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Abstract Grona styracifolia is a photophilous legume that contains abundant flavonoids with multiple pharmacological activities, which is used to cure urethral and biliary calculus in China for thousands of years. The authentication of the rate-limiting enzymes involved in the flavonoids biosynthesis pathway enabled a better understanding of the molecular aspect of quality formation and modulation of this medicinal herb. In this study, the chemical distribution characteristics and content of flavonoids in different tissues of Grona styracifolia were analyzed using ultraperormance liquid chromatography coupled with Q-TOF mass spectrometry and showed that active flavonoids were primarily synthesized and stored in the leaves. Subsequently, RNA sequencing (RNA-seq)-based transcriptome profiling of the different tissues revealed that the flavonoids biosynthesis in the leaves was the most active. Meanwhile, 27 full-length transcripts inferred encoding vital enzymes involved in the flavonoids biosynthesis were preliminarily excavated. Finally, four CHSs, four CHIs, and one FNSII were successfully characterized by heterologous expression, which involved in three rate-limiting steps of the flavonoid biosynthetic pathway. In conclusion, these results laid a foundation for further investigation of the molecular mechanism of the biosynthesis and modulation of active flavonoids in Grona styracifolia.

Abstract Background Species in genus Amomum always have important medicinal and economic values. Classification of Amomum using morphological characters has long been a challenge because they exhibit high similarity. The main goals of this study were to mine genetic markers from cp genomes for Amomum species identification and discover their evolutionary history through comparative analysis. Results Three species Amomum villosum, Amomum maximum and Amomum longipetiolatum were sequenced and annotated for the complete chloroplast (cp) genomes, and the cp genomes of A. longipetiolatum and A. maximum were the first reported. Three cp genomes exhibited typical quadripartite structures with 163,269-163,591 bp in length. Each genome encodes 130 functional genes including 79 protein-coding, 26 tRNAs and 3 rRNAs genes. 113-152 SSRs and 99 long repeats were identified in the three cp genomes. By designing specific primers, we amplified the highly variable loci and the mined genetic marker ccsA exhibited a relatively high species identification resolution in Amomum. The nonsynonymous and synonymous substitution ratios (Ka/Ks) in Amomum and Alpinia showed that most genes were subjected to a purifying selection. Phylogenetic analysis revealed the evolutionary relationships of Amomum and Alpinia species and proved that Amomum is paraphyletic. In addition, the sequenced sample of A. villosum was found to be a hybrid, becoming the first report of natural hybridization of this genus. Meanwhile, the high-throughput sequencing-based ITS2 analysis was proved to be an efficient tool for interspecific hybrid identification and with the help of the chloroplast genome, the hybrid parents can be also be determined. Conclusion The comparative analysis and mined genetic markers of cp genomes were conducive to species identification and evolutionary relationships of Amomum.

Abstract Background The large genus Ficus comprises approximately 800 species, most of which possess high ornamental and ecological values. However, its evolutionary history remains largely unknown. Plastome (chloroplast genome) analysis had become an essential tool for species identification and for unveiling evolutionary relationships between species, genus and other rank groups. In this work we present the plastomes of ten Ficus species. Results The complete chloroplast (CP) genomes of eleven Ficus specimens belonging to ten species were determined and analysed. The full length of the Ficus plastome was nearly 160 kbp with a similar overall GC content, ranging from 35.88 to 36.02%. A total of 114 unique genes, distributed in 80 protein-coding genes, 30 tRNAs, and 4 rRNAs, were annotated in each of the Ficus CP genome. In addition, these CP genomes showed variation in their inverted repeat regions (IR). Tandem repeats and mononucleotide simple sequence repeat (SSR) are widely distributed across the Ficus CP genome. Comparative genome analysis showed low sequence variability. In addition, eight variable regions to be used as potential molecular markers were proposed for future Ficus species identification. According to the phylogenetic analysis, these ten Ficus species were clustered together and further divided into three clades based on different subgenera. Simultaneously, it also showed the relatedness between Ficus and Morus. Conclusion The chloroplast genome structure of 10 Ficus species was similar to that of other angiosperms, with a typical four-part structure. Chloroplast genome sizes vary slightly due to expansion and contraction of the IR region. And the variation of noncoding regions of the chloroplast genome is larger than that of coding regions. Phylogenetic analysis showed that these eleven sampled CP genomes were divided into three clades, clustered with species from subgenus Urostigma, Sycomorus, and Ficus, respectively. These results support the Berg classification system, in which the subgenus Ficus was further decomposed into the subgenus Sycomorus. In general, the sequencing and analysis of Ficus plastomes, especially the ones of species with no or limited sequences available yet, contribute to the study of genetic diversity and species evolution of Ficus, while providing useful information for taxonomic and phylogenetic studies of Ficus.

Abstract Background Helicteres angustifolia has long been used in Chinese traditional medicine. It has multiple pharmacological benefits, including anti-inflammatory, anti-viral and anti-tumor effects. Its main active chemicals include betulinic acid, oleanolic acid, helicteric acid, helicterilic acid, and other triterpenoid saponins. It is worth noting that some acylated triterpenoids, such as helicteric acid and helicterilic acid, are characteristic components of Helicteres and are relatively rare among other plants. However, reliance on natural plants as the only sources of these is not enough to meet the market requirement. Therefore, the engineering of its metabolic pathway is of high research value for enhancing the production of secondary metabolites. Unfortunately, there are few studies on the biosynthetic pathways of triterpenoids in H. angustifolia, hindering its further investigation. Results Here, the RNAs of different groups treated by metabolic stimulation were sequenced with an Illumina high-throughput sequencing platform, resulting in 121 gigabases of data. A total of 424,824 unigenes were obtained after the trimming and assembly of the raw data, and 22,430 unigenes were determined to be differentially expressed. In addition, three oxidosqualene cyclases (OSCs) and four Cytochrome P450 (CYP450s) were screened, of which one OSC (HaOSC1) and one CYP450 (HaCYPi3) achieved functional verification, suggesting that they could catalyze the production of lupeol and oleanolic acid, respectively. Conclusion In general, the transcriptomic data of H. angustifolia was first reported and analyzed to study functional genes. Three OSCs, four CYP450s and three acyltransferases were screened out as candidate genes to perform further functional verification, which demonstrated that HaOSC1 and HaCYPi3 encode for lupeol synthase and β-amyrin oxidase, which produce corresponding products of lupeol and oleanolic acid, respectively. Their successful identification revealed pivotal steps in the biosynthesis of acylated triterpenoids precursors, which laid a foundation for further study on acylated triterpenoids. Overall, these results shed light on the regulation of acylated triterpenoids biosynthesis.