Title: A plasma membrane-bound putative endo-1,4-beta -D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis
Abstract: Article1 October 1998free access A plasma membrane-bound putative endo-1,4-β-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis Frédéric Nicol Frédéric Nicol Laboratoire de Biologie Cellulaire, Institut National de Recherche Agronomique, Route de Saint-Cyr, F-78026 Versailles, Cedex, France Search for more papers by this author Isabelle His Isabelle His Université de Rouen, CNRS UPRESA 6307, Faculté des Sciences, F-76821 Mont-Saint-Aignan, Cedex, France Search for more papers by this author Alain Jauneau Alain Jauneau Université de Rouen, CNRS UPRESA 6307, Faculté des Sciences, F-76821 Mont-Saint-Aignan, Cedex, France Search for more papers by this author Samantha Vernhettes Samantha Vernhettes Laboratoire de Biologie Cellulaire, Institut National de Recherche Agronomique, Route de Saint-Cyr, F-78026 Versailles, Cedex, France Search for more papers by this author Hervé Canut Hervé Canut Centre de Physiologie Végétale de l'Université Paul Sabatier, U.A. CNRS No. 241, 118, Route de Narbonne, F-31062 Toulouse, Cedex, France Search for more papers by this author Herman Höfte Herman Höfte Laboratoire de Biologie Cellulaire, Institut National de Recherche Agronomique, Route de Saint-Cyr, F-78026 Versailles, Cedex, France Search for more papers by this author Frédéric Nicol Frédéric Nicol Laboratoire de Biologie Cellulaire, Institut National de Recherche Agronomique, Route de Saint-Cyr, F-78026 Versailles, Cedex, France Search for more papers by this author Isabelle His Isabelle His Université de Rouen, CNRS UPRESA 6307, Faculté des Sciences, F-76821 Mont-Saint-Aignan, Cedex, France Search for more papers by this author Alain Jauneau Alain Jauneau Université de Rouen, CNRS UPRESA 6307, Faculté des Sciences, F-76821 Mont-Saint-Aignan, Cedex, France Search for more papers by this author Samantha Vernhettes Samantha Vernhettes Laboratoire de Biologie Cellulaire, Institut National de Recherche Agronomique, Route de Saint-Cyr, F-78026 Versailles, Cedex, France Search for more papers by this author Hervé Canut Hervé Canut Centre de Physiologie Végétale de l'Université Paul Sabatier, U.A. CNRS No. 241, 118, Route de Narbonne, F-31062 Toulouse, Cedex, France Search for more papers by this author Herman Höfte Herman Höfte Laboratoire de Biologie Cellulaire, Institut National de Recherche Agronomique, Route de Saint-Cyr, F-78026 Versailles, Cedex, France Search for more papers by this author Author Information Frédéric Nicol1, Isabelle His2, Alain Jauneau2, Samantha Vernhettes1, Hervé Canut3 and Herman Höfte1 1Laboratoire de Biologie Cellulaire, Institut National de Recherche Agronomique, Route de Saint-Cyr, F-78026 Versailles, Cedex, France 2Université de Rouen, CNRS UPRESA 6307, Faculté des Sciences, F-76821 Mont-Saint-Aignan, Cedex, France 3Centre de Physiologie Végétale de l'Université Paul Sabatier, U.A. CNRS No. 241, 118, Route de Narbonne, F-31062 Toulouse, Cedex, France The EMBO Journal (1998)17:5563-5576https://doi.org/10.1093/emboj/17.19.5563 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Endo-1,4-β-D-glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes. In higher plants, potential substrates in vivo are xyloglucan and non-crystalline cellulose in the cell wall. Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion. Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells. KORRIGAN (KOR) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall. The KOR gene was isolated and encodes a membrane-anchored member of the EGase family, which is highly conserved between mono- and dicotyledonous plants. KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane–cell wall interface. KOR mRNA was found in all organs examined, and in the developing dark-grown hypocotyl, mRNA levels were correlated with rapid cell elongation. Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR-mRNA levels. However, reduced KOR-mRNA levels were observed in det2, a mutant deficient for brassinosteroids. Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose–hemicellulose network in the expanding cell wall. Introduction Endo-1,4-β-D-glucanases (EGases) constitute a large, ubiquitous family of enzymes that hydrolyse 1,4-β linkages adjacent to unsubstituted glucose residues (Henrissat et al., 1989; Brummell et al., 1994). Microbial EGases can degrade crystalline cellulose, and are intensively studied and used in industrial processes. Plant EGases form a separate subclass (type E2, Brummell et al., 1994), lack a cellulose-binding domain and are unable to hydrolyse crystalline cellulose. In vitro activity has been observed with various 1,4-β-linked glucan polymers such as xyloglucans or the artificial substrate carboxy-methyl cellulose (CMC). However, the in vivo substrates remain to be determined. All higher plant species investigated express multiple EGases, for instance in Arabidopsis thaliana at least a dozen different family members can be found in sequence databases (F.Nicol and H.Höfte, unpublished data). It is not known to what extent different family members vary in their biochemical properties. The expression of most plant EGases is tightly regulated, suggesting a role in plant development. Some EGases are expressed specifically during fruit ripening or leaf abscission (Cass et al., 1990; Tucker and Milligan, 1991; Lashbrook et al., 1994; Ferrarese et al., 1995; Del Campillo and Bennett, 1996). The expression of other EGases is highest in expanding cells which may be indicative of a role in cell wall polysaccharide assembly or rearrangements in the primary cell wall. A role for EGases in cell expansion has been debated over the past 20 years, and despite the vast body of literature, the issue remains controversial (Hoson and Nevins, 1989; Inouhe and Nevins, 1991; Wu et al., 1996; Brummell et al., 1997). To understand the mechanism of plant cell expansion, it should be noted that plant cells are surrounded by a polymeric cell wall which imposes constraints on the cell expansion process. The wall must resist the extreme tensile forces exerted by the osmotic pressure of the protoplast and at the same time, in a growing cell, it must be able to expand in a coordinated fashion without losing its integrity. Wall expansion involves an initial stress relaxation (Cosgrove, 1997), followed by the biosynthesis of new polymers and their regulated incorporation into the wall structure. The molecular mechanisms underlying these processes are poorly understood. Primary cell walls of dicotyledonous plants are composed of three major classes of polysaccharides: cellulose, xyloglucan and pectin (Carpita and Gibeaut, 1993). Cellulose microfibrils are composed of 1,4-β-D-glucan polymers synthesized by putative cellulose–synthase complexes which can be visualized as rosettes on freeze-fracture images of the plasma membrane. Genes encoding the catalytic subunit of cellulose synthase were recently identified in cotton and A.thaliana (Pear et al., 1996; Arioli et al., 1998). The matrix polysaccharides, xyloglucans and pectins, are synthesized in the Golgi apparatus and secreted into the cell wall (Staehelin and Moore, 1995). Since the biosynthetic enzymes have not been purified nor the genes cloned, little is known about their biosynthesis. Also, the mechanisms regulating synthesis and secretion of matrix polysaccharides during cell expansion remain largely unknown. Upon release into the apoplast, xyloglucans bind tightly to the nascent cellulose microfibrils through hydrogen bonds (Hayashi and Maclachlan, 1984; Hayashi, 1989). Electron microscopic evidence indicates that xyloglucan molecules form cross-bridges between cellulose microfibrils, thereby establishing a load-bearing cellulose–xyloglucan network (McCann et al., 1990). Studies on tomato cell cultures adapted to the inhibitor of cellulose deposition, 2,6-dichlorobenzonitrile (DCB), indicate that cellulose–hemicellulose on the one hand, and pectins on the other hand, form two independent networks in the primary wall. DCB-adapted cell walls have greatly reduced cellulose contents. As a result, xyloglucans cannot bind to cellulose and are secreted into the medium. Surprisingly, in the absence of a cellulose–hemicellulose network, the wall integrity and strength is maintained by a Ca2+-linked network of acidic pectins, as shown by the extreme susceptibility of these walls to Ca2+-chelators. Wall stress relaxation may involve slippage or hydrolysis of the load-bearing xyloglucan cross-bridges (Cleland, 1971; Albersheim, 1975; Hayashi and Maclachlan, 1984) and it has frequently been suggested that EGases, together with other cell-wall proteins, such as expansins and xyloglucan-endo-transglycosylases, may play a role in this process. To our knowledge, this work represents the first demonstration of an essential role for a member of the EGase family in plant cell expansion. The gene was cloned from an A.thaliana dwarf mutant, korrigan (kor), showing a defect in the elongation of all non-tip-growing cells examined. KOR encodes an evolutionarily conserved integral membrane protein which is primarily located in the plasma membrane. KOR-transcript levels in the hypocotyl correlated positively with cell elongation and were not affected in mutants overproducing free auxin or ethylene, or mutants deficient in gibberellins. In contrast, a mutant deficient in brassinosteroids showed reduced KOR-mRNA levels. The observed structural alterations in the mutant cell wall suggest a role for KOR in the correct assembly of the cellulose–hemicellulose network in the expanding cell wall. Results kor, a novel cell elongation mutant kor was found as a single allele in a screen for short hypocotyl mutants among dark-grown T-DNA or chemically mutagenized seedlings. Despite the relatively large scale of the screen, no other alleles for KOR were identified although several alleles were found for other loci like PROCUSTE (eight alleles, H.Höfte, unpublished data and Desnos et al., 1996) and BOTERO (six alleles, H.Höfte, unpublished data). The segregation in the progeny of kor heterozygous plants showed a 3:1 ratio (1136 wild-type and 386 kor seedlings among 1522 seeds sown; χ2 = 0.031; P <0.05) which demonstrates that the mutant phenotype segregates as a single, recessive, nuclear locus. The genetic map position of the locus was deduced from the localization of the cloned KOR gene (see below) on two yeast artificial chromosomes (CIC4G5 and CIC8D5) containing the microsatellite marker nga129, which maps to the bottom of chromosome V (position 81.7). The hypocotyl of 7-day-old dark-grown kor seedlings was four times shorter (Figures 1C and 2A) than that of the wild-type, but elongation kinetics were unchanged. For both the mutant and wild-type, the hypocotyl elongated between day 2 and 7, after which a plateau was reached (Figure 2A). kor hypocotyls grown in white light were only slightly shorter than those of the wild-type (Figures 1A and 2A). Root growth was also reduced in the mutant compared with wild-type (Figures 1C and 2B) and the difference was most pronounced in the light. In light conditions the root growth rate of the wild-type was significantly higher (0.19 cm/day) than in dark-grown plants (0.12 cm/day). The size of all other organs investigated was also reduced in kor, including stems, rosette leaves, flowers and siliques (Figure 1B and D). Despite the reduced size, mutant plants were fully fertile. No differences in size between the mutant and wild-type were observed for tip-growing cells such as trichomes and root hairs (data not shown). Furthermore, the normal Mendelian segregation ratio for the mutant phenotype suggested that pollen tube elongation was unaffected. Figure 1.kor mutant phenotype. (A) Seven-day-old light-grown wild-type (left) and korrigan (right) seedlings. (B) Six week-old greenhouse-grown plants: wild-type (left) and korrigan (right). (C) Seven-day-old dark-grown wild-type (left), korrigan (middle) and kor transformed with the 8.5 kb complementing genomic fragment (right) seedlings. (D) Fifteen-day-old light-grown wild-type (left) and korrigan (right) seedlings. (E) Seven-day-old light-grown wild-type roots. (F) Seven-day-old light-grown korrigan roots. Calcofluor-stained transverse sections through hypocotyls of 7-day-old dark-grown (G) wild-type and (H) korrigan seedlings. Arrows indicate wall separations often observed in the kor mutant. Scale bar represents 65 μm. Download figure Download PowerPoint Figure 2.Reduced hypocotyl and root growth in kor mutants. (A) Hypocotyl and (B) root lengths were measured as a function of the number of days after transfer to the growth chamber. Download figure Download PowerPoint Scanning electron microscopy (SEM) of mutant dark-grown hypocotyls revealed an irregular surface (Figure 3J) consisting of epidermal cells with irregular shapes. Wide cells alternated with narrow cells and some cells had collapsed entirely or had failed to expand. Although 7-day-old dark-grown kor hypocotyls showed a length comparable with that of wild-type light-grown hypocotyls (Figure 2A), the apical hook, the closed cotyledons and the epidermal differentiation pattern (Gendreau et al., 1997) indicated that the mutation did not affect other aspects of skotomorphogenic development. Besides the epidermis, all other hypocotyl cells were also affected in the mutant as shown in transverse sections (Figure 3C and G) compared with the wild-type (Figure 3B and F). No changes in radial organization could be observed. However, all cells, including those in the central cylinder, showed increased radial expansion. Similar observations were made for the root (Figure 1E and F). In light-grown kor hypocotyls (Figure 3D), cell shape was also different from the wild-type (Figure 3A), but differences were less pronounced than in the dark (Figure 3E and H). Figure 3.A cell elongation defect in kor mutants. Scanning electron micrographs (SEM) of 7-day-old (A, D) light-grown seedlings and (E, H) 7-day-old dark-grown seedlings. (B, C) Transverse sections of 7-day-old light-grown wild-type (B) and kor (C) and (F, G) dark-grown wild-type (F) and kor (G) hypocotyls. (I, J) SEM of 7-day-old light-grown wild-type (I) and kor (J) cotyledons. Arrows indicate the unexpanded epidermal cells. Scale bar represents 100 μm. Download figure Download PowerPoint In kor cotyledons, cell shape was highly variable. Areas with almost fully expanded, jigsaw puzzle-like cells alternated with islands of poorly expanded cells with a smooth surface (Figure 3I and J), suggesting a stochastic expression of the mutant phenotype. KOR encodes a member of the endo-1,4-β-D-glucanase family The T-DNA segregated as a single locus and could not be separated genetically from the kor mutation, indicating a close linkage (<3 cM). Southern analysis of DNA extracted from kor and wild-type plants, digested with several enzymes and probed with DNA fragments corresponding to either the left or right T-DNA border revealed a single, simple T-DNA insertion. Plant DNA flanking the right border was isolated and used to obtain genomic clones. Complementation tests were carried out with an 8.5 kb DNA fragment, presumably containing the entire gene, cloned into a binary T-DNA vector carrying a hygromycin-resistance gene. Plants heterozygous for kor were transformed by in planta infiltration (Bechtold et al., 1993). All primary transformants had a wild-type phenotype in the light. The T2 progeny obtained after selfing of one of these transformants segregated for kor with a 1:15 ratio (65 kor and 1167 wild-type seedlings of 1232 seeds sown; χ2 = 0.12; P <0.05) which was expected for an unlinked insertion complementing the mutant phenotype. In addition, all phenotypically mutant T2 seedlings were hygromycin-sensitive, whereas all hygromycin-resistant seedlings had a wild-type phenotype in the light and in the dark. The transformation efficiency of plants homozygous for kor was much less efficient and yielded only a single transformant. This transformant showed a wild-type phenotype. The T2 progeny from this transformant was 100% kanamycin-resistant, confirming the homozygous state of the T-DNA tagged kor mutation, and segregated the wild-type phenotype in a 3:1 ratio (315 wild-type and 95 kor seedlings of 410 seeds sown; χ2 = 0.75; P <0.05). All wild-type plants were also hygromycin resistant, indicating the presence of the transgene. Figure 1C shows that the presence of one copy of the transgene completely restores the phenotype of dark-grown seedlings homozygous for the kor mutation to wild-type. The adult phenotype was also entirely complemented in the transformant (data not shown). All these data indicate that the 8.5 kb fragment had complemented the mutation and therefore should contain the KOR gene. A 2.3 kb cDNA clone was isolated using the right border T-DNA flanking fragment, and the sequence was compared with 3 kb of genomic sequence flanking the right border (DDJB/EMBL/GenBank accession No. AF073875). The genomic sequence contained five Open Reading Frames (ORFs) corresponding to the cDNA sequence interrupted by four introns (Figure 4A) with consensus splice-donor and acceptor sites. The first ATG codon is most likely to be the initiator codon, since it is preceded by an in-frame stop codon at −46 bp. The T-DNA is inserted 200 bp upstream of the initiator ATG, presumably in the promoter. The cDNA sequence is 100% identical to an A.thaliana sequence present in the public databases (DDJB/EMBL/GenBank accession No. U37702). The predicted amino acid sequence of 622 residues contains eight potential N-glycosylation sites and is similar over almost its entire length to EGases from plants and bacteria. For instance, the amino acid sequence is 43.3% identical to avocado Cel1 sequence and 44.4% identical to pea EGL1. In the bacterial enzyme CelD, site-directed mutagenesis and chemical modification techniques have identified four amino acids (D198/201, H516 and E555) as being potentially important in catalysis (Chauvaux et al., 1992). As in other plant EGases, these four residues are also conserved in the KOR sequence (Figure 4C). KOR is highly similar (81.8% identity) to Cel3, a recently identified membrane-bound EGase family member in tomato (Brummell et al., 1997). In contrast to all other plant EGases, the sequences of KOR and Cel3 do not contain a predicted cleavable signal peptide. Instead, the mature protein is predicted to be a type II integral membrane protein anchored in the membrane by a stretch of highly hydrophobic amino acids located in the N-terminus (Von Heijne, 1986; Figure 4A and B). The N-terminal stretch of 100 amino acids distinguishes KOR and Cel3 from other plant EGases. Interestingly, this sequence is highly conserved between dicots and monocots The sequence shows 94% identity between KOR and Cel3, and one rice EST encodes a polypeptide showing 82% identity to this segment in KOR (Figure 4C). KOR, Cel3 and the rice EST are more similar to each other than to other plant EGases suggesting a functional specialization preceding the divergence between monocots and dicots in evolution (Figure 5). KOR and Cel3 also can be distinguished from other EGases by the presence of a C-terminal extension of 30 amino acids rich in prolines (Figure 4A). Figure 4.KOR, a putative membrane-bound endo-1,4-β-D-glucanase. (A) Domain structure: unique restriction sites in the gene sequence are shown, introns are represented by black arrows, the T-DNA is inserted 200 bp upstream of the ATG-initiation codon. The predicted N-terminal cytosolic tail, the membrane anchor and the extracellular EGase domain and the proline-rich C-terminal domain are indicated. (B) Hydrophobicity plot according to Kyte and Doolittle (1982). (C) Alignment of KOR with plant and bacterial EGases: AvoCel1, Tomcel3 and celD are the amino acids sequences of a soluble EGase from avocado without the signal peptide, a membrane-bound EGase from tomato, an EGase from Clostridium thermocellum, respectively. The predicted amino acid sequence of a rice EST is included. ⊠represents the residues essential for catalytic activity identified in celD which are also conserved in KOR (Asp198, Asp201, H516, Glu555); *indicates the eight predicted glycosylation sites. The thick black line overlines the predicted transmembrane domain in KOR (Von Heijne, 1986). The accession numbers are as in Figure 5. Download figure Download PowerPoint Figure 5.KOR represents a distinct, evolutionarily conserved subclass of plant EGases. Similarity tree (Genetics Computer Group- version 9.0) of type E EGases after removal of the predicted signal sequences. This family consists of two subgroups whereby subgroup E2 possesses both microbial and plant members. Sequences and DDJB/EMBL/GenBank accession numbers are: korrigan (U37702), tomcel3 (X97189), rice (EST; D46633), tomcel1 (U13054), pepper cel1 (X87323), BAC (M57400), Atglucanase (U17888), EGL1 (L41046), tomcel2 (U13055), peppercel3 (X83711), peppercel2 (X97190), poplarcel1 (D32166), avocel1 (M17634), tomcel4 (U20590), avocel2 (X55790), CstcelZ (X55299) and CtcelD (X04584). Download figure Download PowerPoint KOR is an integral membrane protein To determine the intracellular localization of KOR, a polyclonal rabbit antiserum was raised against a chimeric protein produced in Escherichia coli, composed of bacterial glutathione-S-transferase fused to the 65 N-terminal amino acids of KOR. Immunoblotting with the immuno-purified antiserum demonstrated the presence of a 70 kDa protein in wild-type leaf microsomes but not in soluble fractions (Figure 6A, lanes 1 and 2). No cross-reacting band was detected in the mutant, demonstrating the specificity of the antiserum (Figure 6A; lanes 3 and 4). After treatment of the microsomes with Na2CO3 (pH 11), a procedure known to strip peripheral proteins from membranes, the cross-reacting protein remained in the membrane fraction (Figure 6B). As a control, the soluble binding protein (BIP), present in the reticulum lumen (Denecke et al., 1993), was recovered in the soluble fraction (Figure 6C). These results show that KOR is an integral membrane protein. Figure 6.KOR is an integral membrane protein. (A) Immunoblot of wild-type (lanes 1 and 2) and kor (lanes 3 and 4) leaf microsomes (lanes 1 and 3) and supernatant (lanes 2 and 4). (B) Immunoblot of wild-type leaf microsomes before and after Na2CO3 (pH 11) extraction. A polyclonal rabbit antiserum raised against the N-terminus of KOR (anti-NKOR) was used in (A) and (B) as primary antibody. (C) Immunoblot of wild-type leaf microsomes before and after Na2CO3 (pH 11) extraction, with a polyclonal rabbit antiserum against the soluble ER-localized protein BIP as primary antiserum (Höfte et al., 1992). Download figure Download PowerPoint KOR is preferentially located in the plasma membrane Microsomal membrane vesicles prepared from an A.thaliana suspension culture were separated using two consecutive rounds of free-flow electrophoresis (Canut et al., 1988). A first separation yielded three peaks (Figure 7A), respectively enriched for (1) plasma membranes, (2) endoplasmic reticulum (ER) membranes and (3) tonoplast markers. Fractions 1 and 3 were pooled and subjected to a second separation, yielding fractions 4 and 6 (Figure 7B) that were highly enriched for plasma membrane and tonoplast, respectively (Table I). Fraction 2 was also subjected to a further round of purification giving fraction 5 (Figure 7C), which was further enriched for the ER marker. Essentially immunoblotting localized KOR to the plasma membrane-enriched fraction (Figure 7D), no cross-reacting protein was detected in the ER fraction. A faint 70 kDa band could be detected in the tonoplast fraction together with some smaller molecular weight bands. These bands were reproducibly present in different experiments and might be the result of the proteolytic cleavage of the protein in the vacuole. Figure 7.KOR is primarily located in the plasma membrane. Separation of microsomal membranes by free-flow electrophoresis. (A) Separation profiles of microsomal membranes. (B) Profile obtained after a second separation round of pooled fractions 1 and 3 and (C) after a second separation round of fraction 2. Profiles were monitored at 280 nm. Fractions 4 and 6 are respectively enriched for plasma membrane and tonoplast markers as summarized in Table I. (D) Immunoblot analysis of membrane fractions (50 μg protein/lane) with the same rabbit anti-NKOR primary antiserum as in Figure 8. Download figure Download PowerPoint Table 1. Enrichment of free-flow electrophoresis fractions for plasma membrane- and tonoplast-associated ATPase activities Marker enzymes (fractions) 4 6 Total ATPase activity (nmoles Pi/ min.mg protein) 1297 231 Inhibition (%) by KNO3 (tonoplast) −1 89 Inhibition (%) by NaN3 (mitochondria) 1 12 Inhibition (%) by V2O5 (plasma membrane) 86 15 Fractions are as in Figure 7. Cell wall alterations in kor Using microscopy, the walls of mutant and wild-type were investigated on transverse hypocotyl sections of 7-day-old dark-grown seedlings. Transmission electron microscopy (TEM) showed that fixed mutant cell walls were invariably thicker than wild-type walls. This was true both for the external epidermal wall (0.9 ± 0.38 μm [n = 156] in kor versus 0.56 ± 0.31 μm [n = 162] in the wild-type) and cortical walls (0.37 ± 0.19 μm [n = 10] in kor versus 0.14 ± 0.05 μm [n = 10] in the wild-type). The distribution of polysaccharides was visualized by periodic acid-thiocarbohydrazyde-silver proteinate (PATAg) staining (Thiéry, 1967; Roland and Vian, 1991). In epidermal walls three domains could be distinguished including an intensely stained cuticle, a poorly stained outer layer and a strongly stained inner layer. Wild-type walls had a regular surface and visible layers of material could be distinguished in the innermost layer (Figure 8A). In the external epidermal wall of kor, the staining was much more irregular, i.e. highly electron-dense deposits were visible at the cytoplasmic side of the wall (see white arrows on Figure 8B), whereas in other areas staining was practically absent. In mutant cortical cell walls irregular fibrillar material could be observed (Figure 8D) compared with the wild-type (Figure 8C). Figure 8.Cell wall defects in kor mutants. Transmission electron microscopy of transverse hypocotyl sections. (A, B) Outer epidermal and (C, D) cortical cell walls sections of 7-day-old dark-grown (A, C) wild-type and (B, D) korrigan hypocotyls. Sections were stained for polysaccharides with PATAg. Note the irregular surface and the absence of stratified microfibrils in the inner part of the mutant wall. White arrows indicate aggregates of highly electron-dense material frequently observed at the cytoplasmic side of mutant walls. (E, F, G, H) Outer epidermal sections of 7-day-old dark-grown (E, G) wild-type and (F, H) korrigan hypocotyls submitted (G, H) or not (E, F) to a CDTA extraction. Xyloglucans were visualized with polyclonal anti-XG-antibodies and polysaccharides stained by the PATAg method. C, cytoplasm; Cu, cuticle. Scale bar represents 0.6 μm. Download figure Download PowerPoint To visualize cellulosic material, sections were stained with calcofluor (Figure 1G and H). In the wild-type a regular distribution of fluorescent material could be observed in epidermal and cortical cell walls. Separation of cortical walls could be observed occasionally, but exclusively at cell corners. In the mutant, walls of adjacent cortical cells were separated at numerous places distributed over the entire cell surface (Figure 1H, arrows), and the fluorescent staining was more irregular. Xyloglucan (XG) epitopes were visualized in external epidermal walls with a polyclonal anti-XG antiserum. Gold particles could be observed throughout the wall section for both wild-type and mutant (Figure 8E and F). To assess the contribution of the cellulose–xyloglucan network to the integrity of the wall, an extraction was carried out with the Ca2+-chelator CDTA, a procedure known to selectively extract Ca2+-bridged pectates from the wall (Jauneau et al., 1992). In the wild-type, CDTA extraction caused the wall to swell, but with the preservation of the overall wall structure (Figure 8G). Fibrillar material decorated with antibodies could be seen in the innermost half of the wall. CDTA-treated kor mutant walls contained XG in amounts comparable with that observed in the wild-type as indicated by the gold-labeling; however, CDTA not only caused mutant walls to swell, but invariably resulted in the separation of the most recently deposited material from the rest of the wall. Disordered fibrillar material could be observed in the ruptured areas (Figure 8H). Regulation of KOR-mRNA levels Northern blot analysis using a probe specific for KOR detected a single transcript of 2 kb in wild-type plants. The mRNA was found in all orga