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Post-transcriptional regulation of ascorbate peroxidase during light adaptation of Euglena gracilis
Plant Science 165 (2003) 233 /238 www.elsevier.com/locate/plantsci
Post-transcriptional regulation of ascorbate peroxidase during light adaptation of Euglena gracilis Rapolu Madhusudhan a, Takahiro Ishikawa a,*, Yoshihiro Sawa a, Shigeru Shigeoka b, Hitoshi Shibata a a
Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan b Department of Food and Nutrition, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan Received 3 February 2003; received in revised form 10 March 2003; accepted 31 March 2003
Abstract When Euglena cells are grown heterotrophically in the dark, the chloroplast development stops at the proplastid stage. Upon exposure to light, the cells rapidly develop chloroplasts and the photosynthesis resumes. Here, we have investigated the regulation of cytosolic ascorbate peroxidase (APX) in Euglena gracilis during light adaptation of dark-grown cells. Both the activity and protein levels of APX increased by nearly fourfold in about 24 h of illumination. Northern hybridization with a partial cDNA of Euglena APX as the probe revealed a constant level of APX transcripts during the light adaptation. Similarly, cycloheximide almost completely inhibited APX induction whereas transcription inhibitors did not have a significant effect, suggesting that the light induction of APX is post-transcriptionally regulated. APX induction was abolished when the development of chloroplast was suppressed by norflurazon, which inhibits the carotenoid synthesis. However, treatment of the dark-grown cells with H2O2 or methyl viologen did not induce APX. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ascorbate peroxidase; Euglena gracilis ; Light adaptation; Post-transcriptional regulation
1. Introduction The photosynthetic electron transport inadvertently generates superoxide radical (O2+ ), which is spontaneously or enzymatically dismutated into H2O2. In higher plants, the H2O2 generated in chloroplasts is detoxified in situ by two isoenzymes of ascorbate peroxidase (APX; EC 1.11.1.11), one soluble in stroma and the other bound to the thylakoid membrane [1,2]. Higher plants also contain APX isoenzymes distributed in cytosol, microbody, and mitochondria [2]. Thus, the higher plants seem to have evolved a strategy of scavenging the H2O2 at the site of its generation.
Abbreviations: AOS, active oxygen species; APX, ascorbate peroxidase; AsA, ascorbate; GSH, glutathione; MV, methyl viologen; NF, norflurazon. * Corresponding author. Fax: /81-852-32-6092. E-mail address: [email protected] (T. Ishikawa).
In contrast to higher plants, eukaryotic algae have restricted cellular distribution of APX isoenzymes. In Euglena , which lacks catalase, APX is localized exclusively in cytosol but not in any other organelle including the chloroplast [3]. Similarly, Chlorella vulgaris contains APX localized only in cytosol [4], whereas in Chlamydomonas reinhardtii APX occurs exclusively in stroma of chloroplasts [5]. Enzymes that comprise the ascorbate/ glutathione (AsA /GSH) cycle, such as the monodehydroascorbate reductase, dehydroascorbate reductase, and GSH reductase, are also found only in the cytosol of Euglena [6]. Due to the restricted localization of APX, the natural diffusion of H2O2 might be important in its detoxification in algae [7]. Further, the photosynthetic apparatus of Euglena is significantly resistant to H2O2 compared to that of higher plants. The tolerance to H2O2 results from the insensitivity of fructose-1,6-/ sedoheptulose-1,7-biphosphatase, NADP -glyceraldehyde-3-phosphate dehydrogenase, and ribulose-5-phosphate kinase of Calvin cycle to H2O2 of up to 1 mM
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concentration [8]. Although these findings are suggestive of a central role of the cytosolic APX in scavenging H2O2 in Euglena , there could be other peroxidases as well, such as the thioredoxin peroxidase, in chloroplast and other organelles as it has been reported in cyanobacteria [9]. In order to understand the role of the cytosolic APX in relation to photosynthesis in Euglena , we have studied APX expression during the development of chloroplast. When Euglena is heterotrophically grown in the dark, the chloroplast development stops at the stage of a small proplastid. On exposure to light, the proplastids develop into mature chloroplasts, in both dividing and nondividing cells, within a period of 3 /4 days. This dramatic process, which has been extensively studied [10,11], offers an interesting stage to study the photo-oxidative stress. Earlier we showed that during the light adaptation of Euglena , AsA and GSH levels increase by 7- and 4.5-fold, respectively [6]. In this paper, we report the induction of cytosolic APX during the early period of light adaptation of dark-grown Euglena cells.
2. Materials and methods 2.1. Strains and culture Euglena gracilis strain Z, maintained by regular subculturing, was grown in Koren /Hutner medium [12] under continuous light from fluorescent lamps (FL40S-PG; Panasonic, Osaka) at a photosynthetic photon flux density of 24 mmol m 2 s 1 at 26 8C for 6 days, by which time the stationary phase was reached. For dark adaptation of the cells, referred to as the ‘darkgrown cells’ in the text, the cultures were grown in the dark in the same nutrient medium to late stationary phase (about 6 days). A bleached mutant of Euglena , SM-ZK strain, was also grown under the same culture conditions. Cell density was counted using a hemocytometer. 2.2. Assay of APX activity Euglena cells were collected by brief centrifugation and suspended in three volumes of an ice-cold buffer (50 mM potassium phosphate (pH 7.0), 1 mM EDTA, and 1 mM AsA) and disrupted by sonication. The cell lysate was centrifuged at 100,000 /g at 4 8C for 30 min and the supernatant was used for the assay of APX activity and immunoblot analyses. APX activity was assayed as described previously [13]. Briefly, 750 ml of the reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 0.4 mM AsA, 0.1 mM H2O2, and the enzyme. The oxidation of AsA was followed by the decrease in absorbance at 290 nm (o /2.80 mM 1
cm 1). The protein concentration was measured with Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). 2.3. Estimation of chlorophyll Euglena cells collected from 1 ml of culture by brief centrifugation were extracted at room temperature with 1 ml of 80% (v/v) acetone by vigorous vortex mixing and centrifuged at 10,000 /g for 5 min. Optical density of the clear supernatant was measured at 645 and 663 nm after appropriate dilution with 80% (v/v) acetone. The chlorophyll contents were estimated using the following formulas: chlorophyll a (mg ml 1) /12.7 /OD663/ 2.69 /OD645; Chlorophyll b (mg ml1)/22.9 / OD645/4.68 /OD663. 2.4. Immunoblot analysis Proteins were separated on a 12.5% polyacrylamide gel by the standard SDS-PAGE and transblotted onto a polyvinylidene difluoride (PVDF) membrane (Immobilon; Millipore, Bedford, MA) with the transfer buffer of Towbin et al. [14] using a semi-dry electroblot apparatus (Taitec, Saitama, Japan). The membrane was incubated with a monoclonal antibody (EAP1) raised against purified Euglena APX [13] and the immunocomplexes were detected with horseradish peroxidase-conjugated goat anti-mouse IgG (Cappel; Organon Technika, Durham, NC) as the secondary antibody and 4-methoxy-1-naphthol (Aldrich, Milwaukee, WI) as the chromogenic substrate. 2.5. Northern analysis Twenty micrograms of total RNA isolated from Euglena cells using ISOGEN reagent (Nippongene, Toyama, Japan) was separated by electrophoresis through a 1.2% agarose gel containing 2.2 M formaldehyde, and blotted onto a nylon membrane (Hybond N ; Amersham Pharmacia, Buckinghamshire, England). A partial cDNA sequence of Euglena APX containing 1326 nucleotides (accession No. AB077953) was labeled with 32P by random priming using Bca BEST polymerase (Takara, Kyoto, Japan) and used to hybridize RNA-blot membranes. The hybridization was carried out in Church-phosphate buffer [15] at 65 8C for 16 h. The membrane was then washed with 0.1 /SSC containing 0.1% SDS at 65 8C for 1 h and the autoradiography was carried out with a phosphorimager (BAS 1500; Fuji Film, Tokyo, Japan). 2.6. Stress conditions to study APX expression in Euglena Six-day-old photosynthetic vegetative cells were exposed for 6 h to 4 8C (cold), or 42 8C temperature (heat),
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Fig. 1. Synthesis of chlorophyll and induction of APX activity in dark-grown Euglena cells upon illumination. (A, B) Dark-grown Euglena cells were illuminated at three different light intensities ((m) 120 mmol m 2 s 1; (') 60 mmol m 2 s 1; (j) 24 mmol m 2 s 1) and the time-course of (A) chlorophyll content and (B) APX activity were measured. Continuous dependence of (C) chlorophyll synthesis and (D) APX induction on light during the adaptation of Euglena . The light (120 mmol m 2 s 1) was turned off between 12 and 24 h. The broken lines show APX activity or the chlorophyll content during the intervening period of darkness (mean9/S.D.; n/3).
light intensity of 1200 mmol m 2 s 1 (high light), or treated with 30% (w/v) polyethylene glycol, 100 mM methyl viologen (MV), or 1 mM H2O2. After the treatment, the cells were analyzed for the activity, protein, and transcript levels of APX as described above. The cell viability was determined by Trypan Blue dye exclusion test.
raised against purified Euglena APX [13]. As shown in Fig. 2A, immunoblot analysis of the light adapting Euglena showed an increase in the level of APX protein that was parallel with the increase in the activity, suggesting that the induction of activity was due to de novo synthesis of APX protein. We have isolated a partial cDNA fragment of Euglena APX by immuno-
3. Results 3.1. Induction of APX during light adaptation Euglena cells heterotrophically grown in the dark were shifted to growth under light. Upon illumination, the chlorophyll content of the cells increased steadily, after an initial lag period of about 6 h, for over 48 h reaching a maximum of about 10 /12 mg 106 cells, which was comparable to that of the green cells grown under continuous light (Fig. 1A). However, APX activity was induced much rapidly after an initial lag period (Fig. 1B). Under an illumination of 120 mmol m 2 s 1, the activity increased by over 3.5-fold reaching the maximum in about 24 h. An intensity of 60 mmol m 2 s 1 being optimum, a lower light intensity caused a longer lag period. Further, both the synthesis of chlorophyll and the induction of APX activity were continuously dependent on light during the early adaptation period since the chlorophyll content and APX activity ceased to increase during an intervening period of darkness, which, however, resumed following the reillumination (Fig. 1C and D). To estimate APX protein levels, we used a monoclonal antibody (EAP1)
Fig. 2. Changes in the protein and transcript levels of APX during the light adaptation of Euglena . Dark-grown Euglena cells were illuminated at an intensity of 120 mmol m 2 s 1. Samples were taken out at the indicated times and analyzed by immunoblot and Northern hybridization. (A) Immunoblot analyses. Soluble proteins (approximately 20 mg) extracted from equal number of cells of each sample were separated by SDS-PAGE, electrophoretically blotted onto a membrane, and detected by hybridization with a monoclonal antibody (EAP1) raised against Euglena APX. (B) Northern analyses. Twenty micrograms of total RNA extracted from each sample was electrophoressed through a formaldehyde-containing agarose gel, capillary blotted onto a nylon membrane, and hybridized with 32P-labeled partial cDNA of Euglena APX. Ethidium bromide staining of the ribosomal RNA is shown for the equality of loading.
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screening of Euglena cDNA library (accession No. AB077953). The cDNA sequence contained 1326 nucleotides with an open reading frame encoding for 296 amino acids that encompassed most of the active site region including the distal and proximal His-containing regions for binding the heme ligand. The authenticity of the cDNA sequence was further confirmed by the presence of several of the internal peptide-fragment sequences that were identified previously in the native enzyme [13]. Using radio-labeled partial cDNA as the probe, we estimated APX transcript levels during the light adaptation. In contrast to the immunoblot results, however, Northern hybridization revealed constant levels of APX transcripts (Fig. 2B). These data suggest that the induction of APX during the light adaptation is effected through a post-transcriptional mechanism.
3.2. Effect of transcriptional and translational inhibitors To further support the hypothesis that APX induction is post-transcriptionally regulated, we tested the effect of transcriptional and translational inhibitors on APX induction. As shown in Table 1, the presence of 50 mM cycloheximide, which blocks protein synthesis by inhibiting peptidyl transferase, suppressed APX induction by over 90% during the light adaptation. On the other hand, neither actinomycin D nor a-amanitin, which inhibits RNA polymerase-I and -II, respectively, had a significant effect on APX induction. RNA polymerases II and III of Euglena have been shown to be inhibited, in vitro, by a-amanitin with IC50 of 30 nM and 18 mM, respectively [16]. These results clearly suggest that APX gene in Euglena is constitutively expressed, and APX level is regulated by a lightdependent post-transcriptional mechanism.
3.3. Dependence of APX induction on chloroplast development We then investigated whether APX induction is dependent on the development of chloroplast. We used norflurazon (NF) to suppress the chloroplast development during the light adaptation. NF is an inhibitor of carotenoid biosynthesis, which leads to photobleaching of chlorophyll [17]. Interestingly, the presence of 100 mM NF almost completely abolished the cytosolic APX induction along with the suppression of chlorophyll synthesis (Fig. 3A). Similarly, a bleached mutant strain of Euglena (SM-ZK) that has a defect in the development of chloroplast did not exhibit the light induction of APX. APX activity and protein levels of this strain were constitutively low and quite comparable to those of dark-grown wild-type Euglena (Fig. 3A and B). On the other hand, APX transcripts in the mutant strain were as abundant as in the wild-type cells (Fig. 3C). These observations indicate that APX induction is dependent on the development of chloroplast. In contrast, in greening mustard plant, APX induction has been reported to be mediated by the photoreceptor phytochrome [18].
Table 1 Effect of transcriptional and translational inhibitors on induction of APX Relative induction (%) Control Actinomycin D (50 mM) Amanitin (10 mM) Cycloheximide (50 mM)
100 91.9 90.3 8.0
The inhibitors were added to dark-grown Euglena cells at the indicated final concentration, and the cells were exposed to light at an intensity of 120 mmol m 2 s 1 for 24 h, and APX activities per unit cell count were measured and expressed as percentage relative to that of control cultures to which no inhibitors were added. Data represent the mean of values from three independent experiments. The S.D.s were within 5% of the mean values.
Fig. 3. Dependence of APX induction in Euglena on the development of chloroplast. (A) APX activity and chlorophyll content were estimated after illuminating the dark-grown Euglena cultures at a photon flux density of 120 mmol m 2 s 1 for 24 h. For NF treatment, the inhibitor was added at a final concentration of 100 mM and preincubated for 1 h in the dark prior to illumination. SM-ZK mutant cultures were grown under continuous light of the same intensity. (B) Immunoblot analyses. (C) Northern hybridization. Please see the legend to Fig. 2 for experimental details of the immunoblot and Northern analyses.
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3.4. Effect of redox conditions on APX induction
4. Discussion
As the data from the above experiments suggested that the induction of APX is dependent on the development of chloroplast, we verified whether superoxide or H2O2 could trigger the induction of APX, since these active oxygen species (AOS) are generated during the course of photosynthesis [19]. Dark-grown Euglena cells were incubated with 1 mM H2O2, or 100 mM MV for 24 h in the dark. However, these treatments could not mimic the effect of illumination in that no induction of APX activity was observed (Fig. 4). Moreover, treatment of dark-grown Euglena cells with H2O2 above 1 mM or MV above 0.1 mM concentration severely affected the cell viability (data not shown). During the light adaptation of Euglena , AsA and GSH levels increase by 7- and 4.5-fold, respectively, even prior to the onset of the development of chloroplast reaching the maximum in the first 7 h of illumination [6]. To make sure whether the elevated levels of these reducing species in turn induce APX activity, we incubated the dark-grown cells with Lgalactono-g-lactone and N -acetylcystein, which are precursors for AsA and GSH, respectively [20]. Feeding of Euglena cells with these precursors results in several fold increase in their respective final products [20]. However, APX activity remained unchanged in these experiments (Fig. 4). These results suggest that redox species are not involved in APX induction in Euglena . In addition to light, several other environmental factors, such as drought, salt, chilling, and air pollutants, have also been known to upregulate APX expression in higher plants [21]. In this study, we also examined the effect of heat, high light, cold, osmotic, and oxidative stress conditions, described in Section 2, on the level of APX in Euglena . However, no significant response of APX was found under these conditions (data not shown).
In this paper, we describe the induction of cytosolic APX during the early period of light adaptation of darkgrown Euglena cells. Our data clearly show that APX induction is regulated by a post-transcriptional mechanism. The process of light adaptation involves a rapid development of proplastid into a mature and fully functional chloroplast [11]. It has been known that chloroplasts are the major source of AOS [19]. Because Euglena does not possess catalase, and if the cytosolic APX were to scavenge the H2O2 generated in the chloroplast, a prior induction of the cytosolic APX ahead of the chloroplast development (Fig. 1) would be a logical event in the adaptation to the light regime. Both AsA and GSH, which are important components of the AsA /GSH cycle, increase by 7- and 4.5-fold, respectively, within the first 8 h of illumination, even before the onset of the development of chloroplast [6]. The H2O2 tolerance of Calvin cycle enzymes of Euglena , coupled with the rapid diffusivity of H2O2 [7,8], also might facilitate the rapid development of chloroplast and the resumption of photosynthesis. Based on the recent information on the regulation of AOS-scavenging enzymes in higher plants, we investigated several potential candidates that might mediate the light response in Euglena . Our data from the use of oxidative and reducing agents (Fig. 4) indicate, at least, that the induction of APX during the light adaptation is not a result of the accompanying redox changes in the cell. In contrast, the cellular redox status in higher plants is an important factor in the regulation of APX expression under high light condition. For example, in spinach, the transcript levels of cytosolic APX markedly increase in response to high light stress and MV treatment [21]. Karpinski et al. [22] have reported that H2O2 accumulation and/or redox status of the plastoquinone pool might be essential for the transcriptional induction of cytosolic APX in Arabidopsis during high light intensity. Although our experiments did not reveal the exact factor that mediates the induction of APX, our data single out the dependence of APX induction on the development of chloroplast. The process of light adaptation of Euglena cells entails a host of structural and metabolic events [10]. For example, the nuclear-geneencoded small subunit of ribulose-1,5-bisphosphate carboxylase/oxigenase (rbcS) and the light-harvesting chlorophyll a /b binding protein (LHCPII) are expressed coordinately [23,24]. Interestingly, the protein levels of both rbcS and LHCPII increase upon illumination whereas their mRNA levels remain unchanged. It appears that the post-transcriptional control is a common mechanism of gene regulation in Euglena [25]. On the other hand, during the greening in higher plants, the expression of both the above-mentioned genes is transcriptionally regulated [26,27]. Kishore and Schwartz-
Fig. 4. Effect of cellular redox status on APX activity in dark-grown Euglena cells. L-Galactono-g-lactone (L-GAL), N -acetyl cysteine (NAC), H2O2, or MV was added to dark-grown Euglena cultures at the indicated final concentrations. After 24 h of incubation in the dark, APX activity was assayed. A sample of the culture was incubated under light for 24 h at a photon flux density of 120 mmol m 2 s 1 (light) (mean9/S.D.; n/3).
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bach [24] showed that the light-dependent synthesis of LHCPII is regulated at the level of polypeptide chain elongation. These authors concluded that the signaling for the polypeptide chain elongation requires a ‘lightinduced effector molecule’ that is either synthesized within the chloroplast or related to chloroplast development, because the amount of polysome-associated LHCPII mRNA in both the wild-type and bleached mutant were almost equal. The light-induced expression of APX described in this study might involve the similar signaling as in the case of LHCPII, because APX level in SM-ZK-bleached mutant showed no significant response to light, whereas the transcript levels of the enzyme in both the wild-type and bleached mutant were quite comparable (Fig. 3C). A detailed investigation on the role of photosynthetic electron transport in the development of chloroplast during the adaptation of dark-grown Euglena may throw some light on the underlying regulation mechanism.
Acknowledgements R.M. gratefully acknowledges a fellowship from the Ministry of Education, Science, Sports, and Culture of Japan.
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[8] T. Takeda, A. Yokota, S. Shigeoka, Resistance of photosynthesis to hydrogen peroxide in algae, Plant Cell Physiol. 36 (1995) 1089 / 1095. [9] H. Yamamoto, C. Miyake, K.-J. Dietz, K.-I. Tomizawa, N. Murata, A. Yokota, Thioredoxin peroxidase in the Cyanobacterium synechocystis sp. PCC 6803, FEBS Lett. 447 (1999) 269 /273. [10] J.A. Schiff, S.D. Schwartzbach, Photocontrol of chloroplast development in Euglena , in: D.E. Buetow (Ed.), The Biology of Euglena , vol. III, Academic Press, New York, 1982, pp. 313 /352. [11] S.D. Schwartzbach, Photocontrol of organelle biogenesis in Euglena , Photochem. Photobiol. 51 (1990) 231 /254. [12] L.E. Koren, S.H. Hutner, High-yield media for photosynthesizing Euglena gracilis Z, J. Protozool. 14 (1967) 17. [13] T. Ishikawa, T. Takeda, H. Kohno, S. Shigeoka, Molecular characterization of Euglena ascorbate peroxidase using monoclonal antibody, Biochim. Biophys. Acta 1290 (1996) 69 /75. [14] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA 76 (1979) 4350 /4354. [15] G.M. Church, W. Gilbert, Genomic sequencing, Proc. Natl. Acad. Sci. USA 81 (1984) 1991 /1995. [16] K.H. Falchuk, B. Mazus, E. Ber, L. Ulpino-Lobb, B.L. Vallee, Zinc deficiency and the Euglena gracilis chromatin: formation of an a-amanitin-resistant RNA polymerase II, Biochemistry 24 (1985) 2576 /2580. [17] D. Chamovitz, I. Pecker, J. Hirschberg, The molecular basis of resistance to the herbicide norflurazon, Plant Mol. Biol. 16 (1991) 967 /974. [18] B. Thomsen, H. Drumm-Herrel, H. Mohr, Control of the appearance of ascorbate peroxidase (EC 1.11.1.11) in mustard seedling cotyledons by phytochrome and photooxidative treatments, Planta 186 (1992) 600 /608. [19] C.H. Foyer, M. Lelandais, K.J. Kunert, Photo-oxidative stress in plants, Physiol. Plant 92 (1994) 696 /717. [20] S. Shigeoka, Y. Nakano, S. Kitaoka, The biosynthetic pathway of L-ascorbic acid in Euglena gracilis Z, J. Nutr. Sci. Vitaminol. 25 (1979) 299 /307. [21] K. Yoshimura, Y. Yabuta, T. Ishikawa, S. Shigeoka, Expression of spinach ascorbate peroxidase isoenzymes in response to oxidative stresses, Plant Physiol. 123 (2000) 223 /233. [22] S. Karpinski, H. Reynolds, B. Karpinska, G. Wingsle, G. Creissen, P. Mullineaux, Systemic signaling and acclimation in response to excess excitation energy in Arabidopsis , Science 284 (1999) 654 /657. [23] M. Keller, R.L. Chan, L.H. Tessier, J.-H. Weil, P. Imbault, Posttranscriptional regulation by light of the biosynthesis of Euglena ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, Plant Mol. Biol. 17 (1991) 73 /82. [24] R. Kishore, S.D. Schwartzbach, Translational control of the synthesis of the Euglena light harvesting chlorophyll a /b binding protein of photosystem II, Plant Sci. 85 (1992) 79 /89. [25] J. Canaday, L.H. Tessier, P. Imbault, F. Paulus, Analysis of Euglena gracilis alpha -, beta - and gamma -tubulin genes: introns and pre-mRNA maturation, Mol. Genet. Genomics 265 (2001) 153 /160. [26] C. Fiebig, G. Link, 5?-Upstream cis -elements and binding factor(s) potentially involved in light-regulated expression of a Brassica napus rbcS gene, Curr. Genet. 21 (1992) 161 /168. [27] A. Batschauer, E. Mosinger, K. Kreuz, I. Dorr, K. Apel, The implication of a plastid-derived factor in the transcriptional control of nuclear genes encoding the light-harvesting chlorophyll a /b protein, Eur. J. Biochem. 154 (1986) 625 /634.
Post-transcriptional regulation of ascorbate peroxidase during light adaptation of Euglena gracilis Rapolu Madhusudhan a, Takahiro Ishikawa a,*, Yoshihiro Sawa a, Shigeru Shigeoka b, Hitoshi Shibata a a
Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane, 690-8504, Japan b Department of Food and Nutrition, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan Received 3 February 2003; received in revised form 10 March 2003; accepted 31 March 2003
Abstract When Euglena cells are grown heterotrophically in the dark, the chloroplast development stops at the proplastid stage. Upon exposure to light, the cells rapidly develop chloroplasts and the photosynthesis resumes. Here, we have investigated the regulation of cytosolic ascorbate peroxidase (APX) in Euglena gracilis during light adaptation of dark-grown cells. Both the activity and protein levels of APX increased by nearly fourfold in about 24 h of illumination. Northern hybridization with a partial cDNA of Euglena APX as the probe revealed a constant level of APX transcripts during the light adaptation. Similarly, cycloheximide almost completely inhibited APX induction whereas transcription inhibitors did not have a significant effect, suggesting that the light induction of APX is post-transcriptionally regulated. APX induction was abolished when the development of chloroplast was suppressed by norflurazon, which inhibits the carotenoid synthesis. However, treatment of the dark-grown cells with H2O2 or methyl viologen did not induce APX. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ascorbate peroxidase; Euglena gracilis ; Light adaptation; Post-transcriptional regulation
1. Introduction The photosynthetic electron transport inadvertently generates superoxide radical (O2+ ), which is spontaneously or enzymatically dismutated into H2O2. In higher plants, the H2O2 generated in chloroplasts is detoxified in situ by two isoenzymes of ascorbate peroxidase (APX; EC 1.11.1.11), one soluble in stroma and the other bound to the thylakoid membrane [1,2]. Higher plants also contain APX isoenzymes distributed in cytosol, microbody, and mitochondria [2]. Thus, the higher plants seem to have evolved a strategy of scavenging the H2O2 at the site of its generation.
Abbreviations: AOS, active oxygen species; APX, ascorbate peroxidase; AsA, ascorbate; GSH, glutathione; MV, methyl viologen; NF, norflurazon. * Corresponding author. Fax: /81-852-32-6092. E-mail address: [email protected] (T. Ishikawa).
In contrast to higher plants, eukaryotic algae have restricted cellular distribution of APX isoenzymes. In Euglena , which lacks catalase, APX is localized exclusively in cytosol but not in any other organelle including the chloroplast [3]. Similarly, Chlorella vulgaris contains APX localized only in cytosol [4], whereas in Chlamydomonas reinhardtii APX occurs exclusively in stroma of chloroplasts [5]. Enzymes that comprise the ascorbate/ glutathione (AsA /GSH) cycle, such as the monodehydroascorbate reductase, dehydroascorbate reductase, and GSH reductase, are also found only in the cytosol of Euglena [6]. Due to the restricted localization of APX, the natural diffusion of H2O2 might be important in its detoxification in algae [7]. Further, the photosynthetic apparatus of Euglena is significantly resistant to H2O2 compared to that of higher plants. The tolerance to H2O2 results from the insensitivity of fructose-1,6-/ sedoheptulose-1,7-biphosphatase, NADP -glyceraldehyde-3-phosphate dehydrogenase, and ribulose-5-phosphate kinase of Calvin cycle to H2O2 of up to 1 mM
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00164-X
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concentration [8]. Although these findings are suggestive of a central role of the cytosolic APX in scavenging H2O2 in Euglena , there could be other peroxidases as well, such as the thioredoxin peroxidase, in chloroplast and other organelles as it has been reported in cyanobacteria [9]. In order to understand the role of the cytosolic APX in relation to photosynthesis in Euglena , we have studied APX expression during the development of chloroplast. When Euglena is heterotrophically grown in the dark, the chloroplast development stops at the stage of a small proplastid. On exposure to light, the proplastids develop into mature chloroplasts, in both dividing and nondividing cells, within a period of 3 /4 days. This dramatic process, which has been extensively studied [10,11], offers an interesting stage to study the photo-oxidative stress. Earlier we showed that during the light adaptation of Euglena , AsA and GSH levels increase by 7- and 4.5-fold, respectively [6]. In this paper, we report the induction of cytosolic APX during the early period of light adaptation of dark-grown Euglena cells.
2. Materials and methods 2.1. Strains and culture Euglena gracilis strain Z, maintained by regular subculturing, was grown in Koren /Hutner medium [12] under continuous light from fluorescent lamps (FL40S-PG; Panasonic, Osaka) at a photosynthetic photon flux density of 24 mmol m 2 s 1 at 26 8C for 6 days, by which time the stationary phase was reached. For dark adaptation of the cells, referred to as the ‘darkgrown cells’ in the text, the cultures were grown in the dark in the same nutrient medium to late stationary phase (about 6 days). A bleached mutant of Euglena , SM-ZK strain, was also grown under the same culture conditions. Cell density was counted using a hemocytometer. 2.2. Assay of APX activity Euglena cells were collected by brief centrifugation and suspended in three volumes of an ice-cold buffer (50 mM potassium phosphate (pH 7.0), 1 mM EDTA, and 1 mM AsA) and disrupted by sonication. The cell lysate was centrifuged at 100,000 /g at 4 8C for 30 min and the supernatant was used for the assay of APX activity and immunoblot analyses. APX activity was assayed as described previously [13]. Briefly, 750 ml of the reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 0.4 mM AsA, 0.1 mM H2O2, and the enzyme. The oxidation of AsA was followed by the decrease in absorbance at 290 nm (o /2.80 mM 1
cm 1). The protein concentration was measured with Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). 2.3. Estimation of chlorophyll Euglena cells collected from 1 ml of culture by brief centrifugation were extracted at room temperature with 1 ml of 80% (v/v) acetone by vigorous vortex mixing and centrifuged at 10,000 /g for 5 min. Optical density of the clear supernatant was measured at 645 and 663 nm after appropriate dilution with 80% (v/v) acetone. The chlorophyll contents were estimated using the following formulas: chlorophyll a (mg ml 1) /12.7 /OD663/ 2.69 /OD645; Chlorophyll b (mg ml1)/22.9 / OD645/4.68 /OD663. 2.4. Immunoblot analysis Proteins were separated on a 12.5% polyacrylamide gel by the standard SDS-PAGE and transblotted onto a polyvinylidene difluoride (PVDF) membrane (Immobilon; Millipore, Bedford, MA) with the transfer buffer of Towbin et al. [14] using a semi-dry electroblot apparatus (Taitec, Saitama, Japan). The membrane was incubated with a monoclonal antibody (EAP1) raised against purified Euglena APX [13] and the immunocomplexes were detected with horseradish peroxidase-conjugated goat anti-mouse IgG (Cappel; Organon Technika, Durham, NC) as the secondary antibody and 4-methoxy-1-naphthol (Aldrich, Milwaukee, WI) as the chromogenic substrate. 2.5. Northern analysis Twenty micrograms of total RNA isolated from Euglena cells using ISOGEN reagent (Nippongene, Toyama, Japan) was separated by electrophoresis through a 1.2% agarose gel containing 2.2 M formaldehyde, and blotted onto a nylon membrane (Hybond N ; Amersham Pharmacia, Buckinghamshire, England). A partial cDNA sequence of Euglena APX containing 1326 nucleotides (accession No. AB077953) was labeled with 32P by random priming using Bca BEST polymerase (Takara, Kyoto, Japan) and used to hybridize RNA-blot membranes. The hybridization was carried out in Church-phosphate buffer [15] at 65 8C for 16 h. The membrane was then washed with 0.1 /SSC containing 0.1% SDS at 65 8C for 1 h and the autoradiography was carried out with a phosphorimager (BAS 1500; Fuji Film, Tokyo, Japan). 2.6. Stress conditions to study APX expression in Euglena Six-day-old photosynthetic vegetative cells were exposed for 6 h to 4 8C (cold), or 42 8C temperature (heat),
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Fig. 1. Synthesis of chlorophyll and induction of APX activity in dark-grown Euglena cells upon illumination. (A, B) Dark-grown Euglena cells were illuminated at three different light intensities ((m) 120 mmol m 2 s 1; (') 60 mmol m 2 s 1; (j) 24 mmol m 2 s 1) and the time-course of (A) chlorophyll content and (B) APX activity were measured. Continuous dependence of (C) chlorophyll synthesis and (D) APX induction on light during the adaptation of Euglena . The light (120 mmol m 2 s 1) was turned off between 12 and 24 h. The broken lines show APX activity or the chlorophyll content during the intervening period of darkness (mean9/S.D.; n/3).
light intensity of 1200 mmol m 2 s 1 (high light), or treated with 30% (w/v) polyethylene glycol, 100 mM methyl viologen (MV), or 1 mM H2O2. After the treatment, the cells were analyzed for the activity, protein, and transcript levels of APX as described above. The cell viability was determined by Trypan Blue dye exclusion test.
raised against purified Euglena APX [13]. As shown in Fig. 2A, immunoblot analysis of the light adapting Euglena showed an increase in the level of APX protein that was parallel with the increase in the activity, suggesting that the induction of activity was due to de novo synthesis of APX protein. We have isolated a partial cDNA fragment of Euglena APX by immuno-
3. Results 3.1. Induction of APX during light adaptation Euglena cells heterotrophically grown in the dark were shifted to growth under light. Upon illumination, the chlorophyll content of the cells increased steadily, after an initial lag period of about 6 h, for over 48 h reaching a maximum of about 10 /12 mg 106 cells, which was comparable to that of the green cells grown under continuous light (Fig. 1A). However, APX activity was induced much rapidly after an initial lag period (Fig. 1B). Under an illumination of 120 mmol m 2 s 1, the activity increased by over 3.5-fold reaching the maximum in about 24 h. An intensity of 60 mmol m 2 s 1 being optimum, a lower light intensity caused a longer lag period. Further, both the synthesis of chlorophyll and the induction of APX activity were continuously dependent on light during the early adaptation period since the chlorophyll content and APX activity ceased to increase during an intervening period of darkness, which, however, resumed following the reillumination (Fig. 1C and D). To estimate APX protein levels, we used a monoclonal antibody (EAP1)
Fig. 2. Changes in the protein and transcript levels of APX during the light adaptation of Euglena . Dark-grown Euglena cells were illuminated at an intensity of 120 mmol m 2 s 1. Samples were taken out at the indicated times and analyzed by immunoblot and Northern hybridization. (A) Immunoblot analyses. Soluble proteins (approximately 20 mg) extracted from equal number of cells of each sample were separated by SDS-PAGE, electrophoretically blotted onto a membrane, and detected by hybridization with a monoclonal antibody (EAP1) raised against Euglena APX. (B) Northern analyses. Twenty micrograms of total RNA extracted from each sample was electrophoressed through a formaldehyde-containing agarose gel, capillary blotted onto a nylon membrane, and hybridized with 32P-labeled partial cDNA of Euglena APX. Ethidium bromide staining of the ribosomal RNA is shown for the equality of loading.
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screening of Euglena cDNA library (accession No. AB077953). The cDNA sequence contained 1326 nucleotides with an open reading frame encoding for 296 amino acids that encompassed most of the active site region including the distal and proximal His-containing regions for binding the heme ligand. The authenticity of the cDNA sequence was further confirmed by the presence of several of the internal peptide-fragment sequences that were identified previously in the native enzyme [13]. Using radio-labeled partial cDNA as the probe, we estimated APX transcript levels during the light adaptation. In contrast to the immunoblot results, however, Northern hybridization revealed constant levels of APX transcripts (Fig. 2B). These data suggest that the induction of APX during the light adaptation is effected through a post-transcriptional mechanism.
3.2. Effect of transcriptional and translational inhibitors To further support the hypothesis that APX induction is post-transcriptionally regulated, we tested the effect of transcriptional and translational inhibitors on APX induction. As shown in Table 1, the presence of 50 mM cycloheximide, which blocks protein synthesis by inhibiting peptidyl transferase, suppressed APX induction by over 90% during the light adaptation. On the other hand, neither actinomycin D nor a-amanitin, which inhibits RNA polymerase-I and -II, respectively, had a significant effect on APX induction. RNA polymerases II and III of Euglena have been shown to be inhibited, in vitro, by a-amanitin with IC50 of 30 nM and 18 mM, respectively [16]. These results clearly suggest that APX gene in Euglena is constitutively expressed, and APX level is regulated by a lightdependent post-transcriptional mechanism.
3.3. Dependence of APX induction on chloroplast development We then investigated whether APX induction is dependent on the development of chloroplast. We used norflurazon (NF) to suppress the chloroplast development during the light adaptation. NF is an inhibitor of carotenoid biosynthesis, which leads to photobleaching of chlorophyll [17]. Interestingly, the presence of 100 mM NF almost completely abolished the cytosolic APX induction along with the suppression of chlorophyll synthesis (Fig. 3A). Similarly, a bleached mutant strain of Euglena (SM-ZK) that has a defect in the development of chloroplast did not exhibit the light induction of APX. APX activity and protein levels of this strain were constitutively low and quite comparable to those of dark-grown wild-type Euglena (Fig. 3A and B). On the other hand, APX transcripts in the mutant strain were as abundant as in the wild-type cells (Fig. 3C). These observations indicate that APX induction is dependent on the development of chloroplast. In contrast, in greening mustard plant, APX induction has been reported to be mediated by the photoreceptor phytochrome [18].
Table 1 Effect of transcriptional and translational inhibitors on induction of APX Relative induction (%) Control Actinomycin D (50 mM) Amanitin (10 mM) Cycloheximide (50 mM)
100 91.9 90.3 8.0
The inhibitors were added to dark-grown Euglena cells at the indicated final concentration, and the cells were exposed to light at an intensity of 120 mmol m 2 s 1 for 24 h, and APX activities per unit cell count were measured and expressed as percentage relative to that of control cultures to which no inhibitors were added. Data represent the mean of values from three independent experiments. The S.D.s were within 5% of the mean values.
Fig. 3. Dependence of APX induction in Euglena on the development of chloroplast. (A) APX activity and chlorophyll content were estimated after illuminating the dark-grown Euglena cultures at a photon flux density of 120 mmol m 2 s 1 for 24 h. For NF treatment, the inhibitor was added at a final concentration of 100 mM and preincubated for 1 h in the dark prior to illumination. SM-ZK mutant cultures were grown under continuous light of the same intensity. (B) Immunoblot analyses. (C) Northern hybridization. Please see the legend to Fig. 2 for experimental details of the immunoblot and Northern analyses.
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3.4. Effect of redox conditions on APX induction
4. Discussion
As the data from the above experiments suggested that the induction of APX is dependent on the development of chloroplast, we verified whether superoxide or H2O2 could trigger the induction of APX, since these active oxygen species (AOS) are generated during the course of photosynthesis [19]. Dark-grown Euglena cells were incubated with 1 mM H2O2, or 100 mM MV for 24 h in the dark. However, these treatments could not mimic the effect of illumination in that no induction of APX activity was observed (Fig. 4). Moreover, treatment of dark-grown Euglena cells with H2O2 above 1 mM or MV above 0.1 mM concentration severely affected the cell viability (data not shown). During the light adaptation of Euglena , AsA and GSH levels increase by 7- and 4.5-fold, respectively, even prior to the onset of the development of chloroplast reaching the maximum in the first 7 h of illumination [6]. To make sure whether the elevated levels of these reducing species in turn induce APX activity, we incubated the dark-grown cells with Lgalactono-g-lactone and N -acetylcystein, which are precursors for AsA and GSH, respectively [20]. Feeding of Euglena cells with these precursors results in several fold increase in their respective final products [20]. However, APX activity remained unchanged in these experiments (Fig. 4). These results suggest that redox species are not involved in APX induction in Euglena . In addition to light, several other environmental factors, such as drought, salt, chilling, and air pollutants, have also been known to upregulate APX expression in higher plants [21]. In this study, we also examined the effect of heat, high light, cold, osmotic, and oxidative stress conditions, described in Section 2, on the level of APX in Euglena . However, no significant response of APX was found under these conditions (data not shown).
In this paper, we describe the induction of cytosolic APX during the early period of light adaptation of darkgrown Euglena cells. Our data clearly show that APX induction is regulated by a post-transcriptional mechanism. The process of light adaptation involves a rapid development of proplastid into a mature and fully functional chloroplast [11]. It has been known that chloroplasts are the major source of AOS [19]. Because Euglena does not possess catalase, and if the cytosolic APX were to scavenge the H2O2 generated in the chloroplast, a prior induction of the cytosolic APX ahead of the chloroplast development (Fig. 1) would be a logical event in the adaptation to the light regime. Both AsA and GSH, which are important components of the AsA /GSH cycle, increase by 7- and 4.5-fold, respectively, within the first 8 h of illumination, even before the onset of the development of chloroplast [6]. The H2O2 tolerance of Calvin cycle enzymes of Euglena , coupled with the rapid diffusivity of H2O2 [7,8], also might facilitate the rapid development of chloroplast and the resumption of photosynthesis. Based on the recent information on the regulation of AOS-scavenging enzymes in higher plants, we investigated several potential candidates that might mediate the light response in Euglena . Our data from the use of oxidative and reducing agents (Fig. 4) indicate, at least, that the induction of APX during the light adaptation is not a result of the accompanying redox changes in the cell. In contrast, the cellular redox status in higher plants is an important factor in the regulation of APX expression under high light condition. For example, in spinach, the transcript levels of cytosolic APX markedly increase in response to high light stress and MV treatment [21]. Karpinski et al. [22] have reported that H2O2 accumulation and/or redox status of the plastoquinone pool might be essential for the transcriptional induction of cytosolic APX in Arabidopsis during high light intensity. Although our experiments did not reveal the exact factor that mediates the induction of APX, our data single out the dependence of APX induction on the development of chloroplast. The process of light adaptation of Euglena cells entails a host of structural and metabolic events [10]. For example, the nuclear-geneencoded small subunit of ribulose-1,5-bisphosphate carboxylase/oxigenase (rbcS) and the light-harvesting chlorophyll a /b binding protein (LHCPII) are expressed coordinately [23,24]. Interestingly, the protein levels of both rbcS and LHCPII increase upon illumination whereas their mRNA levels remain unchanged. It appears that the post-transcriptional control is a common mechanism of gene regulation in Euglena [25]. On the other hand, during the greening in higher plants, the expression of both the above-mentioned genes is transcriptionally regulated [26,27]. Kishore and Schwartz-
Fig. 4. Effect of cellular redox status on APX activity in dark-grown Euglena cells. L-Galactono-g-lactone (L-GAL), N -acetyl cysteine (NAC), H2O2, or MV was added to dark-grown Euglena cultures at the indicated final concentrations. After 24 h of incubation in the dark, APX activity was assayed. A sample of the culture was incubated under light for 24 h at a photon flux density of 120 mmol m 2 s 1 (light) (mean9/S.D.; n/3).
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bach [24] showed that the light-dependent synthesis of LHCPII is regulated at the level of polypeptide chain elongation. These authors concluded that the signaling for the polypeptide chain elongation requires a ‘lightinduced effector molecule’ that is either synthesized within the chloroplast or related to chloroplast development, because the amount of polysome-associated LHCPII mRNA in both the wild-type and bleached mutant were almost equal. The light-induced expression of APX described in this study might involve the similar signaling as in the case of LHCPII, because APX level in SM-ZK-bleached mutant showed no significant response to light, whereas the transcript levels of the enzyme in both the wild-type and bleached mutant were quite comparable (Fig. 3C). A detailed investigation on the role of photosynthetic electron transport in the development of chloroplast during the adaptation of dark-grown Euglena may throw some light on the underlying regulation mechanism.
Acknowledgements R.M. gratefully acknowledges a fellowship from the Ministry of Education, Science, Sports, and Culture of Japan.
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