| Abstract: | Previously, we reported that although the Arabidopsis (Arabidopsis thaliana) Xyloglucan Endotransglucosylase-Hydrolase31 (XTH31) has predominately xyloglucan endohydrolase activity in vitro, loss of XTH31 results in remarkably reduced in vivo xyloglucan endotransglucosylase (XET) action and enhanced Al resistance. Here, we report that XTH17, predicted to have XET activity, binds XTH31 in yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitations assays and that this interaction may be required for XTH17 XET activity in planta. XTH17 and XTH31 may be colocalized in plant cells because tagged XTH17 fusion proteins, like XTH31 fusion proteins, appear to target to the plasma membrane. XTH17 expression, like that of XTH31, was substantially reduced in the presence of aluminum (Al), even at concentrations as low as 10 µm for 24 h or 25 µm for just 30 min. Agrobacterium tumefaciens-mediated transfer DNA insertion mutant of XTH17, xth17, showed low XET action and had moderately shorter roots than the wild type but was more Al resistant than the wild type. Similar to xth31, xth17 had low hemicellulose content and retained less Al in the cell wall. These data suggest a model whereby XTH17 and XTH31 may exist as a dimer at the plasma membrane to confer in vivo XET action, which modulates cell wall Al-binding capacity and thereby affects Al sensitivity in Arabidopsis.Soil acidity (pH < 5.5) affects about 40% of the world’s arable land (von Uexküll and Mutert, 1995) and more than 50% of land that is potentially arable, particularly in the tropics and subtropics (Kochian et al., 2004; Eticha et al., 2010). Al is the most growth-limiting factor for crop production on acid soils worldwide (Foy, 1988; Kochian, 1995), especially when the pH drops below 5 (Eswaran et al., 1997).To survive in an Al-toxic environment, Al-resistant plants have evolved two mechanisms to cope with Al toxicity. One is to restrict Al uptake from the root, while the other is to cope with internalized Al (Taylor, 1991; Kochian et al., 2004). The latter is usually employed by Al-accumulating species such as Hydrangea macrophylla (Ma et al., 1997a) and buckwheat (Fagopyrum
esculentum; Ma et al., 1997b). In most cases, secretion of the organic acid anions is the most important Al exclusion mechanism (Kochian, 1995; Ryan et al., 2001; Ma and Furukawa, 2003), although it does not explain all the Al resistance in some plants such as signalgrass (Brachiaria decumbens Stapf cv Basilisk; Wenzl et al., 2001), maize (Zea mays; Piñeros et al., 2005), buckwheat (Zheng et al., 2005), rice (Oryza sativa; Ma et al., 2005; Yang et al., 2008), or Fagopyrum tataricum (Yang et al., 2011a). Therefore, it is possible that for some plant species (such as buckwheat), Al tolerance is a combination of mechanisms including organic anion efflux.Recently, evidence has accumulated that the cell wall, especially the hemicellulose component, may impact Al resistance. For example, Al induces significant changes in the hemicellulose fraction of wheat (Triticum aestivum; Tabuchi and Matsumoto, 2001), triticale (× Triticosecale Wittmack; Liu et al., 2008), rice (Yang et al., 2008), and Arabidopsis (Arabidopsis thaliana; Zhu et al., 2012), especially the Al-sensitive cultivars. Moreover, we found that Arabidopsis hemicellulose is not only very sensitive to Al stress (the content of hemicellulose increased quickly under Al stress), but is also the principal binding site for Al (Yang et al., 2011b). Furthermore, loss of Xyloglucan Endotransglucosylase-Hydrolase31 (XTH31) function resulted in lower xyloglucan content and cell wall Al-binding capacity in Arabidopsis (Zhu et al., 2012). Thus, xyloglucan may be a major Al-binding site in Arabidopsis, and any effects leading to xyloglucan modifications may regulate Al sensitivity.XTHs are a family of enzymes that play principal roles in the construction and restructuring of the load-bearing cross links among cellulose microfibrils (Osato et al., 2006) through catalyzing the molecular grafting or hydrolyzing of the xyloglucans to form the framework (Fry et al., 1992; Nishitani and Tominaga, 1992; Okazawa et al., 1993; Rose et al., 2002). There are 33 identified XTH genes in the Arabidopsis genome, and one-third occur as clusters resulting from genome duplication (Blanc et al., 2000; Yokoyama and Nishitani, 2001); XTH1-11 are classified in subgroup 1, XTH12-26 are in subgroup 2, and XTH27-33 are in subgroup 3 (Rose et al., 2002). Each member of the XTH gene family is likely regulated by specific cues and committed to cell wall dynamics specific to certain tissues or cell types (Nishitani, 2002; Becnel et al., 2006; Osato et al., 2006). For example, XTH27 is involved in the cell wall modification of tracheary elements at a specific stage of rosette leaf development and is essential for tertiary vein development (Matsui et al., 2005), whereas XTH31 is involved in cell wall modification and cell elongation through modulating xyloglucan endotransglucosylase (XET) action under Al stress (Zhu et al., 2012). However, XTH31 is an XTH for which xyloglucan endohydrolase (XEH) activity has been predicted (Baumann et al., 2007), and in our previous report, we demonstrated that XTH31 produced heterologously in Pichia
pastoris has high XEH activity but low XET activity in vitro (Zhu et al., 2012), which is in accordance with Kaewthai et al. (2013), who reported that XTH31 is a predominant hydrolase using the in vitro activity assays and enzyme product analysis, as well as the use of a fluorogenic substrate in vivo. Unexpectedly, however, the xth31 mutant has very low XET action and activity (Zhu et al., 2012). One possible explanation for this result is that XTH31 may interact with and be required for activity of XET-active XTHs.In this study, we demonstrate that XTH17 can bind to XTH31 in vitro and in vivo and that a transfer DNA (T-DNA) insertional mutant of XTH17 has elevated Al resistance and exhibits a phenotype very similar to xth31. Together, these data are consistent with the interpretation that XTH17 and XTH31 may interact with each other and thereby contribute to Al-inhibited XET action in Arabidopsis. |
