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First published online April 4, 2002; 10.1105/tpc.010402 American Society of Plant Biologists The Arabidopsis TONNEAU2 Gene Encodes a Putative Novel Protein Phosphatase 2A Regulatory Subunit Essential for the Control of the Cortical Cytoskeleton
a Station de Génétique et Amélioration des Plantes, Centre de Versailles, F78026 Versailles Cedex, France 1 To whom correspondence should be addressed. E-mail bouchez{at}versailles.inra.fr; fax 33-1-30-83-33-19
In Arabidopsis ton2 mutants, abnormalities of the cortical microtubular cytoskeleton, such as disorganization of the interphase microtubule array and lack of the preprophase band before mitosis, markedly affect cell shape and arrangement as well as overall plant morphology. We present the molecular isolation of the TON2 gene, which is highly conserved in higher plants and has a vertebrate homolog of unknown function. It encodes a protein similar in its C-terminal part to B'' regulatory subunits of type 2A protein phosphatases (PP2As). We show that the TON2 protein interacts with an Arabidopsis type A subunit of PP2A in the yeast two-hybrid system and thus likely defines a novel subclass of PP2A subunits that are possibly involved in the control of cytoskeletal structures in plants.
Plants possess unique features in many aspects of development compared with animals. At the cellular level, plants are characterized by specific features such as the presence of a pecto-cellulosic cell wall, continuous cytoplasmic connections through the plasmodesmata, and the lack of cell motility during morphogenesis (Kaplan and Hagemann, 1991  ). Reflecting these unique characteristics, the highly dynamic cortical arrays of microtubules (MTs) and actin filaments have adopted various specialized arrangements in plants. The organization of cortical arrays is coordinated tightly with other cellular events, and the cortical cytoskeleton, plasmalemma, and cell wall operate as a continuum (Wyatt and Carpita, 1993). The cortical cytoskeleton reorganizes steadily during all stages of the plant cell life and plays a crucial role in governing the orientation of both cell division and expansion (Cyr and Palevitz, 1995). In contrast to animals, fungi, and protists, in land plants the establishment of division planes depends on (1) the positioning of the preprophase band (PPB), a transient cortical ring of MTs that precisely foretells the position of the cell division plane at the G2/prophase transition, and (2) guidance of the phragmoplast to this predetermined cortical site during cytokinesis. During interphase, arrays of parallel MTs encircle the cell at the cortex. The positioning of MTs within these cortical arrays appears to involve both MT dynamicity (nucleation, growth/shrinkage, stabilization, and severing) and translocation, but the relative roles of these events and the proteins involved are unknown.
To understand the molecular mechanisms underlying MT arrangements, mutants impaired in MT functions are essential, and a number of proteins involved in MT organization or dynamics have been identified through a genetic approach (Azimzadeh et al., 2001
The Arabidopsis zwichel trichome-branching mutation affects the kinesin-like calmodulin binding protein (Oppenheimer et al., 1997
The Arabidopsis fass/tonneau mutants were isolated originally from visual genetic screens for mutations affecting seedling body organization (Mayer et al., 1991
Mutations at these loci drastically change plant shape, resulting in thick, dwarf seedlings and plantlets. However, the general body pattern (i.e., the number and relative positions of organs) is not altered, and mutants eventually produce highly compressed inflorescences when grown in vitro (Mayer et al., 1991
At the cellular level, ton mutants are altered markedly in cell size, shape, and arrangement. This appears to be related to a defect in cell elongation and a random orientation of division planes. Cell differentiation seems mostly unaffected, and most cell types are present, including cell structures such as stomata that result from asymmetrical divisions. Cells of ton mutants have abnormal organization of the interphase cortical cytoskeleton and are unable to form PPBs (Traas et al., 1995
Thus, the ton mutations uncouple histogenesis (oriented cell divisions and cell shape changes) and morphogenesis from organogenesis and provide new insights into basic processes of plant development. The elucidation of the molecular bases of these mutations should provide key information on the molecular control of the cytoskeleton and cell division/elongation in plants (Lloyd, 1995
Isolation and Genetic Characterization of ton2 Mutants A total of 16 independent alleles of the ton2 mutation were identified in several visual screens for Arabidopsis morphological mutants affected in cell elongation. Mutant alleles ton2-13 and ton2-14 were isolated from a population of T-DNA insertion lines in ecotype Wassilewskija (Ws). Other ton2 alleles were recovered from ethyl methanesulfonate–mutagenized populations in Columbia (Col), Landsberg erecta, and Ws ecotypes; among those, mutants ton2-5 and ton2-12, both in the Col background, were chosen for further analysis. All ton2 mutations segregate as single recessive Mendelian traits and are allelic to the previously described fass (Mayer et al., 1991 ) and gordo (Fisher et al., 1996) mutations.
ton2-5 and ton2-13 show extreme phenotypes with a highly compressed apical–basal axis and presumably correspond to a complete loss of function, whereas ton2-12 and ton2-14 have milder phenotypes with less pronounced morphological alterations and better elongation (Figures 1A to 1D)
. In vitro, plants reach a maximum height of
ton2 Mutations Affect the Organization of the Cortical Microtubular Cytoskeleton It was demonstrated previously that ton mutant cells show abnormalities in the organization of the cortical microtubular cytoskeleton (Traas et al., 1995 ; McClinton and Sung, 1997). In this study, we examined the dynamic organization of MTs in ton2 mutants using a newly developed fusion protein, the green fluorescent protein–microtubule binding domain (GFP-MBD) reporter protein (Marc et al., 1998). The GFP-MBD reporter protein enables the labeling of MTs in vivo by a noninvasive method, which allows us to follow MT rearrangements during mitosis and interphase. Ws lines expressing a GFP-MBD marker were obtained by in planta transformation. One morphologically normal line was selected and then crossed to heterozygous ton2-13 (strong allele) and ton2-14 (weak allele) lines. Examples of confocal imaging of wild-type and mutant cells are shown in Figure 2
.
Division figures were difficult to observe in mutant roots because of their shape and thickness, and they were observed mainly from leaf primordia at the apex. Together, our results confirm the absence of observable PPBs in premitotic cells of the mutants (Figures 2A to 2C). The mitotic arrays of MTs are not affected visibly in ton2 mutants, and division proceeds normally, as judged from the sequence of events observed with the GFP-MBD marker. Spindles have a normal appearance (Figures 2D to 2F), as do the phragmoplasts, which grow centrifugally, as expected (Figures 2G to 2I). However, the guidance of the phragmoplast is clearly affected, as shown by its erratic growth (Figure 2I). Oblique, misshapen phragmoplasts often are observed in mutant cells. Ultimately, they attach to random sites at the cortex, generating the irregularly shaped cells observed in the mutants. We observed no difference in the MT organization of weak and strong alleles in dividing cells. There also was no difference in the duration of the cell cycle phases between wild-type and weak and strong allele cells.
GFP-MBD expression also was observed in hypocotyl cells to examine MT interphase arrays in the mutant background (so-called interphase MTs, even if these cells are not strictly in G1 but are in G0 phase). In wild-type epidermal hypocotyl cells of 7-day-old seedlings, cortical MTs have a parallel and transverse alignment (Figure 2J). In contrast to wild-type seedlings, ton2 mutant epidermal hypocotyl cells are more irregular in shape, radially swollen, and only partially elongated compared with wild-type cells, especially in the strong allele (Figures 2K and 2L). In hypocotyl cells of ton2-13 (strong allele), interphase MTs have mostly parallel arrays whose orientation is not well fixed, with cells in the same area having a transverse, oblique, or longitudinal MT array orientation (Figure 2L). In ton2-14 (weak allele), these defects are less pronounced, and most MTs are oriented obliquely with respect to the growth axis (Figure 2K). This parallel organization is in contrast to the organization of cortical MTs in root epidermal cells observed previously in random arrays (Traas et al., 1995
Molecular Characterization of the TON2 Locus The TON2 cDNA is 1.75 kb long and contains a single open reading frame of 1440 bp, which encodes a predicted polypeptide of 480 residues. The TON2 gene contains 11 introns and 12 exons (Figure 3) . In the ton2-13 strong allele, the single T-DNA insertion is located at the very beginning of intron 6. The right border of the T-DNA is associated with a 37-bp deletion of the genomic DNA and a 16-bp insertion of unknown origin. This mutation presumably gives rise to a fusion translation product of 222 amino acids composed of the first 209 residues of the TON2 protein and 23 residues of T-DNA origin.
The molecular defects in the ton2-5, ton2-14, and ton2-12 alleles were characterized by sequencing of reverse transcriptase–mediated (RT)–PCR-amplified cDNA and genomic DNA fragments. Compared with wild-type parental sequences, all alleles carry mutations in the same gene (Figure 3). In the ton2-5 strong allele, a nonsense mutation (C960T in cDNA clone YAY132) causes the premature termination of translation after Leu-267 (Gln-268 to Stop) (Figure 3). Therefore, the N-terminal part of the protein (up to residue 267) presumably is not enough for the correct translation and/or activity of the protein. Interestingly, mutations in both weak alleles (ton2-12 and ton2-14) affect the same splice site, the donor site of intron 1 (Figure 4) . In both mutants, two major transcripts were revealed by RT-PCR experiments that are expected to give nonfunctional truncated polypeptides comprising (part of) exon I with small extensions (Figure 4). However, in both weak mutants, an alternative misspliced transcript of lower abundance can be detected using specific RT-PCR primers (Figure 4). This second misspliced transcript results in a 10–amino acid truncation of the wild-type predicted polypeptide (Gly-38 to Met-47). This transcript most likely is responsible for the leaky phenotype of the ton2-12 and ton2-14 alleles. Whether the weak mutant phenotype is caused by a low abundance of transcript and protein, a partial functionality of the deleted polypeptide, or both is not known at present.
The TON2 Gene Is Expressed Ubiquitously
The TON2 Protein Has Strong Similarities to a Regulatory Subunit of PP2A
The C-terminal part of TON2 shows significant similarity to the C terminus of a 72-kD human protein (PR72) (Figure 6B)
. PR72 was purified originally from rabbit skeletal muscle as a component of the trimeric form of Ser/Thr PP2A (Hendrix et al., 1993
In addition, the TON2 protein is similar over its entire length to vertebrate proteins of unknown function (Figure 6A), showing 57% similarity (36% identity) to human (Figure 6B) and mouse homologs. Partial EST sequences indicate that an ortholog exists in Danio rerio (Figure 6A). With respect to the mammalian polypeptide, all plant TON2 proteins possess an N-terminal extension of variable length (22 to 31 amino acids; 24 for Arabidopsis) and sequence (Figure 6B). Software available for the prediction of organellar or secretion targeting signals failed to attribute a clear functional significance to this N-terminal extension. All of these results indicate that the plant TON2 proteins and their vertebrate homologs define a novel subclass of PP2A B'' subunits (Figure 6A).
The TON2 Protein Interacts with an Arabidopsis Type A Subunit of PP2A in the Yeast Two-Hybrid System
The full-length TON2 protein clearly is able to interact with the entire A-type subunit AtA  , as is the genuine B-type regulatory subunit AtB' . This interaction is abolished either when the C-terminal part of TON2 (from Glu-269) is deleted or when a truncated form of the A subunit lacking the last 193 amino acids (AtA ) is used (Table 1). These results suggest that TON2 could act as a B-type regulatory subunit in PP2A enzymatic complexes. As expected from sequence data, the C-terminal part of the protein is necessary for interaction with other subunits.
For more than a decade, Arabidopsis mutants have been used to characterize the molecular bases of development in flowering plants, and several mutants impaired in cytoskeleton organization have been isolated. To address the question of the control of MT organization in plants, we have undertaken the isolation of the TON2 gene. The ton2 mutation affects the cortical MT cytoskeleton and has been shown to perturb a basic mechanism(s) involved in both cell elongation and cell division. Here, we report the identification of the TON2 gene. The TON2 protein is similar to a type B'' PP2A regulatory subunit and interacts in the two-hybrid system with known PP2A subunits. We also characterized in greater detail the MT organization of this mutant.
The use of the GFP-MBD MT reporter protein allowed us to visualize accurately dynamic changes in the organization of the MT cytoskeleton in living cells of ton2 and wild-type plants. Time-lapse observations of cell division in GFP-MBD–expressing plants confirm unambiguously the absence of the MT PPB in both strong and weak mutant alleles, in agreement with tubulin-immunolabeling studies from McClinton and Sung (1997)
Thus, the ton2 phenotype reinforces hypotheses linking the PPB and the division site. It also confirms that PPBs are not required for mitosis per se, the progression of which is normal in mutant plants (McClinton and Sung, 1997
To determine whether PPB formation is the main target of the ton2 mutation or if the mutation also primarily affects interphase MTs, we observed MT organization in nondividing cells. In hypocotyls of both ton2 strong and weak mutant alleles, most epidermal cells were found to form parallel arrays of MTs. This ability to align parallel arrays of MTs favors the idea that the loss of growth anisotropy in the ton2 mutant results only from the mispositioning of division planes caused by the absence of the PPB. However, two other observations prove that interphase cortical MT organization is a primary defect of the ton2 mutation. (1) In agreement with previous studies (Traas et al., 1995
It is surprising that in hypocotyl epidermal cells of the ton2 mutant, MTs are able to form parallel arrays even in strong allele cells in which TON2 most likely is nonfunctional. This phenomenon was not observed in root epidermal cells. We propose that the alignment of cortical MTs in hypocotyl cells may be a consequence of cell shape and/or wall architecture during the late stages of the elongation process. For example, cellulose microfibrils, which are the strongest component of the primary wall, could be involved in providing spatial cues for cortical MT organization during the late elongation phase. For instance, MT organization is inhibited after treatment of tobacco cells with the cellulose synthase inhibitor isoxaben (Fisher and Cyr, 1998
These observations favor a role for TON2 in organizing the cortical microtubular network. How this is achieved remains to be determined. TON2 may act directly on the MT arrays or indirectly by influencing the actin cytoskeleton. Indeed, actin microfilaments are aligned in the cell cortex parallel to MTs (Palevitz, 1987
Cortical MT arrays are of major importance for plant morphogenesis, but their dynamics and interrelation remain poorly understood. To our knowledge, TON2 is the only plant protein identified that is essential for the organization of both arrays, and its molecular identification provides the first clue to the regulatory processes of such cellular structures. The C-terminal part of the TON2 protein is highly similar to members of the B'' regulatory subunit family of type 2A Ser/Thr protein phosphatases. Two major structurally distinct families of Ser/Thr phosphatases, PP1/PP2A and PP2C, are present in plants as in animals. Additional studies also suggest the presence of other types of phosphatases in Arabidopsis (Andreeva et al., 1998
The B-type subunits have been shown to modulate the activity, substrate specificity, and subcellular localization of the PP2A complex (Goldberg, 1999 As expected for a bona fide PP2A B-type subunit, we observed a clear interaction between TON2 and an A-type subunit from Arabidopsis in the yeast two-hybrid system. Together with sequence similarity, these results support TON2's involvement in the formation of a PP2A complex, which presumably controls the phosphorylation status of proteins important for the structure of the plant cortical cytoskeleton.
In yeast and animals, PP2A activity regulates a wide range of cell processes (Virshup, 2000
The finding that TON2 likely encodes a PP2A regulatory subunit provides molecular evidence of a key role for the phosphorylation/dephosphorylation of proteins in the control of cortical MT organization. The target of such phosphatase activity is not known at present, although a possible candidate could be the protein(s) encoded by the TON1 locus (Nacry et al., 1998
Most basic processes that determine the architecture of cortical MT arrays remain unknown. As suggested by Baskin and Wilson (1997)
Plant Material and Growth Conditions Arabidopsis thaliana plants were grown in vitro as described previously (Nacry et al., 1998 ). T-DNA insertion lines were obtained by vacuum infiltration transformation (Bechtold et al., 1993) using Agrobacterium tumefaciens strain MP5-1 (Bouchez et al., 1993). Ethyl methanesulfonate mutants were obtained from a screen of individual M2 families derived from ethyl methanesulfonate–mutagenized seed of Columbia ecotype (Santoni et al., 1994).
Green Fluorescent Protein–Microtubule Binding Domain Marker Lines and Confocal Imaging Roots, hypocotyls, and flower primordia of plants expressing the GFP-MBD fusion protein were mounted in low-melting-point agarose (0.4% in water) and viewed directly using a Leica TCS-NT confocal laser scanning microscope (Leica, Heidelberg, Germany) with an argon/krypton laser (Omnichrome, Chino, CA) and an acousto-optical tunable filter (AOTF) for excitation. GFP fluorescence was excited with the 488-nm line and collected through a bandpass filter (BP 530/30). Slow-scan (220 lines/sec) images (1024 x 1024 pixels) were generated using a x40/1.00-0.50 PL FLUO-TAR objective.
Molecular Cloning Techniques and Sequence Analysis
Reverse Transcriptase–Mediated Polymerase Chain Reaction Polymerase chain reaction (PCR) was performed using 5 µL of the diluted cDNA sample with 250 pmol of the primers in a 25-µL reaction. PCR conditions were as follows: 94°C for 2 min; 28 cycles of 94°C for 15 sec, 58°C for 20 sec, and 72°C for 45 sec; and a final elongation step of 5 min at 72°C. APRT1- and TON2-specific primers were used in separate reactions. Five microliters of each reaction were electrophoresed on an agarose gel. Primers used for PCR were as follows: Ton2Nco (5'-CCGAATTCCCCATGGCTAGCGGATCTA- GCGATGGTGAGA-3'), Ton2F0 (5'-GCTTTGGGTTAGAAATCTACG- AC-3'), Ton2F2 (5'-AATCAAATGTATTGCCGCATAGCTT-3'), Ton2R1 (5'-GCACAGCGATCCACTCATATCTT-3'), and Ton2R3 (5'-ATAGCA- GCCAAATCATCAGCGTTTA-3'). For APRT1, primers 5'-TCCCAG- AATCGCTAAGATTGCC-3' and 5'-CCTTTCCCTTAAGCTCTG-3' were used.
Yeast Two-Hybrid Assays
Plasmids pGBT9 and pGAD424 (Clontech) containing cDNAs encoding Arabidopsis type 2A protein phosphatase subunits A
Accession Numbers
We are grateful to Gerd Jürgens and Ulrike Mayer (University of Tübingen, Germany), Jérôme Giraudat (CNRS, Gif-sur-Yvette, France), Richard Cyr (Pennsylvania State University), and Sabine Rundle (Western Carolina University) for kindly providing materials used in this work. We thank Josette Rousse and Anna Christodoulidou for their help and Herman Höfte for critical reading of the manuscript. J.A. was the recipient of a fellowship from the French Ministry for Research. This work was supported by a grant from the French Ministry for Research (Action Concertée Incitative Biologie du Développement Grant 47).
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010402. Received September 17, 2001; accepted December 17, 2001.
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Abstract
INTRODUCTION
1.5 to 3 cm at maturity for strong and weak alleles, respectively. Some almost morphologically normal, although sterile, flowers are observed in weak alleles (
