Journal of Plant Physiology 171 (2014) 1625–1633

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Physiology

Cytokinin oxidase/dehydrogenase overexpression modifies antioxidant defense against heat, drought and their combination in Nicotiana tabacum plants a ˇ Zuzana Lubovská a,b , Jana Dobrá a , Helena Storchová , Nad’a Wilhelmová a , Radomíra Vanková a,∗ a b

Institute of Experimental Botany AS CR, Rozvojová 263, 16502 Prague 6, Czech Republic Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Viniˇcná 5, 128 44 Prague 2, Czech Republic

a r t i c l e

i n f o

Article history: Received 29 April 2014 Received in revised form 13 June 2014 Accepted 14 June 2014 Available online 12 August 2014 Keywords: Cytokinin Drought Heat Antioxidant enzymes

a b s t r a c t Cytokinins (CKs) as well as the antioxidant enzyme system (AES) play important roles in plant stress responses. The expression and activity of antioxidant enzymes (AE) were determined in drought, heat and combination of both stresses, comparing the response of tobacco plants overexpressing the main cytokinin degrading enzyme, cytokinin oxidase/dehydrogenase, under the control of root-specific WRKY6 promoter (W6:CKX1 plants) or constitutive promoter (35S:CKX1 plants) and the corresponding wild-type (WT). Expression levels as well as activities of cytosolic ascorbate peroxidase, catalase 3, and cytosolic superoxide dismutase were low under optimal conditions and increased after heat and combined stress in all genotypes. Unlike catalase 3, two other peroxisomal enzymes, catalase 1 and catalase 2, were transcribed extensively under control conditions. Heat stress, in contrast to drought or combined stress, increased catalase 1 and reduced catalase 2 expression in WT and W6:CKX1 plants. In 35S:CKX1, catalase 1 expression was enhanced by heat or drought, but not under combined stress conditions. Mitochondrial superoxide dismutase expression was generally higher in 35S:CKX1 plants than in WT. Genes encoding for chloroplastic AEs, stromatal ascorbate peroxidase, thylakoidal ascorbate peroxidase and chloroplastic superoxide dismutase, were strongly transcribed under control conditions. All stresses down-regulated their expression in WT and W6:CKX1, whereas more stress-tolerant 35S:CKX1 plants maintained high expression during drought and heat. The achieved data show that the effect of down-regulation of CK levels on AES may be mediated by altered habit, resulting in improved stress tolerance, which is associated with diminished stress impact on photosynthesis, and changes in source/sink relations. © 2014 Elsevier GmbH. All rights reserved.

Introduction Drought is a frequent stress that reduces plant biomass production and crop yield. Plant responses to this stress are associated with stomata closure and photosynthesis inhibition (Rivero et al., 2009). In nature, drought is often accompanied by heat stress. Contrary

Abbreviations: AES, antioxidant enzyme system; AE, antioxidant enzyme; APX, ascorbate peroxidase; cAPX, cytosolic APX; ERD, early responsive to dehydration stress; CAT, catalase; CK, cytokinin; CKX, cytokinin oxidase/dehydrogenase; tAPX, thylakoidal APX; sAPX, stromatal APX; ROS, reactive oxygen species; RWC, relative water content; SOD, superoxide dismutase; WT, wild type. ∗ Corresponding author. Tel.: +420 225106427; fax: +420 225106456. E-mail addresses: [email protected] (Z. Lubovská), [email protected] ˇ (J. Dobrá), [email protected] (H. Storchová), [email protected] (N.-0.1. Wilhelmová), [email protected] (R. Vanková). http://dx.doi.org/10.1016/j.jplph.2014.06.021 0176-1617/© 2014 Elsevier GmbH. All rights reserved.

to drought, heat induces transient stomatal opening, and respiration (Rizhsky et al., 2002; Wahid et al., 2007). Thus, the combined stress response differs from those to single stresses. From 1833 transcripts influenced by drought and/or heat, only 77 transcripts were affected under stress combination in a similar way as in single stresses (Rizhsky et al., 2004). Drought and heat are associated with an oxidative stress triggered by the enhanced production of reactive oxygen species (ROS). The main source of ROS are photosynthetic and respiratory electron transport chains, photosystem I and II, overexcited chlorophyll, NADPH oxidase, fatty acid ␤-oxidation, glycolate oxidase, oxalate oxidase, xanthine oxidase, peroxidase and amine oxidase (Mittler, 2002). ROS exhibit several functions, including stress signal transduction (Gechev et al., 2006). On the other hand, they may disrupt cell redox homeostasis (Scandalios, 1993). In order to maintain the delicate equilibrium of ROS concentrations, plants evolved

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antioxidant systems. The main antioxidant enzymes (AE) are ascorbate peroxidase (APX), catalase (CAT), and superoxide dismutase (SOD). The APX is a component of the ascorbate-glutathione cycle in chloroplasts, mitochondria, microsomes and cytosol, where it maintains an appropriate level of H2 O2 (Shigeoka et al., 2002). APX has a high affinity to H2 O2 , in the micromolar and submicromolar range. In Arabidopsis, seven genes coding for APX have been identified (Panchuk et al., 2002). Two chloroplastic APX enzymes, the thylakoid-bound and stromatal, originate from one gene in tobacco (Yoshimura et al., 2002). CAT scavenges H2 O2 in millimolar concentrations, especially in peroxisomes (Mittler, 2006). In most plant species, three genes coding for CAT subunits are present. CAT subunits form various homo- and heterotetramers (Zimmermann et al., 2006). SOD catalyses dismutation of superoxide into oxygen and H2 O2 in chloroplasts, mitochondria, peroxisomes, and cytosol. There are three types of SOD isoenzymes associated to different metal cofactors – Mn (MnSOD), Fe (FeSOD), or Cu and Zn (CuZnSOD). Seven SOD genes have been found in Arabidopsis (Kliebenstein et al., 1998). Cytokinins (CKs) are plant hormones indispensable for the regulation of growth and development (Spíchal, 2012). They also play an important role in stress responses (Ha et al., 2012). Exogenously applied CKs were found to act like stress protectants (Metwally et al., 1997; Rulcová and Pospíˇsilová, 2001). Accordingly, the elevation of endogenous CK levels, achieved by overexpression of CK biosynthetic gene isopentenyltransferase under stress- or senescence-inducible promoter, resulted in improved drought as well as heat tolerance (Rivero et al., 2007, 2009; Zhang et al., 2010; Merewitz et al., 2012; Xu et al., 2009). However, plants with diminished endogenous CK content, caused by overexpression of cytokinin oxidase/dehydrogenase (CKX), the gene encoding the main enzyme for CK degradation, are also ´ highly tolerant to drought or heat (Mytinová et al., 2010; Werner et al., 2010; Nishiyama et al., 2011; Macková et al., 2013). Tobacco plants overexpressing AtCKX1 under the constitutive promoter 35S (35S:CKX1) exhibit an enlarged root system, but stunted shoot phenotype (Werner et al., 2003; Cortleven et al., 2011). In order to avoid negative effects of CKX overexpression on shoot growth, the root-specific promoter WRKY6 was used (Werner et al., 2010). The transgenic line W6:CKX1 displayed a shoot habit quite similar to WT and maintained an enhanced root system. The W6:CKX1 plants were found to be more tolerant to drought than WT (Werner et al., 2010). The activity of the WRKY6 promoter was found to be suppressed under stress conditions (Macková et al., 2013). CKs exhibit cross-talk with the antioxidant system. They are able to scavenge superoxide (Gidrol et al., 1994), inhibit lipoxygenase activity (Swamy and Suguna, 1992) and directly interact with NO (Liu et al., 2013). Exogenous CKs increased CAT and APX activity during dark-induced senescence (Zavaleta-Mancera et al., 2007) and enhanced CAT and SOD activity after heat stress (Liu and Huang, 2002). Modulation of endogenous CK levels was reported to affect the antioxidant system under both control and stress conditions ´ et al., 2010; Cortleven and Valcke, 2012). (Mytinová As AEs contribute significantly to stress tolerance, we evaluated their interplay with CKs under drought and/or heat. Antioxidant transcript levels, as well as enzyme activities, were estimated in tobacco overexpressing CKX1 under the control of either constitutive 35S or root-specific WRKY6 promoters.

Materials and methods Plant material, cultivation and stress conditions Two transgenic lines of Nicotiana tabacum L. cv. Samsun NN overexpressing a gene for CKX1 from Arabidopsis thaliana (AtCKX1)

either under the root-specific promoter WRKY6 (W6:CKX1) or the constitutive promoter 35S (35S:CKX1) and the corresponding wild˝ type (WT) were kindly donated by Prof. Thomas Schmulling and Dr. Tomas Werner, Free University Berlin, Germany. Seeds were sown in sterilized soil (Garden Substrate B; Raˇselina Sobˇeslav, Czech Republic; pot volume 350 mL) without added fertilizer. Plants were cultivated in a growth chamber (Sanyo MLR 350H, Japan) at 25/23 ◦ C, 16 h photoperiod at 130 ␮mol m−2 s−1 and air humidity 80%. Six-week old plants were subjected to 8-day water withdrawal (cessation of watering at air humidity 35%). Heat was applied for 2 h at 40 ◦ C. Stress combination was achieved by application of the heat at the end of drought period. Upper, middle and lower leaves, and roots were harvested. Protein extraction Soluble proteins were extracted from 2 g of frozen leaves or roots homogenized with Ultra-turrax in 10 mL buffer (0.1 M Tris–HCl, 1 mM dithiotreitol, 1 mM Na2 EDTA, 1% Triton X-100, 5 mM ascorbic acid, pH 7.8). For isolation of membrane associated proteins, samples were after 2-min ultrasonic treatment incubated on ice in the dark for 30 min, centrifuged (20,000 × g, 10 min, 4 ◦ C), and filtered. Samples for spectrophotometrical determination of superoxide dismutase (SOD) activity were desalted by passing through Sephadex G-25 (18 × g, 2 min, 4 ◦ C). Samples were frozen in liquid nitrogen and stored at −70 ◦ C. Extract for catalase (CAT) native electrophoresis was isolated from 1 g of frozen leaves homogenized with Ultra-turrax in 10 mL buffer (0.1 M Tris–HCl, 20% glycerol, 30 mM DTT, pH 8) (Frugoli et al., 1996). The homogenate was processed as described above. Protein content was determined according to Bradford (1976). Total enzyme activity Ascorbate peroxidase (APX) and SOD activities were assayed spectrophotometrically in stirred cells maintained at 25 ◦ C (spectrophotometer U-3300, Hitachi, Japan). Total APX activity was determined by monitoring the decrease of absorbance at 290 nm (A290 ) according to Nakano and Asada (1981). Total SOD activity was measured at 470 nm according to Ukeda et al. (1997). One unit of SOD activity was defined as amount of the enzyme required for 50% inhibition of the rate of reduction of sodium 3,3 -{-[(phenylamino)carbonyl]-3,4-tetrazolium}-bis(4methoxy-6-nitro)benzene sulfonic acid hydrate (XTT). CAT activity was determined polarographically (Hansatech Instruments, UK) according to Thomas et al. (1998). Native polyacrylamide electrophoresis The isoenzymes were separated by native polyacrylamide gel electrophoresis (PAGE) according to Laemmli (1970). Electrophoresis ran at 4 ◦ C on gels with a ratio of acrylamide:bisacrylamide of 75:2. To visualize APX activity 20 ␮g of sample was subjected to electrophoretic separation at voltage 300 V for 90 min using 12.5% polyacrylamide resolving with 7.5% stacking gel (Mittler and Zilinskas, 1993). For CAT separation, 4 ␮g of sample ran in 7.5% resolving and 6% stacking gel for 16 h under 80 V (Zimmermann et al., 2006). Determination of SOD isoenzymes (20 ␮g of sample) was carried out on 12% polyacrylamide resolving gel and 6% stacking gel at 20 mA for 2 h. Isoforms with different cofactors were identified with an aid of specific inhibitors (Beauchamp and Fridovich, 1971). Activities in gels were analyzed densitometrically using Multi Gauge software (Fuji film, Science Lab 2002, Japan).

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Fig. 1. Relative water content (RWC) of middle leaves (A), dehydrin ERD1 expression levels in upper, middle and lower leaves and roots (B) of WT, W6:CKX1, and 35S:CKX1 plants. C – control (hydrated) conditions; D – drought stress (8-day dehydration); H – heat (40 ◦ C for 2 h); D + H – combined stress (8-day dehydration + 40 ◦ C for 2 h). Data represent mean values + SD from three biological repetitions. Symbol * indicates significant difference (p ≤ 0.05) between particular stress treatment and control conditions in the corresponding part and genotype. Symbol + indicates significant (p ≤ 0.05) difference between transgenic plants and WT in particular part of plant under given conditions.

RNA isolation, qRT-PCR Total RNA from leaf and root tips was extracted using RNeasy Plant Mini Kit (Qiagen, Germany). DNaseI-treated RNA (DNAfreeTM DNase Treatment and Removal Reagents, Ambion, USA) was transcribed with M-MLV Reverse Transcriptase RNase H Minus, Point Mutant (Promega, USA) according to the manufacturer’s protocol. Primers were designed by software Primer3 (Koressaar and Remm, 2007). Quantitative PCR was run in Light Cycler 1.2 (Roche, Switzerland) using FastStart DNAPLUS SYBR GreenI (Roche, Switzerland) in the following program: 5 min at 95 ◦ C, then 45 cycles of 10 s at 95 ◦ C, 10 s at appropriate annealing temperature (Table S1), and 10 s at 72 ◦ C. The transcript levels were normalized using the Act9 gene as a reference (Havlová et al., 2008). Determination of relative water content (RWC) Cut middle leaves were weighed (fresh mass, FM), saturated overnight with water in the beakers (water saturated mass, SM), and dried at 88 ◦ C for 24 h (dry mass, DM). RWC was calculated as: RWC (%) = (FM − DM)/(SM − DM) × 100 (Weatherley, 1950). Data analysis Results from three independent biological experiments were evaluated by analysis of variance using the Statistica 10 program (StatSoft, USA). All data were normalized by square root transformation. Data for leaves and roots were analyzed separately.

Statistically important differences were proved by post hoc Tukey test (p < 0.05). Significant correlations among variables were identified on the basis of Spearman correlation coefficient (p < 0.001). Data were also tested by principal component analysis (PCA).

Results Characterization of plants under control and stress conditions Wild-type (WT) tobacco and two transgenic lines overexpressing AtCKX1 under 35S or WRKY6 promoters were used to investigate the impact of modulated endogenous CK levels on the antioxidant enzyme system (AES) under drought, heat and their combination. The WT and W6:CKX1 plants had similar shoot habits, however, the W6:CKX1 had larger root systems and after seven weeks were two internodes shorter. The 35S:CKX1 plants developed smaller and thicker leaves, shorter internodes and considerably enhanced root system in comparison to WT. Fifty days after germination, WT and 35S:CKX1 plants had 12–14 leaves, while W6:CKX1 had 10–13. Under control conditions, expression of AtCKX1 was in W6:CKX1 plants high in roots and moderate in senescent leaves. All stress treatments were associated with its rapid decrease in roots (Fig. S1A). As the expression of AtCKX1 in the 35S:CKX1 plants was significantly higher than that of W6:CKX1 under all tested conditions (Fig. S1B), WRKY6 promoter effect is shown in more detail in the insert in Fig. S1A. The impact of stresses on the physiological state of plants was evaluated by determination of RWC and expression level of the dehydration marker gene (ERD1). The 35S:CKX1 plants had significantly higher RWC both after 8-day drought and after combined

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Fig. 2. Effect of drought or/and heat on ascorbate peroxidase expression in upper, middle, and lower leaves and roots of WT, W6:CKX1, and 35S:CKX1. (A) cytosolic (cAPX), (B) stromatal (sAPX), (C) thylakoidal (tAPX). Other designation as described in Fig. 1.

stresses in comparison with the other genotypes (Fig. 1A). Dehydrin ERD1 expression was very low in all genotypes under optimal conditions and heat (Fig. 1B). Drought, both alone or in combination with heat, significantly increased ERD1 expression in all genotypes, albeit to a lower extent in 35S:CKX1 (Fig. 1B). Ascorbate peroxidase Expression of three APX genes, cytosolic (cAPX), stromatal (sAPX) and thylakoidal (tAPX) was examined. Primers for chloroplastic APX transcripts were designed to distinguish between the sAPX and tAPX which arose from the same pre-mRNA. In all genotypes, cAPX expression was markedly enhanced by heat, but not by drought, in both shoots and roots (Fig. 2A). The elevation of expression was the lowest in 35S:CKX1 plants. An expression gradient increasing from upper to lower leaves appeared in all genotypes. The application of heat at the end of the drought period stimulated cAPX expression in WT and W6:CKX1 less than heat alone (Fig. 2A). Both chloroplastic APX transcripts were abundant in leaves of all genotypes under control conditions. The APX transcripts were also quite abundant in roots, especially in W6:CKX1. The sAPX expression in W6:CKX1 leaves was similar to that in WT (Fig. 2B). All tested stresses down-regulated the sAPX transcription in WT and W6:CKX1 plants. In contrast, 35S:CKX1 maintained expression under drought or heat. The stress combination decreased sAPX expression in mature and old leaves of this CKX1 overexpressor. Transcription of tAPX decreased after all applied stresses in both leaves and roots of WT and W6:CKX1 compared to the control conditions (Fig. 2C). It diminished after heat in 35S:CKX1 plants. The

tAPX transcription exhibited a gradient in favor of upper leaves in all genotypes and under all conditions (Fig. 2C). The activity of APX isoforms was detected on zymograms. Isoforms were numbered according to their mobility from the slowest (APX1) to that with the highest (APX9) (Fig. 3A). The most abundant isoform was APX5. Spectrophotometrically-determined total APX activity showed an increase in WT after drought and combined stresses, and in W6:CKX1 after all stress treatments. It was not significantly elevated in 35S:CKX1 (Fig. S2A). Catalase Three genes coding the CAT subunits have been named CAT1-3, in accordance with genes in Nicotiana plumbaginifolia (Willekens et al., 1994). Heat activated CAT1 expression in all genotypes. The expression was decreased after drought and combined stresses in WT and W6:CKX1 leaves (Fig. 4A). In contrast, CAT1 increased after drought in 35S:CKX1. An expression gradient of CAT1 in favor of upper leaves was observed in all genotypes after heat, in 35S:CKX1 after all stress treatments. The expression of CAT1 was not detected in roots of any genotype under any treatment. In contrast to CAT1, the CAT2 expression was repressed by heat in all lines. Combined drought and heat also decreased CAT2 expression in all genotypes. Drought did not affect significantly CAT2 transcripts in the 35S:CKX1 leaves, while elevation was observed in WT and W6:CKX1 middle and lower leaves (Fig. 4B). Expression of CAT3 was up-regulated slightly by drought (except of 35S:CKX1) and strongly by heat alone or in combination with drought in all genotypes (Fig. 4C). Low CAT3 expression was detected in the roots under all treatments.

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in comparison to control conditions. In control and single stress treated 35S:CKX1 middle leaves, FeSOD expression was higher than in WT. After heat, FeSOD expression was higher in middle and lower leaves of both transgenic plants in comparison to WT. The FeSOD transcripts were found in roots of all genotypes (Fig. 5B). A substantial decrease in CuZnSOD expression was caused by drought in all lines, predominantly in upper leaves (Fig. 5C). In contrast, a strong promotion of CuZnSOD expression was observed following heat stress alone in all genotypes, after combined stresses only in transgenic plants. The heat induction was the most profound in 35S:CKX1 plants and in the WT roots. The activity of individual SOD isoforms was visualized using native electrophoresis. Six SOD isoforms detected were named according to their cofactor and mobility from the slowest to the most rapid: MnSOD1, FeSOD1-2, CuZnSOD1-3 (Fig. 3C). Protein MnSOD1 was synthesized from MnSOD mRNA. According to image analysis of zymograms, MnSOD1 activity was lower in 35S:CKX1 leaves than in WT under all conditions. The activity of MnSOD1 showed a decreasing gradient from upper to lower leaves in WT under all conditions. Low MnSOD1 activity was detected in roots (Fig. 6). Total SOD activity was measured spectrophotometrically (Fig. S2C). Relationships among expression levels of antioxidant enzymes

Fig. 3. Densitograms, zymograms, zymograms with edited colors and diagrams of (A) ascorbate peroxidase (APX), (B) catalase (CAT), and (C) superoxide dismutase (SOD). The activity of all individual isoforms was detected in representative mixed samples of WT tobacco leaves and roots. Relative mobility is marked in brackets.

Bands displaying CAT activity identified on zymograms were numbered according to their mobility in the gel, from the isoform with the lowest mobility, CAT1, to CAT9 with the highest one (Fig. 3B). The numbering of the CAT proteins does not correspond to the gene numbers. No significant differences in the activity of particular isoforms or in total CAT activity, measured polarographically, were observed among variants. A weak CAT activity was detectable in roots of all genotypes. In 35S:CKX1 plants, a gradient of CAT activity descending from upper to lower leaves was observed (Fig. S2B).

Relationships between AE expression levels were evaluated by correlation analysis and principal component analysis (PCA). Pearson’s coefficient on square root transformed data (not shown) displayed significant correlations between the same pairs of transcripts as Spearman coefficient on non-transformed data (Fig. 7, Tables S2 and S3), with only two exceptions. When analyzed parametrically, AtCKX1 and CAT3 expression did not correlate, probably due to excessively low values in some variants. In the text below only Spearman coefficient has been employed. In leaves, a weak positive correlation was found between the expression of AtCKX1 and CAT1 (correlation coefficient 0.48), and between AtCKX1 and MnSOD (0.52). Expression of FeSOD and CAT1 negatively correlated with ERD1 (−0.52 and −0.51, respectively). The FeSOD and CAT1 transcripts correlated positively (0.57) (Table S2). A positive correlation was found among expression levels of antioxidant chloroplastic enzymes. Stromatal APX strongly correlated with thylakoidal APX (0.87), these two genes correlated with FeSOD (0.57 and 0.56, respectively). Expression of both chloroplastic APX genes correlated with CAT2 (0.64 and 0.69, respectively). Transcripts of enzymes with general stress protective functions correlated positively – CuZnSOD with cAPX and CAT3 (0.62 and 0.38, respectively) (Table S2). cAPX negatively correlated with CAT2 (−0.54). PCA clustered together all chloroplastic isoforms (sAPX, tAPX, FeSOD), and also cAPX, CAT3 and CuZnSOD (Fig. S3). In roots, the expression of cAPX correlated with CuZnSOD (0.64), sAPX with FeSOD (0.61) and sAPX with CAT3 (0.65) (Table S3).

Superoxide dismutase Discussion Expression of three SOD genes was investigated, i.e. mitochondrial MnSOD, chloroplastic FeSOD, and cytosolic CuZnSOD. The expression of MnSOD declined after all stress treatments in WT leaves and after combined stresses in leaves of W6:CKX1 and 35S:CKX1 (Fig. 5A). Under all stresses, MnSOD transcripts were more abundant in 35S:CKX1 leaves in comparison to the other genotypes. MnSOD was highly transcribed in roots in all tested variants, generally not affected by stress treatment (Fig. 5A). Expression of FeSOD showed a descending gradient from upper toward lower leaves in WT and W6:CKX1 under control conditions and after heat (Fig. 5B). In WT and W6:CKX1 leaves, transcription of FeSOD decreased markedly after drought and combined stresses

Our expression study of nine genes encoding components of the AES in tobacco with modulated CK content under drought and heat, alone and in combination, indicated a complex regulation of AES at the level of gene expression, influenced by CKs (Fig. 7). In dependence on the abiotic stress defense strategy, both down-regulation (Werner et al., 2010; Macková et al., 2013) as well as up-regulation of CK content (Metwally et al., 1997; Rivero et al., 2007; Merewitz et al., 2012) may have positive effects on the stress tolerance. Drought tolerance of plants with lowered CK content seems to be associated with enhanced root system, slower growth rates and altered leaf morphology (Werner et al., 2010; Nishiyama

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Fig. 4. Expression levels of catalase genes in upper, middle and lower leaves and roots of WT, W6:CKX1 and 35S:CKX1. (A) CAT1, (B) CAT2, (C) CAT3. Other designation as described in Fig. 1.

et al., 2011). On the other hand, stress- or senescence-induced CK up-regulation seems to diminish negative stress effects by stabilizing photosynthesis. The root-specific promoter WRKY6, in combination with CKX1, enables plants to utilize both approaches. High CKX1 expression under control conditions enhances root growth, whilst the promoter switch-off under stress conditions contributes to CK elevation during the early stages of the stress response (Macková et al., 2013). We evaluated the effect of CK modulation on the plant stress tolerance. As a marker of the stress impact we chose EARLY RESPONSIVE TO DEHYDRATION STRESS 1 (ERD1), which belongs to dehydration-inducible genes with fast and strong response (Kiyosue et al., 1994, Vivek et al., 2013). Lower expression of ERD1 in drought stressed 35S:CKX1 plants seems to reflect their higher stress tolerance, which is in accordance with the report of Tran et al. (2004), who did not find elevation of ERD1 expression in transgenic plants with enhanced stress tolerance. The impact of CK modulation on AES was tested under both control and stress conditions. The assayed AEs may be grouped according to their functions: (1) chloroplastic enzymes – sAPX, tAPX, FeSOD; (2) enzymes with general stress defense function – cAPX, CAT3, cytosolic CuZnSOD; (3) peroxisome protecting enzymes – CAT1, CAT2 and CAT3; and (4) mitochondrial MnSOD, enzyme removing ROS produced during respiration (Figs. 7 and S3). The chloroplastic enzymes protect individual components of the photosynthetic apparatus. The tAPX is functionally associated with photosystem I (Yabuta et al., 2002; Asada, 2006). Chloroplastic FeSOD is responsible for the elimination of superoxide produced in photosystem I and protects both photosystems (Van Camp et al., 1997; Zhang et al., 2011). The enzyme sAPX scavenges H2 O2 in stroma, where it serves as the second defense after primary protection by tAPX and FeSOD (Asada, 2006). The expression

patterns of chloroplastic AEs were highly coordinated, as indicated by correlation coefficients and PCA (Figs. 7 and S3). Under optimal conditions, the principal ROS source in plants during daylight is electron transport in the chloroplasts. All chloroplastic AEs were strongly transcribed in leaves of all genotypes under control conditions. Surprisingly, expression of sAPX was also relatively high in roots, particularly in W6:CKX1. Expression of chloroplastic APX genes was previously observed in roots of spinach (Yoshimura et al., 2002), but their role in roots has not yet been elucidated. Stress conditions bring about changes in the metabolic activity of plants. The photosynthetic activity is depressed during both drought (Rivero et al., 2009) as well as elevated temperature (Allakhverdiev et al., 2008). A decrease in sAPX and tAPX expression in all stress conditions was observed in WT and W6:CKX1. This down-regulation could reflect a diminished ROS production in their chloroplasts as a result of reduced photosynthesis. Our data are in accordance with Rizhsky et al. (2002) who found diminished sAPX expression in tobacco after heat and de Carvalho et al. (2013) who reported decrease in chloroplastic APX transcripts after drought. In spite of the fact that exogenous CKs as well as enhanced endogenous CK levels (Rivero et al., 2009) were reported to increase the activity of AEs, we detected under stress conditions higher sAPX expression in 35S:CKX1 plants which have diminished CK content. Potential explanation may be that sAPX expression reflects predominantly plant stress tolerance. This assumption is in accordance with strong positive effect of elevated proline content on both stress tolerance and chloroplastic APX expression in Swingle citrumelo (de Carvalho et al., 2013). Positive correlation of FeSOD expression with stress tolerance was suggested already by Van Camp et al. (1996a). Our results showing higher FeSOD expression in both

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Fig. 5. Expression levels of superoxide dismutase genes in upper, middle and lower leaves and roots of WT, W6:CKX1 and 35S:CKX1. (A) mitochondrial MnSOD, (B) chloroplastic FeSOD, (C) cytosolic CuZnSOD. Other designation described in Fig. 1.

CKX1 over-expressors (especially in more resistant 35S:CKX1) are in accordance with this conclusion. Enzymes participating in stress protection, i.e. cAPX, CAT3 and cytosolic CuZnSOD, were expressed to a relatively low extent under control conditions. Under optimal conditions, their transcription was comparable in WT and both transgenic lines which indicated that overexpression of AtCKX1 did not induce any oxidative stress. The expression profiles of these genes showed a similar response to applied stresses in all genotypes, as indicated both by strong correlation between cAPX and CuZnSOD and a weaker one between CAT3 and CuZnSOD (Table S2), and by PCA, grouping all these three transcripts (Fig. S2). Heat and stress combination activated CAT3, CuZnSOD and especially cAPX. These results are in line with the

reported stimulation of cAPX expression in heat stressed tobacco (Rizhsky et al., 2002) and Arabidopsis (Panchuk et al., 2002). The peroxisomal enzymes CATs, are present in plants in several isoforms. In tobacco three genes were detected – CAT1, CAT2, and CAT3 (Willekens et al., 1994). We found extensive transcription of CAT1 and CAT2, unlike CAT3, in leaves under control conditions. Our data accord with the report of Luna et al. (2005) on wheat and Du et al. (2008) on Arabidopsis. Drought and heat promote photorespiration, which is associated with production of H2 O2 . Subsequently, the demand for antioxidant defense in peroxisomes increases. CAT1 expression was stimulated by heat stress, being repressed by drought and combined stresses in WT and W6:CKX1 plants. Mild up-regulation of CAT2 expression

Fig. 6. The activity of MnSOD1 in upper , middle and lower leaves and roots of WT, W6:CKX1 and 35S:CKX1. Other designation as described in Fig. 1.

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Acknowledgements We thank Prof. David Morris for critical reading and language ˝ and Dr. Tomas editing. We also thank Prof. Thomas Schmulling Werner for the seeds of both transgenic lines and the corresponding WT. This work was supported by the Czech Science Foundation, project no. 206/09/2062 and Ministry of Education of the Czech Republic grant no. LD14120. Appendix A. Supplementary data

Fig. 7. Schematic representation of the relationships among the expression profiles of individual antioxidant enzymes in tobacco. Solid and dashed lines represent positive and negative correlations, respectively, with coefficient > |0.5|. (1) Chloroplastic transcripts, sAPX, tAPX and FeSOD, were correlated. (2) Transcript profiles of antioxidant enzymes in chloroplasts and peroxisomes followed similar trends. (3) CAT transcript levels were not inter-related, (4) Cytosolic CuZnSOD correlated with another stress protective enzyme, cAPX. (5) MnSOD exhibited positive correlation with AtCKX1 expression.

was observed in drought, the other stresses had adverse effects. The CAT3 expression was strongly stimulated by all stresses, predominantly by their combination. CAT3 seems to provide majority of H2 O2 scavenging in peroxisomes under stress conditions. Our results are in accordance with Du et al. (2008) who found different regulation of individual CAT isoforms by drought, cold, oxidative stress and ABA application. Thus, different expression profiles as well as the lack of correlation among them indicate different function and regulation of CAT isoforms. Mitochondrial MnSOD removes superoxide generated by electron leakage in a respiratory electron transport chain (Bowler et al., 1989; Van Camp et al., 1996b). The gradient in MnSOD activity in favor of upper leaves found in WT agrees with the negative correlation between the expression as well as the activity of MnSOD and leaf age reported by Priault et al. (2007). It is known that both expression and activity of MnSOD in tobacco are stimulated by exogenous application of sucrose (Bowler et al., 1989). Taking into account that CKs control sink/source distribution of sugars, it is possible to anticipate that CKs can regulate MnSOD by modulation of carbohydrate metabolism via sink/source dynamics (Cowan et al., 2005; Roitsch and Ehneß, 2000). The potential CK function is also indicated by the coincidence of lower CK content as well as reduced concentration of soluble sugars in 35S:CKX1 plants (Werner et al., 2008) and their lower MnSOD1 activity (Fig. 6). In contrast to low MnSOD1 activity, MnSOD transcription was high in 35S:CKX1. The discrepancy between the activity and transcript levels were reported also for other AEs, e.g. APX (de Campos et al., 2011). The lack of correspondence may be given by transcript stability, posttranscriptional or post-translational regulations (de Carvalho et al., 2013). It may be, however, also caused by inhibition of MnSOD enzymatic activity in 35S:CKX1 plants. In conclusion, tobacco with down-regulated CK content differed in their stress tolerance as well as in AES responses compared to WT. Enhanced stress tolerance was reflected by better preservation of photosynthetic activity under stress conditions, as indicated by higher expression of photosynthesis-related antioxidant genes. The defense mediated by AES exerts a high plasticity. It is specifically regulated under various stress conditions, exhibiting differences between the response to a single stress and to combined stresses. The application of heat at the end of drought, which strongly deepened the stress severity, further decreased the expression of chloroplast and mitochondria related genes, while the expression of cytosol related genes and CAT3 was enhanced. The impact of down-regulation of CK levels on AES seems to be indirect, mediated by the positive effect on plant stress tolerance or on photosynthesis.

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