Plant Science 214 (2014) 74–87 Contents lists available at ScienceDirect Plant Science jo u r n al homep age: www.elsevier.com/locate/plantsci The knockdown of chloroplastic ascorbate peroxidases reveals its regulatory role in the photosynthesis and protection under photo-oxidative stress in rice Andréia Caverzan a,b , Aurenivia Bonifacio d , Fabricio E.L. Carvalho d , Claudia M.B. Andrade b,1 , Gisele Passaia a , Mariana Schünemann a , Felipe dos Santos Maraschin c , Marcio O. Martins d , Felipe K. Teixeira a , Rafael Rauber b , Rogério Margis b,e , Joaquim Albenisio Gomes Silveira d , Márcia Margis-Pinheiro a,∗ a Department of Genetics, Federal University of Rio Grande do Sul, Brazil b Biotechnology Center, Federal University of Rio Grande do Sul, Brazil c Department of Botany, Federal University of Rio Grande do Sul, Brazil d Department of Biochemistry and Molecular Biology, Federal University of Ceará, Brazil e Department of Biophysics, Federal University of Rio Grande do Sul, Brazil a r t i c l e i n f o Article history: Received 19 July 2013 Received in revised form 12 September 2013 Accepted 1 October 2013 Available online 8 October 2013 Keywords: Oryza sativa Rice APX Ascorbate peroxidase Photosynthesis Oxidative stress a b s t r a c t The inactivation of the chloroplast ascorbate peroxidases (chlAPXs) has been thought to limit the effi- ciency of the water–water cycle and photo-oxidative protection under stress conditions. In this study, we have generated double knockdown rice (Oryza sativa L.) plants in both OsAPX7 (sAPX) and OsAPX8 (tAPX) genes, which encode chloroplastic APXs (chlAPXs). By employing an integrated approach involving gene expression, proteomics, biochemical and physiological analyses of photosynthesis, we have assessed the role of chlAPXs in the regulation of the protection of the photosystem II (PSII) activity and CO 2 assimila- tion in rice plants exposed to high light (HL) and methyl violagen (MV). The chlAPX knockdown plants were affected more severely than the non-transformed (NT) plants in the activity and structure of PSII and CO 2 assimilation in the presence of MV. Although MV induced significant increases in pigment content in the knockdown plants, the increases were apparently not sufficient for protection. Treatment with HL also caused generalized damage in PSII in both types of plants. The knockdown and NT plants exhibited differences in photosynthetic parameters related to efficiency of utilization of light and CO 2 . The knock- down plants overexpressed other antioxidant enzymes in response to the stresses and increased the GPX activity in the chloroplast-enriched fraction. Our data suggest that a partial deficiency of chlAPX expres- sion modulate the PSII activity and integrity, reflecting the overall photosynthesis when rice plants are subjected to acute oxidative stress. However, under normal growth conditions, the knockdown plants exhibit normal phenotype, biochemical and physiological performance. © 2013 Elsevier Ireland Ltd. All rights reserved. Abbreviations: APX, ascorbate peroxidase; AsA, ascorbate; cAPX, cytosol APX; CAT, catalase; chlAPX, chloroplastic APX; Ci, intercellular concentration of CO2; E, transpi- ration; ETR, apparent electron transport rate; EXC, energy excess; Fv/Fm, potential quantum yield of photosystem II; GPX, glutathione peroxidase; gs , stomatal conductance; H2O2, hydrogen peroxide; HL, high light; Jmax, maximum photosynthetic electron transport; mAPX, peroxisome/glyoxysomes APX; miAPX, mitochondria APX; MV, methyl violagen; NPQ, non-photochemical quenching; NT, non-transformed; PET, photosynthetic electron transport; PN–Ci, photosynthesis depending on the intercellular con- centration of CO2; PN–PPFD, photosynthesis depending on light intensity; PN, net CO2 assimilation; PPFD, photosynthetic photon flux density; PQ, plastoquinone pool; Pr , photorespiration; PSI, photosystem I; PSII, photosystem II; qP, photochemical quenching; R d , light respiration; Rn, dark respiration; ROS, reactive oxygen species; RT-qPCR, quantitative real-time PCR; sAPX, stroma APX; SOD, superoxide dismutase; tAPX, thylakoid APX; TBARS, thiobarbituric acid-reactive substances; Vcmax, maximum Rubisco carboxylation rate; Fv/F ′ m , actual quantum yield of photosystem II. ∗ Corresponding author at: Universidade Federal do Rio Grande do Sul, Avenida Bento Gonc ¸ alves, 9500, CEP: 915910-970, Porto Alegre, RS, Brazil. Tel.: +55 51 3308 9814. E-mail addresses: marcia.margis@ufrgs.br, marcia.p.margis@gmail.com (M. Margis-Pinheiro). 1 Present address: Department of Chemistry, Federal University of Mato Grosso, Brazil. 0168-9452/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2013.10.001