Enhanced electrostatic interactions in tomato cell suspensions.
Sankaran, A. K., Nijsse, J., Bialek, L., Shpigelman, A., Hendrickx, M. E. & Van Loey, A. M. (2015). Food Hydrocolloids, 43, 442-450.
The natural consistency of processed tomato products arises from cell wall particles and the interactions between them. In this study, ion exchange resins were used to investigate these interactions. Two types of resins were used, a hydrogen form cation exchange resin and an anion resin in the hydroxide form. Serum phase of tomato suspensions were treated with either a cationic or an anionic resin to exchange various ionic compounds with hydrogen and hydroxide ion respectively. The treated serum was then reconstituted to the tomato pulp and the suspension was re-suspended with shear. The linear storage modulus varied with the different types of resin treatment. Samples treated with the anion exchange resin resulted in a higher modulus than the untreated tomato puree and the puree treated with the cation resin. Effect of the resins was dependent on the concentration of resin used. The anion treated sample resulted in a network formation which was quite sensitive to pH and was attributed to long range electrostatic interactions caused by protein–pectin interactions. Using Infrared spectroscopy the conformational changes in the protein structure as a result of resin treatment was detected by analyzing the amide-I and amide-II regions
Cell separation in kiwifruit without development of a specialised detachment zone.
Prakash, R., Hallett, I. C., Wong, S. F., Johnston, S. L., O’Donoghue, E. M., McAtee, P. A., Seal, A. G., Atkinson, R. G. & Schröder, R. (2017). BMC Plant Biology, 17(1), 86.
Background: Unlike in abscission or dehiscence, fruit of kiwifruit Actinidia eriantha develop the ability for peel detachment when they are ripe and soft in the absence of a morphologically identifiable abscission zone. Two closely-related genotypes with contrasting detachment behaviour have been identified. The ‘good-peeling’ genotype has detachment with clean debonding of cells, and a peel tissue that does not tear. The ‘poor-peeling’ genotype has poor detachability, with cells that rupture upon debonding, and peel tissue that fragments easily. Results: Structural studies indicated that peel detachability in both genotypes occurred in the outer pericarp beneath the hypodermis. Immunolabelling showed differences in methylesterification of pectin, where the interface of labelling coincided with the location of detachment in the good-peeling genotype, whereas in the poor-peeling genotype, no such interface existed. This zone of difference in methylesterification was enhanced by differential cell wall changes between the peel and outer pericarp tissue. Although both genotypes expressed two polygalacturonase genes, no enzyme activity was detected in the good-peeling genotype, suggesting limited pectin breakdown, keeping cell walls strong without tearing or fragmentation of the peel and flesh upon detachment. Differences in location and amounts of wall-stiffening galactan in the peel of the good-peeling genotype possibly contributed to this phenotype. Hemicellulose-acting transglycosylases were more active in the good-peeling genotype, suggesting an influence on peel flexibility by remodelling their substrates during development of detachability. High xyloglucanase activity in the peel of the good-peeling genotype may contribute by having a strengthening effect on the cellulose-xyloglucan network. Conclusions: In fruit of A. eriantha, peel detachability is due to the establishment of a zone of discontinuity created by differential cell wall changes in peel and outer pericarp tissues that lead to changes in mechanical properties of the peel. During ripening, the peel becomes flexible and the cells continue to adhere strongly to each other, preventing breakage, whereas the underlying outer pericarp loses cell wall strength as softening proceeds. Together these results reveal a novel and interesting mechanism for enabling cell separation.
Introduction and characterization of charged functional domains into an esterified pectic homogalacturonan by a citrus pectin methylesterase and comparison of its modes of action to other pectin methylesterase isozymes.
Kim, Y., Williams, M. A., Luzio, G. A. & Cameron, R. G. (2017). Food Hydrocolloids, 69, 422-431.
One of the four pectin methylesterase types isolated from Citrus sinensis var. Valencia fruit was used to demethylesterify a model homogalacturonan (HG) to 30%, 50% and 70% degree of methylesterification (DM) at pH 4.5 and 7.0, respectively. Introduced demethylesterified blocks (DMBs) were released by a limited endo-polygalacturonase (EPG) digestion, separated and quantified by HPAEC. Average DMB size (BS) and number of such blocks per molecule (BN) differed depending on final DM and reaction pH (P < 0.05). BS and BN were significantly higher in 30% DM HG than 50 and 70 DMs. pH 4.5 series showed significantly larger BS compared to pH 7.5 series (P < 0.01). Distribution of DMBs released by limited EPG digest was predicted by mathematical modeling and in silico modeled processive (degree of processivity = 10), multiple attack mode of action best explains the experimental block distributions. Absolute degree of blockiness (DBabs) obtained from exhaustive EPG digestions, displayed a linear relationship with DM regardless of reaction pH (P < 0.001). Significant correlation coefficients between BS, BN, DBabs, and DM manifested the effectiveness of the block information gained from both EPG digestion to estimate DMB distribution pattern (P < 0.05). However, comparison of block distribution information of three isozymes revealed that difference in block pattern could be manifested by parameters from limited EPG digest (BS, BN ) but not by those from exhaustive digest (DBabs). The results suggested the possibility to control BS and to customize specific population of demethylesterified pectin molecules using PME isozymes from Valencia orange.
Partial purification of a polygalacturonase from a new Aspergillus sojae mutant and its application in grape mash maceration.
Yıldız, S., Mata‐Gómez, M. A., Tarı, C. & Rito‐Palomares, M. (2017). International Journal of Food Science & Technology, 52(3), 834-842.
The use of polygalacturonase (PG) preparations in winemaking promotes the release of phenolic compounds. A PG from a new source, Aspergillus sojae mutant, was semi-purified and tested for grape mash maceration. Crude extract (CE), a commercial pectinase, and two high PG activity semi-purified preparations, FI and FII, were applied for maceration at PG activity of 3.5 U g-1 of grape for 46 h. Enzyme-assisted maceration significantly (P < 0.05) increased the total phenolic content from 255.8 to 916.3 ± 5.2, 5732.9 ± 9.9, 563.4 ± 6.7 and 620.6 ± 18.4 mg L-1 for CE, commercial pectinase, FI and FII, respectively. The content of individual phenolics such as gallic, protocatechuic, chlorogenic and p-coumaric acids was improved. Principal component and hierarchical clustering analyses suggested that CE has a better performance upon the release of phenols. Semi-purified preparations acted similar to commercial pectinase. These findings open an opportunity for the potential use of PG from the mutant strain as an alternative macerating enzyme.
Characterization and comparison of polysaccharides from Lycium barbarum in China using saccharide mapping based on PACE and HPTLC.
Wu, D. T., Cheong, K. L., Deng, Y., Lin, P. C., Wei, F., Lv, X. J., Long, Z. R., Zhao, J., Ma, S. C. & Li, S. P. (2015). Carbohydrate polymers, 134, 12-19.
Water-soluble polysaccharides from 51 batches of fruits of L. barbarum (wolfberry) in China were investigated and compared using saccharide mapping, partial acid hydrolysis, single and composite enzymatic digestion, followed by polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) analysis and high performance thin layer chromatography (HPTLC) analysis, respectively. Results showed that multiple PACE and HPTLC fingerprints of partial acid and enzymatic hydrolysates of polysaccharides from L. barbarum in China were similar, respectively. In addition, results indicated that β-1,3-glucosidic, α-1,4-galactosiduronic and α-1,5-arabinosidic linkages existed in polysaccharides from L. barbarum collected in China, and the similarity of polysaccharides in L. barbarum collected from different regions of China was pretty high, which are helpful for the improvement of the performance of polysaccharides from L. barbarum in functional/health foods area. Furthermore, polysaccharides from Panax notoginseng, Angelica sinensis, and Astragalus membranaceus var. mongholicus were successfully distinguished from those of L. barbarum based on their PACE fingerprints. These results were beneficial to improve the quality control of polysaccharides from L. barbarum and their products, which suggested that saccharide mapping based on PACE and HPTLC analysis could be a routine approach for quality control of polysaccharides.
Remodeling of pectin and hemicelluloses in tomato pericarp during fruit growth.
Guillon, F., Moïse, A., Quemener, B., Bouchet, B., Devaux, M. F., Alvarado, C. & Lahaye, M. (2017). Plant Science, 257, 48-62.
Tomato fruit texture depends on histology and cell wall architecture, both under genetic and developmental controls. If ripening related cell wall modifications have been well documented with regard to softening, little is known about cell wall construction during early fruit development. Identification of key events and their kinetics with regard to tissue architecture and cell wall development can provide new insights on early phases of texture elaboration. In this study, changes in pectin and hemicellulose chemical characteristics and location were investigated in the pericarp tissue of tomato (Solanum lycopersicon var Levovil) at four stages of development (7, 14 and 21 day after anthesis (DPA) and mature green stages). Analysis of cell wall composition and polysaccharide structure revealed that both are continuously modified during fruit development. At early stages, the relative high rhamnose content in cell walls indicates a high synthesis of rhamnogalacturonan I next to homogalacturonan. Fine tuning of rhamnogalacturonan I side chains appears to occur from the cell expansion phase until prior to the mature green stage. Cell wall polysaccharide remodelling also concerns xyloglucans and (galacto)glucomannans, the major hemicelluloses in tomato cell walls. In situ localization of cell wall polysaccharides in pericarp tissue revealed non-ramified RG-I rich pectin and XyG at cellular junctions and in the middle lamella of young fruit. Blocks of non-methyl esterified homogalacturonan are detected as soon as 14 DPA in the mesocarp and remained restricted to cell corner and middle lamella whatever the stages. These results point to new questions about the role of pectin RGI and XyG in cell adhesion and its maintenance during cell expansion.
Boron bridging of rhamnogalacturonan‐II is promoted in vitro by cationic chaperones, including polyhistidine and wall glycoproteins.
Chormova, D. & Fry, S. C. (2016). New Phytologist, 209(1), 241-251.
Dimerization of rhamnogalacturonan-II (RG-II) via boron cross-links contributes to the assembly and biophysical properties of the cell wall. Pure RG-II is efficiently dimerized by boric acid B(OH)3 in vitro only if nonbiological agents for example Pb2+ are added. By contrast, newly synthesized RG-II domains dimerize very rapidly in vivo. We investigated biological agents that might enable this. We tested for three such agents: novel enzymes, borate-transferring ligands and cationic ‘chaperones’ that facilitate the close approach of two polyanionic RG-II molecules. Dimerization was monitored electrophoretically. Parsley shoot cell-wall enzymes did not affect RG-II dimerization in vitro. Borate-binding ligands (apiose, dehydroascorbic acid, alditols) and small organic cations (including polyamines) also lacked consistent effects. Polylysine bound permanently to RG-II, precluding electrophoretic analysis. However, another polycation, polyhistidine, strongly promoted RG-II dimerization by B(OH)3 without irreversible polyhistidine–RG-II complexation. Likewise, partially purified spinach extensins (histidine/lysine-rich cationic glycoproteins), strongly promoted RG-II dimerization by B(OH)3 in vitro. Thus certain polycations, including polyhistidine and wall glycoproteins, can chaperone RG-II, manoeuvring this polyanionic polysaccharide domain such that boron-bridging is favoured. These chaperones dissociate from RG-II after facilitating its dimerization, indicating that they act catalytically rather than stoichiometrically. We propose a natural role for extensin–RG-II interaction in steering cell-wall assembly.
Structure characterization, chemical and enzymatic degradation, and chain conformation of an acidic polysaccharide from Lycium barbarum L.
Liu, W., Liu, Y., Zhu, R., Yu, J., Lu, W., Pan, C., Yao, W. & Gao, X. (2016). Carbohydrate Polymers, 147, 114-124.
An acidic polysaccharide, named as p-LBP, was isolated from Lycium barbarum L. by water extraction and purified by decoloration, ion exchange chromatography, dialysis and gel chromatography, successively. The primary structure analysis was determined by HPAEC-PAD, HPSEC, FT-IR, GC–MS, and NMR. The results showed p-LBP was a homogeneous heteropolysaccharide as a pectin molecule with an average molecular weight of 64 kDa p-LBP was an approximately 87 nm hollow sphere in 0.05 mol/L sodium sulfate solution determined by HPSEC-MALLS, DLS and TEM. A discussion of degradation patterns gave the detailed structural information of p-LBP. Therefore, the results from degraded fragments elucidated that the backbone of p-LBP was formed by → 4-7alpha;-GalpA-(1 →, repeatedly. Partial region was connected by → 4-α-GalpA-(1 → and → 2-α-Rhap-(1 →, alternatively. On the C-4 of partial → 2-α-Rhap-(1 → residues existed branches forming by → 4-β-Galp-(1 →, → 3-β-Galp-(1 → or → 5-α-Araf-(1 →, while on the C-6 of partial → 3-β-Galp-(1 → residues existed secondary branches forming by terminal-α-Araf, terminal-β-Galp or → 3-α-Araf-(1 →.
Macrophages treated with non-digestible polysaccharides reveal a transcriptionally unique phenotype.
Tang, Y., Govers, C., Wichers, H. J. & Mes, J. J. (2017). Journal of Functional Foods, 36, 280-289.
Dietary non-digestible polysaccharides (NDPs) might promote intestinal health via immuno-modulation. Immunomodulatory effects of NDP are most likely brought about by antigen processing cells such as macrophages that populate the intestine, although the mechanisms are still poorly understood. We validated the in vitro model of M1 and M2 macrophages to mimic the intestinal inflammatory and tolerant macrophages using literature and microarray-derived gene markers. All these markers were used to characterise the macrophage phenotype following NDP stimulation. This identified an alternative subset, termed M(NDP), which commonly modulated a set of 126 genes, involved in migration, metabolic processes, cell cycle, and inflammatory immune function. This gene-based analysis for macrophage subsets provides an additional tool to characterise NDP bioactivity for their in vivo potential.
Stomatal opening involves polar, not radial, stiffening of guard cells.
Carter, R., Woolfenden, H., Baillie, A., Amsbury, S., Carroll, S., Healicon, E., Sovatzoglou, S., Braybrook, S., Gray, J. E., Hobbs, J., Morris, R. J. & Morris, R. J. (2017). Current Biology, 27(19), 2974-2983.
It has long been accepted that differential radial thickening of guard cells plays an important role in the turgor-driven shape changes required for stomatal pore opening to occur. This textbook description derives from an original interpretation of structure rather than measurement of mechanical properties. Here we show, using atomic force microscopy, that although mature guard cells display a radial gradient of stiffness, this is not present in immature guard cells, yet young stomata show a normal opening response. Finite element modeling supports the experimental observation that radial stiffening plays a very limited role in stomatal opening. In addition, our analysis reveals an unexpected stiffening of the polar regions of the stomata complexes, both in Arabidopsis and other plants, suggesting a widespread occurrence. Combined experimental data (analysis of guard cell wall epitopes and treatment of tissue with cell wall digesting enzymes, coupled with bioassay of guard cell function) plus modeling lead us to propose that polar stiffening reflects a mechanical, pectin-based pinning down of the guard cell ends, which restricts increase of stomatal complex length during opening. This is predicted to lead to an improved response sensitivity of stomatal aperture movement with respect to change of turgor pressure. Our results provide new insight into the mechanics of stomatal function, both negating an established view of the importance of radial thickening and providing evidence for a significant role for polar stiffening. Improved stomatal performance via altered cell-wall-mediated mechanics is likely to be of evolutionary and agronomic significance.
Altered lignification in mur1-1 a mutant deficient in GDP-L-fucose synthesis with reduced RG-II cross linking.
Voxeur, A., Soubigou-Taconnat, L., Legée, F., Sakai, K., Antelme, S., Durand-Tardif, M., Lapierre, C. & Sibout, R. (2017). PloS one, 12(9), e0184820.
In the plant cell wall, boron links two pectic domain rhamnogalacturonan II (RG-II) chains together to form a dimer and thus contributes to the reinforcement of cell adhesion. We studied the mur1-1 mutant of Arabidopsis thaliana which has lost the ability to form GDP-fucose in the shoots and show that the extent of RG-II cross-linking is reduced in the lignified stem of this mutant. Surprisingly, MUR1 mutation induced an enrichment of resistant interunit bonds in lignin and triggered the overexpression of many genes involved in lignified tissue formation and in jasmonic acid signaling. The defect in GDP-fucose synthesis induced a loss of cell adhesion at the interface between stele and cortex, as well as between interfascicular fibers. This led to the formation of regenerative xylem, where tissue detachment occurred, and underlined a loss of resistance to mechanical forces. Similar observations were also made on bor1-3 mutant stems which are altered in boron xylem loading, leading us to suggest that diminished RG-II dimerization is responsible for regenerative xylem formation.
Etiolated Seedling Development Requires Repression of Photomorphogenesis by a Small Cell-Wall-Derived Dark Signal.
Sinclair, S. A., Larue, C., Bonk, L., Khan, A., Castillo-Michel, H., Stein, R. J., Grolimund, D., Begerow, D., Neumann, U., Haydon, M. J. & Krämer, U. (2017). Current Biology, In Press.
Etiolated growth in darkness or the irreversible transition to photomorphogenesis in the light engages alternative developmental programs operating across all organs of a plant seedling. Dark-grown Arabidopsis de-etiolated by zinc (dez) mutants exhibit morphological, cellular, metabolic, and transcriptional characteristics of light-grown seedlings. We identify the causal mutation in TRICHOME BIREFRINGENCE encoding a putative acyl transferase. Pectin acetylation is decreased in dez, as previously found in the reduced wall acetylation2-3 mutant, shown here to phenocopy dez. Moreover, pectin of dez is excessively methylesterified. The addition of very short fragments of homogalacturonan, tri-galacturonate, and tetra-galacturonate, restores skotomorphogenesis in dark-grown dez and similar mutants, suggesting that the mutants are unable to generate these de-methylesterified pectin fragments. In combination with genetic data, we propose a model of spatiotemporally separated photoreceptive and signal-responsive cell types, which contain overlapping subsets of the regulatory network of light-dependent seedling development and communicate via a pectin-derived dark signal.