GR1 And NTRA Regulate Pollen Tube Growth Of On The Stigma In Arabidopsis

Pollination of angiosperms includes pollen adhesion and hydration on stigma,germination of pollen tubes,polar growth of pollen tubes in style,pollen tubes reaching the micropyle to release sperm cells,then,the sperm cells fused with egg cell and central cell.Pollination recognition is an essential step in the process of sexual reproduction of angiosperms.During pollination,the recognition of pollen?tube?and stigma determines the hydration and germination of pollen on stigma and the growth of pollen tubes on stigma.In Arabidopsis,GR1 encodes glutathione reductase localized in cytoplasma,and NTRA encodes NADPH-dependent thioredoxin reductase localized in cytoplasma and mitochondrion.Both GR1 and NTRA are involved in the regulation of the redox level of cells,which might have functional redundancy.In this study,we obtained a double mutant of gr1 ntra,and investigated the pollen phenotypes of the single mutant and the double mutant.The gr1-1 and ntra-1 mutants are both T-DNA insertion mutants.The T-DNAs in gr1-1and ntra-1 were inserted in the third intron of GR1 and in the first exon of NTRA,respectively.qRT-PCR analysis revealed that the expression levels of GR1 and NTRA were significantly downregulated in gr1-1 and ntra-1,respectively.Then we observed the transmission efficiencies of the male gametes of gr1,ntra,gr1/+ntra/-,and gr1/-ntra/+.The male gamete transmission efficiencies in gr1 and ntra are normal,but they are zero in gr1/+ntra/-and gr1/-ntra/+,indicating that gr1 and ntra double mutations disturb the normal function of male gametophyte.To further dissect the roles in GR1 and NTRA regulating male gametophyte development,we observered the pollen phenotypes of the wild-type and the mutant plants.Scanning electron microscopy,DAPI staining,Alexander staining,and FDA staining did not show significant abnormalities of the pollen morophology and viability of the single and the double mutants.Furthermore,no obvious abnormalities were observed for the pollen germination in vitro of the wild-type,single mutant and double mutant.We then pollinated the wild-type,single-mutant,and double-mutant pollen on the wild-type stigmas to observe pollen hydration and germination in vivo.There was no significant difference of the pollen hydration between the mutant and the wild-type.The heterozygous double mutant pollens could germinate on the stigma,but lots pollen tubes showed retarded growing on the surface of the stigma and could not reach the transmitting tissue,which suggests that GR1 and NTRA regulate the growth of pollen tubes on the stigma.To further investigate whether the ingredients of the cell wall in mutant pollen was changed,we stained the cellulose of pollen wall using the fluorescent dye S4B.There were no significant difference after S4B staining among the wild-type,single mutants and double mutants pollen.Mito Tracker?Deep Red FM labeling revealed that the number of pollen containing abnormal mitochondria in single and double pollen was significantly increased compared with that of the wild type.However,the ratio of pollen without mitochondria was much higher than those of the wild type and the single mutants.GR1 and NTRA participate in the regulation of intracellular redox levels,we detected the reactive oxygen species with CM-H₂DCFDA staining.The levels of the intracellular reactive oxygen species in pollens of the double mutants were increased significantly compared with that in the wild type.Our results indicate that GR1 and NTRA functions in regulating the growth of pollen tubes on the stigma,which might be involved in the regulation of intracellular redox levels in pollen.However,the signaling pathway for GR1 and NTRA regulating pollen tube growth on the stigma remains to be studied.