Functional Characterization Of Arabidopsis Pollen-specific Expressed LRXs In Pollen Germination And Pollen Tube Growth

In the higher plants,the male gametophyte-pollen germinated pollen tube,and the tube as a medium to transport two sperm cells to the female gametophyte-ovule,followed by sperm cells combined with the egg cell and the central cell to complete the fertilization.In this process,normal pollen germination and pollen tube growth are critical.In this study,the functions of four Arabidopsis pollen-specific expressed Leucine-Rich Repeats/Extensins(LRXs)-LRX8,LRX9,LRX10 and LRX11 in pollen germination and pollen tube growth were characterized by a variety of biological methods including reverse genetics,cell biology,molecular biology and biochemistry.LRXs are a class of cell wall proteins that are named due to harboring an Leucine-Rich Repeats(LRR)domain and an Extensin(EXT)domain.RT-PCR and GUS staining analysis showed that LRX8–11 displayed a consistent expression pattern in mature tricellular pollen grains and pollen tubes.We performed a detailed phenotypic analysis of the mutants including four single mutants,six double mutants,four triple mutants and a quadruple mutant.In the vegetative growth,all the mutants have no significant defects compared with the wild type.In the reproductive growth,single mutants have no significant difference compared with the wild type,and among double mutants only lrx8 lrx9 and lrx8 lrx10 showed a slight decrease in silique seed set,while the triple mutants and quadruple mutant displayed markedly reduced seed set than that of the wild type.Reciprocal cross experiment between the quadruple mutant and wild type suggested that the decline of seed-setting rate was due to the defects of male gametophyte.Further the genetic analysis revealed that the male gametophyte transmission efficiency of the mutants were significantly decreased.Pollen vitality and nuclear composition of all mutants were normal using alexander,DAPI and FDA staining.In vitro,pollen germination experiment showed that the triple mutants and quadruple mutant had different degrees of pollen rupture and pollen tubemalformations.In vivo,the pollen tubes of the mutants grew slower in the stylar canal than that of the wild type.GFP-fused LRX8,LRX10,and LRX11 were found to be localized to pollen tube cytoplasm and cell walls.An immunohistochemical analysis of pollen tube cell wall polysaccharides showed an increase in the amount of rhamnogalacturonan I in the subapical walls of pollen tubes of the lrx9 lrx10 lrx11 and lrx8 lrx9 lrx11 mutants,and decrease in the content of fucosylated xyloglucans in lrx8lrx9 lrx11 compared to wild-type plants.Moreover,the callose content in the apical walls of pollen tubes increased in the lrx8 lrx9 lrx11 mutant.The genomic DNA of LRX1,which functions in root hair,partially complemented the pollen germination rate and silique seed set of lrx8 lrx9 lrx10.In summary,LRX8-11 may be involved in the pollen grains and pollen tubes cell wall structure assembly and cell morphology maintenance.LRX8-11 gene knockout affected the pollen tubes cell wall polysaccharide deposition,leading to pollen rupture and pollen tube deformity.Therefore LRX8-11 played an important role in pollen germination and pollen tube growth.Our study not only enriched the function of cell wall proteins,but also laid a foundation for further analysis of the molecular mechanism about pollen germination and pollen tube growth.