The Study Of AtFIM4 And AtFIM5 Regulating Pollen Germination And Pollen Tube Polar Growth In Arabidopsis

Pollen germination and pollen tube growth are indispensable in sexual reproduction of higher plants.Microfilament cytoskeleton plays a critical role in this process.Pollen tube is a typical example of polar growth cells,in which the microfilament cytoskeleton had been widely researched.Actin bundles are necessary for pollen tube polar growth.The process of bundles formation is regulated by many kinds of actin-binding proteins(ABPs).Fimbrins,also known as plastins,are the only CH domain-containing actin bundling protein identified in plants so far.Five fimbrin-like genes are present in the Arabidopsis thaliana genome,termed AtFIM1-5.AtFIM4 and AtFIM5,which are abundant in pollen tubes,had been shown to organized filaments into higher-order actin structures.However,the relationship of the two genes' functions has remained unknown.Therefore,the study of AtFIM4 and AtFIM5 in pollen tube is not only of great significance to reveal the regulation mechanism of the microfilament cytoskeleton in plants,but also helpful to understand the key role of the microfilament cytoskeleton in sexual reproduction in plants.Here,we functionally investigated the regulation of actin-binding proteins AtFIM4 and AtFIM5 in pollen tubes,and explored the functional links between the two genes.The main results are as follows:(1)Bioinformatics analysis showed that AtFIM4 and AtFIM5 had overlapping and distinct activities,but they belonged to different branches in the phylogenetic tree,indicating that they may had different functions while preserving family conservatism.(2)Compared with wild-type Col-0,the phenotypes of T-DNA insertion single mutants were not significantly different,but the double mutants were significantly different.As follows:pollen germination rate was decreased,pollen tube growth was inhibited,the deformity ratio was increased,the length of silique becomed shorter,and the actin bundles in pollen tubes were decreased.Results all showed that AtFIM4 and AtFIM5 were critical for the maintenance of microfilament cytoskeleton in pollen tubes,and loss of AtFIM4 and AtFIM5 functions could inhibit the pollen tube growth.(3)The expression patterns of AtFIM4 and AtFIM5 in pollen tubes were shown by the histochemical staining of GUS(?-glucuronidase).Compared with AtFIM4 only expressed in pollen tube growth stage,AtFIM5 also expressed in pollen gains.(4)By the biochemical experiments in vitro,we demonstrated that the abilities of binding and bundling actin of AtFIM4 were stronger than AtFIM5.In addition,AtFIM4 and AtFIM5 could inhibit the depolymerization of microfilaments by Lat B(latrunculin B).In order to observe the mechanisms of actin bundles formation,we used laser scanning confocal microscopy.Results showed that AtFIM4 and AtFIM5 could both bind actin in vitro.AtFIM4 generated a loose network structure,whereas AtFIM5 generated tight actin bundles.(5)In order to explore the key sites of the AtFIM5 amino acid sequences in the actin binding function,we constructed a single site mutant protein AtFIM5-461 and a double sites mutant protein AtFIM5-589,and carried out a series of biochemical experiments in vitro.Indicating that AtFIM5-461 have no efficient effects on the ability of AtFIM5 binding actin,whereas AtFIM5-5 89 enhanced the ability to a certain extent.In addition,AtFIM5-461 and AtFIM5-589 did not effect the function of AtFIM5 in generating distinct actin structures.In summary,the effects of AtFIM4 and AtFIM5 on pollen germination and pollen tube growth in Arabidopsis were studied by molecular biology,cell biology and biochemistry in vitro.Our study showed that AtFIM4 and AtFIM5 play important roles in the formation of microfilament cytoskeleton in pollen tubes,which is necessary for the polar growth of the cells.In addition,we also explored the functional contact and division of the two genes in regulating pollen germination and pollen tube growth.