Hydrogen peroxide pretreatment of roots enhanced ... - Springer Link

Acta Physiol Plant (2013) 35:1905–1913 DOI 10.1007/s11738-013-1228-7

ORIGINAL PAPER

Hydrogen peroxide pretreatment of roots enhanced oxidative stress response of tomato under cold stress ¨ zlem Darcansoy I˙s¸ eri • Didem Aksoy Ko¨rpe O Feride Iffet Sahin • Mehmet Haberal

•

Received: 13 September 2012 / Revised: 20 December 2012 / Accepted: 22 January 2013 / Published online: 5 February 2013 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2013

Abstract In the view of physiological role of H2O2, we investigated whether exogenous H2O2 application would affect short-term cold response of tomato and induce acclimation. Pretreatments were performed by immersing roots into 1 mM H2O2 solution for 1 h when transferring seedlings from seedling substrate to soil (acclimated group). Cold stress (3 °C for 16 h) caused significant reduction in relative water content (RWC) of control and non-acclimated (distilled water treated) groups when compared with unstressed plants. H2O2 promoted maintenance of relatively higher RWC under stress. Anthocyanin level in leaves of acclimated plants under cold stress was significantly higher than that of unstressed control and non-acclimated plants. Malondialdehyde (MDA) levels demonstrated low temperature induced oxidative damage to control and nonacclimated plants. MDA remained around unstressed conditions in acclimated plants, which demonstrate that H2O2 acclimation protected tissues against cold induced lipid peroxidation. H2O2 acclimation caused proline accumulation in roots under cold stress. Ascorbate peroxidase (APX) activity in roots of cold stressed and unstressed H2O2 acclimated plants increased when compared with control Communicated by H. Li. ¨ . D. ˙Is¸ eri (&) D. A. Ko¨rpe F. I. Sahin M. Haberal O Institute of Transplantation and Gene Sciences, Baskent University, Kazan, Ankara, Turkey e-mail: [email protected] F. I. Sahin Department of Medical Genetics, Faculty of Medicine, Baskent University, Ankara, Turkey M. Haberal Department of Surgery, Faculty of Medicine, Baskent University, Ankara, Turkey

and non-acclimated plants, with highest increase in roots of acclimated plants under cold stress. CAT levels in roots of acclimated plants also increased, whereas levels remained unchanged in unstressed plants. Endogenous H2O2 levels significantly increased in roots of control and non-acclimated plants under cold stress. On the other hand, H2O2 content in roots of acclimated plants was significantly lower than control and non-acclimated plants under cold stress. The results presented here demonstrated that H2O2 significantly enhanced oxidative stress response by elevating the antioxidant status of tomato. Keywords Acclimation Antioxidant response Hydrogen peroxide Low temperature stress Lycopersicon esculentum Mill

Introduction Hydrogen peroxide is a major reactive oxygen species (ROS) generated as a result of oxidative stress in plants. Oxidative stress arises from imbalance of metabolism and generation of ROS, and the extent of oxidative stress in a cell is determined by the amounts of superoxide, hydrogen peroxide, and hydroxyl radicals. Electron transport causes generation of H2O2 during metabolic processes, such as photosynthesis and mitochondrial respiration. It is scavenged in plant cells by a complex network of antioxidants and antioxidant enzymes. Ascorbate peroxidase (APX), glutathione peroxidase (GPX) and catalase (CAT) detoxify hydrogen peroxide. ROS balance in plant cells may be disturbed by a variety of abiotic and biotic environmental stimuli such as high light, drought, low and high temperature, and mechanical stress (reviewed by Apel and Hirt 2004).

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There is now growing evidence that H2O2 functions as a signaling molecule to induce a range of molecular, biochemical and physiological responses in plants. For the first time, Prasad et al. (1994a) demonstrated chilling induced oxidative damage in maize seedlings, and dual role of changes in cellular H2O2 at low temperature treatments with its accumulation during both acclimation (14 °C for 3 days in the dark) and non-acclimation. They showed that during acclimation, its early transient accumulation signaled the induction of antioxidant enzymes (e.g. CAT3). However, in non-acclimated seedlings, it accumulated to damaging levels at 4 °C. In the same study, the exogenous root pretreatment of seedlings with H2O2 at both 27 and 4 °C prior to 7 days chilling treatment at 4 °C had also found to have similar protective effects against chilling induced damage. In maize, the effect of exogenously applied H2O2 on salt stress acclimation was also demonstrated with regard to plant growth, lipid peroxidation, and activity of antioxidant enzymes (SOD, APX, guaiacol peroxidase, glutathione reductase and CAT) in leaves and roots (de Azevedo Neto et al. 2005). In tomato, Orozco-Ca´rdenas et al. (2001) showed that H2O2 acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. In addition involvement of H2O2 signalling in copper tolerance of tomato was also reported (Wang et al. 2010b). In tobacco, spraying of leaves with H2O2 provided protection against oxidative stress generated by high light stress or aminotriazole by inducing CAT, APX, and guaiacol peroxiadases (Gechev et al. 2002). Recently, foliar application of H2O2 was also shown to induce tolerance of creping bent grass to heat stress by reducing oxidative damage (Larkindale and Huang 2004). In addition, foliar hydrogen peroxide pretreatment at low concentrations (5–15 mM) was shown to improve the tolerance of warmseason grasses to chilling stress (Wang et al. 2010a). In terms of plant survival, effect of pretreatment with H2O2 leaf spraying was demonstrated by reduced deleterious effects of salinity on seedling growth and lipid peroxidation (Gondim et al. 2012). Important evidence on H2O2 acclimation was obtained with rice seedlings pre-treated with various levels of H2O2. Under salt and heat stresses, survival of more green leaf tissue, higher quantum yield for photosystem II, and increased oxygen scavenging enzyme activities than in nontreated controls were demonstrated (Uchida et al. 2002). Yu et al. (2002) demonstrated enhanced chilling tolerance of mung bean seedlings and increase of glutathione in due 200 mM H2O2 pretreatment. Furthermore, moderately higher levels of H2O2, as a byproduct of choline oxidase catalyzed glycine betaine (GB) synthesis in transgenic tomato which express codA gene, and synthesize choline oxidase, was postulated to activate the H2O2-inducible protective mechanism, resulting in improved chilling and oxidative tolerances in these plants (Park et al. 2004).

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Acta Physiol Plant (2013) 35:1905–1913

Cold acclimation is a process by which plants acquire tolerance to low temperatures upon pre-exposure to low non-freezing temperatures. However, many crops including tomato are chilling sensitive, although sensitivity of genotypes show variation depending on the mineral and nutrient supply, photoperiod of low temperature exposure and ecotypes (Martin and Ort 1985; Yakır et al. 1986; Starck et al. 2000; Chinnusamy et al. 2007). Tomato is also sensitive to cold acclimation (Chinnusamy et al. 2007). Tomato has economic importance in Turkey, with almost 10 million metric tons of production, placing Turkey in the fourth rank (FAO). However, seasonal temperature variations affect growth and yields. In the view of physiological role H2O2 and effect of foliar application in acclimation phenomenon and stress tolerance (Gechev et al. 2002; Larkindale and Huang 2004; de Azevedo Neto et al. 2005; Wang et al. 2010a), we addressed the question if exogenous root application of H2O2 would affect short-term cold response of tomato (Lycopersicon esculentum Mill.), and induce acclimation. The H2O2 pretreatment was performed by immersing roots of tomato into 1 mM H2O2 (Prasad et al. 1994a; Gechev et al. 2002; Larkindale and Huang 2004) solution for 1 h during seedling substrate to soil transplant of 4–5 leaf seedlings. Cold stress was applied at 3 °C for 16 h, and the effects of H2O2 pretreatment were assessed by determining lipid peroxidation, RWC, chlorophyll, carotenoid, anthocyanin contents, and proline accumulation. The involvement of enzymatic antioxidant system was assessed by following changes of two H2O2 scavenging enzymes, APX and CAT.

Materials and methods Plant material and treatments Lycopersicon esculentum Mill. cv. H2274 (MayAgro Seed Corporation, Turkey) seeds were used in the experiments. Seeds were sown into cell plants containing seedling substrate (Klasman-Deilmann GmbH, Germany), and grown for 25 days in a plastic greenhouse (Institute of Transplantation and Gene Sciences, Baskent University, KazanAnkara, Turkey) under controlled conditions (27–30 °C, 60–65 % relative humidity, photoperiod of 16/8 light/ dark). Plants with 4–5 true leaves were chosen for treatments. Roots of acclimated plants were immersed into 1 mM H2O2 for 1 h at 28 °C, whereas non-acclimated were immersed into distilled water (dH2O) for 1 h at 28 °C prior to soil transplanting to individual pots containing an animal-based fertilizer (soil:fertilizer; 2:1). Roots of H2O2 treatment groups were rinsed with dH2O several times for the removal of H2O2. Control group was directly transplanted to soil. After 4 days (27–30 °C, 60–65 % relative


humidity, photoperiod of 16/8 light/dark), acclimated, nonacclimated, and control plants were further separated into two groups as cold stress and, unstressed. Short term cold stressed plants were incubated at 3 °C for 16 h, whereas unstressed plants were incubated at 28 °C for 16 h in the growth chamber. 16 h of stress treatment was applied at dark period after 8 h of light treatment. Roots and leaves of all plants were immediately harvested at the end of 16-h period for analysis. Relative water content Fresh (FW), dry (DW) and turgid (TW) weights of plants were assessed for the evaluation of water cogent. Relative water content was calculated according to the formula (Smart and Bingham 1974): Relative water content (RWC; %) = [(FW - DW) (TW - DW)-1] 9 100 Lipid peroxidation Lipid peroxidation was assayed by determining malondialdehyde (MDA) amount, a product of lipid peroxidation (Ohkawa et al. 1979). In brief, leaf (0.2 g) and root (0.1 g) samples were homogenized in liquid nitrogen, and the homogenates were suspended in 5 % (w/v) tricholoroacetic acid (TCA; Merck) in a ratio of 0.2 g:1 mL. Samples were centrifuged and supernatant was mixed with 1:1 volume of 0.5 % (w/v) thiobarbituric acid (TBA; Sigma) in 20 % (w/v) TCA. Following 96 °C, 25 min incubation, samples were cooled to room temperature and centrifuged. Absorbance of the supernatant was recorded at 532 and 600 nm against 0.5 % (w/v) TBA in 20 % (w/v) TCA. After subtracting the nonspecific absorbance at 600 nm, MDA concentration was calculated by its extinction coefficient of 155 mM-1 cm-1. Chlorophyll, carotenoid, and anthocyanin amount For the determination of chlorophyll a and b, total chlorophyll (a ? b), and total carotenoid (xanthophylls and carotenes; x ? c) amounts, small pieces of leaves (1 cm2) were put in pure acetone (Merck), and incubated at 4 °C for 4 days. Absorbance was measured at 470, 644.8 and 661.6 nm, and concentration of a, b, and x ? c in leaves was calculated according to Lichtenthaler and Buschmann (2001). Anthocyanin content of leaves and roots (1 cm2) were determined by crashing tissue samples in 1 mL of 79 % (v/v) methyl alcohol (Merck) and 1 % HCl (Merck) (v/v) solution. Samples were incubated at 4 °C for 4 days and absorbance of the sample solution was measured at 530 and 657 nm (e & 34,300 M-1 cm-1) (Mancinelli 1990; Giusti and Wrolstad 2001).

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Proline levels Proline amount were determined according to method described by Bates et al. (1973). In brief, 0.2 g leaf and 0.1 g root samples were homogenized with mortar and pestle in liquid nitrogen, and the homogenates were suspended in 1 mL of 3 % (w/v) sulphosalicylic acid (Merck). After centrifugation, 0.1 mL of supernatant was transferred into a solution of 0.2 mL acid ninhydrin (Merck), 0.2 mL of 96 % (v/v) acetic acid (Merck), and 0.1 mL of 3 % (w/v) sulphosalicylic acid. Samples were incubated for a 1 h at 96 °C, and 1 mL of toluene (Merck) was added. After centrifugation, upper phase was transferred into quartz cuvettes and absorbance was recorded at 520 nm against toluene. Proline amount was calculated by using proline (Fluka, USA) standard curve. Enzyme assays For determination of APX activity (Nakano and Asada 1987), leaf (0.2 g) and root (0.1 g) samples were homogenized with mortar and pestle in liquid nitrogen, and the homogenates were suspended in 50 mM Tris–HCl buffer (pH 7.2) containing 2 % (w/v) polyvinylpoly pyrrolidone (Sigma), 2 mM ascorbate (Sigma) and 1 mM EDTA (Applichem, USA) (0.2 g:1 mL). After centrifugation, total protein amount in supernatants was determined according to Bradford (1976). One hundred microgram of total protein was added to assay solution (50 mM potassium phosphate buffer with 2.5 mM ascorbate; 1 mL total assay medium), and reaction was initiated by the addition of 100 lL of 10 mM H2O2. Decrease in the absorbance of ascorbate was recorded at 290 nm for 3 min against assay solution (e = 2.8 mM-1 cm-1). For determination of CAT activity (Chance and Mahly 1995), leaf (0.2 g) and root (0.1 g) samples were homogenized in liquid nitrogen, and the homogenates were suspended in suspension buffer (0.2 g:1 mL). After centrifugation, total protein amount in supernatants were determined according to Bradford method. One hundred microgram of total protein was added to 100 mM potassium phosphate buffer (pH 7.0; 1 mL total assay medium), and reaction was initiated by the addition of 100 lL of 100 mM H2O2. Decrease in the absorbance of H2O2 was recorded at 240 nm for 3 min against assay solution (e = 39.4 mM-1 cm-1). Endogenous hydrogen peroxide Leaf (0.2 g) and root (0.1 g) samples were homogenized by mortar and pestle in liquid nitrogen, and the homogenates were suspended in 100 mM potassium phosphate buffer (pH 6.8) in a ratio of 1 g:3 mL. The method of Bernt and

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Bergmeyer (1974) was used to determine the H2O2 content. Homogenates were centrifuged at 18,000 g for 20 min at 4 °C, and the supernatant was mixed with peroxidase reagent containing 40 lg/mL peroxidase (Sigma) and 0.005 % (w/v) o-dianizide (Sigma) in 83 mM potassium phosphate buffer (pH 7.0) in a ratio of 1:5. After incubation at 30 °C, the reaction was terminated with 1 N perchloric acid (Sigma), and the samples were centrifuged at 5,000g for 10 min. Absorbance of the supernatant was measured at 436 nm, and H2O2 content was calculated using its extinction coefficient of 39.4 mM-1 cm-1. Statistical analysis Statistical analyses were performed using SPSS 11.5 software (SPSS Inc., USA). All data are expressed as mean ± standard error of the means (SEM) of five biological replicates in enzyme assays, lipid peroxidation, hydrogen peroxide and proline contents, and of 4 for pigment amounts and RWC. Mean differences between control, non-acclimated, and acclimated plants at stress and unstressed temperatures were statistically evaluated by one-way ANOVA analysis at the 0.05 levels and post hoc Tukey’s analyses were carried out to find groups whose mean differences were significant.

82 80

RWC (%)

78 76

a

74

b

c

72

a, c

70 68

a,b, c

66 64

Control dw 28°C 28°C

H2O2 28°C

Control 3°C

dw 3°C

H2O2 3°C

Fig. 1 Changes in relative water content (RWC). Standard error of the means were derived from four biological replicates. RWC of 28 °C control, 3 °C control and acclimated groups (represented by a); 28 and 3 °C non-acclimated groups (represented by b), and 28 °C acclimated and 3 °C control and non-acclimated groups (represented by c) significantly differ (p \ 0.05)

Table 1 Leaf and root anthocyanin levels Anthocyanin (nmol/cm2) ± SEM*

28 °C

Control

Results

Root

5.53 ± 0.69a

4.87 ± 0.57

5.8 ± 0.7b

5.28 ± 1.23

Acclimated

7.05 ± 0.56

5.97 ± 0.93

Control

7.26 ± 0.67

4.81 ± 0.89

Non-acclimated

7.14 ± 0.55

4.92 ± 1.21

Acclimated

8.04 ± 0.52a,b

4.61 ± 0.72

Non-acclimated 3 °C

Leaf

*

Short-term low temperature induced changes in RWC To test the effects of short-term cold stress and exogenous H2O2 application at physiological level, we evaluated relative water, chlorophyll, carotenoid, and anthocyanin contents of control, non-acclimated, and acclimated plants in comparison to 28 °C incubation groups (Fig. 1). Sixteen hours of cold stress caused significant reduction in RWC of control and non-acclimated treatment groups when compared with unstressed plants. Hydrogen peroxide acclimation promoted maintenance of relatively higher RWC under cold stress. Furthermore, cold induced leaf wilting and curling, correlated with lower RWC, seemed to be higher in control and non-acclimated plants when compared with the symptoms in acclimated group (observational interpretation). A slight decrease in total leaf chlorophyll amount of cold stressed plants was observed with a concomitant increase in chlorophyll a to b ratio, and decrease in total chlorophyll to carotenoid ratio, however, the changes were not statistically significant. Anthocyanin level in leaves of acclimated plants under cold stress was significantly higher than that of unstressed control and nonacclimated plants (Table 1).

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SEM (standard error of the means) were derived from four biological replicates Superscript letters represent significant difference between leaf anthocyanin of both of 28 °C control and non-acclimated, and 3 °C acclimated group according to one-way ANOVA analysis and post hoc Tukey’s analysis (p \ 0.05)

H2O2 acclimation provided protection against cold induced oxidative damage in roots and leaves of tomato Lipid peroxidation was quantified by measuring MDA levels under cold stress (Fig. 2). Root MDA levels, which were in between 13 and 15 nmol gFW-1 in roots of unstressed plants, significantly increased to *25 nmol gFW-1 in roots of control, and non-acclimated plants under cold stress. Similarly, significant increments in leaf MDA levels of control and non-acclimated upon cold treatment (from 15–16 to 22–23 nmol gFW-1) demonstrated low temperature induced oxidative damage to leaf tissue. MDA levels in roots and leaves of acclimated plants remained around unstressed conditions, which demonstrate that H2O2 acclimation protected roots and leaves of tomato plants against cold induced lipid peroxidation.


Root

MDA (nmol/gFW)

25

Leaf

20 15

a

f

b g

c

2.0

a,b, f,g, c,e h,j

1.8

d,e i,j

h

10 5

proline (mg/gFW)

a,b, c,d f,g, h,i

30

1909

1.6

Root

a,b

Leaf

1.4 1.2 1.0

a

0.8

b

0.6 0.4 0.2 0.0

0

Control 28°C

dw 28°C

H2O2 28°C

Control 3°C

dw 3°C

H2O2 3°C

Fig. 2 Malondialdehyde (MDA) levels in roots and leaves of control, non-acclimated, and acclimated plants at 3 and 28 °C. Standard error of the means was derived from five biological replicates. Same letters represent significant difference between groups (p \ 0.05): a root MDA of 28 °C control, 3 °C control, and 3 °C dw groups. b Root MDA of 28 °C dw, 3 °C control, and 3 °C dw groups. c Root MDA of 28 °C acclimated, 3 °C control, and 3 °C dw groups. d Root MDA of 3 °C control and 3 °C acclimated groups. e Root MDA of 3 °C dw and 3 °C acclimated groups. f Leaf MDA of 28 °C control, 3 °C control, and 3 °C dw groups. g Leaf MDA of 28 °C dw, 3 °C control, and 3 °C dw groups. h Leaf MDA of 28 °C acclimated, 3 °C control, and 3 °C dw groups. i Leaf MDA of 3 °C control and 3 °C acclimated groups. j Leaf MDA of 3 °C dw and 3 °C acclimated groups

H2O2 acclimation caused proline accumulation in roots of tomato under cold stress To investigate possible contribution of proline accumulation to H2O2 acclimation of tomato plants to cold stress, we measured free proline levels in roots and leaves of stressed and unstressed plants. Around 0.7 mg gFW-1 proline in roots of control and non-acclimated plants increased to 1.6 mg gFW-1 in roots of cold stressed acclimated plants (Fig. 3). However, there was not any significant proline accumulation in roots of cold stressed control and nonacclimated plants. Leaf proline levels also remained unchanged among all treatment groups. H2O2 acclimation induced changes in activities of APX and CAT Figure 4 demonstrates H2O2 induced changes in activities of APX, CAT, and endogenous hydrogen peroxide levels. The APX activity in roots of cold stressed and unstressed acclimated plants increased when compared with control and non-acclimated plants. Similar alterations were also observed in leaf tissues though the increments were less pronounced in comparison to roots. Highest response was observed in roots of acclimated plants under cold stress (approximately 4-fold with respect to both control and nonacclimated plants). CAT levels in roots of acclimated plants also increased when compared with other groups, whereas levels remained unchanged in roots of unstressed

Control 28°C

dw 28°C

H2O2 28°C

Control 3°C

dw 3°C

H2O2 3°C

Fig. 3 Proline accumulation in roots and leaves. Standard error of the means was derived from five biological replicates. Proline in roots of 28 °C control and 3 °C acclimated groups (represented by a), and 28 °C non-acclimated and 3 °C acclimated groups (represented by b) significantly differ (p \ 0.05)

plants. However, in contrast to APX activity, no significant alteration was observed in leaf CAT activities of different treatment groups. Endogenous hydrogen peroxide levels significantly increased in roots of control and non-acclimated plants under cold stress. On the other hand, hydrogen peroxide content in roots of acclimated plants was significantly lower when compared with control and non-acclimated plants under cold stress. However, levels remained unchanged in the case of unstressed plants. In leaf tissue although alterations were observed, these were insignificant.

Discussion Low temperature stress above 0 °C has been termed as chilling stress. It results primarily in decreased membrane viscosity, retarded metabolism, and delayed energy dissipation leading to radical formation and oxidative stress (reviewed by Beck et al. 2004). Chilling immediately causes mechanical constraints, changes in activities of macromolecules, and reduced osmotic potential (Xiong et al. 2002). Plants exposed to chilling stress often show water-stress symptoms because of chilling induced inhibition of water uptake and water loss. Water loss is primarily related to loss of membrane properties or transition of membranes from a normal fluid state to a restricted, less fluid, semicrystalline state (Wright 1974). RWC is a quantitative indication of water status of plants. Hydrogen peroxide acclimation promoted maintenance of relatively higher RWC under cold stress (Fig. 1). The most common visible symptom is wilting during and after the low temperature exposure. Leaf wilting and curling correlated with lower RWC, seemed to be less pronounced in acclimated group (observational interpretation).

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H2O2 (nmol/gFW)

CAT activity (nmoleH 2O2 /min/mg protein)

APX activity (umoleAsA/min/mg protein)

1910 Root

Leaf

7

a,b,c,d,e

6 5 4 3 2

a,b,c a

1

f

b g

d

f

f,g, h,i

e i

h

0

160 140

28°C

120 100

a

28°C

28°C

3°C

c

d

b

80

a,b,c,d,e 3°C e

60 40 20 0 45 40 35 30 25 20 15 10 5 0

Control 28°C

dw 28°C

H2O2 28°C

Control dw 3°C 3°C

a,b,c a

Control 28°C

a,b,d

b

dw 28°C

H2O2 3°C

c,d

H2O2 28°C

Control 3°C

dw 3°C

H2O2 3°C

Fig. 4 Ascorbate peroxidase (APX), catalase (CAT) activity, and endogenous hydrogen peroxide levels in roots and leaves of control, non-acclimated, and acclimated plants at 3 and 28 °C. Standard error of the means was derived from five biological replicates. a–e Significant difference between root treatment groups, and f–i represent significant difference between leaf treatment groups (p \ 0.05). Significantly different groups, which were determined by post hoc analysis, were represented by the same letters

Anthocyanin level in leaves of acclimated plants under cold stress was significantly higher than that of unstressed control and non-acclimated plants (Table 1). Anthocyanin biosynthesis may be induced by environmental factors, and production and localization in root, stem and leaf tissues may develop resistance to abiotic stresses (Chalker-Scott 1999). In the case of control and non-acclimated groups, 16 h short-term cold stress at dark period may not cause high anthocyanin accumulation. In concordance, there is increase in leaf anthocyanin levels of control and nonacclimated plants though the levels are statistically not significant as in the case of acclimated group. Recently, anthocyanin accumulation was demonstrated in leaves of rice seedlings upon H2O2 accumulation after 48 h of abscisic acid treatment (Huang et al. 2008).

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Cytosolic proline helps to maintain osmotic adjustment and membrane stability, and reduces the disruptive effects of ROS as a free radical scavenger (Mansour 1998). Because proline accumulates in plants at low temperatures (Gilmour et al. 2000; Yadegari et al. 2007; Koç et al. 2010), we investigated possible contribution of proline accumulation to H2O2 acclimation. In case of our study, proline accumulation in roots of cold stressed acclimated plants was observed (Fig. 3). It may be result of (1) total impact of H2O2 and cold induced stimuli, or (2) combinatory effects of H2O2 and cold induced pathways; in either case which were not alone sufficient to stimulate significant proline levels. Proline accumulation could be due to either de novo synthesis or decreased degradation or both. Proline is synthesized via either the glutamate or ornithine pathways, localized in different cellular compartments, and accumulation is often achieved through different mechanisms depending on the species, developmental stage, and stress factor (reviewed by Szabados and Savoure0 2009). Glutamate is converted to proline by two successive reductions catalyzed by pyrroline-5- carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR), respectively. In the alternative ornithine pathway, ornithine is transaminated to P5C by Orn-d-aminotransferase (OAT). In the study of Yang et al. (2009), rapid proline accumulation in maize induced by exogenous H2O2 treatment was shown to be a combined result of the sequential activation of the glutamate and ornithine pathways (i.e. ornithine pathway activation being a delayed response), and the inhibition of the proline degradation pathway. Osmotic stress induces P5CS1 expression through ABA responsive (ABRE) element, and H2O2 is part of ABA signaling, and ABA-regulated responses like proline accumulation (Verbruggen and Hermans 2008; Verslues et al. 2007; Szabados and Savoure0 2009). ABA-insensitive 1 (ABI1) controlled regulatory pathway is also important in transcriptional regulation. In addition, P5CS2 can be activated also by ROS signals P5CS activity is under metabolic control as well (Szabados and Savoure0 2009). Control of proline accumulation is a multifactorial process which involves cellular compartmentalization. Osmotic stress, cold induced ROS accumulation, and H2O2 treatment might act in concert stimulating different pathways for proline accumulation. Cold induced oxidative damage in both root and leaf tissues of tomato were demonstrated in terms of accumulating MDA levels (Fig. 2). H2O2 acclimation provided protection against oxidative stress in both root and leaf tissues. The extent of oxidative stress in a cell is determined by the amounts of superoxide, hydrogen peroxide and hydroxyl radicals. The balance of superoxide dismutase (SOD), APX, and CAT activities is important for suppression of toxic ROS levels. APX activity in roots and


leaves of cold stressed and unstressed H2O2 acclimated plants increased, and root APX of cold stressed plants was considerably higher (Fig. 4). In a study of Morita et al. (1999), exposure to 0.01 mM H2O2 for 4 h resulted in a significant increase in cytosolic APX mRNA of suspension cultures of germinating rice. Furthermore, treatment of soybean cells with exogenous H2O2 resulted in the alteration of cellular APX transcription levels (Lee et al. 1999). These reports support the idea that cellular APX gene expression is regulated in response to cellular H2O2 levels. In our study, APX appeared to be induced by H2O2 treatment as evidenced by both root and leaf activities of unstressed acclimated plants. On the other hand, induction of CAT activity appeared to be enhanced by H2O2 treatment. APX has a higher affinity for H2O2 (lM range) than CAT (mM range). Moreover, ascorbate–glutathione cycle is found in all compartments of the cells, whereas CAT is only present in peroxisomes (Mittler 2002). Conclusively, APX plays a more crucial role in controlling the ROS level although, CAT is indispensible when high levels of ROS are produced (Willekens et al. 1997) as in the case of cold induced oxidative damage. In fact, cold induced ROS is also supported by higher endogenous H2O2 levels upon cold stress, but not exogenous H2O2 application, in roots of control and non-acclimated plants (Fig. 4). Guan et al. (2000) presented that H2O2 plays an important intermediary role in the ABA signal transduction pathway leading to the induction of the maize Cat1 gene. In another study, time course experiments indicated that high concentrations of H2O2 induced Cat1 and Cat2 gene expression to higher levels, and in less time, than lower H2O2 concentrations (Polidoros et al. 1999). In addition, protein DNA interactions in the ARE motif and the U2 snRNA homologous regions of the Cat1 promoter were observed, suggesting that ARE may play a role in the high induction of Cat1 under oxidative stress. Osmotic stress and cold induced ROS accumulation together with H2O2 treatment might cause more pronounced effects at protein level. In the study of Kerdnaimongkol et al. (1997), acclimation of tomato seedlings at 14 °C for 48 h induced chilling tolerance, and catalase activity was shown to increase in acclimated plants. The increase in catalase activity was correlated to low temperature induced oxidative stress in these seedlings, and which could, in turn, provide protection from more severe oxidative stress during exposure to damaging low temperatures. Furthermore, in the same study, diurnal variation in the chilling sensitivity of tomato seedlings was examined by either dark or light acclimation. The results revealed that although catalase activity remained higher at all stages of chilling for seedlings transferred to low temperatures following the light period compared to the dark period, there was not any time-dependent variation in catalase activity of after light period seedlings. In our

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study, 16 h of stress treatment was applied at dark period after 8 h of light treatment, which might have manifested low temperature induced damage, and correlated to the faster recovery of leaf gas exchange following chilling after an 8-h light period. Park et al. (2004) demonstrated that exogenous dark application of H2O2 or glycine betaine (GB) improved chilling tolerance of tomato plants. They have reported that although endogenous levels of H2O2 in the H2O2 treated plants became similar to those in the water treated plants within 2 h after exogenous application, as the chilling stress was prolonged, both H2O2- and GB treated plants always maintained lower amounts of H2O2, and higher catalase activity than those treated with water. In a recent study, they also demonstrated that during day 1 of chilling stress, catalase activity increased by up to 57 and 18 % in the GB treated and control plants, respectively. The expression pattern for the catalase gene (CAT1) was also shown to well correlate with its enzyme activity (Park et al. 2006). GB-enhanced chilling tolerance, which was also demonstrated with long-term plant growth, was correlated to the induction of H2O2-mediated enhanced catalase expression and catalase activity. In concordance, changes in APX and CAT activities seemed to restore H2O2 content in roots of acclimated plants as the levels were significantly lower in comparison to control and nonacclimated plants under cold stress. However, the basal non-stress H2O2 levels in acclimated and non-acclimated plants were similar which may be correlated to unchanged catalase levels in these plants (Fig. 4). Although the issue was not addressed in the experimental setups, the overall success of a plant to cope with low temperature also depends on the capacity and speed of recovery after normal growth temperatures are restored. The issue has been discussed in the previous reports in terms of physiology, antioxidant enzymes, photosynthesis, and acclimation. For example, chilling tolerance of maize has been correlated with the rate of increase of root length in the cold period (Sowinski and Kro´likowski 1995). In maize, 1 or 2 weeks of subsequent growth under optimum conditions were sufficient for the complete efficient recovery of photosystem II and chlorophyll content (Hola´ et al. 2007). However, high intensity illumination was shown to enhance chilling damage of photosynthesis, and delayed the recovery of photosynthesis in tomato (Martin and Ort 1985). Prasad et al. (1994b) demonstrated that exogenous root H2O2 treatment of maize seedlings at 27 °C protect mitochondria from irreversible oxidative damage, and upon over 3 days of recovery, the mitochondria of treated seedlings showed altered mitochondrial respiration pathway, recovered cytochrome oxidase activity, regained ability to generate mitochondrial ATP, and the antioxidant enzyme activities remained at or above control levels. In addition, after the 7 days chilling treatment at 4 °C, the

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H2O2 treated maize seedlings were transferred to the greenhouse for 10 days, and the survival data indicated that 58 % of the H2O2 treated and 2 % of the non-acclimated seedlings (Prasad et al. 1994a). In another study of Nayyar et al. (2005), Ca2? or abscisic acid acclimated chickpea to chilling stress accumulated cryoprotective solutes, and during recovery, solute levels in plants remained considerably higher. Although the role of H2O2 as a signaling molecule in biotic and abiotic defense response is documented, exact molecular network has not been fully elucidated yet. Response to cold and stress repair may take place by activation of LEA-like class of stress responsive genes with DRE/CRT and ABRE cis elements, whose ectopic expression may induce cold acclimation (reviewed by Xiong et al. 2002; Chinnusamy et al. 2007). Calcium signal amplification through mechano-sensitive or ligand activated Ca2? channels, and phospholipid signaling might be involved via ABA-dependent and -independent pathways. For example, Guan et al. (2000) reported ABA induced H2O2 production and its involvement in CAT1 gene expression in maize. Direct sensing of H2O2 by signaling proteins via oxidation is another proposed model, and there is also evidence on the link of H2O2 with MAP kinase pathway in plants (Xiong and Zhu 2002). At cellular level, cold stress increases ROS production, which, in turn, results in altered antioxidant response. The extent of the physiological and biochemical changes may vary depending on the cold tolerance/sensitivity. Although previous reports pointed out the manifestation of cold induced injury by exogenous foliar application of H2O2 in different species and in tomato, in this report, we investigated whether practical, short root application of H2O2 may decrease cold induced damage and improve cold response of tomato. The results presented here demonstrated that 1 h exogenous application of H2O2 when transferring the seedlings from seedling substrate to soil, may significantly enhance oxidative stress response and tolerance by elevating the antioxidant status of tomato as evidenced by proline accumulation and altered APX and CAT activity. ¨ . D. ˙Is¸ eri participated in experiAuthor contribution O mental design, experiments, data evaluation, writing of the manuscript. D. A. Ko¨rpe participated in experimental design and experiments. F. I. Sahin participated in data evaluation, statistical analysis and writing of the manuscript. M. Haberal participated in data evaluation, writing and reviewing of the manuscript. Acknowledgments This study was approved by Baskent University Institutional Review Board (Project no: DA11/06), and supported by Baskent University Research Fund.

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