Role of xyloglucan in primary cell walls.

The plant cell wall is a highly complex and dynamic structure made up of cellulose, hemicelluloses, pectins, lignin, and numerous proteins and inorganic molecules (Carpita and McCann, 2000). The hemicellulose xyloglucan (XyG) is found in the cell walls of all vascular plants and is the major hemicellulose in the primary cell wall of eudicots and nongraminaceous monocots, where it is believed to play an important role in cell wall structure and function. Models of the plant cell wall postulate that XyG functions to cross-link adjacent cellulose microfibrils to form a cellulose-XyG network that constitutes the major load-bearing structure of the primary cell wall (Keegstra et al., 1973; Obel et al., 2007; O’Neill and York, 2003). It is also thought to act as a spacer preventing cellulose microfibrils from aggregating (Thompson, 2005). In this issue of The Plant Cell, Cavalier et al. (pages 1519–1537) show that XyG provides some degree of mechanical strength to the primary cell wall, but the consequences of a lack of any detectable XyG in Arabidopsis seedlings is not catastrophic, as would be expected if it were a critical component of the load-bearing capacity of the expanding primary cell wall. The authors employ state-of-the art methodology to assess XyG content in XyG xylotransferase mutants, showing that the XyG xylotransferases XXT1 and XXT2 are required for XyG biosynthesis and opening new questions about the role of XyG in primary cell walls that challenge current thinking in this important area of fundamental plant biology. XyG consists of a b-(1/4)-glucan backbone substituted with a-(1/6)-xylosyl residues in a regular pattern, as well as occasional galactosyl or fucosyl residues. A number of enzymes participate in XyG biosynthesis, including b-(1/4)-glucan synthase, a-fucosyltransferases, b-galactosyltransferases, and a-xylosyltransferases. Arabidopsis contains seven genes that encode putative XyG xylosyltransferases (XXTs), at least two of which (XXT1 and XXT2) exhibit activities capable of forming XyG from appropriate substrates in vitro (Faik et al., 2002; Cavalier and Keegstra, 2006; Fauré et al., 2007). Recent work by Zabotina et al. (2008) showed that XXT5 in Arabidopsis is involved in XyG biosynthesis in vivo, since mutant plants have altered XyG composition. In their work, Cavalier et al. set out to examine the putative role of XXT1 and XXT2 in XyG biosynthesis in more detail through the isolation and characterization of single and double T-DNA insertional mutants of these two genes. First, the authors obtained the xxt1 and xxt2 single mutant T-DNA insertion lines, which were crossed to obtain the xxt1 xxt2 double mutant. RT-PCR analysis confirmed the absence of the corresponding transcripts from the single and double mutant plants. The gross phenotype of the single mutant lines was indistinguishable from that of wild-type plants, whereas the double mutant plants grew more slowly, were smaller, and showed severely shortened and abnormal root hairs (see figure), which could be complemented by expression of either 35Spro:XXT1 or 35Spro:XXT2. Biochemical analyses of the mutant plants suggested that XXT1 and XXT2 encode active XXTs that participate in XyG biosynthesis in Arabidopsis. Surprisingly, despite a moderate abnormal phenotype, the double mutant plants were found to contain no trace of detectable XyG. Since this result was unexpected, the authors went to great lengths to assess XyG content of the mutant lines and confirm its absence in the double mutant. Four different types of analysis were conducted, all of which suggested that the double mutant lacks any detectable XyG: (1) oligosaccharide mass profiling using MALDI-TOF mass spectrometry, (2) immunohistochemistry using XyG-directed antibodies, (3) glycosyl linkage analysis, and (4) analysis of cell wall polysaccharides digested with the enzyme preparation Driselase, which provides a diagnostic indicator of XyG. The lack of XyG in the double mutant, despite expression of XXT5 (previously shown to participate in