Effects of osmotic stress on antioxidant enzymes activities in leaf discs of P_SAG12-IPT modified gerbera

Effects of osmotic stress on antioxidant enzymes activities in leaf discs of P SAG12 - IPT modified gerbera 1 3 first-author 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhjzhu@zju.edu.cn †E-mail:zhjzhu@zju.edu.cn zhjzhu@zju.edu.cn 1 2 Key Laboratory of Horticultural Plant Development and Biotechnology, Department of Horticulture, Zhejiang University, Hangzhou 310029, China Key Laboratory of Horticultural Plant Development and Biotechnology, Department of Horticulture, Zhejiang University, Hangzhou 310029, China Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China Department of Horticulture, Zhejiang Forestry University, Lin’an 311300, China Department of Horticulture, Zhejiang Forestry University, Lin’an 311300, China Leaf senescence is often caused by water deficit and the chimeric gene P SAG12 - IPT is an auto-regulated gene delaying leaf senescence. Using in vitro leaf discs culture system, the changes of contents of chlorophylls, carotenoids, soluble protein and thiobarbituric acid reactive substance (TBARS) and antioxidant enzymes activities were investigated during leaf senescence of P SAGl2 - IPT modified gerbera induced by osmotic stress compared with the control plant (wild type). Leaf discs were incubated in 20%, 40% (w/v) polyethylene glycol (PEG) 6 000 nutrient solution for 20 h under continuous light [130 µmol/(m 2 ·s)]. The results showed that the contents of chlorophylls, carotenoids and soluble protein were decreased by osmotic stress with the decrease being more pronounced at 40% PEG, but that, at the same PEG concentration the decrease in the transgenic plants was significantly lower than that in the control plant. The activities of superoxide dismutase (SOD), catalases (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and dehydroascorbate reductase (DHAR) were stimulated by PEG treatment. However, the increases were higher in P SAG12 - IPT transgenic plants than in the control plants, particularly at 40% PEG treatment. Lipid peroxidation (TBARS content) was increased by PEG treatment with the increase being much lower in transgenic plant than in the control plant. It could be concluded that the increases in the activities of antioxidant enzymes including SOD, CAT, APX, GPX and DHAR were responsible for the delay of leaf senescence induced by osmotic stress. Antioxidant enzymes Gerbera Leaf disc Leaf senescence Osmotic stress P SAG12 - IPT INTRODUCTION 1 Leaf senescence always adversely affects higher plant production in agriculture (Dong et al., 2006 2006 ). Senescence devalues ornamental plants and foliar vegetables during transportation, storage and on shelves (Li et al., 2006 2006 ; Toit et al., 2004 2004 ). Therefore, manipulation of leaf senescence may result in agricultural benefits. Senescence is regulated by several factors including plant hormones such as abscisic acid (ABA), ethylene, and cytokinin. ABA and ethylene have been shown to promote senescence whereas cytokinin application inhibits leaf senescence (Gan and Amasino, 1997 1997 ). The IPT gene encodes a bacterial isopentenyl transferase which catalyses the rate limiting step in cytokinin biosynthesis. The IPT gene under the control of a chalcone synthase promoter has been reported to increase chlorophyll levels and stem thickness, and delay flowering onset and flower development in tobacco plants (Wang et al., 1997 1997 ). In order to control leaf senescence, an autoregulatory senescence inhibition system, in which a highly senescence specific promoter SAG12 from Arabidopsis is fused to IPT , has been developed (Gan and Amasino, 1995 1995 ). This fusion directs the expression of IPT in leaves at the onset of senescence and subsequently increases the cytokinin level that prevents the leaves from senescing, and high cytokinin level resulting in down regulation of the senescence specific promoter that prevents cytokinin from accumulating too high, otherwise that will interfere with normal plant development. In modified tobacco, senescence was delayed by IPT expression under control of the senescence-specific SAG12 promoter in leaves under elevated atmospheric CO 2 (Ludewig and Sonnewald, 2000 2000 ). The IPT gene under control of the senescence-specific SAG12 promoter from Arabidopsis also significantly delayed developmental and postharvest leaf senescence in mature heads of transgenic lettuce (McCabe et al., 2001 2001 ). Among ornamentals, introduction of the IPT gene into petunia, linked to senescence-associated SAG promoters from Arabidopsis , has been reported to inhibit senescence of the lower leaves, although transgenic lines show stronger symptoms of nutritional deficiency (Jandrew and Clark, 2001 2001 ). Environmental factors, such as drought, induce plant leaf senescence (Guo and Gan, 2005 2005 ). Reactive oxygen species (ROS) have been linked to stress-induced or normal cell death (Pastori and Rio, 1997 1997 ). ROS are a group of very reactive, short-lived chemicals produced during normal metabolism or after an oxidative reaction. ROS include superoxide anion, hydrogen peroxide, hydroxyl radical (Sun, 1990 1990 ). ROS, at low concentrations, have been implicated in the regulation of several physiological processes such as proliferation (Shibanuma et al., 1990 1990 ), differentiation (Allen and Balin, 1989 1989 ), senescence (de Haan et al., 1996 1996 ), and apoptosis (Mignotte and Vayssiere, 1998 1998 ). At high concentrations, ROS are highly cytotoxic by inducing DNA damage, lipid peroxidation, and protein degradation (Sun, 1990 1990 ). Antioxidant defense systems have been developed in aerobic cells to counteract the damaging effects of ROS. Plants possess antioxidant defense systems comprised of enzymatic and non-enzymatic components, which normally maintain ROS balance within the cell. For instance, they use a diverse array of enzymes like superoxide dismutase (SOD), catalases (CAT) and peroxidases as well as low molecular mass antioxidants (glutathione and ascorbate) to scavenge different types of ROS (Foyer et al., 1994 1994 ). SOD catalyses the dismutation of superoxide to hydrogen peroxide and oxygen. However, hydrogen peroxide is also toxic to cells and has to be further detoxified by CAT and/or peroxidases to water and oxygen. The antioxidant enzymes’ activities play an important role in scavenging ROS and therefore their improvement could increase the ability of stress tolerance and delay the senescence (Alscher et al., 2002 2002 ). P SAG12 - IPT is a chimeric gene associated with cytokinin synthesis and therefore inhibits leaf senescence. However, whether the changes of antioxidant enzyme activities are involved in the regulation of leaf senescence in P SAGl2 - IPT modified gerbera is unknown. Using in vitro leaf discs culture system, the changes of antioxidant enzymes activities were investigated during leaf senescence of P SAGl2 - IPT modified gerbera induced by osmotic stress. The objectives are to know whether antioxidant enzymes activities are involved in the regulation of leaf senescence in P SAGl2 - IPT modified gerbera and clarify the physiological mechanisms of senescence delay induced by a chimeric gene P SAGl2 - IPT . MATERIALS AND METHODS 1 Plant materials and growth conditions 2 Gerbera plants, Gerbera jamesonii ‘Yuyan’, which are P SAG12 - IPT transgenic plants obtained through particle bombardment transformation, and control plants, were cultured in mixed media with peat, perlite and vermiculite in the proportion of 3, 1 and 1, respectively. The plastic pot was 4 L in volume, 18 cm in diameter and 18 cm in height. The concentration of nutrient elements were as follows: 5.5 mmol/L K + , 3.0 mmol/L Ca 2+ , 1.0 mmol/L Mg 2+ , 11.25 mmol/L NO 3 − , 1.5 mmol/L NH 4 + , 1.25 mmol/L H 2 PO 4 − , 1.25 mmol/L SO 4 2− , 35 μmol/L Fe, 5.0 μmol/L Mn, 4.0 μmol/L Zn, 0.75 μmol/L Cu, 30 μmol/L B and 0.5 μmol/L Mo. Fe was added as Fe-EDTA. The plants were grown in a greenhouse. The air temperature was set at 20 °C (night) and 30 °C (day), relative humidity ranged from 60% to 80% and light intensity was 500 µmol/(m 2 ·s) at noon. The photoperiod was day 14 h and night 10 h. The plant materials were cultivated under the growth conditions until flowering. Leaf discs (11 mm diameter) were prepared from fully expanded leaves using a cork borer. Leaf surface was cleaned with water and immediately transferred into a medium (5 mmol/L HEPES, 10 mmol/L KCl, 1.5 mmol/L CaCl 2 , pH 6.0) (Huguet-Robert et al., 2003 2003 ). Osmotic treatments 2 Osmotic treatments were applied by adding polyethylene glycol (PEG) 6 000 at specified concentrations, 0, 20%, 40% (w/v) to the medium (Huguet-Robert et al., 2003 2003 ) and pH was readjusted to 6.0. Leaf discs were placed in Petri dishes with covers, with each containing 15 discs in 20 ml of stress medium, placed under continuous light [130 µmol/(m 2 ·s) at the leaf discs level], shaken continuously at 20 °C and incubated for 20 h. All treatments were replicated three times concurrently with control assays performed with leaf discs incubated in flasks containing 20 ml of reference medium. Enzyme extraction 2 For enzyme assays, leaf discs were ground with 4 ml ice-cold 50 mmol/L HEPES buffer (pH 7.8) containing 0.2 mmol/L EDTA, 2% PVPP and 2 mmol/L ascorbate. The homogenates were centrifuged at 4 °C for 20 min at 12 000×g and the resulting supernatants were used for determination of enzymatic activities (Zhu et al., 2000 2000 ). All spectrophotometric analyses were conducted on a SHIMADZU UV-2410PC spectrophotometer. Determination of enzymatic activities 2 SOD activity was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (Stewart and Bewley, 1980 1980 ). CAT activity was measured using the reduction in absorbance at 240 nm due to the decrease of extinction of H 2 O 2 (Patra et al., 1978 1978 ). Guaiacol peroxidase (GPX) activity was measured by the increase in absorbance at 470 nm due to guaiacol oxidation (Nickel and Cunningham, 1969 1969 ). Ascorbate peroxidase (APX) activity was measured by the decrease in absorbance at 290 nm as ascorbate was oxidized (Nakano and Asada, 1981 1981 ). The assay of dehydroascorbate reductase (DHAR) activity was carried out by measuring the increase in absorbance at 265 nm due to reduced ascorbate formation (Nakano and Asada, 1981 1981 ). Determination of lipid peroxidation 2 Lipid peroxidation was estimated by measuring the concentration of thiobarbituric acid reactive substances (TBARS) (Shalata and Tal, 1998 1998 ), the reaction mixture in a total volume of 4 ml containing 1 ml of extracts, 3 ml of 2% (w/v) TBA (2-thiobarbituric acid) made in 20% trichloroacetic acid (TCA). The mixture was heated at 95 °C for 30 min and then quickly cooled on ice. After centrifugation at 10 000×g for 10 min, the absorbance of the supernatant was measured at 532 nm. A correction of non-specific turbidity was made by subtracting the absorbance value taken at 600 nm. The lipid peroxides were expressed as (nmol TBARS)/(g DW) by using an extinction coefficient of 155 (mmol/L) −1 ·cm −1 (Shalata and Tal, 1998 1998 ). Determination of soluble protein content 2 Soluble protein concentration was measured using bovine serum albumin as standard at 595 nm according to the method of Bradford ( 1976 1976 ). Determination of chlorophylls and carotenoids content 2 Contents of chlorophyll (a and b) and carotenoids were determined in alkaline acetone extracts (Lichtenthaler, 1987 1987 ). RESULTS 1 Chlorophylls and carotenoids 2 Fig. 1 1 indicates that the contents of chlorophyll a, chlorophyll b and total chlorophylls in leaf discs were significantly decreased by osmotic stress (incubation in PEG solution) with the decrease being more pronounced in 40% PEG than in 20% PEG. However, at the same PEG concentration the decrease in transgenic plants was significantly lower than that in the control plants, especially at 40% PEG. The contents of carotenoids in leaf discs were reduced by PEG treatment, especially in the control plants at 40% PEG treatment. Soluble protein content 2 The soluble protein content in leaf discs was decreased by PEG treatment with the decrease at 40% PEG being higher than that at 20% PEG. However, the decrease induced by PEG in modified plants was significantly lower than that in the control plants, especially at 40% PEG (Fig. 2 2 ). Antioxidant enzyme activities 2 After osmotic stress, the changes of activities of SOD, CAT, APX, GPX and DHAR had similar tendency. The activities of SOD, CAT, APX, GPX and DHAR were stimulated by PEG treatment. However, the increases were higher in P SAG12 - IPT transgenic plants than in the control plants, particularly at 40% PEG treatment (Fig. 3 3 ). Lipid peroxidation 2 As an indicator of lipid peroxidation, TBARS concentration was measured. The TBARS concentration in the leaf discs was increased by osmotic stress with the increase being higher at 40% PEG than at 20% PEG. However, this increase was significantly higher in the control plants than in transgenic plants (Fig. 4 4 ). DISCUSSION 1 Genotypic differences in drought tolerance could be attributed to the ability of plants to growth (Turtola et al., 2006 2006 ). Leaf senescence can be affected by a complex array of factors and manipulating any one of these factors will result in either promotion or prevention of leaf senescence, for example, water deficit promotes leaf senescence (Thomas et al., 1995 1995 ). In many studies, PEG 6 000 is often used as an inducer of water deficit (osmotic stress) and promotes plant senescence (Behera et al., 2003 2003 ). In the present experiment, adding PEG in the incubation medium of leaf discs induced leaf senescence, indicated by the considerable decrease of chlorophylls and soluble protein content (Figs. 1 1 and 2 2 ). However, the decreases of chlorophylls and soluble protein content in P SAG12 - IPT modified gerbera plants were significantly reduced compared to the control plants, indicating that leaf senescence was significantly delayed by the particle bombardment transformation of the chimeric gene P SAG12 - IPT . Even under optimal conditions, many metabolic processes produce ROS. The production of toxic oxygen derivatives is increased as a result of all types of abiotic or biotic stresses. Plants possess efficient systems for scavenging active oxygen species that protect them from destructive oxidative reactions (Foyer et al., 1994 1994 ). As part of this system, antioxidant enzymes are key elements in the defense mechanisms. Many changes have been observed in the activities of antioxidant enzymes in plants under osmotic stress. Superoxide dismutase and ascorbate peroxidase activities were found to be enhanced in osmotically stressed leaf discs of rape plants compared with the control (Aziz and Larher, 1998 1998 ). An increase in ascorbate peroxidase, superoxide dismutase and glutathione reductase activities was also observed in the drought tolerant wheat cultivar when the leaves were subjected to osmotic stress in vitro (Lascano et al., 2001 2001 ). The results of the present experiment showed that the activities of SOD, CAT, APX, GPX and DHAR in leaf discs were stimulated by PEG treatment (osmotic stress) with the increases in leaf discs of P SAG12 - IPT transgenic plants being higher than those of control plants, particularly at 40% PEG treatment. Higher activities of SOD, CAT, APX, GPX and DHAR in leaf discs of P SAG12 - IPT transgenic plants with PEG treatment coincided with decrease in the TBARS concentration, suggesting that oxidative damage induced by osmotic stress could be alleviated by transformation of the chimeric gene P SAG12 - IPT . Such protection system against oxidative stress may play a crucial role in preventing leaves from senescence induced by osmotic stress. This result was consistent with the observation of Dertinger et al.( 2003 2003 ). 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