The Organization Pattern of Root Border-Like Cells of Arabidopsis Is Dependent on Cell Wall Homogalacturonan

Border-like cells are released by Arabidopsis (Arabidopsis thaliana) root tips as organized layers of several cells that remain attached to each other rather than completely detached from each other, as is usually observed in border cells of many species. Unlike border cells, cell attachment between border-like cells is maintained after their release into the external environment. To investigate the role of cell wall polysaccharides in the attachment and organization of border-like cells, we have examined their release in several well-characterized mutants defective in the biosynthesis of xyloglucan, cellulose, or pectin. Our data show that among all mutants examined, only quasimodo mutants (qua1-1 and qua2-1), which have been characterized as producing less homogalacturonan, had an altered border-like cell phenotype as compared with the wild type. Border-like cells in both lines were released as isolated cells separated from each other, with the phenotype being much more pronounced in qua1-1 than in qua2-1. Further analysis of border-like cells in the qua1-1 mutant using immunocytochemistry and a set of anti-cell wall polysaccharide antibodies showed that the loss of the wild-type phenotype was accompanied by (1) a reduction in homogalacturonan-JIM5 epitope in the cell wall of border-like cells, confirmed by Fourier transform infrared microspectrometry, and (2) the secretion of an abundant mucilage that is enriched in xylogalacturonan and arabinogalactan-protein epitopes, in which the cells are trapped in the vicinity of the root tip.Higher plants rely on their roots to acquire water and other nutrients in the soil to grow and develop (Esau, 1977). At the tip of every growing root is a conical covering consisting of several layers of cells called the root cap that plays a major role in root protection and its interaction with the rhizosphere (Rougier, 1981; Baluška et al., 1996; Barlow, 2003).Root tips of most plant species produce a large number of cells programmed to separate from the root cap and to be released into the external environment (Hawes et al., 2003). This process occurs through the action of cell wall-degrading enzymes that solubilize the interconnections between root cap peripheral cells, causing the cells to separate from each other and from the root as populations of single cells (Hawes et al., 2003). Because of their specific position at the interface between root and soil, these living cells are defined as root border cells. It has been shown that the number of these cells per root varies between plant families: from about 100 (e.g. the Solanaceae family) to several thousands (e.g. 10,000 or more for the Pinaceae; Hawes et al., 2003). It has also been suggested that species of the Brassicaceae family including Arabidopsis (Arabidopsis thaliana) do not produce border cells (Hawes et al., 2003). Indeed, the Arabidopsis root tip does not produce isolated border cells per se, but it does produce and release cells that remain attached to each other, forming a block of several cell layers called border-like cells (Vicré et al., 2005; Fig. 1). This also occurs in other Brassicaceae species, including rapeseed (Brassica napus), mustard (Brassica juncea), and Brussels sprout (Brassica oleracea gemmifera), indicating that such an organization might be specific to this family (Driouich et al., 2007).Open in a separate windowFigure 1.Morphological phenotypes of root tips showing border-like cells (BLC) of the wild type and cell wall mutants of Arabidopsis. Wild-type Columbia (Col O; A), wild-type Wassilewskija (Ws; B), mur3 (C), mur2-1 (D), kor1 (E), rsw1 (F), epc1-1 (G), arad1-1 (H), qua1-1 (I), and qua2-1 (J) are shown. Border-like cells are released from the root tip in organized cell layers (arrows) in the wild type and in all mutants examined with the exception of qua1-1 and qua2-1. Note also that border-like cell organization is similar between Columbia and Wassilewskija. M, Mucilage. Bars = 20 μm (A, B, D–H, and J) or 50 μm (C and I).The unique organization pattern of Arabidopsis border-like cells (e.g. they do not disperse individually) suggests that they might have a specific cell wall composition and/or structure that makes them resistant to cell wall-hydrolyzing enzymes or that the enzymes are not present or not functional (Driouich et al., 2007). The only information on cell wall composition of Arabidopsis border-like cells was obtained from immunocytochemical studies, in which it has been shown that the cell wall of border-like cells is rich in pectic homogalacturonan and arabinogalactan-proteins, two wall polymers believed to be involved in cell adhesion in plants (Vicré et al., 2005). Based on this observation, we postulated that pectic polysaccharides of the cell wall may serve as a glue to cement border-like cells together, leading to that particular organization (Vicré et al., 2005).The cell wall of higher plants comprises mainly polysaccharides and proteoglycans. Cell wall polysaccharides are assembled into complex macromolecules, including cellulose, hemicellulose, and pectin. Cellulose forms microfibrils, which constitute an ordered, fibrous phase, whereas pectin and hemicellulose form an amorphous matrix phase surrounding the microfibrils (Cosgrove, 1997). Pectins constitute a highly complex family of cell wall polysaccharides, including homogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II. Homogalacturonan domains consist of α-d-(1→4)-GalUA residues, which can be methyl esterified, acetylated, and/or substituted with β-(1→3)-Xyl residues to form xylogalacturonan (Schols et al., 1995; Willats et al., 2001; Vincken et al., 2003). Deesterified blocks of homogalacturonan can be cross-linked by calcium, leading to the formation of a gel that is believed to be involved in cell adhesion (Jarvis et al., 2003). Rhamnogalacturonan I consists of a backbone of up to 100 repeats of the disaccharide α-(1→4)-GalUA-(1→2)-rhamnose, which carries complex and variable side chains. The rhamnose residues are commonly substituted with polymeric β-(1→4)-linked d-galactosyl residues and/or α-(1→5)-linked l-arabinosyl residues (Ridley et al., 2001). Rhamnogalacturonan II is a highly complex but conserved molecule consisting of a homogalacturonan-like backbone substituted with four different side chains containing specific sugars (O''Neill et al., 2004).Xyloglucan is the major hemicellulosic polysaccharide of the primary wall of dicotyledonous plants, and it consists of a β-d-(1→4)-glucan backbone to which are attached side chains containing xylosyl, galactosyl-xylosyl, or fucosyl-galactosyl-xylosyl residues. Xyloglucan is the principal polysaccharide that cross-links the cellulose microfibrils. The xyloglucan-cellulose network forms a major load-bearing structure that contributes to the control of cell expansion (Hayashi, 1989; Cosgrove, 1999).Glycoproteins, such as arabinogalactan-proteins, are also present in the cell wall matrix (Showalter, 1993; Seifert and Roberts, 2007). Arabinogalactan-proteins are highly glycosylated members of the Hyp-rich glycoprotein superfamily. Many of these glycoproteins, the so-called classical arabinogalactan-proteins, are anchored to the plasma membrane by a glycosylphosphatidylinositol anchor and have the potential to bind both cell wall components (Immerzeel et al., 2006) and cytosolic cortical microtubules (Schultz et al., 2002; Sardar et al., 2006; Nguema-Ona et al., 2007). These proteoglycans have been implicated in many aspects of plant life, including cell expansion, cell signaling and communication, embryogenesis, and wound response (Johnson et al., 2003; Seifert and Roberts, 2007; Driouich and Baskin, 2008).Although cell-to-cell interaction is a fundamental feature of plant growth and development, the molecular bases of intercellular adhesion and its loss are not fully understood (Roberts et al., 2002; Jarvis et al., 2003; Willats et al., 2004). This study aims at investigating the role of cell wall polysaccharides in cell attachment and the organization of border-like cells in Arabidopsis. To this end, we took advantage of the recent characterization of several Arabidopsis mutants affected in the biosynthesis of different classes of cell wall polysaccharides, including pectin, xyloglucan, and cellulose. We thus examined the pattern of border-like cells released by the root tip of selected Arabidopsis mutants using microscopy and immunocytochemistry. These mutants are (1) quasimodo1-1 (qua1-1) and qua2-1 (Bouton et al., 2002; Mouille et al., 2007), ectopically parting cells1-1 (epc1-1; Singh et al., 2005), and arabinan deficient1-1 (arad 1-1; Harholt et al., 2006), which all have been reported to be possibly affected in pectin biosynthesis; (2) murus2-1 (mur2-1) and mur3, which make altered xyloglucan (Vanzin et al., 2002; Madson et al., 2003); and (3) radially swollen1 (rsw1) and korrigan1 (kor1), which are affected in cellulose biosynthesis (Arioli et al., 1998; Nicol et al., 1998).Our data show that the organization of border-like cells had a wild-type phenotype in all of the mutants examined except in qua1-1 and qua2-1. In both of these mutants, border-like cells had lost the wild-type phenotype, as they were released as single cells separated from each other. This phenotype was far more pronounced in qua1-1 than in qua2-1. Further analysis of qua1-1 using immunocytochemistry and Fourier transform infrared microspectrometry showed a substantial loss of homogalacturonan content in border-like cells. In addition, border-like cells in the qua1-1 mutant secreted an abundant mucilage enriched in xylogalacturonan and arabinogalactan-protein epitopes.