Differential expression of plastome-encoded ndh genes in ... · NDH-H, -J and -K were strongly increased in bundle- sheath plastids as compared to mesophyll plastids. This increase

Planta(1996) 199:276 281 P l a ~ t ~

(c) Springer-Verlag 1996

Differential expression of plastome-encoded ndh genes in mesophyll and bundle-sheath chloroplasts of the C4 plant Sorghum bicolor indicates that the complex I-homologous NAD(P)H-plastoquinone oxidoreductase is involved in cyclic electron transport Andreas Kubicki, Edgar Funk, Peter Westhoff, Klaus Steinmiiller

lnstitut fiir Entwicklungs- und Molekularbiologie der Pflanzen, Heinrich-Heine-Universitiit, Universit~itsstrasse 1, D-40225 Diisseldorf, Germany

Received: 19 September 1995/Accepted: 14 November 1995

Abstract. Cyanobacteria and plastids harbor a putative NAD(P)H- or ferredoxin-plastoquinone oxidoreductase that is homologous to the NADH-ubiquinone oxido- reductase (complex I) of mitochondria and eubacteria. The enzyme is a multimeric protein complex that consists of at least 11 subunits (NDH-A-K) and is localized in the stroma lamellae of the thylakoid membrane system. We investigated the expression of the different subunits of the enzyme in mesophyll and bundle-sheath chloroplasts of Sorghum bicolor [L.] Moench, a C4 plant of the NADP- malic enzyme type. The relative amounts of the subunits NDH-H, -J and -K were strongly increased in bundle- sheath plastids as compared to mesophyll plastids. This increase was accompanied by enhanced transcript levels for all subunits except NDH-I. Because the main function of the protein complexes in the thylakoid membranes of bundle-sheath chloroplasts (photosystem I, cytochrome b6/J:complex and ATPase) is the generation of ATP for C02 fixation via cyclic electron transport, we conclude that the NAD(P)H/ferredoxin-plastoquinone oxidoreduc- tase is an essential component of the cyclic electron- transport pathway in chloroplasts.

Key words: C4 photosynthesis - Cyclic electron transport - NAD(P)H-plastoquinone-oxidoreductase Sorghum

Introduction

The plastid chromosomes of angiosperms contain eleven reading frames (ndhA K) that are homologous to genes for the mitochondrial NADH-ubiquinone oxidoreduc- tase, also called complex I of the respiratory chain (Sugiura 1992). Therefore, it has been suggested that plas- tids harbor an NAD(P)H dehydrogenase with the putative activity of an NAD(P)H-plastoquinone oxidoreductase (Ohyama et al. 1988; Marder and Barber 1989). However,

Correspondence to: K. Steinmiiller; FAX: 49(211)8114871; E-mail: [email protected]

so far, the activity of such an enzyme in chloroplasts has never been demonstrated nor has the enzyme been iso- lated.

Cyanobacteria also contain eleven ndh genes that share a high sequence similarity with the homologous plastidial genes (Ellersiek and Steinmiiller 1992), and the purification of a subcomplex of the enzyme from Synechocystis sp. PCC6803 has been reported (Berger et al. 1993a). Studies on a Synechocystis mutant that carries a defective ndhB gene have shown that the enzyme can donate reduction equivalents to the photosynthetic electron-transport chain at the level of plastoquinone (Mi et al. 1992); and recently it has been demonstrated that the enzyme participates in a ferredoxin-dependent as well as in an NADPH-dependent cyclic electron- transport pathway around PSI in cyanobacteria (Mi et al. 1995).

Less is known about the enzyme from plastids. All ndh genes are transcribed into stable mRNA species (Mat- subayashi et al. 1987; Steinmiiller et al. 1989; Kanno and Hirai 1993). However, so far only three subunits, NDH-H, -I and -K have been identified by immunoblot analysis (Nixon et al. 1989; Lin and Wu 1990; Berger et al. 1993b). Both polypeptides were found in the stromal thylakoids which are considered as the membrane domain where cyclic electron transport takes place (Anderson 1992). This suggests that the plastidial NAD(P)H dehydrogenase is also involved in cyclic electron transport.

In order to investigate this possibility, we have ana- lysed the expression of the ndh genes in mesophyll and bundle-sheath plastids of the C4 plant Sorghum bicolor. While mesophyll chloroplasts of C~ plants of the NADP- malic enzyme type like maize and Sorghum contain the typical arrangement of grana and stroma thylakoids, and produce NADPH and ATP via linear electron flow, the thylakoid membrane system of bundle-sheath plastids is composed almost entirely of stroma lamellae. In the rudi- mentary granal bundle-sheath chloroplasts of maize, low levels of PSII activity can be detected; however, the com- pletely agranal bundle-sheath plastids of Sorghum lack any measurable PSII activity (Woo et al. 1970; Meierhoff and Westhoff 1993).

A. Kubicki et al.: Differential expression of ndh genes

O n the o ther hand, bund le - shea th ch lo rop las t s have to genera te A T P for CO2 fixat ion via the Ca lv in -Benson cycle and it has been shown tha t this A T P d e m a n d is fulfilled by cyclic e lec t ron t r a n s p o r t ( C h a p m a n et al. 1980; Leegood et al. 1981). Thus, if the N A D ( P ) H dehydrogen- ase is an essential c o m p o n e n t of the cyclic e lec t ron- t rans- po r t p a t h w a y in plast ids, one m a y expect tha t the relat ive a m o u n t of the enzyme is increased in bund le - shea th ch lo rop las t s as c o m p a r e d to mesophyl l ch loroplas ts .

Materials and methods

Plant material. Sorghum bicolor (L.) Moench cv. TX430 (Pioneer Hi-Breed, Plainview, Tex., USA) was grown in soil for 7-9 d.

Isolation of mesophyll and bundle-sheath chloroplasts. The isolation of plastids was carried out as described in Kubicki et al. (1994).

Preparation of antibodies. Plastid-DNA fragments from tobacco (NDH-H) or rice (NDH-J and -K) were subcloned into the expres- sion vectors pGEMEX1 or 2 (Promega, Heidelberg, Germany) yielding the plasmids pGX-H, pGX-1275RJ and pGX-758RKI as documented in Table 1. The plasmids were transformed into JM 109(DE3) and the expression of the fusion proteins was analysed by SDS-gel electrophoresis. After lysis of the bacteria, the recom- binant proteins were purified by two washings with 3.5 M urea and preparative SDS-electrophoresis. The proteins were then eluted from the gel and used for the immunisation of rabbits. The anti- bodies were generated by Eurogentec (Seraing, Belgium). Antisera against PSII-B (CP47), cytochrome f and PSI-D are described in Oswald et al. (1990) or were kindly provided by R. Nechustai (Department of Botany, The Hebrew University of Jerusalem, Israel) or N. Nelson (Roche Institute of Molecular Biology, Nutley, NJ, USA).

Protein electrophoresis and Western blotting. The SDS-polyacry- lamide electrophoresis was carried out according to Laemmli (1970). Western blots were prepared by transferring the proteins onto polyvinlidene difluoride membranes (PVDF; Millipore, Eschborn, Germany) and incubating the membranes with antibody solutions. Bound antibodies were detected by using the ECL Western-blotting analysis system from Amersham Buchler (Braunschweig, Germany).

Construction of hybridization probes for hybridization. Fragments containing all ndh genes (except ndhC) were subcloned from primary clones of the rice plastid DNA clone bank (Shimada et al. 1989) in pBluescript KS (Stratagene, Heidelberg, Germany) using standard cloning methods (Maniatis et al. 1982). The probe for ndhC was

277

obtained from maize plastid DNA (Steinmiiller et al. 1989). The exact locations of the subclones are listed in Table 2.

Northern analysis and quantification of transcript levels. The RNA from mesophyll and bundle-sheath cells was isolated as described earlier (Kubicki et al. 1994). The RNA was glyoxylated, separated on an agarose gel and then transferred onto nylon membranes (Biodyne A; Pall, Dreieich, Germany). Hybridization probes for single ndh genes were generated by in vitro transcription of the respective plasmid clones (Table 2) into antisense RNA in the presence of a-[32p]UTP (Amersham Buchler). The hybridization was carried out in 250 mM sodium phosphate (pH 7.2), 7% SDS and 2.5 mM Na/EDTA at 650C (Church and Gilbert 1984). The blots were washed three times in 1 • SSC (standard sodium citrate buffer; 0.15 M NaC1, 0.015 M Na3-citrate; pH 7.2) 0.1% SDS and then three times in 0.5 • SSC, 0.1% SDS at 65~ For the quantification of transcript levels, 100 ng RNA from each type of chloroplast was dotted onto nylon membranes and hybridized with the antisense RNA transcript. After a short exposure to X-ray film to localize the signals, the dots were excised from the membrane and counted in the Beckman scintillation counter LS 5000 CE (Beckman, Mfinehen, Germany).

Chlorophyll determination. The concentration of chlorophyll was determined according to Arnon (1949).

Results

Relative amounts o f N D H proteins are highly increased in bundle-sheath plastids compared to mesophyll plastids. In o rde r to ob ta in an t ibod ies agains t different N D H pro- teins, par t s of the read ing frames of N D H - H from tobacco and of N D H - J and -K from rice p las t id D N A were c loned into the express ion vector p G E M E X and the reby fused to the gene 10 of bac t e r i ophage T7 (Table 1). W h e n expressed in Escherichia coli, the cons t ruc ts led to high a m o u n t s of fusion pro te ins tha t were purif ied and used to genera te ant ibodies .

In tac t mesophyl l and bund le - shea th ch lo rop las t s f rom Sorghum were i so la ted by a new m e t h o d tha t reduces c ros s - con tamina t ion (Kubick i et al. 1994). By measur ing the activit ies of the mesophyl l -speci f ic N A D P - m a l a t e de- hydrogenase and the bundle-shea th-spec i f ic N A D P - m a l i c enzyme, the degree of c r o s s - c o n t a m i n a t i o n was ca lcu la ted to a m o u n t to 3% for bo th p repa ra t ions . The pro te ins were separa ted by SDS-gel e lec t rophores is and one gel was s ta ined with Coomass i e br i l l iant b lue to con t ro l aga in the pur i ty of the p r e p a r a t i o n s (Fig. 1, lane A). The mesophy l l

Table 1. Characteristics of NDH-H, -J and -K and the fusion constructs used for expression in E. coli. The sequences are from Shinozaki et al. 1986 (tobacco) and Hiratsuka et al. 1989 (rice)

NDH-H NDH-J NDH-K

Source tobacco rice rice Amino acids 393 159 246 Molecular mass 45487 18627 27681 Fusion construct pGX-H pGX-1275RJ pGX-758RKI Subcloned plastid DNA 123672 124910 47145-48420 48420-49178 fragment Restriction sites for PstI SalI BgllI - HindlII BgllI excission from plastid DNA Restriction sites for NsiI - SalI BamHI HindlII BamHI cloning in pGEMEX (pGEMEX1) (pGEMEX1) (pGEMEX2) Amino acids covered 38-393 (90%) 19-159 (88%) 45-246 (82%) in fusion construct (per cent of reading frame)

278

Table 2. Subclones of maize and rice plastid DNA used as gene-specific hybridization probes. The numeration of the sequences is according to Steinmiiller et al. 1989 (maize ndhC) and Hiratsuka et al. 1989 (rice ndh genes)

A. Kubicki et at.: Differential expression of ndh genes

Genes Subclone Fragment Nucleotides

ndhC (maize) pM169HHI HindIII 205-373 ndhK (rice) pR469HB HindIII - BglII 48709-49178 ndhJ (rice) pR376SB SnaB1 - BgIII 48044-48420 ndhH (rice) pR638EEI EcoRI 112993-113631 ndhA (rice) pR396HS HindIII SalI 110785-111181 ndht (rice) pR592DHI DraI - HincII 109916-110508 ndhG (rice) pR317ED EcoRI - DraI 109599-109916 ndhE (rice) pR353SE SspI - EcoRI 108551--108904 psaC (rice) pR668HSI HincII SspI 107883-108551 ndhD (rice) pR494PH PstI - HincII 107389-107883 ndhF (rice) pR895PS PstI - Spel 102325-103220 ndhB (rice) pR998HB HindIII BamHI 86913-87911

A B C D E F G

Fig. 1A-G. Western analysis of the expression of NDH proteins in leaves of Sorghum bicoIor. The proteins of mesophyll (M) and bundle sheath (B) chloroplasts were separated by SDS-gel electrophoresis (5 gg chlorophyll per lane). One gel was stained with Coomassie brilliant blue (A); proteins of other gels were transferred to polyvinylidene difluoride membranes and tested with antibodies against PSII-B (B), cytochrome f (C), PSI-D (D), NDH-H (E), NDH-K (F) and NDH-J (G)

preparation does not contain bundle-sheath plastids as can be seen by the distribution of the large and the small subunit of ribulose-bisphosphate carboxylase at 55 and 14kDa, respectively, which are dominant proteins of the bundle sheath chloroplasts. Other gels were then used for Western analysis to allow a semi-quantitative deter- mination of the expression of NDH-proteins. Antibodies against PSI and PSII subunits, as well as an antibody against a subunit of the cytochrome b6 / f complex, were included as controls.

As expected, the antibody against the PSII subunit PSII-B reacted only with mesophyll chloroplasts demon- strating that this chloroplast preparation is free from bundle-sheath plastids (Fig. 1, lane B). The antibodies against cytochrome f (Fig. 1, lane C) and PSI-D (Fig. 1, lane D) gave signals of comparable intensity with both chloroplast preparations. In contrast, the signals of the three antibodies against the N D H proteins H, J and K were very weak in the mesophyll; however, these signals were strongly increased in the bundle-sheath plastid prep- aration (Fig. 1, lanes E, F, G). All signals of the N D H proteins appear at molecular sizes which are in good agreement with the molecular masses deduced from se- quence analysis of the genes, i.e. NDH-H: 45 kDa, NDH-J: 18kDa and NDH-K: 29kDa (compare Table 1).

Transcript levels for all ndh 9enes are enhanced in bundle- sheath chloroplasts compared to mesophyll plastids. The differentiation of mesophyll and bundle-sheath cells is accompanied by the differential expression of genes en- coding components of the photosynthetic electron-trans- port chain and the Calvin-Benson cycle (Oswald et al. t990; Meierhoff and Westhoff 1993). The differential ex- pression of plastome-encoded genes is mainly regulated at the level of RNA abundance (Kubicki et al. 1994). To investigate, whether the expression of ndh genes is also regulated by mRNA abundance, the transcript levels of ndh genes were determined.

The 11 ndh genes of the plastid chromosome are ar- ranged in four transcriptional units that are shown in Fig. 2. According to Kanno and Hirai (1993) ndh genes are cotranscribed with several other genes. One cluster com- prises six ndh genes (ndhH-A-I-G-E and D) together with a component for PSI, the psaC gene (Fig. 2A). Another cluster contains the genes ndhC-K-J together with genes for subunits of the ATPase (Fig. 2B). The ndhB gene is part of a transcription unit that contains genes for two ribosomal proteins (Fig. 2C) while probably only ndhF (Fig. 2D) is transcribed monocistronically (no transcript was found by Kanno and Hirai 1993).

All ndh genes and the psaC gene were subcloned from rice or maize plastid DNA in the vector pBluescript KS to

A. Kubicki et al.: Differential expression of ndh genes

allow the generation of antisense RNA as a hybridization probe. The clones and their positions are listed in Table 2. Northern blots containing equal amounts of RNA from mesophyll and bundle-sheath chloroplasts were prepared and tested with the different labelled RNA probes. Con- trol hybridizations were carried out with psaA and psbB. Figure 3 shows that the transcript levels for the ndh genes A, B, D, E, F, G, H and I were increased in bundle-sheath plastids as compared to mesophyll plastids. Interestingly, the mRNA levels for the two genes encoding PSI subunits, psaA and psaC, were also higher in this plastid type. In contrast, the mRNA concentration for psbB was clearly higher in mesophyll plastids.

279

In order to quantify more accurately the concentrations of ndh transcripts in mesophyll and bundle-sheath plas- tids, RNA from both plastid types was dotted onto a nylon membrane and hybridized with the gene-specific hybridization probes. The amount of bound radioactive probe was then determined by liquid scintillation count- ing and the relative abundance of transcripts for each gene was calculated. Figure 4 shows that the transcript levels for all ndh genes, except ndhI, were increased by a factor of two to five in the bundle-sheath chloroplasts as com- pared to mesophyll plastids. In contrast, the m R N A con- centration for psbA was reduced in the bundle-sheath chloroplasts.

l I m m A

trnV n

m n

c [ ] I 1

ndhF

D [i! ii ', !ili!ii:i!i! i ii ii iiiiil , ,kb , /

Fig. 2A-D. Arrangement of ndh genes in transcription units as described by Kanno and Hirai (1993) for the rice plastid chromo- some. A The ndhH-A-I-G-E operon; B the ndhC-K-J operon; C the ndhB operon; D ndhF. Open boxes within the genes ndhA, ndhB, trnV and rpsl2 represent introns. Bars below the ndh genes indicate the positions of the gene fragments that were subcloned and used for the hybridization of the Northern and the dot blots

Discussion

The energy requirements for C3 photosynthesis are three ATP and two N A D P H molecules for each molecule of CO2 fixed. Since ATP and N A D P H are produced in stoichiometric amounts during linear electron flow, it is generally assumed that the extra ATP is generated by cyclic electron transport driven by PSI (Allen 1983). In C4 plants of the NADP-mal ic enzyme type the energy demand is even higher. For mesophyll chloroplasts the A T P / N A D P H ratio is 3:2, as for C3 plastids; however, bundle-sheath chloroplasts need an additional two mol- ecules of ATP for CO2 fixation (Hatch 1987). Therefore, bundle-sheath plastids have to establish a high capacity for cyclic electron transport.

According to the classical view, cyclic electron trans- port involves the action of PSI and the cytochrome b6/f- complex (Gimmler 1977). However, the part of the cyclic pathway from PSI back to the cytochrome b6/f-complex is not well defined. Bendall and co-workers have sugges- ted that ferredoxin does not reduce cytochrome b 6 directly

kb

5.1

3.5

2.0

1.6

0 .95

0.56

Fig. 3. Northern analysis of the expression of the ndh genes. The RNA from mesophyll (M) and bundle-sheath (B) chloroplasts was isolated and separated by agarose gel electrophoresis (7 lag RNA per lane). The RNA was transferred to nylon membranes and tested with gene-specific antisense RNA probes against the ndh genes

280

:f Relative amounts of transcripts [ ] Mesophyll [ ] Bundle Sheath 4 -

3 -

2-

1-

psbA H A I G E psaC D F B C K J

Fig. 4. Quantitative determination of the levels of transcripts for the different ndh genes. The RNA from mesophyll and bundle-sheath chloroplasts was dotted onto nylon membranes and hybridized with gene-specific antisense RNA probes against psbA, psaC and the 11 ndh genes. The amount of each transcript in mesophyll chloroplasts was set as one. The mean values from three different experiments are shown

and that a ferredoxin-plastoquinone oxidoreductase is involved (Bendall and Manasse 1995). Since in cyanobac- teria the NADPH-plastoquinone oxidoreductase is part of a ferredoxin-dependent and an NADPH-dependent cyclic electron-transport pathway around PSI (Mi et al. 1995), we have investigated whether the enzyme is also involved in cyclic electron transport in chloroplasts.

Our results demonstrate that in Sorghum the expres- sion of three different subunits of the NAD(P)H dehydro- genase is increased in the PSII-deficient bundle-sheath chloroplasts as compared to mesophyll plastids. Since both plastids contain PSI and the cytochrome b6/f com- plex in similar amounts (Oswald et al. 1990; Fig. 1, lanes 3,4), the increase in the amount of the NDH subunits indicates a relative increase of the NAD(P)H dehydro- genase in bundle-sheath plastids. We conclude from that finding that the enzyme is an essential component of the cyclic electron-transport pathway in bundle-sheath chloroplasts as well as in the chloroplasts of C3 plants. This conclusion is in agreement with the function of the enzyme as a proton pump supporting the transthylakoid pH gradient and thus serving the generation of ATP for CO2 fixation. In mesophyll plastids of C~ plants and in C3 plastids the amount of the enzyme is lower than in bundle-sheath chloroplasts because these plastids produce ATP also by linear electron transport.

Cyclic electron transport is very sensitive to a correct balance between the input of electrons and the extraction of electrons by the different reductive processes present in chloroplasts (Allen 1983). By inhibiting PSII activity with 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU) it has been shown that electrons derived from PSII usually pro- vide the necessary electrons for this redox poising (Arnon and Chain 1975). While in the rudimentary granal bundle- sheath chloroplasts of maize the residual PSII may deliver the electrons, it is not clear how reduction equivalents are fed into the cyclic electron-transport pathway in the agranal bundle-sheath chloroplasts of Sorghum which lack PSI! completely.

This question becomes even more complicated when one considers the molecular structure of the NAD(P)H dehydrogenase. The enzymes of cyanobacteria and chloroplasts are multimeric protein complexes that con- sist of at least 11 different proteins, as can be deduced from

A. Kubicki et al.: Differential expression of ndh genes

the gene content of both genomes (Ellersiek and Stein- m/filler 1992; Sugiura 1992). In contrast, the NADH- ubiquinone oxidoreductase of E. coli is composed of 14 different subunits and is considered to represent a minimal form of all complex I-homologous enzymes. The E. coli enzyme can be fragmented into three subfragments: an NADH-oxidizing fragment which contains three subunits, a connecting fragment of four subunits, and a membrane fragment with seven subunits. (Friedrich et al. 1995; Leifet al. 1995). The NAD(P)H dehydrogenases of cyanobacteria and chloroplasts differ from this typical complex I organ- ization in that they lack homologues of the subunits that make up the NADH-oxidizing fragment (Berger et al. 1993a). We have therefore proposed that the immediate electron donor for these enzymes is ferredoxin (Friedrich et al. 1995). From this view, the enzyme is probably identical to the ferredoxin-plastoquinone oxidoreductase identified by Bendall and Manasse (1995).

On the other hand, the demand for a correct redox poising of the cyclic pathway requires that an NADPH- oxidizing activity must be closely associated with the enzyme. Moreover, the experiments with the ndhB-defec- rive mutant of Synechocystis sp. PCC6803 have shown that in cyanobacteria electron flow from NADPH to the plastoquinone pool proceeds through the enzyme (Mi et al. 1995). We have proposed that ferredoxin-NADPH oxidoreductase (FNR) operating in reverse direction may represent this activity (Friedrich et al. 1995), since it has been shown that FNR is involved in the reduction of the plastoquinone pool in the dark (Mills et al. 1979). But the amount of FNR is reduced in bundle-sheath chloroplasts as measured by rocket immunoelectrophoresis or Western analysis (Broglie et al. 1984; and data not shown). Yet recently it has been shown that at least two different forms of FNR enzymes exist in leaves, a leaf-type FNR being involved in the photoreduction of NADP and a root-type FNR which mediates reverse electron flow from NADPH to ferredoxin (Jin et al. 1994). Since the amino acid se- quences of both FNRs of rice share only a similarity of 49% (Aoki and Ida 1994), and the antibodies used for the immunodetection experiments were directed against the leaf-type FNR, the root-type FNR is a likely candidate for the enzyme that oxidizes NADPH in cyclic electron trans- port in chloroplasts. However, it cannot be ruled out that the plastidial NAD(P)H dehydrogenase contains as yet unidentified subunits which mediate the oxidation of NAD(P)H. Therefore, the designation of the enzyme as an NAD(P)H-plastoquinone- or a ferredoxin-plastoquinone oxidoreductase has to be postponed until the enzyme has been purified and its activity has been unambiguously determined.

We thank Prof. Dr. M. Sugiura (Center for Gene Research, Nagoya University, Chikusa, Nagoya, Japan) for the rice plastid DNA clone bank, Pioneer Hi-Bred Inc. for Sorghum seeds and the Deutsche Forschungsgemeinschaft for financial support (SFB189).

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