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

Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91phox) 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 gp91phox/NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca2+. However, the significance of Ca2+ 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 Ca2+ ionophore that induces Ca2+ influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca2+ 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 Ca2+ binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.

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 plants, NOX family members, which are called respiratory burst oxidase homolog (rboh) proteins, have been identified as gp91 phox /NOX2 homologs in various plant species, including rice, Arabidopsis, tomato, potato, and tobacco (10,(13)(14)(15)(16)(17). However, the only homolog for the regulatory subunits that has been identified in plants is that for Rac. In total, 10 rboh genes (AtrbohA-J) have been identified in the genome of A. thaliana (18). atrbohC/root hair-defective2 (rhd2) mutant was defective in ROS accumulation at the apex of the root hair bulge (19), and a atrbohD atrbohF double mutant was defective in abscisic acid-induced ROS production in stomatal guard cells (20) and pathogen-induced ROS production (21). These genetic studies suggested that Atrboh proteins are significant in ROS production. We focused on the AtrbohD gene in the current study because it has an important role in ROS production in response to biotic and abiotic stress, and its contribution to ROS production in leaves is known to be greater than that of the AtrbohF gene (18,20,21).
Plant rboh proteins have two putative EF-hand motifs (18). An immobilized synthetic peptide including the EF-hand motifs of AtrbohF (formerly RbohA) was shown to bind 45 Ca 2ϩ (16), suggesting an important role for Ca 2ϩ binding in its activation. However, neither the biochemical activity nor the physiological function of the EF-hand motifs has been thoroughly studied. Phosphorylation also has been implicated in the activation of rboh, as the protein kinase inhibitor K-252a inhibits elicitor-induced ROS production (6,22,23), and the protein serine/threonine phosphatase inhibitor calyculin A (CA) induces ROS production (24 -28).
The molecular mechanisms for this activation by Ca 2ϩ and phosphorylation have remained unclear, as plant rboh proteins have been difficult to express in systems in which ROS production can be monitored in real time. To address this problem, we applied a heterologous expression system using the mammalian cell line human embryonic kidney (HEK) 293T to express AtrbohD. We also measured the affinity for Ca 2ϩ and the ␣-helical content of the putative EF-hand region of AtrbohD by fluorescence and circular dichroism (CD) spectroscopy. We found that AtrbohD possesses ROS-producing activity, which is induced by ionomycin. AtrbohD-dependent ROS production requires a conformational change in the EF-hand region as a result of Ca 2ϩ binding, and direct phosphorylation of AtrbohD activated ROS production and enhanced ionomycin-induced ROS production. Our results suggest that both the conformational change in the EF-hand region induced by Ca 2ϩ binding to the EF-hand motifs and direct phosphorylation synergistically activate AtrbohD to produce ROS.
Cell Culture and Transfection-HEK293T cells were maintained at 37°C in 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) or DMEM nutrient mixture F-12 HAM (Sigma) supplemented with 10% fetal bovine serum. HEK293T cells were transiently transfected with pcDNA3.1-FLAG:AtrbohD or an empty pcDNA3.1 vector using FuGENE6 (Roche Applied Science) or GeneJuice transfection reagent (Novagen) according to the manufacturer's protocol. Similar results were obtained regardless of the culture medium and the transfection reagents used.
Measurement of ROS Production-ROS production was measured by the peroxidase-dependent luminol-amplified chemiluminescence technique based on a method described by Bánfi et al. (29). In brief, HEK293T cells were plated in 96-well plates 18 h before transfection. After transfection, the cells were incubated for 48 h, washed with Hanks' balanced salt solution (HBSS) containing 1.26 mM Ca 2ϩ , and measurements were made in HBSS supplemented with 6 units/ml horseradish peroxidase (Wako, Osaka, Japan) and 250 M luminol (Nacalai Tesque, Kyoto, Japan). To mobilize Ca 2ϩ uptake, 1 M ionomycin (Calbiochem) was applied to the cells (unless otherwise stated). Chemiluminescence was measured every minute at 37°C using a microplate luminometer LB96V (EG&G Berthold). ROS production was expressed as relative luminescence units per second (RLU/s). Data are presented as the average of three samples in a representative experiment. We independently replicated this experiment more than five times.
Expression and Purification of Recombinant AtrbohD Proteins Containing EF-hand Motifs-The E. coli strain Rosetta2 (DE3) (Novagen) harboring the pET-26b(ϩ)-based expression vector was cultured in LB medium at 37°C. When the cultures reached an optical density at 600 nm of 0.8, 1.0 mM isopropyl-␤-D-thiogalactopyranoside was added for another 6 h. The cells were harvested by centrifugation, resuspended in buffer A (20 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA), and disrupted by sonication. After centrifugation, the supernatant was incubated at 90°C for 30 min with 1 mM Ca 2ϩ to precipitate the E. coli proteins, centrifuged again, and the supernatant was treated with 3% (v/v) trichloroacetic acid to precipitate the Ca 2ϩ -depleted recombinant proteins (30). The precipitate was redissolved in 1 M Tris base solution and dialyzed against buffer A. The protein was then purified on a DEAE-Toyopearl 650 M column (Tosoh, Japan) and eluted stepwise with buffer A supplemented with 100, 200, 300, or 400 mM NaCl. The eluted protein was concentrated using VivaSpin (Sartorius) and run through a Superdex 75 HR 10/30 column (GE Healthcare). The purity of the peptides was confirmed by the presence of a single band on SDS-PAGE.
Fluorescence Spectroscopy-Fluorescence spectra were recorded at room temperature (ϳ20°C) using a Shimadzu RF-5300PC spectrofluorimeter, with excitation at 280 nm and emission at 310 nm. The pCa value at the half-maximal change in fluorescence (pCa 50 ) was determined by plotting the relative fluorescence change against the pCa and fitting a sigmoidal dose-response (variable response) curve by nonlinear regression using GraphPad Prism Version 3.00 for Windows (Graph-Pad, San Diego, CA).
CD Measurements-Far-ultraviolet (UV) CD spectra were acquired at room temperature (ϳ20°C) using a Jasco J-720 spectropolarimeter, which was set for a 200 -260-nm wavelength range, 0.2-nm step resolution, 100-nm⅐min Ϫ1 scan speed, 2-s response time, 1-nm bandwidth, and eight scans per sample. Samples of the 20 M AtrbohD EF-hand region dissolved in 100 mM KCl and 20 mM MOPS-KOH (pH 7.0) were measured in the metal-free apo state, and with the addition of 2 mM CaCl 2 or 2 mM MgCl 2 in the metal-bound state. The ␣-helical content of the protein was calculated using the CONTIN program (31).
In Vivo Labeling and Immunoprecipitation-Cells that had been transfected with pcDNA3.1-FLAG: AtrbohD or empty pcDNA3.1 vector 48 h earlier were washed and incubated with phosphate-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 18.5 MBq/ml [ 32 P]orthophosphate (GE Healthcare) for 4 h. The labeled cells were washed with HBSS buffer, incubated for 10 min in HBSS in the presence or absence of 0.1 M CA, and then solubilized in 50 mM HEPES (pH 7.5), 0.1% (v/v) Triton X-100, 0.5 M NaCl, 5 mM EDTA, and complete protease inhibitor mixture (Roche Applied Science). The 32 P-labeled FLAG:AtrbohD was immunoprecipitated with anti-FLAG antibody (Sigma) and protein G-Sepharose overnight at 4°C. The beads were boiled in sample buffer before separating the proteins by SDS-PAGE and visualizing them by autoradiography.

AtrbohD-dependent ROS Production in Transfected HEK293T cells-
To analyze ROS-producing enzyme activity and the regulatory mechanisms of AtrbohD, we used expression in a heterologous system, HEK293T cells. This cell line is derived from mammalian HEK293 cells, which lack gp91 phox /NOX2 and NOX5 (29,32); this allowed us to quantitatively monitor exogenous Ca 2ϩdependent NADPH oxidase activity in real-time. When HEK293T cells were transiently transfected with FLAG:AtrbohD, the amount of recombinant protein expressed was proportional to the concentration of transfected DNA (Fig. 1A, lower panel). We measured the production of ROS in HEK293T cells transfected with FLAG: AtrbohD and empty vector by a peroxidase-dependent luminol-amplified chemiluminescence signal (33). The chemiluminescence signal detected in FLAG:AtrbohD-transfected HEK293T cells was significant, and increased in proportion to the amount of transfected FLAG:AtrbohD (Fig. 1A, upper  panel). These results demonstrated that AtrbohD expressed in HEK293T cells possessed ROS-producing enzyme activity.
ROS-producing Activity of AtrbohD is Ca 2ϩ -dependent-We added ionomycin, a Ca 2ϩ ionophore, which induces Ca 2ϩ influx into the cell, to FLAG:AtrbohD-transfected and empty vector-transfected cells, to examine the effect of Ca 2ϩ on AtrbohD activity. Ionomycin induced rapid transient ROS production in FLAG:AtrbohD-transfected cells, but not in empty vector-transfected cells (Fig. 1B). The level of ionomycin-induced ROS production increased in proportion to the amount of transfected DNA (Fig. 1B). To examine the dependence of ionomycin-induced ROS production on NADPH oxidase activity attributable to AtrbohD, we pretreated the transfected cells with DPI, which inhibits NADPH oxidase activity by binding to the redox center of flavoproteins (34). DPI inhibited ionomycin-induced ROS production in a dose-dependent manner (Fig.  1C). The addition of SOD, which is a scavenger of the superoxide anion radical, to the FLAG:AtrbohD-transfected cells drastically reduced the ionomycin-induced chemiluminescence signal (Fig. 1D), suggesting that the main ROS produced by AtrbohD are superoxide anion radicals. These results are consistent with physiological evidence that superoxide production in vivo is associated with a flavin-containing NOX-like activity (35)(36)(37)(38). To test the Ca 2ϩ dependence of ionomycin-induced ROS production, AtrbohD-transfected HEK293T cells were pretreated with increasing concentrations of CaCl 2 from 0 to 1.26 mM. No ROS were produced in the absence of extracellular Ca 2ϩ , and ROS production increased in proportion to the concentration of extracellular Ca 2ϩ (Fig. 1E), showing that ROS production by AtrbohD is a Ca 2ϩ -dependent enzyme activity.
Ca 2ϩ Binding to the Two Putative EF-hand Motifs-The two putative EF-hand motifs in the N-terminal, cytosolic region of AtrbohD have been suggested to bind Ca 2ϩ based on amino acid sequence homologies. However, Ca 2ϩ binding to the EFhand motifs has not been directly demonstrated. We measured the affinity of Ca 2ϩ binding to a recombinant protein, WT-EF, which consists of 83 amino acids containing the two putative EF-hand motifs of AtrbohD ( Fig. 2A), using fluorescence spectroscopy. The effect of Ca 2ϩ concentration (pCa ϭ Ϫlog 10 [Ca 2ϩ ]) on the normalized fluorescence attributed to Tyr residues is shown in Fig. 2B. The pCA 50 value for WT-EF was 4.0 Ϯ 0.03, indicating that AtrbohD is a Ca 2ϩ -binding protein.
In addition, we found that the second EF-hand motif of AtrbohD has a non-bidentate Asn in the twelfth (-Z) coordinating position ( Fig. 2A, arrow), in contrast to the bidentate Asp/ Glu at the same position in most EF-hand motifs. This residue is critical for Ca 2ϩ chelation (30,39); we therefore hypothesized that the first EF-hand motif was likely to make a greater contribution to Ca 2ϩ binding by AtrbohD than the second EF-hand motif. To test this hypothesis, we compared the affinity for Ca 2ϩ of WT-EF with the mutants E277Q-EF and N321E-EF ( Fig. 2A), which had substitutions at the twelfth (-Z) position in the first and second EF-hand motifs, respectively. We predicted that the E277Q mutation would reduce the affinity for Ca 2ϩ because of the substitution of a bidentate Glu by a non-bidentate Gln in the first EF-hand motif. Conversely, we predicted that the N321E mutation would increase the affinity for Ca 2ϩ because of the substitution of a non-bidentate Asn by a bidentate Glu in the second EF-hand motif (Fig. 2A). The affinities of N321E-EF and E277Q-EF for Ca 2ϩ were higher and lower, respectively, than that of WT-EF, as we expected. As an Asp residue at the first (X) position in a Ca 2ϩ -binding site is known to be critical for Ca 2ϩ chelation (39), we also measured the affinity for Ca 2ϩ of other recombinant proteins with point mutations at this position in the first (D266A-EF) and second (D310A-EF) EF-hand motifs. In agreement with our predictions, the affinities of both D266A-EF and D310A-EF for Ca 2ϩ were lower than that of WT-EF ( Fig. 2A). In addition, the D266A mutation reduced the affinity for Ca 2ϩ to a greater extent than the D310A mutation (Fig. 2B); this is consistent with our hypothesis that the first EF-hand motif contributes more than the second EF-hand motif in AtrbohD for Ca 2ϩ binding.
Ca 2ϩ -induced Conformational Changes in the EF-hand Region-To examine the effect of Ca 2ϩ binding to the two EFhand motifs on the conformation of the EF-hand region, we measured far-UV CD to estimate the ␣-helical content of WT-EF, E277Q-EF, and N321E-EF. The CD spectra of these recombinant proteins showed two negative minima at around 208 and 222 nm, which is typical of ␣-helical proteins (Fig. 2C). The relative ␣-helical contents of WT-EF, E277Q-EF, and N321E-EF in their Ca 2ϩ bound and apo states were 1.85, 1.41, and 1.57, respectively (Fig. 2D). This indicated that WT-EF underwent a greater change in secondary structure on binding Ca 2ϩ than N321E-EF, while the affinity of WT-EF for Ca 2ϩ was lower than that of N321E-EF (Fig. 2B). E277Q-EF underwent a smaller conformational change than WT-EF on binding Ca 2ϩ , which is consistent with it having a low affinity for Ca 2ϩ . Mg 2ϩ binding also increased the ␣-helical content of the three recombinant proteins, but to a lesser extent than Ca 2ϩ binding, indicating that Ca 2ϩ binding to the EF-hand region of AtrbohD could cause a significant conformational change in its secondary structure (Fig. 2D).
Effects of Mutations in the EF-hand Motif on ROS Production by AtrbohD-To examine the effects of the mutations in the EF-hand motifs on ionomycin-induced ROS production, we expressed FLAG:AtrbohD proteins carrying point mutations (E277Q-AtrbohD and N321E-AtrbohD) in HEK293T cells, and measured the production of ROS. The mutant proteins were expressed at levels comparable to wild type, indicating that the mutations had no effect on protein expression (Fig. 3A). However, the E277Q mutation completely abolished ionomycin-induced ROS production (Fig. 3B). A similar result was obtained with a FLAG:D266A-AtrbohD mutant and a deletion mutant, FLAG:⌬328-AtrbohD, that lacked the N-terminal region containing the two EF-hand motifs (data not shown). These results indicated that ionomycin-induced activation of AtrbohD is mediated by Ca 2ϩ binding to its EF-hand region.
Although the affinity of N321E-EF for Ca 2ϩ was higher than that of WT-EF (Fig. 2B), ROS production by FLAG:N321E-AtrbohD was much lower than that of FLAG:AtrbohD (Fig. 3B), suggesting that the correct conformational change in the EFhand region upon Ca 2ϩ binding, rather than the level of the affinity for Ca 2ϩ , is the important factor for ROS production by AtrbohD.

Regulation of ROS Production by AtrbohD through Protein Phosphorylation-
The protein phosphatase inhibitor CA is known to induce ROS production (24 -28). We therefore tested its effect on ROS production in FLAG:AtrbohDtransfected cells in the absence of ionomycin, and found it induced ROS production (Fig. 4A), but at a much lower level and over a much longer time period than ionomycin. We observed CA-induced ROS production in FLAG:E277Q-AtrbohDtransfected cells that did not produce ROS in response to ionomycin (Fig. 4B). These results suggested that the mechanisms by which ionomycin and CA induce ROS production differ. We also observed a rapid initial phase of CA-induced ROS production in FLAG:AtrbohDtransfected cells. This might be due to trace amounts of Ca 2ϩ influx (Fig.  4A), as it was eliminated in FLAG: E277Q-AtrbohD transfected cells (Fig. 4B).
To investigate the phosphorylation of AtrbohD in vivo, HEK293T cells were transfected with FLAG-AtrbohD or empty vector and labeled with [ 32 P]orthophosphate. Protein extracts were immunoprecipitated with anti-FLAG antibody, separated by SDS-PAGE, and visualized by autoradiography. We detected a phosphorylated protein with an apparent molecular mass of 105 kDa, which corresponded to the size predicted for FLAG:AtrbohD, in FLAG:AtrbohD-transfected cells, but not in control cells transfected with the empty vector (Fig. 4D, left  panel). This protein appeared more heavily phosphorylated when the cells were treated with CA (Fig. 4D,  right panel). These results suggested that AtrbohD is directly phosphorylated, and that this phosphorylation activates its ROS-producing enzyme activity.
Synergistic Effect of CA and Ionomycin on ROS Production by AtrbohD-Having shown that AtrbohD has a ROS-producing enzyme activity that is regulated by phosphorylation and Ca 2ϩ binding to EF-hand motifs, we assessed the   interaction of the regulation of ROS production by phosphorylation and Ca 2ϩ , by simultaneously treating cells with CA and ionomycin. Pretreatment of FLAG:AtrbohD-transfected cells with CA dramatically enhanced the transient ROS production induced by ionomycin (Fig. 5). The FLAG:E277Q-AtrbohD mutant, which was not activated by ionomycin (Fig. 3), did not show this enhanced activation in the presence of both CA and ionomycin (data not shown). Taken together, our results suggest that the conformational change in the EF-hand region induced when Ca 2ϩ binds to the EF-hand region and direct phosphorylation of AtrbohD synergistically activate its ROSproducing activity.

DISCUSSION
The AtrbohD gene has been implicated in plant defense response signaling and abscisic acid-induced stomatal closure signaling via ROS production, based on the phenotype of lossof-function mutants and sequence homology to mammalian gp91 phox /NOX2 (20,21,40). However, the activation mechanism of the NADPH oxidase activity of AtrbohD had not been clear, because of the lack of a plant expression system for the real-time monitoring of ROS production by rboh. Using a heterologous expression system, we have shown that AtrbohD possesses ROS-producing activity that is rapidly and transiently  activated by ionomycin and synergistically activated by CA and ionomycin (Fig. 5). The activation of AtrbohD by CA and ionomycin was massive and immediate, suggesting that this protein could be a key to rapid signaling, as is required for plant defense and stomatal closure.
It was suggested that the EF-hand motif of AtrbohF (formerly RbohA) binds to Ca 2ϩ (16), but was not characterized quantitatively, and its physiological significance remained unclear. We have determined the Ca 2ϩ binding affinity of the two putative EF-hand motifs in AtrbohD by employing Ca 2ϩ titration experiments and Ca 2ϩ -dependent fluorescence changes (Fig.  2B). The affinity of WT-EF for Ca 2ϩ was lower than that of most EF-hand-containing Ca 2ϩ sensor proteins. The relatively low Ca 2ϩ affinity of WT-EF (pCa 50 ϭ 4.0 Ϯ 0.03) is likely to be due to the truncation of the EF-hand region, which consists of two EF-hand motifs; in fact, other truncated proteins, such as troponin C and Ca 2ϩ -binding protein 40, which have only the EF-hand motifs, also generally show lower affinities for Ca 2ϩ (41,42). We also observed that treating HEK293T cells with 1 M ionomycin elevated [Ca 2ϩ ] cyt from ϳ10 Ϫ8 to 10 Ϫ7 M using Fluo-4 NW Calcium Assay kits (Invitrogen) (data not shown), suggesting that AtrbohD is activated by Ca 2ϩ as well as being an EF-hand containing Ca 2ϩ sensor protein, or the affinity for Ca 2ϩ of AtrbohD may become greater by phosphorylation.
The E277Q mutation in the first EF-hand motif reduced the affinity for Ca 2ϩ (Fig. 2B), as a result of losing a chelating oxygen atom and the hydrogen bond network. The two oxygen atoms on the side chain of the Glu at the twelfth position in the Ca 2ϩ -binding site coordinate bidentately with one Ca 2ϩ . By contrast, the N321E mutation in the second EF-hand motif increased affinity for Ca 2ϩ (Fig. 2B). This mutation increases the chelating residues and stabilizes the hydrogen bond network (43), even though the Pro residue at the second position in the second EF-hand motif of AtrbohD is not part of this network. Interestingly, the N321E-EF mutant had an increased affinity for Ca 2ϩ , but FLAG:N321E-AtrbohD produced lower levels of ROS (Figs. 2B and 3B). There are two possible reasons why increasing the Ca 2ϩ sensitivity of AtrbohD could inhibit its enzyme function. One is that the small change in the secondary structure on Ca 2ϩ binding, demonstrated in the N321-EF mutant (Fig. 2D), might not be sufficient in N321E-AtrbohD for the Ca 2ϩ -dependent activation of the AtrbohD. The other possibility is that the high affinity of N321E-AtrbohD for Mg 2ϩ might inhibit the Ca 2ϩ -dependent activation of AtrbohD. As N321E-EF had a high affinity not only for Ca 2ϩ but also for Mg 2ϩ , Mg 2ϩ binding might interfere with the Ca 2ϩ binding to the EF-hand region under physiological conditions. CA induced ROS production and enhanced the phosphorylation of AtrbohD (Fig. 4, A-C), suggesting that direct phosphorylation of AtrbohD activates its ROS-producing enzyme activity. Several studies have suggested that this phosphorylation is mediated by calcium-dependent protein kinases (CDPKs). Ectopic expression of an Arabidopsis CDPK enhanced NADPH oxidase activity and stimulated an oxidative burst in tomato protoplasts (44). Recently, Benschop et al. (45) showed that seven different residues in AtrbohD were phosphorylated in response to elicitors, using mass spectrometry and quantitative phospho-proteomics. Nühse et al. (46) showed that phosphorylation at Ser 343/347 was a major effect induced by elicitors and was required for the activation of AtrbohD. These results suggest that CDPK might phosphorylate AtrbohD. Potato CDPK has been shown to phosphorylate potato StrbohB and to induce ROS production; as StrbohB was phosphorylated in the process of mounting defense responses to pathogens, its phosphorylation by CDPK might be an important step in defense signaling (47). As the rise in [Ca 2ϩ ] cyt occurs prior to ROS production in defense signaling (5-7) and CA and ionomycin synergistically activate the AtrbohD (Fig. 5), we suggest there are two possible mechanisms for the Ca 2ϩ -triggered activation of rboh proteins: Ca 2ϩ directly activates AtrbohD by inducing conformational changes in the EF-hand region, or Ca 2ϩ indirectly activates it via phosphorylation mediated by CDPKs.
We suggest that the conformational change in the EF-hand region on Ca 2ϩ binding is important for activating ROS production by AtrbohD (Figs. 2C, 2D, and 3). In human NOX5, the intramolecular interaction between the regulatory N terminus containing four EF-hand motifs and the catalytic C terminus is regulated in a Ca 2ϩ -dependent manner and leads to a Ca 2ϩinduced conformational change at the N terminus and activation of the enzyme (48). The activation of AtrbohD by Ca 2ϩ binding to the EF-hand motifs might also be mediated by such an intramolecular interaction, although outside the EF-hand motifs, the amino acid sequences in the N-terminal regions of AtrbohD and NOX5 differ. We also showed that CA enhanced ionomycin-induced ROS production (Fig. 5). Perhaps phosphorylated AtrbohD, induced by CA treatment, can undergo greater conformational changes than non-phosphorylated AtrbohD, such that intramolecular interactions between the N-terminal and C-terminal regions of AtrbohD, and thus its activation, might be enhanced by CA and ionomycin together.
Translocation of the cytosolic components of mammalian phagocyte NADPH oxidase, including p47 phox , p67 phox , p40 phox , and Rac, is necessary for its activation (49). By contrast, homologs of p47 phox , p40 phox , and p67 phox have not been found in the Arabidopsis genome (50). HEK293T cells also lack p47 phox and p67 phox (29), yet AtrbohD could be activated in them. Thus, plant rboh proteins do not appear to need these additional components for activation, in common with NOX5, which can also operate independently of these cytosolic proteins (29,48). This is consistent with results using gel active stain assays that found NADPH oxidase activity in membrane extracts from tomato and tobacco in the absence of additional cytosolic components (11). However, we cannot exclude a role for Rac homologs in the activation of AtrbohD in plant cells. In plants, members of a family of Rac-like small GTP-binding proteins, named Rho-related small GTPases (ROPs), are known to be involved in the regulation of ROS production during defense responses (26,(51)(52)(53). SUPERCENTIPEDE1 (SCN1), which is a Rho GTPase GDP dissociation inhibitor, controls the production of ROS by AtrbohC/RHD2 via ROP GTPase (3,54). It has also been suggested that AtrbohC/RHD2 is required for ROP2dependent ROS production (55). The heterologous expression system used in this study would be a useful tool to investigate the function of ROP/RAC in plant rboh-dependent ROS production.