Stress-induced reactive oxygen species compartmentalization, perception and signalling

Abstract

Reactive oxygen species (ROS) are essential for life and are involved in the regulation of almost all biological processes. ROS production is critical for plant development, response to abiotic stresses and immune responses. Here, we focus on recent discoveries in ROS biology emphasizing abiotic and biotic stress responses. Recent advancements have resulted in the identification of one of the first sensors for extracellular ROS and highlighted waves of ROS production during stress signalling in Arabidopsis. Enzymes that produce ROS, including NADPH oxidases, exhibit precise regulation through diverse post-translational modifications. Discoveries highlight the importance of both amino- and carboxy-terminal regulation of NADPH oxidases through protein phosphorylation and cysteine oxidation. Here, we discuss advancements in ROS compartmentalization, systemic ROS waves, ROS sensing and post-translational modification of ROS-producing enzymes and identify areas where foundational gaps remain.

Figure 1.. Biphasic ROS production and cell-to-cell communication.

1. Stimuli induce rapid initial apoplastic ROS production by NADPH oxidases (NOXs) and peroxidases (PER). 2. Apoplastic ROS can enter into the cytosol via aquaporins. 3. ROS then activates Ca²⁺ influx into the cytosol from the apoplast and vacuole. 4. Ca²⁺ binds to NOXs or protein kinases, which amplifies NOX activation. 5. Apoplastic ROS accumulation frequently coincides with extracellular alkalization by inhibition of H⁺-ATPases. 6. NOX-dependent ROS can restrict symplastic signaling by modulating callose deposition at plasmodesma. 7. After initial rapid systemic signaling, a second ROS burst with contributions from the chloroplast may affect nuclear gene expression.

Figure 2.. ROS perception by the HPCA1 receptor.

Left: The flagellin receptor complex and ROS receptor HPCA1 in the absence of pathogen perception. Right: Perception of the immunogenic flagellin epitope flg22 results in FLS2-BAK1 complex formation, inhibition of the H⁺-ATPase resulting in alkalization of the apoplast, and activation of NOX-induced ROS production. The increase in extracellular pH enables cysteine modification of HPCA1’s hydrogen peroxidase (HP) domain, increasing HPCA1’s kinase activity and downstream ROS signalling.

Figure 3.. Conservation of the NOX C-terminus across plants and conservation of critical residues in RBOHD and human NOX5.

(a) Conservation of plant NOX homologs when comparing full length, N-terminal (residues 1–376) or C-terminal regions (residues 680–920). Percent amino acid (AA) similarity was compared using AtRBOHD as a reference across 112 plant NOXs. (b) Sliding window of average amino acid similarity displayed in red with a 95% confidence interval in light red along the C-termini for plant NOX homologs. AtRBOHD was used as a reference with a 7 amino acid sliding window. Regions known to be regulated via posttranslational modifications (PTMs) are highlighted in grey and displayed by weblogos with modified residues differentially colored. (c) Structural models of (left) AtRBOHD C-terminus (residues 613–920) and (right) HsNOX5 β-isoform C-terminus (residues 401–719). Residues are labelled with different colours, based on the type of post-translational modifications (PTMs) underlying their regulatory role. Positive and negative regulatory outputs of the PTMs are specified by up- and down-arrows, respectively. Red label indicates positive regulation by phosphorylation, blue indicates negative regulation by phosphorylation, black specify ubiquitination, purple specify S-nitrosylation and orange indicates persulfidation. Residues highlighted with light blue were not found to be post-translationally modified, but were experimentally shown to modulate ROS producing activity.