ISSN 1021-4437, Russian Journal of Plant Physiology, 2013, Vol. 60, No. 5, pp. 623–632. © Pleiades Publishing, Ltd., 2013. 4 623 1 INTRODUCTION Tribulus terrestris L. is a flowering plant of the fam- ily Zygophyllaceae, native to warm temperate and tropical regions of the Old World in southern Europe, southern Asia, throughout Africa, and Australia. T. terrestris plant extracts contain saponins, fla- vonoids, amides, and alkaloids [1]. The biologically active furostanol saponin fraction of this plant finds application in the contemporary medicine as a com- 1 This text was submitted by the authors in English. ponent 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 concentra- tions 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-pol- luted agricultural soils, because possess a significant EDTA Reduces Heavy Metal Impacts on Tribulus terrestris Photosynthesis and Antioxidants 1 Y. Markovska a , M. Geneva b , P. Petrov b , M. Boychinova b , I. Lazarova c , I. Todorov b , and I. Stancheva b a Faculty of Biology, Sofia University “St. Kliment Ohridski”, 8 D. Tzankov Blvd., 1164 Sofia, Bulgaria b 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 c Faculty of Pharmacy, Department of Chemistry, Medical University of Sofia, Bulgaria Received August 21, 2012 Abstract—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 CO 2 for enhanced CO 2 assimilation and providing NADPH for the antioxidant system. Antioxidants, such as ascorbate, reduced glutathione, and flavonoids, increased more in the shoots of T. ter- restris after the addition of EDTA. The activities of guaiacol peroxidase, catalase, and the enzymes of the ascorbate–glutathione cycle enhanced significantly in the presence of EDTA. Increased activities of antiox- idant 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 α-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, protot- ribestin, 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: 10.1134/S1021443713050117 Abbreviations: AGC—ascorbate–glutathione cycle; ASC— ascorbic acid; CAT—catalase; DHASC—dehydroascorbic acid; GPX—glutathione peroxidase; GR—glutathione reductase; GSH—reduced; GSSG—oxidized glutathione, GST—glu- tathione S-transferase; HM—heavy metal; NADP-ME— NADP-malic enzyme; PAL—phenylalanine ammonia-lyase; PEPC—phosphoenolpyruvate carboxylase; PPDK—pyruvate orthophosphate dikinase; SOD—superoxide dismutase. RESEARCH PAPERS