ABSTRACT
Cinnamomum cassia (L.) J.Presl, a tropical aromatic evergreen tree belonging to the Lauraceae family, is commonly used in traditional Chinese medicine. It is also a traditional spice used worldwide. However, little is currently known about the extent of the genetic variability and population structure of C. cassia. In this study, 71 individuals were collected from seven populations across two geographical provinces in China. Nine morphological features, three chemical components, and single nucleotide polymorphism (SNP) markers were used in an integrated study of C. cassia germplasm variations. Remarkable genetic variation exists in both phenotypic and chemical compositions, and certain traits, such as leaf length, leaf width, volatile oil content, and geographic distribution, are correlated with each other. One-year-old C. cassia seedling leaf length, leaf width, elevation, and volatile oil content were found to be the main contributors to diversity, according to principal component analysis (PCA). Three major groupings were identified by cluster analysis based on the phenotypic and volatile oil data. This was in line with the findings of related research using 1,387,213 SNP markers; crucially, they all demonstrated a substantial link with geographic origin. However, there was little similarity between the results of the two clusters. Analysis of molecular variance (AMOVA) revealed that the genetic diversity of C. Cassia populations was low, primarily among individuals within populations, accounting for 95.87% of the total. Shannon's information index (I) varied from 0.418 to 0.513, with a mean of 0.478 (Na=1.860, Ne =1.584, Ho =0.481, He =0.325, and PPB =86.04%). Genetic differentiation across populations was not significant because natural adaptation or extensive exchange of seeds among farmers between environments, thus maintaining the relationship. Following a population structure analysis using the ADMIXTURE software, 71 accessions were found to be clustered into three groups, with 38% of them being of the pure type, a finding that was further supported by PCA. Future breeding strategies and our understanding of the evolutionary relationships within the C. cassia population would benefit greatly from a thorough investigation of phenotypic, chemical, and molecular markers.
ABSTRACT
Scutellaria barbata is a traditional Chinese herb medicine and a major source of bioactive clerodane diterpenoids. However, barely clerodanes have been isolated from the closely related S. baicalensis. Here we assembled a chromosome-level genome of S. barbata and identified three class II clerodane diterpene synthases (SbarKPS1, SbarKPS2 and SbaiKPS1) from these two organisms. Using in vitro and in vivo assays, SbarKPS1 was characterized as a monofunctional (-)-kolavenyl diphosphate synthases ((-)-KPS), while SbarKPS2 and SbaiKPS1 produced major neo-cleroda-4(18),13E-dienyl diphosphate with small amount of (-)-KPP. SbarKPS1 and SbarKPS2 shared a high protein sequence identity and formed a tandem gene pair, indicating tandem duplication and sub-functionalization probably led to the evolution of monofunctional (-)-KPS in S. barbata. Additionally, SbarKPS1 and SbarKPS2 were primarily expressed in the leaves and flowers of S. barbata, which was consistent with the distribution of major clerodane diterpenoids scutebarbatine A and B. In contrast, SbaiKPS1 was barely expressed in any tissue of S. baicalensis. We further explored the downstream class I diTPS by functional characterizing of SbarKSL3 and SbarKSL4. Unfortunately, no dephosphorylated product was detected in the coupled assays with SbarKSL3/KSL4 and four class II diTPSs (SbarKPS1, SbarKPS2, SbarCPS2 and SbarCPS4) when a phosphatase inhibitor cocktail was included. Co-expression of SbarKSL3/KSL4 with class II diTPSs in yeast cells did not increase the yield of the corresponding dephosphorylated products, either. Together, these findings elucidated the involvement of two class II diTPSs in clerodane biosynthesis in S. barbata, while the class I diTPS is likely not responsible for the subsequent dephosphorylation step.
ABSTRACT
MAIN CONCLUSION: We identify two ferruginol synthases and a 11-hydroxyferruginol synthase from a traditional Chinese medicinal herb Isodon lophanthoides and propose their involvement in two independent abietane diterpenoids biosynthetic pathways. Isodon lophanthoides is a traditional Chinese medicinal herb rich in highly oxidized abietane-type diterpenoids. These compounds exhibit a wide range of pharmaceutical activities, yet the biosynthesis is barely known. Here, we describe the screening and functional characterization of P450s that oxidize the abietane skeleton abietatriene. We mainly focused on CYP76 family and identified 12 CYP76AHs by mining the RNA-seq data of I. lophanthoides. Among the 12 CYP76AHs, 6 exhibited similar transcriptional expression features as upstream diterpene synthases, including root or leaf-preferential expression pattern and highly MeJA inducibility. These six P450s were considered as first-tier candidates and functionally characterized in yeast and plant cells. In yeast assays showed that both CYP76AH42 and CYP76AH43 were ferruginol synthases hydroxylating the C12 position of abietatriene, whereas CYP76AH46 was characterized as a 11-hydroxyferruginol synthase which catalyzes two successive oxidations at C12 and C11 of abietatriene. Heterologous expression of three CYP76AHs in Nicotiana benthamiana resulted in the formation of ferruginol. qPCR analysis showed CYP76AH42 and CYP76AH43 were mainly expressed in the root, which was consistent with the distribution of ferruginol in the root periderms. CYP76AH46 was primarily expressed in the leaves where barely ferruginol or 11-hydroxyferruginol was detected. In addition to distinct organ-specific expression pattern, three CYP76AHs exhibited different genomic structures (w or w/o introns), low protein sequence identities (51-63%) and were placed in separate subclades in the phylogenetic tree. These results suggest that the identified CYP76AHs may be involved in at least two independent abietane biosynthetic pathways in the aerial and underground parts of I. lophanthoides.
