Plant Growth Regul (2012) 67:83–92 DOI 10.1007/s10725-012-9668-4
ORIGINAL PAPER
Response of antioxidant systems in oxygen deprived suspension cultures of rice (Oryza sativa L.) Revandy Iskandar Damanik • Mohd Razi Ismail Zulkifli Shamsuddin • Sariam Othman • Abd Mohd Zain • Mahmood Maziah
•
Received: 4 May 2011 / Accepted: 27 January 2012 / Published online: 15 March 2012 Springer Science+Business Media B.V. 2012
Abstract The effect of oxygen deprivation (anoxia) on the antioxidant system in suspension culture of anoxiaintolerant Malaysian rice mutants cells was examined. Abiotic stresses have been reported to adversely affect cell division, damage cellular and organelle membranes. The signaling defense mechanisms, such as molecular and biochemical aspects responding to stress have been proven to be very complex, and still largely untapped. The objective of this study was to determine the potential involvement of activated oxygen species, such as superoxide dismutase, catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase which occur in cells of rice plants exposed to anoxia stress in two Malaysian rice mutants, MR219-4 and MR219-9, and rice cultivar FR13A which is known to be tolerant to anoxia stress during
R. I. Damanik M. Maziah (&) Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia e-mail:
[email protected] M. R. Ismail Institute of Tropicial Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Z. Shamsuddin Department of Land Management, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia S. Othman Rice and Industrial Crop Research Centre MARDI Head Quarters, 50774 Kuala Lumpur, Malaysia A. M. Zain Department of Agrotechnology, Faculty of Agrotechnology and Food Science, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia
5–30 days of exposure. The antioxidative enzymes were decreased for MR219-4 and MR219-9 mutants for CAT and APX activities, and increased in FR13A cultivar starting at 20 days in suspension culture compared to that of control. CAT and APX activities were maintained higher in anoxia condition for all mutants and cultivar. These findings suggested that anoxia stress in suspension cultures induced the level of H2O2 to toxic levels. Keywords Antioxidant enzymes Suspension culture Cultivars Periods of stress Rice Anoxia stress
Introduction The responses to a specific submergence stress may vary with the genotypes, nevertheless, some general reactions occur in all genotypes. In most submergence environments, O2 concentration is usually below air saturation leading to anoxia (no O2) or hypoxia (low O2). The condition has rapid and intense, consequences on cell physiology. The exposure to that conditions leads to energy production become limited and metabolic changes in plant cells, since O2 participates in a broad spectrum ranges from production of ATP though oxidative phosphorylation and respiration, which draws on over 95% of the cellular O2 consumption to cover the energetic needs of the cell (Babcock 1999). Anoxia-tolerant plants are particularly efficient at mobilizing storage polysaccharides when challenged by the higher carbohydrate consumption (Pasteur effect) required by fermentation processes (Perata et al. 1996). Transcription of a-amylase, for instance, could be observed in rice seeds but not in barley or wheat (Perata et al. 1993). Moreover, exogenously supplied sugars could improve energy metabolism and survival (Webb and Armstrong
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1983; Saglio 1985; Perata et al. 1993) as well as restore the mitochondrial ultrastructure of both sensitive and tolerant species under anoxia (Vartapetian et al. 1977). Rice (Oryza sativa L.) is the staple food for over 50% the global population, is a semi aquatic plant, particularly able to survive under the conditions of prolonged oxygen deprivation, at both the seedling and adult stages. However, Most of rice cultivars are not always submergence tolerant, only a few cultivars, such as FR13A, can survive for 10–14 days of complete submergence (Fukao and BaileySerres 2008). The world’s projected demand of rice by 2020 is 880 million tons in proportion to the increased population (Anbazhagan et al. 2009). Although with the increasing population and decreasing land availability, agriculture is suffering from severe damage of biotic and abiotic stresses. Conventional breeding is essential to improve rice but progress is slow due to several barriers (Wang et al. 2005). To overcome the problems, we have noticed that suspension culture of plant cells, tissues or organs under controlled environmental conditions has been developed over five decades. Cultured cells are useful and easily recognized as suitable model systems for investigation of the molecular responses of plants to stimuli that affect growth, and to avoid the complications caused by the process of transporting oxygen from shoots to roots at the whole plant system. Rapidly growing, fine-textured plant cell cultures can provide a homogeneous cell population for many studies in plant biology. Cell cultures constitute an essential tool for developmental, biochemical and molecular biological investigations in the study of programmed cell death (PCD), but remain underexploited in such investigations in plant species (Cotter and Al-Rubeai 1995). The observations made with parsley and soybean suspension cells mutually can be suitable model systems for studying the salicylic acid (SA), 2,6-dichloroisonicotinic acid (INA), and benzothiadiazole (BTH) induced priming for potential activation of cellular plant defense responses (Ryals et al. 1996; Sticher et al. 1997; Dempsey et al. 1999). Moreover, one of the differences between the well-sealed tissue culture vessel and plant submergence (the most extreme form of flooding stress), the restrictions in gas and solute diffusibility commonly encountered in whole tissues (e.g. roots, rhizomes, and tubers) are largely eliminated when working with isolated cells. Considerably, the consequence of plants response to unsuitable in vitro environment (various stresses) was a primary event, which can induce the biochemical changes occurring the production of reactive oxygen species (ROS; such as the superoxide radical, O2 2 , the hydroxyl radical, OH, and hydrogen peroxide, H2O2), which disrupt the normal metabolism through severe oxidative damage to proteins, nucleic acids, and lipids (Park et al. 2004). The
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consequences of ROS formation depend on the intensity of the stress and on the physicochemical conditions (i.e. antioxidant status, redox state and pH) in the cell. Abiotic stresses, such as salt, have been reported to adversely affect cell division in rice roots (Samarajeewa et al. 1999). Under anoxia conditions, plants can generate H2O2 up to toxic levels resulting in oxidative stress. The source of ROS has been primarily related to dysfunctions in electron transport chains and other membrane-associated processes (Elstner and Osswald 1994). To counter the hazardous effects of ROS under stress, plants have evolved a complex antioxidative defense system composed of both antioxidant enzymes and metabolites such as ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), ascorbic acid (AA), glutathione (GSH), oxidized glutathione (GSSG) and Vitamin E (Ahmed et al. 2002; Dewir et al. 2006). The activation of antioxidative enzymes like SOD, CAT, APX and GR are the most important components in the scavenging system of the harmful effect of ROS in chloroplast and mitochondria (Lee et al. 2007). SOD, the first enzyme in detoxifying process, converts superoxide anion radicals (•O2-) to hydrogen peroxide (H2O2), CAT convert H2O2 to water and oxygen, APX is the most important peroxidase in H2O2 detoxification, that utilize either H2O2 or O2 to oxidized a wide variety of molecules. The efficiently combination action of SOD and CAT is critical in mitigating the effects of oxidative stress, which play an important role in regulating the concentrations of ROS. SOD removes O2- by disproportionating it to H2O2 and O2 and exists in three isoforms. These isoforms include a copperzinc containing protein, sensitive to CN- and predominantly presented in chloroplast, a CN-insensitive Mn-SOD predominantly associated with mitochondria and a CN-insensitive Fe-SOD located in chloroplast (Ushimaru et al. 1999). The role of GR and GSH in the H2O2 scavenging in plant cells has been well established in Halliwell–Asada pathway (Bray et al. 2000). GR is an important factor involved in the recycling of reduced GSH, providing a constant intracellular level of GSH, therefore, elevating the total quantity of GSH thus reducing the stress injury (Kocsy et al. 2001; Hung et al. 2005). The objective of this study was to determine the potential involvement of activated oxygen species which occur in cells of rice plants treated with anoxia stress in intolerant two Malaysian rice mutants, MR219-4 and MR219-9, and compared with cultivar FR13A which is known to be tolerant to anoxia stress. In this condition, the study is expected to understand the tolerant mechanisms to anoxia stress and to clarify the contribution of activities of antioxidant enzymes to tolerant anaerobiosis development. This study will also examine the growth and activity of some antioxidative enzymes in rice suspension culture
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treated with anoxia condition during 5–30 days. Simultaneously, non enzymatic lipid peroxidation product malondialdehyde (MDA) was also determined.
Materials and methods Plant materials and culture conditions The study was conducted using two Malaysian rice (Oryza sativa L.) mutants, MR219-4 and MR219-9, and FR13A cultivar, which is known to be tolerant to submergence (IRRI 1988). All those of them were provided by Malaysian Agricultural Research and Development Institute (MARDI) Seberang Perai, Malaysia. MR219-4 and MR219-9 mutants are two potential rice mutants that were generated from MR219 as a result of laboratory and glass house selection. Mature seeds of the two selected Malaysian rice mutants and FR13A cultivar were dehusked and surface sterilized with 70% (v/v) ethanol for 2 min followed by 40% (v/v) sodium hypochlorite for another 20 min shaking. After rinsing five times with sterile distilled water, the sterilized seeds were used for callus induction. Callus induction Rice callus induction was following the method of (Htwe et al. 2011). The mature seeds were placed on MS medium (Murashige and Skoog, 1962) supplemented with 2,4 D (2.21 mg l-1), sucrose (30 g l-1), casein hydrolysate (0.4 g l-1) and gelrite (2.75 g l-1). The pH of the media was set to 5.8 prior to autoclaving at 121C for 25 min. The cultures were incubated at 25C ± 2C under dark condition. After 2 weeks of culture, the primary callus was removed from the scutellum, and subcultured every 2 weeks in the same media for up to 8 weeks. Establishment of rice cell suspension cultures Friable callus was selected and transfer to initiate cell suspension cultures in liquid MS media with the same composition and plant hormone supplements as the callus induction media. This cell line was maintained in the dark and subcultured every 7 days. About 10 ml of the fine fraction was cultured in 40 ml of liquid MS media in a 150 ml Erlenmeyer flask. The suspension cultures were incubated on a rotary shaker (120 rpm) at 25 ± 2C under dark condition. Treatment for oxygen deprivation (anoxic) condition Fourteen-days-old aerobically grown cultures were taken out and sieved through a 500 lm sterile nylon. Then each
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of the 10 ml of the culture was added to 40 ml ‘degas’ (oxygen-depleted medium) fresh medium. Some cultures were made and kept anoxic by flushing the newly inoculated flasks (media ? flasks) with Nitrogen gas (oxygen free) for 10 min and sealed directly with aluminum foil and plastic wrap. The oxygen concentration of the culture medium was detected with dissolved oxygen meter. The remainder of the cultures were kept grown under aerobic condition (control). Determination of growth of rice cell suspension cultures For the determination of growth kinetics and changes in antioxidant activity, suspension cultures were grown for 0, 5, 10, 15, 20, 25, and 30 days postsubculture without transferring to the fresh medium (based on the treatment of the stress time periods at Table 1). For fresh weight (FW) determination, cell suspension cultures of rice were separated from culture media, filtered (No 1 filter paper; Macherey–Nagel, Germany) by vacuum filtration, and washed once with distilled water, kept on filter paper for a few minutes to remove excess water and weighed. The cells were then dried in an oven at 60C to a constant weight (about 48 h) for dry weight (DW) determination. The data obtained were expressed in milligrams of cells per 50 ml of culture media. Preparation of crude enzyme extracts Three replicated of rice cells were collected, frozen and powdered in liquid N2. The powdered cells were homogenized with ice cold 0.05 M sodium phosphate buffer (pH 7.8) containing 0.1 mM ethylenediaminetetraacetic acid (EDTA). Na2 and 2% (w/v) insoluble polyvinyl polypyrrolidone (PVPP), in the ratio of 5 ml extraction buffer: 1 g FW. The homogenates were then centrifuged at 10,0009g for 25 min at 4C and supernatants were used for the determination of protein and antioxidant enzyme activities. Protein concentration of the enzyme extract was determined according to Bradford (1976), using bovine serum albumin as the standard. ROS scavenging enzyme assays All spectrophotometric analyses were conducted at 25C on a UV:visible light spectrophotometer (UV-2602, Labomed, Inc. USA). The assay for superoxide dismutase (SOD, EC 1.15.1.1) activity was estimated spectrophotometrically as the inhibition of photochemical reduction of nitroblue-tetrazolium (NBT) at 560 nm (Stewart and Bewley 1980). The reaction mixture contained enzyme extract, 390 mM L-Methionine, 2.25 mM NBT, 1 mM EDTA, 7% Na2CO3,
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86 Table 1 Growth of rice suspension cultures subjected to different periods of anoxia condition
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Culture condition
Aerobic
Cultivars
MR219-4
Stress time (d)
0
0.0787 ± 0.004
0.0063 ± 0.001jkl
15
0.1770 ± 0.003
h
0.0133 ± 0.001ef
20
0.6453 ± 0.008b
25
0.2170 ± 0.008
f
0.0133 ± 0.003ef
0.1147 ± 0.003
k
0.0067 ± 0.003jk
0
0.1197 ± 0.003k
15 20
e
0.2610 ± 0.003 0.2903 ± 0.006d
0.0203 ± 0.002d 0.0203 ± 0.001d
25
0.4917 ± 0.004c
0.0343 ± 0.002c
30
a
u
0.0063 ± 0.001jkl 0.0020 ± 0.0q
0.0106 ± 0.0
5
0.0630 ± 0.0p
10
0.0639 ± 0.0
p
0.0070 ± 0.0jk
0.0719 ± 0.0
o
0.0130 ± 0.0f
0.0912 ± 0.0
m
0.0430 ± 0.0b
25
0.0640 ± 0.0
p
0.0210 ± 0.0d
30
0.0320 ± 0.0s
0.0050 ± 0.0lm
0.0040 ± 0.0mno
0.0473 ± 0.002
qr
0.0037 ± 0.001mnop
0.0510 ± 0.002
q
0.0033 ± 0.001nopq
0.0933 ± 0.006
m
0.0060 ± 0.002kl
20
0.1920 ± 0.009
g
0.0107 ± 0.001gh
25 30
0.1343 ± 0.005j 0.0280 ± 0.003st
0.0107 ± 0.001gh 0.0023 ± 0.001pq
5
0.0470 ± 0.006qr
0.0033 ± 0.001nopq
5
0.1047 ± 0.003
l
0.0077 ± 0.001ij
0.1550 ± 0.003
i
0.0093 ± 0.001hi
20
0.1603 ± 0.006
i
0.0117 ± 0.001fg
25
0.2193 ± 0.009f
15
30
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0.6530 ± 0.002
0.0107 ± 0.002gh
0
10
0.05 M sodium phosphate buffer (pH 7.8), and 60 lM riboflavin. The riboflavin was added last. The reaction mixture was placed 30 cm from light source (about 15 W fluorescent lamps) for 10 min, and the decrease in the absorbance was recorded at 560 nm. The non-irradiated reaction mixture served as control and the value was deducted from that of A560. One unit of SOD activity was
0.0107 ± 0.001gh
10
15
FR13A
0.0030 ± 0.0opq m
0.0967 ± 0.004
10
Values are given as averages of three replicate experiments ± SD, anoxic condition started at 5 days for all cultivars. Treatments with at least one letter the same are not significantly different according to Duncan’s test
0.0420 ± 0.0
r
0.0503 ± 0.001a
5
20
MR219-9
0.0030 ± 0.0opq 0.0047 ± 0.001lmn
15
MR219-4
0.0450 ± 0.0qr n
30
Anoxic
DW (mg/50 ml medium)
0.0650 ± 0.006
10
FR13A
FW (mg/50 ml medium)
p
5
MR219-9
Growth parameters
5 10
0.0463 ± 0.005 0.0102 ± 0.0
qr
u
0.0800 ± 0.002
0.0147 ± 0.001e 0.0037 ± 0.001mnop 0.0030 ± 0.0opq
n
0.0050 ± 0.0lm
15
0.0820 ± 0.0
n
0.0050 ± 0.0lm
20
0.0790 ± 0.0n
0.0050 ± 0.0lm
25
0.0318 ± 0.0
st
0.0030 ± 0.0opq
0.0259 ± 0.0
t
0.0040 ± 0.0mno
30
defined as the amount of enzyme which caused 50% inhibition of the initial rate of reaction in the absence of enzyme, expressed in units mg protein -1. Catalase (CAT, EC 1.11.1.6) activity was assayed by measuring the initial rate of disappearance of H2O2 (Bergmeyer 1970). Extraction mixture contained 300 ll of enzyme extract, 0.5 ml of 10 mM H2O2 and 600 ll of
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30 mM potassium phosphate buffer (pH 7.0) and the decrease in absorbance was recorded at 240 nm for 30 s. The enzyme activity was calculated as lmol H2O2 decomposed min-1 mg protein-1 by using extinction coefficient (€ = 36 lM-1 cm-1). Ascorbate peroxidase (APX, EC 1.11.1.11) activity was measured following the method by Nakano and Asada (1981). The assay mixture contained 100 ll of enzyme extract, 600 ll 0.1 mM EDTA in 0.05 M sodium phosphate (pH 7.0), and 400 ll 0.5 mM AA. The reaction started with addition of 400 ll of 3% H2O2 and the absorbance decreased was recorded after 1 min at 290 nm. The concentration of APX was calculated using extinction coefficient (€ = 2.8 mM-1 cm-1). One unit of APX was defined as 1 nmol ml-1 ascorbate oxidized per minute. Glutathione reductase (GR, EC 1.6.4.2) activity was estimated following the method by Goldberg and Spooner (1983). The assay mixture contained of 100 ll enzyme extract, 2.5 ml of 0.1 mM EDTA in 0.05 M sodium phosphate (pH 7.0), and 100 ll 0.5 mM GSSG. After 5 min 50 ll of 9.6 mM NADH was added and mixed thoroughly. The absorbance decreased was recorded at 290 nm at an interval 1 min. The expression of 1 unit of GR activity is nmol GSH reduced per minute, calculated using extinction coefficient (€ = 6.22 mM-1 cm-1). Lipid peroxidation The crude extracts were also measured for lipid peroxidation capacity. Lipid peroxidation was determined by measuring the amount of MDA formation using the thiobarbituric acid method described by Dionisio-Sese and Tobita (1998). For every 1 ml of the crude extract, 4 ml of 20% (w/v) trichloroacetic acid (TCA) containing 0.5% (w/v) thiobarbituric acid (TBA) was added. The mixture was heated at 95C for 30 min and then cooled quickly on an ice bath. The mixture was centrifuged for 15 min at 10,0009g and the absorbance of the supernatant was measured at 532 nm. Measurements were corrected for unspecific turbidity by subtracting the absorbance at 600 nm. The concentration of MDA was calculated by using an extinction coefficient of 155 mM-1 cm-1. Statistical analysis All the data obtained were subjected to a two-way analysis of variance (ANOVA), and the mean differences were compared by least significant differences (LSD) post hoc analysis using SPSS 11.5 for windows software. Comparisons with p \ 0.05 were considered significantly different. In all the figures, the spread of values is shown as error bars representing standard deviation of the means.
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Results and discussion Growth of rice suspension cultures In growth of rice suspension cultures, differential response in fresh and DW of the cultures were measured over the experimental stress period (0–30 days) (Table 1). The results showed that there were significant differences in fresh and DW of rice suspension cultures for all stress period and between the cultivars. The fresh and DW of rice suspension cultures increased at day 20 for both MR219-4 mutant and FR13A cultivar, but for MR219-9 mutant showed an increase at day 30 for FW and 25 for DW. FW of the cell suspension culture was increased gradually after 5 days and reached the lowest in the anoxia stress of the two intolerant mutants MR219-4 and MR219-9 at 20 and 30 days by 70 and 93% respectively compared to aerobic (untreated) condition. Interestingly, FW was significantly increased in the tolerant cultivar FR13A treated with anoxia stress at 10 and 15 days by 25 and 14% respectively compared to aerobic condition, and decreased progressively with the longer stress period (Table 1). However, in DW were decreased for all cultivars starting at 5 days period of anoxic stress (Table 1). The observed increased in FW might be due to the combination of the growth and development of the cells as a result of 30 days postsubculture without transferring to the fresh medium (‘‘Materials and methods’’), especially for tolerant cultivar FR13A which maybe related to capacity to protect themselves from anoxia stress. Effect of anoxia condition on antioxidant enzymes activities In order to determine the differential effect of anoxia condition, the activities of the enzymes in anoxia condition of rice suspension cultures were measured and any changes in those activities when cells were made anoxic. The pattern of SOD activity varies depending on the cultivar and the period of anoxia stress. There was a significant difference in SOD activities among cultivars as shown in Fig. 1. SOD activities were decreased in the aerobic suspension cultures for all cultivars when compared with the control (0 day) except for FR13A cultivar which was significantly increased by 129% higher for SOD activity at 10 days. In anoxia condition, SOD activity was increased starting at 5 days, after that its activity decreased gradually until 30 days in anoxia condition for all mutants and cultivar, when compared with the aerobic condition, except for FR13A cultivar was increased again at 25 days in anoxia condition (Fig. 1). A significant increased in SOD activity, a key enzyme in response to anoxia condition, may have
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Fig. 1 Superoxide dismutase activity in rice cultivars suspension culture at different days of anoxic stress. Values are mean ± standard deviations based on three independent assays for each determination. Different letters indicated significant differences at (p \ 0.05) according to LSD post hoc analysis
Fig. 2 Catalase activity in rice cultivars suspension culture at different days of anoxic stress. Values are mean ± standard deviations based on three independent assays for each determination. Different letters indicated significant differences at (p \ 0.05) according to LSD post hoc analysis
resulted due to the formation of O2- and toxic levels of H2O2 (Saher et al. 2005). There was a significant decrease in the aerobic suspension culture for CAT activity starting at 5 days for all mutants and cultivar, except for MR219-9 mutant and FR13A cultivar where CAT activity started to increase again at 20 days, when compared with the control (0 day) (Fig. 2). CAT activity was significantly increased in anoxia
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condition for MR219-4 and MR219-9 mutants compared to aerobic condition started from 5 days and strongly increased for FR13A cultivar by almost eight times higher. However, after 10 days of culture in anoxia condition, CAT activity was decreased for MR219-9 mutant and 20 days for MR219-4 mutant and FR13A cultivar compared to aerobic condition. Regarding the data obtained, there was a concerted action between SOD and CAT at 20
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and 25 days for MR219-4 and at 30 days for MR219-9 in anoxic condition, indicated simultaneously phase of the plant cells to protect against the stress. Compared with our previous reports have shown that SOD and CAT did not participate in active H2O2 reduction at the same period of submergence treatment (0–12 days), except for flood-tolerant FR13A cultivar (Damanik et al. 2010). Apparently, the action of SOD and CAT is localized in almost all compartments of the plant cell and critical in mitigating the effects of oxidative stress, since SOD acts on the superoxide anion converting it to another reactive intermediate (H2O2) and CAT acts on H2O2 converting it to water and oxygen (Mate´s 2000; Blokhina et al. 2003). H2O2 is produced during different metabolic processes such as photorespiration in chloroplasts (Henzler and Steudle 2000). H2O2 has been considered as an essential signal involved in plant defense against biotic and abiotic stresses and affects the integrity of cells because it produces highly ROS such as hydroxyl radical (HO-) which attacks protein, lipids and nucleic acids (Foyer et al. 1994). Thus, by means of the enzymes SOD and CAT activities, the cells are keep low levels of toxic compounds (Peng and Kuc 1992). APX activity (Fig. 3) of MR219-4 and MR219-9 mutants showed a significant decreased starting at 5 days in aerobic condition compared to control, although APX activity of FR13A cultivar significantly increased starting at 15 days in aerobic condition. The results also showed that APX activity of the intolerant mutants MR219-4 and MR219-9 had lower values compared to FR13A cultivar (Fig. 3). We found that
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there were a similar trends for CAT and APX activities in anoxic condition for all cultivars (Figs. 2, 3), hence the possibility that CAT and APX activities were play a significant role in the fine regulation of ROS concentration in the cell through activation of H2O2 in response for anoxia condition. The H2O2 scavenging system represented by APX and CAT is more important in partitioning tolerance than SOD as reported in oxidative stressed wheat varieties (Lafitte et al. 2007). CAT is considered as a key enzyme removing toxic hydrogen peroxide, and APX have a complementary duty (Malecka et al. 2009). Glutathione reductase activity had shown significant decrease for all cultivars in aerobic conditions, except for FR13A cultivar when compared with the control (0 days) (Fig. 4). MR219-4 mutant had increased GR activity at 20 and 25 days under anoxia condition relative to aerobic condition, but MR219-9 mutant had decreased starting at 10 days. For FR13A cultivar had increased starting at 5 days in anoxia condition, but decreased after 15 days in anoxia condition for GR activity. Higher GR activities observed under the periods of anoxia stress indicates that mechanism of antioxidant defense was enhanced in the maintenance of a high GSH/ GSSG ratio in stressed plants yielding AA. This AA produced by non enzymatic dispropotionation of MDHA (monodehydroascorbate reductase) was used by APX to directly detoxify H2O2 (Kocsy et al. 2001; Shanker et al. 2004). Maintenance of high GSH/GSSG ratio and GR activity also play an important role in salt, desiccation, and drought tolerance as found in tomato and wheat (Shalata
Fig. 3 Ascorbate peroxidase activity in rice cultivars suspension culture at different days of anoxic stress. Values are mean ± standard deviations based on three independent assays for each determination. Different letters indicated significant differences at (p \ 0.05) according to LSD post hoc analysis
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Fig. 4 Glutathione reductase activity in rice cultivars suspension culture at different days of anoxic stress. Values are mean ± standard deviations based on three independent assays for each determination. Different letters indicated significant differences at (p \ 0.05) according to LSD post hoc analysis
Fig. 5 Malondialdehyde content in rice cultivars suspension culture at different days of anoxic stress. Values are mean ± standard deviations based on three independent assays for each determination. Different letters indicated significant differences at (p \ 0.05) according to LSD post hoc analysis
et al. 2001; Kocsy et al. 2004). The activity of APX and GR is important in determining the efficiency of stress tolerance. APX is the first enzyme in ascorbate–glutathione cycle, and its major function is catalyzing the H2O2 to H2O, while GR is the last step in the pathway, playing a essential role in protection against oxidative stress by maintaining a reduced GSH level (Blokhina et al. 2003). As anoxia stress increased both APX and GR activities, this maybe explain
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the effective removal of H2O2 by the activity of the ascorbate–glutathione cycle.
Effect of anoxia condition on lipid peroxidation Lipid peroxidation levels were measured as the content of MDA produced in plants exposed to oxygen deprivation
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conditions, are given in Fig. 5. There was a significant different in MDA production started at 10 days in aerobic condition for MR219-4 and MR219-9 mutants compared to control (0 days). The tolerant FR13A cultivar had much more MDA production than intolerant MR219-4 and MR219-9 mutants, at 20 days in aerobic condition. Moreover, the application of 15 days of anoxia condition, had the highest of MDA production by 34% for MR219-4 mutant when compared with the aerobic condition. The content of MDA for MR219-9 mutant and FR13A cultivar showed a similar trend from 5 days in anoxia condition, both reached the highest level at 20 days by 49 and 48%, respectively, compared to the aerobic condition (Fig. 5). The content of MDA is produced in plants exposed to adverse environmental conditions and is a reliable indicator of free radical formation in the tissue (Parida et al. 2004). Our results showed that there was significant difference in MDA content among aerobic and anoxia conditions, and the results indicated the extended periods of anoxic condition increased lipid peroxidation (Fig. 5). It can be suggested that MR219-4, MR219-9 mutants and FR13A cultivar tend to upregulate their antioxidant system to detoxify ROS generated during stress, with the opposite of higher level of MDA in anoxia condition. H2O2 plays a dual role in plants: at low concentration it acts as a messenger involving in signaling and triggering tolerance against various abiotic stresses, but at high concentrations it causes oxidative stress which leads to a loss of protein function, membrane integrity, and to PCD (Asada 1999; Lin and Kao 2000). Under anoxia condition a decrease in membrane integrity is a symptom of injury, and it can be measured as changes in the sensitive molecules unsaturated fatty acids, such as the lipid content and composition, can be reflected of stress-induced damage at cellular level (Jain et al. 2001; Blokhina et al. 2003). Boscolo et al. (2003) and Hodges et al. (1999) observed that carbohydrates and even some protein oxidation rather than lipid peroxidation are known to undergo decomposition and produce MDA as an end-product suggesting that the target of oxidative stress varies depending on the plant species, type and intensity of stress factors. Further research is needed to resolve the physiological relevance of the signaling mechanisms in ROS detoxifying-enzymes (SOD, CAT, APX and GR) and to determine the specific role(s) of ROS in signaling pathways under oxygen deprivation. Acknowledgments This research was supported by Graduate Research Fund (GRF) of Universiti Putra Malaysia (UPM).
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