Thioredoxin Regulates The Pollen Germination On The Stigma In Arabidopsis

In angiosperm,pollination is an important process for sexual reproduction during which a series of events occur,including pollen adhesion,hydration and germination on the stigma,the polarized growth of pollen tubes across the stigmatic tissues,pollen tubes guiding to micropyles and embryo sac.The interaction between pollen and stigma is crucial for the germination of the pollen on the stigma during pollination.Up to now,only several factors implicated in pollen-stigma interaction have been identified,such as the peroxisomal membrane protein,S locus cysteine-rich protein and the pollen coat protein.However,the mechanism of the interaction between pollen and stigma is still poorly understood.In our previous study,we isolated a mutant of an Arabidopsis SnRK1 complex subunit KIN??.There were no obvious differences of pollen germination between the KIN?? mutant and wild type in vitro.However,the adhesion,hydration and germination of the KIN?? mutant pollen grains on the stigma were compromised.KIN?? protein harbors a glycogen binding domain(GBD)and four cystathionine ?-synthase(CBS)domains.To investigate the molecular mechanism of KIN?? regulating pollen germination on the stigma,we identified a protein interacting with KIN?? named NUCLEUS THIOREDOXIN1(NRX1).NRX1 is localized in the nucleus and cytoplasm,and showed typical activity of Trx.Bimolecular fluorescence complementation analysis showed KIN?? interacts with NRX1 in cytoplasm,indicating KIN?? may regulate the activity of NRX1.To test this suggestion,we measured the activity of disulfide reduction in the wild type and the 35S::ami RNA-KIN?? transgenic lines of Arabidopsis.We found the activity of disulfide reduction in the 35S::amiRNA-KIN?? transgenic lines decreased significantly compared with that of the wild type plants.These results suggest KIN?? positively regulates the activity of disulfide reduction in Arabidopsis.We isolated two Arabidopsis T-DNA insertion mutants of NRX1,nrx1-1 and nrx1-2.The T-DNAs were inserted in the second exon of NRX1 gene in nrx1-1 mutant and 3'UTR in nrx1-2 mutant,respectively.In the progenies of the nrx1-1/+ and nrx1-2/+ heterozygotic mutant plants,the segregation ratio of wild type to heterozygous to homozygous plants was about 1:1.68:0.78 and 1:1.64:0.77,respectively;instead of the normal separation ratio 1:2:1.Reciprocal cross analysis between the wild-type plant and the heterozygous mutants showed that only 71% and 69% of transmission occurred via the male gametophytes in nrx1-1;and nrx1-2,respectively.These results suggested that some of the male gametophytes in nrx1 mutant lines were defective.No obvious difference of mature pollen grains in viability and morphology was detected between nrx1 mutants and the wild type analyzed by Alexander staining and scanning electron microscope,respectively.Additionally,the pollen adhesions of the nrx1 mutant and the wild type are nearly equal.Furthermore,in vitro pollen germination analysis revealed a similar germination ratio for the nrx1 mutants and wild type.However,only 58% and 54% pollen grains of the nrx1-1 and nrx1-2 can germinate on the wild-type stigma,respectively,which is lower than that of the wild type(85% germination ratio).These results suggest that NRX1 plays roles in the pollen germination on the stigma.GUS staining analysis in Arabidopsis proNRX1::NRX1-GUS transgenic lines revealed that NRX1 was expressed in seedlings,leaves,pollen grains,papilla cell of stigma.We detected the endogenous levels of H2O2 and superoxide in two-week-old seedlings of the wild type and the nrx1 mutants by 3,3'-diaminobenzidine and nitroblue tetrazolium staining,respectively.We found that the root of the nrx1 mutants accumulated more H2O2 and superoxide than that of the wild type.These results suggest that NRX1 participates in the regulation of ROS levels in Arabidopsis seedlings.Our results revealed that KIN?? may positively regulate the NRX1 activity of disulfide reduction by interaction,and NRX1 functions for the pollen germination on the stigma in Arabidopsis.NRX1 was involved in the regulation of ROS level in Arabidopsis seedlings.However,the mechanism of NRX1 regulating pollen germination on the stigma needs future study.