| Honttuynia cordata Thunb. is a species of the genus Houttuynia in ancient Saururaceae family. The plants used as traditional Chinese medicine, are rich in monoterpenes which possess a variety of pharmacological activities, such as antioxidant, anti-bacteria, and anti-viruses. Based on the ecological role, a defense response of monoterpenes were induced by UV-B radiation, and related signaling pathways, which regulating the gene of monoterpene synthases, were investigated. Then, signal compounds were applied to accumulate monoterpenes in H. cordata. In addition, optimization of potassium for proper growth was also tested. Therefore, this study was conducted to promote monoterpene production and biomass together. The main results of this study are as following:Oxidative damage, antioxidant enzymes and monoterpenes were evaluated in H. cordata after exposure with elevated UV-B radiation. Results showed that the shock of UV-B induced oxidative damage according to the increase of H2O2and malondialdehyde. However, a recovery could be found after two days. Catalase, superoxide dismutase, and peroxidase were stimulated to their maximum activities on the first, second, and third day, respectively. Monoterpenes showed rapid accumulation and might play a considerable role as antioxidant agents. Nevertheless, only four individuals mainly contributed to the substantial increase of total monoterpene contents,4-terpineol was quickly induced to the peak on the first day, and β-ocimene, y-terpinene and D-limonene reached the maximum on the second day. This clearly suggests that not only antioxidant enzymes but also monoterpenes play their role in rapid and multiple-phase defense strategy against UV-B-induced oxidative stress in H. cordata. Therefore, multiple signalling pathways may be involved in the regulation of monoterpene biosynthesis under stresses.Mono-TPS, HcTPSl and HcTPS2were isolated from H. cordata by using rapid amplification of cDNA ends （RACE）. Full length cDNA of HcTPSl and HcTPS2are1886bp and1962bp, and encode558AA and584AA, respectively. Both amino-acid sequences are dominated by hydrophobic regions, and contain the transit peptide, the highly conserved DDxxD motif and RR （degenerated RRx8W） motif. HcTPS1and （?）TPS2belong to TPS-g andTPS-b subfamilies, respectively. Multiple clones of HcTPS1and HcTPS2were isolated from H. cordata accessions w01-99, w01-86, and x10-1. Various mutations, including single nucleotide polymorphisms （SNP）, insertion/deletion, intron detention, and exon loss are observed in HcTPS1and HcTPS2.44and33SNP sites are noted in HcTPS1and HcTPS2, respectively. HcTPS1and HcTPS2have insertion and deletion insertion and deletion mutations, respectively, and premature stop codon block the protein translation. Because the change of cleavage site, partial intron sequences are observed in the cDNA of HcTPS2. Two exons are lost in different clones of HcTPS2. Therefore, the mutations of10-TPS might be extensive in H. cordata.Monoterpene contents and the expression levels of HcTPSl and HcTPS2in two chemotypes of H. cordata were investigated. The Through4week pre-culture in darkness and wounding after1week pre-culture, the browning rate of leaf explants decreased significantly and resulted in efficient regeneration （20.64±5.94adventitious buds per explant） on the induction medium. In vitro plantlets of chemotype myrcene accessions w01-99, w01-46, w01-71, and chemotype decanal accessions w01-4, w01-86, and x10-1were obtained by using the high-frequency induction technique. GC-MS analysis indicates that total monoterpene contents of w01-99, w01-46and w01-71are higher than that of w01-86, and x10-1. Interestingly, no terpenoids were detected. However, the relative transcript levels of HcTPSl and HcTPS2in w01-86and x10-1were significantly higher than that of w01-99, w01-46, w01-71. This suggests that some other targets, such as gene mutation, carbon flux and posttranscriptional processes might play an important role in regulation of monoterpene biosynthesis in H. cordata.Multiple signalling pathways mediating UV-B-induced monoterpene biosynthesis in H. cordata were investigated by employing the inhibitors of signal transduction pathways. Before exposure with elevated UV-B radiation, the plants were sprayed with the inhibitors of ethylene, salicylic acid, jasmonate, nitric oxide, hydrogen peroxide and calcium ion. Results showed that the dosage of UV-B radiation was within the capacity of H. cordata according to the changes of morphology and malondialdehyde content. foliar application of the inhibitors of salicylic acid, jasmonate, nitric oxide, hydrogen peroxide and calcium ion choud reduce the induction of UV-B-induced HcTPSl expression. Therefore these signalling pathways might be involved in regulation of monoterpene biosynthesis in H. cordata. But ethylene inhibitor, AgNO3resulted in oxidative damage to the plants, and induced the defense response of monoterpenes. Thus it is not well known whether ethylene pathway is involved in regulation of UV-B-induced monoterpene biosynthesis in H. cordata.Effects of the foliar application of signal molecules or donors, salicylic acid, methyl jasmonate, ethephon （an ethylene-releasing compound）, sodium nitroprusside （a nitric oxide-releasing compound） and CaC12on monoterpene biosynthesis and lipid peroxidation in H. cordata were evaluated. Results showed that the optimizing concentrations of salicylic acid, methyl jasmonate, ethephon, sodium nitroprusside, and CaC12for monoterpene production are0.01,0.1,0.1,0.1, and1mM because of the maximum yield of monoterpenes and the little adverse effect on H. cordata plants, even some treatments could increase the resistance against oxidative stress. The maximum yield of monoterpenes were generally observed on4d or8d after foliar application.Optimization of potassium for proper growth and physiological response of H. cordata were investigated. Sterile plantlets were cultured in media with different potassium levels, and parameters related to growth, foliar potassium, water and chlorophyll contents, photosynthesis, transpiration, H2O2contents and antioxidative enzyme activities were determined after a month. Results showed that1.28mM potassium was the optimum for H. cordata as highest values of dry weight, shoot height, root length and number were obtained at this concentration. The optimum potassium concentration resulted in the maximum net photosynthetic rate which could be associated with the highest chlorophyll content rather than limited stomatal conductance. The supply of surplus potassium resulted in higher content of foliar potassium, but negatively correlated with the biomass. Both potassium starvation （0mM） and high potassium （>1.28mM） could lead to water loss through high transpiration rate and low water absorption, respectively, and resulted in H2O2accumulation and increased activities of catalase and peroxidase, which suggested induction of oxidative stress. Moreover, H. cordata showed the minimum of H2O2content and the maximum of superoxide dismutase activitiy on1.28mM potassium, implying its role in inducing tolerance against oxidative stress. Total monoterpene content only had a minor share （9.85%） of essential oil in the control treatment （0mM）, while it showed a remarkable increase in the treatments supplemented with potassium and10.26mM K resulted in the maximum total monoterpenes. The optimum potassium concentration resulted in lower total monoterpene conent. However, the optimum potassium could make sure the optimal growth of plants for signal strategies in the commercial production of H. cordataThe role of monoterpenes in the essential oils of H. cordata was assessed by antimicrobial activitiy and antioxidant activity assays. Disc diffusion method was employed for the determination of antimicrobial activities. Results indicated that monoterpene components, such as β-pinene, terpinolene, y-terpinene, D-limonene, and4-terpinenol had great potential against bacteria tested, and partly contributed to the antimicrobial capability of essential oils. ABTS+and β-Carotene bleaching assays indicate that the essential oils have hydrogen-donating capacities and acceptable activities against lipid peroxidation, respectively, which may be significantly associated with total monoterpenes according to correlation analysis. Although differential antioxidant capacities are noted, monoterpene individuals observed in the essential oils generally show great antioxidant activities. This suggests that the induction of monoterpene biosynthesis in H. cordata may be very important for medicinal uses. |
