Differential patterns of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets

Patterns of accumulation of singlet oxygen, superoxide radical and hydrogen peroxide

The transfer of plantlets after 3 weeks of growth to control and treatment media, as described in Methods, was designed to compare plantlets at the same developmental and physiological stages. As previously described in numerous studies of sugar effects in plants, mannitol treatment was used as osmotic control. Moreover, we previously showed that the deleterious effects of atrazine on Arabidopsis plantlets followed the same dose-response curve and the same time dependence in the absence or presence of 80 mM mannitol [16, 17]. It was also verified that, in accordance with previous studies [26], exogenous sugar treatment resulted in increased levels of endogenous soluble sugars in Arabidopsis plantlets (data not shown).

At the end of treatments, plantlets were specifically stained for singlet oxygen, superoxide radical, and hydrogen peroxide. Hideg et al. [27] described some limitations in the use of vacuum infiltration of ROS probes and reagents with excised leaves or leaf segments from pea, spinach or tobacco. However, vacuum infiltration has been successfully used on whole Arabidopsis thaliana plantlets under various experimental conditions [28–30]. Moreover, under the conditions of the present work, whatever the dye used and therefore the ROS detected, the non-stressed plantlets, transferred to 80 mM mannitol or 80 mM sucrose media, presented expected responses related to ROS production (Fig. 1, 2 and 3; Additional files 1, 2 and 3). Plantlets that were transferred for 12 h on mannitol medium presented the same ROS levels as three-week-old plantlets prior to transfer (Fig. 1, 2 and 3; Additional files 1, 2 and 3).

Detection and quantification of singlet oxygen (¹O₂) were performed with the specific Singlet Oxygen Sensor Green^® reagent [31]. For atrazine-containing treatments (MA and SA), green fluorescence indicating primary events of ¹O₂ accumulation was detected in cotyledons as soon as after 12 hours of treatment (Fig. 1 and Additional file 1). Tolerance treatment (SA) maintained a low level of ¹O₂ in cotyledons throughout the treatment, while atrazine treatment (MA) strongly increased ¹O₂ production in cotyledons and leaves from 12 to 72 hours of treatment. The presence of sucrose in herbicide-containing medium thus appeared to prevent accumulation of ¹O₂ generated by atrazine.

Superoxide radical (O₂^.-) detection and quantification were performed using the nitroblue tetrazolium (NBT) staining method. The levels of superoxide radical staining after 12 hours of transfer (Fig. 2 and Additional file 2) were quite similar in the absence (M or S) or presence (MA or SA) of 10 μM atrazine. However, the time-course revealed constant levels of O₂^.- in control plantlets (M), while a strong blue coloration appeared in plantlets treated with sucrose (S). This increase was more visible in young leaves. Superoxide radical levels in atrazine-treated plantlets (MA) decreased from 24 hours of treatment. The combination of sucrose plus atrazine (SA) led to an intermediate state with slight coloration maintained in young leaves throughout the treatment. Low levels of O₂^.-, relatively to the mannitol control, were also observed when a drop of 10 μM atrazine solution was directly applied to leaf tissue (data not shown).

H₂O₂ detection and quantification were performed using the 3,3'-diaminobenzidine (DAB) staining method [32]. Polymerization of DAB, visible as a brown precipitate in the presence of H₂O₂, was detected under all conditions. No coloration was observed when infiltration was carried out in the presence of ascorbic acid, thus confirming the H₂O₂ specificity of DAB staining, in accordance with previous reports [33–36]. Figure 3 and Additional file 3 summarize the time-course of H₂O₂ accumulation. From 24 hours of transfer, control (M) and sucrose-treated (S) plantlets exhibited a much weaker level of H₂O₂ than plantlets of atrazine-containing treatments (MA and SA). No variation of H₂O₂ accumulation was detected in the presence of mannitol, whereas H₂O₂ content decreased in sucrose-treated plantlets. In contrast, atrazine in the absence or presence of sucrose tended to increase progressively H₂O₂ levels until 72 hours of treatment. This increase could be detected as early as the fourth hour of atrazine treatment (data not shown). Likewise, an immediate increase of H₂O₂ levels was also observed when a drop of 10 μM atrazine solution was directly applied to leaf tissue (data not shown).

Patterns of singlet oxygen quenching mechanisms

Another group of genes exhibited induction of expression under atrazine conditions, whereas they were less induced or not differentially expressed under sucrose-atrazine conditions. Activation of these genes might reflect stress signalling due to high ¹O₂ content in atrazine treated-cells, as revealed by ROS detection ((Fig. 1 and Additional file 1). Some of these genes belonged to carotenoid biosynthesis pathways, such as Zeta-carotene desaturase ZDS1 (At3g04870), beta-carotene hydroxylase (At4g25700) or 4-hydroxyphenylpyruvate dioxygenase HPD (At1g06570) (Table 1). Carotenoids, which are known to act in chloroplasts as accessory pigments in light harvesting, can detoxify ¹O₂ and triplet chlorophyll and dissipate excess excitation energy [9].

Higher induction by atrazine treatment was also found for the violaxanthin de-epoxidase precursor (At1g08550) gene, which is involved in the xanthophyll cycle (Table 1). Together with carotenoids, zeaxanthin, synthesized from violaxanthin via the xanthophyll cycle, protects chloroplasts by accepting excitation energy from singlet chlorophyll [42]. Two genes involved in the shikimate (shikimate kinase, At3g26900) and terpenoid pathways (geranylgeranyl pyrophosphate synthase, At4g36810), which are essential for tocopherol synthesis [43], were also induced by the herbicide and not differentially expressed by the tolerance treatment (SA) (Table 1). The antioxidant tocopherol is known to scavenge oxygen free radicals, lipid peroxy radicals and ¹O₂ [44]. Finally, the presence of atrazine alone was found to induce the At3g55610 gene, which is involved in proline synthesis, with a higher intensity than under conditions of combination with sucrose (Table 1). Proline is also known to be an ¹O₂ quencher [45].

Patterns of superoxide radical scavenging mechanisms

Excess of superoxide radical caused by numerous environmental stresses is detoxified by superoxide dismutase (SOD) enzymes and converted into H₂O₂. Seven isoenzymes have been identified, differing by their metal cofactor (Fe, Mn, or Cu and Zn), in Arabidopsis thaliana [46]. Transcriptome profiling was carried out after 24 hours of treatment [18]. Measurements of enzyme activities at different times of treatment showed that modifications were most contrasted after 48 hours of treatment (data not shown). Thus, given the potential delay between transcription and protein synthesis, modifications of global SOD activities after 48 hours of treatment were compared with modifications of SOD-encoding transcript levels after 24 hours of treatment.

SOD activity (Fig. 4) was decreased by atrazine treatment (MA) in comparison to the mannitol control (M). In contrast, addition of sucrose in the presence of atrazine (SA) maintained a functional level of SOD activity equivalent to that of the mannitol control. Since sucrose alone was found to increase SOD activity, it thus seemed that sucrose might balance the negative effect of atrazine in the situation of SA treatment.

Potential origin of hydrogen peroxide accumulation in the presence of atrazine

Patterns of hydrogen peroxide scavenging mechanisms

In order to investigate the efficiency of hydrogen peroxide scavenging mechanisms, global H₂O₂-scavenging enzyme activities and transcript levels of related genes were analysed. As explained above, modifications of enzyme activities after 48 hours of treatment were compared with modifications of transcript levels after 24 hours of treatment.

H₂O₂ can be principally scavenged by two different ways: ascorbate-glutathione cycles and catalases, which play important roles in plant defence and senescence. Ascorbate-glutathione cycles are catalysed by a set of four enzymes: ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), glutathione-dependent dehydroascorbate reductase (DHAR), and glutathione reductase (GR) [47].

The five enzymes belonging to H₂O₂-scavenging mechanisms presented two different profiles of global activity according to the different treatments. The majority of enzymes involved in ascorbate-glutathione cycles (APX, DHAR and MDAR) were differentially affected by the different treatments. Activity of these three enzymes was significantly reduced by addition of atrazine, while sucrose treatment had an opposite effect and significantly increased these activities (Fig. 5a, b, c). The tolerance condition (SA) succeeded to limit repressive effects of the herbicide and maintained enzyme activities at the control level. The fourth enzyme of the ascorbate-glutathione cycles, GR, did not present any significant variation of activity between the different treatments (Fig. 5d). Finally, catalase exhibited slightly lower activity under conditions of sucrose plus atrazine, when compared to control and atrazine-containing medium (Fig. 5e).

Dehydroascorbate reductase (DHAR) is a key component of the ascorbate recycling system. DHAR recycles dehydroascorbate into ascorbate by using reduced glutathione as a reductant. Two functional DHAR genes, among three that are encoded in the Arabidopsis thaliana genome, plus a putative gene, were represented in the microarray (Table 5). The cytosolic DHAR2 (At1g75270) and putative DHAR (At5g36270) genes exhibited high induction in the combined presence of sucrose and atrazine, and, respectively, a slight repression or no variation in the presence of atrazine in comparison to control condition. In contrast to the repressive effects of atrazine, which were associated with a decrease of DHAR activity, the increase of DHAR transcript levels in the combined presence of sucrose and atrazine was not associated with an increase of global DHAR enzyme activity (Fig. 5b).

Reduction of monodehydroascorbate by monodehydroascorbate reductase (MDAR) is also an important step in ascorbate recycling. Among the five MDAR genes present in the microarray, only cytosolic MDAR2 (At5g03630) exhibited differential expression patterns according to the treatment applied. While atrazine repressed its expression, the protective combination of sucrose and atrazine upregulated it (Table 5). Atrazine was also found to decrease global MDAR activity, while sucrose plus atrazine treatment resulted in maintenance of MDAR activity relatively to the mannitol control (Fig. 5c).

Glutathione serves as a reductant in oxidation-reduction processes, such as recycling of oxidised ascorbate by dehydroascorbate reductase [48]. Reduction of oxidised glutathione is catalysed by glutathione reductase (GR), which requires NADPH. Among the two isoenzymes present in the microarray, only the cytosolic glutathione reductase GR1 (At3g24170) was found to be induced by sucrose-atrazine and sucrose treatments, while no variation of expression was detected in the presence of atrazine (Table 5). These variations of expression were not associated with changes of global GR activity, since no significant difference of activity was observed between treatments (Fig. 5d).

Characterisation of the impact of atrazine on ROS patterns

ROS patterns appear to depend strongly on the nature and intensity of stress conditions applied to plants [50]. It is therefore of great importance to characterise ROS accumulation kinetics associated with a particular stress, and not to rely on expected effects. Thus, while, as expected, atrazine inhibition of photosystem II was associated with ¹O₂ accumulation [7] (Fig. 1 and Additional file 1), decrease of superoxide radical levels and increase of H₂O₂ levels were also observed (Figs. 2, 3 and Additional files 2, 3). This disagreed with the proposed, but experimentally unproven, accumulation of superoxide radical by triazine treatment in Arabidopsis leaves [51]. It was however coherent with inhibition of photosynthetic activity and of the Mehler reaction, whereby superoxide radical is formed by reduction of oxygen at the PSI site [52]. Atrazine binding to D1 protein of PSII and inhibition of electron feeding to PSI were indeed likely to decrease superoxide radical production by blocking the Mehler reaction.

The induction of H₂O₂ accumulation by atrazine was all the more surprising as it occurred rapidly after transfer to atrazine (Fig. 3 and Additional file 3) and in the absence or in the presence of sucrose, which by itself had a negative effect on H₂O₂ accumulation. This is, to our knowledge, the first demonstration of rapid in vivo H₂O₂ accumulation under conditions of atrazine treatment. The negative effect of sucrose on H₂O₂ accumulation was consistent with the previously-described repression of protein and lipid catabolism, including a number of oxidase-based processes, by soluble sugars [18, 53]. In contrast, atrazine by itself was found to induce a number of genes encoding oxidases, the most highly induced being a gene encoding a proline oxidase (Table 4). Since this induction occurred prior to significant impairment of photosystems and phototrophic growth [18], it could not be ascribed to a situation of metabolic starvation. Activation of protein and lipid catabolism and of oxidase-based processes has been reported to occur under conditions of carbohydrate limitation or starvation [54, 55]. In this context, it was extremely interesting that, in the presence of exogenous sucrose, i.e. in a situation of carbohydrate optimum, atrazine was able to induce a number of oxidase-encoding genes and other genes typical of carbohydrate-limitation response, such as the gene encoding isovaleryl-CoA dehydrogenase [18, 56].

Numerous abiotic stressors, including xenobiotics, are known to produce oxidative stress in photosynthetic organisms. This is the case for benzoxazolinone [57], metronidazole, dinoterb [58], acetochlor [59], copper [60], wounding [61] and high light [6]. Studies on these stresses mainly focus on the effects of a single ROS and rarely consider the effects of ROS combination. However, ROS are chemically distinct and selectively perceived for the fine control of adjusting antioxidants and photosynthesis to different environmental stress conditions [62]. Indeed, cross-talk between ¹O₂ and H₂O₂ has been clearly demonstrated by Laloi et al. [50], who suggested antagonistic interactions between ¹O₂ and H₂O₂ with a reduction of ¹O₂-mediated cell death and stress signalling response by H₂O₂ content. In contrast, the present condition of atrazine treatment, which eventually leads to plantlet death, was characterised by high ¹O₂, high H₂O₂ and low superoxide radical levels. Laloi et al. [63], who described antagonistic effects between ¹O₂, and H₂O₂, modulated H₂O₂ levels in Arabidopsis transgenic plants at the plastid level. It was thus possible that non-antagonistic effects of H₂O₂ and ¹O₂ under conditions of atrazine treatment were due to differences of ROS localisation.

Finally, among the set of 29 induced transcription factors that have been characterized as ¹O₂-specific by Gadjev et al. [64], only one was slightly induced (data not shown) during the course of atrazine treatment despite the high accumulation of singlet oxygen (Fig. 1 and Additional file 1). The analysis of ¹O₂ responses by Gadjev et al. [64] was based on studies of the Arabidopsis conditional flu mutant [65]. It was thus clear that other signals than ¹O₂ were perceived by atrazine-treated plantlets or that atrazine-induced ¹O₂ accumulation involved other processes and responses than flu-mutant-dependent ¹O₂ accumulation [50, 64, 65]. However, full characterisation of the signalling events associated with xenobiotic exposure in plants remains to be carried out.

Impairment of antioxidant defences in the presence of atrazine

Atrazine-treated plantlets were characterised by low O₂^.- levels and high H₂O₂ levels, in contrast with sucrose-treated atrazine-tolerant plants, which showed high O₂^.- and high H₂O₂ levels. These differences of ROS patterns were associated with striking differences of gene expressions and enzyme activities involved in ROS-scavenging pathways.

Thus, atrazine sensitivity was associated with down-regulation of key players of H₂O₂ scavenging. Among the four enzymes involved in ascorbate-glutathione cycles, which are essential to remove large amounts of H₂O₂ generated by stress [48, 66], three enzymes (APX, MDAR and DHAR) exhibited a significant decrease of global activities in atrazine-treated plantlets. Moreover, this repression was correlated with a global down-regulation of typical corresponding transcripts (APX1, sAPX, DHAR2, and MDAR2), which, conversely, have already been shown to undergo important induction during responses to several environmental abiotic stresses. APX1, a cytosolic enzyme, has previously been described as a central component of the reactive oxygen gene network of Arabidopsis [67]. Involvement of sAPX in response to oxidative stress has also been reported by transcriptional induction in the presence of H₂O₂, methylviologen, FeCl₃ or UV treatments in soybean seedlings [68]. Finally, Yoshida et al. [69] reported the importance of DHAR2 under conditions of ozone treatment, with higher sensitivity to ozone in a DHAR2-deficient mutant, probably due to insufficient recycling of ascorbate.

Consequently, repression of these transcripts and decrease of the corresponding enzyme activities in the presence of atrazine might accentuate the effects of H₂O₂ accumulation by reduction of ascorbate recycling, thus leading to disruption of antioxidant mechanisms and propagation of atrazine injuries. It was thus clear that the effects of atrazine at transcript level [18] had actual negative consequences on biochemical defences and could be involved in xenobiotic sensitivity. This is strong evidence that xenobiotic sensitivity may be linked to gene regulation effects in plants. Correlatively, the situation of sucrose-induced tolerance was characterised by the lifting of atrazine repression, in the case of APX1 and sAPX, or by the induction by sucrose-atrazine combination, in the case of DHAR2 and MDAR2. These positive effects on transcript levels were associated with maintenance of the corresponding enzyme activities at control levels. Although ROS can mediate induction of protective proteins involved in the stability of specific mRNAs [70], they can also cause RNA oxidative damages and induce protein inactivation and degradation [71]. Increase of transcript levels was therefore likely to be an adaptive response to ensure protein synthesis under stress conditions resulting in higher protein turnover.

The decline of O₂^.- levels in atrazine-treated plantlets, which, as explained above, could be ascribed to inhibition of electron transfer through PSI, was associated with a general repression of transcripts encoding the different isoenzymes of SOD and with a decrease of the global activity of this O₂^.--scavenging enzyme family, thus indicating that atrazine-treated cells responded to the low superoxide radical situation. Association of low O₂^.- and high H₂O₂ may be a cause for the ill-adapted response of anti-oxidant defences in atrazine-treated plantlets, thus suggesting that further work should be carried out on the adaptation of organisms to fluctuations of ROS combinations.

Mechanisms of sucrose-induced tolerance to singlet oxygen

In contrast with non-induction of H₂O₂-scavenging systems, atrazine-treated plantlets seemed to be able to sense the increase of ¹O₂ levels and induce some genes potentially involved in ¹O₂ quenching (Table 1). Thus, atrazine-treated plantlets, in the absence or presence of sucrose showed increased expression of 4-Hydroxyphenylpyruvate dioxygenase (HPD) gene (At1g06570), which could be involved in the maintenance of the photoprotective role of carotenoids. The At5g06690 gene, encoding a chloroplastic thioredoxin, which is a potential ¹O₂-quencher [41], was also induced in atrazine-treated plantlet. However, generally, the thioredoxin gene family was negatively affected by atrazine treatment, with 7 genes among 12 significantly repressed by atrazine. Correlatively, ten of these twelve genes showed lifting of repression or significant induction in the combined presence of sucrose and atrazine. Nevertheless, most of these genes encoded extraplastidial thioredoxins or thioredoxins of unknown localisation (Table 1). The link of thioredoxin gene family differential expression with efficient ¹O₂ quenching in the presence of atrazine plus sucrose (Fig. 1 and Additional file 1) was thus difficult to ascertain. On one hand, several studies have shown the efficiency of thioredoxins in maintenance of cellular reductant environment and in cytoprotective mechanisms [37–40]. On the other hand, efficient ¹O₂ quenching in the case of PSII inhibition by atrazine would require the involvement of chloroplastic TRXs. Two TRX genes, At3g06730 and At5g06690, have been described as encoding chloroplastic TRXs (Table 1). Expression of these two genes showed contrasted patterns in the presence of atrazine or in the presence of sucrose plus atrazine, with At3g06730 being repressed by atrazine, and At5g06690 being induced by atrazine, whereas the presence of sucrose and atrazine resulted in a return to baseline levels. Thus, further work would be required to analyse the physiological significance of this different pattern, and whether the At3g06730 gene product may play an important role in atrazine responses. Further work would also be required to characterise the potential importance of At1g69880 and At2g17420 TRX genes, which are induced by sucrose and by sucrose plus atrazine, in sucrose-induced tolerance.

Plant material and growth conditions

Seeds of Arabidopsis thaliana (ecotype Colombia, Col0) were surfaced-sterilized in bayrochlore/ethanol (1/1, v/v), rinsed in absolute ethanol and dried overnight. Germination and growth were carried out under axenic conditions in square Petri dishes. After seeds were sowed, Petri dishes were placed at 4°C for 48 h in order to break dormancy and homogenize germination and transferred to a control growth chamber at 22°C under a 16 h light period regime at 85 μmol m^-2 s^-1 for 3 weeks. Growth medium consisted of 0.8% (w/v) agar in 1× Murashige and Skoog (MS) basal salt mix (M5519, Sigma-Aldrich) adjusted to pH 5.7. Plantlets were then transferred to fresh MS agar medium containing 80 mM mannitol (M, control), 80 mM mannitol and 10 μM atrazine (MA, lethal treatment), 80 mM sucrose (S, sugar treatment) and 80 mM sucrose and 10 μM atrazine (SA, tolerance treatment).

Chlorophyll and carotenoid extraction and quantification

Pigments were extracted by pounding aerial parts of seedlings in 80% acetone, and absorbance of the resulting extracts was measured at 663 nm, 646 nm and 470 nm. Levels of chlorophyll and total carotenoids (xanthophylls and carotenes) were determined from the equations given by Lichtenthaler and Wellburn [72]. Measurements were done on 3 replicas of 5–10 pooled seedlings each.

Singlet oxygen staining

Three week-old plantlets were transferred for 12, 24, 48 or 72 hours to the different control and treatment media described above (M, S, MA and SA). Plantlets, prior to the transfer and at the end of the treatment, were immersed and infiltrated in the dark under vacuum with a solution of 100 μM Singlet Oxygen Sensor Green^® reagent (SOSG) (S36002, Invitrogen) [31] in 50 mM phosphate potassium buffer (pH 7.5). Infiltrated plantlets were then placed again on control and treatment media during 30 minutes in the light before being photographed under the microscope. Following excitation at 480 nm, the fluorescence emission at 530 nm was then detected by an Olympus BX41 spectrofluorometer coupled with a camera. The presence of red chlorophyll autofluorescence from chloroplasts did not alter the green fluorescence of SOSG. The infiltration method was chosen in order to measure singlet oxygen levels after the different times of treatment. Image analysis and quantification of level fluorescence were performed using the ImageJ software [73]. Experiments were repeated four times on at least 15 plantlets.

Superoxide radical staining

The nitroblue tetrazolium (NBT) (N6876, Sigma-Aldrich) staining method of Rao and Davis [74] was modified as follows for in situ detection of superoxide radical. Three week-old plantlets were transferred for 12, 24, 48 or 72 hours to the different control and treatment media described above (M, S, MA and SA). Plantlets, prior to the transfer and at the end of the treatment, were immersed and infiltrated under vacuum with 3.5 mg ml^-1 NBT staining solution in potassium phosphate buffer (10 mM) containing 10 mM NaN₃. After infiltration, stained plantlets were bleached in acetic acid-glycerol-ethanol (1/1/3) (v/v/v) solution at 100°C during 5 min. Plantlets were then stored in a glycerol-ethanol (1/4) (v/v) solution until photographs were taken. O₂^.- was visualized as a blue color produced by NBT precipitation. A modified version of previously described assays for superoxide quantification was used [75, 76]. Briefly, NBT-stained plantlets were ground in liquid nitrogen, the formazan content of the obtained powder was solubilized in 2 M KOH-DMSO (1/1.16) (v/v), and then centrifuged for 10 min at 12,000 g. The A₆₃₀ was immediately measured, and compared with a standard curve obtained from known amounts of NBT in the KOH-DMSO mix. Experiments were repeated four times on at least 15 plantlets.

Hydrogen peroxide staining

The H₂O₂ staining agent, 3,3'diaminobenzidine (DAB) (D5637, Sigma-Aldrich), was dissolved in H₂O and adjusted to pH 3.8 with KOH. The DAB solution was freshly prepared in order to avoid auto-oxidation [32]. Three week-old plantlets were transferred for 12, 24, 48 or 72 hours to the different control and treatment media described above (M, S, MA and SA). Plantlets, prior to the transfer and at the end of the treatment, were immersed and infiltrated under vacuum with 1.25 mg ml^-1 DAB staining solution. Stained plantlets were then bleached in acetic acid-glycerol-ethanol (1/1/3) (v/v/v) solution at 100°C during 5 min, and then stored in glycerol-ethanol (1/4) (v/v) solution until photographs were taken. H₂O₂ was visualized as a brown color due to DAB polymerization. Quantification of H₂O₂ contents was determined using the method of Kotchoni et al. (2006) [77]. The DAB-stained plantlets were ground in liquid nitrogen. The resulting powder was homogenized in 0.2 M HClO₄, and then centrifuged for 10 min at 12,000 g. The A₄₅₀ was immediately measured and compared with a standard curve containing known amounts of H₂O₂ in 0.2 M HClO₄-DAB. Experiments were repeated four times on at least 15 plantlets. The specificity of DAB staining towards H₂O₂ was assessed in control infiltrations in the presence of 10 mM ascorbic acid.

Enzyme activities

Three week-old plantlets were transferred for 48 hours to the different control and treatment media described above (M, S, MA and SA). Whole plantlets (100 mg FW) were ground in liquid nitrogen to extract total proteins. The powder obtained was suspended in 500 μl of extraction buffer containing 50 mM phosphate buffer (pH 7.5), 1% (w/v) polyvinylpyrrolidone (PVP), 0.5% (v/v) Triton X-100, 1 mM EDTA and a cocktail of protease inhibitors (P9599, Sigma-Aldrich). In the specific case of APX activity measurement, the plant powder was suspended in 50 mM Hepes (pH 7) buffer containing 0.5 mM ascorbate, 0.5% (v/v) Triton X-100 and 1% (w/v) PVP. After centrifugation (15 min, 10,000 g), the supernatant was recovered and a second extraction of the pellet was identically realized. The two supernatants were pooled and constituted the total protein extract that was immediately used for enzyme activity measurement.

Superoxide dismutase (SOD) activity (EC 1.15.1.1) was determined using the method of Beauchamp and Fridovich [78] that spectrophotometrically measures inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm. One unit of SOD activity was defined as the amount of enzyme required to inhibit the reduction rate of NBT by 50%. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.5), 10 mM methionine, 2 μM riboflavin, 0.1 mM EDTA, 70 μM NBT and enzyme sample. Reactions were carried out at 25°C under a light intensity of about 120 μmol m^-2 s^-1 for 10 min.

Ascorbate peroxidase (APX) activity (EC 1.11.1.11) was measured according to Nakano and Asada [79] by monitoring the rate of hydrogen peroxide-dependent oxidation of ascorbate at 290 nm (E = 2.8 mM^-1 cm^-1). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7), 0.5 mM ascorbic acid, 0.1 mM H₂O₂, 1 mM EDTA and enzyme sample.

Dehydroascorbate reductase (DHAR) activity (EC 1.8.5.1) was measured as described by Hossain and Asada [80]. DHAR was assayed spectrophotometrically by monitoring the increase in absorbance at 265 nm due to ascorbate formation (E = 14 mM^-1 cm^-1). The reaction mixture, freshly prepared in N₂-saturated buffer, consisted of 50 mM potassium phosphate buffer (pH 7), 0.5 mM dehydroascorbate, 5 mM reduced glutathione, 1 mM EDTA and enzyme sample. Correction was made for non-enzymatic reduction rate of DHA in absence of protein extract.

Monodehydroascorbate reductase (MDAR) activity (EC 1.6.5.4) was measured as described by Hossain et al. [81]. MDAR was assayed spectrophotometrically by following the decrease in absorbance at 340 nm due to NADH oxidation (E = 6.2 mM^-1 cm^-1). The reaction mixture consisted of 50 mM buffer TES (pH 7.5), 0.1 mM NADH, 2.5 mM ascorbate, ascorbate oxidase (1 U ml^-1) (Curcubita enzyme (EC 1.10.3.3), A0157, Sigma-Aldrich) and enzyme sample.

Glutathione reductase (GR) activity (EC 1.6.4.2) was measured as described by Smith et al. [82] following spectrophotometrically the disappearance of NADPH at 340 nm (E = 6.2 mM^-1 cm^-1). The reaction mixture contained 50 mM Hepes-NaOH buffer (pH 7.5), 0.5 mM oxidized glutathione, 0.25 mM NADPH, 0.5 mM EDTA and enzyme sample.

Catalase (CAT) activity (EC 1.11.1.6) was measured spectrophotometrically at 250 nm by following the disappearance of H₂O₂ (E = 39.4 mM^-1 cm^-1) in a reaction mixture containing 50 mM potassium phosphate buffer (pH 7) and protein extract. The reaction of dismutation was initiated by the addition of H₂O₂ (10 mM) as described by Aebi [83].

Transcriptome profiling

Gene expression data were extracted from the transcriptomic profiling experiment registered as E-MEXP-411 in ArrayExpress [18, 84]. Genes with a Bonferroni P-value higher than 5% were considered as being not differentially expressed as described by Lurin et al. [85]. Differentially expressed genes are those genes showing at least one P-value ≤ 0.05 after Bonferroni correction, in one of the MA/M, SA/M or S/M comparisons [18]. This P-value corresponds to genes whose Log₂(ratio) was greater than 0.73 or lower than -0.73 (corresponding to 1.6586-fold changes). This transcriptomic experiment compared the RNA profiles of three-week-old MS-grown plantlets transferred for 24 hours to the different control and treatment media described above (M, S, MA and SA).

Statistical analysis

Statistical analysis was carried out with the Minitab^® 15.1.1.0 software (Minitab SARL, Paris, France). The non-parametrical Mann-Whitney test was used for the different comparisons of means. Means that were not significantly different (P > 0.05) show the same letter in graph representations.