Cross-linking of soluble extensin in isolated cell walls

The extensin component of primary cell walls has generally been considered to be an intrinsically insoluble cell wall glycoprotein. Recent data have established that cell wall extensin is in fact secreted in a soluble monomeric form which slowly becomes insolubilized in the cell wall probably through the oxidative formation of isodityrosine cross-links. We now show that isolated cell walls from aerated root slices of Daucus carota have the capacity to insolubilize extensin through the formation of isodityrosine. This in vitro cross-linking is specific for the extensin glycoprotein, as other wall proteins are not cross-linked by the isolated wall system. Although extensin can be cross-linked in solution by peroxidase and H₂O₂, dityrosine and not isodityrosine is the phenolic cross-link formed. Wall-catalyzed cross-linking of soluble extensin is inhibited by l-ascorbate, and both the initial rate and total extent of cross-linking are inhibited by acidic pH in the physiological range (pH 4 to 6). We suggest several mechanisms by which acid might inhibit cross-linking and propose that cytoplasmic factors (ascorbate and/or hydrogen ions) may regulate the solubility of extensin in vivo.     

Oxidative coupling of tyrosine and ferulic acid residues: Intra- and extra-protoplasmic occurrence,predominance of trimers and larger products,and possible role in inter-polymeric cross-linking

I discuss the range of oxidative phenolic coupling products formed from the tyrosine residues of cell wall glycoproteins and from the feruloyl residues of wall polysaccharides possibly by the action of peroxidases and/or laccases. In the cases of both tyrosine- and ferulate-coupling, the coupling products are not confined to dimers but include trimers and probably higher oligomers, which are sometimes predominant. Thus, some previous assays, in which specifically dimers were monitored, will have underestimated the extent of phenolic coupling. The possibility is discussed that some of the phenolic coupling products, in both glycoproteins and polysaccharides, are inter-polymeric and that they may therefore act as cross-links in the cell wall. The limitations in the evidence for this hypothesis are stressed. The sub-cellular site of oxidative phenolic coupling is discussed. In-vivo radiolabelling of cultured maize cells with [¹⁴C]cinnamate has shown that, especially in young, rapidly growing cultures, much oxidative coupling of feruloyl-arabinoxylans occurs within the endomembrane system, before secretion of the polysaccharides into the cell wall. Appreciable feruloyl coupling within the cell wall depended on the supply of H₂O₂ and on culture age. The situation with tyrosine coupling in glycoproteins is also debated. Although peroxidase activity has long been known to occur in the endomembrane system, the recent finding of intraprotoplasmic feruloyl coupling provided the first evidence that peroxidases (and/or laccases) may act in this sub-cellular location in vivo. I draw attention to the distinction between peroxidase action (in vivo) and activity (assayed in vitro), and to the unknown origin of H₂O₂ within the endomembrane system.     

Prooxidant capacity of phenolic acids defines antioxidant potential

Phenolic acids derived from vegetables, fruits and beverages are considered abundant sources of natural antioxidants consumed in the human diet. In addition to having well-known antioxidant activity, phenolic acids also exhibit pro-oxidant activity under selected conditions. We hypothesized that the availability of extracellular H₂O₂ derived from phenolic acid autoxidation will diffuse across cell membranes to participate as a messenger molecule to activate intracellular redox signaling in response to oxidative stress. We report on the relative activity of structurally different phenolic acids to generate specific changes in the extracellular - intracellular H₂O₂ flux that induces intracellular redox signaling corresponding to a function to reduce intracellular oxidative stress. HyPer-3 methodology was used to measure increases in intracellular H₂O₂ in differentiated Caco-2 intestinal cells in response to phenolic acid autoxidation and changes in extracellular H₂O₂ production. The potential for different phenolic acids to autoxidize and generate H₂O₂ was dependent on the structure and concentration of phenolic acid. Activation of nuclear factor erythroid 2-related factor (Nrf2) cell signaling was enhanced (p < 0.05) by phenolic acid induced H₂O₂ production, and mitigated when present along with catalase (p < 0.05), or, alternatively by blocking aquaporin 3 (AQP3) function (p < 0.05) using DFP00173 as the AQP3 inhibitor. The relative capacity of phenolic acids to generate H₂O₂ via autoxidation was structure specific and corresponded to the level of Nrf2 cell signaling in differentiated Caco-2 epithelial cells. The Nrf2-Keap1 response paralleled the extent of reduced oxidative stress observed in differentiated Caco-2 cells determined by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.     

Changes in phenolic metabolism in salicylic acid-treated shoots of Cistus heterophyllus

The exogenous application of salicylic acid (SA) not only protects plants against stress, but also enhances their growth and productivity. In this study, proliferating shoots of Cistus heterophyllus subsp. carthaginensis, an endangered plant species, were incubated in the presence of 0, 10, 100, and 1,000 μM SA for a period of 2 months. Overall growth, phenylpropanoid metabolism and antioxidant capacity were then determined. At low SA concentration, the efficiency of photosystem II (PSII) and shoot growth remained stable, while chlorophyll and carotenoid levels increased. Furthermore, there were no major changes in the levels of H₂O₂ in the different treatments (less than 10 % compared with the control), but an increase in lipid peroxidation, proline content and free and bound SA concentrations was observed in 100 μM SA-treated shoots. SA treatments resulted in increased activities of phenylalanine ammonia lyase (EC 4.3.1.24) and soluble peroxidases (EC 1.11.1.7), which strongly correlated with the decrease in soluble flavanols and the increase of proanthocyanidins, whereas cell wall-bound peroxidases exhibited a SA-concentration-dependent down-regulation. The results provided evidence that the differences in SA-induced changes in phenolic metabolism, especially the oxidation of flavanols by soluble peroxidases, could serve as a backup defence system contributing to a reduction in oxidative cellular damage, as suggested by the high anti-lipid oxidation activity displayed by Cistus extracts.     

Ataxia telangiectasia mutated inhibits oxidative stress-induced apoptosis by regulating heme oxygenase-1 expression

Ataxia telangiectasia (AT) is caused by mutational inactivation of the ataxia telangiectasia mutated (Atm) gene, which is involved in DNA repair. Increased oxidative stress has been shown in human AT cells and neuronal tissues of Atm-deficient mice. Heme oxygenase-1 (HO-1) is an inducible antioxidant enzyme and protects cells against oxidative stress. The purpose of this study is to determine whether ATM induces antioxidant enzyme HO-1 and protects cells from oxidative stress-mediated apoptosis by driving the activation of PKC-δ and NF-κB, by increasing cell viability, and by downregulating DNA fragmentation and apoptotic indicators (apoptosis-inducing factor and cleaved caspase-3). AT fibroblasts stably transfected with human full-length ATM cDNA (YZ5 cells) or the empty vector (MOCK cells) were treated with H₂O₂ as a source of reactive oxygen species (ROS). As a result, transfection with ATM inhibited ROS-induced cell death and DNA fragmentation in MOCK cells. Transfection with ATM induced expression of HO-1 which was mediated by PKC-δ and NF-κB in H₂O₂-treated MOCK cells. ZnPP, an HO-1 inhibitor, and transfection with HO-1 siRNA increased ROS levels and apoptosis, whereas hemin, an HO-1 activator, reduced ROS levels and apoptosis in H₂O₂-treated YZ5 cells. Rottlerin, a PKC-δ inhibitor, inhibited NF-κB activation and HO-1 expression in H₂O₂-treated YZ5 cells. MOCK cells showed increased cell death, DNA fragmentation, and apoptotic indicators compared to YZ5 cells exposed to H₂O₂. In addition, transfection with p65 siRNA increased ROS levels and DNA fragmentation, but decreased HO-1 protein levels in H₂O₂-treated YZ5 cells. In conclusion, ATM induces HO-1 expression via activation of PKC-δ and NF-κB and inhibits oxidative stress-induced apoptosis. A loss of HO-1 induction may explain why AT patients are vulnerable to oxidative stress.     

Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space

Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions.     

Degradation of isolated tomato cell walls by purified polygalacturonase in vitro

Cell wall preparations from green pericarp of normal and mutant Neverripe (Nr) and ripening inhibitor (rin) tomato (Lycopersicon esculentum Mill.) fruit were all equally degraded in vitro by a cell wall-bound protein extract from ripe normal tomatoes.     

Response of photosynthesis and chlorophyll fluorescence to acute ozone stress in tomato (Solanum lycopersicum Mill.)

The crop sensitivity to ozone (O₃) is affected by the timing of the O₃ exposure, by the O₃ concentration, and by the crop age. To determine the physiological response to the acute ozone stress, tomato plants were exposed to O₃ at two growth stages. In Experiment I (Exp. I), O₃ (500 μg m^?3) was applied to 30-d-old plants (PL30). In Experiment II (Exp. II), three O₃ concentrations (200, 350, and 500 μg m^?3) were applied to 51-d-old plants (PL51). The time of the treatment was 4 h (7:30–11:30 h). Photosynthesis and chlorophyll fluorescence measurements were done 4 times (before the exposure; 20 min, 20 h, and 2–3 weeks after the end of the treatment) using a LI-COR 6400 photosynthesis meter. The stomatal pore area and stomatal conductance were reduced as the O₃ concentration increased. Ozone induced the decrease in the photosynthetic parameters of tomato regardless of the plant age. Both the photosystem (PS) II operating efficiency and the maximum quantum efficiency of PSII photochemistry declined under the ozone stress suggesting that the PSII activity was inhibited by O₃. The impaired PSII contributed to the reduced photosynthetic rate. The greater decline of photosynthetic parameters was found in the PL30 compared with the PL51. It proved the age-dependent ozone sensitivity of tomato, where the younger plants were more vulnerable. Ozone caused the degradation of photosynthetic apparatus, which affected the photosynthesis of tomato plants depending on the growth stage and the O₃ concentration.     

Oxygen Inhibition of Nitrate Reductase Biosynthesis in Detached Corn Leaves via Inhibition of Total Soluble Protein Synthesis

Detached first leaves of 3-day-old corn seedlings (Zea mays L. W64AxW183E) were incubated with nitrate in air or 100% O₂ in the light. Nitrate accumulation in the leaves was not depressed by O₂. NADH:nitrate reductase activity and enzyme protein, as measured with an enzyme-linked immunosorbent assay, increased in parallel during the 8 h nitrate treatment in air, but in O₂ the levels of enzyme activity and protein were depressed. NADH:nitrate reductase mRNA levels were the same in the air-and O₂-treated leaves. Total soluble protein levels in leaves were slightly depressed by O₂ and shifting from O₂ to an air environment increased the protein level. Incorporation of [³⁵S]methionine during nitrate treatment revealed that total soluble protein and nitrate reductase protein synthesis were both depressed by the O₂ environment relative to air, but both recovered when leaves were shifted from O₂ to air. Although O₂ accelerated inactivation of nitrate reductase in vitro, the in vivo inactivation rate appeared to be too low to account for the depressed level of nitrate reductase activity in O₂-treated leaves. We concluded that O₂ inhibition of nitrate reductase biosynthesis in detached corn leaves was largely due to inhibition of total soluble protein synthesis at the level of translation.     

Involvement of phenolic acids in disease resistance of potato tubers from CEPA-treated plants

Treatment of vegetative parts of potato plants two weeks before the harvest with 0.2% 2-chloroethylphosphonic acid (CEPA) delayed the sprouting of tubers and increased the resistance of tubers to infections caused byPhytophthora infestans, Erwinia carotovora andFusarium spp. during the storage period. Levels of free, soluble ester- and glycoside-bound phenolic acids and cell wall-bound phenolics were determined in cortical parenchyma of tubers (periderm). The enhancement of phenolic acids in tubers from treated plants was caused primarily by the increase in the contents of free vanillic, caffeic andp-hydroxybenzoic acids and cell wall-bound ferulic, vanillic andp-coumaric acids.     

In vitro characterization of tomato fruit softening : the use of enzymically active cell walls

Cell wall isolated from pericarp of normal tomato (Lycopersicon esculentum Mill. cv `Rutgers') fruit released pectic polymers in a reaction apparently mediated by wall-bound polygalacturonase that appears with the onset of ripening. Release was negligible in wall preparations from normal green and the ripening mutant rin fruit. Pectin solubilization was most extensive at pH 2.5 with a less significant peak at 5.5. Brief exposure to low (1.5) or high (7.5) pH resulted in reduction of autolytic activity, which was also inhibited by high temperature, Ca²⁺, and treatments employed to dissociate protein from cell wall. Uronic acid solubilization was significantly enhanced by 150 millimolar NaCl and by increasing temperature within the physiological range. These data indicate that the release of polyuronide from isolated cell walls is enzymic and may provide a convenient and reliable system for the study of softening metabolism.     

Suppression of Acid Invertase Activity by Antisense RNA Modifies the Sugar Composition of Tomato Fruit

cDNA for an acid invertase (EC 3.2.1.26 [EC] ) of tomato (Lycopersiconesculentum Mill.) fruit was introduced into tomato plants underthe control of the cauliflower mosaic virus 35S promoter inthe antisense orientation. The antisense gene effectively suppressedthe invertase activity in soluble and cell wall fractions fromripening fruits. The sucrose content of fruits of the transformantswas markedly increased, while the hexose content was reduced.These results indicate that acid invertase is one of main determinantsof the sugar composition of tomato fruit. The invertase activityin the cell wall fraction of the leaf tissues of the transformantswas not suppressed to the same extent as that in the solublefraction. Wounding of the control leaf tissues induced invertaseactivity in both soluble and cell wall fractions. The inductionof activity in the soluble fraction was suppressed by the antisensegene, while that in the cell wall fraction was unaffected. Thesefindings suggest that mRNA for some other invertase, in particular,the mRNA for a cell wall-bound invertase, was present in leaves. ¹Present address: Plant Breeding and Genetics Research Laboratory,Japan Tobacco Inc., 700 Higashibara, Toyoda, Iwata, Shizuoka,438 Japan. ²Present address: National Institute of Agrobiological Resources,Kannondai, Tsukuba, Ibaraki, 305 Japan.     

Physiological,structural and molecular traits activated in strawberry plants after inoculation with the plant growth‐promoting bacterium Azospirillum brasilense REC3

The plant growth‐promoting strain REC3 of Azospirillum brasilense, isolated from strawberry roots, prompts growth promotion and systemic protection against anthracnose disease in this crop. Hence, we hypothesised that A. brasilense REC3 can induce different physiological, structural and molecular responses in strawberry plants. Therefore, the aim of this work was to study these traits activated in Azospirillum‐colonised strawberry plants, which have not been assessed until now. Healthy, in vitro micropropagated plants were root‐inoculated with REC3 under hydroponic conditions; root and leaf tissues were sampled at different times, and oxidative burst, phenolic compound content, malondialdehyde (MDA) concentration, callose deposition, cell wall fortification and gene expression were evaluated. Azospirillum inoculation enhanced levels of soluble phenolic compounds after 12 h post‐inoculation (hpi), while amounts of cell wall bound phenolics were similar in inoculated and control plants. Other early responses activated by REC3 (at 24 hpi) were a decline of lipid peroxidation and up‐regulation of strawberry genes involved in defence (FaPR1), bacterial recognition (FaFLS2) and H₂O₂ depuration (FaCAT and FaAPXc). The last may explain the apparent absence of oxidative burst in leaves after bacterial inoculation. Also, REC3 inoculation induced delayed structural responses such as callose deposition and cell wall fortification (at 72 hpi). Results showed that A. brasilense REC3 is capable of exerting beneficial effects on strawberry plants, reinforcing their physiological and cellular characteristics, which in turns contribute to improve plant performance.     

Products Released from Enzymically Active Cell Wall Stimulate Ethylene Production and Ripening in Preclimacteric Tomato (Lycopersicon esculentum Mill.) Fruit

Enzymically active cell wall from ripe tomato (Lycopersicon esculentum Mill.) fruit pericarp release uronic acids through the action of wall-bound polygalacturonase. The potential involvement of products of wall hydrolysis in the induction of ethylene synthesis during tomato ripening was investigated by vacuum infiltrating preclimacteric (green) fruit with solutions containing pectin fragments enzymically released from cell wall from ripe fruit. Ripening initiation was accelerated in pectin-infiltrated fruit compared to control (buffer-infiltrated) fruit as measured by initiation of climacteric CO₂ and ethylene production and appearance of red color. The response to infiltration was maximum at a concentration of 25 micrograms pectin per fruit; higher concentrations (up to 125 micrograms per fruit) had no additional effect. When products released from isolated cell wall from ripe pericarp were separated on Bio-Gel P-2 and specific size classes infiltrated into preclimacteric fruit, ripening-promotive activity was found only in the larger (degree of polymerization >8) fragments. Products released from pectin derived from preclimacteric pericarp upon treatment with polygalacturonase from ripe pericarp did not stimulate ripening when infiltrated into preclimacteric fruit.     

A catalytic antioxidant metalloporphyrin blocks hydrogen peroxide-induced mitochondrial DNA damage

Reactive oxygen species (ROS) have been implicated as the cause of cumulative damage to DNA, proteins and lipids that can ultimately result in cell death. A common problem when measuring oxidative DNA damage has been the introduction of modifications in the native state of the molecule by many DNA isolation methods. We circumvented this problem by employing direct PCR (DPCR) of whole cell lysates. DPCR of mouse lung fibroblasts performed better than PCRs containing template acquired by phenol/chloroform extraction or a commercially available genomic DNA isolation kit. We investigated the direct use of whole cell preparations in the polymerase chain reaction (PCR) to detect hydrogen peroxide (H₂O₂)-mediated DNA damage. We observed a concentration-dependent decrease in amplification efficiency of a 4.3 kb mitochondrial (mt)DNA target in H₂O₂-treated mouse lung fibroblasts (MLFs). At low doses the efficiency of amplification returns to control levels over 24 h. We detected no change in amplification efficiency of a plasmid control containing our mtDNA target under any of the culture conditions employed in these studies. Treatment of MLFs with the catalytic antioxidant manganese(III) meso-tetrakis(4-benzoic acid)porphyrin (MnTBAP) attenuates the effects of H₂O₂ exposure. When quantitated with an external standard the use of DPCR in tandem with a PCR amplification efficiency assay provides a powerful approach to assess oxidative mtDNA damage.     

Continuous production of extracellular antioxidants in suspension cells attenuates the oxidative burst detected in plant microbe interactions

Suspension cells of Solanacearum tuberosum and Nicotiana tabacum placed in fresh buffer rapidly produce and maintain significant pools of extracellular antioxidants. The extracellular antioxidant was detected by first adding a known amount of exogenous H₂O₂ to samples and then immediately measuring the remaining H₂O₂. The difference between the amount added and amount remaining was used to determine the antioxidant capacity of the sample. This extracellular antioxidant pool attenuates levels of hydrogen peroxide produced during plant–bacterial interactions. When tobacco cells were inoculated with an isolate Pseudomonas syringae pv. syringae that causes a hypersensitive response much of the antioxidant capacity had been expended neutralizing the oxidative burst characteristic of such plant–microbe interactions. After a brief delay, the levels of extracellular phenolics increased commensurate to antioxidative capacity in freshly transferred cells within 2–4 h. The strong UV absorbance of these extracellular phenolics within 250 and 350 nm was used to follow oxidation upon reaction with H₂O₂. This extracellular antioxidant pool is an important consideration in cell suspension studies of the plant–microbe oxidative burst. This study demonstrates that the true magnitude and timing of the oxidative burst in cell suspensions is masked by extracellular antioxidants.     

Molecular identification for epigallocatechin-3-gallate-mediated antioxidant intervention on the H2O2-induced oxidative stress in H9c2 rat cardiomyoblasts

Background

Epigallocatechin-3-gallate (EGCG) has been documented for its beneficial effects protecting oxidative stress to cardiac cells. Previously, we have shown the EGCG-mediated cardiac protection by attenuating reactive oxygen species and cytosolic Ca²⁺ in cardiac cells during oxidative stress and myocardial ischemia. Here, we aimed to seek a deeper elucidation of the molecular anti-oxidative capabilities of EGCG in an H₂O₂-induced oxidative stress model of myocardial ischemia injury using H9c2 rat cardiomyoblasts.

Results

Proteomics analysis was used to determine the differential expression of proteins in H9c2 cells cultured in the conditions of control, 400 μM H₂O₂ exposure for 30 min with and/or without 10 to 20 μM EGCG pre-treatment. In this model, eight proteins associated with energy metabolism, mitochondrial electron transfer, redox regulation, signal transduction, and RNA binding were identified to take part in EGCG-ameliorating H₂O₂-induced injury in H9c2 cells. H₂O₂ exposure increased oxidative stress evidenced by increases in reactive oxygen species and cytosolic Ca²⁺ overload, increases in glycolytic protein, α-enolase, decreases in antioxidant protein, peroxiredoxin-4, as well as decreases in mitochondrial proteins, including aldehyde dehydrogenase-2, ornithine aminotransferase, and succinate dehydrogenase ubiquinone flavoprotein subunit. All of these effects were reversed by EGCG pre-treatment. In addition, EGCG attenuated the H₂O₂-induced increases of Type II inositol 3, 4-bisphosphate 4-phosphatase and relieved its subsequent inhibition of the downstream signalling for Akt and glycogen synthase kinase-3β (GSK-3β)/cyclin D1 in H9c2 cells. Pre-treatment with EGCG or GSK-3β inhibitor (SB 216763) significantly improved the H₂O₂-induced suppression on cell viability, phosphorylation of pAkt (S473) and pGSK-3β (S9), and level of cyclin D1 in cells.

Conclusions

Collectively, these findings suggest that EGCG blunts the H₂O₂-induced oxidative effect on the Akt activity through the modulation of PIP3 synthesis leading to the subsequent inactivation of GSK-3β mediated cardiac cell injury.     

Time course study on accumulation of cell wall-bound phenolics and activities of defense enzymes in tomato roots in relation to <Emphasis Type="Italic">Fusarium</Emphasis> wilt

Major cell wall-bound phenolic compounds were detected and identified in roots of tomato at different stages of growth. Alkaline hydrolysis of the cell wall material of the root tissues yielded ferulic acid as the major bulk of the phenolic compounds. Other phenolic compounds identified were 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin and 4-coumaric acid. All the six phenolic acids were higher in very early stage of plant growth. Ferulic acid, 4-hydroxybenzoic acid and 4-coumaric acid exhibited a decreasing trend up to 60 days and then the content of these phenolic acids increased somewhat steadily towards the later stage of growth. Total phenolics, phenylalanine ammonia-lyase (PAL) activity and peroxidase (POD) activity were in tandem match with the occurrence pattern of the phenolic acids. Ferulic acid showed highest antifungal activity against tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici. The results of this study may be interpreted to seek an explanation for high susceptibility of tomato plants at flowering stage to Fusarium wilt. It may also be concluded that greater amounts of ferulic acid in combination with other phenolics and higher level of PAL and POD activities after 60 days of growth may have a role in imparting resistance against Fusarium wilt at a late stage of plant growth.