Plasma membrane-generated ROS and their possible contribution to leaf cell growth of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) MSC16 mitochondrial mutant

Reactive oxygen species (ROS) generally regarded as harmful products of oxygenic metabolism causing oxidative stress and cell damage are also important for control and regulation of biological processes. ROS can be generated by various enzymatic activities and removed by an array of ROS-scavenging molecules in the cell. In plants, the generation of ROS initiated by the plasma membrane NADPH oxidase can be used for controlled polymer breakdown leading to cell wall loosening during extension growth. The mosaic (MSC16) mitochondrial mutant of cucumber (Cucumis sativus L.) has marked phenotypic changes, including a slower growth rate which partially may result from disturbed leaf carbon and energy metabolism and ROS/antioxidants equilibrium. Cytochemical localization of H₂O₂ in leaf cells showed lower total level of H₂O₂ particularly in the apoplast of MSC16 leaf cells as compared to WT. The activity of plasma membrane NADPH oxidase (EC 1.6.3.1) was about 30% lower in plasmalemma vesicles isolated from MSC16 leaf tissue as compared to WT. The total foliar ascorbate pool (reduced and oxidized) was about 35% higher in MSC16 compared to WT leaves due to an increased content of the oxidized form. About 3% of the whole-leaf ascorbate was localized in the apoplast but in MSC16 it was considerably more reduced. We conclude that the lower apoplastic ROS content caused by decreased activity of plasma membrane NADPH oxidase and lower amounts of H₂O₂ in the apoplast may also contribute to altered growth of the MSC16 cucumber mutant.     

Zinc‐induced oxidative stress in Verbascum thapsus is caused by an accumulation of reactive oxygen species and quinhydrone in the cell wall

Oxidative stress is one aspect of metal toxicity. Zinc, although unable to perform univalent oxido‐reduction reactions, can induce the oxidative damage of cellular components and alter antioxidative systems. Verbascum thapsus L. plants that were grown hydroponically were exposed to 1 and 5 mM Zn²⁺. Reactive oxygen species (ROS) accumulation was demonstrated by the fluorescent probe H₂DCFDA and EPR measurements. The extent of zinc‐induced oxidative damage was assessed by measuring the level of protein carbonylation. Activities and isoform profiles of some antioxidant enzymes and the changes in ascorbate and total phenolic contents of leaves and roots were determined. Stunted growth because of zinc accumulation, preferentially in the roots, was accompanied by H₂O₂ production in the leaf and root apoplasts. Increased EPR signals of the endogenous oxidant quinhydrone, ^?CH₃ and ^?OH, were found in the cell walls of zinc‐treated plants. The activities of the antioxidative enzymes ascorbate peroxidase (APX) (EC 1.11.1.11), soluble superoxide dismutase (SOD) (EC 1.15.1.1), peroxidase (POD), (EC 1.11.1.7) and monodehydroascorbate reductase (EC 1.6.5.4) were increased; those of glutathione reductase (EC 1.6.4.2), dehydroascorbate reductase (EC 1.8.5.1) and ascorbate oxidase (AAO) (EC 1.10.3.3) were decreased with zinc treatment. Zinc induced a cell‐wall‐bound SOD isoform in both organs. Leaves accumulated more ascorbate and phenolics in comparison to roots. We propose a mechanism for zinc‐promoted oxidative stress in V. thapsus L. through the generation of charge transfer complexes and quinhydrone because of phenoxyl radical stabilisation by Zn²⁺ in the cell wall. Our results suggest that the SOD and APX responses are mediated by ROS accumulation in the apoplast. The importance of the POD/Phe/AA (ascorbic acid) scavenging system in the apoplast is also discussed.     

Axial changes in apoplast properties in the elongation zone of maize mesocotyl

Changes in extensibility of cell walls and composition of apoplastic solution along the elongation zone were studied in mesocotyls of etiolated seedlings of maize (Zea mays L.). It was found that plastic and elastic extensibility of cell walls was much greater in the cells with a high rate of elongation. Basipetal decrease in hydrogen peroxide concentration in the apoplast (from 5.1 to 2.0 ??M) was detected. We determined the activity of cell wall enzymes participating in H₂O₂ metabolism and found that in basal direction, potential ability of these enzymes to decompose H₂O₂ rises stronger than the ability to produce it. We found a basipetal decrease in polyamine oxidase activity, an increase in oxalate oxidase activity, and a rise in the ratio between peroxidase and NADH-oxidase activities of peroxidases. IAA (10^?6 M) promoted elongation of mesocotyl segments, induced a steady elevation of H₂O₂ content in the apoplast, an increase in NADH-oxidase activity of peroxidases, and a transient decrease in oxalate oxidase activity. Treatment with ABA (10^?4 M) suppressed elongation of mesocotyl segments, induced a transient elevation of H₂O₂ content in the apoplast, and a decrease in oxalate oxidase activity. It was shown that the main metabolites of apoplastic solution are glucose (20?C30 mM), fructose (6?C7 mM), malic acid (3 mM), and amino acids, namely, Asp, Glu, Asn, Gln, Ala, Val, Ser, Thr, and Phe. In basal direction, we observed a decrease in the content of glucose (from 30 to 20 mM), inositol (from 0.24 to 0.08 mM), and total amino acids (from 5.5 to 3.3 mM), whereas concentration of orthophosphate (3 mM) and malate (3 mM) did not change significantly. A relationship between the detected changes in the apoplast composition and basipetal decrease in the elongation rate of mesocotyl cells is discussed.     

Oxidative coupling of a feruloyl-arabinoxylan trisaccharide (FAXX) in the walls of living maize cells requires endogenous hydrogen peroxide and is controlled by a low-M<Subscript>r</Subscript> apoplastic inhibitor

Feruloyl-polysaccharides can be oxidatively coupled in isolated cell walls by peroxidase plus exogenous H₂O₂ in vitro, but the extent to which similar reactions may occur in the apoplast in vivo was unclear. Numerous cellular factors potentially control feruloyl coupling in vivo, and their net controlling influence is not readily studied in vitro. Therefore, we have monitored apoplastic feruloyl coupling in cultured maize cells in vivo using a radiolabelled model substrate, 5-O-feruloyl-α-L-arabinofuranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-D-xylose (FAXX). FAXX was expected to permeate the wall and to undergo reactions analogous to those normally exhibited by apoplastic feruloyl-polysaccharides in vivo. Little difference was found between the fates of [feruloyl−¹⁴C]FAXX and [pentosyl−³H]FAXX, indicating negligible apoplastic hydrolase or transferase activities. Very little radioactivity entered the protoplasm. Maize cells that had recently been washed in fresh medium were able to bind most of the FAXX (90%) in their cell walls, regardless of the age of the culture. During wall-binding, the [¹⁴C]feruloyl groups were converted to [¹⁴C]dehydrodiferulates and larger coupling products, as revealed by TLC after alkaline hydrolysis. As expected for an oxidative reaction, wall-binding was delayed by added anti-oxidants (ascorbate, ferulate, sinapate, chlorogenate or rutin). It was also completely inhibited by iodide, an H₂O₂-scavenger, indicating a role for peroxidase rather than oxidase. The observations indicate that oxidative coupling of feruloyl groups occurred within the cell wall, dependent on endogenous apoplastic H₂O₂ and wall-localised peroxidase, in vivo. Cells that had not recently been washed in fresh medium were much less able to bind FAXX, indicating the presence in the apoplast of an endogenous inhibitor of oxidative coupling. This inhibitor was of low M_r, was destroyed by heating, and remained in the aqueous phase (pH ≈3.5) when shaken with ethyl acetate. Its effectiveness was not altered by ascorbate oxidase. It is thus a small, heat-labile, hydrophilic inhibitor (not ascorbate) which we suggest plays a natural role in the control of wall cross-linking, and thus potentially in the control of cell growth.     

Oxidation of vacuolar and apoplastic phenolic substrates by peroxidase: Physiological significance of the oxidation reactions

Phenolic components and peroxidases are localized in vacuoles. Vacuolar peroxidase can oxidize phenolics when H₂O₂ is formed in vacuoles or tonoplasts, or when H₂O₂ formed outside of vacuoles is diffused into the organelles. In a mixture of phenolics containing a good and a poor substrate for peroxidase, a radical transfer reaction is possible from the radicals of the good substrate to the poor substrate, resulting in the enhancement of oxidation of the poor substrate. Phenoxyl radicals formed by peroxidase-dependent reactions are reduced by ascorbate in vacuoles. So, as long as ascorbate is present in vacuoles, the accumulation of oxidation products of phenolics is not significant. This suggests that ascorbate/phenolics/peroxidase systems in the vacuoles can scavenge H₂O₂. During aging, some phenolics are accumulated in vacuoles and the apoplast, and the accumulated phenolics are oxidized to brown components by peroxidase-dependent reactions. The brown components can produced O₂ ^? and H₂O₂ by autooxidation. The significance and the mechanisms of browning are discussed in tobacco leaves and onion scales.

     

Pathogenic infection and the oxidative defences in plant apoplast

Summary The structural and functional continuum of the plant apoplast is the first site of contact with a pathogen and plays a crucial role in initiation and coordination of many defence responses. In this paper, we present an overview of the involvement of the plant apoplast in plant-pathogen interactions. The process of infection of French bean (Phaseolus vulgaris L.) plants byColletotrichum lindemuthianum is analysed. The ultrastructural features of plant defence responses to fungal infection are then compared with those observed in plants or cell suspensions treated with various elicitors. Changes in cell walls and in whole plant cells responding to infection seem to be highly similar in all systems used. Model systems of French bean and white lupin (Lupinus albus L.) are then utilised to provide some biochemical characteristics of oxidative reactions in the apoplast evoked by elicitor treatment. The species specificity of various mechanisms generating reactive oxygen species is discussed, and some details of pH-dependent H₂O₂-generating activity of peroxidases are demonstrated. As its exocellular nature is an important feature of the oxidative burst, the major consequence of this event, i.e., the oxidative cross-linking of wall components during the papilla formation and strengthening of the walls, is analysed. Finally, the possible involvement of other wall-associated and developmentally regulated H₂O₂-generating mechanisms, like amine and oxalate oxidases, in plant defence is demonstrated. It is concluded that under stress conditions, such apoplastic mechanisms might be employed to increase plants' chances of survival.Abbreviations HR hypersensitive response - IWF intercellular washing fluid - OxO oxalate oxidase - ROS reactive oxygen species - YE elicitor preparation from yeast cell walls     

Oxidation of dehydroascorbic acid and 2,3-diketogulonate under plant apoplastic conditions

The rate of l-ascorbate catabolism in plants often correlates positively with the rate of cell expansion. The reason for this correlation is difficult to explore because of our incomplete knowledge of ascorbate catabolism pathways. These involve enzymic and/or non-enzymic oxidation to dehydroascorbic acid (DHA), which may then be hydrolysed to 2,3-diketogulonate (DKG). Both DHA and DKG were susceptible to further oxidation under conditions of pH and H₂O₂ concentration comparable with the plant apoplast. The kinetics of their oxidation and the identity of some of the products have been investigated here. DHA, whether added in pure form or generated in situ by ascorbate oxidation, was oxidised non-enzymically to yield, almost simultaneously, a monoanion (cyclic-oxalyl-threonate; cOxT) and a dianion (oxalyl-threonate; OxT). The monoanion was resistant to periodate oxidation, showing that it was not oxalic threonic anhydride. The OxT population was shown to be an interconverting mixture of 3-OxT and 4-OxT, differing in pK_a. The 3-OxT appeared to be formed earlier than 4-OxT, but the latter predominated at equilibrium. DKG was oxidised by H₂O₂ to two partially characterised products, one of which was itself further oxidised by H₂O₂ to yield threonate. The possible occurrence of these reactions in the apoplast in vivo and the biological roles of vitamin C catabolites are discussed.     

Methyl jasmonate-induced antioxidant defence in root apoplast from sunflower seedlings

We have investigated the physiological functions of the rapid generation of reactive oxygen species (ROS) and the implication of the antioxidant enzymes in the apoplast and symplast of roots of sunflower (Helianthus annuus L.) seedlings exposed to methyl jasmonate (MeJA, 50 μM). MeJA-elicited roots showed a fast increase in ROS content, followed by a marked increase in the activity of H₂O₂-scavenging enzymes, guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and catalase (CAT). The mechanisms responsible for MeJA-induced H₂O₂ accumulation was investigated further by studying both the production and scavenging of H₂O₂ in the extracellular matrix. Peroxidases active against (2,2′-azino-bis-[3-ethylbenzthiazoline-6-sulfonic acid], ABTS) and guaiacol were found in the apoplastic fluid, and proved to be ionically and covalently associated with sunflower cell walls, although only the peroxidase activities of the soluble apoplastic fractions and those ionically linked to the cell wall were correlated with the accumulation of the H₂O₂ detected. The results indicated that H₂O₂ accumulation is a complex and highly regulated event requiring the time-dependent stimulation and down-regulation of differently located enzymes, some of which are involved in H₂O₂ generation and degradation. It is concluded that exogenous MeJA may be involved in the oxidative stress processes by regulating antioxidant enzyme activities.     

Simulating ozone detoxification in the leaf apoplast through the direct reaction with ascorbate

 This paper presents a mathematical model which enables the semi-quantification of ozone (O₃) detoxification, based upon the direct reaction of the pollutant with ascorbate (ASC) located in the aqueous matrix associated with the cell wall (i.e. the apoplast). The model describes the uptake of ozone into the leaf and its direct reaction with ASC, taking into consideration the regeneration of dehydroascorbic acid in the cytosol, the rate of replenishment of cell wall ASC and the distribution of ASC between sub-cellular compartments – based upon the permeability of biomembranes to the neutral species, ascorbic acid and the pH of various sub-cellular compartments. The importance of various physico-chemical characteristics (e.g. stomatal conductance, mesophyll cell wall thickness and tortuosity, chloroplast volume, apoplast pH, ASC:O₃ reaction stoichiometry) in mediating the flux of ozone to the plasmalemma is analysed. Model simulations, supported by experimental observations, suggest that the ASC concentration in the leaf apoplast is high enough to scavenge a significant proportion of the O₃ taken up into the leaf interior, under environmentally relevant conditions. However, there is considerable variation between taxa in the potential degree of protection afforded by apoplastic ASC, emphasizing the need for an improved understanding of the reaction chemistry of O₃ in the cell wall. Received: 13 May 1999 / Accepted: 5 August 1999     

The apoplastic antioxidant enzymatic system in the wood-forming tissues of trees

The complete apoplastic enzymatic antioxidant system, composed by class I ascorbate peroxidases (class I APXs), class III ascorbate peroxidases (class III APXs), ascorbate oxidases (AAOs), and other class III peroxidases (PRX), of wood-forming tissues has been studied in Populus alba, Citrus aurantium, and Eucalyptus camaldulensis. The aim was to ascertain whether these enzymatic systems may regulate directly (in the case of APXs), or indirectly (in the case of AAOs), apoplastic H₂O₂ levels in lignifying tissues, whose capacity to produce and to accumulate H₂O₂ is demonstrated here. Although class I APXs are particularly found in the apoplastic fraction of P. alba (poplar), and class III APXs are particularly found in the apoplastic fraction of C. aurantium (bitter orange tree), the results showed that the universal presence of AAO in the extracellular cell wall matrix of these woody species provokes the partial or total dysfunction of apoplastic class I and class III APXs, and of the whole plethora of non-enzymatic redox shuttles in which ascorbic acid (ASC) is involved, by the competitive and effective removal of ASC. In fact, the redox state (ASC/ASC+DHA) in intercellular wash fluids (IWFs) of these woody species was zero, and thus strongly shifted towards DHA (dehydroascorbate), the oxidized product of ASC. This imbalance of the apoplastic antioxidant enzymatic system apparently results in the accumulation of H₂O₂ in the apoplast of secondary wood-forming tissues, as can be experimentally observed. Furthermore, it is hypothesized that since AAO uses O₂ to remove ASC, it could regulate O₂ availability in the lignifying xylem and, thorough this mechanism, AAO could also control the activity of NADPH oxidase (the enzyme responsible for H₂O₂ production in lignifying tissues) at substrate level, by controlling the tension of O₂. That is, the presence of AAO in the extracellular cell wall matrix appears to be essential for finely tuning the oxidative performance of secondary wood-forming tissues.     

The activity of antioxidative enzymes,contents of H<Subscript>2</Subscript>O<Subscript>2</Subscript> and of ascorbate in tomato leaves of cultivars more or less sensitive to infection with <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

The aim of the present studies was to compare H₂O₂ and ascorbate contents as well as peroxidase (PO) and catalase (CAT) activities in leaves of less susceptible cultivar Perkoz and more susceptible Corindo after B. cinerea infection. Increase in H₂O₂ contents in both Perkoz and Corindo cytosol was observed, however, it appeared earlier in the less susceptible cultivar. The increase in PO activity in the cytosol fraction was observed 48 hours after infection in both cultivars but it was greater in the less susceptible Perkoz. No significant differences between the tested cultivars were observed in ascorbate peroxidase (APX) activity and in reduced and oxidated ascorbate contents. PO activity was thoroughly analyzed in the apoplast fraction. It was measured with syringaldazine (S), tetramethylbenzidine (TMB) and ferulic acid (FA)—substrates characteristic of isoenzymes involved in lignification and stiffening of a cell wall. Increase in PO activity with these substrates was observed earlier in cultivar Perkoz than in cultivar Corindo. Similarly, increase in PO activity with NADH appeared significantly earlier in cultivar Perkoz. Apoplastic PO was separated with DEAE Sepharose and two fractions binding and non-binding were obtained. Binding PO fraction was significantly more active especially with S, TMB and NADH after B. cinerea infection. The increase in the enzyme activity was mostly observed in cultivar Perkoz. Binding PO was separated by electrophoresis on acrylamide gel and revealed six enzymatic forms from which three were much more active after infection in cultivar Perkoz. The obtained results suggest that cell wall strengthening mediated by apoplast PO is a key factor responsible for different resistance of tomato cultivars Perkoz and Corindo to B. cinerea infection.     

Cytochemistry and reactive oxygen species: a retrospective

This retrospective reviews the methodology we have developed over several decades for detecting reactive oxygen species (ROS), using the activated polymorphonuclear leukocyte (PMN) as the paradigm of a cell which vigorously generates ROS through activation of NADPH oxidase. In the seventies, the sites of ROS generation by PMN were not clear from biochemical data, and we sought to develop new methods for the cytochemical localization of O^·– ₂, H₂O₂, and the H₂O₂-myeloperoxidase (MPO)-halide system. The H₂O₂-MPO-halide system in phagocytosing cells was localized at the fine structural level by our development of 3,3-diaminobenzidine (DAB) as a cytochemical probe for detecting peroxidase activities. Using DAB and exogenous H₂O₂, we confirmed that azurophil granules discharged MPO into the phagosome, and using particles coated with DAB and relying on endogenous H₂O₂ to yield oxidized DAB, H₂O₂ was localized to phagolysosomes. The subcellular sites of H₂O₂ generation were shown using cerium ions which react with H₂O₂ and precipitate electron opaque cerium perhydroxides (Ce(OH)₂OOH and Ce(OH)₃OOH). The results suggested that NADPH oxidase is associated with the plasmalemma, and that the enzyme enters the phagosome along with the invaginating plasmalemma, accounting for the presence of H₂O₂ in the phagosome. As O^·– ₂ is the major product of NADPH oxidase, its detection was of some importance. Based on the concept that O^·– ₂ oxidizes Mn²⁺ to Mn³⁺, and Mn³⁺ oxidizes DAB, a medium containing DAB-Mn²⁺ was used to localize sites of O^·– ₂ production in stimulated PMN. The localizations were, as expected, similar to those for H₂O₂. These techniques have been of considerable usefulness and in general provide the foundation for cytochemistry of ROS in other systems.Presented at the 36th Symposium of the Society for Histochemistry, 22 September 1994, Heidelberg, Germany     

Reactive oxygen species are involved in regulation of pollen wall cytomechanics

Production and scavenging of reactive oxygen species (ROS) in somatic plant cells is developmentally regulated and plays an important role in the modification of cell wall mechanical properties. Here we show that H₂O₂ and the hydroxyl radical (^?OH) can regulate germination of tobacco pollen by modifying the mechanical properties of the pollen intine (inner layer of the pollen wall). Pollen germination was affected by addition of exogenous H₂O₂, ^?OH, and by antioxidants scavenging endogenous ROS: superoxide dismutase, superoxide dismutase/catalase mimic Mn‐5,10,15,20‐tetrakis(1‐methyl‐4‐pyridyl)21H, 23H‐porphin, or a spin‐trap α‐(4‐pyridyl‐1‐oxide)‐N‐tert‐butylnitrone, which eliminates ^?OH. The inhibiting concentrations of exogenous H₂O₂ and ^?OH did not decrease pollen viability, but influenced the mechanical properties of the wall. The latter were estimated by studying the resistance of pollen to hypo‐osmotic shock. ^?OH caused excess loosening of the intine all over the surface of the pollen grain, disrupting polar growth induction. In contrast, H₂O₂, as well as partial removal of endogenous ^?OH, over‐tightened the wall, impeding pollen tube emergence. Feruloyl esterase (FAE) was used as a tool to examine whether H₂O₂‐inducible inter‐polymer cross‐linking is involved in the intine tightening. FAE treatment caused loosening of the intine and stimulated pollen germination and pollen tube growth, revealing ferulate cross‐links in the intine. Taken together, the data suggest that pollen intine properties can be regulated differentially by ROS. ^?OH is involved in local loosening of the intine in the germination pore region, while H₂O₂ is necessary for intine strengthening in the rest of the wall through oxidative coupling of feruloyl polysaccharides.     

Generation of Hydroxyl Radical in Isolated Pea Root Cell Wall, and the Role of Cell Wall-Bound Peroxidase, Mn-SOD and Phenolics in Their Production

The hydroxyl radical produced in the apoplast has been demonstratedto facilitate cell wall loosening during cell elongation. Cellwall-bound peroxidases (PODs) have been implicated in hydroxylradical formation. For this mechanism, the apoplast or cellwalls should contain the electron donors for (i) H₂O₂ formationfrom dioxygen; and (ii) the POD-catalyzed reduction of H₂O₂to the hydroxyl radical. The aim of the work was to identifythe electron donors in these reactions. In this report, hydroxylradical (·OH) generation in the cell wall isolated frompea roots was detected in the absence of any exogenous reductants,suggesting that the plant cell wall possesses the capacity togenerate ·OH in situ. Distinct POD and Mn-superoxidedismutase (Mn-SOD) isoforms different from other cellular isoformswere shown by native gel electropho-resis to be preferably boundto the cell walls. Electron paramagnetic resonance (EPR) spectroscopyof cell wall isolates containing the spin-trapping reagent,5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO),was used for detection of and differentiation between ·OHand the superoxide radical (O₂^–·). The data obtainedusing POD inhibitors confirmed that tightly bound cell wallPODs are involved in DEPMPO/OH adduct formation. A decreasein DEPMPO/OH adduct formation in the presence of H₂O₂ scavengersdemonstrated that this hydroxyl radical was derived from H₂O₂.During the generation of ·OH, the concentration of quinhydronestructures (as detected by EPR spectroscopy) increased, suggestingthat the H₂O₂ required for the formation of ·OH in isolatedcell walls is produced during the reduction of O₂ by hydroxycinnamicacids. Cell wall isolates in which the proteins have been denaturated(including the endogenous POD and SOD) did not produce ·OH.Addition of exogenous H₂O₂ again induced the production of ·OH,and these were shown to originate from the Fenton reaction withtightly bound metal ions. However, the appearance of the DEPMPO/OOHadduct could also be observed, due to the production of O₂^–·when endogenous SOD has been inactivated. Also, O₂^–·was converted to ·OH in an in vitro horseradish peroxidase(HRP)/H₂O₂ system to which exogenous SOD has been added. Takentogether with the discovery of the cell wall-bound Mn-SOD isoform,these results support the role of such a cell wall-bound SODin the formation of ·OH jointly with the cell wall-boundPOD. According to the above findings, it seems that the hydroxycinnamicacids from the cell wall, acting as reductants, contribute tothe formation of H₂O₂ in the presence of O₂ in an autocatalyticmanner, and that POD and Mn-SOD coupled together generate ·OHfrom such H₂O₂.     

Changes in the ascorbate metabolism of apoplastic and symplastic spaces are associated with cell differentiation

Ascorbate levels and redox state, as well as the activities of the ascorbate related enzymes, have been analysed both in the apoplastic and symplastic spaces of etiolated pea (Pisum sativum L.) shoots during cellular differentiation. The ascorbate pool and the ascorbate oxidizing enzymes, namely ascorbate oxidase and ascorbate peroxidase, were present in both pea apoplast and symplast, whereas ascorbate free radical reductase and dehydroascorbate reductase were only present in the symplastic fractions. During cell differentiation the ascorbate redox enzymes changed in different ways, since a decrease in ascorbate levels, ascorbate peroxidase and ascorbate free radical reductase occurred from meristematic to differentiated cells, whereas ascorbate oxidase and dehydroascorbate reductase increased. The activity of secretory peroxidases has also been followed in the apoplast of meristematic and differentiating cells. These peroxidases increased their activity during differentiation. This behaviour was accompanied by changes in their isoenzymatic profiles. The analysis of the kinetic characteristics of the different peroxidases present in the apoplast suggests that the presence of ascorbate and ascorbate peroxidase in the cell wall could play a critical role in regulating the wall stiffening process during cell differentiation by interfering with the activity of secretory peroxidases.     

Oxidative reactions in axenically seedling roots of olive (Olea europaea, L.)

The production of reactive oxygen species (ROS) plays important roles in the life cycle and in the stress response and defence mechanisms of plants. Various enzyme systems are involved in the formation of ROS in the apoplast, including plasmalemma NADPH oxidase and apoplastic peroxidases. The production of O ₂ ^·? and apoplastic peroxidase and exogenous NADH oxidation activities are all strongly dependent on the age of roots??the younger the root, the greater the activity. Apoplastic production of ROS is shown in the root by using specific histochemical probes, this ROS production is growing zone dependent. In the present study, using olive seedlings, differences were also observed between cultivars, especially in O ₂ ^·? production by the Verdial cultivar which was well above that of other cultivars studied. In all the cultivars, treatment of roots with methyl jasmonate (MeJA) or methyl salicylate (MeSA) increased O ₂ ^·? production. Similar results were observed for peroxidase activity, but not for the oxidation of exogenous NADH which was either unaffected (MeJA) or even partially inhibited (MeSA). A conclusion was that MeJA or MeSA induced apoplastic production of ROS does not use exogenous NADH. Treatment with diphenylene iodonium (DPI) reduced the formation of O ₂ ^·? , but affected neither peroxidase nor NADH oxidation activities. Cyanide inhibited O ₂ ^·? production and peroxidase and NADH oxidation activities. Treatment with MnCl₂ had a strong stimulatory effect on peroxidase and NADH oxidation activities, but much less on O ₂ ^·? production. Finally, azide greatly reduced all activities, but especially O ₂ ^·? production. Together, these results indicate a relationship between oxidative activities and the processes of root growth, and that those activities are also dependent on the cultivar, as well as an involvement of peroxidases and plasmalemma NADPH oxidase in apoplast ROS production which is sensitive to DPI, azide, and cyanide but relatively insensitive to MnCl₂, while exogenous NADH oxidation is linked to peroxidase activity.     

Expression of a Trichoderma reesei β-1,4 endo-xylanase in tall fescue modifies cell wall structure and digestibility and elicits pathogen defence responses

An endo-xylanase from Trichoderma reesei (xyn2) has been expressed in tall fescue targeted to the vacuole, apoplast or Golgi, constitutively under the control of the rice actin promoter, and to the apoplast under the control of a senescence enhanced gene promoter. Constitutive xylanase expression in the vacuole, apoplast, and golgi, resulted in only a small number of plants with low enzyme activities and in reduced plant growth in apoplast, and golgi targeted plants. Constitutive expression in the apoplast also resulted in increased levels of cell wall bound hydroxycinnamic acid monomers and dimers, but no significant effect on cell wall xylose or arabinose content. In situ constitutive xylanase expression in the Golgi also resulted in increased ferulate dimers. However, senescence induced xylanase expression in the apoplast was considerably higher and did not affect plant growth or the level of monomeric hydroxycinnamic acids or lignin in the cell walls. These plants also showed increased levels of ferulate dimers, and decreased levels of xylose with increased levels of arabinose in their cell walls. While the release of cell wall hydroxycinnamic acids on self digestion was enhanced in these plants in the presence of exogenously applied ferulic acid esterase, changes in cell wall composition resulted in decreases in both tissue digestibility and cellulase mediated sugar release. In situ detection of H₂O₂ production mediated by ethylene release in leaves of plants expressing apoplast xylanase could be leading to increased dimerisation. High-level xylanase expression in the apoplast also resulted in necrotic lesions on the leaves. Together these results indicate that xylanase expression in tall fescue may be triggering plant defence responses analogous to foliar pathogen attack mediated by ethylene and H₂O₂.     

Generation of superoxide anion and hydrogen peroxide at the surface of plant cells

In addition to well-known cell wall peroxidases, there is now evidence for the presence of this enzyme at the plasma membrane of the plant cells (surface peroxidase). Both are able to catalyze, through a chain of reactions involving the superoxide anion, the oxidation of NADH to generate hydrogen peroxide. The latter is oxidized by other wall-bound peroxidases to convert cinnamoyl alcohols into radical forms, which, then polymerize to generate lignin. However, there are other enzymes at the surface of plasma membranes capable of generating hydrogen peroxide (cell wall polyamine oxidase), superoxide anion (plasma membrane Turbo reductase), or both (plasma membrane flavoprotein?). These enzymes utilize NAD(P)H as a substrate. The Turbo reductase and the flavoprotein catalyze the univalent reduction of Fe³⁺ and then of O₂ to produce Fe²⁺ and \(O_2^{\bar \cdot } \) , respectively. The superoxide anion, in the acidic environment of the cell wall, may then dismutate to H₂O₂. These superoxide anion- and hydrogen peroxide-generating systems are discussed in relation to their possible involvement in physiological and pathological processes in the apoplast of plant cells.     

Rheological analysis of viscoelastic cell wall changes in maize coleoptiles as affected by auxin and osmotic stress

The effects of auxin and osmotic stress on elongation growth of maize (Zea mays L.) coleoptile segments are accompanied by characteristic changes in the extensibility of the growth-limiting cell walls. At full turgor auxin causes growth by an increase in wall extensibility (wall looseining). Growth can be stopped by an osmotically produced step-down in turgor of 0.45 MPa. Under these conditions auxin causes the accumulation of a potential for future wall extension which is released after restoration of full turgor. Turgor reduction causes a reversible decrease in wall extensibility (wall stiffening) both in the presence and absence of auxin. These changes in vivo are correlated with corresponding changes in the rheological properties of the cell walls in vitro which can be traced back to specific modifications in the shape of the hysteretic stress-strain relationship. The longitudinally load-bearing walls of the coleoptile demonstrate almost perfect viscoelasticity as documented by a nearly closed hysteresis loop. Auxin-mediated wall loosening causes an increase of loop width and thus affects primarily the amount of hysteresis in the isolated wall. In contrast, turgor reduction by osmotic stress reduces loop length and thus affects primarily the amount of viscoelastic wall extensibility. Pretreatment of segments with anoxia and H₂O₂ modify the hysteresis loop in agreement with the conclusion that the wall-stiffening reaction visualized under osmotic stress in vivo is an O₂-dependent process in which O₂ can be substituted by H₂O₂. Cycloheximide specifically inhibits auxin-mediated wall loosening without affecting wall stiffening, and this is mirrored in specific changes of the hysteresis loop. Corroborating a previous in vivo study (Hohl et al. 1995, Physiol. Plant. 94: 491–498) these results show that cell wall stiffening in vivo can also be demonstrated by Theological measurements with the isolated cell wall and that this process can be separated from cell wall loosening by specific changes in the shape of the hysteresis loop.     

Plasma membrane-generated reactive oxygen intermediates and their role in cell growth of plants

Reactive oxygen species (ROS) produced as intermediates in the reduction of O2 to H2O (superoxide radical, hydrogen peroxide, hydroxyl radical), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry can also play useful roles if it takes place outside of the protoplast. In plants, the production of these ROS initiated by the plasma membrane NAD(P)H oxidase can be used for controlled polymer breakdown leading to wall loosening during extension growth. Backbone cleavage of cell wall polysaccharides can be accomplished by hydroxyl radicals produced from hydrogen peroxide and superoxide in a reaction catalyzed by cell wall peroxidase. Growing plant organs such as coleoptiles or roots of maize seedlings produce these ROS specifically in the apoplast of actively growing tissues, e.g. in the epidermis of the coleoptile and the growing zone of the root. Auxin promotes the release of hydroxyl radicals when inducing elongation growth. Experimental generation of hydroxyl radicals in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of ROS or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which the generation of hydroxyl radicals, initiated by the plasma membrane NAD(P)H oxidase, plays a central role.