Abstract

Cyclase‐associated proteins (CAP) are multifunctional proteins involved in Ras‐cAMP signalling and regulation of the actin cytoskeleton. It has recently been demonstrated that over‐expression of AtCAP1 in transgenic arabidopsis plants causes severe morphological defects owing to loss of actin filaments. To test the generality of the function of AtCAP1 in plants, transgenic tobacco plants over‐expressing an arabidopsis CAP (AtCAP1) under the regulation of a glucocorticoid‐inducible promoter were produced. Over‐expression of AtCAP1 in transgenic tobacco plants led to growth abnormalities, in particular a reduction in the size of leaves. Morphological alterations in leaves were the result of reduced elongation of epidermal and mesophyll cells.

Received: 4 October 2002; Returned for revision: 29 November 2002; Accepted: 27 December 2002    Published electronically: 13 February 2003

INTRODUCTION

The sessile nature of higher plants prevents them escaping from unfavourable environments, thus adaptability and developmental plasticity are critical features. Although plant growth is influenced by environmental factors, it appears that the intrinsic size of plant organs is determined by internal developmental factors. Previous studies have focused primarily on the role of polarized cell elongation in determining organ morphology (Kropf et al., 1998). However, the way in which intrinsic organ size is genetically controlled and the nature of the developmental regulators involved in the control of plant organ size are not well understood.

Actin is the major constituent protein of microfilaments in eukaryotic cells. In plants, actin filaments have been implicated in cytokinesis, cell expansion and development (Kost et al., 1999; Baluska et al., 2001). Polymerization and depolymerization of actin filaments are tightly regulated, both spatially and temporally, enabling cells to remodel their cytoskeleton rapidly in response to endogenous cues or external signals (Kost et al., 1999).

Cyclase‐associated protein (CAP) is a multifunctional protein, the amino‐terminal domain of which is involved in the cAMP‐signalling pathway, while the carboxyl‐terminal domain is required for nutritional responses and cytoskeleton organization (Fedor‐Chaiken et al., 1990; Field et al., 1990; Gerst et al., 1991; Mintzer and Field, 1994). CAP proteins have an actin monomer binding activity and induce depolymerization of F‐actin (Freeman et al., 1995; Barrero et al., 2002).

In drosophila, act up (a CAP homologue) was shown to be required to prevent excessive accumulation of actin filaments in the eye disc leading to normal eye development (Benlali et al., 2000). Furthermore, Baum et al. (2000) reported that loss of endogenous CAP in drosophila caused loss of cell polarity during oogenesis.

Recently, we have isolated cDNA clones encoding a putative cotton CAP homologue (GhCAP) (Kawai et al., 1998) as well as an arabidopsis CAP homologue (AtCAP1) (Barrero et al., 2002). Over‐expression of AtCAP1 in arabidopsis led to reduced organ size owing to a decrease in both cell number and cell expansion. AtCAP1 interacted with actin filaments in vivo and caused loss of F‐actin in tobacco Bright Yellow 2 (BY‐2) suspension‐cultured cells (Barrero et al., 2002). In the present study, we demonstrate that over‐expression of AtCAP1 in transgenic tobacco plants causes morphological alterations, which are mainly associated with reduced cell expansion of epidermal and mesophyll cells in leaf tissues.

MATERIALS AND METHODS

Plasmid construction

The binary transformation vector pTA7002 containing the two‐component glucocorticoid system (Aoyama and Chua, 1997) was digested with XhoI and SpeI (Takara, Tokyo, Japan). The AtCAP1 coding sequence was isolated from pBSAtCAP1 (Barrero et al., 2002) following digestion with XhoI and SpeI, and was cloned into pTA7002 to give rise to pTA‐AtCAP1.

Generation of transgenic plants

pTA‐AtCAP1 was introduced into Agrobacterium tumefaciens (EHA105) (Barrero et al., 2002) and then into Nicotiana tabacum using the leaf disc transformation method (Gallois and Marinho, 1995). To confirm that each line contained the AtCAP1 construct, genomic DNA was isolated from the potential lines by homogenizing one young leaf using the Nucleon Phytopure DNA extraction kit (Amersham Pharmacia, Piscataway, NJ, USA) and following manufacturer’s instructions. PCR reactions using oligonucleotide primers directed to the 5′ and 3′ ends of AtCAP1 were performed. T2 and T3 seeds were collected from these lines and grown on MS medium (Murashiga and Skoog, 1994) for subsequent experiments. All plants were grown under light at 26 °C.

Immunoblot analysis

For immunoblot analysis, SDS‐PAGE (polyacryamide gel electrophoresis) was carried out in 12 % polyacrylamide slab gels. Polypeptides were transferred onto polyvinylidene difluoride membranes (Immobilon P; Millipore Corp., Bedford, MA, USA) and reacted with antibodies. A polyclonal antibody against the carboxyl‐terminal region (residues 466‐476) of AtCAP1 was raised in rabbit, and a mouse anti‐actin monoclonal (clone 4) antibody (ICN Biomedicals, Inc., Costa Mesa, CA, USA) was used. All antibodies were detected using an enhanced chemiluminescence kit (ECL‐PLUS; Amersham Pharmacia) and were detected on X‐ray film (Fuji Co., Tokyo, Japan).

Morphological observations

Leaves were numbered from the first leaf that emerged after the cotyledons to the last leaf. The appearance of a leaf (1 mm long) was defined as the initiation of a leaf primordium, as defined by Kim et al. (1998). The definitions of directions within each leaf blade were those originally suggested by Tsuge et al. (1996).

Induction of expression of AtCAP1 in tobacco transgenic plants was as described previously (Aoyama and Chua, 1997; Barrero et al., 2002) using the glucorticoid dexamethasone (DEX). To make morphological comparisons between DEX‐treated transgenic and control plants, seeds were subjected to cold treatment (4 °C) for 1 week to break dormancy and to synchronize germination. Plants thus obtained were grown in DEX‐free medium until the first two pairs of rosette leaves had partially expanded (2–3 d). Plants were then transferred to a new medium containing DEX and grown for 10 d at 26 °C under light. Third leaves were removed and the dimensions of the blade and petiole were recorded. For observation of epidermal and palisade cells, leaf pieces were cut from the central portions of leaf blades. Samples were submerged in water and then placed in a vacuum for few seconds until intercellular air spaces were filled. Samples were placed on a glass slide and photographed under bright‐field illumination to obtain paradermal images of the layers of leaf cells. Average epidermal cell area was determined by measuring the total area of epidermal cells on a photograph and then dividing this area by the number of epidermal cells in the photograph.

RESULTS

Expression of AtCAP1 in transgenic tobacco plants

Transgenic tobacco lines were obtained that possessed a chimeric AtCAP1 gene under a GAL4 promoter, which is recognized by the GVG transcription factor upon activation with DEX (Aoyama and Chua, 1997) (Fig. 1A). Two independent transgenic tobacco plants, NS6 and NS11, with different levels of expression of the AtCAP1 protein, and an empty‐vector control line, V5 (Fig. 1B), were chosen for further analysis.

Accumulation of AtCAP1 was tightly regulated by DEX treatment; no AtCAP1 protein was detected in transgenic plants grown in a DEX‐free medium. In contrast, high levels of AtCAP1 were accumulated in transgenic plants grown on a medium containing DEX (Fig. 1B). NS11 plants accumulated AtCAP1 protein at high levels, whereas the protein was accumulated at low levels in NS6 plants.

Over‐expression of AtCAP1 led to a reduction in organ size and affected elongation of leaf cells. To characterize the morphological effects of AtCAP1 over‐expression in tobacco plants, young tobacco seedlings with two partially expanded leaves but prior to the establishment of the third leaf primordium (defined as the appearance of a leaf 1 mm long; Kim et al., 1998) were treated with DEX (1 µm) for 7 d to induce accumulation of AtCAP1 protein. All anatomical comparisons were performed on the third leaf. A typical transgenic tobacco plant over‐expressing AtCAP1 has cotyledons and leaves that are reduced in size, but a main root of normal length, as compared with those of empty‐vector control plants (Fig. 2A). When grown on DEX‐free medium, over‐expression lines were not phenotypically different from empty‐vector control plants (data not shown), suggesting that only when AtCAP1 is expressed does a reduction in leaf size occur. Tobacco transgenic plants over‐expressing AtCAP1 showed a reduction in leaf blade width and length, as well as in petiole length (Fig. 2B; Table 1). NS11 plants over‐expressing AtCAP1 at high levels showed the severest phenotype, with a 63·6 and 66 % reduction of leaf blade width and length, respectively. Petiole lengths in these plants were severely reduced (61·2 %) as compared with those of empty‐vector controls (Table 1). These results suggest that over‐expression of AtCAP1 correlates with leaf organ size reduction, in accordance with our findings for transgenic arabidopsis plants (Barrero et al., 2002).

To determine whether the reduction in leaf organ size in NS11 transgenic tobacco plants was attributable to a change in cell expansion or a change in cell number, an anatomical analysis of leaves of NS11 plants was undertaken, comparing them with leaves of empty‐vector control plants treated with DEX (Table 2). Epidermal cells of NS11 transgenic plants were smaller than those of empty‐vector controls (Fig. 2C and D; Table 2). The areas of abaxial epidermal and palisade cells in NS11 transgenic plants were only 39·6 and 70·5 % of those of control plants, respectively (Table 2). These results suggest that a decrease in cell size directly affected and accounted for most of the reduction in organ size observed in NS11 plants over‐expressing AtCAP1, and that only a small proportion (less than 5 %) of the total organ size reduction might be the result of decreased cell number (Tables 1 and 2; Barrero et al., 2002).

DISCUSSION

CAP proteins have been shown to have actin‐binding activity in yeast (Freeman et al., 1995; Zelicof et al., 1996; Lila and Drubin, 1997), Dictyostelium (Gottwald et al., 1996), mushroom (Zhou et al., 1998), mammals (Freeman and Field, 2000) and arabidopsis (Barrero et al., 2002). A knock‐out mutant for the CAP gene in drosophila showed an increased actin filament cytoskeleton and disruption in cell polarity, suggesting that CAP function prevents excessive actin filament polymerization that is required for normal eye disc development and proper oocyte polarity (Baum et al., 2000; Benlali et al., 2000). We have recently generated sense arabidopsis transgenic plants over‐expressing a chimeric AtCAP1 gene under the control of a glucocorticoid‐inducible promoter to analyse the effect of increased CAP activity on plant development. Over‐expression of AtCAP1 in transgenic arabidopsis plants was correlated with a reduction in leaf size owing to both a decrease in cell number and reduced cell expansion. We demonstrated that AtCAP1 interacted with actin in vitro and in vivo, and its over‐expression caused the loss of actin filaments (Barrero et al., 2002). To analyse the generality of AtCAP1 function in plants, transgenic tobacco plants over‐expressing AtCAP1 were generated. Over‐expression of AtCAP1 in transgenic tobacco plants led to a reduction in leaf size. Anatomical studies on the third leaf showed that the observed leaf size reduction was mainly due to reduced expansion of epidermal and palisade cells. This result contrasts with previous findings in arabidopsis (Barrero et al., 2002). Such a difference can be attributed to both structural differences among CAP proteins and their high specificity in binding to interacting proteins (Kawamukai et al., 1991, 1992). Although AtCAP1 and the cotton CAP (GhCAP; Kawai et al., 1998) are very similar (Barrero et al., 2002), GhCAP failed to rescue cytoskeleton‐related defects of budding yeast CAP‐deficient cells (unpubl. res.). In addition, Kawamukai et al. (1991, 1992) showed that the fission yeast CAP only binds the fission yeast adenylyl cyclase, and fails to interact with the budding yeast adenylyl cyclase, and vice versa. These findings indicate that a highly specific interaction may occur between CAPs and adenylyl cyclase proteins.

A number of reports have indicated that cell elongation might be F‐actin‐dependent. Thimann and Biradivolu (1994) reported that elongation of oat coleoptile cells decreased by almost 50 % after cytochalasin D‐induced loss of F‐actin. Recently, Dong et al. (2001) reported that over‐expression of actin‐depolymerizing factor (AtADF1) in arabidopsis resulted in the disappearance of thick actin cables in different cell types, causing growth reduction of cells and organs. Moreover, when arabidopsis plants were treated with latrunculin B, smaller plants were obtained with a cell expansion defect; these plants resembled genetic dwarfs plants (Baluska et al., 2001). Our results suggest that AtCAP1 over‐expression in either arabidopsis (Barrero et al., 2002) or transgenic tobacco plants is correlated with a reduction in leaf size. Further studies into the effect of over‐expression of AtCAP1 in other plant tissues, such as reproductive organs, may increase our understanding of the role of CAP proteins in plant organ development.

ACKNOWLEDGEMENTS

We thank Drs Maki Kawai, Csaba Koncz, Takashi Aoyama, Nam‐Hai Chua, Hirokazu Tsukaya, Fumi Kumagai and Seiichiro Hasezawa for their help and for the gift of material. This work was supported by the Research for the Future from the Japan Society for the Promotion of Science.

Fig. 1. Expression of AtCAP1 in transgenic tobacco lines following DEX treatment. A, Schematic representation of the glucocorticoid‐inducible construct. 35S, Cauliflower mosaic virus 35S promoter; GVG, chimeric GVG transcription factor; E9, pea rbcS‐E9 poly A addition sequences; UAS6, six copies of the DNA binding sites for GAL4; AtCAP1, AtCAP1 coding sequence; 3A, pea rbc‐3A poly A addition sequence. Arrows indicate direction of transcription. B, Protein gel blot detection of AtCAP1. Tobacco plants were grown on MS plates for 7 d and then treated with DEX (1 µm) for 10 d. Protein was extracted from shoot tissues and 20 µg protein per line was loaded in duplicate gels. Results of tobacco empty‐vector line (V5) and transgenic plants over‐expressing AtCAP1 (NS6 and NS11) are shown.

Fig. 1. Expression of AtCAP1 in transgenic tobacco lines following DEX treatment. A, Schematic representation of the glucocorticoid‐inducible construct. 35S, Cauliflower mosaic virus 35S promoter; GVG, chimeric GVG transcription factor; E9, pea rbcS‐E9 poly A addition sequences; UAS6, six copies of the DNA binding sites for GAL4; AtCAP1, AtCAP1 coding sequence; 3A, pea rbc‐3A poly A addition sequence. Arrows indicate direction of transcription. B, Protein gel blot detection of AtCAP1. Tobacco plants were grown on MS plates for 7 d and then treated with DEX (1 µm) for 10 d. Protein was extracted from shoot tissues and 20 µg protein per line was loaded in duplicate gels. Results of tobacco empty‐vector line (V5) and transgenic plants over‐expressing AtCAP1 (NS6 and NS11) are shown.

Fig. 2. Anatomical comparison of transgenic tobacco plants over‐expressing AtCAP1. A, Plants grown on DEX‐free medium for 7 d were transferred to a 1 µm DEX medium and grown for 7 d. B, Leaves of the empty‐vector line (V5) and transgenic tobacco plants (NS11). The leaves in each row are, from left to right, the cotyledon and leaves. Epidermal cells of the third leaf of empty‐vector line (V5) (C) and of transgenic tobacco plants over‐expressing AtCAP1 (NS11) (D) shown in B. Bar = 100 µm.

Fig. 2. Anatomical comparison of transgenic tobacco plants over‐expressing AtCAP1. A, Plants grown on DEX‐free medium for 7 d were transferred to a 1 µm DEX medium and grown for 7 d. B, Leaves of the empty‐vector line (V5) and transgenic tobacco plants (NS11). The leaves in each row are, from left to right, the cotyledon and leaves. Epidermal cells of the third leaf of empty‐vector line (V5) (C) and of transgenic tobacco plants over‐expressing AtCAP1 (NS11) (D) shown in B. Bar = 100 µm.

Table 1.

Comparison of leaves from transgenic tobacco plants over‐expressing AtCAP1 and empty‐vector controls

Plant line Width of leaf blade (mm) Relative width (%) Length of leaf blade (mm) Relative length (%) Length of leaf petiole (mm) Relative length (%) 
V5 (n = 4) 22·5 ± 2·7 100·0 25·0 ± 1·9 100·0 9·0 ± 2·1 100·0 
NS11 (n = 4) 12·0 ± 0·5 36·4 8·5 ± 0·4 34·0 3·5 ± 0·9 38·8 
Plant line Width of leaf blade (mm) Relative width (%) Length of leaf blade (mm) Relative length (%) Length of leaf petiole (mm) Relative length (%) 
V5 (n = 4) 22·5 ± 2·7 100·0 25·0 ± 1·9 100·0 9·0 ± 2·1 100·0 
NS11 (n = 4) 12·0 ± 0·5 36·4 8·5 ± 0·4 34·0 3·5 ± 0·9 38·8 

V5, Tobacco empty‐vector line; NS11, transgenic plants over‐expressing AtCAP1.

Observations were made on third leaves 10 d after DEX (1 µm) treatment of 7‐d‐old plants.

Data are means (± s.d.) for n (n = 4) tobacco transgenic seedlings.

Table 2.

Comparison of leaf cell area of transgenic tobacco plants over‐expressing AtCAP1 and empty‐vector controls

Plant line Abaxial epidermal cell area (µm2Relative area (%) Palisade cell area (µm2Relative area (%) 
V5 1003·5 ± 822·6 100·0 257·8 ± 201·9 100·0 
NS11 396·7 ± 760·4 39·6 181·7 ± 227·0 70·5  
Plant line Abaxial epidermal cell area (µm2Relative area (%) Palisade cell area (µm2Relative area (%) 
V5 1003·5 ± 822·6 100·0 257·8 ± 201·9 100·0 
NS11 396·7 ± 760·4 39·6 181·7 ± 227·0 70·5  

V5, Tobacco empty‐vector line; NS11, transgenic plants over‐expressing AtCAP1.

Observations were made on third leaves 10 d after DEX (1 µm) treatment of 7‐d‐old plants.

Data are means (± s.d.) for more than 500 cells per leaf.

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Author notes

1Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi 1‐1‐1, Bunkyo‐ku, Tokyo 113‐0032, Japan and 2Iwate Biotechnology Center, Kitakami, Iwate 024‐0003, Japan

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