title: Protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Arabidopsis NADPH oxidases and may function as a trigger for the positive feedback regulation of Ca2+ and reactive oxygen species.
abstract: Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in signalling and development. Given the high toxicity of ROS, their production is tightly regulated. In Arabidopsis, respiratory burst oxidase homologue F (AtrbohF) encodes NADPH oxidase. Here we characterised the activation of AtRbohF using a heterologous expression system. AtRbohF exhibited ROS-producing activity that was synergistically activated by protein phosphorylation and Ca2+. The two EF-hand motifs of AtRbohF in the N-terminal cytosolic region were crucial for its Ca2+-dependent activation. AtrbohD and AtrbohF are involved in stress responses. Although the activation mechanisms for AtRbohD and AtRbohF were similar, AtRbohD had significantly greater ROS-producing activity than AtRbohF, which may reflect their functional diversity, at least in part. We further characterised the interrelationship between Ca2+ and phosphorylation regarding activation and found that protein phosphorylation-induced activation was independent of Ca2+. In contrast, K-252a, a protein kinase inhibitor, inhibited the Ca2+-dependent ROS-producing activity of AtRbohD and AtRbohF in a dose-dependent manner, suggesting that protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Rboh. Positive feedback regulation of Ca2+ and ROS through AtRbohC has been proposed to play a critical role in root hair tip growth. Our findings suggest that Rboh phosphorylation is the initial trigger for the plant Ca2+-ROS signalling network.
authors: Department of Applied Biological Science, Tokyo University of Science, Chiba, Japan.
authors: Sachie
authors: S
authors: Kimura
authors: Hidetaka
authors: H
authors: Kaya
authors: Tomoko
authors: T
authors: Kawarazaki
authors: Goro
authors: G
authors: Hiraoka
authors: Eriko
authors: E
authors: Senzaki
authors: Masataka
authors: M
authors: Michikawa
authors: Kazuyuki
authors: K
authors: Kuchitsu
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D061207:Calcium Ionophores / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D025461:Feedback, Physiological; D057809:HEK293 Cells; D006801:Humans; D015759:Ionomycin / Q000378:metabolism; D008387:Marine Toxins; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010080:Oxazoles / Q000378:metabolism; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D029681:Arabidopsis Proteins; D061207:Calcium Ionophores; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D017382:Reactive Oxygen Species; D015759:Ionomycin; C059041:calyculin A; D019255:NADPH Oxidases; D002118:Calcium