Arabidopsis cDNA Research Articles

Anther-specific coumarate CoA ligase-like gene from Nicotiana sylvestris expressed during uninucleate microspore development

An anther-specific cDNA clone from Nicotiana sylvestris /NsCL-lk/, showing similarity to coumarate CoA ligases, was isolated and characterized. Northern blot and in situ hybridization analyses demonstrated transcript accumulation in tapetal cells during short period of anther development correlated with tetrad dissolution and uninucleate microspore development. An Arabidopsis cDNA clone /AtCL-lk/ homologous to NsCL-lk was isolated after screening of Arabidopsis flower cDNA library with NsCL-lk as a heterologous probe. RT-PCR analysis detected AtCL-lk transcripts only in young flower buds but not in other plant organs and seedlings. Sequence analysis of NsCL-lk and AtCL-lk shows significant similarity to coumarate CoA ligase protein sequences. The phylogenetic study suggests that the anther-specific CL-like genes represent a separate group in the family of adenylate-forming enzymes, close to the group of coumarate CoA ligases. The putative function of described CL-like enzymes in anther development is discussed.

T-DNA tagging in Brassica napus as an efficient tool for the isolation of new promoters for selectable marker genes.

A simple strategy to identify and isolate new promoters suitable for driving the expression of selectable marker genes is described. By employing a Brassica napus hypocotyl transformation protocol and a promoterless gus::nptII tagging construct, a series of 20 kanamycin-resistant tagged lines was produced. Most of the regenerated plants showed hardly any GUS activity in leaf, stem and root tissues. However, expression was readily restored in callus tissue induced on in vitro leaf segments. Genomic sequences upstream of the gus::nptII insertions were isolated via plasmid rescue. Three clones originating from single copy T-DNA lines were selected for further evaluation. The rescued plasmids were cloned as linear fragments in binary vectors and re-transformed to Brassica napus hypocotyl and Solanum tuberosum stem segments. The new sequences maintained their promoter activity, demonstrated by transient and stable GUS activity after transformation. Furthermore, the promoters provided sufficient expression of the nptII gene to yield transgenic plants when using kanamycin as selective agent. Database searching (BLASTN) revealed that the promoters have significant homology with three Arabidopsis BAC clones, one Arabidopsis cDNA and one Brassica napus cDNA. The results presented in this paper illustrate the strength of combined methods for identification, isolation and testing of new plant promoters.

Generation of Arabidopsis protein chips for antibody and serum screening.

Protein array technology has emerged as a new tool to enable ordered screening of proteins for expression and molecular interactions in high throughput. Besides classical solid-phase substrates, such as micro-titre plates and membrane filters, protein arrays have recently been devised with chip-sized supports. Several applications on protein chips have been described, but to our knowledge no studies using plant protein chips were published so far. The aim of this study was to generate Arabidopsis protein chips and to demonstrate the feasibility of the protein chip technology for the investigation of antigen-antibody interactions. Therefore, Arabidopsis cDNAs encoding 95 different proteins were cloned into a GATEWAY-compatible Escherichia coli expression vector. RGS-His6-tagged recombinant proteins were purified in high throughput and robotically arrayed onto glass slides coated either with a nitrocellulose based polymer (FAST slides) or polyacrylamide (PAA slides). Using an anti-RGS-His6 antibody all proteins were detected on the chips. The detection limit was ca. 2-3.6 fmol per spot on FAST slides or 0.1-1.8 fmol per spot on PAA slides. The Arabidopsis protein chips were used for the characterisation of monoclonal antibodies or polyclonal sera. We were able to show that a monoclonal anti-TCP1 antibody and anti-MYB6 and anti-DOF11 sera bound specifically to their respective antigens and did not cross-react with the other 94 proteins including other DOF and MYB transcription factors on the chips. To enable screening of antibodies or other interacting molecules against thousands of Arabidopsis proteins in future, we generated an ordered cDNA expression library and started with high-throughput cloning of full-length cDNAs with GATEWAY technology.

Use of SAGE technology to reveal changes in gene expression in Arabidopsis leaves undergoing cold stress.

The genes expressed within an organism determine its biological characteristics. Various internal or external factors can modulate these gene expression patterns, which then elicit physiological or pathological changes. We have characterized the global gene expression patterns of Arabidopsis leaves by serial analysis of gene expression (SAGE). A total of 21,280 SAGE tags were sequenced and 12,049 unique tags were identified. Among these, only 3367 tags (27.9%) were matched to the Arabidopsis cDNA or EST database. Functional analysis of annotated tags indicated that a significant proportion of the genes expressed in normal leaves were involved in energy and metabolism, especially in photosynthesis. To systematically analyze differential gene expression profiles under cold stress, a similar SAGE tag library from cold-treated leaves was constructed and analyzed. A comparison of the tags derived from the cold-treated leaves with those identified in the normal leaves revealed 272 differentially expressed genes (P<0.01): 82 genes were highly expressed in the normal leaves and 190 genes were highly expressed in the cold-treated leaves. After cold stress, in general, many of the genes involved in cell rescue/defense/cell death/aging, protein synthesis, metabolism, transport facilitation, and protein destination were induced. They included various COR genes, lipid transfer protein genes, alcohol dehydrogenase, beta-amylase and many novel genes. By comparison, down-regulated genes were mostly photosynthesis related genes involved in energy metabolism. The expression patterns of several cold responsive transcripts identified by SAGE were confirmed by northern analysis. The results presented here will provide valuable information for understanding the mechanisms of the freezing tolerance of plants.

Identification of anther-specific genes in a cruciferous model plant, Arabidopsis thaliana, by using a combination of Arabidopsis macroarray and mRNA derived from Brassica oleracea

Arabidopsis thaliana, a member of the Brassicaceae, is a model plant whose genome was the first higher plant genome to be sequenced. Because of the small size of the flowers, it is difficult to dissect and separate reproductive organs (anthers and pistils) at different developmental stages in A. thaliana. In order to perform genome-wide identification of anther-specific genes in A. thaliana, an Arabidopsis cDNA macroarray was hybridized to cDNA derived from anthers and pistils of another crucifer, Brassica oleracea. After scanning the signal intensity for each clone, and cluster analysis, 52 anther-specific genes were identified. These clones contained several anther-specific genes that have already been characterized, as well as novel anther-specific genes. In RT-PCR analysis with mRNA of A. thaliana and B. oleracea, the expression pattern of one-third of the clones was similar to that determined by cDNA macroarray. This system of heterologous hybridization analysis (Arabidopsis cDNA macroarray vs Brassica tissue-specific mRNA) should be applicable to other model species and their close relatives.

Differential phosphorylation activities of CDK-activating kinases in Arabidopsis thaliana

Activation of cyclin-dependent kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop by a CDK-activating kinase (CAK). Here we isolated an Arabidopsis cDNA ( CAK4At) whose predicted product shows a high similarity to vertebrate CDK7/p40 MO15. Northern blot analysis showed that expressions of the four Arabidopsis CAKs ( CAK1At–CAK4At) were not dependent on cell division. CAK2At- and CAK4At-immunoprecipitates of Arabidopsis crude extract phosphorylated CDK and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II with different preferences. These results suggest the existence of differential mechanisms in Arabidopsis that control CDK and CTD phosphorylation by multiple CAKs.

Molecular characterization of an Arabidopsis acyl-coenzyme a synthetase localized on glyoxysomal membranes.

In higher plants, fat-storing seeds utilize storage lipids as a source of energy during germination. To enter the beta-oxidation pathway, fatty acids need to be activated to acyl-coenzyme As (CoAs) by the enzyme acyl-CoA synthetase (ACS; EC 6.2.1.3). Here, we report the characterization of an Arabidopsis cDNA clone encoding for a glyoxysomal acyl-CoA synthetase designated AtLACS6. The cDNA sequence is 2,106 bp long and it encodes a polypeptide of 701 amino acids with a calculated molecular mass of 76,617 D. Analysis of the amino-terminal sequence indicates that acyl-CoA synthetase is synthesized as a larger precursor containing a cleavable amino-terminal presequence so that the mature polypeptide size is 663 amino acids. The presequence shows high similarity to the typical PTS2 (peroxisomal targeting signal 2). The AtLACS6 also shows high amino acid identity to prokaryotic and eukaryotic fatty acyl-CoA synthetases. Immunocytochemical and cell fractionation analyses indicated that the AtLACS6 is localized on glyoxysomal membranes. AtLACS6 was overexpressed in insect cells and purified to near homogeneity. The purified enzyme is particularly active on long-chain fatty acids (C16:0). Results from immunoblot analysis revealed that the expression of both AtLACS6 and beta-oxidation enzymes coincide with fatty acid degradation. These data suggested that AtLACS6 might play a regulatory role both in fatty acid import into glyoxysomes by making a complex with other factors, e.g. PMP70, and in fatty acid beta-oxidation activating the fatty acids.

Cloning and Characterization of Mitochondrial 5-Formyltetrahydrofolate Cycloligase from Higher Plants

5-Formyltetrahydrofolate cycloligase (5-FCL) catalyzes the conversion of 5-formyltetrahydrofolate (5-CHO-H(4)PteGlu(n)) to 5,10-methenyltetrahydrofolate and is considered to be the main means whereby 5-CHO-H(4)PteGlu(n) is metabolized in mammals, yeast, and bacteria. 5-CHO-H(4)PteGlu(n) is known to occur in plants and to be highly abundant in leaf mitochondria. Genomics-based approaches identified Arabidopsis and tomato cDNAs encoding proteins homologous to 5-FCLs of other organisms but containing N-terminal extensions with the features of mitochondrial targeting peptides. These homologs were shown to have 5-FCL activity by characterizing recombinant enzymes produced in Escherichia coli and by functional complementation of a yeast fau1 mutation with the Arabidopsis 5-FCL cDNA. The recombinant Arabidopsis enzyme is active as a monomer, prefers the penta- to the monoglutamyl form of 5-CHO-H(4)PteGlu(n), and has kinetic properties broadly similar to those of 5-FCLs from other organisms. Enzyme assays and immunoblot analyses indicated that 5-FCL is located predominantly if not exclusively in plant mitochondria and that the mature, active enzyme lacks the putative targeting sequence. Serine hydroxymethyltransferase (SHMT) from plant mitochondria was shown to be inhibited by 5-CHO-H(4)PteGlu(n) as are SHMTs from other organisms. Since mitochondrial SHMT is crucial to photorespiration, 5-FCL may help prevent 5-CHO-H(4)PteGlu(n) from reaching levels that would inhibit this process. Consistent with this possibility, 5-FCL activity was far higher in leaf mitochondria than root mitochondria.

Comparative proteome bioinformatics: Identification of a whole complement of putative protein tyrosine kinases in the model flowering plant Arabidopsis thaliana

Phosphorylation by protein tyrosine kinases is crucial to the control of growth and development of multicellular eukaryotes, including humans, and it also seems to play an important role in multicellular prokaryotes. A plant tyrosine-specific kinase has not been identified yet; hence, plants have been suggested to share with unicellular eukaryote yeast a tyrosine phosphorylation system where a limited number of stress proteins are tyrosyl-phosphorylated only by a few dual-specificity (serine/threonine and tyrosine) kinases. However, preliminary evidence obtained so far suggests that tyrosine phosphorylation in plants depends on the developmental conditions. Since sequencing of the genome of the model flowering plant Arabidopsis thaliana has been recently completed, we have performed a bioinformatic screening of the whole Arabidopsis proteome to identify a model complement of bona fide protein tyrosine kinases. In silico analyses suggest that < 4% of Arabidopsis kinases are tyrosine-specific kinases, whose gene expression has been assessed by a preliminary polymerase chain reaction screening of an Arabidopsis cDNA library. Finally, immunological evidence confirms that the number of Arabidopsis proteins specifically phosphorylated on tyrosine residues is much higher than in yeast.

Role of gibberellins and of the RGA and GAI genes in controlling nitrate assimilation in Arabidopsis thaliana

Screening of an Arabidopsis cDNA library for complementation of a yeast mutant affected in the regulation of nitrogen metabolism led to the isolation of the RGA and GAI cDNAs that were also known to be involved in plant response to gibberellins. This raised the question of RGA and GAI being also involved in controlling nitrogen metabolism in plants. To address this issue we studied whether loss of function ( rga2, gai-t6) or gain of function mutations ( gai-1) in RGA or GAI or in both ( rga24 gai-t6) genes had any impact on nitrate assimilation in Arabidopsis plants grown in the greenhouse or in vitro. In addition, as the sole known plant function of RGA and GAI was their implication in gibberellin signal transduction, we analysed the effects of gibberellin treatment on nitrate assimilation of wild-type plants or of the gibberellin-deficient ga1-3 mutant. In most cases, no difference in expression of the NIA1, NIA2 (encoding nitrate reductase, NR EC 1.6.6.1), NII (encoding nitrite reductase, NiR EC 1.7.7.1), GS1 and GS2 genes (encoding cytosolic and chloroplastic glutamine synthetases EC 6.3.1.2) could be detected in the different genetic backgrounds or after GA treatment of wild-type plants or GA-deficient plants. This absence of effect is also in general supported by similar NR and NiR activities and nitrate or nitrogen content in the different plants. In conclusion, our studies show that RGA, GAI and gibberellins do not act as major factors controlling nitrate assimilation in Arabidopsis at the vegetative stage although they may have, in some instances, an effect on nitrate assimilation genes.

COI1 links jasmonate signalling and fertility to the SCF ubiquitin-ligase complex in Arabidopsis.

Jasmonates (JAs) regulate Arabidopsis thaliana wound and defence responses, pollen development, and stress-related growth inhibition. Significantly, each of these responses requires COI1, an F-box protein. Other F-box proteins interact with SKP1 and cullin proteins to form SCF complexes that selectively recruit regulatory proteins targeted for ubiquitination. To determine whether COI1 also functions in an SCF complex, we have characterized Arabidopsis proteins that bind to COI1. An Arabidopsis cDNA expression library was screened in yeast for clones that produce proteins which can bind to COI1. We recovered two SKP1 homologues and a histone deacetylase. The Arabidopsis F-box protein TIR1 interacted with SKP1 proteins, but not with the histone deacetylase. Mutant COI1 proteins revealed that the F-box is required for interaction with SKP1s, but that sequences in leucine-rich repeat domains are required for interaction with the histone deacetylase. Epitope-tagged COI1 was introduced into Arabidopsis plants and cell cultures. Co-immunoprecipitation experiments confirmed the interaction in planta of COI1 with SKP1-like proteins and histone deacetylase, and also indicated that COI1 interacted with cullin. These results suggest that COI1 forms an SCFCOI1 complex in vivo. COI1 is therefore expected to form a functional E3-type ubiquitin ligase in plants and to regulate expression of jasmonate responsive genes, possibly by targeted ubiquitination of a histone deacetylase.

Cloning and characterization of Arabidopsis homologues of the animal CstF complex that regulates 3' mRNA cleavage and polyadenylation.

The 3' cleavage and polyadenylation of mRNAs has been studied in detail in animals and yeast, but not in plants. Aimed at elucidating the regulation of mRNA 3' end formation in plants, three Arabidopsis cDNAs encoding homologues of the animal proteins CstF-64, CstF-77 and CstF-50 that form the cleavage stimulating factor of the polyadenylation machinery have been cloned. It is shown experimentally that the N-terminal domain of the Arabidopsis CstF-64 homologue binds the mRNA 3' non-coding region in an analogous manner to the animal protein. It is also shown that the Arabidopsis CstF-64 and CstF-77 homologues strongly interact with each other in a similar way to their animal counterparts. These results imply that these Arabidopsis homologues belong to the polyadenylation machinery of nuclear mRNAs.

Molecular characterization of a heteromeric ATP-citrate lyase that generates cytosolic acetyl-coenzyme A in Arabidopsis.

Acetyl-coenzyme A (CoA) is used in the cytosol of plant cells for the synthesis of a diverse set of phytochemicals including waxes, isoprenoids, stilbenes, and flavonoids. The source of cytosolic acetyl-CoA is unclear. We identified two Arabidopsis cDNAs that encode proteins similar to the amino and carboxy portions of human ATP-citrate lyase (ACL). Coexpression of these cDNAs in yeast (Saccharomyces cerevisiae) confers ACL activity, indicating that both the Arabidopsis genes are required for ACL activity. Arabidopsis ACL is a heteromeric enzyme composed of two distinct subunits, ACLA (45 kD) and ACLB (65 kD). The holoprotein has a molecular mass of 500 kD, which corresponds to a heterooctomer with an A(4)B(4) configuration. ACL activity and the ACLA and ACLB polypeptides are located in the cytosol, consistent with the lack of targeting peptides in the ACLA and ACLB sequences. In the Arabidopsis genome, three genes encode for the ACLA subunit (ACLA-1, At1g10670; ACLA-2, At1g60810; and ACLA-3, At1g09430), and two genes encode the ACLB subunit (ACLB-1, At3g06650 and ACLB-2, At5g49460). The ACLA and ACLB mRNAs accumulate in coordinated spatial and temporal patterns during plant development. This complex accumulation pattern is consistent with the predicted physiological needs for cytosolic acetyl-CoA, and is closely coordinated with the accumulation pattern of cytosolic acetyl-CoA carboxylase, an enzyme using cytosolic acetyl-CoA as a substrate. Taken together, these results indicate that ACL, encoded by the ACLA and ACLB genes of Arabidopsis, generates cytosolic acetyl-CoA. The heteromeric organization of this enzyme is common to green plants (including Chlorophyceae, Marchantimorpha, Bryopsida, Pinaceae, monocotyledons, and eudicots), species of fungi, Glaucophytes, Chlamydomonas, and prokaryotes. In contrast, all known animal ACL enzymes have a homomeric structure, indicating that a evolutionary fusion of the ACLA and ACLB genes probably occurred early in the evolutionary history of this kingdom.

A Putative Role for the Vacuolar Calcium/Manganese Proton Antiporter AtCAX2 in Heavy Metal Detoxification

Abstract: Regulation of uptake and compartmentation of metal ions is important for the maintenance of metal ion homeostasis. To identify mechanisms involved in the protection of plants from Mn toxicity, wild‐type yeast was transformed with an Arabidopsis cDNA library and transformants were screened on toxic Mn concentrations. Wild‐type yeast could not grow in the presence of 30 mM MnSO4, while two transformants carrying variants of the same gene were able to grow. Database searches revealed that the isolated cDNAs correspond to AtCAX2, previously described as a vacuolar calcium‐proton antiporter. Since no other genes could be identified, AtCAX2 might represent a major function permitting Mn detoxification in this suppressor screen. Furthermore, yeast transformed with the two AtCAX2 cDNAs showed increased sensitivity towards hydrogen peroxide, pointing to a limited availability of cytoplasmic Mn in the presence of AtCAX2 activity. The open reading frames of the cDNA encoded polypeptides that have a 42 and a 92 amino acids shorter N‐terminal region relative to the predicted full‐length coding region of AtCAX2. In contrast to both truncated cDNAs, the full‐length clone was unable to confer Mn resistance to yeast, indicating that, similar to AtCAX1, AtCAX2 also carries an autoinhibitory N‐terminal domain regulating the activity of AtCAX2.

Identification of an Unusual MYB-type Transcription Factor That Binds to Promoter of H-Protein of Glycine Decarboxylase

Expression of the H-protein of the mitochondrial glycine decarboxylase (GDC) complex is controlled at the transcriptional level in a light-dependent and tissue-specific manner. We have identified a protein binding site, HPP-1, within the region of the gdcH promoter that appears to control expression of this gene. Gel shift experiments suggested that the HPP-1 site contains a core DNA sequence of the hexameric palindrome, AATATT. An Arabidopsis cDNA coding for a DNA binding protein, HPPBF-1, was cloned that binds to this same HPP-1 site and has a binding requirement for the same AATATT core sequence. HPPBF-1 belongs to a small class of DNA binding factors that includes the initiator binding proteins, IBPl and BPF-1, as well as the telomeric proteins RTBPl and AtTRP1. These factors are unusual in that they contain a single C-terminal MYB-type DNA binding motif. This binding motif contains the same LKDKW DNA interaction region noted in other MYB factors. RNA blot analysis suggested that the mRNA for HPPBF-1 is present in all organs at varying levels.

Characterization of plant proteins that interact with cowpea mosaic virus '60K' protein in the yeast two-hybrid system.

Cowpea mosaic virus (CPMV) replication occurs in close association with small membranous vesicles in the host cell. The CPMV RNA1-encoded 60 kDa nucleotide-binding protein ('60K') plays a role in the formation of these vesicles. In this study, five cellular proteins were identified that interacted with different domains of 60K using a yeast two-hybrid search of an Arabidopsis cDNA library. Two of these host proteins (termed VAP27-1 and VAP27-2), with high homology to the VAP33 family of SNARE-like proteins from animals, interacted specifically with the C-terminal domain of 60K and upon transient expression colocalized with 60K in CPMV-infected cowpea protoplasts. eEF1-beta, picked up using the central domain of 60K, was also found to colocalize with 60K. The possible role of these host proteins in the viral replicative cycle is discussed.

Characterization of CAX4, an Arabidopsis H(+)/cation antiporter.

Ion compartmentalization is essential for plant growth and development. The Arabidopsis open reading frames for CAX1, CAX2, and CAX3 (cation exchangers 1, 2, and 3) were previously identified as transporters that may modulate ion fluxes across the vacuolar membrane. To understand the diversity and role of H(+)/cation transporters in controlling plant ion levels, another homolog of the CAX genes, CAX4, was cloned from an Arabidopsis cDNA library. CAX4 is 53% identical to CAX1 at the amino acid level, 42% identical to CAX2, and 54% identical to CAX3. CAX4 transcripts appeared to be expressed at low levels in all tissues and levels of CAX4 RNA increased after Mn(2+), Na(+), and Ni(2+) treatment. An N-terminal CAX4-hemagglutinin fusion appeared to localize to both yeast and plant vacuolar membranes. When expressed in yeast, CAX4, like CAX3, failed to suppress the Ca(2+) sensitivity of yeast strains deficient in vacuolar Ca(2+) transport. Several modifications to CAX4 allowed the protein to transport Ca(2+). Addition of amino acids to the N terminus of CAX4 and CAX3 caused both transporters to suppress the sensitivity of yeast strains deficient in vacuolar Ca(2+) transport. These findings suggest that CAX transporters may modulate their ion transport properties through alterations at the N terminus.

Functional annotation of a full-length Arabidopsis cDNA collection.

Full-length complementary DNAs (cDNAs) are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We isolated 155,144 RIKEN Arabidopsis full-length (RAFL) cDNA clones. The 3'-end expressed sequence tags (ESTs) of 155,144 RAFL cDNAs were clustered into 14,668 nonredundant cDNA groups, about 60% of predicted genes. We also obtained 5' ESTs from 14,034 nonredundant cDNA groups and constructed a promoter database. The sequence database of the RAFL cDNAs is useful for promoter analysis and correct annotation of predicted transcription units and gene products. Furthermore, the full-length cDNAs are useful resources for analyses of the expression profiles, functions, and structures of plant proteins.

Functional Cloning and Characterization of a Plant Efflux Carrier for Multidrug and Heavy Metal Detoxification

We have identified a detoxifying efflux carrier from Arabidopsis using a functional cloning strategy. A bacterial mutant, KAM3, is deficient in multidrug resistance and does not survive on medium containing norfloxacin. After transformation of KAM3 cells with an Arabidopsis cDNA library, transformants were selected for restored growth on the toxic medium. One cDNA clone that complemented KAM3 encodes a novel protein with twelve putative transmembrane domains and contains limited sequence homology to a multidrug and toxin efflux carrier from bacteria. We named this Arabidopsis protein AtDTX1 (for Arabidopsis thaliana Detoxification 1). A large gene family of at least 56 members encoding related proteins was identified from the Arabidopsis genome. Further functional analysis of AtDTX1 protein in KAM3 mutant demonstrated that AtDTX1 serves as an efflux carrier for plant-derived alkaloids, antibiotics, and other toxic compounds. Interestingly, AtDTX1 was also capable of detoxifying Cd(2+), a heavy metal. Further experiments suggest that AtDTX1 is localized in the plasma membrane in plant cells thereby mediating the efflux of plant-derived or exogenous toxic compounds from the cytoplasm.

Arabidopsis CAP regulates the actin cytoskeleton necessary for plant cell elongation and division.

An Arabidopsis cDNA (AtCAP1) that encodes a predicted protein of 476 amino acids highly homologous with the yeast cyclase-associated protein (CAP) was isolated. Expression of AtCAP1 in the budding yeast CAP mutant was able to rescue defects such as abnormal cell morphology and random budding pattern. The C-terminal domain, 158 amino acids of AtCAP1 possessing in vitro actin binding activity, was needed for the regulation of cytoskeleton-related defects of yeast. Transgenic plants overexpressing AtCAP1 under the regulation of a glucocorticoid-inducible promoter showed different levels of AtCAP1 accumulation related to the extent of growth abnormalities, in particular size reduction of leaves as well as petioles. Morphological alterations in leaves were attributable to decreased cell size and cell number in both epidermal and mesophyll cells. Tobacco suspension-cultured cells (Bright Yellow 2) overexpressing AtCAP1 exhibited defects in actin filaments and were unable to undergo mitosis. Furthermore, an immunoprecipitation experiment suggested that AtCAP1 interacted with actin in vivo. Therefore, AtCAP1 may play a functional role in actin cytoskeleton networking that is essential for proper cell elongation and division.