ФИЗИОЛОГИЯ РАСТЕНИЙ, 2013, том 60, № 5, с. 691-699
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
ARSENIC-INDUCED CHANGES IN GROWTH AND ANTIOXIDANT METABOLISM OF FENUGREEK1
© 2013 D. Talukdar
Plant Cell and Stress Biology laboratory, Department of Botany, R.P.M. College, University of Calcutta, Uttarpara, West Bengal, India Received August 2, 2012
The effects of arsenic (As) on growth and antioxidant metabolism of fenugreek (Trigonella foenum-graecum L. cv. Azad) plants were studied using 10, 20, and 30 mg As/kg of soil in a pot experiment under controlled conditions. The length and dry weights of shoots and roots, photosynthetic traits, and grain yield components exhibited a significant increase over control (0 mg As/kg) at As20 but decreased markedly at As30. The cause of this completely reverse response of plant growth between As20 and As30 was investigated in the backdrop of H2O2 metabolism by analyzing responses of three prominent antioxidant enzymes, namely superoxide dis-mutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) along with cellular ascorbate pool and its redox state. Despite a significant increase in the H2O2 content in both As20 and As30 plants, the former, unlike As30 plants, did not experience any type of As-induced oxidative stress (membrane lipid peroxidation, electrolyte leakage). Normal to high levels of leaf APX, CAT, and redox pool of ascorbate effectively balanced the elevated SOD activity at As20, maintaining the H2O2 concentration higher than control but obviously in favor of As20 plant growth. By contrast, soil amendment with phosphorus (200 mg P/kg) at As30 prevented As-induced oxidative stress through the reduction of the H2O2 level even below As0. The increased enzyme activity was mainly due to the induction of unique Cu/Zn, Fe, and Mn isoforms of SOD and APX-3/APX-4 and/or their increased expression in coordination with CAT. The detection of novel isoforms suggests a strong response of H2O2-metabolizing enzymes against As-induced oxidative stress in fenugreek.
Keywords: Trigonella foenum-graecum - arsenic - hydrogen peroxide - antioxidant defense - isozymes -growth - oxidative stress
DOI: 10.7868/S0015330313050138
INTRODUCTION
The metalloid arsenic (As) is a widely distributed environmental toxicant. The impact of irrigation with high As-contaminated groundwater on soil and crop has drawn huge attention due to As transfer to the food chain via the groundwater-plant-soil system [1, 2]. Major crops, such as cereals and legumes, grown in As-contaminated fields accumulate substantial amounts of As in their edible parts that may pose huge health risks [3, 4].
Accumulating findings suggest that plant exposure to As leads to ROS generation through the conversion
1 This text was submitted by the author in English.
Abbreviations: APX - ascorbate peroxidase; AsA - ascorbate (reduced); CAT - catalase; Chl - chlorophyll; DHA - dehy-droascorbate; NBT - nitroblue tetrazolium; NEM - N',N-ethyl-maleimide; PPFD - photosynthetic photon flux density; SOD -superoxide dismutase.
Corresponding authors: Dibyendu Talukdar. Plant Cell and Stress Biology laboratory Department of Botany, R.P.M. College, University of Calcutta, Uttarpara, Hooghly 712258, Wfest Bengal, India. Fax: +91033-2663-4155; e-mail: dibyendutalukdar9@gmail.com
of arsenate to highly toxic arsenite [5]. The ascorbate (AsA) is an extremely important non-enzymatic antioxidant, which regulates scavenging of H2O2, a predominant ROS [6-8]. Among the enzymatic compounds, SOD constitutes the first line of defense against ROS, but it generates H2O2 during dismutation of superoxide radicals mainly by the action of its membrane-bound Cu/Zn isoforms [6]. This H2O2 is readily scavenged by ascorbate peroxidase (APX) exclusively using AsA as its co-factor within the AsA-GSH cycle and by catalases (CAT) outside this cycle [6]. These three prominent H2O2-metabolizing enzymes hold key in controlling H2O2 level during the onset of oxidative stress in plants [9, 10].
Fenugreek (Trigonella foenum-graecum L.) is cultivated as an annual legume in several countries for diverse edible and medicinal purposes [11, 12]. However, vast areas of As-contaminated regions have been used for fenugreek cultivation without performing any assessment of its toxic effect [11]. In a recent study, Talukdar [4] observed that As treatment significantly decreased seed germination and early seedling growth of
fenugreek. Sporadic reports are available regarding major reduction in the growth, nutrients, and yield of different legumes due to exposure to As [2, 5, 13-15]. Being chemically similar to phosphate (P), As competes with P for the uptake system and inhibits growth [14, 15]. Despite increasing exposure to As, legumes in general have drawn little attention regarding As effect compared to cereals and fern species. Furthermore, our understanding of the growth and antioxidant response of fenugreek plants to ROS, particularly H2O2, during As exposure is extremely limited. The present investigation was, therefore, performed to investigate: (1) the effects of As on plant growth and seed yield parameters, (2) the effect of P application to soil prior to seed sowing, and (3) As-induced oxidative stress and alteration in the activities of three major H2O2-metab-olizing enzymes, SOD, APX, and CAT, as well as the AsA redox status in leaves and roots of As-treated fenugreek plants. In-gel activity of enzymes was determined by native PAGE analysis under control and As-treated conditions.
MATERIALS AND METHODS
Experimental design and As treatment. Dry, uniform, healthy, and equal sized seeds of fenugreek (Trigonellafoenum-graecum L. cv. Azad) were surface-sterilized in 1% mercuric chloride for 15 min followed by 70% ethanol wash for 5 min, washed three times in sterile distilled water, and then placed in Petri dishes containing sterile half-strength MS medium for germination under 14-h photoperiod at 25°C. Germinated seeds were immediately transplanted to plastic pots of equal sizes (20 cm in height and 20 cm in diameter) with six seedlings per pot. Air-dried soil (5 kg) was put in each pot. The soil (top soil of 0-20 cm of Alfisols sampled from Gangetic West Bengal, India, 15 m above sea level, 88.35° E/22.67° N, clay-alluvial brown soil, pH 6.3, soil : water ratio of 1 : 2.5, organic matter 5.17 g/kg soil, Kjeldahl N 750 mg/kg soil, POlsen 14.18 mg/kg soil, available K 77.67 mg/kg, available S 13.80 mg/kg, available As 0.18 mg/kg soil [16]) was mixed, air-dried, and ground to a particle size of <2 mm. Two sets of experiment were designed. In the first set, pot soil was amended with arsenic (as NaAsO2) at the rates of 0 (control treatment), 10, 20, and 30 mg As/kg (As0, As10, As20, and As30, respectively). In the second set, 200 mg phosphorus (P)/kg soil was mixed (as KH2PO4) with each of the four As treatment (As0P200, As10P200, As20P200, and As30P200). Although initially four levels of P (25, 50, 100, and 200 mg/kg soil) were tested with above said four doses of As, the addition of 200 mg/kg soil at As30 only was found to exert significant effect on plant growth and yield traits and thus was selected for the present study. All these treatments were applied and incorporated into the soil before seed sowing. Each treatment had four replicates. The pots were arranged in a completely
randomized way, and the plants were grown in a controlled environment (temperature 27 ± 2°C, relative humidity 70 ± 3%, 14-h photoperiod, PPFD - 200 ^mol/(m2 s)). The plants were ultimately thinned to one per pot. Water content of the soil was maintained at 80% of water-holding capacity. Nine weeks after sowing, shoots and roots were harvested separately. Roots were thoroughly washed with tap water to remove adhering contaminants. Immediately after harvest, shoot and root lengths were measured. Dry weight was determined after drying shoots (leaves + stems + pods) and roots at 70°C for 48 h. Plant height, pod number per plant, 1000 seed weight (g), and seed yield per plant (g) were recorded at harvest.
Chemical analysis. Plant samples were appropriately labeled and dried in an air-oven at 105°C for 24 h. Then a portion (1 g) of the ground samples were digested on a sand bath with a tri-acid mixture (HNO3 : H2SO4 : HClO4 = 10 : 1 : 4, by volume) to obtain a clear digest. The As content in the digested samples was measured by the use of AAS (AA-400, "Perkin Elmer", United States) following the manufacturer's instruction. A standard reference material of tomato leaves (Item number 1573a, National Institute of Standards and Technology, United States) was analyzed in the same procedure as a part of the quality assurance/quality control protocol.
Photosynthetic rate. Leaf photosynthetic rate was assayed following the methods of Coombs et al. [17] using a portable photosynthesis system (LI-6400XT, "LI-COR", United States).
Estimation of chlorophyll and carotenoid contents. Leaf Chl and carotenoid contents were determined by the method of Lichtenthaler [18]. Leaf tissue (50 mg) was homogenized in 10 mL of chilled acetone (80%) and centrifuged at 4000 g for 12 min. Absorbance of the supernatant was recorded at 663, 647, and 470 nm for Chl a, Chl b, and carotenoids, respectively.
Estimation of ascorbic acid. Reduced (AsA) and oxidized ascorbate (DHA) contents were determined by the method of Law et al. [19].
Antioxidant enzyme assay. Fresh tissue (250 mg) was homogenized in 1 mL of 50 mM K-phosphate buffer (pH 7.8) containing 1 mM EDTA, 1 mM DTT, and 2% (w/v) polyvinyl pyrrolidone (PVP) using a chilled mortar and pestle kept in ice bath. The homo-genate was centrifuged at 15000 g at 4°C for 30 min. The clear supernatant was used for enzyme assays. For measuring APX activity, the tissue was separately ground in homogenizing medium containing 2.0 mM AsA in addition to the other ingredients. All assays were done at 25°C with at least four replicates. Soluble protein content was determined according
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