Synergistic Activation of the Arabidopsis NADPH Oxidase AtrbohD by Ca2+ and Phosphorylation

Abstract

Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91 phox ) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91 phox /NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca 2؉ . However, the significance of Ca 2؉ binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca 2؉ ionophore that induces Ca 2؉ influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca 2؉ binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca 2؉ binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.Photosynthetic plants have developed various mechanisms to cope with oxidative stress, such as the production of antioxidants and enzymes that scavenge reactive oxygen species (ROS).3 Plants are also equipped with mechanisms for producing ROS in response to internal and external stimuli. ROS production is induced during many physiological processes, including stress responses, cell growth, hormonal responses, stomatal closure, and disease resistance (see Refs. 1-4 and references therein).ROS production is induced in plants in response to recognition of pathogenic signals, such as pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) or elicitors. Elicitor-induced ROS production is preceded by a rapid increase in the cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) (5-7) and is inhibited both by Ca 2ϩ chelators such as EGTA and BAPTA, and by Ca 2ϩ channel blockers such as La 3ϩ (6,8). The overexpression of rice two-pore channel 1 (OsTPC1), which is a putative voltage-gated Ca 2ϩ channel, enhanced elicitor-induced ROS production (9). Elicitor-induced ROS production is also inhibited by diphenylene iodonium (DPI), which is known to inhibit NADPH oxidase activity (6, 10). NADPH oxidase activity in the microsomal membrane fraction from tomato and tobacco was activated by adding Ca 2ϩ in vitro (11), suggesting that elicitor-induced ROS production by plant NADPH oxidase might be dependent on Ca 2ϩ . In mammalian phagocytes, ROS production is mediated by the NADPH-dependent phagocytic oxidase (phox) complex, which consists of the catalytic subunit gp91 phox /NADPH oxidase (NOX) 2, together with the regulatory subunits p22 phox , p40 phox , p47 phox , p67 phox , and the small GTP-binding protein Rac (12). In...

Reactive Oxygen Species-Driven Transcription in Arabidopsis under Oxygen Deprivation

“…Moreover, transient changes in Ca 2+ during low O 2 was found to be involved in ADH regulation (Sedbrook et al, 1996;Subbaiah et al, 1994aSubbaiah et al, , 1994b. A connection between NADPH-oxidase-dependent ROS production and Ca 2+ oscillation has been suggested in Arabidopsis, where Ca 2+ likely binds to the EF hands of the N-terminal region of NADPH oxidase and, together with phosphorylation, promotes ROS production (Ogasawara et al, 2008;Takeda et al, 2008;Suzuki et al, 2011). Whether and how this mechanism could in part overlap with N-end rule pathway is still unknown.…”

Dual Roles of Reactive Oxygen Species and NADPH Oxidase RBOHD in an Arabidopsis-Alternaria Pathosystem

“…Expression of the rbohF gene was not induced by the infection, providing additional evidence that RBOHD plays a more significant role in the response of Arabidopsis to a necrotrophic fungus attack than RBOHF. We find it interesting that RBOHD is not only regulated at the level of transcription but also is under tight posttranslational control through synergistic activation by phosphorylation or Ca 2+ binding (Benschop et al, 2007;Nü hse et al, 2007;Ogasawara et al, 2008). Conformational changes regulated by two Ca 2+ -binding EF-hand motifs and induction of the S 343/347 phosphorylation sites are both necessary for a full RBOHD-dependent oxidative burst.…”

“…In addition, transcript accumulation of a tobacco gp91 phox homolog (NtrbohD) was observed in the same catalase antisense plants after exposure to high light . DPI efficiently inhibits the activity of Arabidopsis RBOHD in a heterologous expression system (Ogasawara et al, 2008).…”

Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus

“…The measurement of ROS production in HEK293T cells was performed essentially as described previously (11,61). See SI Experimental Procedures for details.…”