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Arabidopsis CAP1 – a Key Regulator of Actin Organisation and Development

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Arabidopsis CAP1 – a Key Regulator of Actin Organisation and Development Research Article 2609 Arabidopsis CAP1 – a key regulator of actin organisation and development Michael J. Deeks1,*, Cecília Rodrigues1,2,*, Simon Dimmock1,*, Tijs Ketelaar1, Sutherland K. Maciver3, Rui Malhó2 and Patrick J. Hussey1,‡ 1The Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, Durham University, South Road, Durham, DH1 3LE, UK 2Universidade de Lisboa, Faculdade Ciências, Instituto Ciência Aplicada e Tecnologia, Lisbon, Portugal 3Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, George Square, Edinburgh, EH8 9XD, UK *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected]) Accepted 15 May 2007 Journal of Cell Science 120, 2609-2618 Published by The Company of Biologists 2007 doi:10.1242/jcs.007302 Summary Maintenance of F-actin turnover is essential for plant cell also show synthetic phenotypes when combined with morphogenesis. Actin-binding protein mutants reveal that mutants of the Arp2/3 complex pathway, which further plants place emphasis on particular aspects of actin suggests a contribution of CAP1 to in planta actin biochemistry distinct from animals and fungi. Here we dynamics. In yeast, CAP interacts with adenylate cyclase show that mutants in CAP1, an A. thaliana member of the in a Ras signalling cascade; but plants do not have Ras. cyclase-associated protein family, display a phenotype that Surprisingly, cap1 plants show disruption in plant establishes CAP1 as a fundamental facilitator of actin signalling pathways required for co-ordinated organ dynamics over a wide range of plant tissues. Plants expansion suggesting that plant CAP has evolved to attain homozygous for cap1 alleles show a reduction in stature plant-specific signalling functions. and morphogenetic disruption of multiple cell types. Pollen grains exhibit reduced germination efficiency, and cap1 pollen tubes and root hairs grow at a decreased rate and to Supplementary material available online at a reduced length. Live cell imaging of growing root hairs http://jcs.biologists.org/cgi/content/full/120/15/2609/DC1 reveals actin filament disruption and cytoplasmic disorganisation in the tip growth zone. Mutant cap1 alleles Key words: Actin, CAP, Arabidopsis Journal of Cell Science Introduction cerevisiae cap mutants or vice-versa (Kawamukai et al., 1992). Cyclase-associated protein (CAP) was identified in S. CAP isoforms from other species are also unable to cerevisiae as an interactor of adenylate cyclase (AC) (Field et complement S. cerevisiae AC activation (Matviw et al., 1992; al., 1990). Mutations in CAP/SRV2 not only affect the Vojtek and Cooper, 1993; Yu et al., 1994; Zelicof et al., 1993) regulation of AC by Ras (Fedor-Chaiken et al., 1990; Shima et and have been argued to operate in their own species-specific al., 2000) but also cause actin organisational phenotypes signalling pathways (Hubberstey and Mottillo, 2002). The (Vojtek et al., 1991). Investigations into the biochemical cross-species association of apparently independent signalling activity of CAP in the context of the actin cytoskeleton has and cytoskeletal activities might reflect an as yet unidentified defined CAP as an actin-binding protein (ABP) capable of functional integration of the two roles (Vojtek and Cooper, associating with monomeric actin and facilitating actin 1993). treadmilling (Balcer et al., 2003; Mattila et al., 2004). The C- In addition to S. cerevisiae and S. pombe, CAP mutants have terminus of S. cerevisiae CAP is required in vivo and in vitro been identified and characterised in Drosophila (Baum et al., for the majority of cytoskeletal functions (Gerst et al., 1991; 2000; Benlali et al., 2000), in Dictyostelium (Noegel et al., Mattila et al., 2004), while the N-terminus regulates AC 1999) and in mammals, where RNAi suppression of CAP activation in vivo. The functional division between signalling function has been performed (Bertling et al., 2004). and actin organisation has led to CAP being considered a Phenotypes shared by these mutants are reductions in polarised bifunctional protein. cell morphology and cell motility coinciding with CAP is conserved over a wide range of organisms. Cross- disorganisation of actin-rich structures. At the level of tissue species complementation experiments have shown that organisation the cap phenotypes reveal a requirement for CAP heterologous CAP can consistently complement S. cerevisiae in multicellular developmental signalling pathways. In CAP-dependent cytoskeletal functions but not AC activation. Dictyostelium, CAP is required to perpetuate the cAMP relay The N-terminus of S. cerevisiae CAP is required to expose AC signal to organise fruitbody formation (Noegel et al., 2004), binding sites to Ras (Shima et al., 2000). S. pombe also requires and in Drosophila CAP is essential for Hedgehog-mediated eye CAP for AC activity (Kawamukai et al., 1992), but S. pombe development (Benlali et al., 2000). AC is not activated by the Ras pathway. CAP in S. pombe must Homologues of CAP have been identified in plants (Barrero facilitate AC activation in a novel fashion and, consequently, et al., 2002; Kawai et al., 1998). The single Arabidopsis the N-terminus of S. pombe CAP cannot complement S. isoform has been shown to have the ability to bind actin and 2610 Journal of Cell Science 120 (15) to complement the cytoskeletal defects of CAP-deficient yeast 3 (Barrero et al., 2002), which suggests that plant CAP proteins T-DNA have the potential to regulate the actin cytoskeleton, but the cap1-1 endogenous role of CAP in plant cells has remained uncharacterised. The plant actin network is required for a variety of processes 1 2 including the regulation of transpiration, pathogen defence cap1-2 responses, and (most visibly) growth and development T-DNA 500 bp (reviewed by Hussey et al., 2006). Disruption of actin 4 polymerisation by drugs (Baluska et al., 2001), and by some loss-of-function, gain-of-function and misexpression actin Fig. 1. Location of the T-DNA inserts in Arabidopsis CAP1 mutants (Gilliland et al., 2002; Kandasamy et al., 2002; (At4g34490). Translated sections of exons of Arabidopsis CAP1 are Nishimura et al., 2003) results in dwarf plants with restricted represented by boxes, and introns are represented by horizontal lines. and uncoordinated cell expansion phenotypes. Sequenced plant T-DNAs are not drawn to scale. Primer 1 (CAP28F) combined with genomes contain homologues of many ABPs, some of which primer 2 (CAP28R) was capable of amplifying CAP1 cDNA from have been shown to modulate actin behaviour in planta. With azygous plants but not from cap1-1 or cap1-2 homozygote plants the exception of AIP1 (Ketelaar et al., 2004a), most plant ABP (see Fig. 2). Products could be amplified with primers 1 and 2 mutants and suppression constructs affect the morphogenesis combined with T-DNA primers (3 and 4, respectively) using the appropriate homozygote plant genomic template, but not from a of only a variable subset of cell types. The tissue-specific cDNA template, which suggests the absence of processed CAP1:T- nature of formin phenotypes (Deeks et al., 2005; Ingouff et al., DNA fusion transcripts in homozygote mutant plants. 2005; Yi et al., 2005) and profilin (McKinney et al., 2001) can be considered to be a symptom of large gene families with the potential for genetic redundancy, but the relatively mild phenotypes of components of the Arp2/3 complex (Mathur et Mutant plants homozygous for cap1 alleles have severely al., 2003) together with the unexpectedly severe Arabidopsis reduced stature (Fig. 2B). Rosette diameters of mutant plants AIP1 phenotype suggests that plants place functional emphasis measured at 22 days after germination (DAG) are reduced upon individual classes of ABPs in a pattern that differs from compared with wild-type controls (20.7, 14.3 and 15.3 mm for animals and fungi. Here, we show that the Arabidopsis WT, cap1-1 and cap1-2, respectively; n>30 for all lines) homologue of CAP (CAP1) is essential for the development of although the mean number of rosette organs is equal. Root multiple cell types and that null mutant phenotypes of these growth is also impaired in cap1 seedlings, with a 44% tissues correlate with actin organisational defects. Moreover, reduction in primary root length compared with wild-type deactivation of CAP1 alters the growth behaviour of multiple plants after a 5-day growth period on vertical plates. Wild-type organs in a novel fashion resulting in curled inflorescences and and cap1 plants grown in parallel initiated inflorescences meandering roots consistent with CAP1 contributing to the simultaneously but differed in rates of inflorescence growth Journal of Cell Science function of plant-specific signalling pathways. (Fig. 2B). At 35 DAG wild-type, cap1-1 and cap1-2 inflorescences measured a mean height of 139.9, 89.9 and 90 Results mm, respectively. Inflorescences of cap1 plants produce floral Disruption of CAP1 affects plant development buds at a slower mean rate than wild-type inflorescences, The biological role of the actin-binding protein CAP1 was contributing to height differences. Epidermal peels taken from investigated through the characterisation of T-DNA insertion synchronous stem internodes of cap1 and wild-type alleles SALK_112802 (designated cap1-1) and GABI-KAT inflorescences show a reduction in cell elongation (Fig. 2C,D). 453G08 (cap1-2; Fig. 1). Plants homozygous for the insertion alleles were identified among segregating populations using Mutant cap1 pollen grains show reduced fertility genomic PCR. RT-PCR designed to amplify full-length CAP1 Comparison of microarray expression analysis experiments demonstrated an absence of CAP1 transcript in cDNA highlights maturing pollen grains as a major site of CAP1 generated from cap1-1 homozygote and cap1-2 homozygote expression. The viability of pollen with mutant cap1 alleles RNA templates (Fig. 2A). No truncated CAP1 mRNA was was assessed in vitro. Pollen grains and the tubes they produce detected in mutant plants. provide a convenient model to study highly polar growth Plants homozygous for either cap1-1 or cap1-2 showed a processes.
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