A Peroxidase-Dependent Apoplastic Oxidative Burst in Cultured Arabidopsis Cells Functions in MAMP-Elicited Defense    

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 (H 2 O 2 ) 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 H 2 O 2 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 H 2 O 2 -mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl 2 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.

Reactive oxygen species and plant resistance to fungal pathogens

“…Assays with ferrous ion oxidation (FOX) are based on the spectrophotometrical detection of peroxide-mediated oxidation of Fe 2+ to Fe 3+ ions that forms a complex with xylenol orange. The FOX method was used on cultured suspension cells (Boubakri et al, 2013;O'Brien et al, 2012b) or on incubation medium of leaf explants (Bellincampi et al, 2000). Other methods to determine H 2 O 2 spectrophotometrically in plant samples include the use of resorcinol/titanium oxalate (Becana et al, 1986) or ABTS (2,2 0 -Azino-bis-(3-ethylbenthiazoline-6-sulfonate)) (formation of blue colour) (Messner and Boll, 1994).…”

“…When Arabidopsis tissue culture lines generated from FBP1 antisense plants are treated with ROS inhibitors (azide and DPI) about half of the MAMP-induced H 2 O 2 can be accounted for by a peroxidase-generated reaction while the rest is likely to depend on NADPH oxidases and other sources. The expression of MAMPelicited genes including MYB51, CYP79B2, and CYP81F2 and the two cysteine-rich defence-related peptides PDF2.2 and PDF2.3 are decreased in the antisense cell lines (O'Brien et al, 2012b). The importance of peroxidase-mediated ROS formation was studied in Arabidopsis T-DNA insertion lines impaired in the expression of the PRX33 or PRX34 mRNAs.…”

Salicylic acid-induced superoxide generation catalyzed by plant peroxidase in hydrogen peroxide-independent manner

“…2) suggests that there is possibility that SA but not IAA contributes to the alkaline-responsive oxidative burst in plant defense mechanisms associated with microbe-associated molecular patterns, by involving the extracellular peroxidases as proposed by the group of Bolwell. [34][35][36][37] …”

Changes in Antioxidant Enzyme Activities of European Mistletoe (Viscum album L. subsp. Album) Leaves as a Response to Environmental Stress Caused by Pollution of the Atmosphere by Nitrogen Dioxide

“…•-and H 2 O 2 are often deactivated already in the apoplastic fraction and this is closly connected with cell wall reconstrution [45]. Peroxidases participate in the process of the cell wall remodeling during oxidative stress [45,46].…”

“…Peroxidases participate in the process of the cell wall remodeling during oxidative stress [45,46]. Activities of special peroxidases responsible for lignifi cation process and for cell wall strengthening are often higher under stress [47].…”