Indian Journal of Biochemistry & Biophysics Vol. 48, October 2011, pp 346-352
Role of antioxidant and anaerobic metabolism enzymes in providing tolerance to maize (Zea mays L.) seedlings against waterlogging Vishal Chugh, Narinder Kaur and Anil K Gupta* Department of Biochemistry, Punjab Agricultural University, Ludhiana 141004, India Received 28 February 2011; revised 29 July 2011 The present investigation was undertaken to identify the possible mode of mechanism that could provide tolerance to maize (Zea mays L.) seedlings under waterlogging. Using cup method, a number of maize genotypes were screened on the basis of survival of the seedlings kept under waterlogging. Two tolerant (LM5 and Parkash) and three susceptible (PMH2, JH3459 and LM14) genotypes were selected for the present study. Activities of antioxidant and ethanolic fermentation enzymes and content of hydrogen peroxide (H2O2), glutathione and ascorbic acid were determined in roots of these genotypes after 72 h of waterlogging. Waterlogging treatment caused decline in activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) in all the genotypes. However, only susceptible genotypes showed slight increase in glutathione reductase (GR) activity. Significant reduction in APX/GR ratio in susceptible genotypes might be the cause of their susceptibility to waterlogging. The tolerant seedlings had higher GR activity than susceptible genotypes under unstressed conditions. Stress led to decrease in H2O2 and increase in glutathione content of both tolerant and susceptible genotypes, but only tolerant genotypes exhibited increase in ascorbic acid under waterlogging conditions. In the tolerant genotypes, all the enzymes of anaerobic metabolism viz. alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and pyruvate decarboxylase (PDC) were upregulated under waterlogging, whereas in susceptible genotypes, only ADH was upregulated, suggesting that efficient upregulation of entire anaerobic metabolic machinery is essential for providing tolerance against waterlogging. The study provides a possible mechanism for waterlogging tolerance in maize. Keywords: Zea mays, Maize, Waterlogging, Antioxidative enzymes, Ethanolic fermentation, Antioxidants
Higher plants are aerobic organisms requiring oxygen for their life. Plants may experience low oxygen availability (hypoxia) or total absence of oxygen (anoxia), due to waterlogging of soil or anatomical structures of some tissues whose histological properties severely limit the permeability to oxygen1. Waterlogging is one of the most serious constraints for maize (Zea mays L.) production in South-Asia and many other parts of the world. In South-East Asia, about 15% of total maize growing area is affected by floods and waterlogging problem. In India also, waterlogging is one of the most serious constraints for maize production and productivity. Out of total 6.6 mha area under maize, about 2.5 mha is affected by waterlogging causing losses in production of 25-30% almost every year2. —————— *Corresponding author E-mail: [email protected]
Fax: 91-161-2400945; Tel: 09872452820 Abbreviations: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ANPs, anaerobic proteins; APX; ascorbate peroxidase; CAT, catalase; GR, glutathione reductase; PDC, pyruvate dehydrogenase; POX, peroxidase; ROS, reactive oxygen species.
Waterlogging changes plant metabolic activity. One of the root metabolic features affected by waterlogging conditions is the antioxidant system. Waterlogging generates oxidative stress and promotes the production of reactive oxygen species (ROS) including superoxide (O2-), singlet oxygen, hydroxyl anion (OH-) and hydrogen peroxide (H2O2) which can be detrimental to proteins, lipids and nucleic acids3,4. H2O2 accumulation under hypoxic conditions has been shown in the roots and leaves of barley (Hordeum vulgare)5 and wheat roots3. In plants, enzymatic and non-enzymatic defense systems are involved in ROS scavenging and detoxification. In enzymatic defense system, superoxide dismutase (SOD) constitutes the first line of defense against ROS by dismutating O2- to H2O2. When plant roots are subjected to waterlogging conditions, SOD activity increases in barley roots5 and remains unaffected in tomato6 and wheat roots3. H2O2 is decomposed by peroxidase (POX) and catalase (CAT). The ascorbate-glutathione cycle is an important and efficient enzymatic defense system for decomposing H2O2 and maintaining the balance of antioxidants.
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This cycle involves several enzymes including ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DAR) and glutathione reductase (GR). APX is the first enzyme in this pathway and its major function is converting H2O2 to H2O. GR is the last step in the pathway playing a crucial role in protection against oxidative stress by maintaining reduced glutathione level7. The anaerobic response of maize root cells studied using two-dimensional electrophoresis has revealed that a set of about 20 anaerobic proteins (ANPs) are synthesized during low oxygen treatment, while synthesis of normal aerobic proteins is drastically repressed. Many of these induced proteins have been subsequently identified as enzymes of the glycolytic and fermentative pathways8. Alcohol dehydrogenase (ADH) is a major anaerobic protein expressed under hypoxic/anoxic conditions. Its activity is critical for the recycling of NADH for the continuation of glycolytic pathway9. Pyruvate decarboxylase (PDC), glyceraldehyde-3-phosphate dehydrogenase, aldehyde dehydrogenase (ALDH) and sucrose synthase10 are the other important ANPs. Development of excess moisture tolerant genotypes would be an ideal and affordable approach, suitable for poor maize growing farmers in waterlogging prone marginal areas. In this study, we have examined whether antioxidant defence enzymes and anaerobic proteins be exploited as markers to identify waterlogging tolerant and susceptible genotypes or are involved in waterlogging tolerance in maize by studying the status of antioxidants and antioxidants and anaerobic enzymes during seedling growth of tolerant and susceptible genotypes under waterlogging conditions.
technique (Cup method) was standardized against waterlogging stress. Disposable plastic cups (250 cm3) were used to grow maize seedlings. Cups were filled with 220 cm3 of its volume with mixture of farm yard manure (FYM) and siphoned soil (1:1 V/V) and filled cups were placed in plastic trays (40 × 28 × 6 cm). After 7 days of normal growth, when second leaf was fully expanded, the seedlings were subjected to waterlogging conditions by filling the cups with water 3 cm above the surface of the soil and this water level was maintained in the cups till seedling death. This screening experiment was repeated three-times and on the basis of survival of the seedlings under stress, the genotypes were selected as tolerant and susceptible (Fig. 1a & b). Five genotypes (LM5, Parkash, PMH2, JH3459 and LM14) were selected for further study, of which LM5 and Parkash were found tolerant and PMH2, JH3459 and LM14 were found susceptible to waterlogging stress. For further experiments, these five genotypes were grown by the above method for 7 days and after which 72 h waterlogging treatment was given. On the other hand, control plants were provided normal moisture throughout the experiment. After 72 h waterlogging, the roots were washed free
Materials and Methods Screening and plant material
The germplasm of maize was provided by the Maize Section, Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana. The seeds were surface-sterilized with 0.1% mercuric chloride for 5 min and then washed with double-distilled water before use. Experiments were conducted in a plant growth chamber (NSW-193 CALTAN) under controlled temperature of 30 ± 2°C in the dark (12 h) and continuous illumination (12 h) of 200 µmol m-2 s-1 photosynthetically active radiation (PAR). For screening of the germplasm, a
Fig. 1—Effect of waterlogging (72 h) (a): on the growth of maize seedlings; and (b): on root system in maize seedlings
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from soil and used immediately for extraction and assay of the enzymes and estimation of non-enzymatic antioxidants. Extraction and assay of antioxidant and anaerobic enzyme activities
All extractions were conducted in three replicates at 4°C. For enzyme extraction, seedlings from three different cups were taken separately. Each cup constituted one independent replicate. Enzymes were extracted at 4°C. SOD, POX and GR were extracted by homogenizing the samples in 0.1 M phosphate buffer (pH 7.5) containing 1% polyvinylpyrrolidone (PVP), 1 mM EDTA and 10 mM β-mercaptoethanol. CAT and APX were extracted with 0.05 M phosphate buffer (pH 7.5) containing 1% PVP11. Both the homogenates were centrifuged at 10000 g for 20 min and the supernatant was used for assaying. All the enzymes were assayed at 30°C. Components of enzyme assay system, except the enzyme were preincubated at 30°C for 20 min before starting the reaction. Assay of GR contained 0.2 ml of 200 mM potassium phosphate buffer (pH 7.5), 0.1 ml MgCl2 (1.5 mM), 0.1 ml EDTA (0.2 mM), 0.2 ml NADPH (0.025 mM) and 0.2 ml of enzyme extract, followed by 0.2 ml of oxidized glutathione (0.25 mM) in a quartz cuvette12. Decrease in absorbance at 340 nm after an interval of 30 s upto 3 min was recorded. The molar extinction coefficient for NADPH is 6.22 mM¯¹ cm¯¹. GR activity was expressed as nmoles of NADP+ formed min¯¹g¯¹ of fresh weight (FW). Assay system of SOD contained 1.4 ml of 100 mM Tris HCl buffer (pH 8.2), 0.5 ml of 6 mM EDTA, 1 ml of 6 mM pyrogallol and 0.1 ml of enzyme extract was added13. Change in absorbance was recorded at 420 nm after an interval of 30 s upto 3 min. A unit of enzyme activity was defined as the amount of enzyme causing 50% inhibition of auto-oxidation of pyrogallol observed in the blank. Activity of CAT was determined by taking 1.8 ml of 50 mM sodium phosphate buffer (pH 7.5) to which 0.2 ml of enzyme extract was added. The reaction was initiated by adding 1 ml H2O2 and utilization of H2O2 was recorded at an interval of 30 s for 3 min by measuring the decrease in absorbance at 240 nm14. The molar extinction coefficient for H2O2 is 0.0394 mM-1 cm-1. CAT activity was expressed as µmoles of H2O2 decomposed min-1g-1 of FW.
Activity of APX was assayed by taking 1 ml of 50 mM sodium phosphate buffer (pH 7.0), 0.8 ml of 0.5 mM ascorbic acid, 0.2 ml of enzyme extract and 1 ml of H2O2 solution in total volume of 3 ml15. Absorbance was recorded at 290 nm in a spectrophotometer after an interval of 30 s upto 3 min. The molar extinction coefficient of mono dehydroascorbic acid is 2.8 mM-1 cm-1. APX activity was expressed as nmoles of mono dehydro ascorbic acid formed min-1g-1 of FW. For extraction of ADH and ALDH, tissue (0.1 g) was crushed with 2 ml of 100 mM HEPES buffer having 2 mM dithiothreitol (pH 6.5) to fine powder under liquid nitrogen in pre-chilled pestle and mortar. After centrifugation at 10000 g for 15 min at 4°C, supernatant was taken for assay. The activity of ADH and ALDH was determined by the methods described elsewhere16,17. Root tissue (0.1 g) was crushed with 50 mM sodium phosphate buffer (pH 6.5) containing 50 µM thiamine pyrophosphate (TPP), 5 mM MgCl2 and 5 mM β-mercaptoethanol under liquid nitrogen. Homogenate was centrifuged at 10000 g for 15 min at 4oC. Supernatant was used for assaying PDC activity18. Protein content was estimated by the method of Lowry et al19. Extraction and estimation of non-enzymatic antioxidants
Tissue (0.3 g) was homogenized with 2 ml of ice cold sodium phosphate buffer (pH 7.0) using liquid nitrogen. Homogenate was centrifuged at 10000 g for 20 min and H2O2 content was estimated in supernatant20. For estimation of reduced ascorbic acid, tissue (0.1 g) was crushed in 1.5 ml of 5% ice cold metaphosphoric acid and then centrifuged at 10000 g for 10 min. Ascorbic acid content was estimated in the supernatant21. Tissue (0.1 g) was ground and homogenized in 2 ml of 5% sulphosalicylic acid and mixture was centrifuged at 10000 g for 15 min. Glutathione was estimated using 5,5’-dithiobis-2-nitrobenzoic acid (DTNB), NADPH and GR22. Results Changes in activities of enzymatic and nonenzymatic antioxidants and enzymes of anaerobic metabolism were determined after 72 h of waterlogging in the roots at 2-leaf stage in LM5 and Parkash (tolerant) and LM14, PMH2 and JH3459 (susceptible) seedlings.
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Changes in antioxidant enzymes
Waterlogging caused significant decrease in the SOD activity in both tolerant as well as susceptible genotypes (Table 1). Maximum of 8.8-fold decrease was observed in Parkash genotype though other genotypes also showed average 4-fold decrease in their activities with respect to control. The activity of CAT significantly decreased by 2.6-fold and 1.4-fold in Parkash and LM5 and 1.6, 3.5 and 2.6-fold in PMH2, JH3459 and LM14, respectively. Imposition of waterlogging stress led to decline in the activity of APX in both tolerant and susceptible genotypes, however, the reduction was greater in susceptible genotypes than in tolerant ones. For example, an average of 2-fold decrease was observed in susceptible genotypes in comparison with about 1.5-fold in tolerant genotypes under stressed conditions.
In control seedlings, GR activity was greater in tolerant genotypes than susceptible genotypes. However, after waterlogging a in GR activity was observed in tolerant genotypes, but the susceptible genotypes showed slight increase in the activity of GR in comparison with control seedlings (Table 1). Changes in H2O2, glutathione and ascorbic acid
Except PMH2 and JH3459, the remaining genotypes showed decline in H2O2 content under waterlogging when compared with the controlled samples (Table 1). PMH2 showed 1.2-fold increase in H2O2 content under waterlogging. All the genotypes showed increase in glutathione content after waterlogging treatment. Maximum 3.2-fold increase was observed in PMH2. Tolerant genotypes showed increase in ascorbic acid content
Table 1—Changes in antioxidant enzymes, antioxidants, H2O2 and anaerobic enzymes after 72 h of waterlogging treatment in maize roots in tolerant (LM5 and Parkash) and susceptible (PMH2, JH3459 and LM14) genotypes LM5 PARKASH Antioxidant __________________ ___________________ enzymes Control Stressed Control Stressed
PMH2 ___________________ Control Stressed
JH3459 __________________ Control Stressed
LM14 _____________________ Control Stressed
2475.8±20.5 2053.6±39.2 2774.2±24.2 1557.1±14.3a 2715.6±34.4 1502.7±75.9a 3250.0±35.7 1528.6±14.3a 2302.3±86.6 1183.9±69.6a (236.7±7.4) (313.7±30.9) (262.3±8.8) (173.5±4.4)a (102.1±5.7) (210.4±18.1)a (135.6±13.5) (232.0±5.1)a (135.2±8.9) (126.9±16.3) 409.9±8.5 294.8±14.8 503.3±15.9 150.6±6.2a (44.1±5.1) (37.1±2.8) (43.2±2.3) (13.4±1.9)a
Antioxidants and H2O2 H2O2
Glutathione 6.0±1.2 Ascorbic 62.5±3.0 acid Anaerobic enzymes
548±16.0 634±15.9a 522±8.4 792±19.3a (189.8±8.3) (352.6±14.1) (114.1±12.2) (159.9±3.4)a
758±12.9 758±25.2 (133.1±12.7) (114.9±18.2)
Values represent mean ± SD of three replicates. Values without parentheses represent enzyme activity g-1 fresh weight (FW). Values within parentheses represent specific activity mg-1 protein. One enzyme unit of superoxide dismutase (SOD) is the quantity of enzyme which inhibits its activity by 50%. Glutathione reductase (GR) and ascorbate peroxidase (APX) activities are expressed as nmoles of product formed min-1 g-1 FW, catalase (CAT) activity as µmoles of H2O2 decomposed min-1 g-1 FW, alcohol dehydrogenase (ADH) activity as nmoles of NAD formed min-1 g-1 FW, aldehyde dehydrogenase (ALDH) activity is as nmoles of NADH formed min-1 g-1 FW and pyruvate decarboxylase (PDC) activity as nmoles of NAD formed min-1 g-1 FW. Glutathione and ascorbic acid content is expressed in nmoles g-1FW. H2O2 content is expressed in µmoles g-1 FW. a- significant at 1% level as compared to control. (Student’s t-test).
INDIAN J. BIOCHEM. BIOPHYS., VOL. 48, OCTOBER 2011
(about 2-fold for LM5 and 1.5 for Parkash), but in susceptible genotypes, it was either decreased (3.5-fold in PMH2) or remained unchanged in comparison with control (Table 1). Changes in anaerobic enzymes
In comparison with control, significant increase was observed in ADH activity in tolerant as well as susceptible genotypes under waterlogging. Maximum 9.8-fold increase was observed in JH3459 and minimum 2.6-fold increase was observed for LM14 genotype under waterlogging conditions (Table 1). Response of ALDH was different in tolerant and susceptible genotypes. A significant increase in ALDH was observed in tolerant genotypes only, however, susceptible genotypes showed decrease in activity with maximum 2.6-fold decrease in LM14 under stressed conditions. The activity of PDC also increased in tolerant genotypes after waterlogging stress, but it either decreased (PMH2 and LM14) or remained unchanged (JH3459) in susceptible genotypes (Table 1). Discussion Anoxia-induced changes in plant roots represent combined effects of two stresses – anoxic stress itself and oxidative stress, which occur during soil waterlogging. The major cause of oxidative stress is disability of the scavenging system to metabolize the toxic active oxygen due to either increased ROS formation or decreased activity of the scavenging enzymes. SOD disproportionates superoxide to H2O2 and dioxygen and this class of enzyme is found in almost all aerobic organisms. Intracellular level of H2O2 is regulated by a wide range of enzymes, the most important being CAT and POX. In addition the ascorbate-glutathione cycle is also important. In our experiments, we found a significant decrease in activity and specific activity of SOD in roots of all the genotypes, susceptible as well as tolerant (Table 1). A similar decrease in SOD was observed in barley leaves when subjected to soil waterlogging23. An excessive accumulation of superoxide due to reduced activity of SOD under waterlogging has been reported in corn leaves24. A decline in activity of SOD in wheat roots under anoxia has also been reported3. The ascorbate-glutathione cycle is an effective system in the detoxification of H2O2. The activity of APX and GR is important in determining the efficiency of this pathway. Waterlogging caused decline in activity of APX in both susceptible and
tolerant genotypes, whereas GR activity was decreased only in tolerant genotypes (Table 1). This caused imbalance in APX to GR ratio in susceptible genotypes which declined from 7.4 to 10.4 in control seedlings to 2.1 to 4.3 in waterlogged genotypes. In tolerant genotypes, the APX/GR ratio showed increasing/unchanged trend under waterlogging conditions. Significant reduction in APX/GR ratio might be the cause of susceptibility of maize genotypes to waterlogging. A greater decrease in APX activity in the roots of creeping bent grass, susceptible to waterlogging in comparison with tolerant genotypes has been reported25. APX activity could serve as criteria for evaluating the waterlogging tolerance of tomato and egg plant roots6. Waterlogging does not affect the GR activity in tomatoes and egg plant6 and bent grass roots25, whereas inhibition of GR activity is also reported in wheat roots under waterlogging stress24. Waterlogging treatment caused decrease in CAT activity in tolerant as well as susceptible genotypes. Downregulation of expression of peroxisomal catalase A in Saccharomyces cerevisiae under anoxia conditions has been reported26. The activity of this enzyme in Oryza sativa seedlings germinated under water for 6 days is also found to decline significantly27. A decreased H2O2 content in roots of pigeonpea plants after 2 days waterlogging suggests that this reduction could be due to shift from aerobic respiration to fermentation, as in non-green tissues mitochondrial electron transfer chain is the main site for ROS production28. This might be the reason for the decline of H2O2 in majority of maize genotypes under waterlogging conditions (Table 1). A significant increase was observed in glutathione content of tolerant as well as susceptible genotypes. But only tolerant genotypes showed increase in ascorbic acid content and a decrease was observed in susceptible genotypes due to waterlogging (Table 1). An increase in ascorbic acid and glutathione content has been reported in tolerant plants only29. Also, an investigation on the antioxidant defense system in the roots of wheat seedlings under hypoxia or whole plant anoxia has revealed a significant increase in the reduced form of ascorbate and glutathione30. Under oxygen deficiency, ATP formation through oxidative phosphorylation is inhibited and ATP has to be produced through fermentation31. These changes lead to low oxygen consumption. Waterlogging caused a significant increase in activities as well as
CHUGH et al.: ROLE OF ANTIOXIDANT AND ANAEROBIC METABOLISM ENZYMES
specific activity of ADH, ALDH and PDC in tolerant genotypes, however, in susceptible genotypes only ADH activity increased, the other two enzymes were either decreased or unaffected (Table 1). Plants which are more waterlogging tolerant have been reported to possess more effective alcoholic fermentation pathway32. Ethanol is the main end product of anaerobic metabolism in plants. High ADH activity may be required to prevent accumulation of potentially toxic acetaldehyde33. During alcoholic fermentation, ADH is responsible for the recycling of NAD+ needed for the glycolysis to continue. Relationship between survival percentage and acetaldehyde production after submergence is significantly negative in rice plants34. Studies of adh1 mutants of maize and Arabidopsis have shown that ADH activity is necessary for short-term waterlogging resistance35. PDC is perhaps the most important branching point in anaerobic metabolism and catalyzes decarboxylation of pyruvate, yielding CO2 and acetaldehyde (precursor of ethanol). It has been suggested to be a rate-limiting enzyme for ethanol synthesis from pyruvate under anoxic conditions33,36. High level of ADH and PDC may be required to prevent the accumulation of potentially toxic acetaldehyde33. The increased PDC activity results in an increased carbon flow through the ethanol pathway with increase in both acetaldehyde and ethanol pool sizes. This ultimately results in increased ATP and NAD+ production. Overexpression of ADH or PDC has been shown to improve low-oxygen stress resistance in Arabidopsis37. Sustained ATP production through ethanolic fermentation, unlike lactic acid fermentation is beneficial as it does not lead to cytoplasmic acidosis. Expression analysis studies have also revealed the increased transcription of genes that are involved in the alcoholic (pyruvate decarboxylase 1, Pdc1, Pdc2 and adh1) and lactic acid fermentation pathways (ldh1), when external oxygen concentration is decreased to 5%38. This can be interpreted as a pre-adaptation that allows continued energy production during a subsequent period of anoxia. Accumulated ethanol in the roots can be remetabolized to acetaldehyde and then converted to acetate and acetyl-CoA with the enzymes ALDH and acetyl-CoA synthase39. Overexpression of a stress inducible acetaldehyde dehydrogenase gene (Ath-ALDH3) appears to be a constitutive
detoxification mechanism that limits acetaldehyde accumulation and oxidative stress, thus revealing a novel pathway of detoxification in plants40. In conclusion, the results suggested that a better alcoholic fermentation system might be more important for the survival of seedlings under hypoxia conditions than antioxidant system. This was reflected by increase in ethanolic fermentative enzymes (i.e. PDC, ADH and ALDH) in tolerant genotypes LM5 and Parkash. The results imply that root system of tolerant genotypes is more efficient in converting ethanol to acetaldehyde and finally detoxifying the toxic acetaldehyde to acetate under waterlogging conditions. The study provides a mechanism which involves efficient upregulation of entire anaerobic metabolic machinery for providing tolerance against waterlogging. References 1 2
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