The Arabidopsis GPI-anchored protein COBL11 is necessary for regulating pollen tube integrity.

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.

AtFH5-labeled secretory vesicles-dependent calcium oscillation drives exocytosis and stepwise bulge during pollen germination.

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.

RAD51C-RAD51D interplays with MSH5 and regulates crossover maturation in rice meiosis.

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.

AtMAC stabilizes the phragmoplast by crosslinking microtubules and actin filaments during cytokinesis.

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.

Actin: Static and Dynamic Studies.

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.

Controlling the Gate: The Functions of the Cytoskeleton in Stomatal Movement.

Stomata are specialized epidermal structures composed of two guard cells and are involved in gas and water exchange between plants and the environment and pathogen entry into the plant interior. Stomatal movement is a response to many internal and external stimuli to increase adaptability to environmental change. The cytoskeleton, including actin filaments and microtubules, is highly dynamic in guard cells during stomatal movement, and the destruction of the cytoskeleton interferes with stomatal movement. In this review, we discuss recent progress on the organization and dynamics of actin filaments and microtubule network in guard cells, and we pay special attention to cytoskeletal-associated protein-mediated cytoskeletal rearrangements during stomatal movement. We also discuss the potential mechanisms of stomatal movement in relation to the cytoskeleton and attempt to provide a foundation for further research in this field.

OsFH3 Encodes a Type II Formin Required for Rice Morphogenesis.

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.

Plant multiscale networks: charting plant connectivity by multi-level analysis and imaging techniques.

In multicellular and even single-celled organisms, individual components are interconnected at multiscale levels to produce enormously complex biological networks that help these systems maintain homeostasis for development and environmental adaptation. Systems biology studies initially adopted network analysis to explore how relationships between individual components give rise to complex biological processes. Network analysis has been applied to dissect the complex connectivity of mammalian brains across different scales in time and space in The Human Brain Project. In plant science, network analysis has similarly been applied to study the connectivity of plant components at the molecular, subcellular, cellular, organic, and organism levels. Analysis of these multiscale networks contributes to our understanding of how genotype determines phenotype. In this review, we summarized the theoretical framework of plant multiscale networks and introduced studies investigating plant networks by various experimental and computational modalities. We next discussed the currently available analytic methodologies and multi-level imaging techniques used to map multiscale networks in plants. Finally, we highlighted some of the technical challenges and key questions remaining to be addressed in this emerging field.

Profilin promotes formin-mediated actin filament assembly and vesicle transport during polarity formation in pollen.

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.

AtFH14 crosslinks actin filaments and microtubules in different manners.

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.

Secretory Vesicles Targeted to Plasma Membrane During Pollen Germination and Tube Growth.

Pollen germination and pollen tube growth are important biological events in the sexual reproduction of higher plants, during which a large number of vesicle trafficking and membrane fusion events occur. When secretory vesicles are transported via the F-actin network in proximity to the apex of the pollen tube, the secretory vesicles are tethered and fused to the plasma membrane by tethering factors and SNARE proteins, respectively. The coupling and uncoupling between the vesicle membrane and plasma membrane are also regulated by dynamic cytoskeleton, proteins, and signaling molecules, including small G proteins, calcium, and PIP2. In this review, we focus on the current knowledge regarding secretory vesicle delivery, tethering, and fusion during pollen germination and tube growth and summarize the progress in research on how regulators and signaling molecules participate in the above processes.

Cryo-EM Structure of Actin Filaments from Zea mays Pollen.

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.

An Auxin Transport Inhibitor Targets Villin-Mediated Actin Dynamics to Regulate Polar Auxin Transport.

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.

Actin Polymerization Mediated by AtFH5 Directs the Polarity Establishment and Vesicle Trafficking for Pollen Germination in Arabidopsis.

The process of pollen germination is crucial for flowering plant reproduction, but the mechanisms through which pollen grains establish polarity and select germination sites are not well understood. In this study, we report that a formin family protein, AtFH5, is localized to the vesicles and rotates ahead of Lifeact-mEGFP-labeled actin filaments during pollen germination. The translocation of AtFH5 to the plasma membrane initiates the assembly of a collar-like actin structure at the prospective germination site prior to germination. Genetic and pharmacological evidence further revealed an interdependent relationship between the mobility of AtFH5-labeled vesicles and the polymerization of actin filaments: vesicle-localized AtFH5 promotes actin assembly, and the polymerization and elongation of actin filaments, in turn, is essential for the mobility of AtFH5-labeled vesicles in pollen grains. Taken together, our work revealed a molecular mechanism underlying the polarity establishment and vesicle mobility during pollen germination.

Reversible Double Nucleophilic Substitution Reaction inside Single-Crystal MOF Tuned Remarkable Magnetic Behavior.

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.

OsFH15, a class I formin, interacts with microfilaments and microtubules to regulate grain size via affecting cell expansion in rice.

Grain size is an important agronomic trait determining rice yield and is mainly restricted by spikelet hull size. However, it remains largely unknown how the spikelet hull size is regulated. In this study, OsFH15, a class I formin protein in Oryza sativa, was found to be able to regulate the size of cells and spikelet hull. OsFH15-Cas9 and OsFH15-RNAi mutants had decreased grain size with reduced cell length, cell width and cell area of inner epidermal cells of the lemma compared with wild-type plants. By contrast, OsFH15-overexpressed plants had increased grain size with larger cells, as well as more abundant microtubules (MTs) and actin filaments (AFs) arrays. OsFH15 was mainly expressed in shoot apical meristem (SAM), spikelets, spikelet hulls and seeds in rice. In vitro biochemical experiments showed that OsFH15 can efficiently nucleate actin polymerization with or without profilin, can cap the barbed end of AFs, and can bind and bundle both AFs and MTs. OsFH15 can also crosslink AFs with MTs, and preferentially bind MTs to AFs. These results demonstrated that OsFH15 played an important role in grain-size control by affecting cell expansion through regulating AFs and MTs.

LlFH1-mediated interaction between actin fringe and exocytic vesicles is involved in pollen tube tip growth.

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.

A Processive Arabidopsis Formin Modulates Actin Filament Dynamics in Association with Profilin.

Formins are conserved regulators of actin cytoskeletal organization and dynamics that have been implicated to be important for cell division and cell polarity. The mechanism by which diverse formins regulate actin dynamics in plants is still not well understood. Using in vitro single-molecule imaging technology, we directly observed that the FH1-FH2 domain of an Arabidopsis thaliana formin, AtFH14, processively attaches to the barbed end of actin filaments as a dimer and slows their elongation rate by 90%. The attachment persistence of FH1-FH2 is concentration dependent. Furthermore, by use of the triple-color total internal reflection fluorescence microscopy, we found that ABP29, a barbed-end capping protein, competes with FH1-FH2 at the filament barbed end, where its binding is mutually exclusive with AtFH14. In the presence of different plant profilin isoforms, FH1-FH2 enhances filament elongation rates from about 10 to 42 times. Filaments buckle when FH1-FH2 is anchored specifically to cover slides, further indicating that AtFH14 moves processively on the elongating barbed end. At high concentration, AtFH14 bundles actin filaments randomly into antiparallel or parallel spindle-like structures; however, the FH1-FH2-mediated bundles become thinner and longer in the presence of plant profilins. This is the direct demonstration of a processive formin from plants. Our results also illuminate the molecular mechanism of AtFH14 in regulating actin dynamics via association with profilin.

Enhanced Catalysis Activity in a Coordinatively Unsaturated Cobalt-MOF Generated via Single-Crystal-to-Single-Crystal Dehydration.

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.

The functions of the cytoskeleton and associated proteins during mitosis and cytokinesis in plant cells.

In higher plants, microtubule (MT)-based, and actin filament (AF)-based structures play important roles in mitosis and cytokinesis. Besides the mitotic spindle, the evolution of a band comprising cortical MTs and AFs, namely, the preprophase band (PPB), is evident in plant cells. This band forecasts a specific division plane before the initiation of mitosis. During cytokinesis, another plant-specific cytoskeletal structure called the phragmoplast guides vesicles in the creation of a new cell wall. In addition, a number of cytoskeleton-associated proteins are reportedly involved in the formation and function of the PPB, mitotic spindle, and phragmoplast. This review summarizes current knowledge on the cytoskeleton-associated proteins that mediate the cytoskeletal arrays during mitosis and cytokinesis in plant cells and discusses the interaction between MTs and AFs involved in mitosis and cytokinesis.