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Nuclear and mitochondrial genes encoding cytochrome c oxidase subunits respond differently to the same metabolic factors
Plant Physiology and Biochemistry 41 (2003) 689–693 www.elsevier.com/locate/plaphy
Original article
Nuclear and mitochondrial genes encoding cytochrome c oxidase subunits respond differently to the same metabolic factors Graciela C. Curi, Elina Welchen, Raquel L. Chan, Daniel H. Gonzalez * Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, CC 242 Paraje El Pozo, 3000 Santa Fe, Argentina Received 10 September 2002; accepted 5 March 2003
Abstract We have identified in the Arabidopsis thaliana Heyhn. nuclear genome a gene encoding COX6a, a homologue of subunit 6a of the mitochondrial cytochrome c oxidase (COX, E.C. 1.9.3.1) from animals and fungi. The expression of this gene and of the previously identified nuclear and mitochondrial genes, respectively, that encode subunit 6b (COX6b) and subunit 2 of the same enzyme was analyzed by northern blot. The analysis indicated that incubation of plants in solutions containing metabolizable sugars produced an increase in transcript levels for the nuclear genes but not for the mitochondrial one. The effect of carbohydrates showed the same time-dependence for COX6a and COX6b. Incubation of plants in solutions with different nitrogen sources also produced changes in expression. The behavior of the nuclear genes was very similar to that previously observed for genes encoding cytochrome c and cytochrome oxidase subunit 5b, suggesting the operation of a common regulatory mechanism for components of the mitochondrial respiratory chain encoded in the nucleus, but not for their counterparts encoded in the mitochondria. Regulation of the expression of mitochondrial genes, if any, may operate at a different level. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Cytochrome c oxidase; Gene expression; Metabolic regulation; Mitochondrion
1. Introduction Mitochondrial biogenesis involves the coordinated expression of genes present in separate genomes. This is especially valid for respiratory chain components, which participate in electron transfer processes sometimes as part of the same multisubunit complex. In plants, the respiratory chain ends up in two different pathways that transfer electrons from ubiquinone to O2 [1]. The cyanide-resistant or alternative pathway is composed of a single oxidase encoded by a small number of homologous nuclear genes [22,24]. The cyanidesensitive or cytochrome c-dependent pathway is similar to that found in other organisms and involves cytochrome c reductase, cytochrome c, and cytochrome c oxidase (COX). It is now well established that most mitochondrial components show enhanced expression in flowers [5,7,8,11,26].
Abbreviations: COX; cytochrome c oxidase. * Corresponding author. E-mail address: [email protected] (D.H. Gonzalez). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S0981-9428(03)00093-7
This fits well with the fact that the number of mitochondria per cell increases considerably during flower development [12]. In situ hybridization studies have shown that the expression in flowers is cell-specific, and that there is a good correlation in the expression of several mitochondrial genes [19] and the nuclear gene encoding cytochrome c [18], suggesting the existence of coordinated expression mechanisms. Besides this organ- or cell-type-specific expression, we have observed that cytochrome c and COX subunit 5b transcript levels are modified by illumination, incubation of plants in solutions containing metabolizable sugars or different nitrogen sources [5,6,25]. A pertinent question is then if this behavior is also observed for other genes encoding respiratory chain components, particularly those that participate in the cytochrome c-dependent pathway. To address this, we have analyzed the expression of Arabidopsis thaliana genes encoding COX subunits 6a, 6b and 2. Our results indicate that COX6a and COX6b genes, as other nuclear genes encoding components of the cytochrome c-dependent respiratory pathway, are subject to regulation through carbohydrate and nitrogen compounds, while the mitochondrial gene encoding subunit 2 shows a different behavior.
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Fig. 1. Effect of carbohydrates on the expression of COX6a, COX6b and COX2 genes. (A) Dark-adapted Arabidopsis plants were incubated in darkness for 12 h in Murashige and Skoog medium containing 3% (w/v) of different carbohydrates, as indicated above each lane; (B) dark-adapted Arabidopsis plants were incubated in darkness in Murashige and Skoog medium containing 3% (w/v) sucrose during 0, 6, 12, 18, 24 or 48 h, as indicated above each lane. After incubation, total RNA was extracted and analyzed as described in Section 4. Beside each blot, a graph showing the relative intensities of the signals corrected for differences in rRNA is shown.
2. Results COX is a multimeric complex composed of several different subunits [2,9], two or three of them (depending on the species) encoded by the mitochondrial genome [20] and the rest encoded in the nucleus [10,14,16]. In silico analysis of genomic and Expressed Sequence Tag Arabidopsis sequences deposited in data banks indicated the existence of nuclear genes and transcripts representing homologues of structural subunits COX5b (two genes), COX6a and COX6b (three genes) from other organisms. COX6a, not described previously from plants, is encoded by a single nuclear gene located on chromosome 4 (AGI gene code At4g37830; accession number NM_119944 for the predicted mRNA). We have previously shown that the expression of genes encoding cytochrome c and COX5b is influenced by carbon and nitrogen compounds. To study if other genes encoding COX components are influenced in a similar way, we have incubated dark-adapted whole plants with their roots immersed in different carbohydrate solutions and analyzed transcript levels of the nuclear COX6a and COX6b and the mitochondrial COX2 genes in aerial parts. Under the conditions employed, it is likely that the COX6b-1 probe detects transcripts from the three COX6b genes. The results indicate that plants incubated in either 3% (w/v) fructose or sucrose possess higher transcript levels for the nuclear genes than plants incubated in the presence of mannitol, a nonmetabolizable compound included in the medium to exclude
the effect of changes in osmotic conditions (Fig. 1A). Mannose, which enters the cells but is not significantly metabolized beyond mannose-6-P, produced only a slight activating effect. This suggests that the effect of sugars is associated with their metabolic function as we have previously described for other genes [25]. When the same samples were hybridized with the COX2 probe, transcript levels remained almost constant with all the treatments assayed. Further analysis of transcript levels at different times after the inclusion of sucrose indicated that, for the nuclear genes, a noticeable effect could be observed after 6 h of incubation, with a further increase after 18 h (Fig. 1B). In the presence of mannitol, transcript levels were similar as those observed at the beginning of the experiment, indicating that the results reflect a time-dependent induction by sugars, rather than a negative effect of mannitol on expression (not shown). Once again, COX2 transcript levels remained almost invariant along the sucrose treatment even after 48 h of incubation (Fig. 1B). The experiments described above were performed with plants kept in darkness to avoid any possible effect of photosynthesis-dependent sugar accumulation on expression. To address this point, we have also analyzed the effect of illumination of plants on transcript levels. The results showed that there is a slight increase in transcript levels noticeable after 12 h of illumination of dark-adapted plants, which is more pronounced for COX6b than for COX6a (Fig. 2). COX2 transcript levels remained unchanged. The
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effect on expression. Neither compound caused perceptible changes in COX2 transcript levels. 3. Discussion
Fig. 2. Combined effect of light and sucrose on COX6a, COX6b and COX2 gene expression. Dark-adapted Arabidopsis plants were incubated for 12 h either in darkness or under illumination in Murashige and Skoog medium with or without 3% (w/v) sucrose. The incubation medium without sucrose contained an equivalent amount of mannitol. After incubation, total RNA was extracted and analyzed as described in Section 4. Beside each blot, a graph showing the relative intensities of the signals corrected for differences in rRNA is shown.
combined effect of light and sucrose was analyzed by incubating dark-adapted plants with either mannitol or sucrose, in darkness or under illumination. The results clearly show that sugars produce a more pronounced increase in transcript levels than illumination (Fig. 2). In addition, in the presence of sucrose the effect of light was almost abolished. We have also incubated plants in solutions containing different amounts of nitrogen sources in the form of either nitrate or ammonium. The inclusion of 0.5 or 5 mM NH4Cl produced an increase in transcript levels of the two nuclear genes examined (Fig. 3). Potassium nitrate had a smaller
Fig. 3. Effect of the nitrogen source on the expression of COX6a, COX6b and COX2 genes. Dark-adapted Arabidopsis plants were incubated for 12 h in darkness in nitrogen-free Murashige and Skoog medium with the addition of either NH4Cl or KNO3 at the concentrations, in mM, indicated above each lane. No nitrogen source was added to samples labeled “C”. After incubation, total RNA was extracted and analyzed as described in Section 4. Beside each blot, a graph showing the relative intensities of the signals, corrected for differences in rRNA, is shown.
The respiratory chain of plant mitochondria ends in two different terminal oxidases [1,22]. While it is well established that expression of the alternative oxidase is regulated by different factors, such as reactive oxygen species and metabolites [17,21–24], little is known about the regulation of the expression of components of the cytochrome c-dependent pathway. Studies from our laboratory have shown that transcript levels for sunflower and Arabidopsis cytochrome c and Arabidopsis COX subunit 5b are influenced by illumination, carbohydrate feeding and the availability of nitrogen [5,6,25], while another nuclear COX gene, COX5c, shows a different behavior [4]. In the present study, we have analyzed the expression of Arabidopsis genes encoding additional COX subunits, namely COX6a, COX6b and COX2, after subjecting plants to similar treatments. We have obtained evidence of a coordinated control of gene expression, with metabolizable carbohydrates producing an increase in transcript levels of the nuclear genes under study. This can be taken as an indication that these genes respond to the same signal produced by carbohydrate accumulation or metabolization. The above considerations are also applicable to the effect of nitrogen availability on expression. On the other hand, no changes were observed in mitochondrial COX2 transcript levels. It should be emphasized that COX2 transcript levels were analyzed in total cellular RNA and not in RNA from isolated mitochondria. This means that any changes, either specific for COX2 or for the whole set of mitochondrial transcripts, would have been detected in this way. We conclude then that the COX2 mitochondrial gene, and possibly other mitochondrial COX genes, are not regulated by metabolites in a way similar to nuclear COX genes. Regulation at the translational or posttranslational level, however, cannot be ruled out. In conclusion, we describe here that carbon and nitrogen availability act in concert to regulate the expression of nuclear genes encoding COX components. We have obtained evidence for the coordinated expression of these genes with others studied previously. We have also observed that a mitochondrial gene encoding another subunit of the enzyme does not respond to the same signals. Our future research will focus on the molecular mechanisms involved in the coordination of expression. 4. Methods 4.1. Plant material and growth conditions A. thaliana Heyhn. ecotype Columbia (Col-0) was purchased from Lehle Seeds (Tucson, AZ). Plants were grown on soil in a growth chamber at 22–24 °C under long day
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G.C. Curi et al. / Plant Physiology and Biochemistry 41 (2003) 689–693
photoperiods (16 h of illumination by a mixture of coolwhite and GroLux fluorescent lamps) at an intensity of approximately 200 µmol m–2 s–1. Plants used for the different treatments were grown in pots covered with nylon nets during 3 weeks and then transferred to darkness for 48 h. After carefully washing off the soil from the roots, plants attached to the nylon net were transferred to Petri dishes containing Murashige and Skoog medium [13] with different additions as indicated. For the analysis of the effect of nitrogen compounds, nitrogen-free Murashige and Skoog medium was used.
D.H. Gonzalez are members of Consejo Nacional de Investigaciones Científicas y Técnicas, E. Welchen is a fellow of the same institution. References [1]
[2] [3]
4.2. cDNA clones EST clones for COX6a (clone 278C5T7, accession number AA650680) and COX6b-1 (AGI gene code At1g22450; clone 145J18T7; accession number T46836) were obtained from the Arabidopsis Biological Resource Center (Ohio State University) [15] and used as probes. A probe for the mitochondrial COX2 gene was obtained by PCR using oligonucleotides 5'-GGCGGATCCAATGATTGTTCTAAAATG-3' and 5'-GGCGAATTCGGCATGATTAGTTCCACA-3' on total DNA prepared from A. thaliana leaves.
[4]
[5]
[6]
[7]
4.3. RNA isolation and analysis Total RNA was isolated as described by Carpenter and Simon [3]. For northern blot analysis, specific amounts of RNA were electrophoresed through 1.5% (w/v) agarose/6% formaldehyde gels. The integrity of the RNA and equality of RNA loading were verified by ethidium bromide staining. RNA was transferred to Hybond-N nylon membranes (Amersham Corp., Buckinghamshire, UK) and hybridized overnight at 65 °C to 32P-labeled probes in buffer containing 6× SSC, 0.1% (w/v) polyvinylpirrolidone, 0.1% (w/v) BSA, 0.1% (w/v) Ficoll and 0.2% (w/v) SDS. Filters were washed with SSC (twofold concentration) plus 0.1% (w/v) SDS at 65 °C (four times, 15 min each), dried and exposed to Kodak BioMax MS films. To check the amount of total RNA loaded in each lane, filters were then re-probed with a 25S rDNA from Vicia faba under similar conditions as those described above, except that hybridization was performed at 62 °C. For quantification, autoradiographs were scanned and band intensities were determined with image analysis software. Values were corrected with those obtained for rRNA controls. Acknowledgements We gratefully acknowledge the Arabidopsis Biological Resource Center at the Ohio State University, Columbus, OH, for providing us with the Expressed Sequence Tag clones. We also thank Dr. Kimitaka Yakura, Kanazawa University, Japan, for sending us a Vicia faba rDNA clone. This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas and Agencia Nacional de Promoción Científica y Tecnológica (Argentina). R.L. Chan and
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M.D. Brownleader, J.B. Harborne, P.M. Dey, Carbohydrate metabolism: primary metabolism of monosaccharides, in: P.M. Dey, J.B. Harborne (Eds.), Plant Biochemistry, Academic Press, San Diego, 1997, pp. 111–141. R.A. Capaldi, Structure and function of cytochrome c oxidase, Annu. Rev. Biochem. 59 (1990) 569–596. C.D. Carpenter, A.E. Simon, Preparation of RNA, in: J. MartinezZapater, J. Salinas (Eds.), Methods in Molecular Biology, vol. 82, Arabidopsis Protocols, Humana Press Inc., Totowa, NJ, 1998, pp. 85–89. G.C. Curi, R.L. Chan, D.H. Gonzalez, Genes encoding cytochrome c oxidase subunit 5c from sunflower (Helianthus annuus L.) are regulated by nitrate and oxygen availability, Plant Sci. 163 (2002) 897–905. S.A. Felitti, R.L. Chan, G.M. Gago, E.M. Valle, D.H. Gonzalez, Expression of sunflower cytochrome c mRNA is tissue-specific and controlled by nitrate and light, Physiol. Plant. 99 (1997) 342–347. S.A. Felitti, D.H. Gonzalez, Carbohydrates modulate the expression of the sunflower cytochrome c gene at the mRNA level, Planta 206 (1998) 410–415. V. Heiser, A. Brennicke, L. Grohmann, The plant mitochondrial 22 kDa (PSST) subunit of respiratory chain complex I is encoded by a nuclear gene with enhanced transcript levels in flowers, Plant Mol. Biol. 31 (1997) 1195–1204. J. Huang, F. Struck, D.F. Matzinger, C.S. Levings III, Flowerenhanced expression of a nuclear-encoded mitochondrial respiratory protein is associated with changes in mitochondrion number, Plant Cell 6 (1994) 439–448. L. Jänsch, V. Kruft, U.K. Schmitz, H.P. Braun, New insights into the composition, molecular mass and stoichiometry of the protein complexes of plant mitochondria, Plant J. 9 (1996) 357–368. K. Kadowaki, N. Kubo, K. Ozawa, A. Hirai, Targeting presequence acquisition after mitochondrial gene transfer to the nucleus occurs by duplication of existing targeting signal, EMBO J. 15 (1996) 6652–6661. V. Landschütze, B. Müller-Röber, L. Willmitzer, Mitochondrial citrate synthase from potato: predominant expression in mature leaves and young flower buds, Planta 196 (1995) 756–764. S.L.J. Lee, H.E. Warmke, Organelle size and number in fertile and T-cytoplasmic male-sterile corn, Am. J. Bot. 60 (1979) 141–148. T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473–497. T. Nakagawa, M. Maeshima, K. Nakamura, T. Asahi, Molecular cloning of a cDNA for the smallest nuclear-encoded subunit of sweet potato cytochrome c oxidase: analysis with the cDNA of the structure and import into mitochondria of the subunit, Eur. J. Biochem. 191 (1990) 557–561. T. Newman, F.J. deBruijn, P. Green, K. Keegstra, H. Kende, L. McIntosh, J. Ohlrogge, N. Raikhel, S. Somerville, M. Thomashow, E. Retzel, C. Somerville, Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones, Plant Physiol. 106 (1994) 1241–1255. K. Ohtsu, M. Nakazono, N. Tsutsumi, A. Hirai, Characterization and expression of the genes for cytochrome c oxidase subunit VIb (COX6b) from rice and Arabidopsis thaliana, Gene 264 (2001) 233–239. D.M. Rhoads, L. McIntosh, Salicylic acid regulation of respiration in higher plants: alternative oxidase expression, Plant Cell 4 (1992) 1131–1139.
G.C. Curi et al. / Plant Physiology and Biochemistry 41 (2003) 689–693 [18] K. Ribichich, M.F. Tioni, R.L. Chan, D.H. Gonzalez, Cell-type specific expression of plant cytochrome c mRNA in developing flowers and roots, Plant Physiol. 125 (2001) 1603–1610. [19] C. Smart, F. Monéger, C.J. Leaver, Cell-specific regulation of gene expression in mitochondria during anther development in sunflower, Plant Cell 6 (1994) 811–825. [20] M. Unseld, J.R. Marienfeld, P. Brandt, A. Brennicke, The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366 924 nucleotides, Nat. Genet. 15 (1997) 57–61. [21] G.C. Vanlerberghe, L. McIntosh, Signals regulating the expression of the nuclear gene encoding alternative oxidase of plant mitochondria, Plant Physiol. 111 (1996) 589–595. [22] G.C. Vanlerberghe, L. McIntosh, Alternative oxidase: from gene to function, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48 (1997) 703–734.
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[23] A.M. Wagner, A role for active oxygen species as second messengers in the induction of alternative oxidase gene expression in Petunia hybrida cells, FEBS Lett. 368 (1995) 339–342. [24] A.M. Wagner, K. Krab, The alternative respiration pathway in plants: role and regulation, Physiol. Plant. 95 (1995) 318–325. [25] E. Welchen, R.L. Chan, D.H. Gonzalez, Metabolic regulation of genes encoding cytochrome c and cytochrome c oxidase subunit Vb in Arabidopsis, Plant Cell Environ. 25 (2002) 1605–1615. [26] E. Zabaleta, V. Heiser, L. Grohmann, A. Brennicke, Promoters of nuclear-encoded respiratory chain complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen-specific expression, Plant J. 15 (1998) 49–59.
Original article
Nuclear and mitochondrial genes encoding cytochrome c oxidase subunits respond differently to the same metabolic factors Graciela C. Curi, Elina Welchen, Raquel L. Chan, Daniel H. Gonzalez * Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, CC 242 Paraje El Pozo, 3000 Santa Fe, Argentina Received 10 September 2002; accepted 5 March 2003
Abstract We have identified in the Arabidopsis thaliana Heyhn. nuclear genome a gene encoding COX6a, a homologue of subunit 6a of the mitochondrial cytochrome c oxidase (COX, E.C. 1.9.3.1) from animals and fungi. The expression of this gene and of the previously identified nuclear and mitochondrial genes, respectively, that encode subunit 6b (COX6b) and subunit 2 of the same enzyme was analyzed by northern blot. The analysis indicated that incubation of plants in solutions containing metabolizable sugars produced an increase in transcript levels for the nuclear genes but not for the mitochondrial one. The effect of carbohydrates showed the same time-dependence for COX6a and COX6b. Incubation of plants in solutions with different nitrogen sources also produced changes in expression. The behavior of the nuclear genes was very similar to that previously observed for genes encoding cytochrome c and cytochrome oxidase subunit 5b, suggesting the operation of a common regulatory mechanism for components of the mitochondrial respiratory chain encoded in the nucleus, but not for their counterparts encoded in the mitochondria. Regulation of the expression of mitochondrial genes, if any, may operate at a different level. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Cytochrome c oxidase; Gene expression; Metabolic regulation; Mitochondrion
1. Introduction Mitochondrial biogenesis involves the coordinated expression of genes present in separate genomes. This is especially valid for respiratory chain components, which participate in electron transfer processes sometimes as part of the same multisubunit complex. In plants, the respiratory chain ends up in two different pathways that transfer electrons from ubiquinone to O2 [1]. The cyanide-resistant or alternative pathway is composed of a single oxidase encoded by a small number of homologous nuclear genes [22,24]. The cyanidesensitive or cytochrome c-dependent pathway is similar to that found in other organisms and involves cytochrome c reductase, cytochrome c, and cytochrome c oxidase (COX). It is now well established that most mitochondrial components show enhanced expression in flowers [5,7,8,11,26].
Abbreviations: COX; cytochrome c oxidase. * Corresponding author. E-mail address: [email protected] (D.H. Gonzalez). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S0981-9428(03)00093-7
This fits well with the fact that the number of mitochondria per cell increases considerably during flower development [12]. In situ hybridization studies have shown that the expression in flowers is cell-specific, and that there is a good correlation in the expression of several mitochondrial genes [19] and the nuclear gene encoding cytochrome c [18], suggesting the existence of coordinated expression mechanisms. Besides this organ- or cell-type-specific expression, we have observed that cytochrome c and COX subunit 5b transcript levels are modified by illumination, incubation of plants in solutions containing metabolizable sugars or different nitrogen sources [5,6,25]. A pertinent question is then if this behavior is also observed for other genes encoding respiratory chain components, particularly those that participate in the cytochrome c-dependent pathway. To address this, we have analyzed the expression of Arabidopsis thaliana genes encoding COX subunits 6a, 6b and 2. Our results indicate that COX6a and COX6b genes, as other nuclear genes encoding components of the cytochrome c-dependent respiratory pathway, are subject to regulation through carbohydrate and nitrogen compounds, while the mitochondrial gene encoding subunit 2 shows a different behavior.
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Fig. 1. Effect of carbohydrates on the expression of COX6a, COX6b and COX2 genes. (A) Dark-adapted Arabidopsis plants were incubated in darkness for 12 h in Murashige and Skoog medium containing 3% (w/v) of different carbohydrates, as indicated above each lane; (B) dark-adapted Arabidopsis plants were incubated in darkness in Murashige and Skoog medium containing 3% (w/v) sucrose during 0, 6, 12, 18, 24 or 48 h, as indicated above each lane. After incubation, total RNA was extracted and analyzed as described in Section 4. Beside each blot, a graph showing the relative intensities of the signals corrected for differences in rRNA is shown.
2. Results COX is a multimeric complex composed of several different subunits [2,9], two or three of them (depending on the species) encoded by the mitochondrial genome [20] and the rest encoded in the nucleus [10,14,16]. In silico analysis of genomic and Expressed Sequence Tag Arabidopsis sequences deposited in data banks indicated the existence of nuclear genes and transcripts representing homologues of structural subunits COX5b (two genes), COX6a and COX6b (three genes) from other organisms. COX6a, not described previously from plants, is encoded by a single nuclear gene located on chromosome 4 (AGI gene code At4g37830; accession number NM_119944 for the predicted mRNA). We have previously shown that the expression of genes encoding cytochrome c and COX5b is influenced by carbon and nitrogen compounds. To study if other genes encoding COX components are influenced in a similar way, we have incubated dark-adapted whole plants with their roots immersed in different carbohydrate solutions and analyzed transcript levels of the nuclear COX6a and COX6b and the mitochondrial COX2 genes in aerial parts. Under the conditions employed, it is likely that the COX6b-1 probe detects transcripts from the three COX6b genes. The results indicate that plants incubated in either 3% (w/v) fructose or sucrose possess higher transcript levels for the nuclear genes than plants incubated in the presence of mannitol, a nonmetabolizable compound included in the medium to exclude
the effect of changes in osmotic conditions (Fig. 1A). Mannose, which enters the cells but is not significantly metabolized beyond mannose-6-P, produced only a slight activating effect. This suggests that the effect of sugars is associated with their metabolic function as we have previously described for other genes [25]. When the same samples were hybridized with the COX2 probe, transcript levels remained almost constant with all the treatments assayed. Further analysis of transcript levels at different times after the inclusion of sucrose indicated that, for the nuclear genes, a noticeable effect could be observed after 6 h of incubation, with a further increase after 18 h (Fig. 1B). In the presence of mannitol, transcript levels were similar as those observed at the beginning of the experiment, indicating that the results reflect a time-dependent induction by sugars, rather than a negative effect of mannitol on expression (not shown). Once again, COX2 transcript levels remained almost invariant along the sucrose treatment even after 48 h of incubation (Fig. 1B). The experiments described above were performed with plants kept in darkness to avoid any possible effect of photosynthesis-dependent sugar accumulation on expression. To address this point, we have also analyzed the effect of illumination of plants on transcript levels. The results showed that there is a slight increase in transcript levels noticeable after 12 h of illumination of dark-adapted plants, which is more pronounced for COX6b than for COX6a (Fig. 2). COX2 transcript levels remained unchanged. The
G.C. Curi et al. / Plant Physiology and Biochemistry 41 (2003) 689–693
691
effect on expression. Neither compound caused perceptible changes in COX2 transcript levels. 3. Discussion
Fig. 2. Combined effect of light and sucrose on COX6a, COX6b and COX2 gene expression. Dark-adapted Arabidopsis plants were incubated for 12 h either in darkness or under illumination in Murashige and Skoog medium with or without 3% (w/v) sucrose. The incubation medium without sucrose contained an equivalent amount of mannitol. After incubation, total RNA was extracted and analyzed as described in Section 4. Beside each blot, a graph showing the relative intensities of the signals corrected for differences in rRNA is shown.
combined effect of light and sucrose was analyzed by incubating dark-adapted plants with either mannitol or sucrose, in darkness or under illumination. The results clearly show that sugars produce a more pronounced increase in transcript levels than illumination (Fig. 2). In addition, in the presence of sucrose the effect of light was almost abolished. We have also incubated plants in solutions containing different amounts of nitrogen sources in the form of either nitrate or ammonium. The inclusion of 0.5 or 5 mM NH4Cl produced an increase in transcript levels of the two nuclear genes examined (Fig. 3). Potassium nitrate had a smaller
Fig. 3. Effect of the nitrogen source on the expression of COX6a, COX6b and COX2 genes. Dark-adapted Arabidopsis plants were incubated for 12 h in darkness in nitrogen-free Murashige and Skoog medium with the addition of either NH4Cl or KNO3 at the concentrations, in mM, indicated above each lane. No nitrogen source was added to samples labeled “C”. After incubation, total RNA was extracted and analyzed as described in Section 4. Beside each blot, a graph showing the relative intensities of the signals, corrected for differences in rRNA, is shown.
The respiratory chain of plant mitochondria ends in two different terminal oxidases [1,22]. While it is well established that expression of the alternative oxidase is regulated by different factors, such as reactive oxygen species and metabolites [17,21–24], little is known about the regulation of the expression of components of the cytochrome c-dependent pathway. Studies from our laboratory have shown that transcript levels for sunflower and Arabidopsis cytochrome c and Arabidopsis COX subunit 5b are influenced by illumination, carbohydrate feeding and the availability of nitrogen [5,6,25], while another nuclear COX gene, COX5c, shows a different behavior [4]. In the present study, we have analyzed the expression of Arabidopsis genes encoding additional COX subunits, namely COX6a, COX6b and COX2, after subjecting plants to similar treatments. We have obtained evidence of a coordinated control of gene expression, with metabolizable carbohydrates producing an increase in transcript levels of the nuclear genes under study. This can be taken as an indication that these genes respond to the same signal produced by carbohydrate accumulation or metabolization. The above considerations are also applicable to the effect of nitrogen availability on expression. On the other hand, no changes were observed in mitochondrial COX2 transcript levels. It should be emphasized that COX2 transcript levels were analyzed in total cellular RNA and not in RNA from isolated mitochondria. This means that any changes, either specific for COX2 or for the whole set of mitochondrial transcripts, would have been detected in this way. We conclude then that the COX2 mitochondrial gene, and possibly other mitochondrial COX genes, are not regulated by metabolites in a way similar to nuclear COX genes. Regulation at the translational or posttranslational level, however, cannot be ruled out. In conclusion, we describe here that carbon and nitrogen availability act in concert to regulate the expression of nuclear genes encoding COX components. We have obtained evidence for the coordinated expression of these genes with others studied previously. We have also observed that a mitochondrial gene encoding another subunit of the enzyme does not respond to the same signals. Our future research will focus on the molecular mechanisms involved in the coordination of expression. 4. Methods 4.1. Plant material and growth conditions A. thaliana Heyhn. ecotype Columbia (Col-0) was purchased from Lehle Seeds (Tucson, AZ). Plants were grown on soil in a growth chamber at 22–24 °C under long day
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G.C. Curi et al. / Plant Physiology and Biochemistry 41 (2003) 689–693
photoperiods (16 h of illumination by a mixture of coolwhite and GroLux fluorescent lamps) at an intensity of approximately 200 µmol m–2 s–1. Plants used for the different treatments were grown in pots covered with nylon nets during 3 weeks and then transferred to darkness for 48 h. After carefully washing off the soil from the roots, plants attached to the nylon net were transferred to Petri dishes containing Murashige and Skoog medium [13] with different additions as indicated. For the analysis of the effect of nitrogen compounds, nitrogen-free Murashige and Skoog medium was used.
D.H. Gonzalez are members of Consejo Nacional de Investigaciones Científicas y Técnicas, E. Welchen is a fellow of the same institution. References [1]
[2] [3]
4.2. cDNA clones EST clones for COX6a (clone 278C5T7, accession number AA650680) and COX6b-1 (AGI gene code At1g22450; clone 145J18T7; accession number T46836) were obtained from the Arabidopsis Biological Resource Center (Ohio State University) [15] and used as probes. A probe for the mitochondrial COX2 gene was obtained by PCR using oligonucleotides 5'-GGCGGATCCAATGATTGTTCTAAAATG-3' and 5'-GGCGAATTCGGCATGATTAGTTCCACA-3' on total DNA prepared from A. thaliana leaves.
[4]
[5]
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[7]
4.3. RNA isolation and analysis Total RNA was isolated as described by Carpenter and Simon [3]. For northern blot analysis, specific amounts of RNA were electrophoresed through 1.5% (w/v) agarose/6% formaldehyde gels. The integrity of the RNA and equality of RNA loading were verified by ethidium bromide staining. RNA was transferred to Hybond-N nylon membranes (Amersham Corp., Buckinghamshire, UK) and hybridized overnight at 65 °C to 32P-labeled probes in buffer containing 6× SSC, 0.1% (w/v) polyvinylpirrolidone, 0.1% (w/v) BSA, 0.1% (w/v) Ficoll and 0.2% (w/v) SDS. Filters were washed with SSC (twofold concentration) plus 0.1% (w/v) SDS at 65 °C (four times, 15 min each), dried and exposed to Kodak BioMax MS films. To check the amount of total RNA loaded in each lane, filters were then re-probed with a 25S rDNA from Vicia faba under similar conditions as those described above, except that hybridization was performed at 62 °C. For quantification, autoradiographs were scanned and band intensities were determined with image analysis software. Values were corrected with those obtained for rRNA controls. Acknowledgements We gratefully acknowledge the Arabidopsis Biological Resource Center at the Ohio State University, Columbus, OH, for providing us with the Expressed Sequence Tag clones. We also thank Dr. Kimitaka Yakura, Kanazawa University, Japan, for sending us a Vicia faba rDNA clone. This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas and Agencia Nacional de Promoción Científica y Tecnológica (Argentina). R.L. Chan and
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