibit a tight link between major transcriptional changes and stages. Co-expressed gene clusters revealed an obvious over-representation of genes associated with cell division and expansion at the initiation stage of bulbils (T1). Transcriptional changes of hormone-related genes highly coincided with hormone levels, indicating that bulbil initiation and growth are coordinately controlled by multiple phytohormones. In particular, localized auxin is transiently required to trigger bulbil initiation, and be further depleted or exported from bulbils to promote growth by up-regulation of genes involved in auxinconjugation and efflux. The sharp increase in supply of sucrose and an enhanced trehalose-6-phophate pathway at T1 were observed, suggesting that sucrose probably functions as a key signal and promotes bulbil initiation. Analysis of the expression of transcription factors (TFs) predicated 149 TFs as stage-specifically expressed; several T1-specific TFs (from Aux/IAA, E2F, MYB, and bHLH families) have been shown to play key roles in triggering bulbil formation. Together, our work provides a crucial angle for in-depth understanding of the molecular programs underlying yam's unique bulbil development processes. Stage-specific gene sets can be queried to obtain key candidates regulating bulbil growth, serving as valuable resources for further functional research.

Transciptome analysis reveals flavonoid biosynthesis regulation and simple sequence repeats in yam (Dioscorea alata L.) tubers.

BACKGROUND: Yam (Dioscorea alata L.) is an important tuber crop and purple pigmented elite cultivar has recently become popular because of associated health benefits. Identifying candidate genes responsible for flavonoid biosynthesis pathway (FBP) will facilitate understanding the molecular mechanism of controlling pigment formation in yam tubers. Here, we used Illumina sequencing to characterize the transcriptome of tubers from elite purple-flesh cultivar (DP) and conventional white-flesh cultivar (DW) of yam. In this process, we also designed high quality molecular markers to assist molecular breeding for tuber trait improvement. RESULTS: A total of 125,123 unigenes were identified from the DP and DW cDNA libraries, of which about 49.5% (60,020 unigenes) were annotated by BLASTX analysis using the publicly available protein database. These unigenes were further annotated functionally and subject to biochemical pathway analysis. 511 genes were identified to be more than 2-fold (FDR < 0.05) differentially expressed between the two yam cultivars, of which 288 genes were up-regulated and 223 genes were down-regulated in the DP tubers. Transcriptome analysis detected 61 unigenes encoding multiple well-known enzymes in the FBP. Furthermore, the unigenes encoding chalcone isomerase (CHS), flavanone 3-hydroxylase (F3H), flavonoid 3'-monooxygenase (F3'H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and flavonol 3-O-glucosyltransferase (UF3GT) were found to be significantly up-regulated in the DP, implying that these genes were potentially associated with tuber color formation in this elite cultivar. The expression of these genes was further confirmed by qRT-PCR. Finally, 11,793 SSRs were successfully identified with these unigenes and 6,082 SSR markers were developed using Primer 3. CONCLUSIONS: This study provides the first comprehensive transcriptomic dataset for yam tubers, which will significantly contribute to genomic research of this and other related species. Some key genes associated with purple-flesh trait were successfully identified, thus providing valuable information about molecular process of regulating pigment accumulation in elite yam tubers. In the future, this information might be directly used to genetically manipulate the conventional white-fleshed tuber cultivars to enable them to produce purple flesh. In addition, our SSR marker sets will facilitate identification of QTLs for various tuber traits in yam breeding programs.

[Breeding of new Curcuma wenyujin variety "Wenyujin No. 1"].

In order to breed and spread a new cultivar of Curcuma wenyujin, the C. wenyujin germplasm resources were investigated in authentic regions. Better varieties were chosen by comparing the yield, economic characters and quality differences between different cultivars. The results showed that the character of new selected cultivar was stable, the yield of zedoary, turmeric and curcuma was reached 313.7, 177.9, 91.2 kg per 667 m2, respectively, it increased 11.6%, 10.2%, 14.2% comparing with farmer varieties. The volatile oil contents in zedoary and turmeric was 4.0%, 3.0%, respectively. The target ingredients (germacrone) content was stable. It is demonstrated that the new cultivar "Wenyujin No. 1" has value for extension at authentic regions.

Anti-hepatitis B virus and cytotoxic diterpenoids from Isodon lophanthoides var. gerardianus.

Four new diterpenoides, isolophanthins A-D (1-4) together with seven known abietane diterpenoides (5-11), have been isolated from Isodon lophanthoides var. gerardianus. The new diterpenoides were elucidated by spectroscopic analysis. Some of them showed significant activities against HBsAg and HBeAg of hepatitis B virus in Hep G 2.2.15 cells, as well as the human tumor cell lines, HL-60, A-549, MOLT-4, and BEL-7402.

[Analysis on adaptive area of Curcuma wenyujin based on GIS].

OBJECTIVE: This stdudy was to determine the suitable area for Curcuma wenyujin in Wenzhou, and expand its growing region, based the growth characteristic of Curcuma wenyujin. METHODS: The 13 main factors including climate, soil and other environmental conditions were selected. The best genuine production area was regarded as the goal area in similar calculation. The grid data was calculated from the station and sampling sites by Inverse Distance Interpolation on GIS. After normolizing to the grid data, the dimension effect was removed. on this basis, calculating the absolute distance of every cell to the the goal cell. Wenzhou region was divided into best suitable area, suitable area, second suitable area and unsuitable area by clustering analyzing. RESULT: Most of area in Wenzhou are suit for Curcuma wenyujin, best suitable area, suitable area, second suitable area and unsuitable area reach 98% area of Wenzhou. CONCLUSION: This result give important reference value of Curcuma wenyujin breeding in Wenzhou.

[Study on geo-herbalism of Curcuma wenyujin based on ecological factors].

OBJECTIVE: To reveal the effects of ecological factors on geo-herbalism,by researching the correlation between yields or qualities of Curcuma wenyujin and ecological factors. METHODS: All measured dates were divided into three components, meteorological factors, soil conditions, the yields and qualities of geo-herbals, and analyzed through canonical correlation using SPSS software. RESULTS: The results showed that there were two canonical correlation models to explain correlation between meteorological factors and yields or qualities of medicinal materials, as well as soil conditions and yields or qualities, and both of canonical correlations reached the significant or extremely significant level. The analysis indicated that main ecological factors affecting the yields or qualities of medicinal materials were average temperature from May to June,the days of high temperature (over 35 degrees C) and average rainfall from August to October, annual average rainfall, altitude, available potassium content in soil. CONCLUSIONS: This study has tentatively made clear which ecological factors to affect the yields and qualities of Curcuma wenyujin; and provided some basis for revealing geo-herbalism of Curcuma wenyujin.

Physiological and transcriptomic analyses of cadmium stress response in Dendrobium officinale seedling.

Dendrobium officinale is an economically important Chinese herb with ornamental and medicinal values. However, the mechanisms by which D. officinale adapts to cadmium (Cd) stress is unknown. Here, physiological changes in D. officinale roots and leaves exposed to increasing levels of Cd stress (CdSO4 concentration of 2, 5, 9, 14 mg L-1) were analyzed at 7, 15, 30, and 45 days after treatment. The Cd stress of 14 mg L-1 significantly increased the levels of antioxidants and induced malondialdehyde and proline accumulation (P < 0.05). Cd subcellular distribution showed that Cd sequestration into soluble fraction is the major detoxification mechanism in D. officinale roots. Subsequently, the transcriptome profile of D. officinale roots treated with 14 mg L-1 Cd for 15 and 30 days was analyzed. Compared to control, 2,469 differentially expressed genes (DEGs) were identified, comprising 1,486 up-regulated genes and 983 down-regulated genes. The DEGs associated with metabolic pathways for Cd uptake, transportation and detoxification were analyzed. Several processes such as metal transporter, sulfate glutathione metabolism, cell wall metabolism, phenylpropanoid metabolism were identified to be important for Cd stress adaptation. More genes were expressed at 15 days after treatment compared to 30 days. WRKY, Trihelix, NF-YC, MYB, bZIP and bHLH transcription factors were over-expressed at both time points. Furthermore, candidate genes from the glutathione metabolism pathway were identified, and qRT-PCR analysis of ten DEGs indicated a high coorelation with RNA-seq expression profiles. Our findings provide significant information for further research of Cd stress responsive genes functions in D. officinale, especially the genes from the glutathione metabolism pathway.

[Genetic diversity of Dioscorea alata based on ISSR analysis].

This article assessed the genetic relationship and genetic diversity in Dioscorea alata. Twenty samples were examined to identify their original plants, and analyzed by ISSR markers. The results showed that 20 samples were classified into three different plants, such as D. alata, D. persimilis, and D. fordii. There was significant difference in genetic similarity coefficient between D. alata and D. persimi as well as D. fordii. There was distinct differences in D. alata, the genetic similarity coefficient was resulted from 0.672 9 to 0.990 7. With UPGMA clustering method, 16 samples of D. alata could be divided into 4 groups. After comparing samples with the phenotypic characteristics of original plants, it showed that the color and the number of tuber were the most important characteristics of judging the genetic relationship of D. alata. It is concluded that the genetic variation of Dioscorea spp is significant, especially the genetic diversity in D. alata were in a high level. This article supplied a molecular biologic support for distinguishing Dioscorea spp, and also provided basis for breeding of D. alata.

[Study on genetic diversity of Curcuma wenyujin from various habitats by SRAP].

OBJECTIVE: The DNA fingerprints of Curcuma wenyujin from various habitats were generated by using SRAP markers to find the feasibility in analyzing their relationship. METHODS: The DNA polymorphism of Curcuma wenyujin from various habitats were detected by SRAP molecular markers. RESULTS: We had screened five pairs of primer combinations, and built the DNA fingerprints of Curcuma wenyujin from various habitats. The phylogenetic clustering results revealed that the genetic difference in Curcuma wenyujin from various habitats were little. CONCLUSION: SRAP markers may offer some evidence for protecting and exploiting of Curcuma wenyuji.

Chemical constituents from aerial part of Curcuma wenyujin.

OBJECTIVE: To investigate the chemical constituents from aerial part of Curcuma wenyujin. METHOD: Compounds were isolated by repeated column chromatography on silica gel. Their structures were elucidated on the basis of spectral analysis and comparison with literature data. RESULT: Six compounds were isolated and identified as codonolactone (1), voleneol (2), octacosanoic acid (3), beta-sitosterol (4), mangdesisterol (5), and daucosterol (6). CONCLUSION: Compounds 1, 2, and 5 were isolated from the plant for the first time.

[Study on effects of seed stock, cultivating density on the yields and volatile oil of Curcuma wenyujin].

OBJECTIVE: According to the study on effects of cultivating density and seed stock on the yield and volatile oil, to provide a scientific basis for standardized cultivating measures. METHODS: The yield and the content of volatile oil at harvest were counted and analyzed the variance by SPSS. RESULTS: The effect of cultivating density on the yields from 3 different medicinal parts of medical material was significant. The effect of seed stock on the yield of Rhizoma wenyujin concisa and Rhizoma wenyujin curcumae was significant, but to the yield of Radix wenyujin curcumae was very little. The mutual effect between cultivating density and seed stock was also very little. CONCLUSION: The cultivating density play an important role in the yields of Curcuma wenyujin.

[Study on growth characteristics of Curcuma wenyujin].

OBJECTIVE: To study on growth characteristics of Curcuma wenyujin, and provide theoretical basis for the development of high-quality and high-yield medical material. METHOD: The morphological change of the plant was observed periodically, the content of volatile oil and dry matter in leaves, rhizome, root tuber was determine. RESULT: The growth of C. wenyujin could be divided into 5 stages, i. e. seed germination, seedling, leaf growth, rhizome expansion, accumulation of dry matter, respectively. Before the stage of rhizome expansion, over 70% dry matter was accumulated in the aerial part of the plant, and during the stage of leaf growth, the maximum increase rate of dry matter in aerial part was 3.90 g/p/d. During the stage of rhizome expansion, the ratio of dry matter of rhizome increased quickly and reached above 33% , and the increase rate of dry matter of rhizome rise up to 3.83 g/p/d, in the end of the stage, the content of volatile oil in the rhizome also rose up to 1.20 mL x 100 g(-1). CONCLUSION: During the whole growth stage, there are two growth centers, when the two curves of dry matter increase of aerial part and rhizome intersect, it is regarded as a signal that the growth transformed form the aerial part to rhizome. When the rate of dry matter from rhizome rise, the content of volatile oil in rhizome rises quickly with the increase of dry matter in rhizome. The optimal harvest time is in mid-December.

Insights from the Cold Transcriptome and Metabolome of Dendrobium officinale: Global Reprogramming of Metabolic and Gene Regulation Networks during Cold Acclimation.

Plant cold acclimation (CA) is a genetically complex phenomenon involving gene regulation and expression. Little is known about the cascading pattern of gene regulatroy network and the link between genes and metabolites during CA. Dendrobium officinale (DOKM) is an important medicinal and ornamental plant and hypersensitive to low temperature. Here, we used the large scale metabolomic and transcriptomic technologies to reveal the response to CA in DOKM seedlings based on the physiological profile analyses. Lowering temperature from 4 to -2°C resulted in significant increase (P < 0.01) in antioxidant activities and electrolyte leakage (EL) during 24 h. The fitness CA piont of 0°C and control (20°C) during 20 h were firstly obtained according to physiological analyses. Subsequently, massive transcriptome and metabolome reprogramming occurred during CA. The gene to metabolite network demonstrated that the CA associated processes are highly energy demanding through activating hydrolysis of sugars, amino acids catabolism and citrate cycle. The expression levels of 2,767 genes were significantly affected by CA, including 153-fold upregulation of CBF transcription factor, 56-fold upregulation of MAPKKK16 protein kinase. Moreover, the gene interaction and regulation network analysis revealed that the CA as an active process, was regulated at the transcriptional, post-transcriptional, translational and post-translational levels. Our findings highligted a comprehensive regulatory mechanism including cold signal transduction, transcriptional regulation, and gene expression, which contributes a deeper understanding of the highly complex regulatory program during CA in DOKM. Some marker genes identified in DOKM seedlings will allow us to understand the role of each individual during CA by further functional analyses.

Characterizing diversity based on nutritional and bioactive compositions of yam germplasm (Dioscorea spp.) commonly cultivated in China.

Yams (Dioscorea spp.) are widely cultivated as edible resources and medical materials in China. Characterizing chemical compositions in yam germplasm is crucial to determine their diversity and suitability for food and medicine applications. In this study, a core germplasm containing 25 yam landraces was used to create an effective classification of usage by characterizing their nutritive and medicinal compositions. All studied landraces exhibited high contents of starch from 60.7% to 80.6% dry weight (DW), protein (6.3-12.2% DW), minerals (especially Mg 326.8-544.7 mg/kg DW), and essential amino acids. Allantoin and dioscin varied considerably, with values of 0.62-1.49% DW and 0.032-0.092% DW, respectively. The quality variability of 25 yam landraces was clearly separated in light of UPGMA clustering and principal component analysis (PCA). Using an eigenvalue ≥1 as the cutoff, the first three principal components accounted for most of the total variability (62.33%). Classification was achieved based on the results of the measured parameters and principal component analysis scores. The results are of great help in determining appropriate application strategies for yam germplasm in China.