RESEARCH PAPER Tetracycline accumulates in Iberis sempervirens L. through apoplastic transport inducing oxidative stress and growth inhibition G. Di Marco 1 , A. Gismondi 1 , L. Canuti 1 , M. Scimeca 2 , A. Volpe 2 & A. Canini 1 1 Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy 2 Department of Biomedicine and Prevention, University of Rome ‘Tor Vergata’, Rome, Italy Keywords Antibiotic bioaccumulation; apoplastic transport; confocal microscopy; environmental contamination; oxidative stress; tetracycline; transmission electron microscopy. Correspondence A. Canini, Department of Biology, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica 1, 00133 Roma, Italy. E-mail: canini@uniroma2.it Editor E. Pichersky Received: 16 July 2013; Accepted: 23 July 2013 doi:10.1111/plb.12102 ABSTRACT Environmental antibiotic contamination is due mainly to improper and illegal disposal of these molecules that, yet pharmacologically active, are excreted by humans and animals. These compounds contaminate soil, water and plants. Many studies have reported the bioaccumulation of antibiotics in plants and their negative effects on photosynthesis, cell growth and oxidative balance. Therefore, the principal objective of this paper was the study of antibiotic accumulation sites in plants and its uptake modality. Iberis sempervirens L., grown in soil and in agar in the presence or absence of tetracycline, were used as a model system. Using confocal and transmission electron microscopy, we demonstrated that tetracycline was absorbed and propagated in plants through apoplastic transport and also accumulated in intercellular spaces. Tetracy- cline was rarely detected inside cells (in cytoplasm and mitochondria where, coherent to its pharmacological activity, it probably affected ribosomes), except in stomata. Moreover, we verified and clarified further the phytotoxic effects of tetracycline on plants. We observed that the antibiotic induced a large reduction in plant growth and development and inhibition of photosynthetic activity. As tetracycline may lead to oxidative stress in plants, plant cells tried to balance this disequilibrium by increasing the amount and activity of some endogenous enzyme antioxidant agents (superoxide dismutase 1 and catalase) and levels of antiradical secondary metabolites. INTRODUCTION Over the last 50 years, public awareness of the long-term adverse effects of antibiotics on human health and ecology has greatly increased, despite scientific evidence (Halling-Sørensen et al. 1998; Zuccato et al. 2006; Cox & Wright 2013; Xue et al. 2013). Tetracycline (TC) is a broad-spectrum antibiotic largely employed in veterinary and human medicine for the treatment and control of a wide variety of bacterial infections (Baguer et al. 2000). Environmental contamination with pharmaceuti- cal residues is determined in different ways, but essentially is due to wastewater discharge and agricultural application of sewage sludge and animal manure (Hirsch et al. 1999). In par- ticular, intensive animal farming implies considerable use of antimicrobial drugs. These compounds are given to livestock as ‘medicated’ feed additives in order to prevent diseases (Kolpin et al. 2002). Since only a small percentage of antibiotics are absorbed or degraded through metabolism, they are excreted with urine and faeces, as a mixture of bioactive molecules, in sewage systems and consequently in the environment (Carballa et al. 2004; Glassmeyer et al. 2005). However, the introduction of such chemicals in the environment can be also associated with animal waste application in field manuring because anti- biotics generally remain stable for a long time during storage (Halling-Sørensen et al. 1998; Jørgensen & Halling-Sørensen 2000). These molecules contaminate the soil, plants and waters, causing problems to aquatic organisms, animals, microorgan- isms, humans and plants (Kumar et al. 2005b). In fact, antibi- otics can induce development of bacterial resistance (mutation in genes of ‘resistance’, transfer of resistance genes among microorganisms, increase in selection pressure for resistant organisms) and alterations in microbial communities, with consequent alteration of nutrient cycles and decomposition of organic matter (Jensen 2001). In humans, antibiotics can cause chronic or acute toxic effects or allergic reactions, e.g. intoler- ance, urticaria, anaphylactic shock, bronchial asthma and angioedema, because of overdose or low-level prolonged expo- sure (Sungpyo & Aga 2007). Finally, antibiotics can affect plants through inhibition of photosynthesis, consistent decreases in growth, reduction in leaf length, reduction in root number and elongation and an increase in levels of reactive oxygen species (ROS) (Kapustka 1997). It is reported that plants grown on contaminated soils absorb antibiotics and accumulate them in different organs, but the uptake modality, the storage sites and the processing of these drugs in cells are not yet documented. Moreover, the major concern about anti- biotic pollution in plants is relative contamination of the food supply and consequent health risks (Migliore et al. 1996, 2010; Kumar et al. 2005a; Dolliver et al. 2007). The aim of this work was to study the interaction between Iberis sempervirens L. and TC. We chose this plant as a model system because it belongs to Cruciferae, like Arabidopsis thaliana (L.) Heynh, the most Plant Biology © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands 1 Plant Biology ISSN 1435-8603