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- Materials and Methods
Primary plant cell walls are dynamic cellular compartments that provide the mechanical strength and the cell adhesion that underpins both the defined morphologies and growth capacities of cells, meristems and organs. Primary cell walls are largely comprised of diverse sets of complex polysaccharides. These polysaccharides are currently classed as microfibrillar cellulose and the structurally complex hemicellulose and pectic groups of polymers (O’Neill & York, 2003). The pectic group of polymers are envisaged to form a matrix surrounding and interacting with a cellulose–hemicellulose network and influencing a wide range of functions, including cell wall assembly, cell wall porosity, cell extension and mechanical properties in addition to cell adhesion. Pectins contain a number of structurally diverse polysaccharide domains that include homogalacturonan (HG), rhamnogalacturonan-I (RG-I), rhamnogalacturonan-II and xylogalacturonan (Ridley et al., 2001; Willats et al., 2001; Caffell & Mohnen, 2009). How these pectic domains are integrated in both structural and functional terms is not well understood.
Rhamnogalacturonan-I polymers, which include a rhamnogalacturonan backbone and 1,4-galactan and 1,5-arabinan as major structural motifs, are highly heterogeneous sets of polysaccharides displaying considerable structural variation in both cell and taxonomic contexts (Willats et al., 2001; Caffell & Mohnen, 2009; Verhertbruggen et al., 2009). Two monoclonal antibodies to 1,4-galactan and 1,5-arabinan motifs – LM5 and LM6, respectively – have been instrumental tools for exploring RG-I structure and heterogeneity in cell wall contexts and have been indicative of complex and perhaps separate functional roles for galactan- and arabinan-rich RG-I polymers in plant systems, although what these roles are in mechanistic terms is as yet uncertain (Willats et al., 1999; McCartney et al., 2000, 2003; Ulvskov et al., 2005). Recent work with a widened set of arabinan-directed monoclonal antibodies has emphasized that 1,5-arabinan and metabolically related structures are highly dynamic components of primary cell walls (Verhertbruggen et al., 2009).
At the carrot root meristem, the LM6 arabinan epitope is associated with walls of the central meristematic cells and there is a switch in relative abundance of the LM5 galactan and LM6 arabinan epitopes associated with a switch from cell proliferation to cell elongation and differentiation (Willats et al., 1999). At the surface of the Arabidopsis root apex, the LM5 galactan epitope has been shown to mark the transition zone, where the cells leave the meristem and begin to elongate (McCartney et al., 2003). In such studies the LM6 arabinan epitope is associated with meristematic regions of root apices (Willats et al., 2001; Verhertbruggen & Knox, 2007). Although in some studies reduced levels of pectic arabinan are not associated with growth phenotypes (Skjot et al., 2002; Harholt et al., 2006), a functional association of pectic arabinan with meristematic regions has been demonstrated in series of experiments involving overexpression of microbial arabinanases that resulted in serious defects to cell and meristem development (Oomen et al., 2002; Skjot et al., 2002; Borkhardt et al., 2005). It has also been suggested that pectic arabinan has a role in maintaining cell–cell linkages (Iwai et al., 2001; Orfila et al., 2001; Peňa & Carpita, 2004) and cell adhesion has long been established to be important in the organization, differentiation state and tissue patterning within meristems (van den Berg et al., 1997). In this context, cell wall structures and properties are clearly of high importance in meristems, where the specification and generation of cellular properties is crucial for successful organ initiation, growth and development. Although there is some knowledge of the hormonal regulation of factors that influence cell walls in A. thaliana (Sánchez-Rodríguez et al., 2010) there is little known about the pathways that can influence the composition of the hemicellulose and pectin classes of polysaccharides during meristem and cell development.
The root meristem is the source of all cells and patterning in the root, and the processes involved in its specification and function have been studied extensively. The phytohormones auxin, ethylene and cytokinin are known to play important roles in this. Auxin has a response maximum in the meristem, and is necessary for maintaining the potency of the stem cell niche and its fast dividing progenitor cells (Grieneisen et al., 2007). Cytokinin is involved in regulating the position of the transition zone, with exogenous application resulting in markedly less cell elongation (Ioio et al., 2007). Ethylene is known to be involved in the regulation of mitotic activity within the quiescent centre (Ortega-Martínez et al., 2007) and the spatial distribution of auxin within the root meristem (Růžička et al., 2007).
Detection of the LM5 galactan epitope at the Arabidopsis seedling root surface as cells begin to elongate has been shown to be responsive to phytohormone signals correlating with impacts upon cell elongation (McCartney et al., 2003). However, the specification of pectic arabinan, as imaged with LM6 arabinan monoclonal antibody, has not yet been placed in any physiological or signalling networks. Here we demonstrate that abscisic acid (ABA) signalling, which has recently been shown to have an impact on meristem functions (Zhang et al., 2010), leads to an increase in the detection of the LM6 arabinan epitope at the surface of Arabidopsis root apices and that this is dependent upon the ABI4 transcription factor.