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The stiffening of the cell walls observed during physiological softening of pears.

ABSTRACT: The Young's modulus of the primary cell walls of pears decreases linearly during the pre-harvest on-tree maturation and increases during postharvest storage, and does not correlate with firmness of fruit. The determination of mechanical properties of cell walls is indispensable for understanding the mechanism of physiological softening and deterioration of quality of fruits during postharvest storage. The Young's modulus of the primary cell walls from pear fruit (Pyrus communis L., cultivars 'Conference' and 'Xenia') during pre-harvest maturation and postharvest storage in an ambient atmosphere at 2 °C followed by shelf life was studied using atomic force microscopy (AFM). The results were related to the firmness of fruits, galacturonic acid content in water, chelator, sodium carbonate and insoluble pectin fractions, polygalacturonase and pectin methylesterase activities. The Young's modulus of the primary cell walls decreased linearly during the last month of pre-harvest maturation from 3.2 ± 1.8 to 1.1 ± 0.7 MPa for 'Conference' and from 1.9 ± 1.2 to 0.2 ± 0.1 MPa for 'Xenia' which correlated with linear firmness decrease. During postharvest storage the cell wall Young's modulus increased while firmness continued to decrease. Correlation analysis for the entire period of the experiment showed a lack of straightforward relation between the Young's modulus of primary cell walls and fruit firmness. The Young's modulus of cell walls correlated negatively either with galacturonic acid content in sodium carbonate soluble pectin ('Conference') or with insoluble pectin fractions ('Xenia') and positively with polygalacturonase activity. It was therefore evidenced that covalently linked pectins play the key role for the stiffness of fruit cell walls. Based on the obtained results, the model explaining the fruit transition from firm and crispy to soft and mealy was proposed.

SUBMITTER: Zdunek A

PROVIDER: S-EPMC4722064 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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The stiffening of the cell walls observed during physiological softening of pears.

Zdunek Artur A Kozioł Arkadiusz A Cybulska Justyna J Lekka Małgorzata M Pieczywek Piotr M PM

Planta 20151026 2

<h4>Main conclusion</h4>The Young's modulus of the primary cell walls of pears decreases linearly during the pre-harvest on-tree maturation and increases during postharvest storage, and does not correlate with firmness of fruit. The determination of mechanical properties of cell walls is indispensable for understanding the mechanism of physiological softening and deterioration of quality of fruits during postharvest storage. The Young's modulus of the primary cell walls from pear fruit (Pyrus co ...[more]

PMID: 26498014

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Project description:Background and aimsThe programmed softening occurring during fruit development requires scission of cell wall polysaccharides, especially pectin. Proposed mechanisms include the action of wall enzymes or hydroxyl radicals. Enzyme activities found in fruit extracts include pectate lyase (PL) and endo-polygalacturonase (EPG), which, in vitro, cleave de-esterified homogalacturonan in mid-chain by β-elimination and hydrolysis, respectively. However, the important biological question of whether PL exhibits action in vivo had not been tested.MethodsWe developed a method for specifically and sensitively detecting in-vivo PL products, based on Driselase digestion of cell wall polysaccharides and detection of the characteristic unsaturated product of PL action.Key resultsIn model in-vitro experiments, pectic homogalacturonan that had been partially cleaved by commercial PL was digested to completion with Driselase, releasing an unsaturated disaccharide ('ΔUA-GalA'), taken as diagnostic of PL action. ΔUA-GalA was separated from saturated oligogalacturonides (EPG products) by electrophoresis, then subjected to thin-layer chromatography (TLC), resolving ΔUA-GalA from higher homologues. The ΔUA-GalA was confirmed as 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→4)-d-galacturonic acid by NMR spectroscopy. Driselase digestion of cell walls from ripe fruits of date (Phoenix dactylifera), pear (Pyrus communis), rowan (Sorbus aucuparia) and apple (Malus pumila) yielded ΔUA-GalA, demonstrating that PL had been acting in vivo in these fruits prior to harvest. Date-derived ΔUA-GalA was verified by negative-mode mass spectrometry, including collision-induced dissociation (CID) fragmentation. The ΔUA-GalA:GalA ratio from ripe dates was roughly 1:20 (mol mol-1), indicating that approx. 5 % of the bonds in endogenous homogalacturonan had been cleaved by in-vivo PL action.ConclusionsThe results provide the first demonstration that PL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.

Project description:Changes in extracellular matrix stiffness impact a variety of biological processes including cancer progression. However, cells also actively remodel the matrices they interact with, dynamically altering the matrix mechanics they respond to. Further, cells not only react to matrix stiffness, but also have a distinct reaction to matrix viscoelasticity. The impact of cell-driven matrix remodeling on matrix stiffness and viscoelasticity at the microscale remains unclear, as existing methods to measure mechanics are largely at the bulk scale or probe only the surface of matrices, and focus on stiffness. Yet, establishing the impact of the matrix remodeling at the microscale is crucial to obtaining an understanding of mechanotransduction in biological matrices, and biological matrices are not just elastic, but are viscoelastic. Here, we advanced magnetic probe-based microrheology to overcome its previous limitations in measuring viscoelasticity at the cell-size-scale spatial resolution within 3D cell cultures that have tissue-relevant stiffness levels up to a Young's modulus of 0.5 kPa. Our magnetic microrheometers exert controlled magnetic forces on magnetic microprobes within reconstituted extracellular matrices and detect microprobe displacement responses to measure matrix viscoelasticity and determine the frequency-dependent shear modulus (stiffness), the loss tangent, and spatial heterogeneity. We applied these tools to investigate how microscale viscoelasticity of collagen matrices is altered by fibroblast cells as they contract collagen gels, a process studied extensively at the macroscale. Interestingly, we found that fibroblasts first soften the matrix locally over the first 32 hours of culture, and then progressively stiffen the matrix thereafter. Fibroblast activity also progressively increased the matrix loss tangent. We confirmed that the softening is caused by matrix-metalloproteinase-mediated collagen degradation, whereas stiffening is associated with local alignment and densification of collagen fibers around the fibroblasts. This work paves the way for the use of measurement systems that quantify microscale viscoelasticity within 3D cell cultures for studies of cell-matrix interactions in cancer progression and other areas.

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The stiffening of the cell walls observed during physiological softening of pears.

Publications

The stiffening of the cell walls observed during physiological softening of pears.

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