Function And Mechanism Of Arabidopsis Profilin During Pollen Tube Growth

Pollen tube growth is a crucial step for double fertilization in flowering plants. It’s characterized by polarized tip elongation that relies on a population of highly dynamic actin filaments. The arrangement and dynamics of actin filaments is tightly regulated by a collection of different actin binding proteins. Profilin is an ubiquitous actin binding protein that form a 1:1 complex with G-actin, regulating both actin polymerization and depolymerization. However, the role of profilin in regulating actin dynamics in pollen tubes remains largely unclear. The present study selected reproductive Arabidopsis PRF4 and PRF5 to dissect the mechanisms by which profilins regulate pollen tube tip growth.Observations showed no obvious differences in germination between wild type and profilin mutants. However, the pollen tube growth rate of profilin mutants was lower than that of wild type and the morphology of prf4 prf5 double mutant pollen tubes was abnormal. These observations indicated that PRF4 and PRF5 redundantly regulate polarized pollen tube growth.To dissect the mechanisms by which profilins regulate pollen tube tip growth, actin cytoskeleton in the pollen tubes of profilin mutants was examined. Observations showed that the amount of F-actin was decreased and the arrangement of actin filaments was disordered in apical and subapical region of profilin mutant pollen tubes. Live cell imaging showed that actin filaments labeled by Lifeact-EGFP continuously emerge from the apical membrane of prf4 prf5 pollen tubes, but they cannot elongate effectively. Subapical actin structures have been suggested to play an important role in vesicle accumulation. Consistent with this, transport vesicles labeled by YFP-RabA4b were more diffusely localized in prf4 prf5 pollen tubes. In addition, the transport velocity was decreased and the composition of cell wall was altered. These results demonstrate that PRF4 and PRF5 promote actin assembly in apical region of pollen tubes. PRF5 was then selected as the representative profilin to generate the PLP (poly-L-proline) binding deficient mutant PRF5Y6A·Both F-actin amount in the apical-subapical region and pollen tube growth rate of transgenic plants (pgPRF5y6A;prf4 prf5) cannot restore to that of prf4. Biochemical assays showed that PRF5-actin complex can be effectively used by formin5, but PRF5Y6A-actin complex cannot. These results demonstrate that profilin’s role in driving actin polymerization may be mediated by formin.To investigate the role of profilins in regulating actin dynamics, germination of pollen grains and growth of pollen tubes were compared between wild type and profilin mutants under the treatment of LatB. It was shown that both the germination of pollen grains and the growh of pollen tubes were less sensitive to LatB treatment in profilin mutants. These observations indicated that profilins promote actin turnover. The actin binding deficient mutant PRF5K89E cannot rescue the phenotype of prf5 pollen tubes, but PRF5Y6A can. This suggests that profilins promote actin turnover by sequestering actin monomers and this activity is independent of PLP binding.In addition, N-terminal GFP fusions of PRF4 and PRF5 distributed evenly in pollen tubes, suggesting that profilin-actin complexes are uniformly localized in the cytoplasm of pollen tubes.This study demonstrates that evenly distributed PRF4 and PRF5 are key regulators of pollen tube growth. They promote actin turnover through the G-actin binding activity whereas drive actin polymerization dependently on formin, which may represent the key pathway to polymerize actin filaments in pollen tube tips.