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• W1973050962 abstract "Polypeptides encoded by plastid ndhgenes form a complex (Ndh) which could reduce plastoquinone with NADH. Through a terminal oxidase, reduced plastoquinone would be oxidized in chlororespiration. However, isolated Ndh complex has low activity with plastoquinone and no terminal oxidase has been found in chloroplasts, thus the function of Ndh complex is unknown. Alternatively, thylakoid hydroquinone peroxidase could oxidize reduced plastoquinone with H2O2. By immunoaffinity chromatography, we have purified the plastid Ndh complex of barley (Hordeum vulgareL.) to investigate the electron donor and acceptor specificity. A detergent-containing system was reconstructed with thylakoid Ndh complex and peroxidase which oxidized NADH with H2O2 in a plastoquinone-dependent process. This system and the increases of thylakoid Ndh complex and peroxidase activities under photooxidative stress suggest that the chlororespiratory process consists of the sequence of reactions catalyzed by Ndh complex, peroxidase (acting on reduced plastoquinone), superoxide dismutase, and the non-enzymic one-electron transfer from reduced iron-sulfur protein (FeSP) to O2. When FeSP is a component of cytochrome b 6·fcomplex or of the same Ndh complex, O2 may be reduced with NADH, without requirement of light. Chlororespiration consumes reactive species of oxygen and, eventually, may decrease their production by lowering O2 concentration in chloroplasts. The common plastoquinone pool with photosynthetic electron transport suggests that chlororespiratory reactions may poise reduced and oxidized forms of the intermediates of cyclic electron transport under highly fluctuating light intensities. Polypeptides encoded by plastid ndhgenes form a complex (Ndh) which could reduce plastoquinone with NADH. Through a terminal oxidase, reduced plastoquinone would be oxidized in chlororespiration. However, isolated Ndh complex has low activity with plastoquinone and no terminal oxidase has been found in chloroplasts, thus the function of Ndh complex is unknown. Alternatively, thylakoid hydroquinone peroxidase could oxidize reduced plastoquinone with H2O2. By immunoaffinity chromatography, we have purified the plastid Ndh complex of barley (Hordeum vulgareL.) to investigate the electron donor and acceptor specificity. A detergent-containing system was reconstructed with thylakoid Ndh complex and peroxidase which oxidized NADH with H2O2 in a plastoquinone-dependent process. This system and the increases of thylakoid Ndh complex and peroxidase activities under photooxidative stress suggest that the chlororespiratory process consists of the sequence of reactions catalyzed by Ndh complex, peroxidase (acting on reduced plastoquinone), superoxide dismutase, and the non-enzymic one-electron transfer from reduced iron-sulfur protein (FeSP) to O2. When FeSP is a component of cytochrome b 6·fcomplex or of the same Ndh complex, O2 may be reduced with NADH, without requirement of light. Chlororespiration consumes reactive species of oxygen and, eventually, may decrease their production by lowering O2 concentration in chloroplasts. The common plastoquinone pool with photosynthetic electron transport suggests that chlororespiratory reactions may poise reduced and oxidized forms of the intermediates of cyclic electron transport under highly fluctuating light intensities. the plastid complex with activity NADH dehydrogenase and homologous to mitochondrial complex I potassium ferricyanide iron-sulfur protein p-dihydroxybenzene (hydroquinone) plastoquinone reduced plastoquinone superoxide dismutase photosystem polyacrylamide gel electrophoresis The existence of chlororespiration, or respiration in chloroplasts, was first proposed in Chlamydomonas (1.Bennoun P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4352-4356Crossref PubMed Google Scholar) and later in higher plants (2.Garab G. Lajkó F. Mustárdy L. Marton L. Planta (Heidelb.). 1989; 179: 349-358Crossref PubMed Scopus (108) Google Scholar). However, little progress has been made toward the elucidation of the mechanism of chlororespiration and, consequently, of its functional role. The discovery of ndhgenes in chloroplasts (3.Shinozaki K. Ohme M. Tanaka M. Wakasugi T. Hayashida N. Matsubayashi T. Zaita N. Chunwongse J. Obokata J. Yamaguchi-Shinozaki K. Ohto C. Torazawa K. Meng B.Y. Sugita M. Deno H. Kamogashira T. Yamada K. Kusuda J. Takaiwa F. Kato A. Tohdoh N. Shimada H. Sugiura M. EMBO J. 1986; 5: 2043-2049Crossref PubMed Scopus (1906) Google Scholar, 4.Ohyama K. Fukuzawa H. Kohchi T. Shirai H. Sano T. Sano S. Umesono K. Shiki Y. Takeuchi M. Chang Z. Aota N.S. Inokuchi H. Ozeki H. Nature. 1986; 322: 572-574Crossref Scopus (1196) Google Scholar) suggested that a plastid NAD(P)H dehydrogenase complex (similar to complex I of the mitochondrial respiratory chain, EC 1.6.5.3) (5.Weiss H. Friedrich T. Hofhaus G. Preiss D. Eur. J. Biochem. 1991; 197: 563-576Crossref PubMed Scopus (431) Google Scholar) would participate in chloroplast respiration. In fact, in higher plants, ndh transcripts are edited (6.Freyer R. López C. Maier R.M. Martin M. Sabater B. Kossel H. Plant Mol. Biol. 1995; 29: 679-684Crossref PubMed Scopus (50) Google Scholar) for appropriate translation, several ndh encoded polypeptides have been found in thylakoids (7.Berger S. Ellersiek U. Westhoff P. Steinmuller K. Planta (Heidelb.). 1993; 190: 25-31Crossref Scopus (80) Google Scholar, 8.Martı́n M. Casano L.M. Sabater B. Plant Cell Physiol. 1996; 37: 293-298Crossref PubMed Scopus (87) Google Scholar, 9.Kubicki A. Funk E. Westhoff P. Steinmüller K. Planta (Heidelb.). 1996; 199: 276-281Crossref Scopus (90) Google Scholar, 10.Catalá R. Sabater B. Guéra A. Plant Cell Physiol. 1997; 38: 1382-1388Crossref PubMed Scopus (43) Google Scholar, 11.Burrows P.A. Sazanov L.A. Svab Z. Maliga P. Nixon P.J. EMBO J. 1998; 17: 868-876Crossref PubMed Scopus (403) Google Scholar) and a thylakoid-bound high molecular weight complex with NAD(P)H dehydrogenase activity has been identified (11.Burrows P.A. Sazanov L.A. Svab Z. Maliga P. Nixon P.J. EMBO J. 1998; 17: 868-876Crossref PubMed Scopus (403) Google Scholar, 12.Cuello J. Quiles M.J. Albacete M.E. Sabater B. Plant Cell Physiol. 1995; 36: 265-271Crossref Scopus (43) Google Scholar, 13.Sazanov L. Burrows P.A. Nixon P.J. Biochem. Soc. Trans. 1996; 24: 739-743Crossref PubMed Scopus (44) Google Scholar, 14.Quiles M.J. Albacete M.E. Sabater B. Cuello J. Plant Cell Physiol. 1996; 37: 1134-1142Crossref Scopus (20) Google Scholar). Western assays with appropriate antibodies and microsequencing indicated (15.Sazanov L. Burrows P.A. Nixon P.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1319-1324Crossref PubMed Scopus (169) Google Scholar) that the NADH complex contains ndh encoded NDH polypeptides (Ndh complex).1 By analogy with mitochondria, in chlororespiration, the plastid Ndh complex would transfer electrons from NADH to a quinone (presumably plastoquinone, PQ) whose reduced form would in turn be oxidized by oxygen by a terminal oxidase. Lower PQ reduction in mutant tobacco plants defective in ndh genes (11.Burrows P.A. Sazanov L.A. Svab Z. Maliga P. Nixon P.J. EMBO J. 1998; 17: 868-876Crossref PubMed Scopus (403) Google Scholar, 16.Kofer W. Koop H.-U. Wanner G. Steinmüller K. Mol. Gen. Genet. 1998; 258: 166-173Crossref PubMed Scopus (144) Google Scholar) points that the Ndh complex is responsible for PQ reduction. However, due to problems of solubility of substrate, plastid Ndh complex can hardly be assayed with PQ (15.Sazanov L. Burrows P.A. Nixon P.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1319-1324Crossref PubMed Scopus (169) Google Scholar) and is, therefore, commonly assayed by reduction of ferricyanide (FeCN) with NADH. Moreover, there are doubts as to whether the plastid Ndh complex is involved in cyclic electron transport in chloroplasts, in chlororespiration, or both processes (15.Sazanov L. Burrows P.A. Nixon P.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1319-1324Crossref PubMed Scopus (169) Google Scholar). While non-photosynthetic parasitic plants lack both genes for proteins of the photosynthetic machinery and ndh genes (17.de Pamphilis C.W. Palmer J.D. Nature. 1990; 348: 337-339Crossref PubMed Scopus (240) Google Scholar, 18.Haberhausen G. Zetsche K. Plant Mol. Biol. 1994; 24: 217-222Crossref PubMed Scopus (60) Google Scholar), no plant has been found containing ndh genes but lacking genes for photosynthetic machinery, which suggests that the function ofndh gene products is related to photosynthesis. Until now, only the black pine, a photosynthetic plant with plastid genes for photosynthetic machinery, lacks functional ndh genes (19.Wakasugi T. Tsudzuki J. Ito S. Nakashima K. Tsudzuki T. Sugiura M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9794-9798Crossref PubMed Scopus (411) Google Scholar). On the other hand, no clear terminal oxidase has been found in chloroplasts (20.Bennoun P. Biochim. Biophys. Acta. 1994; 1186: 59-66Crossref Scopus (127) Google Scholar), which raises further questions regarding chlororespiration and the role of ndh genes and the Ndh complex. The level of the NDH-A polypeptide, encoded by thendhA gene, increased in thylakoid when barley leaves received high irradiances or were incubated under high oxygen concentrations (8.Martı́n M. Casano L.M. Sabater B. Plant Cell Physiol. 1996; 37: 293-298Crossref PubMed Scopus (87) Google Scholar), which suggested that ndh genes and the Ndh complex may be involved in the protection against photooxidative stress. Most photosynthetic O2 uptake by drought-stressed leaves (a condition which produces photooxidative stress) is due to a one-electron transfer to O2 forming superoxide anion radical (O⨪2) (21.Biehler K. Fock H. Plant Physiol. 1996; 112: 265-272Crossref PubMed Scopus (229) Google Scholar). The subsequent action of superoxide dismutase (SOD, EC 1.15.1.1) converts O⨪2 to H2O2, which is finally consumed in a peroxidase-catalyzed reaction. Hydroquinone peroxidases (EC 1.11.1.7) have been identified in chloroplasts (22.Laloue H. Weber-Lofti F. Lucau-Danila A. Guillemaut P. Plant Physiol. Biochem. 1997; 35: 341-346Google Scholar, 23.Zapata J.M. Sabater B. Martı́n M. Phytochem. 1998; 48: 1119-1123Crossref Scopus (28) Google Scholar). A peroxidase solubilized from barley thylakoid with Triton X-100 (23.Zapata J.M. Sabater B. Martı́n M. Phytochem. 1998; 48: 1119-1123Crossref Scopus (28) Google Scholar) oxidizes the reduced form of plastoquinone (PQH2). Thus, the catalytic activities of Ndh complex, peroxidase (acting on PQH2), and SOD and the non-enzymic one-electron transfer from a reduced iron-sulfur protein (FeSP) to O2 could be responsible for electron transport from NADH to O2 in chlororespiration. In the dark, O2 may be reduced with NADH if FeSP is a component of the cytochrome b 6·f complex or of the same Ndh complex. To provide biochemical support for this hypothesis, we have reconstructed, with immunopurified Ndh complex and peroxidase isolated from barley thylakoid, an in vitro system which (in a detergent-containing medium) oxidizes NADH with H2O2 in a PQ-dependent reaction. The properties of this in vitro system and the effects of different treatments of leaves on enzyme activities described herein suggest that chlororespiration may protect against photooxidative stress and could regulate the proportion of reduced and oxidized forms of the intermediates of cyclic electron transport to optimize cyclic photophosphorylation. Barley plants (Hordeum vulgare L. cv. Hassan) were grown on vermiculite at 23 °C under a 16-h photoperiod of 100 μmol of photon m−2 s−1 white light as described (24.Casano L.M. Martı́n M. Sabater B. Plant Physiol. 1994; 106: 1033-1039Crossref PubMed Scopus (96) Google Scholar). Unless otherwise stated, in the present work, we have used primary leaves of 14-day-old plants. Photooxidative stress was induced by incubating leaf segments in higher irradiance (300 μmol of photon m−2 s−1) in the presence of either 21 or 100% O2. PQ and other chemicals were from Sigma. Intact chloroplasts were isolated as described (8.Martı́n M. Casano L.M. Sabater B. Plant Cell Physiol. 1996; 37: 293-298Crossref PubMed Scopus (87) Google Scholar). The pellet of chloroplasts was washed with buffer E (50 mm potassium phosphate, 1 mm l-ascorbic acid, 1 mm EDTA, 0.33 m sorbitol, pH 7.5) (10 ml/1 g of original leaves) to obtain a preparation of chloroplasts free from soluble or mitochondrial fractions (8.Martı́n M. Casano L.M. Sabater B. Plant Cell Physiol. 1996; 37: 293-298Crossref PubMed Scopus (87) Google Scholar, 10.Catalá R. Sabater B. Guéra A. Plant Cell Physiol. 1997; 38: 1382-1388Crossref PubMed Scopus (43) Google Scholar, 23.Zapata J.M. Sabater B. Martı́n M. Phytochem. 1998; 48: 1119-1123Crossref Scopus (28) Google Scholar). To obtain high percentages (>80%) of intact chloroplasts, all steps from leaf segments to pellet of washed chloroplasts were performed at 0–5 °C in no more than 45 min. The chloroplast pellet was resuspended for osmotic shock in buffer E without sorbitol (buffer H) at 0–4 °C with gentle shaking during 6 min and then centrifuged for 15 min at 4,500 × g. The thylakoid pellet was resuspended to approximately 2 mg of protein/ml in buffer H and used for solubilization of thylakoid-bound NADH dehydrogenase or peroxidase activities. The membranous fraction was washed with Triton X-100 (0.2 mg of detergent/mg of protein) and gently stirred for 15 min. After centrifugation at 10,000 × g for 20 min, NADH dehydrogenase was solubilized from the pellet, which was resuspended in 20 mm Tris-HCl, pH 7.5, 5 mm EDTA, by adding 10% (w/v) Triton X-100 to make a final concentration of 2% (5 mg of detergent/mg of protein) and gently stirred for 30 min. The suspension was centrifuged at 105,000 × g for 45 min. The supernatant (around 4 mg of protein/ml, about 40% of original thylakoid proteins) was the NADH dehydrogenase-solubilized fraction. Peroxidase was solubilized from the thylakoid membranous fraction by adding 10% (w/v) Triton X-100 to make a final 1.5% solution and gently stirred for 45 min. The suspension was centrifuged at 20,000 × g for 30 min. The supernatant (around 0.9 mg of protein/ml) contained the peroxidase solubilized from thylakoid. In other experiments, NADH dehydrogenase activities were assayed in whole leaf extracts obtained as follows: 10 3-cm leaf segments were homogenized with a mortar and pestle in 2 ml of buffer H and centrifuged at 500 × g for 10 min. Triton X-100 was added to supernatant to make a final 2% (w/v) solution (5 mg of detergent/mg of protein) and gently stirred for 30 min. The suspension was centrifuged at 20,000 × g for 30 min. Supernatants contained 0.7 to 1.3 mg of protein/ml. All the solubilization procedures were performed at 4 °C. Antibodies against NDH-A protein (8.Martı́n M. Casano L.M. Sabater B. Plant Cell Physiol. 1996; 37: 293-298Crossref PubMed Scopus (87) Google Scholar) were purified from rabbit antiserum by (NH4)2SO4 precipitation and affinity chromatography on a protein A-Sepharose column (25.$$$$$$ ref data missingGoogle Scholar) and then covalently coupled to an Affi-Gel hydrazide-agarose gel according to the manufacturer (Bio-Rad) and packed into a 5-ml column. Five to six ml of the NADH dehydrogenase-solubilized fraction were passed through the immunoaffinity column three times. After washings (25.$$$$$$ ref data missingGoogle Scholar), the Ndh complex was eluted with 50 mm glycine-HCl, pH 2.5, 0.1% Triton X-100 (w/v), and 0.15 m NaCl. Fractions (1 ml each) were immediately neutralized with 1 m Tris-HCl, pH 9.0. As described (8.Martı́n M. Casano L.M. Sabater B. Plant Cell Physiol. 1996; 37: 293-298Crossref PubMed Scopus (87) Google Scholar), antibodies against NDH-A do not react with any mitochondrial protein. Native PAGE was carried out at 5 °C (usually with 50–100-μg protein samples) in a linear gradient gel of 3–10% polyacrylamide (2.5% bisacrylamide) in the same way as SDS-PAGE with the exception that gels contained 0.1% (w/v) Triton X-100 instead of SDS (26.Kuonen D.R. Roberts P.J. Cottingham I.R. Anal. Biochem. 1986; 153: 221-226Crossref PubMed Scopus (47) Google Scholar). For immunoblot analyses, after electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Millipore). Immunocomplex with antibody prepared against the NDH-A polypeptide encoded by the ndhA gene (8.Martı́n M. Casano L.M. Sabater B. Plant Cell Physiol. 1996; 37: 293-298Crossref PubMed Scopus (87) Google Scholar) was detected with the peroxidase Western blotting analysis system (Roche Molecular Biochemicals). In other experiments, after native PAGE, the immunopurified Ndh complex band was electroeluted and polypeptides were separated by denaturating SDS-PAGE in a linear gradient gel of 12–16% polyacrylamide. After membrane blotting, the presence of NDH-A polypeptide was determined by antibody against the NDH-A polypeptide encoded by the ndhA gene (8.Martı́n M. Casano L.M. Sabater B. Plant Cell Physiol. 1996; 37: 293-298Crossref PubMed Scopus (87) Google Scholar). For zymograms, NADH dehydrogenase activity was detected by incubation of gel slices for 20–30 min at 30 °C in darkness in 50 mm potassium phosphate, pH 8.0, 1 mmNa2-EDTA, 0.2 mm NADH, and 0.5 mg/ml nitro blue tetrazolium. In controls without NADH, no color developed. Bands were scanned with a UVP Easy Digital Image analyzer to comparatively quantify activity values which were expressed as percentages of the reference activity (usually that of 7-day-old plants). NADH:FeCN oxidoreductase activity was assayed at 30 °C by measuring the reduction of FeCN at 420 nm (extinction coefficient: 1.03 mm−1 cm−1) and the oxidation of NADH at 340 nm (extinction coefficient: 6.22 mm−1 cm−1) in a Beckman DU-650 spectrophotometer. The reaction mixture, with a final volume of 1.0 ml, included 50 mm potassium phosphate, pH 7.5, 1 mm Na2EDTA, 0.2 mm NADH, 1 mm FeCN, and variable enzyme preparations. The rate was determined from linear absorbance decrease between 45 and 240 s. Control values, obtained without protein, were subtracted. No detectable transformation of one substrate was observed in controls without the other substrate. In assays with PQ (usually 0.4 mm) as electron acceptor, preincubation and detergent treatments were performed as described under “Results.” The spectrophotometric assay of peroxidase was performed at 30 °C in a 1.0-ml assay volume containing 0.5 mm p-dihydroxybenzene (hydroquinone, HQ), 0.1 mmH2O2, 50 mm potassium phosphate buffer, pH 7.5. After mixing, the enzymatic reaction was initiated by adding 10 μl of enzyme. The oxidation of HQ was recorded as the increase in absorbance at 250 nm (extinction coefficient: 19 mm−1 cm−1) over a time period of 2 min. The absorbance increases were always linear with respect to time. Appropriate controls were subtracted (23.Zapata J.M. Sabater B. Martı́n M. Phytochem. 1998; 48: 1119-1123Crossref Scopus (28) Google Scholar). Specific activities are expressed as micromoles of NADH or HQ consumed per min/mg of protein. Protein concentration was quantified by the Bradford method (27.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (218585) Google Scholar) with a Protein Assay Kit (Bio-Rad) using bovine serum albumin as a standard. All reported results were reproduced at least three times. When appropriate, standard deviations are indicated by bars in figures. NADH dehydrogenase preparations solubilized from barley thylakoid still contained about 18% peroxidase activity. To obtain a peroxidase-free preparation, the above fraction was purified with a column containing antibody against NDH-A covalently bound to agarose. Most of the protein eluted with washing solutions, and the protein retained by immunoaffinity was subsequently eluted with an acid solution as shown in Fig. 1. Fractions 10 to 12 were pooled and were found to be almost free of peroxidase activity (<0.3%). When assayed as NADH:FeCN oxidoreductase, the specific activity of pooled fractions (about 5.5 μmol min−1 mg of protein−1) was in the range of the purified plastid and mitochondrial complex I (15.Sazanov L. Burrows P.A. Nixon P.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1319-1324Crossref PubMed Scopus (169) Google Scholar). With regard to the fraction resuspended from thylakoid with Triton X-100, immunoaffinity achieved 100-fold purification of the activity with a recovery of 22%. The activity of purified fractions on NADPH was only 3.6% of their activity on NADH, which is in accordance with previous results for the activities investigated in barley (14.Quiles M.J. Albacete M.E. Sabater B. Cuello J. Plant Cell Physiol. 1996; 37: 1134-1142Crossref Scopus (20) Google Scholar) and pea (15.Sazanov L. Burrows P.A. Nixon P.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1319-1324Crossref PubMed Scopus (169) Google Scholar) and indicates that the NADH dehydrogenase activity of pure thylakoid Ndh complex was essentially NADH-specific, although crude thylakoid preparations also contain NADPH dehydrogenase activities (14.Quiles M.J. Albacete M.E. Sabater B. Cuello J. Plant Cell Physiol. 1996; 37: 1134-1142Crossref Scopus (20) Google Scholar). NADH dehydrogenase activity of purified Ndh complex was 100% inhibited after 30 min incubation with NDH-A antibody, confirming that the activity of purified fraction was due to the Ndh complex that includes the NDH-A polypeptide. After native electrophoresis, zymograms of whole leaf extract and protein solubilized from thylakoid showed several NADH dehydrogenase bands (Fig. 2 A), while immunopurified Ndh complex showed a single activity band co-migrating with first thylakoid and second whole extract low migrating bands. Western assays showed that immunopurified Ndh complex and the co-migrating bands of whole leaf extract and thylakoid contained the NDH-A polypeptide (Fig. 2 B). At least 9 polypeptide bands (sizes ranging between 17 and 70 kDa) were detected with Coomassie staining (not shown) after SDS-PAGE electrophoresis of immunopurified Ndh complex. Western assay after SDS-PAGE (Fig. 2 C) indicated that only a polypeptide of 35 kDa of the Ndh complex reacted with NDH-A antibody. It must be noted that, of several NADH dehydrogenase bands detected in thylakoid and whole leaf extract, a single (low migrating and high activity) band contained the NDH-A polypeptide (Fig. 2 A, lane Thylak). The identification of the low migrating NADH dehydrogenase activity band corresponding to thylakoid Ndh complex allows us to comparatively quantify this activity from zymograms of whole leaf extracts with NADH and nitro blue tetrazolium. According to Fig. 2 A, activity of Ndh complex accounts for about two-thirds of the whole NADH dehydrogenase activity solubilized from washed thylakoid (about 50 nmol of NADH oxidized min−1 mg of protein−1, assayed as NADH:FeCN oxidoreductase). Immunoblots (not shown) with antibody against the 70-kDa NDH-F polypeptide (10.Catalá R. Sabater B. Guéra A. Plant Cell Physiol. 1997; 38: 1382-1388Crossref PubMed Scopus (43) Google Scholar) produced similar results to those shown in Fig. 2, B and C, for NDH-A antibody, indicating that our purified Ndh complex also included NDH-F polypeptide. Although band intensities in Fig. 2, A and B,roughly agree with the amount of protein loaded and Ndh purity in each fraction, quantitative conclusions cannot be deduced by comparing leaf, thylakoid, and purified Ndh complex bands, because NADH-DH lane signal was saturating and stability of activity (and probably of the same Ndh complex) was lower in purified and thylakoid fractions than in whole leaf fraction. In standard spectrophotometric assays, without detergent added to the incubation mixture, the activity of purified Ndh complex with PQ as electron acceptor is only 5% of the activity with FeCN as electron acceptor (both measured as the rate of NADH oxidation). The activity with PQ increased to 45% of the activity with FeCN when the reaction mixture containing 0.2% (w/v) deoxycholate was preincubated during 10 min at 4 °C prior to the addition of NADH. Measurements of absorbance changes at 262 nm due to PQ reduction (extinction coefficient 18.9 mm−1 cm−1) indicated an equimolar reaction between NADH and PQ. Activities with PQ were lower after preincubation with higher or lower deoxycholate concentrations. Apparently, hydrophobic detergent-containing particles include the Ndh complex in a state accessible enough to NADH and PQ, and provide an efficient system to assay NADH:PQ oxidoreductase activity. Hydroquinone peroxidase activity in the Triton X-100-solubilized fraction of thylakoid changed during the development of the primary leaf of barley. It reached a maximum (about 0.3 μmol of HQ oxidized min−1 mg of thylakoid protein−1) around 14 days after sowing (Fig.3), at the beginning of senescence soon after full expansion. When Ndh complex activity was quantified after zymographic assays with whole leaf extract it also reached a maximum in 14-day-old primary leaves, although changes were less pronounced than for peroxidase. For comparison, NADH dehydrogenase activity due to Ndh complex (see Fig. 2 A and discussion therein) is about 35 nmol of NADH oxidized min−1 mg of thylakoid protein−1 in 14-day-old leaves (220% in Fig. 3). To investigate the possible role of the Ndh complex and peroxidase activities in the protection against photooxidative stress, activity levels were measured in response to photooxidative treatments of leaf segments in both 7-day-old expanding leaves and 14-day-old fully expanded leaves. A typical zymogram of the NADH dehydrogenase activity of Ndh complex is shown in Fig.4 A. When compared with the levels in freshly detached leaf segments (To) or leaf segments incubated during 20 h with growth light (100 μmol of photon m−2 s−1), Ndh complex activity of leaf segments incubated for 20 h with relative photooxidative light (PhL) (300 μmol of photon m−2 s−1) increased approximately 100% in 14-day-old leaves and approximately 30% in 7-day-old leaves (Fig. 4, A and B). With relative photooxidative light under a 100% oxygen atmosphere, Ndh complex activity increased up to 3.5-fold in 14-day-old leaves and almost 2-fold in 7-day-old leaves. Photooxidative treatments also increased peroxidase activity (Fig. 4 C) but, in contrast to the Ndh complex, the relative increases of peroxidase were higher in 7-day-old leaves than in 14-day-old leaves. Western assays with antibody against NDH-A (8.Martı́n M. Casano L.M. Sabater B. Plant Cell Physiol. 1996; 37: 293-298Crossref PubMed Scopus (87) Google Scholar) showed increases of NDH-A polypeptide similar to those of Ndh complex activity (Fig. 4, A andB) in response to the same photooxidative treatments. The low solubilities of PQ and PQH2 make it difficult to assay them as substrates of, respectively, Ndh complex and peroxidase. Additionally, PQH2, which is obtained by reduction of commercial PQ with dithionite, is unstable. Although indirect estimations suggested that the barley thylakoid peroxidase uses PQH2 as substrate (23.Zapata J.M. Sabater B. Martı́n M. Phytochem. 1998; 48: 1119-1123Crossref Scopus (28) Google Scholar), measurements of this activity were difficult to reproduce. To overcome these difficulties, we developed an assay system designed to detect whether the PQH2 produced by reduction of PQ (with NADH in the reaction catalyzed by Ndh complex) is oxidized by H2O2 in the reaction catalyzed by peroxidase. Essentially, the system measures NADH oxidation by H2O2 dependent on the presence of Ndh complex, PQ, and peroxidase according to the sequence of reactions,NADH+H++PQ →Ndh complex NAD++PQH2Equation 1 H2O2+PQH2 →Peroxidase 2 H2O+PQEquation 2 In chloroplasts, the Ndh complex, PQ·PQH2 and peroxidase are immersed in the hydrophobic environment of thylakoid membrane, thus their functions are hardly reproducible in water mixtures commonly used for spectrophotometric assays. We have stated above that a 10-min preincubation at 4 °C of the reaction mixture of Ndh complex (including 0.2% deoxycholate and PQ but not NADH) increased the rate of PQ oxidation approximately 9-fold. In order to test appropriate controls, we carried out a partial purification of the peroxidase. When solubilized from thylakoid membrane by 1.5% Triton X-100, the peroxidase preparation contained 10% of NADH dehydrogenase activity. Taking advantage of the low stability of Ndh complex activity, a fraction of Triton-solubilized peroxidase between 40 and 70% (NH4)2SO2 was desalted with a Centricon 10 concentrator (Amicon). The resulting peroxidase preparation showed a specific activity of 3.8 μmol of HQ oxidized min−1 mg of protein−1, which was essentially free of NADH dehydrogenase activity (<0.1%). As found for non-purified fraction (23.Zapata J.M. Sabater B. Martı́n M. Phytochem. 1998; 48: 1119-1123Crossref Scopus (28) Google Scholar), partially purified peroxidase showed a single HQ peroxidase activity band in zymograms and was free of other peroxidase or oxidase activities. Preincubation of partially purified peroxidase at 4 °C with 0.2% deoxycholate did not significantly affect the activity of the enzyme in standard assays with HQ and H2O2 as substrates. The complete system reconstructed in vitro was assayed as follows: 40 mm potassium phosphate buffer, pH 7.5, H2O to make a final volume of 1.0 ml in the reaction mixture, 2 mg of deoxycholate, 4 μg of purified Ndh complex, 11 μg of partially purified peroxidase (resuspended in buffer H containing 1.5% Triton X-100), and variable concentrations of PQ were preincubated during 10 min at 4 °C. The reaction was started by adding NADH to make a final concentration of 0.2 mm (0.1 ml of a 2 mm solution) and 20 μl of pure water (controls) or 10 mm H2O2. The final concentrations of PQ were: 0, 0.04, 0.1, 0.2, and 0.4 mm. In the absence of H2O2 (left side of Fig. 5) but with PQ in the assay mixture, absorbance decreas" @default.
• W1973050962 created "2016-06-24" @default.
• W1973050962 creator A5003169683 @default.
• W1973050962 creator A5003960030 @default.
• W1973050962 creator A5026581953 @default.
• W1973050962 creator A5078701084 @default.
• W1973050962 date "2000-01-01" @default.
• W1973050962 modified "2023-09-30" @default.
• W1973050962 title "Chlororespiration and Poising of Cyclic Electron Transport" @default.
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