TY - JOUR
AU - Perl, Avihai
AB - Abstract Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is overexpressed in plants under abiotic and biotic stress conditions that mediate oxidative stress. To study its biological role and its ability to confer stress resistance in plants, we tried to obtain transgenic plants overexpressing citrus (Citrus sinensis) PHGPx (cit-PHGPx). All attempts to obtain regenerated plants expressing this enzyme constitutively failed. However, when the enzyme’s catalytic activity was abolished by active site-directed mutagenesis, transgenic plants constitutively expressing inactive cit-PHGPx were successfully regenerated. Constitutive expression of enzymatically active cit-PHGPx could only be obtained when transformation was based on non-regenerative processes. These results indicate that overexpression of the antioxidant enzyme PHGPx interferes with shoot organogenesis and suggests the involvement of reactive oxygen species (ROS) in this process. Using transgenic tobacco (Nicotiana tabacum) leaves obtained from plants transformed with a β-estradiol-inducible promoter, time-dependent induction of cit-PHGPx expression was employed. A pronounced inhibitory effect of cit-PHGPx on shoot formation was found to be limited to the early stage of the regeneration process. Monitoring the ROS level during regeneration revealed that upon cit-PHGPx induction, the lowest level of ROS correlated with the maximal level of shoot inhibition. Our results clearly demonstrate the essential role of ROS in the early stages of in vitro shoot organogenesis and the possible involvement of PHGPx in maintaining ROS homeostasis at this point. Introduction Reactive oxygen species (ROS) such as superoxide, H2O2 and lipid hydroperoxides are toxic cellular metabolites which are rapidly detoxified by various enzymatic and non-enzymatic scavengers. At the same time, they serve as signaling molecules regulating important biological processes in both animal and plant cells (Halliwell 2006). In plants, they have been shown to control biotic and abiotic stress responses, programmed cell death, hormone signaling, cellular differentiation, growth and development (Potikha et al. 1999, Mittler et al. 2004, Gechev et al. 2006). Thus, the ROS-scavenging systems need to maintain a redox homeostasis, above which the different signals can be detected. In many cases, thiol-containing proteins serve as transducers, which are either activated or inactivated in response to ROS (Foyer and Noctor 2005). A major candidate as a ROS-level regulator is the phospholipid hydroperoxide glutathione peroxidase (PHGPx) which serves as a detoxifying and signaling enzyme. PHGPx, a member of the glutathione peroxidase (GPx) family, is a unique antioxidant enzyme that can directly reduce phospholipid hydroperoxides and complex hydroperoxy lipids which comprise the biomembrane lipid layers, in addition to H2O2 and other organic hydroperoxides which are substrates of the rest of the GPx family. Accordingly, PHGPx is considered crucial for protecting membranes from oxidative stress (Maiorino et al. 1990). In addition to using the glutathione system for its regeneration, PHGPx can also utilize a large variety of other reducing compounds, including cysteine (Cys) and Cys-containing proteins (Ursini et al. 1997). In plants, the thioredoxin-regenerating system has been shown to be much more efficient than the glutathione system, and therefore the plant PHGPx is in fact a thioredoxin peroxidase as well (Herbette et al. 2002, Jung et al 2002). This latter characteristic could be related to the finding that all currently known plant PHGPxs that reveal sequence homology to animal PHGPxs possess Cys as the catalytic residue in their active site instead of selenocysteine found in most animal PHGPxs (for a review, see Herbette et al. 2007). Mammalian PHGPx isoforms are involved in controlling fundamental cellular processes, and many of their physiological functions have been thoroughly investigated. Deletion of the complete gene in mice leads to early embryogenic lethality (Imai et al. 2003). In addition to protection from cell death caused by free radical initiation and membrane lipid peroxidation, PHGPx plays a variety of specific roles by serving as a thiol peroxidase and regulating ROS signaling. Hence, PHGPx prevents apoptosis by reducing cardiolipin in the mitochondria, mediates inflammation defense mechanisms by regulating the activity of lipoxygenase and cyclooxygenase, and is highly expressed in germ cells where it plays a central role in spermatogenesis (Brigelius-Flohé 1999, Imai and Nakagawa 2003, Conrad et al. 2007). Recently, the role of PHGPx has also been demonstrated in murine embryogenic development (Savaskan et al. 2007). In plants, isolation of the tobacco (Nicotiana tabacum) and citrus (Citrus sinensis) PHGPx genes (Criqui et al. 1992, Holland et al. 1993), and the functional characterization of its encoded enzyme in citrus (Ben-Hayyim et al. 1993, Holland et al. 1994, Beeor-Tzahar et al. 1995, Eshdat et al. 1997), led to the identification of PHGPx family genes, all involved in biotic and abiotic stress responses mediated by oxidative stress (Depège et al. 1998, Agrawal et al. 2002, Rodriguez Milla et al. 2003, Navrot et al. 2006). As a ubiquitous enzyme, PHGPx has been shown to be expressed in different plant tissues, different compartments and during plant development (Mullineaux et al. 1998, Yang et al. 2005, Yang et al. 2006). However, only a limited number of studies attempting to understand the physiological role of plant PHGPx in ROS scavenging (Yang et al. 2005), cell death suppression (Chen et al. 2004) and cell cycle regulation (Kadota et al. 2005) have been reported to date. Using ‘loss or gain of function’ studies, it was also demonstrated in Arabidopsis (Arabidopsis thaliana) that PHGPx plays a role in H2O2 scavenging, signal transduction and photooxidative stress tolerance (Miao et al. 2006, Chang et al. 2009). To establish the biological role of plant PHGPx further, we tried to obtain transgenic plants overexpressing the citrus PHGPx (cit-PHGPx). We have found (Faltin et al. 2004) that it was impossible to regenerate tobacco plants constitutively overexpressing cit-PHGPx, suggesting that this enzyme may have a detrimental effect during in vitro shoot differentiation. We therefore focused on understanding the role of PHGPx during plant regeneration, and the correlation between ROS level and successful shoot regeneration. Our results suggest that proper maintenance of redox homeostasis is crucial for successful regeneration in the early stages of shoot organogenesis. Results Transformation attempts using 35S, RD29A and patatin promoters Unexpectedly, all early attempts by our laboratories to obtain transgenic plants of tobacco, tomato (Solanum lycopersicum) and potato (Solanum tuberosum) that constitutively overexpress cit-PHGPx using the 35S promoter failed. To follow the transformation process, tobacco leaves were inoculated with Agrobacterium carrying pCAMBIA-cit-PHGPx which contains, separately, both the cit-PHGPx and β-glucoronidase (GUS) expression cassettes. No GUS expression was observed following these transformation attempts: GUS expression was only observed when transformation was performed in the absence of the cit-PHGPx cassette (Fig. 1). Moreover, transformation experiments carried out with various constructs harboring inducible promoters, such as that of the RD29A gene (which is induced by salt, drought and cold stresses) and the promoter of the class I patatin gene B33 [which is induced by sucrose (Suc) and proline (Pro)], also failed. Fig. 1 View largeDownload slide GUS expression in tobacco leaves transformed by Agrobacterium carrying a pCambia GUS-expressing cassette lacking cit-PHGPx (PHGPx [−]) or containing a cit-PHGPx-expressing cassette (PHGPx [+]), after 5, 9 or 15 d of cultivation on regeneration medium. For experimental data see text. Fig. 1 View largeDownload slide GUS expression in tobacco leaves transformed by Agrobacterium carrying a pCambia GUS-expressing cassette lacking cit-PHGPx (PHGPx [−]) or containing a cit-PHGPx-expressing cassette (PHGPx [+]), after 5, 9 or 15 d of cultivation on regeneration medium. For experimental data see text. Expression of enzymatically inactive cit-PHGPx Since the above results suggested that continuous expression of cit-PHGPx may interfere with completion of the transformation process, we examined whether the increased enzymatic activity of cit-PHGPx abolishes Agrobacterium-mediated transformation of this gene. Transformation was therefore performed using the 35S-Ser41cit-PHGPx plasmid in which a point mutation, leading to substitution of the catalytic residue Cys41 with serine (Ser)41, yielded enzymatically inactive Ser41cit-PHGPx, similarly to what was also shown for Chinese cabbage (Jung et al. 2002). This enzymatic inactivation was demonstrated by measuring the activity of the soluble fraction of recombinant Escherichia coli expressing the native Cys41cit-PHGPx (Faltin et al. 1998) or the mutated cit-Ser41cit-PHGPx. The activity of the native and the mutated enzymes was measured with either H2O2 or phosphatidylcholine hydroperoxide as substrates and by using glutathione or thioredoxin for regeneration. Specific activity of 43.5 U mg−1 protein could be measured for phosphatidylcholine hydroperoxide, and none for H2O2, while using glutathione. An activity of 219 and 291 U mg−1 was determined for both substrates, respectively, while using thioredoxin. The mutated enzyme did not exhibit any noticeable activity in any of the described enzymatic assays. As shown in Fig. 2, transformation of tobacco leaves using the modified 35S-Ser41cit-PHGPx yielded numerous regenerated shoots (Fig. 2A), whereas no shoots were observed when native cit-PHGPx was used (Fig. 2B). Western blot analysis of the regenerated plantlets transformed with the mutated Ser41cit-PHGPx showed that they indeed overexpress the Ser41-containing protein (Fig. 2C). Fig. 2 View largeDownload slide Transformation of tobacco with mutated inactive cit-PHGPx. Agrobacterium-mediated transformation of tobacco leaves carrying: (A) 35S-Ser41cit-PHGPx plasmid (containing the Ser41 codon by site-directed mutagenesis) or (B) the 35S-cit-PHGPx plasmid (containing the Cys41 codon). (C) Western blot analysis of leaves taken from several transgenic tobacco plants expressing the mutated Ser41cit-PHGPx (C41S-PHGPx) (M, the 20 kDa cit-PHGPX. C, tobacco leaf extract). A high expression level of Ser41cit-PHGPx (third lane from the right) was observed for the transformed leaves presented in A. The relative location of endogenic tobacco PHGPx is marked, based on a separate Western blot analysis in which a mixture of cit-PHGPx and tobacco leaf extract was stained subsequently with anti-tobacco PHGPx and anti-cit-PHGPx antibodies; cross-reactivity was observed between cit-PHGPx antibodies and the tobacco PHGPx (for details see Materials and Methods). Fig. 2 View largeDownload slide Transformation of tobacco with mutated inactive cit-PHGPx. Agrobacterium-mediated transformation of tobacco leaves carrying: (A) 35S-Ser41cit-PHGPx plasmid (containing the Ser41 codon by site-directed mutagenesis) or (B) the 35S-cit-PHGPx plasmid (containing the Cys41 codon). (C) Western blot analysis of leaves taken from several transgenic tobacco plants expressing the mutated Ser41cit-PHGPx (C41S-PHGPx) (M, the 20 kDa cit-PHGPX. C, tobacco leaf extract). A high expression level of Ser41cit-PHGPx (third lane from the right) was observed for the transformed leaves presented in A. The relative location of endogenic tobacco PHGPx is marked, based on a separate Western blot analysis in which a mixture of cit-PHGPx and tobacco leaf extract was stained subsequently with anti-tobacco PHGPx and anti-cit-PHGPx antibodies; cross-reactivity was observed between cit-PHGPx antibodies and the tobacco PHGPx (for details see Materials and Methods). cit-PHGPx transformation in non-regenerative plant systems Successful transformation with cit-PHGPx-containing vectors was achieved by using systems in which a regeneration stage does not exist in the transformation process. BY-2 cells were successfully transformed with cit-PHGPx using the constitutive 35S promoter as well as the inducible promoters RD29A and patatin. As shown in Fig. 3, constitutive expression of cit-PHGPx was detected with the 35S vector (Fig. 3A1), with patatin promoter following induction with 100 mM Pro (Fig. 3A4), and with RD29A following induction with 0.2 M NaCl and 0.05 mM ABA (Fig. 3A7). Expression of cit-PHGPx could even be identified with the latter two promoters in the absence of induction (Fig. 3A2, A6). cit-PHGPx overexpression was also obtained when potato calli, produced from leaf segments, were transformed with 35S-cit-PHGPx (Fig. 3A10). Fig. 3 View largeDownload slide Western blot analysis using anti-cit-PHGPx antibodies for identification of the expression level of cit-PHGPx in various non-regenerative plant systems transformed with plasmids carrying cit-PHGPx fused to different promoters. (A) Transformation of tobacco BY-2 cells (1–7) and of potato cells (9–10) with: (1) the 35S promoter; (2–4) the patatin promoter, without induction (2), with plasmid devoid of cit-PHGPx (3, control) and with 100 mM Pro induction (4); (5–7) the RD29A promoter, with plasmid devoid of cit-PHGPx (5), without induction (6) and with 0.2 M NaCl containing 0.05 mM ABA (7); (8) marker (M) of cit-PHGPx; (9–10) potato cell transformation with plasmid devoid of cit-PHGPx (9, control), and with plasmid carrying the 35S promoter and cit-PHGPx (10). (B) Transformation of tobacco leaves cultivated on callus induction medium. (1) Marker (M) of cit-PHGPx; (2) transformed tobacco callus using the 35S promoter (six different callus clones); (3) transformed tobacco callus using a plasmid carrying the Cys41Ser-mutated cit-PHGPx (C41S-PHGPx) and 35S promoter (three different clones). (C) In planta transformation of Arabidopsis using the 35S promoter (10 different transformed plants). The solid arrow indicates the location of cit-PHGPx and the dashed arrow indicates the location of tobacco-PHGPx (see Fig. 2). Fig. 3 View largeDownload slide Western blot analysis using anti-cit-PHGPx antibodies for identification of the expression level of cit-PHGPx in various non-regenerative plant systems transformed with plasmids carrying cit-PHGPx fused to different promoters. (A) Transformation of tobacco BY-2 cells (1–7) and of potato cells (9–10) with: (1) the 35S promoter; (2–4) the patatin promoter, without induction (2), with plasmid devoid of cit-PHGPx (3, control) and with 100 mM Pro induction (4); (5–7) the RD29A promoter, with plasmid devoid of cit-PHGPx (5), without induction (6) and with 0.2 M NaCl containing 0.05 mM ABA (7); (8) marker (M) of cit-PHGPx; (9–10) potato cell transformation with plasmid devoid of cit-PHGPx (9, control), and with plasmid carrying the 35S promoter and cit-PHGPx (10). (B) Transformation of tobacco leaves cultivated on callus induction medium. (1) Marker (M) of cit-PHGPx; (2) transformed tobacco callus using the 35S promoter (six different callus clones); (3) transformed tobacco callus using a plasmid carrying the Cys41Ser-mutated cit-PHGPx (C41S-PHGPx) and 35S promoter (three different clones). (C) In planta transformation of Arabidopsis using the 35S promoter (10 different transformed plants). The solid arrow indicates the location of cit-PHGPx and the dashed arrow indicates the location of tobacco-PHGPx (see Fig. 2). To determine whether the expression of cit-PHGPx in putatively transformed tissue interferes with organogenesis or with cell division, tobacco leaf discs were cultured following transformation on callus-inducing medium in order to promote division of transgenic cells at the transformation site, rather than on regeneration medium to induce organogenesis of transgenic shoots. Using both the native 35S-cit-PHGPx and the mutated 35S-Ser41cit-PHGPx, expression of cit-PHGPx was obtained with both vectors (Fig. 3B), in contrast to the results obtained when the leaves were cultured on regeneration-inducing medium. Constitutive expression of cit-PHGPx was obtained by transforming Arabidopsis plants ‘in planta’ using the infiltration method. As many as 90% of the T1 seeds germinated and selected on kanamycin exhibited cit-PHGPx overexpression (Fig. 3C). Plant transformation with cit-PHGPx mediated by the β-estradiol-inducible pER8 vector Transgenic tobacco plants were successfully regenerated by employing the leaf disc method, using the inducible pER8-cit-PHGPx vector. This vector (Zuo et al. 2000) contains the transcription activator XVE, which is strictly regulated by estrogen. Transgenic lines expressing cit-PHGPx following induction with β-estradiol (Fig. 4A) were selected for further experiments. No expression of cit-PHGPx could be detected in non-induced transgenic plants (Fig. 4A). The rate of cit-PHGPx expression in transformed leaf discs following β-estradiol induction was rapid, reaching maximal levels already after 1 d (Fig 4B). Fig. 4 View largeDownload slide Western blot analysis using anti-cit-PHGPx antibodies for identification of the expression level of cit-PHGPx in a strictly induced regenerative system using the pER8 plasmid. (A) Level of cit-PHGPx in different plantlets obtained from transformed tobacco leaf discs following induction with β-estradiol for 48 h. (B) Kinetics of the cit-PHGPx expression level following induction with β-estradiol for up to 3 d. Fig. 4 View largeDownload slide Western blot analysis using anti-cit-PHGPx antibodies for identification of the expression level of cit-PHGPx in a strictly induced regenerative system using the pER8 plasmid. (A) Level of cit-PHGPx in different plantlets obtained from transformed tobacco leaf discs following induction with β-estradiol for 48 h. (B) Kinetics of the cit-PHGPx expression level following induction with β-estradiol for up to 3 d. Effect of cit-PHGPx expression level on transformed plant regeneration Leaf discs taken from three representative pER8-cit-PHGPx-transgenic tobacco plants—GPx2 which expressed the highest level of cit-PHGPx, GPx23 which expressed cit-PHGPx at 15% of the level in GPx2 and GPx4 which did not express any detectable cit-PHGPx (Fig. 5)—were placed on agar regeneration medium in the presence or absence of 5 μM β-estradiol. Fig. 5 View largeDownload slide Shoot regeneration in transformed tobacco lines expressing different levels of cit-PHGPx. Leaf discs from three different cit-PHGPx-transformed tobacco lines were cultivated on regeneration medium in the absence (−) or presence (+) of β-estradiol. Western blot analysis revealed hardly detectable cit-PHGPx expression of <2% in GPx4, and 15% in GPx23, compared with Gpx2. Fig. 5 View largeDownload slide Shoot regeneration in transformed tobacco lines expressing different levels of cit-PHGPx. Leaf discs from three different cit-PHGPx-transformed tobacco lines were cultivated on regeneration medium in the absence (−) or presence (+) of β-estradiol. Western blot analysis revealed hardly detectable cit-PHGPx expression of <2% in GPx4, and 15% in GPx23, compared with Gpx2. Leaf discs taken from GPx2 did not produce any shoots while cultured on induction medium, while those taken from GPx23 produced several meristematic zones which did not develop further to yield leaf or shoot structures. Leaf discs taken from GPx4 yielded an average 30.7 shoots per explant, similar to the yield obtained from all leaf discs grown in the absence of β-estradiol (Fig. 5). Inhibition of shoot formation by cit-PHGPx induction during the regeneration process To identify the developmental stage at which cit-PHGPx inhibits the regeneration of shoots from pER8-cit-PHGPx-transformed leaf discs, ectopic expression of the enzyme was induced at different time points during the regeneration process (see Materials and Methods). Fig. 6A a shows the number of shoots obtained when the leaf discs from GPx2 were first cultivated on solid regeneration medium and then transferred, on alternate days, to plates containing 5 μM β-estradiol, up to a total of 21 d. Leaf discs that were exposed to β-estradiol on day 1 and day 3 of culture on regeneration medium produced 1–3 shoots per explant only. Discs exposed to β-estradiol on day 5 and day 7 of the regeneration process exhibited a smaller inhibitory effect, yielding 5–10 shoots per explant. For discs exposed to β-estradiol on day 10, no inhibition of shoot regeneration was observed. Fig. 6 View largeDownload slide Dependence of shoot formation on cit-PHGPx induction time and level. (A) The number of shoots obtained in GPx2 transgenic tobacco leaf discs cultivated for 21 d on solid regeneration medium: (a) leaf discs were pre-cultivated on β-estradiol (−) medium for different periods and then transferred to β-estradiol (+) medium on the specified day; (b) leaves were cultivated on β-estradiol (−) medium but were pulse-induced with β-estradiol for 24 h on the specified day. (B) Western blot of the GPx2 ectopic expression level in transgenic tobacco leaf discs taken at day 0 (A), following continuous (a) or 24 h pulse (b) β-estradiol induction. Data are presented as means ± SE (n = 16). For details see text. Fig. 6 View largeDownload slide Dependence of shoot formation on cit-PHGPx induction time and level. (A) The number of shoots obtained in GPx2 transgenic tobacco leaf discs cultivated for 21 d on solid regeneration medium: (a) leaf discs were pre-cultivated on β-estradiol (−) medium for different periods and then transferred to β-estradiol (+) medium on the specified day; (b) leaves were cultivated on β-estradiol (−) medium but were pulse-induced with β-estradiol for 24 h on the specified day. (B) Western blot of the GPx2 ectopic expression level in transgenic tobacco leaf discs taken at day 0 (A), following continuous (a) or 24 h pulse (b) β-estradiol induction. Data are presented as means ± SE (n = 16). For details see text. Fig. 6Ab shows the number of shoots obtained when the leaf discs were first cultivated on solid regeneration medium, then transferred, on alternate days, to plates supplemented with β-estradiol for 24 h and, finally, transferred back to plates containing regeneration medium without inducer. Shoot inhibition exhibited a ‘U-shaped’ curve in which maximal inhibition was obtained when the 24 h β-estradiol pulse was given between days 3 and 5 of the regeneration process. Kinetic measurements of cit-PHGPx expression levels in pER8-cit-PHGPx-transformed leaf discs that were cultivated on β-estradiol-containing regeneration medium for 21 d were determined by Western blot analysis. cit-PHGPx expression reached its maximal level 24 h after exposure to β-estradiol (Fig. 6Ba), followed by a gradual decrease to 77% of its maximal level on day 6 and 29% of its maximal level on day 14 (Fig. 6Ba). When the cit-PHGPx level was determined in discs cultivated on regenerating medium and induced by only a 24 h pulse of β-estradiol, cit-PHGPx decreased to as low as 8% of its maximal level within 6 d after induction, and almost no expression was observed after 2 weeks (Fig. 6Bb). Effect of cit-PHGPx level on ROS production during the regeneration process Production of ROS and superoxides, determined by 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) and nitroblue tetrazolium (NBT), respectively, was monitored during cultivation of leaf discs taken from GPx2 on regeneration medium in the presence or absence of β-estradiol (Fig. 7). As shown in Fig. 7A1, the ROS level in GPx2 leaf discs cultivated on regeneration medium in the absence of β-estradiol (non-induced) decreased slightly during the first 2 d, stabilized on days 3 and 4, and then increased markedly, up to 9-fold, reaching a plateau on day 10. A similar observation was obtained by NBT staining of the leaf discs (Fig. 7A2): a prominent increase in superoxide production was observed at the cut edges during the same cultivation time period. The same result was obtained when ROS and superoxide levels were determined for the non-expressing cit-PHGPx line (GPx4) cultivated in both the presence and absence of inducer (data not shown). Fig. 7 View largeDownload slide ROS and superoxide production in leaf discs taken from GPx2 during 15 d of cultivation on regeneration medium in the presence or absence of β-estradiol. ROS production was fluorometrically determined using carboxy-H2DCFDA, while superoxide production was histochemically monitored by formazan deposition after staining the explants with NBT. (A1) Fluorescence at 525 nm; (A2) NBT staining. (B) Relative fluorescence of leaf discs, cultivated for 7 d on regeneration medium and then transferred to the same medium containing 5 μM β-estradiol for an additional 4 d. Data are presented as means ± SE (n = 22). For experimental details see text. Fig. 7 View largeDownload slide ROS and superoxide production in leaf discs taken from GPx2 during 15 d of cultivation on regeneration medium in the presence or absence of β-estradiol. ROS production was fluorometrically determined using carboxy-H2DCFDA, while superoxide production was histochemically monitored by formazan deposition after staining the explants with NBT. (A1) Fluorescence at 525 nm; (A2) NBT staining. (B) Relative fluorescence of leaf discs, cultivated for 7 d on regeneration medium and then transferred to the same medium containing 5 μM β-estradiol for an additional 4 d. Data are presented as means ± SE (n = 22). For experimental details see text. A completely different pattern of ROS levels was observed for the cit-PHGPx-transformed leaf discs which were cultivated on β-estradiol-containing medium. ROS production by the induced cit-PHGPx-transformed discs gradually declined throughout the first 4 d of cultivation, to a lower level than that obtained for discs cultivated in the absence of β-estradiol (Fig. 7A1), and remained low throughout cultivation. A similar observation was obtained for superoxide levels determined by NBT staining (Fig. 7A2). To establish that overproduction of cit-PHGPx indeed scavenges ROS in the leaf discs, GPx2 discs cultivated on regeneration medium (lacking β-estradiol) were transferred on day 7, at which time overexpression of cit-PHGPx does not inhibit regeneration, to a medium containing β-estradiol. The ROS level was measured 4 d later. cit-PHGPx overexpression resulted in a noticeable reduction of ROS in the leaf discs, compared with discs which were not induced with β-estradiol (Fig. 7B). Similar results were obtained when the cit-PHGPx-transformed discs were transferred from regeneration medium to β-estradiol-containing medium on day 10, the day when ROS reach maximal levels (data not shown). To demonstrate further the role of ROS in the regeneration process, Mn2+ ions, shown to inhibit the generation of superoxides in BY-2 tobacco cells (Kawano et al. 2002), were examined for their effect on the regeneration process. Following tobacco leaf disc cultivation on regeneration medium containing 5, 10 or 20 mM Mn2+, NBT staining revealed inhibition of superoxide production already at 5 mM Mn2+ (Fig. 8), with no effect on shoot regeneration. However, the number of shoots per explant decreased to 43 and 3% after cultivation with 10 mM and 20 mM MnSO4, respectively. Fig. 8 View largeDownload slide NBT staining of tobacco leaf discs cultivated for 17 d on regeneration medium containing different concentrations of MnSO4. Fig. 8 View largeDownload slide NBT staining of tobacco leaf discs cultivated for 17 d on regeneration medium containing different concentrations of MnSO4. Discussion Although ROS have become the focus of many studies due to their dual function as toxic and signaling molecules, very little has been reported regarding the requirement and involvement of ROS during plant differentiation in vitro. By ectopic expression of cit-PHGPx during regeneration of micropropagated tobacco plants, we could demonstrate the essential role of ROS in shoot organogenesis. In accordance with our finding that cit-PHGPx is indeed capable of specifically reducing phospholipid hydroperoxides via the glutathione-regenerating system (Eshdat et al. 1997), as well as H2O2 when utilizing thioredoxin, we set out to obtain transgenic plants that overexpress cit-PHGPx in an attempt to increase the plant’s antioxidative capacity and to improve its stress tolerance. Supporting evidence for this assumption was our success in regenerating antisense cit-PHGPx tobacco plants that exhibited increased sensitivity to methyl viologen, implying the involvement of cit-PHGPx in ROS scavenging (unpublished results). In contrast, all of our attempts to regenerate a variety of transgenic plants (tobacco, potato and tomato) that constitutively overexpress cit-PHGPx failed. Attempts to use the inducible promoters, patatin and RD29A, which turned out to be leaky, also failed. Successful transformation of cit-PHGPx was obtained when carried out in tobacco and potato cell cultures, tobacco calli originating from leaf discs cultured on media promoting only cell division and not shoot regeneration, and Arabidopsis plants using the floral-dip method. Thus, transformation was obtained in plant systems in which regeneration was not required, strongly suggesting that overexpression of cit-PHGPx could hamper the regeneration and differentiation process occurring during plantlet development. Indeed, regenerated transgenic tobacco plants were obtained by using the non-leaky and strictly β-estradiol inducible pER8 vector. Moreover, when transformation was performed using the Ser41-mutated cit-PHGPx gene, leading to constitutive expression of enzymatically inactive cit-PHGPx, transgenic plants, expressing the mutated protein, were obtained. This result indicates that enhanced enzymatic activity of PHGPx plays a dominant role in the inhibition of plant regeneration. We therefore further focused on the effect of enhanced PHGPx activity on scavenging of ROS during plant regeneration and on determination of the developmental stage at which the enzyme is involved. The absence of GUS expression following Agrobacterium transformation with the pCambia plasmid, containing both GUS and cit-PHGPx (Fig. 1), was the first indication that the inhibitory involvement of cit-PHGPx occurs in the early stages of the regeneration process. By inducing cit-PHGPx overexpression with β-estradiol at different time points during the regeneration process, the critical stage at which cit-PHGPx inhibition of organogenesis occurs was identified. We have found that the foremost inhibitory effect of cit-PHGPx is limited to the first 5 d of cultivation on regeneration-inducing medium and gradually decreases afterwards. The 24 h pulse induction experiment further showed that the maximal inhibition is between days 3 and 5. As expected from previous studies (Attfield and Evans 1991), we observed meristematic clusters on tobacco leaf discs cultivated on regeneration medium as early as day 7, and first shoot regeneration on day 15; similar results were obtained for non-induced transgenic tobacco leaf discs. On the other hand, β-estradiol induction of cit-PHGPx overexpression in the transgenic leaf discs caused a dramatic decrease in the appearance of meristematic clusters. Thus, the inhibitory effect of PHGPx overexpression is associated with the stage by which meristematic cluster/primordium are formed, suggesting that ROS are needed for the differentiation of shoot primordia. Indeed some of the physiological processes occurring during primordial development (Fleming 2005) are known to be ROS dependent, such as cell wall loosening (Schopfer and Liszkay 2006), cell growth (Gapper and Dolan 2006) and cell cycle progression (Jiang and Feldman 2005). It is well documented that the redox state is a key determinant of cell fate. Cells at the G1–S transition phase of the cell cycle, a redox-sensitive stage, may stop proliferating and switch to differentiation or programmed cell death (den Boer and Murray 2000). As we could not observe any notable changes in the proliferation rate of transgenic callus cells overexpressing cit-PHGPx, it is tempting to assume that overexpression of PHGPx interrupts the proliferating cells to switch to a differentiation pathway. Alternatively, PHGPx might interfere with differentiation by suppressing programmed cell death, an integral part of many developmental processes (Gadjev et al. 2008). The anti-apoptotic activity of PHGPx was also demonstrated for mammalian PHGPx and plant PHGPx (Chen et al. 2004, Nakagawa 2004). Recent studies have linked the activity of auxin in root and shoot development with changes in redox balance. High levels of auxin are correlated with the generation of ROS, and it was suggested that redox sensing could be responsible for the arrest of the stem cells in the quiescent center in G1 (Veit 2004, Jiang and Feldman 2005). By monitoring ROS production during the regeneration process, we have observed a marked increase in ROS production in non-induced transgenic leaf discs at the stage of meristematic primordium appearance, starting on day 7. Indeed, NBT staining was localized in this area. When cit-PHGPx was expressed following β-estradiol induction, lower ROS values were measured on days 2–4 of culture compared with non- cit-PHGPx-expressing tissue (Fig. 7A1). In contrast to the non-induced leaf discs, no increase in ROS from day 7 on was observed, which can be correlated with and associated with the absence of shoot primordium formation. These results suggest a correlation between the reduction in ROS level and the inhibitory effect of cit-PHGPx on shoot regeneration during a critical time window (days 3–5, Fig. 7A1) in the differentiation process. Hence, PHGPx overexpression may reduce ROS below a critical threshold level necessary for primordium development. The finding that seedling development of constitutively cit-PHGPX-transformed Arabidopsis was not affected by the enzyme expression suggests that the steady-state level of ROS depends on metabolic pathways which differ between diverse tissues and cells. Our attempts to overcome the inhibitory effect of cit- PHGPx overexpression, by exogenous supplementation of free radical donors such as methyl viologen (10 and 100 nM) or riboflavin (5 and 50 μM) at the onset of the regeneration process, were unsuccessful. This might reflect the difficulties in targeting the exogenous ROS to specific cells within the tissue or to the precise subcellular site at the optimal concentration and time window necessary to compensate cit-PHGPx activity. Supportive evidence for the conclusion that a certain level of ROS is indeed required for shoot regeneration is demonstrated by the result showing the ability of Mn2+ ions to inhibit shoot formation. It has already been shown that Mn2+ ions can act catalytically as scavengers of either superoxide or H2O2 by redox reactions between Mn2+ and Mn3+ status (Horsburgh et al. 2002), and demonstrated in BY-2 cells as a potential inhibitor of superoxides following its extracellular supplementation at a concentration of 5 mM Mn2+ (Kawano et al. 2002). Transgenic plants overexpressing other ROS-scavenging enzymes, such as ascorbate peroxidase and superoxide dismutase which are not known to exhibit enzymatic activity towards lipid hydroperoxides, were reported several years ago (Allen et al. 1997, Lee et al. 2007). The absence of reports on the negative effect of these enzymes on the transformation rate suggests that the negative effect of cit-PHGPx overexpression described herein may be unique to this enzyme’s specific function. Moreover, since transgenic tobacco plants were obtained by transforming the tomato PHGPx gene under the control of a constitutive promoter (Chen et al. 2004), it is suggested that differences in the substrate specificity of PHGPx isoforms, in either the same plant species or among different plants, may affect the mode of action and the micro-environmental activity of the enzyme and of its physiological impact. In fact, the 71% identity that exists between the tomato PHGPx and cit-PHGPx, and the differences in their specific activity (Faltin et al. 1998, Chen et al. 2004) towards phospholipid hydroperoxides, suggest diverse structure–function relationship that may explain the different behavior of these two enzymes during regeneration. Hence, it is very likely that phospholipid hydroperoxides are a unique group among ROS whose concentration serves as a significant factor in the process of plant shoot regeneration. Indeed, oxylipin metabolites originating from hydroperoxy fatty acids, the oxygenation product of lipoxygenase and members of cytochrome P450 which are also localized in developing primordia, are active as signaling molecules in developmental and defense-related processes (Howe and Schilmiller 2002, Miyoshi et al. 2004, Vellosillo et al. 2007). PHGPx was described as a moonlighting enzyme in mammals (Puglisi et al. 2005); in yeast, it serves as a sensor and transducer of stress response to H2O2, regulates the protein repair activity of methionine sulfoxide reductase and protects glutamine synthetase from inactivation via oxidative stress, in addition to its general peroxide-scavenging activity (Delaunay et al. 2002, Kho et al. 2006, Lee et al. 2007). Further studies should be carried out to reveal the mechanism by which cit-PHGPx is involved in shoot regeneration. One possibility evolved from this study shows the ability of this enzyme to affect the steady-state level of ROS by scavenging either H2O2 or phospholipids. It is also possible that cit-PHGPx is affecting regeneration by modulating the activities of redox-sensitive thiol-proteins, particularly those involved in signal transduction pathways. In fact PHGPx has already been shown to function as a redox transducer in ABA and drought stress signaling (Miao et al. 2006). The dramatic effect of cit-PHGPx on regeneration may point to the physiological significance of PHGPx in plants, which was mainly attributed so far to ascorbate peroxidase (Foyer and Noctor 2005). The availability of a method that enables modification of the level of ROS within differentiating plant cells at desired time points may shed light on as yet unknown mechanisms regulating differentiation processes in plants. Manipulation of ROS production at specific time points could perhaps also be utilized to control cell differentiation in processes such as genetic transformation. Our results clearly demonstrate the essential role of ROS in the early stages of shoot organogenesis and the possible involvement of PHGPx in maintaining ROS homeostasis at this point in the process. It is suggested that uncontrolled reduction of free radicals, by employing antioxidants that scavenge ROS, can lead to the disruption of metabolic pathways that are essential for tissue and organ differentiation. Materials and Methods Vector construction Several vectors, all harboring the coding sequence for cit-PHGPx (also termed csa), were constructed as follows. 35S-cit-PHGPx plasmid was constructed by inserting the PCR product of cit-PHGPx into an expression cassette (Shaul and Galili 1992) between the cauliflower mosaic virus 35S promoter followed by the Ω translation enhancer and the OSC terminator using SalI and EcoRI restriction sites. RD29A-cit-PHGPx and Pat-cit-PHGPx plasmids were constructed by replacing the 35SΩ promoter of 35S-cit-PHGPx with the promoter of the stress-inducible RD29A gene or the promoter of the class I patatin B33 gene, respectively. The RD29A promoter (−862/75) was amplified from the genomic DNA of A. thaliana, using the primers 5′-CGGGATCCGGAGCCATAGATGCAATTCAATC-3′ and 5′-ACGCGTCGACAAGATTTTTTTCTTTCCAATAGAAGTAATC-3′, and cloned between BamHI and SalI restriction sites of 35S-cit-PHGPx. The patatin promoter (Perl et al. 1991) was cloned between the SmaI and SalI restriction sites. 35S-Ser41cit-PHGPx was obtained by oligonucleotide-directed site-specific mutagenesis of the 35S-cit-PHGPx plasmid according to Kunkel et al. (1987), using 35S-cit-PHGPx as a template. The mutation was inserted with the primer 5′-TGTTGCATCACGT CTGGCTTGACCAATTC-3′, changing nucleotide 125 (TGT to TCT) and leading to replacement of Cys41 with Ser. All of these cloning cassettes were cloned into the binary vector pPZP111 between SmaI and XbaI (Hajdukiewicz et al. 1994). pCambia-cit-PHGPx was obtained after cloning the 35S-cit-PHGPx cassette into the multiple cloning site of pCAMBIA 2301 which contains a GUS reporter cassette. pER8-cit-PHGPx was constructed by cloning cit-PHGPx into the binary vector pER8 (Zuo et al. 2000), kindly provided by Professor N. H. Chua (Rockefeller University New York, NY, USA). This vector is strictly regulated by β-estradiol. cit-PHGPx was inserted between XhoI and SpeI in the multiple cloning site of pER8. All of the vectors were electroporated into A. tumefaciens EHA105, and were used for the transformation experiments. Plant material, propagation and transformation Several plant sources were used for the transformation experiments. Except when otherwise noted, plant material was cultured and maintained under a cool light fluorescent tube (16/8 h photoperiod at 42 μmol photons m−2 s−1) at 25°C. BY-2 cell cultures were maintained and transformed as previously described (Shaul et al. 1996). Shoot and stem segments (1–2 cm) from in vitro grown shoots of S. tuberosum cv. Desiree were placed horizontally on potato callus induction medium containing solidified [0.25% (w/v) Gelrite] Murashige and Skoog (MS) basal salts supplemented with 3% (w/v) Suc, 2 mg l−1 2,4-D and 0.2 mg l−1 6-benzylaminopurine (pH 5.8). Calli were resuspended in liquid potato callus induction medium and subcultured every 10 d. Transformation was carried out by co-cultivation (48 h) of the potato calli with Agrobacterium harboring the 35S-cit-PHGPx plasmid and subsequent selection on solidified potato callus induction medium containing paromomycin (75 mg l−1) and claforan (500 mg l−1). In vitro grown tobacco (N. tabacum cv. SR1) leaves were wounded using a scalpel and co-cultivated with Agrobacterium containing the 35S-cit-PHGPx plasmid. Following co-cultivation (48 h), leaves were transferred to selection on callus-inducing MS medium containing 3% Suc, 1 mg l−1 2,4-D, 2 mg l−1 naphthaleneacetic acid, 100 mg l−1 kanamycin and 500 mg l−1 claforan. The transformed calli, developed around the wounded area, were routinely subcultured on a medium containing 3 mg l−1 naphthaleneacetic acid, 1 mg l−1 6-benzylaminopurine and 100 mg l kanamycin. Tobacco leaves were transformed with Agrobacterium carrying the specified plasmid by the leaf disc method (Horsch et al. 1985). The leaves were cutivated on regeneration medium (MS medium containing 3% Suc, 2 mg l−1 kinetin, 0.8 mg l−1 IAA and 1 mg l−1 zeatin), and selected with either hygromycin (30 mg l−1) for pER8-cit-PHGPx transformants or kanamycin (100 mg l−1) for 35S-cit-PHGPx, RD29A-cit-PHGPx, Pat-cit-PHGPx, 35S-Ser41cit-PHGPx and pCambia-cit-PHGPx transformants. To enable elongation of putative pER8-cit-PHGPx-transformed shoots, regeneration medium containing only 15 mg l−1 hygromycin was used. Arabidopsis thaliana (ecotype Columbia) was transformed by the infiltration method according to Clough and Bent (1998). Transformants were selected on kanamycin, and seeds from T1 were collected for further analysis. Induction of cit-PHGPx expression in transformed plant material Plant cit-PHGPx-transformants were induced as follows. BY-2 cell cultures. Liquid cultures of BY-2 cells transformed with the Pat-cit-PHGPx construct were maintained in liquid culture as described (Shaul et al. 1996). On day 4 after subculturing, 100 mM Pro was added to the culture medium. Cells were harvested 24 and 48 h later. Similarly, the expression of cit-PHGPx was induced in BY-2 cells transformed with the RD29A-cit-PHGPx construct by the addition of 0.2 M NaCl and 0.05 mM ABA. Cells were harvested 8, 24 and 48 h after induction. Tobacco leaf discs. Induction of cit-PHGPx expression in tobacco leaf discs was carried out by two different methods. (i) Leaves were detached from putative in vitro pER8-cit- PHGPx-transformed plants and were brushed with a solution of 10 μM β-estradiol [containing 0.01% (v/v) Tween-20 in water]. Leaves were placed in a Petri dish for 2 d on a wetted filter paper prior to their extraction for cit-PHGPx-expression assays. (ii) Leaf discs were taken from pER8-cit-PHGPx-transformed plants and placed on solidified MS medium supplemented with 3% Suc and 5 μM β-estradiol for 24–48 h prior to cit- PHGPx assay. cit-PHGPx induction at different time points during the regeneration process In an attempt to characterize the stage of regeneration at which inhibition by cit-PHGPx overexpression occurs, two different experiments were conducted using leaves from pER8- cit-PHGPx-transgenic plants. Leaf discs (1.3 cm in diameter) were excised from in vitro grown sterile shoots and transferred to solid regeneration medium. On each alternate day for the following 10 d, discs were transferred onto plates containing the same medium with the addition of 5 μM β-estradiol. Discs were either maintained on this medium until the end of the experiment or moved back after 24 h to regeneration medium lacking β-estradiol. After a total cultivation of 21 d, the number of regenerated shoots was counted. ROS determination Relative ROS level was determined in leaf discs taken from pER8-cit-PHGPx-transformed tobacco plants which were cultivated on solidified regeneration medium in the presence or absence of β-estradiol for 15 d. Two different methods were used to determine ROS qualitatively and semi-quantitatively, respectively. Superoxide radicals were detected histochemically with NBT according to Fryer et al. (2002). Leaf discs were infiltrated and incubated for 10 min in 10 mM phosphate buffer pH 7.8 containing 10 mM sodium azide, followed by 30 min incubation in the same solution containing 0.1% (w/v) NBT. O2−· decomposition by 10 mM MnCl2 or O2− generation by 10 mM H2O2 were used as controls. The fluorescent dye carboxy-H2DCFDA (Molecular Probes, Eugene, OR, USA) was used to determine ROS such as H2O2, peroxyl radicals and peroxynitrite anions. ROS were measured semi-quantitatively using carboxy-H2DCFDA, as modified from Lu and Higgins (1998), Govrin and Levine (2000) and Belenghi et al. (2004), on leaf discs (0.7 cm in diameter) taken from a highly expressing line (GPx2) and a non-expressing line (GPx4) of in vitro pER8-cit-PHGPx-transformed tobacco plant shoots. The discs, taken at different time points during cultivation, were washed briefly with 100 mM phosphate buffer (pH 7.4) and incubated for 30 min in the dark in 24-well plates, to which 1 ml of 5 μM carboxy-H2DCFDA in the same buffer was added. The total fluorescent emission (at 525 nm) was measured with a microplate fluorescent reader (FL600 Bio-Tek, Bio Tek Instruments, Winooski, VT, USA; excitation at 485 nm). The ROS level was also measured by the same procedure in GPx2 leaf discs cultivated on regeneration medium for 7 d and then transferred to inducing medium for an additional 4 d. To study the effect of Mn2+ ions on ROS scavenging, non-induced transformed tobacco leaf discs taken from GPx2 were cultivated on regeneration medium containing 5, 10 or 20 mM MnSO4. Superoxide radicals were detected after 17 d using the NBT method. The number of shoots regenerated after 21 d was also determined. All fluorescence measurements were normalized according to Royall and Ischiropoulos (1993). Twenty-two discs were taken to obtain an average value for each fluorescence measurement. Equivalent experiments were also performed at pH 6.2 and 8.0 to determine the effect of pH on the measurements. GUS staining Tobacco leaves were stained for histochemical analysis of GUS activity (Janssen and Gardner 1989) at 5, 9 and 15 d following their co-cultivation with Agrobacterium carrying pCambia-cit-PHGPx. Images were obtained with a Leica MZ FL III microscope equipped with a Leica DC200 camera (Leica Microsystems, Wetzlar, Germany). Protein extraction and Western blot Protein extracts from all plant material were obtained by grinding the plant tissue in liquid nitrogen and solubilizing in SDS–Laemmli buffer (Ben-Hayyim et al. 1993). In the BY-2 cell protein analysis, the cells were collected by filtration and 10 mg ml−1 DNase was added to the solubilized sample to reduce solution viscosity. Gel electrophoresis and Western blots were performed as described previously (Hazebrouck et al. 2000). Polyclonal antibodies against cit-PHGPx were raised in rabbits as we described before (Ben-Hayyim et al. 1993), and were used for cit-PHGPx identification in Western blot analysis. Anti-tobacco PHGPx antibodies, kindly provided by Professor D. Inzé (Ghent University, Belgium), were used for the endogenous detection of tobacco PHGPx. cit-PHGPx obtained from transformed E. coli cells served as a marker for the electrophoretic analysis. Images were captured with a CCD camera (FluorChem 8800, Alpha Innotech, CA, USA) and protein quantification was determined by densitometry using Alpha Innotech software. Enzymatic assay cit-PHGPx, as well as the mutated Ser41cit-PHGPx cloned in the Bluescript vector, were expressed in E. coli as described before (Beeor-Tzahar et al. 1995). The enzymatic activity of each of the proteins was measured in extracts obtained from the recombinant bacteria using the substrates phosphatidylcholine hydroperoxide (2 μM; pH 8.5) and H2O2 (100 μM; pH 7.5), and the regenarating substrate glutathione (Faltin et al. 1998). When thioredoxin (5 μM) was employed as the regenerating agent, the reaction was carried out at pH 7.5 using thioredoxin reductase (0.3 U ml−1) instead of glutathione reductase. One unit of PHGPx enzymatic activity is defined as the amount of enzyme which oxidizes 1 nmol of NADPH min−1. Sequence data mentioned in this article can be found in the GenBank/EMBL data libraries under the following accession numbers: X66377 (cit-PHGPx), A08215 (B33) and D13044 (RD29A). Funding This work was supported by the US–Israel Binational Agricultural Research and Development Fund. Abbreviations Abbreviations cit-PHGPx citrus phospholipid hydroperoxide glutathione peroxidase Cys cysteine C41S Cys41 in cit-PHGPx replaced by Ser GPx glutathione peroxidase GUS β -glucuronidase carboxy-H2 DCFDA 5-(and-6)-carboxy-2′,7′- dichlorodihydrouorescein diacetate MS Murashige and Skoog NBT nitroblue tetrazolium Pro proline ROS reactive oxygen species Ser serine Suc sucrose. 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TI - Glutathione Peroxidase Regulation of Reactive Oxygen Species Level is Crucial for In Vitro Plant Differentiation
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcq082
DA - 2010-06-08
UR - https://www.deepdyve.com/lp/oxford-university-press/glutathione-peroxidase-regulation-of-reactive-oxygen-species-level-is-iYls7CeJC0
SP - 1151
EP - 1162
VL - 51
IS - 7
DP - DeepDyve
ER -