The Functional Analysis Of A Transmembrane Protein TMEM165d In Pollen Development In Arabidopsis Thaliana

Pollen development is a complex process which involves in vast array of biological events,in which a large number of genes are participated.Pollen is the male germ cell,and its abnormal development is one of the most universal causes of male infertility in many higher plants.Therefore,the development of functional pollens plays a very important role in this species continuation.The supply of nutrients is one of the key factors in ensuring pollen development.However,the molecular basis of the relevant transporters that mediate these nutrients is not still clear.Magnesium(Mg2+)is the most abundant divalent cation in Arabidopsis thaliana cells and acts a critical role in many physiological processes.Due to its especially chemical characteristics,organisms are supposed to evolve unique transporters for Mg2+ transportation.Up to now,there are five families containing potential candidates of Mg2+ transporters in Arabidopsis,including Cobalt resistance(CorA),Mg2+/H+exchanger,Mg2+ channels,P-type ATPase,and magnesium transporte E(MgtE).Three CorA family members,AtMGT4,AtMGT5 and AtMGT9,sub-located at endomembrane systems,have been found the roles in the male reproductive process of Arabidopsis thaliana.However,the candidates in the plasma membrane control the Mg2+ flux across the plasma membrane are still unknown.In this study,we used Arabidopsis thaliana as the main experimental materials with the combined methods of histochemical staining,fluorescent protein labeling,and plant molecular genetics,and found a transmembrane protein TMEM165d involving in pollen development.We also analyzed the potential mechanisms of this gene in this process.The main results were summarized as follows.1.Disruption of TMEM165d gene expression resulted in abortion of half of the mature pollen grains in heterozygous mutant +/tmem165d,and no homozygous mutant plant was obtained in the progeny of selfed +/tmem165d plants.These initial results suggest that TMEM165d may be involved in seed formation.2.Approximately 50%of the mature pollen grains from +/tmem165d heterozygous plant were aborted.Hoechst 33342 staining of pollen grains of WT and the +/tmem165d plants respectively.Fluorescent signaling of the pollens excited by UV were observed under a fluorescence microscope.Nearly 50%pollens in+/tmem165d mutants had disperse fluorescence and did not show unambiguous nuclei,while the vast majority of pollens in the wild type showed bright nucleus fluorescences.3.TMEM165d was transformed into the mesophyll protoplasts of Arabidopsis thaliana,and the GFP fluorescent signal was detected in the plasma membrane under the fluorescence microscopy,indicating TMEM165d protein was localized in the plasma membrane of a cell.4.Transmembrane protein TMEM165d can compensate for the growth phenotype of MM281 at low Mg2+ concentration.Negative control pTrc99A can not replenish the growth phenotype of MM281 at low Mg2+ concentration.Positive control MGT10 is a highly compatible transporter that has been demonstrated.The experiment results show that,TMEM165d can complement the defect in magnesium uptake of MM281 mutant strains,suggesting the role of TMEM165d as Mg2+transporter.In summary,the thesis reported that a new gene TMEM165d controls plant male reproduction,and that TMEM165d might function as an Mg2+ transporter conducting absorption and transport of Mg2+,the nutrient needed for pollen development.Therefore,the findings reveals a new mechanism of pollen development,and provides theoretical guidance and experimental basis for the optimizing breeding of crops.