Alfonso Carreras

Localization of Nitric-oxide Synthase in Plant Peroxisomes

The presence of nitric-oxide synthase (NOS) in peroxisomes from leaves of pea plants (Pisum sativum L.) was studied. Plant organelles were purified by differential and sucrose density gradient centrifugation. In purified intact peroxisomes a Ca2+-dependent NOS activity of 5.61 nmol ofl-[3H]citrulline mg−1 protein min−1 was measured while no activity was detected in mitochondria. The peroxisomal NOS activity was clearly inhibited (60–90%) by different well characterized inhibitors of mammalian NO synthases. The immunoblot analysis of peroxisomes with a polyclonal antibody against the C terminus region of murine iNOS revealed an immunoreactive protein of 130 kDa. Electron microscopy immunogold-labeling confirmed the subcellular localization of NOS in the matrix of peroxisomes as well as in chloroplasts. The presence of NOS in peroxisomes suggests that these oxidative organelles are a cellular source of nitric oxide (NO) and implies new roles for peroxisomes in the cellular signal transduction mechanisms.

Cellular and Subcellular Localization of Endogenous Nitric Oxide in Young and Senescent Pea Plants

The cellular and subcellular localization of endogenous nitric oxide (NO˙) in leaves from young and senescent pea (Pisum sativum) plants was studied. Confocal laser scanning microscopy analysis of pea leaf sections with the fluorescent probe 4,5-diaminofluorescein diacetate revealed that endogenous NO˙ was mainly present in vascular tissues (xylem and phloem). Green fluorescence spots were also detected in the epidermal cells, palisade and spongy mesophyll cells, and guard cells. In senescent leaves, NO˙ generation was clearly reduced in the vascular tissues. At the subcellular level, by electron paramagnetic resonance spectroscopy with the spin trap Fe(MGD)2 and fluorometric analysis with 4,5-diaminofluorescein diacetate, NO˙ was found to be an endogenous metabolite of peroxisomes. The characteristic three-line electron paramagnetic resonance spectrum of NO˙, with g = 2.05 and aN = 12.8 G, was detected in peroxisomes. By fluorometry, NO˙ was also found in these organelles, and the level measured of NO˙ was linearly dependent on the amount of peroxisomal protein. The enzymatic production of NO˙ from l-Arg (nitric oxide synthase [NOS]-like activity) was measured by ozone chemiluminiscence. The specific activity of peroxisomal NOS was 4.9 nmol NO˙ mg−1 protein min−1; was strictly dependent on NADPH, calmodulin, and BH4; and required calcium. In senescent pea leaves, the NOS-like activity of peroxisomes was down-regulated by 72%. It is proposed that peroxisomal NO˙ could be involved in the process of senescence of pea leaves.

Nitrosative stress in plants.

Nitrosative stress has become a usual term in the physiology of nitric oxide in mammalian systems. However, in plants there is much less information on this type of stress. Using olive leaves as experimental model, the effect of salinity on the potential induction of nitrosative stress was studied. The enzymatic l‐arginine‐dependent production of nitric oxide (NOS activity) was measured by ozone chemiluminiscence. The specific activity of NOS in olive leaves was 0.280 nmol NO mg−1 protein min−1, and was dependent on l‐arginine, NADPH and calcium. Salt stress (200 mM NaCl) caused an increase of the l‐arginine‐dependent production of nitric oxide (NO), total S‐nitrosothiols (RSNO) and number of proteins that underwent tyrosine nitration. Confocal laser scanning microscopy analysis using either specific fluorescent probes for NO and RSNO or antibodies to S‐nitrosoglutathione and 3‐nitrotyrosine, showed also a general increase of these reactive nitrogen species (RNS) mainly in the vascular tissue. Taken together, these findings show that in olive leaves salinity induces nitrosative stress, and vascular tissues could play an important role in the redistribution of NO‐derived molecules during nitrosative stress.

Metabolism of Reactive Nitrogen Species in Pea Plants Under Abiotic Stress Conditions

Nitric oxide (*NO) is a key signaling molecule in different physiological processes of animals and plants. However, little is known about the metabolism of endogenous *NO and other reactive nitrogen species (RNS) in plants under abiotic stress conditions. Using pea plants exposed to six different abiotic stress conditions (high light intensity, low and high temperature, continuous light, continuous dark and mechanical wounding), several key components of the metabolism of RNS including the content of *NO, S-nitrosothiols (RSNOs) and nitrite plus nitrate, the enzyme activities of l-arginine-dependent nitric oxide synthase (NOS) and S-nitrosogluthathione reductase (GSNOR), and the profile of protein tyrosine nitration (NO(2)-Tyr) were analyzed in leaves. Low temperature was the stress that produced the highest increase of NOS and GSNOR activities, and this was accompanied by an increase in the content of total *NO and S-nitrosothiols, and an intensification of the immunoreactivity with an antibody against NO(2)-Tyr. Mechanical wounding, high temperature and light also had a clear activating effect on the different indicators of RNS metabolism in pea plants. However, the total content of nitrite and nitrate in leaves was not affected by any of these stresses. Considering that protein tyrosine nitration is a potential marker of nitrosative stress, the results obtained suggest that low and high temperature, continuous light and high light intensity are abiotic stress conditions that can induce nitrosative stress in pea plants.

Involvement of Reactive Nitrogen and Oxygen Species (RNS and ROS) in Sunflower-Mildew Interaction

Nitric oxide (.NO) has been shown to participate in plant response against pathogen infection; however, less is known of the participation of other NO-derived molecules designated as reactive nitrogen species (RNS). Using two sunflower (Helianthus annuus L.) cultivars with different sensitivity to infection by the pathogen Plasmopara halstedii, we studied key components involved in RNS and ROS metabolism. We analyzed the superoxide radical production, hydrogen peroxide content, l-arginine-dependent nitric oxide synthase (NOS) and S-nitrosoglutathione reductase (GSNOR) activities. Furthermore, we examined the location and contents of .NO, S-nitrosothiols (RSNOs), S-nitrosoglutathione (GSNO) and protein 3-nitrotyrosine (NO(2)-Tyr) by confocal laser scanning microscopy (CLSM) and biochemical analyses. In the susceptible cultivar, the pathogen induces an increase in proteins that undergo tyrosine nitration accompanied by an augmentation in RSNOs. This rise of RSNOs seems to be independent of the enzymatic generation of .NO because the l-arginine-dependent NOS activity is reduced after infection. These results suggest that pathogens induce nitrosative stress in susceptible cultivars. In contrast, in the resistant cultivar, no increase of RSNOs or tyrosine nitration of proteins was observed, implying an absence of nitrosative stress. Therefore, it is proposed that the increase of tyrosine nitration of proteins can be considered a general biological marker of nitrosative stress in plants under biotic conditions.

Dual regulation of cytosolic ascorbate peroxidase (APX) by tyrosine nitration and S-nitrosylation

Post-translational modifications (PTMs) mediated by nitric oxide (NO)-derived molecules have become a new area of research, as they can modulate the function of target proteins. Proteomic data have shown that ascorbate peroxidase (APX) is one of the potential targets of PTMs mediated by NO-derived molecules. Using recombinant pea cytosolic APX, the impact of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO), which are known to mediate protein nitration and S-nitrosylation processes, respectively, was analysed. While peroxynitrite inhibits APX activity, GSNO enhances its enzymatic activity. Mass spectrometric analysis of the nitrated APX enabled the determination that Tyr5 and Tyr235 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Residue Cys32 was identified by the biotin switch method as S-nitrosylated. The location of these residues on the structure of pea APX reveals that Tyr235 is found at the bottom of the pocket where the haem group is enclosed, whereas Cys32 is at the ascorbate binding site. Pea plants grown under saline (150mM NaCl) stress showed an enhancement of both APX activity and S-nitrosylated APX, as well as an increase of H2O2, NO, and S-nitrosothiol (SNO) content that can justify the induction of the APX activity. The results provide new insight into the molecular mechanism of the regulation of APX which can be both inactivated by irreversible nitration and activated by reversible S-nitrosylation.

Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings

Nitric oxide (NO) and related molecules such as peroxynitrite, S-nitrosoglutathione (GSNO), and nitrotyrosine, among others, are involved in physiological processes as well in the mechanisms of response to stress conditions. In sunflower seedlings exposed to five different adverse environmental conditions (low temperature, mechanical wounding, high light intensity, continuous light, and continuous darkness), key components of the metabolism of reactive nitrogen species (RNS) and reactive oxygen species (ROS), including the enzyme activities L-arginine-dependent nitric oxide synthase (NOS), S-nitrosogluthathione reductase (GSNOR), nitrate reductase (NR), catalase, and superoxide dismutase, the content of lipid hydroperoxide, hydrogen peroxide, S-nitrosothiols (SNOs), the cellular level of NO, GSNO, and GSNOR, and protein tyrosine nitration [nitrotyrosine (NO2-Tyr)] were analysed. Among the stress conditions studied, mechanical wounding was the only one that caused a down-regulation of NOS and GSNOR activities, which in turn provoked an accumulation of SNOs. The analyses of the cellular content of NO, GSNO, GSNOR, and NO2-Tyr by confocal laser scanning microscopy confirmed these biochemical data. Therefore, it is proposed that mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a down-regulation of GSNOR activity, while NO2-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants.

High temperature triggers the metabolism of S-nitrosothiols in sunflower mediating a process of nitrosative stress which provokes the inhibition of ferredoxin–NADP reductase by tyrosine nitration

High temperature (HT) is considered a major abiotic stress that negatively affects both vegetative and reproductive growth. Whereas the metabolism of reactive oxygen species (ROS) is well established under HT, less is known about the metabolism of reactive nitrogen species (RNS). In sunflower (Helianthus annuus L.) seedlings exposed to HT, NO content as well as S-nitrosoglutathione reductase (GSNOR) activity and expression were down-regulated with the simultaneous accumulation of total S-nitrosothiols (SNOs) including S-nitrosoglutathione (GSNO). However, the content of tyrosine nitration (NO(2) -Tyr) studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and by confocal laser scanning microscope was induced. Nitroproteome analysis under HT showed that this stress induced the protein expression of 13 tyrosine-nitrated proteins. Among the induced proteins, ferredoxin-NADP reductase (FNR) was selected to evaluate the effect of nitration on its activity after heat stress and in vitro conditions using 3-morpholinosydnonimine (SIN-1) (peroxynitrite donor) as the nitrating agent, the FNR activity being inhibited. Taken together, these results suggest that HT augments SNOs, which appear to mediate protein tyrosine nitration, inhibiting FNR, which is involved in the photosynthesis process.

Tyrosine nitration provokes inhibition of sunflower carbonic anhydrase (β-CA) activity under high temperature stress.

Protein tyrosine nitration is a post-translational modification (PTM) mediated by reactive nitrogen species (RNS) and it is a new area of research in higher plants. Previously, it was demonstrated that the exposition of sunflower (Helianthus annuus L.) seedlings to high temperature (HT) caused both oxidative and nitrosative stress. The nitroproteome analysis under this stress condition showed the induction of 13 tyrosine-nitrated proteins being the carbonic anhydrase (CA) one of these proteins. The analysis of CA activity under high temperature showed that this stress inhibited the CA activity by a 43%. To evaluate the effect of nitration on the CA activity in sunflower it was used 3-morpholinosydnonimine (SIN-1) (peroxynitrite donor) as the nitrating agent. Thus the CA activity was inhibited by 41%. In silico analysis of the pea CA protein sequence suggests that Tyr(205) is the most likely potential target for nitration.

Functional analysis of superoxide dismutases (SODs) in sunflower under biotic and abiotic stress conditions. Identification of two new genes of mitochondrial Mn-SOD

Superoxide dismutases (SODs) are a family of metalloenzymes that catalyse the disproportionation of superoxide radicals into hydrogen peroxide and oxygen. In sunflower (Helianthus annuus L.) seedlings, two new Mn-SOD isozymes, designated as I and II, were identified. However, no evidence for a Fe-SOD was found. Both Mn-SOD I and Mn-SOD II have a cleaved sequence of 14 residues that target the mitochondrion with a probability of 81% and 95%, respectively. The gene expression of these new mitochondrial Mn-SODs as well as the previously reported cytosolic and chloroplastic CuZnSODs was analyzed by real-time quantitative reverse transcription-PCR. This was done in the main organs (roots, hypocotyls, and cotyledons) of sunflower seedlings and also under biotic (infection by the pathogen Plasmopara halstedii) and abiotic stress conditions, including high and low temperature and mechanical wounding. Both CuZn-SODs had a gene expression of 1000-fold higher than that of mitochondrial Mn-SODs. And the expression of the Mn-SOD I was approximately 12-fold higher than that of Mn-SOD II. The Mn-SOD I showed a significant modulation in response to the assayed biotic and abiotic stresses even when it had no apparent oxidative stress, such as low temperature. Thus, it is proposed that the mitochondrial Mn-SOD I gene could act as an early sensor of adverse conditions to prevent potential oxidative damage.