Zhihui Gao

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

Whole-tree Agarwood-Inducing Technique: An Efficient Novel Technique for Producing High-Quality Agarwood in Cultivated Aquilaria sinensis Trees

Agarwood is the fragrant resin-infused wood derived from the wounded trees of Aquilaria species. It is a valuable non-timber forest product used in fragrances and as medicine. Reforestation for Aquilaria trees in combination with artificial agarwood-inducing methods serves as a way to supply agarwood and conserve of wild Aquilaria stock. However, the existing agarwood-inducing methods produce poor-quality agarwood at low yield. Our study evaluated a novel technique for producing agarwood in cultivated Aquilaria trees, called the whole-tree agarwood-inducing technique (Agar-Wit). Ten different agarwood inducers were used for comparison of Agar-Wit with three existing agarwood-inducing methods. For Aquilaria trees treated with these ten inducers, agarwood formed and spread throughout the entire tree from the transfusion point in the trunk to the roots and branches of the whole tree. Agarwood yield per tree reached 2,444.83 to 5,860.74 g, which is 4 to 28 times higher than that by the existing agarwood-inducing methods. Furthermore, this agarwood derived from Agar-Wit induction was found to have a higher quality compared with the existing methods, and similar to that of wild agarwood. This indicates Agar-Wit may have commercial potential. Induction of cultivated agarwood using this method could satisfy the significant demand for agarwood, while conserving and protecting the remaining wild Aquilaria trees.

Selection and validation of reference genes for studying stress-related agarwood formation of Aquilaria sinensis.

AbstractAgarwood is a high-valued woody material for medicine, perfume, and incense production in Asia, Middle East, and Europe. The wild resources of agarwood-producing tree species, e.g., Aquilaria sinensis have been greatly threatened. The formation of agarwood is considered to be associated with the plant stress and defensive responses, thus it would be urgent and significant to investigate the molecular mechanism of these species responding to a variety of stresses. This is the first report regarding the reference gene selection of Aquilaria species for studying the molecular mechanism of stress-related agarwood production. Candidate reference genes were selected according to previous reports and the sequences were obtained from the 454 EST library of A. sinensis. To obtain the robust genes, we applied three independent programs depending on distinct assumptions and combined these results by a rank aggregation algorithm. The result supports tubulin, ribosomal protein, and glyceraldehyde-3-phosphate dehydrogenase to be the most stable reference genes for quantification of target gene expression in the overall samples examined. Validation of these genes through normalizing the expression of a terpene synthase demonstrated that these three genes are reliable. The selective usage of three algorithms based on their characteristics was underlined. However, more robust genes could be identified if the results of all algorithms were combined by a proper method such as the rank aggregation algorithm. Key message Reference genes which are critical in gene expression studies are recommended for future molecular studies of stress response and agarwood production in the endangered Aquilaria and other tree species.

Identification of conserved and novel microRNAs in Aquilaria sinensis based on small RNA sequencing and transcriptome sequence data

Agarwood is in great demand for its high value in medicine, incense, and perfume across Asia, Middle East, and Europe. As agarwood is formed only when the Aquilaria trees are wounded or infected by some microbes, overharvesting and habitat loss are threatening some populations of agarwood-producing species. Aquilaria sinensis is such a significant economic tree species. To promote the production efficiency and protect the resource of A. sinensis, it would be critical to reveal the regulation mechanisms of stress-induced agarwood formation. MicroRNAs (miRNAs), a key gene expression regulator involved in various plant stress response and metabolic processes, might function in agarwood formation, but no report concerning miRNAs in Aquilaria is available. In this study, the small RNA high-throughput sequencing and 454 transcriptome data were adopted to identify both conserved and novel miRNAs in A. sinensis. Deep sequencing showed that the small RNA (sRNA) population of A. sinensis was complex and the length of sRNAs varied. By in silico analysis of the small RNA deep sequencing data and transcriptome data, we discovered 27 novel miRNAs in A. sinensis. Based on the mature miRNA sequence conservation, we identified 74 putative conserved miRNAs from A. sinensis and 10 of them were confirmed with hairpin forming precursor. Interestingly, a novel miRNA sequence was determined to be the miRNA of asi-miR408, but with accumulation much higher than asi-miR408. The expression levels of ten stress-responsive miRNAs were examined during the time-course after wound treatment. Eight were shown to be wound-responsive. This not only shows the existence of miRNAs in this Asian economically significant tree species but also indicated its critical role in stress-induced agarwood formation. The highly accumulated miRNA of asi-miR408 implied miRNAs would be functional as well as miRNAs in plants.

Hydrogen peroxide induces vessel occlusions and stimulates sesquiterpenes accumulation in stems of Aquilaria sinensis

Agarwood is highly valuable resinous and fragrant heartwood, produced principally from tropical tree species in the genus Aquilaria, which is used widely in countries of the Middle East, Southeast Asia and Japan. Generally, healthy trees will not produce agarwood, but wounding of the tree initiates the production of agarwood. In this study, the pruning of actively growing saplings of Aquilaria sinensis resulted in hydrogen peroxide (H2O2) burst, which was followed by formation of vessel occlusions and sesquiterpene biosynthesis in the pruned stems. Treatment of the pruned stems with scavenger of H2O2 (ascorbate, AsA) greatly reduced the amount of H2O2 released, the number of vessel occlusions, and the amount of sesquiterpenes produced. In addition, exogenous H2O2 also induced A. sinensis plants to form vessel occlusions and produce sesquiterpenes as pruning treatment. The results indicated that H2O2 may be an important post-wounding signal in A. sinensis that leads to the induction of vessel occlusions formation and sesquiterpene biosynthesis, and thus H2O2 might play a vital role in agarwood formation in pruned stems of A. sinensis.

The Sesquiterpene Biosynthesis and Vessel-Occlusion Formation in Stems of Aquilaria sinensis (Lour.) Gilg Trees Induced by Wounding Treatments without Variation of Microbial Communities

As widely recognized, agarwood formation in Aquilaria trees is induced by external wounding. Because agarwood usually harbors specific microbes, the function of microbes in agarwood formation has been debated for almost a century. In this study, two wounding methods, the burning-chisel-drilling method (BCD) and the whole-tree agarwood-inducing method (Agar-Wit), were used under the non-contamination of environmental microorganisms. After pyrosequencing the small rRNA subunits of the wounds induced by the BCD and Agar-Wit, no substantial variation was observed either in fungal and bacterial enrichment and diversity or in the relative abundances of taxa. By contrast, significant variations in fungal and bacterial communities were detected following the partial tree pruning (PTP)-wounding. The wound-induced sesquiterpene biosynthesis and vessel-occlusion formation, however, were found to be similar in all types of wounded trunks. We thus infer that wounding in the absence of variations in microbial communities may induce agarwood formation. This result does not support the long-standing notion that agarwood formation depends on microbes.

Compositions and antifungal activities of essential oils from agarwood of Aquilaria sinensis (Lour.) Gilg induced by Lasiodiplodia theobromae (Pat.) Griffon. & Maubl

A composicao e atividade antimicrobiana dos oleos essenciais obtidos de madeira de agar originaria de Aquilaria sinensis (Lour.) Gilg induzido por agente biologico da madeira de agar, Lasiodiplodia theobromae (F), foram caracterizadas e comparadas com madeira de agar selvagem (W) e arvores saudaveis nao inoculadas (H) como controles positivo e negativo, respectivamente. A composicao quimica de F foi investigada usando cromatografia gasosa-espectrometria de massas (GC-MS). O oleo essencial de F mostrou uma composicao similar de W, sendo rico em sesquiterpenos e constituintes aromaticos. No entanto, o oleo essencial de H era abundante em alcanos. Os oleos essenciais de F e W mostraram ser inibidores mais potentes de L. theobromae, Fusarium oxysporum, e Candida albicans do que o oleo essencial de H. Nossas descobertas demonstram pela primeira vez que o oleo essencial obtido da madeira de agar originado de A. sinensis induzido por L. theobromae teve uma alta similaridade com o oleo essencial da madeira de agar selvagem, tanto em composicao quimica como em atividade antimicrobiana. Alem disso, a estrategia de madeira de agar induzida por fungos pode ser potencialmente aplicada em madeira de agar e producao de oleo essencial em arvores do genero Aquilaria. The composition and antimicrobial activity of essential oils obtained from agarwood originating from Aquilaria sinensis (Lour.) Gilg induced by a biological agent of agarwood, Lasiodiplodia theobromae (F), were characterized and compared to those from wild agarwood (W) and uninoculated healthy trees (H) as positive and negative control, respectively. The chemical composition of F was investigated using gas chromatography-mass spectrometry (GC-MS). The essential oil of F showed a similar composition to that of W, being rich in sesquiterpenes and aromatic constituents. However, the essential oil of H was abundant in alkanes. Essential oils of F and W were more potent inhibitors of L. theobromae, Fusarium oxysporum, and Candida albicans than the essential oil of H. Our findings demonstrate for the first time that the essential oil obtained from the agarwood originating from A. sinensis induced by L. theobromae had a high similarity to that of the essential oil of wild agarwood, both in chemical composition and in antimicrobial activity. Furthermore, the strategy of agarwood induced by fungi could be potentially applied in agarwood and essential oil production in Aquilaria trees.

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.

A Review of Quality Assessment and Grading for Agarwood

Abstract Agarwood is an important non-timber forest product widely used in religious and cultural activities as perfume and fragrance and as traditional medicine in Asia. The high value of agarwood and the inflated consuming market have led to constant rising of the prices. In general, the price of the agarwood is determined according to its quality, which can be divided into different grades. But up to now, there is not any standard quality grading system which could be used overwhelmingly throughout the agarwood producing, commerce and consumption. Therefore, we reviewed agarwood in diversified grading indexes, systems and methods.

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.