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. Pollen derived from mutant plants showed a consistent co-segregating pleiotropic phenotype (n=70 and 65, reduction in the rate of germination after 24 hours of respectively). The absence of the phenotype in heterozygotes incubation in growth medium when compared to wild-type and F1 plants derived from backcrosses defines the cap1-1 and pollen (Fig. 3A-C). The growth rates of tubes successfully cap1-2 alleles as recessive. F1 plants from crosses between produced by mutant pollen grains were compared with wild- cap1-1 and cap1-2 homozygotes confirmed allelism. All type tubes 5 hours after the initiation of germination (Fig. 3E) identified aspects of the phenotype are present in both cap1-1 and were found to grow at a mean speed of approximately 1.0 and cap1-2 homozygote plants. The influence of maternal ␮m per minute, almost one-third of the rate of wild-type genotype on seedling and plant growth behaviour was growth (2.8 ␮m per minute). After 24 hours of growth in vitro negligible, as plants descended from outcrosses with cap1 mutant pollen tubes do not reach the same terminal lengths as homozygote plants as the maternal parent appeared normal. wild-type tubes (Fig. 3D). Moreover, cap1 homozygote plants descended from The growth phenotype of cap1 pollen tubes was confirmed heterozygote parents exhibited all phenotypic aspects. in vivo using pollen grains germinated on intact flowers. Arabidopsis cyclase-associated protein 1 2611

Emasculated wild-type stigmas were fertilised with either wild-type or cap1 pollen and after a minimum of 4 hours were dissected, fixed and stained with aniline blue to assess pollen viability. After 4 hours, wild-type pollen tubes had traversed most of the style, whereas mutant pollen tubes had yet to penetrate the stigma (Fig. 4). Mutant tube penetration was beginning to occur by 5.5 hours, by which time pioneering wild-type tubes were in contact with ovules (Fig. 4). Flowers observed 24 hours after pollination showed that mutant pollen tubes were capable of eventually reaching ovules after a sufficient growth period. If the poor viability of mutant pollen is due to the debilitating effect of the male gametophyte inheriting a cap1 mutant allele, then the transmission frequency of cap1 alleles within segregating populations is likely to be reduced. The frequency of F2 cap1-1 and cap1-2 homozygotes from F1 heterozygote parents is low (3% compared with an expected value of 25%). Progeny of heterozygote plants were not observed to have an increase in mortality to explain the absence of homozygotes, which suggests that a fertility problem was causing the low frequency of cap1 mutants. Genotyping of F1 plants generated by crosses between wild-type plants and cap1 heterozygotes shows that the fertility of cap1 pollen is reduced: wild-type plants fertilised with pollen from cap1 heterozygote plants show a cap1 transmission frequency of 1.2% (n=85). By contrast, the reciprocal cross shows a transmission frequency of 44% (n=41). This indicates that pollen grains with a cap1 genotype display a fertilisation handicap when competing against wild-type pollen generated by the same parent, as an ovule is nearly 50 times more likely to be fertilised by a pollen grain inheriting a wild-type allele.

F-actin is disrupted in cap1 mutants Mutant cap1 lines were crossed with plants carrying

Journal of Cell Science GFP:FABD2, a construct consisting of the second actin- binding domain of fimbrin fused to GFP under the control of the CaMV 35S promoter, to identify possible actin cytoskeletal disruption associated with the developmental abnormalities of cap1 mutants. Observing the pollen tube cytoskeleton with Fig. 2. RT-PCR shows that CAP1 is absent in plants homozygous for GFP:FABD2 was found to be prohibited as the CaMV 35S alleles cap1-1 and cap1-2 (A). The full-length CAP1 transcript can promoter does not stimulate expression within the be amplified from plants with a wild-type CAP1 allele (1), but not from plants homozygous for either cap1-1 or cap1-2 (2). Control gametophyte. Instead, we compared the actin arrays of root individuals are azygous plants from populations segregating cap1-1 hairs, a sporophytic tissue used as a model for tip growth. Root or cap1-2. Primer combinations are illustrated in Fig. 1. All plants hairs of cap1 homozygotes are severely shortened, bulbous, were successful templates for amplifying the control GAPC waved and occasionally branched when compared with wild- transcript (upper gel). Plants homozygous for cap1 alleles show type root hairs grown in equivalent conditions (Fig. 5A,B). growth deficiencies when compared with wild-types (B), the most Mean growth speed is reduced to 0.36 ␮m per minute for obvious of which is a reduction in the rate of inflorescence mutant hairs compared with 0.79 ␮m per minute for wild-type development (plants were photographed at 53 DAG; bar, 5 cm). hairs. Epidermal peels taken from primary inflorescences between the Elongating wild-type root hairs contain a population of second and third developing silique at 42 DAG demonstrate that longitudinally oriented actin cables within the shank of the hair wild-type cells (C) are further elongated than equivalent cap1 cells ␮ (D). Individual primary inflorescences chosen for comparison bore that disperse approximately 10 m from the growing tip (Fig. equal numbers of mature lateral organs. Wild-type GFP:FABD2 5C). Dynamic F-actin populations at the tip are essential for primary inflorescence epidermis cells (E) have a parallel directing and facilitating Golgi-derived vesicle fusion to the arrangement of fine actin cables along the axis of cell expansion. plasma membrane of the growth zone. Induced stabilisation of Mutant epidermis (F) contains shorter F-actin bundles poorly aligned F-actin and subsequent invasion of the apical clear zone by with respect to the axis of growth. Bars, 200 ␮m. actin bundles has been found to correlate with cessation of growth (Ketelaar et al., 2004a; Ketelaar et al., 2004b). Growing hairs lacking CAP1 contain short F-actin bundles that 5C,D). Frequently, the cortical F-actin aggregates extend to the congregate at the cell cortex (Fig. 5D). These F-actin bodies very tip of growing cap1 root hairs (Fig. 5E,F). The absence often appear as aggregates rather than defined bundles (Fig. of organised long actin cables coincides with an apparent 2612 Journal of Cell Science 120 (15)

Fig. 3. Wild-type pollen grains incubated for 24 hours in vitro in the presence of pollinated stigmas germinate and develop tubes (A). Pollen from cap1-1 and cap1-2 plants shows a visibly lower frequency of germination and reduced tube development (B). Analysis of larger numbers of pollen grains (C) shows that the germination rate of mutant pollen grains is less than half that of wild- type grains (n>800 for all genotypes). The mean length of pollen tubes after 24 hours (D) is similarly reduced (n>180). The growth speed of pollen tubes was compared at 5 hours after exposing the pollen grains to germination medium (E). The mean speed of mutant grains is reduced to nearly a third that of wild-type (n=32 for wild-type, n=17 for cap1).

reduction in long range transport. Labeling of mitochondria Morphological abnormalities associated with F-actin using mitotracker red showed that the wild-type pattern of disruption also occur in cap1 cell types where expansion is reverse fountain streaming of organelles (supplementary localised to ‘diffuse’ zones of cell wall. The epidermal cells material Movie 1) is not apparent in immature cap1-1 and of cap1 inflorescences are shorter with respect to the cap1-2 root hairs (supplementary material Movie 2). Growing longitudinal axis of the inflorescence than wild-type cells mutant root hairs often contain large zones of cytoplasm (Fig. 2C,D) and contain a relatively sparse population of between the growing tip and central vacuole that are depleted poorly aligned F-actin bundles. Trichome cells of the leaf in F-actin relative to the accumulations at the cortex (Fig. 5D). epidermis also grow in a diffuse manner (Schwab et al., 2003) Journal of Cell Science

Fig. 4. The in vitro growth behaviour of pollen grains of different genotypes was confirmed in vivo. Flowers were pollinated and after incubation were dissected and stained with aniline blue. After 4 hours (left panel) large numbers of wild-type pollen grains have germinated and produced intensely staining callose deposits. Many wild-type pollen tubes have grown the length of the style tissue and are beginning to enter the ovary. A minority of cap1 pollen grains show signs of germination at this time point, and only auto-fluorescence from vascular tissue is visible within the style. Stigmas stained at 5.5 hours (centre panel) after pollination with wild-type pollen contain a significant number of pollen tubes developing callose plugs (white arrows) within the ovary, and some tubes are contacting ovules. A greater proportion of mutant pollen grains are germinating but tube growth is still retarded. Stigmas stained after 24 hours of pollen tube growth (right panel) shows that some mutant pollen tubes do eventually contact ovules. Bars, 200 ␮m. Arabidopsis cyclase-associated protein 1 2613

and are sensitive to disruptions in actin turnover (Mathur et al., 1999; Szymanski et al., 1999). Arabidopsis leaf trichome cells exposed to actin depolymerising drugs or produced by plants homozygous for null alleles of components of the Arp2/3 complex and associated signalling pathway display a ‘distorted’ phenotype consisting of bloating and twisting of trichome stalks and branches. Trichomes from cap1 plants display a weak distorted phenotype: cap1 trichome branches are mildly twisted, and stalk inter-branch zones are often excessively elongated (Fig. 6A,B). The angle between trichome branches is also affected in a large proportion of trichomes that otherwise would have a wild-type appearance (Fig. 6A-C). Comparison of developing trichomes expressing GFP:FABD2 identified frequent excessive accumulation of F-actin in the core of elongating branches (Fig. 6D,E) that does not resemble the cohesive network of longitudinally aligned cables observed in the wild-type. This unusual F-actin array correlates with branches with diameters broader than those of wild-type branches of a comparable age. The phenotype is prevalent in trichomes between developmental stages 4 and 5, where branches are undergoing rapid expansion, but excessive F-actin accumulation within central cytoplasmic regions can be identified in trichomes as young as developmental stage 2. The central bundles of cap1 trichomes show some resistance to the Fig. 5. When compared with wild-type root hairs (A), cap1 root hairs (B) are short, action of the actin-deploymerising drug bulbous, and occasionally waved or branched (Bars, 200 ␮m). Growing wild-type latrunculin B (data not shown), which possibly hairs expressing GFP:FABD2 (C) have longitudinal actin cables within the proximal cytoplasm aligned with the axis of growth. F-actin forms a diffuse Journal of Cell Science explains the absence of enhanced sensitivity of cap1 trichome morphogenesis to latrunculin B dynamic network at the growing distal end of the hair that regulates vesicle fusion to the tip. In cap1 growing hairs (D) the diffuse tip network is replaced by F-actin treatment (see supplementary material Fig. S2). aggregates (brightly labelled by GFP:FABD2), which can be observed at the very The redistribution of F-actin in cap1 mutant tip of the hair. Bars, 20 ␮m. Long actin bundles are absent from the central regions trichomes is the reverse of microfilament of the cytoplasm and instead F-actin can be found in shorter accumulations redistribution in mutant root hairs, which restricted largely to the cell cortex. Imaging of the very tip of these growing root suggests fundamental differences in the hairs shows the presence of GFP:fimbrin in bright aggregates at the cortex (F) in cytoskeletal organisation of tip growing and zones normally free of F-actin (E; bars, 5 ␮m). diffusely growing plant cells.

Synthetic phenotypes are produced between CAP1 arp2 single mutants making ARP2 epistatic with respect to alleles and the Arp2/3 complex pathway CAP1 during trichome development. This is expected as The cap1 mutant alleles were crossed with previously mutants of Arp2/3 complex components resemble trichomes characterised null alleles of SCAR2 and ARP2 (Basu et al., grown in the presence of high concentrations of actin 2005; Le et al., 2003) to search for novel synthetic depolymerising drugs, and therefore may represent an actin phenotypes that would reveal new functional roles for CAP1 phenotypic zenith of morphological distortion. Double and the Arp2/3 pathway during plant development. Null mutants of either arp2-1/cap1-1 or arp2-1/cap1-2 showed a alleles of the Arp2/3 activator SCAR2 display a weak greater reduction in plant stature and a more severe inhibition distorted trichome phenotype (Basu et al., 2005; Zhang et al., of root hair elongation (Fig. 7B). The root hairs of the double 2005). The scar2-1/cap1-1 and scar2-1/cap1-2 double mutant are arrested at the stage of bulge expansion at the homozygotes show an enhanced trichome phenotype with surface of the root epidermis and do not initiate tip growth. increased trichome distortion greater than either single Drug studies have previously shown that bulge formation is mutant (Fig. 7A), which suggests that the actin-binding not as dependent upon the actin cytoskeleton as the later stage proteins SCAR2 and CAP1 act in parallel to control trichome of tip growth (Baluska et al., 2000) explaining the arrest of branch expansion. No further synthetic phenotypes were the ABP double at this stage. Under standard growth detected in other tissues. The distorted shapes of arp2- conditions arp2 root hair development has previously been 1/cap1-1 and arp2-1/cap1-2 trichomes are similar to those of described as normal (Mathur et al., 2003) and no 2614 Journal of Cell Science 120 (15)

Fig. 7. Mutants homozygous for both cap1-1/cap1-2 and scar2-1 show an enhancement of the distorted phenotype greater than either single mutant (A), with increased bulging and twisting of branches and inter-branch zones (ii). Mutants homozygous for both cap1- 1/cap1-2 and arp2-1 have root hairs that do not progress beyond bulges on the surface of trichoblasts (B), unlike the cap1 single Journal of Cell Science mutants, which initiate tip growth. Root hairs of an arp2-1 homozygote at the same magnification are shown as a control. Bars, 200 ␮m. Fig. 6. ESEM images of rosette leaves demonstrates that the majority of Columbia ecotype wild-type leaf trichomes have three branches (A). Mutant trichomes (B) show abnormal branch angles, twisted CAP mutants are affected in co-ordination of organ branches, and expanded inter-branch zones [e.g. see trichome growth labelled (i)]. Bars, 500 ␮m. The most prevalent aspect of the mutant In addition to being retarded in length, cap1 inflorescences curl trichome phenotype is an increased variation in branch angles. A during bolting and exhibit alterations in the direction of histogram (C) divided into bins of 10 degrees from 0 to 240 shows expansion that create ‘kinks’ in the stem (Fig. 8A compared that the majority of wild-type three-prong branches are separated by with Fig. 8B). The changes in growth angle occur at nodes, an angle of approximately 120°. Trichomes of mutant plants show a creating corners at points of lateral organ development. wider spread of angles with extremes of 23 and 220 degrees. The Zigzagging of the inflorescence has been reported in number of measurements was 90 for all lines and all trichomes were gravitropic mutants, but cap1 inflorescences remain taken from the sixth rosette leaf at 16 DAG. Imaging of GFP:FABD2 in wild-type trichomes (D) and cap1 trichomes (E) aged between gravitropic. Also unlike gravitropic mutants, cap1 secondary developmental stages 4 to 5 shows that F-actin in mutant trichomes inflorescences regularly achieve 360 degree loops relative to accumulates within the core of expanding branches (E; bars, 20 ␮m). the vector of primary inflorescence expansion (Fig. 8B). The looping process begins with secondary inflorescence heads bending downwards the gravity vector. At any one moment in time 37% of cap1 inflorescence heads are at an angle lower morphological abnormalities were identified in arp2-1 single than the gravitational horizon (n=307). In the same mutant root hairs grown as controls in parallel with double environmental conditions only 1% of wild-type inflorescence mutants. Despite the absence of severe synergistic heads grow at an angle below the same threshold (n=279). phenotypes in the double mutants, the additive arp2/cap1 root 6.5% of cap1 inflorescence heads over a period of 7 days hair phenotype shows that the Arp2/3 pathway has a achieved a complete 360 degree rotation relative to the axis of fundamental role in root hair tip growth that is only revealed their own stem. The inflorescence rotation rarely exceeds one in a cap1 background. complete loop and is a temporary phenomenon; affected Arabidopsis cyclase-associated protein 1 2615

the interpretation of these signals in target tissues indicating an involvement of CAP in as yet unknown plant signalling pathways.

Discussion Endogenous CAP1 is required for Arabidopsis actin organisation Disruption of the CAP1 gene causes phenotypes in multiple tissues that correlate with disturbances in the actin cytoskeleton. Elongating epidermal cells, tip growing cells, and trichomes show severe morphological abnormalities and unusual aggregation of F-actin. We have shown that the Arabidopsis CAP homologue is an actin monomer binding protein (supplementary material Fig. S1), and recent work has shown that CAP1 directly accelerates the exchange of ADP for ATP by actin monomers (Staiger and Blanchoin, 2006; Chaudhry et al., 2007) filling a functional space in actin biochemistry left by the absence of plant profilin nucleotide exchange activity. Drug studies have long shown that cap1 plant phenotypes are compatible with a suppression of actin biochemistry – sequestering, capping, or stabilising actin (with latrunculin B, cytochalasin D, or jaspokinolide, respectively) produces a similar suite of defects. A previous study has shown that the overexpression of plant CAP disassembles F-actin arrays in vivo and causes severe growth defects (Barrero et al., 2002), which is surprising when considering that the overexpression of CAP in other organisms does not result in gross phenotypic abnormalities. We have established that this potential for CAP1 to influence actin biochemistry is affirmed Fig. 8. Wild-type inflorescences (A) remain relatively straight during by the function of endogenous plant CAP in actin-dependent bolting. Inflorescences from cap1 mutants exhibit curls and kinks at nodes (B). Some young inflorescences perform almost a complete growth processes. rotation during early expansion. Pedicles supporting flowers or growing siliques are also curled. The root systems of wild-type Reconciling CAP1 biochemistry with the CAP1 plants (C) grown on the surface of solidified agar medium extend phenotype

Journal of Cell Science radially. Root systems of cap1 plants (D) fail to efficiently colonise The visible disruption to the Arabidopsis actin cytoskeleton the agar surface. Comparison of an individual wild-type root and resulting from absence of CAP1 consists of an F-actin re- associated lateral roots (E) with a cap1 root and its associated laterals arrangement into short bundles or aggregates that retain an (F) shows that cap1 roots are excessively curled and looped. Bars, unusual position within their respective cells types. Short 2 mm. bundles congregate to form a dense actin ‘core’ in expanding trichome branches while in growing root hairs actin bundles diminish and withdraw to the cell cortex. The formations of F- inflorescences uncurl hours to days after loop completion actin adopted in cells lacking functional CAP vary from (supplementary material Movie 4). The pedicles of floral organism to organism. In S. cerevisiae the appearance of the organs are also susceptible to curling (Fig. 8B) but these F-actin arrays of dividing cells undergoes only a subtle distortions are permanent. Time-lapse recording of wild-type alteration, as both actin patches and cables remain intact. Actin and cap1 plants revealed that cap1 inflorescences do not patch distribution is perturbed (Field et al., 1990) and the ASH1 undergo rotational circumnutation movements (supplementary mRNA polar marker is not anchored correctly after being material Movies 3, 4) but instead cap1 inflorescence-heads transported along actin cables (Baum et al., 2000) possibly oscillate at irregular intervals within the vertical plane. An indicating a subtle cable defect. Animal cells with knocked analogous phenotype can be observed in roots; cap1 roots are down CAP amass large arrays of stable F-actin and lose unable to grow in a straight line across the horizontal surface dynamic F-actin arrays associated with lamellipodia (Baum et of agar medium (Fig. 8D,F), yet remain gravitropic. al., 2000; Benlali et al., 2000; Bertling et al., 2004; Rogers et Microscopic analysis did not reveal twisting of epidermal cell al., 2003). These re-arrangements can be interpreted as files within affected organs, and the chirality of inflorescence evidence supporting biochemical observations that some CAP curls occurs randomly between individual plants and between isoforms can sequester actin monomers in vitro and thus inflorescences of the same plant. These aspects of the cap1 prevent excessive polymerisation (Freeman et al., 1995; phenotype indicate a loss of coordination in organ expansion. Gieselmann and Mann, 1992; Gottwald et al., 1996). Plant F- Bending of organs is achieved in plants by simultaneous actin does not homogenously accumulate in the absence of differential expansion of opposed cell layers. Loss of CAP as cap1 root hairs appear devoid of the organised bundles circumnutation movements and initiation of novel curling that amass in the shank of wild-type root hairs behind the motion can result from either corruption of growth signals or growing tip. These observations suggest a more complex role 2616 Journal of Cell Science 120 (15)

for CAP1 in the turnover of actin filaments, possibly relating chemotactic signal (Noegel et al., 2004) and to respond to the to the observed in vitro activity of accelerating actin monomer same signal by stimulating cytoskeletal based motility (Noegel nucleotide exchange either directly (Moriyama and Yahara, et al., 1999). Recently, SRV2 of yeast was shown to have a 2002; Chaudhry et al., 2007) or through interplay with ADF remarkable association with the cytoskeleton during Ras- (Mattila et al., 2004; Chaudhry et al., 2007). mediated signalling to apoptotic-like pathways (Gourlay and The critical state of actin dynamics depends upon the Ayscough, 2006). Suppression of actin dynamics leads to Ras balance of actin-binding protein activity. Overexpression of activation, which in turn activates adenylate cyclase. One of the both plant CAP1 and AIP1 (Barrero et al., 2002; Ketelaar et consequences of this pathway is further actin rearrangements, al., 2007) mimic the phenotypic effects of reduced expression possibly via PKA effectors downstream from cAMP signalling of these respective proteins (this study) (Ketelaar et al., (Gourlay and Ayscough, 2006). CAP is needed to transduce the 2004a). A sub-nominal level of CAP1 protein is likely to signal from Ras to adenylate cyclase, and the actin-binding reduce the in vivo concentration of ATP-actin monomers, domain of CAP is required for this process. In this instance while an increase in CAP1 concentration or activity could CAP appears to offer an input to the pathway dependent upon enhance CAP1 sequestration of actin monomers from the G- the actin-binding domain, even feasibly acting as some form actin pool. Any imbalance in ABP activity impacts upon the of sensor to the free G-actin pool. As the effectors of the efficacy of actin turnover and, consequently, on cytoskeletal- signalling pathway are very likely to include other actin- driven cell growth. binding proteins, the cytoskeletal phenotypic effects of CAP knockouts in yeast are dependent on CAP signalling activities, Arabidopsis CAP1 is required for signalling totally integrating the two roles and confusing any phenotypic The curling of cap1 inflorescences and roots indicates a role distinction. for CAP1 in coordinating the expansion of tissues. The curling The concept of integration invites a model for CAP of cap1 organs is distinct from the twisting observed in mutants function based upon feedback from the actin cytoskeleton. such as spiral or lefty, which show defects in microtubule Latrunculin B treatment is known to exaggerate the organisation as the cap1 curls have no consistent chirality, and gravitropically stimulated bending of roots (Hou et al., 2004) the turns of roots are not associated with visible twisting of through an uncharacterised mechanism. The roots of cap1 epidermal cell files. However, the inflorescence curling plants also respond in an exaggerated fashion to phenomenon has a common pattern of behaviour: cap1 gravistimulation (P.J.H., unpublished). The meandering inflorescences always initiate a curl by turning towards the behaviour of cap1 roots and the curling of inflorescences gravity vector. Recovery to a vertical position closes the curl, might result from an overcompensation to internal cues aimed and the process is then rapidly reversed over the course of a at maintaining a controlled angle of tissue expansion. CAP1 few hours to re-straighten the inflorescence. Such movement could feasibly be required to monitor and respond to the requires the simultaneous differential expansion of many cell status of the G-actin pool in expanding cells. Rapid dynamics files, suggesting the involvement of an intercellular signal that could provoke the perpetuation of an intercellular is either misinterpreted or misdirected in the absence of CAP1. compensatory signal via CAP1 to expand other cell files in

Journal of Cell Science The interpretation of mutant phenotypes in understanding an antagonistic manner and stimulate direct intracellular the signalling role of CAP is complicated by the multiple suppression of actin turnover. consequences of cytoskeletal disturbance. Clonal analysis of Drosophila eye discs shows a requirement for CAP in Conclusion signalling processes to organise photoreceptor differentiation In conclusion, Arabidopsis CAP1 is essential for healthy (Benlali et al., 2000). Rather than directly transducing a signal growth, but unlike AIP1 its deactivation is not lethal to plant in the manner of SRV2, Drosophila CAP was hypothesised to life. The cap1 mutants could reveal aspects of the in vivo perturb hedgehog signalling by causing morphological behaviour of other ABPs, both through the observation of F- abnormalities across the surface of the eye disc leading to the actin formation in affected tissues and through double-mutant physical disruption of morphogen distribution. Therefore analysis. The accumulation of F-actin at the cortex of affected dissection of the plant CAP signalling phenotype should cells suggests the presence of so far unidentified F-actin always be considered in light of the effects of actin disruption machinery at sites of intense exocytosis. Arabidopsis cap1 is on morphogenesis and other basal cell processes such as also required for the coordination of tissue expansion in the exocytosis and endocytosis. Attempting to separate signalling manner of a component of an intercellular signalling pathway. and cytoskeletal activities through domain analysis must also be approached with caution. In S. cerevisiae, where this Materials and Methods analysis was first performed, the ‘cytoskeletal’ phenotypes Recombinant protein purification were never fully complemented using only the C-terminus For actin-binding studies, the full-length transcript of CAP1 was cloned into a Gateway derivative of pGEX-4T-1 to form a translational fusion with GST at the (Gerst et al., 1991) and recently the transduction of signals N-terminus of CAP1. Recombinant protein was expressed in E. coli strain BL21 from Ras to adenylate cyclase within a cell-death pathway was DE3 pLysS Rosetta 2. Cultures were grown to OD600 0.8 and induced using IPTG found to be more reliant upon the C-terminus of SRV2 than the (final concentration 1 mM) at 37°C for 2.5 hours or overnight at 14°C. Cells expressing GST-tagged proteins were resuspended in PBS, pH 7.3 and lysed using N-terminus (Gourlay and Ayscough, 2006). a freeze-thaw cycle. Cell supernatant was incubated with glutathione sepharose 4B The two biological activities of CAP have long been according to the manufacturer’s instructions (Amersham, UK) and washed three considered independent functions, but evidence is times with PBS. accumulating that interaction with actin monomers could be Actin purification coupled with participation in signalling pathways. In Rabbit skeletal muscle actin was purified as described previously (Spudich and Dictyostelium, CAP is required to perpetuate the cAMP Watt, 1971), and as later modified (Winder et al., 1995). Briefly, rabbit muscle Arabidopsis cyclase-associated protein 1 2617

acetone powder was mixed with buffer G (2 mM Tris pH 8.0, 0.2 mM ATP, 0.5 mM Genotyping DTT, 0.2 mM CaCl2, 1 mM NaN3). After a 30 minute incubation and spin, the DNA from plants was prepared as described by (Edwards et al., 1991). All PCR supernatant was filtered. MgCl2 was added to a final concentration of 2 mM and experiments used RedTaq (Bioline) polymerase in accordance with manufacturer’s KCl was added to 0.8 M. After polymerisation the F-actin was pelleted for 2 hours instructions. The CAP1 wild-type allele was amplified using primers: at 50,000 g. Actin was resuspended in G-buffer and dialysed for 2 days, then CAP1-1 For: 5Ј-CTCCTGACTTCGCCATC-3Ј centrifuged at 50,000 g. The top two-thirds of the supernatant was gel-filtered using CAP1-1 Rev: 5Ј-GCAACCTGAACAAGTAACTATA-3Ј sephacryl S300 to isolate actin monomers. CAP1-2 For: 5Ј-CTCGTACCAGTAAACCGGCCTT-3Ј ADP-actin was generated from purified ATP-actin monomers on the day of use CAP1-2 Rev: 5Ј-CCGTGAAAACAACACCCACTTT-3Ј. by incubation with yeast 20 U/ml hexokinase and 1 mM glucose in G-buffer for 3 The amplification of the T-DNA insertion alleles was achieved with primers: hours (Pollard, 1986). 0.1 M ADP (Sigma) stock solution was also treated with CAP1-1 Rev and LBb1 (5Ј-GCGTGGACCGCTTGCTGCAACT-3Ј) hexokinase and glucose to remove ATP contaminants. CAP1-2 Rev and cap1-2TDNA (5Ј-CCCATTTGGACGTGAATGTAGA-3Ј).

Actin-binding assays RT-PCR For actin-depletion assays, G-actin was added to 225 ␮l of depletion buffer (10 mM Total RNA was isolated from homozygous mutant and azygous plants using the Tris pH 7.5, 0.2 mM CaCl2, 0.5 mM DTT, 0.2 mM ADP, 1 mM MgCl2, 100 mM RNeasy Plant Mini Kit (Qiagen). The total RNA was DNase treated with RNase- KCl) to make a final solution of 3 ␮M. 25 ␮l (12.5 ␮l bead volume) of glutathione free DNase (Promega) following the manufacturer’s instructions. The cDNA first- sepharose beads (Amersham) coated with either GST or GST-CAP1 were strand was synthesised using 5 pmol of oligo(dT) 12-18mer and 200 units of immediately added to the actin solution to make a 9 ␮M bait protein suspension. SuperscriptTMII RNaseH-Reverse Transcriptase (Invitrogen), according to the Beads were incubated with the actin for either 5 minutes or 30 seconds with gentle manufacturer’s instructions. The RNA was removed by addition of 2 units of agitation. Following incubation, beads were briefly spun to the bottom of the tube Ribonuclease H (Promega) and incubation at 37°C for 20 minutes. 1 ␮l of the and 100 ␮l of supernatant removed and mixed with 2ϫ SDS loading buffer. Samples reaction mixture was used as a template for the PCR reaction with BioRed Taq DNA were run on an 8% polyacrylamide SDS gel and stained using Coomassie solution. polymerase (Bioline). Native gels were polymerised at a final concentration of 10% Protogel CAP28F: 5Ј-GGAATCCATATGGAAGAGGATTTGATTAAGCGCCTT-3Ј acrylamide/bisacrylamide mix (National Diagnostics). 0.2 mM ADP (pre-treated CAP28R: 5Ј-GGAATCCATATGTTAGGCACCTGAATGCGAGACCGGTGTTG- with 20 U/ml hexokinase and 1 mM glucose) was present in both gel and running 3Ј. buffer (25 mM Tris, 200 mM glycine, 0.5 mM DTT). Recombinant GST-CAP1 and Control primers to Arabidopsis glyceraldehyde-3-phosphate dehydrogenase C GST were removed from glutathione beads using elution buffer (10 mM reduced were: glutathione, 50 mM Tris-HCl, pH 8.0) and dialysed for 5 hours with 4 changes of GAPCFOR: 5Ј-CACTTGAAGGGTGGTGCCAAG-3Ј G-buffer. Combinations of ADP-actin (final concentration 1 ␮M), GST-CAP1 (5 GAPCREV: 5Ј-CCTGTTGTCGCCAACGAAGTC-3Ј. ␮M) and GST (5 ␮M) were assembled in G-buffer with a total volume of 20 ␮l and incubated on ice for five minutes before loading onto the native gel. Environmental scanning electron microscope (ESEM) preparation Plant lines The plant material (leaves from homozygous mutant and azygous plants, from each Arabidopsis seed was sterilised using 5% bleach (BDH) for 25 minutes with gentle T-DNA line) was prepared for ESEM by fixation (PBS, 1% glutaldehyde, 0.1% (v/v) agitation followed by 4 washes with water. Seeds were plated on to half-strength Tween) and dehydration by an ethanol series, followed by critical point drying with Murashige and Skoog salts (Sigma) with 0.8% plant agar. After germination all carbon dioxide. Samples were imaged using a Philips XL30 ESEM in low vacuum plants were grown either on compost or half MS plates in 16 hours light (at 22°C) mode (0.4 Torr). and 8 hours dark (at 18°C). Salk T-DNA lines (Alonso et al., 2003) were created by SIGNAL (the Salk Institute Genomic Analysis Laboratory) and supplied by This work was supported by the Biotechnology and Biological NASC (Nottingham, UK). GABI KAT line 453G08 (Rosso et al., 2003) was created Sciences Research Council (M.J.D., S.D., T.K., P.J.H.) and the FCT and supplied by the Max Planck Institute for Plant Breeding Research (Cologne, Germany). Portugal, SFRH/BD/8760/2002 (C.R., R.M.). We thank Steve Winder for purified actin, and Christopher Staiger and Laurent Blanchoin for Pollen assays personal communications concerning the biochemistry of Arabidopsis Journal of Cell Science Wild-type and cap1 pollen was germinated in vitro as previously described CAP1. (Krishnakumar and Oppenheimer, 1999). 100 ␮l aliquots of agarose pollen germination medium [1 mM CaCl, 1 mM Ca(NO3)2, 1 mM MgCl2, 0.01% boric acid, 18% sucrose, 0.5% agarose, pH 6.0] were solidified on microscope slides to References produce a smooth coating. A 5 ␮l drop of liquid germination medium (without Alonso, J. M., Stepanova, A. N., Leisse, T. J., Kim, C. J., Chen, H., Shinn, P., agarose) was applied to the centre of the slide, and mature wild-type or cap1 pollen Stevenson, D. K., Zimmerman, J., Barajas, P., Cheuk, R. et al. (2003). Genome- was released into the liquid from open pollen sacs. Two mature pollinated stigmas wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653-657. were placed on the slide within 5 mm of the samples. Samples were left either for Balcer, H. I., Goodman, A. L., Rodal, A. A., Smith, E., Kugler, J., Heuser, J. E. and 5 hours or overnight in a closed humid environment within standard growth room Goode, B. L. (2003). Coordinated regulation of actin filament turnover by a high- conditions. After application of coverslips, pollen tubes were observed using a Zeiss molecular-weight Srv2/CAP complex, cofilin, profilin, and Aip1. Curr. Biol. 13, 2159- Axioskop microscope with 40ϫ objective, and images were captured using a video 2169. camera (Roper Scientific) controlled by Openlab 3 software (Improvision, UK). Baluska, F., Salaj, J., Mathur, J., Braun, M., Jasper, F., Samaj, J., Chua, N. H., For in vivo growth assays, wild-type and cap1 pollen was used to fertilise WT Barlow, P. W. and Volkmann, D. (2000). Root hair formation: F-actin-dependent tip growth is initiated by local assembly of profilin-supported F-actin meshworks stigmas. After a specific period of germination within standard growth room accumulated within expansin-enriched bulges. Dev. Biol. 227, 618-632. conditions, stigmas were prepared as described (Jiang et al., 2005) with minor Baluska, F., Jasik, J., Edelmann, H. G., Salajova, T. and Volkmann, D. (2001). modifications. Fertilised carpels were dissected longitudinally to bisect the septum. Latrunculin B-induced plant dwarfism: plant cell elongation is F-actin-dependent. Dev. The dissected tissue was fixed in a 3:1 ethanol:acetic acid solution for 2 hours. The Biol. 231, 113-124. samples were then washed with water and incubated with 10 M NaOH for 2 hours. Barrero, R. A., Umeda, M., Yamamura, S. and Uchimiya, H. (2002). 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