Cellular ascorbic acid regulates the activity of major peroxidases in the apical poles of germinating white spruce (Picea glauca) somatic embryos.

In previous studies we have reported that applications of ascorbic acid (ACS) enhance the conversion frequency of white spruce somatic embryos by "rescuing" structurally disorganized meristems and inducing cell proliferation in the apical poles [C. Stasolla, E.C. Yeung, Ascorbic acid improves the conversion of white spruce somatic embryos, In Vitro Cell. Dev. Biol. Plant 35 (1999) 316-319]. In order to determine if the role played by this metabolite during embryo conversion is mediated by cellular peroxidases, the activity of guaiacol-, ferulic acid-, and ascorbic acid-dependent peroxidases were measured in the apical poles of germinating embryos with altered ASC levels. Changes in the endogenous ASC pool were achieved by treating the embryos with exogenously supplied ASC, L-galactono-gamma-lactone (GL) the last precursor of the de novo biosynthesis of ASC, and lycorine (L), an inhibitor of the last reaction leading to the synthesis of ASC. Our studies demonstrate the existence of a negative correlation between cellular ASC levels and activities of both guaiacol and ferulic acid peroxidases in root and shoot apices. A depletion of cellular ASC enhanced the rate of both guaiacol and ferulic acid oxidation at the apical poles of the embryos and resulted in meristem abortion. In contrast, the activity of guaiacol and ferulic acid peroxidases decreased below control levels if the endogenous ASC content of the embryos was experimentally increased. Fluctuations of total peroxidase activity following alterations in ASC pool were also confirmed by histochemical staining and in vitro studies. Overall our results suggest that a threshold of ASC level must be maintained in the apical poles of germinating embryos in order to inhibit peroxidase activities from cross-linking cell wall components and preventing post-embryonic growth.     

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.     

Zonal changes in ascorbate and hydrogen peroxide contents,peroxidase, and ascorbate-related enzyme activities in onion roots

Onion (Allium cepa) roots growing hydroponically show differential zonal values for intra- (symplastic) and extra- (apoplastic) cellular ascorbate (ASC) and dehydroascorbate (DHA) contents and for related enzyme activities. In whole roots, ASC and DHA concentrations were higher in root apex and meristem and gradually decreased toward the root base. Guaiacol peroxidase, ASC peroxidase, monodehydroascorbate oxidoreductase, DHA reductase, catalase, and glutathione reductase activities showed differential activity patterns depending on the zone of the root and their apoplastic or symplastic origin. An in vivo staining of peroxidase activity also revealed a specific distribution pattern along the root axis. Using electron microscopy, hydrogen peroxide was found at different locations depending on the root zone but was mainly located in cell walls from epidermal and meristematic cells and in cells undergoing lignification. A balanced control of all of these molecules seems to exist along the root axis and may be directly related to the mechanisms in which the ASC system is involved, as cell division and elongation. The role of ASC on growth and development in relation to its presence at the different zones of the root is discussed.     

Redox state of ascorbic acid in the apoplast of stems of Kalanchoë daigremontiana

The aqueous phase of cell walls in stems of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie (apoplast) contained ascorbic acid (AA) and dehydroascorbic acid (DHA). Ratios of AA/(AA + DHA) were 0.31 ± 0.12 (SD, n = 4), whereas those of whole stems (tissues plus apoplast) were >0.9. The amounts of (AA + DHA) in the stems were 1970 ± 190 (SD, n = 4) nmol g⁻¹ fresh weight and those in the apoplast were 14 ± 2 (SD, n = 4) nmol g⁻¹ fresh weight of stems. Ratios of AA/(AA + DHA) differed in different tissues of the stems. The ratios of AA/(AA + DHA) of apoplast plus symplast were in the following order: pith ⋍ epidermis plus cortex > vascular bundle system, and those of apoplast were: pith > epidermis plus cortex > vascular bundle system. Ratios of AA/(AA + DHA) in the apoplast of the different tissues decreased to about 1/3 of the original values after wounding, while the amounts of (AA + DHA) remained largely unaffected. In contrast, soluble apoplastic peroxidase activities increased 30- to 70-fold on wounding. Hydrogen peroxide infiltrated into stems caused a rapid oxidation of AA. Coniferyl alcohol was oxidized by peroxidase in intercellular washing fluid and by cell wall-bound peroxidase. The oxidation of coniferyl alcohol by peroxidase in intercellular washing fluid was completely inhibited as long as AA was present in reaction mixtures. The oxidation of the coniferyl alcohol by cell wall-bound peroxidase was partially inihibited by AA and the degree of inhibition was dependent upon the concentration of AA. The possible functions of AA in the apoplast are discussed in relation to the control of peroxidase-dependent oxidation of phenolics.     

Components of apoplastic ascorbate use in Betula pendula leaves exposed to CO2 and O3 enrichment

Here, the aim was to estimate loads imposed on the apoplastic ascorbate (ASC) pool by enzymatic and nonenzymatic reactions in Betula pendula exposed to doubled CO2 and O3 concentrations in open-top chambers. Leaf apoplastic extracts were analysed for peroxidase and oxidase activities in vitro, using different substrates. Partial loads in vivo were deduced using measured kinetic constants and substituted-enzyme catalysis approaches. Ascorbate use in O3 scavenging was calculated using measured stomatal conductances and ASC concentrations. Under elevated O3, stomatal conductance and O3 uptake were higher. O3 fluxes to the plasmalemma were levelled off by higher apoplastic ASC concentrations. The effect of CO2 enrichment on ASC concentrations under elevated O3 was minor. Under ambient O3, the ascending hierarchy of ASC users was: peroxidases, O3 scavenging, oxidases, coniferyl alcohol re-reduction. Under elevated O3, ASC use in O3 scavenging was higher than by oxidases. The redox state of ASC was not depressed by O3; there was no leaf injury. The cell wall/plasmalemma/cytosol system in birch had sufficient capacity to maintain ASC redox status in the apoplast, without necessity to restrict O3 uptake by stomatal closure.     

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     

Licorice compounds glycyrrhizin and 18beta-glycyrrhetinic acid are potent modulators of bile acid-induced cytotoxicity in rat hepatocytes

The accumulation of hydrophobic bile acids results in cholestatic liver injury by increasing oxidative stress, mitochondrial dysfunction, and activation of cell signaling pathways. Licorice root and its constituents have been utilized as antihepatotoxic agents. The purpose of this study was to evaluate the potential modulation by a primary component of licorice root, glycyrrhizin (GL), and its metabolite, 18beta-glycyrrhetinic acid (GA), in a hepatocyte model of cholestatic liver injury. Preincubation of fresh rat hepatocyte suspensions with GL or GA reduced glycochenodeoxycholic acid (GCDC)-dependent reactive oxygen species generation, with GA more potent than GL. Interestingly, GL and GA had opposing effects toward GCDC-induced cytotoxicity; GA prevented both necrosis and apoptosis, whereas GL enhanced apoptosis. GCDC promoted activation of caspase 10, caspase 3, and PARP; all were inhibited by GA but not GL. Induction of apoptosis by GCDC was also associated with activation of JNK, which was prevented by GA. Activation of caspase 9 and dissipation of mitochondrial membrane potential were prevented by GA but not GL. In liver mitochondrial studies, GL and GA were both potent inhibitors of the mitochondrial permeability transition, reactive oxygen species generation, and cytochrome c release at submicromolar concentrations. Results from this study suggest that GL exhibits pro-apoptotic properties, whereas GA is a potent inhibitor of bile acid-induced apoptosis and necrosis in a manner consistent with its antioxidative effect.     

Swelling of Root Cell Walls as an Indicator of Their Functional State

The swelling capacity of cell walls isolated from different parts of lupine root was investigated. The water content in fragments of intact roots (Q) and swelling coefficient of standardized samples of cell walls (K^cw) were determined, and the dependences of Q and Kcw on the distance from the root tip (L) were plotted. It was shown that the change in Q value along the stretch of the lupine root reaches its maximum at distances of 1.5-6 cm or 7-12 cm from the root tip in 7-day-old and 14-day-old seedlings, respectively, whereas the K^cw value distribution over the root length is virtually invariable. In the radial direction, both the Q and K^cw values in cortex tissues are about twice higher than in the central cylinder. In our opinion, the changes of both Q and K^cw in the radial direction are associated with different degrees of cross-linking between polymer chains in cell wall structures of root cortex and central cylinder. The results of measurement of the K^cw value are consistent with the widely accepted mechanisms of water transport in roots in the radial direction. These data show that water transport through apoplast to the border between the cortex and central cylinder is accompanied by an increase in the resistance to water flow. Among other factors, this increase is due to a greater degree of cross-linking between cell wall polymers in the central cylinder. The results of measurement of the swelling coefficient of standardized cell wall samples in water and in 10 mM KCl at different pH values show that the swelling capacity of root cell walls varies according to the physicochemical properties of synthetic ion exchangers. Cell walls shrink (cell wall volume decreases) as ion concentration in solution increases and pH decreases. This causes an increase in the hydraulic resistance (or a decrease in the hydraulic conductivity) of apoplast. It was concluded that swelling is determined by the physicochemical properties of the cell wall, whereas the change in the swelling capacity induced by variation of external or internal conditions is an element of the mechanism of regulation of volume water flow in roots.     

Ascorbate transport from the apoplast to the symplast in intact leaves

Infiltration of reduced ascorbate (ASC) into the leaves of Betula pendula Roth and subsequent measurement of its loss therein after incubation allowed us to follow ascorbate transport from apoplast to symplast in intact leaves. All of the ascorbate extracted from the native apoplast was in fully oxidized form, dehydroascorbate (DHA). When 5 m M of ASC was infiltrated into the leaves, its intense decay occurred, but only 55% of ASC lost was recovered in apoplast as DHA. When ASC was added to the freshly extracted intercellular washing fluid (IWF), ASC oxidation occurred as well. However, all oxidized ASC was recovered as DHA, indicating that further decomposition of DHA did not occur. Similarly, all of the ASC infiltrated into the leaves was found therein either as ASC or DHA after incubation of leaves for up to 60 min. On this base the ascorbate infiltrated into the leaves and not recovered in the IWF was interpreted as ascorbate taken up into the symplast. The calculated uptake rates of ascorbate at different ASC concentrations followed saturation kinetics with the maximum uptake rate of 300 nmol m⁻² plasma membrane (PM) area min⁻¹ and Michaelis constant of 12.8 m M . The uptake of ascorbate was significantly inhibited by the addition of dithiothreitol or by PM H⁺ ATPase inhibitor erythrosin B. Thus, our results support the previous observations that DHA is preferably transported from the apoplastic to the cytoplasmic side of the membrane and show that this process is dependent upon PM proton gradient.     

The Effects of Ascorbate on Root Regeneration in Seedling Cuttings of Tomato

The changes in ascorbate (ASC) and dehydroascorbate (DHA) levels and the activities of ascorbate metabolising enzymes were examined during adventitious root formation in cuttings of tomato (Lycopersicon esculentum Mill. cv. Paw) seedlings. The effects of ASC, DHA and the immediate ascorbate precursor – galactono-γ-lactone (GalL) supplemented to the culture medium on the rooting response, ascorbate content and the activities of the ASC-metabolising enzymes were also investigated. The cuttings treated with abovementioned compounds formed more roots then control plants. However, in contrast to the number of regenerated organs, the elongation of newly formed roots was markedly inhibited. Treatment with auxin (IAA) resulted in a similar phenotype. The inhibitor of auxin polar transport-TIBA (2,3,5-triiodobenzoic acid) effectively blocked rooting. The inhibitory effect of TIBA was reversed by auxin and ASC treatments, while DHA and GalL were ineffective. Both auxin and ASC stimulated cell divisions in an area of pericycle layer of TIBA-treated rooting zones, that enabled cuttings to form roots in the presence of the inhibitor of auxin polar transport. It has been found that the first stages of rooting, preceding the emergence of roots, are accompanied by an increase in endogenous content of ASC with a peak in the 3rd day of rooting. Subsequent stages, when elongation of newly formed roots occurs, are characterised by low level of ASC. The activities of the ascorbate peroxidase (APX), ascorbate oxidase (AOX), ascorbate free radical reductase (AFRR) and dehydroascorbate reductase (DHR) increased in the first 3 days of root formation. The initial period of rooting was also accompanied by the increase of the hydrogen peroxide content and the activities of catalase (CAT) and guaiacol peroxidase (GPX) in the rooting zones. IAA, ASC, DHA as well as Gal stimulated the APX activity, however the rise of the enzyme's activity induced by ASC, DHA and Gal was reversed by TIBA, which was found to inhibit APX. Only exogenous IAA was able to maintain the high level of APX activity in the TIBA-treated cuttings. AOX was strongly affected by ASC and GalL – treatments, its activity increased in the cuttings grown on the media containing ASC in the absence as well as in the presence of TIBA. On the other hand, GalL-dependent stimulation of its activity was suppressed if TIBA was present in a rooting medium.     

Methodological approaches for studying apoplastic redox activity: 2. Regulation of peroxidase activity

This work is devoted to the study of mechanisms of substrate regulation of extracellular peroxidase (ECPOX) activity at a distant stress (wounding) signal transmission from aboveground organ (leaf) of wheat (Triticum aestivum L., cv. Kazanskaya Yubileinaya) seedlings to the roots. Along with the high dianizidine peroxidase activity, the extracellular solution manifested 3,4-dihydrooxi-L-phenylalanine peroxidase, ascorbate peroxidase, and catalase activities. Dianizidine peroxidases were represented by several isoforms and had broad substrate specificity. It was found that ECPOX was released from the roots into the growing solution and its activity in the solution increased with root growth. Excision of the apical leaf parts in seedlings induced a sharp activation of root ECPOX in the growing solution. The interaction between ECPOX substrates at oxidation in two- and three-component systems is demonstrated. The role of ECPOX in the control of ROS balance in the plant cell apoplast might be determined by competitive and complementary interactions between different peroxidase substrates. Such substrate-substrate regulation of peroxidase activities may be important for stress-induced oxidative burst in plant cells.     

水分胁迫诱导玉米叶片质外体产生H2O2机理

通过组织化学染色、电镜观察、酶活性分析对水分胁迫诱导玉米叶片质外体产生H2O2进行了研究。结果表明:水分胁迫能够诱导玉米叶片内源ABA的积累,ABA参与了水分胁迫诱导的玉米叶片H2O2的产生,质膜NADPH氧化酶、细胞壁过氧化物酶(POD)以及质外体多胺氧化酶(PAO)是水分胁迫诱导玉米细胞在质外体产生H2O2的来源,其中质膜NADPH氧化酶是主要来源;内源ABA的积累参与了水分胁迫激活的质膜NADPH氧化酶、细胞壁POD和质外体PAO活性的提高。研究认为,水分胁迫诱导玉米细胞在质外体产生H2O2可能是由于水分胁迫下内源ABA的积累通过激活质膜NADPH氧化酶、细胞壁POD以及质外体PAO的活性而实现的。     

Activity of Enzymes Related to H2O2 Generation and Metabolism in Leaf Apoplastic Fraction of Tomato Leaves Infected with Botrytis cinerea

Hydrogen peroxide generation rates of uninfected and infected leaves of two tomato (Lycopersicon esculentum) cultivars showing differential susceptibility to Botrytis cinerea were determined. The superoxide anion, hydroxyl radical, ascorbate contents and changes in NADH peroxidase, superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities in the apoplast fraction were analysed. Infected leaves had an increased hydrogen peroxide level. It was greater and generally occurred earlier in plants of the less susceptible cv. Perkoz than in those of the more susceptible cv. Corindo. Induction of nitrotetrazolium blue reducing activity and SOD levels in apoplast were higher in cv. Perkoz 24 h after inoculation. In the controls, NADH peroxidase activity in apoplast was higher in the more susceptible cv. Corindo, but after infection it increased faster and to a higher level in the less susceptible cv. Perkoz. NADH oxidation was inhibited by only 15% by a specific inhibitor DPI (diphenylene‐iodonium) but was completely inhibited by KCN and NaN₃. Similar increases in APX activity after 48 h and a small increase in catalase activities were observed in both cultivars soon after infection. These results indicate that resistance of tomato plants to infection by the necrotrophic fungus B. cinerea may result from early stimulation of hydrogen peroxide and superoxide radical generations by NADH peroxidase and SOD in apoplastic space, and they confirm the important role of their enhanced production in apoplastic spaces of plants.     

In vitro salt tolerance of cell wall enzymes from halophytes and glycophytes

The halophyte Suaeda maritima grows optimally in high concentrations(40–60% seawater) of salt. In these conditions the concentrationof salt in the apoplast of the leaves is at least 500 mM, aconcentration which severely inhibits the activity of cytoplasmicenzymes of both glycophytes and halophytes. The in vitro salttolerance of a number of cell wall enzymes was assayed in thepresence of a range of concentrations of NaCl. There was nosignificant inhibition of the activity of galactosidase, glucosidase,peroxidase or xyloglucan endo-transglycosylase extracted fromSuaeda maritima by in vitro concentrations of NaCl up to atleast 1 M. In vitro salt tolerance of cell wall enzymes wasnot restricted to the halophyte, similar enzymes from the non-halophilicrelative Kochia tricophylla, and from the glycophytes Vignaradiata and Cicer arietinum, were inhibited little, or not atall, by the same concentrations of salt. Pectin esterase wassomewhat less tolerant, but activity at 500 mM NaCl was stillgreater than at 0 mM NaCl in both Suaeda and Vigna. It is concludedthat these enzymes of the cell wall compartment are much moresalt-tolerant than cytoplasmic enzymes of higher plants. Theresults are discussed in relation to conditions thought to pertainin the apoplast. Key words: Apoplast, cell wall enzymes, halophyte, salt tolerance, Suaeda maritima     

Antioxidant Systems and O2.−/H2O2 Production in the Apoplast of Pea Leaves. Its Relation with Salt-Induced Necrotic Lesions in Minor Veins

The present work describes, for the first time, the changes that take place in the leaf apoplastic antioxidant defenses in response to NaCl stress in two pea (Pisum sativum) cultivars (cv Lincoln and cv Puget) showing different degrees of sensitivity to high NaCl concentrations. The results showed that only superoxide dismutase, and probably dehydroascorbate reductase (DHAR), were present in the leaf apoplastic space, whereas ascorbate (ASC) peroxidase, monodehydroascorbate reductase (MDHAR), and glutathione (GSH) reductase (GR) seemed to be absent. Both ASC and GSH were detected in the leaf apoplastic space and although their absolute levels did not change in response to salt stress, the ASC/dehydroascorbate and GSH to GSH oxidized form ratios decreased progressively with the severity of the stress. Apoplastic superoxide dismutase activity was induced in NaCl-treated pea cv Puget but decreased in NaCl-treated pea cv Lincoln. An increase in DHAR and GR and a decrease in ASC peroxidase, MDHAR, ASC, and GSH levels was observed in the symplast from NaCl-treated pea cv Lincoln, whereas in pea cv Puget an increase in DHAR, GR, and MDHAR occurred. The results suggest a strong interaction between both cell compartments in the control of the apoplastic ASC content in pea leaves. However, this anti-oxidative response does not seem to be sufficient to remove the harmful effects of high salinity. This finding is more evident in pea cv Lincoln, which is characterized by a greater inhibition of the growth response and by a higher rise in the apoplastic hydrogen peroxide content, O(2)(.-) production and thiobarbituric acid-reactive substances, and CO protein levels. This NaCl-induced oxidative stress in the apoplasts might be related to the appearance of highly localized O(2)(.-)/H(2)O(2)-induced necrotic lesions in the minor veins in NaCl-treated pea plants. It is possible that both the different anti-oxidative capacity and the NaCl-induced response in the apoplast and in the symplast from pea cv Puget in comparison with pea cv Lincoln contributes to a better protection of pea cv Puget against salt stress.     

Changes in intracellular and apoplastic peroxidase activity, ascorbate redox status, and root elongation induced by enhanced ascorbate content in Allium cepa L

Onions (Allium cepa L.) treated with external ascorbic acid or with the immediate precursor of its synthesis L-galactono-gamma-lactone show a stimulated elongation rate of the roots and an increase in the number of new radicles appearing at the bulb base. Treatment with both molecules resulted in an enhanced accumulation of ascorbate and dehydroascorbate along the root axis, but the distribution of these redox forms was not uniform along the root, as detected in intracellular (symplastic) and extracellular (apoplastic) compartments. Thus, those radicular zones metabolically more active, such as the meristem and the elongation zone, accumulated the highest amount of both redox forms of ascorbate. On the other hand, ascorbate and L-galactono-gamma-lactone also stimulated cytosolic glucose-6-phosphate dehydrogenase activity and inhibited peroxidase activity as deduced from in vivo and in vitro experiments. Differences were also found when comparing apoplastic and symplastic activities. These results are compatible with the idea of an ascorbate-mediated stimulation of root growth by inhibiting cell wall stiffening and increasing root metabolism.     

Salinity up-regulates the antioxidative system in root mitochondria and peroxisomes of the wild salt-tolerant tomato species Lycopersicon pennellii

The effect of salinity on the antioxidative system of root mitochondria and peroxisomes of a cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species L. pennellii (Lpa) was studied. Salt stress induced oxidative stress in Lem mitochondria, as indicated by the increased levels of lipid peroxidation and H(2)O(2). These changes were associated with decreased activities of superoxide dismutase (SOD) and guaiacol peroxidases (POD) and contents of ascorbate (ASC) and glutathione (GSH). By contrast, in mitochondria of salt-treated Lpa plants both H(2)O(2) and lipid peroxidation levels decreased while the levels of ASC and GSH and activities of SOD, several isoforms of ascorbate peroxidase (APX), and POD increased. Similarly to mitochondria, peroxisomes isolated from roots of salt-treated Lpa plants exhibited also decreased levels of lipid peroxidation and H(2)O(2) and increased SOD, ascorbate peroxidase (APX), and catalase (CAT) activities. In spite of the fact that salt stress decreased activities of antioxidant enzymes in Lem peroxisome, oxidative stress was not evident in these organelles.     

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.     

The application of ascorbate or its immediate precursor, galactono-1,4-lactone, does not affect the response of nitrogen-fixing pea nodules to water stress

Nitrogen fixation in legumes is dramatically inhibited by abiotic stresses, and this reduction is often associated with oxidative damage. Although ascorbate (ASC) has been firmly associated with antioxidant defence, recent studies have suggested that the functions of ASC are related primarily to developmental processes. This study examines the hypothesis that ASC is involved in alleviating the oxidative damage to nodules caused by an increase in reactive oxygen species (ROS) under water stress. The hypothesis was tested by supplying 5 mM ASC to pea plants (Pisum sativum L.) experiencing moderate water stress (ca. −1 MPa) and monitoring plant responses in relation to those experiencing the same water stress without ASC. A supply of exogenous ASC increased the nodule ASC+dehydroascorbate (DHA) pool compared to water-stressed nodules without ASC, and significantly modulated the response to water stress of the unspecific guaiacol peroxidase (EC 1.11.1.7) in leaves and nodules. However, ASC supply did not produce recovery from water stress in other nodule antioxidant enzymes, nodule carbon and nitrogen enzymes, or nitrogen fixation. The supply of the immediate ASC precursor, galactono-1,4-lactone (GL), increased the nodule ASC+DHA pool, but also failed to prevent the decline of nitrogen fixation and the reduction of carbon flux in nodules. These results suggest that ASC has a limited role in preventing the negative effects of water stress on nodule metabolism and nitrogen fixation.     

The GLABRA2 homeodomain protein directly regulates CESA5 and XTH17 gene expression in Arabidopsis roots

Arabidopsis root hair formation is determined by the patterning genes CAPRICE ( CPC ), GLABRA3 ( GL3 ), WEREWOLF ( WER ) and GLABRA2 ( GL2 ), but little is known about the later changes in cell wall material during root hair formation. A combined Fourier-transform infrared microspectroscopy–principal components analysis (FTIR-PCA) method was used to detect subtle differences in the cell wall material between wild-type and root hair mutants in Arabidopsis. Among several root hair mutants, only the gl2 mutation affected root cell wall polysaccharides. Five of the 10 genes encoding cellulose synthase ( CESA1 – 10 ) and 4 of 33 xyloglucan endotransglucosylase ( XTH1 – 33 ) genes in Arabidopsis are expressed in the root, but only CESA5 and XTH17 were affected by the gl2 mutation. The L1-box sequence located in the promoter region of these genes was recognized by the GL2 protein. These results indicate that GL2 directly regulates cell wall-related gene expression during root development.