Pectin methylesterase selectively softens the onion epidermal wall yet reduces acid-induced creep

Abstract

De-esterification of homogalacturonan (HG) is thought to stiffen pectin gels and primary cell walls by increasing calcium cross-linking between HG chains. Contrary to this idea, recent studies found that HG de-esterification correlated with reduced stiffness of living tissues, measured by surface indentation. The physical basis of such apparent wall softening is unclear, but possibly involves complex biological responses to HG modification. To assess the direct physical consequences of HG de-esterification on wall mechanics without such complications, we treated isolated onion (Allium cepa) epidermal walls with pectin methylesterase (PME) and assessed wall biomechanics with indentation and tensile tests. In nanoindentation assays, PME action softened the wall (reduced the indentation modulus). In tensile force/extension assays, PME increased plasticity, but not elasticity. These softening effects are attributed, at least in part, to increased electrostatic repulsion and swelling of the wall after PME treatment. Despite softening and swelling upon HG de-esterification, PME treatment alone failed to induce cell wall creep. Instead, acid-induced creep, mediated by endogenous α-expansin, was reduced. We conclude that HG de-esterification physically softens the onion wall, yet reduces expansin-mediated wall extensibility.

Keywords: Atomic force microscopy; biomechanics; expansin; homogalacturonan; nanoindentation; onion (Allium cepa) epidermis; pectin methylesterase; plant cell wall mechanics; tensile testing; wall hydration.

Fig. 1.

De-esterification of HG in onion epidermal walls by PME. (A) Immunolabeling of the inner surface of onion epidermal cell walls after 2 h in HEPES buffer ±PME. JIM7 and LM19 antibodies detect HG of high and low esterifcation, respectively. Paired images ±PME were captured with identical light intensities and camera exposure settings. Scale bar=100 µm. Representative results from two independent replicates. (B) Methanol release from onion epidermal wall as a function of PME treatment time. Mean ±SE of two independent replicates.

Fig. 2.

PME effect on AFM nanoindentation. Force–indentation measurements performed on onion epidermal walls submerged in 20 mM pH 7.5 HEPES buffer alone (A) and in buffer containing 50 µg ml^–1 PME (B) at time 0 h and 3 h. The representative extend and retract curves have modulus values close to the average value of each treatment. (C) Modulus calculated by the Sneddon model using retract curves. Values are mean ±SE (23≤n≤24). Letters indicate statistical significance based on one-way ANOVA with post-hoc Tukey test (P<0.01).

Fig. 3.

PME effect on tensile mechanics of onion epidermal walls. (A) Force–extension curves of control (solid) and PME-treated (dashed) walls. For each data set, the first stretch is the upper curve which contains both plastic and elastic components, while the second stretch is the bottom curve containing only the elastic component. These are representative curves with compliance values close to the average. (B) Statistical summary of elastic and plastic compliances (mean ±SE; n=15). Student’s t-test (paired, two-tail) was used to assess statistical significance (*P<0.05). The experiment was repeated four times with similar results.

Fig. 4.

Effect of 100 mM CaCl₂ and MgCl₂ on elastic and plastic compliances of walls pre-treated with buffer ±PME. Values are means ±SEM (9≤n≤17). Letters indicate statistical difference in one-way ANOVA with post-hoc Tukey test (P<0.05). Replicated three times for MgCl₂ and twice for CaCl₂.

Fig. 5.

Zeta potential of onion wall fragments treated with PME or BSA (box and whiskers plot). Wall fragments were suspended in 20 mM HEPES pH 7.0. Three biological samples with statistical analysis by Student’s t-test.

Fig. 6.

Cell wall swelling caused by PME treatment. (A) Onion scale cross-sections stained with toluidine blue; the wall thickness is marked by black arrows (top). Scale bar=20 μm. The binary image was generated using ImageJ; the red line shows the selection for wall thickness measurement (bottom). (B) Wall thickness comparisons at 0 h and 3 h, ±PME treatment. Values are means ±SE (n=20). Student’s t-test (paired, two-tail) was used to assess statistical significance (**P<0.01).

Fig. 7.

PME treatment reduces cellulose microfibril resolution by AFM imaging. (A) Peak force error image of an onion epidermal wall surface in HEPES buffer. Scale bar=20 nm. Distinct cellulose microfibrils and bundles are well resolved by AFM. (B) The same area scanned after PME treatment. The resolution of cellulose microfibrils is reduced while fibrils in the underlying lamella were obscured. Similar results were observed with six biological replicates.

Fig. 8.

Effect of acid pH and PME pre-treatment on stress relaxation spectra of onion epidermal walls. Each curve is the average of 7–8 data sets. Boxed regions show statistically significant difference in relaxation rate. Student’s t-test was used to evaluate statistical significance (*boxed region different at P<0.05). (A) Walls in acidic (pH 4.5) buffer have greater stress relaxation than walls in neutral (pH 6.8) buffer. (B) Stress relaxation spectra of walls after 3 h incubation in 20 mM HEPES pH 7.5 ±PME.

Fig. 9.

Influence of PME treatment on cell wall creep. (A) Addition of 100 μg ml^–1 PME in pH 7.5 HEPES buffer does not induce creep. Walls were incubated in pH 7.5 buffer while clamped on the constant force extensometer. At the time indicated by the arrow, the buffer was exchanged with fresh buffer containing PME. (B) Acid-induced creep of walls pre-treated for 16 h with buffer (solid line) is substantially greater than creep of walls pre-treated for 16 h with PME (dashed line). Curves are averages of six (PME) and nine (buffer- and PME-pre-treated) replicates.