Plant Growth Regulation

, Volume 53, Issue 2, pp 107–115

Oxidative stress and antioxidant activity as the basis of senescence in chrysanthemum florets

Authors

    • Floriculture SectionNational Botanical Research Institute
  • J. Chatterjee
    • Floriculture SectionNational Botanical Research Institute
  • S. K. Datta
    • Floriculture SectionNational Botanical Research Institute
Original Paper

DOI: 10.1007/s10725-007-9208-9

Cite this article as:
Chakrabarty, D., Chatterjee, J. & Datta, S.K. Plant Growth Regul (2007) 53: 107. doi:10.1007/s10725-007-9208-9

Abstract

Stems of chrysanthemum (Chrysanthemum morifolium Ramat.) cv. Maghi were harvested when half of the buds showed colour and were put in distilled water at 21°C. Flowers showed visible senescence symptoms after 12–15 d. Reactive oxygen species (ROS) concentration and lipid peroxidation increased from young floret stage to the senescent stage. Activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD) and catalase (CAT) showed uniform increases from young floret through to the mature stage and thereafter, declined. Among the SOD isoforms, Fe-SOD and Cu/Zn-SOD were induced during the onset of senescence. Similarly different isoforms of APX and glutathione reductase (GR) also appeared during the senescence process. The capacity of the antioxidative defence system increased during the onset of senescence but the imbalance between ROS production and antioxidant defences ultimately led to oxidative damage. It is proposed that a decrease in the activity of a number of antioxidant enzymes that normally prevent the build up of free radicals can at least partially account for the observed senescence of chrysanthemum florets.

Keywords

Antioxidant enzymesChrysanthemumFloretsSenescenceReactive oxygen species

Abbreviations

APX

Ascorbate peroxidase

CAT

Catalase

GR

Glutathione reductase

LOX

Lipoxygenase

MDA

Malondialdehyde

G-POD

Guaiacol peroxidase

ROS

Reactive oxygen species

RWC

Relative water content

SOD

Superoxide dismutase

Introduction

Reactive oxygen species such as superoxide radical, hydrogen peroxide and hydroxyl radical have a role in lipid peroxidation, membrane damage and consequently in leaf senescence. Free radicals have been involved in programmed cell death, both in animal and in plant cells (Dhindsa et al. 1981). Plants possess a well defined antioxidant defence mechanisms which eliminate hazardous free radicals (Larson 1988). Antioxidant protection involves compounds such as carotenoids, ascorbic acid, α-tocopherol, glutathione, phenolics and flavonoids (Schöner and Krause 1990) and a battery of enzymatic systems including catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), glutathione peroxidase (GPX), (glutathione-S-transferase) GST and the Halliwell-Asada Pathway (or the ascorbate-glutathione cycle). The ascorbate-glutathione cycle involves four enzymes: ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) enzymes (Bowler et al. 1992; Halliwell 1987). It has been shown that during leaf senescence, proteins, phospholipids and pigments may be degraded by free radicals as free radical scavenging declines (Prochazkova et al. 2001).

Florets/petals are the organs which primarily determine the commercial longevity of flowers and as a consequence it is beneficial to study the physiological, biochemical, and genetic processes that occur during floret senescence. Most of the early work on flower senescence focused on ethylene sensitive plants. Chrysanthemum is an ethylene insensitive plant where lipid peroxidation and membrane damage are involved in flower deterioration (Chrysanthemum morifolium) (Bartoli et al. 1995). However, little information is available on the actual role of oxidative stress and the protective enzymatic systems, and corresponding isoenzymes, in relation to floret senescence in chrysanthemum.

The objective of the present study was to examine variation, in oxidative stress and the antioxidant enzyme activity in the florets of chrysanthemum during senescence.

Materials and methods

Plant material

Chrysanthemum cv. Maghi (spray type) were used as experimental material. Stems (approx. 8 buds per stem) were harvested when half of the buds showed colour and reached 1.5 cm in diameter. The leaves from the lower 1/3rd portion of the stem were removed. The basal 2–3 cm portion of the stem was recut under water and placed in distilled water. Antibiotic ampicillin was added at a concentration of 100 mg l−1 to prevent infection. The vase-life parameters were evaluated at 21°C and 16 h illumination (36 μmol m−2 s−1) under laboratory condition. Bud development (mean of all the buds on the stem, based on a numerical scale) was arbitrarily divided into five different developmental stages—stage 1, 0 day after harvest; stage 2, 5 days after harvest; stage 3, 10 days after harvest; stage 4, 15 days after harvest and stage 5, 20 days after harvest (Fig. 1). All the parameters were studied using the florets from outer three whorls.
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Fig. 1

The stages of flower head development in Chrysanthemum morifolium Ramat. cv. Maghi were arbitrarily divided into five different developmental stages—stage 1, 0 day after harvest; stage 2, 5 days after harvest; stage 3, 10 days after harvest; stage 4, 15 days after harvest and stage 5, 20 days after harvest

Leakage of ions

Leakage of ions from the florets was measured according to Sairam et al. (1997) and expressed as MSI percentage. Florets (0.2 g) were placed in 20 ml of deionized water in two different 100 ml Erlenmeyer flasks. One flask was kept in water bath at constant temperature of 40°C for 30 min and its conductivity (C1) was measured with a conductivity meter (DiST 3 Conductivity meter, Hanna make, Portugal). The second flask was kept in a boiling water bath (100°C) for 10 min and conductivity was recorded (C2). MSI was expressed in percentage using the formula: [1–(C1/C2)] × 100.

Relative water content (RWC)

Floret RWC was determined and calculated from the following relationship: (WfreshWdry)/(WturgidWdry) × 100, where Wfresh is the sample fresh weight, Wturgid is the sample turgid weight after saturating with distilled water for 24 h at 4°C, and Wdry is the oven-dry (70°C for 48 h) weight of the sample (Weatherley 1950).

ROS measurement

ROS production was measured by the reduction of sodium, 3,-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) in the presence of O2·−, with some modifications (Able et al. 1988). Leaves (1 g) were homogenized in 1 ml of 50 mM Tris-HCl buffer (pH 7.5) and centrifuged at 14,000g for 20 min. The reaction mixture contained 50 mM Tris–HCl buffer (pH 7.5), 50 μl proteins and 0.5 mM XTT. The reduction of XTT was determined at 470 nm for 4 min. Corrections were made for the background absorbance in the presence of 50 units SOD. O2·− production rate was calculated using an extinction coefficient of 2.16 × 104 M−1 cm−1. H2O2 was measured spectrophotometrically after reaction with potassium iodide (Alexieva et al. 2001).

Antioxidant enzyme assay

For determination of antioxidant enzyme activities, 0.5 g of florets was homogenized in 1.5 ml of respective extraction buffer in a liquid nitrogen pre-chilled mortar and pestle. The homogenate was filtered through four layers of cheesecloth and centrifuged at 22,000g for 20 min at 4°C. The supernatant was re-centrifuged again at 22,000g for 20 min at 4°C for determination of antioxidant enzyme activities. Protein concentration of the enzyme extract was determined according to Bradford (1976).

Superoxide dismutase (EC 1.15.1.1) activity was assayed by monitoring the inhibition of photochemical reduction of nitro-blue tetrazolium (NBT) according to the method of Bayer and Fridovich (1987). Florets were homogenized in 1 ml cold 100 mM K-phosphate buffer (pH 7.8) containing 0.1 mM ethylenediamine tetraacetic acid (EDTA), 1% (w/v) polyvinyl-pyrrolidone (PVP) and 0.5% (v/v) Triton X-100. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT as monitored at 560 nm. For the determination of APX, florets were homogenized in 100 mM sodium phosphate buffer (pH 7.0) containing 5 mM ascorbate, 10% glycerol and 1 mM EDTA. APX (EC 1.11.1.11) activity was determined in 1 ml reaction mixture containing 50 mM K-phosphate (pH 7.0), 0.1 mM ascorbate (extinction coefficient, 2.8 mM−1 cm−1), 0.3 mM H2O2. The decrease in absorbance was recorded at 290 nm for 3 min (Chen and Asada 1989). For determination of CAT and G-POD florets were homogenized in 100 mM sodium phosphate buffer (pH 7.0) containing 1 mM EDTA under liquid nitrogen. Catalase (EC 1.11.1.6) activity was determined by following the consumption of H2O2 (extinction coefficient, 39.4 mM−1 cm−1) at 240 nm for 3 min (Aebi 1974). POD (EC 1.11.1.7). The activity was measured by following the change of absorption at 436 nm due to guaiacol oxidation (extinction coefficient, 6.39 mM−1 cm−1) following Pütter (1974). The activity was assayed for 5 min in a reaction solution composed of 50 mM K-phosphate buffer (pH 7.0), 20.1 mM guaiacol, 12.3 mM H2O2 and the required amount of enzyme extract from leaves. GR (EC 1.6.4.2) activity was assayed by following the reduction of 5, 5′- Dithio-bis (2-nitrobenzoic acid) (DTNB) at 412 nm (extinction coefficient, 13.6 mM−1 cm−1) with modifications as described by Smith et al. (1988). The assay mixture (1 ml) contained 100 mM K-phosphate buffer (pH 7.5), 1 mM oxidized glutathione and 0.1 mM NADPH and 100 μl of enzyme extract.

Native PAGE and activity stain

Native polyacrylamide gel electrophoresis (PAGE) was performed at 4°C, 180 V, following Laemmli (1970). For SOD, POD, APX and GR, the enzyme solutions were subjected to native PAGE with 10% polyacrylamide gel. SDS was omitted from the PAGE. Activity stain for each enzyme was carried out as follows. APX activity was detected by the procedure described by Mittler and Zilinskas (1993). The gel equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing 2 mM ascorbate for 30 min was incubated in a solution composed of 50 mM sodium phosphate (pH 7.0), 4 mM ascorbate and 2 mM H2O2 for 20 min. The gel was washed in the buffer for 1 min and submerged in a solution of 50 mM sodium phosphate buffer (pH 7.8) containing 28 mM TEMED and 2.45 mM NBT for 10−20 min with gentle agitation in the presence of light. SOD activity was detected by the procedure described by Beauchamp and Fridovich (1971). The gel equilibrated with 50 mM K-phosphate buffer (pH 7.8) containing 2.8 × 10−5 M riboflavin, 0.028 M N,N,N′,N′-tetramethyl ethylenediamine (TEMED) for 30 min. The gel was washed in distilled water for 1 min and submerged in a same solution (mentioned above) containing 2.45 mM NBT for 10–20 min with gentle agitation in the presence of light. For POD, gel was incubated in 25 mM potassium buffer (pH 7.0) for 15 min to lower the pH and then gel was submerged again in a freshly prepared solution containing 18 mM guaiacol and 25 mM H2O2 in 25 mM K-phosphate buffer (pH, 7.0), till the POD activity-containing band was visualized (Fielding and Hall 1978). GR activity staining was adapted from Anderson et al. (1995) and was carried out in 0.4 mM NADPH, 3.4 mM oxidized glutathione (GSSG), 1.2 mM 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 0.3 mM 2,6-dichlorophenol-indophenol (DPIP) and 50 mM Tris-HCl buffer (pH 7.75).

Measurement of lipid peroxidation and LOX activity

Malondialdehyde (MDA) concentration was determined by the thiobarbituric acid (TBA) reaction as described by Heath and Packer (1968). LOX activity was determined according to Axerold et al. (1981).

Statistical analysis

All experiments were repeated three times with three replicates per treatment. Data were subjected to Duncan’s multiple range test using SAS program (Version 6.12, SAS Institute Inc., Cary, USA).

Results and discussion

Fresh weight and diameter of the capitulum increased until stage 3 and decreased thereafter (Table 1). Relative water content (RWC) recorded at different stages of floret senescence declined significantly at stage 4 and stage 5 (Table 1). Significant increase in MDA concentration was observed after stage 3 (Fig. 2). MDA, a decomposition product of polyunsaturated fatty acids hydroperoxides, has been frequently described as a suitable biomarker for lipid peroxidation (Bailly et al. 1996). This increase in lipid peroxidation over the senescence period was supported by the high degree of membrane deterioration expressed as a decrease in MSI. A steady decrease in MSI% upon the progression of floret senescence indicates a gradual loss of membrane’s stability (Table 1). This hypothesis was also confirmed by the higher LOX activity with senescence (Fig 2). The cause of lipid peroxidation may be due in part to LOX activity, which oxidizes liberated membrane fatty acids. During senescence marked changes occur in the biochemical and biophysical properties of cell membranes. This results from losses of membrane phospholipids, increases in neutral lipids, increases in the sterol to phospholipid ratio, and increases in the saturation:unsaturation index of fatty acids (Lesham 1992; Thompson et al. 1998). Membrane polyunsaturated fatty acids are prone to oxidation either by enzymatic means (LOX) or through autoxidative events (non-enzyme catalyzed). Increases in lipid peroxidation, usually determind from changes in MDA concentration, accompanies the increase in LOX activity while the products of peroxidation are considered to perturb membrane function (Leverentz et al. 2002). It has already been reported that LOX activity may promote senescence through oxidative damage membrane as seen in day lily (Panavas and Rubinstein 1998) and gladiolus (Peary and Prince 1990; Hossain et al. 2006). Paulin et al. (1986) suggested that transformation of lipids leads to membrane breakdown, free radicals are then produced by peroxidation and these free radicals promote the burst of oxidative stress.
Table 1

Changes in morphological and physiological parameters during five different floral developmental stages of Chrysanthemum morifolium Ramat. cv. Maghi

Parameters

Stage 1

Stage 2

Stage 3

Stage 4

Stage 5

Fresh weight of the flower head (g)

0.76bz

0.99b

1.69a

1.40a

1.39a

Diameter of the flower head (cm)

1.68c

2.58b

3.7a

3.5a

3.5a

Relative water content of the floret (%)

83.36a

82.04a

86.25a

65.78b

61.85b

Membrane stability index of the floret (%)

57.82a

59.35a

58.83a

44.43b

36.67b

Stage 1, 0 day after harvest; stage 2, 5 days after harvest; stage 3, 10 days after harvest; stage 4, 15 days after harvest and stage 5, 20 days after harvest

zMean separation within rows by Duncan’s multiple range test at 5% level

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Fig. 2

Changes in the level of MDA concentration (A) and LOX activity (B) in florets of Chrysanthemum morifolium Ramat. cv. Maghi at five different stages of floral development and senescence. Assays were carried out as described in methods. Stage 1, 0 day after harvest; stage 2, 5 days after harvest; stage 3, 10 days after harvest; stage 4, 15 days after harvest and stage 5, 20 days after harvest. *Values followed by the same letters are not significantly different according to Duncan’s multiple range test at 5% level

ROS concentration (H2O2 concentration and O2·−) increased with age of sampling (Fig 3A, B). Singlet oxygen, superoxide radical and H2O2 are reactive oxygen species that are generated when plant tissues are exposed to a variety of environmental stresses. Among the different ROS, only H2O2 is relatively stable and able to penetrate the plasma membrane as an uncharged molecule. Recently, H2O2, in addition to being toxic, it is now regarded as a signal molecule and a regulator of gene expression (Hung et al. 2005). The most potentially deleterious effect of H2O2 under these conditions, is that at higher concentrations it can trigger genetically programmed cell suicide. Soybean cell cultures where H2O2 activated a hypersensitive cell death mechanism (Levine et al. 1994). Hydroperoxyl radicals (HO2·−) that are formed from O2·− by protomation in aqueous solution can cross biological membranes and subtract hydrogen atoms from polyunsaturated fatty acids and lipid hydroperoxides, thus initiating lipid peroxidation (Halliwell and Gutteridge 1989). Our results suggest that increased oxidative stress as indicated by an increase in O2·− concentration with plant age results in increased lipid degradation or lipid peroxidation and is reflected in an increase in MDA concentration which is further manifested by a decline in MSI% as seen herein senescent florets.
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Fig. 3

Changes in the concentration of ROS activity (O2·− and H2O2) in florets of Chrysanthemum morifolium Ramat. cv. Maghi at five different stages of floral development and senescence. Assays were carried out as described in methods. Stage 1, 0 day after harvest; stage 2, 5 days after harvest; stage 3, 10 days after harvest; stage 4, 15 days after harvest and stage 5, 20 days after harvest. *Values followed by the same letters are not significantly different according to Duncan’s multiple range test at 5% level

Since higher concentration of endogenous H2O2 would stimulate senescence, it is important to determine the activity of enzymes that serve to regulate concentration of H2O2. Total SOD activity increased to its maximum at stage 3 and thereafter, it declined (Table 2). Non-denaturing PAGE coupled with activity localization revealed six SOD isozymes (Fig. 4A). Incubation of the gels in 2 mM potassium cyanide or 5 mM H2O2 before staining for SOD activity indicated isozymes SOD-1 to be Fe-SOD, SOD-2, SOD-3, SOD-4 and SOD-5 to be Mn-SOD, and isozymes SOD-6 to be Cu/Zn-SOD (data not shown). Three classes of SODs have been identified in plants on the basis of their metal cofactor. Fe-SODs are found only in plastids, Mn-SODs are found mainly in mitochondria as well as peroxisome and Cu/Zn-SODs are located in plastids, cytosol, apoplast, and peroxisomes (Bowler et al. 1994). Mn-SOD and Cu/Zn-SOD were more prominent at stage 3, while an Fe-SOD was induced in senescent florets. This may imply that during chrysanthemum floret senescence, mitochondria/peroxisomes are the major site of superoxide radical formation and, that Mn-SOD was the major isoform responsible for superoxide radical scavenging that constitutively appeared during floret development (particularly during floret senescence), while chromoplastic Fe-SOD and cytosolic/peroxisomal Cu/Zn-SOD may play crucial roles in superoxide radical scavenging during senescence. Synthesis of new antioxidant isozymes, with altered kinetic properties, may be more beneficial in ROS metabolism compared to simple enhancement of existing enzyme activities (Edwards et al. 1994; Rao et al. 1996).
Table 2

Changes in the level of antioxidant enzyme activities in the florets of five different floral developmental stages of Chrysanthemum morifolium Ramat. cv. Maghi

Antioxidant enzyme activities

Stage 1

Stage 2

Stage 3

Stage 4

Stage 5

Superoxide dismutase (unit mg−1 protein)

0.13cz

0.18c

0.44a

0.29b

0.29b

Ascorbate peroxidase (μmol min−1 mg−1 protein)

2.4c

4.47b

5.92b

11.5a

5.13b

Catalase (μmol min−1 mg−1 protein)

0.2c

1.34b

5.54a

5.77a

0.54c

Guaiacol peroxidase (μmol min−1 mg−1 protein)

20.84d

192.14c

663.28a

670.81a

459.8b

Glutathione reductase (μmol min−1 mg−1 protein)

94.43b

32.39c

44.31c

117.33a

124.31a

Stage 1, 0 day after harvest; stage 2, 5 days after harvest; stage 3, 10 days after harvest; stage 4, 15 days after harvest and stage 5, 20 days after harvest

zMean separation within rows by Duncan’s multiple range test at 5% level

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Fig. 4

Superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD) and glutathione reductase (GR) in-gel assay of proteins from florets of Chrysanthemum morifolium Ramat. cv. Maghi at five different stages of floral development and senescence. Samples were electrophoresed on a 10% native polyacrylamide gel. 150 μg proteins were loaded per each well. Assays were carried out as described in methods. The different isoforms are numbered from cathode to anode. Stage 1, 0 day after harvest; stage 2, 5 days after harvest; stage 3, 10 days after harvest; stage 4, 15 days after harvest and stage 5, 20 days after harvest

Estimation of APX activity also showed a varying pattern with senescence, high at stage 4 and decreased thereafter (Table 2). Examination of APX in chrysanthemum florets revealed six isozymes expressed differentially during the different stages of senescence (Fig. 4B). The large increase of APX activity during senescence contributed to stress tolerance. Surprisingly, floret APX activity due to APX-1, APX-2, APX-3, APX-5 and APX-6 were not detectable during early floret development. These isozymes appeared only at the onset of senescence. APX plays a key role in the ascorbate-glutathione cycle by reducing H2O2 to water using the reducing power of ascorbate and producing monodehydroascorbate (MDHA) (Asada 1992). Our results show that during the senescence process, as a result of an oxidative burst florets accumulate H2O2 which in turn provoked APX enzyme expression during late senescence, while the loss of APX activity resulted in higher concentration of H2O2. The latter may act as a signal molecule for PCD. Similar result have also been reported by Hossain et al. 2006 and they suggest that down regulation of APX activity was a prerequisite for inducing senescence in Gladiolus cv. ‘Snow Princess’ tepal which in turn enhances the concentration of endogenous H2O2.

Catalase activity steadily increased during vase life and highest activity was observed at stage 4 before it declined (Table 2). Similarly total peroxidase activity was uniformly increased in young to mature florets and then declined (Table 2; Fig 4C). It has been suggested that senescence brings about important alterations in the reactive oxygen metabolism of peroxisomes which are mainly characterized by the disappearance of CAT activity and an overproduction of H2O2 and O2·− (del Rio et al. 1998). This suggests that floret senescence was associated with the reverse metabolic transition of leaf peroxisomes to glyoxysomes, with the channelling of acetyl-CoA through the glyoxylate cycle (del Rio et al. 1998; McCarthy et al. 2001). Recently a role has been proposed for peroxisomes as a source of ROS which are involved in signal transduction pathways leading to specific gene expression (Corpas et al. 2001). Similarly, the senescence induced post-translational activation of H2O2-producing Mn-SOD, could have a significant contribution in peroxisomes as an additional source of H2O2 (del Rio et al. 2003). The senescence-induced H2O2 leaking from peroxisomes might act in the cytosol as a second messenger in signal transduction pathways.

The activity of GR remained high even when the florets showed clear visible senescence symptoms (Table 2). Similar results have also been reported by Bailly et al. 2001 during senescence of Iris tepals. We also observed five GR isoforms in chrysanthemum florets. GR-1, GR-3 and GR-5 isoforms have been clearly detected during onset of senescence (Fig. 4D). GR is involved in the recycling of reduced glutathione, providing a constant intracellular concentration of GSH (Calbert and Mannervik 1985), the main cell antioxidant (Meister 1981; Alscher 1989; Reed 1990). Elevated concentration of GSH are associated with the increase in oxidative stress tolerance (Broadbent et al. 1995). However, the relationship between the activity of GR isoforms and senescence, if any, is not clear (Bailly et al. 2001). However, we suggest that GR activity is responding to oxidative stress resulting from ROS accumulation which can only be partly counteracted by the peroxisomal ascorbate-glutathione cycle. High GR activity was also observed during early floret development. Recently it has been reported that there is a relationship between the amount of GSH synthesized and flowering in Arabidopsis(Ogawa et al. 2001).

Senescence can lead to the loss of membrane permeability due to the oxidation of existing membrane components, for example, lipid peroxidation, as measured by MDA concentration, increases during senescence. The combined decrease of SOD APX and CAT at stage 5 resulted in high superoxide and high H2O2 concentrations. It may directly, or indirectly, via the formation of free radicals, be involved in the last stages of senescence and cell death in chrysanthemum florets.

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© Springer Science+Business Media B.V. 2007