PnNAC2 promotes the biosynthesis of Panax notoginseng saponins and induces early flowering.

KEY MESSAGE: PnNAC2 positively regulates saponin biosynthesis by binding the promoters of key biosynthetic genes, including PnSS, PnSE, and PnDS. PnNAC2 accelerates flowering through directly associating with the promoters of FT genes. NAC transcription factors play an important regulatory role in both terpenoid biosynthesis and flowering. Saponins with multiple pharmacological activities are recognized as the major active components of Panax notoginseng. The P. notoginseng flower is crucial for growth and used for medicinal and food purposes. However, the precise function of the P. notoginseng NAC transcription factor in the regulation of saponin biosynthesis and flowering remains largely unknown. Here, we conducted a comprehensive characterization of a specific NAC transcription factor, designated as PnNAC2, from P. notoginseng. PnNAC2 was identified as a nuclear-localized protein with transcription activator activity. The expression profile of PnNAC2 across various tissues mirrored the accumulation pattern of total saponins. Knockdown experiments of PnNAC2 in P. notoginseng calli revealed a significant reduction in saponin content and the expression level of pivotal saponin biosynthetic genes, including PnSS, PnSE, and PnDS. Subsequently, Y1H assays, dual-LUC assays, and electrophoretic mobility shift assays (EMSAs) demonstrated that PnNAC2 exhibits binding affinity to the promoters of PnSS, PnSE and PnDS, thereby activating their transcription. Additionally, an overexpression assay of PnNAC2 in Arabidopsis thaliana witnessed the acceleration of flowering and the induction of the FLOWERING LOCUS T (FT) gene expression. Furthermore, PnNAC2 demonstrated the ability to bind to the promoters of AtFT and PnFT genes, further activating their transcription. In summary, these results revealed that PnNAC2 acts as a multifunctional regulator, intricately involved in the modulation of triterpenoid saponin biosynthesis and flowering processes.

[DNA barcode screening, identification of germplasm resources, and analysis of genetic diversity of Anemarrhena asphodeloides].

Anemarrhena asphodeloides is a common medicinal material used in clinical prescriptions and Chinese patent medicine. In this study, the Illumina platform was used to obtain the chloroplast genome sequences of seven kinds of A. asphodeloides from different areas. The specific DNA barcodes were screened by comparative genomics analysis, and the DNA barcodes were used to identify the germplasm resources and analyze the genetic diversity of A. asphodeloides samples from different areas in China. All the seven chloroplast genomes had a ring structure. The total length was 156 801-156 930 bp, and 113 genes were annotated, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. The comparative genomics analysis showed that rps16, trnG-GCC, atpF, rpoB, ycf3, rpl16, ndhF, trnS-GCU＿trnG-GCC, petN-psbM, and ndhF-rpl32 were potential candidates for specific DNA barcodes of A. asphodeloides. In this study, the second intron of ycf3 and atpF intron sequences with a sequence length of 700-800 bp and easy amplification were selected for polymerase chain reaction(PCR) amplification and sequencing of 594 samples from 26 areas. The sequence analysis showed that six and eight haplotypes of ycf3 and atpF sequences could be identified, respectively, and 17 haplotypes could be identified by combined analysis of the two sequences, which were named Hap1-Hap17. The haplotype diversity(H_d), nucleotide diversity(P_i), and genetic distance of A. asphodeloides in 26 populations were 0.68, 0.93×10~(-3), and 0-0.003 1, respectively, indicating that the genetic diversity within the species of A. asphodeloides is rich. The intermediary adjacent network analysis showed that Hap5 was the oldest haplotype, which was mainly distributed in Yixian county of Baoding, Hebei province, Hequ county of Xinzhou, Shanxi province, and Xiangfen county of Linfen, Shanxi province. This study has important guiding significance for the identification of A. asphodeloides species, the protection and development of germplasm resources, and the identification of production areas, and it provides a research basis for further revealing the genetic evolution law of A. asphodeloides.

A Novel Evidence Combination Method Based on Improved Pignistic Probability.

Evidence theory is widely used to deal with the fusion of uncertain information, but the fusion of conflicting evidence remains an open question. To solve the problem of conflicting evidence fusion in single target recognition, we proposed a novel evidence combination method based on an improved pignistic probability function. Firstly, the improved pignistic probability function could redistribute the probability of multi-subset proposition according to the weight of single subset propositions in a basic probability assignment (BPA), which reduces the computational complexity and information loss in the conversion process. The combination of the Manhattan distance and evidence angle measurements is proposed to extract evidence certainty and obtain mutual support information between each piece of evidence; then, entropy is used to calculate the uncertainty of the evidence and the weighted average method is used to correct and update the original evidence. Finally, the Dempster combination rule is used to fuse the updated evidence. Verified by the analysis results of single-subset proposition and multi-subset proposition highly conflicting evidence examples, compared to the Jousselme distance method, the Lance distance and reliability entropy combination method, and the Jousselme distance and uncertainty measure combination method, our approach achieved better convergence and the average accuracy was improved by 0.51% and 2.43%.

Population identification and genetic diversity analysis of Fritillaria ussuriensis (Fritillaria) based on chloroplast genes atpF and petB.

The chloroplast genomes of five Fritillaria ussuriensis materials from different production areas were comparatively analyzed, atpF and petB were screened as specific DNA barcodes, and the population identification and genetic diversity of F. ussuriensis were analyzed based on them. The F. ussuriensis chloroplast genome showed a total length of 151 515-151 548 bp with a typical tetrad structure and encoded 130 genes. atpF and petB were used to amplify 183 samples from 13 populations, and they could identify 6 and 9 haplotypes, respectively. Joint analysis of the two sequences revealed 18 haplotypes, named H1-H18, with the most widely distributed and most abundant being H4. Ten haplotypes were unique for 7 populations that they could be used to distinguish from others. Haplotype diversity and nucleotide diversity were 0.99 and 2.09 × 10-3, respectively, indicating the genetic diversity was relatively rich. The results of the intermediary adjacency network showed that H5 was the oldest haplotype, and stellate radiation was centered around it, indicating that population expansion occurred in genuine production areas. This study lays a theoretical foundation for the population identification, genetic evolution, and breed selection of F. ussuriensis.

PnMYB4 negatively modulates saponin biosynthesis in Panax notoginseng through interplay with PnMYB1.

Saponins are the main triterpenoid ingredients from Panax notoginseng, a well-known Chinese medicine, and are important sources for producing drugs to prevent and treat cerebrovascular and cardiovascular diseases. However, the transcriptional regulatory network of saponin biosynthesis in P. notoginseng is largely unknown. In the present study we demonstrated that one R2R3-MYB transcription factor, designated PnMYB4, acts as a repressor of saponin accumulation. Suppression of PnMYB4 in P. notoginseng calli significantly increased the saponin content and the expression level of saponin biosynthetic genes. PnMYB4 directly bound to the promoters of key saponin biosynthetic genes, including PnSS, PnSE, and PnDS, to repress saponin accumulation. PnMYB4 and the activator PnMYB1 could interacted with PnbHLH, which is a positive regulator of saponin biosynthesis, to modulate the biosynthesis of saponin. PnMYB4 competed with PnMYB1 for binding to PnbHLH, repressing activation of the promoters of saponin structural genes induced by the PnMYB1-PnbHLH complex. Our study reveals that a complex regulatory module of saponin biosynthesis is associated with positive and negative MYB transcriptional regulators and provides a theoretical basis for improving the content of saponins and efficacy of P. notoginseng.