научная статья по теме EDTA REDUCES HEAVY METAL IMPACTS ON TRIBULUS TERRESTRIS PHOTOSYNTHESIS AND ANTIOXIDANTS Биология

Текст научной статьи на тему «EDTA REDUCES HEAVY METAL IMPACTS ON TRIBULUS TERRESTRIS PHOTOSYNTHESIS AND ANTIOXIDANTS»

ФИЗИОЛОГИЯ РАСТЕНИЙ, 2013, том 60, № 5, с. 661-670

ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ

УДК 581.1

EDTA REDUCES HEAVY METAL IMPACTS ON Tribulus terrestris PHOTOSYNTHESIS AND ANTIOXIDANTS1

© 2013 Y. Markovska*, M. Geneva**, P. Petrov**, M. Boychinova**, I. Lazarova***,

I. Todorov**, I. Stancheva**

* Faculty of Biology, Sofia University "St. Kliment Ohridski", Sofia, Bulgaria ** Department of Plant Mineral Nutrition and Water Relation, Institute of Plant Physiology and Genetics,

Bulgarian Academy of Sciences, Sofia, Bulgaria *** Faculty of Pharmacy, Department of Chemistry, Medical University of Sofia, Bulgaria

Received August 21, 2012

The effects of EDTA application to heavy metal-polluted soil on phytoextraction of heavy metals, leaf anatomy, gas exchange parameters, enzyme activities of C4 carbon cycle, antioxidant defense, and active compounds of Tribulus terrestris L. were evaluated. The addition of EDTA to the soil polluted with Cd and Pb markedly increased dry weight and Pb, Zn, and Cd contents in shoots. Plants responded to the action of EDTA by an increased stomatal conductance, photosynthetic and transpiration rates, water use efficiency, chlorophyll and carotenoid contents. The activities of C4 carbon cycle enzymes simultaneously increased, thus concentrating CO2 for enhanced CO2 assimilation and providing NADPH for the antioxidant system. Antioxidants, such as ascorbate, reduced glutathione, and flavonoids, increased more in the shoots of T. terrestris after the addition of EDTA. The activities of guaiacol peroxidase, catalase, and the enzymes of the ascorbate-glu-tathione cycle enhanced significantly in the presence of EDTA. Increased activities of antioxidant enzymes suggest that they have some additive functions in the mechanism of metal tolerance. EDTA application lowered the activity of phenylalanine ammonia-lyase and the content of total phenols, MDA, hydrogen peroxide, dehydroascorbate, and lipid-soluble antioxidant capacity expressed as a-tocopherol. Increased levels of total radical-scavenging activity are in correspondence with the activity of water-soluble antioxidant compounds in T. terrestris tissues. The content of furostanol saponins protodioscin, prototribestin, and rutin increased as a result of EDTA addition. The results obtained allowed us to assume that applied EDTA reduced a negative heavy metal impact on puncture vine photosynthesis and antioxidant potential.

Keywords: Tribulus terrestris - C4 carbon cycle enzymes - EDTA - heavy metals - leaf anatomy - antioxidant capacity - furostanol saponins

DOI: 10.7868/S0015330313050114

INTRODUCTION

Tribulus terrestris L. is a flowering plant of the family Zygophyllaceae, native to warm temperate and tropical regions of the Old World in southern Europe, southern Asia, throughout Africa, and Australia.

1 This text was submitted by the authors in English.

Abbreviations: AGC - ascorbate—glutathione cycle; ASC - ascorbic acid; CAT - catalase; DHASC - dehydroascoibic acid; GPX - glutathione peroxidase; GR - glutathione reductase; GSH - reduced glutathion; GSSG - oxidized glutathione, GST - glutathione S-transferase; HM - heavy metal; NADP-ME - NADP-malic enzyme; PAL - phenylalanine ammonia-lyase; PEPC - phosphoe-nolpyruvate carboxylase; PPDK - pyruvate orthophosphate diki-nase; SOD - superoxide dismutase.

Corresponding authors: Ira Stancheva. Department of Plant Mineral Nutrition and Water Relation, Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Acad. G. Bonchev str., Bl. 21, 1113 Sofia, Bulgaria. Fax: +359-2-873-9952, e-mail: ira_stancheva@abv.bg

T. terrestris plant extracts contain saponins, flavonoids, amides, and alkaloids [1]. The biologically active furostanol saponin fraction of this plant finds application in the contemporary medicine as a component of drugs effective in treatment impotence and other sexual disorders [2].

T. terrestris grown on industrially polluted soil showed good ability for Cd, Pb, and Zn accumulation and could be used for phytoremediation of polluted soils [3]. The plant biomass production and the heavy metal (HM) concentration in the harvestable biomass are important factors for the practical efficiency of phytoextraction [4]. One strategy to achieve the higher HM removal efficiency is to enhance the concentrations of soluble HMs in the soil with the application of complexing agents [5]. EDTA is widely investigated due to its high complexing capability towards Pb, Cu, Cd, and Zn. That leads to an increase in the metal uptake by plants like Brassica juncea, Helianthus annuus,

and Zea mays [6]. The medicinal plants could be grown as alternatives to edible crops in HM-polluted agricultural soils, because possess a significant phy-toextraction potential, and metal content in essential oils prepared from them is negligible [7].

Puncture vine is characterized as NADP-malic enzyme C4 plant with chlorenchymatous vascular bundle sheath in the leaves and low CO2-compensation point [8]. The information about HM influence on the activities of carboxylating enzymes responsible for the various pathways of CO2 assimilation is scarce. The enzymes phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), and pyruvate orthophosphate dikinase (PPDK) participate in the process of CO2 concentrating in C4 photosynthetic pathway, but their functions appear to be more important for plants under stress growth conditions [9]. The production of NADPH in reactions catalyzed by NADP-ME and Krebs cycle probably helps to establish the redox balance in the early-activated antioxidant defense mechanism [10].

Oxidative stress due to the heavy metal toxicity resulted in the increase in the activities of enzymes that participate in ROS quenching [10]. Not only activities of antioxidant enzymes, such as superoxide dismutase (SOD), peroxidases, catalases (CAT), and enzymes of the ascorbate-glutathione cycle (AGC), but the level of low-molecular quenchers (ascorbic acid, glu-tathione, and tocopherols) is changed during heavy metal treatment [11].

The aim of this study was to evaluate the effects of applied EDTA on the HM phytoextraction, leaf anatomy, gas-exchange parameters, furostanol saponins and also to establish the possible antioxidant mechanisms that could operate in T. terrestris grown in heavy metal-polluted soil. The changes in the activities of the enzymes of C4 carbon cycle (PEPC, NADP-ME, and PPDK) are traced to evaluate their role for NADPH biosynthesis and the activation of antioxidant defense system under conditions of HM pollution. The effects of heavy metal accumulation on plant productivity and quantity of furostanol saponins are tested in order to use this medicinal plant as an alternative crop for purification of polluted agricultural soils.

MATERIALS AND METHODS

Plant material and sampling. Tribulus terrestris L. plants were grown from seeds (kindly provided by Dr. Ivan Salamon from Preshov University in Slovakia) in a climatic chamber at a 12-h photoperiod, a day/night temperature of 25/18°C, and photon flux density of 95 ^mol/(m2 s) until the 21st day. The seedlings were transferred to 5-kg plastic pots (2 plants per pot) and grown for 3 months until early fruit ripeness stage on the soil/sand substrate in the ratio 3 : 1. All pots were adjusted daily to 60% water holding capacity. There were three replicates of each treatment.

The polluted soil was taken from the field near waste depository of Kremikovtzi ferrous metallurgical combine and have the following agrochemical characteristics: pH

(H2O) = 7.94, soil total mobile nitrogen (N-N O- +

N-NH+) - 10 mg/kg, P2O5 - 536.8 mg/kg, K2O -308 mg/kg soil. The following contents of studied HMs (^g/g dry wt) were measured: Cd - 14, Cu - 27, Pb -142, and Zn - 207. Because the Bulgarian permissible limit concentrations (PLC) at pH (H2O) = 7.94 are Cd < 3.0, Cu < 260, Pb < 80, and Zn < 340 (^g/g dry wt) the soils were heavily polluted with Cd and Pb (the content of Cd and Pb exceeded PLC by 4.6 and 1.8 times, respectively).

Two formulations of foliar fertilizers (Agroleaf®, "Scotts Company", United States) were applied to both treatments by spraying (0.5% solution): (1) Agroleaf® total - N : P : K = 20 : 20 : 20 + chelated microelements, was applied twice during the vegetative growth stage with 20-day intervals until the seed formation phase; (2) Agroleaf® with high P - N : P : K = 12 : 52 : 5 + chelated microelements, was applied at full blossoming and seed formation stages.

The following treatments were studied: (1) foliar fed control plants grown on HM-polluted soil (HM); (2) foliar fed plants grown on HM-polluted soil with the addition of 3 mmol/kg soil of EDTA disodium salt (HM + EDTA). According to Huang et al. [12], the appropriate EDTA concentration used as a chelate in phytoremediation for field trials is 1.3-6.7 mmol/kg soil. That is why we choose EDTA in the rate of 3 mmol/kg soil for our pot experiments with soil substrate.

Heavy metal analysis. The plant samples were digested in a solution containing HNO3 and HClO4, 3 : 1 (v/v). The samples were heated at 200° C to evaporate them to dryness. The residue was taken up in 25 mL of 1 N HCl. Metal concentration were determined on the inductively-coupled Plasma Mass Spectrometer (CCD Simultaneus ICP OES, "Varian", Austria).

Light microscopy. Samples from the central area of the leaf blade were taken and fixed in 3% glutaralde-hyde in 0.2 M phosphate buffer (pH 7.2) and embedded in low-viscosity Spurr epoxy resin. Semi-thin paradermal sections (ultramicrotome, "Tesla", Czech Republic) were stained with 0.01% (w/v) toluidine blue and observed under a light microscope ("Carl Zeiss", Germany). Microscopic images of the cross sections of the leafwere captured and saved on a digital image processor (667 EI Camino Real, "International Micro-Vision", United States). For the statistical analysis, leaf thickness was evaluated in cross sections obtained from four leaves per treatment an

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