LÍLIAN MARIA VINCIS PEREIRA SANGLARD THE NEGATIVE IMPACTS OF ARSENIC ON PHOTOSYNTHESIS OF RICE LEAVES ARE ALLEVIATED BY SILICON SUPPLEMENTATION, WITHOUT UP-REGULATION OF THE ANTIOXIDANT CAPACITY Dissertation submitted to Federal University of Viçosa, as part of the requirements for obtaining of Magister Scientiae degree in Plant Physiology. VIÇOSA MINAS GERAIS – BRASIL 2014 LÍLIAN MARIA VINCIS PEREIRA SANGLARD THE NEGATIVE IMPACTS OF ARSENIC ON PHOTOSYNTHESIS OF RICE LEAVES ARE ALLEVIATED BY SILICON SUPPLEMENTATION, WITHOUT UP-REGULATION OF THE ANTIOXIDANT CAPACITY Dissertation submitted to Feder al University of Viçosa, as part of the requirements for obtaining of Magister Scientiae degree in Plant Physiology. ATTESTED IN: February 26 th, 2014. __________________________________ ____________________________________ Wagner Luiz Araújo Kelly da Silva Coutinho Detmann ___________________________________ Fábio Murilo DaMatta (Adviser) To my mother Dayse and my father Ferdinand and my sister Letícia, for all their love, sacrifice and constant support, always present in my heart With all my love to my grandfather Magela and my grandmother Ada ii ACKNOWLEDGMENTS I thank God for everything. Thanks are also due to the Federal University of Viçosa (UFV), especially to the Plant Biology Department, the graduate program in Plant Physiology and the Coordination of Improvement of Higher Education (CAPES) for granting a scholarship. I am indebted to Professor Fábio Murilo DaMatta for his valuable advice, guidance, patience, friendship and opportunity despite my limitations and difficulties. Thanks are also due to Professors Wagner Araújo, Juraci Oliveira, Adriano Nunes Nesi, Marcelo Rogalski and Raimundo Barros for their contributions to my professional education. I am grateful to all my colleagues in the Graduate Program in Plant Physiology at UFV, especially my friends Kelly, Leandro, Mariela, Alice, Samuel, Paulo, Alyne, Paulo Cavatte and Medina. I am also grateful to the following members of undergraduate programs at UFV: Josimar, Lucas and Rodrigo, for their help and encouragement during this time. Thanks also go to Carlos Raimundo, Geraldo (Marreco), Osvaldo, Reginaldo, Rogério, Zé Antônio, Mercês and Luciene for their help and collaboration. Thanks to my friends Carol, Marina, Natália, Kallyne, Alberto, Fred and Tales for their friendship and collaboration. Last but not least, thanks are due to all my colleagues who contributed to this work and were not mentioned here. iii BIOGRAPHY LÍLIAN MARIA VINCIS PEREIRA SANGLARD was born in Itaperuna-RJ, Brazil, on May 29th, 1990. In 2012, she graduated in Biology at Federal University of Viçosa, Viçosa-MG, Brazil. In March 2012, she began her Master Scientiae course in the Plant Physiology Program at the Federal University of Viçosa, Viçosa-MG, Brazil. iv CONTENT RESUMO…………………………………………………………………………...…. vi ABSTRACT……………………..……………………………………………………. vii The negative impacts of arsenic on photosynthesis of rice leaves are alleviated by silicon supplementation, without up-regulation of the antioxidant capacity. 1. Introduction……………………………………………………………….……. 1 2. Materials and Methods…………………………………………………………. 2 2.1. Plant material, growth conditions and experimental design……………. 2 2.2. Si and As concentrations………………………………………..……… 3 2.3. Photosynthetic measurements…………………………………...……… 3 2.4. Biochemical assays……………………………………….…………….. 5 2.5. Statistical analysis………………………………………………………. 6 3. Results……...……………………………………………………………………… 6 3.1. Si concentrations are increased, whereas As concentrations are decreased upon Si supplementation……………………………………………….. 6 3.2. The negative effects of As on photosynthetic rates may be reversed by Si in a genotype- and time-dependent manner…………………………….. 7 3.3. Photochemical events are minimally affected by Si and As levels…..… 8 3.4. The antioxidant capacity increases upon As addition, particularly in plants not treated with Si……………………………………………….. 8 4. Discussion………………….....…………………………………………………… 9 5. References............................................................................................................... 12 v RESUMO SANGLARD, Lílian Maria Vincis Pereira, M.Sc., Universidade Federal de Viçosa, fevereiro de 2014. Impactos negativos do arsênico na fotossíntese em folhas de arroz são mitigados pela suplementação de silício, sem aumento da capacidade antioxidante. Orientador: Fábio Murilo DaMatta. O silício (Si) desempenha um papel importante em mitigar vários estresses abióticos, possivelmente por minimizar o dano oxidativo induzido por estresse. Em arroz (Oryza sativa), acredita-se que o arsênio (As) possa causar estresse oxidativo compartilhando com o Si a mesma via de entrada nas raízes. Apesar dos grandes avanços na compreensão dos mecanismos de absorção de As e como estes podem ser afetados por Si, os mecanismos fisiológicos pelos quais o Si pode mitigar a toxicidade do As em plantas ainda precisam ser esclarecidos. Neste trabalho, avaliaram-se os efeitos isolados e combinados de Si e arsenito [As(III)] em plantas de arroz, usando um genótipo selvagem e o mutante lsi1 (low silicon rice 1) defectivo para a absorção de Si, no que diz respeito às trocas gasosas e parâmetros de fluorescência da clorofila a e aos danos celulares. A presença de As(III) levou a uma diminuição da fixação de carbono, fato não relacionado com limitações estomáticas e fotoquímicas, mas associada à diminuição da condutância mesofílica. Esta redução pôde ser revertida ao longo do tempo de forma considerável pela presença do Si, nos dois genótipos. Entretanto, os efeitos benéficos do Si em plantas tratadas com As(III) não apresentaram relação direta com o aumento da regulação da capacidade antioxidante. A fertilização com Si pode ser importante tanto na tentativa de diminuir as concentrações de As(III) quanto para melhorar o desempenho fotossintético de plantas de arroz contaminadas com As, o que pode resultar em uma melhor produtividade da cultura, além de promover aumento da segurança alimentar. vi ABSTRACT SANGLARD, Lílian Maria Vincis Pereira, M.Sc., Universidade Federal de Viçosa, February, 2014. The negative impacts of arsenic on photosynthesis of rice leaves are alleviated by silicon supplementation, without up-regulation of the antioxidant capacity. Adviser: Fábio Murilo DaMatta. Silicon (Si) plays important roles in alleviating various abiotic stresses, possibly by counteracting stress-induced oxidative damage. In rice (Oryza sativa), arsenic (As) is believed to cause oxidative stress and to share the Si transport pathway for entry into roots. Despite much progress in understanding the mechanisms underlying the uptake of As and how they can be affected by Si, the physiological mechanisms through which Si might alleviate As toxicity in plants remain to be elucidated. We combined gas exchange and chlorophyll fluorescence measurements with analysis of the activity of the antioxidant system to examine the effects of Si nutrition on photosynthetic performance and oxidative stress in rice plants (a wild-type cultivar and its lsi1 mutant defective in Si uptake) challenged with arsenite. Arsenite treatment led to an impairment of carbon fixation that was unrelated to stomatal and photochemical limitations but, rather, was associated with decreased mesophyll conductance. This impairment could be reverted to a considerable extent by Si in a time- and genotype- dependent manner. The ameliorative effects of Si on As-treated plants were uncoupled from any noticeable up-regulation of the antioxidant capacity. We identified Si nutrition as an important target in attempts to not only decrease As concentrations but also to improve the photosynthetic performance of rice plants challenged with As, which may ultimately result in better crop yield coupled with enhanced food safety. vii 1. Introduction Pollution with transition metals and metalloids is an increasing environmental problem, and arsenic (As), in particular, is highly toxic for all forms of life, including plants [1]. Plants take up arsenate [As(V)], the predominant form of As in aerated soils; however, in reducing environments such as in paddy soils, arsenite [As(III)] becomes the predominant chemical species of As [2]. The main pathway of As(V) uptake in plants is through phosphate transporters, whereas As(III) is believed to be taken up through the nodulin 26-like intrinsic aquaporin channels [3]. The accumulation of As in plants negatively impacts their morphology and physiology by inhibiting root and shoot growth, generating reactive oxygen species (ROS), decreasing photosynthetic activity and altering carbohydrate and amino acid metabolism [4,5,6]. Plants exhibit a range of As detoxification mechanisms, including metal transport, chelation and sequestration [1,7]. In addition, activation of enzymatic antioxidants, including superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPX), as well as antioxidant molecules, such as ascorbate (AsA) and glutathione (GSH), has been reported to neutralise As-mediated oxidative stress [8]. Failure of the antioxidant defence system may result in damage when metabolites and components of the cellular machinery react with ROS, resulting in lipid peroxidation and oxidation of proteins and nucleic acids [9], thus ultimately impairing photosynthesis and plant growth [10]. Silicon (Si) is not considered an essential element for higher plants, although it has been proven to be beneficial for improving the growth and crop yields of some plant species, such as rice [11]. Studies addressing Si nutrition have extensively demonstrated the ability of Si to alleviate biotic and abiotic stresses in a wide variety of plant species [12,13], including toxicity associated with elements such as aluminium [14], cadmium [15] and arsenic [16]. The ameliorative effect of Si on plants suffering from abiotic stresses is believed to occur, to a large extent, through counteracting oxidative stress via modulating antioxidant enzymes [17]. In rice roots, two genes encoding Si transporters (Lsi1 and Lsi2) have been identified to date [18,19]. These Si transporters are associated with the great ability of rice to actively take up Si and could explain the high Si concentrations observed in this species [11]. Intriguingly, it has been observed that high levels of Lsi1 and Lsi2 1