JXB Advance Access originally published online on March 21, 2006
Journal of Experimental Botany 2006 57(6):1373-1379; doi:10.1093/jxb/erj113
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RESEARCH PAPER |
Subcellular localization of Strboh proteins and NADPH-dependent O2–-generating activity in potato tuber tissues
1Plant Pathology Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
2Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
3Laboratory of Defense in Plant–Pathogen Interactions, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
* To whom correspondence should be addressed. E-mail: hyoshiok{at}agr.nagoya-u.ac.jp
Received 1 September 2005; Accepted 10 January 2006
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Abstract |
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Rapid generation of reactive oxygen species (ROS) at the cell surface has been implicated in plant defence responses. Genetic evidence indicates that a plant NADPH oxidase (Rboh; respiratory burst oxidase homologue) is associated with oxidative burst. However, there is not enough physiological evidence of Rboh localization available yet. Isozyme-specific antibodies against potato StrbohA and StrbohB (St; Solanum tuberosum) were prepared to investigate the localization of these proteins. Immunoblot analyses using potato microsomal proteins revealed that StrbohA was expressed constitutively at a low level, whereas the accumulation of StrbohB protein was induced by the cell wall elicitor of the potato pathogen Phytophthora infestans. It is demonstrated here that StrbohA and StrbohB are distributed in plasma membrane fractions which have been separated by sucrose density-gradient centrifugation using their specific antibodies. Green fluorescent protein-tagged Strboh proteins were also located on the plasma membrane by transient expression assay in onion epidermal cells. Additionally, NADPH-dependent
-generating activities in plasma membrane fractions were diphenylene iodonium-sensitive and NaN3-insensitive. These data suggest that StrbohA and StrbohB are predominantly localized on the plasma membrane and regulate ROS production in defence signalling. Key words: NADPH oxidase, Rboh, reactive oxygen species, subcellular localization
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The production of reactive oxygen species (ROS), called the oxidative burst, is one of the earliest events detected during incompatible interactions between plants and pathogens (Doke, 1983
; Lamb and Dixon, 1997). Roles for ROS have been proposed in the induction of an intracellular signalling pathway (Desikan et al., 1999; Grant et al., 2000) and in the activation of systemic-acquired resistance (Alvarez et al., 1998; Park et al., 1998). Several lines of evidence indicate that the NADPH oxidase seems to play a pivotal role in defence responses. Diphenylene iodonium (DPI), an inhibitor of the neutrophil NADPH oxidase, blocks the oxidative burst in plant cells (Levine et al., 1994; Auh and Murphy, 1995; Dwyer et al., 1996).
Plant NADPH oxidases called Rbohs (respiratory burst oxidase homologue, a homologue of gp91phox, which is a catalytic subunit of phagocyte NADPH oxidase) have been isolated from various plant species (Groom et al., 1996; Keller et al., 1998; Torres et al., 1998; Amicucci et al., 1999; Yoshioka et al., 2001; Yoshie et al., 2005). The Arabidopsis genome contains 10 Atrboh paralogues (Kwak et al., 2003). These plant Rboh proteins are predicted to have six transmembrane-spanning domains that correspond to those identified in gp91phox, and carry an N-terminal extension comprising two EF-hand motifs, suggesting that the calcium ion (Ca2+) regulates its activity. Expression analyses of Rbohs indicate that some of them are induced by elicitor or abiotic stress and others accumulate constitutively (Desikan et al., 1998; Yoshioka et al., 2001, 2003; Simon-Plas et al., 2002; Kwak et al., 2003; Yoshie et al., 2005). Genetic studies indicate that Rboh is a key regulator of ROS production and displays pleiotropic functions in plants (Sagi et al., 2004; Torres and Dangl, 2005). Arabidopsis atrbohD/atrbohF mutant does not induce ROS production against pathogen infection (Torres et al., 2002) and abscisic acid-mediated stomatal closure (Kwak et al., 2003), and the atrbohC mutant has a defect in root hair formation by decreased levels of ROS production (Foreman et al., 2003). Tobacco cells transformed with antisense constructs of NtrbohD lose ROS production to an elicitor treatment (Simon-Plas et al., 2002). NbrbohA/NbrbohB-silenced Nicotiana benthamiana plants show a reduced oxidative burst and reduced disease resistance to Phytophthora infestans (Yoshioka et al., 2003).
In potato plants, treatment of tubers with hyphal wall components (HWC) from P. infestans causes a rapid and transient accumulation of H2O2 (phase I), followed by a massive oxidative burst 6–9 h after the treatment (phase II). RNA gel blot analyses indicate that StrbohA is constitutively expressed at a low level, whereas StrbohB is up-regulated during the phase II burst. DPI blocks both bursts, while pretreatment of the protein synthesis inhibitor cycloheximide with the tuber abolishes only the second burst. These data suggest that StrbohA and StrbohB contribute to phase I and phase II bursts, respectively (Yoshioka et al., 2001).
Oxidative burst is known to induce a rapid increase in cytosolic free calcium [Ca2+]cyt during defence to initiate the signalling pathway. Application of H2O2 to suspension-cultured cells causes a rapid [Ca2+]cyt increase (Lecourieux et al., 2002). The [Ca2+]cyt levels may rise via Ca2+ channels located on the plasma membrane and endomembranes. Plasma membrane ion channels are rapidly activated by pathogen infection or elicitor treatment. In particular, extracellular alkalinization, Ca2+ influx, and effluxes of K+ and Cl– lead to depolarization of the plasma membrane (Scheel, 1998). Extracellular Ca2+ appears to be crucial to induction of plant defence against pathogens (Yang et al., 1997; Scheel, 1998). It was hypothesized that oxidative burst on the plasma membrane contributes to the extracellular Ca2+ influx. However, subcellular localization of Rboh proteins is not well documented. To investigate the localization of StrbohA and StrbohB proteins, anti-StrbohA and anti-StrbohB antibodies, respectively, were prepared. Immunoblot analysis using the fractions separated by sucrose density-gradient centrifugation from potato tuber tissues indicated that both StrbohA and StrbohB were distributed in the plasma membrane fractions. Fusion proteins of Strbohs and green fluorescent protein (GFP) were expressed transiently in onion epidermal cells and confirmed plasma membrane localization by confocal laser-scanning microscopy. These data suggest that StrbohA and StrbohB proteins are localized on the plasma membrane and regulate ROS production in defence signalling.
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Materials and methods |
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Plant materials, HWC elicitor, and treatment
Tubers of the potato cultivar Rishiri carrying the R1 gene were stored at 4 °C until use. Tuber discs were prepared 20 mm in diameter and 2 mm thick. These discs were aged for 18 h and treated with 100 µl of water or HWC elicitor (0.1 mg ml–1). HWC elicitor was prepared from the mycelium of P. infestans (Doke and Tomiyama, 1980
). In all experiments, the treated discs were incubated at 20 °C in the dark.
Membrane preparation from potato tubers
Potato tuber discs were homogenized in 50 mM MOPS-KOH buffer (pH 7.6), containing 0.5 M sorbitol, 5 mM EDTA, 5 mM EGTA, 20 mM 2-mercaptoethanol, 50 mM ß-glycerophosphate, 1 mM Na3VO4, 100 mM NaF, 10% polyclar VT, 0.1 mM AEBSF, and 1 µM E-64, for 1 min in a blender. The homogenate was filtered through four layers of gauze. The filtrate was centrifuged at 10 000 g for 15 min and the resulting supernatant fraction centrifuged at 150 000 g for 20 min. The pellet was suspended in buffer A [20 mM TRIS-HCl buffer (pH 7.5) containing 0.25 M sucrose, 5 mM 2-mercaptoethanol, 10 mM ß-glycerophosphate, 1 mM Na3VO4, 10 mM NaF, 0.1 mM AEBSF, and 1 µM E-64] with a Teflon homogenizer. The suspension was centrifuged at 150 000 g for 20 min and the pellet was resuspended in buffer A. For sucrose density-gradient centrifugation, microsomes were suspended in buffer B [20 mM potassium phosphate buffer (pH 7.5) containing 5 mM 2-mercaptoethanol, 0.1 mM AEBSF, and 1 µM E-64], overlaid on 11 ml of 20–50% (w/w) sucrose gradient in buffer B, and centrifuged in an SW41 rotor at 100 000 g for 18 h. The gradient was separated into 18 fractions. Protein concentration was determined using the Bradford assay (Bradford, 1976) with bovine albumin as a standard.
Preparation of recombinant proteins and antibody preparation
An 837-bp-long fragment from StrbohA N-terminal and a 603-bp-long fragment from StrbohB N-terminal were subcloned into pET-30a (+) expression vector (Novagen) for antigen preparation or into pGEX-4T-1 expression vector (Amersham Biosciences) for determination of antibody specificity. The resulting proteins were used to immunize rabbits at weekly intervals. Polyclonal antibodies against plasma membrane (PM) aquaporin (Ohshima et al., 2001), endoplasmic reticulum (ER) luminal protein (Bip) (Kobae et al., 2004), and vacuolar membrane H+-ATPase subunit A were prepared as previously (Matsuura-Endo et al., 1992).
Immunoblotting
Samples with equal amounts of proteins were used in the experiments shown in Figs 1 and 2 or with equal volumes of fractions for those shown in Figs 3 and 5. They were subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Probing and detection of immunoblot were performed as described in the ECL Western-Blotting detection kit (Amersham Biosciences).
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O2– production
NADPH oxidase was assayed in membrane by a modified assay using sulfophenyl, carboxanilide-2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) instead of cytochrome c as a substrate (Sang et al., 2001
). The reaction mixture contained 0.3 mM XTT, 20 µM ATP, 3 µM GTP (
) S, 0.02% (w/v) Triton X-100, 20 mM TRIS-HCl (pH 7.5), and a 20 µl fraction with or without various inhibitors in a total volume of 0.5 ml. The reaction was initiated in a microcuvette at room temperature by addition of 80 µM NADPH or buffer for NADPH minus control, and scanned by the change in A470 over 5 min in a dual-beam spectrophotometer.
Expression of a fusion protein of green fluorescent protein and Strboh in onion epidermal cells
PCR products of StrbohA and StrbohB added to the 4 bp sequences (CACC) on the N-terminal were subcloned into pENTRTM/D-TOPO® (Invitrogen). The StrbohA and StrbohB sequences were subcloned into pUGW5 vector (Kobae et al., 2004) by Gateway® LR recombination reaction system (Invitrogen). A transient transformation was performed as described by Chiu et al. (1996). Onion epidermal cell segments were peeled and placed on the MS medium plate [1x MS salts (Wako), 0.3% phytagelTM (Sigma)]. Plasmid DNAs (0.83 µg) were introduced to onion epidermal segments using a pneumatic particle gun (PDS-1000/He; Bio-Rad). The conditions of bombardment were a vacuum of 28 inches of Hg, helium pressure of 1350 psi, and 6 cm of target distance using 1.6 µm gold microcarriers. After bombardment, tissues were incubated for 20–22 h at room temperature. Green fluorescence was observed using a fluorescence microscope or by FITC-filtered visual inspection under a confocal laser-scanning microscope (Carl Zeiss LSM 510).
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Results |
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Strboh protein accumulation in potato tuber tissues treated with HWC elicitor
For preparation of StrbohA and StrbohB antibodies, N-terminal regions just before the first EF hand motif for the antigen were used. The regions were not conserved between these proteins. Immunoblot analysis showed that the anti-StrbohB antibody detected only StrbohB N-terminal recombinant proteins (Fig. 1). On the other hand, the anti-StrbohA antibody slightly cross-reacted with StrbohB N-terminal recombinant proteins. However, the antibody detected only 102 kDa StrbohA proteins in immunoblot analysis using the microsomal fraction (Fig. 2).
Changes in protein accumulation levels of Strbohs in microsomal proteins from potato tubers treated with water or HWC elicitor were estimated by immunoblot analyses (Fig. 2). The anti-StrbohA and anti-StrbohB antibodies detected 102 kDa and 83 kDa proteins, respectively, in good agreement with the estimated molecular mass of these proteins. StrbohA proteins accumulated constitutively at a low level despite the treatments, whereas accumulation of StrbohB proteins was induced, peaking 9 h after treatment with HWC elicitor (Fig. 2). The peak of StrbohB protein accumulation corresponded to that of the phase II oxidative burst in potato tubers treated with HWC elicitor (Yoshioka et al., 2001).
Intracellular distribution of StrbohA and StrbohB
The microsomal fraction prepared from potato tubers was further separated by sucrose density-gradient centrifugation. Then the distribution of StrbohA, StrbohB, and the marker enzymes such as aquaporin for plasma membrane, V-ATPase A subunit for vacuolar membrane, and Bip for ER was examined by immunoblot analysis (Fig. 3). In non-elicited tissues, StrbohA proteins were detected in the fractions corresponding to PM aquaporin (Fig. 3A). In the tissues treated with HWC elicitor for 9 h, both StrbohA and StrbohB proteins were also detected in the fractions corresponding to PM aquaporin (Fig. 3B). Bip was detected in two distinct molecular weight bands. The distribution pattern of the lower bands was similar to that of vacuolar membrane proteins, and the upper bands were divided into low- and high-density areas. It is known that Bip distributes in low-density fractions near to vacuolar membrane fractions under Mg2+-free conditions (Kobae et al., 2004). To prepare the microsomal fraction from potato tubers 5 mM EDTA was used, suggesting that immunodetected bands in low molecular positions are Bip in potato tubers. The possibility cannot be excluded that the upper bands detected with anti-Bip may also be Bip in potato tubers. However, the distribution pattern is different from that of the StrbohA and StrbohB proteins. Therefore, these data indicate that both StrbohA and StrbohB are distributed in the plasma membrane fractions.
Subcellular localization of Strboh and GFP fusion proteins expressed in onion epidermal cells
To confirm plasma membrane localization of Strboh proteins, Strboh:GFP fusion proteins were transiently expressed in onion epidermal cells. The GFP sequence was fused at the C-terminus of Strboh under the control of Cauliflower mosaic virus 35S promoter and bombarded in an onion epidermal segment. After 20–22 h, the localization of GFP fusion proteins was observed by fluorescence microscopy or confocal laser-scanning microscopy. In the case of expression of the GFP control, green fluorescence was observed by fluorescence microscopy throughout the cytoplasm and nucleus. In the cells expressed for StrbohA:GFP or StrbohB:GFP, the fluorescence was observed equally over the whole cell (Fig. 4A). Confocal imaging of GFP fluorescence revealed that Strboh:GFP fusion protein was located on the plasma membrane, whereas only GFP protein was seen throughout the cytoplasm (Fig. 4B).
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Intracellular distribution of NADPH-dependent O2–-generating activities
Because treatment of potato tubers with HWC elicitor caused a massive oxidative burst 6–9 h after treatment (Yoshioka et al., 2001
), intracellular distribution of NADPH-dependent -generating activities from potato tubers treated with HWC elicitor for 9 h were examined. -generation was detected by XTT-reducing activities. The distribution of NADPH oxidase activities was paralleled by the distribution of immunostained bands of StrbohB and PM aquaporin (Fig. 5A). NADPH oxidase is DPI-sensitive and NaN3 (a general inhibitor of haem protein)-insensitive. The effects of these inhibitors on the activity were confirmed using fraction no. 12. Addition of 100 U ml–1 of superoxide dismutase (SOD) or 50 µM of DPI resulted in suppression of NADPH oxidase activity; on the other hand, 10 mM of NaN3 did not change the activity (Fig. 5B). |
Discussion |
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Immunoblot analyses indicated that antibodies raised against StrbohA and StrbohB were detecting 102 kDa and 83 kDa proteins, respectively, in good agreement with the estimated molecular mass of these proteins (Fig. 2). Here it is shown that StrbohA accumulated constitutively at a low level, whereas StrbohB was induced by HWC peaking 9 h after the treatment (Fig. 2). This peak of StrbohB protein accumulation corresponds to the maximum point of phase II burst in potato tubers in response to HWC elicitor (Yoshioka et al., 2001
).
Next, immunoblot analyses in fractions obtained by sucrose density-gradient centrifugation were performed to examine the distribution of the StrbohA and StrbohB proteins. Both proteins were distributed in fractions corresponding to that of PM aquaporin (Fig. 3). Observation of onion epidermal cells expressing transiently Stroboh:GFP fusion proteins under confocal laser-scanning microscopy showed GFP fluorescence only on the plasma membrane (Fig. 4). Furthermore, it was confirmed that the distribution of NADPH oxidase activities in the fractions from sucrose density-gradient centrifugation was paralleled by the distribution of immunostained bands of StrbohB and PM aquaporin (Fig. 5). These lines of evidence indicate that both StrbohA and StrbohB proteins are localized on the plasma membrane. NADPH oxidase in phagocytic cells makes an enzymatic complex and comprises two membrane-associated proteins (p22phox and gp91phox, also known as Nox2) and cytosolic components (p67phox, p47phox, and Rac2, a small G protein). Although homologues of p22phox, p67phox, and p47phox have not been identified from any plants and have not been found in the Arabidopsis genome sequence, Rac homologues are isolated from several plants and participate in the oxidative burst. A constitutive active mutant of OsRac1 involves generation of H2O2 in rice cells and plants (Kawasaki et al., 1999). OsRac1 includes a prenylation motif on the C-terminal region, which is required for plasma membrane localization (Ono et al., 2001). OsRac1 might associate with the plasma membrane and respond to external stimuli activating Rboh directly or indirectly.
A rapid Ca2+ influx is known to be induced by elicitor treatments during the oxidative burst. Pharmacological experiments indicate that apoplastic Ca2+ influx is important in the oxidative burst (Baker et al., 1993; Miura et al., 1995, 1999; Chandra and Low, 1997). Recently, it was reported that overexpression of OsTPC1, a putative voltage-gated Ca2+-permeable channel, which localizes to plasma membrane, enhances an elicitor-induced oxidative burst (Kurusu et al., 2005). As referred to above, Ca2+ influx across the plasma membrane may contribute to the activation process of NADPH oxidase in the plasma membrane. In animal cells, Nox5 in testis, spleen, and lymph nodes (Bánfi et al., 2001) and Duox1 and Duox2 in the thyroid (Dupuy et al., 1999, 2000), which are longer forms of Nox2, have EF hand motifs in their N-terminal regions and appear to be regulated by Ca2+. Corresponding with these observations, Rboh EF hands could bind Ca2+ (Keller et al., 1998) and the in-gel NADPH oxidase activities of tobacco and tomato plasma membranes in SDS-polyacrylamide gel are activated by addition of Ca2+ and inhibited by EGTA (Sagi and Fluhr, 2001).
It is also suggested that [Ca2+]cyt regulates an oxidative burst via calcium-dependent protein kinase (CDPK). CDPK is a serine-threonine protein kinase, which carries a calmodulin-like domain at the C-terminal region and allows a response to Ca2+ signalling. Membrane-bound CDPK is activated by Cf-9/Avr9 interaction in the tobacco plant prior to the oxidative burst (Romeis et al., 2000), and ectopic expression of Arabidopsis CDPK in tomato protoplast elevates plasma membrane-associated NADPH oxidase activity (Xing et al., 2001). Transient expression of the constitutive active form of NtCDPK2 in N. benthamiana provides oxidative burst-mediated cell death against hypo-osmotic stress (Ludwig et al., 2005). These data suggest that CDPK is involved in the activation process of NADPH oxidase.
Presumably, ROS generated on the plasma membrane are released to the apoplast, induce oxidative crosslinking of glycoproteins, and strengthen the cell wall against secondary infection (Bradley et al., 1992), simultaneously activating the Ca2+ channel to increase the level of [Ca2+]cyt. Ca2+ may function not only as an inducer of the oxidative burst, but also as a signalling molecule downstream of the oxidative burst. It is shown that Ca2+ influx induced by cryptogein in Nicotiana plumbaginifolia cells activates NADPH oxidase, which in turns leads to an increase in [Ca2+]cyt mainly from extracellular Ca2+ influx (Lecourieux et al., 2002). In stomatal closure, ROS induced by abscisic acid, which provoke expression of the genes for AtrbohF and AtrbohD in guard cells, activate the plasma membrane Ca2+ channel (Pei et al., 2000). Taken together, the present results support the idea that plant NADPH oxidases may function at the plasma membrane to regulate various cellular responses. However, the possibility cannot be ruled out that the isozymes of Strboh, which have not been identified, might localize to the endomembranes.
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Acknowledgements |
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We thank Dr T Nakagawa of Shimane University for providing the pUGW5 vector. This work was supported in part by a Grant-in-Aid for Scientific Research (S) (14104004) from the Ministry of Education, Science and Culture of Japan and also by a Grant-in-Aid for Scientific Research on Priority Area (A) from the Research for the Future Program of the Japan Society for the Promotion of Science.
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