A population of dynamic apical actin filaments is required for rapid polarized pollen tube growth. However, the cellular mechanisms driving their assembly remain incompletely understood. It was ...postulated that formin is a major player in nucleating apical actin assembly, but direct genetic and cytological evidence remains to be firmly established. Here we found that both Arabidopsis formin 3 (AtFH3) and formin 5 (AtFH5) are involved in the regulation of apical actin polymerization and actin array construction in pollen tubes, with AtFH3 playing a more dominant role. We found that both formins have plasma membrane (PM) localization signals but exhibit distinct PM localization patterns in the pollen tube, and loss of their function reduces the amount of apical actin filaments. Live-cell imaging revealed that the reduction in filamentous actin is very likely due to the decrease in filament elongation. Furthermore, we found that the rate of tip-directed vesicle transport is reduced and the pattern of apical vesicle accumulation is altered in formin loss-of-function mutant pollen tubes, which explains to some extent the reduction in pollen tube elongation. Thus, we provide direct genetic and cytological evidence showing that formin is an important player in nucleating actin assembly from the PM at pollen tube tips.
Summary
Actin is an ancient conserved protein that is encoded by multiple isovariants in multicellular organisms. There are eight functional actin genes in the Arabidopsis genome, and the precise ...function and mechanism of action of each isovariant remain poorly understood. Here, we report the characterization of ACT11, a reproductive actin isovariant. Our studies reveal that loss of function of ACT11 causes a delay in pollen germination, but enhances pollen tube growth. Cytological analysis revealed that the amount of filamentous actin decreased, and the rate of actin turnover increased in act11 pollen. Convergence of actin filaments upon the germination aperture was impaired in act11 pollen, consistent with the observed delay of germination. Reduction of actin dynamics with jasplakinolide suppressed the germination and tube growth phenotypes in act11 pollen, suggesting that the underlying mechanisms involve an increase in actin dynamics. Thus, we demonstrate that ACT11 is required to maintain the rate of actin turnover in order to promote pollen germination and maintain the normal rate of pollen tube growth.
Significance Statement
Actin is normally encoded by multiple isovariants in multicellular organisms, and actin isovariants have been shown to be functionally non‐equivalent. However, exactly how individual actin isovariants contribute to actin dynamics remains to be fully elucidated. Here, we demonstrate that ACT11 is required to maintain the rate of actin turnover in pollen to promote pollen germination and maintain the normal rate of tube growth.
Cell-to-cell communication in plants is mediated by plasmodesmata (PD) whose permeability is tightly regulated during plant growth and development. The actin cytoskeleton has been implicated in ...regulating the permeability of PD, but the underlying mechanism remains largely unknown. Recent characterization of PD-localized formin proteins has shed light on the role and mechanism of action of actin in regulating PD-mediated intercellular trafficking. In this mini-review article, we will describe the progress in this area.
Srv2p/CAP1 is an essential regulator of actin turnover, but its exact function in regulating actin polymerization, particularly the contribution of its actin nucleotide exchange activity, remains ...incompletely understood. We found that, although Arabidopsis CAP1 is distributed uniformly in the cytoplasm, its loss of function has differential effects on the actin cytoskeleton within different regions of the pollen tube. Specifically, the F-actin level increases in the shank but decreases in the apical region of cap1 pollen tubes. The reduction in apical F-actin results mainly from impaired polymerization of membrane-originated actin within cap1 pollen tubes. The actin nucleotide exchange activity of CAP1 is involved in apical actin polymerization. CAP1 acts synergistically with pollen ADF and profilin to promote actin turnover in vitro, and it can overcome the inhibitory effects of ADF and synergize with profilin to promote actin nucleotide exchange. Consistent with its role as a shuttle molecule between ADF and profilin, the cytosolic concentration of CAP1 is much lower than that of ADF and profilin in pollen. Thus, CAP1 synergizes with ADF and profilin to drive actin turnover in pollen and promote apical actin polymerization in pollen tubes in a manner that involves its actin nucleotide exchange activity.
The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, but the underlying mechanism for ...this cytoskeletal reorganization remains largely unclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2 does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actin filaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actin depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive to ABA. Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filament reorganization and stomatal closure mainly through phosphorylation of ADF.
Tight regulation of gene transcription and mRNA splicing is essential for plant growth and development. Here we demonstrate that a plant-specific protein, EMBRYO DEFECTIVE 1579 (EMB1579), controls ...multiple growth and developmental processes in Arabidopsis. We demonstrate that EMB1579 forms liquid-like condensates both in vitro and in vivo, and the formation of normal-sized EMB1579 condensates is crucial for its cellular functions. We found that some chromosomal and RNA-related proteins interact with EMB1579 compartments, and loss of function of EMB1579 affects global gene transcription and mRNA splicing. Using floral transition as a physiological process, we demonstrate that EMB1579 is involved in FLOWERING LOCUS C (FLC)-mediated repression of flowering. Interestingly, we found that EMB1579 physically interacts with a homologue of Drosophila nucleosome remodeling factor 55-kDa (p55) called MULTIPLE SUPPRESSOR OF IRA 4 (MSI4), which has been implicated in repressing the expression of FLC by forming a complex with DNA Damage Binding Protein 1 (DDB1) and Cullin 4 (CUL4). This complex, named CUL4-DDB1MSI4, physically associates with a CURLY LEAF (CLF)-containing Polycomb Repressive Complex 2 (CLF-PRC2). We further demonstrate that EMB1579 interacts with CUL4 and DDB1, and EMB1579 condensates can recruit and condense MSI4 and DDB1. Furthermore, emb1579 phenocopies msi4 in terms of the level of H3K27 trimethylation on FLC. This allows us to propose that EMB1579 condensates recruit and condense CUL4-DDB1MSI4 complex, which facilitates the interaction of CUL4-DDB1MSI4 with CLF-PRC2 and promotes the role of CLF-PRC2 in establishing and/or maintaining the level of H3K27 trimethylation on FLC. Thus, we report a new mechanism for regulating plant gene transcription, mRNA splicing, and growth and development.
Polarized tip growth is a fundamental cellular process in many eukaryotes. In this study, we examined the dynamic restructuring of the actin cytoskeleton and its relationship to vesicle transport ...during pollen tip growth in Arabidopsis. We found that actin filaments originating from the apical membrane form a specialized structure consisting of longitudinally aligned actin bundles at the cortex and inner cytoplasmic fila- ments with a distinct distribution. Using actin-based pharmacological treatments and genetic mutants in combination with FRAP （fluorescence recovery after photobleaching） technology to visualize the transport of vesicles within the growth domain of pollen tubes, we demonstrated that cortical actin filaments facilitate tip-ward vesicle transport. We also discovered that the inner apical actin filaments prevent backward movement of vesicles, thus ensuring that sufficient vesicles accumulate at the pollen tube tip to support the rapid growth of the pollen tube. The combinatorial effect of cortical and internal apical actin filaments perfectly explains the generation of the inverted ＂V＂ cone-shaped vesicle distribution pattern at the pollen tube tip. When pollen tubes turn, apical actin filaments at the facing side undergo depolymerization and repolymerization to reorient the apical actin structure toward the new growth direction. This actin restructuring precedes vesicle accumulation and changes in tube morphology. Thus, our study provides new insights into the functional relationship between actin dynamics and vesicle transport during rapid and directional pollen tube growth.
Here, we demonstrate that
Formin 2 (AtFH2) localizes to plasmodesmata (PD) through its transmembrane domain and is required for normal intercellular trafficking. Although loss-of-function
mutants ...have no overt developmental defect, PD's permeability and sensitivity to virus infection are increased in
plants. Interestingly, AtFH2 functions in a partially redundant manner with its closest homolog AtFH1, which also contains a PD localization signal. Strikingly, targeting of Class I formins to PD was also confirmed in rice, suggesting that the involvement of Class I formins in regulating actin dynamics at PD may be evolutionarily conserved in plants. In vitro biochemical analysis showed that AtFH2 fails to nucleate actin assembly but caps and stabilizes actin filaments. We also demonstrate that the interaction between AtFH2 and actin filaments is crucial for its function in vivo. These data allow us to propose that AtFH2 regulates PD's permeability by anchoring actin filaments to PD.
Regulation of actin dynamics is a central theme in cell biology that is important for different aspects of cell physiology. Villin, a member of the villin/gelsolin/fragmin superfamily of proteins, is ...an important regulator of actin. Villins contain six gelsolin homology domains （G1-G6） and an extra headpiece domain. In contrast to their mammalian counterparts, plant villins are expressed widely, implying that plant villins play a more general role in regulating actin dynamics. Some plant villins have a defined role in modifying actin dynamics in the pollentube; most of their in vivo activities remain to be ascertained. Recently, our understanding of the functions and mechanisms of action for plant villins has progressed rapidly, primarily due to the advent of Arabidopsis thaliana genetic approaches and imaging capabilities that can visualize actin dynamics at the single filament level in vitro and in living plant cells. In this review, we focus on discussing the biochemical activities and modes of regulation of plant villins. Here, we present current understand- ing of the functions of plant villins. Finally, we highlight some of the key unanswered questions regarding the functions and regulation of plant villins for future research.
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