Pollen Actin Research Articles

Methods for Determining Relative Average Number of Channels per Maize Starch Granule and Digestion of Raw Granules of Mutant Maize Cultivars by Amyloglucosidase

AbstractSix methods were investigated for determining the relative average numbers of channels per granule in populations of starch granules. The first method involved digestion of raw starch granules with amyloglucosidase. There were differences in the rates of digestion of starch of the same mutation in two different inbred‐line backgrounds, but we concluded that a better method was needed. Three methods involved measuring either all channel proteins or one specific protein. Extraction of granules using 70% 2‐propanol containing 1% 2‐mercaptoethanol (ME) was successful in removing the majority of external surface proteins (<31 kDa). Further extraction of starch granules using 0.1% SDS containing 0.2% ME, pH 8, removed channel proteins without extracting matrix proteins. By measuring SDS‐PAGE band intensities of protein clusters at 31–97.4 kDa or a marker protein at ≈42 kDa, methods to quantify relative average degrees of channelization (RADC) in different maize backgrounds were developed. Using either method, Oh43 had the highest RADC and B73 had the lowest RADC among the inbred lines examined. Another method involves extraction of total proteins using 2% SDS containing 0.2% ME from granules with the majority of surface proteins removed. The extract is subjected to SDS‐PAGE and Western blotting, followed by location and quantification of the actin band using antibodies to maize pollen actin. These four methods give somewhat different results for six inbred lines of corn with a relatively narrow range of RADC. In an attempt to relate them to the actual number of observed pores (channel openings to the surface), granules were treated with α‐amylase to enlarge the pores, with and without prior treatment with a protease. We concluded that this treatment revealed something about the nature of the granules and the action of α‐amylase on them, but it was not an indication of the number of channels present. The outcome of this research provides evidence that the number of channels in corn starch granules varies with the genetic makeup of the parent plant. We concluded that measuring granule actin is the preferred method.

The late pollen actins are essential for normal male and female development in Arabidopsis

In angiosperms the late pollen actins (LPAs) are strongly expressed in mature pollen and pollen tubes and at much lower levels in ovules. Four Arabidopsis lines with homozygous knockout mutations in the four individual LPA genes displayed normal flowers, pollen, and seed set. However, when all four LPAs were silenced simultaneously with a single RNA interference (RNAi) construct targeting the 3'UTR of each mRNA, obvious reproductive defects were observed. Western analysis of various Late Pollen actin RNA interference (LPRi) epialleles showed total LPA protein and RNA expression levels were knocked down from 0% to 95% compared to wild-type levels. Reciprocal crosses with the RNAi lines demonstrated that lowered LPA expression was associated with defects in both male and female fertility. Strong epialleles showed significant reductions in normal silique and seed production and were nearly sterile. Dissection of the siliques from moderate LPRi epialleles revealed many unfertilized ovules, increased numbers of aborted seeds, and decreased numbers of healthy seeds. Microscopic analysis of LPRi pollen indicated that the pollen shape and size were normal, but pollen germinated poorly. While multiple LPA genes may have some functional redundancy, the combined expression of multiple LPA genes appears essential to normal male and female reproductive development.

Actins from plant and animal sources tend not to form heteropolymers in vitro and function differently in plant cells.

Pollen and skeletal muscle actins were purified and labeled with fluorescent dyes that have different emission wavelengths. Observation by electron microscopy shows that the fluorescent actins are capable to polymerize into filamentous actin in vitro, bind to myosin S-1 fragments, and have a critical concentration similar to unlabeled actin, indicating that they are functionally active. The globular actins from two sources were mixed and polymerized by the addition of ATP and salts. The copolymerization experiment shows that when excited by light of the appropriate wavelength, both red actin filaments (pollen actin) and green actin filaments (muscle actin) can be visualized under the microscope, but no filaments exhibiting both green and red colors are detected. Furthermore, coprecipitations of labeled pollen actin with unlabeled pollen and skeletal muscle actin were performed. Measurements of fluorescent intensity show that the amount of labeled pollen actin precipitating with pollen actin was much higher than that with skeletal muscle actin, indicating that pollen and muscle actin tend not to form heteropolymers. Injection of labeled pollen actin into living stamen hair cells results in the formation of normal actin filaments in transvacuolar strands and the cortical cytoplasm. In contrast, labeled skeletal muscle actin has detrimental effects on the cellular architecture. The results from coinjection of the actin-disrupting reagent cytochalasin D with pollen actin show that overexpression of pollen actin prolongs the displacement of the nucleus and facilitates the recovery of the nuclear position, actin filament architecture, and transvacuolar strands. However, muscle actin perturbs actin filaments when injected into stamen hair cells. Moreover, nuclear displacement occurs more rapidly when cytochalasin D and muscle actin are coinjected into the cell. It is concluded that actins from plant and animal sources behave differently in vitro and in vivo and that they are functionally not interchangeable.

Regulation of gelsolin to plant actin filaments and its distribution in pollen

The effect of plasma gelsolin on plant microfilaments and its localization in plant cells were investigated. The results by using ultracentrifugation and electron microscopy showed that plant microfilaments could be severed into shorter fragments by gelsolin in a Ca(2+)-dependent manner. By measuring the binding ability of plasma gelsolin to pollen actin using the method of immunoprecipitation, it was shown that pollen actin could bind gelsolin at a ratio of 2.0±0.21 in the presence of Ca(2+). Addition of EGTA could disassociate the actin-gelsolin complexes, reducing the ratio to 1.2±0.23, and the addition of PIP(2) could further reduce the ratio to 0.8±0.1. The results indicate that plant actin has similar binding properties with plasma gelsolin as that of animal actin. By Western blotting we identified the existence of gelsolin in lily pollen. The results of immunolocalization of gelsolin in pollen and pollen tube showed that gelsolin was mainly localized at the germinal furrow in pollen grains and at the cytoplasm in pollen tube, especially in the tip region.

Multiple conserved 5' elements are required for high-level pollen expression of the Arabidopsis reproductive actin ACT1.

The Arabidopsis thaliana actin gene family comprises eight genes, which are divided into two ancient classes, vegetative and reproductive. We dissected various 5' elements and the conserved expression pattern of the reproductive actin gene ACT1, which is the most strongly expressed pollen actin gene. A basal construct containing only 310 bp of sequence upstream of the major transcriptional start site showed essentially full promoter activity in pollen and ovules. Further truncations of the 5'-flanking region and two different 10 bp replacements within a 55 bp conserved domain each caused a several-fold reduction in mature pollen expression. Intron L, located in the 5'-untranslated region (5'-UTR) was also required for high-level expression of pollen and organ primordia, and it had the properties of an enhancer. Pollen expression was not preserved when intron L was precisely replaced by intron L2 from the vegetatively expressed actin gene ACT2. ACT1 reporter gene constructs were strongly expressed in both pollen and ovules of tobacco and in the pollen of rice. Promoter-reporter fusions of the most distantly related Arabidopsis reproductive actin gene ACT1 showed strong expression in tobacco pollen and ovules indistinguishable from that directed by ACT1. Thus, multiple conserved cis-sequence elements within the 5'-flanking region and 5'-UTR of ACT1 direct high levels of reproductive tissue-specific expression.

The actin cytoskeleton is a target of the self-incompatibility response in Papaver rhoeas.

The integration of signals received by a cell, and their transduction to targets, is essential for all cellular responses. The cytoskeleton has been identified as a major target of signalling cascades in both animal and plant cells. Self-incompatibility (SI) in Papaver rhoeas involves an allele-specific recognition between stigmatic S-proteins and pollen, resulting in the inhibition of incompatible pollen. This highly specific response triggers a Ca(2+)-dependent signalling cascade in incompatible pollen when a stigmatic S-protein interacts with it. It has been demonstrated recently that SI induces dramatic alterations in the organization of the pollen actin cytoskeleton. This implicates the actin cytoskeleton as a key target for the SI-stimulated signals. The cytological alterations to the actin cytoskeleton that are triggered in response to SI are described here and there seem to be several stages that are distinguishable temporally. Evidence was obtained that F-actin depolymerization is also stimulated. The current understanding that the actin cytoskeleton is a target for the signals triggered by the SI response is discussed. It is suggested that these F-actin alterations may be Ca(2+)-mediated and that this could be a mechanism whereby SI-induced tip growth inhibition is achieved. The potential for actin-binding proteins to act as key mediators of this response is discussed and the mechanisms that may be responsible for effecting these changes are described. In particular, the parallels between sustained actin rearrangements during SI and in apoptosis of animal cells are considered.

The actin cytoskeleton is a target of the self-incompatibility response in Papaver rhoeas

The integration of signals received by a cell, and their transduction to targets, is essential for all cellular responses. The cytoskeleton has been identified as a major target of signalling cascades in both animal and plant cells. Self‐incompatibility (SI) in Papaver rhoeas involves an allele‐specific recognition between stigmatic S‐proteins and pollen, resulting in the inhibition of incompatible pollen. This highly specific response triggers a Ca2+‐dependent signalling cascade in incompatible pollen when a stigmatic S‐protein interacts with it. It has been demonstrated recently that SI induces dramatic alterations in the organization of the pollen actin cytoskeleton. This implicates the actin cytoskeleton as a key target for the SI‐stimulated signals. The cytological alterations to the actin cytoskeleton that are triggered in response to SI are described here and there seem to be several stages that are distinguishable temporally. Evidence was obtained that F‐actin depolymerization is also stimulated. The current understanding that the actin cytoskeleton is a target for the signals triggered by the SI response is discussed. It is suggested that these F‐actin alterations may be Ca2+‐mediated and that this could be a mechanism whereby SI‐induced tip growth inhibition is achieved. The potential for actin‐binding proteins to act as key mediators of this response is discussed and the mechanisms that may be responsible for effecting these changes are described. In particular, the parallels between sustained actin rearrangements during SI and in apoptosis of animal cells are considered.

AtFim1 is an actin filament crosslinking protein from Arabidopsis thaliana

ATFIM1 is a widely expressed gene in Arabidopsis thaliana that encodes a putative actin filament-crosslinking protein, AtFim1, belonging to the fimbrin/plastin class of actin-binding proteins. In this report we have used bacterially expressed AtFim1 and actin isolated from Zea mays pollen to demonstrate that AtFim1 functions as an actin filament-crosslinking protein. AtFim1 binds pollen actin filaments (F-actin) in a calcium-independent manner, with an average dissociation constant (Kd) of 0.55+/-0.21 microM and with a stoichiometry at saturation of 1:4 (mol AtFim1 : mol actin monomer). AtFim1 also crosslinks pollen F-actin by a calcium-independent mechanism, in contrast to crosslinking of plant actin by human T-plastin, a known calcium-sensitive actin-crosslinking protein. When micro-injected at high concentration into living Tradescantia virginiana stamen hair cells, AtFim1 caused cessation of both cytoplasmic streaming and transvacuolar strand dynamics within 2-4 min. Using the 'nuclear displacement assay' as a measure of the integrity of the actin cytoskeleton in living stamen hair cells, we demonstrated that AtFim1 protects actin filaments in these cells from Z. mays profilin (ZmPRO5)-induced depolymerization, in a dose-dependent manner. The apparent ability of AtFim1 to protect actin filaments in vivo from profilin-mediated depolymerization was confirmed by in vitro sedimentation assays. Our results indicate that AtFim1 is a calcium-independent, actin filament-crosslinking protein that interacts with the actin cytoskeleton in living plant cells.

Polymerization of fluorescent analogue of plant actinin vitro andin vivo

Maize pollen actin has been labeled with Oregon Green 488 iodoacetamide. A yield of 3 mg fluorescent actin analogue has been obtained from 10 mg of maize pollen actin, which is 99% in purity and the dye/protein ratio is 72%. In the presence of Mg2+ and K+, the fluorescent actin analogue polymerized into filamentsin vitro. Green fluorescent filaments were observed when the fluorescent actin was introduced into living plant cells by microinjection, indicating that the fluorescent actin analogue functions similarly to the native actin.

From pollen actin to crop male sterility

Actin plays an important role in the life activity of animal and plant cells. Pollen cells have plenty of actin whose structure and characteristics are very similar to the animal actin. The nucleotide sequence and amino acid sequence of plant actin gene are very similar to those of the animal gene. The content of pollen actin from male sterile plants is much more lower than that from its maintainer plants. The expression of actin gene is organ-specific during the plant development. The expression quantity of actin gene in pollen is much more higher than those from root, stem and leaf. The expression plasmid of the anti-sense actin gene was constructed, transferred to the protoplasts of wheat and tomato to inhibit the expression of actin gene in pollen and thus the male sterile plants of wheat and tomato were obtained. The actin in pollens from the transgenic plants was reduced significantly, whereas the pistil was not affected. This study might pave a new way to breeding male sterile lines for the application of hybrid vigor of wheat and tomato.

Signalling and the Cytoskeleton of Pollen Tubes of Papaver rhoeas

Self-incompatibility (SI) is a genetically controlled system used by many flowering plants to prevent self-pollination, often by the inhibition of pollen tube growth. The importance of cytosolic free calcium, [Ca2+]i, for the regulation of pollen tube growth is well known. We have established, using calcium imaging, that the SI response in Papaver rhoeas L. pollen involves a calcium-mediated intracellular signalling pathway. Tip growth of cells is dependent upon a typical configuration of the actin cytoskeleton, which is controlled by actin binding proteins. In animal cells, the actin-binding protein, profilin, is thought to act as a key intermediate between signalling pathways and actin rearrangements. Profilin is an abundant component of pollen. To better understand the signalling cascades that modulate pollen tip growth and actin dynamics, we are investigating a possible signalling role for profilin. We have demonstrated that profilin modulates the phosphorylation of pollen proteins in vitro. This implicates a role for profilin in altering protein kinase or phosphatase activity. Furthermore, we demonstrate for the first time that profilin from pollen can be phosphorylated in vitro. This provides compelling evidence that profilin interacts with signalling pathways in angiosperms. Finally, we demonstrate that in the SI response, the actin cytoskeleton of incompatible pollen tubes is dramatically rearranged. Our data strongly support a role for the cytoskeleton and actin-binding proteins interacting with signalling pathways involved in the regulation of pollen tube growth.

Aluminum Modifies the Viscosity of Filamentous Actin Solutions as Measured by Optical Displacement Microviscometry

A microtechnique has been developed that is capable of measuring the viscosity of filamentous actin (F-actin) solutions. This method, called optical displacement microviscometry (ODM), was utilized to determine the changes in viscosity of solutions of rabbit muscle, human platelet, and maize pollen actin when measured in the absence and presence of aluminum. Measurements demonstrated that the viscosity of the different actin solutions decreased with aluminum concentration. In contrast, increases in viscosity were observed when aluminum was added to F-actin solutions containing filamin (chicken gizzard), a protein that bundles actin filaments. Confocal fluorescence imaging of pure actin solutions in the presence of aluminum showed a disrupted actin network composed of fragmented actin filaments in the form of small aggregates. In contrast, in the presence of filamin, aluminum promoted the formation of thicker actin filaments. These measurements demonstrate that aluminum can affect actin filaments differentially depending on the presence of an actin-binding protein. In addition, a strong correlation is observed between the changes in viscosity as measured by ODM and the thickness and assembled state of bundles of actin filaments.

Latrunculin B has different effects on pollen germination and tube growth.

The actin cytoskeleton is absolutely required for pollen germination and tube growth, but little is known about the regulation of actin polymer concentrations or dynamics in pollen. Here, we report that latrunculin B (LATB), a potent inhibitor of actin polymerization, had effects on pollen that were distinct from those of cytochalasin D. The equilibrium dissociation constant measured for LATB binding to maize pollen actin was determined to be 74 nM. This high affinity for pollen actin suggested that treatment of pollen with LATB would have marked effects on actin function. Indeed, LATB inhibited maize pollen germination half-maximally at 50 nM, yet it blocked pollen tube growth at one-tenth of that concentration. Low concentrations of LATB also caused partial disruption of the actin cytoskeleton in germinated maize pollen, as visualized by light microscopy and fluorescent-phalloidin staining. The amounts of filamentous actin (F-actin) in pollen were quantified by measuring phalloidin binding sites, a sensitive assay that had not been used previously for plant cells. The amount of F-actin in maize pollen increased slightly upon germination, whereas the total actin protein level did not change. LATB treatment caused a dose-dependent depolymerization of F-actin in populations of maize pollen grains and tubes. Moreover, the same concentrations of LATB caused similar depolymerization in pollen grains before germination and in pollen tubes. These data indicate that the increased sensitivity of pollen tube growth to LATB was not due to general destabilization of the actin cytoskeleton or to decreases in F-actin amounts after germination. We postulate that germination is less sensitive to LATB than tube extension because the presence of a small population of LATB-sensitive actin filaments is critical for maintenance of tip growth but not for germination of pollen, or because germination is less sensitive to partial depolymerization of the actin cytoskeleton.

Actin Purified from Maize Pollen Functions in Living Plant Cells.

A vast array of actin binding proteins (ABPs), together with intracellular signaling molecules, modulates the spatiotemporal distribution of actin filaments in eukaryotic cells. To investigate the complex regulation of actin organization in plant cells, we designed experiments to reconstitute actin-ABP interactions in vitro with purified components. Because vertebrate skeletal [alpha]-actin has distinct and unpredictable binding affinity for nonvertebrate ABPs, it is essential that these in vitro studies be performed with purified plant actin. Here, we report the development of a new method for isolating functional actin from maize pollen. The addition of large amounts of recombinant profilin to pollen extracts facilitated the depolymerization of actin filaments and the formation of a profilin-actin complex. The profilin-actin complex was then isolated by affinity chromatography on poly-L-proline-Sepharose, and actin was selectively eluted with a salt wash. Pollen actin was further purified by one cycle of polymerization and depolymerization. The recovery of functional actin by this rapid and convenient procedure was substantial; the average yield was 6 mg of actin from 10 g of pollen. We undertook an initial physicochemical characterization of this native pollen actin. Under physiological conditions, pollen actin polymerized with kinetics similar in quality to those for vertebrate [alpha]-actin and had a critical concentration for assembly of 0.6 [mu]M. Moreover, pollen actin interacted specifically and in a characteristic fashion with several ABPs. Tradescantia cells were microinjected and used as an experimental system to study the behavior of pollen actin in vivo. We demonstrated that purified pollen actin ameliorated the effects of injecting excess profilin into live stamen hair cells.

Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.

In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group.

Interaction of pollen F-actin with rabbit muscle myosin and its subfragments (HMM, S1)

Actin purified from maize pollen grains can be polymerized into F-actin which increased the ATPase activities of proteolytic fragments (HMM, S1) of rabbit muscle myosin. The values of Kapp is 232 μM for HMM and 290 μM for S1, which are six- and seven-fold higher than those of rabbit muscle F-actin under the same conditions. Pollen actin and rabbit muscle myosin form hybrid actomyosin showing increase in viscosity and turbidity of solution. Viscosity and turbidity of the actomyosin dropped and then increased again with addition of ATP. Polymerized pollen actin can be decorated in vitro with both rabbit muscle HMM and S1 to form an arrowhead-shaped structure like that observed in living plant cells. The results show that pollen actin is similar to muscle actin at a qualitative level. But there are differences between them at a quantitative level.

Polymerization of Actin from Maize Pollen.

Here we describe the in vitro polymerization of actin from maize (Zea mays) pollen. The purified actin from maize pollen reported in our previous paper (X. Liu, L.F. Yen [1992] Plant Physiol 99: 1151-1155) is biologically active. In the presence of ATP, KCl, and MgCl2 the purified pollen actin polymerized into filaments. During polymerization the spectra of absorbance at 232 nm increased gradually. Polymerization of pollen actin was evidently accompanied by an increase in viscosity of the pollen actin solution. Also, the specific viscosity of pollen F-actin increased in a concentration-dependent manner. The ultraviolet difference spectrum of pollen actin is very similar to that of rabbit muscle actin. The activity of myosin ATPase from rabbit muscle was activated 7-fold by the polymerized pollen actin (F-actin). The actin filaments were visualized under the electron microscope as doubly wound strands of 7 nm diameter. If cytochalasin B was added before staining, no actin filaments were observed. When actin filaments were treated with rabbit heavy meromyosin, the actin filaments were decorated with an arrowhead structure. These results imply that there is much similarity between pollen and muscle actin.

Identification of profilin as an actin-binding protein in higher plants.

Profilin is a low molecular weight protein involved in the organization of the mammalian and protozoan cytoskeleton as well as in signal transduction. In this study, profilin is identified as an actin-binding protein in higher plants which is present in monocot and dicot angiosperms. Birch pollen profilin and actin can be copurified as a complex, and purified recombinant birch profilin can be used as an affinity matrix to obtain birch pollen actin. The binding of 125I-labeled recombinant birch pollen profilin to plant and animal actins can be blocked by profilin-specific antibodies that react with different epitopes of birch profilin. One of the blocking antibodies was raised against the 25 COOH-terminal amino acids indicating the importance of this region in the profilactin complex formation.

Purification and Characterization of Actin from Maize Pollen

Pollen is an excellent source of actin for biochemical and physiological studies of the actomyosin system in higher plants. We have developed an efficient method to prepare relatively high levels of actin from the pollen of maize (Zea mays L.). The procedures of purification include acetone powder preparation, saturated ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, a cycle of polymerization-depolymerization, and Sephacryl S-200 gel filtration. The average yield of actin is 19 milligrams per 100 grams of pollen grains extracted. This is comparable with those of Acanthamoeba castellanii and human platelets. The purified pollen actin is electrophoretically homogeneous and its molecular mass is 42 kilodaltons. The amino acid composition and circular dichroism spectrum of pollen actin are identical to those of muscle actin. The actin purified from pollen is able to polymerize to F-actin. The pollen F-actin activated the activity of the muscle myosin ATPase sevenfold.

Cyclical transformations of the actin cytoskeleton of hyacinth pollen subjected to recurrent vapour-phase hydration and dehydration

Transformations of the actin cytoskeleton of the pollen of Hyacinthus orientalis during cycles of vapour-phase hydration and dehydration have been examined using a non-fixation, DMSO permeabilisation method for TRITC-phalloidin staining, coupled with microwave stabilisation. In freshly shed pollen actin appears: a) at the plasmalemma in the form of extended, thin fibrils co-oriented with the cellulosic microfibrils of the contiguous intine; b) as a sheath investing the generative cell; c) as spicules around the vegetative nucleus; and d) in scattered spicules in the cytoplasm. During hydration in 85–95% relative humidity (RH), actin from all of these sites is progressively translated into a system of extended fibrils in the vegetative cell, concurrently with the onset of movement in the cytoplasm. Dehydration at 5–7% RH reverses this process, actin accumulating in rodlets, spicules or larger fusiform bodies in close association with the generative cell and vegetative nucleus, and also in the cytoplasm. The fibril system initially present at the plasmalemma is not restored. After ten cycles of hydration and dehydration 3.6% of the grains remained germinable. The ecological significance of the findings is noted, and the possibility that the observed transformations result from variation in the Ca 2+ concentration in the cytosol as the water content of the cytosol changes is considered.