The dried rhizomes of Coptis chinensis have been extensively used in heat clearing, dampness drying, fire draining, and detoxification by virtue of their major bioactive components, benzylisoquinoline alkaloids (BIAs). However, C. teeta and C. chinensis are occasionally interchanged, and current understanding of the molecular basis of BIA biosynthesis in these two species is limited. Here, berberine, coptisine, jatrorrhizine, and palmatine were detected in two species, and showed the highest contents in the roots, while epiberberine were found only in C. chinensis. Comprehensive transcriptome analysis of the roots and leaves of C. teeta and C. chinensis, respectively, identified 53 and 52 unigenes encoding enzymes potentially involved in BIA biosynthesis. By integrating probable biosynthetic pathways for BIAs, the jatrorrhizine biosynthesis ill-informed previously was further characterized. Two genes encoding norcoclaurine/norlaudanosoline 6-O-methyltransferases (Cc6OMT1 and Cc6OMT2) and one gene encoding norcoclaurine-7OMT (Ct7OMT) catalyzed enzymatically O-methylate (S)-norcoclaurine at C6 that yield (S)-coclaurine, along with a smaller amount of O-methylation occurred at C7, thereby forming its isomer (isococlaurine). In addition, scoulerine 9-OMT (CtSOMT) was determined to show strict substrate specificity, targeting (S)-scoulerine to yield (S)-tetrahydrocolumbamine. Taken together, the integration of the transcriptome and enzyme activity assays further provides new insight into molecular mechanisms underlying BIA biosynthesis in plants and identifies candidate genes for the study of synthetic biology in microorganisms.

Dactylicapnos scandens (D. Don) Hutch (Papaveraceae) is a well-known traditional Chinese herb used for treatment of hypertension, inflammation, bleeding and pain for centuries. Although the major bioactive components in this herb are considered as isoquinoline alkaloids (IQAs), little is known about molecular basis of their biosynthesis. Here, we carried out transcriptomic analysis of roots, leaves and stems of D. scandens, and obtained a total of 96,741 unigenes. Based on gene expression and phylogenetic relationship, we proposed the biosynthetic pathways of isocorydine, corydine, glaucine and sinomenine, and identified 67 unigenes encoding enzymes potentially involved in biosynthesis of IQAs in D. scandens. High performance liquid chromatography analysis demonstrated that while isocorydine is the most abundant IQA in D. scandens, the last O-methylation biosynthesis step remains unclear. Further enzyme activity assay, for the first time, characterized a gene encoding O- methyltransferase (DsOMT), which catalyzes O-methylation at C7 of (S)-corytuberine to form isocorydine. We also identified candidate transcription factor genes belonging to WRKY and bHLH families that may be involved in the regulation of IQAs biosynthesis. Taken together, we first provided valuable genetic information for D. scandens, shedding light on candidate genes involved in IQA biosynthesis, which will be critical for further gene functional characterization.

Plant-derived terpenes are effective in treating chronic dysentery, rheumatism, hepatitis, and hyperlipemia. Thus, understanding the molecular basis of terpene biosynthesis in some terpene-abundant Chinese medicinal plants is of great importance. Abundant in mono- and sesqui-terpenes, Rhodomyrtus tomentosa (Ait.) Hassk, an evergreen shrub belonging to the family Myrtaceae, is widely used as a traditional Chinese medicine. In this study, (+)-α-pinene and β-caryophyllene were detected to be the two major components in the leaves of R. tomentosa, in which (+)-α-pinene is higher in the young leaves than in the mature leaves, whereas the distribution of β-caryophyllene is opposite. Genome-wide transcriptome analysis of leaves identified 138 unigenes potentially involved in terpenoid biosynthesis. By integrating known biosynthetic pathways for terpenoids, 7 candidate genes encoding terpene synthase (RtTPS1-7) that potentially catalyze the last step in pinene and caryophyllene biosynthesis were further characterized. Sequence alignment analysis showed that RtTPS1, RtTPS3 and RtTPS4 do not contain typical N-terminal transit peptides (62–64aa), thus probably producing multiple isomers and enantiomers by terpenoid isomerization. Further enzyme activity in vitro confirmed that RtTPS1-4 mainly produce (+)-α-pinene and (+)-β-pinene, as well as small amounts of (−)-α-pinene and (−)-β-pinene with GPP, while RtTPS1 and RtTPS3 are also active with FPP, producing β-caryophyllene, along with a smaller amount of α-humulene. Our results deepen the understanding of molecular mechanisms of terpenes biosynthesis in Myrtaceae.

Background Polygonatum kingianum Coll. et Hemsl. is an important plant in Traditional Chinese Medicine. The extracts from its tubers are rich in polysaccharides and other metabolites such as saponins. It is a well-known concept that growing medicinal plants in semi-arid (or drought stress) increases their natural compounds concentrations. This study was conducted to explore the morpho-physiological responses of P. kingianum plants and transcriptomic signatures of P. kingianum tubers exposed to mild, moderate, and severe drought and rewatering. Results The stress effects on the morpho-physiological parameters were dependent on the intensity of the drought stress. The leaf area, relative water content, chlorophyll content, and shoot fresh weight decreased whereas electrolyte leakage increased with increase in drought stress intensity. A total of 53,081 unigenes were obtained; 59% of which were annotated. We observed that 1352 and 350 core genes were differentially expressed in drought and rewatering, respectively. Drought stress driven differentially expressed genes (DEGs) were enriched in phenylpropanoid biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism, and stilbenoid diarylheptanoid and gingerol biosynthesis, and carotenoid biosynthesis pathways. Pathways such as plant-pathogen interaction and galactose metabolism were differentially regulated between severe drought and rewatering. Drought reduced the expression of lignin, gingerol, and flavonoid biosynthesis related genes and rewatering recovered the tubers from stress by increasing the expression of the genes. Increased expression of carotenoid biosynthesis pathway related genes under drought suggested their important role in stress endurance. An increase in starch and sucrose biosynthesis was evident from transcriptomic changes under drought stress. Rewatering recovered the drought affected tubers as evident from the contrasting expression profiles of genes related to these pathways. P. kingianum tuber experiences an increased biosynthesis of sucrose, starch, and carotenoid under drought stress. Drought decreases the flavonoids, phenylpropanoids, gingerol, and lignin biosynthesis. These changes can be reversed by rewatering the P. kingianum plants. Conclusions These results provide a transcriptome resource for P. kingianum and expands the knowledge on the effect of drought and rewatering on important pathways. This study also provides a large number of candidate genes that could be manipulated for drought stress tolerance and managing the polysaccharide and secondary metabolites’ contents in P. kingianum.

Gastrodia elata BI. is an important cultivated medicinal plant in China. To analyze the genetic diversity and evolutionary relationship of the germplasm resources of G. elata, speci c Single nucleotide polymorphism (SNP) markers were developed. SLAF analysis was used to compare 28 samples of the same G. elata cultivar. Plants from 4 different varieties or different habitats were collected to explore intraspeci c variation and to lay a foundation for resource protection. This will facilitate improved variety breeding in future. In this study, Single nucleotide polymorphism (SNP) genetic variation and differentiation of G. elata f. glauca, G. elata f. viridis, and G. elata f. elata were analyzed using Speci c-Locus Ampli ed Fragment Sequencing (SLAF-seq). A total of 75.95M reads with an average sequencing depth of 19.32 × and a mean Q30 of 91.71% were obtained. Based on the 19,675 polymorphic SLAF tags, 60,238 SNPs were identi ed and a subset of 22,737 SNPs with minor allele frequency > 0.05 and integrity > 0.5 were selected. A model-based analysis divided the accessions into two groups, wild type G. elata f. glauca and G. elata f. viridis groups. Phylogenetic analysis also clustered the samples into the two major groups. G. elata has high genetic diversity. Population diversity was highest in G. elata f. elata and lowest in G. elata f. viridis. Analysis of molecular variance (AMOVA) revealed signi cant variations within individuals (92.23%). This study provides new insights into the genetic variation and differentiation of G. elata, which can be exploited to improve existing commercial cultivars.

Gastrodia elata BI. is an important cultivated medicinal plant in China. To analyze the genetic diversity and evolutionary relationship of the germplasm resources of G. elata, specific Single nucleotide polymorphism (SNP) markers were developed. SLAF analysis was used to compare 28 samples of the same G. elata cultivar. Plants from 4 different varieties or different habitats were collected to explore intraspecific variation and to lay a foundation for resource protection. This will facilitate improved variety breeding in future. In this study, Single nucleotide polymorphism (SNP) genetic variation and differentiation of G. elata f. glauca, G. elata f. viridis, and G. elata f. elata were analyzed using Specific-Locus Amplified Fragment Sequencing (SLAF-seq). A total of 75.95M reads with an average sequencing depth of 19.32 × and a mean Q30 of 91.71% were obtained. Based on the 19,675 polymorphic SLAF tags, 60,238 SNPs were identified and a subset of 22,737 SNPs with minor allele frequency > 0.05 and integrity > 0.5 were selected. A model-based analysis divided the accessions into two groups, wild type G. elata f. glauca and G. elata f. viridis groups. Phylogenetic analysis also clustered the samples into the two major groups. G. elata has high genetic diversity. Population diversity was highest in G. elata f. elata and lowest in G. elata f. viridis. Analysis of molecular variance (AMOVA) revealed significant variations within individuals (92.23%). This study provides new insights into the genetic variation and differentiation of G. elata, which can be exploited to improve existing commercial cultivars.