ORCID IDs: 0000-0002-6213-9791 (D.Q.); 0000-0002-5178-0700 (Y.X.)Functional divergence in paralogs is an important genetic source of evolutionary innovation. Actin-depolymerizing factors (ADFs) are among the most important actin binding proteins and are involved in generating and remodeling actin cytoskeletal architecture via their conserved F-actin severing or depolymerizing activity. In plants, ADFs coevolved with actin, but their biochemical properties are diverse. Unfortunately, the biochemical function of most plant ADFs and the potential mechanisms of their functional divergence remain unclear. Here, in vitro biochemical analyses demonstrated that all 11 ADF genes in Arabidopsis thaliana exhibit opposing biochemical properties. Subclass III ADFs evolved F-actin bundling (B-type) function from conserved F-actin depolymerizing (D-type) function, and subclass I ADFs have enhanced D-type function. By tracking historical mutation sites on ancestral proteins, several fundamental amino acid residues affecting the biochemical functions of these proteins were identified in Arabidopsis and various plants, suggesting that the biochemical divergence of ADFs has been conserved during the evolution of angiosperm plants. Importantly, N-terminal extensions on subclass III ADFs that arose from intron-sliding events are indispensable for the alteration of D-type to B-type function. We conclude that the evolution of these N-terminal extensions and several conserved mutations produced the diverse biochemical functions of plant ADFs from a putative ancestor.

ADF5 promotes stomatal closure by regulating actin filament dynamics, and members of the ABF/AREB transcription factor family may serve as potential upstream regulators of ADF5 in the drought stress/ABA signaling pathway.

The regulation of pollen development and pollen tube growth is a complicated biological process that is crucial for sexual reproduction in flowering plants. Annexins are widely distributed from protists to higher eukaryotes and play multiple roles in numerous cellular events by acting as a putative “linker” between Ca2+ signaling, the actin cytoskeleton and the membrane, which are required for pollen development and pollen tube growth. Our recent report suggested that downregulation of the function of Arabidopsis annexin 5 (Ann5) in transgenic Ann5-RNAi lines caused severely sterile pollen grains. However, little is known about the underlying mechanisms of the function of Ann5 in pollen. This study demonstrated that Ann5 associates with phospholipid membrane and this association is stimulated by Ca2+ in vitro. Brefeldin A (BFA) interferes with endomembrane trafficking and inhibits pollen germination and pollen tube growth. Both pollen germination and pollen tube growth of Ann5-overexpressing plants showed increased resistance to BFA treatment, and this effect was regulated by calcium. Overexpression of Ann5 promoted Ca2+-dependent cytoplasmic streaming in pollen tubes in vivo in response to BFA. Lactrunculin (LatB) significantly prohibited pollen germination and tube growth by binding with high affinity to monomeric actin and preferentially targeting dynamic actin filament arrays and preventing actin polymerization. Overexpression of Ann5 did not affect pollen germination or pollen tube growth in response to LatB compared with wild-type, although Ann5 interacts with actin filaments in a manner similar to some animal annexins. In addition, the sterile pollen phenotype could be only partially rescued by Ann5 mutants at Ca2+-binding sites when compared to the complete recovery by wild-type Ann5. These data demonstrated that Ann5 is involved in pollen development, germination and pollen tube growth through the promotion of endomembrane trafficking modulated by calcium. Our results provide reliable molecular mechanisms that underlie the function of Ann5 in pollen.

Dynamics of the actin cytoskeleton are essential for pollen germination and pollen tube growth. ACTIN-DEPOLYMERIZING FACTORs (ADFs) typically contribute to actin turnover by severing/depolymerizing actin filaments. Recently, we demonstrated that Arabidopsis subclass III ADFs (ADF5 and ADF9) evolved F-actin-bundling function from conserved F-actin-depolymerizing function. However, little is known about the physiological function, the evolutional significance, and the actin-bundling mechanism of these neofunctionalized ADFs. Here, we report that loss of ADF5 function caused delayed pollen germination, retarded pollen tube growth, and increased sensitive to latrunculin B (LatB) treatment by affecting the generation and maintenance of actin bundles. Examination of actin filament dynamics in living cells revealed that the bundling frequency was significantly decreased in adf5 pollen tubes, consistent with its biochemical functions. Further biochemical and genetic complementation analyses demonstrated that both the N- and C-terminal actin-binding domains of ADF5 are required for its physiological and biochemical functions. Interestingly, while both are atypical actin-bundling ADFs, ADF5, but not ADF9, plays an important role in mature pollen physiological activities. Taken together, our results suggest that ADF5 has evolved the function of bundling actin filaments and plays an important role in the formation, organization, and maintenance of actin bundles during pollen germination and pollen tube growth.

Vacuolar H؉ -ATPase (V-ATPase) is a membrane-bound multisubunit enzyme complex composed of at least 14 different subunits. The complex regulates the physiological processes of a cell by controlling the acidic environment, which is necessary for certain activities and the interaction with the actin cytoskeleton through its B and C subunits in both humans and yeast. Arabidopsis V-ATPase has three B subunits (AtVAB1, AtVAB2, and AtVAB3), which share 97.27% sequence identity and have two potential actin-binding sites, indicating that these AtVABs may have crucial functions in actin cytoskeleton remodeling and plant cell development. However, their biochemical functions are poorly understood. In this study, we demonstrated that AtVABs bind to and co-localize with F-actin, bundle F-actin to form higher order structures, and stabilize actin filaments in vitro. In addition, the AtVABs also show different degrees of activities in capping the barbed ends but no nucleating activities, and these activities were not regulated by calcium. The functional similarity and differences of the AtVABs implied that they may play cooperative and distinct roles in Arabidopsis cells.