The Mechanism Of Regulating Pollen Tube Growth By Arabidopsis MICROTUBULE-DESTABILIZING PROTEIN25

The germination and growth of pollen tube play a crucial role in plant gamogenesis. Pollen grains germinate and produce pollen tubes on the stigma of the pistil, which grow through the style and are guided to the micropylar. Then the growth of pollen tubes is arrested and the tube tips rupture to release the sperm cells. Therefore, the positive and negative regulation of pollen tube growth have important physiological significances. Actin filaments play critical roles in regulation of pollen tube growth. The organization and dynamics of actin fimaments are regulated by many developmental signals and environmental cues. However, the regulation mechanisms of actin filament organization and dynamics are poorly understood.In this study, we investigated the actin filament orgnization and dynamics in Arabidopsis pollen tubes and the role of a calcium-binding and microtubule-destabilizing protein (MDP25) in negative regulation of pollen tube growth by modulating the actin cytoskeleton in the subapical region.The data from the Affymetrix AG and ATH1GeneChip arrays in the Genevestigator database (http://www.genevestigator.ethz.ch) indicate that MDP25is expressed in pollen. GUS staining aslo revealed that MDP25was expressed in the pollen and pollen tubes. Statistical analysis indicated that the growth rate of mdp25-1pollen tubes was significantly higher than that of wild-type. Furthermore, aniline blue staining showed that mdp25-1pollen tube grow faster than wild-type pollen tube.Recombinant MDP25was expressed and purified from Escherichia coli. Cosedimentation assay showed that recombinant MDP25directly binds F-actin in vitro. Confocal observation and TIRFM assay revealed that MDP25is capable of severing actin filaments and the severing activity of MDP25is significantly enhanced in the presence of Ca2+in vitro.Sub-cellular localization assay showed that MDP25-GFP was mainly localized in the plasma membrane at the shank region of the pollen tube, but was absent at the membrane of the subapical and apex regions. A fluorescence microscopy assay showed that the mutated MDP25protein (Ca2+-binding site) is predominantly localized to the plasma membrane along the whole length of the pollen tube. The F-actin-severing frequency was significantly decreased and high density of actin filaments was observed in the subapical region of pollen tubes from the mdp25-1mutant.MDP25K7A and MDP25K18A exhibited similar actin filament binding and severing activity as wild-type MDP25. MDP25N5A maintained normal function on microtubules, however lost its actin filament binding and severing activity. Genetic evidence demonstrates that MDP25regulates pollen tube elongation by regulating actin filaments, but not by its microtubule-destabilizing activity.Our study suggests that Arabidopsis MDP25is a novel actin binding protein (ABP) regulated by Ca2+and MDP25is involved in negative regulation of pollen tube growth by modulating the actin cytoskeleton in the subapical region via its F-actin-severing activity.