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First published online July 9, 2004; 10.1104/pp.104.041921 Plant Physiology 135:1685-1696 (2004) © 2004 American Society of Plant Biologists
The Salt-Stress Signal Transduction Pathway That Activates the gpx1 Promoter Is Mediated by Intracellular H2O2, Different from the Pathway Induced by Extracellular H2O2[w]Department of Fruit-Tree Breeding and Molecular Genetics, ARO, The Volcani Center, Bet Dagan 50250, Israel (O.A.-K., Y.G.-D., R.G., G.B.-H.); and Department of Biology, Faculty of Science and Science Education, University of Haifa-Oranim, Tivon 36006, Israel (S.L.-Y.)
Several genes encoding putative glutathione peroxidase have been isolated from a variety of plants, all of which show the highest homology to the phospholipid hydroperoxide isoform. Several observations suggest that the proteins are involved in biotic and abiotic stress responses. Previous studies on the regulation of gpx1, the Citrus sinensis gene encoding phospholipid hydroperoxide isoform, led to the conclusion that salt-induced expression of gpx1 transcript and its encoded protein is mediated by oxidative stress. In this paper, we describe the induction of gpx1 promoter:uidA fusions in stable transformants of tobacco (Nicotiana tabacum) cultured cells and plants. We show that the induction of gpx1 by salt and oxidative stress occurs at the transcriptional level. gpx1 promoter analysis confirmed our previous assumption that the salt signal is transduced via oxidative stress. We used induction of the fusion construct to achieve better insight into, and to monitor salt-induced oxidative stress. The gpx1 promoter responded preferentially to oxidative stress in the form of hydrogen peroxide, rather than to superoxide-generating agents. Antioxidants abolished the salt-induced expression of gpx1 promoter, but were unable to eliminate the induction by H2O2. The commonly employed NADPH-oxidase inhibitor diphenyleneiodonium chloride and catalase inhibited the H2O2-induced expression of gpx1 promoter, but did not affect its induction by salt. Our results led us to conclude that salt induces oxidative stress in the form of H2O2, its production occurs in the intracellular space, and its signal transduction pathway activating the gpx1 promoter is different from the pathway induced by extracellular H2O2.
Gluthatione peroxidases (GPXs) are a family of isozymes that use glutathione to reduce hydrogen peroxide (H2O2) and organic and lipid hydroperoxides, thereby protecting cells against oxidative damage (Flohé and Günzler, 1984  ). Several forms of these enzymes have been described in great detail in mammalian systems (Ursini et al., 1995). The classical cytosolic GPX is highly active in reducing H2O2 and in its native form is composed of homotetramers. The other isoform, phospholipid hydroperoxide glutathione peroxidase (PHGPX), is active as a monomer and has a higher affinity to lipid hydroperoxides than to H2O2. The mammalian PHGPX isoforms and most of the mammalian GPXs are selenoproteins characterized by the presence of the rare amino acid residue selenoCys in their catalytic site (Brigelius-Flohe, 1999; Arthur, 2000).
In recent years, several genes encoding putative GPXs have been isolated from a variety of plants, including citrus, tobacco, tomato, sunflower, spinach, pea, rice, barley, Chinese cabbage, Avena fatua, and Arabidopsis (Criqui et al., 1992
Several observations suggest that plant PHGPXs are involved in biotic and abiotic stress responses. Increased levels of expression of PHGPX protein and of mRNA were induced by exposure to salt (Avsian-Kretchmer et al., 1999
In previous studies intended to understand the regulation of gpx1 (formerly named csa), a citrus gene encoding PHGPX, we examined its expression under salt and oxidative stress. We showed that salt treatment increases the amount of PHGPX with a concomitant decrease in overall APX activity (Gueta-Dahan et al., 1997
The salt-induced increase in steady-state mRNA accumulation may be regulated at the transcriptional and post-transcriptonal levels. In alfalfa, both types of regulation were found in different genes induced by salt (Winicov and Krishnan, 1996
In this study, we describe the structure of a citrus gene encoding PHGPX and analyze its promoter's pattern of expression. The isolated promoter fused to uidA was inserted into tobacco plants and cultured cells.
gpx1 Genomic Clone and Its Promoter
Isolation of a genomic clone corresponding to the cDNA of gpx1 revealed a gene comprised of six exons and five introns (Fig. 1A). Four of the introns were shorter than 200 bp, and the length of intron number 4 was 610 bp. All the introns were spliced following the consensus sequence, having GT and AG at their 5' and 3' end, respectively. The putative polyadenylation signal (AATAAA) is located 18 bp upstream of the poly(A) site (Joshi, 1987a
We identified several other consensus elements on the gpx1 promoter. The core sequence ACGT, which is characteristic for ABRE motifs (Uno et al., 2000 ), exists at positions –129, –234, and –1,011. Only the one at position –129 has the consensus sequence of a G-box (CACGTG; Choi et al., 2000), whereas the two other sequences do not have any of the predicted context. A CE1-like sequence, CACCG, which is suggested to be involved in ABA-induction, is found at position –201 (Niu et al., 2002). A sequence similar to the mammalian binding site for the factor NF-kB (GGGATATTTC), which is related to the oxidative stress response, was identified at position –1,417 with one mismatch (Scandalios et al., 1997). A cis-acting regulatory element involved in methyl jasmonate (MeJ)-responsiveness, CGTCA, which is also part of the as-1 element (Johnson et al., 2001), was found in the minus strand of the DNA at position –176. Several copies of a novel 6-bp CGCG box (Yang and Poovaiah, 2002), which responds to a variety of stresses including salt, MeJ, and H2O2, were located at positions –322, –275, and –150. A heat shock-responsive element consisting of tri-nucleotide repeats arranged in alternating, palindromic orientation separated by 2 bp was identified at position –447 (Guilfoyle, 1997). The WUN-motif aAATTtcct, described as a wound-responsive element in Brassica oleracea, was found at position –1075. A stress-responsive element (STRE) is characterized by the core sequence AGGGG (Ruis and Koller, 1997) and was found at position –402. The consensus sequence RSTGACTMANN, which is present in various ROS-modulated plant gene promoters (Kim et al., 2003), was found at position –547 with one mismatch. Several AT-rich regions exist in the gpx1 promoter, located at –620 to –724 (94% AT), –940 to –964 (96% AT), and –1,078 to –1,118 (88% AT).
The expression of GUS by the full-length promoter and two additional deletion constructs was analyzed in tobacco plants to determine tissue specificity and in BY-2 tobacco cells for quantitative measurements of the induction.
Transgenic tobacco plants containing each one of the three gpx1 promoter:uidA constructs were analyzed for uidA expression of following salt and sorbitol treatments. Data presented in Figure 2 show the patterns of GUS staining in roots and leaves of seedlings transformed with the full-length promoter, which were transferred to a medium containing 0.1 M NaCl or 0.2 M sorbitol for 2 to 4 d. No GUS activity was observed in control roots (Fig. 2A). In roots of plants exposed to NaCl for 48 h, GUS activity was observed in distinct cells but not in the root tips (Fig. 2B). After 72 h, this activity was exhibited all along the roots but expanded to the root hairs only after 96 h (Fig. 2, C and D). In roots treated with sorbitol, only the root-tip was stained after 48 h (Fig. 2E), and roots treated for 96 h were stained all over, similar to those treated with NaCl. In control leaves of seedlings germinating on wet filter paper, no GUS activity was observed (Fig. 2F), but when these seeds were germinated and grown in vitro under sterile conditions on Murashige and Skoog medium without any hormones, a background of blue staining was often observed (not shown).
In leaves, the overall intensity of GUS staining increased with the duration of the stress and different leaves from the same seedling were stained at different intensities (Fig. 2, G and H). There was no difference in the pattern of staining among NaCl, KCl, and sorbitol treated seedlings (data not shown). The staining of GUS expression in leaves of NaCl-treated seedlings was quite evenly spread over the whole leaf area, whereas in many leaves of Na2SO4-treated seedlings, the pattern of staining showed more pronounced GUS activity around the large veins and less intense activity throughout the leaf area (Fig. 2, I and J). Wounding leaves by squeezing them with forceps resulted in high GUS activity in the wounded leaf, as well as in the nonwounded leaves and roots, which was detected as early as 24 h following the treatment (Fig. 2, K and L). We analyzed GUS activity in homozygous plants transformed with the two deletion constructs. No activity could be detected in salt-grown plants transformed with the construct containing only the 0.5-kb fragment of the gpx1 promoter. GUS activity could be detected in roots and leaves of salt-grown plants transformed with the 1-kb fragment of the gpx1 promoter, but in most of the plants the intensity of the staining was somewhat lower than that observed with the full-length promoter (data not shown).
Transformation of BY-2 tobacco cells results in a large number of transformation events, exhibiting a large variation in transformants. The use of cells enables a better quantification of GUS activity and more uniform exposure to stress. The various stresses were usually imposed overnight to 24 h, as this was found the time point to obtain maximal GUS activity. Exposing cells to both NaCl and H2O2 for 4, 7, and 17 h resulted in 0%, 40%, and 90% to 100% of the activity obtained after exposure for 24 h, respectively. GUS activity was determined in control and treated cells by the fluorometric method, as explained in "Materials and Methods." NaCl, sorbitol, and mannitol induced high levels of GUS activity (Fig. 3), suggesting an osmotic effect. KCl was as effective as NaCl in inducing GUS activity, but Na2SO4 was less so. GUS activity induced by the addition of 100 mM CaCl2 or 200 mM NaNO3 was similar to that induced by Na2SO4, and almost no activity was induced by 200 mM choline chloride (data not shown). It is of interest that ABA and heat treatment (24 h at 37°C), which induce a high level of gpx1 transcript (Avsian-Kretchmer et al., 1999
We have previously shown that direct oxidative stress induced by tert-butyl hydroperoxide (tBH), but not by H2O2, increases gpx1-specific mRNA levels to the same extent as NaCl (Avsian-Kretchmer et al., 1999 ). Results presented in Figure 4 show that H2O2 induced high activity of GUS driven by uidA fused to the gpx1 promoter, even higher than that observed with tBH. It should be noted that cells recovered well from the H2O2 shock, as evidenced by the virtual lack of growth inhibition when they were subcultured for further growth. Lower GUS activity was obtained following treatment with Glc and Glc oxidase. We tested the effects of paraquat and menadione as sources for the generation of superoxide. Paraquat, at a concentration of 3 µM, inhibits the growth of cultured citrus and tobacco cells by about 50% and fails to increase the level of specific gpx1 mRNA or its encoded protein (Gueta-Dahan et al., 1997). At this concentration it induced a very low level of GUS activity (Fig. 4). We treated BY-2 tobacco cells with menadione, at concentrations ranging from 5 to 25 µM and obtained low to total inhibition of growth. At a concentration of 10 µM, which inhibited growth by about 50%, it also induced a very low level of GUS activity, comparable to that obtained with paraquat. Salicylic acid (0.1–1 mM), which has been shown to generate superoxide in tobacco cell suspension culture (Kawano and Muto, 2000), did not induce GUS expression.
The level of induction of GUS activity reflects the degree of stress imposed on the tissue; 0.1 M NaCl induced a much lower level of GUS activity than 0.2 M NaCl (Fig. 5). Preloading tobacco cells with the antioxidant N-acetyl Cys (NAC) decreased NaCl-induced GUS activity. Loading cells with NAC prior to the addition of NaCl reduced GUS activity more than the simultaneous addition of both, and loading with a higher concentration of NAC resulted in better protection.
Mn2+ and Zn2+ ions have been shown to be efficient scavengers of ROS in tobacco cell suspension (Kawano et al., 2002 ). As shown in Figure 6, the addition of 5 mM Mn2+ had no effect on GUS activity, but preloading cells with Mn2+ ions strongly inhibited the NaCl-induced expression of GUS. The action of Mn2+ as an antioxidant in our system was independently proved by applying the fluorometric method and the dye 5-(and-6)-carboxy-2',7'- dichlorodihydrofluorescein diacetate. We observed a very low fluorescence in control cells which was dramatically increased by salt, whereas in overnight Mn2+-preloaded cells, the addition of salt did not result in an increase of fluorescence, and the level observed was even below that of the control cells (see supplemental data). The addition of Zn2+ alone induced a high expression of GUS, and the addition of Zn2+ and NaCl resulted in a very high level of GUS activity, which was basically the sum of the two stresses. The effects of either Mn2+ or Zn2+ were rather specific, since other bivalent ions, such as Co2+ or Ni2+, did not induce any GUS activity by themselves, and they had a minor effect on the activity induced by NaCl (data not shown). Sorbitol-induced GUS activity was inhibited by NAC and Mn2+ in the same way as NaCl-induced activity (data not shown). It should be noted that Mn2+ failed to inhibit H2O2-induced GUS activity (Fig. 6). It has been suggested that Zn2+, but not Mn2+, acts as an inhibitor of the putative NADPH oxidase in BY-2 tobacco cells (Kawano et al., 2002). To test whether Zn2+-induced GUS activity involves NADPH oxidase, we tested the effect of two known inhibitors of this enzyme, diphenyleneiodonium chloride (DPI) and immidazole. Both inhibitors induced GUS activity when applied alone, although not to he same extent, and as in the case of Zn2+, their induced activity increased upon addition of NaCl (Fig. 7A). In contrast, DPI and Zn2+ dramatically inhibited the activity of GUS induced by H2O2 (Fig. 7B). We analyzed the ability of imidazole, Zn2+ and DPI to produce H2O2 in BY-2 tobacco cells by the fluorometric method using 5-(and-6)-carboxy-2',7'- dichlorodihydrofluorescein diacetate. None of these agents showed any activity against a positive control obtained by applying NaCl (data not shown). There is no correlation between the extent of induced GUS activity and the degree of growth inhibition obtained by the various chemical agents. Growth of cells was 50% inhibited by 0.2 M NaCl or 10 mM H2O2, and a similar inhibition was obtained by 5 mM Mn2+. The addition of Zn2+ inhibited the growth of cells by about 80%, whereas very few cells survived the treatment with DPI.
To test whether the inability of DPI to inhibit NaCl-induced expression of gpx1 is due to the intracellular production of H2O2, we added catalase to the cell suspension prior to the addition of either salt or H2O2. The addition of catalase dramatically reduced the H2O2-induced expression of gpx1 but had no effect or even increased the NaCl-induced expression. The addition of catalase 30 min after the addition of H2O2 had only a minor effect on GUS activity (Fig. 8). In parallel, we measured the capacity of the BY-2 cell suspension to remove H2O2 from the medium. Following the addition of 10 to 40 mM H2O2, only about 20% to 25% could be detected in the medium after 2 min, and the level was below 5% after 5 min. Addition of MeJ, which may be involved in signal transduction pathways induced by wounding (Ryan and Moura, 2002 ), resulted in a moderate level of GUS activity in tobacco cells at a concentration of 200 µM (Fig. 3).
We tested the ability of the two deletion constructs to induce GUS activity under a variety of stresses. In tobacco cells transformed with the construct containing the 1-kb fragment of the promoter, GUS activities induced by salt, sorbitol, H2O2, tBH, and Zn2+ were similar to those obtained with the full-length promoter. No GUS activity could be induced in tobacco cells transformed with the 0.5-kb fragment of the promoter.
Although genes encoding plant PHGPXs have been found in many species, their function in vivo has not been clearly resolved. Recent reports indicate that the encoded proteins are mainly active as thioredoxin-dependent peroxidases rather than GPXs (Herbette et al., 2002 ; Jung et al., 2002). The expression of most of the genes has been found to be induced under various stress conditions, where oxidative stress is most probably the common secondary effect. Our intensive studies on the regulation of citrus PHGPX clearly demonstrate that its expression under salt stress is mediated via oxidative stress (Avsian-Kretchmer et al., 1999). To better understand the transduction of salt stress to oxidative stress, we analyzed the regulation of this gene by activating its promoter fused to a reporter gene.
The sequence of the gene gpx1 shows a structure similar to the Arabidopsis gene encoding PHGPX (NM_117229). It consists of six exons and all of the five introns are spliced at conventional sequences. The homologous genes from human (GPX4) and pig heart contain seven exons (Brigelius-Flohe et al., 1994
Several putative regulatory elements were identified in the promoter region. The fact that the construct containing the 0.5-kb promoter fragment fused to uidA showed no GUS activity under any of the applied treatments suggests that the cis-acting elements found in the region up to –390 are not sufficient for the regulation of gpx1 expression. These elements include the CGCG box and the core sequences of ABRE motifs. Although osmotic stress induced a high level of GUS activity (Fig. 3), we could not detect drought-responsive-element motif found in drought or the cold-induced RD29A and COR78 genes, which are independent of ABA in Arabidopsis (Stockinger et al., 1997
Of all the consensus elements identified on the promoter, the STRE at position –402 and the cis-acting element responsive to ROS identified at position –547 may serve as important putative cis-acting elements in regulating gpx1 promoter activity. Both factors exist in the 1- and 1.5-kb promoter fragments, the region which is required for driving GUS activity. The STRE has been characterized in promoters of stress-induced yeast genes. Two copies were located on the catalase T promoter, which was shown to respond to a variety of stresses such as essential nutrient starvation (nitrogen, carbon, sulfur, and phosphorus), external low pH, ethanol, osmotic, and oxidative stress (Ruis and Koller, 1997
Similar levels of GUS activity were induced by NaCl, KCl, sorbitol, and mannitol, suggesting that the gpx1 promoter is actually activated by osmotic stress. However, the lower induction of GUS activity obtained in the presence of other salts, such as NaNO3 and Na2SO4, at iso-osmolar concentrations and the lack of specificity for any particular ion indicate that additional factors play a role in this response. We suggest that both osmotic and salt stresses are transduced to an oxidative stress and that the response of the reporter gene is correlated with the degree of oxidative stress imposed. This degree might change with the permeability of the various ions and their distribution within the cells. The fact that salt- and osmotic stress-induced GUS activity were eliminated by preloading the cells with antioxidants, such as NAC and Mn2+ ions (Figs. 5 and 6), provides further support for this notion and strengthens our previous model that salt stress induces gpx1 expression via the production of ROS (Avsian-Kretchmer et al., 1999
Northern-blot analysis of ABA-treated citrus cells showed a marked increase in the level of the gpx1 transcript, which was interpreted to be mediated by ROS (Avsian-Kretchmer et al., 1999
The failure of ABA to activate the gpx1 promoter appears to be in contradiction with recent reports indicating that ABA signaling is mediated by ROS production. It has been shown that ABA treatment increases the production of superoxide in maize leaves (Jiang and Zhang, 2002
The most effective ROS in inducing gpx1 expression seems to be H2O2, which, when added into the medium, needs to be at the rather high concentration of 10 mM (Fig. 4). Lower concentrations of H2O2 (1–5 mM) were less effective (data not shown). The mixture of Glc/Glc oxides, which generate H2O2, induced low GUS activity, most probably due to the low concentration of H2O2 produced under these conditions. It is of interest that the activation induced by paraquat and menadione were rather poor, although both reagents were applied at concentrations which imposed stress, as evidenced by inhibition of cell growth. Paraquat and menadione generate superoxide, which is subsequently dismutated by superoxide dismutase to produce H2O2. In citrus cells, it has been clearly shown that paraquat, but not H2O2, enhances the scavenging capacity of both superoxide dismutase and ascorbate peroxidase (APX; Gueta-Dahan et al., 1997
In animal systems, Mn2+ ions have been shown to have a protective effect against H2O2-induced cell and tissue injury (Ledig et al., 1991 The lack of activation of gpx1 promoter by the combination of H2O2 and DPI or Zn2+ versus the high induction by each of these reagents alone is puzzling. If we accept that DPI and Zn2+indeed inhibit NADPH oxidase, we have to assume that: (1) NADPH oxidase takes part in the transduction of H2O2 signal to the activation of gpx1 promoter; and (2) gpx1 promoter also responds to a deprivation of ROS (a stress developed in the presence of DPI, or Zn2+), and that the required level of ROS, which is supplied by NADPH oxidase in the absence of the inhibitors, can be directly derived from H2O2, resulting in a much lower stress (Fig. 7). Alternatively, it is possible that both DPI and Zn2+ induce an unknown NADPH oxidase-independent pathway leading to the induction of gpx1 promoter, which is negatively regulated by H2O2 levels. Despite the fact that NaCl stress produces H2O2, there are differences in the responses of H2O2- and NaCl-induced expression of gpx1 promoter to antioxidants, DPI, and catalase (Figs. 7 and 8). The most likely explanation would be that added H2O2 acts as an extracellular signal, which is transduced via a component in the plasma membrane, possibly NADPH oxidase, whereas the H2O2 mediating the salt signal is formed inside the cell. Our suggestions and conclusions from the experiments described here are summarized in a schematic model (Fig. 9). It is proposed that preloaded Mn2+ ions are able to act as antioxidant for the salt and sorbitol signal, when H2O2 is produced inside the cells, and fail to abolish this signal when it is perceived from the extracellular space (Fig. 6). It seems that the intracellular salt-induced production of H2O2 occurs throughout the cytosol and is not restricted to any of the organelles, as indicated by the specific staining with the dye 5-(and-6)-carboxy-2',7'- dichlorodihydrofluorescein diacetate (supplemental data, see www.plantphysiol.org).
The failure of catalase to reduce H2O2-induced GUS activity when added 30 min after the addition of H2O2 reflect the fact that H2O2 is already absent from the medium and that a short exposure is required for the induction of gpx1 promoter.
Salt activation of gpx1 promoter in leaves and roots of transformed tobacco plants confirms our previous studies showing increased levels of PHGPX protein in leaves and roots of salt-induced citrus plants and cells (Gueta-Dahan et al., 1997
The transformation of plants revealed that wounding is a very strong inducer of gpx1. Studies on the regulation of catalase gene expression in maize have suggested that wound-induced expression may be mediated by MeJ and share a common signal transduction but may also be regulated in a MeJ-independent pathway mediated by ROS (Guan and Scandalios, 2000 Our analysis of the gpx1 promoter shows that the expression of this gene can monitor the degree of oxidative stress imposed on the cells and gives better insight into the nature of the oxidative stress induced by salt. We have shown that oxidative stress is indeed a mediator in the salt signal and that in the induction of gpx1 it acts at the transcriptional level. It is suggested that salt-induced ROS are predominantly formed as H2O2 and that this process occurs inside the cell. It is likely that NADPH oxidase is involved in the signaling pathway activating the gpx1 promoter by exogenously added H2O2, but not in the signaling pathway activated by NaCl, and that inhibition of its activity imposes stress conditions.
Plant Material and Growth Conditions
Tobacco (Nicotiana tobacuum) var XHFD8 (Bourgin, 1978
Valencia (Citrus sinensis) genomic library in Lambda GEM-12 (Promega, Madison, WI) was kindly donated by Dr. A. Koltunow (Commonwealth Scientific and Industrial Research Organization, Australia). The amplified library was screened at high stringency with a 32P-labeled probe derived from the 5' end of citrus gpx1 cDNA digested with SacI (Sambrook et al., 1989
We isolated a 1.6-kb fragment upstream to the 5' end of the coding sequence of gpx1 by PCR using the forward primer c3-17 (5'-TTATGTCGACTTAGTCTTGTGGCTGGG-3') from position –1,526 to –1,510 and the reverse primer c3-16 (5'-TTGGATCCATTGTATGATCCGATCTAG-3') from position 129 to 111 and to which SalI and BamHI sites were added, respectively. The bold letters represent full homology to the genomic DNA and the italic letters represent the nonhomologous addition used to create a restriction enzyme site (BamHI or SalI underlined) at the ends of the PCR product. The PCR product was obtained by using Pwo DNA polymerase (Boeringer, Mannheim, Germany) and purified. It was digested by BamHI and SalI and ligated to a pUC19 vector digested with the same enzymes to form the plasmid 63-3. The insert was sequenced to verify its identity to the genomic clone. Two promoter deletion constructs were prepared (see Fig. 1B). The first deletion resulted in a 1,003-bp promoter fragment that was obtained as follows: Plasmid 63-3 was digested with PstI and SpeI to give two products of 600 and 3,700 bp. The bigger product was isolated from a gel and purified, and the cohesive ends were blunted using Klenow (Beoringer). The fragment was then self-ligated using T4 ligase (Beoringer) to form the plasmid 63-2. The second deletion resulted in a 515-bp promoter fragment which was obtained as follows: Plasmid 63-3 was digested with HphI, which produced several fragments of which one fragment of the size of 1.5 kb is composed of a vector fragment downstream to the polylinker cloning sites and a promoter sequence 5' upstream to the ATG. All other fragments were considerably smaller. DNA fragments were precipitated in ethanol and the cohesive ends were blunted by a fill-in reaction as described above. The 1.5-kb fragment was purified and cut with BamHI at the multiple cloning sites to yield two fragments, a 520-bp fragment of the promoter and about 1,000 bp of the vector. The 520-bp fragment was purified and ligated to pUC19 previously digested with BamHI and HincII to form the plasmid 63-1.
Insert of plasmids 63-3, 63-2, and 63-1 were cut out by BamHI and HindIII and further cloned into the Agrobacterium binary vector pBI101-GUSInt previously digested with the same enzymes to form plasmids pBI63-3, pBI63-2, and pBI63-1, respectively. The pBI101-GUSInt vector was designed to exclude the expression of the fused promoter:uidA gene in Agrobacterium and thus eliminate possible background (Gollop et al., 2002
Transformation of Tobacco Plants
Transformation of BY-2 Tobacco Cell Line
Analysis of GUS Activity in Tobacco Seedlings
Analysis of GUS Activity in Tobacco Cells Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AJ582678 and X66377. Received March 2, 2004; returned for revision April 20, 2004; accepted May 2, 2004.
[w] The online version of this article contains Web-only data.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.041921. * Corresponding author; e-mail vhgozal{at}agri.gov.il; fax 972–3–9669583.
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ABSTRACT
RESULTS
-Glucuronidase (GUS) activity in the transformed tissues showed that gpx1 is regulated at the transcriptional level by salt and oxidative stress but not by ABA. Our experiments led to the conclusion that the expression of gpx1 is not tissue specific, and that the oxidative stress induced by salt is essentially in the form of H2O2, which is produced in the intracellular space. NADPH oxidase is involved in the signal transduction activating the gpx1 promoter by H2O2 induction but not in salt-induced activation of this promoter. 
