[Content changes of triterpene saponins in crude and sweated Dipsacus asper:an iTRAQ-based analysis].

The present study aimed to explore the mechanism of the sweating of Dipsacus asper on content changes of triterpene sa-ponins by detecting the total triterpene saponins and the index component asperosaponin Ⅵ in the crude and sweated D. asper, and analyzing the differentially expressed proteins by isobaric tags for relative and absolute quantification(iTRAQ) combined with LC-MS/MS. After sweating, the content of total triterpene saponins decreased manifestly, while that of asperosaponin Ⅵ increased significantly. As revealed by the iTRAQ-LC-MS/MS analysis, 140 proteins with significant differential expression were figured out, with 50 up-regulated and 90 down-regulated. GO analysis indicated a variety of hydrolases, oxido-reductases, and transferases in the differential proteins. The results of activity test on two differentially expressed oxido-reductases were consistent with those of the iTRAQ-LC-MS/MS analysis. As demonstrated by the analysis of enzymes related to the triterpene saponin biosynthesis pathway, two enzymes(from CYP450 and UGT families, respectively, which are involved in the structural modification of triterpene saponins) were significantly down-regulated after sweating. The findings suggested that sweating of D. asper presumedly regulated triterpene saponins by affecting the expression of downstream CYP450 s and UGTs in the biosynthesis pathway of triterpene saponins of D. asper.

Gibberellin disturbs the balance of endogenesis hormones and inhibits adventitious root development of Pseudostellaria heterophylla through regulating gene expression related to hormone synthesis.

The adventitious roots of some plants will develop into tuberous roots which are widely used in many traditional Chinese medicines, including Pseudostellaria heterophylla. If adventitious root development is inhibited, the yield of Chinese medicinal materials will be reduced. Gibberellic acid is an important phytohormone that promotes plant growth and increases the resistance to drought, flood or disease. However, the effects of gibberellic acid on adventitious roots of Pseudostellaria heterophylla are not clear. Here, we reports GA3 suppressed adventitious root development of Pseudostellaria heterophylla by disturbing the balance of endogenesis hormones. By detecting the contents of various endogenous hormones, we found that the development of adventitious roots negatively correlated with the content of CA3 in tuberous roots. Exogenous GA3 treatment decreased the diameter of adventitious roots, but increased the length of adventitious roots of Pseudostellaria heterophylla. In contrast, blocking the biosynthesis of GA3 suppressed stem growth and promoted the xylem of tuberous roots development. Moreover, exogenous GA3 treatment resulted in imbalance of endogenesis hormones by regulating their synthesis-related genes expression in xylem of tuberous roots. These results suggest GA3 broke the established distribution of hormones by regulating synthesis, transport and biological activation of hormones to activate the apical meristem and suppress lateral meristem. Regulating GA3 signaling during adventitious roots development would be one of the possible ways to increase the yield of P. heterophylla.

The Biosynthesis of Heterophyllin B in Pseudostellaria heterophylla From prePhHB-Encoded Precursor.

Plant cyclic peptides (CPs) are a large group of small molecule metabolites found in a wide variety of plants, including traditional Chinese medicinal plants. However, the majority of plant CPs have not been studied for their biosynthetic mechanisms, including heterophyllin B (HB), which is one of the characteristic chemical components of Pseudostellaria heterophylla. Here, we screened the precursor gene (prePhHB) of HB in P. heterophylla and functionally identified its correctness in vivo and in vitro. First, we developed a new method to screen the precursors of HB from 16 candidate linear peptides. According to transcriptome sequencing data, we cloned the genes that encoded the HB precursor peptides and confirmed that the prePhHB-encoded precursor peptide could enzymatically synthesize HB. Next, we generated the transgenic tobacco that expressed prePhHB, and the results showed that HB was detected in transgenic tobacco. Moreover, we revealed that prePhHB gene expression is positively correlated with HB accumulation in P. heterophylla. Mutations in the prePhHB gene may influence the accumulation of HB in P. heterophylla. These results suggest that HB is ribosomally synthesized and posttranslationally modified peptide (RiPP) derived from the precursor gene prePhHB-encoded precursor peptide, and the core peptide sequence of HB is IFGGLPPP in P. heterophylla. This study developed a new idea for the rapid identification of Caryophyllaceae-type CP precursor peptides via RNA-sequencing data mining.

Association between dipsacus saponin VI level and diversity of endophytic fungi in roots of Dipsacus asperoides.

Dipsacus asperoides contains multiple pharmacologically active compounds. The principal are saponins. The plant can be cultivated, but it contains lower levels of bioactive compounds than the plant in the wild. It may be the reason to exploit the endophytic fungi that colonize the plant roots in order to produce bioactive compounds. However, the endophytic fungi of D. asperoides have not been analyzed in detail. In this study, we isolated and identified 46 endophytic fungal strains from the taproots, lateral roots and leaves, and we used morphological and molecular biological methods to assign them into 15 genera: Fusarium sp., Ceratobasidium sp., Chaetomium sp., Penicillium sp., Aspergillus sp., Talaromyces sp., Cladosporium sp., Bionectria sp., Mucor sp., Trichoderma sp., Myrothecium sp., Clonostachys sp., Ijuhya sp., Leptosphaeria sp. and Phoma sp. Taproots contained abundant endophytic fungi, the numbers of which correlated positively with level of dipsacus saponin VI. Primary fermentation of several endophytic fungal strains from taproots showed that Fusarium, Leptosphaeria, Ceratobasidium sp. and Phoma sp. can produce the triterpenoid saponin. These results may guide efforts to sustainably produce bioactive compounds from D. asperoides.

Correction: De Novo Sequencing and Assembly Analysis of the Pseudostellaria heterophylla Transcriptome.

[This corrects the article DOI: 10.1371/journal.pone.0164235.].

De Novo Sequencing and Assembly Analysis of the Pseudostellaria heterophylla Transcriptome.

Pseudostellaria heterophylla (Miq.) Pax is a mild tonic herb widely cultivated in the Southern part of China. The tuberous roots of P. heterophylla accumulate high levels of secondary metabolism products of medicinal value such as saponins, flavonoids, and isoquinoline alkaloids. Despite numerous studies on the pharmacological importance and purification of these compounds in P. heterophylla, their biosynthesis is not well understood. In the present study, we used Illumina HiSeq 4000 sequencing platform to sequence the RNA from flowers, leaves, stem, root cortex and xylem tissues of P. heterophylla. We obtained 616,413,316 clean reads that we assembled into 127, 334 unique sequences with an N50 length of 951 bp. Among these unigenes, 53,184 unigenes (41.76%) were annotated in a public database and 39, 795 unigenes were assigned to 356 KEGG pathways; 23,714 unigenes (8.82%) had high homology with the genes from Beta vulgaris. We discovered 32, 095 DEGs in different tissues and performed GO and KEGG enrichment analysis. The most enriched KEGG pathway of secondary metabolism showed up-regulated expression in tuberous roots as compared with the ground parts of P. heterophylla. Moreover, we identified 72 candidate genes involved in triterpenoids saponins biosynthesis in P. heterophylla. The expression profiles of 11 candidate unigenes were analyzed by quantitative real-time PCR (RT-qPCR). Our study established a global transcriptome database of P. heterophylla for gene identification and regulation. We also identified the candidate unigenes involved in triterpenoids saponins biosynthesis. Our results provide an invaluable resource for the secondary metabolites and physiological processes in different tissues of P. heterophylla.