Abstract
Hydrogen peroxide (H2O2) functions as an important signaling molecule in plants during biotic interactions. However, the extent to which H2O2 accumulates during these interactions and its implications in the development of disease symptoms is unclear. In this work, we provide a step-by-step optimized protocol for in situ quantification of relative H2O2 concentrations in wheat leaves infected with the pathogenic bacterium Pseudomonas syringae pv. atrofaciens (Psa), either alone or in the presence of the beneficial bacterium Herbaspirillum seropedicae (RAM10). This protocol involved the use of 3-3′diaminobenzidine (DAB) staining method combined with image processing to conduct deconvolution and downstream analysis of the digitalized leaf image. The application of a linear regression model allowed to relate the intensity of the pixels resulting from DAB staining with a given concentration of H2O2. Decreasing H2O2 accumulation patterns were detected at increasing distances from the site of pathogen infection, and H2O2 concentrations were different depending on the bacterial combinations tested. Notably, Psa-challenged plants in presence of RAM10 accumulated less H2O2 in the leaf and showed reduced necrotic symptoms, pointing to a potential role of RAM10 in reducing pathogen-triggered H2O2 levels in young wheat plants.
Highlights
Accumulation of reactive oxygen species (ROS) is a common plant response to pathogens, having many and often contrasting functions depending on the plant-pathogen system under study (González-Bosch, 2018)
Infiltration with pv. atrofaciens strain 2213 (Psa) in wheat leaves caused the development of dried brown, necrotic lesions surrounded by chlorosis after 24 h both in Psa and RAM10 + Psa seedlings (Figure 1A)
Around 75% of the diseased leaf area in Psa leaves was composed of necrotic tissue, which appeared in form of a dried brown area, presumably as a result of the onset a hypersensitive response at the site of pathogen entrance
Summary
Accumulation of reactive oxygen species (ROS) is a common plant response to pathogens, having many and often contrasting functions depending on the plant-pathogen system under study (González-Bosch, 2018). Any type of ROS has specific biochemical characteristics and most of them are extremely unstable (Mittler, 2017). Wheat Leaf H2O2 Quantification is relatively more stable having a half-life time of more than 1 ms, and is considered the predominant ROS involved in cellular signaling (Cerný et al, 2018). ROS regulate numerous immune responses to invading microorganisms, including both the hypersensitive and programmed cell death responses, the cross-linking of cell wall proteins, the deposition of callose or the activation of redox-sensitive genes. ROS, participate in cell-to cell signal transduction to systemic tissues, where localized ROS bursts can induce defenses to prepare (or “prime”) plants for future challenges (Torres et al, 2006; Noctor et al, 2018)
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