Discovering a uniform functional trade-off of the CBC-type 2,3-oxidosqualene cyclases and deciphering its chemical logic.

Many functionally promiscuous plant 2,3-oxidosqualene cyclases (OSCs) have been found, but complete functional reshaping is rarely reported. In this study, we have identified two new plant OSCs: a unique protostadienol synthase (AoPDS) and a common cycloartenol synthase (AoCAS) from Alisma orientale (Sam.) Juzep. Multiscale simulations and mutagenesis experiments revealed that threonine-727 is an essential residue responsible for protosta-13 (17),24-dienol biosynthesis in AoPDS and that the F726T mutant completely reshapes the native function of AoCAS into a PDS function to yield almost exclusively protosta-13 (17),24-dienol. Unexpectedly, various native functions were uniformly reshaped into a PDS function by introducing the phenylalanine → threonine substitution at this conserved position in other plant and non-plant chair-boat-chair-type OSCs. Further computational modeling elaborated the trade-off mechanisms of the phenylalanine → threonine substitution that leads to the PDS activity. This study demonstrates a general strategy for functional reshaping by using a plastic residue based on the decipherment of the catalytic mechanism.

Two cardenolide glycosides from the seed fairs of Asclepias curassavica and their cytotoxic activities.

Two cardenolide glycosides, corotoxigenin 3-O-[ß-D-glucopyranosyl-(1→4)-6-deoxy-ß-D-glucopyranoside] (1) and coroglaucigenin 3-O-[ß-D-glucopyranosyl-(1→4)-6-deoxy-ß-D-glucopyranoside] (2), were isolated from the seed fairs of Asclepias curassavica. The structures of 1-2 were determined based on the combination of the analysis of their MS, NMR spectroscopic data and acid hydrolysis. The inhibitory effects of compounds 1 and 2 on human colorectal carcinoma cells (HCT116), non-small cell lung carcinoma cells (A549) and hepatic cancer cells (SMMC-7721) were evaluated. The results showed that both compounds 1 and 2 significantly inhibited the viability, proliferation, and migration of A549, HCT116 and SMMC-7721 cells, suggesting that compounds 1 and 2 can be applied in the treatment of lung, colon and liver cancers in clinical practice. This study may not only provide a scientific basis for clarifying the active ingredients in A. curassavica, but also help to understand its antitumor activity, which can promote the application of A. curassavica in clinical treatment of various cancers.

Genome-wide identification and analysis of AP2/ERF transcription factors related to camptothecin biosynthesis in Camptotheca acuminata.

Camptotheca acuminata produces camptothecin (CPT), a monoterpene indole alkaloid (MIA) that is widely used in the treatment of lung, colorectal, cervical, and ovarian cancers. Its biosynthesis pathway has attracted significant attention, but the regulation of CPT biosynthesis by the APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors (TFs) remains unclear. In this study, a systematic analysis of the AP2/ERF TFs family in C. acuminata was performed, including phylogeny, gene structure, conserved motifs, and gene expression profiles in different tissues and organs (immature bark, cotyledons, young flower, immature fruit, mature fruit, mature leaf, roots, upper stem, and lower stem) of C. acuminata. A total of 198 AP2/ERF genes were identified and divided into five relatively conserved subfamilies, including AP2 (26 genes), DREB (61 genes), ERF (92 genes), RAV (18 genes), and Soloist (one gene). The combination of gene expression patterns in different C. acuminata tissues and organs, the phylogenetic tree, the co-expression analysis with biosynthetic genes, and the analysis of promoter sequences of key enzymes genes involved in CPT biosynthesis pathways revealed that eight AP2/ERF TFs in C. acuminata might be involved in CPT synthesis regulation, which exhibit relatively high expression levels in the upper stem or immature bark. Among these, four genes (CacAP2/ERF123, CacAP2/ERF125, CacAP2/ERF126, and CacAP2/ERF127) belong to the ERF-B2 subgroup; two genes (CacAP2/ERF149 and CacAP2/ERF152) belong to the ERF-B3 subgroup; and two more genes (CacAP2/ERF095 and CacAP2/ERF096) belong to the DREB-A6 subgroup. These results provide a foundation for future functional characterization of the AP2/ERF genes to enhance the biosynthesis of CPT compounds of C. acuminata.

bHLH transcription factor SmbHLH92 negatively regulates biosynthesis of phenolic acids and tanshinones in Salvia miltiorrhiza.

Objective: Salvia miltiorrhiza is a valuable herbal medicine with tanshinone and phenolic acid as the main biological active ingredients. The biosynthetic regulation of these bioactive compounds is controlled by a set of transcription factors (TFs). The basic helix-loop-helix (bHLH) transcription factor plays an important role in various physiological and biochemical processes in plants. However, research on bHLH TFs regulating phenolic acid or tanshinone biosynthesis in S. miltiorrhiza is limited. Methods: qRT-PCR was used for gene expression analysis. The subcellular localization of SmbHLH92 was detected by SmbHLH92-GFP transient transformation into tobacco leaves, and its fluorescence was observed using a confocal laser scanning microscope. The transcriptional activity of SmbHLH92 was confirmed in the AH109 yeast strain. RNA interference hairy roots of SmbHLH92-RNAi transgenic lines were obtained through Agrobacterium-mediated genetic transformation. Ultra performance liquid chromatography (UPLC) was used to detect the changes of phenolic acids and tanshinones. Results: SmbHLH92 is a bHLH transcription factor that is highly expressed in the root and phloem of S. miltiorrhiza. The subcellular localization and transcriptional activity of SmbHLH92 indicated that SmbHLH92 was located in the nucleus and may be a transcription factor. RNA interference (RNAi) of SmbHLH92 in hairy roots of S. miltiorrhiza significantly increased the accumulation of phenolic acid and tanshinone. Quantitative RT-PCR (RT-qPCR) analysis showed the transcription level of genes encoding the key enzymes involved in the phenolic acid and tanshinone biosynthetic pathways was increased in the hairy roots of the SmbHLH92-RNAi transgenic line, comparing with the control line. Conclusion: These data indicate that SmbHLH92 is a negative regulator involved in the regulation of phenolic acid and tanshinone biosynthesis in S. miltiorrhiza.

Genome-wide analysis of LBD(lateral organ boundaries domain) gene family in Cannabis sativa of traditional Chinese medicine hemp seed / 中国中药杂志

LBD(lateral organ boundaries)transcription factors play an important role in the regulation of plant growth, development and secondary metabolism. In order to explore the function of LBD genes in cannabis, the Cannabis sativa genome and transcriptome were used to identify the C. sativa LBD gene family, and analyzed their expression patterns. Our results showed that the cannabis LBD contains 32 members, which were divided into two major categories, seven sub-families. Class Ⅰ was divided into 5 sub-families, named Class Ⅰ_a to Class Ⅰ_e, while Class Ⅱ was divided into 2 sub-families, including Class Ⅱ_a and Class Ⅱ_b. Analysis showed that the number of amino acids encoded LBDs was between 172 and 356, and the isoelectric point was between 4.92 and 9.43. The mole-cular weight of LBD was between 18 862.92 Da and 40 081.33 Da, and most members are located in the nucleus. Chromosome positioning of LBD showed that 32 members were unevenly distributed on 10 chromosomes of C. sativa LBD transcription factor domain, gene structure and motifs are relatively conservative, and the characteristics of different class members are similar. The upstream promoter region of the gene contains a variety of cis-acting elements related to plant hormones and environmental factors, C. sativa LBD genes have different expression patterns in the stems, leaves, and flowers of ZYS varieties(low tetrahydrocannabinol, high cannabidiol). The members of the LBD gene family are mainly expressed in the flowers and stems of ZYS varieties, while members expressed in the leaves very few; Class Ⅱ members CsLBD21 and CsLBD23 are expressed in flowers and stems, and CsLBD8 and CsLBD18 are expressed in flowers, stems and leaves. These genes may participate in the growth and development of cannabis and affect the biosynthesis of cannabinoids. This study laid the foundation for the subsequently functional research of the cannabis LBD gene family.

Selection and validation of reference genes for normalization of quantitative real-time reverse transcription PCR analysis in Poria cocos (Schw.) Wolf (Fuling).

BACKGROUND: Quantitative real-time reverse transcription PCR (qRT-PCR) requires a stable internal control to avoid misinterpretation of data or errors for gene expression normalization. However, there are still no validated reference genes for stable internal control in Poria cocos (Schw.) Wolf (Fuling). This study aims to validate the reference genes of P. cocos. METHODS: This study firstly collected the 14 candidate reference genes by BLASTP from the genome of P. cocos for qRT-PCR analysis to determine the expression levels of 14 housekeeping genes (GAPDH, MAPK, ß-Act, RPB2, RPB1-1, RPB1-2, his3-1, his3-2, APT, SAMDC, RP, ß-Tub, EIF, and CYP) under different temperatures and in response to different plant hormones (indole-3-acetic acid, abscisic acid, 6-benzylaminopurine, methyl jasmonate, and gibberellic acid), and the threshold cycle (Ct) values. The results were analyzed by four programs (i.e., geNorm, NormFinder, BestKeeper, and RefFinder) for evaluating the candidate reference genes. RESULTS: SAMDC, his3-2, RP, RPB2, and his3-1 were recommended as reference genes for treating P. cocos with indole-3-acetic acid, abscisic acid, 6-benzylaminopurine, methyl jasmonate, and gibberellic acid, respectively. Under different temperatures RPB2 was the most stable reference gene. CYP was the most stable gene for all 90 samples by RefFinder. CONCLUSION: SAMDC, his3-2, RP, RPB2, and his3-1 were evaluated to be suitable reference genes for P. cocos following different treatments. RPB2 was the most stable reference gene under different temperatures and CYP was the most stable gene in the mycelia under all six evaluated conditions.

[Progress in the study of Velvet and LaeA proteins and their relation to the development and bioactive compounds in medicinal fungi].

The medicinal fungi, which are of great importance in traditional medicine, are facing the problems of wild resources scarcity and low concentration of bioactive compounds. Velvet family and LaeA global regulator play a vital role in secondary metabolism and developmental programs, which are found in a wide variety of fungi ranging from Chytridiomycota to Basidiomycota. This review elaborates the structures and functions between Velvet family and LaeA protein. The Velvet family which shares the Velvet protein domain, including VeA (Velvet), VelB (Velvet like B), VosA (viability of spores A) and VelC (Velvet like C), acts on the regulation function is secondary metabolism and developmental programs such as asexual and sexual development. Furthermore, the function is affected by environmental factors such as light and temperature. LaeA protein which owns S-adenosylmethionine-dependent methyltransferase domain, coordinately regulates development and secondary metabolism by regulating and modifying the Velvet proteins. The regulation of LaeA is mediated by light receptor proteins. Therefore, clarifying the mechanism of Velvet and LaeA proteins in medicinal fungi will pave the way for nurturing medicinal fungi and improving production of bioactive compounds.