Overexpression of transcription factor FaMYB63 enhances salt tolerance by directly binding to the SOS1 promoter in Arabidopsis thaliana.

Salinity is a pivotal abiotic stress factor with far-reaching consequences on global crop growth, yield, and quality and which includes strawberries. R2R3-MYB transcription factors encompass a range of roles in plant development and responses to abiotic stress. In this study, we identified that strawberry transcription factor FaMYB63 exhibited a significant upregulation in its expression under salt stress conditions. An analysis using yeast assay demonstrated that FaMYB63 exhibited the ability to activate transcriptional activity. Compared with those in the wild-type (WT) plants, the seed germination rate, root length, contents of chlorophyll and proline, and antioxidant activities (SOD, CAT, and POD) were significantly higher in FaMYB63-overexpressing Arabidopsis plants exposed to salt stress. Conversely, the levels of malondialdehyde (MDA) were considerably lower. Additionally, the FaMYB63-overexpressed Arabidopsis plants displayed a substantially improved capacity to scavenge active oxygen. Furthermore, the activation of stress-related genes by FaMYB63 bolstered the tolerance of transgenic Arabidopsis to salt stress. It was also established that FaMYB63 binds directly to the promoter of the salt overly sensitive gene SOS1, thereby activating its expression. These findings identified FaMYB63 as a possible and important regulator of salt stress tolerance in strawberries.

ATP-binding cassette protein ABCC8 promotes anthocyanin accumulation in strawberry fruits.

Anthocyanins are important health-promoting flavonoid compounds that substantially contribute to fruit quality. Anthocyanin biosynthesis and most regulatory mechanisms are relatively well understood. However, the functions of anthocyanin transport genes in strawberry fruit remain unclear. In this study, a gene encoding an ATP-binding cassette (ABC) protein of type C, ABCC8, was isolated from strawberry fruits. qRT-PCR analysis demonstrated that the transcript levels of FvABCC8 were the highest and were strongly correlated with anthocyanin accumulation during strawberry fruit ripening. Transient overexpression and RNAi of FvABCC8 led to an increase and decrease in anthocyanin content in strawberry fruits, respectively. Moreover, the ABCC8 promoter was activated by MYB and bHLH transcription factors MYB10, bHLH33, and MYC1. Sucrose enhanced anthocyanin accumulation in FvABCC8-overexpressing Arabidopsis, particularly at higher concentrations. FvABCC8-overexpressing lines were less sensitive to ABA during seed germination and seedling development. These results suggest that strawberry vacuolar anthocyanin transport may be mediated by the ABCC transporter ABCC8, the expression of which may be regulated by transcription factors MYB10, bHLH33, and MYC1.

R2R3-MYB transcription factor FaMYB5 is involved in citric acid metabolism in strawberry fruits.

The citrate content of strawberry fruits affects their organoleptic quality. However, little is known about the transcriptional regulatory mechanisms of citric acid metabolism in strawberry fruits. In this study, the R2R3-MYB transcription factor FaMYB5 was identified and placed in the R2R3-MYB subfamily. FaMYB5 is found in the nucleus and shows tissue- and stage-specific expression levels. Citric acid content was positively correlated with FaMYB5 transcript levels. Upregulated FaMYB5 increased citric acid accumulation in transient FaMYB5-overexpressing strawberry fruits, whereas transient RNA silencing of FaMYB5 in strawberry fruits resulted in a reduction of citric acid content. The role of FaMYB5 was verified using stable transgenic NC89 tobacco. Furthermore, a yeast one-hybrid assay revealed that FaMYB5 influences citric acid accumulation by binding to the FaACO (aconitase), FaGAD (glutamate decarboxylase), and FaCS2 (citrate synthase) promoters. Dual-luciferase assays were used to demonstrate that FaMYB5 could activate FaCS2 expression and repress the transcription levels of FaACO and FaGAD. This study identified important roles of FaMYB5 in the regulation of citric acid metabolism and provided a potential target for improving strawberry fruit taste in horticultural crops.

The R2R3-MYB transcription factor FaMYB63 participates in regulation of eugenol production in strawberry.

The biosynthetic pathway of volatile phenylpropanoids, including 4-allyl-2-methoxyphenol (eugenol), has been investigated in petunia (Petunia hybrida). However, the regulatory network for eugenol accumulation in strawberry (Fragaria × ananassa Duch.) fruit remains unclear. Here, an R2R3-type MYB transcription factor (TF; FaMYB63) was isolated from strawberry by yeast one-hybrid (Y1H) screening using the promoter of the FaEGS1 (eugenol synthase 1 [EGS 1]) gene, which encodes the enzyme responsible for the last step in eugenol biosynthesis. FaMYB63 is phylogenetically distinct from other R2R3-MYB TFs, including FaEOBІІ (EMISSION OF BENZENOID II [EOBII]), which also participates in regulating eugenol biosynthesis in strawberry receptacles. Reverse transcription quantitative PCR (RT-qPCR) assays showed that the expression of FaMYB63 was tissue-specific and consistent with eugenol content through strawberry fruit development, was repressed by abscisic acid, and was activated by auxins (indole-3-acetic acid). Overexpression and RNA interference-mediated silencing of FaMYB63 resulted in marked changes in the transcript levels of the biosynthetic genes FaEGS1, FaEGS2, and FaCAD1 (cinnamyl alcohol dehydrogenase 1 [CAD1]) and, thereby, the accumulation of eugenol. Electrophoretic mobility shift, Y1H, GUS activity, and dual-luciferase activity assays demonstrated that the transcript levels of FaEOBІІ and FaMYB10 were regulated by FaMYB63, but not the other way around. Together, these results demonstrate that FaMYB63 directly activates FaEGS1, FaEGS2, FaCAD1, FaEOBІІ, and FaMYB10 to induce eugenol biosynthesis during strawberry fruit development. These findings deepen the understanding of the regulatory network that influences eugenol metabolism in an edible fruit crop.

FvMYB24, a strawberry R2R3-MYB transcription factor, improved salt stress tolerance in transgenic Arabidopsis.

Salinity is one of the major environmental stresses that limit crop growth and productivity. In this study, the FvMYB24 gene that encodes an R2R3-type MYB transcription factor was cloned and characterized. An expression analysis showed that FvMYB24 had a tissue- and stage-specific profile and was induced by salt treatment. Arabidopsis plants that overexpressed transgenic FvMYB24 exhibited a higher germination rate, fresh weight, chlorophyll content, and longer root length than the wild type (WT) under salt stress. The transgenic plants had higher activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) and the accumulation of proline, while these plants accumulated lower amounts of malondialdehyde (MDA) compared with the WT. Furthermore, our results also revealed that the overexpression of FvMYB24 up-regulated the expression of several stress-related genes (AtSOS1, AtSOS2, AtSOS3, AtSOD, AtPOD, AtCAT1, AtNHX1, and AtLEA3) in response to salt stress, thus, enhancing the tolerance of transgenic Arabidopsis. An analysis of the cis-acting elements in the SOS1, SOS2, and SOS3 promoters revealed MYB-binding sites. However, FvMYB24 could only bind to the SOS1 promoter to mediate salt tolerance but not to the SOS2 and SOS3 promoters. These findings suggest that FvMYB24 could potentially be used as a positive regulator in transgenic plant breeding to improve the tolerance of strawberry plants to salt.

Volatile constituents and ellagic acid formation in strawberry fruits of selected cultivars.

Strawberries (Fragaria × ananassa Duch.) are considered a functional food and pleasing fruit in China, mainly because of their high concentration of ellagic acid (EA) and their aroma. A total of 127 volatile compounds were identified by HS-SPME-GC-MS. Changes in volatile constituents and EA were investigated in 50 strawberry cultivars in the red-ripening stage and in 6 cultivars, including 'Benihoppe', 'Snow White', 'Yanli', 'Kaorino', 'Tokun', and 'Xiaobai', at four developmental stages. The results indicated that the components and amounts of volatile compounds and EA markedly varied among and within cultivars. Through multivariate statistical analysis of the volatile compounds, 50 cultivars were divided into 4 clusters. Aromatic components that affected the cluster formation of cultivars were detected. Volatile compounds varied quantitatively among the 6 varieties during the developmental stages, and distinct changes were observed in both red-turning fruits and red-ripening fruits compared with white fruits. Except for 'Xiaobai', which showed the highest EA content at the red-ripening stage, the other 5 cultivars exhibited the highest EA level at the large green fruit stage. Partial least squares-discriminant analysis (PLS-DA) of the profiles of volatile compounds indicated that large green fruits were characterized by EA and aldehydes; white fruits were characterized by ketones and alkanes; and red-ripening fruits were characterized by esters, acids, furans, and alcohols. The results contribute new and important information to breeding programs and the desirable cultivation of strawberry production.

Time-series transcriptomic analysis reveals novel gene modules that control theanine biosynthesis in tea plant (Camellia sinensis).

Theanine (thea) is a unique non-protein amino acid in tea plant (Camellia sinensis) and one of the most important small molecular compounds for tea quality and health effects. The molecular mechanism that maintains thea biosynthesis is not clear but may be reflected in complicated biological networks as other secondary metabolites in plants. We performed an integrative transcriptomic analysis of tea seedlings bud and leave over the time-course of ethylamine (EA) treatment that activated thea pathway. We identified 54 consistent differentially expressed genes (cDEGs, 25 upregulated and 29 downregulated) during thea activation. Gene Ontology (GO) functional enrichment analysis of upregulated genes and downregulated genes showed that they may function as a cascade of biological events during their cooperative contribution to thea biosynthesis. Among the total cDEGs, a diversity of functional genes (e.g., enzymes, transcription factors, transport and binding proteins) were identified, indicating a hierarchy of gene control network underlying thea biosynthesis. A gene network associated with thea biosynthesis was modeled and three interconnected gene functional modules were identified. Among the gene modules, several topologically important genes (e.g., CsBCS-1, CsRP, CsABC2) were experimentally validated using a combined thea content and gene expression analysis. Collectively, we presented here for the first time a comprehensive landscape of the biosynthetic mechanism of thea controlled by a underling gene network, which might provide a theoretical basis for the identification of key genes that contribute to thea biosynthesis.

Kiwifruit Genome Database (KGD): a comprehensive resource for kiwifruit genomics.

Kiwifruit (Actinidia spp.) plants produce economically important fruits containing abundant, balanced phytonutrients with extraordinarily high vitamin C contents. Since the release of the first kiwifruit reference genome sequence in 2013, large volumes of genome and transcriptome data have been rapidly accumulated for a handful of kiwifruit species. To efficiently store, analyze, integrate, and disseminate these large-scale datasets to the research community, we constructed the Kiwifruit Genome Database (KGD; http://kiwifruitgenome.org/). The database currently contains all publicly available genome and gene sequences, gene annotations, biochemical pathways, transcriptome profiles derived from public RNA-Seq datasets, and comparative genomic analysis results such as syntenic blocks and homologous gene pairs between different kiwifruit genome assemblies. A set of user-friendly query interfaces, analysis tools and visualization modules have been implemented in KGD to facilitate translational and applied research in kiwifruit, which include JBrowse, a popular genome browser, and the NCBI BLAST sequence search tool. Other notable tools developed within KGD include a genome synteny viewer and tools for differential gene expression analysis as well as gene ontology (GO) term and pathway enrichment analysis.

The complete chloroplast genome of Akebia trifoliata (Lardizabalaceae), a traditional herb in China.

Akebia trifoliata, commonly known as 'Bayuezha' in China, has been widely used as traditional Chinese medicinal herbs with a long history. In the present study, the complete chloroplast genome of A. trifoliata was sequenced using Illumina high-throughput sequencing approach. The length of the complete chloroplast genome is 157,952 bp with 38.7% GC content. It contains 131 genes, including 86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis indicated that A. trifoliata was closely related to another Lardizabalaceae species, Akebia quinata, which further confirms traditional species classification.

The tea plant reference genome and improved gene annotation using long-read and paired-end sequencing data.

Tea is a globally consumed non-alcohol beverage with great economic importance. However, lack of the reference genome has largely hampered the utilization of precious tea plant genetic resources towards breeding. To address this issue, we previously generated a high-quality reference genome of tea plant using Illumina and PacBio sequencing technology, which produced a total of 2,124 Gb short and 125 Gb long read data, respectively. A hybrid strategy was employed to assemble the tea genome that has been publicly released. We here described the data framework used to generate, annotate and validate the genome assembly. Besides, we re-predicted the protein-coding genes and annotated their putative functions using more comprehensive omics datasets with improved training models. We reassessed the assembly and annotation quality using the latest version of BUSCO. These data can be utilized to develop new methodologies/tools for better assembly of complex genomes, aid in finding of novel genes, variations and evolutionary clues associated with tea quality, thus help to breed new varieties with high yield and better quality in the future.

Chromosome-scale genome assembly of kiwifruit Actinidia eriantha with single-molecule sequencing and chromatin interaction mapping.

BACKGROUND: Kiwifruit (Actinidia spp.) is a dioecious plant with fruits containing abundant vitamin C and minerals. A handful of kiwifruit species have been domesticated, among which Actinidiaeriantha is increasingly favored in breeding owing to its superior commercial traits. Recently, elite cultivars from A. eriantha have been successfully selected and further studies on their biology and breeding potential require genomic information, which is currently unavailable. FINDINGS: We assembled a chromosome-scale genome sequence of A. eriantha cultivar White using single-molecular sequencing and chromatin interaction map-based scaffolding. The assembly has a total size of 690.6 megabases and an N50 of 21.7 megabases. Approximately 99% of the assembly were in 29 pseudomolecules corresponding to the 29 kiwifruit chromosomes. Forty-three percent of the A. eriantha genome are repetitive sequences, and the non-repetitive part encodes 42,988 protein-coding genes, of which 39,075 have homologues from other plant species or protein domains. The divergence time between A. eriantha and its close relative Actinidia chinensis is estimated to be 3.3 million years, and after diversification, 1,727 and 1,506 gene families are expanded and contracted in A. eriantha, respectively. CONCLUSIONS: We provide a high-quality reference genome for kiwifruit A. eriantha. This chromosome-scale genome assembly is substantially better than 2 published kiwifruit assemblies from A. chinensis in terms of genome contiguity and completeness. The availability of the A. eriantha genome provides a valuable resource for facilitating kiwifruit breeding and studies of kiwifruit biology.

A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarp of red-centered kiwifruit Actinidia chinensis cv. Hongyang.

Many Actinidia cultivars are characterized by anthocyanin accumulation, specifically in the inner pericarp, but the underlying regulatory mechanism remains elusive. Here we report two interacting transcription factors, AcMYB123 and AcbHLH42, that regulate tissue-specific anthocyanin biosynthesis in the inner pericarp of Actinidia chinensis cv. Hongyang. Through transcriptome profiling analysis we identified five MYB and three bHLH transcription factors that were upregulated in the inner pericarp. We show that the combinatorial action of two of them, AcMYB123 and AcbHLH42, is required for activating promoters of AcANS and AcF3GT1 that encode the dedicated enzymes for anthocyanin biosynthesis. The presence of anthocyanin in the inner pericarp appears to be tightly associated with elevated expression of AcMYB123 and AcbHLH42. RNA interference repression of AcMYB123, AcbHLH42, AcF3GT1 and AcANS in 'Hongyang' fruits resulted in significantly reduced anthocyanin biosynthesis. Using both transient assays in Nicotiana tabacum leaves or Actinidia arguta fruits and stable transformation in Arabidopsis, we demonstrate that co-expression of AcMYB123 and AcbHLH42 is a prerequisite for anthocyanin production by activating transcription of AcF3GT1 and AcANS or the homologous genes. Phylogenetic analysis suggests that AcMYB123 or AcbHLH42 are closely related to TT2 or TT8, respectively, which determines proanthocyanidin biosynthesis in Arabidopsis, and to anthocyanin regulators in monocots rather than regulators in dicots. All these experimental results suggest that AcMYB123 and AcbHLH42 are the components involved in spatiotemporal regulation of anthocyanin biosynthesis specifically in the inner pericarp of kiwifruit.

Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality.

Tea, one of the world's most important beverage crops, provides numerous secondary metabolites that account for its rich taste and health benefits. Here we present a high-quality sequence of the genome of tea, Camellia sinensis var. sinensis (CSS), using both Illumina and PacBio sequencing technologies. At least 64% of the 3.1-Gb genome assembly consists of repetitive sequences, and the rest yields 33,932 high-confidence predictions of encoded proteins. Divergence between two major lineages, CSS and Camellia sinensis var. assamica (CSA), is calculated to ∼0.38 to 1.54 million years ago (Mya). Analysis of genic collinearity reveals that the tea genome is the product of two rounds of whole-genome duplications (WGDs) that occurred ∼30 to 40 and ∼90 to 100 Mya. We provide evidence that these WGD events, and subsequent paralogous duplications, had major impacts on the copy numbers of secondary metabolite genes, particularly genes critical to producing three key quality compounds: catechins, theanine, and caffeine. Analyses of transcriptome and phytochemistry data show that amplification and transcriptional divergence of genes encoding a large acyltransferase family and leucoanthocyanidin reductases are associated with the characteristic young leaf accumulation of monomeric galloylated catechins in tea, while functional divergence of a single member of the glutamine synthetase gene family yielded theanine synthetase. This genome sequence will facilitate understanding of tea genome evolution and tea metabolite pathways, and will promote germplasm utilization for breeding improved tea varieties.

Transcriptomic and phytochemical analysis of the biosynthesis of characteristic constituents in tea (Camellia sinensis) compared with oil tea (Camellia oleifera).

BACKGROUND: Tea plants (Camellia sinensis) are used to produce one of the most important beverages worldwide. The nutritional value and healthful properties of tea are closely related to the large amounts of three major characteristic constituents including polyphenols (mainly catechins), theanine and caffeine. Although oil tea (Camellia oleifera) belongs to the genus Camellia, this plant lacks these three characteristic constituents. Comparative analysis of tea and oil tea via RNA-Seq would help uncover the genetic components underlying the biosynthesis of characteristic metabolites in tea. RESULTS: We found that 3,787 and 3,359 bud genes, as well as 4,042 and 3,302 leaf genes, were up-regulated in tea and oil tea, respectively. High-performance liquid chromatography (HPLC) analysis revealed high levels of all types of catechins, theanine and caffeine in tea compared to those in oil tea. Activation of the genes involved in the biosynthesis of these characteristic compounds was detected by RNA-Seq analysis. In particular, genes encoding enzymes involved in flavonoid, theanine and caffeine pathways exhibited considerably different expression levels in tea compared to oil tea, which were also confirmed by quantitative RT-PCR (qRT-PCR). CONCLUSION: We assembled 81,826 and 78,863 unigenes for tea and oil tea, respectively, based on their differences at the transcriptomic level. A potential connection was observed between gene expression and content variation for catechins, theanine and caffeine in tea and oil tea. The results demonstrated that the metabolism was activated during the accumulation of characteristic metabolites in tea, which were present at low levels in oil tea. From the molecular biological perspective, our comparison of the transcriptomes and related metabolites revealed differential regulatory mechanisms underlying secondary metabolic pathways in tea versus oil tea.

Transcriptome profiling of fruit development and maturation in Chinese white pear (Pyrus bretschneideri Rehd).

BACKGROUND: Pear (Pyrus spp) is an important fruit species worldwide; however, its genetics and genomic information is limited. Combining the Solexa/Illumina RNA-seq high-throughput sequencing approach (RNA-seq) with Digital Gene Expression (DGE) analysis would be a powerful tool for transcriptomic study. This paper reports the transcriptome profiling analysis of Chinese white pear (P. bretschneideri) using RNA-seq and DGE to better understand the molecular mechanisms in fruit development and maturation of Chinese white pear. RESULTS: De novo transcriptome assembly and gene expression analysis of Chinese white pear were performed in an unprecedented depth (5.47 gigabase pairs) using high-throughput Illumina RNA-seq combined with a tag-based Digital Gene Expression (DGE) system. Approximately, 60.77 million reads were sequenced, trimmed, and assembled into 90,227 unigenes. These unigenes comprised 17,619 contigs and 72,608 singletons with an average length of 508 bp and had an N50 of 635 bp. Sequence similarity analyses against six public databases (Uniprot, NR, and COGs at NCBI, Pfam, InterPro, and KEGG) found that 61,636 unigenes can be annotated with gene descriptions, conserved protein domains, or gene ontology terms. By BLASTing all 61,636 unigenes in KEGG, a total of 31,215 unigenes were annotated into 121 known metabolic or signaling pathways in which a few primary, intermediate, and secondary metabolic pathways are directly related to pear fruit quality. DGE libraries were constructed for each of the five fruit developmental stages. Variations in gene expression among all developmental stages of pear fruit were significantly different in a large amount of unigenes. CONCLUSION: Extensive transcriptome and DGE profiling data at five fruit developmental stages of Chinese white pear have been obtained from a deep sequencing, which provides comprehensive gene expression information at the transcriptional level. This could facilitate understanding of the molecular mechanisms in fruit development and maturation. Such a database can also be used as a public information platform for research on molecular biology and functional genomics in pear and other related species.

Metabolic profiling of strawberry (Fragaria x ananassa Duch.) during fruit development and maturation.

Strawberry (Fragaria × ananassa Duch), a fruit of economic and nutritional importance, is also a model species for fleshy fruits and genomics in Rosaceae. Strawberry fruit quality at different harvest stages is a function of the fruit's metabolite content, which results from physiological changes during fruit growth and ripening. In order to investigate strawberry fruit development, untargeted (GC-MS) and targeted (HPLC) metabolic profiling analyses were conducted. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were employed to explore the non-polar and polar metabolite profiles from fruit samples at seven developmental stages. Different cluster patterns and a broad range of metabolites that exerted influence on cluster formation of metabolite profiles were observed. Significant changes in metabolite levels were found in both fruits turning red and fruits over-ripening in comparison with red-ripening fruits. The levels of free amino acids decreased gradually before the red-ripening stage, but increased significantly in the over-ripening stage. Metabolite correlation and network analysis revealed the interdependencies of individual metabolites and metabolic pathways. Activities of several metabolic pathways, including ester biosynthesis, the tricarboxylic acid cycle, the shikimate pathway, and amino acid metabolism, shifted during fruit growth and ripening. These results not only confirmed published metabolic data but also revealed new insights into strawberry fruit composition and metabolite changes, thus demonstrating the value of metabolomics as a functional genomics tool in characterizing the mechanism of fruit quality formation, a key developmental stage in most economically important fruit crops.

[Effect of several physiochemical factors on cell growth and isoflavone accumulation of Pueraria lobata cell suspension culture].

OBJECTIVE: To illustrate the effects of several physiochemical factors on cell growth and isoflavone accumulation of Pueraria lobata cell suspension cultures. METHOD: High performance liquid chromatography and plant tissue culture were applied. RESULT AND CONCLUSION: Cell growth and isoflavone accumulation were significantly stimulated in P. lobata cell suspension cultures by the increase of the sucrose concentration. Maintaining the pH value at the range over 5. 4 to 5. 8 was most suitable for isoflavone accumulation in P. lobata cell suspension cultures. Cell dried weight and isoflavone accumulation decreased sharply with the increase of the treated concentration of active carbon, while XAD-4 significantly stimulated cell growth and isoflavone accumulation.

Separation and determination of isoflavonoids in several kudzu samples by high-performance capillary electrophoresis (HPCE).

Pueraria lobata is a rich source of isoflavonoids. The detection and identification of isoflavonoid components from root, stem, leaf, callus and cell samples, is very important for the best, safest and most efficacious use of kudzu as a medicinal plant, and for the studies on quantitative analysis in the secondary metabolism of isoflavonoids. In this paper, a simple, rapid and precise high-performance capillary electrophoresis (HPCE) method with diode array detection (DAD) has been developed for separation and determination of isoflavonoids in several kudzu samples. The isoflavonoids could be well separated within 15 min in a 40 cm length capillary at a separation voltage of 15kV in a 30 mmol L(-1) borax buffer (pH9.29), and this proposed method demonstrated excellent reproducibility and accuracy with relative standard deviations of less than 5% for isoflavonoid content (n = 5) of different kudzu samples. The relationship between peak areas and isoflavone concentrations, in a specified working range with linear response, was determined by first-order polynomial regression over the range 0.05-0.5 mg mL(-1) for puerarin and 2.5-50 microg mL(-1) for 3'-methoxypuerarin, daidzin and daidzein, respectively, and quantitative evaluation of those four main isoflavonoid components was determined by ultraviolet absorption at lambda = 192 nm. The differences were also illustrated by comparison of the determination of isoflavonoid components from kudzu root, stem, leaf samples and plant tissue cultures in vitro.

Identification of isoflavonoids in several kudzu samples by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

Pueraria lobata is a rich source of isoflavonoids. The detection and identification of isoflavonoid components from Pueraria radix (RP), callus and cell cultures, is very important for the safest and most effective use of kudzu as a medicinal plant, and for the studies on quantitative analysis and secondary metabolism of isoflavonoids in vitro cultures. Liquid chromatography is coupled with negative and positive electrospray ionization (ESI) tandem mass spectrometry (MS-MS), and photodiode array detection is used to characterize and detect isoflavonoids in root, callus, and cell samples of P. lobata. Characteristic product ions of aglycones, O-glucosides, and C-glucosides were obtained from the full-scan ESI-MS chromatography of the major peaks and the MS-MS spectra of the protonated ions. Five major components of puerarin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, and daidzein are detected and identified from the methanolic extract of P. lobata callus cultures. The major isoflavonoid components of P. lobata cell suspension cultures are identified as puerarin, daidzin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, genistein-8-C-glucoside-6"-O-malonylester, and daidzein, on the basis of ESI-MS and MS-MS spectra analysis. Likewise, puerarin, daidzin, genistein-6"-O-malonylester, 3'-methoxypuerarin, and daidzein are detected and identified from RP. Of those isoflavonoid components detected, daidzin-6"-O-acetylester is a new isoflavonoid glucoside and is for the first time detected from P. lobata cultures in vitro.