Nitro-Oleic Acid in Seeds and Differently Developed Seedlings of Brassica napus L.

Similar to animals, it has recently been proven that nitro-fatty acids such as nitro-linolenic acid and nitro-oleic acid (NO2-OA) have relevant physiological roles as signalling molecules also in plants. Although NO2-OA is of great therapeutic importance, its presence in plants as a free fatty acid has not been observed so far. Since Brassica napus (oilseed rape) is a crop with high oleic acid content, the abundance of NO2-OA in its tissues can be assumed. Therefore, we quantified NO2-OA in B. napus seeds and differently developed seedlings. In all samples, NO2-OA was detectable at nanomolar concentrations. The seeds showed the highest NO2-OA content, which decreased during germination. In contrast, nitric oxide (•NO) levels increased in the early stages of germination and seedling growth. Exogenous NO2-OA treatment (100 µM, 24 h) of Brassica seeds resulted in significantly increased •NO level and induced germination capacity compared to untreated seeds. The results of in vitro approaches (4-Amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) fluorescence, •NO-sensitive electrode) supported the •NO liberating capacity of NO2-OA. We observed for the first time that Brassica seeds and seedlings contain free NO2-OA which may be involved in germination as an •NO donor as suggested both by the results of exogenous NO2-OA treatment of seeds and in vitro approaches. Due to their high NO2-OA content, Brassica sprouts can be considered as a good source of dietary NO2-OA intake.


Introduction
Nitro-fatty acids (NO 2 -FA) as endogenous signal molecules in animals and humans have gained great attention, since these nitrated lipid derivatives exert relevant bioactivity in association with anti-thrombotic, cytoprotective, and anti-inflammatory processes (recently reviewed in Ref. [1]). The addition reaction of nitric oxide (•NO) and •NO-derived higher oxides of nitrogen (peroxynitrite, nitrogen dioxide) with conjugated double bond-containing, unsaturated fatty acids results in the formation of NO 2 -FA; although, the in vivo mechanism is still unknown [2]. During the first proposed mechanism, radical hydrogen abstraction from a bis-allylic carbon takes place resulting in the formation of an alkyl radical which is followed by the formation of a peroxyl radical via double bond rearrangement and molecular oxygen insertion. The insertion of nitrogen dioxide (•NO 2 ) and the consequent formation of a non-electrophilic nitroalkane-alkene product is also possible [3]. The second mechanism includes

Characterization of the Synthesized NO 2 -OA Standard
The synthesized compound was identified as (E)-9-nitrooctadec-9-enoic acid based on the identical 1 H and 13 C NMR data with those reported in the literature [25]. This was assured by mass spectrometric measurements where the measured molecule ion mass was shown to be m/z 326.5, calculated neutral molecule mass was 327.4589 Da, molecular formula C 18 H 33 NO 4 ( Figure 1A,B).

Calibration
From the synthesized standard a stock standard was prepared with methanol (HPLC grade) and working solutions were prepared (50-2500 ng/mL) by diluting the stock standard. Each solution was injected three times, the precision (Relative Standard Deviation, RSD, %) of the calibration measurements ranged between 1.2093-2.3633%. Calibration showed linear regression, the R 2 value was 0.9986, the limit of detection (LOD, S/N = 3.3) was 0.1184 nmol/mL, and the limit of quantitation (LOD, S/N = 10) was 0.3588 nmol/mL ( Figure 2).

NO 2 -OA Content of Brassica napus at the Seed and Seedling Stages
Seeds and seedlings of Brassica napus are remarkably rich in the unsaturated fatty acid, oleic acid, which implies the possibility of the presence of NO 2 -OA in Brassica as well. Figure 3A represents Brassica napus seeds and seedlings at day 0, and 2nd, 4th, and 7th day after sowing. Therefore, we performed the analyses of NO 2 -OA concentrations and the quantitative data are presented in Table 1, while the mean NO 2 -OA concentrations with standard errors are shown in Figure 3. Ion chromatograms of the reference standard NO 2 -OA and of the 7-day-old Brassica napus seedlings are presented in Figure 4. Further chromatograms are presented as Figure S1. In samples, one further unidentified isomer of NO 2 -OA can be detected with higher retention time.    Brassica napus seeds contained a notably high amount of NO 2 -OA compared to seedlings (Table 1, Figure 3B). During the early phase of seedling growth (2nd day), the high NO 2 -OA content of Brassica seeds decreased by 78%, and it continued to decline in the following two days (4th day). At the 7th day of seedling growth, the seedlings showed an increased NO 2 -OA content compared to the 2-and 4-day-old seedlings, but it was only approximately 40% of the NO 2 -OA content of the seed. Separated analysis of the shoot and root material indicated that both organs of 7-day-old Brassica napus seedlings contained NO 2 -OA in similar quantities (Table 1). In Arabidopsis tissues, NO 2 -OA could not be detected, but NO 2 -Ln content was 2.9-fold higher in the seeds than in the 14-day-old seedlings ( [6], Table 2). The relatively high NO 2 -Ln and NO 2 -OA contents of Arabidopsis and Brassica seeds ( Table 1,  Table 2) indicate the involvement of NO 2 -FA in seed germination possibly as endogenous •NO donors [16]. To examine this hypothesis, we detected •NO levels in Brassica seeds and seedlings (2nd, 7th days) ( Figure 3C). Compared to seeds, on the 2nd day the •NO content increased by almost 5-fold. The opposite changes in NO 2 -OA and •NO levels suggest that following the induction of germination NO 2 -OA as endogenous donor in the seed may release NO resulting in the decrease in its own free level and concomitant •NO accumulation. Furthermore, it is important to note, that the measured NO 2 -OA concentrations in Brassica tissues are in the nanomolar range (Table 1, Figure 3B), that is an order of magnitude higher than the picomolar NO 2 -Ln concentrations of Arabidopsis seedlings, cell suspension, pea roots, leaves and rice leaves ( [6,7], Table 2). In case of NO 2 -OA supplemented tomato cell suspension, the endogenous NO 2 -OA content was similar to that of the untreated Brassica napus seeds and seedlings ( [8,9], Table 2). From the above comparisons, it can be concluded that the oleic acid content of Brassica napus seeds and seedlings is susceptible to physiological nitration making the plant a rich source of NO 2 -OA.

Exogenous NO 2 -OA Treatment of Brassica Seeds Positively Influences •NO Levels and Germination Capacity
The 24-hour-long treatment with 100 µM NO 2 -OA caused 10-fold increment in the endogenous •NO level of seeds ( Figure 5A,B), while NO 2 -OA at 50 µM or 500 µM concentrations did not cause significant changes in •NO levels compared to controls (50 or 500 µM DMSO, respectively). The significant effect of 100 µM NO 2 -OA on •NO level prevailed also on the 2nd day after sowing, since the •NO level of NO 2 -OA-supplemented seedlings was 4.7 times that of the control (100 µM DMSO, Figure 5A,B). The NO 2 -OA-induced DAF-FM fluorescence was quenched by the •NO scavenger cPTIO ( Figure 5B) indicating that the alterations in DAF fluorescence correspond to •NO level changes. Oleic acid (OA) treatments applied as controls did not influence •NO levels either in seeds or in 2-day-old seedlings ( Figure 5A). The germination percentages were altered in accordance with •NO levels, since 100 µM NO 2 -OA treatment doubled the percentage of germinated seeds compared to control (100 µM DMSO) ( Figure 5C). DMSO alone at 50 µM or 100 µM concentrations had no effect on seed germination, although in the presence of 500 µM DMSO germination percentage of Brassica seeds slightly decreased compared to control (0 µM DMSO). Moreover, OA at 50 or 100 µM concentration exerted no effect on germination of B. napus seeds ( Figure 5C). The effect of DMSO could dominate in case of 500 µM NO 2 -OA or 500 µM OA treatments, since in these seeds the germination ability was weaker than in control and •NO levels remained at control level ( Figure 5A). These data clearly indicate that exogenous NO 2 -OA can result in the long-term increase of endogenous •NO level in Brassica napus seeds and seedlings, i.e., it may act as a •NO donor. The fact that nitro-fatty acids may act as •NO donors in plants has been previously raised [16]. It was revealed that NO 2 -Ln has the ability to release in vivo, in leaves and roots of old Arabidopsis plants [26], in Arabidopsis seedlings [16], and in Arabidopsis cell-suspension cultures [27]. Contrary to NO 2 -Ln and to NO 2 -OA in our study, exogenous application of NO 2 -OA (0.5, 5, 10, 12.5, 25, or 50 µM, 1 h or 6 h) did not increase •NO level in tomato cell suspension as was recently reported by Di Palma et al. [8,9]. Contradiction of the results can be explained by that here higher concentration of NO 2 -OA (100 µM) were applied for longer time period (24 h) than in the studies of Di Palma et al. [8,9].

NO 2 -OA Releases •NO in Vitro
Additionally, the •NO donor nature of NO 2 -Ln was proved by using several different in vitro approaches (DAF fluorescence, oxyhaemoglobin method, ozone chemiluminescence, [27]. In this work, we performed in vitro tests to support the hypothesis regarding the •NO donor role of NO 2 -OA ( Figure 6). Spectrofluorometric measurement of DAF-FM-associated fluorescence revealed that the sample containing 10 µM NO 2 -OA liberated a relatively small amount of •NO during the 80 min period, while the same dosage of OA did not induce •NO level increase compared to the blind sample containing only buffer and the fluorophore ( Figure 6A). Moreover, NO 2 -OA liberated •NO in a concentration-dependent manner and the fluorescence increase could be quenched by cPTIO. Elevating doses of OA did not increase •NO levels ( Figure 6B).
Using the more sensitive •NO electrode, we could quantify •NO liberation in NO 2 -OA solution (pH 5.8) within 5 min reaching its maximum (~30 nM •NO) after 20 min incubation. The produced •NO concentrations (20-30 nM) quantified by ISO-NOP electrode in this study are similar to those measured by •NO autoanalyzer in the case of 100 µM NO 2 -Ln or 80 µM NO 2 -LA [27]. The same concentration of OA showed no relevant •NO releasing capacity in the solution ( Figure 6C). These in vitro data support the •NO donor character of NO 2 -OA in solutions and the degree of •NO liberation is similar to other NO 2 -FAs. However, the same dosage of S-nitrosoglutathione (GSNO) or sodium nitroprusside (SNP) produced approx. 10-fold higher •NO concentration in solutions following 20 min incubation in light (data not shown) indicating that NO 2 -FAs (including NO 2 -OA) •NO donor capacity is much lower compared to "classical •NO donors". In our experimental system, NO 2 -OA (100 µM, 24 h) treatment of Brassica seeds also promoted germination, presumably through the induction of high •NO levels. In vitro tests revealed that the concentration of liberated •NO is relatively low (Figure 6), but in seeds NO 2 -OA treatment caused intense •NO formation ( Figure 5). Therefore, we assume that beyond direct •NO emission, secondary signal processes may also be activated in the presence of NO 2 -OA leading to the activation of •NO metabolic routes in seeds and seedlings. It is well known, that •NO attenuates seed dormancy and promotes germination and we are beginning to recognize also the molecular mechanisms of •NO action [28].

Plant Material and Growing Conditions
Experiments were carried out on Brassica napus L. (cv. GK Gabriella) seedlings. The seeds were obtained from the Cereal Research Non-profit Ltd., Szeged, Hungary. Brassica seeds were surface sterilised in 70% (v/v) ethanol and 5% (v/v) sodium hypochlorite, then placed on moistened filter paper in Petri dishes (9 cm diameter, 30 seeds/Petri dish). Germination took place under controlled conditions (150 µmol m −2 s −1 photon flux density, 12 h/12 h light/dark cycle, relative humidity 55-60% and temperature 25 ± 2 • C). For the NO 2 -OA analysis, the samples were taken at the 2nd, 4th and 7th day after sowing. Additionally, seeds after imbibition, at the early stage of germination (day 0) were also sampled for NO 2 -OA quantification. Plant material (5 g) was collected, frozen in liquid nitrogen and stored at -80 • C until the analyses.

Synthesis and Structure Determination of 9-Nitro-Oleic Acid Standard
Using the slightly modified method of Woodcock et al. [29], 9-nitro-oleic acid was synthesized. Bromononanoic acid was used as starting material for allylization to gain 9-bromononanoic acid allyl ester.
This compound was nitrated using silver nitrite.
In the next step, 10-hydroxy-9-nitro-octadecanoic acid allyl ester was synthesized by the addition of nonyl aldehyde and 1,8-diazabicyclo [5.4.0]undec-7-ene. This compound was acylated with acetic anhydride in the presence of p-toluenesulfonic acid to gain 10-acetoxy-9-nitro-octadecanoic acid allyl ester. Deacylation in the presence of Na 2 CO 3 led to the synthesis of 9-nitro-oleic acid allyl ester. The final product 9-nitro-oleic acid was gained by a catalytic hydrolysis using palladium tetrakis(triphenylphosphine). Purification was carried out by flash chromatography on silica gel (Merck, 40-63 µm) using a gradient of 0.5% acetic acid. Fractions with similar compositions were combined and the combined fraction containing a spot with remarkable absorption at 254 nm and with an R f approximately 0.5 (silica gel plate, eluent CHCl 3 -MeOH 95:5) was subjected to NMR identification and purity check.
1 H (500.1 MHz) and 13 C (125.6 MHz) NMR spectra were recorded in CDCl 3 on a Bruker Avance DRX-500 spectrometer. The peaks of the residual solvent were taken as reference points. The compound was identified by comparison of its chemical shifts with literature data [25,30]. Mass spectrometric identification was performed on an API 2000 triple quadrupole tandem mass spectrometer (MDS Sciex, Toronto, ON, Canada) equipped with electrospray ion source. Mass spectrometric measurement was carried out by direct infusion in Q1 MS scan type in negative mode. Flow rate was set to 40 µL/min, scan range was set from m/z 100 to 1000, ion source temperature was set to 100 • C. The nebulizer gas was set to 18 psi. Measured molecule ion mass shown to be m/z 326.5, calculated neutral molecule mass was 327.4589 Da.

LC-MS Quantification of NO 2 -OA in Brassica Seeds and Seedlings
For the quantification of NO 2 -OA, 5 g of fresh plant material (seeds, whole seedlings or separated root and shoot) was used and the analysis was conducted by LC-MS. Mass spectrometry measurement was performed using single ion monitoring (SIM) on an API 2000 triple quadrupole tandem mass spectrometer (MDS Sciex, Toronto, ON, Canada) equipped with electrospray ion source. The nebulizer and heater gas was nitrogen, generated from a Peak NM20Z nitrogen generator (Peak Scientific Instruments Ltd., Scotland, UK) coupled with an Atlas Copco SF 4FF compressor. The nebulizer gas was set to 30 psi, the heater gas was set to 80 psi. The ion source temperature was set to 300 • C. Measurement was in negative mode. The voltage volumes were adjusted to m/z 326.5, the collision energy was set to −5 V, focusing potential to −330 V, declustering potential to −61 V and entrance potential to −10 V. HPLC separation was performed with Shimadzu HPLC system (Kyoto, Kyoto Prefecture, Japan): DGU-20A3 degasser, CBM-20A controller, two LC-20AD pumps, SIL-20A HT autosampler, CTO-20AC column thermostat, SPD-20A UV-Vis detector, using Kinetex F5 (100 × 4.6 mm, 2.6 µm, 100Ä) (Phenomenex, Inc., Torrance, CA, USA). Elution was carried out with the gradient system of H 2 O-methanol (0 min: H 2 O-methanol 2:8, 0.6 min: 2:8, 1.5 min: 0:1, 3 min: 0:1, 3.2 min: 2:8, 8.2 min: 2:8). The oven was set to 40 • C and the flow rate was 600 µL/min. The retention time of NO 2 -OA was 4.32 min. Data acquisition was performed with Analyst software (ver. 1.6.3). Quantification of nitro-oleic acid was carried out by using the synthesized standard (see in Section 2.2).
Plant material was extracted with pure MeOH (HPLC grade) in a VWR Ultrasonic Cleaner USC 300D (capacity: 1 L, internal dimension: W × D × H 240 × 135 × 100 mm) ultrasonic bath for 10 min, with dual half-wave sound with sweep, frequency was set to 45 kHz, ultrasonic power to 80 W, temperature was set to 25 • C, and filtered through a syringe filter (PTFE, 0.45 µm pore size, Labex Ltd.), the first half ml of the filtrate was thrown into the waste. From each plant part, three parallel samples were prepared, and each sample was injected three times.

NO 2 -OA Treatment of Brassica napus Seeds
The synthesized NO 2 -OA or OA was dissolved in dimethyl sulfoxide (DMSO) in order to obtain a stock solution (10 mM). The NO 2 -OA or OA stock was diluted with distilled water to the final concentrations (50, 100, 500 µM). Control solutions were prepared by measuring the volume of DMSO corresponding to the stock solutions (indicated as 0, 50, 100, or 500 DMSO). Brassica napus seeds (30 seeds per treatment) were incubated in NO 2 -OA, in OA or in control solutions for 24 h on an orbital shaker and then were placed on moist filter paper in Petri dishes (20 seeds/Petri dish). Sets of seeds were treated with NO 2 -OA or control solutions in the presence of 800 µM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Germination took place as indicated above. Nitric oxide levels were detected in seeds prior to germination (day 0), and in seedlings 2 days after sowing (2nd day). Germination percentages (%) were also calculated.

Spectrofluorometric Determination of •NO Levels
•NO liberation from NO 2 -OA solutions were detected by fluorescence spectrophotometry (Ref. [27] with modifications). Reaction mixtures (2 mL final volume) containing 0 (blind) 10, 25, 35, 50, 125 µM NO 2 -OA or the same concentrations of OA, plus 2 µM DAF-FM and Tris-HCl (pH 7.4) buffer were incubated at room temperature in the dark for several time periods (5-80 min) and the emitted fluorescence was recorded by a spectrofluorimeter (Hitachi F-4500, Hitachi Ltd., Tokyo, Japan). Excitation wavelength was set at 485 nm and emissions were measured at 515 nm. The NO 2 -OA-induced fluorescence was quenched by the addition of 100 µM cPTIO.

Measurement of •NO Concentration by •NO-Specific Electrode
The •NO -sensitive electrode (ISO-NOP, 2 mm, World Precision Instruments Inc., Sarasota, FL, USA) was calibrated using a method based on S-nitroso-N-acetylpenicillamine (SNAP) decomposition to •NO in the presence of copper [32]. Two mL of NO 2 -OA or OA (both at 50 µM concentration) solutions were prepared in a 5-mL glass bottle and were measured immediately after preparation. To ensure constant mixing of the solution a magnetic stirrer was applied during the measurement. •NO concentration (nM) was calculated from a standard curve.

Statistical Analysis
All results are shown as mean ± SE. Data were statistically evaluated by the Holm-Sidak method (One-way ANOVA, P ≤ 0.001) using SigmaPlot 12.

Conclusions
Following successful standard synthesis and method optimization, we have been the first to observe that Brassica seeds and seedlings contain free NO 2 -OA. Exogenous treatment of Brassica seeds with NO 2 -OA promoted germination and increased endogenous •NO level suggesting that NO 2 -OA may be involved in germination as an •NO donor. The •NO liberating capacity of NO 2 -OA was proved also by in vitro approaches (spectrofluorometric detection of DAF-FM fluorescence and •NO-sensitive electrode). Due to their relatively high NO 2 -OA concentrations, Brassica sprouts can be considered as a good source of dietary NO 2 -OA intake in addition to their nutrient, mineral and vitamin content. Therefore, future studies should quantify the NO 2 -OA content of additional Brassica species and food plants.