Peroxisomal polyamine oxidase and NADPH-oxidase cross-talk for ROS homeostasis which affects respiration rate in Arabidopsis thaliana

Abstract

Homeostasis of reactive oxygen species (ROS) in the intracellular compartments is of critical importance as ROS have been linked with nearly all cellular processes and more importantly with diseases and aging. PAs are nitrogenous molecules with an evolutionary conserved role in the regulation of metabolic and energetic status of cells. Recent evidence also suggests that polyamines (PA) are major regulators of ROS homeostasis. In Arabidopsis the backconversion of the PAs spermidine (Spd) and spermine to putrescine and Spd, respectively, is catalyzed by two peroxisomal PA oxidases (AtPAO). However, the physiological role of this pathway remains largely elusive. Here we explore the role of peroxisomal PA backconversion and in particular that catalyzed by the highly expressed AtPAO3 in the regulation of ROS homeostasis and mitochondrial respiratory burst. Exogenous PAs exert an NADPH-oxidase dependent stimulation of oxygen consumption, with Spd exerting the strongest effect. This increase is attenuated by treatment with the NADPH-oxidase blocker diphenyleneiodonium iodide (DPI). Loss-of-function of AtPAO3 gene results to increased NADPH-oxidase-dependent production of superoxide anions ([Formula: see text] ), but not H2O2, which activate the mitochondrial alternative oxidase pathway (AOX). On the contrary, overexpression of AtPAO3 results to an increased but balanced production of both H2O2 and [Formula: see text] . These results suggest that the ratio of [Formula: see text] /H2O2 regulates respiratory chain in mitochondria, with PA-dependent production of [Formula: see text] by NADPH-oxidase tilting the balance of electron transfer chain in favor of the AOX pathway. In addition, AtPAO3 seems to be an important component in the regulating module of ROS homeostasis, while a conserved role for PA backconversion and ROS across kingdoms is discussed.

Keywords: Arabidopsis; NADPH-oxidase; ROS homeostasis; polyamine oxidases; polyamines; respiration.

FIGURE 1

Effect of exogenous polyamines on oxygen consumption rate in Arabidopsis. Arabidopsis Col-0 seedlings were treated with 1 mM Put, Spd, or Spm for a period of 10 min, and oxygen consumption rate was estimated by a Clark-type electrode. Data are the means of three independent experiments ±SD. Asterisks indicate statistical significant differences (***P < 0.001).

FIGURE 2

Effect of exogenous Spd and DPI on ROS homeostasis in Arabidopsis plants. (A) Plants were treated with 1 mM Spd for a period of 10 min and H₂O₂ or ${\mathrm{O}}_{\mathrm{2}}^{\mathrm{•}\mathrm{-}}$ were estimated by in situ detection methods. Scale bars, 2.8 cm. (B) Relative pixel intensity of the brownish (detection of H₂O₂) or bluish (detection of ${\mathrm{O}}_{\mathrm{2}}^{\mathrm{•}\mathrm{-}}$ ) adduct formed after application of 1 mM Spd. AU, arbitrary units. Data are the means of three independent experiments ±SD. Asterisks indicate statistical significant differences (***P < 0.001; **P < 0.01).

FIGURE 3

Combined effect of exogenous Spd and ASA, CAT or SOD on oxygen consumption rate of Arabidopsis plants and dose-dependent response of NADPH-oxidase activity by Spd. (A) Oxygen consumption rate in plants incubated in the respective medium for 10 min. Data are the means of three independent experiments ±SD. Asterisks indicate statistical significant differences (***P < 0.001; *P < 0.05). For Spd+SOD treatment P value is indicated. (B) Effect of exogenous Spd on NADPH-oxidase activity in Col-0 Arabidopsis plants. Plants were treated with 0.1, 0.2, 0.5, 1, and 5 mM Spd for a period of 10 min and relative pixel intensity of lane profile after application of 1 mM Spd was assessed.

FIGURE 4

In situ ROS in WT, S-AtPAO3, and Atpao3 Arabidopsis plants. (A) In situ ROS detection in WT, S-AtPAO3 and Atpao3 plants. Data are from a single representative experiment, repeated three times, and densitometric analysis. Data are the means of three different positions on a leave. Asterisks indicate statistical significant differences from the Col-0 (***P < 0.001). Scale bars, 2.8 cm. (B) Native electrophoresis and activity staining of SOD and densitometric analysis of isoenzymes. (C) Native electrophoresis and activity staining of APX and densitometric analysis.

FIGURE 5

Oxygen consumption rate in Col-0, S-AtPAO3 and Atpao3 Arabidopsis plants. (A) Oxygen consumption rate in Col-0, S-AtPAO3 and Atpao3 plants. Rate of oxygen consumption was determined in untreated plants and plants treated with the AOX blocker SHAM, exogenous Spd and DPI. Data are the means of three different positions on a leave. Asterisks indicate statistical significant differences from the S-AtPAO3 control (***P < 0.001). (B) Western blot of AOX immunoreactive protein levels in Col-0, S-AtPAO3 and Atpao3 plants. Data are from a single representative experiment, repeated three times. (C) Densitometric analysis of AOX immunoreactive protein in (B). Data are from a single representative experiment, repeated three times.