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.

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 (NO(2)-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 NO(2)-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 NO(2)-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants.

Protein targets of tyrosine nitration in sunflower (Helianthus annuus L.) hypocotyls.

Tyrosine nitration is recognized as an important post-translational protein modification in animal cells that can be used as an indicator of a nitrosative process. However, in plant systems, there is scant information on proteins that undergo this process. In sunflower hypocotyls, the content of tyrosine nitration (NO(2)-Tyr) and the identification of nitrated proteins were studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and proteomic approaches, respectively. In addition, the cell localization of nitrotyrosine proteins and peroxynitrite were analysed by confocal laser-scanning microscopy (CLSM) using antibodies against 3-nitrotyrosine and 3'-(p-aminophenyl) fluorescein (APF) as the fluorescent probe, in that order. The concentration of Tyr and NO(2)-Tyr in hypocotyls was 0.56 micromol mg(-1) protein and 0.19 pmol mg(-1) protein, respectively. By proteomic analysis, a total of 21 nitrotyrosine-immunopositive proteins were identified. These targets include proteins involved in photosynthesis, and in antioxidant, ATP, carbohydrate, and nitrogen metabolism. Among the proteins identified, S-adenosyl homocysteine hydrolase (SAHH) was selected as a model to evaluate the effect of nitration on SAHH activity using SIN-1 (a peroxynitrite donor) as the nitrating agent. When the hypocotyl extracts were exposed to 0.5 mM, 1 mM, and 5 mM SIN-1, the SAHH activity was inhibited by some 49%, 89%, and 94%, respectively. In silico analysis of the barley SAHH sequence, characterized Tyr448 as the most likely potential target for nitration. In summary, the present data are the first in plants concerning the content of nitrotyrosine and the identification of candidates of protein nitration. Taken together, the results suggest that Tyr nitration occurs in plant tissues under physiological conditions that could constitute an important process of protein regulation in such a way that, when it is overproduced in adverse circumstances, it can be used as a marker of nitrosative stress.

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.

In vivo antifungal activity of the essential oil of Bupleurum gibraltarium against Plasmopara halstedii in sunflower.

The antifungal activity of the essential oil of the aerial parts of Bupleurum gibraltarium was evaluated against Plasmopara halstedii. Fungus spores were inoculated in sunflower seedlings, previously treated with several essential oil solutions, and the sporulation percentage was measured after an 11-day treatment. The oil at a concentration of 5.0 mL/L clearly inhibited the fungus sporulation. The contact between fungus sporangia and essential oil was minimized, so it seems that the oil pretreatment could activate the defense response of the sunflower seedlings against the pathogen invasion. The main compounds in the oil were sabinene (31.1%), alpha-pinene (15.6%), and 2,3,4-trimethylbenzaldehyde (10.9%), among a total of 65 components identified.