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The Expression of Subunits of the Mitochondrial Complex I-Homologous NAD(P)H-Plastoquinone- during Plastid Development Martina Fischer, Edgar Funk and Klaus Steinmüller Institut für Entwicklungs- und Molekularbiologie der Pflanzen. Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany Z. Naturforsch. 52c, 481-486 (1997); received May 12/May 28, 1997 Development, Etioplast, NAD(P)H-Plastoquinone-Oxidoreductase Plastids contain a NAD(P)H-plastoquinone-oxidoreductase which is homologous to the eubacterial and mitochondrial NADH-ubiquinone-oxidoreductase (complex I), but the meta­ bolic function of the is still not yet understood. The enzyme consists of at least eleven subunits (NDH-A-K), which are all encoded in the plastid chromosome. In this study we have investigated the tissue-specific and -dependent expression of the subunits NDH-H and NDH-K in maize, rice and mustard by western blot analysis. No NDH- were found in root tissue, indicating that the presence of the enzyme is confined to leaf plastids. Analysis of the expression during the light-dependent development from etioplasts to chloro­ plasts showed that high amounts of NDH-H and -K are present in etioplasts. The same result was found for subunits of the ATPase. In contrast, components of the photosynthetic (PSII-B, cytochrome f and PSI-D) accumulated only after illumination. In an second investigation, the expression of NDH-proteins along the natural chloroplast develop­ mental gradient from proplastids to in light-grown maize leaves was analysed. NDH-H and NDH-K as well as the ATPase were present at the youngest stages of chloro­ plast development, while the massive accumulation of subunits of the and the cytochrome Z>6//-complex took place in older leaf sections. We conclude from these studies that a functional NAD(P)H-plastoquinone-oxidoreductase is present in etioplasts and devel­ oping plastids. We suggest that the enzyme serves the generation of a proton gradient across the prothylakoid membrane that is necessary for integration into the membrane at developmental stages where a functional photosynthetic electron transport chain is not yet operating.

Introduction Firstly, the enzyme may be part of a so called chlo- and chloroplasts of higher rorespiratory pathway which serves the oxidation harbor an NAD(P)H-plastoquinone-oxidoreduc- of surplus reduction equivalents generated via the tase that is homologous to the NADH-ubiqui­ oxidative pentose phosphate pathway during none-oxidoreductase (complex I) of bacteria and starch degradation in the dark (Bennoun, 1982). mitochondria (Friedrich et al., 1995). This enzyme Secondly, the enzyme may be a component of the is composed of at least eleven subunits (NDH-A cyclic electron transport pathway around pho­ to K), which are all encoded in the plastid chromo­ tosystem I. Support for this hypothesis comes from some (Sugiura, 1992). It has been shown that the the finding that in the bundle sheath plastids of enzyme is located on the stromal system C4-plants, which rely on cyclic electron transport (Berger et al., 1993). to fullfil their ATP requirements for C 02 fixation, In bacteria and mitochondria, complex I oxi­ the relative amounts of NDH-proteins are dizes NADH and forms the first proton-pumping increased as compared to mesophyll plastids (Kub- complex of the respiratory chain (Friedrich et al., icki et al., 1996). Thirdly, the enzyme may be in­ 1995), however the function of the homologous volved in the detoxification of deleterious oxygen plastidial enzyme has not yet been clarified. So far, species in senescent chloroplasts or under condi­ three possible functions have been proposed. tions of photooxidative stress (Martin et al., 1996). For a better understanding of the function of the NAD(P)H-plastoquinone-oxidoreductase in plas­ tid metabolism, we have investigated, Reprint requests to PD Dr. Klaus Steinmüller. whether the enzyme is expressed in other Fax: (0211) 81-14871. organs, i.e. roots, and studied the expression of the

0939-5075/97/0700-0481 $ 06.00 © 1997 Verlag der Zeitschrift für Naturforschung. All rights reserved. D 482 M. Fischer et al. ■ Expression of Subunits of the NAD(P)H-Plastoquinone-Oxidoreductase enzyme during light-dependent chloroplast de­ lyvinyidene difluoride (PVDF, Immobilon P, velopment. Millipore. Eschborn, FRG) membranes according to Kyhse-Anderson (1984). The membranes were incubated with the different antisera listed below Materials and Methods and antigen-antibody complexes were detected The plants were obtained from the following with the ECL Western-blotting analysis system sources: maize (Zea mays, cv. B73 from Klein- from Amersham Buchler (Braunschweig, FRG). wanzlebener Saatzucht, Einbeck, FRG), rice (Or- The sources of the antibodies were: tobacco yza sativa, cv. Lido from Societe Italiana Sementi. NDH-H and rice NDH-K (Kubicki et al., 1996) Italy) and mustard (Sinapis alba from Carl Sper­ and PSII-B (Oswald et al., 1990). The ling & Co., Lüneburg, FRG). antibodies against spinach ATPase, PSI-D and For the analysis of the light-dependent expres­ cytochrome f were gifts from H. Strotmann sion of NDH-proteins, maize and rice were grown (Heinrich-Heine-University, Düsseldorf, FRG), at 26 °C in darknes for 7 or 8 days, respectively. R. Nechushtai (Hebrew University, Jerusalem, Is­ Mustard was grown at 21 °C for 4 days. The plants rael) and N. Nelson (Roche Institute, Nutley, were then illuminated for up to 24 hours with N. J., USA), respectively. white fluorescent lamps. For the analysis of the ex­ Protein concentrations were determined accord­ pression along the developmental gradient from ing to Bensadoun and Weinstein (1976). proplastids to chloroplasts in monocotyledous plants, maize was grown under greenhouse condi­ Results tions for 12 days with additional illumination (14 h per day), provided by a combination of sodium The antisera against NDH-H and NDH-K were and mercury high-pressure vapour lamps. At har­ obtained against fusion proteins expressed in E. vest, the first and second leaves were discarded coli, which contained only parts of the reading and the third leaf with the enclosed younger leaves frames of ndhH from tobacco and ndhK from rice was cut into six sections (0-1, 1-2, 2-4, 4-6, 6- (Kubicki et al., 1996). Initially, the antisera were 8 and 8-rest cm) starting from the the first nodium tested against total membrane preparations from as described in Martineau and Taylor (1985). different mono- and dicotyledonous plant species. Intact plastids were prepared by the homogeni- Specific signals of the expected molecular masses zation of leaf material in medium A as described were obtained with the NDH-H antibody for in Herrmann (1982) in a Waring blendor. The ho- maize, rice and mustard and with the NDH-K anti­ mogenate was filtered trough 80 (.im nylon gaze body for maize and rice. These plants were there­ and three layers of miracloth. The plastids were fore chosen for further analysis. collected by centrifugation (2 min, 6000 x g), re­ Since the expression of NDH-proteins has been suspended in a small volume of medium A and studied so far only in chloroplasts, we analysed, if then layered upon a percoll density step gradient NDH-H and NDH-K are also present in roots. Af­ (40/85%) as described in Bartlett et al. (1982). ter western analysis of root plastids from maize Broken and intact plastids were separated by cen­ and rice no specific signals were detectable for trifugation for 10 min at 10000 x g in the Sorvall both subunits (data not shown). Thus the expres­ HB-4 rotor (Sorvall Dupont de Nemours, Bad sion of NDH-proteins is restricted to leaves. Homburg, FRG). The band of intact plastids at the We then studied the expression of NDH-H and - 40/85% percoll interphase was recovered from the K in leaves during the light-dependent conversion gradient, washed two times in 10 mM 4-(2-hydro- of etioplasts to chloroplasts. To allow a compari­ xyethyl)-l-piperazine ethanesulfonic acid (Hepes)/ son of the expression of NDH-proteins to other KOH. pH 7.8, 330 mM sorbitol and then stored components of the photosynthetic electron trans­ at -20 °C. port chain, the concentrations of subunits of pho­ The plastids were solubilized in Laemmli's fi­ tosystem I (PSI-D) and II (PSII-B), the cyto­ nal sample buffer and separated by electrophore­ chrome b6/f complex (cytochrome f) and the sis trough a 12.5% polyacrylamide gel (Laemmli, ATPase (a- and ß-subunit) were monitored in par­ 1970). The proteins were then transferred to po- allel. The kinetics of protein accumulation after M. Fischer et al. ■ Expression of Subunits of the NAD(P)H-Plastoquinone-Oxidoreductase 483 the illumination of dark grown maize, rice and 0 4 8 16 24 h light mustard plants, respectively, are shown in Figs 1, 2 and 3. In all three plant species tested, NDH-H and NDH-K were found in etioplasts. In maize and rice, the concentrations of both NDH-proteins did not change significantly after illumination (Figs. 1 and 2), while in mustard, the relative amount of NDH-H increased (Fig. 3). A similar result was obtained for the a- and ß-subunits of the ATPase. In contrast to the subunits of the NAD(P)H-plastoquinone-oxidoreductase and the ATPase, subunit D of I and subunit B of photosystem II were not detectable in eti­ oplasts. For cytochrome f different kinetics of ac­ cumulation were found in the three plant species. PS"-B While in mustard cytochrome / was detectable t f e only after 16 hours of illumination (Fig. 3), maize Fig. 2. Western blot analysis of the expression of NDH- and rice etioplasts contained cytochrome f. A sim­ H and NDH-K, the a- and ß- subunits of the ATPase, ilar difference between mono- and dicotyledonous and components of the photosynthetic electron trans­ plant species in the accumulation of cytochrome / port chain (PSI-D, Cyt./, PSII-B) during the greening of etiolated 8 day old rice plants. The plants were illumi­ and other subunits of the cytochrome b6/f complex nated for 4, 8, 16 to 24 hours. Each lane contains 50 ng has been reported for wheat and peas by Takabe protein. The two weak bands underlying the PSII-B sig­ et al. (1989). nal are from a preceeding incubation with the ATPase antibody. A gradient of plastid development from pro- plastids to chloroplasts is formed in leaves of mon- 0 4 8 16 24 h light

0 4 8 16 24 h light

NDH-K PSI-D

ATPase -H Cyt. f

PSII-B PSI-D

Cyt. f Fig. 3. Western blot analysis of the expression of NDH- H, the a- and ß- subunits of the ATPase, and compo­ nents of the photosynthetic electron transport chain PSII-B (PSI-D, Cyt. f PSII-B) during the greening of 4 day old mustard plants. The plants were illuminated for 4, 8, 16 to 24 hours. Each lane contains 50 |xg protein. Fig. 1. Western blot analysis of the expression of NDH- H and NDH-K, the a- and ß- subunits of the ATPase, and components of the photosynthetic electron trans­ ocotyledonous plants grown under a natural day/ port chain (PSI-D, Cyt. /, PSII-B) during the greening of night regime. Originating from the basal meristem, etiolated 7 day old maize leaves. The plants were illumi­ the plastids differentiate via several typical devel­ nated for 4, 8, 16 to 24 hours. Each lane contains 50 jag protein. The a- and ß-subunits of the ATPase run to­ opmental stages, i.e. proplastids, amyloplasts, gether in one band. amoeboid plastids and prochloroplasts to chloro- 484 M. Fischer et al. • Expression of Subunits of the NAD(P)H-Plastoquinone-Oxidoreductase plasts (Whatley, 1977; Leech, 1984). To analyse, if Taylor. 1985; Takabe et al., 1986). A direct compar­ the expression of the NAD(P)H-plastoquinone- ison of these experiments is diffcult, because dif­ oxidoreductase is coupled to this development, ferent plant species were used and different pro­ plastids of different sections of the third leaf of a teins were analysed. However it has been shown 12 day old maize plant were isolated and then in other studies, that has to be present tested by immunoblot analysis. NDH-H as well as for the accumulation of the chlorophyll-binding the a- and ß-subunits of the ATPase were found subunits of both photosystems (Kreuz et al., 1986; in the second leaf section only 1 cm apart from Sutton et al., 1987). In agreement with these find­ the basal meristem (Fig. 4). Their concentrations ings are experiments which demonstrate that the increased up to the fourth leaf section and then photosynthetic electron transport chain develops gradually. In general, photosystem I activity can 1 2 3 4 5 6 be observed before a functional photosystem II is formed, and the whole electron transport from water to NADP reaches it’s full activity only sev­ eral hours after the illumination of etioplasts (Wellburn, 1982; Baker, 1984; Ohashi et al., 1989). On the other hand, it has long been known that the ATPase is present in etioplasts (Lockshin et al., 1971; Wellburn, 1977), but no functional im­ portance has been ascribed to this observation. We have extended these studies to another pro­ tein complex of the thylakoid membrane, the NAD(P)H-plastoquinone oxidoreductase. We found that the subunits NDH-H and NDH-K ac­ cumulate in etioplasts to high amounts. Moreover Fig. 4. Western blot analysis of the accumulation of these proteins become synthesized at the earliest NDH-H, the a- and ß- subunits of the ATPase and com­ stages of natural plastid development in the light. ponents of the photosynthetic electron transport chain Etioplasts and developing plastids are “heterotro- (PSI-D, Cyt. /, PSII-B) within sections of the third leaf of a 12 day old maize plant grown under a day/night phic” and rely on the import of energy regime. The sections 1-6 were: 0-1, 1-2, 2-4, 4-6, 6- and carbon skeletons for growth, which is medi­ 8 and 8-rest cm. Each lane contains 50 j.ig of etio-chloro- ated by specific carriers in the plastid envelope plast protein. (Wellburn, 1982; Flügge and Heldt, 1991). Eti­ oplasts and developing chloroplasts build up volu­ remained constant. PSI-D was also detectable in minous internal membrane systems, the prolam- the second leaf section, but the main accumulation mellar body with protruding prothylakoids and the of this protein took place in sections 4 and 5 where , respectively. This development of in­ the concentrations of the NAD(P)H-plastoqui- ternal membranes requires the synthesis of none-oxidoreductase and the ATPase had already nuclear and plastid encoded proteins, the import reached their maximal level. Low amounts of cyto­ of cytoplasmatically synthesized proteins into the chrome / and PSII-B were detectable in the third and the insertion of proteins into the leaf section, but, like for PSI-D. the highest prothylakoid or growing thylakoid membranes. amounts were found in older leaf sections. All of these processes are dependent on an suffi­ cient energy supply in the form of ATP (De Boer Discussion and Weisbeek, 1991; Cohen et al., 1995). However, The light- and developmental-dependent expres­ the integration or translocation of certain proteins sion of proteins involved in chloroplast metabolism into the growing thylakoid membrane requires in has been studied extensively with two experimental addition a proton gradient across the membrane. systems, the etioplast-chloroplast conversion and This has been shown for example for the light- the developmental gradient within leaves of mono- harvesting /b -binding proteins cotyledonous cereals (Leech, 1982; Martineau and (LHCP), the Rieske iron-sulfur protein and the M. Fischer et al. ■ Expression of Subunits of the NAD(P)H-Plastoquinone-Oxidoreductase 485 subunits of the so-called oxygen-evolving subcom­ by oxidizing reduction equivalents imported into plex of photosystem II (Cline et al., 1992; Cohen the etioplast generates a proton gradient and en­ et al., 1995). On the other hand, etioplasts and de­ ables the integration and the assembly of internal veloping chloroplasts posess no functional photo­ thylakoid membrane proteins. A functional AT­ synthetic electron transport chain and are thus not Pase would then be required for the counterregu­ able to build up a proton gradient by light-driven lation of this gradient. electron transport. Our experiments show that the NAD(P)H-plastoquinone-oxidoreductase and the Acknowledgements ATPase are the only major protein complexes of the prothylakoid membrane. We therefore suggest This research was supported by the Deutsche that the NAD(P)H-plastoquinone-oxidoreductase Forschungsgemeinschaft (A2/SFB189).

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