Abstract
Xyloglucan is widely believed to function as a tether between cellulose microfibrils in the primary cell wall, limiting cell enlargement by restricting the ability of microfibrils to separate laterally. To test the biomechanical predictions of this "tethered network" model, we assessed the ability of cucumber (Cucumis sativus) hypocotyl walls to undergo creep (long-term, irreversible extension) in response to three family-12 endo-β-1,4-glucanases that can specifically hydrolyze xyloglucan, cellulose, or both. Xyloglucan-specific endoglucanase (XEG from Aspergillus aculeatus) failed to induce cell wall creep, whereas an endoglucanase that hydrolyzes both xyloglucan and cellulose (Cel12A from Hypocrea jecorina) induced a high creep rate. A cellulose-specific endoglucanase (CEG from Aspergillus niger) did not cause cell wall creep, either by itself or in combination with XEG. Tests with additional enzymes, including a family-5 endoglucanase, confirmed the conclusion that to cause creep, endoglucanases must cut both xyloglucan and cellulose. Similar results were obtained with measurements of elastic and plastic compliance. Both XEG and Cel12A hydrolyzed xyloglucan in intact walls, but Cel12A could hydrolyze a minor xyloglucan compartment recalcitrant to XEG digestion. Xyloglucan involvement in these enzyme responses was confirmed by experiments with Arabidopsis (Arabidopsis thaliana) hypocotyls, where Cel12A induced creep in wild-type but not in xyloglucan-deficient (xxt1/xxt2) walls. Our results are incompatible with the common depiction of xyloglucan as a load-bearing tether spanning the 20- to 40-nm spacing between cellulose microfibrils, but they do implicate a minor xyloglucan component in wall mechanics. The structurally important xyloglucan may be located in limited regions of tight contact between microfibrils.
Highlights
Since the 1970s, xyloglucan (XyG) has been a central figure in theories relating primary cell wall architecture to plant cell enlargement (Keegstra et al, 1973; Labavitch and Ray, 1974; Hayashi, 1989)
Any molecular model of the primary cell wall leads to predictions and expectations about how it will respond to mechanical forces, what kinds of enzymes and biochemical processes might be involved in its expansive growth, and how new polymers could be integrated into its structure
The enzymes are small onedomain proteins (25 kD, approximately 4 nm in diameter), lack a separate carbohydrate-binding domain, and differ in substrate specificities. Their size is below the porosity limit of primary cell walls (Carpita et al, 1979; Baron-Epel et al, 1988) and less than the spacing between cellulose microfibrils, so they should have access to polysaccharides in the hydrated matrix between microfibrils
Summary
Since the 1970s, xyloglucan (XyG) has been a central figure in theories relating primary cell wall architecture to plant cell enlargement (Keegstra et al, 1973; Labavitch and Ray, 1974; Hayashi, 1989). Et al (1999) operationally identified three XyG domains: a xyloglucanase-accessible domain that includes the hypothetical tethers, loops, and free strands between microfibrils; a second domain tightly bound to the surface of cellulose microfibrils (defined as KOH soluble but inaccessible to xyloglucanase); and a third XyG domain only released upon complete wall digestion and thought to be trapped inside or between cellulose microfibrils This “tethered network model” is based on numerous, but indirect, results, including the extractability and binding behavior of XyG as well as microscopy indicating that XyG is present in the spaces between microfibrils (Hayashi and Maclachlan, 1984; Hayashi et al, 1987; McCann et al, 1990; Pauly et al, 1999). The tethered network model inevitably emphasizes the hypothetical XyG tethers between microfibrils as key determinants of cell wall mechanics and as points for control of cell wall expansion. There are good grounds for reexamining the role of XyG in cell wall mechanics
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