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First published online 17 July 2007
doi: 10.1242/jcs.007302

Journal of Cell Science 120, 2609-2618 (2007)
Published by The Company of Biologists 2007
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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,{ddagger}

1 The Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, Durham University, South Road, Durham, DH1 3LE, UK
2 Universidade de Lisboa, Faculdade Ciências, Instituto Ciência Aplicada e Tecnologia, Lisbon, Portugal
3 Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, George Square, Edinburgh, EH8 9XD, UK


Figure 1
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  		Fig. 1. Location of the T-DNA inserts in Arabidopsis CAP1 (At4g34490). Translated sections of exons of Arabidopsis CAP1 are represented by boxes, and introns are represented by horizontal lines. T-DNAs are not drawn to scale. Primer 1 (CAP28F) combined with primer 2 (CAP28R) was capable of amplifying CAP1 cDNA from azygous plants but not from cap1-1 or cap1-2 homozygote plants (see Fig. 2). Products could be amplified with primers 1 and 2 combined with T-DNA primers (3 and 4, respectively) using the appropriate homozygote plant genomic template, but not from a cDNA template, which suggests the absence of processed CAP1:T-DNA fusion transcripts in homozygote mutant plants.

 

Figure 2
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  		Fig. 2. RT-PCR shows that CAP1 is absent in plants homozygous for alleles cap1-1 and cap1-2 (A). The full-length CAP1 transcript can 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 individuals are azygous plants from populations segregating cap1-1 or cap1-2. Primer combinations are illustrated in Fig. 1. All plants were successful templates for amplifying the control GAPC transcript (upper gel). Plants homozygous for cap1 alleles show growth deficiencies when compared with wild-types (B), the most obvious of which is a reduction in the rate of inflorescence development (plants were photographed at 53 DAG; bar, 5 cm). Epidermal peels taken from primary inflorescences between the second and third developing silique at 42 DAG demonstrate that wild-type cells (C) are further elongated than equivalent cap1 cells (D). Individual primary inflorescences chosen for comparison bore equal numbers of mature lateral organs. Wild-type GFP:FABD2 primary inflorescence epidermis cells (E) have a parallel arrangement of fine actin cables along the axis of cell expansion. Mutant epidermis (F) contains shorter F-actin bundles poorly aligned with respect to the axis of growth. Bars, 200 µm.

 

Figure 3
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  		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).

 

Figure 4
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  		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 µm.

 

Figure 5
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  		Fig. 5. When compared with wild-type root hairs (A), cap1 root hairs (B) are short, bulbous, and occasionally waved or branched (Bars, 200 µm). Growing wild-type hairs expressing GFP:FABD2 (C) have longitudinal actin cables within the proximal cytoplasm aligned with the axis of growth. F-actin forms a diffuse 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 aggregates (brightly labelled by GFP:FABD2), which can be observed at the very tip of the hair. Bars, 20 µm. Long actin bundles are absent from the central regions of the cytoplasm and instead F-actin can be found in shorter accumulations restricted largely to the cell cortex. Imaging of the very tip of these growing root hairs shows the presence of GFP:fimbrin in bright aggregates at the cortex (F) in zones normally free of F-actin (E; bars, 5 µm).

 

Figure 6
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  		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 branches, and expanded inter-branch zones [e.g. see trichome labelled (i)]. Bars, 500 µm. The most prevalent aspect of the mutant trichome phenotype is an increased variation in branch angles. A histogram (C) divided into bins of 10 degrees from 0 to 240 shows that the majority of wild-type three-prong branches are separated by an angle of approximately 120°. Trichomes of mutant plants show a wider spread of angles with extremes of 23 and 220 degrees. The number of measurements was 90 for all lines and all trichomes were taken from the sixth rosette leaf at 16 DAG. Imaging of GFP:FABD2 in wild-type trichomes (D) and cap1 trichomes (E) aged between developmental stages 4 to 5 shows that F-actin in mutant trichomes accumulates within the core of expanding branches (E; bars, 20 µm).

 

Figure 7
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  		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 mutants, which initiate tip growth. Root hairs of an arp2-1 homozygote at the same magnification are shown as a control. Bars, 200 µm.

 

Figure 8
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  		Fig. 8. Wild-type inflorescences (A) remain relatively straight during bolting. Inflorescences from cap1 mutants exhibit curls and kinks at nodes (B). Some young inflorescences perform almost a complete rotation during early expansion. Pedicles supporting flowers or growing siliques are also curled. The root systems of wild-type plants (C) grown on the surface of solidified agar medium extend radially. Root systems of cap1 plants (D) fail to efficiently colonise the agar surface. Comparison of an individual wild-type root and associated lateral roots (E) with a cap1 root and its associated laterals (F) shows that cap1 roots are excessively curled and looped. Bars, 2 mm.

 

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© The Company of Biologists Ltd 2007