Exploring The Role Of NPH Pollen-Stigma Interaction In Arabidopsis

The sexual reproduction of angiosperms plays important roles in plant genetics and breeding.In the initial stage of double fertilization,the pollen falls on the surface of the pistil stigma.Upon the pollen-stigma recognition,the pollen will adhere,hydrate and germinate,and the pollen tube will grow.Then the pollen tube will extend through the stigma into the style,and the sperm cells enter the female gametophyte along the pollen tube.The double fertilization process is affected by the interaction between the male and tne female reproductive systems.At the initial stage of double fertilization,the pollination process of Arabidopsis is regulated by the interaction of pollen and stigma.The factors involved in the pollen-stigma interaction include: factors in the pollen tissue and factors in the stigma tissue.Studies have shown that the adhesion of Arabidopsis pollen in regulated by the waxy layer and certain cell wall components on the surface of the stigma.The abnormality of lipids and protein from the pollen coat causes male sterility in Arabidopsis.Studies have shown that many Arabidopsis male sterile mutants are caused by the deletion or the abnormal expression of some genes in the pollen coat,However the pollen coat is developed from the tapetum.It remains unclear whether genes of pollen itself in important for pollen-stigma interaction.In this paper,Arabidopsis plants with mutation on NPH gene was to the functions of NPH gene in the interaction between pollen and stigma during hydration process.Through the research on the genetic expression pattern of NPH gene,the analysis of localization,the genetic complementation and the role of calcium ion in pollen germination,this study revealed the important role of calcium ion in the pollen hydration and germination processes.The main experimental results were summarized as following:1.Genetic screening of nph mutant: The Arabidopsis T-DNA insertion mutant library constructed in our laboratory was screened out for the male sterile phenotype and the mutant nph with hydration defects in the pollen was identified.We constructed the p NPH::LAT52-EGFP vector and obtained the plants transgenic,in order that we can identify homozygous or heterozygosis plants by fluorescence and distinguish mutant pollen from wild-type pollen.2.nph phenotypic analysis: Alexander staining showed that nph pollen the morphology and viability were normal,and there was no difference in the tissue of pollen coat under transmission electron microscope.The pollen could not hydrate under the normal growth humidity of Arabidopsis,but it could be complete hydration under high humidity.The pollen tube in vitro germination experiment showed that the mutant pollen can not germinate.3.Gene localization: TAIL-PCR technique obtains the flanking sequence of T-DNA,and also proves that the NPH mutation is a T-DNA single copy insertion,and the NPH gene is determined to be an important gene in the calcium ion pathway.The complementation vector was constructed for recovery experiments.The results showed that the pollen of the restorer plants could normally hydrate and germinate,and pollen tubes were produced.If indicates that the defective phenotype exhibited by the mutant is indeed caused of the mutation of the NPH gene.4.NPH protein subcellular localization: Bioinformatics analysis indicated that the NPH gene encodes a calcium channel protein.Detection of the pro LAT52::NPH-GFP transgenic plants revealed that the NPH protein was localized in the cytoplasm.5.The calcium ion indicator shows the distribution of calcium ions: The calcium ion indicator is transferred to the mutant plant,and the accumulation and the concentration gradient of calcium ions can be observed using laser microscopy.The results show that at the tip of the pollen tube,calcium ions will dynamically aggregate with the elongation of the pollen tube,indicating that calcium ions do participate in the growth of pollen tubes.6.Calcium channel drug inhibition treatment: by adding a certain concentration of calcium channel inhibitor in pollen germination medium,for the pollen culture experiment,we proves that the addition of calcium channel inhibitor in vitro can inhibit pollen germination and pollen tube growth,indicating that the germination of pollen and the growth of pollen tubes require the participation of calcium ions.