Modulation Of Nitric Oxide In Tea Pollen Germination And Pollen Tube Growth Under Low Temperature

Tea tree pollen germination was influenced by many factors. Relevent study was showed the effect of plant-growth regulator and microelements on the germination percentage of tea tree pollen. Further more, low temperature effect in many kinds of plant pollen germination have reported.This study based on previous studies, application of different agentia of nitric oxide (NO) and cGMP to regulate the germination of tea tree pollen and pollen tube growth under low temperature, for explore NO regulate tea pollen germination and pollen tube growth in an effective way under low temperature. The Research Result as followed.Fresh pollen grains from Camellia sinensis were incubated in the normal temperature (25℃) and exposed to low temperature(4℃) for up to3h. The results showed that low temperature significantly inhibited pollen germination and tube growth. Under normal temperature, the effects of different concentration of SNP (0,50,100,200,1000μM/L) and DEA NONOate (0,25,50,75,100μM/L) on pollen germination and pollen tube growth were investigated. The results showed that both pollen germination and tube growth were inhibited not only by exogenous SNP but also by exogenous DEA NONOate, and that the effects of both SNP and DEA NONOate were dose dependent. Furthermore, NO-scavenger (cPTIO) and an inhibitor of NOS (L-NNA) can abolished the inhibitive effects of both SNP and DEA NONOate on pollen germination and tube growth. SNP and DEA NONOate enhanced the reduction of both pollen germination and tube growth induced by the low temperature; cPTIO and L-NNA reversed this reduction effect.To research the distribution and generation of NO in pollen grain and tube by using laser confocal microscopy techniques and hemoglobin method. The result showed that NO generation in pollen grain and tube under low temperature was higher than under normal temperature. cPTIO and L-NNA largely suppressed endogenous NO production in pollen grain and tube, however, SNP and DEA NONOate can promote endogenous NO production both in low temperature and normal temperature. We used the method of hemoglobin assay; the results showed that NOS activity was significantly enhanced by low temperature.ODQ using inhibitors of the enzyme guanylate cyclase cause a decrease in the level of cGMP. The PDE inhibitor Viagra causes an increase in the level of cGMP by inhibiting the mechanism of cGMP hydrolysis. The result showed that80μM Viagra can enhanced the inhibition of pollen germination and tube growth induced by low temperature, however,50μM ODQ had counter-productive. In the presence of100μM SNP or25μM DEA, the results of pollen germination percent and pollen tube length were lower than that those of pollen grown with50μM ODQ at the same treatment. Furthermore, treatments with200μMcPTIO or300μM L-NNA under cold environment compare with added80μM viagra at the same treatments; pollen germination percent and pollen tube length are both reduced. High performance liquid chromatography was used to investigate the content of cGMP in pollen, the results showed that cGMP level in pollen of C. sinensis was relatively upon exposure to low temperature and DEA NONOate, but the level was significantly inhibited by L-NNA. These data suggest that cGMP is required, at least partly, for low temperature-and NO-inhibited pollen germination and tube growth.Ninhydrin was used to assay proline (Pro) content and results showed that proline content was raise by low temperature and exogenous nitric oxide. The stimulation of Pro accumulation by cold temperature and NO was reduced by L-NNA and cPTIO. Further research was according to ODQ and Viagra, the result showed that ODQ treatment not only can decrease Pro content in tea pollen but also can reverse Pro content raised induced by low temperature, SNP and DEA NONOate. While, Viagra treatment not only can enhanced Pro content in tea pollen but also can reverse Pro content decreased induced by L-NNA and cPTIO.The experimental results showed that NO and cGMP signal molecules were increased in tea tree pollen under low temperature, and involed in low temperature-inhibited pollen germination and pollen tube growth. The increase of NO and cGMP promote the pollen proline content.