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NADPH Thioredoxin Reductase C Is Involved in Redox Regulation of the Mg-Chelatase I Subunit in Arabidopsis thaliana Chloroplasts
Molecular Plant 7, 1252–1255, July 2014
LETTER TO THE EDITOR
NADPH Thioredoxin Reductase C Is Involved in Redox Regulation of the Mg-Chelatase I Subunit in Arabidopsis thaliana Chloroplasts subunit of Mg-chelatase was shown to be redox-regulated in Arabidopsis, TRX f being involved in this regulation (Ikegami et al., 2007). Further analyses confirmed the involvement of Trx f, and Trx m, in CHLI redox regulation in pea, but found that additional redox systems may be involved (Luo et al., 2012). Stenbaek and Jensen (2010) reported NTRC stimulation of CHLI activity; however, no interaction of NTRC and CHLI in yeast two-hybrid assays nor in vitro NTRC stimulation of Mg-chelatase activity were observed (Luo et al., 2012). Therefore, the implication of NTRC in CHLI redox regulation remains uncertain. Here, we have analyzed the possible function of NTRC in CHLI redox regulation in Arabidopsis by a combination of in vitro and in vivo approaches. To analyze the redox regulation of CHLI in vitro, Histagged mature forms of both Arabidopsis CHLI isoenzymes were expressed in Escherichia coli and a polyclonal antibody was raised against CHLI-1, which efficiently cross-reacted with both CHLI-1 and CHLI-2 (Figure 1A). Both isoforms showed redox-dependent electrophoretic mobility; the upper band, enriched by DTT treatment, corresponded to the reduced form, whereas the lower one, enriched by CuCl2 treatment, corresponded to the oxidized form (Figure 1A). RT–qPCR, in agreement with Genevestigator data (Supplemental Figure 1A and 1B), showed higher level CHLI-1 expression indicating that CHLI-1 is the most relevant form of the enzyme and, thus, it was analyzed in more detail in this work. In vitro activity assays showed that NTRC, with NADPH as reductant, promoted a higher activation of CHLI-1 ATPase activity than TRX f1 and TRX x, both of them assayed with a small amount of DTT (10 μM) as reductant (Figure 1B). To study further the function of NTRC on the redox regulation of CHLI-1, we took advantage of the redox-dependent differential electrophoretic mobility of CHLI. NTRC and NADPH increased the amount of the reduced form of the enzyme more efficiently than TRX x (Figure 1C). No reduction of CHLI-1 was observed when NTRC was replaced by mutant variants of the enzyme at © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/ssu032, Advance Access publication 21 March 2014 Received 23 December 2013; accepted 18 March 2014
Downloaded from http://mplant.oxfordjournals.org/ at Monash University on December 5, 2014
Dear Editor, The synthesis of tetrapyrroles, including chlorophylls, is central for chloroplast function. The metabolic pathway of tetrapyrrole biosynthesis in Arabidopsis is initiated with the formation of amino levulinic acid (ALA), which is converted by a series of common reactions to protoporphyrin IX (Proto IX) (Tanaka et al., 2011). Then the pathway diverges into two branches: the synthesis of heme/bilin and chlorophylls. The insertion of Mg2+ into Proto IX, catalyzed by Mg-chelatase, is the first committed reaction of the chlorophyll branch and is considered a key step for the regulation of the whole pathway. Mg-chelatase is a heterotrimeric enzyme composed of subunits CHLI, CHLD, and CHLH, the reaction mechanism of which has been established. It is a two-step process consisting in the Mg-ATPdependent activation of the enzyme, which implies the formation of a ternary complex of subunits CHLI and CHLD with ATP-Mg2+, and Mg2+ chelation, which is catalyzed by CHLH driven by ATP hydrolysis, CHLI providing ATPase activity to the complex (Tanaka et al., 2011). In Arabidopsis, CHLH and CHLD are encoded by single genes, whereas two genes, CHLI-1 and CHLI-2, encode the two isoforms of CHLI. The pathway of chlorophyll biosynthesis needs to be tightly regulated to adjust chlorophyll synthesis to the demand of photosynthetic cells and to avoid the phototoxicity of chlorophyll intermediates. Redox regulation constitutes a reversible mechanism for the rapid adaptation of enzyme activity which has an important contribution to chloroplast function. In chloroplasts, redox regulation relies on reducing power, provided by ferredoxin (Fd) reduced by the photosynthetic electron transport chain, a Fd-dependent thioredoxin reductase (FTR), and a complex set of thioredoxins (TRXs). This notion of chloroplast redox regulation was modified by the finding of NADPHdependent thioredoxin reductase C (NTRC) (Serrato et al., 2004), which acts as an alternative pathway allowing the use of NADPH for redox regulation in plastids (Cejudo et al., 2012; Kirchsteiger et al., 2012; Pulido et al., 2010). The Arabidopsis NTRC knockout mutant displays a characteristic pale green leaf color (Serrato et al., 2004), which suggests the involvement of NTRC in redox regulation of chlorophyll synthesis. Indeed, NTRC-dependent regulation of two enzymes of the pathway, glutamyl-transfer RNA reductase1 (GluTR1) and MgP methyltransferase (CHLM), has been shown (Richter et al., 2013). Moreover, the CHLI
Molecular Plant
Letter to the Editor
1253
(A) Western blot analysis of purified recombinant CHLI-1 and CHLI-2 (50 ng of protein) treated or not with 0.25 mM CuCl2 or 10 mM DTT, under non-reducing conditions. Red, reduced; ox, oxidized. (B) Effect of NTRC, TRX f1, and TRX x on CHLI ATPase activity. Assays were performed as indicated in the supplementary information per triplicate and mean values ± SE are shown. (C) Western blot analysis showing the effect of wild-type or mutant variants C380S and C140S of NTRC and TRX x on the redox status of pre-oxidized CHLI-1. Molecular mass markers (kDa) are indicated on the left. Red, reduced; ox, oxidized. (D) Confocal microscopy micrographs of mesophyll cells of Nicotiana benthamiana leaves agro-infiltrated with the indicated constructs. Red, chlorophyll auto-fluorescence; yellow, YFP fluorescence. (E) In vivo redox status of CHLI determined by MMPEG24 thiol labeling. Numbers on the right indicate shifted bands. The numbers below the blot represent quantification of the shifted bands, expressed as a percentage of the total CHLI protein.
the NTR (C140S) or the TRX (C380S) domains (Figure 1C), showing that NTRC is able to reduce CHLI-1 in vitro. Two additional approaches were used to confirm the function of NTRC in redox regulation of CHLI in vivo.
Bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana leaves expressing CHLI-1, fused to the N-terminal part of YFP (CHLI-1–NYFP), and NTRC, fused to the C-terminal part of YFP (NTRC–C YFP), showed no
Downloaded from http://mplant.oxfordjournals.org/ at Monash University on December 5, 2014
Figure 1 Analysis of the Function of NTRC on CHLI Redox Regulation.
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Molecular Plant
Letter to the Editor
(Supplemental Figure 4). However, the level of CHLI proteins in the ntrc mutant and, to a lower extent, in the trxf1 mutant was clearly decreased as compared with wild-type plants, suggesting that the impairment of the redox regulation of CHLI causes the instability of the protein. It should be noted that the chlorophyll-deficient phenotype of the ntrc mutant is not exclusively due to the impairment of NTRC-dependent redox regulation of CHLI. ALA feeding experiments shows accumulation of Mg-Proto IX and only a slight increase in Proto IX in leaves of the ntrc mutant (Stenbaek et al., 2008), suggesting that the impairment of Mg-chelatase activity is not the main reason for the chlorophyll-deficient phenotype of the ntrc mutant. It was shown that Mg-protoporphyrin monomethyl ester cyclase was stimulated by NTRC and 2-Cys PRX—an effect attributed to the H2O2 scavenging activity of this system (Stenbaek et al., 2008). Furthermore, other enzymes of the pathway catalyzing either initial steps, such as GluTR1, or final steps, such as CHLM, are regulated by NTRC (Richter et al., 2013). Altogether, these results emphasize the relevant function of NTRC in redox regulation in chlorophyll biosynthesis, regulation being exerted at different levels of the pathway.
SUPPLEMENTARY DATA Supplementary data are available at Molecular Plant Online.
FUNDING This work was supported by European Regional Development Fund, co-financed grants from the Spanish Ministry of Science and Innovation (BIO2010–15430) and Junta de Andalucía (BIO-182 and CVI-5919). J.M.P.-R. was funded by the Juan de la Cierva program from the Spanish Ministry of Education, while M.G. was recipient of a predoctoral fellowship from Junta de Andalucía.
Acknowledgments We thank Alicia Orea for technical assistance with confocal microscopy, Jesse D. Woodson and Joanne Chory (The Salk Institute for Biological Studies, San Diego, USA) for the gift of cs and gun5 seeds, and Sebastian Schornack (Sainsbury Laboratory, Cambridge, UK) for providing the BiFC vectors. No conflict of interest declared.
Juan Manuel Pérez-Ruiza,2, Manuel Guineaa,b,2, Leonor Puerto-Galána, and Francisco Javier Cejudoa,1
Downloaded from http://mplant.oxfordjournals.org/ at Monash University on December 5, 2014
fluorescence in either single combination, while co-infiltration of both constructs resulted in a chloroplast-localized yellow fluorescence (Figure 1D) showing the interaction of NTRC with CHLI-1 in vivo. The NTRC–CHLI-1 interaction was confirmed by the reverse combination of fusions CHLI-1–C YFP and NTRC–NYFP (Supplemental Figure 2). In both cases, strong YFP signals appeared as dots, but whether this distribution indicates subcompartmentalization of the NTRC–CHLI-1 interaction is not yet known. A more direct knowledge of the redox regulation of CHLI was addressed by determining the actual redox state of these enzymes in vivo with the alkylating reagent methylmaleimide-(polyethylene glycol)24 (MMPEG24), which binds reduced thiol groups adding 1.2 kDa. Figure 1E shows a Western blot analysis of CHLI from leaf extracts that were treated with MMPEG24 in which up to four predominant shifted bands could be detected. Overall, the reduced form of CHLI was predominant in all the lines analyzed, as bands 1 and 2 were more abundant than bands 3 and 4. In wild-type plants, bands 1 and 2 account for ~83% of the total CHLI protein, as compared to bands 3 and 4, representing ~17% (Figure 1E). In contrast, bands 3 and 4 account for ~32% of the total CHLI protein in the ntrc mutant indicating a lower level of CHLI reduction in this mutant (Figure 1E). While the pattern of bands observed in extracts from the trxx mutant was very similar to that of the wild-type, the trxf1 mutant showed a low intensity of band 1, which might be indicative of the implication of Trx f1 in CHLI redox regulation. These results suggest that the NTRC-dependent redox regulation of CHLI might contribute to the chlorophylldeficient phenotype of the ntrc mutant. If this is the case, it should be expected that mutants deficient in this subunit of Mg-chelatase present a chlorophyll-deficient phenotype comparable to that of the ntrc mutant. This possibility was analyzed in Arabidopsis mutants showing mild and severe deficiency of CHLI. To that end, the Arabidopsis line (SALK_067450) with a T-DNA insertion in the 5’-untranslated region of the CHLI-1 gene, chli1.1 mutant, was isolated. This mutant showed slightly lower levels of CHLI than the wild-type plants in contrast with the severe deficiency shown by the cs mutant (Supplemental Figure 3A). In accordance with the level of CHLI proteins, the leaf chlorophyll content of the chli1.1 mutant was comparable to the ntrc mutant, while the cs mutant showed a more dramatic decrease (Supplemental Figure 3B and 3C). The gun5 mutant, which is deficient in the CHLH subunit of the Mg-chelatase, here included for comparative purposes, displayed chlorophyll content similar to that of the chli1.1 and ntrc mutants (Supplemental Figure 3B and 3C). Finally, we tested whether NTRC deficiency has any effect on the level of CHLI. Similar levels of CHLI-1 and CHLI-2 transcripts were detected in wild-type, trxf1, and trxx mutants, while the ntrc mutant contained slightly increased CHLI-1 transcripts
Molecular Plant a Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-Consejo Superior de Investigaciones Científicas, 41092-Sevilla, Spain b Present address: Umea Plant Science Centre, Department of Plant Physiology, Umea University, S-901 87 Umea, Sweden 1 To whom correspondence should be addressed. E-mail [email protected], tel. +34 954489511, fax +34 954461065. 2 These authors contributed equally to this work.
References
Ikegami, A., Yoshimura, N., Motohashi, K., Takahashi, S., Romano, P.G., Hisabori, T., Takamiya, K., and Masuda, T. (2007). The CHLI1 subunit of Arabidopsis thaliana magnesium chelatase is a target protein of the chloroplast thioredoxin. J. Biol. Chem. 282, 19282–19291. Kirchsteiger, K., Ferrández, J., Pascual, M.B., González, M., and Cejudo, F.J. (2012). NADPH thioredoxin reductase C is localized in plastids of photosynthetic and nonphotosynthetic tissues and is involved in lateral root formation in Arabidopsis. Plant Cell. 24, 1534–1548.
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Luo, T., Fan, T., Liu, Y., Rothbart, M., Yu, J., Zhou, S., Grimm, B., and Luo, M. (2012). Thioredoxin redox regulates ATPase activity of magnesium chelatase CHLI subunit and modulates redox-mediated signaling in tetrapyrrole biosynthesis and homeostasis of reactive oxygen species in pea plants. Plant Physiol. 159, 118–130. Pulido, P., Spinola, M.C., Kirchsteiger, K., Guinea, M., Pascual, M.B., Sahrawy, M., Sandalio, L.M., Dietz, K.J., González, M., and Cejudo, F.J. (2010). Functional analysis of the pathways for 2-Cys peroxiredoxin reduction in Arabidopsis thaliana chloroplasts. J. Exp. Bot. 61, 4043–4054. Richter, A.S., Peter, E., Rothbart, M., Schlicke, H., Toivola, J., Rintamäki, E., and Grimm, B. (2013). Posttranslational influence of NADPH-dependent thioredoxin reductase C on enzymes in tetrapyrrole synthesis. Plant Physiol. 162, 63–73. Serrato, A.J., Pérez-Ruiz, J.M., Spinola, M.C., and Cejudo, F.J. (2004). A novel NADPH thioredoxin reductase, localized in the chloroplast, which deficiency causes hypersensitivity to abiotic stress in Arabidopsis thaliana. J. Biol. Chem. 279, 43821–43827. Stenbaek, A., and Jensen, P.E. (2010). Redox regulation of chlorophyll biosynthesis. Phytochemistry. 71, 853–859. Stenbaek, A., Hansson, A., Wulff, R.P., Hansson, M., Dietz, K.J., and Jensen, P.E. (2008). NADPH-dependent thioredoxin reductase and 2-Cys peroxiredoxins are needed for the protection of Mg-protoporphyrin monomethyl ester cyclase. FEBS Lett. 582, 2773–2778. Tanaka, R., Kobayashi, K., and Masuda, T. (2011). Tetrapyrrole metabolism in Arabidopsis thaliana. Arabidopsis Book. 9, e0145.
Downloaded from http://mplant.oxfordjournals.org/ at Monash University on December 5, 2014
Cejudo, F.J., Ferrández, J., Cano, B., Puerto-Galán, L., and Guinea, M. (2012). The function of the NADPH thioredoxin reductase C-2-Cys peroxiredoxin system in plastid redox regulation and signalling. FEBS Lett. 586, 2974–2980.
Letter to the Editor
LETTER TO THE EDITOR
NADPH Thioredoxin Reductase C Is Involved in Redox Regulation of the Mg-Chelatase I Subunit in Arabidopsis thaliana Chloroplasts subunit of Mg-chelatase was shown to be redox-regulated in Arabidopsis, TRX f being involved in this regulation (Ikegami et al., 2007). Further analyses confirmed the involvement of Trx f, and Trx m, in CHLI redox regulation in pea, but found that additional redox systems may be involved (Luo et al., 2012). Stenbaek and Jensen (2010) reported NTRC stimulation of CHLI activity; however, no interaction of NTRC and CHLI in yeast two-hybrid assays nor in vitro NTRC stimulation of Mg-chelatase activity were observed (Luo et al., 2012). Therefore, the implication of NTRC in CHLI redox regulation remains uncertain. Here, we have analyzed the possible function of NTRC in CHLI redox regulation in Arabidopsis by a combination of in vitro and in vivo approaches. To analyze the redox regulation of CHLI in vitro, Histagged mature forms of both Arabidopsis CHLI isoenzymes were expressed in Escherichia coli and a polyclonal antibody was raised against CHLI-1, which efficiently cross-reacted with both CHLI-1 and CHLI-2 (Figure 1A). Both isoforms showed redox-dependent electrophoretic mobility; the upper band, enriched by DTT treatment, corresponded to the reduced form, whereas the lower one, enriched by CuCl2 treatment, corresponded to the oxidized form (Figure 1A). RT–qPCR, in agreement with Genevestigator data (Supplemental Figure 1A and 1B), showed higher level CHLI-1 expression indicating that CHLI-1 is the most relevant form of the enzyme and, thus, it was analyzed in more detail in this work. In vitro activity assays showed that NTRC, with NADPH as reductant, promoted a higher activation of CHLI-1 ATPase activity than TRX f1 and TRX x, both of them assayed with a small amount of DTT (10 μM) as reductant (Figure 1B). To study further the function of NTRC on the redox regulation of CHLI-1, we took advantage of the redox-dependent differential electrophoretic mobility of CHLI. NTRC and NADPH increased the amount of the reduced form of the enzyme more efficiently than TRX x (Figure 1C). No reduction of CHLI-1 was observed when NTRC was replaced by mutant variants of the enzyme at © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/ssu032, Advance Access publication 21 March 2014 Received 23 December 2013; accepted 18 March 2014
Downloaded from http://mplant.oxfordjournals.org/ at Monash University on December 5, 2014
Dear Editor, The synthesis of tetrapyrroles, including chlorophylls, is central for chloroplast function. The metabolic pathway of tetrapyrrole biosynthesis in Arabidopsis is initiated with the formation of amino levulinic acid (ALA), which is converted by a series of common reactions to protoporphyrin IX (Proto IX) (Tanaka et al., 2011). Then the pathway diverges into two branches: the synthesis of heme/bilin and chlorophylls. The insertion of Mg2+ into Proto IX, catalyzed by Mg-chelatase, is the first committed reaction of the chlorophyll branch and is considered a key step for the regulation of the whole pathway. Mg-chelatase is a heterotrimeric enzyme composed of subunits CHLI, CHLD, and CHLH, the reaction mechanism of which has been established. It is a two-step process consisting in the Mg-ATPdependent activation of the enzyme, which implies the formation of a ternary complex of subunits CHLI and CHLD with ATP-Mg2+, and Mg2+ chelation, which is catalyzed by CHLH driven by ATP hydrolysis, CHLI providing ATPase activity to the complex (Tanaka et al., 2011). In Arabidopsis, CHLH and CHLD are encoded by single genes, whereas two genes, CHLI-1 and CHLI-2, encode the two isoforms of CHLI. The pathway of chlorophyll biosynthesis needs to be tightly regulated to adjust chlorophyll synthesis to the demand of photosynthetic cells and to avoid the phototoxicity of chlorophyll intermediates. Redox regulation constitutes a reversible mechanism for the rapid adaptation of enzyme activity which has an important contribution to chloroplast function. In chloroplasts, redox regulation relies on reducing power, provided by ferredoxin (Fd) reduced by the photosynthetic electron transport chain, a Fd-dependent thioredoxin reductase (FTR), and a complex set of thioredoxins (TRXs). This notion of chloroplast redox regulation was modified by the finding of NADPHdependent thioredoxin reductase C (NTRC) (Serrato et al., 2004), which acts as an alternative pathway allowing the use of NADPH for redox regulation in plastids (Cejudo et al., 2012; Kirchsteiger et al., 2012; Pulido et al., 2010). The Arabidopsis NTRC knockout mutant displays a characteristic pale green leaf color (Serrato et al., 2004), which suggests the involvement of NTRC in redox regulation of chlorophyll synthesis. Indeed, NTRC-dependent regulation of two enzymes of the pathway, glutamyl-transfer RNA reductase1 (GluTR1) and MgP methyltransferase (CHLM), has been shown (Richter et al., 2013). Moreover, the CHLI
Molecular Plant
Letter to the Editor
1253
(A) Western blot analysis of purified recombinant CHLI-1 and CHLI-2 (50 ng of protein) treated or not with 0.25 mM CuCl2 or 10 mM DTT, under non-reducing conditions. Red, reduced; ox, oxidized. (B) Effect of NTRC, TRX f1, and TRX x on CHLI ATPase activity. Assays were performed as indicated in the supplementary information per triplicate and mean values ± SE are shown. (C) Western blot analysis showing the effect of wild-type or mutant variants C380S and C140S of NTRC and TRX x on the redox status of pre-oxidized CHLI-1. Molecular mass markers (kDa) are indicated on the left. Red, reduced; ox, oxidized. (D) Confocal microscopy micrographs of mesophyll cells of Nicotiana benthamiana leaves agro-infiltrated with the indicated constructs. Red, chlorophyll auto-fluorescence; yellow, YFP fluorescence. (E) In vivo redox status of CHLI determined by MMPEG24 thiol labeling. Numbers on the right indicate shifted bands. The numbers below the blot represent quantification of the shifted bands, expressed as a percentage of the total CHLI protein.
the NTR (C140S) or the TRX (C380S) domains (Figure 1C), showing that NTRC is able to reduce CHLI-1 in vitro. Two additional approaches were used to confirm the function of NTRC in redox regulation of CHLI in vivo.
Bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana leaves expressing CHLI-1, fused to the N-terminal part of YFP (CHLI-1–NYFP), and NTRC, fused to the C-terminal part of YFP (NTRC–C YFP), showed no
Downloaded from http://mplant.oxfordjournals.org/ at Monash University on December 5, 2014
Figure 1 Analysis of the Function of NTRC on CHLI Redox Regulation.
1254
Molecular Plant
Letter to the Editor
(Supplemental Figure 4). However, the level of CHLI proteins in the ntrc mutant and, to a lower extent, in the trxf1 mutant was clearly decreased as compared with wild-type plants, suggesting that the impairment of the redox regulation of CHLI causes the instability of the protein. It should be noted that the chlorophyll-deficient phenotype of the ntrc mutant is not exclusively due to the impairment of NTRC-dependent redox regulation of CHLI. ALA feeding experiments shows accumulation of Mg-Proto IX and only a slight increase in Proto IX in leaves of the ntrc mutant (Stenbaek et al., 2008), suggesting that the impairment of Mg-chelatase activity is not the main reason for the chlorophyll-deficient phenotype of the ntrc mutant. It was shown that Mg-protoporphyrin monomethyl ester cyclase was stimulated by NTRC and 2-Cys PRX—an effect attributed to the H2O2 scavenging activity of this system (Stenbaek et al., 2008). Furthermore, other enzymes of the pathway catalyzing either initial steps, such as GluTR1, or final steps, such as CHLM, are regulated by NTRC (Richter et al., 2013). Altogether, these results emphasize the relevant function of NTRC in redox regulation in chlorophyll biosynthesis, regulation being exerted at different levels of the pathway.
SUPPLEMENTARY DATA Supplementary data are available at Molecular Plant Online.
FUNDING This work was supported by European Regional Development Fund, co-financed grants from the Spanish Ministry of Science and Innovation (BIO2010–15430) and Junta de Andalucía (BIO-182 and CVI-5919). J.M.P.-R. was funded by the Juan de la Cierva program from the Spanish Ministry of Education, while M.G. was recipient of a predoctoral fellowship from Junta de Andalucía.
Acknowledgments We thank Alicia Orea for technical assistance with confocal microscopy, Jesse D. Woodson and Joanne Chory (The Salk Institute for Biological Studies, San Diego, USA) for the gift of cs and gun5 seeds, and Sebastian Schornack (Sainsbury Laboratory, Cambridge, UK) for providing the BiFC vectors. No conflict of interest declared.
Juan Manuel Pérez-Ruiza,2, Manuel Guineaa,b,2, Leonor Puerto-Galána, and Francisco Javier Cejudoa,1
Downloaded from http://mplant.oxfordjournals.org/ at Monash University on December 5, 2014
fluorescence in either single combination, while co-infiltration of both constructs resulted in a chloroplast-localized yellow fluorescence (Figure 1D) showing the interaction of NTRC with CHLI-1 in vivo. The NTRC–CHLI-1 interaction was confirmed by the reverse combination of fusions CHLI-1–C YFP and NTRC–NYFP (Supplemental Figure 2). In both cases, strong YFP signals appeared as dots, but whether this distribution indicates subcompartmentalization of the NTRC–CHLI-1 interaction is not yet known. A more direct knowledge of the redox regulation of CHLI was addressed by determining the actual redox state of these enzymes in vivo with the alkylating reagent methylmaleimide-(polyethylene glycol)24 (MMPEG24), which binds reduced thiol groups adding 1.2 kDa. Figure 1E shows a Western blot analysis of CHLI from leaf extracts that were treated with MMPEG24 in which up to four predominant shifted bands could be detected. Overall, the reduced form of CHLI was predominant in all the lines analyzed, as bands 1 and 2 were more abundant than bands 3 and 4. In wild-type plants, bands 1 and 2 account for ~83% of the total CHLI protein, as compared to bands 3 and 4, representing ~17% (Figure 1E). In contrast, bands 3 and 4 account for ~32% of the total CHLI protein in the ntrc mutant indicating a lower level of CHLI reduction in this mutant (Figure 1E). While the pattern of bands observed in extracts from the trxx mutant was very similar to that of the wild-type, the trxf1 mutant showed a low intensity of band 1, which might be indicative of the implication of Trx f1 in CHLI redox regulation. These results suggest that the NTRC-dependent redox regulation of CHLI might contribute to the chlorophylldeficient phenotype of the ntrc mutant. If this is the case, it should be expected that mutants deficient in this subunit of Mg-chelatase present a chlorophyll-deficient phenotype comparable to that of the ntrc mutant. This possibility was analyzed in Arabidopsis mutants showing mild and severe deficiency of CHLI. To that end, the Arabidopsis line (SALK_067450) with a T-DNA insertion in the 5’-untranslated region of the CHLI-1 gene, chli1.1 mutant, was isolated. This mutant showed slightly lower levels of CHLI than the wild-type plants in contrast with the severe deficiency shown by the cs mutant (Supplemental Figure 3A). In accordance with the level of CHLI proteins, the leaf chlorophyll content of the chli1.1 mutant was comparable to the ntrc mutant, while the cs mutant showed a more dramatic decrease (Supplemental Figure 3B and 3C). The gun5 mutant, which is deficient in the CHLH subunit of the Mg-chelatase, here included for comparative purposes, displayed chlorophyll content similar to that of the chli1.1 and ntrc mutants (Supplemental Figure 3B and 3C). Finally, we tested whether NTRC deficiency has any effect on the level of CHLI. Similar levels of CHLI-1 and CHLI-2 transcripts were detected in wild-type, trxf1, and trxx mutants, while the ntrc mutant contained slightly increased CHLI-1 transcripts
Molecular Plant a Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-Consejo Superior de Investigaciones Científicas, 41092-Sevilla, Spain b Present address: Umea Plant Science Centre, Department of Plant Physiology, Umea University, S-901 87 Umea, Sweden 1 To whom correspondence should be addressed. E-mail [email protected], tel. +34 954489511, fax +34 954461065. 2 These authors contributed equally to this work.
References
Ikegami, A., Yoshimura, N., Motohashi, K., Takahashi, S., Romano, P.G., Hisabori, T., Takamiya, K., and Masuda, T. (2007). The CHLI1 subunit of Arabidopsis thaliana magnesium chelatase is a target protein of the chloroplast thioredoxin. J. Biol. Chem. 282, 19282–19291. Kirchsteiger, K., Ferrández, J., Pascual, M.B., González, M., and Cejudo, F.J. (2012). NADPH thioredoxin reductase C is localized in plastids of photosynthetic and nonphotosynthetic tissues and is involved in lateral root formation in Arabidopsis. Plant Cell. 24, 1534–1548.
1255
Luo, T., Fan, T., Liu, Y., Rothbart, M., Yu, J., Zhou, S., Grimm, B., and Luo, M. (2012). Thioredoxin redox regulates ATPase activity of magnesium chelatase CHLI subunit and modulates redox-mediated signaling in tetrapyrrole biosynthesis and homeostasis of reactive oxygen species in pea plants. Plant Physiol. 159, 118–130. Pulido, P., Spinola, M.C., Kirchsteiger, K., Guinea, M., Pascual, M.B., Sahrawy, M., Sandalio, L.M., Dietz, K.J., González, M., and Cejudo, F.J. (2010). Functional analysis of the pathways for 2-Cys peroxiredoxin reduction in Arabidopsis thaliana chloroplasts. J. Exp. Bot. 61, 4043–4054. Richter, A.S., Peter, E., Rothbart, M., Schlicke, H., Toivola, J., Rintamäki, E., and Grimm, B. (2013). Posttranslational influence of NADPH-dependent thioredoxin reductase C on enzymes in tetrapyrrole synthesis. Plant Physiol. 162, 63–73. Serrato, A.J., Pérez-Ruiz, J.M., Spinola, M.C., and Cejudo, F.J. (2004). A novel NADPH thioredoxin reductase, localized in the chloroplast, which deficiency causes hypersensitivity to abiotic stress in Arabidopsis thaliana. J. Biol. Chem. 279, 43821–43827. Stenbaek, A., and Jensen, P.E. (2010). Redox regulation of chlorophyll biosynthesis. Phytochemistry. 71, 853–859. Stenbaek, A., Hansson, A., Wulff, R.P., Hansson, M., Dietz, K.J., and Jensen, P.E. (2008). NADPH-dependent thioredoxin reductase and 2-Cys peroxiredoxins are needed for the protection of Mg-protoporphyrin monomethyl ester cyclase. FEBS Lett. 582, 2773–2778. Tanaka, R., Kobayashi, K., and Masuda, T. (2011). Tetrapyrrole metabolism in Arabidopsis thaliana. Arabidopsis Book. 9, e0145.
Downloaded from http://mplant.oxfordjournals.org/ at Monash University on December 5, 2014
Cejudo, F.J., Ferrández, J., Cano, B., Puerto-Galán, L., and Guinea, M. (2012). The function of the NADPH thioredoxin reductase C-2-Cys peroxiredoxin system in plastid redox regulation and signalling. FEBS Lett. 586, 2974–2980.
Letter to the Editor