Ultraviolet-B-induced Flavonoid Accumulation In Betlua Pendula Leaves Is Dependent Upon Nitrate Reductase-mediated Nitric Oxide Signaling

In the atmosphere any changes of Ultraviolet-B (UV-B) radiation will have great biological effects on the ecosystem, and enhanced Ultraviolet-B radiation may have a significant impact on both physiological and biochemical processes and other aspects on forest plants. Nitric oxide generation and flavonoid accumulation are two early reactions of plants to UV-B irradiation. However, the number of detailed studies to date addressing the source of UV-B-triggered NO generation and the role of NO in UV-B-induced flavonoid accumulation is quite low, especially with tree species. In this article, the effects of UV-B on NO generation and flavonoid accumulation, the role of nitratre reductase (NR) in UV-B-induced NO generation and flavonoid accumulation in the leaves of Betula pendula Roth (silver birch) seedlings were examined. The underlying mechanisms of UV-B-triggered NO generation were investigated. The followings are the main results of the experiments:1. The effects of UV-B radiation on endogenous NO levels and flavonoid contents in silver birch seedlings were examined. The results showed that UV-B irradiation significantly enhanced NO levels and flavonoid contents of the seedlings. The highest NO level and flavonoid content were obtained10h and3d after UV-B treatment, being3.7and1.0folds of the control. The data indicated that NO burst and flavonoid accumulation were two early responses of silver birch seedlings. Moreover, the results showed that UV-B-induced NO burst more early than flavonod accumulation in the seedlings.2. The NO specific scavenger2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) was used as the inhibitor of NO signaling pathway to examine functions of NO in UV-B-induced flavonod accumulation in silver birch seedlings. The data showed that pretreatment of the seedlings with cPTIO not only suppressed the UV-B-triggered NO generation but also the UV-B-induced flavonoid accumulation, which suggest that NO signaling is essential for UV-B-induced flavonoid biosynthesis in the seedlings.3. The responses of nitrate reductase (NR) activity and the expression levels of NIAl and NIA2genes in leaves of silver birch seedlings to UV-B-irradiation were examined. The data show that UV-B irradiation stimulates NR activity and induces up-regulation of NIAl but does not affect NIA2expression during UV-B-triggered NO generation. The results suggest that UV-B radiation stimulates NR activity in the seedlings and the up-regulation of NIAl gene may be responsible for UV-B stimulated NR activity.4. In order to assay the roles of NR in UV-B-triggered NO generation, we determined the effects of NR inhibitors TUN and Gin on NO levels of the seedlings under UV-B irradiation. The data showed that pretreatment of the seedlings with TUN and Gin not only inhibited the UV-B-stimulated NR activity but also suppressed the UV-B-triggered NO levels in the seedlings, which suggest that NR plays important roles in UV-B-triggered NO generation in the seedlings.5. To further investigate the role of NR-mediated NO generation in UV-B stimulated flavonoid accumulation, we examined the effects of NR inhibitors on flavonoid contents in the seedlings under UV-B irradiation. The results showed that pretreatment of the seedlings with TUN and GIn significantly suppressed the UV-B-induced flavnoid accumulation in the seedlings. Moreover, the suppression of TUN and Glin on UV-B-induced flavnoid accumulation can be reversed by exogenous application of NO via its donor SNP. Together, the data indicated the involvement of the NR-mediated NO signaling in UV-B-induced flavonoid accumulation in silver birch seedlings.Taken together, the results of our present work not only demonstrated that UV-B radiation triggered NO-dependent signaling pathway in the leaves of Betula pendula Roth (silver birch) seedlings, but also indicated that the endogenous NO signaling triggered by UV-B radiation was essential for flavonoid accumulation in the seedlings. Moreover, our results suggest that NR that was encoded by NIAl gene was the main source of NO generation in Betula pendula leaves under UV-B irradiation. The results provided helpful evidences for understanding the mechanisms of nitric oxide generation and flavonoid accumulation to UV-B irradiation and suggest a new strategy to reduce the hazard of UV-B radiation to trees by enhancing flavonoid accumulation in plants through regulating NR activity in plants, which provides a new tool to enhance plant resistance to UV-B stress.