A peroxidase-dependent apoplastic oxidative burst in cultured Arabidopsis cells functions in MAMP-elicited defense.

Jose A O'Brien,G Paul Bolwell,Puneet Souda,Arsalan Daudi,Vernon S Butt,Julian P Whitelegge,Frederick M Ausubel,Paul Finch

doi:10.1104/pp.111.190140

Abstract

Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.

Highlights

Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS)
By generating Arabidopsis cell culture lines in which the expression of two genes encoding cell wall peroxidases, PRX33 and PRX34, is knocked down by heterologous expression of an antisense heterologous French bean peroxidase cDNA, we have shown that these peroxidases are required for a variety of MAMPactivated responses, including the production of an oxidative burst and the expression of a variety of MAMP-activated genes
While PRX33 was not detected in nonelicited cultures, the relatively strong up-regulation of PRX33 (24-fold) following elicitation compared with PRX34 (3-fold) suggests that it may play an important role after the which primarily inhibits peroxidases but not NADPH oxidases, blocks MAMP-mediated responses in Arabidopsis tissue culture cells, whereas diphenylene iodonium (DPI), which preferentially inhibits NADPH oxidases, does not

Summary

Introduction

Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of patterntriggered immunity in tissue culture cells. The activation of receptors leading to rapid responses such as Ca2+ and H+ influx and K+ efflux were first characterized in tissue cultures (Bolwell and Daudi, 2009) These studies have contributed significantly to our understanding of the underlying biochemistry leading to the generation of reactive oxygen species (ROS), including hydrogen peroxide (H2O2), superoxide, and nitric oxide, as antimicrobial factors or as intracellular or intercellular. Because there are several isoforms of the main subunit of the NADPH protein complex encoded by separate genes (rboh genes), it has been difficult to assess whether NADPH oxidases play an essential role in the plant defense response, recently, rbohF loss-of-function mutants were shown to be more susceptible to P. syringae (Chaouch et al, 2012)

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