Mass spectrometry imaging and single-cell transcriptional profiling reveal the tissue-specific regulation of bioactive ingredient biosynthesis in Taxus leaves.

Taxus leaves provide the raw industrial materials for taxol, a natural antineoplastic drug widely used in the treatment of various cancers. However, the precise distribution, biosynthesis, and transcriptional regulation of taxoids and other active components in Taxus leaves remain unknown. Matrix-assisted laser desorption/ionization-mass spectrometry imaging analysis was used to visualize various secondary metabolites in leaf sections of Taxus mairei, confirming the tissue-specific accumulation of different active metabolites. Single-cell sequencing was used to produce expression profiles of 8846 cells, with a median of 2352 genes per cell. Based on a series of cluster-specific markers, cells were grouped into 15 clusters, suggesting a high degree of cell heterogeneity in T. mairei leaves. Our data were used to create the first Taxus leaf metabolic single-cell atlas and to reveal spatial and temporal expression patterns of several secondary metabolic pathways. According to the cell-type annotation, most taxol biosynthesis genes are expressed mainly in leaf mesophyll cells; phenolic acid and flavonoid biosynthesis genes are highly expressed in leaf epidermal cells (including the stomatal complex and guard cells); and terpenoid and steroid biosynthesis genes are expressed specifically in leaf mesophyll cells. A number of novel and cell-specific transcription factors involved in secondary metabolite biosynthesis were identified, including MYB17, WRKY12, WRKY31, ERF13, GT_2, and bHLH46. Our research establishes the transcriptional landscape of major cell types in T. mairei leaves at a single-cell resolution and provides valuable resources for studying the basic principles of cell-type-specific regulation of secondary metabolism.

Development of Chloroplast Microsatellite Markers and Evaluation of Genetic Diversity and Population Structure of Cutleaf Groundcherry (Physalis angulata L.) in China.

Cutleaf groundcherry (Physalis angulata L.), an annual plant containing a variety of active ingredients, has great medicinal value. However, studies on the genetic diversity and population structure of P. angulata are limited. In this study, we developed chloroplast microsatellite (cpSSR) markers and applied them to evaluate the genetic diversity and population structure of P. angulata. A total of 57 cpSSRs were identified from the chloroplast genome of P. angulata. Among all cpSSR loci, mononucleotide markers were the most abundant (68.24%), followed by tetranucleotide (12.28%), dinucleotide (10.53%), and trinucleotide (8.77%) markers. In total, 30 newly developed cpSSR markers with rich polymorphism and good stability were selected for further genetic diversity and population structure analyses. These cpSSRs amplified a total of 156 alleles, 132 (84.62%) of which were polymorphic. The percentage of polymorphic alleles and the average polymorphic information content (PIC) value of the cpSSRs were 81.29% and 0.830, respectively. Population genetic diversity analysis indicated that the average observed number of alleles (Na), number of effective alleles (He), Nei's gene diversity (h), and Shannon information indices (I) of 16 P. angulata populations were 1.3161, 1.1754, 0.1023, and 0.1538, respectively. Moreover, unweighted group arithmetic mean, neighbor-joining, principal coordinate, and STRUCTURE analyses indicated that 203 P. angulata individuals from 16 populations were grouped into four clusters. A molecular variance analysis (AMOVA) illustrated the considerable genetic variation among populations, while the gene flow (Nm) value (0.2324) indicated a low level of gene flow among populations. Our study not only provided a batch of efficient genetic markers for research on P. angulata but also laid an important foundation for the protection and genetic breeding of P. angulata resources.

Integrated mass spectrometry imaging and single-cell transcriptome atlas strategies provide novel insights into taxoid biosynthesis and transport in Taxus mairei stems.

Taxol, which is a widely used important chemotherapeutic agent, was originally isolated from Taxus stem barks. However, little is known about the precise distribution of taxoids and the transcriptional regulation of taxoid biosynthesis across Taxus stems. Here, we used MALDI-IMS analysis to visualize the taxoid distribution across Taxus mairei stems and single-cell RNA sequencing to generate expression profiles. A single-cell T. mairei stem atlas was created, providing a spatial distribution pattern of Taxus stem cells. Cells were reordered using a main developmental pseudotime trajectory which provided temporal distribution patterns in Taxus stem cells. Most known taxol biosynthesis-related genes were primarily expressed in epidermal, endodermal, and xylem parenchyma cells, which caused an uneven taxoid distribution across T. mairei stems. We developed a single-cell strategy to screen novel transcription factors (TFs) involved in taxol biosynthesis regulation. Several TF genes, such as endodermal cell-specific MYB47 and xylem parenchyma cell-specific NAC2 and bHLH68, were implicated as potential regulators of taxol biosynthesis. Furthermore, an ATP-binding cassette family transporter gene, ABCG2, was proposed as a potential taxoid transporter candidate. In summary, we generated a single-cell Taxus stem metabolic atlas and identified molecular mechanisms underpinning the cell-specific transcriptional regulation of the taxol biosynthesis pathway.

Investigation of the role of TmMYB16/123 and their targets (TmMTP1/11) in the tolerance of Taxus media to cadmium.

The toxicity and stress caused by heavy metal contamination has become an important constraint to the growth and flourishing of trees. In particular, species belonging to the genus Taxus, which are the only natural source for the anti-tumor medicine paclitaxel, are known to be highly sensitive to environmental changes. To investigate the response of Taxus spp. to heavy metal stress, we analyzed the transcriptomic profiles of Taxus media trees exposed to cadmium (Cd2+). In total, six putative genes from the metal tolerance protein (MTP) family were identified in T. media, including two Cd2+ stress inducible TMP genes (TmMTP1, TmMTP11 and Taxus media). Secondary structure analyses predicted that TmMTP1 and TmMTP11, which are members of the Zn-CDF and Mn-CDF subfamily proteins, respectively, contained six and four classic transmembrane domains, respectively. The introduction of TmMTP1/11 into the ∆ycf1 yeast cadmium-sensitive mutant strain showed that TmMTP1/11 might regulate the accumulation of Cd2+ to yeast cells. To screen the upstream regulators, partial promoter sequences of the TmMTP1/11 genes were isolated using the chromosome walking method. Several myeloblastosis (MYB) recognition elements were identified in the promoters of these genes. Furthermore, two Cd2+-induced R2R3-MYB TFs, TmMYB16 and TmMYB123, were identified. Both in vitro and in vivo assays confirmed that TmMTB16/123 play a role in Cd2+ tolerance by activating and repressing the expression of TmMTP1/11 genes. The present study elucidated new regulatory mechanisms underlying the response to Cd stress and can contribute to the breeding of Taxus species with high environmental adaptability.

Complete Plastome of Physalis angulata var. villosa, Gene Organization, Comparative Genomics and Phylogenetic Relationships among Solanaceae.

Physalis angulata var. villosa, rich in withanolides, has been used as a traditional Chinese medicine for many years. To date, few extensive molecular studies of this plant have been conducted. In the present study, the plastome of P. angulata var. villosa was sequenced, characterized and compared with that of other Physalis species, and a phylogenetic analysis was conducted in the family Solanaceae. The plastome of P. angulata var. villosa was 156,898 bp in length with a GC content of 37.52%, and exhibited a quadripartite structure typical of land plants, consisting of a large single-copy (LSC, 87,108 bp) region, a small single-copy (SSC, 18,462 bp) region and a pair of inverted repeats (IR: IRA and IRB, 25,664 bp each). The plastome contained 131 genes, of which 114 were unique and 17 were duplicated in IR regions. The genome consisted of 85 protein-coding genes, eight rRNA genes and 38 tRNA genes. A total of 38 long, repeat sequences of three types were identified in the plastome, of which forward repeats had the highest frequency. Simple sequence repeats (SSRs) analysis revealed a total of 57 SSRs, of which the T mononucleotide constituted the majority, with most of SSRs being located in the intergenic spacer regions. Comparative genomic analysis among nine Physalis species revealed that the single-copy regions were less conserved than the pair of inverted repeats, with most of the variation being found in the intergenic spacer regions rather than in the coding regions. Phylogenetic analysis indicated a close relationship between Physalis and Withania. In addition, Iochroma, Dunalia, Saracha and Eriolarynx were paraphyletic, and clustered together in the phylogenetic tree. Our study published the first sequence and assembly of the plastome of P. angulata var. villosa, reported its basic resources for evolutionary studies and provided an important tool for evaluating the phylogenetic relationship within the family Solanaceae.

Role of female-predominant MYB39-bHLH13 complex in sexually dimorphic accumulation of taxol in Taxus media.

Taxus trees are major natural sources for the extraction of taxol, an anti-cancer agent used worldwide. Taxus media is a dioecious woody tree with high taxol yield. However, the sexually dimorphic accumulation of taxoids in T. media is largely unknown. Our study revealed high accumulation of taxoids in female T. media trees using a UPLC-MS/MS method. Thereafter, many differential metabolites and genes between female and male T. media trees were identified using metabolomic and transcriptomic analyses, respectively. Most of the taxol-related genes were predominantly expressed in female trees. A female-specific R2R3-MYB transcription factor gene, TmMYB39, was identified. Furthermore, bimolecular fluorescence complementation and yeast two-hybrid assays suggested the potential interaction between TmMYB39 and TmbHLH13. Several taxol biosynthesis-related promoter sequences were isolated and used for the screening of MYB recognition elements. The electrophoretic mobility shift assay indicated that TmMYB39 could bind to the promoters of the GGPPS, T10OH, T13OH, and TBT genes. Interaction between TmMYB39 and TmbHLH13 transactivated the expression of the GGPPS and T10OH genes. TmMYB39 might function in the transcriptional regulation of taxol biosynthesis through an MYB-bHLH module. Our results give a potential explanation for the sexually dimorphic biosynthesis of taxol in T. media.

Development and Application of a Cultivar-Specific Sequence-Characterized Amplified Region (SCAR) Marker for the Detection of Chrysanthemum morifolium Ramat. 'Daboju'.

Chrysanthemummorifolium Ramat. 'Daboju' is a C. morifolium cultivar with important ornamental and medicinal values, and is often used in the treatment of colds, blurred vision, dizziness, and itchy skin. As the morphological characteristics of C. morifolium 'Daboju' are very similar to those of other C. morifolium cultivars, they are often confused in practice. However, the medicinal value and practical use of C. morifolium depends on using the correct rapid and accurate identification of C. morifolium 'Daboju' and its differentiation from other, morphologically similar C. × morifolium cultivars. Twenty-one polymorphic start codon-targeted (SCoT) primers were amplified in 21 distinct C. morifolium cultivars. One cultivar-specific DNA marker was developed with the aim of the rapid and accurate identification of C. morifolium 'Daboju' and its differentiation from other, similar C. morifolium cultivars. Twenty-one polymorphic start codon-targeted (SCoT) primers were amplified in 21 distinct C. morifolium cultivars. One cultivar-specific 385-bp amplicon (named SCoT36-385), amplified only in C. morifolium 'Daboju' (and in all samples of this cultivar), was identified, cloned, and sequenced. Subsequently, a sequence-characterized amplified region (SCAR) marker (named DBJF/DBJR), generating a 360-bp amplicon, was developed from SCoT36-385 and tested for amplification in all 21 C. morifolium cultivars, ten C. morifolium 'Daboju' populations, and different simulated adulterations of 'Daboju' with other cultivars. The primers amplified the specific 360-bp-long DNA fragment in all the tested C. morifolium 'Daboju' samples but failed in the absence of 'Daboju'. The detection limit of the SCAR primer pair (DBJF/DBJR) was 100 pg of DNA extracted from C. morifolium 'Daboju'. Hence, this SCAR marker has a very high detection sensitivity, and can be used for accurate and rapid identification of C. morifolium 'Daboju'. It can play an important role in ensuring the quality of medicinal preparations and protecting C. morifolium 'Daboju' germplasm resources in breeding programs and in identifying lines generated from this cultivar.

Development, Identification, and Application of a Germplasm Specific SCAR Marker for Dendrobium officinale Kimura et Migo.

The stems of Dendrobium officinale have been used as a rare and valuable Chinese tonic medicine, known as "Tiepi Fengdou", since the Qing dynasty. Because of the increased market demand and continued exploitation of this plant, the reserves of wild D. officinale resources have been depleted, and D. officinale products on the market are being increasingly adulterated. Such changes have strongly affected the sustainable utilization of this valuable medicinal plant resource and the development of related industries. In this study, a species-specific DNA marker was developed for the rapid and accurate authentication of D. officinale. In total, 36 start codon-targeted (SCoT) polymorphism primers were screened in 36 definite Dendrobium species, and a distinct species-specific DNA amplicon (SCoT13-215) for D. officinale was obtained. After the sequence was cloned and sequenced, a sequence-characterized amplified region marker was developed (named SHF/SHR) and validated through PCR amplification of all 38 Dendrobium samples. The marker's specificity for D. officinale was confirmed through the consistent amplification of a clear 197-bp band. This SCAR marker can be used to rapidly, effectively, and reliably identify D. officinale among various Dendrobium species and may play an important role in ensuring the quality of medicinal preparations and protecting the germplasm of this important medicinal species.

Complete chloroplast genomes of four Physalis species (Solanaceae): lights into genome structure, comparative analysis, and phylogenetic relationships.

BACKGROUND: Physalis L. is a genus of herbaceous plants of the family Solanaceae, which has important medicinal, edible, and ornamental values. The morphological characteristics of Physalis species are similar, and it is difficult to rapidly and accurately distinguish them based only on morphological characteristics. At present, the species classification and phylogeny of Physalis are still controversial. In this study, the complete chloroplast (cp) genomes of four Physalis species (Physalis angulata, P. alkekengi var. franchetii, P. minima and P. pubescens) were sequenced, and the first comprehensive cp genome analysis of Physalis was performed, which included the previously published cp genome sequence of Physalis peruviana. RESULTS: The Physalis cp genomes exhibited typical quadripartite and circular structures, and were relatively conserved in their structure and gene synteny. However, the Physalis cp genomes showed obvious variations at four regional boundaries, especially those of the inverted repeat and the large single-copy regions. The cp genomes' lengths ranged from 156,578 bp to 157,007 bp. A total of 114 different genes, 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes, were observed in four new sequenced Physalis cp genomes. Differences in repeat sequences and simple sequence repeats were detected among the Physalis cp genomes. Phylogenetic relationships among 36 species of 11 genera of Solanaceae based on their cp genomes placed Physalis in the middle and upper part of the phylogenetic tree, with a monophyletic evolution having a 100% bootstrap value. CONCLUSION: Our results enrich the data on the cp genomes of the genus Physalis. The availability of these cp genomes will provide abundant information for further species identification, increase the taxonomic and phylogenetic resolution of Physalis, and assist in the investigation and utilization of Physalis plants.

Bioactive compounds induced in Physalis angulata L. by methyl-jasmonate: an investigation of compound accumulation patterns and biosynthesis-related candidate genes.

KEY MESSAGE: We employed both metabolomic and transcriptomic approaches to explore the accumulation patterns of physalins, flavonoids and chlorogenic acid in Physalis angulata and revealed the genes associated with the biosynthesis of bioactive compounds under methyl-jasmonate (MeJA) treatment. Physalis angulata L. is an annual Solanaceae plant with a number of medicinally active compounds. Despite the potential pharmacological benefits of P. angulata, the scarce genomic information regarding this plant has limited the studies on the mechanisms of bioactive compound biosynthesis. To facilitate the basic understanding of the main chemical constituent biosynthesis pathways, we performed both metabolomic and transcriptomic approaches to reveal the genes associated with the biosynthesis of bioactive compounds under methyl-jasmonate (MeJA) treatment. Untargeted metabolome analysis showed that most physalins, flavonoids and chlorogenic acid were significantly upregulated. Targeted HPLC-MS/MS analysis confirmed variations in the contents of two important representative steroid derivatives (physalins B and G), total flavonoids, neochlorogenic acid, and chlorogenic acid between MeJA-treated plants and controls. Transcript levels of a few steroid biosynthesis-, flavonoid biosynthesis-, and chlorogenic acid biosynthesis-related genes were upregulated, providing a potential explanation for MeJA-induced active ingredient synthesis in P. angulata. Systematic correlation analysis identified a number of novel candidate genes associated with bioactive compound biosynthesis. These results may help to elucidate the regulatory mechanism underlying MeJA-induced active compound accumulation and provide several valuable candidate genes for further functional study.

Tissue-specific study across the stem of Taxus media identifies a phloem-specific TmMYB3 involved in the transcriptional regulation of paclitaxel biosynthesis.

Taxus stem barks can be used for extraction of paclitaxel. However, the composition of taxoids across the whole stem and the stem tissue-specificity of paclitaxel biosynthesis-related enzymes remain largely unknown. We used cultivated Taxus media trees for analyses of the chemical composition and protein of major stem tissues by an integrated metabolomic and proteomic approach, and the role of TmMYB3 in paclitaxel biosynthesis was investigated. The metabolomic landscape analysis showed differences in stem tissue-specific accumulation of metabolites. Phytochemical analysis revealed that there is high accumulation of paclitaxel in the phloem. Ten key enzymes involved in paclitaxel biosynthesis were identified, most of which are predominantly produced in the phloem. The full-length sequence of TmMYB3 and partial promoter sequences of five paclitaxel biosynthesis-related genes were isolated. Several MYB recognition elements were found in the promoters of TBT, DBTNBT and TS. Further in vitro and in vivo investigations indicated that TmMYB3 is involved in paclitaxel biosynthesis by activating the expression of TBT and TS. Differences in the taxoid composition of different stem tissues suggest that the whole stem of T. media has potential for biotechnological applications. Phloem-specific TmMYB3 plays a role in the transcriptional regulation of paclitaxel biosynthesis, and may explain the phloem-specific accumulation of paclitaxel.

Comparative Metabolomic and Proteomic Analyses Reveal the Regulation Mechanism Underlying MeJA-Induced Bioactive Compound Accumulation in Cutleaf Groundcherry ( Physalis angulata L.) Hairy Roots.

Cutleaf groundcherry ( Physalis angulata L.) is an annual plant with a number of medicinal ingredients. However, studies about the secondary metabolism of P. angulata are very limited. An integrated metabolome and proteome approach was used to reveal the variations in the metabolism associated with bioactive compounds under methyl-jasmonate (MeJA) treatment. Application of MeJA to the hairy roots could significantly increase the accumulation of most active ingredients. A targeted approach confirmed the variations in physalins D and H between MeJA treatment and the controls. Increases in the levels of a number of terpenoid backbone biosynthesis and steroid biosynthesis related enzymes, cytochrome P450 monooxygenases and 3ß-hydroxysterioid dehydrogenase might provide a potential explanation for the MeJA-induced active ingredient synthesis. Our results may contribute to a deeper understanding of the regulation mechanism underlying the MeJA-induced active compound accumulation in P. angulata.

Development of Species-Specific SCAR Markers, Based on a SCoT Analysis, to Authenticate Physalis (Solanaceae) Species.

Physalis is an important genus in the Solanaceae family. It includes many species of significant medicinal value, edible value, and ornamental value. However, many Physalis species are easily confused because of their similar morphological traits, which hinder the utilization and protection of Physalis resources. Therefore, it is necessary to create fast, sensitive, and reliable methods for the Physalis species authentication. Intended for that, in this study, species-specific sequence-characterized amplified region (SCAR) markers were developed for accurate identification of the closely related Physalis species P. angulata, P. minima, P. pubescens, and P. alkekengi var. franchetii, based on a simple and novel marker system, start codon targeted (SCoT) marker. A total of 34 selected SCoT primers yielded 289 reliable SCoT loci, of which 265 were polymorphic. Four species-specific SCoT fragments (SCoT3-1404, SCoT3-1589, SCoT5-550, and SCoT36-520) from Physalis species were successfully identified, cloned, and sequenced. Based on these selected specific DNA fragments, four SCAR primers pairs were developed and named ST3KZ, ST3MSJ, ST5SJ, and ST36XSJ. PCR analysis of each of these primer pairs clearly demonstrated a specific amplified band in all samples of the target Physalis species, but no amplification was observed in other Physalis species. Therefore, the species-specific SCAR primer pairs developed in this study could be used as powerful tools that can rapidly, effectively, and reliably identify and differentiate Physalis species.

High-Density Genetic Map Construction and Stem Total Polysaccharide Content-Related QTL Exploration for Chinese Endemic Dendrobium (Orchidaceae).

Plants of the Dendrobium genus are orchids with not only ornamental value but also high medicinal value. To understand the genetic basis of variations in active ingredients of the stem total polysaccharide contents (STPCs) among different Dendrobium species, it is of paramount importance to understand the mechanism of STPC formation and identify genes affecting its process at the whole genome level. Here, we report the first high-density single-nucleotide polymorphism (SNP) integrated genetic map with a good genome coverage of Dendrobium. The specific-locus amplified fragment sequencing (SLAF-seq) technology led to identification of 7,013,400 SNPs from 1,503,626 high-quality SLAF markers from two parents (Dendrobium moniliforme ♀ × Dendrobium officinale ♂) and their interspecific F1 hybrid population. The final genetic map contained 8, 573 SLAF markers, covering 19 linkage groups (LGs). This genetic map spanned a length of 2,737.49 cM, where the average distance between markers is 0.32 cM. In total, 5 quantitative trait loci (QTL) related to STPC were identified, 3 of which have candidate genes within the confidence intervals of these stable QTLs based on the D. officinale genome sequence. This study will build a foundation up for the mapping of other medicinal-related traits and provide an important reference for the molecular breeding of these Chinese herb.

[Application of DNA molecular marker technologies in study of medicinal Physalis species].

As traditional Chinese medicinal herbs, Physalis plants have a variety of pharmacological activities, such as anti-inflammatory, anti-oxidant, and anti-cancer effects, and have been used for the treatment of malaria, rheumatism, hepatitis, asthma, and cancer. In addition to the medicinal value, many Physalis species are also the high-grade nutrition health care fruits, can be made canned and candied etc. In the study, the application progress of DNA molecular marker technologies in medicinal Physalis plants in recent years was reviewed, in order to provide an important molecular technical basis for the identification, classification and rational development and protection of medicinal Physalis resources.

Development of SCoT-Based SCAR Marker for Rapid Authentication of Taxus Media.

Taxus media is an important species in the family Taxaceae with high medicinal and commercial value. Overexploitation and illegal trade have led T. media to a severe threat of extinction. In addition, T. media and other Taxus species have similar morphological traits and are easily misidentified, particularly during the seedling stage. The purpose of this study is to develop a species-specific marker for T. media. Through a screening of 36 start codon targeted (SCoT) polymorphism primers, among 15 individuals of 4 Taxus species (T. media, T. chinensis, T. cuspidate and T. fuana), a clear species-specific DNA fragment (amplified by primer SCoT3) for T. media was identified. After isolation and sequencing, a DNA sequence with 530 bp was obtained. Based on this DNA fragment, a primer pair for the sequence-characterized amplified region marker was designed and named MHSF/MHSR. PCR analysis with primer pair MHSF/MHSR revealed a clear amplified band for all individuals of T. media but not for T. chinensis, T. cuspidate and T. fuana. Therefore, this marker can be used as a quick, efficient and reliable tool to identify T. media among other related Taxus species. The results of this study will lay an important foundation for the protection and management of T. media as a natural resource.

Molecular identification of species of Physalis (Solanaceae) using a candidate DNA barcode: the chloroplast psbA-trnH intergenic region.

Physalis L., an important genus of the family Solanaceae, includes many commercially important edible and medicinal species. Traditionally, species identification is based on morphological traits; however, the highly similar morphological traits among species of Physalis make this approach difficult. In this study, we evaluated the feasibility of using a popular DNA barcode, the chloroplast psbA-trnH intergenic region, in the identification of species of Physalis. Thirty-six psbA-trnH regions of species of Physalis and of the closely related plant Nicandra physalodes were analyzed. The success rates of PCR amplification and sequencing of the psbA-trnH region were 100%. MEGA V6.0 was utilized to align the psbA-trnH sequences and to compute genetic distances. The results show an apparent barcoding gap between intra- and interspecific variations. Results of both BLAST1 and nearest-distance methods prove that the psbA-trnH regions can be used to identify all species examined in the present study. In addition, phylogenetic analysis using psbA-trnH data revealed a distinct boundary between species. It also confirmed the relationship between species of Physalis and closely related species, as established by previous studies. In conclusion, the psbA-trnH intergenic region can be used as an efficient DNA barcode for the identification of species of Physalis.

Succinyl-proteome profiling of Dendrobium officinale, an important traditional Chinese orchid herb, revealed involvement of succinylation in the glycolysis pathway.

BACKGROUND: Lysine succinylation is a ubiquitous and important protein post-translational modification in various eukaryotic and prokaryotic cells. However, its functions in Dendrobium officinale, an important traditional Chinese orchid herb with high polysaccharide contents, are largely unknown. RESULTS: In our study, LC-MS/MS was used to identify the peptides that were enriched by immune-purification with a high-efficiency succinyl-lysine antibody. In total, 314 lysine succinylation sites in 207 proteins were identified. A gene ontology analysis showed that these proteins are associated with a wide range of cellular functions, from metabolic processes to stimuli responses. Moreover, two types of conserved succinylation motifs, '***Ksuc******K**' and '****EKsuc***', were identified. Our data showed that lysine succinylation occurred on five key enzymes in the glycolysis pathway. The numbers of average succinylation sites on these five enzymes in plants were lower than those in bacteria and mammals. Interestingly, two active site amino acids residues, K103 and K225, could be succinylated in fructose-bisphosphate aldolase, indicating a potential function of lysine succinylation in the regulation of glycolytic enzyme activities. Furthermore, the protein-protein interaction network for the succinylated proteins showed that several functional terms, such as glycolysis, TCA cycle, oxidative phosphorylation and ribosome, are consisted. CONCLUSIONS: Our results provide the first comprehensive view of the succinylome of D. officinale and may accelerate future biological investigations of succinylation in the synthesis of polysaccharides, which are major active ingredients.

Identification and analysis of genes associated with the synthesis of bioactive constituents in Dendrobium officinale using RNA-Seq.

Dendrobium officinale L. is an important traditional herb with high commercial value in China. Several bioactive constituents, including polysaccharides and alkaloids, reportedly make major contributions toward the excellent medicinal effect of D. officinale. In this study, the contents of polysaccharides and alkaloids in various organs of D. officinale were measured and compared. We took advantage of transcriptomes from four organs to explore biological mechanisms in the organ-specific distribution of active ingredients in D. officinale. Based on Kyoto Encyclopedia of Genes and Genomes pathways, unigenes related to the enzymes involved in fructose and mannose metabolism and unigenes associated with putative upstream elements of the alkaloid biosynthetic pathway were identified. A large number of candidates, including 35 full-length glycosyltransferase genes and 49 full-length P450 genes, were also identified based on the transcriptome data, and the organ-specific expression pattern of these genes was determined. Furthermore, differential expression of all candidate genes was analyzed in two Dendrobium species, D. nobile L. and D. officinale. The data will supply important clues to exploit useful genes involved in polysaccharide and alkaloid synthesis.