An NADPH-Oxidase/Polyamine Oxidase Feedback Loop Controls Oxidative Burst Under Salinity

Abstract

The apoplastic polyamine oxidase (PAO) catalyzes the oxidation of the higher polyamines spermidine and spermine, contributing to hydrogen peroxide (HO) accumulation. However, it is yet unclear whether apoplastic PAO is part of a network that coordinates the accumulation of reactive oxygen species (ROS) under salinity or if it acts independently. Here, we unravel that NADPH oxidase and apoplastic PAO cooperate to control the accumulation of HO and superoxides (O) in tobacco (Nicotiana tabacum). To examine to what extent apoplastic PAO constitutes part of a ROS-generating network, we examined ROS accumulation in guard cells of plants overexpressing or down-regulating apoplastic PAO (lines S2.2 and A2, respectively) or down-regulating NADPH oxidase (line AS-NtRbohD/F). The HO-specific probe benzene sulfonyl-HO showed that, under salinity, HO increased in S2.2 and decreased in A2 compared with the wild type. Surprisingly, the O-specific probe benzene sulfonyl-So showed that O levels correlated positively with that of apoplastic PAO (i.e. showed high and low levels in S2.2 and A2, respectively). By using AS-NtRbohD/F lines and a pharmacological approach, we could show that HO and O accumulation at the onset of salinity stress was dependent on NADPH oxidase, indicating that NADPH oxidase is upstream of apoplastic PAO. Our results suggest that NADPH oxidase and the apoplastic PAO form a feed-forward ROS amplification loop, which impinges on oxidative state and culminates in the execution of programmed cell death. We propose that the PAO/NADPH oxidase loop is a central hub in the plethora of responses controlling salt stress tolerance, with potential functions extending beyond stress tolerance.

Deregulation of apoplastic polyamine oxidase affects development and salt response of tobacco plants

“…Furthermore, total phenolics seem to be developmentally regulated with S-ZmPAO containing significantly higher total phenolics content (Fig. 6), consistent with the higher ROS load (Gémes et al, 2016;Moschou et al, 2008aMoschou et al, , 2008b. However, there is no further accumulation of phenolics post-salt treatment consistent with the recent results that salt tolerance of the halophyte Limonium delicatulum is more associated with antioxidant enzyme activities than phenolic compounds (Souid et al, 2016).…”

“…The S-ZmPAO plants showed the highest mRNA levels of APXc, at 6 h post-NaCl treatment ( Fig. 5E; 6 h) in accordance with the higher ROS levels in this genotype, while the AS-ZmPAO showed the lowest mRNA levels (Moschou et al, 2008b;Gémes et al, 2016). Under control conditions CAT1 mRNA levels did not differ among the three genotypes, whereas a profound increase of CAT1 mRNA levels was observed 1 h post-treatment with NaCl in all three genotypes ( Fig.…”

“…On the contrary, the cytoplasmic APXc mRNA levels show a positive correlation with the ROS levels post-treatment with NaCl, and seems to be leading antioxidant enzyme in the AS-ZmPAO plants (Fig. 5E) (Gémes et al, 2016;Moschou et al, 2008aMoschou et al, , 2008b. The results suggest that PAO generates H 2 O 2 that signals the expression of key enzymes in ROS scavenging/homeostasis, during the initial stages of salinity.…”

“…Considering that SOD is a H 2 O 2 producing enzyme, the increased SOD levels in AS-ZmPAO may reflect a homeostatic adjustment towards sustainment of H 2 O 2 levels, while the increase of SOD in S-ZmPAO could reflect the increased O 2 .-content ( Fig. 5A-D; Gémes et al, 2016;Moschou et al, 2008aMoschou et al, , 2008b. Furthermore, total phenolics seem to be developmentally regulated with S-ZmPAO containing significantly higher total phenolics content (Fig.…”

“…NADPH-oxidase and the apoplastic PAO constitute a feedforward ROS amplification loop, which impinges on oxidative state and culminates in the execution of programmed cell death (PCD). The data suggest that this loop is a central hub in the plethora of responses controlling salt stress tolerance, with potential functions extending beyond stress tolerance (Gémes et al, 2016).…”