Abstract
Xyloglucan endotransglucosylase (XET) activity, which cuts and re-joins hemicellulose chains in the plant cell wall, contributing to wall assembly and growth regulation, is the major activity of XTH proteins. During purification, XTHs often lose XET activity which, however, is restored by treatment with certain cold-water-extractable, heat-stable polymers (CHPs), e.g. from cauliflower florets. It was not known whether the XTH-activating factor (XAF) present in CHPs works by promoting (e.g. allosterically) XET activity or by re-solubilising sequestered XTH proteins. We now show that XTHs in dilute solution bind to diverse surfaces (e.g. glass and cellulose), and that CHPs can re-solubilise the bound enzyme, re-activating it. Cell walls prepared from cauliflower florets, mung bean shoots and Arabidopsis cell-suspension cultures each contained endogenous, tightly bound, inactive XTHs, which were likewise rapidly solubilised (within 0.5h) and thus activated by cauliflower XAF. We present a convenient quantitative assay for XAF acting on the native sequestered XTHs of Arabidopsis cell walls; using this assay, we show that CHPs from all plants tested possess XAF activity. The XAF activity of diverse CHPs does not correlate with their conductivity, showing that this activity is not a simple ionic effect. The XAF action of cauliflower CHPs was augmented by NaCl, although NaCl alone was much less effective than a CHP solution of similar conductivity, confirming that the cauliflower polymers did not simply exert a salt effect. We suggest that XAF is an endogenous regulator of XET action, modulating cell-wall loosening and/or assembly in vivo.
Highlights
In dicot primary cell walls, the major tension-bearing structure, limiting cell expansion, is widely proposed to include a xyloglucan–cellulose complex (Fry, 1989; Hayashi, 1989; McCann et al, 1990; Passioura and Fry, 1992; Fenwick et al, 1999; Park and Cosgrove, 2015)
XTH24 is inactivated by binding to surfaces; cold water-extractable heat-stable polymer (CHP) reactivates it by solubilisation
XTH24 is not expected to be unusual among the 33 xyloglucan endotransglucosylase/hydrolase (XTH) of Arabidopsis thaliana; it is highly expressed in young, growing tissues, e.g. hypocotyls (Campbell and Braam, 1999; Jamet et al, 2009) and floral parts especially the stamens
Summary
In dicot primary cell walls, the major tension-bearing structure, limiting cell expansion, is widely proposed to include a xyloglucan–cellulose complex (Fry, 1989; Hayashi, 1989; McCann et al, 1990; Passioura and Fry, 1992; Fenwick et al, 1999; Park and Cosgrove, 2015). Enzymes of xyloglucan metabolism are of interest to our understanding of the control of wall strength and extensibility. The transient cleavage and rearrangement of xyloglucan chains is achieved by GH16-family enzymes named xyloglucan endotransglucosylase/hydrolases (XTHs), which are ubiquitous in land plants. The major activity of most XTHs is xyloglucan endotransglucosylase (XET; EC 2.4.1.207; Baydoun and Fry, 1989; Smith and Fry, 1991; Farkaš et al, 1992; Fry et al, 1992; Nishitani and Tominaga, 1992), though a few exhibit predominantly xyloglucan endohydrolase activity (XEH; Shi et al, 2015). The XET reaction involves the covalent ‘cutting and pasting’ of xyloglucan chains, which has been shown by in-vivo density labelling to occur during — and probably contribute to the mechanism of — cell-wall assembly (Thompson et al, 1997) and loosening (Thompson and Fry, 2001). Roles for XET activity in growth control, both stimulatory and inhibitory, have been demonstrated multiple times (Osato et al, 2006; Miedes et al, 2013; Maris et al, 2009; Liu et al, 2007; Van Sandt et al, 2007)
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