Rhamnogalacturonan-II cross-linking of plant pectins via boron bridges occurs during polysaccharide synthesis and/or secretion

Rhamnogalacturonan-II (RG-II), a domain of plant cell wall pectins, is able to cross-link with other RG-II domains through borate diester bridges. Although it is known to affect mechanical properties of the cell wall, the biochemical requirements and lifecycle of this cross-linking remain unclear. We developed a PAGE methodology to allow separation of monomeric and dimeric RG-II and used this to study the dynamics of cross-linking in vitro and in vivo. Rosa cells grown in medium with no added boron contained no RG-II dimers, although these re-appeared after addition of boron to the medium. However, other Rosa cultures which were unable to synthesize new polysaccharides did not show dimer formation. We conclude that RG-II normally becomes cross-linked intraprotoplasmically or during secretion, but not post-secretion

Boron bridging of rhamnogalacturonan-II is promoted in vitro by cationic chaperones, including polyhistidine and wall glycoproteins

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 Pb(2+) 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

Oxalyltransferase, a plant cell-wall acyltransferase activity, transfers oxalate groups from ascorbate metabolites to carbohydrates

In the plant apoplast, ascorbate is oxidised, via dehydroascorbic acid, to O-oxalyl esters [oxalyl-l-threonate (OxT) and cyclic oxalyl-l-threonate (cOxT)]. We tested whether OxT and cOxT can donate the oxalyl group in transacylation reactions to form oxalyl-polysaccharides, potentially modifying the cell wall. [oxalyl-14 C]OxT was incubated with living spinach (Spinacia oleracea) and Arabidopsis cell-suspension cultures in the presence or absence of proposed acceptor substrates (carbohydrates). In addition, [14 C]OxT and [14 C]cOxT were incubated in vitro with cell-wall enzyme preparations plus proposed acceptor substrates. Radioactive products were monitored electrophoretically. Oxalyltransferase activity was detected. Living cells incorporated oxalate groups from OxT into cell-wall polymers via ester bonds. When sugars were added, [14 C]oxalyl-sugars were formed, in competition with OxT hydrolysis. Preferred acceptor substrates were carbohydrates possessing primary alcohols e.g. glucose. A model transacylation product, [14 C]oxalyl-glucose, was relatively stable in vivo (half-life >24 h), whereas [14 C]OxT underwent rapid turnover (half-life ~6 h). Ionically wall-bound enzymes catalysed similar transacylation reactions in vitro with OxT or cOxT as oxalyl donor substrates and any of a range of sugars or hemicelluloses as acceptor substrates. Glucosamine was O-oxalylated, not N-oxalylated. We conclude that plants possess apoplastic acyltransferase (oxalyltransferase) activity that transfers oxalyl groups from ascorbate catabolites to carbohydrates, forming relatively long-lived O-oxalyl-carbohydrates. The findings increase the range of known metabolites whose accumulation in vivo indicates vitamin C catabolism. Possible signalling roles of the resulting oxalyl-sugars can now be investigated, as can the potential ability of polysaccharide oxalylation to modify the wall's physical properties

Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening

AbstractSmall molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes’ biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme–cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors – especially compounds that generate singlet oxygen (1O2) e.g., riboflavin (IC50 29μM), retinoic acid, eosin (IC50 27μM) and erythrosin (IC50 36μM). The riboflavin effect was light-dependent, supporting 1O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (−)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40μM mycophenolic acid and (−)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads

A nanostructural view of the cell wall disassembly process during fruit ripening and postharvest storage by atomic force microscopy

Background: The mechanical properties of parenchyma cell walls and the strength and extension of adhesion areas between adjacent cells, jointly with cell turgor, are main determinants of firmness of fleshy fruits. These traits are modified during ripening leading to fruit softening. Cell wall modifications involve the depolymerisation of matrix glycans and pectins, the solubilisation of pectins and the loss of neutral sugars from pectin side chains. These changes weaken the cell walls and increase cell separation, which in combination with a reduction in cell turgor, bring about textural changes. Atomic force microscopy (AFM) has been used to characterize the nanostructure of cell wall polysaccharides during the ripening and postharvest storage of several fruits. This technique allows the imaging of individual polymers at high magnification with minimal sample preparation. Scope and approach: This paper reviews the main features of the cell wall disassembly process associated to fruit softening from a nanostructural point of view, as has been provided by AFM studies. Key findings and conclusions: AFM studies show that pectin size, ramification and complexity is reduced during fruit ripening and storage, and in most cases these changes correlate with softening. Postharvest treatments that improve fruit quality have been proven to preserve pectin structure, suggesting a clear link between softening and pectin metabolism. Nanostructural characterization of cellulose and hemicellulose during ripening has been poorly explored by AFM and the scarce results available are not conclusive. Globally, AFM could be a powerful tool to gain insights about the bases of textural fruit quality in fresh and stored fruits

Glycosylinositol phosphorylceramides from <i>Rosa cell</i> cultures are boron-bridged in the plasma membrane and form complexes with rhamnogalacturonan II

Boron (B) is essential for plant cell-wall structure and membrane functions. Compared with its role in cross-linking the pectic domain rhamnogalacturonan II (RG-II), little information is known about the biological role of B in membranes. Here, we investigated the involvement of glycosylinositol phosphorylceramides (GIPCs), major components of lipid rafts, in the membrane requirement for B. Using thin-layer chromatography and mass spectrometry, we first characterized GIPCs from Rosa cell culture. The major GIPC has one hexose residue, one hexuronic acid residue, inositol phosphate, and a ceramide moiety with a C(18) trihydroxylated mono-unsaturated long-chain base and a C(24) monohydroxylated saturated fatty acid. Disrupting B bridging (by B starvation in vivo or by treatment with cold dilute HCl or with excess borate in vitro) enhanced the GIPCs’ extractability. As RG-II is the main B-binding site in plants, we investigated whether it could form a B-centred complex with GIPCs. Using high-voltage paper electrophoresis, we showed that addition of GIPCs decreased the electrophoretic mobility of radiolabelled RG-II, suggesting formation of a GIPC–B–RG-II complex. Last, using polyacrylamide gel electrophoresis, we showed that added GIPCs facilitate RG-II dimerization in vitro. We conclude that B plays a structural role in the plasma membrane. The disruption of membrane components by high borate may account for the phytotoxicity of excess B. Moreover, the in-vitro formation of a GIPC–B–RG-II complex gives the first molecular explanation of the wall–membrane attachment sites observed in vivo. Finally, our results suggest a role for GIPCs in the RG-II dimerization process

The oxidation of dehydroascorbic acid and 2,3-diketogulonate by distinct reactive oxygen species

l-Ascorbate, dehydro-l-ascorbic acid (DHA), and 2,3-diketo-l-gulonate (DKG) can all quench reactive oxygen species (ROS) in plants and animals. The vitamin C oxidation products thereby formed are investigated here. DHA and DKG were incubated aerobically at pH 4.7 with peroxide (H2O2), 'superoxide' (a ∼50 : 50 mixture of [Formula: see text] and [Formula: see text]), hydroxyl radicals (•OH, formed in Fenton mixtures), and illuminated riboflavin (generating singlet oxygen, 1O2). Products were monitored electrophoretically. DHA quenched H2O2 far more effectively than superoxide, but the main products in both cases were 4-O-oxalyl-l-threonate (4-OxT) and smaller amounts of 3-OxT and OxA + threonate. H2O2, but not superoxide, also yielded cyclic-OxT. Dilute Fenton mixture almost completely oxidised a 50-fold excess of DHA, indicating that it generated oxidant(s) greatly exceeding the theoretical •OH yield; it yielded oxalate, threonate, and OxT. 1O2 had no effect on DHA. DKG was oxidatively decarboxylated by H2O2, Fenton mixture, and 1O2, forming a newly characterised product, 2-oxo-l-threo-pentonate (OTP; '2-keto-l-xylonate'). Superoxide yielded negligible OTP. Prolonged H2O2 treatment oxidatively decarboxylated OTP to threonate. Oxidation of DKG by H2O2, Fenton mixture, or 1O2 also gave traces of 4-OxT but no detectable 3-OxT or cyclic-OxT. In conclusion, DHA and DKG yield different oxidation products when attacked by different ROS. DHA is more readily oxidised by H2O2 and superoxide; DKG more readily by 1O2 The diverse products are potential signals, enabling organisms to respond appropriately to diverse stresses. Also, the reaction-product 'fingerprints' are analytically useful, indicating which ROS are acting in vivo

Nitrogen Application Can Be Reduced without Affecting Carotenoid Content, Maturation, Shelf Life and Yield of Greenhouse Tomatoes

Tomatoes (Solanum lycopersicum L.) of the variety Elpida were grown under standard Mediterranean greenhouse conditions during the spring season at three different nitrogen levels (low 6.4, standard 12.8, high 25.9 mM/plant), which were replicated during two consecutive years. Application of high nitrogen significantly increased the colour index a* (p p ≤ 0.001) or by super-optimal temperatures in the second year of experimentation (10 days, p ≤ 0.001). The colour indices L* and a* and the hue angle (a/b*) were positively correlated with the sum of total carotenoids, while differences in b* depended on the year of cultivation. The sustainability of this type of tomato production can be improved by reducing the nitrogen supply to less than the current standard practice, with minimal risk or negative effects on yield and quality of tomatoes