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

After 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.

After enzymatic de-esterification without added calcium, the onion epidermal wall swells and 1 3 becomes softer, as assessed by nanoindentation and tensile plasticity tests, yet exhibits reduced 1 4 expansin-mediated creep. De-esterification of homogalacturonan (HG) is thought to stiffen pectin gels and primary cell 1 7 walls by increasing calcium crosslinking between HG chains. Contrary to this idea, recent 1 8 studies found that HG de-esterification correlated with reduced stiffness of living tissues, 1 9 measured by surface indentation. The physical basis of such apparent wall softening is unclear, 2 0 but possibly involves complex biological responses to HG modification. To assess the direct 2 1 physical consequences of HG de-esterification on wall mechanics without such complications, 2 2 we treated isolated onion (Allium cepa) epidermal walls with pectin methylesterase (PME) and 2 3 assessed wall biomechanics with indentation and tensile tests. In nanoindentation assays, PME 2 4 softened the wall (reduced the indentation modulus). In tensile force/extension assays, PME 2 5 increased plasticity (but not elasticity). These softening effects are attributed to increased 2 6 electrostatic repulsion and swelling of the wall after PME treatment. Despite softening and 2 7 swelling upon HG de-esterification, PME treatment alone failed to induce cell wall creep.

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Instead, acid-induced creep, mediated by endogenous α -expansin, was reduced. We conclude 2 9 that HG de-esterification physically softens the onion wall, but reduces expansin-mediated wall This study attempts to resolve some perplexing and apparently contradictory results concerning 4 2 the influence of pectin de-esterification on the mechanics and extensibility of growing cell walls.

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Pectins are acidic polysaccharides that constitute a large proportion of the cell wall in primary indentation assays measure out-of-plane wall stiffness while tensile assays measure in-plane 1 1 0 stiffness. As shown below, PME does soften the wall in some (but not all) respects, yet does not 1 1 1 display loosening activity and in fact diminishes the loosening action of endogenous expansins. White onion bulbs (Allium cepa), ~15 cm in diameter, were purchased from local grocery stores. The 5th scale, with the 1st being the outermost fleshy scale, was used to make epidermal peels. piperazineethanesulfonic acid), pH 7.5, with 0.01% (v/v) Tween 20 for 15 min to eliminate the Creep and stress relaxation assays were used to assess PME's ability to induce cell wall wall is extended and then held at constant length, whereas the creep assay measures the time-  For the creep experiments, wall strips were clamped at 0.1 N tension in neutral buffer and 3 5 3 after the length stabilized the buffer was swapped for one containing PME. Length remained 3 5 4 nearly constant for the duration of the experiment (90 min) and was not increased by PME 3 5 5 addition (Fig. 9A). Thus we did not find evidence of PME-mediated wall loosening in either 3 5 6 creep or stress relaxation assays.

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We also tested whether PME pretreatment affected acid-induced extension, mediated by the increase of wall hydration, tensile plasticity, and nanoindentation by PME, expansin-3 6 5 mediated creep was reduced. We conclude that PME may selectively soften the epidermal wall 3 6 6 under calcium-limited conditions, but we found no evidence for wall loosening by PME. As detailed in the introduction, this study was initiated to resolve some of the confusion and 3 6 9 speculation surrounding the action of PME on cell wall stiffness and extensibility (Levesque- Tremblay et al., 2015;Peaucelle et al., 2008). Our results show that -even in our simplified, 3 7 1 cell-free system (isolated outer epidermal walls from onion) -a more nuanced appreciation of the 3 7 2 complexity of wall biomechanics and the action of PME is needed to unpack this issue. Thus, 3 7 3 measured by nanoindentation ( Fig. 2) or by tensile plasticity (Fig. 3), PME softened the onion 3 7 4 cell wall, yet it did not change tensile elasticity nor did it loosen the wall, assayed as the ability 3 7 5 to induce cell wall creep (Fig. 9). Indeed, despite its selective softening and hydrating actions, The results of the current study are relevant to understanding: (1) PME effects on wall 3 8 8 mechanics, (2) the relationships of different biomechanical assays to each other and to growth, and (3) the relationships between wall structure and various biomechanical properties. These three points are discussed below.
3 9 1 PME effects on wall mechanics 3 9 2 After PME treatment, the electrostatic potential of the onion wall (measured as zeta potential) 3 9 3 became more negative, as expected for an enzyme that unmasks carboxylate groups of 3 9 4 methylesterified HG (Moustacas et al., 1986). It is likely that cell wall swelling, hence greater 3 9 5 wall hydration, after PME treatment resulted from increased electrostatic repulsion of negatively-3 9 6 charged HG chains (MacDougall et al., 2001;Ryden et al., 2000) and these effects in turn 3 9 7 contributed to increases in wall plasticity in the tensile test. This later point is supported by the 3 9 8 fact that MgCl 2 , which was used to reduce electrostatic fields within the wall, negated the PME  Hydration also influences wall extensibility in some conditions. For instance, wall mechanisms for such interference (Ricard, 1987). A more detailed look at the effects of The greater electrostatic charge after PME treatment amplified the sensitivity of the cell wall compliances of buffer-treated walls, whereas after PME-treatment calcium addition substantially 4 1 5 reduced wall plasticity (but not elasticity) (Fig. 4). These observations suggest the possibility that interactions between cell wall polymers is required to assess the relative contributions of these 4 2 7 different biophysical mechanisms to wall plasticity.

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Because the experiments in the current study imposed large changes in HG methyl stiffness relates to other wall properties is considered next.

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Relating different biomechanical assays to each other and to growth 4 3 6 One striking conclusion from this study is that different measures of wall biomechanics are not 4 3 7 closely coupled to one another. Thus, PME action softened the wall, as measured by indentation, results thus do not support the concept that PME has direct wall loosening activity. Various These results with PME are consistent with another recent study likewise showing that wall  For instance, treatment with pectate lyase reduced wall stiffness as measured by nanoindentation, Relating wall structure to various biomechanical properties 4 5 4 Many conceptual models of primary cell wall structure have been proposed since the 1970s, wall mechanics, yet rarely has biomechanics been used to test the validity of these models, which 4 5 7 can be viewed as graphical hypotheses in need of experimental testing. One such test by Park into a load-bearing network by long xyloglucan tethers. A recent study using onion epidermal The current study of PME action indicates that electrostatics and hydration affect selective 4 6 7 aspects of wall mechanics. Because PME does not cut the HG backbone, its biomechanical HG. This leads to charge repulsion of HG chains and swelling of the wall, which in turn affects 4 7 0 the indentation properties. The increase in tensile plasticity may result from increased hydration, 4 7 1 but higher electrostatic charge density within the wall may also contribute more directly. Despite 4 7 2 PME-induced changes in nano-indentation, tensile elasticity was insensitive to HG esterification 4 7 3 and to calcium cross linking. This is a remarkable result and suggests that static tensile forces are  Concluding remarks 4 7 8 By use of isolated epidermal wall strips to explore the physical consequences of PME action, we wall plasticity, however, did not translate into a more extensible cell wall, as measured by cell HG. The multidimensionality of wall biomechanics offers a rich path for gaining greater insights 4 9 0 into cell wall structure.    C  h  a  r  a  c  t  e  r  i  z  a  t  i  o  n  o  f  l  o  n  g  -t  e  r  m  e  x  t  e  n  s  i  o  n  o  f  i  s  o  l  a  t  e  d  c  e  l  l  w  a  l  l  s  f  r  o  m  g  r  o  w  i  n  g  5  1  3  c  u  c  u  m  b  e  r  h  y  p  o  c  o  t  y  l  s  .  P  l  a  n  t  a  1  7  7  ,  1  2  1  -1  3  0  .  5  1  4  C  o  s  g  r  o  v  e  D  J  .  2  0  1  6  .  C  a  t  a  l  y  s  t  s  o  f  p  l  a  n  t  c  e  l  l  w  a  l  l  l  o  o  s  e  n  i  n  g  .  F  1  0  0  0  R  e  s  5  ,  D  o  i  5  1  5  1  0  .  1  2  6  8  8  /  f  1  1  0  0  0  r  e  s  e  a  r  c  h  .  1  7  1  8  0  .  1  2  6  8  1 .  p  h  i  c  a  l  5  5  9  T  r  a  n  s  a  c  t  i  o  n  s  o  f  t  h  e  R  o  y  a  l  S  o  c  i  e  t  y  A  :  M  a  t  h  e  m  a  t  i  c  a  l  ,  P  h  y  s  i  c  a  l  a  n  d  E  n  g  i  n  e  e  r  i  n  g  S  c  i  e  n  c  e  s  2  4  9  ,  3  2  1  -3  8  7  .  5  6  0  L  o  p  e  z  -S  a  n  c  h  e  z  P  ,  M  a  r  t  i  n  e  z  -S  a  n  z  M  ,  B  o  n  i  l  l  a  M  R  ,  S  o  n  n  i  F  ,  G  i  l  b  e  r  t  E  P  ,  G  i  d  l  e  y  M  J  .  2  0  2  0  .  N  a  n  o  s  t  r  u  c  t  u  r  e  5  6  1  a  n  d  p  o  r  o  v  i  s  c  o  e  l  a  s  t  i  c  i  t  y  i  n  c  e  l  l  w  a  l  l  m  a  t  e  r  i  a  l  s  f  r  o  m  o  n  i  o  n  ,  c  a  r  r  o  t  a  n  d  a  p  p  l  e  :  R  o  l  e  s  o  f  p  e  c  t  i S  m  i  t  h  e  r  s  E  T  ,  L  u  o  J  ,  D  y  s  o  n  R  J  .  2  0  1  9  .  M  a  t  h  e  m  a  t  i  c  a  l  p  r  i  n  c  i  p  l  e  s  a  n  d  m  o  d  e  l  s  o  f  p  l  a  n  t  g  r  o  w  t  h  m  e  c  h  a  n  i  c  s  :  6  0  7  f  r  o  m  c  e  l  l  w  a  l  l  d  y  n  a  m  i  c  s  t  o  t  i  s  s  u  e  m  o  r  p  h  o  g  e  n  e  s  i  s  .  J  E  x  p  B  o  t  7  0  ,  3  5  8  7  -3  6  0  0  .  6  0  8  S  n  e  d  d  o  n  I  N  .  1  9  6  5  .  T  h  e  r  e  l  a  t  i  o  n  b  e  t  w  e  e  n  l  o  a  d  a  n  d  p  e  n  e  t  r  a  t  i  o  n  i  n  t  h  e  a  x  i  s  y  m  m  e  t  r  i  c  b  o  u  s  s  i  n  e  s  q  p  r  o  b  l  e  m  6  0  9  f  o  r  a  p  u  n  c  h  o  f  a  r  b  i  t  r  a  r  y  p  r  o  f  i  l  e  .  I  n  t  e  r  n  a  t  i  o  n  a  l  J  o  u  r  n  a  l  o  f  E  n  g  i  n  e  e  r  i  n  g  S  c  i  e  n  c  e  3  ,  4  7  -5  7 .  v  a  n  o  v  i  t  s  G  ,  M  a  c  D  o  u  g  a  l  l  A  J  ,  S  m  i  t  h  A  C  ,  R  i  n  g  S  G  .  2  0  0  4  .  M  a  t  e  r  i  a  l  p  r  o  p  e  r  t  i  e  s  o  f  c  o  n  c  e  n  t  r  a  t  e  d  p  e  c  t  i  n  6  5  5  n  e  t  w  o  r  k  s  .  C  a  r  b  o  h  y  d  r  R  e  s  3  3  9  ,  1  3  1  7  -1  3  2 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 ± SEM; n = 15). Student's t-test (paired, twotail) was used to assess statistical significance (* p < 0.05). Experiment was repeated four times with similar results.  Fig. 4. Effect of CaCl 2 and MgCl 2 on elastic and plastic compliances of walls pretreated 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 Mg and twice for Ca. Values are means ± SEM (n = 20). Student's ttest (paired, two-tail) was used to assess statistical significance (**-p < 0.01).  Fig. 8. Effect of acid pH and PME pretreatment 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 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 pretreated for 16 h with buffer (solid) is substantially greater than creep of walls pretreated for 16 h with PME (dash). Curves are averages of 6 (PME) and 9 (buffer-and PME-pretreated) replicates.