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 crosslinking 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.HighlightAfter enzymatic de-esterification without added calcium, the onion epidermal wall swells and becomes softer, as assessed by nanoindentation and tensile plasticity tests, yet exhibits reduced expansin-mediated creep.

Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero‐transglucosylation

“…However, the addition of heat‐denatured Equisetum extracts strongly increased the CXE activity of Pichia ‐produced Ef HTG (Figure 4a; graphs iii and iv, pale grey bars), especially when natural cellulose I was the donor substrate (Figure 4a; graph iv): for example, adding denatured root extracts approximately trebled the CXE activity (Figure 4a; graph iv). The short treatment in boiling water (approximately 5 sec) was clearly sufficient to inactivate enzymes such as HTG and XTHs (Figure 4a; graphs i and ii), but may not have inactivated co‐extracted expansins, whose activity has been reported to withstand boiling in methanol (approximately 65°C; McQueen‐Mason et al ., 1992; Fry, 1994; Wang et al ., 2020). A bacterial and a fungal expansin even kept more than two‐thirds of their activities after water‐boiling for 5 min (Wang et al ., 2014).…”

Saccharide Analysis of Onion Outer Epidermal Walls

“…This study concerns monosaccharide analysis of the outer periclinal wall of onion scale epidermis. This wall is ~7 μm thick (9) and comprised of many lamellae in which cellulose is organized in highly anisotropic 2D networks (10) that when averaged across the total thickness of the wall can appear to be isotropic in some cases (11) and slightly anisotropic in other cases (12,13), depending on developmental state. Because of the simplicity of preparing epidermal strips, this material has frequently been studied for optical and mechanical analyses (14)(15)(16)(17).…”

“…Although the monosaccharide composition of onion parenchyma walls has been well studied (21)(22)(23)(24)(25)(26), there is less information about onion epidermal walls and none as far as we know specifically focused on the outer periclinal wall, which has been the subject of detailed mechanics, microscopy and spectroscopy studies recently (9,11,20,(27)(28)(29)(30)(31)(32)(33). In preliminary attempts to measure total sugars in this material by the phenol/sulfuric acid method (34), we discovered that a colored reaction product persistently interfered with this colorimetric method.…”

“…This relatively homogeneous wall preparation has been used to assess the role of different wall components in wall mechanics by a combination of selective enzymatic digestions and imaging by atomic force microscopy (20). Although the monosaccharide composition of onion parenchyma walls has been well studied (21–26), there is less information about onion epidermal walls and none as far as we know specifically focused on the outer periclinal wall, which has been the subject of detailed mechanics, microscopy and spectroscopy studies recently (9, 11, 20, 27–33). In preliminary attempts to measure total sugars in this material by the phenol/sulfuric acid method (34), we discovered that a colored reaction product persistently interfered with this colorimetric method.…”

Protocols for Isolating and Characterizing Polysaccharides From 1 Plant Cell Walls: A Case Study Using Rhamnogalacturonan-II

“…Early models of the cell wall predicted that the cellulosehemicellulose scaffold was embedded in a pectin matrix that itself had only a limited effect on wall properties. However, there is now an increasing awareness that pectins are a dynamic type of polysaccharide that associate with cellulose and hemicelluloses to determine wall charge, porosity, rigidity and hydration [5][6][7][8].…”