Sophie Kneeshaw, Rumana Keyani, Valérie Delorme-Hinoux, Lisa Imrie, Gary J. Loake, Thierry Le Bihan, Jean-Philippe Reichheld, and Steven H. Spoel

PNAS August 1 2017; vol.114; no.31: 8414–8419 (PLANT BIOLOGY)

Significance

Cellular accumulation of reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) is associated with stress responses as well as aging. The reactive nature of ROS marks these molecules as a serious threat to cell integrity. Consequently, eukaryotic cells deploy numerous antioxidant enzymes that detoxify ROS to protect them from ROS-induced damage to proteins. Although the importance of antioxidant enzymes is well understood, how these proteins avoid becoming damaged in the hostile, ROS-rich environments in which they function remains unknown. We show that in plant cells the oxidoreductase Nucleoredoxin 1 (NRX1) protects antioxidant enzymes such as catalase from ROS-induced oxidation. Importantly, this protective effect of NRX1 boosted the H₂O₂ detoxification capacity of catalase, thereby protecting the plant cell from oxidative stress.

Abstract

Cellular accumulation of reactive oxygen species (ROS) is associated with a wide range of developmental and stress responses. Although cells have evolved to use ROS as signaling molecules, their chemically reactive nature also poses a threat. Antioxidant systems are required to detoxify ROS and prevent cellular damage, but little is known about how these systems manage to function in hostile, ROS-rich environments. Here we show that during oxidative stress in plant cells, the pathogen-inducible oxidoreductase Nucleoredoxin 1 (NRX1) targets enzymes of major hydrogen peroxide (H₂O₂)-scavenging pathways, including catalases. Mutant nrx1 plants displayed reduced catalase activity and were hypersensitive to oxidative stress. Remarkably, catalase was maintained in a reduced state by substrate-interaction with NRX1, a process necessary for its H₂O₂-scavenging activity. These data suggest that unexpectedly H₂O₂-scavenging enzymes experience oxidative distress in ROS-rich environments and require reductive protection from NRX1 for optimal activity.

See: http://www.pnas.org/content/114/31/8414.abstract.html?etoc

Figure 1: NRX1 negatively regulates plant immune responses. (A) Plants were infected with Psm ES4326 (5 × 10⁵ cells) for the indicated times. Expression of the NRX1 gene was analyzed and normalized against UBQ5. Error bars represent SD (n = 3). (B) WT plants were infiltrated with or without Psm ES4326 (5 × 10⁵ cells). Total protein was denatured and alkylated to capture true mixed disulphide intermediates between NRX1 and its substrates, separated by SDS/PAGE in the presence or absence of DTT, and analyzed by Western blot against NRX1. Indicated are free NRX1 monomer, mixed disulfide intermediates (NRX1–substrate), and total NRX1. (C) Expression of NRX1 was analyzed in WT, nrx1-1, nrx1-2, and 35S::Flag-NRX1 (in nrx1-1) plants. Gene expression was normalized against UBQ5 (error bars represent SD; n = 3) and immunoblotting performed with anti-NRX1 and anti-HSP90 antibodies. (D) SA-dependent PR-1 and PR-2 gene expression. Error bars represent SD (n = 3). (E) Plants were mock-treated or sprayed with 0.5 mM SA 24 h before infection with Psm ES4326 (5 × 10⁶ cells). Growth of Psm ES4326 was assessed after 3 d. Cfu, colony-forming units. Error bars represent statistical 95% confidence limits (n = 8). Asterisks indicate statistically significant differences compared with the WT (Tukey–Kramer ANOVA test; α = 0.05, n = 8). (F) Lower leaves were infiltrated with avirulent P. syringae pv. tomato DC3000/avrRpt2 (5 × 10⁷) or 10 mM MgSO₄. After 2 d, upper leaves were infected with Psm ES4326 (5 × 10⁶ cells) and pathogen growth assessed after 3 d. Cfu, colony-forming units. Error bars represent statistical 95% confidence limits (n = 8). Asterisks indicate statistically significant differences compared with the WT (Tukey–Kramer ANOVA test; α = 0.05, n = 8). (G) Plants were infected with Psm ES4326 (5 × 10⁶ cells) and pathogen growth assessed after 5 d. Cfu, colony-forming units. Error bars represent statistical 95% confidence limits (n = 8). *P < 0.05 for statistical differences with WT and nrx1; **P < 0.05 for statistical differences with WT, ics1, and nrx1 ics1 (Tukey–Kramer ANOVA test; α = 0.05, n = 8).