The knockdown of chloroplastic ascorbate peroxidases reveals its regulatory role in the photosynthesis and protection under photo-oxidative stress in rice

Abstract

The inactivation of the chloroplast ascorbate peroxidases (chlAPXs) has been thought to limit the efficiency 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 CO2 assimilation 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 CO2 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 CO2. The knockdown 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 expression 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.

Keywords: APX; AsA; Ascorbate peroxidase; CAT; Ci; E; ETR; EXC; F(v)/F(m); GPX; H(2)O(2); HL; J(max); MV; NPQ; NT; Oryza sativa; Oxidative stress; P(N); P(N)–Ci; P(N)–PPFD; P(r); PET; PPFD; PQ; PSI; PSII; Photosynthesis; R(d); R(n); ROS; RT-qPCR; Rice; SOD; TBARS; V(cmax); actual quantum yield of photosystem II; apparent electron transport rate; ascorbate; ascorbate peroxidase; cAPX; catalase; chlAPX; chloroplastic APX; cytosol APX; dark respiration; energy excess; g(s); glutathione peroxidase; high light; hydrogen peroxide; intercellular concentration of CO(2); light respiration; mAPX; maximum Rubisco carboxylation rate; maximum photosynthetic electron transport; methyl violagen; miAPX; mitochondria APX; net CO(2) assimilation; non-photochemical quenching; non-transformed; peroxisome/glyoxysomes APX; photochemical quenching; photorespiration; photosynthesis depending on light intensity; photosynthesis depending on the intercellular concentration of CO(2); photosynthetic electron transport; photosynthetic photon flux density; photosystem I; photosystem II; plastoquinone pool; potential quantum yield of photosystem II; qP; quantitative real-time PCR; reactive oxygen species; sAPX; stomatal conductance; stroma APX; superoxide dismutase; tAPX; thiobarbituric acid-reactive substances; thylakoid APX; transpiration.