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Originally published In Press as doi:10.1074/jbc.M708106200 on January 23, 2008

J. Biol. Chem., Vol. 283, Issue 14, 8885-8892, April 4, 2008
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Synergistic Activation of the Arabidopsis NADPH Oxidase AtrbohD by Ca2+ and Phosphorylation*

Yoko Ogasawara{ddagger}1, Hidetaka Kaya{ddagger}§1, Goro Hiraoka{ddagger}, Fumiaki Yumoto||, Sachie Kimura{ddagger}, Yasuhiro Kadota{ddagger}, Haruka Hishinuma{ddagger}, Eriko Senzaki{ddagger}, Satoshi Yamagoe**, Koji Nagata, Masayuki Nara{ddagger}{ddagger}, Kazuo Suzuki**, Masaru Tanokura, and Kazuyuki Kuchitsu{ddagger}§2

From the {ddagger}Department of Applied Biological Science and the §Genome & Drug Research Center, Tokyo University of Science, Noda, Chiba 278-8510, Japan, the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan, the ||Department of Physiology II, Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan, the **Department of Bioactive Molecules, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan, and the {ddagger}{ddagger}Laboratory of Chemistry, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Ichikawa, Chiba 272-0827, Japan

Received for publication, September 28, 2007 , and in revised form, December 26, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 Ca2+ concentration ([Ca2+]cyt) (5–7) and is inhibited both by Ca2+ chelators such as EGTA and BAPTA, and by Ca2+ channel blockers such as La3+ (6, 8). The overexpression of rice two-pore channel 1 (OsTPC1), which is a putative voltage-gated Ca2+ 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 Ca2+ in vitro (11), suggesting that elicitor-induced ROS production by plant NADPH oxidase might be dependent on Ca2+.

In mammalian phagocytes, ROS production is mediated by the NADPH-dependent phagocytic oxidase (phox) complex, which consists of the catalytic subunit gp91phox/NADPH oxidase (NOX) 2, together with the regulatory subunits p22phox, p40phox, p47phox, p67phox, 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 gp91phox/NOX2 homologs in various plant species, including rice, Arabidopsis, tomato, potato, and tobacco (10, 13–17). 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 45Ca2+ (16), suggesting an important role for Ca2+ 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 Ca2+ 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 Ca2+ and the {alpha}-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 Ca2+ 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 Ca2+ binding to the EF-hand motifs and direct phosphorylation synergistically activate AtrbohD to produce ROS.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructs—We amplified the coding region of AtrbohD (Arabidopsis Genome Initiative [AGI] code At5g47910, accession no. AF055357 [GenBank] , amino acid residues 1–921) from a cDNA library derived from seedlings of A. thaliana, and subcloned it into the pcDNA3.1(-) vector (Invitrogen) to express AtrbohD with the FLAG epitope tag at its 5'-end. We also generated mutants of AtrbohD (E277Q-AtrbohD and N321E-AtrbohD) using point mutant primers and the mega-primer PCR method. For protein expression in Escherichia coli, we amplified the cDNA encoding WT-EF (corresponding to amino acid residues 250–332 and including the two putative EF-hand motifs) by PCR, and subcloned the cDNA into the pET-26b(+) vector (Novagen) at NdeI and BamHI restriction sites. We generated mutants of WT-EF (D266A-EF, E277Q-EF, D310A-EF, and N321E-EF). The primer sequences used were as follows: AtrbohD forward, 5'-ATTGAATTCATGAAAATGAGACGAGGCAA-3', and reverse 5'-ATTGGTACCCTAGAAGTTCTCTTTGTGGA-3'; WT-EF forward, 5'-CATATGAGCGATGAAAGCTTTGATGCC-3', and reverse, 5'-GGATCCTTACTGGTTTGGTGCTTGTAACAG-3'.

Cell Culture and Transfection—HEK293T cells were maintained at 37 °C in 5% CO2 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 Ca2+, and measurements were made in HBSS supplemented with 6 units/ml horseradish peroxidase (Wako, Osaka, Japan) and 250 µM luminol (Nacalai Tesque, Kyoto, Japan). To mobilize Ca2+ uptake, 1 µM 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(+)-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-β-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 Ca2+ to precipitate the E. coli proteins, centrifuged again, and the supernatant was treated with 3% (v/v) trichloroacetic acid to precipitate the Ca2+-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 (~20 °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 (pCa50) 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).


Figure 1
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  		FIGURE 1.
AtrbohD produces ROS in HEK293T cells. A, HEK293T cells were transiently transfected with FLAG:AtrbohD. ROS production was proportional to the concentration of transfected FLAG:AtrbohD (upper panel). FLAG:AtrbohD was detected by Western blotting with anti-FLAG antibodies (lower panel). B, after baseline measurements of ROS production for 5 min, in cells transfected with different concentrations of FLAG: AtrbohD, 1 µM ionomycin was added. C, cells transfected with 100 ng of FLAG:AtrbohD were pretreated with different concentrations of DPI. D, 5 min after inducing ROS with ionomycin, 800 units/ml SOD was added to FLAG:AtrbohD-transfected cells. E, effect of extracellular Ca2+ concentration on ROS production induced by 1 µM ionomycin. FLAG:AtrbohD-transfected cells were incubated in HBSS with increasing concentrations of extracellular Ca2+. ROS production was measured by chemiluminescence and expressed as relative luminescence units (RLU). Results given are means ± S.E. (n = 3).

 
CD Measurements—Far-ultraviolet (UV) CD spectra were acquired at room temperature (~20 °C) using a Jasco J-720 spectropolarimeter, which was set for a 200–260-nm wavelength range, 0.2-nm step resolution, 100-nm·min–1 scan speed, 2-s response time, 1-nm bandwidth, and eight scans per sample. Samples of the 20 µM 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 CaCl2 or 2 mM MgCl2 in the metal-bound state. The {alpha}-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 [32P]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 µM 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 32P-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 gp91phox/NOX2 and NOX5 (29, 32); this allowed us to quantitatively monitor exogenous Ca2+-dependent 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 Ca2+-dependent—We added ionomycin, a Ca2+ ionophore, which induces Ca2+ influx into the cell, to FLAG:AtrbohD-transfected and empty vector-transfected cells, to examine the effect of Ca2+ 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 (35–38). To test the Ca2+ dependence of ionomycin-induced ROS production, AtrbohD-transfected HEK293T cells were pretreated with increasing concentrations of CaCl2 from 0 to 1.26 mM. No ROS were produced in the absence of extracellular Ca2+, and ROS production increased in proportion to the concentration of extracellular Ca2+ (Fig. 1E), showing that ROS production by AtrbohD is a Ca2+-dependent enzyme activity.

Ca2+ 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 Ca2+ based on amino acid sequence homologies. However, Ca2+ binding to the EF-hand motifs has not been directly demonstrated. We measured the affinity of Ca2+ 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 Ca2+ concentration (pCa = –log10[Ca2+]) on the normalized fluorescence attributed to Tyr residues is shown in Fig. 2B. The pCA50 value for WT-EF was 4.0 ± 0.03, indicating that AtrbohD is a Ca2+-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 Ca2+ chelation (30, 39); we therefore hypothesized that the first EF-hand motif was likely to make a greater contribution to Ca2+ binding by AtrbohD than the second EF-hand motif. To test this hypothesis, we compared the affinity for Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ were higher and lower, respectively, than that of WT-EF, as we expected. As an Asp residue at the first (X) position in a Ca2+-binding site is known to be critical for Ca2+ chelation (39), we also measured the affinity for Ca2+ 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 Ca2+ were lower than that of WT-EF (Fig. 2A). In addition, the D266A mutation reduced the affinity for Ca2+ 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 Ca2+ binding.

Ca2+-induced Conformational Changes in the EF-hand Region—To examine the effect of Ca2+ binding to the two EF-hand motifs on the conformation of the EF-hand region, we measured far-UV CD to estimate the {alpha}-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 {alpha}-helical proteins (Fig. 2C). The relative {alpha}-helical contents of WT-EF, E277Q-EF, and N321E-EF in their Ca2+ 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 Ca2+ than N321E-EF, while the affinity of WT-EF for Ca2+ was lower than that of N321E-EF (Fig. 2B). E277Q-EF underwent a smaller conformational change than WT-EF on binding Ca2+, which is consistent with it having a low affinity for Ca2+.Mg2+ binding also increased the {alpha}-helical content of the three recombinant proteins, but to a lesser extent than Ca2+ binding, indicating that Ca2+ 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:{Delta}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 Ca2+ binding to its EF-hand region.

Although the affinity of N321E-EF for Ca2+ 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 EF-hand region upon Ca2+ binding, rather than the level of the affinity for Ca2+, is the important factor for ROS production by AtrbohD.


Figure 2
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  		FIGURE 2.
Ca2+ binding activities and Ca2+-induced changes in the physicochemical properties of the EF-hand region of AtrbohD. A, amino acid sequences of wild-type and mutant EF-hand regions of AtrbohD. Both EF-hand motifs are underlined. The Ca2+-binding sites in the first and second EF-hand motifs are in boxes. The consensus Ca2+-binding site is shown: first (X), third (Y), fourth (Z), seventh (#), ninth (–X) = Ca2+ ligand; twelfth (-Z) = Ca2+ ligand, a bidentate Glu or Asp; fifth (G) = Gly; eighth (I) = Ile or other aliphatic residues are found at this position; *, any residue. An arrow indicates the non-bidentate Asn residue in the second EF-hand motif of AtrbohD. B, effect of mutations in the putative EF-hand motifs on Ca2+ affinity. Ca2+-dependent fluorescence was measured by fluorescence spectroscopy. Filled circle, WT-EF; open diamond, D266A-EF; filled diamond, E277Q-EF; filled square, N321E-EF; open square, D310A-EF. C, CD spectra of EF-hand regions in apo states (line), Mg2+-bound states (dashed line), and Ca2+-bound states (dotted line). D, relative helical content of AtrbohD EF-hand regions; white, apo states; gray, Mg2+-bound states; black, Ca2+-bound states.

 
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:AtrbohD-transfected 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-AtrbohD-transfected 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:AtrbohD-transfected cells. This might be due to trace amounts of Ca2+ 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 [32P]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 Ca2+ binding to EF-hand motifs, we assessed the interaction of the regulation of ROS production by phosphorylation and Ca2+, 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 Ca2+ binds to the EF-hand region and direct phosphorylation of AtrbohD synergistically activate its ROS-producing activity.


Figure 3
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  		FIGURE 3.
Effect of mutations in the EF-hand motifs on ionomycin-induced ROS production. HEK293T cells were transiently transfected with 100 ng of FLAG:AtrbohD, FLAG:E277Q-AtrbohD, or FLAG:N321E-AtrbohD. A, expressed proteins were detected by Western blotting with anti-FLAG antibodies. Comparable protein loading was confirmed by comparison with blots for β-actin. B, after making baseline measurements for 5 min, 1 µM ionomycin was added.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 loss-of-function mutants and sequence homology to mammalian gp91phox/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.


Figure 4
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  		FIGURE 4.
Effects of phosphatase inhibition on AtrohD ROS production and phosphorylation. A and B, effect of phosphatase inhibitor CA on ROS production by FLAG:AtrbohD or FLAG:E277Q-AtrbohD. After making baseline measurements for 5 min, 0.1 µM CA was added to the cells. C, FLAG:AtrbohD-transfected or empty vector-transfected HEK293T cells were radiolabeled with [32P]orthophosphate in vivo. Proteins were immunoprecipitated with anti-FLAG antibodies, separated by SDS-PAGE, and detected by autoradiography (left panel). FLAG:AtrbohD-transfected cells were radiolabeled with [32P]orthophosphate and then either treated with 0.1 µM CA for 10 min (right panel) or not treated. Arrowheads indicate the molecular mass of FLAG: AtrbohD (105 kDa).

 


Figure 5
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  		FIGURE 5.
Synergistic induction of AtrbohD by CA and ionomycin. HEK293T cells were transiently transfected with 100 ng of FLAG:AtrbohD. After making baseline measurements for 5 min, 0.1 µM CA was added to the cells, and 10 min later, either 1 µM ionomycin was added to the cells, or they were not treated.

 
It was suggested that the EF-hand motif of AtrbohF (formerly RbohA) binds to Ca2+ (16), but was not characterized quantitatively, and its physiological significance remained unclear. We have determined the Ca2+ binding affinity of the two putative EF-hand motifs in AtrbohD by employing Ca2+ titration experiments and Ca2+-dependent fluorescence changes (Fig. 2B). The affinity of WT-EF for Ca2+ was lower than that of most EF-hand-containing Ca2+ sensor proteins. The relatively low Ca2+ affinity of WT-EF (pCa50 = 4.0 ± 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 Ca2+-binding protein 40, which have only the EF-hand motifs, also generally show lower affinities for Ca2+ (41, 42). We also observed that treating HEK293T cells with 1 µM ionomycin elevated [Ca2+]cyt from ~10–8 to 10–7 M using Fluo-4 NW Calcium Assay kits (Invitrogen) (data not shown), suggesting that AtrbohD is activated by Ca2+ as well as being an EF-hand containing Ca2+ sensor protein, or the affinity for Ca2+ of AtrbohD may become greater by phosphorylation.

The E277Q mutation in the first EF-hand motif reduced the affinity for Ca2+ (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 Ca2+-binding site coordinate bidentately with one Ca2+. By contrast, the N321E mutation in the second EF-hand motif increased affinity for Ca2+ (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 Ca2+, but FLAG:N321E-AtrbohD produced lower levels of ROS (Figs. 2B and 3B). There are two possible reasons why increasing the Ca2+ sensitivity of AtrbohD could inhibit its enzyme function. One is that the small change in the secondary structure on Ca2+ binding, demonstrated in the N321-EF mutant (Fig. 2D), might not be sufficient in N321E-AtrbohD for the Ca2+-dependent activation of the AtrbohD. The other possibility is that the high affinity of N321E-AtrbohD for Mg2+ might inhibit the Ca2+-dependent activation of AtrbohD. As N321E-EF had a high affinity not only for Ca2+ but also for Mg2+, Mg2+ binding might interfere with the Ca2+ 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 Ser343/347 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 [Ca2+]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 Ca2+-triggered activation of rboh proteins: Ca2+ directly activates AtrbohD by inducing conformational changes in the EF-hand region, or Ca2+ indirectly activates it via phosphorylation mediated by CDPKs.

We suggest that the conformational change in the EF-hand region on Ca2+ 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 Ca2+-dependent manner and leads to a Ca2+-induced conformational change at the N terminus and activation of the enzyme (48). The activation of AtrbohD by Ca2+ 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 p47phox, p67phox, p40phox, and Rac, is necessary for its activation (49). By contrast, homologs of p47phox, p40phox, and p67phox have not been found in the Arabidopsis genome (50). HEK293T cells also lack p47phox and p67phox (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, 51–53). 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 ROP2-dependent 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.


   FOOTNOTES
 
* This work was supported by a Grant-in-Aid for Young Scientists (B) (19770035) from Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to H. K.), by a Grant-in-Aid for Plant Graduate Student from Nara Institute of Science and Technology, supported by MEXT of Japan (to S. K.), in part by a Grant-in-Aid for Japanese Society for the Promotion of Science Fellows (18-6801, to Y. K.), and in part by Grants-in-Aid for Scientific Research (B) (19370023) from MEXT of Japan (to K. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Dept. of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan. Tel.: 81-4-7122-9404; Fax: 81-4-7123-9767; E-mail: kuchitsu{at}rs.noda.tus.ac.jp.

3 The abbreviations used are: ROS, reactive oxygen species; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CA, calyculin A; [Ca2+]cyt, cytosolic free Ca2+ concentration; CD, circular dichroism; DPI, diphenylene iodonium; EGTA, ethyleneglycol-bis(β-aminoethyl)-N,N,N',N'-tetraacetic acid; gp91phox, 91-kDa glycoprotein subunit of the phagocyte NADPH oxidase; HEK, human embryonic kidney; MOPS, 3-morpholinopropanesulfonic acid; NOX, NADPH oxidase; rboh, respiratory burst oxidase homolog; SOD, superoxide dismutase; CDPK, calcium-dependent protein kinases. Back


   ACKNOWLEDGMENTS
 
We thank L. Dolan, S. Takeda, E. Bell, T. Kurusu, and T. Hayashi for critical reading of the manuscript, and Y. Miyauchi, A, Morita, M. Ikekita, Y. Takakusagi, K. Sakaguchi, R. Takasawa, and S. Tanuma for materials and advice.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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