The Effect Of UV-B Radiation On Pollen Germination, Pollen Tube Growth And Pollen Tube Wall Construction Of Qingzhuan And Its Relationship With H₂O₂ And NO

Previous studies have shown that ultraviolet-B(UV-B)radiation inhibits pollen germination and tube growth via promoting generation of hydrogen peroxide(H2O2)and nitric oxide(NO)in angiosperm plants,but the effect of UV-B radiation on pollen germination and tube growth and its relationship with H2O2 and NO are not clear in gymnosperm plants.In addition,previous studies also show that both H2O2 and NO are required for the polarized growth of pollen tubes,which is contradicted to the inhibitive role of H2O2 and NO on pollen germination and tube growth under UV-B radiation,but the reason for this controversy is not clear.Meanwhile,the polarized growth of pollen tubes depends on the targeted transport and distribution of pollen wall components,but whether H2O2 and NO regulate the polarized growth of pollen tubes via affecting pollen wall construction is not clear.Based on the above situation,the gymnosperm Picea(Picea wilsonii)pollens were used in this study to determine the role of H2O2 and NO in pollen germination and tube growth and their relationship with pollen wall construction under normal growth condition and UV-B radiation.The main findings are as follows:(1)With the increases of UV-B dose,pollen germination rate was redued,pollen tube length was gradually shorted,and tube tip width was enhanced,indicating that UV-B radiation inhibits pollen germination and pollen tube growth in a dose-dependent manner in Picea wilsonii.0.4 W m-2 UV-B was selected as the optimal dose used in the following experiments.(2)H2O2 in pollen tubes grown under normal condition has a gradient distribution with highest level in the pollen tips.H2O2 scavenger ascorbic acid(ASC)and NADPH oxidase inhibitor diphenyl iodide(DPI)not only reduced the level of H2O2 and eliminated its gradient distribution in pollen tubes,but also significantly inhibited pollen germination and pollen tube growth,indicating that the maintaining the proper level of H2O2 and its gradient distribution in pollen tubes are required for the polarized growth of pollen tubes.UV-B radiation induced H2O2 generation throughout the pollen tube and disrupted the gradient distribution of H2O2 in pollen tube tips;while ASC and DPI significantly reduced H2O2 level and attenuated UV-B-inhibited pollen germination and tube growth,indicating that UV-B inhibits pollen germination and tube growth via promoting H2O2 production and disrupting its gradient distribution in pollen tube tips.Combined together,these results suggest that keeping the suitable level of H2O2 and its gradient distribution in pollen tubes is required for the polarized growth of pollen tubes.Meanwhile,the significantly inhibitive role of DPI on H2O2 production in pollen tubes also suggests that NADPH oxidases are the main enzymatic source of H2O2 in pollens of Picea.(3)NO in pollen tubes grown under normal condition uniformly distributed in pollen tubes outside the tube tips.NO scavenger c-PTIO and nitrate reductase inhibitor sodium tungstate not only could significantly reduce the NO levels in pollen tubes,but also inhibited pollen germination and tube growth,suggesting that keeping the proper level of NO in pollen tubes is required for its polarized growth.UV-B radiation induced NO production throughout the pollen tubes,while c-PTIO and tungstate reduced NO level and rescued the inhibitive effect of UV-B on pollen germination and tube growth,indicating that UV-B inhibits pollen germination and polarized growth of tubes via promoting NO production and disrupting its normal distribution pattern.Taken together,these results indicate that keeping the appropriate level of NO and its normal distribution pattern in the pollen tubes is required for the polarized growth of pollen tubes.In addition,the inhibitive role of tungstate on NO generation in pollen tubes,also suggest that NO in pollens of Picea is mainly sourced from nitrate reductases.(4)Cellulose deposited uniformly in the cell wall of pollen tubes grown under normal condition,but cellulose deposition was reduced by scavenging or inhibiting synthesis of H2O2 and NO.UV-B radiation induced accumulation of cellulose in the extreme apex of pollen tubes,but this UV-B effect was inhibited by scavenging or inhibiting synthesis of H2O2 and NO.These results indicate that both H2O2 and NO regulate cellulose distribution in cell wall of pollen tubes,which may be one of the reasons for their regulation of polarized growth of pollen tubes.(5)Compared to the graduall decreases of callose diposition from the base to the top of cell wall of pollen tubes grown under normal condition,callose accumulated at the tip of pollen tubes afer scavenging or inhibiting synthesis of H2O2 and NO.UV-B radiation also induced callose diposition in the extreme apex of pollen tubes,but this UV-B effect was prevented by scavenging or inhibiting synthesis of H2O2 and NO.These results indicate that H2O2 and NO regulate the polarized growth of pollen tubes via controlling callose distribution in the cell wall of pollen tubes.(6)Deesterified pectins mainly deposited along the tube shank and less accumulated in the tube tip in the pollen tubes grown under normal condition,but ASC and tungstate treatment enhanced their deposition in the tube tip and reduced accumulation along the tube shank.Under UV-B radiation,deesterified pectins were accumulated in the tube tip and depleted from the tube shank,but they were found only in the basal site near the germinating aperture or depleted from the tube tip after ASC and tungstate application.These results show that H2O2 and NO may regulate the polarized growth of pollen tubes via regulating deesterified pectins distribution in the cell wall of pollen tubes.(7)After ASC and tungstate application,esterified pectins were found only in the basl site and tube shank,instead of only accumulating at the tip of pollen tubes grown under normal condition.However,esterified pectins distributed uniformly in the cell wall of pollen tubes under UV-B radiation,but ASC and tungstate application induced their accumulation in the apex and subapical region of pollen tubes under UV-B radiation.These results suggest that the deposition of esterified pectins in cell wall of pollen tubeswere also regulated by H2O2 and NO,which may be one of the reasons for H2O2 and NO to regulate the polarized growth of pollen tubes.In summary,keeping the appropriate levels of H2O2 and NO and their specific distribution patterns in pollen tubes are all necessary for the proper distribution of pollen wall components such as cellulose,callose,deesterified pectins and estified pectins,so it is required for the polarized growth of pollen tubes.UV-B-inhibitedpolarized growth of pollen tubes is caused by the promotion of H2O2 and NO production,disruption of their specific distribution patterns and subsequently disturbing the proper deposition of pollen wall components in in pollen tubes of Picea wilsonii.