Plants contain highly divergent actin isovariants

Actin protein isovariants have been identified in animals with distinct cytoplasmic or muscle specific patterns of expression. Analysis of vascular plant actin gene sequences suggests that an even greater diversity should exist within the plant actin protein families, but previous studies on plant proteins have not demonstrated the presence of multiple actin isovariants. Antibodies recognizing a conserved amino-terminal plant actin peptide, a family of plant actin peptides from a variable region, and two monoclonal antibodies to conserved epitopes within animal actins were used to identify isovariants of soybean actin resolved by two-dimensional isoelectric focusing (IEF) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Approximately six to eight actin isovariants with pI values ranging from 5.1 to 5.8 have been identified from soybean hypocotyls, stems, leaves, and roots with varying amounts of most isovariants present in all four organs. Acidic isovariants were present in much higher levels in leaves and stems. Antisera with lambda-class actin specificity detected a subset of three isovariants in all organs examined. One monoclonal and one antipeptide antisera are shown to react well with a wide variety of plant actin isovariants. Similar patterns of actin isovariants were detected in the distant angiosperms, Arabidopsis, petunia, and maize. It is likely that many of these diverse classes of isovariants have been preserved throughout vascular plant evolution and reflect the ancient diversity within plant actin gene families. The extreme difference among isovariants implies the presence of a complex actin-based cytoskeletal system in plants.     

Both vegetative and reproductive actin isovariants complement the stunted root hair phenotype of the Arabidopsis act2-1 mutation

The ACT2 gene, encoding one of eight actin isovariants in Arabidopsis, is the most strongly expressed actin gene in vegetative tissues. A search was conducted for physical defects in act2-1 mutant plants to account for their reduced fitness compared with wild type in population studies. The act2-1 insertion fully disrupted expression of ACT2 RNA and significantly lowered the level of total actin protein in vegetative organs. The root hairs of the act2-1 mutants were 10% to 70% the length of wild-type root hairs, and they bulged severely at the base. The length of the mutant root hairs and degree of bulging at the base were affected by adjusting the osmolarity and gelling agent of the growth medium. The act2-1 mutant phenotypes were fully rescued by an ACT2 genomic transgene. When the act2-1 mutation was combined with another vegetative actin mutation, act7-1, the resulting double mutant exhibited extensive synergistic phenotypes ranging from developmental lethality to severe dwarfism. Transgenic overexpression of the ACT7 vegetative isovariant and ectopic expression of the ACT1 reproductive actin isovariant also rescued the root hair elongation defects of the act2-1 mutant. These results suggest normal ACT2 gene regulation is essential to proper root hair elongation and that even minor differences may cause root defects. However, differences in the actin protein isovariant are not significant to root hair elongation, in sharp contrast to recent reports on the functional nonequivalency of plant actin isovariants. Impairment of root hair functions such as nutrient mining, water uptake, and physical anchoring are the likely cause of the reduced fitness seen for act2-1 mutants in multigenerational studies.     

Class-specific interaction of profilin and ADF isovariants with actin in the regulation of plant development

Two ancient and highly divergent actin-based cytoskeletal systems have evolved in angiosperms. Plant genomes encode complex actin and actin binding protein (ABP) gene families, most of which are phylogenetically grouped into gene classes with distinct vegetative or constitutive and reproductive expression patterns. In Arabidopsis thaliana, ectopic expression of high levels of a reproductive class actin, ACT1, in vegetative tissues causes severe dwarfing of plants with aberrant organization of most plant organs and cell types due to a severely altered actin cytoskeletal architecture. Overexpression of the vegetative class actin ACT2 to similar levels, however, produces insignificant phenotypic changes. We proposed that the misexpression of the pollen-specific ACT1 in vegetative cell types affects the dynamics of actin due to its inappropriate interaction with endogenous vegetative ABPs. To examine the functionally distinct interactions among the major classes of actins and ABPs, we ectopically coexpressed reproductive profilin (PRF4) or actin-depolymerizing factor (ADF) isovariants (e.g., ADF7) with ACT1. Our results demonstrated that the coexpression of these reproductive, but not vegetative, ABP isovariants suppressed the ectopic ACT1 expression phenotypes and restored wild-type stature and normal actin cytoskeletal architecture to the double transgenic plants. Thus, the actins and ABPs appear to have evolved class-specific, protein-protein interactions that are essential to the normal regulation of plant growth and development.     

Trypanosoma cruzi: Multiple actin isovariants are observed along different developmental stages

The expression and biological role of actin during the Trypanosoma cruzi life cycle remains largely unknown. Polyclonal antibodies against a recombinant T. cruzi actin protein were used to confirm its expression in epimastigotes, trypomastigotes, and amastigotes. Although the overall levels of expression were similar, clear differences in the subcellular distribution of actin among the developmental stages were identified. The existence of five actin variants in each developmental stage with distinct patterns of expression were uncovered by immunoblotting of protein extracts separated 2D-SDS gels. The isoelectric points of the actin variants in epimastigotes ranged from 4.45 to 4.9, whereas they ranged from 4.9 to 5.24 in trypomastigotes and amastigotes. To determine if the actin variants found could represent previously unidentified actins, we performed a genomic survey of the T. cruzi GeneDB database and found 12 independent loci encoding for a diverse group of actins and actin-like proteins that are conserved among trypanosomatids.     

Functional design in the actin cytoskeleton

Changes in cell shape, anchorage and motility are all associated with the dynamic reorganisation of the architectural arrays of actin filaments that make up the actin cytoskeleton. The relative expression of these functionally different actin filament arrays is intimately linked to the pattern of contacts that a cell develops with its extracellular substrate. Cell polarity is acquired by the development of an asymmetric pattern of substrate contacts, effected in a specific, site-directed manner by the delivery of adhesion-site modulators along microtubules.     

Functional homologs of fungal metallothionein genes from Arabidopsis.

Metallothioneins (MTs) are cysteine-rich proteins required for heavy metal tolerance in animals and fungi. Two cDNAs encoding proteins with homology to animal and fungal MTs have been isolated from Arabidopsis. The genes represented by these cDNAs are referred to as MT1 and MT2. When expressed in an MT-deficient (cup1 delta) mutant of yeast, both MT1 and MT2 complemented the cup1 delta mutation, providing a high level of resistance to CuSO4 and moderate resistance to CdSO4. Although the MT-deficient yeast was not viable in the presence of either 300 microM CuSO4 or 5 microM CdSO4, cells expressing MT1 were able to grow in medium supplemented with 3 mM CuSO4 and 10 microM CdSO4, and those expressing MT2 grew in the presence of 3 mM CuSO4 and 100 microM CdSO4. In plants, MT1 mRNA was more abundant in roots and dark-grown seedlings than in leaves. In contrast, MT2 mRNA accumulated more in leaves than in either roots or darkgrown seedlings. MT2 mRNA was strongly induced in seedlings by CuSO4, but only slightly by CdSO4 or ZnSO4. However, MT1 mRNA was induced by CuSO4 in excised leaves that were submerged in medium. These results indicated that Arabidopsis MT genes are involved in copper tolerance. Plants also synthesized metal binding phytochelatins (poly[gamma-glutamylcysteine]glycine) when exposed to heavy metals. The results presented here argue against the hypothesis that phytochelatins are the sole molecules involved in heavy metal tolerance in plants. We conclude that Arabidopsis MT1 and MT2 are functional homologs of yeast MT.     

Functional analysis of actin fibrils in Physarum polycephalum

Summary Fluorochromed heavy meromyosin (TRITC-HMM) was microinjected as a molecular probe into small sandwich-plasmodia of Physarum polycephalum with the aim to demonstrate the spatial morphology and to analyze the dynamic activity of the fibrillar actin system in the living state. The plasmodia display different fibrillar organizations with a polygonal arrangement in the front region (FR) and a parallel or helical arrangement along protoplasmic veins in the intermediate (IR) and uroid region (UR). Quantitative evaluations by measuring the total length, lifetime, dynamic activity, long-term stability and optical density of fibrils reveal distinct differences between the three plasmodial regions: The total length (FR = 27.1 ± 18.5 m, IR = 24.8 ± 12.9 m, UR= 12.3 ± 4.7 m), the lifetime (FR = 12.2 ± 3.4 min, IR=10.5 ± 3.7 min, UR = 6.0 ± 3.4 min), and the dynamic activity as measured in length changes per min (FR = 17.9 ± 11.3 m, IR = 13.1 ± 3.9 m, UR = 8.3 ± 3.9 m) distinctly decrease from the front to the uroid region. On the other hand, the greatest stability as determined by lifetime changes in length (FR = -2.4 ± 16.2 m, IR = 0.3 ± 10.1 m, UR = -6.6 ± 8.9 m) and the highest optical density as expressed in grey-values (FR = 57.0 ± 14.1 gv, IR = 115.6 ± 26.1 gv, UR 62.5 ± 8.1 gv) were found for actomyosin fibrils of the intermediate region. The morphological and physiological data of the present paper are discussed with respect to the biological significance of the fibrillar microfilament system in Physarum polycephalum.     

Functional sorting of actin isoforms in microvascular pericytes

We characterized the form and distribution of muscle and nonmuscle actin within retinal pericytes. Antibodies with demonstrable specificities for the actin isoforms were used in localization and immunoprecipitation experiments to identify those cellular domains that were enriched or deficient in one or several actin isoforms. Living pericyte behavior was monitored with phase-contract video microscopy before fixation to identify those cellular areas that might preferentially be stained with either of the fluorescent antiactins or phallotoxins. Antibody and phallotoxin staining of pericytes revealed that nonmuscle actin is present within membrane ruffles, pseudopods, and stress fibers. In contrast, muscle actin could be convincingly localized in stress fibers, but not within specific motile areas of pericyte cytoplasm. To confirm and quantitatively extend the results obtained by fluorescence microscopy, nonionic and ionic detergents were used to selectively extract the motile or immobilized (stress fiber-containing) regions of biosynthetically labeled pericyte cytoplasm. Immunoprecipitated actins that were present within these discrete cellular domains were subjected to isoelectric focusing in urea-polyacrylamide gels before fluorographic analysis. Scanning laser densitometry of the focused actins could not reveal any detectable alpha-actin within those beta- and gamma-actin-enriched motile regions extracted with nonionic detergents. Moreover, when pericyte stress fibers are completely dissolved by ionic detergent lysis, three actin isoforms can be quantified to be present in a ratio of 1:2.75:3 (alpha:beta:gamma). These biochemical findings on biosynthetically labeled and immunoprecipitated pericyte actins confirm the fluorescent localization studies. While the regulatory events governing this actin sorting are unknown, it seems possible that such events may play important roles in controlling cell shape, adhesion, or the promotion of localized cell spreading.     

Functional differentiation of endosomes in Arabidopsis cells

Endocytosis plays an important role in plant physiology, but how endocytic organelles are organized remains unknown. We present the evidence that endosomes are functionally differentiated in Arabidopsis cells. Two types of Rab5-related GTPases are localized on distinct population of endosomes in a partially overlapping manner. Ara7 and Rha1 are on an early type of endosomes with AtVamp727, where recycling of plasma membrane proteins occurs. In contrast, the plant-unique Rab5, Ara6, resides on distinct endosomes with the prevacuolar SNAREs. Partially overlapping localization of Ara6 and Ara7/Rha1 with reciprocal gradients suggests maturation of endosomes from one to the other.     

The plant-specific actin binding protein SCAB1 stabilizes actin filaments and regulates stomatal movement in Arabidopsis

Microfilament dynamics play a critical role in regulating stomatal movement; however, the molecular mechanism underlying this process is not well understood. We report here the identification and characterization of STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1), an Arabidopsis thaliana actin binding protein. Plants lacking SCAB1 were hypersensitive to drought stress and exhibited reduced abscisic acid-, H(2)O(2)-, and CaCl(2)-regulated stomatal movement. In vitro and in vivo analyses revealed that SCAB1 binds, stabilizes, and bundles actin filaments. SCAB1 shares sequence similarity only with plant proteins and contains a previously undiscovered actin binding domain. During stomatal closure, actin filaments switched from a radial orientation in open stomata to a longitudinal orientation in closed stomata. This switch took longer in scab1 plants than in wild-type plants and was correlated with the delay in stomatal closure seen in scab1 mutants in response to drought stress. Our results suggest that SCAB1 is required for the precise regulation of actin filament reorganization during stomatal closure.     

Functional synergy of actin filament cross-linking proteins

The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.     

Functional bioinformatics for Arabidopsis thaliana

The authors would     

Functional bioinformatics for Arabidopsis thaliana

MOTIVATION: The genome of Arabidopsis thaliana, which has the best understood plant genome, still has approximately one-third of its genes with no functional annotation at all from either MIPS or TAIR. We have applied our Data Mining Prediction (DMP) method to the problem of predicting the functional classes of these protein sequences. This method is based on using a hybrid machine-learning/data-mining method to identify patterns in the bioinformatic data about sequences that are predictive of function. We use data about sequence, predicted secondary structure, predicted structural domain, InterPro patterns, sequence similarity profile and expressions data. RESULTS: We predicted the functional class of a high percentage of the Arabidopsis genes with currently unknown function. These predictions are interpretable and have good test accuracies. We describe in detail seven of the rules produced.     

Green fluorescent protein-mTalin causes defects in actin organization and cell expansion in Arabidopsis and inhibits actin depolymerizing factor's actin depolymerizing activity in vitro

Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that GFP-mTalin colocalizes with the actin cytoskeleton, its effect on actin dynamics and cell expansion has not been studied in detail. We created Arabidopsis (Arabidopsis thaliana) plants harboring alcohol inducible GFP-mTalin constructs to assess the effect of GFP-mTalin expression in vivo. We focused on the growing root hair as this is a model cell for studying cell expansion and root hair tip growth that requires a highly dynamic and polar actin cytoskeleton. We show that alcohol inducible expression of GFP-mTalin in root hairs causes severe defects in actin organization, resulting in either the termination of growth, cell death, and/or changes in cell shape. Fluorescence recovery after photobleaching experiments demonstrate that the interaction of GFP-mTalin and actin filaments is highly dynamic. To assess how GFP-mTalin affects actin dynamics we performed cosedimentation assays of GFP-mTalin with actin on its own or in the presence of the actin modulating protein, actin depolymerizing factor. We show that that GFP-mTalin does not affect actin polymerization but that it does inhibit the actin depolymerizing activity of actin depolymerizing factor. These observations demonstrate that GFP-mTalin can affect cell expansion, actin organization, and the interaction of actin binding proteins with actin.     

Functional analysis of N-terminal domains of Arabidopsis chlorophyllide a oxygenase.

Higher plants acclimate to various light environments by changing the antenna size of a light-harvesting photosystem. The antenna size of a photosystem is partly determined by the amount of chlorophyll b in the light-harvesting complexes. Chlorophyllide a oxygenase (CAO) converts chlorophyll a to chlorophyll b in a two-step oxygenation reaction. In our previous study, we demonstrated that the cellular level of the CAO protein controls accumulation of chlorophyll b. We found that the amino acids sequences of CAO in higher plants consist of three domains (A, B, and C domains). The C domain exhibits a catalytic function, and we demonstrated that the combination of the A and B domains regulates the cellular level of CAO. However, the individual function of each of A and B domain has not been determined yet. Therefore, in the present study we constructed a series of deleted CAO sequences that were fused with green fluorescent protein and overexpressed in a chlorophyll b-less mutant of Arabidopsis thaliana, ch1-1, to further dissect functions of A and B domains. Subsequent comparative analyses of the transgenic plants overexpressing B domain containing proteins and those lacking the B domain determined that there was no significant difference in CAO protein levels. These results indicate that the B domain is not involved in the regulation of the CAO protein levels. Taken together, we concluded that the A domain alone is involved in the regulatory mechanism of the CAO protein levels.     

Functional interdependence between septin and actin cytoskeleton

Background  

Septin2 is a member of a highly conserved GTPase family found in fungi and animals. Septins have been implicated in a diversity of cellular processes including cytokinesis, formation of diffusion barriers and vesicle trafficking. Septin2 partially co-localises with actin bundles in mammalian interphase cells and Septin2-filamentmorphology depends upon an intact actin cytoskeleton. How this interaction is regulated is not known. Moreover, evidence that Septin2 is remodelled or redistributed in response to other changes in actin organisation is lacking.