Exploratory Researches Of The Interplay Between Programmed Cell Death And Induced Sesquiterpenes Of Aquilaria Sinensis

Agarwood is the resinous wood portion of some Aquilaria and Gyrinops trees. It possessed various biological activities such as sedative, analgesic and digestive, which is used to treat rheumatism, spasm of bronchus and respiration, abdominal cramp, diarrhea nausea, vomiting, anxiety and so on. Aquilaria sinensis （Lour.） Gilg is one of the most important plant resources for producing agarwood in China as well as the only certified source for agarwood listed in China Pharmacopoeia. It is mainly distributed in Hainan, Guangdong, Guangxi, Yunnan, and Fujian provinces. Due to the over harvest, the wildlife resources have been almost destroyed. As early as 1998, it was included in the "International Trade in Endangered Species Convention" （CITES） Appendix Ⅱ. In 1999, it was listed as category Ⅱ protected plant in the "List of Wild Plants under State Protection". In addition, it was recognized on the IUCN red list as vulnerable status in 2000. With the sharply increasing use of agarwood in medicine, essential oil, and smoked incense, the natural resources are gradually exhausted. Thus, the conflict between agarwood supply and demand contradiction is prominent, which heavily influenced the agarwood industry development. It is an urgent problem to speed up the production and improve the quality for agarwood. To uncover the mechanism of agarwood formation is the key point to solve the above problems. Defense reaction of A. sinensis has been considered as the essential mechanism to form agarwood. However, it is still hard to explain why agarwood could be constantly produced after the tree was wounded or infected. In this research, we studied the significance of sesquiterpene biosynthetic metabolism and programmed cell death （PCD） for agarwood formation. And the relation between them was firstly explored. Data derived from present study will provide theoretical support for the research of agarwood formation mechanism and give a foundation to improve agarwood-induced technique.1. Suspension cell culture was_established, which was the ideal material to study the mechanism. A. sinensis callus induced by stem tips were used to establish the suspension cell system. The results showed that the most suitable medium for callus induction and subculture was MS+2.0mg·L^-1 NAA+1.0mg·L^-1 6-BA. After 12 times of subculture, the energetic and loose callus, which were appropriate for cell suspension culture, were cultured and shook in liquid medium MS+2.0mg·L-1 NAA+1.0mg·L-1 6-BA+500.0 mg·L^-1 casein hydrolysate to establish the suspension cell system. The growth curve of suspension cells showed a "S" type. At the beginning of the culture, cell density increased slowly; during 4 to 6 days, suspension cells reached logarithmic growth period; during 7 to 12 days, suspension cells reached platform period; after 12 days, cell density and activity went down obviously. Agarwood sesquiterpenes were not detected in the suspension cells during the growth period, however, they could be detected in MeJA treated suspension cells. Using TUNEL, FDA-PI staining, DNA damage, q-PCR to detect PCD, PCD was triggered by the inner habitat stress after 24 culturing days. With q-PCR detection, gene expression of sesquiterpene biosynthesis was increased, which could enhance the sesquiterpene accumulation. In this study, stably and active growing suspension cell system was established, which was a proper system during 7-12 d to study the mechanism of agarwood sesquiterpene formation, and additionally provided a potential way to generate agarwood sesquiterpenes through application of cell culture.2/The novel full-length cDNAs of AsAACT, AsHMGS. AsDXR, AsHDR were isolated in this work. Additionally, the expression patterns of nine key genes （AsAACT, AsHMGS, AsHMGR, AsDXS, AsDXR, AsHDR, AsFPS, ASS1 and ASS2） of A. sinensis were analyzed in different stress treatment of suspension cells. Cloning and analyzing AsAACT, AsHMGS, AsDXR, AsHDR gene from A. sinensis provided basic information for study the function and expression regulation of those genes in terpenoid biosynthesis. The expression of nine key genes （AsAACT, AsHMGS, AsHMGR, AsDXS, AsDXR, AsHDR, AsFPS, ASS1 and ASS2） of A. sinensis were analyzed in the treatment of suspension cells in methyl jasmonate （MeJA） and salicylic acid （SA）. In addition, the transcriptome data of different treated A. sinensis trees were also analyzed. At last, AsAACT, AsFPS, ASS1 and ASS2 were screened as reference standards in the subsequent experimental detection.3. H₂O₂ could induced PCD obviously, and sesquiterpene production increased with the enhancement of PCD degree. PCD was triggered after 3 h H₂O₂ treatment. Nuclear shrinkage and DNA damage appeared during 6-12 h treatment. Gene expression of MCP1, MCP2, Cyt-c was increased after 24 h treatment. The nuclear became apoptotic bodies after 48 h treatment. Moreover,0.058% α-humulene was detected at 3 h of H₂O₂ treatment. Gene expression of FPS, ASS1, ASS2 was up-regulated at 6 h of treatment. 0.244% α-humulene and 0.521% δ-guaiene were detected at 12 h of treatment. After 24 h of treatment, α-guaiene, α-humulene, δ-guaiene could all be detected.4. MeJA could induced sesquiterpene production obviously, and the induced sesquiterpenes could obviously promote PCD in suspension cells of A. sinensis. The relation between PCD and sesquiterpene was uncovered using A. sinensis cell suspension with different concentration MeJA. With 2.5 μM～25μM MeJA treatment, the level of PCD was lower and sesquiterpene production was firstly increased then decreased. With 50 μM～100 μM MeJA treatment, sesquiterpenes produced fast and PCD occurred obviously. With 250 μM～500 μM MeJA treatment, the sesquiterpene biosynthesis was slower and PCD was not obvious. Caspase inhibitor Z-VAD-FMK, one of PCD inhibitors, could slow down the degree of PCD occurrence, but could not totally stop the process of PCD. Under MeJA induction, the induced sesquiterpenes produced, and caspase enzyme could obviously slow down the gene expression related to PCD, and partly decreased the expression of sesquiterpene biosynthetic genes. These results could preliminarily deduce the relation between sesquiterpenes and PCD.5. Both guaiene and a-humulene could stimulate PCD of A. sinensis cell suspensions, which indicated that induced sesquiterpenes, as secondary wound signals, could promote PCD occurrence. PCD could be detected in suspension cell of A. sinensis after guaiene or α-humulene treatment. PCD showed more easily induced by guaiene than α-humulene. With the increased sesquiterpene concentration, the degree of PCD occurrence continuously improved, which gives a good explanation of the relation between sesquiterpnene and PCD.In this research, we firstly put forward an idea that PCD participate in agarwood formation, and the relation between PCD and sesquiterpene biosynthesis was firstly explored. From our study, we conclude that sesquiterpene biosynthesis and PCD could form a positive cycle, which give an explanation of constantly agarwood production. Data derived from present study will provide theoretical support for the research of agarwood formation mechanism.