Abscisic acid-induced actin reorganization in guard cells of dayflower is mediated by cytosolic calcium levels and by protein kinase and protein phosphatase activities

In guard cells of open stomata under daylight, long actin filaments are arranged at the cortex, radiating out from the stomatal pore. Abscisic acid (ABA), a signal for stomatal closure, induces rapid depolymerization of cortical actin filaments and the slower formation of a new type of actin that is randomly oriented throughout the cell. This change in actin organization has been suggested to be important in signaling pathways involved in stomatal closing movement, since actin antagonists interfere with normal stomatal closing responses to ABA. Here we present evidence that the actin changes induced by ABA in guard cells of dayflower (Commelina communis) are mediated by cytosolic calcium levels and by protein phosphatase and protein kinase activities. Treatment of guard cells with CaCl2 induced changes in actin organization similar to those induced by ABA. Removal of extracellular calcium with EGTA inhibited ABA-induced actin changes. These results suggest that Ca2+ acts as a signal mediator in actin reorganization during guard cell response to ABA. A protein kinase inhibitor, staurosporine, inhibited actin reorganization in guard cells treated with ABA or CaCl2, and also increased the population of cells with long radial cortical actin filaments in untreated control cells. A protein phosphatase inhibitor, calyculin A, induced fragmentation of actin filaments in ABA- or CaCl2-treated cells and in control cells, and inhibited the formation of randomly oriented long actin filaments induced by ABA or CaCl2. These results suggest that protein kinase(s) and phosphatase(s) participate in actin remodeling in guard cells during ABA-induced stomatal closure.     

ARP2/3 complex‐mediated actin dynamics is required for hydrogen peroxide‐induced stomatal closure in Arabidopsis

Multiple cellular events like dynamic actin reorganization and hydrogen peroxide (H₂O₂) production were demonstrated to be involved in abscisic acid (ABA)‐induced stomatal closure. However, the relationship between them as well as the underlying mechanisms remains poorly understood. Here, we showed that H₂O₂ generation is indispensable for ABA induction of actin reorganization in guard cells of Arabidopsis that requires the presence of ARP2/3 complex. H₂O₂‐induced stomatal closure was delayed in the mutants of arpc4 and arpc5, and the rate of actin reorganization was slowed down in arpc4 and arpc5 in response to H₂O₂, suggesting that ARP2/3‐mediated actin nucleation is required for H₂O₂‐induced actin cytoskeleton remodelling. Furthermore, the expression of H₂O₂ biosynthetic related gene AtrbohD and the accumulation of H₂O₂ was delayed in response to ABA in arpc4 and arpc5, demonstrating that misregulated actin dynamics affects H₂O₂ production upon ABA treatment. These results support a possible causal relation between the production of H₂O₂ and actin dynamics in ABA‐mediated guard cell signalling: ABA triggers H₂O₂ generation that causes the reorganization of the actin cytoskeleton partially mediated by ARP2/3 complex, and ARP2/3 complex‐mediated actin dynamics may feedback regulate H₂O₂ production.     

Osmo-sensitive and stretch-activated calcium-permeable channels in Vicia faba guard cells are regulated by actin dynamics

In responses to a number of environmental stimuli, changes of cytoplasmic [Ca(2+)](cyt) in stomatal guard cells play important roles in regulation of stomatal movements. In this study, the osmo-sensitive and stretch-activated (SA) Ca(2+) channels in the plasma membrane of Vicia faba guard cells are identified, and their regulation by osmotic changes and actin dynamics are characterized. The identified Ca(2+) channels were activated under hypotonic conditions at both whole-cell and single-channel levels. The channels were also activated by a stretch force directly applied to the membrane patches. The channel-mediated inward currents observed under hypotonic conditions or in the presence of a stretch force were blocked by the Ca(2+) channel inhibitor Gd(3+). Disruption of actin filaments activated SA Ca(2+) channels, whereas stabilization of actin filaments blocked the channel activation induced by stretch or hypotonic treatment, indicating that actin dynamics may mediate the stretch activation of these channels. In addition, [Ca(2+)](cyt) imaging demonstrated that both the hypotonic treatment and disruption of actin filaments induced significant Ca(2+) elevation in guard cell protoplasts, which is consistent with our electrophysiological results. It is concluded that stomatal guard cells may utilize SA Ca(2+) channels as osmo sensors, by which swelling of guard cells causes elevation of [Ca(2+)](cyt) and consequently inhibits overswelling of guard cells. This SA Ca(2+) channel-mediated negative feedback mechanism may coordinate with previously hypothesized positive feedback mechanisms and regulate stomatal movement in response to environmental changes.     

Cortical actin filaments in guard cells respond differently to abscisic acid in wild-type and abi1-1 mutant Arabidopsis

Cortical actin filaments in guard cells of Commelina communis L. show signal-specific organization during stomatal movements [S.-O. Eun and Y. Lee (1997) Plant Physiol 115: 1491–1498; S.-O. Eun and Y. Lee (2000) Planta 210: 1014–1017]. To study the roles of actin in signal transduction, it is advantageous to use Arabidopsis thaliana (L.) Heynh., an excellent model plant with numerous well-characterized mutants. Using an immunolocalization technique, we found that actin deployments in guard cells of A. thaliana were basically identical to those in C. communis: actin proteins were assembled into radial filaments under illumination, and were disassembled by ABA. In addition, we examined actin organization in an ABA-insensitive mutant (abi1-1) to test the involvement of protein phosphatase 2C (PP2C) in the control of actin structure. A clear difference was observed after ABA treatment, namely, neither stomatal closing nor depolymerization of actin filaments was observed in guard cells of the mutant. Our results indicate that PP2C participates in ABA-induced actin changes in guard cells. Received: 23 June 2000 / Accepted: 20 October 2000     

The role of ARPC4 in tip growth and alignment of the polar axis in filaments of Physcomitrella patens

When the actin related protein 2/3 (Arp2/3) complex member arpc4 was deleted in Physcomitrella patens (moss), the resulting null mutant (Deltaarpc4) was viable and revealed no gross changes during morphogenesis of filaments into gametophores. However, we observed a striking reduction of filamentous tip growth, resulting in smaller, denser colonies. Although polar responses of Deltaarpc4 filaments to unilateral white light were unaffected, these mutant filaments were defective in their response to polarized white light. These observations strongly suggest a specific role of the Arp2/3 complex as a downstream target for signals regulating oriented tip growth. Insertion of YFP-ARPC4 into Deltaarpc4 rescued the mutant phenotypes and localized ARPC4 exclusively to the tip cell of filaments, the site of actin dynamics and polarized growth. The ability of Deltaarpc4 to perform some but not all cellular responses will allow the study of its function in orientation of tip growth in response to directional cues (e.g. light) in a viable but mutated background.     

The putative Arabidopsis arp2/3 complex controls leaf cell morphogenesis

The evolutionarily conserved Arp2/3 complex has been shown to activate actin nucleation and branching in several eukaryotes, but its biological functions are not well understood in multicellular organisms. The model plant Arabidopsis provides many advantages for genetic dissection of the function of this conserved actin-nucleating machinery, yet the existence of this complex in plants has not been determined. We have identified Arabidopsis genes encoding homologs of all of the seven Arp2/3 subunits. The function of the putative Arabidopsis Arp2/3 complex has been studied using four homozygous T-DNA insertion mutants for ARP2, ARP3, and ARPC5/p16. All four mutants display identical defects in the development of jigsaw-shaped epidermal pavement cells and branched trichomes in the leaf. These loss-of-function mutations cause mislocalization of diffuse cortical F-actin to the neck region and inhibit lobe extension in pavement cells. The mutant trichomes resemble those treated with the actin-depolymerizing drug cytochalasin D, exhibiting stunted branches but dramatically enlarged stalks due to depolarized growth suggesting defects in the formation of a fine actin network. Our data demonstrate that the putative Arabidopsis Arp2/3 complex controls cell morphogenesis through its roles in cell polarity establishment and polar cell expansion. Furthermore, our data suggest a novel function for the putative Arp2/3 complex in the modulation of the spatial distribution of cortical F-actin and provide evidence that the putative Arp2/3 complex may activate the polymerization of some types of actin filaments in specific cell types.     

Dynamics of vacuoles and actin filaments in guard cells and their roles in stomatal movement

Vacuoles and actin filaments are important cytoarchitectures involved in guard cell function. The changes in the morphology and number of vacuoles and the regulation of ion channel activity in tonoplast of guard cells are essential for stomatal movement. A number of studies have investigated the regulation of ion channels in animal and plant cells; however, little is known about the regulating mechanism for vacuolar dynamics in stomatal movement. Actin filaments of guard cells are remodelling with the changes in the stomatal aperture; however, the dynamic functions of actin filaments in stomatal movement remain elusive. In this paper, we summarize the recent developments in the understanding of the dynamics of actin filaments and vacuoles of guard cells during stomatal movement. All relevant studies suggest that actin filaments might be involved in stomatal movement by regulating vacuolar dynamics and the ion channels in tonoplast. The future study could be focused on the linker protein mediating the interaction between actin filaments and tonoplast, which will provide insights into the interactive function of actin and vacuole in stomatal movement regulation.     

DISTORTED2 encodes an ARPC2 subunit of the putative Arabidopsis ARP2/3 complex

Arabidopsis trichomes are unicellular, branched structures that have highly constrained requirements for the cytoskeleton. The 'distorted group' genes function downstream from microtubule-based branch initiation, and are required during the actin-dependent phase of polarized stalk and branch expansion. Of the eight known 'distorted group' genes, a subset encode homologs of ARP2/3 complex subunits. In eukaryotic cells, the seven-protein ARP2/3 complex nucleates actin filament networks that push on the plasma membrane and organelles. In plants cells, the existence and function of an ARP2/3 complex is unclear. In this paper, we report that DISTORTED2 (DIS2) encodes a paralogue of the ARP2/3 complex subunit ARPC2. DIS2 has ARPC2 activity, based on its ability to rescue the growth defects of arpc2 (arc35Delta) null yeast cells. Like known ARPC2s, DIS2 physically interacts with ARPC4. Mutations in DIS2 cause a distorted trichome phenotype, defects in cell-cell adhesion, and a modest reduction in shoot FW. The actin cytoskeleton in dis2 trichomes is extensive, but developing branches fail to generate and maintain highly organized cytoplasmic actin bundles.     

ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H(2)O(2) have been identified as essential components of the complex signalling network inducing stomatal closure in response to the phytohormone abscisic acid (ABA). A close inter-relationship exists between ABA and the spatial and temporal production and action of both NO and H(2)O(2) in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H(2)O(2) production. Stomatal closure induced by H(2)O(2) is inhibited by the removal of NO with NO scavenger, and both ABA and H(2)O(2) stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H(2)O(2) synthesis nor does NO treatment induce H(2)O(2) production in guard cells. Tungstate inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates NO production in response to nitrite in vitro and in response to H(2)O(2) and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H(2)O(2) synthesis. In the NR double mutant nia1, nia2 both ABA and H(2)O(2) fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H(2)O(2) are not impaired. Importantly, we show that in the NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and stomatal closure to ABA are severely reduced, indicating that endogenous H(2)O(2) production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H(2)O(2) and NO-induced stomatal closure.     

Signalling mechanisms in the regulation of vacuolar ion release in guard cells

Pharmacological agents were used to investigate the possible involvement of actin in signalling chains associated with abscisic acid (ABA)-induced ion release from the guard cell vacuole, a process which is absolutely essential for stomatal closure. Effects on the ABA-induced transient stimulation of tonoplast efflux were measured, using (86)Rb in isolated guard cells of Commelina communis, together with effects on stomatal apertures. In the response to 10 microm ABA (triggered by Ca(2+) influx rather than internal Ca(2+) release), jasplakinolide (stabilizing actin filaments) and latrunculin B (depolymerizing actin filaments) had opposite effects. Both closure and the vacuolar efflux transient were inhibited by jasplakinolide but enhanced by latrunculin B. At 10 microm ABA prevention of mitogen-activated protein (MAP) kinase activation by PD98059 partially inhibited closure and reduced the efflux transient. By contrast, latrunculin B inhibited the efflux transient at 0.1 microm ABA (involving internal Ca(2+) release rather than Ca(2+) influx). The results suggest that 10 microm ABA activates Ca(2+)-dependent vacuolar ion efflux via a Ca(2+)-permeable influx channel which is maintained closed by interaction with F-actin. A MAP kinase is also involved, in a chain similar to that postulated for Ca(2+)-dependent gene expression in cold acclimation.     

Actin Filaments Modulate Both Stomatal Opening and Inward K+-Channel Activities in Guard Cells of Vicia faba L

Actin antagonists have previously been shown to alter responses of Commelina communis stomata to physiological stimuli, implicating actin filaments in the control of guard cell volume changes (M. Kim, P.K. Hepler, S.-O. Eun, K.S. Ha, Y. Lee [1995] Plant Physiol 109: 1077-1084). Since K+ channels in the guard cell play an important role in stomatal movements, we examined the possible regulation of K+-channel activities by the state of actin polymerization. Agents affecting actin polymerization altered light-induced stomatal opening and inward K+-channel activities measured by patch clamping in Vicia faba. Cytochalasin D, which induces depolymerization of actin filaments, promoted light-induced stomatal opening and potentiated the inward K+ current in guard cell protoplasts. Phalloidin, a stabilizer of filamentous actin, inhibited both light-induced stomatal opening and inward K+ current. Inward K+-channel activities in outside-out membrane patches showed responses to these agents that support results at the whole-cell current level, suggesting that cytochalasin D facilitates and phalloidin inhibits K+ influx in intact guard cells, thus resulting in enhancement and inhibition of stomatal opening, respectively. To our knowledge, this is the first report that provides evidence that actin filaments may regulate an important physiological process by modulating the activities of ion channels in plant cells.     

Actin-related protein2/3 complex component ARPC1 is required for proper cell morphogenesis and polarized cell growth in Physcomitrella patens

The actin-related protein2/3 (Arp2/3) complex functions as a regulator of actin filament dynamics in a wide array of eukaryotic cells. Here, we focus on the role of the Arp2/3 complex subunit ARPC1 in elongating tip cells of protonemal filaments of the moss Physcomitrella patens. Using RNA interference (RNAi) to generate loss-of-function mutants, we show dramatic defects in cell morphology manifested as short, irregularly shaped cells with abnormal division patterns. The arpc1 RNAi plants lack the rapidly elongating caulonemal cell type found in wild-type protonemal tissue. The absence of this cell type prevents normal bud formation even in response to cytokinin treatment and results in filamentous colonies lacking leafy gametophores. In addition, arpc1 protoplasts show an increased sensitivity to osmotic shock and are defective in their ability to properly establish a polarized outgrowth during regeneration from a single cell. This failure of arpc1 protoplasts to undergo proper tip growth is rescued by ARPC1 overexpression and is phenocopied in wild-type protoplasts treated with Latrunculin B, a potent inhibitor of actin polymerization. We show in moss that ARPC1, and by inference the Arp2/3 complex, plays a critical role in controlling polarized growth and cell division patterning through its regulation of actin dynamics at the cell apex.     

CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca(2+)-permeable channels and stomatal closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca²⁺ in guard cell ion channel regulation. However, genetic mutants in Ca²⁺ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca²⁺-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca²⁺ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca²⁺-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca²⁺-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca²⁺ oscillation experiments revealed that Ca²⁺-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca²⁺-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca²⁺-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.     

Heterotrimeric G-protein regulation of ROS signalling and calcium currents in Arabidopsis guard cells

Heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits are important signalling agents in both animals and plants. In plants, G proteins modulate numerous responses, including abscisic acid (ABA) and pathogen-associated molecular pattern (PAMP) regulation of guard cell ion channels and stomatal apertures. Previous analyses of mutants deficient in the sole canonical Arabidopsis Gα subunit, GPA1, have shown that Gα-deficient guard cells are impaired in ABA inhibition of K(+) influx channels, and in pH-independent activation of anion efflux channels. ABA-induced Ca(2+) uptake through ROS-activated Ca(2+)-permeable channels in the plasma membrane is another key component of ABA signal transduction in guard cells, but the question of whether these channels are also dependent on Gα for their ABA response has not been evaluated previously. We used two independent Arabidopsis T-DNA null mutant lines, gpa1-3 and gpa1-4, to investigate this issue. We observed that gpa1 mutants are disrupted both in ABA-induced Ca(2+)-channel activation, and in production of reactive oxygen species (ROS) in response to ABA. However, in response to exogenous H(2)O(2) application, I(Ca) channels are activated normally in gpa1 guard cells. In addition, H(2)O(2) inhibition of stomatal opening and promotion of stomatal closure are not disrupted in gpa1 mutant guard cells. These data indicate that absence of GPA1 interrupts ABA signalling between ABA reception and ROS production, with a consequent impairment in Ca(2+)-channel activation.     

MICAL2 fine-tunes Arp2/3 for actin branching

The ARP2/3 complex promotes branched actin networks, but the importance of specific subunit isoforms is unclear. In this issue, Galloni, Carra, et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202102043) show that MICAL2 mediates methionine oxidation of ARP3B, thus destabilizing ARP2/3 complexes and leading to disassembly of branched actin filaments.

Remodeling of branched actin networks enables cell protrusion and sensing of the environment and is essential for cell motility. Migrating cells such as fibroblasts, immune cells, and metastatic cancer cells rely on actin dynamics to generate pushing, pulling, and squeezing forces to propel themselves. Therefore, studying the processes regulating assembly and disassembly of actin filaments is key to understanding cell locomotion in health and disease. One of the most important catalyzers of actin assembly is the Arp2/3 complex, which drives lamellipodia formation and cell protrusion. Arp2/3-generated actin networks are also important for endocytic trafficking, membrane remodeling during vesicle internalization, cargo sorting, and membrane excision (1). The seven-protein ARP2/3 complex contains two unconventional actin-related proteins (ARP2 and ARP3) and five additional subunits (ARPC1–5). Mammals express two isoforms of three of the subunits (ARP3/ARP3B, ARPC1A/ARPC1B, and ARPC5/ARPC5L), resulting in functional diversity depending on the specific isoforms incorporated into the ARP2/3 complex; however, despite some intriguing roles described in muscle development (2) and platelet function (3), little is known about the biological significance of these isoforms.The nucleation activity of ARP2/3 complex is regulated at multiple levels to ensure that new actin generation is spatially and temporally controlled. Activation is controlled by Wiskott Aldrich Syndrome Protein (WASP)–family proteins, which are themselves part of multi-protein complex machines (4). WASP-family protein complexes detect multiple inputs such as membrane phospholipids, protein–protein interactions, or post-translational modifications, and act as signaling hubs to regulate branched actin nucleation. Other proteins, such as cortactin or coronin, also modulate branch stability in an antagonistic manner (5). ARP2/3 can be post-translationally modified by phosphorylation and interaction with negative regulators, whereas actin itself is regulated by targeted oxidation of methionine residues (6). How these feedback loops that control ARP2/3 activity are coordinated with cell function is an intense area of research.Molecule interacting with CasL (MICAL) proteins have emerged as important mediators of targeted protein oxidation (6). MICAL proteins (MICAL1–3) are flavin adenine dinucleotide–binding monooxygenases capable of oxidizing target proteins (including actin), either directly or through generation of diffusible H₂O₂, which in turn oxidizes proteins in close proximity. Actin oxidation occurs on two methionine residues (Met44 and Met47), resulting in F-actin disassembly and increased cofilin-mediated F-actin severing. Although actin is the best characterized MICAL substrate, there remains the intriguing possibility of the existence of additional targets that regulate cytoskeleton dynamics.In this issue, Galloni, Carra, et al. evaluated the ability of ARP2/3 complexes, containing either ARP3 or the ARP3B isoform (i.e., isocomplexes), to promote actin assembly, and determined isoform-specific differences in their activity and molecular regulation (7). As a model system, the authors used HeLa cells infected with vaccinia virus to study actin branching, given that this virus induces actin tail nucleation in the host cells. They noticed that in cells lacking ARP3, the localization of GFP-ARP3 or GFP-ARP3B to actin tails was comparable, and both isoforms were similarly incorporated into ARP2/3 complexes (Fig. 1). However, the length of the actin tails in ARP3B-expressing cells was shorter than in ARP3-expressing counterparts. Given that ARP3 and ARP3B isocomplexes were equivalent in their ability to induce actin polymerization in vitro, these data pointed to a faster disassembly rate as the potential cause underlying shorter actin tails in ARP3B-expressing cells. Indeed, by tracking photoactivatable actin to study its dynamics, the researchers confirmed that the rate of filament disassembly was faster in ARP3B-expressing cells.Open in a separate windowFigure 1.Vaccinia virus surfs on the outside of the cell, forming an actin tail in the cytoplasm that aids its propulsion. Arp2/3 complex is involved in initiating the branched actin structures and shows slow dissociation from the branches when it is stabilized by the linker protein cortactin. When an Arp2/3 complex containing the ARP3B isoform of ARP3 forms, the dissociation is enhanced, as ARP3B is subject to oxidation by MICAL2, which is recruited to branches by coronin, causing cortactin displacement and rapid branch dissociating leading to shorter actin tails.To identify the molecular basis for the differences between ARP3 and ARP3B, the authors tested a series of ARP3 and ARP3B chimeric proteins, which revealed the importance of ARP3B amino acids 281–418 in mediating the functional differences with ARP3. In particular, Met293 was essential for ARP3B to generate short actin tails. Given that MICAL enzymes promote actin filament disassembly through oxidation of actin Met44 and Met47, Galloni, Carra, et al. decided to investigate the possibility that MICAL-induced oxidation of Met293 in ARP3B inhibits ARP3B activity. Fluorescently tagged MICAL2, but not MICAL1, was recruited to vaccinia-induced actin tails at a position relatively distant from the virus itself, similar to the actin-binding protein coronin (8). Down-regulation of MICAL2, but not MICAL1, increased actin tail stability and suppressed the short actin tail phenotype induced by ARP3B overexpression. Using an antibody raised against oxidized Met293, the researchers confirmed that ARP3B oxidation was reduced following MICAL2 knockdown. Recruitment of MICAL2 to actin tails was dependent on coronin 1C expression, and silencing of coronin 1C resulted in actin filament stabilization and reversal of ARP3B-induced actin tail shortening comparable to MICAL2 knockdown. Thus, coronin 1C recruitment of MICAL2 results in ARP3B oxidation on Met293, leading to dissociation of ARP2/3B isocomplexes and consequent actin networks destabilization.Interestingly, the authors noted that the actin nucleation promoting factor cortactin, which stabilizes ARP2/3-mediated branch points along actin filaments, was required for actin tail destabilization in ARP3B overexpressing cells but was not necessary for localization of coronin 1C or MICAL2 to actin tails. One possibility is that cortactin supports local MICAL2-mediated oxidation of ARP3B at branch points to induce filament de-branching, rather than bulk actin filament depolymerization that would result from direct actin oxidation. Since MICAL proteins are directed to specific cytoskeleton locations by interacting with Myosin 5A (9) and Myosin 15 (10), the consequences of MICAL activity on actin cytoskeleton organization and function may be fine-tuned by specific MICAL subcellular localization and interacting partners.Given that actin binds directly to the catalytic monooxygenase and calponin homology domains of MICAL proteins to increase enzyme activity and promote methionine oxidation, it is not entirely surprising that the actin-related ARP3B protein can be oxidized by MICAL2. However, the location of Met293 in ARP3B is not analogous to the Met44 or Met47 residues of actin, which raises questions regarding the mechanism of ARP3B oxidation by MICAL2. Structural modeling of the MICAL3–actin complex positions the actin loop containing Met44 and Met47 near the enzyme active site (11). ARP3B may interact with MICAL2 differently to bring Met293 close to the active site for direct oxidation, or H₂O₂ produced by MICAL2 might diffuse and oxidize highly concentrated nearby proteins. If this second possibility were true, then it is also possible that additional protein targets (e.g., coronin 1C, cortactin, additional ARP2/3 subunits) might also be oxidized on Met or Cys residues. Since the effects of MICAL1 on actin are counteracted via reduction of the oxidized Met residues by the sulfoxide reductase enzyme SelR (12), it remains to be determined if ARP3B can be similarly reactivated.     

Functional surfaces on the p35/ARPC2 subunit of Arp2/3 complex required for cell growth, actin nucleation, and endocytosis

The Arp2/3 complex is comprised of seven evolutionarily conserved subunits and upon activation by WASp or another nucleation promoting factor nucleates the formation of actin filaments. These events are critical for driving a wide range of cellular processes, including motility, endocytosis, and intracellular trafficking. However, an in depth understanding of the Arp2/3 complex activation and nucleation mechanism is still lacking. Here, we used a mutagenesis approach in Saccharomyces cerevisiae to dissect the structural and functional roles of the p35/ARPC2 subunit. Using integrated alleles that target conserved and solvent-exposed residues, we identified surfaces on p35/ARPC2 required for cell growth, actin organization, and endocytosis. In parallel, we purified the mutant Arp2/3 complexes and compared their actin assembly activities both in the presence and in the absence of WASp. The majority of alleles with defects mapped to one face of p35/ARPC2, where there was a close correlation between loss of actin nucleation and endocytosis. A second site required for nucleation and endocytosis was identified near the contact surface between p35/ARPC2 and p19/ARPC4. A third site was identified at a more distal conserved surface, which was critical for endocytosis but not nucleation. These findings pinpoint the key surfaces on p35/ARPC2 required for Arp2/3 complex-mediated actin assembly and cellular function and provide a higher resolution view of Arp2/3 structure and mechanism.     

Stomatal morphology and physiology explain varied sensitivity to abscisic acid across vascular plant lineages

Abscisic acid (ABA) can induce rapid stomatal closure in seed plants, but the action of this hormone on the stomata of fern and lycophyte species remains equivocal. Here, ABA-induced stomatal closure, signaling components, guard cell K⁺ and Ca²⁺ fluxes, vacuolar and actin cytoskeleton dynamics, and the permeability coefficient of guard cell protoplasts (P_f) were analyzed in species spanning the diversity of vascular land plants including 11 seed plants, 6 ferns, and 1 lycophyte. We found that all 11 seed plants exhibited ABA-induced stomatal closure, but the fern and lycophyte species did not. ABA-induced hydrogen peroxide elevation was observed in all species, but the signaling pathway downstream of nitric oxide production, including ion channel activation, was only observed in seed plants. In the angiosperm faba bean (Vicia faba), ABA application caused large vacuolar compartments to disaggregate, actin filaments to disintegrate into short fragments and P_f to increase. None of these changes was observed in the guard cells of the fern Matteuccia struthiopteris and lycophyte Selaginella moellendorffii treated with ABA, but a hypertonic osmotic solution did induce stomatal closure in fern and the lycophyte. Our results suggest that there is a major difference in the regulation of stomata between the fern and lycophyte plants and the seed plants. Importantly, these findings have uncovered the physiological and biophysical mechanisms that may have been responsible for the evolution of a stomatal response to ABA in the earliest seed plants.

Physiological and biophysical evidence for insensitivity of stomata to abscisic acid in ferns and lycophytes supports stomatal responsiveness to abscisic acid evolved after the divergence of ferns.     

Actin filaments of guard cells are reorganized in response to light and abscisic acid.

We recently showed that treatment with actin antagonists perturbed stomatal behavior in Commelina communis L. leaf epidermis and therefore suggested that dynamic changes in actin are necessary for signal responses in guard cells (M. Kim, P.K. Hepler, S.O. Eun, K.-S. Ha, Y. Lee [1995] Plant Physiol 109: 1077-1084). Here we show that actin filaments of guard cells, visualized by immunofluorescence microscopy, change their distribution in response to physiological stimuli. When stomata were open under white-light illumination, actin filaments were localized in the cortex of guard cells, arranged in a pattern that radiates from the stomatal pore. In marked contrast, for guard cells of stomata closed by darkness or by abscisic acid, the actin organization was characterized by short fragments randomly oriented and diffusely labeled along the pore site. Upon abscisic acid treatment, the radial pattern of actin arrays in the illuminated guard cells began to disintegrate within a few minutes and was completely disintegrated in the majority of labeled guard cells by 60 min. Unlike actin filaments, microtubules of guard cells retained an unaltered organization under all conditions tested. These results further support the involvement of actin filaments in signal transduction pathways of guard cells.     

CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca2+- Permeable Channels and Stomatal Closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca²⁺ in guard cell ion channel regulation. However, genetic mutants in Ca²⁺ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca²⁺-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca²⁺ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca²⁺-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca²⁺-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca²⁺ oscillation experiments revealed that Ca²⁺-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca²⁺-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca²⁺-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.     

拟南芥微丝加帽蛋白β亚基参与壳梭孢素诱导的气孔开放过程

气孔是植物响应外源信号,与环境进行水分和气体交换的门户。由外源信号引起的保卫细胞微丝骨架动态变化在气孔运动中发挥重要作用,但是具体的精确调节机制仍不清楚。微丝结合蛋白家族(ABPs) 是微丝动态组装最直接的调控者,它们的作用不容忽视。本文运用反向遗传学,以微丝结合蛋白—加帽蛋白 (CP) β-亚基 (CPB) 突变体cpb-3为实验材料,探究其在壳梭孢素 (FC)诱导气孔开放中的作用。结果发现:离体叶片干燥3 h,cpb-3突变体的叶片失水率为63.45%,明显高于野生型的48.99%。气孔开度测量及激光共聚焦显微镜观察发现,cpb-3突变体的气孔开放程度以及微丝动态重排对FC分子更敏感。气孔开度相比野生型增大了20% (P<0.05),含辐射状微丝排布的保卫细胞数量比例增幅达到58.3%,比对照组高出18.5%。此外,非损伤微测技术记录保卫细胞Ca²⁺、K⁺等跨膜运输动态,FC处理下,cpb-3突变体保卫细胞中Ca²⁺外流速度升至212.86 pmol cm^-2s^-1,野生型仅为68.76 pmol cm^-2s^-1,明显快于野生型。且K⁺内流也有相同表现。综上表明,微丝加帽蛋白CP的β亚基CPB可能通过调节保卫细胞微丝骨架动态重排以及离子流动,在FC诱导的气孔运动中发挥重要的作用。