Interaction Between Nitric Oxide And Hydrogen Peroxide Signaling In UV-B-Induced Flavoniod Accumulation Of Hypericum Perforatum Cells

Medicinal plants and other natural plant is an important source of active substances.Although plants are renewable resources, but due to cultural difficulties, slow growth, as well as unplanned logging of natural substances in recent years, some species are becoming more difficult to obtain in sufficient amounts to meet increasing demands. Plant cell cultures offer promising alternatives for the production of the useful secondary metabolites because totipotency enables plant cells to produce the chemicals in vitro. However, the numbers of compounds that are producible commercially by cell culture technology are still very few. One of the main limitations is the low productivity of the desired compounds.UV-B radiation (280-320 nm), an integral component of sunlight, has diverse effects on plants. UV-B can affect the growth, development, reproduction, and survival of many plants. Exposure to low levels of UV-B induces multiple responses of plants. The rapid accumulation of the UV-absorbing substances such as flavonoid, is one of the early reactions of plants to UV-B. Therefore, UV-B has been widely used as an effective strategy for improving the secondary metabolite production of the cultured plant cells. However, the mechanism of UV-B-induced secondary metabolite production is largely unknown. Although the accumulation of flavonoid is one of the most effective protection responses of plants to UV-B, the underlying mechanisms of UV-B perception and signal transduction are very poorly understood.In this study, we utilized the H. perforatum cells to study the function of UV-B in enhancing flavonoid synthesis in H. perforatum cells. Furthermore, we studied the signal transduction mechanism during the above-mentioned process. The main results are summarized as follows:1,Establishing and optimizing the H. perforatum cell line and culture conditionsThe plant cell line for the study was induced from the young stems of Hypericum perforatum L. with MS medium (Murashige and Skoog, 1962).supplement with 1.0 mg L^-1 of a-naphthaleneacetic acid (NAA), 2.0 mg L^-1 of indole acetic acid (IAA), 0.2 mg L^-1 of kinetin, 20 g L^-1 sucrose and 8 g L ^-1 agar. The callus line had been in culture for 14 months by the time of this study. The suspension culture of the cell line was initiated from the callus culture on a liquid medium similar to that for the callus culture, and we got the best suspension culture condition: MS medium with hormones (1.5×10^-5 mol·L^-1 NAA + 10^-6 mol·L^-1 BA) and 3% sucrose, the medium was adjusted to pH 5.8 and then sterilized at 121℃for 20 min before use, the culture temperature is 25℃, shaker's rating speed is 100 rpm, and the suspension culture was subcultured every2 weeks.2,Effects of UV-B on flavonoid synthesize in H. perforatum cells.Five-day-old cells treated with UV-B were harvested as the time indicated to determine flavonoid production and biomass. The result shows than although UV-B irradiation does not affect the cell growth, it can signicantly enhance flavonoid production of Hypericum perforatum L. suspension cells. The results suggest that Hypericum perforatum cell is an appropriate system for studying the effects of UV-B on flavonoidaccumulation.3,Dependence of UV-induced flavonoid production on NOThe results show that UV-B treatment stimulates NO generation of Hypericum perforatum cells. NO scavenger cPTIO inhibits not only the UV-B-induced NO generation but also the UV-B-triggered flavonoid accumulation, which suggests that NO might be essential for UV-B-induced flavonoid production. This conclusion gains further support from result that the UV-B-induced flavonoid accumulation can be suppressed by NOS inhibitor PBITU.4,Involvement of H₂O₂ in UV-induced flavonoid accumulationThe results show that UV-B irradiation stimulates hydrogen peroxide (H₂O₂) generation of the cells. CAT and DPI not only inhibit the UV-B-triggered H₂O₂ generation but also suppress the UV-B-induced flavonoid accumulation of the cells, which implies the involvement of H₂O₂ in UV-B-induced flavonoid production.5,Interaction between NO and H₂O₂ in inducing flavonoid productionIt suggests that NO per se is sufficient for flavonoid production but H₂O₂ alone is not sufficient for triggering flavonoid production of the cells, although it is necessary for UV-B-induced flavonoid accumulation. Interestingly, contents of flavonoids of the cells treated with both NO and H₂O₂ are significantly enhanced as compared to those of the cells treated with NO alone, although H₂O₂ per se does not increase flavonoid production of the cells. The results demonstrate the interaction between NO and H₂O₂ in flavonoid production.6,Mutually amplifying reactions between NO and H₂O₂ signalingTreatment of NO scavenger suppresses not only UV-B-induced NO production but also the UV-B-triggered H₂O₂ generation. And CAT inhibits both the UV-B-induced H₂O₂ generation and NO production. Furthermore, external application of NO stimulates H₂O₂ levels of the cells, whereas treatment of H₂O₂ activates NO-synthase-like activities and enhances NO levels of the cells. Together, the data demonstrate a special catalyzing loop between NO and H₂O₂ in Ginkgo biloba cells during UV-B irradiation.7,The synergy of NO and H₂O₂ in flavonoid production is dependent on their mutually amplifying reactionsThe results show that the inhibitors could block the catalyzing loop between NO and H₂O₂, and the synergistic action between NO and H₂O₂ on flavonoid production is also suppressed by the inhibitors. Thus, the data suggest that the synergy between NO and H₂O₂ on flavonoid production might be dependent on their catalyzing loop.8,NO and H₂O₂ mediate the UV-induced flavonoid accumulation through different mechanismCHS is the first committed gene of flavonoid glycoside biosynthetic pathway, the result showes that the UV-B-induced CHS expression is significantly inhibited by cPTIO but not affected by CAT, which strongly suggests that NO and H₂O₂ may mediate the UV-B-induced flavonoid accumulation of the cells through different mechanism.