Metabolic responses in root nodules of Phaseolus vulgaris and Vicia sativa exposed to the imazamox herbicide.

Alterations on growth, amino acids metabolism and some antioxidant enzyme activities as result of imazamox treatment were examined in determinate and indeterminate nodules, formed by Phaseolus vulgaris and Vicia sativa, respectively. Young seedlings of both legumes were inoculated with their respective microsymbionts and grown under controlled conditions. At vegetative growth, plants were treated with imazamox (250µM) in the nutrient solution and harvested 7days after. Imazamox was mainly accumulated in V. sativa where concentrations were more than six fold higher than those detected in P. vulgaris. Nodule dry weight and total nitrogen content were reduced by the herbicide treatment: the highest decrease of nodule biomass (50%) and nitrogen content (40%) were registered in V. sativa and P. vulgaris, respectively. The concentration of branched-chain amino acids (BCAA) did not change in neither determinate nor indeterminate nodules even though the acetohydroxyacid synthase activity decreased in root and nodules of both symbioses with the herbicide application. Based on this last result and taking into account that total free amino acids increased in roots but not in nodules of common vetch, a possible BCAA translocation from root to nodule could occur. Our results suggest that the maintenance of BCAA balance in nodule become a priority for the plant in such conditions. The involvement of activities glutathione-S-transferase, guaiacol peroxidase and superoxide dismutase in the response of the symbioses to imazamox are also discussed.

Involvement of abscisic acid in the response of Medicago sativa plants in symbiosis with Sinorhizobium meliloti to salinity.

Legumes are classified as salt-sensitive crops with their productivity particularly affected by salinity. Abcisic acid (ABA) plays an important role in the response to environmental stresses as signal molecule which led us to study its role in the response of nitrogen fixation and antioxidant metabolism in root nodules of Medicago sativa under salt stress conditions. Adult plants inoculated with Sinorhizobium meliloti were treated with 1 µM and 10 µM ABA two days before 200 mM salt addition. Exogenous ABA together with the salt treatment provoked a strong induction of the ABA content in the nodular tissue which alleviated the inhibition induced by salinity in the plant growth and nitrogen fixation. Antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were induced by ABA pre-treatments under salt stress conditions which together with the reduction of the lipid peroxidation, suggest a role for ABA as signal molecule in the activation of the nodular antioxidant metabolism. Interaction between ABA and polyamines (PAs), described as anti-stress molecules, was studied being detected an induction of the common polyamines spermidine (Spd) and spermine (Spm) levels by ABA under salt stress conditions. In conclusion, ABA pre-treatment improved the nitrogen fixation capacity under salt stress conditions by the induction of the nodular antioxidant defenses which may be mediated by the common PAs Spd and Spm that seems to be involved in the anti-stress response induced by ABA.

Salicylic acid improves the salinity tolerance of Medicago sativa in symbiosis with Sinorhizobium meliloti by preventing nitrogen fixation inhibition.

In this work we have investigated the contribution of pretreatment with 0.1 and 0.5mM salicylic acid (SA) to the protection against salt stress in root nodules of Medicago sativa in symbiosis with Sinorhizobium meliloti. SA alleviated the inhibition induced by salinity in the plant growth and photosynthetic capacity of M. sativa-S. meliloti symbiosis. In addition, SA prevented the inhibition of the nitrogen fixation capacity under salt stress since nodule biomass was not affected by salinity in SA pretreated plants. Antioxidant enzymes peroxidase (POX), superoxide dismutase (SOD), ascorbate peroxidase (APX), dehidroascorbate reductase (DHAR) and glutathione reductase (GR), key in the main pathway that scavenges H2O2 in plants, were induced by SA pretreatments which suggest that SA may participate in the redox balance in root nodules under salt stress. Catalase activity (CAT) was inhibited around 40% by SA which could be behind the increase of H2O2 detected in nodules of plants pretreated with SA. The accumulation of polyamines (PAs) synthesized in response to salinity was prevented by SA which together with the induction of 1-aminocyclopropane-l-carboxylic acid (ACC) content suggest the prevalence of the ethylene signaling pathway induced by SA in detriment of the synthesis of PAs. In conclusion, SA alleviated the negative effect of salt stress in the M. sativa-S. meliloti symbiosis through the increased level of nodule biomass and the induction of the nodular antioxidant metabolism under salt stress. The H2O2 accumulation and the PAs inhibition induced by SA in nodules of M. sativa suggest that SA activates a hypersensitive response dependent on ethylene.

Effect of some abiotic factors on the biological activity of Gluconacetobacter diazotrophicus.

AIMS: The effect of some abiotic factors, dryness, heat and salinity on the growth and biological activity of Gluconacetobacter diazotrophicus, and the influence of a salt stress on some enzymes involved in carbon metabolism of these bacteria is studied under laboratory conditions. METHODS AND RESULTS: Strain PAL-5 of G. diazotrophicus was incubated under different conditions of drying, heat and salinity. Cells showed tolerance to heat treatments and salt concentrations, and sensitivity to drying conditions. Higher NaCl dosage of 150 and 200 mmol l -1 limited its growth and drastically affected the nitrogenase activity and the enzymes glucose dehydrogenase, alcohol dehydrogenase, fumarase, isocitrate dehydrogenase and malate dehydrogenase. CONCLUSIONS: Gluconacetobacter diazotrophicus, despite its endophytic nature, tolerated heat treatments and salinity stress, but its nitrogenase activity and carbon metabolism enzymes were affected by high NaCl dosage. SIGNIFICANCE AND IMPACT OF THE STUDY: The investigation of the biological activity of G. diazotrophicus in response to different abiotic factors led to more knowledge of this endophyte and may help to clarify pathways involved in its transmission into the host plant.

Renal tubulointerstitial damage caused by persistent proteinuria is attenuated in AT1-deficient mice: role of endothelin-1.

Using angiotensin II (AngII) type 1A receptor-deficient mice [AT1(-/-)], in which we induced protein overload nephropathy, we explored the potential implication of AngII and endothelin-1 (ET-1) in the tubulointerstitial damage because of persistent proteinuria. At day 7, AT1(-/-) showed marked proteinuria to a similar extent to that of wild-type mice (WT). However, at day14, AT1(-/-) had significantly less proteinuria, renal damage, transforming growth factor-beta, and matrix mRNA expression and mortality. AT1(-/-) also showed a significant diminution in the activation of the transcriptional factors nuclear factor-kappaB and AP-1. Unexpectedly, AT1(-/-) had a higher interstitial infiltration than WT. The administration of the angiotensin-converting enzyme inhibitor quinapril to WT caused a marked improvement in proteinuria and renal lesions, resembling that seen in untreated AT1(-/-). However, the interstitial infiltration persisted in AT1(-/-) when treated with quinapril. Because ET-1 may participate in the recruitment of mononuclear cells, we also studied the implication of this peptide. AT1(-/-) had a significantly higher ET-1 expression in tubular epithelial cells than WT. The administration of the dual ETA/ETB antagonist bosentan to AT1(-/-) considerably reduced the interstitial infiltrates. Bosentan also exerted a beneficial effect on proteinuria, renal lesions, and mortality in WT. These data show that in overload nephropathy, proteinuria and renal lesions are, to a large extent, AngII-dependent. The up-regulation of ET-1 in tubular epithelial cells in AT1(-/-), associated with interstitial infiltrates, suggests that the combination of drugs interfering with both vasopeptides may be of therapeutic interest in renal diseases with severe proteinuria and tubulointerstitial damage.

Activation of NF-kappaB in tubular epithelial cells of rats with intense proteinuria: role of angiotensin II and endothelin-1.

The mechanisms by which persistent proteinuria induces interstitial inflammation and fibrosis are not well known, although nuclear factor-kappaB (NF-kappaB), which regulates the transcription of many genes involved in renal injury, could be implicated. In rats with intense proteinuria, we studied the renal activation of NF-kappaB as well as the potential involvement of the vasoactive hormones angiotensin II (Ang II) and endothelin-1 (ET-1). Uninephrectomized Wistar-Kyoto rats receiving 1 g/d of BSA had proteinuria but no renal morphological lesions at day 1. By contrast, tubular atrophy and/or dilation and mononuclear cell infiltration were observed after 8 or 28 days of BSA administration, coinciding with maximal proteinuria. In relation to control uninephrectomized rats, the renal cortex of nephritic rats showed an increment in the activation of NF-kappaB at all time periods studied. By in situ Southwestern histochemistry, NF-kappaB activity was mainly localized in proximal tubules, interstitial mononuclear cells, and, to a lesser extent, the glomeruli. The administration of the ACE inhibitor quinapril plus the ET(A)/ET(B) receptor antagonist bosentan during 28 days to BSA-overloaded animals diminished proteinuria, renal lesions, and NF-kappaB activity more markedly than single drugs. Cultured tubular epithelial cells exposed to BSA revealed an intense NF-kappaB activation in a time- and dose-dependent manner. Incubation of cells with receptor antagonists of Ang II (AT(1): losartan and AT(2): PD-123,319) or ET-1 (ET(A): BQ123 and ET(B): IRL1038) inhibited significantly the BSA-induced NF-kappaB activity (90%, 75%, 90%, and 60% of inhibition versus basal, respectively). Our results show that overload proteinuria causes NF-kappaB activation in tubular epithelial cells both in vivo and in vitro. The vasoactive peptides Ang II and ET-1 appear to be implicated in this effect. The results reveal a novel mechanism of perpetuation of renal damage induced by persistent proteinuria.

Androgen deprivation facilitates acetylcholine-induced relaxation by superoxide anion generation.

The aim of the present study was to assess the role of superoxide anions in the relaxation induced by acetylcholine (ACh) in aortic segments from male rats, and to investigate if their production is altered by sex hormone deprivation. In segments precontracted with 10 nmol/l noradrenaline, ACh (0.1 nmol/l-10 micromol/l) induced concentration-dependent relaxation, which was greater in segments from castrated compared with control animals. ACh-induced relaxation was abolished in segments from control rats, and reduced in those from castrated rats, by 0.1 mmol/l N(G)-nitro-L-arginine methyl ester (L-NAME; a nitric oxide synthase inhibitor). Indomethacin (1 micromol/l; a cyclo-oxygenase blocker) decreased the ACh-induced relaxation in arteries from control males only. Incubation of segments with superoxide dismutase (SOD; 100 units/ml; a superoxide anion scavenger) enhanced and reduced relaxation in segments from control and castrated animals respectively. For arteries from castrated animals, the presence of SOD plus L-NAME abolished such responses. In these arteries, incubation with L-NAME abolished the relaxation caused by ACh when the segments were precontracted with 30 mmol/l KCl. In segments obtained from castrated rats and pretreated with L-NAME, 1 mmol/l tetraethylammonium or 0.4 micromol/l charybdotoxin [blockers of Ca(2+)-sensitive and large-conductance Ca(2+)-sensitive (BK(Ca)) K(+) channels respectively] abolished the relaxation induced by ACh. These results suggest that ACh generates endothelial NO and superoxide anions from the arterial wall in both control and castrated animals; these agents negatively modulate ACh-induced relaxation in control rats by destruction of NO, and positively modulate ACh-induced relaxation in castrated rats by activation of BK(Ca) channels.

Gender differences in the endothelial regulation of alpha2-adrenoceptor-mediated contraction in the rat aorta.

The aim of this study was to determine the possible influence of sex hormones on the contractile responses induced by clonidine, an agonist of alpha2-adrenoceptors, as well as the endothelial modulation of these responses. For this purpose, thoracic aorta segments from male (control and castrated) and female (in oestrous phase and ovariectomized) rats were used. In intact segments from the four groups of rats, clonidine (0.01-10 micromol/l) induced concentration-dependent contractions, which were increased by the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (0.1 mmol/l) or by endothelium removal, but were reduced by 1 micromol/l yohimbine (an alpha2-adrenoceptor antagonist) in all animals and by 1 micromol/l indomethacin (a cyclo-oxygenase inhibitor) in control males only. The rank order of the magnitude of the maximal response was: oestrous females>ovariectomized females>control males>castrated males, whereas the sensitivity to clonidine (EC50 value) was similar in all animals. In endothelium-denuded segments, the rank order was: oestrous females=control males>ovariectomized females=castrated males. These results suggest that: (1) the presence of oestrogen or androgen increases the contraction caused by alpha2-adrenoceptor activation with clonidine; (2) endothelium negatively modulates the response to this agonist in the four groups of rats, due to endothelial NO release (entirely in females and in part in males); (3) androgen also seems to modulate the response by stimulating the release of an endothelial contracting factor, probably a prostanoid; and (4) the endothelium of males has a greater capacity than that of comparable females for negative regulation of the tension generated by the underlying vascular smooth muscle.

Effect of age on the vasorelaxation elicited by cromakalim. Role of K+ channels and cyclic GMP.

The objective of this study was to investigate whether age induces changes on vasodilator response induced by cromakalim, an ATP-sensitive K+ (K(ATP)) channel opener, as well as the underlying mechanism involved in this possible alteration. For this purpose, aortic segments from young (3-5 months) and old (3 years) rabbits were used, which were precontracted with noradrenaline (NA, 1 microM). The vasodilator response induced by cromakalim (0.01-100 microM) was reduced in intact segments from old rabbits, and endothelium removal reduced and did not modify this effect in young and old animals, respectively. In both groups of animals, glibenclamide (10 microM), a blocker of K(ATP) channels, significantly reduced the response elicited by cromakalim, which was not modified by the large conductance Ca2+-activated K+ channel blocker charybdotoxin (ChTX, 0.4 microM). Acetylcholine (ACh, 10 microM) and sodium nitroprusside (SNP, 100 microM) induced a lesser vasodilator effect in aortic segments from old compared with young rabbits. In segments precontracted with NA, 10 microM ACh or 100 microM SNP similarly increased cGMP levels in both groups of animals. However, basal cGMP level was reduced in segments from old rabbits. Incubation with 8-bromo-cGMP (100 microM) increased the response induced by cromakalim in both groups of animals, reaching similar maximum values in young and old rabbits. The response induced by cromakalim plus 8-bromo-cGMP was markedly decreased by glibenclamide and unmodified by ChTx in both types of animals. These results suggest that aging decreases the vasodilator response to cromakalim, mechanism in which appears to be involved the maintained low cGMP levels observed in old rabbits, and that this messenger modulates the degree of K(ATP) channel activation.