Plant Soil (2015) 391:19–32 DOI 10.1007/s11104-015-2395-7

REGULAR ARTICLE

Genotypic differences in antioxidant response to phosphorus deficiency in Brassica napus Shuisen Chen & Hua Zhao & Guangda Ding & Fangsen Xu

Received: 27 July 2014 / Accepted: 20 January 2015 / Published online: 14 February 2015 # Springer International Publishing Switzerland 2015

Abstract Background and aims The effect of phosphorus (P) deficiency on plant growth and development depends on the ability of plants to adapt to induced stress. Oxidative stress may play a vital role in P deficient Brassica napus and the antioxidant ability of this plant may be an important mechanism for adapting to P deficiency. The present study aims to better understand the correlation between high P use efficiency and antioxidative ability by comparing the activity of antioxidant enzymes and lipid peroxidation in the leaves and roots of two B. napus genotypes. Methods The response of the antioxidant defense system of two B. napus genotypes with different P use efficiencies to short-term P-free starvation and longterm low P stress were investigated by analyzing the activities of antioxidative enzymes, the superoxide free radical ion (O2·−) and H2O2 concentration, and the level of lipid peroxidation. Results The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and glutathione reductase (GR) in the leaves and roots were increased in both genotypes under the two P treatments. However, the activities of some of these enzymes exhibited tissue or genotype Responsible Editor: John Hammond. S. Chen : H. Zhao : G. Ding : F. Xu (*) National Key Laboratory of Crop Genetic Improvement and Microelement Research Center, Huazhong Agricultural University, Wuhan 430070, China e-mail: [email protected]

specificity. In the P use-inefficient genotype ‘B104-2’, more O2·− was accumulated and a higher level of lipid peroxidation was induced under P deficiency conditions compared with P use-efficient genotype ‘Eyou Changjia’. The GPX activity in the roots was considerably higher (20- to 30-fold) than in the leaves, whereas the CAT activity in the roots was lower (approximately 3-fold) than in the leaves in both genotypes. Conclusions Compared with P use-inefficient genotype ‘B104-2’, P use-efficient genotype ‘Eyou Changjia’ suffered less oxidative damage and lipid peroxidation, and inhibited O2·− generation more effectively under low P stress. Severe oxidative damage was accompanied with a higher antioxidative enzyme activities in ‘B1042’ under low P stress. Keywords P deficiency . Oxidative stress . Antioxidative enzymes . Lipid peroxidation . Brassica napus . Genotypic difference

Introduction Reactive oxygen species (ROS) are continuously produced during aerobic metabolic processes in plants even under optimum growth conditions. Generally, the ROS concentrations are strictly controlled by antioxidant defense mechanisms that include enzymatic and nonenzymatic ROS-scavenging systems. However, various biotic and abiotic stresses may perturb the balance between the production and quenching of ROS (Moller 2001; Juvany et al. 2013). ROS accumulation can alter normal

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cellular metabolism through peroxidation of membrane lipids, oxidation of proteins and damage of nucleic acids and other cellular components (Ahmad et al. 2010). The peroxidation of membrane lipids affects the stability of membrane under stressed conditions, with higher membrane stability associated with greater abiotic stress tolerance (Mostofa et al. 2014; Gill and Tuteja 2010). In addition to their role in plant stress response, ROS function as cellular signaling molecules to regulate hormone signaling and biotic stress responses (Mittler et al. 2004, 2011; Miller et al. 2008). A transient increase in ROS can activate a plethora of defense-related genes (Munné-Bosch et al. 2013) and trigger redox signaling, which allows plants to acclimate to stressful conditions (Pintó-Marijuan and Munné-Bosch 2014). In addition, ROS signaling is integrated with a variety of signaling networks in plants, including the protein kinase networks, calcium signaling, and cellular metabolic networks (Nakagami et al. 2006; Kobayashi et al. 2007; Mittler et al. 2011). A recent study suggested that ROS may serve as signaling molecules or cause oxidative damage to plant tissues depending on the delicate equilibrium between ROS generation and scavenging (Sharma et al. 2012). Overproduction of ROS in plants is considered to be a common feature of various environmental stresses. Fortunately, plants have developed a wide range of defense systems to mitigate the oxidative damage caused by ROS, including low molecular mass nonenzymatic antioxidants (e.g., ascorbate and reduced glutathione) and antioxidative enzymes, such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7), and glutathione reductase (GR, EC 1.8.1.7) (Apel and Hirt 2004). SOD was served as the first line of defense against oxidative stress that primarily catalyzes the dismutation of O2·− to H2O2 (Bowler et al. 1992; Alscher et al. 2002), and the H2O2 is catalyzed into H2O and O2 by CAT and other peroxidases, APX and GPX (Wang et al. 2009). CAT has low affinity but higher reaction rate in H2O2 removing (Willekens et al. 1997). APX, which uses ascorbate as an electron donor in the ascorbateglutathione cycle, is thought to play the most essential role in H2O2 detoxification in higher plants (Noctor and Foyer 1998). GPX is less specific to the electron donor substrate and can oxidize several substrates in the presence of H2O2. Both APX and GPX have greater affinity for H2O2 and removal of H2O2 at low concentrations

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(Dat et al. 2000). GR catalyzes the reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) by the consumption of NADPH in the last step of the ascorbate-glutathione cycle (Bray et al. 2000). GSH can function directly as a ROS scavenger and together with ascorbate, plays a key role in H2O2 scavenging via the ascorbate-GSH cycle (Sharma et al. 2012). Plants frequently suffer from nutrient deficiency in their natural habitats, especially phosphorus (P) deficiency. The total amount of P in soils may be high, but inorganic phosphate, the main form for plant uptake, is seldom accessible to the roots due to its immobility in the soil. Therefore, P deficiency is considered as a major abiotic stress that limits plant growth. In addition, P deficiency is expected to reduce the rate of electron transfer in the electron transport systems, leading to an accumulation of O2·− and other ROS (Grossman and Takahashi 2001; Juszczuk et al. 2001; Bargaz et al. 2013), and trigger redox changes that induce oxidative stress with a defined pattern in nutrient-specific alterations in metabolism (Kandlbinder et al. 2004). However, plants have evolved several adaptive strategies to cope with P limitations (Plaxton and Tran 2011; Lynch 2011). For example, membrane transporters are induced to improve P acquisition (Schroeder et al. 2013; Ren et al. 2014). Many organic acids and phosphatases are induced to remobilize internal phosphate or are released into the soil to facilitate uptake of inorganic/ organic P (Robinson et al. 2012; Wang et al. 2014). In addition, well-organized antioxidant defense mechanisms are activated to protect cells from oxidative damage. For example, the activities of several antioxidant enzymes were increased in young mulberry leaves in response to P deficiency (Tewari et al. 2007). The response of plant antioxidative systems to abiotic stresses has been well documented. However, research on the antioxidative systems of rapeseed (Brassica napus) in response to P stress is scarce. B. napus is one of the most important oilseed crops worldwide. Significant genotypic differences in P efficiency have been observed among rapeseed cultivars (Zhang et al. 2009; Duan et al. 2009). The present study aims to evaluate the effects of P deficiency on the activity of antioxidant enzymes and lipid peroxidation in the leaves and roots of two B. napus genotypes at the seedling stage and to better understand the correlation between high P use efficiency and antioxidative ability. We investigated the alteration of antioxidant enzyme activities and lipid peroxidation levels in the leaves and roots of

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B. napus P use-efficient (tolerant to P stress) and P useinefficient (sensitive to low P stress) genotypes in response to long-term low P stress and short-term P-free starvation.

Materials and methods Plant material, growth and treatment conditions The B. napus genotypes used in this study, i.e., the P use-efficient ‘Eyou Changjia’ and P use-inefficient ‘B104-2’, were previously screened from 194 oilseed rape cultivars. ‘Eyou Changjia’ exhibited high tolerance to low P stress and had a high P efficiency coefficient (i.e., the biomass or seed yield under low P conditions relative to a normal P supply), and ‘B104-2’ was sensitive to low P conditions (Duan et al. 2009). Seeds were surface sterilized using 10 % (w/v) sodium hypochlorite for 5 min and washed three times in deionized water. The surface-sterilized seeds were germinated on moistened gauze. The plants were grown in an illuminated culture room with a cycle of 16 h/24 °C day and 8 h/ 22 °C night, and a light intensity of 300–320 μmol proton m−2 s−1 and a relative humidity of 65–80%. In addition to the P, the complete basal nutrient solution contained 80.04 mM NH4NO3, 246.47 mM MgSO4, 74.55 mM KCl, 110.99 mM CaCl2, 0.05 mM EDTAFe, and a microelement solution (Hoagland and Arnon 1950). The initial nutrient solution was 1/4 full-strength for the first 5 days after transplanting, which was increased to 1/2 and then full-strength when it was refreshed every 5 days. For the short-term P-free starvation experiment, after transplanting, all seedlings were grown under 200 μM P (higher P, HP) containing 36 μM Na2HPO4 and 144 μM NaH2PO4 for 15 days, then half the seedlings were moved to a P-free nutrient solution (−P, 0 μM P) and the remaining seedlings were maintained under HP conditions and used as the control. The roots and leaves were harvested at 0, 1, 3, and 5 days after the P was removed. Each treatment was replicated three times. After the fresh weights were measured, the samples were immediately chilled in liquid nitrogen and stored at −80 °C until further use. For the long-term low P stress experiment, half of the seedlings were grown in a low P (LP, 5 μM P) nutrient solution containing 1.4 μM Na2HPO4 and 3.6 μM NaH 2 PO 4 for 18 days after transplanting, then

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transferred to HP for additional 2 to 5 days. Control seedlings were consistently grown in HP nutrient solution. Roots and leaves of both genotypes were harvested separately on the 18, 20 (18+R2), and 23 (18+R5)days after transplanting. Each treatment was replicated three times. Both the long-term and short-term experiments, the 1st and 2nd euphylla next to the cotyledon were collected for the leaf samples used for enzyme activities assay and determining the concentration of TBARS, H2O2, and O2·−. Quantitative analyses of tissue P concentration Roots and shoots were harvested separately at each time point as described above. After fresh weights were measured, samples were baked at 105 °C for 15 min and then dried at 70 °C to constant weight. After the dry weight of roots and shoots were measured, a defined weight of roots or shoots was digested in concentrated H2SO4-H2O2 using a digestion block system, and then P concentration was assayed according to the vanadomolybdate method (Westerman 1990), and P content was calculated. Enzyme activity assays Approximately 0.15 g of root or 0.20 g of leaf fresh weight (FW) were ground into a powder using mortar and pestle in liquid N2. The powder was transferred to an Eppendorf tube and 2 mL of ice-cold extraction buffer, which consisted of 100 mM potassium phosphate buffer (pH 7.8), 2 mM EDTA, and 2 % (w/v) PVP-40, was added. The solution was centrifuged at 16,000 g for 15 min (4 °C). The supernatant was used as the crude enzyme extract for the antioxidative enzyme activity assay directly. The SOD activity was assayed by measuring its ability to inhibit the photochemical reduction of nitrobluetetrazolium chloride (NBT) (Giannopolitis and Ries 1977). The reaction buffer (final volume of 2 mL) contained 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM methionine, 75 μM NBT, 2 μM riboflavin, and 50 μL crude enzyme extract. Riboflavin was added and immediately illuminated using three 20-W fluorescent tubes for 15 min, then the light was switched off and the tube was covered with black cloth. The absorbance of the sample at 560 nm was recorded. A reaction that used water instead of the enzyme extract and without illumination was used

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as the control for zeroing. The amount of enzyme that caused 50 % inhibition of the NBT photoreduction rate was defined as one unit of SOD activity (U), expressed as U mg−1 FW. The CAT activity was assayed following the reduction of H2O2 by monitoring the decrease in absorbance at 240 nm and quantifying its molar extinction coefficient (36 M−1 cm−1) (Beers and Sizer 1952; De Azevedo Neto et al. 2006). Briefly, the reaction buffer (final volume of 1.5 mL) contained 100 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 20 mM H2O2, and 50 μL crude enzyme extract. The reaction was initiated by the addition of the enzyme extract and the decrease in absorbance at 240 nm was recorded. The CAT activity was expressed as μmol H2O2 min−1 g−1 FW. The APX activity was measured according to Nakano and Asada (1981). The reaction buffer (final volume of 1.5 mL) contained 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.3 mM ascorbate acid, 0.6 mM H2O2, and 50 μL enzyme extract. H2O2 was added to initiate the reaction and the decreased absorbance at 290 nm was recorded every minute. The APX activity was quantified using the molar extinction coefficient of ascorbate (2.8 mM−1 cm−1), and the result was expressed as μmol ascorbate min−1 g−1 FW. The GPX activity was measured as the oxidation of guaiacol in the presence of H 2 O 2 , during which tetraguaiacol was formed (De Azevedo Neto et al. 2006). The reaction buffer (final volume of 1.5 mL) contained 100 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 5.0 mM guaiacol, 15.0 mM H2O2, and 50 μL crude enzyme extract. The reaction was initiated by the addition of the enzyme extract and the increased absorbance at 470 nm was recorded every 30 s. The GPX activity was quantified using the molar extinction coefficient of tetraguaiacol (26.6 mM−1 cm−1) and the result was expressed as μmol tetraguaiacol min−1 g−1 FW. The GR activity was measured by the oxidation of NADPH (De Azevedo Neto et al. 2006). The reaction buffer (final volume of 1.0 mL) contained 100 mM potassium phosphate buffer (pH 7.8), 1 mM EDTA, 0.1 mM NADPH, 2.0 mM GSSG, and 40 μL enzyme extract. The reaction was initiated by the addition of GSSG and the decreased absorbance at 340 nm was recorded every 30 s. The activity was calculated using the molar extinction coefficient of NADPH

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(6.2 mM−1 cm−1) and expressed as μmol NADPH min−1 g−1 FW. Lipid peroxidation assay The lipid peroxide levels were determined by measuring the amount of pink color thiobarbituric acid reactive substances (TBARS) (Lin and Kao 2000), according to Hernandez et al. (2010) with minor modifications. Briefly, 0.2 g (FW) of root or leaf sample was homogenized using 2 mL of 5 % trichloroacetic acid (TCA) and centrifuged at 12,000 g for 10 min. Then 0.5 mL of 20 % TCA containing 0.5 % (w/v) thiobarbituric acid was added to a 0.5 mL aliquot of the supernatant. The mixture was heated in a boiling water bath for 15 min and then immediately cooled on ice. The cooled sample was centrifuged at 12,000 g for 3 min, and the absorbance was measured at 532 nm. A correction for nonspecific turbidity was made using the absorbance at 600 and 450 nm. The lipid peroxidation level was expressed as nmol TBARS g−1 FW. Superoxide free radical ion (O2·−) and H2O2 measurements Nitrite is formed from hydroxylammonium chloride in the presence of the superoxide free radical ion (O2·−). The nitrite reacts with p-sulfoaniline and αnaphthylamine to form a pink diazo compound that can be determined at 530 nm (Elstner and Heupel 1976). Root or leaf samples 0.20 g (FW) were ground using mortar and pestle in liquid N2. The powder was transferred into an Eppendorf tube, and 1-mL ice-cold 50 mM potassium phosphate buffer (pH 7.8) was added. The solution was shaken and then centrifuged at 8000 g for 15 min (4 °C). The clear supernatant (250 μL) was mixed with 200 μL of 50 mM potassium phosphate buffer (pH 7.8) and 50 μL of 10 mM hydroxylammonium chloride. After incubation at 25 °C for 1 h, 500 μL of 17 mM p-sulfoaniline and 500 μL of 7 mM α-naphthylamine were added immediately and incubated at 25 °C for 20 min. The absorbance of the reaction solution was determined at 530 nm. The O2·− concentration was calculated according to the sodium nitrite curve. The H2O2 concentration was estimated according to Patterson et al. (1984) with minor modifications. Briefly, 0.15 g (FW) of root or leaf was homogenized in 700 μL of cold acetone using a mortar and pestle, the

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homogenate was transferred to an Eppendorf tube and centrifuged at 10,000 g for 10 min (4 °C). Then, 20 μL of 20 % titanium (TiCl2 in concentrated HCl) was mixed with 200 μL of the extract supernatant, and the complex was precipitated by adding 40 μL of concentrated ammonia solution. The precipitate was washed three times with acetone and dissolved in 1 mL of 1 M H2SO4. The absorbance of the solution was measured at 410 nm, and the H2O2 concentration was calculated according to the curve. A known concentration of H2O2 instead of the extract was used to form the standard curve. Statistical analysis A completely randomized factorial design was used, consisting of two genotypes × two treatments × time points with three replicates. Sample variability was expressed as the standard error of the mean. The significance of differences was conducted using SAS 9.2 (SAS Institute, Cary, NC, USA) and SAS PROC ANOVA LSD model p < 0.05 was considered significant.

Results Biomass, tissue P concentration, and lipid peroxidation Roots fresh weights of the two genotypes significantly increased 5 days after P withdrawal, compared with their controls, and P use-efficient genotype had higher root and shoot fresh weights than the P use-inefficient genotype (Fig. 1a). Under short-term P-free starvation, because no P can be absorbed from the solution, both root and shoot P concentration had no significant difference between two genotypes, though P use-efficient genotype ‘Eyou Changjia’ had higher shoot P concentration than ‘B104-2’ before P starvation (Fig. 1b). However, ‘Eyou Changjia’ had a higher shoot P content than ‘B104-2’ (Fig. 1c). Abundance of TBARS was significantly increased in roots of both genotypes under P-free starvation, but there was no significant difference between the two genotypes. However, abundance of TBARS also increased in leaf in ‘B104-2’ under+P condition (Fig. 1d). Long-term low P stress significantly inhibited shoot growth but promoted root growth. The P use-efficient genotype ‘Eyou Changjia’ had a higher roots mass compared with the P use-inefficient

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genotype ‘B104-2’ under low P condition, but these genotypic differences disappeared when P was resupplied for 5 days (Fig. 2a). Tissue P concentration was significantly lower in roots and shoots of both genotypes under low P stress for 18 days, but there were no significant differences between the two genotypes (Fig. 2b). However, ‘Eyou Changjia’ had a higher root total P accumulation as compared with ‘B104-2’ (Fig. 2c). The abundance of TBARS increased considerably both in the roots and leaves of the two genotypes under long-term low P stress. However, the P use-inefficient ‘B104-2’ produced a higher amount of TBARS than the P use-efficient ‘Eyou Changjia’, irrespective of the tissue sampled. The lipid peroxidation level in both genotypes was decreased when the P was resupplied (Fig. 2d).

Superoxide free radical ion (O2·−) and H2O2 O2·− was induced in both genotypes under short-term Pfree starvation. In ‘B104-2’, the O2·− concentration in the root and leaf increased over time to 173.6 nmol O2·− g−1 FW in the root and 140.1 nmol O2·− g−1 FW in the leaf 5 days after P withdrawal. In ‘Eyou Changjia’ however, the O2·− concentration increased rapidly 1 day after P withdrawal to 109.1 nmol O2·− g−1 FW in the root and 103.5 nmol O2·− g−1 FW in the leaf. The O2·− concentration in ‘B104-2’ was greater than ‘Eyou Changjia’, irrespective of the tissue sampled (Fig. 3a, b). The H2O2 concentration could not be detected in the roots and decreased in the leaves of both genotypes in response to short-term P-free starvation (Fig. 3c). Under long-term low P stress for 18 days, the O2·− concentration in the root increased approximately 49.7 % in ‘B104-2’ and 38.4 % in ‘Eyou Changjia’ (Fig. 3d), and approximately 22.3 % and 18.3 % in ‘B104-2’ and ‘Eyou Changjia’ leaves, respectively (Fig. 3e). The O2·− concentration of both genotypes declined when transferred to HP condition, irrespective of the tissue sampled. The P use-inefficient genotype ‘B104-2’ had a higher O 2 ·− concentration compared with the P useefficient genotype ‘Eyou Changjia’, irrespective of the tissue sampled (Fig. 3d, e). The H2O2 level in the root was below the detection limit, but H 2 O 2 in the leaf was significantly higher by 120.6 % in ‘Eyou Changjia’ and 82.8 % in ‘B104-2’ (Fig. 3f).

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Fig. 1 a Fresh weight, b P concentration, c P content, and d TBARS concentration of the seedlings of two Brassica napus genotypes, ‘Eyou Changjia’ (P use efficient) and ‘B104-2’ (P use inefficient) subjected to short-term P-free starvation. Seedlings were grown under high P (+P, 200 μM P) for 15 days after transplanting, and then half of the plants were transferred to the

P-free nutrient solution (−P, 0 μM P) for P starvation. Roots and leaves (or shoots) samples were collected at 0, 1, 3, and 5 days after P starvation, together with the plants under high P, which were used as the control. Values represent the mean±SD of three independent replicates, and bars with different letters show significant differences (ANOVA, LSD, p<0.05)

Enzyme activity under short-term P-free starvation

root SOD activity was higher in ‘Eyou Changjia’ (41.9 %) than in ‘B104-2’ (29.7 %) 1 day after P withdrawal (Fig. 4a). Root CAT activity in ‘B104-2’ increased significantly 1 day after P withdrawal (Fig. 4c). However, root CAT activity in ‘Eyou Changjia’, as well as leaf CAT activity in both genotypes, increased with time in the P-free starvation treatment (Fig. 4c, d). Leaves had higher CAT activity in both genotypes compared with roots,

SOD activity in the roots of both B. napus genotypes increased abruptly 1 day after P withdrawal, and then increased gradually (Fig. 4a). Leaf SOD activity in both genotypes increased with time in the P-free starvation (Fig. 3b). The root and leaf SOD activity in P use-efficient ‘Eyou Changjia’ was lower than in P use-inefficient ‘B104-2’, but the increase in

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Fig. 3 O2·− and H2O2 concentration in the roots and leaves of two Brassica napus genotypes in response to P stress. a Root and b leaf O2·– concentration and c leaf H2O2 concentration under short-term

P-free starvation. d Root and e leaf O2·− concentration and f leaf H2O2 concentration of plants subjected to long-term low P stress. Additional details as described in Figs. 1 and 2

which 3.87-fold and 3.05-fold in ‘B104-2’ and ‘Eyou Changjia’, respectively, 5 days after P withdrawal (Fig. 4c, d). APX activity in roots and leaves of both genotypes increased in the P-free starvation treatment, with roots having higher APX activity compared with leaves in both genotypes (Fig. 4e, f). Root GPX activity increased 41.9 % and 35.2 % in ‘B104-2’ 1 and 5 days after P withdrawal relative to controls, respectively, 32.3 % and 24.0 % in ‘Eyou Changjia’ 1 and 5 days after P withdrawal relative to controls, respectively (Fig. 4g). Leaf GPX activity was not affected by the short-term P-free starvation, but it was considerably higher (1.4to 1.8-fold) in ‘B104-2’ than in ‘Eyou Changjia’

(Fig. 4h). In the P-free starvation treatment, GPX activity in the roots of both genotypes was more than 20-fold higher compared with their leaves (Fig. 4g, h). The root GR activity increased immediately following the shift to P-free starvation, the activity levels increased 34.3 % in ‘Eyou Changjia’ and 19.8 % in ‘B104-2’ 1 day after P withdrawal (Fig. 4i). The leaf GR activity in both genotypes increased with time under P-free starvation. The activity levels increased 19.8 % 1 day after P withdrawal and 48.8 % 5 days after P withdrawal in ‘Eyou Changjia’, and increased 6.0 % 1 day after P withdrawal and 33.7 % 5 days after P withdrawal in ‘B104-2’ (Fig. 4j).

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Fig. 4 Enzyme activities of a, b SOD; c, d CAT; e, f APX; g, h GPX; and i, j GR in the roots and leaves of two Brassica napus genotypes subjected to short-term P-free starvation. Additional details as described in Figs. 1 and 2

28

Enzyme activity under long-term low P stress Under long-term low P stress conditions, root SOD activity was greater in ‘B104-2’ (54.4 %) than in ‘Eyou Changjia’ (42.0 %) as compared with their control (Fig. 5a). By contrast, leaf SOD activity was greater in ‘Eyou Changjia’ (93.9 %) than in ‘B104-2’ (68.9 %) as compared with their control (Fig. 5b). Root and leaf SOD activity decreased after transferring to HP condition (Fig. 5a, b). Root CAT activity was greater in ‘B104-2’, and lower in ‘Eyou Changjia’ (Fig. 5c), but leaf CAT activity was greater in ‘Eyou Changjia’ (584.7 μmol H2O2 min-1 g-1 FW) and ‘B104-2’ (504.7 μmol H2O2 min-1 g-1 FW) under long-term low P stress conditions relative to controls (Fig. 5d). APX and GPX activities in both genotypes were induced by the low P stress, which increased by 142.3 % and 132.8 %, respectively, in ‘B104-2’ roots relative to controls and by 29.6 % and 58.3 %, respectively, in ‘Eyou Changjia’ roots relative to controls (Fig. 5e, g). Leaf APX and GPX activities increased 24.2 % and 69.5 % relative to controls, respectively, in ‘B104-2’ and by 7.6 % and 85.1 % relative to controls, respectively, in ‘Eyou Changjia’ (Fig. 5f, h). CAT, APX, and GPX activities that were induced under long-term low P stress decreased to normal levels 5 days after the HP was resupplied. CAT activity was considerably higher (about 3-fold) in the leaf than in the root, whereas APX and GPX activities were higher in the root of the two genotypes, especially GPX activity (Fig. 5c–h). Under long-term low P stress, root GR activity increased 18.3 % in ‘B104-2’ and 11.5 % in ‘Eyou Changjia’ relative to controls (Fig. 5i), and leaf GR activity of the two genotypes increased approximately 50 % relative to controls (Fig. 5j). The P stress-induced GR activity decreased both in root and leaf tissues when transferred to the HP condition.

Discussion Many environmental stresses induce cellular damage because of the excess ROS. The ROS are produced through a disturbance in electron transport in photosynthesis and respiration, which disrupts the equilibrium between the production and scavenging of ROS (Apel and Hirt 2004). Therefore, stress-tolerant genotypes may, at least in part, depend on the enhancement of the antioxidative defense system, which includes

Plant Soil (2015) 391:19–32

antioxidant compounds and multi-antioxidative enzymes. In the present study, we investigated changes in the O2·− and H2O2 concentrations, the level of lipid peroxidation, as well as the activities of SOD, CAT, APX, GPX, and GR in two B. napus genotypes suffering from long-term low P stress and short-term P-free starvation. The increased production of ROS and antioxidative enzyme activity under the P-deficient conditions suggests that oxidative stress may be an important component in the response of B. napus to P deficiency. Among the numerous subcellular compartments for ROS production, chloroplasts, mitochondria, and peroxisomes are considered to be the main ROS producers in plants in response to abiotic stress (Mittler et al. 2004; Asada 2006; Bose et al. 2013). O2·− is formed in the first step of oxygen reduction and can rapidly be converted to H2O2 by the action of SOD. Surplus O2·− can be converted to the much more reactive ROS, including the hydroxyl radical (OH·) and perhydroxyl radical (HO2·), by energy transfer or electron transfer reactions, which can initiate lipid peroxidation. In the present study, P deficiency induced the production of O2·− and the activity of SOD in both genotypes, under short-term P-free starvation and long-term low P starvation (Fig. 3a, b, d, and e). Though no significant difference in P concentration between the two genotypes under P stress was identified, the P use-inefficient genotype ‘B104-2’ had a higher O2·− concentration but less P content as compared with the P use-efficient genotype ‘Eyou Changjia’. The higher P content and greater biomass in ‘Eyou Changjia’ indicated that ‘Eyou Changjia’ had more effective P uptake systems and suffered less oxidative damage under low P stress condition. Our previous study also showed that P use-efficient genotypes B. napus could mobilize more internal P to produce more biomass under low P stress (Zhang et al. 2009). Although SOD can dismutate O2·− into H2O2 to alleviate the damage of O2·−, H2O2 is still toxic and needs to be eliminated by conversion to H2O. In plants, a series of enzymes are involved in regulating the intracellular H2O2 level. CAT, APX, and GPX are considered as the most common enzymes and play vital roles in this process. In the present study, we observed that the leaf CAT activity in P use-efficient ‘Eyou Changjia’ was higher than that in P use-inefficient ‘B104-2’, regardless of the treatment (Figs. 4d and 5d). However, the opposite was observed in the root (Figs. 4c and 5c). Previous studies on a range of halophytes also showed an increase, decrease, or no change in the CAT activity in

Plant Soil (2015) 391:19–32

29

a

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Time (days)

Eyou Changjia HP

B104-2 LP

Eyou Changjia LP

Fig. 5 Enzyme activities of a, b SOD; c, d CAT; e, f APX; g, h GPX; and i, j GR in the roots and leaves of two Brassica napus genotypes subjected to long-term low P stress. Additional details as described in Figs. 1 and 2

30

response to salt (Jithesh et al. 2006). Our data showed that the CAT activity in the leaf was significantly higher than in the root, irrespective of long-term low P stress or short-term P-free starvation (Figs. 4c–d and 5c–d). However, the APX and GPX activities showed opposite trends, and the root GPX activity was 20-fold higher than the leaf in both genotypes (Figs. 4g–h and 5g–g). Leaf CAT exhibited higher H2O2-scavenging activity in the two B. napus genotypes, in response to low P stress and P-free starvation. A similar pattern of these enzymes has been observed in salt stressed maize (De Azevedo Neto et al. 2006). Under long-term low P stress, leaf H2O2 concentration of ‘Eyou Changjia’ (10.8 μmol g−1 FW) was considerably higher than ‘B104-2’ (9.1 μmol g−1 FW) (Fig. 3f). Higher CAT activity was also observed in ‘Eyou Changjia’ (584.7 μmol H2O2 min−1 g−1 FW) than in ‘B104-2’ (504.7 μmol H2O2 min−1 g−1 FW) (Fig. 5d). Root H2O2 concentrations were below the detection limit, possibly due to the high GPX activity. Our results suggest that CAT was the major enzyme for H2O2 scavenging in leaves, whereas in B. napus roots, CAT coordinated with GPX to play a central role in the H2O2scavenging process. Our findings agree with previous studies where the higher production of H2O2 in Mg- and K-deficient leaves elevated the levels of H2O2-scavenging enzymes, but the ascorbate-dependent H2O2-scavenging enzymes were not affected by P deficiency (Cakmak 1994). In addition, CAT and GPX are the most important enzymes for H2O2 scavenging in salt tolerance in maize (De Azevedo Neto et al. 2006) and barley (Liang et al. 2003). GR is involved in ascorbate-glutathione cycle and play an important role in antioxidative system. A previous study on transgenic tobacco showed that overexpression of GR increased the GSH concentration and also increased tolerance to oxidative stress (Broadbent et al. 1995). GSH is considered as an important nonenzymatic antioxidant involved in nonenzymatic ROSscavenging mechanisms. In a study conducted by Creissen et al. (1999), mutants with decreased GSH contents were hypersensitive to stress. In the present study, we observed increased GR activity in the roots and leaves of both genotypes in response to P deficiency. These results suggest that the nonenzymatic ROSscavenging mechanisms may also play an important role in protecting B. napus from oxidative damage. Generally, lipid peroxidation is considered as a biochemical marker of free radical-mediated oxidative

Plant Soil (2015) 391:19–32

injury. The complex process of lipid peroxidation consists of three stages including initiation, propagation, and termination (Halliwell and Gutteridge 1999). The most notable initiators in the initiation phase of lipid peroxidation are ROS such as OH· and HO2·, but not H2O2 and O2·−. In the present study, P deficiency increased the level of lipid peroxides in the roots and leaves of both B. napus genotypes. Previous studies have demonstrated alleviation of lipid peroxidation through an increase in the reduced forms of ascorbate and GSH, as well as the antioxidant enzyme activities under salt-stress (Shalata et al. 2001). In present study, we also observed the increased GR activity which could produce more GSH. The higher level of lipid peroxidation in P use-inefficient ‘B104-2’ than that in ‘Eyou Changjia’, especially under long-term low P stress (Fig. 2d), may due to the higher production of ROS in ‘B104-2’.

Conclusions In the present study, the SOD, CAT, APX, GPX, and GR activities were assayed, and the lipid peroxidation level, O2·− and H2O2 concentration were measured in two B. napus genotypes with different tolerances to P stress under long-term low P stress and short-term P-free starvation. P use-efficient genotype ‘Eyou Changjia’ has higher P content as compared with P use-inefficient genotype ‘B104-2’. We compared the ROS-scavenging ability of the two B. napus genotypes in response to P deficiency. In the P use-inefficient genotype ‘B104-2’, the elevated level of SOD activity was accompanied by a higher O 2 ·− production under the two P treatments. Furthermore, the higher O2·− induced higher levels of lipid peroxidation in ‘B104-2’ than in the P useefficient genotype ‘Eyou Changjia’, especially under long-term low P stress conditions. CAT was the major enzyme responsible for H2O2 detoxification in the leaf, and GPX coordinated with CAT to scavenge H2O2 in the root. The higher GPX activity in the root indicated that GPX may play a key role in B. napus roots. The ascorbate-glutathione cycle and the nonenzymatic ROS-scavenging mechanism was activated by the P deficiency because of the enhanced APX and GR activities. Based on these results, we hypothesized that P use-efficient genotype ‘Eyou Changjia’ suffered less oxidative damage and

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lipid peroxidation, and inhibited O2·− generation more effectively under low P stress. Acknowledgments This work was supported by the grants from the National Natural Science Foundation (31172019), National Basic Research and Development Program (2011CB100301), China, and teh Fundamental Research Funds for the Central University (2013JC012).

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