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First published online November 3, 2006; 10.1104/pp.106.088393 Plant Physiology 143:213-224 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Knockout of the AtCESA2 Gene Affects Microtubule Orientation and Causes Abnormal Cell Expansion in Arabidopsis1,[C],[OA]Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604 (Z.C., H.Y.); State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (H.C., Y.Z., Z.Z., N.Z., B.Y., Q.X.); State Key Laboratory of Plant Physiology and Biochemistry, Department of Plant Sciences, College of Biological Sciences, China Agricultural University, Beijing 100094, China (L.Z., M.Y.); College of Life Sciences, Shandong Agricultural University, Taian City, Shandong 271018, China (X. Zhao, X. Zhang); and Graduate University of the Chinese Academy of Sciences, Beijing 100101, China (H.C., Y.Z., Z.Z., B.Y.)
Complete cellulose synthesis is required to form functional cell walls and to facilitate proper cell expansion during plant growth. AtCESA2 is a member of the cellulose synthase A family in Arabidopsis (Arabidopsis thaliana) that participates in cell wall formation. By analysis of transgenic seedlings, we demonstrated that AtCESA2 was expressed in all organs, except root hairs. The atcesa2 mutant was devoid of AtCESA2 expression, leading to the stunted growth of hypocotyls in seedlings and greatly reduced seed production in mature plants. These observations were attributed to alterations in cell size as a result of reduced cellulose synthesis in the mutant. The orientation of microtubules was also altered in the atcesa2 mutant, which was clearly observed in hypocotyls and petioles. Complementary expression of AtCESA2 in atcesa2 could rescue the mutant phenotypes. Together, we conclude that disruption of cellulose synthesis results in altered orientation of microtubules and eventually leads to abnormal plant growth. We also demonstrated that the zinc finger-like domain of AtCESA2 could homodimerize, possibly contributing to rosette assemblies of cellulose synthase A within plasma membranes.
Plant cell shape is a key determinant of plant morphogenesis, which is strongly influenced by the organization of the cell wall (Beeckman et al., 2002  ). Cellulose, the predominant fiber of higher plant cell walls, is made of Glc subunits linked by  (1,4)-glycosidic bonds in unbranched, linear chains. In cell walls, individual cellulose molecules combine in ordered parallel rows to form cellulose microfibrils. Primary cell walls contain cellulose microfibrils in a loose, mesh-like array embedded in a relatively soft, gel-like matrix. As cells mature, they may elongate to as much as 10 to 100 times their embryonic lengths. During this elongation, the cellulose meshwork is first loosened and then stretched. Throughout the period of stretching and elongation, new cellulose microfibrils are deposited just at the outer plasma membrane, gradually thickening the wall as it extends and forms the secondary cell wall. Field emission scanning electron microscopy revealed that Arabidopsis (Arabidopsis thaliana) root epidermal cells have typical dicot primary cell wall structure with prominent transverse cellulose microfibrils embedded in pectic substances. Sugimoto et al. (2000) reported that the innermost layer of the Arabidopsis root epidermal cell wall was mainly composed of a transversely aligned fibrous structure that is most likely made of cellulose microfibrils.
The catalytic subunit responsible for elongation of glucan chains, cellulose synthase A (CESA), is believed to be a plasma membrane glycosyltransferase. These enzymes are thought to form complexes of rosettes embedded within the membrane (Taylor et al., 2000
By analysis of tobacco (Nicotiana tabacum) protoplasts and suspension-cultured cells treated with the cellulose synthesis inhibitor isoxaben, Fisher and Cyr suggested that cellulose biosynthesis was required for microtubule organization and stabilization and the cellulose microfibrils and cortical microtubules become coordinated during cell wall formation (Fisher and Cyr, 1998
All cellulose synthases described to date have a number of conserved structural features. It was discovered that phosphorylation of the catalytic subunit plays a key role in regulation of cellulose synthesis (Taylor, 2005 In this study, we report that the AtCESA2 knockout mutant (ecotype Landsberg erecta [Ler]), atcesa2, has severe defects in cell wall formation and microtubule orientation, resulting in abnormal plant growth and development. The function of the zinc finger-like domain of the AtCESA2 gene was also analyzed.
General Phenotypic Characterization of the atcesa2 Mutant
A screen for RING finger mutants led to the identification of atcesa2. The mutant was isolated from an activator (Ac)/dissociation (Ds)-mutagenized population in the Ler background generated as described by Sundaresan et al. (1995) When grown in light, the atcesa2 seedling phenotype deviated from wild type in that the leaves were rolled out and shrunken (Fig. 1, A and B ) and the hypocotyls were much shorter than those of the wild type (Fig. 1, D and E). Hypocotyls of the mutant grown in the dark for 6 d displayed a similar phenotype, but they were 2 times shorter than those of the wild type (Fig. 1C). They also had a reduced apical hook and opening of the cotyledon compared to wild-type seedlings. Mature greenhouse-grown homozygous plants of atcesa2 showed a strong dwarf phenotype with small inflorescence stems with shorter internodes (Fig. 1, F and G).
Mutant plants were almost sterile, although they could produce some seeds when grown directly under fluent air. Root hairs were of normal length and morphology, indicating that atcesa2 did not affect tip-growing cells. This was confirmed by the absence of atcesa2 expression in root hairs and root tips (Fig. 3B). The atcesa2 phenotype was not due to brassinosteroid, auxin, or gibberellin deficiency because the addition of each hormone separately to the growth medium could not rescue the dwarf phenotype (data not shown).
Genetic Analysis and Molecular Identification of the atcesa2 Mutant The atcesa2 mutant was isolated from a pool of Ac/Ds-transformed lines in the Ler background. The F1 progeny of the mutant backcrossed to wild type were grown on Murashige and Skoog medium and scored for segregation: None of the F1 plants had the mutant phenotype. In the F2 generation, by analysis of a total of 3,069 seedlings germinated on a Murashige and Skoog plate, 2,307 seedlings showed a wild-type phenotype, whereas 762 seedlings appeared as mutant phenotypes, which segregated approximately in a 3:1 ratio. No mild phenotype was observed. The segregation ratio of the original heterozygotes was also 3:1 (data not shown). The mutant phenotype was also linked to the kanamycin-resistant phenotype, which had no segregation in the next generation. Together, these data suggest that atcesa2 was a recessive mutation in a single Mendelian locus. Thermal asymmetric interlaced-PCR analysis demonstrated that the Ds was inserted 143 bp upstream of the start codon of the AtCESA2 gene (Fig. 2A ). Sequencing of the PCR products using primers specific to the Ds and to the AtCESA2 gene further confirmed that the Ds was linked to the AtCESA2 gene and no chromosome fragment deletion was found in the insertion region (data not shown). By checking genome sequence information of the insertion region, no coding sequence was found within 3 kb upstream from the insertion site. Northern-blot analysis (Fig. 2B), using the first specific region (236–676 bp from the ATG) of the AtCESA2 gene as the probe, showed no expression of AtCESA2 in the atcesa2 homomutant and reduced expression in the heteromutant, confirming that the AtCESA2 gene was knocked out in the atcesa2 mutant. Analysis of the cellulose content of the atcesa2 mutant revealed that it had about 61% of wild-type cellulose levels in leaves. However, the cellulose content in roots did not differ from wild type (Fig. 2D).
To further confirm that the atcesa2 mutant phenotype was due to mutation of the AtCESA2 gene, the 7.7-kb genomic sequence, including a 2-kb fragment upstream of ATG and a 500-bp fragment downstream of the stop codon of the AtCESA2 wild-type gene, was amplified and cloned into pCambia 1301, which was transformed into atcesa2 homozygotes by Agrobacterium-mediated methods. Twelve T1 progeny with hygromycin resistance were obtained, which all exhibited the wild-type phenotype (Fig. 2C). T2 progeny were grown on Murashige and Skoog medium plus kanamycin or hygromycin. On the kanamycin-containing medium, all seedlings had resistance, including wild-type and mutant seedlings, whereas on the hygromycin-containing medium, resistant seedlings all exhibited the wild-type phenotype. Northern-blot analysis revealed that rescue of the phenotype was due to expression of the AtCESA2 gene by the complementary construct in transgenic plants (Fig. 2C).
Northern blotting showed that AtCESA2 was expressed in all tissues, such as roots, stem, flowers, and cauline leaves (Burn et al., 2002
The size of a plant organ is determined by the rate of cell division and cell expansion and/or elongation. The hypocotyls and stamen filaments of the atcesa2 were clearly shorter than the wild type (Fig. 4, A, B, E, F, and I ). To investigate whether this shorter phenotype was caused by defects in cell expansion or cell division, the epidermal cell number and length of hypocotyls and stamen filaments were measured and counted. Statistical analysis of these data (Fig. 4J) demonstrated that the number of epidermal cells in hypocotyls and stamen filaments of the mutants did not differ significantly from those of wild type. Furthermore, the epidermal cells of the mutant hypocotyls and stamen filaments were much shorter than the wild type (Fig. 4, C, D, G, and H), suggesting that the altered phenotypes were caused by an alteration in cell size rather than cell number. This is consistent with the notion that the mutant phenotype was due to a defective cellulose synthase gene. Knocking out this gene may cause a defect in cellulose synthesis and lead to a disruption in the deposition of microfibrils, which results in alterations in cell wall extensibility and cell expansion.
The atCESA2 Mutation Causes Cell Wall Defects Mainly in Leaves The above results demonstrated that the atcesa2 phenotypes were caused by the loss of function of the AtCESA2 gene. The AtCESA2 gene is a member of the CESA family, which is responsible for cell wall synthesis. To investigate whether the cell wall defects occurred in atcesa2, we first studied transverse sections of the roots, leaves, and stems of wild-type and mutant plants. The overall anatomical features of the mutant roots and vascular bundles were unaltered and the diametric size of the roots of the mutant was almost the same as the wild type (Fig. 5, A and B ). However, the stems of atcesa2 showed a decrease in girth as a result of decreased cell diameter. The whole vascular bundle of the mutant was unaltered relative to wild type (Fig. 5, G and H). The most affected parts of the mutant were the leaves. The overall anatomy of the mutant leaves differed from wild type, curling much more than wild-type leaves (Fig. 5, C and D). The cell morphology of mutant leaves, especially the cell walls, was also significantly altered (Fig. 5F) compared to the wild type (Fig. 5E).
To further investigate the cell and cell wall aberrances, the ultrastructure of mutant and wild-type leaves was observed by transmission electron microscopy. The palisade cells of wild-type leaves were evenly arrayed perpendicularly to the epidermal cells (Fig. 6A ), but the palisade cells in the mutant were arranged parallel to the epidermal cells with the cell shape altered (Fig. 6C). These results were consistent with the outward rolling of atcesa2 leaves. Additionally, the cell walls of the wild-type cells were smooth and complete (Fig. 6B), whereas those of the mutant were shrunken (Fig. 6D) and incomplete (Fig. 6E). No obvious differences in callose levels were observed between the cell walls of wild-type and mutant cells (data not shown). Quantitative analysis revealed that pectin content in the aerial part of mature atcesa2 mutant plants was higher than that in the wild-type control (Fig. 6G).
The atCESA2 Mutation Affects Microtuble Orientation
Microtuble orientation is a key factor controlling cell expansion and elongation. Himmelspach et al. (2003)
AtCESA2 Interacts with Itself through the N-Terminal Zinc-Binding Domain
The Arabidopsis AtCESA zinc finger shows high similarity to RING finger domains, a small zinc-binding domain found in many functionally distinct proteins (Freemont, 2000
The atcesa2 mutant was obtained during our screening of insertion lines for RING finger-containing mutants. Genetic analysis, thermal asymmetric interlaced-PCR, northern-blot, cellulose content assay, and complementation experiments demonstrated that the mutation caused a loss of expression of the AtCESA2 gene. Reduced expression of the AtCESA2 gene by an antisense method showed no obvious phenotype, except reductions in stem length under certain growth conditions (Burn et al., 2002 ). One possibility is that reduced expression of the AtCESA2 gene by antisense gave rise to a mild phenotype compared to the complete knockout of the gene. These results highlight the differences between the two reverse-genetics approaches, reduction of RNA level (gene silencing) versus gene deletion. Scheible and Pauly (2004) have reported in their review that an atCesA2 insertion line has no obvious phenotypes in plant growth and development. But recently, the same author found some phenotypes with more insertion lines and it is believed that different CesA2 insertion mutants probably have very different effects on plant growth (W.R. Scheible, personal communication). We also analyzed two T-DNA knockout lines, SALK_091570 and SALK_125303, of the AtCesA2 gene in the Col background from the Arabidopsis Biological Resource Center. No obvious phenotypes were detected at all stages of plant growth and regeneration (data not shown). Disruption or mutation of the same gene in different backgrounds (ecotypes) of Arabidopsis also could give different phenotypes or no phenotypes due to gene redundancy in different ecotypes (Maloof et al., 2001; Yang and Hua, 2004; Werner et al., 2005). We used the Ler background in our study and previous reports were all from the Col background (Burn et al., 2002; Scheible and Pauly, 2004).
The AtCESA2 gene is one of about 10 currently known members of the cellulose synthase family. Each member may be one catalytic subunit of the rosettes responsible for cellulose synthesis. Several questions regarding CESA function remain to be answered. Are the various CESA isoforms functionally unique or do some isoforms overlap in function? Available data suggest that significant functional specialization has occurred, including differences in gene expression, regulation, and possibly catalytic function. Various CESA expression analyses have been conducted in both monocot and dicot species. Arabidopsis CESA family members have different expression levels, particularly when multiple CESA genes are expressed in the same cell type. AtCESA1, AtCESA3, and AtCESA6 all exhibit very similar expression patterns, being expressed in cells undergoing expansion in tissues such as roots and hypocotyls (Arioli et al., 1998
Expression studies of CESA genes facilitate functional studies using loss-of-function mutants because only genes that are expressed at the same stage or in the same tissue need to be examined for potentially redundant functions. Using AtCESA-promoter::GUS fusions, CESA2 and CESA5, like CESA1, CESA3, and CESA6, were shown to be expressed at the sites of primary wall synthesis (Scheible and Pauly, 2004
Cellulose synthase proteins are components of CESA complexes (rosettes) and catalyze glucan polymerization. Little is understood about rosette assembly, including how CESAs interact with each other or with other components within the complexes. How many types of CESA are present in one rosette and are components of all rosettes the same in different tissues and organs? AtCESA7 and AtCESA8 were the first to be found to interact with each other in vivo (Taylor et al., 2000
The conserved regions at the N terminus of plant CESA proteins contain two putative zinc fingers that show high homology to the RING finger motif and are thought to facilitate protein-protein interactions. Kurek et al. (2002)
Plant cell expansion is driven by internal turgor pressure and restricted by the ability of the cell wall to extend under this pressure. The extensibility of cell walls depends on at least two related events, including the composition of the wall and how its components are bound to one another and the modification of existing wall structures (Darley et al., 2001
Initially, primary cell wall material must be synthesized in a form that is competent to undergo extension. The primary cell wall is essentially a composite structure comprising a framework of cellulose microfibrils embedded in a matrix of other polymers. Most of the polymers are pectin or cross-linking glycan polysaccharides. Alterations in cellulose synthesis can affect cell wall extensibility, which impacts on plant growth. Studies of the effects of cellulose biosynthesis inhibitors revealed that cultured plant cells can survive in the absence of cellulose (Shedletzky et al., 1990
In the microtubule parallelism hypothesis, transverse deposition of cellulose microfibrils along the axes of elongating cells is proposed to be controlled by the transversely oriented cortical microtubules lying beneath the plasma membrane (Giddings and Staehelin, 1991
Plant Growth and Transformation
Seeds of Arabidopsis (Arabidopsis thaliana; ecotype Ler) were surface sterilized with 30% bleach and 0.001% Triton X-100 for 10 min and washed three times with sterile water. Sterile seeds were suspended in 0.2% agarose and plated on Murashige and Skoog medium or Murashige and Skoog plus 1.5% Suc. Plants were vernalized in darkness for 2 d at 4°C and then transferred to a tissue culture room under white fluorescent light (45 µE m–2 s–1, 16-h light/8-h dark cycle) at 22°C. After 2 to 3 weeks, seedlings were potted in soil and placed in a growth chamber at 22°C with 75% humidity under a 16-h light/8-h dark photoperiod. Transgenic plants were generated by Agrobacterium-mediated methods (Bent and Clough, 1998
The whole genomic sequence, including a 2-kb fragment upstream of the ATG and a 500-bp fragment downstream of the stop codon of the AtCESA2 wild-type gene, was amplified by the Expand Long Template PCR system (Roche Diagnostics GmbH; catalog no. 1 681 842). The forward and reverse primers were 5'-TTTCCAgATTTgTTggTggAgCCTTgTAgg-3' and 5'-TTAATAAACCATgCCTgATTCAATggAACCgAg-3', respectively. The PCR product was inserted into the pGEM-T easy vector at the AatII and PstI restriction sites. The insert was subsequently cloned into pCAMBIA 1301 at the SmaI and PstI restriction sites. After confirming sequence accuracy, the construct was transformed into atcesa2 and 12 T2 progeny were obtained. The segregation of these lines was analyzed on Murashige and Skoog, Murashige and Skoog plus kanamycin, and Murashige and Skoog plus hygromycin media.
RNA was isolated using the Qiagen RNeasy kit and 10 µg of total RNA were loaded per lane. To detect the AtCESA2 transcript, blots were probed with an N-terminal fragment (with HindIII digestion [236–676 bp from ATG]) labeled with [
Seedlings were grown on one-half-strength Murashige and Skoog for 2 weeks. Some of the seedlings were transferred into B5 liquid medium and cultured in suspension at 22°C; root parts were harvested after 3 weeks. The other set of seedlings were potted in soil and placed in a growth chamber at 22°C with 75% humidity under a 16-h light/8-h dark photoperiod; aerial parts were harvested after 3 weeks. Samples were ground into fine powder in liquid nitrogen, washed in phosphate buffer (50 mM, pH 7.2) three times. After centrifugation, pellets were extracted three times in 70% ethanol at 70°C for 1 h and dried at 80°C overnight. Dried materials were extracted with an acetic/nitric acid reagent at 100°C, followed by digestion with 67% sulfuric acid. Cellulose content was determined by the phenol-sulfuric acid method (Dubois et al., 1956
For histochemical localization of GUS activity, seedlings were incubated in a solution containing 50 mM sodium phosphate buffer, pH 7.0, 5 mM K3Fe(CN)6, 0.1% Triton X-100, and 1 mM 5-bromo-4-chloro-3-indolyl-
Seedlings were collected and fixed in 2.5% glutaraldehyde in 50 mM NaPO4 buffer, pH 7.4, for 2.5 h at 4°C. Subsequently, they were washed four times for 15 min in 50 mM NaPO4 buffer, pH 7.4, then twice for 15 min in water. The samples were then dehydrated in increasing concentrations of ethanol and embedded in historesin using a kit according to the manufacturer's instructions (Leica). Sections (2-µm-thick) were prepared and stained with 0.05% toluidine blue O in sodium citrate buffer, pH 4.4. Samples were eventually mounted in water for microscopic observation.
Callose was stained in a solution of 0.005% aniline blue in 50% ethanol and observed under confocal microscopy (Zeiss LSM510) at 405 nm with a band-pass 420- to 480-nm emission filter. For pectin quantitative analysis, samples were harvested from the aerial part of 3-week-old soil-grown plants. Measurement of pectin content was performed according to Zhou et al. (2006)
Fresh tissue samples were placed on a specimen stub and secured with Tissue-Tek (Sakura Finetake Europe B.V.) before cryofixing in liquid nitrogen slush. Samples were then sputter coated with gold and viewed in high vacuum mode on a cryostage maintained at 190°C (CT1500 Cryo transfer system; Oxford Instruments) using a JEOL-JSM 5310-LV scanning electron microscope.
Leaf samples were fixed in 4% glutaraldehyde in 100-mM cacodylate buffer for 3 h and then postfixed with 2% osmium tetroxide in 100 mM sodium cacodylate buffer for 1 h at 4°C. Samples were then dehydrated through a series of 30%, 50%, 70%, 90%, and 100% ethanol and finally immersed in propylene oxide prior to infiltration with Spurr's resin (Spurr, 1969
Three-day-old wild-type and atcesa2 seedlings with Map4-GFP expression of wild type and mutants were mounted in water. Samples were imaged using a dip-in long working distance 63x (NA0.9) water-immersion lens (Zeiss). GFP fluorescence was excited with the 488-nm line of an argon/krypton laser. Emission was detected through a 510-nm beam splitter and a 515 to 565 band-pass filter.
For visualization of microtubules within cells of the root elongation zone and the middle region of the hypocotyl, we used the protocol for fixing and immunofluorescent staining of 5-d-old seedling roots and etiolated hypocotyls as described (Sugimoto et al., 2000
The AtCESA2 zinc finger domain, the N-terminal AtCESA2 fragment, and full-length proteins were inserted into pGADT7 and pGBKT7. The yeast (Saccharomyces cerevisiae) strain HF7c (MATa ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3,112, gal4-542, gal80-538 LYS2::GAL1UAS-GAL1TATA-HIS3, URA3::GAL4, 17 mers [x3]-CyC1TATA-LacZ), containing the two reporter genes LacZ and HIS3, was used. Both pGBKT7 and pGADT7 bearing different lengths of AtCESA2 were transformed into yeast cells. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number At4g39350.
We thank Dr. Venkatesan Sundaresan for Ac/Ds insertion lines, the Arabidopsis Biological Resource Center, Ohio State University, for providing the T-DNA insertion lines, and Ms. Yang Sun Chan for excellent scanning electron microscopy/transmission electron microscopy support. Received August 16, 2006; accepted October 31, 2006; published November 3, 2006.
1 This work was supported by the Chinese Ministry of Science and Technology (grant no. 863–2002AA224111/MST 973–2003CB114304), by the Outstanding Youth Project from the Chinese Natural Science Foundation (grant no. 30325030), and by the Chinese Academy of Sciences (grant nos. KSCX2–YW–N–010 and CXTD–S2005–2 to Q.X.). 
2 These authors contributed equally to the paper.
3 Present address: Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Qi Xie (qxie{at}genetics.ac.cn).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.088393 * Corresponding author; e-mail qxie{at}genetics.ac.cn; fax 86–10–64889351.
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TOP
ABSTRACT
RESULTS
-32P]dCTP using a Ready-primed labeling kit (Amersham International). 
