Nitric oxide release from nitro-fatty acids in Arabidopsis roots

ABSTRACT

In recent years, research on the involvement of nitric oxide (NO) in plant systems has remarkably grown. However, most of the interest in this molecule has been focused on its ability to mediate different post-translational modifications (NO-PTM) in biomolecules, mainly nitration and S-nitrosylation of proteins, and its involvement in physiological and stress situations. Nevertheless, very recently the nitration of other molecules such as fatty acids has commanded increasingly greater attention. In the last February issue of Plant Physiology, we again reported on the endogenous occurrence of nitro-fatty acids (NO₂-FAs), specifically nitro-linolenic acid (NO₂-Ln), in the model plant Arabidopsis thaliana. The analysis of the presence of this nitro-fatty acid showed that levels of NO₂-Ln decreased throughout the plant development with the higher levels detected in seeds and young seedlings of this plant. Furthermore, through a transcriptomic analysis by RNA-seq technology applying NO₂-Ln to A. thaliana cell-suspension cultures, we found high induction in the transcriptional expression of several heat-shock proteins (HSPs) and the enzymes ascorbate peroxidase (APX) and methionine sulfoxide reductase (MSR). Based on these findings, the involvement of NO₂-Ln in the NO metabolism was analyzed showing a significant NO formation in roots from 7-day-old Arabidopsis thaliana seedlings and standing out that NO generated from NO₂-Ln could have an important role at the beginning of plant development. Therefore, these findings highlight the importance of these novel NO-derived molecules in plant systems playing a pivotal role in development and in the antioxidant defense response against different abiotic stress conditions.

Nitro-fatty acids (NO₂-FAs) stem from the reaction between NO-derived molecules and non-saturated fatty acids. The result from this interaction yields a family of molecules that are more stable and have a longer half-life than other NO-derived molecules.¹ Among the main properties of NO₂-FAs, the ability to mediate different anti-inflammatory effects in animal systems is noteworthy,^2,3 thus being considered important mediators of cell signaling. Due to the chemical structure of NO₂-FAs, these molecules have different biological properties, including the release of nitric oxide (NO) in aqueous mediums^4-6 and an electrophilic capacity allowing a reversible posttranslational modification.^7,8 Regarding the ability to release NO, basically 2 mechanisms of NO generation from NO₂-FAs have been proposed, including a modified Nef reaction or by a nitroalkene-rearrangement.^4,6,9 Otherwise, the β-carbon adjacent to the nitro group (-NO₂) has a strong electrophilicity, representing a target for nucleophilic addition and thus generating a covalent bond with NO₂-FAs by a reaction specifically termed nitroalkylation. In this sense, this electrophilic character allowing NO₂-FAs to modulate key cellular targets has been widely reported, e.g., by the activation of the peroxisome proliferator-activated receptor (PPAR) or by the inhibition of nuclear factor-kappa B (NF-B).^10,11

Despite the progress made in the research field of NO₂-FAs in animal systems, the investigation of these signaling molecules in plant systems has been scarcely studied. In this respect, Sánchez-Calvo et al. (2013)¹² evaluated the capacity of a nitro-fatty acid, specifically nitro-linolenic acid (NO₂-Ln), for generating NO. The results of this study showed an increase in green fluorescence due to NO release both in leaves and roots of Arabidopsis thaliana plants pre-incubated with NO₂-Ln. Furthermore, pre-incubation of these plants with the NO scavenger cPTIO reduced the NO signal in both organs, suggesting that NO₂-Ln is a NO donor. Additionally, the endogenous presence of nitro-conjugated linoleic acid (NO₂-cLA) in extra-virgin olive oil (EVOO) and protein cysteine adducts of nitro-oleic acid (NO₂-OA) in fresh olives has been reported.¹³ Besides these previous studies, in a recent publication we have again explored the endogenous occurrence of NO₂-Ln in the model plant A. thaliana by mass spectrometry and the possible role of this acid in plant systems through a transcriptomic approach by RNA-seq technology.¹⁴ In this work, we found the endogenous presence of NO₂-Ln in A. thaliana at picomolar levels, suggesting a role in plant signaling, as previously mentioned for animal systems. In this regard, we performed a transcriptomic approach by RNA-seq technology in A. thaliana cell-suspension cultures incubated with NO₂-Ln. The results from this analysis showed, at the transcriptional level, an increase to a high percentage of different heat-shock proteins (HSPs), from which most of them were small HSPs. These findings agree with those of a previous microarray analysis in cultures of human endothelial cells incubated with NO₂-OA¹⁵ displaying a conserved mechanism of action both in animal and plant systems. Through several bioinformatic studies, we found that NO₂-Ln was involved in the response to oxidative-stress situations with a high induction in ascorbate peroxidase (APX) transcripts. Also related to oxidative-stress events, NO₂-Ln was able to induce the transcriptional levels of methionine sulfoxide reductase and alkenal reductase enzymes to set up a defense mechanism against this stress. Therefore, this data set derived from bioinformatic analysis highlights the important signaling role of NO₂-Ln in the plant-defense mechanism against different abiotic-stress situations. In this respect, we sought to confirm the involvement of NO₂-Ln in several abiotic-stress conditions and, for this reason, the levels of NO₂-Ln were analyzed under stress caused by mechanical wounding, cadmium, low temperature, and salinity. In all cases, we detected a significant increase in NO₂-Ln levels, suggesting a potential role for this molecule in setting up a defense mechanism against these adverse abiotic conditions by the induction of several antioxidant systems.

In addition to analyzing the endogenous presence of NO₂-Ln in A. thaliana, we extended our study to an exploration of the levels of NO₂-Ln throughout the development of this plant. In this sense, we found the highest levels of this nitro-fatty acid at the beginning of development (seeds and 14-day-old whole seedlings) with a subsequent decrease at the final stages of this process (adult and senescent leaves). Due to the ability of NO₂-Ln to release NO,¹² the highest levels of NO₂-Ln observed at initial stages of development could contribute to the availability of NO at the beginning of this process, favoring germination and promoting the onset of vegetative development. Because this study was made mainly with leaves from A. thaliana, we analyzed the ability to release NO in the roots of young A. thaliana plants (7-day-old seedlings) pre-incubated with NO₂-Ln, using confocal laser scanning microscopy (CLSM) (Fig. 1). This analysis showed that pre-incubation of 7-day-old roots from A. thaliana with 100 µM of NO₂-Ln prompted a rise in green fluorescence corresponding to an increase in NO generation (Fig. 1, panel C), while this behavior was not detected in plants untreated and pre-incubated with linolenic acid (Ln) (Fig. 1, panels A and B). Furthermore, to confirm that this release of NO was due to NO₂-Ln, 7-day-old seedlings were pre-incubated with the NO scavenger cPTIO followed by the treatment with NO₂-Ln and the green fluorescence returned to endogenous levels of NO (Fig. 1, panel D). Therefore this data set confirms the capacity of NO₂-Ln to release NO in roots and leaves of A. thaliana plants, which could be important at the initial stages of plant development.

Figure 1.

Nitric oxide (NO) released from nitro-linolenic acid (NO₂-Ln) in Arabidopsis root seedlings. Representative images illustrating the CLSM in vivo detection of NO (green color) in primary roots of Arabidopsis seedlings pre-incubated with (B) 100 µM of linolenic acid (Ln), (C) nitro-linolenic acid (NO₂-Ln) or (D) NO₂-Ln in the presence of 100 µM cPTIO. Control samples (A) show untreated endogenous NO content.

On the basis of the results found in our recently published paper together with the ability of NO₂-Ln being a NO donor, the defense mechanisms which this nitro-fatty acid is able to establish may be due to different pathways. On the one hand, NO₂-Ln can release nitric oxide and therefore this nitro-fatty acid could be involved in the wide range of actions that involve NO. Notable among these actions are the relationship with plant development, several biotic and abiotic processes, the involvement in different post-translational modifications (NO-PTM) such as nitration and S-nitrosylation of proteins, and the modulation of the antioxidant response among many other functions. On the other hand, the electrophilic capacity of the adjacent carbon to the nitro group of NO₂-Ln may mediate nitroalkylation events with different protein targets and thus be involved in several cellular pathways, as it is a signaling molecule as has been shown in animal systems (Fig. 2).

Figure 2.

Model of NO₂-Ln signaling. On one hand, NO₂-Ln may release nitric oxide (NO) and therefore it could be involved in the plethora of actions in which NO is involved such as plant development, biotic and abiotic stress conditions, an antioxidant response and the posttranslational modifications mediated by NO (NO-PTM) such as nitration and S-nitrosylation. On the other hand, the electrophilic capacity of NO₂-Ln may mediate nitroalkylation events with different protein targets and therefore be involved in several signaling events. * indicates the electrophilic β-carbon adjacent to the NO₂ group.

Future experiments investigating the potential of NO₂-Ln modulating the NO release, nitroalkylation or other possible mechanisms will hopefully offer insight into how this nitro-fatty acid signaling regulates the important defense mechanism that this molecule is able to establish.

Material and methods

Plant materials and growth conditions

Arabidopsis thaliana ecotype Columbia seeds were surface-sterilized for 5 min in 70% ethanol containing 0.1% SDS, then for 20 min in sterile water containing 20% bleach and 0.1% SDS and finally washed 4 times in sterile water. A. thaliana 7-day-old seedlings were grown in Petri plates according to the method outlined by Mata-Pérez et al. (2016).¹⁴ The plants were grown at 16 h light, 22°C/ 8 h dark, and 18°C under a light intensity of 100 µE m⁻² s⁻¹ for a total of 7 d.

Nitro-linolenic acid (NO₂-Ln) synthesis

Nitro-linolenic acid (NO₂-Ln) was synthesized by a nitroselenation procedure as described elsewhere.¹⁴

Detection of nitric oxide (NO) by confocal laser scanning microscopy (CLSM)

For CLSM detection of NO, A. thaliana seedlings were pre-incubated with 0 and 100 µM of linolenic acid (Ln) and nitro-linolenic acid (NO₂-Ln), and NO was then detected in A. thaliana roots with 10 µM 4-aminomethyl-2′,7′-difluorofluorescein diacetate (DAF-FM DA, Calbiochem) prepared in 10 mM Tris-HCl (pH 7.4), as described elsewhere.¹⁶ As negative controls, samples were pre-incubated for 30 min with 100 µM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger. Samples were examined using a confocal laser scanning microscope (Leica TCS SL, Leica Microsystems, Heidelberg GmbH, Wetzlar, Germany). In all cases, the images obtained by the CLSM system from control and treated A. thaliana seedlings were kept constant during the course of the experiments in order to compile comparable data.

Disclosure of potential of conflicts of interest

No potential conflicts of interest were disclosed.