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 < 0.001) but did not significantly affect yield or quality. The variety exhibited prolonged postharvest storage at room temperature (median survival time of 93 days). The maturation process was delayed by harvest at the breaker stage (2.5 days, 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.

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.

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

Small 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 ((1)O2) e.g., riboflavin (IC50 29 µM), retinoic acid, eosin (IC50 27 µM) and erythrosin (IC50 36 µM). The riboflavin effect was light-dependent, supporting (1)O2 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 (1 µM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 µM 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.

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, monitored by gel electrophoresis, occurs during polysaccharide synthesis and secretion but not post-secretion.

The cell-wall pectic domain rhamnogalacturonan-II (RG-II) is cross-linked via borate diester bridges, which influence the expansion, thickness and porosity of the wall. Previously, little was known about the mechanism or subcellular site of this cross-linking. Using polyacrylamide gel electrophoresis (PAGE) to separate monomeric from dimeric (boron-bridged) RG-II, we confirmed that Pb(2+) promotes H3 BO3 -dependent dimerisation in vitro. H3 BO3 concentrations as high as 50 mm did not prevent cross-linking. For in-vivo experiments, we successfully cultured 'Paul's Scarlet' rose (Rosa sp.) cells in boron-free medium: their wall-bound pectin contained monomeric RG-II domains but no detectable dimers. Thus pectins containing RG-II domains can be held in the wall other than via boron bridges. Re-addition of H3 BO3 to 3.3 µm triggered a gradual appearance of RG-II dimer over 24 h but without detectable loss of existing monomers, suggesting that only newly synthesised RG-II was amenable to boron bridging. In agreement with this, Rosa cultures whose polysaccharide biosynthetic machinery had been compromised (by carbon starvation, respiratory inhibitors, anaerobiosis, freezing or boiling) lost the ability to generate RG-II dimers. We conclude that RG-II normally becomes boron-bridged during synthesis or secretion but not post-secretion. Supporting this conclusion, exogenous [(3) H]RG-II was neither dimerised in the medium nor cross-linked to existing wall-associated RG-II domains when added to Rosa cultures. In conclusion, in cultured Rosa cells RG-II domains have a brief window of opportunity for boron-bridging intraprotoplasmically or during secretion, but secretion into the apoplast is a point of no return beyond which additional boron-bridging does not readily occur.