Strategies of ROS regulation and antioxidant defense during transition from C3 to C4 photosynthesis in the genus Flaveria under PEG-induced osmotic stress

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Physiology

Strategies of ROS regulation and antioxidant defense during transition from C3 to C4 photosynthesis in the genus Flaveria under PEG-induced osmotic stress Baris Uzilday, Ismail Turkan ∗ , Rengin Ozgur, Askim H. Sekmen Department of Biology, Faculty of Science, Ege University, Bornova, 35100 Izmir, Turkey

a r t i c l e

i n f o

Article history: Received 5 February 2013 Received in revised form 15 May 2013 Accepted 17 June 2013 Available online xxx Keywords: Antioxidant defense system Photosynthesis ROS Flaveria C3–C4 intermediate

a b s t r a c t In the present study, we aimed to elucidate how strategies of reactive oxygen species (ROS) regulation and the antioxidant defense system changed during transition from C3 to C4 photosynthesis, by using the model genus Flaveria, which contains species belonging to different steps in C4 evolution. For this reason, four Flaveria species that have different carboxylation mechanisms, Flaveria robusta (C3 ), Flaveria anomala (C3 –C4 ), Flaveria brownii (C4 -like) and Flaveria bidentis (C4 ), were used. Physiological (growth, relative water content (RWC), osmotic potential), and photosynthetical parameters (stomatal conductance (gs ), assimilation rate (A), electron transport rate (ETR)), antioxidant defense enzymes (superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductases(GR)) and their isoenzymes, non-enzymatic antioxidant contents (ascorbate, glutathione), NADPH oxidase (NOX) activity, hydrogen peroxide (H2 O2 ) content and lipid peroxidation levels (TBARS) were measured comparatively under polyethylene glycol (PEG 6000) induced osmotic stress. Under non-stressed conditions, there was a correlation only between CAT (decreasing), APX and GR (both increasing) and the type of carboxylation pathways through C3 to C4 in Flaveria species. However, they responded differently to PEG-induced osmotic stress in regards to antioxidant defense. The greatest increase in H2 O2 and TBARS content was observed in C3 F. robusta, while the least substantial increase was detected in C4 -like F. brownii and C4 F. bidentis, suggesting that oxidative stress is more effectively countered in C4 -like and C4 species. This was achieved by a better induced enzymatic defense in F. bidentis (increased SOD, CAT, POX, and APX activity) and non-enzymatic antioxidants in F. brownii. As a response to PEG-induced oxidative stress, changes in activities of isoenzymes and also isoenzymatic patterns were observed in all Flaveria species, which might be related to ROS produced in different compartments of cells. © 2013 Elsevier GmbH. All rights reserved.

Introduction Drought is a major environmental constraint to plant growth. Under drought stress, plants try to prevent water loss by primarily closing their stomata. However, preventing water loss from leaves also means limiting gas exchange, which comes with a cost in photosynthetic capacity (Chaves et al., 2003). When diffusion of CO2 is decreased, in plants that use C3 photosynthesis, photorespiration increases drastically, further reducing the assimilation capacity of the plant (Bauwe et al., 2010). Loss of coordination between light reactions and the Calvin cycle due to insufficient supply of CO2 causes over-reduction of electron transport chains in chloroplasts, especially under high light conditions. This over-reduction can cause production of reactive oxygen species such as superoxide anion (O2 − ) by the Mehler reaction or

∗ Corresponding author. Fax: +90 232 388 10 36. E-mail address: [email protected] (I. Turkan).

singlet oxygen (1 O2 ) by overexcitation of chlorophyll molecules (Asada, 2006). Following this, Haber-Weiss and Fenton reactions can cause the production of highly reactive hydroxyl radical (HO. ) from O2 − . These ROS are harmful to proteins, nucleic acids and lipids if accumulated over a certain threshold (Apel and Hirt, 2004). To overcome this issue, plants have developed complex antioxidant machinery, including an array of enzymatic [superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX)] and non-enzymatic antioxidants (ascorbate, glutathione, ␤carotene, ␣-tocopherol) (Mittler et al., 2004). These enzymes and low molecular weight antioxidants work in coordination to detoxify ROS. For example, in chloroplasts, O2 − is dismutated to hydrogen peroxide (H2 O2 ) by SOD, H2 O2 produced by this reaction is further detoxified by APX to water completing the water–water cycle and reducing power for APX is supplied by ascorbate. Also, catalases are major ROS detoxifiers in the peroxisomes, where photorespiratory H2 O2 is produced. In addition to CAT, POX also plays vital roles in H2 O2 scavenging (Miller et al., 2010). Efficient use of these antioxidant enzymes is vital for plants to cope with oxidative stress

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that would otherwise be lethal. In addition to their toxic effects, recently, ROS were shown to be important signaling molecules that take a role in growth, development and stress signaling. Because of this attribute, it makes it important to fine-tune the concentration of ROS in a cell to continue its normal functioning (Mittler et al., 2011). Some plants developed additional measures to alleviate the effects of reduced CO2 diffusion on photosynthesis. One of these adaptations is the C4 carboxylation pathway. C4 plants fix the CO2 in mesophyll cells with phosphoenolpyruvate carboxylase (PEPC), and afterwards this CO2 is released in bundle sheath cells, which are specialized cells around vascular tissue. RuBisCO strictly localizes and fixes CO2 in these cells. By this mechanism, the availability of CO2 around RuBisCO is increased and the rate of photorespiration is minimized (Gowik and Westhoff, 2011). Because they can concentrate CO2 , C4 plants tend to have lower stomatal conductance as compared to C3 plants, which increases their water use efficiency (Way, 2012). These distinct characteristics between C3 and C4 plants also can cause differences in the regulation of other metabolic processes. For example, antioxidant defense mechanisms in species like C3 wheat, C3 sunflower, C4 sorghum and C4 maize (which are distant relatives) were investigated by Zhang and Kirkham (1996), Stepien and Klobus (2005) and Nayyar and Gupta (2006). However, using species from different genera may produce artifacts in a comparative study. To overcome this issue, in our previous study, we compared the differences in antioxidant defenses of two Cleome species differing in carboxylation mechanisms (Cleome spinosa – C3 , Cleome gynandra – C4 ) under drought stress (Uzilday et al., 2012). By using two species from the same genus, we tried to minimize the effects of factors other than the differences in carboxylation pathways. Despite the studies mentioned above, how strategies of ROS regulation and the antioxidant defense system changed during transition from C3 to C4 photosynthesis (in C3 –C4 intermediates) is not known. In fact, elucidating these mechanisms in intermediate plants may contribute to our basic understanding of regulation of photosynthesis under stress conditions and it may also help the efforts to engineer C4 plants like C4 rice (Mitchell and Sheehy, 2006). With this aim, we investigated whether there is a correlation in antioxidant defense systems in Flaveria species that use C3 , C3 –C4 intermediates, C4 -like and C4 pathways in conjugation with photosynthetic parameters. In the current study, we used Flaveria species, which are extensively used in studies investigating the evolutionary changes in photosynthetic enzymes and evolution of C4 characteristics (Ku et al., 1991; Lai et al., 2002; McKown and Dengler, 2007; Gowik et al., 2011) such as Flaveria robusta (C3 ), Flaveria anomala (C3 –C4 ), Flaveria brownii (C4 -like), Flaveria bidentis (C4 ) belonging to different steps in C4 evolution. These species show an increasing number of biochemical and anatomical adaptations until a true C4 metabolism is formed. For example, C3 –C4 F. anomala shows a more distinct kranz anatomy with increased vein intensity and bundle sheath size as compared to C3 F. robusta (McKown and Dengler, 2007). In addition, glycine decarboxylase, a key enzyme in photorespiration, is localized only in bundle sheath cells of C3 –C4 intermediate species (Hylton et al., 1988). This change in localization of glycine decarboxylase results in the release of all of the photorespiratory CO2 in the bundle sheath cells (Ku et al., 1991). Further, in C4 -like F. brownii, typical kranz anatomy and activity of coordinated and localized C4 photosynthetic enzymes can be observed (Cheng et al., 1988). However, in this species, metabolic enzymes in the C4 pathway are not adapted to the specialized environment of the C4 leaves, and as a result, the C4 cycle is less efficient compared to a true C4 species such as F. bidentis (Sage and Sage, 2008, Leegood, 2013). This transition from C3 to C4 photosynthesis definitely affects other cellular processes and needs a series of adaptations. In this respect, in this study we

aimed to elucidate the changes in antioxidant defense to examine whether there is a correlation in activities of antioxidant enzymes as there is in photosynthetic enzymes during transition from C3 to C4 . Growth, relative water content (RWC), osmotic potential, conductance (gs ), assimilation rate (A), electron transport rate (ETR)), superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductases (GR) and their isoenzymes, ascorbate (ASC) and glutathione (GSH) contents, NADPH oxidase (NOX) activity, hydrogen peroxide (H2 O2 ) content and lipid peroxidation levels (TBARS) of F. robusta (C3 ), F. anomala (C3 –C4 ), F. brownii (C4 -like), F. bidentis (C4 ) were measured comparatively under PEG-induced osmotic stress. Materials and methods Plant material and PEG treatment Four Flaveria species, including F. robusta, F. anomala, F. brownii and F. bidentis were used in this study. Seeds were sterilized in 1% NaClO (sodium hypochlorite) solution for 20 min and then rinsed five times with distilled sterile water. Seeds were germinated at 25 ◦ C (16/8 h light/dark) for 1 week. After germination, seedlings were transferred into pots filled with perlite. Seedlings were grown for 6 weeks under controlled conditions (16/8 h light/dark at 25 ◦ C, relative humidity 70%, photosynthetic photon flux density of (PAR) 300 ␮mol m−2 s−1 ) and were sub-irrigated every other day within a half-strength Hoagland’s solution. For stress treatment, plants were watered with a solution containing PEG 6000. To prevent osmotic shock PEG concentration was increased gradually. 1%, 3%, 5%, 7% PEG containing Hoagland solution was for first 4 days of treatment, respectively. Following this acclimation, plants were watered with %10 PEG containing solution for another 5 days (total of 9 including the acclimation period). Seedlings were harvested on the 9th day of treatment in the 3 h middle of the photoperiod and then stored at −80 ◦ C until further analysis. Only youngest fully expanded leaves were used for analysis. Growth analysis Five random plants for each group were used for the growth analyses and were separated to shoot and root fractions on the 9th day of treatment. Fresh weights (FW) of seedlings were measured. Leaf relative water content (RWC) After harvest, six leaves (youngest fully expanded leaf) were obtained from Flaveria plants for each species and their FW was determined. The leaves were floated on deionized water for 6 h under low irradiance and then the turgid tissue was quickly blotted to remove excess water and their turgid weights (TW) were determined. DW was determined after leaves were dried in the oven. The relative water content (RWC) was calculated by the following formula: RWC(%) = [(FW − DW)/(TW − DW)] × 100

Measurements of gas exchange and chlorophyll fluorescence parameters Measurements of gas exchange and chlorophyll fluorescence parameters were done using WALZ GFS-3000. Measurements were done under constant conditions; 385 ppm CO2 , 25 ◦ C cuvette

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temperature, 60% relative humidity and saturating light intensity of 1500 PAR in the hours (4 h) middle of the light period.

Leaf osmotic potential Leaf osmotic potential was measured by the Vapro Vapor pressure Osmometer 5520. The data were collected from six sample leaves per replicate.

Antioxidant enzyme extractions and assays All assays were performed at 4 ◦ C. For protein and enzyme extractions, 0.5 g of samples were ground to fine powder by liquid nitrogen and then homogenized in 1.25 mL of 50 mM Tris–HCl, pH 7.8, containing 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2% (w/v) Triton-X100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 2 mM dithiothreitol (DTT). For APX activity determination, 5 mM ascorbate was added into the homogenization buffer and PVP (2% w/v) was used instead of DTT. Samples were centrifuged at 16,000×g for 10 min, and supernatants were used for the determination of protein content and enzyme activities. Total soluble protein contents of the enzyme extracts were determined according to Bradford (1976) using bovine serum albumin as a standard. All spectrophotometric analyses were conducted on a Shimadzu (UV 1600) spectrophotometer. POX (EC1.11.1.7) activity was measured based on the method described by Herzog and Fahimi (1973). The reaction mixture contained 3,3 -diaminobenzidine-tetra hydrochloride dihydrate solution containing 0.1% (w/v) gelatine and 150 mM Na-phosphatecitrate buffer (pH 4.4) and 0.6% H2 O2 . The increase in the absorbance at 465 nm was followed for 3 min. A unit of POX activity was defined as mmol H2 O2 decomposed mL−1 min−1 . CAT (EC 1.11.1.6) activity was estimated according Bergmeyer (1970), which measures the initial rate of disappearance of H2 O2 at 240 nm. The reaction mixture contained 50 mM Na-phosphate buffer (pH 7.0) with 0.1 mM EDTA and 3% H2 O2 . The decrease in the absorption was followed for 3 min and 1 mmol H2 O2 mL−1 min−1 was defined as 1 unit of CAT. APX (EC 1.11.1.11) activity was measured according to Nakano and Asada (1981). The assay depends on the decrease in absorbance at 290 nm as ascorbate was oxidized. The reaction mixture contained 50 mM K-phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM EDTA Na2 , 0.1 mM H2 O2 and 0.1 mL of enzyme extract in a final assay volume of 1 mL. The concentration of oxidized ascorbate was calculated by using extinction coefficient of 2.8 mM−1 cm−1 . One unit of APX was defined as 1 mmol mL−1 ascorbate oxidized min−1 . GR (EC 1.6.4.2) activity was measured according to Foyer and Halliwell (1976). The assay medium contained 25 mM Naphosphate buffer (pH 7.8), 0.5 mM GSSG, and 0.12 mM NADPH. Na4 and 0.1 mL enzyme extract in a final assay volume of 1 mL. NADPH oxidation was followed at 340 nm. Activity was calculated using the extinction coefficient of NADPH (6.2 mM−1 cm−1 ). One unit of GR was defined as 1 mmol mL−1 GSSG reduced min−1 .

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The specific enzyme activity for all enzymes was expressed as in unit mg−1 protein. Identification of antioxidant isoenzymes Samples containing equal amounts of protein were subjected to non-denaturing polyacrylamide gel electrophoresis (PAGE) as described by Laemmli (1970), except that sodium dodecyl sulphate was omitted. For the separation of SOD isoenzymes, samples containing 50 ␮g protein per well were subjected to non-denaturing polyacrylamide PAGE in 4.5% stacking and 12.5% separating gels. SOD activity was detected by photochemical staining with riboflavin and NBT as described by Beuchamp and Fridovich (1973). The unit activity of each SOD isoenzyme was calculated by running a SOD standard from bovine liver (Sigma Chemical Co.). The different types of SOD were differentiated by incubating gels in inhibitors of SOD before staining, such as 2 mM KCN to inhibit Cu/Zn-SOD activity and 3 mM H2 O2 to inhibit Cu/Zn-SOD and Fe-SOD activities as described by Vitória et al. (2001) (Mn-SOD activity is resistant to both inhibitor treatments). CAT isoforms were detected according to Woodbury et al. (1971). The electrophoretic separation was performed on nondenaturating polyacrylamide gels using 10% separating gel. The gels were incubated in 0.01% H2 O2 for 5 min. After incubation, the gels were washed with distilled water twice and incubated for 5 min in staining solution containing 1% FeCl3 and 1% K3 Fe(CN)6 . POX isoforms were detected according to Seevers et al. (1971). The electrophoretic separation was performed on nondenaturating polyacrylamide mini gels using 10% separating gel. The gels were loaded with 20 ␮g protein. The gels were incubated for 30 min at 25 ◦ C in 200 mM Na-acetate buffer (pH 5.0) containing 1.3 mM benzidine and 3% hydrogen peroxide. GR isoforms were detected using 7.5% native PAGE according to Hou et al. (2004). After electrophoresis of the samples containing 50 ␮g protein, GR isoforms were detected by incubating the gels in a solution containing 10 mM Tris–HCl (pH 7.9), 4 mM GSSG, 1.5 mM NADPH.Na4 and 2 mM DTNB for 20 min. After a brief rinse with 50 mM Tris–HCl buffer (pH 7.9), GR activity was negatively stained by 1.2 mM MTT, and 1.6 mM PMS for 5–10 min at room temperature. Identification of NOX isoenzymes NOX isoenzymes were identified by NBT reduction method as described by Sagi and Fluhr (2001). Non-denaturing PAGE was performed at 4 ◦ C in 7.5% polyacrylamide mini gels and 30 ␮g protein was loaded per lane. Gels were stained in 50 mM Tris–HCl buffer (pH 7.4), 0.2 mM NBT, 0.1 mM MgCl2 and 1 mM CaCl2 , in the dark for 20 min. After then, 0.2 mM NADPH. Na4 was added and the appearance of blue formazan bands was observed. Gels were photographed with a Vilber Lourmat gel imaging system and then analyzed with the BioCapt software package (Vilber Lourmat). We originally performed three independent replicates of SOD, CAT, POX, GR and NOX gels.

NADPH oxidase (NOX) activity

Ascorbate (Asc) and glutathione (GSH) contents

NOX (EC 1.6.3. 1) activity was measured according to Jiang and Zhang (2002). The assay medium contained 50 mM Tris–HCl buffer, pH 7.5, 0.5 mM XTT, 100 ␮M NADPH.Na4 and 20 ␮g of protein. After the addition of NADPH, XTT reduction was followed at 470 nm. The corrections of background production were determined in the presence of 50U SOD. Activity was calculated using the extinction coefficient, 2.16 × 104 M−1 cm−1 .

The content of Asc and GSH was determined according to Queval and Noctor (2007). Extractions were performed at 4 ◦ C. 0.1 g of leaf tissue was ground in liquid nitrogen and was extracted with 1 mL 0.2 N HCl. After this, samples were centrifuged at 16,000 g for 10 min. 0.5 mL supernatant was neutralized with approximately 0.4 mL of 0.2 M NaOH in the presence of 50 ␮L of 0.2 M NaH2 PO4 (pH 5.6). The pH of the neutralized acid extracts was between 5 and

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6. Ascorbate content was determined using ascorbate oxidase and following the decrease in absorbance at 265 nm. Glutathione content was determined using the enzyme cycling assay and following the change in absorbance at 340 nm. Determination of H2 O2 content H2 O2 was determined according to Cheeseman (2006) using eFOX reagent. Extraction was done using ice-cold acetone containing 25 mM H2 SO4 . Samples were centrifuged for 5 min at 3000 g at 4 ◦ C. 950 ␮l of eFOX reagent (250 ␮M ferrous ammonium sulphate, 100 ␮M xylenol orange, 100 ␮M sorbitol, 1% ethanol (v/v)) was used for 50 ␮l of supernatant. Reaction mixtures were incubated at room temperature for 30 min and then absorbance at 550 and 800 nm was measured. H2 O2 concentrations were calculated using a standard curve prepared with known concentrations of H2 O2 . Lipid peroxidation The level of lipid peroxidation in samples was determined in terms of thiobarbituric acid reactive substance (TBARS) content according to the method of Madhava Rao and Sresty (2000). Content of malondialdehyde (MDA), which is an end product of lipid peroxidation, was determined using the thiobarbituric acid reaction. MDA concentration was calculated from the absorbance at 532 nm and measurements were corrected for non-specific turbidity by subtracting the absorbance at 600 nm. The concentration of MDA was calculated using an extinction coefficient of 155 mM−1 cm−1 . Statistical analysis The experiments were repeated three times independently, and each data point was the mean of three replicates (n = 6). All data obtained were subjected to a one-way analysis of variance (ANOVA), The Tukey post-test was used to compare the groups of the same plants species (showed by *) and different plant species in the same treatment groups (showed by a, b, c, d). Comparisons with P < 0.05 were considered significantly different. In all of the figures, the spread of values is shown as error bars representing standard errors of the means. Results Growth PEG treatment did not cause a significant decrease in growth of F. bidentis (C4 ), while growth of other Flaveria species was decreased by PEG treatment (Fig. 1 A). Under stress, the same decreasing growth patterns in F. anomala (C3 –C4 ) (9%) and F. robusta (C3 ) (11%) were observed. However, the greatest decrease in growth was observed in F. brownii (C4 like), as compared to other Flaveria species. Relative water content (RWC) RWC of all Flaveria species decreased under stress (Fig. 1B). The greatest decrease was observed in F. anomala (C3 –C4 ) by 30%. In F. robusta (C3 ), F. brownii (C4 like) and F. bidentis, RWC showed a decrease by 8%, 13% and 5%, respectively.

Fig. 1. Physiological parameters of F. robusta (R), F. anomala (A), F. brownii (BR) and F. bidentis (BI) under control (C) and PEG stress (P).

potentials of F. robusta, F. brownii and F. bidentis were increased by 7%, 17% and 21% under PEG-induced stress, respectively. Stomatal conductance Under control conditions, the highest stomatal conductance was measured in F. robusta, whereas the lowest was measured in F. bidentis (Fig. 2 A). Stomatal conductance of all plants decreased as a response to PEG treatment. These decreases were recorded as 44%, 30%, 36%, and 48% for F. robusta, F. anomala, F. brownii and F. bidentis, respectively. Photosynthetic rate (A)

Leaf osmotic potential PEG-induced osmotic stress increased the osmotic potentials of all Flaveria species (Fig. 1C). This enhancement rate in osmotic potential of F. anomala was recorded as 44% under stress. Osmotic

The highest A was recorded in control group of F. bidentis (Fig. 2B). F. anomala showed a sharp decrease in A under stress by 60%, while 28%, 21%, 26% decreases were observed in F. robusta, F. brownii and F. bidentis, respectively.

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(Fig. 3B). While MnSOD activity was increased by 1.4-fold, FeSOD and CuZnSOD were decreased by 36% and 58%, respectively, as compared to control groups. In F. brownii, 3 FeSOD and 2 CuZnSOD isoenzymes were found (Fig. 3B). Total FeSOD activity, was not changed, but activities of FeSOD1 and FeSOD2 disappeared under stress conditions, while FeSOD3 activity was increased 2.8-fold compared to controls. Total CuZnSOD was decreased by 20% and activity of CuZnSOD2 was not detected under osmotic stress. In the case of C4 F. bidentis, 1 MnSOD, 1 FeSOD and 2 CuZnSOD isoenzymes were determined (Fig. 3B). Under osmotic stress, MnSOD activity was enhanced by 2.5-fold. However, the highest activity of SOD isoenzymes was determined as FeSOD in F. bidentis as 81.2% of the total. Total CuZnSOD activity was only increased by 10%, but one of the CuZnSOD bands disappeared after stress treatment. NOX activity NOX activities of all plants were decreased by PEG treatment (Fig. 3D and E). This decrease was more severe in C3 F. robusta and C3 - C4 intermediate F. anomala as 48% and 53%, respectively. In C4 F. bidentis, NOX activity was reduced by 28%, while it decreased by 13% in C4 like F. brownii under PEG treatment. After all treatments, a total of 15 NOX isoenzymes were determined in this study. While NOX5, 9, and 10 activities were decreased in F. anomala, activity of NOX12 was increased in F. brownii by PEG treatment. Activities of H2 O2 scavenging enzymes

Fig. 2. Gas exchange and fluorescence parameters of F. robusta (R), F. anomala (A), F. brownii (BR) and F. bidentis (BI) under control (C) and PEG stress (P).

Electron transport rate (ETR) The ETR of all species was decreased by PEG treatment (Fig. 2C). The most severe decrease of ETR was measured as 45% in F. anomala as compared to the control, while it decreased by 11% and 31% in F. brownii and F. bidentis. O2 −. Related enzyme activities SOD activity Under normal conditions, the highest constitutive level of SOD enzyme activity was recorded in C3 -C4 F. anomala, while the lowest was observed in C4 like F. brownii (Fig. 3 A and C). PEG treatment enhanced the total SOD activities of F. anomala and F. bidentis by 1.2- and 1.8-fold, respectively. However, SOD activity of F. robusta and F. brownii remained unchanged. C3 F. robusta had 4 SOD isoenzymes, which were determined as 2 MnSOD, 1 FeSOD, 1 CuZnSOD (Fig. 3B). Among of these CuZnSOD only appeared after PEG-induced osmotic stress. FeSOD activity was decreased by 36% with stress treatment. A total of four SOD isoenzymes were found and determined as 2 MnSOD, 1 FeSOD and 1 CuZnSOD in C3 –C4 intermediate F. anomala

CAT activity While the highest constitutive level of CAT was observed in F. robusta (C3 ), the lowest level was determined in F. bidentis (C4 ) under normal conditions (Fig. 4 A and C). PEG treatment did not affect the CAT activity in the leaves of F. robusta (C3 ). However, CAT activity in the leaves of F. bidentis was increased by 17% under osmotic stress. By contrast, PEG-induced osmotic stress caused a significant decrease in CAT activities of F. anomala (C3 –C4 ) and F. brownii (C4 like) by 20% and 25%. 3 CAT (CAT1, CAT2, and CAT3) isoenzymes were determined under normal and stress conditions (Fig. 4A). While CAT1 isoenzyme was determined only in F. brownii, it did not appear in other Flaveria species. Moreover, the CAT3 isoenzyme was observed in all four species. The highest intensity of CAT isoenzymes was observed in F. robusta. POX activity Under normal conditions, the highest POX activities were observed in F. robusta (C3 ) and F. bidentis (C4 ), and the lowest POX activities were determined in F. anomala (C3–C4 ) and F. brownii (C4 like) (Fig. 4B and D). While PEG-induced osmotic stress did not affect the POX activity in F. anomala and F. brownii, it increased by 17% and 24% in F. robusta and F. bidentis, respectively, as compared to control groups. 11 POX isoenzymes were determined (Fig. 4B). Intensities of these POX isoenzymes were changed depending species. While POX1 was determined only in C4 F. bidentis, POX5 was determined only in F. brownii. POX2 and POX3 were observed in both F. robusta (C3 ) and F. anomala (C3 –C4 ). POX6 and POX7 were determined only in F. robusta, while POX10, POX11 and POX12 were observed only in F. anomala under control conditions. The intensities of POX8, POX9, and POX13 were increased in Flaveria species exposed to stress. Moreover, new isoenzymes (POX10 in F. anomala and POX 12 and POX14 in F. bidentis) were also determined under PEG-induced osmotic stress.

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Fig. 3. Activities and isoenzymes of O2 − related enzymes of F. robusta (R), F. anomala (A), F. brownii (BR) and F. bidentis (BI) under control (C) and PEG stress (P). Native-PAGE separation of SOD isoenzymes (A), isoenzyme patterns according to inhibition assays (B), total SOD activity and contribution of different types of SOD isoenzymes (C), Native-PAGE separation of NOX isoenzymes (D), total NOX activity (E).

Asada-Halliwell-Foyer cycle APX activity PEG treatment increased APX activities in the leaves of F. robusta, F. anomala and F. bidentis by 1.6-fold, 1.2-fold and 1.2-fold, as compared to their control groups (Fig. 5B). Although the greatest increase (as %) was observed in F. robusta, the highest activity was determined in F. bidentis under PEG treatment. On the other hand, APX activity was decreased by 25% only in F. brownii under stress. GR activity GR enzyme activities of F. robusta, F. anomala and, F. brownii increased with PEG treatment as 17%, 46%, 21%, respectively (Fig. 5A and C). However, a 32% decrease in GR enzyme activity was detected under osmotic stress in F. bidentis. In our study, six GR isoenzymes were determined in all species (Fig. 5A). While activities of GR3 and GR4 isoenzymes increased in F. anomala, activity of GR1 isoenzyme was enhanced by PEG treatment in F. bidentis. ASC content Although the highest constitutive level of ascorbate was detected in F. robusta, its ascorbate content was decreased by 64%

under stress (Fig. 5D). However, ascorbate levels of the other three Flaveria species were induced by PEG treatment. These increases were by 1.5-fold in F. anomala, 2-fold in F. brownii and 1.2-fold in F. bidentis as compared to their controls.

GSH content GSH content increased in C4 F. bidentis and C3 –C4 intermediate F. anomala as 22.35% and 38.9% respectively, under PEG-induced osmotic stress (Fig. 5E). On the other hand, no significant difference in glutathione content of C3 F. robusta and C4 like F. brownii was determined under PEG treatment.

TBARS content TBARS contents of all Flaveria species were increased under osmotic stress (Fig. 6A). The highest TBARS content was observed in F. bidentis due to its high constitutive levels under normal conditions. However, the greatest increase (2.1-fold) in TBARS content was determined in F. robusta, as compared to its control group. TBARS content in F. anomala was measured as 1.5-fold higher as compared to the control, whereas this enhancement was detected as 13% and 35% in F. brownii and F. bidentis, respectively.

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Fig. 4. Activities and isoenzymes of H2 O2 scavenging enzymes of F. robusta (R), F. anomala (A), F. brownii (BR) and F. bidentis (BI) under control (C) and PEG stress (P). Native-PAGE separation of CAT isoenzymes (A), native-PAGE separation of POX isoenzymes, total CAT activity (C), total POX activity (D).

H2 O2 content Osmotic stress caused significant increases in H2 O2 contents of all Flaveria species, except F. brownii (Fig. 6B). The greatest increase in H2 O2 content was observed by 25% in F. robusta. H2 O2 content was increased by 16% in F. bidentis under stress as compared to its control. However, in F. brownii, H2 O2 content was decreased by 12% under the same conditions. Discussion Growth reduction is one of the first signs of stress in plants. There are many reports which indicate the growth of the C3 plants and abiotic stress-sensitive plants is severely reduced under stress conditions. Nayyar and Gupta (2006), who measured the antioxidant response of C3 wheat and C4 maize to drought, also detected severe growth reduction in wheat under stress. Similarly, in our previous study, we observed that C3 C. spinosa showed a greater decrease in terms of growth as compared to C4 C. gynandra (Uzilday et al., 2012). In the present study, four Flaveria species with different carboxylation pathways were subjected to PEG-induced osmotic stress for 9 days, and while C4 F. bidentis did not show any decreases in growth, growth was significantly decreased in C3 F. robusta, C3 –C4 intermediate F. anomala and C4 like F. brownii. RWC, which is commonly used to measure the water statues of plants, showed a significant decrease in C3 wheat as compared to C4 maize under drought stress (Nayyar and Gupta, 2006). In the present study, in accordance with study mentioned above, RWC of all four species were decreased by PEG treatment. The greatest reduction in RWC was observed in the C3 –C4 intermediate F. anomala, which was followed by RWC C4 like F. brownii. While RWC of all Flaveria species was decreased by osmotic stress, their osmotic potentials were increased due to decreased water potentials. This effect was more pronounced in F. anomala (C3 –C4 ), which might be related to high stomatal conductance. Stomatal closure is the first response of all plants to drought stress to prevent transpirational water loss resulting in a decrease

in leaf turgor and/or water potential (Ludlow and Muchow, 1990; Mansfield and Atkinson, 1990; Cornic, 2000). There is a good correlation between stomatal conductance, osmotic potential and RWC (Giorio et al., 1999; Guerfel et al., 2008; Ozfidan et al., 2013), indicating that leaf water status interacts with stomatal conductance and transpirational water loss under drought stress. In this study, stomatal conductance of all plants decreased under stress. However, as in Flaveria trinervia (C4 ) (Dias and Brüggemann, 2007), the effect of drought stress on stomatal conductance proved to be much more dramatic in F. bidentis (C4 ). However, contrary to the results of Dias and Brüggemann (2007), who reported the highest stomatal conductance in C3 plants under stress conditions, we observed that C3 –C4 F. anomala showed the highest stomatal conductance. Enzymes related to ROS regulation and oxidative stress parameters investigated in this study responded differently to PEGinduced osmotic stress in each Flaveria species. These changes are summarized in Table 1 and activities of enzymes and contents of TBARS and H2 O2 are ranked for controlled and PEG stressed conditions in Table 2. SOD, which converts O2 − to H2 O2 is a key component of the plant antioxidant defense system. Stress conditions enhance the SOD activities of various species such as pea (Mittler and Zilinskas, 1994), barley (Pérez-López et al., 2009), and bean (Turkan et al., 2005). Moreover, Stepien and Klobus reported that C3 sunflower had lower SOD activity than C4 maize under stress. However, Zhang and Kirkham (1996) showed that levels of SOD activity increased significantly in sorghum/sunflower under drought stress. In this study, different SOD isoenzymes were enhanced by drought in comparison between C3 and C4 . These differences could be important in response to drought stress. For example, FeSOD isoenzyme activities of C3 –C4 F. anomala and C4 F. bidentis were induced under PEG-induced osmotic stress. However, PEG treatment induced the activity of the CuZnSOD isoenzyme in C3 F. robusta. Moreover, in C4 like F. brownii, while 2 FeSOD and 1 CuZnSOD activity disappeared under stress, FeSOD3 and CuZnSOD1 activity was induced. By this way, activities of disappeared FeSOD bands (FeSOD1 and FeSOD2)

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Fig. 5. Asada-Halliwell-Foyer cycle enzymes and total contents of ascorbate and glutathione of F. robusta (R), F. anomala (A), F. brownii (BR) and F. bidentis (BI) under control (C) and PEG stress (P). Native-PAGE separation of GR isoenzymes (A), total APX activity (B), total GR activity (C), total ascorbate content (D), total glutathione content (E).

were compensated by another FeSOD isoenzyme (FeSOD3). These changes in the pattern of FeSOD isoenzymes might indicate that induced FeSOD3 isoenzyme plays an important role in osmotic stress tolerance. The differences among these three FeSOD isoenzymes need to be addressed to elucidate the specific traits in which makes the FeSOD3 enzyme preferred under stress conditions. NADPH oxidases produce O2 − in cell membranes and have essential roles in signal transduction and perception, which are related to ROS production (Sagi and Fluhr, 2006). In this study, NOX activities of all four species were reduced by stress, whereas this

reduction was more significant in C3 and C3 –C4 intermediate plants, unlike C4 -like and C4 plants. A greater decrease of NOX in C3 and C3 –C4 plants can be related to the progression of oxidative damage in these plants, which might cause a decrease in production of further ROS by this enzyme. We also observed changes in isoenzyme patterns of NOX, which might be related to expression of specific isoenzymes in plasma membranes of different cell types (such as guard cell, mesophyll cell, bundle sheath cell) under stress and/or different types of isoenzymes expressed in response to different signals produced by environmental fluctuations.

Table 1 Changes in parameters related with cellular redox state in control and drought treated plants. Induced activities and increased contents were indicated as (+) whereas decreased activities and contents were indicated as (−) as compared to control groups. SOD: Superoxide dismutases, NOX: NADPH oxidases, CAT: catalase, POX: peroxidase, APX: ascorbate peroxidase, GR, glutathione reductases, ASC: total ascorbate, GSH: total glutathione, TBARS: thiobarbituric acid substances, H2 O2 : hydrogen peroxide.

SOD NOX CAT POX APX GR ASC GSH TBARS H2 O2

F. robusta (C3 )

F. anomala (C3 –C4 )

F. brownii (C4 -like)

F. bidentis (C4 )

No change − No change + + + − No change + +

+ − − No change + + + + + No change

No change − − No change − + + No change + No change

+ − + + + − + + + +

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Fig. 6. Lipid peroxidation (TBARS content) (A) and H2 O2 content (B) of F. robusta (R), F. anomala (A), F. brownii (BR) and F. bidentis (BI) under control (C) and PEG stress (P).

CAT activity is needed to cope with the increase in H2 O2 production due to stress-related photorespiration. CAT mainly localizes in peroxisomes and reduces the toxic levels of H2 O2 . In the present study, under control conditions CAT activity decreased during the transition from C3 to C4 . In this study, C4 F. bidentis was the only Flaveria species that showed an increase in CAT activity with PEG treatment. Similarly, drought stress induced the CAT activity in leaves of C4 maize (Nayyar and Gupta, 2006). However, CAT activities of F. anomala and F. brownii were decreased after being subjected to stress. F. robusta (C3 ) had higher activities of the CAT enzyme under stress than other Flaveria species. However, this high CAT activity seems to be insufficient to catalyze the destruction of H2 O2 as shown by increased lipid peroxidation under PEG-induced osmotic stress. Interestingly, while C3 –C4 intermediate F. anomala and C4 -like F. brownii had two CAT isoenzymes, F. bidentis only had one. This may indicate that, during shift from C3 to C4 , F. bidentis might have lost its CAT (CAT2) isoenzyme due to decreased rates of photorespiration or does not need to express it under conditions used in this experiment. Similar to our findings, analysis of functional category enrichment of RNA-seq data obtained from F. Table 2 List of biochemical parameters ranked according to photosynthetic type of Flaveria. SOD: Superoxide dismutase, CAT: catalase, POX: peroxidase, APX: ascorbate peroxidase, GR: glutathione reductase, NOX: NADPH oxidase, TBARS: thio-barbituric acid substances, H2 O2 : hydrogen peroxide, C3 : Flaveria robusta, C4 : F. bidentis, C3 –C4 : F. anomala, C4 like: F. brownii.

SOD CAT POX APX GR NOX TBARS H2 O2

Control conditions

Stress conditions

C3 –C4 > C3 > C4 > C4 -like C3 = C4 -like > C3 –C4 > C4 C3 = C4 > C3 –C4 > C4 -like C4 > C3 –C4 > C4 -like > C3 C3 –C4 = C4 -like = C4 > C3 C4 = C3 > C3 –C4 > C4 -like C4 > C4 -like = C3 –C4 > C3 C3 = C3 –C4 = C4 -like = C4

C4 > C3 –C4 > C3 > C4 -like C3 > C4 > C3 –C4 = C4 -like C3 = C4 > C3 –C4 > C4 -like C4 > C3 –C4 > C3 > C4 -like C3 –C4 = C4 -like>C3 = C4 C4 > C3 > C3 –C4 = C4 -like C4 > C3 –C4 > C3 > C4 -like C4 = C3 > C3 –C4 = C4 -like

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trinervia (C4 ), F. bidentis (C4 ), F. ramosissima (C3 –C4 ) and F. pringlei (C3 ) showed that ‘redox.dismutases and catalases’ category is significantly over-represented between these species (Gowik et al., 2011). POX, which are also related to growth, development, lignification and suberization processes, scavenge the main part of H2 O2 in chloroplast and cytosols (Asada and Takahashi, 1987). Under stress conditions, enhancement of POX activities have been reported in many studies (Zhang and Kirkham, 1996; Ozkur et al., 2009), hence C3 C. spinosa and C4 C. gynandra were not exceptions (Uzilday et al., 2012). In agreement to these, C3 F. robusta and C4 F. bidentis showed enhancements in POX activities. No differences were detected in POX activities of the C3 –C4 intermediate F. anomala and C4 like F. brownii under osmotic stress. However, differences in isoenzyme patterns were observed in F. anomala, indicating a change in distribution of POX through the cell under PEG-induced osmotic stress. Unlike SOD, which can be differentiated, it is more difficult to interpret the changes in POX isoenzyme patterns. On the other hand, the occurrence of new POX isoenzymes in F. anomala and F. bidentis shows that these isoenzymes play an important role in stress tolerance, whether growth related or ROS scavenging. APX is another key enzyme responsible for scavenging of H2 O2 , which is produced by action of SOD enzymes and Mehler reaction, using ascorbate (ASC) as the electron donor (Asada, 1992, Asada and Takahashi, 1987). In this study, APX activities of F. robusta (C3 ), F. anomala (C3 –C4 ) and F. bidentis (C4 ) were also increased under stress. However, neither F. robusta (C3 ) nor the intermediate species (C3 –C4 ) ever reached a level as high F. bidentis under the same conditions. F. bidentis (C4 ) had a higher APX activity than the other Flaveria species under both normal and osmotic stress, indicating that it has a higher potential to detoxify H2 O2 . Our results agree with those of Stepien and Klobus (2005) and Nayyar and Gupta (2006), who observed increases in APX activity in maize leaves (C4 ) subjected to water stress. On the other hand, when APX activity of F. brownii (C4 like) was compared to those of other Flaveria species, its activity was decreased, similar to its CAT activity. However, it was observed that despite decreased activities of APX and CAT or unchanged POX activity in F. brownii under stress, its H2 O2 levels were significantly lower than other species. This may be attributed to its more effective alternative mechanisms for removal of H2 O2 in F. brownii (C4 like) such as increased ascorbate, which is one of the most important non enzymatic ROS scavengers (Fig. 5D). GR is an enzyme of the ASC–GSH cycle that catalyzes NADPHdependent reduction of oxidized glutathione. GR has a protective role in the plant’s defense against drought stress-induced oxidative damage (Sharma and Dubey, 2005; Uzilday et al., 2012). Parallel to these results, we also observed an increase in GR activities of all Flaveria species except for F. bidentis (C4 ). Although GR activity of these three Flaveria species increased under stress, the greatest oxidative damage (by a 2.1-fold increase in TBARS) was observed in F. robusta as indicated in Fig. 6A, which might be related to sharp decrease in ASC content (Fig. 5D) Nayyar and Gupta (2006) showed that there is a pattern among H2 O2 , lipid peroxidation and ascorbate in their C3 wheat and C4 maize comparison study. Similar to our results, the highest lipid peroxidation and H2 O2 level was detected in C3 wheat while it had the lowest level of ascorbate. Moreover, except C3 F. robusta, all ascorbate contents of Flaveria species increased under stress. H2 O2 decrease of C4 -like F. brownii under stress might be related to scavenging activity of ascorbate which enhanced by 2 fold as compared to control plants. Also F. brownii had the lowest TBARS content, which indicates the severe of ROS damage in lipid oxidation processes and these could be also a result of increasing activity in ascorbate.

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Conclusion Under non-stressed conditions, there was a correlation only among CAT (decreasing), APX and GR (both increasing) and type of carboxylation pathways (through C3 to C4 ). However, plants responded differently to PEG-induced osmotic stress in regards to antioxidant defense. C4 -like F. brownii and C4 F. bidentis were more tolerant to PEG-induced osmotic stress compared to C3 and C3 –C4 intermediate as evident by H2 O2 content and level of lipid peroxidation, which suggests that C4 -like and C4 species are able to prevent damage caused by oxidative stress. This was achieved by a better induced enzymatic defense in F. bidentis (increased SOD, CAT, POX, and APX activity) and non-enzymatic antioxidants (ascorbate) in F. brownii. Increases in ROS production in different compartments of the cell caused changes in activities of isoenzymes and their patterns such as FeSOD in C4 -like F. brownii and POX in C3 –C4 F. anomala and C4 F. bidentis. In addition, we observed lower CAT activity in C3 –C4 , C4 -like and C4 plants as compared to C3 plant, and ultimately only one (not two like other species) CAT isoenzymes was observed in C4 F. bidentis, which might be related to the amount of photorespiratory H2 O2 production in C3 and C4 plants. To our knowledge, this is the first study elucidating the antioxidant defense system of plants with intermediate carboxylation pathways. Acknowledgements We would like to thank Prof. Dr. Rowan Sage (University of Toronto), Prof. Dr. Peter Westhoff (Heinrich Heine University Düsseldorf) and Assist. Prof. Dr. Ferit Kocacinar (Kahramanmaras Sutcu Imam University) for providing seeds of Flaveria species. This work was support by TUBITAK (TBAG 110T289) and Ege University Research Foundation (2011/BI˙ L/009). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.jplph.2013.06.016. References Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 2004;55:371–99. Asada K. Ascorbate peroxidase a hydrogen peroxide scavenging enzyme in plants. Physiol Plant 1992;86:236–41. Asada K. Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol 2006;141:391–6. Asada K, Takahashi M. Production and scavenging of active oxygen in photosynthesis. In: Kyle DJ, editor. Photoinhibition. Amsterdam, North Holland: Elsevier; 1987. p. 227–87. Bauwe H, Hagemann M, Fernie AR. Photorespiration: players, partners and origin. Trends Plant Sci 2010;15:330–6. Bergmeyer N. Methoden der enzymatischen Analyse, vol. 1. Berlin: Akademie Verlag; 1970. p. 636–47. Beuchamp C, Fridovich I. Isoenzymes of superoxide dismutase from wheat germ. Biochim Biophys Acta 1973;317:50–64. Bradford MM. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of the protein–dye binding. Anal Biochem 1976;72:248–54. Chaves MM, Maroco JP, Pereira JS. Understanding plant responses to drought from genes to the whole plant. Funct Plant Biol 2003;30:239–64. Cheeseman JM. Hydrogen peroxide concentrations in leaves under natural conditions. J Exp Bot 2006;57:2435–44. Cheng SH, Moore BD, Edwards GE, Ku MSB. Photosynthesis in Flaveria brownii, a C4 like species Leaf anatomy, characteristics of CO2 exchange compartmentation of photosynthetic enzymes and metabolism of 14 CO2 . Plant Physiol 1988;87:867–73. Cornic G. Drought stress inhibits photosynthesis by decreasing stomatal aperturenot by affecting ATP synthesis. Trends Plant Sci 2000;5:187–8. Dias MC, Brüggemann W. Differential inhibition of photosynthesis under drought stress in Flaveria species with different degrees of development of the C4 syndrome. Photosynthetica 2007;45:75–84.

Foyer CH, Halliwell B. The presence of glutathione and glutathione reductases in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 1976;133:21–5. Giorio P, Soventino G, D’Andria R. Stomatal behaviour, leaf water status and photosynthetic response in field-grown olive trees under water deficit. Agric Water Manage 1999;42:95–104. Gowik U, Westhoff P. The path from C3 to C4 photosynthesis. Plant Physiol 2011;155:56–63. Gowik U, Brautigam A, Weber KL, Weber APM, Westhoff P. Evolution of C4 photosynthesis in the genus Flaveria: how many and which genes does it take to make C4 ? Plant Cell 2011;23:2087–105. Guerfel M, Baccouri O, Boujnah D, Zarrouk M. Changes in lipid composition, water relations and gas exchange in leaves of two young ‘Chemlali’ and ‘Chetoui’ olive trees in response to water stress. Plant Soil 2008;311:121–9. Herzog V, Fahimi H. Determination of the activity of peroxidase. Anal Biochem 1973;55:554–62. Hou WC, Liang HJ, Wang CC, Liu DZ. Detection of glutathione reductase after electrophoresis on native or sodium dodecyl sulfate polyacrylamide gels. Electrophoresis 2004;25:2926–31. Hylton CM, Rawsthorne S, Smith AM, Jones DA, Woolhouse HW. Glycine decarboxylase is confined to the bundle sheath cells of leaves of C3 –C4 intermediate species. Planta 1988;175:452–9. Jiang M, Zhang J. Involvement of plasma membrane NADPH oxidase in abscicic acid-and water stress-induced antioxidant defense in leaves of maize seedlings. Planta 2002;215:1022–30. Ku MSB, Wu J, Dai Z, Scott RA, Chu C, Edwards GE. Photosynthetic and photorespiratory characteristics of Flaveria species. Plant Physiol 1991;96: 518–28. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. Lai LB, Tausta SL, Nelson TM. Differential regulation of transcripts encoding cytosolic NADP-Malic enzyme in C3 and C4 Flaveria species. Plant Physiol 2002;128:140–9. Leegood RC. Strategies for engineering C4 photosynthesis. J Plant Physiol 2013;170:378–88. Ludlow MM, Muchow RC. A critical evaluation of traits for improving crop yields in water-limited environments. Adv Agric 1990;43:107–53. Madhava Rao KV, Sresty TVS. Antioxidative parameters in the seedlings of pigeon pea (Cajanus cajan L, Millspaugh) in response to Zn and Ni stresses. Plant Sci 2000;157:113–28. Mansfield TJ, Atkinson CJ. Stomatal behaviour in water stressed plants. In: Alscher RG, Cumming JR, editors. Stress Responses in Plants: Adaptation and Acclimation Mechanisms. New York: Wiley-Liss; 1990. p. 241–64. McKown AD, Dengler NG. Key innovations in the evolution of Kranz anatomy and C4 vein pattern I Flaveria (Asteraceae). Am J Bot 2007;94:382–99. Miller G, Suzuki N, Ciftci Yılmaz S, Mittler R. Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ 2010;33:453–67. Mitchell PL, Sheehy JE. Supercharcing rice photosynthesis to increase yield. New Phytol 2006;171:688–93. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F. Reactive oxygen network of plants. Trends Plant Sci 2004;9:490–8. Mittler R, Vanderauwera S, Suzuki N, Miller G, Tognetti VB, Vandepoele K, et al. ROS signaling: the new wave? Trends Plant Sci 2011;16:300–9. Mittler R, Zilinskas B. Regulation of pea cytosolic ascorbate peroxidase and other antioxidant enzymes during the progression of drought stress and following recovery from drought. Plant J 1994;5:397–406. Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbatespecific peroxidase in spinach chloroplasts. Plant Cell Physiol 1981;22: 867–80. Nayyar H, Gupta D. Differential sensitivity of C3 and C4 plants to water deficit stress: association with oxidative stress and antioxidants. Environ Exp Bot 2006;58:106–13. Ozfidan C, Turkan I, Sekmen AH, Seckin B. Time course analysis of ABA and nonionic osmotic stress-induced changes in water status, chlorophyll fluorescence and osmotic adjustment in Arabidopsis thaliana wild-type (Columbia) and ABAdeficient mutant (aba2). Environ Exp Bot 2013;86:44–51. Ozkur O, Ozdemir F, Bor M, Turkan I. Physiochemical and antioxidant responses of the perennial xerophyte Capparis ovata Desf. to drought. Environ Exp Bot 2009;66:487–92. ˜ Pérez-López U, Robredo A, Lacuesta M, Sgherri C, Munoz-Rueda A, Navari-Izzo F, et al. The oxidative stress caused by salinity in two barley cultivars is mitigated by elevated CO2 . Physiol Plant 2009;135:29–42. Queval G, Noctor G. A plate reader method for the measurement of NAD, NADP, glutathione, and ascorbate in tissue extracts: application to redox profiling during Arabidopsis rosette development. Anal Biochem 2007;363: 58–69. Sage R, Sage TL. Learning from nature to develop strategies for directed evolution of C4 rice. In: Sheehy JE, Mitchell PL, Hardy B, editors. Charting New Pathways to C4 Rice,. Los Banos, Philipinnes: International Rice Research Institute, World Scientific Publishing; 2008. p. 195–216. Sagi M, Fluhr R. Superoxide production by plant homologues of the gp91phox NADPH oxidase. Modulation of activity by calcium and by tobacco mosaic virus infection. Plant Physiol 2001;126:1281–90. Sagi M, Fluhr R. Production of reactive oxygen species by plant NADPH oxidases. Plant Physiol 2006;141:336–40.

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G Model JPLPH-51771; No. of Pages 11

ARTICLE IN PRESS B. Uzilday et al. / Journal of Plant Physiology xxx (2013) xxx–xxx

Seevers FM, Daly JM, Catedral FF. The role of peroxidase isozymes in resistance to wheat stem rust. Plant Physiol 1971;48:353–60. Sharma P, Dubey RS. Drought induces oxidative stress and enhances the activities of antioxidant enzymes in growing rice seedlings. Plant Growth Regul 2005;46:209–21. Stepien P, Klobus G. Antioxidant defense in the leaves of C3 and C4 plants under salinity stress. Physiol Plant 2005;125:31–40. Turkan I, Bor M, Ozdemir F, Koca H. Differential responses of lipid peroxidation and antioxidants in the leaves of drought-tolerant P. acutifolius Gray and droughtsensitive P. vulgaris L. subjected to polyethylene glycol mediated water stress. Plant Sci 2005;168:223–31.

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Uzilday B, Turkan I, Sekmen AH, Ozgur R, Karakaya HC. Comparison of ROS formation and antioxidant enzymes in Cleome gynandra (C4 ) and Cleome spinosa (C3 ) under drought stress. Plant Sci 2012;182:59–70. Vitória PA, Vitória, Lea PJ, Azevedo RA. Antioxidant enzymes responses to cadmium in radish tissues. Phytochemistry 2001;57:701–10. Way DA. What lies between: the evolution of stomatal traits on the road to C4 photosynthesis. New Phytol 2012;193:291–3. Woodbury W, Spencer AK, Stahman MA. An improved procedure using ferricyanide for detecting catalase isozymes. Anal Biochem 1971;44:301–5. Zhang JX, Kirkham MB. Enzymatic responses of the ascorbate-glutathione cycle to drought in sorghum and sunflower plants. Plant Sci 1996;113:139–47.

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