Subcellular localization of H₂O₂ in plants. H₂O₂ accumulation in papillae and hypersensitive response during the barley—powdery mildew interaction

TLDR

Active oxygen species are central to plant defense, mediating signal transduction, cell wall reinforcement, hypersensitive response, and phytoalexin production, yet existing techniques cannot localize them in intact tissues. The authors used 3,3‑diaminobenzidine, which polymerizes upon contact with H₂O₂ in the presence of peroxidase, enabling in‑vivo, subcellular detection of H₂O₂ in leaf tissues. The method revealed H₂O₂ accumulation in papillae, surrounding haloes, and epidermal cells during barley–powdery mildew interaction, with earliest detection at 6 h under the germ tube, 15 h under the appressorium, and subsequent cross‑linking of proteins that reinforce the apposition and help arrest the pathogen.

Abstract

Active oxygen species (AOS) are believed to have important roles in plants in general and in plant—pathogen interactions in particular. They are believed to be involved in signal transduction, cell wall reinforcement, hypersensitive response (HR) and phytoalexin production, and to have direct antimicrobial effects. Since current methods are inadequate for localizing AOS in intact plant tissue, most studies have been conducted using cell suspension culture/elicitors systems. 3,3‐diaminobenzidine (DAB) polymerizes instantly and locally as soon as it comes into contact with H 2 O 2 in the presence of peroxidase, and it was found that, by allowing the leaf to take up this substrate, in‐vivo and in‐situ detection of H 2 O 2 can be made at subcellular levels. This method was successfully used to detect H 2 O 2 in developing papillae and surrounding haloes (cell wall appositions) and whole cells of barley leaves interacting with the powdery mildew fungus. Thus, H 2 O 2 can be detected in the epidermal cell wall subjacent to the primary germ tube from 6 h after inoculation, and subjacent to the appressorium from 15 h. The earliest time point for observation of H 2 O 2 in relation to epidermal cells undergoing HR is 15 h after inoculation, first appearing in the zones of attachment to the mesophyll cells underneath, and eventually in the entire epidermal cell. Furthermore, it was observed that proteins in papillae and HR cells are cross‐linked, a process believed to be fuelled by H 2 O 2 . This cross‐linking reinforces the apposition, presumably assisting the arrest of the pathogen.