ABSTRACT
Pollen germination is critical for the reproduction of flowering plants. Formin-dependent actin polymerization plays vital roles in vesicle trafficking and polarity establishment during this process. However, how formin-mediated actin assembly is regulated in vivo remains poorly understood. Here, we investigated the function of reproductive profilin 4 and 5 (PRF4 and PRF5) in polarity establishment during pollen germination in Arabidopsis thaliana. Our data showed that the actin filament content was reduced in the prf4 prf5 double mutant and substantially increased in both PRF4- and PRF5-overexpressing pollen grains. By contrast, the positive effect of profilin in promoting actin polymerization was abolished in a formin mutant, atfh5. In addition, the interaction between Arabidopsis formin homology 5 (AtFH5) and actin filaments was attenuated and the trafficking of AtFH5-labeled vesicles was slowed in prf4 prf5 pollen grains. Formation of the collar-like structure at the germination pore was also defective in prf4 prf5 pollen grains as the fast assembly of actin filaments was impaired. Together, our results suggest that PRF4 and PRF5 regulate vesicle trafficking and polarity establishment during pollen germination by promoting AtFH5-mediated actin polymerization and enhancing the interaction between AtFH5 and actin filaments.
Subject(s)
Actin Cytoskeleton/metabolism , Arabidopsis Proteins/metabolism , Cell Cycle Proteins/metabolism , Pollen/cytology , Profilins/metabolism , Actin Cytoskeleton/genetics , Arabidopsis/cytology , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Biological Transport , Cell Cycle Proteins/genetics , Mutation , Plants, Genetically Modified , Pollen/physiology , Profilins/genetics , Time-Lapse ImagingABSTRACT
Meiotic crossovers ensure accurate chromosome segregation and increase genetic diversity. RAD51C and RAD51D play an early role in facilitating RAD51 during homologous recombination. However, their later function in meiosis is largely unknown in plants. Here, through targeted disruption of RAD51C and RAD51D, we generated three new mutants and revealed their later meiotic role in crossover maturation. The rad51c-3 and rad51d-4 mutants showed a mixture of bivalents and univalents and no chromosomal entanglements, whereas rad51d-5 exhibited an intermediate phenotype with reduced chromosomal entanglements and increased bivalent formation compared with knockout alleles. Comparisons of RAD51 loadings and chromosomal entanglements in these single mutants, rad51c-3 rad51d-4, rad51c-3 dmc1a dmc1b, and rad51d-4 dmc1a dmc1b suggest that the retained level of RAD51 in mutants is required for uncovering their function in crossover formation. Reductions in chiasma frequency and later HEI10 foci in these mutants support that crossover maturation requires RAD51C and RAD51D. Moreover, interaction between RAD51D and MSH5 indicates that RAD51 paralogs may cooperate with MSH5 to ensure accurate Holliday junction processing into crossover products. This finding of the role of RAD51 paralogs in crossover control may be conserved from mammals to plants and advances our current understanding of these proteins.
Subject(s)
Oryza , Animals , Oryza/genetics , Oryza/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Meiosis/genetics , Homologous Recombination , MammalsABSTRACT
The phragmoplast, a structure crucial for the completion of cytokinesis in plant cells, is composed of antiparallel microtubules (MTs) and actin filaments (AFs). However, how the parallel structure of phragmoplast MTs and AFs is maintained, especially during centrifugal phragmoplast expansion, remains elusive. Here, we analyzed a new Arabidopsis thaliana MT and AF crosslinking protein (AtMAC). When AtMAC was deleted, the phragmoplast showed disintegrity during centrifugal expansion, and the resulting phragmoplast fragmentation led to incomplete cell plates. Overexpression of AtMAC increased the resistance of phragmoplasts to depolymerization and caused the formation of additional phragmoplasts during cytokinesis. Biochemical experiments showed that AtMAC crosslinked MTs and AFs in vitro, and the truncated AtMAC protein, N-CC1, was the key domain controlling the ability of AtMAC. Further analysis showed that N-CC1(51-154) is the key domain for binding MTs, and N-CC1(51-125) for binding AFs. In conclusion, AtMAC is the novel MT and AF crosslinking protein found to be involved in regulation of phragmoplast organization during centrifugal phragmoplast expansion, which is required for complete cytokinesis.
Subject(s)
Arabidopsis , Cytokinesis , Cytokinesis/physiology , Microtubules/metabolism , Microtubule-Associated Proteins/metabolism , Arabidopsis/metabolism , Actin Cytoskeleton/metabolismABSTRACT
Actins are among the most abundant and conserved proteins in eukaryotic cells, where they form filamentous structures that perform vital roles in key cellular processes. Although large amounts of data on the biochemical activities, dynamic behaviors, and important cellular functions of plant actin filaments have accumulated, their structural basis remains elusive. Here, we report a 3.9 Å structure of the plant actin filament from Zea mays pollen (ZMPA) using cryo-electron microscopy. The structure shows a right-handed, double-stranded (two parallel strands) and staggered architecture that is stabilized by intra- and interstrand interactions. While the overall structure resembles that of other actin filaments, its DNase I binding loop bends farther outward, adopting an open conformation similar to that of the jasplakinolide- or beryllium fluoride (BeFx)-stabilized rabbit skeletal muscle actin (RSMA) filament. Single-molecule magnetic tweezers analysis revealed that the ZMPA filament can resist a greater stretching force than the RSMA filament. Overall, these data provide evidence that plant actin filaments have greater stability than animal actin filaments, which might be important to their role as tracks for long-distance vesicle and organelle transportation.plantcell;31/12/2855/FX1F1fx1.
Subject(s)
Actin Cytoskeleton/chemistry , Pollen/chemistry , Zea mays/chemistry , Actin Cytoskeleton/metabolism , Cryoelectron Microscopy , Hydrophobic and Hydrophilic Interactions , Pollen/metabolism , Protein Conformation , Protein Subunits/chemistry , Zea mays/metabolismABSTRACT
BACKGROUND INFORMATION: In many cellular processes including cell division, the synergistic dynamics of actin filaments and microtubules play vital roles. However, the regulatory mechanisms of these synergistic dynamics are not fully understood. Proteins such as formins are involved in actin filament-microtubule interactions and Arabidopsis thaliana formin 14 (AtFH14) may function as a crosslinker between actin filaments and microtubules in cell division, but the molecular mechanism underlying such crosslinking remains unclear. RESULTS: Without microtubules, formin homology (FH) 1/FH2 of AtFH14 nucleated actin polymerisation from actin monomers and capped the barbed end of actin filaments. However, in the presence of microtubules, quantitative analysis showed that the binding affinity of AtFH14 FH1FH2 to microtubules was higher than that to actin filaments. Moreover, microtubule-bound AtFH14 FH1FH2 neither nucleated actin polymerisation nor inhibited barbed end elongation. In contrast, tubulin did not affect AtFH14 FH1FH2 to nucleate actin polymerisation and inhibit barbed end elongation. Nevertheless, microtubule-bound AtFH14 FH1FH2 bound actin filaments and the bound actin filaments slid and elongated along the microtubules or elongated away from the microtubules, which induced bundling or crosslinking of actin filaments and microtubules. Pharmacological analyses indicated that AtFH14 FH1FH2 promoted crosslinking of actin filaments and microtubules in vivo. Additionally, co-sedimentation and fluorescent dye-labelling experiments of AtFH14 FH2-truncated proteins in vitro revealed the essential motifs of bundling actin filaments or microtubules, which were 63-92 aa and 42-62 aa in the AtFH14 FH2 N-terminal, respectively, and 42-62 aa was the essential motif to crosslink actin filaments and microtubules. CONCLUSIONS AND SIGNIFICANCE: Our results aid in explaining how AtFH14 functions as a crosslinker between actin filaments and microtubules to regulate their dynamics via different manners during cell division. They also facilitate further understanding of the molecular mechanisms of the interactions between actin filaments and microtubules.
Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Formins/metabolism , Microtubules/metabolism , Animals , Cell Division , Protein Binding , RabbitsABSTRACT
The actin cytoskeleton is crucial for plant morphogenesis, and organization of actin filaments (AF) is dynamically regulated by actin-binding proteins. However, the roles of actin-binding proteins, particularly type II formins, in this process remain poorly understood in plants. Here, we report that a type II formin in rice, Oryza sativa formin homolog 3 (OsFH3), acts as a major player to modulate AF dynamics and contributes to rice morphogenesis. osfh3 mutants were semi-dwarf with reduced size of seeds and unchanged responses to light or gravity compared with mutants of osfh5, another type II formin in rice. osfh3 osfh5 mutants were dwarf with more severe developmental defectiveness. Recombinant OsFH3 could nucleate actin, promote AF bundling, and cap the barbed end of AF to prevent elongation and depolymerization, but in the absence of profilin, OsFH3 could inhibit AF elongation. Different from other reported type II formins, OsFH3 could bind, but not bundle, microtubules directly. Furthermore, its N-terminal phosphatase and tensin homolog domain played a key role in modulating OsFH3 localization at intersections of AF and punctate structures of microtubules, which differed from other reported plant formins. Our results, thus, provide insights into the biological function of type II formins in modulating plant morphology by acting on AF dynamics.
Subject(s)
Formins/genetics , Formins/metabolism , Oryza/growth & development , Actin Cytoskeleton/metabolism , Formins/chemistry , Morphogenesis , Mutation , Organ Size , Oryza/genetics , Oryza/metabolism , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Domains , Seeds/genetics , Seeds/growth & development , Seeds/metabolismABSTRACT
Auxin transport inhibitors are essential tools for understanding auxin-dependent plant development. One mode of inhibition affects actin dynamics; however, the underlying mechanisms remain unclear. In this study, we characterized the action of 2,3,5-triiodobenzoic acid (TIBA) on actin dynamics in greater mechanistic detail. By surveying mutants for candidate actin-binding proteins with reduced TIBA sensitivity, we determined that Arabidopsis (Arabidopsis thaliana) villins contribute to TIBA action. By directly interacting with the C-terminal headpiece domain of villins, TIBA causes villin to oligomerize, driving excessive bundling of actin filaments. The resulting changes in actin dynamics impair auxin transport by disrupting the trafficking of PIN-FORMED auxin efflux carriers and reducing their levels at the plasma membrane. Collectively, our study provides mechanistic insight into the link between the actin cytoskeleton, vesicle trafficking, and auxin transport.
Subject(s)
Actins/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/drug effects , Indoleacetic Acids/metabolism , Microfilament Proteins/antagonists & inhibitors , Plant Growth Regulators/metabolism , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Biological Transport/drug effects , Cell Membrane/metabolism , Microfilament Proteins/drug effects , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Mutation , Protein Transport/drug effects , Triiodobenzoic Acids/pharmacologyABSTRACT
Chemical reactions inside single crystals are highly selective but quite challenging. We present herein an octahedral cobalt-oxygen chain-based 3D coordination network with sqc3868 topology, which underwent a reversible double nucleophilic substitution inside a single crystal involving encapsulated DMF molecules and was converted into a topologically highly related frl network, accompanied by magnetic tuning from antiferromagnetism to ferromagnetism. Combined UV-vis, XPS, EPR, and XANES showed most of the Co centers keep a divalent state with less remarkable electronic structure change during the substitution reaction, indicating magnetic tunability mainly comes from a minor change of local geometry of cobalt atoms with large anisotropy.
ABSTRACT
Pollen tube tip growth is an extreme form of polarized cell growth, which requires polarized exocytosis based on a dynamic actin cytoskeleton. However, the molecular basis for the connection between actin filaments and exocytic vesicles is unclear. Here, we identified a Lilium longiflorum pollen-specific formin (LlFH1) and found that it localized at the apical vesicles and plasma membrane (PM). Overexpression of LlFH1 induced excessive actin cables in the tube tip region, and downregulation of LlFH1 eliminated the actin fringe. Fluorescence recovery after photobleaching (FRAP) analysis revealed that LlFH1-labeled exocytic vesicles exhibited an initial accumulation at the shoulder of the apex and coincided with the leading edge of the actin fringe. Time-lapse analysis suggested that nascent actin filaments followed the emergence of the apical vesicles, implying that LlFH1 at apical vesicles could initiate actin polymerization. Biochemical assays showed that LlFH1 FH1FH2 could nucleate actin polymerization, but then capped the actin filament at the barbed end and inhibited its elongation. However, in the presence of lily profilins, LlFH1 FH1FH2 could accelerate barbed-end actin elongation. In addition, LlFH1 FH1FH2 was able to bundle actin filaments. Thus, we propose that LlFH1 and profilin coordinate the interaction between the actin fringe and exocytic vesicle trafficking during pollen tube growth of lily.
Subject(s)
Actins/metabolism , Exocytosis , Lilium/cytology , Lilium/metabolism , Plant Proteins/metabolism , Pollen Tube/growth & development , Pollen Tube/metabolism , Secretory Vesicles/metabolism , Actin Cytoskeleton/metabolism , Cell Membrane/metabolism , Cloning, Molecular , Lilium/growth & development , Polymerization , Protein BindingABSTRACT
The plant hormone auxin plays a central role in plant growth and development. Auxin transport and signaling depend on actin organization. Despite its functional importance, the mechanistic link between actin filaments (F-actin) and auxin intracellular signaling remains unclear. Here, we report that the actin-organizing protein Rice Morphology Determinant (RMD), a type II formin from rice (Oryza sativa), provides a key link. Mutants lacking RMD display abnormal cell growth and altered configuration of F-actin array direction. The rmd mutants also exhibit an inhibition of auxin-mediated cell elongation, decreased polar auxin transport, altered auxin distribution gradients in root tips, and suppression of plasma membrane localization of auxin transporters O. sativa PIN-FORMED 1b (OsPIN1b) and OsPIN2 in root cells. We demonstrate that RMD is required for endocytosis, exocytosis, and auxin-mediated OsPIN2 recycling to the plasma membrane. Moreover, RMD expression is directly regulated by heterodimerized O. sativa auxin response factor 23 (OsARF23) and OsARF24, providing evidence that auxin modulates the orientation of F-actin arrays through RMD. In support of this regulatory loop, osarf23 and lines with reduced expression of both OsARF23 and OsARF24 display reduced RMD expression, disrupted F-actin organization and cell growth, less sensitivity to auxin response, and altered auxin distribution and OsPIN localization. Our findings establish RMD as a crucial component of the auxin-actin self-organizing regulatory loop from the nucleus to cytoplasm that controls rice cell growth and morphogenesis.
Subject(s)
Cell Nucleus/metabolism , Cell Proliferation , Cytoplasm/metabolism , Microfilament Proteins/metabolism , Oryza/metabolism , Plant Proteins/metabolism , Plant Roots/metabolism , Actins/genetics , Actins/metabolism , Cell Nucleus/genetics , Cytoplasm/genetics , Microfilament Proteins/genetics , Oryza/genetics , Plant Proteins/genetics , Plant Roots/geneticsABSTRACT
An actin fringe structure in the subapex plays an important role in pollen tube tip growth. However, the precise mechanism by which the actin fringe is generated and maintained remains largely unknown. Here, we cloned a 2606-bp full-length cDNA encoding a deduced 77-kD fimbrin-like protein from lily (Lilium longiflorum), named FIMBRIN1 (FIM1). Ll-FIM1 was preferentially expressed in pollen and concentrated at actin fringe in the subapical region, as well as in longitudinal actin-filament bundles in the shank of pollen tubes. Microinjection of Ll-FIM1 antibody into lily pollen tubes inhibited tip growth and disrupted the actin fringe. Furthermore, we verified the function of Ll-FIM1 in the fim5 mutant of its closest relative, Arabidopsis thaliana. Pollen tubes of fim5 mutants grew with a larger diameter in early stages but could recover into normal forms in later stages, despite significantly slower growth rates. The actin fringe of the fim5 mutants, however, was impaired during both early and late stages. Impressively, stable expression of fim5pro:GFP:Ll-FIM1 rescued the actin fringe and the growth rate of Arabidopsis fim5 pollen tubes. In vitro biochemical analysis showed that Ll-FIM1 could bundle actin filaments. Thus, our study has identified a fimbrin that may stabilize the actin fringe by cross-linking actin filaments into bundles, which is important for proper tip growth of lily pollen tubes.
Subject(s)
Actin Cytoskeleton/metabolism , Gene Expression Regulation, Plant , Lilium/genetics , Membrane Glycoproteins/genetics , Microfilament Proteins/genetics , Pollen Tube/genetics , Actins/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis/ultrastructure , DNA, Complementary/genetics , Gene Expression , Gene Expression Regulation, Developmental , Lilium/growth & development , Lilium/metabolism , Lilium/ultrastructure , Membrane Glycoproteins/metabolism , Microfilament Proteins/metabolism , Molecular Sequence Data , Mutation , Organ Specificity , Phenotype , Plant Proteins/genetics , Plant Proteins/metabolism , Pollen Tube/growth & development , Pollen Tube/metabolism , Pollen Tube/ultrastructure , Recombinant Fusion Proteins , Sequence Analysis, DNAABSTRACT
Hydrothermal reaction of Co(NO3)2 and terphenyl-3,2",5",3'-tetracarboxyate (H4tpta) generated Co3(OH)2 chains based 3D coordination framework Co3(OH)2(tpta)(H2O)4 (1) that suffered from single-crystal-to-single-crystal dehydration by heating at 160 °C and was transformed into dehydrated Co3(OH)2(tpta) (1a). During the dehydration course, the local coordination environment of part of the Co atoms was transformed from saturated octahedron to coordinatively unsaturated tetrahedron. Heterogenous catalytic experiments on allylic oxidation of cyclohexene show that dehydrated 1a has 6 times enhanced catalytic activity than as-synthesized 1 by using tert-butyl hydroperoxide (t-BuOOH) as oxidant. The activation energy for the oxidation of cylcohexene with 1a catalyst was 67.3 kJ/mol, far below the value with 1 catalysts, which clearly suggested that coordinatively unsaturated Co(II) sites in 1a have played a significant role in decreasing the activation energy. It is interestingly found that heterogeneous catalytic oxidation of cyclohexene in 1a not only gives the higher conversion of 73.6% but also shows very high selectivity toward 2-cyclohexene-1-one (ca. 64.9%), as evidenced in high turnover numbers (ca. 161) based on the open Co(II) sites of 1a catalyst. Further experiments with a radical trap indicate a radical chain mechanism. This work demonstrates that creativity of coordinatively unsaturated metal sites in MOFs could significantly enhance heterogeneous catalytic activity and selectivity.
ABSTRACT
Higher order actin filament structures are necessary for cytoplasmic streaming, organelle movement, and other physiological processes. However, the mechanism by which the higher order cytoskeleton is formed in plants remains unknown. In this study, we identified a novel actin-cross-linking protein family (named CROLIN) that is well conserved only in the plant kingdom. There are six isovariants of CROLIN in the Arabidopsis genome, with CROLIN1 specifically expressed in pollen. In vitro biochemical analyses showed that CROLIN1 is a novel actin-cross-linking protein with binding and stabilizing activities. Remarkably, CROLIN1 can cross-link actin bundles into actin networks. CROLIN1 loss of function induces pollen germination and pollen tube growth hypersensitive to latrunculin B. All of these results demonstrate that CROLIN1 may play an important role in stabilizing and remodeling actin filaments by binding to and cross-linking actin filaments.
Subject(s)
Actins/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Microfilament Proteins/metabolism , Pollen Tube/metabolism , Actins/genetics , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Microfilament Proteins/genetics , Molecular Sequence Data , Pollen Tube/geneticsABSTRACT
Multicellular organisms contain a large number of formins; however, their physiological roles in plants remain poorly understood. Here, we reveal that formin homology 5 (FH5), a type II formin mutated in rice morphology determinant (rmd), plays a crucial role in determining rice (Oryza sativa) morphology. FH5/RMD encodes a formin-like protein consisting of an N-terminal phosphatase tensin (PTEN)-like domain, an FH1 domain, and an FH2 domain. The rmd mutants display a bending growth pattern in seedlings, are stunted as adult plants, and have aberrant inflorescence (panicle) and seed shape. Cytological analysis showed that rmd mutants have severe cell elongation defects and abnormal microtubule and microfilament arrays. FH5/RMD is ubiquitously expressed in rice tissues, and its protein localization to the chloroplast surface is mediated by the PTEN domain. Biochemical assays demonstrated that recombinant FH5 protein can nucleate actin polymerization from monomeric G-actin or actin/profilin complexes, cap the barbed end of actin filaments, and bundle actin filaments in vitro. Moreover, FH5 can directly bind to and bundle microtubules through its FH2 domain in vitro. Our findings suggest that the rice formin protein FH5 plays a critical role in determining plant morphology by regulating actin dynamics and proper spatial organization of microtubules and microfilaments.
Subject(s)
Membrane Proteins/metabolism , Oryza/growth & development , Oryza/genetics , Plant Proteins/metabolism , Actin Cytoskeleton/metabolism , Actins/metabolism , Cell Enlargement , Cloning, Molecular , Gene Expression Regulation, Plant , Membrane Proteins/genetics , Microscopy, Electron, Scanning , Microtubules/metabolism , Mutation , Oryza/metabolism , Plant Proteins/genetics , Seedlings/growth & developmentABSTRACT
Flowering plants rely on the polarized growth of pollen tubes to deliver sperm cells (SCs) to the embryo sac for double fertilization. In pollen, the vegetative nucleus (VN) and two SCs form the male germ unit (MGU). However, the mechanism underlying directional transportation of MGU is not well understood. In this study, we provide the first full picture of the dynamic interplay among microtubules, actin filaments, and MGU during pollen germination and tube growth. Depolymerization of microtubules and inhibition of kinesin activity result in an increased velocity and magnified amplitude of VN's forward and backward movement. Pharmacological washout experiments further suggest that microtubules participate in coordinating the directional movement of MGU. In contrast, suppression of the actomyosin system leads to a reduced velocity of VN mobility but without a moving pattern change. Moreover, detailed observation shows that the direction and velocity of VN's movement are in close correlations with those of the actomyosin-driven cytoplasmic streaming surrounding VN. Therefore, we propose that while actomyosin-based cytoplasmic streaming influences on the oscillational movement of MGU, microtubules and kinesins avoid MGU drifting with the cytoplasmic streaming and act as the major regulator for fine-tuning the proper positioning and directional migration of MGU in pollen.
Subject(s)
Actin Cytoskeleton , Actomyosin , Kinesins , Microtubules , Pollen , Microtubules/metabolism , Actin Cytoskeleton/metabolism , Kinesins/metabolism , Pollen/metabolism , Actomyosin/metabolism , Pollen Tube/metabolism , Pollen Tube/growth & development , Cell Nucleus/metabolism , Arabidopsis/metabolism , Cytoplasmic Streaming , Germination/physiologyABSTRACT
Formins have long been known to regulate microfilaments but have also recently been shown to associate with microtubules. In this study, Arabidopsis thaliana FORMIN14 (AFH14), a type II formin, was found to regulate both microtubule and microfilament arrays. AFH14 expressed in BY-2 cells was shown to decorate preprophase bands, spindles, and phragmoplasts and to induce coalignment of microtubules with microfilaments. These effects perturbed the process of cell division. Localization of AFH14 to microtubule-based structures was confirmed in Arabidopsis suspension cells. Knockdown of AFH14 in mitotic cells altered interactions between microtubules and microfilaments, resulting in the formation of an abnormal mitotic apparatus. In Arabidopsis afh14 T-DNA insertion mutants, microtubule arrays displayed abnormalities during the meiosis-associated process of microspore formation, which corresponded to altered phenotypes during tetrad formation. In vitro biochemical experiments showed that AFH14 bound directly to either microtubules or microfilaments and that the FH2 domain was essential for cytoskeleton binding and bundling. However, in the presence of both microtubules and microfilaments, AFH14 promoted interactions between microtubules and microfilaments. These results demonstrate that AFH14 is a unique plant formin that functions as a linking protein between microtubules and microfilaments and thus plays important roles in the process of plant cell division.
Subject(s)
Actin Cytoskeleton/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Cell Division , Microfilament Proteins/metabolism , Microtubules/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Cloning, Molecular , Gene Knockdown Techniques , Microfilament Proteins/genetics , Mutagenesis, InsertionalABSTRACT
Formins are well-known regulators that participate in the organization of the actin cytoskeleton in organisms. The Arabidopsis thaliana L. genome encodes 21 formins, which can be divided into two distinct subfamilies. However, type II formins have to date been less well characterized. Here, we cloned a type II formin, AtFH16, and characterized its biochemical activities on actin and microtubule dynamics. The results show that the FH1FH2 structure of AtFH16 cannot nucleate actin polymerization efficiently, but can bind and bundle microfilaments. AtFH16 FH1FH2 is also able to bind and bundle microtubules, and preferentially binds microtubules over microfilaments in vitro. In addition, AtFH16 FH1FH2 co-localizes with microtubules in onion epidermal cells, indicating a higher binding affinity of AtFH16 FH1FH2 for microtubules rather than microfilaments in vivo. In conclusion, AtFH16 is able to interact with both microfilaments and microtubules, suggesting that AtFH16 probably functions as a bifunctional protein, and may thus participate in plant cellular processes.
Subject(s)
Actin Cytoskeleton/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Actin Cytoskeleton/ultrastructure , Actins/metabolism , Arabidopsis/ultrastructure , Arabidopsis Proteins/chemistry , Microtubule-Associated Proteins/chemistry , Onions/cytology , Plant Epidermis/cytology , Polymerization , Protein Binding , Protein Multimerization , Protein Structure, Tertiary , Protein Transport , Recombinant Fusion Proteins/metabolismABSTRACT
The actin cytoskeleton is a highly dynamic network in plant cells, which is precisely regulated by numerous actin-binding proteins. Hence, characterizing the biochemical activities of actin-binding proteins is of great importance. Here we describe methods for determining the binding and bundling of microfilaments as well as methods for visualizing microfilaments using fluorescent phalloidin and single-molecule TIRF imaging.
Subject(s)
Actin Cytoskeleton , Actins , Actins/metabolism , Actin Cytoskeleton/metabolism , Microfilament Proteins/metabolism , Coloring Agents/metabolism , Phalloidine/metabolismABSTRACT
Pollen germination is an essential step for delivering sperm cells to the embryo sac for double fertilization in flowering plants. The cytosolic Ca2+ concentration ([Ca2+]cyt) and vesicle dynamics are critical for pollen germination, but their potential correlation in pollen grains is not fully understood. Here, we report that [Ca2+]cyt oscillates periodically at the prospective germination sites during pollen germination. The [Ca2+]cyt is mainly from extracellular Ca2+ ([Ca2+]ext) influx, which implicates the Ca2+-permeable ion channel cyclic nucleotide-gated channel 18 (CNGC18). The [Ca2+]cyt oscillations spatiotemporally correlate with the accumulation of secretory vesicles labeled by a formin protein AtFH5, and disruption of vesicle accumulation inhibits the [Ca2+]cyt oscillations. In turn, the [Ca2+]cyt oscillations promote exocytosis, which leads to stepwise cell extension during pollen germination. Together, these data provide a timeline of vesicle dynamics, calcium oscillation, and exocytosis during pollen germination and highlight the importance of the correlation of these events for pollen germination.
Subject(s)
Arabidopsis , Calcium Signaling , Arabidopsis/metabolism , Pollen Tube/metabolism , Prospective Studies , Calcium/metabolism , Seeds/metabolism , Pollen/metabolism , Secretory Vesicles/metabolism , ExocytosisABSTRACT
Pollen tube integrity is required for achieving double fertilization in angiosperms. The rapid alkalinization factor4/19-ANXUR1/2-Buddha's paper seal 1/2 (RALF4/19-ANX1/2-BUPS1/2)-complex-mediated signaling pathway is critical to maintain pollen tube integrity, but the underlying mechanisms regulating the polar localization and distribution of these complex members at the pollen tube tip remain unclear. Here, we find that COBRA-like protein 11 (COBL11) loss-of-function mutants display a low pollen germination ratio, premature pollen tube burst, and seed abortion in Arabidopsis. COBL11 could interact with RALF4/19, ANX1/2, and BUPS1/2, and COBL11 functional deficiency could result in the disrupted distribution of RALF4 and ANX1, altered cell wall composition, and decreased levels of reactive oxygen species in pollen tubes. In conclusion, COBL11 is a regulator of pollen tube integrity during polar growth, which is conducted by a direct interaction that ensures the correct localization and polar distribution of RALF4 and ANX1 at the pollen tube tip.