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To address the high demand for Pueraria candollei var. mirifica (PM) used as the active ingredient in health products and its difficulty to cultivate in the field, the growth and production of deoxymiroestrol (DME) and isoflavonoid (ISF) phytoestrogens in PM cell suspensions were studied. In a 125-mL shake flask, the cell suspension produced DME [78.7 ± 8.79–116 ± 18.2 μg/g dry weight (DW)] and ISF (140 ± 6.83–548 ± 18.5 μg/g DW), which are the predominant ISF glycosides. While ISF aglycones accumulated in the PM cell suspension cultured in the airlift bioreactor. The DME content was increased to 976 ± 79.6 μg/g DW when the PM cell suspension was cultured in the 5-L scale bioreactor. The production of DME and ISF was enhanced by elicitors including methyl jasmonate (MJ), yeast extract (YE), and chitosan (CHI). MJ produced the highest induction of DME accumulation, while ISF accumulation was the highest with YE treatment. Analysis of catalase activity implied that the elicitors enhanced ROS production, which resulted in the enhancement of DME and ISF production and accumulation in PM cell suspension cultures. PM cell suspension culture is a promising source of beneficial PM phytoestrogens that exhibit bioactivity that may useful for the treatment of menopausal symptoms.

Background Pueraria is a dry root commonly used in Traditional Chinese Medicine or as food and fodder, and tuberous root expansion is an important agronomic characteristic that influences its yield. However, no specific genes regulating tuberous root expansion in Pueraria have been identified. Therefore, we aimed to explore the expansion mechanism of Pueraria at six developmental stages (P1–P6), by profiling the tuberous roots of an annual local variety “Gange No.1” harvested at 105, 135, 165, 195, 225, and 255 days after transplanting. Results Observations of the tuberous root phenotype and cell microstructural morphology revealed that the P3 stage was a critical boundary point in the expansion process, which was preceded by a thickening diameter and yield gain rapidly of the tuberous roots, and followed by longitudinal elongation at both ends. A total of 17,441 differentially expressed genes (DEGs) were identified by comparing the P1 stage (unexpanded) against the P2–P6 stages (expanded) using transcriptome sequencing; 386 differential genes were shared across the six developmental stages. KEGG pathway enrichment analysis showed that the DEGs shared by P1 and P2–P6 stages were mainly involved in pathways related to the “cell wall and cell cycle”, “plant hormone signal transduction”, “sucrose and starch metabolism”, and “transcription factor (TF)”. The finding is consistent with the physiological data collected on changes in sugar, starch, and hormone contents. In addition, TFs including bHLHs, AP2s, ERFs, MYBs, WRKYs, and bZIPs were involved in cell differentiation, division, and expansion, which may relate to tuberous root expansion. The combination of KEGG and trend analyses revealed six essential candidate genes involved in tuberous root expansion; of them, CDC48, ARF, and EXP genes were significantly upregulated during tuberous root expansion while INV, EXT, and XTH genes were significantly downregulated. Conclusion Our findings provide new insights into the complex mechanisms of tuberous root expansion in Pueraria and candidate target genes, which can aid in increasing Pueraria yield.

Background R2R3-MYB transcription factors regulate secondary metabolism, stress responses and development in various plants. Puerarin is a bioactive ingredient and most abundant secondary metabolite isolated from Pueraria lobata . The biosynthesis of puerarin proceeds via the phenylpropanoid pathway and isoflavonoids pathway, in which 9 key enzymes are involved. The expression of these structural genes is under control of specific PtR2R3-MYB genes in different plant tissues. However, how PtR2R3-MYB genes regulates structural genes in puerarin biosynthesis remains elusive. This study mined the PtR2R3-MYB genes involved in puerarin biosynthesis and response to hormone in Pueraria lobata var. thomsonii . Results A total of 209 PtR2R3-MYB proteins were identified, in which classified into 34 subgroups based on the phylogenetic topology and the classification of the R2R3-MYB superfamily in Arabidopsis thaliana . Furtherly physical and chemical characteristics, gene structure, and conserved motif analysis were also used to further analyze PtR2R3-MYBs . Combining puerarin content and RNA-seq data, speculated on the regulated puerarin biosynthesis of PtR2R3-MYB genes and structural genes, thus 21 PtR2R3-MYB genes and 25 structural genes were selected for validation gene expression and further explore its response to MeJA and GSH treatment by using qRT-PCR analysis technique. Correlation analysis and cis-acting element analysis revealed that 6 PtR2R3-MYB genes ( PtMYB039 , PtMYB057 , PtMYB080 , PtMYB109 , PtMYB115 and PtMYB138 ) and 7 structural genes ( PtHID2 , PtHID9 , PtIFS3 , PtUGT069 , PtUGT188 , PtUGT286 and PtUGT297 ) were directly or indirectly regulation of puerarin biosynthesis in ZG11. It is worth noting that after MeJA and GSH treatment for 12–24 h, the expression changes of most candidate genes were consistent with the correlation of puerarin biosynthesis, which also shows that MeJA and GSH have the potential to mediate puerarin biosynthesis by regulating gene expression in ZG11. Conclusions Overall, this study provides a comprehensive understanding of the PtR2R3-MYB and will paves the way to reveal the transcriptional regulation of puerarin biosynthesis and response to phytohormone of PtR2R3-MYB genes in Pueraria lobata var. thomsonii .

Background Pueraria candollei var. mirifica , a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. Results Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica . To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor ( At MYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. Conclusions Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica .

Soil carbon (C) sequestration, as an ecosystem property, may be strongly influenced by invasive plants capable of depositing disproportionately high quantities of chemically distinct litter that disrupt ecosystem processes. However, a mechanistic understanding of the processes that regulate soil C storage in invaded ecosystems remains surprisingly elusive. Here, we studied the impact of the invasion of two noxious nonnative species, Polygonum cuspidatum, which produces recalcitrant litter, and Pueraria lobata, which produces labile litter, on the quantity, molecular composition, and stability of C in the soils they invade. Compared with an adjacent noninvaded old‐field, P. cuspidatum‐invaded soils exhibited a 26% increase in C, partially through selective preservation of plant polymers. Despite receiving a 22% higher litter input, P. lobata‐invaded Pinus stands exhibited a 28% decrease in soil C and a twofold decrease in plant biomarkers, indicating microbial priming of native soil C. The stability of C exhibited an opposite trend: the proportion of C that was resistant to oxidation was 21% lower in P. cuspidatum‐invaded soils and 50% higher in P. lobata‐invaded soils. Our results highlight the capacity of invasive plants to feed back to climate change by destabilizing native soil C stocks and indicate that environments that promote the biochemical decomposition of plant litter would enhance the long‐term storage of soil C. Further, our study highlights the concurrent influence of dominant plant species on both selective preservation and humification of soil organic matter.

Kudzu (Pueraria montana var. lobata), a plant native to Southeastern Asia, has become a major noxious weed covering millions of hectares in the Southern United States. A kudzu patch displaying virus-like symptoms located in Ackerman, northeastern Mississippi (MS), was used as a source for virus isolation and characterization involving mechanical and vector transmission, ultrastructural observation, surveys, Sanger and high-throughput genome sequencing, and sequence analyses. The results revealed the presence of a new potyvirus in infected kudzu, closely related to wisteria vein mosaic virus (WVMV) and provisionally named kudzu chlorotic ring blotch virus (KudCRBV). Genome features and pairwise comparison with six WVMV genomes currently available in GenBank and three additional isolates from MS sequenced in this work suggest that KudCRBV is likely a member of a new species in the genus Potyvirus. Furthermore, under experimental conditions, KudCRBV was successfully transmitted by cotton and potato aphids and mechanically to soybean and beans. A state-wide survey revealed several kudzu patches infected by the virus in northern MS.

Background Pueraria is a widely cultivated medicinal and edible homologous plant in Asia, and its tuberous roots are commonly used in the food, nutraceutical, and pharmaceutical industries. “Gange No. 5” is a local variety of Pueraria montana var. thomsonii (Bentham) M.R. Almeida (PMT) in Jiangxi Province, China. After optimizing its cultivation technique, we shortened the cultivation cycle of this variety from two years to one year, suggesting that the regulatory mechanism of the endogenous hormone system during tuberous root expansion may have changed significantly. In this study, we focused on the molecular mechanisms of endogenous hormones in promoting tuberous root expansion during one-year cultivation of “Gange No. 5”. Results The mid-late expansion period (S4) is critical for the rapid swelling of “Gange No. 5” tuberous roots during annual cultivation. At S4, the number of cells increased dramatically and their volume enlarged rapidly in the tuberous roots, the fresh weight of a single root quickly increased, and the contents of multiple nutrients (total protein, total phenol, isoflavones) and medicinal components (puerarin, puerarin apigenin, and soy sapogenin) were at their peak values. We compared the transcriptomes and metabolomes of S1 (the pre-expansion period), S4, and S6 (the final expansion period), and screened 42 differentially accumulated hormone metabolites and 1,402 differentially expressed genes (DEGs) associated with hormone biosynthesis, metabolism, and signaling. Most Auxin, cytokinins (CKs), jasmonic acids (JAs), salicylic acid (SA), melatonin (MLT), and ethylene (ETH), reached their maximum levels at S1 and then gradually decreased; however, abscisic acid (ABA) appeared in S6, indicating that most of the endogenous hormones may play a key role in regulating the initiation of tuberous root expansion, while ABA mainly promotes tuberous root maturation. Notably, multiple key genes of the ‘Tryptophan metabolism’ pathway (ko00380) were significantly differentially expressed, and COBRA1 , COBRA2 , YUCCA10 , IAA13 , IAA16 , IAA20 , IAA27 , VAN3 , ACAA2 , and ARF were also identified to be significantly correlated with the expansion of “Gange No. 5” tuberous roots. Conclusions Our study has revealed how endogenous hormone regulation affects the expansion of “Gange No. 5” tuberous roots. These findings offer a theoretical foundation for improving the yield of PMT tuberous roots.

Pueraria lobata and its variety P. lobata var. thomsonii are both traditional Chinese medicines that have high nutritional and medical value; whereas another variety, P. lobata var. montana has low nutritional and medicinal value and can cause ecological disasters. The material basis of different nutritional and medicinal values, which are caused by metabolite differences among these varieties, remains to be further clarified. Here, we performed ultra performance liquid chromatography-tandem mass spectrometry based widely targeted metabolome analysis on Pueraria lobata, P. lobata var. thomsonii, and P. lobata var. montana. Among them, a total of 614 metabolites were identified, and distinguished from each other using orthogonal partial least squares discriminant analysis. Our results suggest that the nutritional differences between P. lobata and its varieties can be explained by variations in the abundance of amino acids, nucleotides, saccharides, and lipids; differences in flavonoids, isoflavones, phenolic acids, organic acids, and coumarins contents caused the differences in the medicinal quality of P. lobata and its varieties. Additionally, the key metabolites responsible for the classification of the three Pueraria varieties were identified. This study provides new insights into the underlying metabolic causes of nutritional and medicinal variation in P. lobata and its varieties.

Pueraria lobata is a traditional Chinese herb in which an isoflavone C-glucoside, namely puerarin, has received the utmost interest due to its medicinal properties. To date, the biogenesis of puerarin, especially its C-glucosyl reaction in the pathway, remains poorly understood. Moreover, the transcription factors (TFs) that regulate puerarin biosynthesis in P. lobata have not been reported. Here, we performed phytochemical studies on the different developmental stages of the root, stem, and leaf tissues of two P. lobata cultivars, which suggested that both the roots and stems of P. lobata were the sites of puerarin biosynthesis. RNA-sequencing was conducted with the root and stem tissues of the two cultivars under different stages, and the clean reads were mapped to the recently published genome of P. lobata var. thomsonii, yielding the transcriptome dataset. A detailed analysis of the gene expression data, gene coexpression network, and phylogeny proposed several C-GTs that likely participate in puerarin biosynthesis. The first genome-wide analysis of the whole MYB superfamily in P. lobata presented here identified a total of 123 nonredundant PlMYB genes that were significantly expressed in the analyzed tissues. The phylogenetic analysis of PlMYBs with other plant MYB proteins revealed strong PlMYB candidates that may regulate the biosynthesis of isoflavones, such as puerarin.

Immunoassays are efficient for the phytochemical analysis of various matrices. However, producing an appropriate recombinant antibody for small molecules is challenging, resulting in costly analyses. In this study, we aimed to develop recombinant fragment antigen-binding (Fab) antibodies against miroestrol, a potent phytoestrogen marker of Pueraria candollei. Two expression cassettes of Fab were established for the production of active Fab antibodies using SHuffle® T7 Escherichia coli cells. The orientation of variable fragment heavy chain (VH) and variable fragment light chain (VL) in the expression vector constructs influences the reactivity, stability, and binding specificity of the resultant Fab. Stability testing of antibodies demonstrated that Fab is a more stable form of recombinant antibody than a single-chain variable fragment (ScFv) antibody in all conditions. Based on the obtained Fab, the ELISA specifically detected miroestrol in the range of 39.06–625.00 ng/mL. The intra- and inter-assay precisions were 0.74–2.98% and 6.57–9.76%, respectively. The recovery of authentic miroestrol spiked into samples was 106.70–110.14%, and the limit of detection was 11.07 ng/mL. The results for P. candollei roots and products determined using our developed ELISA with Fab antibody and an ELISA with anti-miroestrol monoclonal antibody (mAb) were consistent (R2 = 0.9758). The developed ELISA can be applied for the quality control of miroestrol derived from P. candollei. Therefore, the appropriate expression platform of Fab resulted in the stable binding specificity of the recombinant antibody and was applicable for immunoassays.Key points• ELISAs with Fab has higher sensitivity than that with ScFv.• Fab is more stable than ScFv.• Fab-based ELISA can be used for miroestrol determination of Pueraria candollei.