Chun Sui

Chemical Constituents of Agarwood Originating from the Endemic Genus Aquilaria Plants

by Huai-Qiong Chena), Jian-He Wei*a), Jun-Shan Yanga), Zheng Zhanga), Yun Yangb), Zhi-Hui Gaoa), Chun Suia), and Bao Gongb) a) Institute of Medicinal Plant Development (Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education), Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, P. R. China (phone: þ86-10-62818841; e-mail: wjianh@263.net) b) Hainan Branch Institute of Medicinal Plant (Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine), Chinese Academy of Medical Sciences & Peking Union Medical College, Wanning 571533, P. R. China

Application of CRISPR/Cas9 in plant biology

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system was first identified in bacteria and archaea and can degrade exogenous substrates. It was developed as a gene editing technology in 2013. Over the subsequent years, it has received extensive attention owing to its easy manipulation, high efficiency, and wide application in gene mutation and transcriptional regulation in mammals and plants. The process of CRISPR/Cas is optimized constantly and its application has also expanded dramatically. Therefore, CRISPR/Cas is considered a revolutionary technology in plant biology. Here, we introduce the mechanism of the type II CRISPR/Cas called CRISPR/Cas9, update its recent advances in various applications in plants, and discuss its future prospects to provide an argument for its use in the study of medicinal plants.

Jasmonic acid is a crucial signal transducer in heat shock induced sesquiterpene formation in Aquilaria sinensis.

Agarwood, a highly valuable resinous and fragrant heartwood of Aquilaria plants, is widely used in traditional medicines, incense and perfume. Only when Aquilaria trees are wounded by external stimuli do they form agarwood sesquiterpene defensive compounds. Therefore, understanding the signaling pathway of wound-induced agarwood formation is important. Jasmonic acid (JA) is a well-characterized molecule that mediates a plant’s defense response and secondary metabolism. However, little is known about the function of endogenous JA in agarwood sesquiterpene biosynthesis. Here, we report that heat shock can up-regulate the expression of genes in JA signaling pathway, induce JA production and the accumulation of agarwood sesquiterpene in A. sinensis cell suspension cultures. A specific inhibitor of JA, nordihydroguaiaretic acid (NDGA), could block the JA signaling pathway and reduce the accumulation of sesquiterpene compounds. Additionally, compared to SA and H2O2, exogenously supplied methyl jasmonate has the strongest stimulation effect on the production of sesquiterpene compounds. These results clearly demonstrate the central induction role of JA in heat-shock-induced sesquiterpene production in A. sinensis.

Comparison of root transcriptomes and expressions of genes involved in main medicinal secondary metabolites from Bupleurum chinense and Bupleurum scorzonerifolium, the two Chinese official Radix bupleuri source species

Radix bupleuri, roots of Bupleurum species, is a widely used traditional Chinese medicine. Here, we compared the root transcriptomes of both Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. A total of 313 483 and 342 263 high quality expressed sequence tags were obtained, respectively. In addition, 17 117 (59.2%) and 19 416 (62.8%) unigenes for B. chinense and B. scorzonerifolium had homologous genes in the opposite dataset. For B. chinense, Kyoto Encyclopedia of Genes and Genomes database (KEGG) annotation identified carbohydrate metabolism, energy metabolism and amino acid metabolism as the three highest groups in the metabolism category. For B. scorzonerifolium, the lipid metabolism group had the most unigenes. Genes that may participate in the biosynthesis of terpenoid, triterpenoid, sterol, lignan and flavonoids were identified according to their annotations. (Tri)terpenoid-related genes were predominantly found in B. chinense. The expressions of certain genes were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in the roots of the two species. A total of 558 putative transcription factors (TFs) and 137 transcriptional regulators (TRs) among 1364 TFs and 327 TRs, and 610 TFs and 129 TRs among 1600 TFs and 323 TRs were specific for B. chinense and B. scorzonerifolium, respectively. Our transcriptome comparison reflects the different types and proportions of metabolites synthesized by the two species. The data, especially, those genes involved in the biosynthesis of particular types of metabolites, will provide the basis for further investigations of the secondary metabolite repertoire of the two Bupleurum species, as well as other species from the genus of Bupleurum.

Development of genomic SSR and potential EST-SSR markers in Bupleurum chinense DC.

Nineteen genomic SSR markers were developed using inter-simple sequence repeat (ISSR)- suppression PCR technique in Bupleurum chinense DC., a widely used Chinese medicinal plant. A total of 126 alleles were detected across 22 individual plants of B. chinense DC. f. octoradiatum (Bunge) Shan et Sheh, with an average of 3 - 13 alleles per locus. The observed heterozygosity (HO) and the expected heterozygosity (HE) values ranged from 0.23 to 1.00 and from 0.29 to 0.92, respectively. Nine loci deviated from Hardy-Weinberg equilibrium (HWE) (P < 0.05) and eight pairs of loci showed significant linkage disequilibrium (LD) (Fisher’s exact test, P < 0.01). The species transferability of these genomic SSR markers was also detected in seven other Bupleurum species. Eight SSR markers were successfully amplified in all tested species. In addition, forty four EST-SSRs which can be amplified with expected sizes were identified from a B. chinense root cDNA library. The genomic SSR markers and potential EST-SSR markers developed in the present study should be useful for genetic diversity and molecular marker assistant selection breeding research in Bupleurum species.

Transcription Factor AsMYC2 Controls the Jasmonate-Responsive Expression of ASS1 Regulating Sesquiterpene Biosynthesis in Aquilaria sinensis (Lour.) Gilg

Sesquiterpenes are one of the most important defensive secondary metabolite components of agarwood. Agarwood, which is a product of the Aquilaria sinensis response to external damage, is a fragrant and resinous wood that is widely used in traditional medicines, incense and perfume. We previously reported that jasmonic acid (JA) plays an important role in promoting agarwood sesquiterpene biosynthesis and induces expression of the sesquiterpene synthase ASS1, which is a key enzyme that is responsible for the biosynthesis of agarwood sesquiterpenes in A. sinensis. However, little is known about this molecular regulation mechanism. Here, we characterized a basic helix-loop-helix transcription factor, AsMYC2, from A. sinensis as an activator of ASS1 expression. AsMYC2 is an immediate-early jasmonate-responsive gene and is co-induced with ASS1. Using a combination of yeast one-hybrid assays and chromatin immunoprecipitation analyses, we showed that AsMYC2 bound the promoter of ASS1 containing a G-box motif. AsMYC2 activated expression of ASS1 in tobacco epidermis cells and up-regulated expression of sesquiterpene synthase genes (TPS21 and TPS11) in Arabidopsis, which was also promoted by methyl jasmonate. Our results suggest that AsMYC2 participates in the regulation of agarwood sesquiterpene biosynthesis in A. sinensis by controlling the expression of ASS1 through the JA signaling pathway.

Agarwood Formation Induced by Fermentation Liquid of Lasiodiplodia theobromae, the Dominating Fungus in Wounded Wood of Aquilaria sinensis

Agarwood is broadly used in incense and medicine. Traditionally, agarwood formation is induced by wounding the trunks and branches of some species of Aquilaria spp., including A. sinensis. As recently evidenced, some fungi or their fermentation liquid may have the potential of inducing agarwood formation. The present study aimed to analyze the fungi isolated from an agarwood-producing A. sinensis tree and subsequently identify the fungi capable of promoting agarwood formation. We identified a total of 110 fungi isolates based on their morphological characteristics and rDNA ITS sequences. These isolates came from four different layers (namely the decomposing layer, agarwood layer, transition layer, and normal layer) near the agarwood formation site of the trunk. According to the experimental results, most of them belonged to Dothideomycetes (81.82%), while the others to Sordariomycetes (13.64%) or Eurotiomycetes (4.55%). Of note, 88 isolates were shown belonging to the species of Lasiodiplodia theobromae that are most frequently isolated from different layers. In addition, when the fermentation liquid of two isolates of L. theobromae (AF4 and AF12) and one isolate of Fusarium solani (AF21) was inoculated into the A. sinensis wood using the Agar-Wit technique, promoted agarwood formation was observed; however, the effect of AF21 did not keep stable in the later test, while AF4 and AF12 still functioned 1 year later. This study may lay a foundation for exploring the underlying mechanism of agarwood formation as well as fungi application in agarwood production.

Trunk surface agarwood-inducing technique with Rigidoporus vinctus: An efficient novel method for agarwood production

Only when Aquilaria spp. or Gyrinops spp. trees are wounded, due to insect attack, or microbial invasion, agarwood can be successfully induced. In the present study, a fungus which can induce agarwood formation efficiently was isolated and a suitable method for its application to induce agarwood formation was developed. Rigidoporus vinctus was isolated from the inner layers from infectious A. sinensis trees. When the fermentation liquid of fungi inoculated back to A. sinensis tree, agarwood was found to be induced. In addition, a novel method called trunk surface agarwood-inducing technique (Agar-Sit) was developed to produce agarwood with R. vinctus. The alcohol soluble extract content of the agarwood, up to 38.9%, far higher than the requirement (10%) in Chinese Pharmacopoeia and the six characteristic compounds of agarwood used as Chinese Medicinal Materials were all detected. Their relative percentages of the sesquiterpenes in the essential oil were 22.76%. This is the first report of the Agar-Sit and also the application of R. vinctus in agarwood induction. According to the results, when the combination of Agar-Sit and R. vinctus is used agarwood can be induced with high yield and good quality.

Identification of WRKY Transcription Factors Related to Saikosaponin Biosynthesis in Adventitious Roots of Bupleurum chinense

Abstract Objective To identify the genes of WRKY transcription factors (TFs) from roots of Bupleurum chinense and genes that potentially regulate saikosaponin (SS) biosynthesis. Methods Firstly, the subfamily cluster analysis was mainly based on Arabidopsis thaliana WRKYs for 27 putative WRKY TFs selected from previous transcriptome sequencing data. Secondly, qPCR was used to screen such genes of WRKY TFs that could be induced by NaCl and PEG6000 in adventitious roots of B. chinense. Meanwhile, saikosaponins (SSs) in treated adventitious roots were determined by HPLC. The roots were collected at 0, 2, 4, 8, 12, 24, 48, and 72 h after treatments, and 120 h only for PEG. Finally, the tissue-specific expression was analyzed on screened genes by qPCR. Results The 27 genes were grouped into three categories: There were nine in Group I, 15 in Group II, and two in Group III. Four genes of WRKYTFs, BCWRKY6, BCWRKY16, BCWRKY32 , and BCWRKY35 were obviously induced by NaCl in adventitious roots of B. chinense , while only BCWRKY32 was induced by PEG. The content of SSs increased at different levels in NaCl and PEG6000 treatment. Three genes including BCWRKY6, BCWRKY32 , and BCWRKY35 , expressed most in roots, were similar to the accumulation pattern of SS. Conclusion The three WRKY genes, BCWRKY6, BCWRKY32 , and BCWRKY35 , may be involved in the biosynthesis of SS.

Zhongdanyaozhi No. 1 and Zhongdanyaozhi No. 2 Are Hybrid Cultivars of Salvia miltiorrhiza with High Yield and Active Compounds Content

Salvia miltiorrhiza Bunge is an important medicinal plant used for the treatment of cardiovascular disease. Intraspecific hybridization between a male sterile line and inbred lines was followed by 39 F1 crossings. Cultivars “Zhongdanyaozhi No. 1” (ZD1) and “Zhongdanyaozhi No. 2” (ZD2) were obtained. In 2012 and 2013 tests in Beijing, the two cultivars were compared with three widely accepted types, SDCK, SXCK and HNCK from Shandong, Shanxi and Henan provinces. The yield of ZD1 and ZD2 exceeded the three CKs by more than 48.2% and 39.2%, respectively; the composition of the two hybrid cultivars was similar to the control, although the content of some compounds varied to some extent. The content of salvianolic acid B and tanshinone II A of both ZD1 and ZD2 could measure up the requirement of Chinese Pharmacopoeia. The former showed no obvious advantage than the three CKs, while the later’s tanshinone II A was 29.6% higher than the three CKs. Taken together, ZD1 is a high yielding and thick-root-type cultivar which is suitable for decoction pieces; while ZD2 is suitable for component especially lipophilic component extraction. ZD1 and ZD2 reported here are the first cultivars obtained by the hybridization of S. miltiorrhiza.