Identification of an H2O2 permeable PIP aquaporin in barley and a serine residue promoting H2O2 transport

A barley (Hordeum vulgare) plasma membrane type aquaporin, HvPIP2;5, was identified as an H₂O₂ permeable aquaporin among 21 barley and rice PIPs examined in the heterologous expression system using Saccharomyces cerevisiae. Four TIPs were also detected as H₂O₂‐transporting aquaporins among 15 barley and rice TIPs. Influx of H₂O₂ into yeast cells expressing HvPIP2;5 was determined with a florescent‐dye‐based assay. Indirect immunofluorescence indicated that the expression of HvPIP2;5 protein was ubiquitous in root tissues, and was also weakly observed in leaf epidermal cells and cells in the vascular bundle. Point mutated variants of HvPIP2;5 were generated by the site‐directed mutagenesis. Growth assays of yeast cells expressing these mutated HvPIP2;5 proteins suggested that Ser‐126 in HvPIP2;5 has a large impact on H₂O₂ transport with a minor influence on the HvPIP2;5‐mediated water transport.     

Botrytis cinerea BcNma is involved in apoptotic cell death but not in stress adaptation

Apoptotic-like programmed cell death (PCD) occurs naturally in fungi during development and might also be induced by external conditions. Candidate apoptotic genes have been characterized in several model fungal species but not in plant pathogenic fungi. Here we report on the isolation and characterization of BcNMA, an orthologue of the human pro-apoptotic gene HtrA2 from the plant pathogen Botrytis cinerea. The predicted BcNma protein shows high homology to the previously characterized Nma111p from Saccharomyces cerevisiae and despite some structural differences it complemented the function of Nma111p in Δnma111 mutant strains. BcNMA-over-expression and mutant strains had enhanced or reduced appearance of apoptotic markers, respectively. However there was no difference in growth response of the wild type and BcNMA-transgenic strains to application of various stresses, and the effect on pathogenicity was marginal in both the over-expression and mutant strains. When considered together these results suggest that although BcNma has a pro-apoptotic activity, it is not a major regulator of apoptosis. The protein probably has additional roles that are unrelated to apoptosis, which lead to the pleotrophic phenotype of the transgenic strains and lack of a clear effect on stress adaptation and pathogenicity.     

Diamine oxidase is involved in H(2)O(2) production in the chalazal cells during barley grain filling

The localization and activities of diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.4.3.4) together with polyamine levels have been investigated in developing grains of barley (Hordeum vulgare L.). DAO (pH 7.5) is present mainly in vascular tissue and its neighbouring cells, namely chalazal cells and nucellar projection, while PAO (pH 6.0) is mainly localized in the chlorenchymatous cells of the crease and at the base of the vascular tissue. Activities of both these enzymes appear to be independently-regulated, as DAO activity increased steadily throughout grain development while PAO activity was higher during the early stages of grain filling, declined thereafter and again increased towards maturity. The maximum activities of DAO coincided with the maximum content of putrescine while the levels of PAO did not seem to be directly correlated with spermidine or spermine contents. Isoelectric focusing (IEF) of DAO and PAO activities revealed the presence of bands at 30 and 45 DPA. The possible involvement of DAO and PAO in the supply of H(2)O(2) to peroxidase-catalysed reactions in the chalazal cells during grain filling is discussed.     

Enhanced lipoxygenase activity is involved in the stress response but not in the harmful lipid peroxidation and cell death of short-term cadmium-treated barley root tip

Root growth inhibition and radial root swelling were the characteristic symptoms of barley root tips after the short-term exposure of roots to 15 and 30 μM Cd. Higher Cd concentrations caused extensive cell death and root growth arrest. Enhanced lipid peroxidation was observed as early as 1 h after the short-term treatment in a Cd concentration-dependent manner. In contrast to lipid peroxidation, the induction of lipoxygenase activity was detected only 3 h after the exposure of roots to 15 or 30 μM Cd. In addition, it was not observed in 60 μM Cd-treated root tips. The highest lipoxygenase activity was detected 6 h after 15 μM Cd treatment in the meristematic and elongation zone of root tip and was probably associated with the radial expansion of cells. Our results indicate that the upregulation of lipoxygenase is an important component of stress response in barley roots to toxic Cd. It is probably involved in the morphological stress response of root tips or/and in the alleviation of Cd-induced toxic alterations in plant cell membranes, but it is not responsible for the Cd-induced harmful lipid peroxidation and cell death.     

Periplasmic cytochrome c3 of Desulfovibrio vulgaris is directly involved in H2-mediated metal but not sulfate reduction

Kinetic parameters and the role of cytochrome c(3) in sulfate, Fe(III), and U(VI) reduction were investigated in Desulfovibrio vulgaris Hildenborough. While sulfate reduction followed Michaelis-Menten kinetics (K(m) = 220 micro M), loss of Fe(III) and U(VI) was first-order at all concentrations tested. Initial reduction rates of all electron acceptors were similar for cells grown with H(2) and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities. The similarities in metal, but not sulfate, reduction with H(2) and lactate suggest divergent pathways. Respiration assays and reduced minus oxidized spectra were carried out to determine c-type cytochrome involvement in electron acceptor reduction. c-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H(2), lactate, or pyruvate. Sulfidogenesis occurred with all three electron donors and effectively oxidized the c-type cytochrome in lactate- or pyruvate-reduced, but not H(2)-reduced cells. Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss. However, sulfate reduction was slowed but not halted when H(2) was the electron donor in the presence of Fe(III) or U(VI). U(VI) loss was still impeded by Fe(III) when H(2) was used. Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals. We further propose that cytochrome c(3) is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate.     

The rhizobial adhesion protein RapA1 is involved in adsorption of rhizobia to plant roots but not in nodulation

The effect of the rhizobium adhesion protein RapA1 on Rhizobium leguminosarum bv. trifolii adsorption to Trifolium pratense (red clover) roots was investigated. We altered RapA1 production by cloning its encoding gene under the plac promoter into the stable vector pHC60. After introducing this plasmid in R. leguminosarum bv. trifolii, three to four times more RapA1 was produced, and two to five times higher adsorption to red clover roots was obtained, as compared with results for the empty vector. Enhanced adsorption was also observed on soybean and alfalfa roots, not related to R. leguminosarum cross inoculation groups. Although the presence of 1 mM Ca2+ during rhizobial growth enhanced adsorption, it was unrelated to RapA1 level. Similar effects were obtained when the same plasmid was introduced in Rhizobium etli for its adsorption to bean roots. Although root colonization by the RapA1-overproducing strain was also higher, nodulation was not enhanced. In addition, in vitro biofilm formation was similar to the wild-type both on polar and on hydrophobic surfaces. These results suggest that RapA1 receptors are present in root but not on inert surfaces, and that the function of this protein is related to rhizosphere colonization.     

The Yd2 resistance to barley yellow dwarf virus is effective in barley plants but not in their leaf protoplasts

The Yd2 gene for "resistance" to barley yellow dwarf virus (BYDV) has been widely used in barley ( Hordeum vulgare ). We have tested Australian isolates of BYDV of varying severity against barley genotypes with and without the Yd2 gene and report here a positive relationship between symptoms and virus levels determined by ELISA. Cultivar Shannon is the result of backcrossing the resistant line CI 3208 to cultivar Proctor, a susceptible line. It appears to be intermediate in reaction to BYDV between Proctor and CI 3208, although it carries the major gene, Yd2. Unlike the whole plant studies, no significant differences were observed with regard to the ability of protoplasts derived from these various genotypes to support BYDV replication. It is therefore demonstrated for the first time that the Yd2 gene is not among the small number of resistance genes which are effective against virus replication in isolated protoplasts.     

Mechanisms of water transport mediated by PIP aquaporins and their regulation via phosphorylation events under salinity stress in barley roots

Water homeostasis is crucial to the growth and survival of plants under water-related stress. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. Here we report the water transport activity and mechanisms for the regulation of barley (Hordeum vulgare) PIP aquaporins. HvPIP2 but not HvPIP1 channels were found to show robust water transport activity when expressed alone in Xenopus laevis oocytes. However, the co-expression of HvPIP1 with HvPIP2 in oocytes resulted in significant increases in activity compared with the expression of HvPIP2 alone, suggesting the participation of HvPIP1 in water transport together with HvPIP2 presumably through heteromerization. Severe salinity stress (200 mM NaCl) significantly reduced root hydraulic conductivity (Lp(r)) and the accumulation of six of 10 HvPIP mRNAs. However, under relatively mild stress (100 mM NaCl), only a moderate reduction in Lp(r) with no significant difference in HvPIP mRNA levels was observed. Sorbitol-mediated osmotic stress equivalent to 100 and 200 mM NaCl induced nearly identical Lp(r) reductions in barley roots. Furthermore, the water transport activity in intact barley roots was suggested to require phosphorylation that is sensitive to a kinase inhibitor, staurosporine. HvPIP2s also showed water efflux activity in Xenopus oocytes, suggesting a potential ability to mediate water loss from cells under hypertonic conditions. Water transport via HvPIP aquaporins and the significance of reductions of Lp(r) in barley plants during salinity stress are discussed.     

An Ustilago maydis gene involved in H2O2 detoxification is required for virulence

The fungus Ustilago maydis is a biotrophic pathogen of maize (Zea mays). In its genome we have identified an ortholog of YAP1 (for Yeast AP-1-like) from Saccharomyces cerevisae that regulates the oxidative stress response in this organism. yap1 mutants of U. maydis displayed higher sensitivity to H(2)O(2) than wild-type cells, and their virulence was significantly reduced. U. maydis yap1 could partially complement the H(2)O(2) sensitivity of a yap1 deletion mutant of S. cerevisiae, and a Yap1-green fluorescent protein fusion protein showed nuclear localization after H(2)O(2) treatment, suggesting that Yap1 in U. maydis functions as a redox sensor. Mutations in two Cys residues prevented accumulation in the nucleus, and the respective mutant strains showed the same virulence phenotype as Deltayap1 mutants. Diamino benzidine staining revealed an accumulation of H(2)O(2) around yap1 mutant hyphae, which was absent in the wild type. Inhibition of the plant NADPH oxidase prevented this accumulation and restored virulence. During the infection, Yap1 showed nuclear localization after penetration up to 2 to 3 d after infection. Through array analysis, a large set of Yap1-regulated genes were identified and these included two peroxidase genes. Deletion mutants of these genes were attenuated in virulence. These results suggest that U. maydis is using its Yap1-controlled H(2)O(2) detoxification system for coping with early plant defense responses.     

Nickel-induced Inhibition of Wheat Root Growth is Related to H2O2 Production, but not to Lipid Peroxidation

Effects of exogenous nickel (Ni: 10 and 200 μM) on growth, mitotic activity, Ni accumulation, H₂O₂ content and lipid peroxidation as well as the activities of various antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GSH-Px) were investigated in wheat roots. A considerable Ni accumulation in the roots occurred at both the concentrations. Although Ni at 10 μM did not have any significant effect on root growth, it strongly inhibited the root growth at 200 μM. Mitotic activity in the root tips was not significantly affected by exposure of the seedlings to 10 μM Ni; however, it was almost completely inhibited at 200 μM treatment. Ni stress did not result in any significant changes in CAT and APX activities as well as lipid peroxidation. However, H₂O₂ concentration increased up to 82% over the control in the roots of seedlings exposed to 200 μM Ni. There was a significant decline in both SOD (50%) and GSH-Px (20–30%) activities in the roots when the seedlings were treated with 200 μM Ni. The results indicated that a strong inhibition of wheat root growth caused by Ni stress was not due to enhanced lipid peroxidation, but might be related to the accumulation of H₂O₂ in root tissue.     

Raphe magnus nucleus is involved in ventilatory but not hypothermic response to CO2.

There is evidence that serotonin [5-hydroxytryptamine (5-HT)] is involved in the physiological responses to hypercapnia. Serotonergic neurons represent the major cell type (comprising 15-20% of the neurons) in raphe magnus nucleus (RMg), which is a medullary raphe nucleus. In the present study, we tested the hypothesis 1) that RMg plays a role in the ventilatory and thermal responses to hypercapnia, and 2) that RMg serotonergic neurons are involved in these responses. To this end, we microinjected 1) ibotenic acid to promote nonspecific lesioning of neurons in the RMg, or 2) anti-SERT-SAP (an immunotoxin that utilizes a monoclonal antibody to the third extracellular domain of the serotonin reuptake transporter) to specifically kill the serotonergic neurons in the RMg. Hypercapnia caused hyperventilation and hypothermia in all groups. RMg nonspecific lesions elicited a significant reduction of the ventilatory response to hypercapnia due to lower tidal volume (Vt) and respiratory frequency. Rats submitted to specific killing of RMg serotonergic neurons showed no consistent difference in ventilation during air breathing but had a decreased ventilatory response to CO(2) due to lower Vt. The hypercapnia-induced hypothermia was not affected by specific or nonspecific lesions of RMg serotonergic neurons. These data suggest that RMg serotonergic neurons do not participate in the tonic maintenance of ventilation during air breathing but contribute to the ventilatory response to CO(2). Ultimately, this nucleus may not be involved in the thermal responses to CO(2).     

The 2H4 molecule but not the T3-receptor complex is involved in suppressor inducer signals in the AMLR system

It is suggested that autologous mixed lymphocyte reaction (AMLR) may play an important role in generating suppressor inducer signals and in down-regulating the immune response following self-major histocompatibility recognition. In the present study, monoclonal antibodies directed at cell surface structures on T4+ cells activated in AMLR were used to define the molecules important in the generation of the suppressor inducer signal. The density of a 200/220-kDa structure, termed 2H4, increased on T4 cells during activation in AMLR and furthermore a strong correlation was observed between the generated suppressor inducer activity of such cells and the density of the 2H4 antigen. More importantly, we showed that treatment of AMLR activated T4 cells with anti-2H4 but not anti-T3 or T4 antibody abolished the suppressor inducer function of these cells. These results suggest that the 2H4 molecule but not the T3-receptor complex plays an important role in generating suppressor inducer signals in the AMLR system.     

Heme oxygenase is involved in H2O2-induced lateral root formation in apocynin-treated rice

Key message

Apocynin is a natural organic compound structurally related to vanillin. We demonstrated that hydrogen peroxide and heme oxygenase participated in apocynin-induced lateral root formation in rice.

Abstract

Apocynin, also known as acetovanillone, is a natural organic compound structurally related to vanillin. Information concerning the effect of apocynin on plants is limited. In this study, we examined the effect of apocynin on lateral root (LR) formation in rice. Treatment with apocynin induced LR formation and increased H₂O₂ production, but had no effect on nitric oxide production. Diphenyleneiodonium chloride, an inhibitor of H₂O₂ generating NADPH oxidase, was effective in reducing apocynin-induced H₂O₂ production and LR formation. Apocynin treatment also increased superoxide dismutase activity and decreased catalase activity. H₂O₂ application was able to increase the number of LRs. Moreover, H₂O₂ production caused by H₂O₂ and apocynin was localized in the root area corresponding to the LR emergence. Treatment with H₂O₂ and apocynin also increased heme oxygenase (HO) activity and induced OsHO1 mRNA expression. Lateral root formation and HO activity induced by H₂O₂ and apocynin were reduced by Zn protoporphyrin IX (the specific inhibitor of HO). Our data suggest that both H₂O₂ and HO are required for apocynin-induced LR formation in rice.     

H2O2 is involved in the dormancy-breaking effect of hydrogen cyanamide in grapevine buds

Hydrogen cyanamide (HC) is widely used to induce the breakage of endodormancy (ED) in grape and other deciduous fruit crop, though its mechanism of action is poorly understood. Applications of HC to grapevine buds produce oxidative stress and transient respiratory disturbances which are related to the breakage of ED. Moreover, since the expression and activity of catalase (Cat) is inhibited by HC, enhancements in the levels of H₂O₂ have also been associated to the breakage of ED in grapevine buds. Here, we reported that increases in H₂O₂ level in HC-treated grapevine buds are due to the inhibition of Cat activity and enhancement of the respiratory activity of buds. In addition, exogenous applications of H₂O₂ partially reproduced the inducing effect of HC in the breakage of ED, thus providing further support for the hypothesis that H₂O₂ mediates the effects of HC. On the other hand, Mit isolated from both control and HC-treated buds respired equally well when NADH was used as a respiratory substrate, but when succinate was used as an electron donor Mit respiration was non-detected, suggesting that the stimulatory effect of HC on bud respiration is related to metabolic alterations leading to increase of the concentration of NADH rather than to changes in Mit functionality.     

Tobacco aquaporin NtAQP1 is involved in mesophyll conductance to CO2 in vivo

Leaf mesophyll conductance to CO(2) (g(m)) has been recognized to be finite and variable, rapidly adapting to environmental conditions. The physiological basis for fast changes in g(m) is poorly understood, but current reports suggest the involvement of protein-facilitated CO(2) diffusion across cell membranes. A good candidate for this could be the Nicotiana tabacum L. aquaporin NtAQP1, which was shown to increase membrane permeability to CO(2) in Xenopus oocytes. The objective of the present work was to evaluate its effect on the in vivo mesophyll conductance to CO(2), using plants either deficient in or overexpressing NtAQP1. Antisense plants deficient in NtAQP1 (AS) and NtAQP1 overexpressing tobacco plants (O) were compared with their respective wild-type (WT) genotypes (CAS and CO). Plants grown under optimum conditions showed different photosynthetic rates at saturating light, with a decrease of 13% in AS and an increase of 20% in O, compared with their respective controls. CO(2) response curves of photosynthesis also showed significant differences among genotypes. However, in vitro analysis demonstrated that these differences could not be attributed to alterations in Rubisco activity or ribulose-1,5-bisphosphate content. Analyses of chlorophyll fluorescence and on-line (13)C discrimination indicated that the observed differences in net photosynthesis (A(N)) among genotypes were due to different leaf mesophyll conductances to CO(2), which was estimated to be 30% lower in AS and 20% higher in O compared with their respective WT. These results provide evidence for the in vivo involvement of aquaporin NtAQP1 in mesophyll conductance to CO(2).     

Myeloperoxidase is involved in H2O2-induced apoptosis of HL-60 human leukemia cells

We examined the mechanism of H(2)O(2)-induced cytotoxicity and its relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to H(2)O(2), and at concentrations up to about 20-25 micrometer, the killing was mediated by apoptosis. There was limited evidence of lipid peroxidation, suggesting that the effects of H(2)O(2) do not involve hydroxyl radical. When HL-60 cells were exposed to H(2)O(2) in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we detected a 12-line electron paramagnetic resonance spectrum assigned to the POBN/POBN(.) N-centered spin adduct previously described in peroxidase-containing cell-free systems. Generation of this radical by HL-60 cells had the same H(2)O(2) concentration dependence as initiation of apoptosis. In contrast, studies with the K562 human erythroleukemia cell line, which is often used for comparison with the HL-60, and with high passaged HL-60 cells (spent HL-60) studied under the same conditions failed to generate POBN(.). Cellular levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase did not explain the differences between these cell lines. Interestingly, the K562 and spent HL-60 cells, which did not generate the radical, also failed to undergo H(2)O(2)-induced apoptosis. Based on this we reasoned that the difference in H(2)O(2)-induced apoptosis might be due to the enzyme myeloperoxidase. Only the apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme. When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid hydrazide, which are inhibitors of myeloperoxidase, they no longer underwent H(2)O(2)-induced apoptosis. Hypochlorous acid stimulated apoptosis in both HL-60 and spent HL-60 cells, indicating that another oxidant generated by myeloperoxidase induces apoptosis and that it may be the direct mediator of H(2)O(2)-induced apoptosis. Taken together these observations indicate that H(2)O(2)-induced apoptosis in the HL-60 human leukemia cell is mediated by myeloperoxidase and is linked to a non-Fenton oxidative event marked by POBN(.).     

The distribution of aquaporin subtypes (PIP1, PIP2 and gamma-TIP) is tissue dependent in soybean (Glycine max) root nodules

BACKGROUND AND AIMS The inner cortical cells (IC-cells) of legume root nodules have been previously shown to regulate the resistance to nodule O2 diffusion by a rapid contraction/expansion mechanism, which controls the volume of intercellular spaces and their occlusion by a liquid phase. The expression of aquaporins in IC-cells was also found to be involved in this nodule O2 diffusion mechanism. The aim of this study was to compare the expression of plasma membrane intrinsic proteins (PIP) aquaporin isoforms with tonoplast intrinsic protein (gamma-TIP) in both IC-cells and adjacent cell types. METHODS: Using immunogold labelling in ultra-thin sections of Glycine max nodules, the expression of two PIP isoforms was observed and compared with the gamma-TIP pattern. KEY RESULTS: The plasma membrane aquaporins PIP1 and PIP2 were expressed more in IC-cells and endodermis than in pericycle and infected cells. The tonoplast aquaporin gamma-TIP has shown a distribution pattern similar to that of the PIPs. CONCLUSIONS: PIPs and gamma-TIP aquaporins are highly expressed in both plasmalemma and tonoplast of nodule IC-cells. This distribution is consistent with the putative role of water fluxes associated with the regulation of nodule conductance to O2 diffusion and the subsequent ATP-dependent nitrogenase activity. In the endodermis, these aquaporins might also be involved in nutrient transport between the infected zone and vascular traces.