Flower-Enhanced Expression of a Nuclear-Encoded Mitochondrial

The Plant Cell, Vol. 6, 439-448, March 1994 O 1994 American Society of Plant Physiologists

Flower-Enhanced Expression of a Nuclear-Encoded Mitochondrial Respiratory Protein 1s Associated with Changes in Mitochondrion Number

Jintai Huang,' Friedhelm Struck,b Dale F. Matzinger,' and Charles S. Levings 111 'I'

a Department of Genetics, Box 7614, North Carolina State University, Raleigh, North Carolina 27695-7614 lnstitut fÜr Genbiologische, Mascheroder Weg 1, D-38124 Braunschweig, Germany

The mitochondrial Rieske iron-sulfur protein is an obligatory component of the respiratory electron transport chain that is encoded by a single-copy gene in mammals and fungi. In contrast, this protein is encoded by a small gene family in dicotyledonous tobacco and monocotyledonous maize. We cloned four cDNAs from tobacco that encode the mitochondrial Rieske iron-sulfur protein. These clones, along with a previously isolated cDNA, represent five independent members of the gene family that can be divided into three subfamilies. All of these genes were derived from the two progenitor species and were expressed in amphidiploid tobacco. The proteins encoded by these five genes are probably functional because they all contain the universally conserved hexyl peptides necessary for the 2Fe-2s cluster formation. The expression of the Rieske protein gene family is differentially regulated; a 6- to 11-fold higher leve1 of steady state transcripts was found in flowers than in leaves, stems, and roots. Members of at least two subfamilies were preferentially expressed in flowers, indicating that they share a common cis-regulatory element(s), which can respond to a flower- specific signal(s). Although -10 times more transcripts occurred in flowers than in leaves, flower and leaf mitochondria contained a similar amount of the Rieske protein. Flowers, however, contained seven times more Rieske proteins than leaves. These results indicated an increase in mitochondrion number in flowers. High-energy demands during anther development might bring about an increase in mitochondrion numbers in flowers and the flower-enhanced expression of the Rieske protein gene family. Our results suggested that nuclear genes encoding mitochondrial respiratory proteins could sense and respond to changes in energy metabolism andlor changes in mitochondrion numbers.

INTRODUCTION

The mitochondrial electron transport chain of eukaryotes con- sists of four major multimeric enzyme complexes, one of which is the ubiquino1:cytochrome c oxidoreductase, commonly re- ferred to as the cytochrome bcl complex or complex III. Electron flow through the cytochrome bcl complex is coupled with a vectorial transmembrane proton translocation, which generates an electrochemical gradient that is subsequently used for the synthesis of ATP by the Fo/Fl ATPase located in the mitochondrial inner membrane (for review, see Trumpower, 1981). The enzyme complex has been purified from bacteria (Yang and Trumpower, 1986), fungi (Siedow et al., 1978; Weiss and Kolb, 1979), plants (Nakajimaet al., 1984; Berry et al., 1991; Braun and Schmitz, 1992), and mammals (Rieske et al., 1964). Although the number of subunits in the complex varies from as few as three in some bacteria to as many as 11 in some higher eukaryotes, all bcl complexes contain a core of three catalytic subunits, including cytochrome b, which has two heme groups, cytochrome cl, and the Rieske iron-sulfur protein (RISP) that contains a nonheme 2Fe-2s cluster.

To whom correspondence should be addressed.

The RlSP was first observed in isolated bcl complexes because of its nonheme iron content and its signature electronic paramagnetic resonance signal at g = 1.90 (Hatefi et al., 1962; Rieske et al., 1964) and was conclusively identified by isolation of this protein from the bcl complex by Rieske (1967). The function of the mitochondrial RlSP in catalyzing electron transfer through the cytochrome bcl complex is described by the Q-cycle (Mitchell, 1976; Trumpower, 1990). In this cycle, it is proposed that the RlSP transfers an electron from a reduced quinol to cytochrome c1 with the concomitant generation of a ubisemiquinone as a substrate for cytochrome b. Reconstitu- tion experiments and genetic analysis have shown that the RlSP is absolutely required for mitochondrial respiration (Trumpower and Edwards, 1979; Beckmann et al., 1989).

In all eukaryotes investigated to date, the mitochondrial RlSP is encoded by a nuclear gene, translated as a precursor protein in the cytoplasm, and post-translationally imported into mitochondria. The nuclear genes encoding the mitochondrial RlSP have been cloned from Neurospora (Harnisch et al., 1985), Saccharomyces cerevisiae (Beckmann et al., 1987), rat (Nishikimi et al., 1989), human (Nishikimi et al., 1990), and cow

440 The Plant Cell

(Usui et al., 1990). In all organisms studied, the mitochondrial RlSP appears to be encoded by a single gene, although multiple forms of mRNA have been suggested (Nishikimi et al., 1989). We have previously cloned cDNAs encoding the mitochondrial RlSP from maize and tobacco and, by using yeast as a model system, identified structures and domains impor- tant for protein function (Huang et al., 1991). Here, we report the isolation and characterization of several cDNAs encoding the mitochondrial RlSP from tobacco. Unlike the RlSP in fungi and mammals, the plant mitochondrial RlSP is encoded by a gene family. All protein products encoded by the tobacco RlSP gene family are potentially functional. We also showed that the expression of the RISP gene family is differentially regulated; both gene transcripts and protein levels increased in flowers. The flower-enhanced RISP gene expression was associated with an increase in mitochondrion number in flowers. Our results suggested that nuclear genes encoding mitochondrial respiratory proteins could sense and respond to changes in energy metabolism and/or changes in mitochondrion numbers in an organ- or tissue-specific manner.

RESULTS

Mitochondrial RlSP 1s Encoded by a Gene Family in Higher Plants

To determine the number of genes coding for the mitochondrial RlSP in tobacco, genomic DNA was isolated from tobacco leaves and characterized by DNA gel blot analysis (Southern, 1975) by using a 987-nucleotide partial RlSPcDNA cloned from a tobacco leaf cDNA library (Huang et al., 1991) as a probe. As many as five bands could be detected using five different restriction enzymes, suggesting that the mitochondrial RlSP is encoded by several genes in tobacco (data not shown). Simi- lar analysis using a 277-nucleotide Sstl-Hindlll fragment from the 3' end of the maize RlSP gene (Huang et al., 1991) as a probe also revealed multiple bands in the maize genomic DNA gel blot (data not shown). Detection of multiple bands by a rel- atively small probe with a number of different restriction enzymes suggested that the mitochondrial RlSP is encoded by more than one gene in maize as well. The sequences of the hybridizing probes are significantly different from that of the tobacco chloroplast RlSPgene (Madueiío et al., 1992), and DNA blot analyses confirmed that the mitochondrial RlSPgene did not cross-hybridize with its chloroplast counterpart (data not shown). These results suggest that the mitochondrial RlSP is encoded by a gene family in higher plants.

Cloning of cDNAs Coding for the Mitochondrial RlSP of Tobacco

A cDNA library was constructed from tobacco flowers (see Methods) and screened by probing with lhe 987-nucleotide

partial cDNA sequence (Huang et al., 1991). Approximately 300 positive isolates were identified in the first round of screening. Twenty of the positive isolates were randomly selected and purified as single plaques by a second round of screening. The 20 clones contained inserts ranging from 0.9 to 1.3 kb in length. These clones were classified as four distinct groups by restriction endonuclease analysis (data not shown). Clones with the longest inserts were chosen from each group and characterized by DNA sequencing analysis. The four cDNA clones-RISP2, RISP3, RISP4, and RISPS-are very similar to each other and to the previously cloned leaf cDNA (Huang et al., 1991), designated here as RISPI. Nevertheless, com- puter analysis of these DNA sequences (data not shown) and their deduced protein sequences (Figure 1) confirmed that each of these five cDNA clones represented a unique member of the gene family. In addition, pairwise sequence comparisons, especially in the N-terminal region, suggested that these five cDNAs fel1 into three subfamilies, with RISPl and RISP2 in one subfamily, RISP3 and RISPS in a second subfamily, and RISP4 representing a third subfamily (Figure 1).

Each sequenced cDNA contains an open reading frame that could encode a polypeptide of 236 to 272 amino acids (Figure 1). The first methionine-encoding codon of the open reading frame in each cDNA, except for RISPI, is most likely the trans- lation initiation codon, because it is preceded by a1 least one in-frame stop codon (data not shown). Comparison of RISP1 and RlSP2 (Figure 1) suggests that RISP2 is a complete cDNA, whereas 14 codons are missing from the N terminus of the RISPl clone. The first 70 amino acids of RISP1, RISP2, RISP3, and RlSP5 are rich in positively charged and hydroxylated residues (Figure 1) and have the potential to form amphipathic a-helices (data not shown). These features are characteristic of mitochondrial presequences (von Heijne, 1986; Hartl et al., 1989). Indeed, import studies in vivo employing reporter proteins have shown that these proteins are specifically localized to mitochondria (J. Huang and C.S. Levings 111, manuscript in preparation). The N-terminal region of RlSP4 is shorter and does not contain any charged residues. Nevertheless, import studies in vivo suggested that RISP4 is also localized to mitochondria (J. Huang and C.S. Levings III, manuscript in preparation). The 5' sequences, including the untranslated leader sequence and the putative presequence coding region of RISP4, are distinct from those of RISP2. Starting from nucleotide 384 (numbered according to the RISP4 sequence), however, RISP4 is identical to the corresponding region of RISP2 at both amino acid sequence (Figure 1) and DNA sequence (data not shown) levels, suggesting that RISP4 arose by a duplication of RISP2 followed by a nonhomologous recom- bination. A genomic clone corresponding to the RISP4 cDNA was isolated from a genomic library, and a 1.7-kb fragment from this clone was characterized by DNA sequencing (D. Cho, J. Huang, and C.S. Levings 111, unpublished results). This 1.7-kb fragment contained most of the coding region of RISP4 and -1 kb of 5'upstream sequence, including the putative recom- bination junction. This result indicated that the RISP4 is a bona fide cDNA rather than a cloning artifact. All five RlSPs contain

Differential Expression of the RISP Gene Family 441

RISP2 MLRIAGRRASSLSRWPVRSVAPSSSAFISANHFSSDDDSSSP-RSISP-SLASVFLHHTRGFSSNS 64RISP1 .....A.............................................. 50RISP5 ...... .K---..--SSAAAR-.....FTR.P.TFT......T..P..T....Q..DQF. ...... 60R1SP3 ...... .K---. .--SSAATR-. . . ..FTR.P.TFT. .....A..P..A....Q. .DQF. ...... 60RISP4 MINFGSCWGLA.VT.NSFSIIS. . . . . . 28

RISP2 VSHAHDMGLVPDLPPTVAAIKNPTSKIVYDEHNHERYPPGDPSKRAFAYFVLTGGRFVYASLVRLL 130RISP1 . .P. ......................................................... .M... 116RISP5 •- p- -QT---S...A........S......DS. ................................. 126RISP3 .-P..QT...S...A....... .S. .....DS. ................................. 126RISP4 .................................................................. 94

RISP2 ILKFVLSMSASKDVLALASLEVDLSSIEPGTTVTVKWRGKPVFIRRRTEDDINLANSVDLGSLRDP 196RISP1 ....................................................S. ............ 182RISP5 ................................................DE. ............... 192RISP3 ................................................ .E. ............... 192RISP4 .................................................................. 160

RISP2 QQDAERVKSPEWLWIGVCTHLGCIPLPNAGDFGGWFCPCHGSHYDISGRIRKGPAPYNLEVPTYS 262RISP1 ........N......................................................... 248RISP5 ........N..................................................M...... 258RISP3 ........N................................................. .L. ..... 258RISP4 .................................................................. 226

RISP2 FLEENKLLIG 272RISP1 .......... 258RISP5 .M........ 268RISP3 .M........ 268RISP4 .......... 236

Figure 1. Alignment of the Deduced Amino Acid Sequences of the Tobacco RISPs.

Amino acids are designated by the single-letter code. Dots represent an amino acid identical to that found in RISP2. The absence of an aminoacid is designated by a dash. The asterisks denote the C termini of the proteins. The two universally conserved hexyl peptides involved in 2Fe-2Scluster formation are bracketed. The nucleotide and deduced amino acid sequences have been submitted to GenBank as accession numbersL16810, RISP2; L16811, RISP3; L16812, RISP4; and L16813, RISPS.

the two universally conserved hexyl peptides CTHLGC andCPCHGS (Figure 1) that are involved in the 2Fe-2S clusterformation (Graham and Trumpower, 1991; Davidson et al.,1992). Together, these studies indicated that all of these RISPsare potentially functional in plant cells.

Mitochondria! RISP Genes from Both Progenitors AreTranscribed in Tobacco

Tobacco (Nicotiana tabacum) is an amphidiploid plant, probablyderived from the two progenitor species N. tomentosiformisand N. sylvestris (Gerstel, 1966; Gray et al., 1974). To determinewhether all of the RISP genes from both progenitors are tran-scribed in amphidiploid tobacco, genomic DNAs isolated fromN. tabacum, N. tomentosiformis, and N. sylvestris were charac-terized by DNA gel blot analysis using cDNA probes. Whena full-length cDNA (RISP2) was used as the probe, three bandswere detected in both N. tomentosiformis and N. sylvestris, andfive bands were detected in N. tabacum (Figure 2A). If the 3.6-kbfragment in the tobacco is a doublet (Figure 2A), the bandingpattern of N. tabacum is the simple sum of the two progenitorspecies. This result indicated that genes coding for the mito-chondrial RISPs from both progenitors are transcribed in

B

13 —

4.6 —3.6 —

2.9 —

2.0— —

Full Length RISP1/2 RISP3/5 RISP4

Figure 2. Genomic DNA Blot Analysis of Tobacco and ProgenitorSpecies.

Fifteen micrograms of genomic DNA of W. tabacum (Tob), N. sylves-tris (Syl), and N. tomentosiformis (Tom) was digested by Hindlll. Themembranes were probed as given below.(A) Full-length cDNA clone RISP2.(B) fl/SP7/fl/SP2-specific probe.(C) fl/SP3/fl/SP5-specific probe.(D) R/SP4-specific probe.Numbers at left indicate the lengths of the detected fragments inkilobases.

442 The Plant Cell

tobacco and that the gross genomic structure of the progenitorgenes is maintained in the amphidiploid tobacco.

To confirm that all progenitor genes are transcribed intobacco, we performed detailed genomic DMA blot analysisusing specific probes. Because the greater similarity betweengenes within the same subfamily makes it difficult to designprobes specific for each individual, we used probes that werespecific only for subfamilies (see Methods). The probe 3.5-4,specific for RISP1 and RISP2, hybridized to one fragment eachin N. tomentosiformis and N. sylvestris but two fragments inN. tabacum (Figure 2B). This hybridization pattern indicatedthat each progenitor contains either RISP1 or RISP2 but notboth and that both progenitor genes are transcribed in the am-phidiploid plant. Similarly, the RISP3- and fl/SP5-specific probe2-3 also hybridized to one fragment in each progenitor but twofragments in N. tabacum, demonstrating that RISP3 was de-rived from one progenitor and RISP5 from the other and thatboth are transcribed in N. tabacum (Figure 2C). The RISP4gene, however, was detected only in N. sylvestris and N. taba-cum. A sequence corresponding to RISP4 is apparently missingfrom N. tomentosiformis (Figure 2D). The identity of the 4.6-kbfragment detected in N. tabacum and N. tomentosiformis isunclear (Figure 2A), although it may represent a gene mem-ber that has been missed in our cloning. These resultsconfirmed that all five progenitor genes cloned are structur-ally maintained and transcribed in amphidiploid tobacco.

Accumulation of RISP Gene Transcripts Is EnhancedSpecifically in Tobacco Flowers

To study the expression and regulation of the RISP gene fam-ily in tobacco, we first investigated their steady state transcriptlevel in different organs. Total RNAs were isolated from flowers,leaves, stems, and roots and characterized by RNA gel blotanalysis. Figure 3A shows a typical RNA blot using thedigoxigenin-11-dUTP-labeled RISP2 as a probe, which can hy-bridize to all five RISP gene transcripts. A diffuse band of ~1.25kb was detected in all four organs (Figure 3A), showing thatall RISP genes have similar sized transcripts. The steady statetranscript level, however, varied greatly among different organs.Low expression was detected in leaves, stems, and roots (Fig-ure 3A). In contrast, expression was much greater in flowers;the steady state mRNA level was 6- to 11-fold higher in flowersthan in all other organs examined (Figure 3A). The increasedaccumulation of RISP transcripts in flowers, compared withleaves, ranged from 3- to 10-fold in gel blot analyses from differ-ent RNA isolations (data not shown); the range in expressionwas probably associated with differences in flower develop-mental stages (J. Huang and C.S. Levings III, unpublishedresults). Figure 3B shows the ethidium bromide (EtBr)-stainedgel used for preparing this RNA blot analysis and demonstratesthe relative loading in each lane. This result showed that theexpression of the mitochondrial RISP gene family is differen-tially regulated with respect to its spatial localization.

1.25 —

Figure 3. RNA Gel Blot Analysis of the RISP Gene Expression inTobacco Organs.

Each lane contains 15 ng of total RNA, as determined by spectra-photometry.(A) An autoradiogram probed with the full-length cDNA clone RISP2,with the apparent molecular length of the transcript given in kilobases.(B) Photograph of the EtBr-stained gel used for preparing the blot anal-ysis shown in (A). The positions of the 26S and 18S rRNA are asindicated.

More Than One RISP Gene Is Preferentially Expressedin Flowers

The high level of steady state transcript of the RISP family inflowers could be due either to the preferential expression ofa specific family member or to the increased expression ofmore than one RISP gene. To discriminate between these pos-sibilities, oligonucleotide probes specific for subfamilies (seeMethods) were employed for a more detailed RNA gel blot anal-ysis. When the oligonucleotide probe RP92M, which is specificfor RISP1 and RISP2, was used for RNA blot analysis, a dif-fuse band of ~1.25 kb was detected in all four organs (Figure4A). Quantitative differences were detected in the steady statetranscript levels; expression was high in flowers but low instems, leaves, and roots. The same blot was stripped andprobed with the oligonucleotide RP92N. Probe RP92N, whichis specific for RISP3 and RISP5, also detected the 1.25-kb bandin all four organs (Figure 4B). As found with RISP1 and RISP2,the expression of RISP3 and RISP5 was higher in flowers thanin other organs. A control blot using an 18S rDNA gene probedemonstrated that similar amounts of RNA were loaded in eachlane (Figure 4C). Even though three different oligonucleotideprobes specific for RISP4 (see Methods) were used to probethe same blot after stripping, no detectable signal was foundin any of the four organs, even after prolonged exposure (datanot shown). The reason for the absence of the RISP4 transcript,as revealed by the RNA gel blot analysis, is unclear at pres-ent, although it may be related to its peculiar gene structure.

Differential Expression of the RISP Gene Family 443

These results showed that the higher accumulation of the RISPgene transcripts in flowers resulted from increased expressionof members of the two RISP subfamilies.

Leaf and Flower Mitochondria Contain SimilarAmounts of RISP, but Flowers Contain More RISPThan Leaves

RNA gel blot analysis indicated that the level of the RISP genetranscripts was 6- to 11-fold higher in flowers than in other or-gans (Figure 3). Immunoblot studies were conducted todetermine if a comparable increase in expression could bedetected at the translational level. Because the transcript levelwas similar in leaves, stems, and roots, a comparison was madebetween leaves and flowers only. Mitochondrial proteins wereseparated by SDS-PAGE, transferred to a nitrocellulose mem-brane, and probed with a polyclonal antiserum raised againstthe tobacco mitochondrial RISP (see Methods). The antise-rum detected a single band of ~22 kD in purified mitochondriafrom both leaves and flowers (Figure 5A, lanes 1 and 2), indi-cating that all of the processed RISPs have a similar molecularsize. The same band was also immunoreactive to the antibodyraised against the Neurospora mitochondrial RISP (data notshown). Moreover, this band was not detected when the preim-mune serum was used in a similar assay (data not shown),demonstrating the specificity of the antiserum. The antiserumalso cross-reacted with protein bands of similar size from wheat,pea, bean, and soybean mitochondria (data not shown). In con-trast to the 10-fold difference detected in the steady statetranscript levels, the amount of the RISP was found in repeated

Mito. ExtractsLf Fl Lf Fl

B

RISPl/2

RISP3/5

18S

Figure 4. RNA Gel Blot Analysis of RISP Subfamily Expression inTobacco Organs.Each lane contains 15 ng of total RNA from the leaf, stem, root, andflower, as determined by spectrophotometry.(A) Gel blot probed with an oligonucleotide specific for RISP1IRISP2.(B) Gel blot probed with an oligonucleotide specific for RISP3IRISP5.(C) Gel blot probed with the 18S rDNA gene of C. maxima.

B

•RISP

- ATPase-oc

— ATPase-(3

Figure 5. Immunoblot Analysis of the RISP, ATPase a Subunit, andATPase p Subunit in Isolated Mitochondria and Total Cellular Extracts.(A) Analysis of RISP. Lanes 1 and 2 contain 30 ng of proteins of puri-fied mitochondria from leaves (Lf) and flowers (Fl), respectively. Lanes3 and 4 contain 15 nL of leaf total cellular extracts (Lf) and 15 nL offlower total cellular extracts (Fl), respectively. The blot was probed bya polyclonal antiserum raised against the tobacco RISP.(B) Analysis of ATPase a subunit. The amount and source of proteinsloaded in each lane are as in (A). The blot was probed with a poly-clonal antiserum raised against the maize ATPase a subunit.(C) Analysis of ATPase (5 subunit. The amount and source of proteinsloaded in each lane are as in (A). The blot was probed with a poly-clonal antiserum raised against the N. plumbaginifolia ATPase p subunit.

analyses to be the same or to differ by no more than 30% be-tween mitochondria from leaves and flowers (Figure 5A, lanes1 and 2). Importantly, when total cellular protein extracts fromleaves and flowers were analyzed on immunoblots, a seven-fold increase in the amount of RISP was found in flowers (Figure5A, lanes 3 and 4). The membrane blot was stained by Pon-ceau S before immunoblot analysis to confirm that similaramounts of protein were loaded (data not shown). This resultindicated that flower cells contain more RISP than leaf cells,even though flower and leaf mitochondria contain similaramounts of RISP. Furthermore, the greater amounts of RISPfound in flower cells than in leaf cells suggest that flowers alsocontain more mitochondria than leaf cells.

To show that flowers indeed contain more mitochondria thanleaves, identical blots, as shown in Figure 5A, were probedwith an antiserum against the mitochondrially encoded ATPasea subunit and the nuclear-encoded ATPase p subunit. As foundwith the RISP, purified mitochondria from leaves and flowerscontained similar amounts of ATPase a subunit (Figure 5B,lanes 1 and 2), whereas considerably more protein was presentin total cellular protein extracts from flowers (Figure 5B, lane4) than from leaves (Figure 5B, lane 3). A similar pattern ofprotein distribution was also observed with the nuclear-encoded ATPase p subunit (Figure 5C). Together, these results

444 The Plant Cell

supported the conclusion that tobacco flowers contain more mitochondria than leaves.

DISCUSSION

The mitochondrial RlSP gene has been cloned and characterized from several species. In all organisms studied, the mitochondrial RlSP is encoded by a single gene, although multiple forms of mRNA have been suggested, probably due to transcriptional or post-transcriptional modification (Nishikimi et al., 1989). We found that the mitochondrial RlSP is encoded by a small gene family in tobacco; this family can be divided into three subfamilies (Figure 1). Analysis of maize genomic DNA indicated that the mitochondrial RlSP is encoded by a gene family in this monocotyledonous diploid species as well. Together, these results suggested that the mitochondrial RlSP is encoded by a small gene family in these two plants, which is in marked contrast to fungi and mammals in which the mitochondrial RlSP is encoded by a single gene. DNA gel blot analyses of tobacco and its progenitors also showed that the gross genomic structures of the progenitor genes are maintained in tobacco and that the five cloned progenitor genes are transcribed in amphidiploid tobacco (Figure 2). The five tobacco RlSPs are very similar to each other in the mature protein region (Figure l), with amino acid identities as high as 99% when comparisons were made within subfamilies. Al- though differences at the nucleotide level are much higher, many of these changes are silent and did not result in amino acid changes. Because a high ratio of silent-to-nonsilent nucleotide substitutions was observed for the RlSP gene family, the maintenance and expression of RlSP progenitor genes in tobacco may occur because of a selective advantage (Pichersky et al., 1986).

The three RlSP subfamilies differ significantly in their presequences. Because mitochondrial presequences often differ substantially in primary amino acid sequences, it is unclear if the presequence differences have any effect on RlSP function in terms of targeting efficiency and submitochondrial localization. Some differences occur in the mature protein sequences of the RISP, especially in the N-terminal region (Figure 1). This region may delineate species specificity and is essential for the RlSP function (Huang et al., 1991). If these sequence het- erogeneities lead to conformational differences in the RISP, it could affect RlSP function per se or the protein’s interaction with other subunits of the cytochrome bc, complex, thereby altering the function of the bc, complex. Variation among subunit isoforms of an enzyme complex provides the opportunity for differential responses to developmental and environmental stimuli in different plant organs and/or tissues. For example, this diversity may furnish a mechanism for the wide occurrence of alloplasmic male sterility in interspecific crosses in plants in which the mitochondrial defects are manifested only in the male reproductive organ.

Consistent with the essential function of the mitochondrial RISP, the gene transcript was detected in every tobacco organ examined (Figure 3). On the other hand, the expression was clearly regulated by an organ- or tissue-specific mechanism(s) (Figure 3). Furthermore, RNA gel blot analyses using subfamily-specific probes showed that at least two of the three subfamilies exhibited the same pattern of elevated expression in flowers (Figure 4), suggesting that these two subfamilies share a common cis-regulatory element(s), which can respond to a signal(s) marking a flower-enhanced expression. Together, these results indicated that the mitochondrial RlSP gene family is constitutively expressed at a basal level in tobacco plants and that the expression is also differentially regulated in an organ- or tissue-specific manner. The plant mitochondrion is functionally more versatile than animal mitochondria (Dennis, 1987), and the diversity in function of plant mitochondria may reflect differences in gene expression in different organs and/or tissues (Newton and Walbot, 1985; Lind et al., 1991; De Paepe et al., 1993) in response to various developmental or environmental stimuli. Gene expression of individual members of the RlSP family in higher plants may be differentially regulated to meet such stimuli.

Microsporogenesis is a high-energy-demanding process that may impose stress on anther development (Warmke and Lee, 1978). Because microspores are nonphotosynthetic and contain only undifferentiated plastids and amyloplasts, energy in the form of ATP required for pollen development must be supplied mainly by mitochondria. In this respect, it is signifi- cant that the steady state transcript level of the RlSP gene increases in flowers. In light of the central role of the RlSP in electron transport and energy production, the flower-enhanced accumulation of the RlSP transcripts may reflect a response of the gene family to the high-energy demand of anther development. Flower mitochondria could meet the high demand for energy either by increasing their metabolic activity to gener- ate more ATP per mitochondrion or by increasing their number per cell so that more ATP is produced.

Severa1 studies have shown that increases in mitochondrion numbers occur in certain reproductive cells. A 20- to 40-fold increase in mitochondrion number has been found in some anther cells in maize (Warmke and Lee, 1978). The increased expression of several mitochondrial genes in maize microspores was proposed to result from a gene dosage effect caused by either the amplification of specific mitochondrial genes or an increase in mitochondrion number (MonBger et al., 1992). Also, mitochondria were reported to be much more abundant in gametophytic cells than in other cell types in N. sylvestfis, a progenitor of tobacco (De Paepe et al., 1993). Im- munoblot analysis showed that flower and leaf mitochondria contained similar amounts of the RlSP (Figure 5A, lanes 1 and 2), even though the level of steady state transcripts was greatly increased in flowers (Figure 3). This apparent discrepancy could be explained if a large increase in mitochondrion number per cell occurs in some flower cells in tobacco. An increase in mitochondrion number predicts greater amounts of RlSP

Differential Expression of the RlSP Gene Family 445

in flowers than in leaves when equal amounts of total cellular extracts are compared. Indeed, comparable with the 10-fold increase in the steady state transcript level, a sevenfold increase in the amount of RlSP was found in flowers versus leaves (Figure 5A, lanes 3 and 4). lmmunoblot analyses of two additional marker proteins, the ATPase a and p subunits, confirmed that flowers contain more mitochondria than leaves (Figure 56 and 5C). Because these two proteins are involved in ATP production that is coupled with respiratory electron transport under physiological conditions, these data are consistent with the idea that the increased expression of the RlSP in tobacco flowers is regulated by an organ- or tissue-specific signal(s) that may be associated with the high-energy demand during anther development. Our study suggests that nuclear genes coding for mitochondrial respiratory proteins can sense and respond to changes in energy metabolism and/or to changes in mitochondrion numbers in an organ- or tissue- specific manner.

METHODS

Plant Materials

Tobacco species used in this study were Nicotiana tabacum cv SC58, N. mmenmsifomis, and N. syhestris. Plants were maintained in a greenhouse, a growth chamber (25% 16-hr light; 16%, 8-hr dark), or in complete darkness (27°C) when etiolated tissues were used.

Construction and Screening of a Flower cDNA Library

Total RNA was isolated as described previously by Logemann et al. (1987) from flowers (stages 1 to 6 Koltunow ei al., 1990) of fertility- restored N. tabacum plants containing a male-sterile cytoplasm. An mRNA-enriched fraction was prepared using an oligo(-dT) cellulose (Stratagene) column as described previously by Aviv and Leder (1972). The flower cDNA library was constructed using the ZAPII cDNA cloning system (Stratagene) essentially as recommended by the manufacturer. The size of the primary library was 1.1 x 106 plaque- forming units. The primary library was amplified once, and the titer of the amplified library was 2.9 x 1Ol0. An aliquot of the amplified library equivalent to five sizes of the primary library (5.5 x 106 plaque-forming units) was screened according to standard procedures (Ausubel et al., 1987; Sambrook et al., 1989) with a partia1 cDNA clone encoding the tobacco Rieske iron-sulfur protein (RISP) (Huang et al., 1991) labeled with U-~~P-~ATP (New England Nuclear-Du Pont) by the polymerase chain reaction (PCR) (Saiki et al., 1985). Positive isolates identified in the first screening were purified as single plaques by a second round of screening. Phagemids were recovered from positive clones by an in vivo excision procedure following the instructions in the Stratagene manual. The cDNA clones were classified into groups based on restriction analysis.

DNA Sequencing

The insert of a cDNA clone in the original vector pBluescript SK- (Stratagene) was subcloned into pBluescript KS- in the opposite

orientation, and a nested, unidirectional deletion was made of each orientation (Heinrich, 1987). Single-stranded DNA was prepared (Vieira and Messing, 1987), and the complete nucleotide sequences of the cDNA clones were determined for both strands by the dideoxy chain termination method (Sanger et al., 1977) using a DNA sequencing kit (Sequenase Version 2.0; U.S. Biochemical Corp.).

DNA lsolation and Blot Analysis

Total genomic DNAs were isolated from leaves of plants grown in the greenhouse (Dellaporta et al., 1983). Fifteen micrograms of DNA was digested with restriction enzymes for 6 to 8 hr, resolved on an 0.8% agarose gel, transferred to nylon membranes (Nytran; Schleicher & Schuell) as described previously by Southern (1975), and fixed to membranes by either baking at 80% for 1 hr or by UV cross-linking with the Spectro Linker (American Bioanalytical, Westbury, NY). The membrane blots were incubated in a solution containing 5 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M NaH,PO,), 50% formamide, 0.1% sar- kosyl, 0.029/0 SDS, and 2% blocking reagent (Boehringer Mannheim) at 42% for 2 to 4 hr. Double-stranded DNA probes labeled with digoxigenin-11-dUTP (Boehringer Mannheim) by PCR were added to the incubation to a final concentration of 20 nglmL, and incubation was continued for 14 to 18 hr. The blots were then washed twice in 2 x SSC, 0.5% SDS, and twice in 0.1 x SSC, 0.1% SDS at 68%. The subfamily-specific probes were subclones derived from the nested, unidirectional deletions (see above) that contained the unique 5' untranslated region of the cDNAs. The subclone 3.54 that is specific for RISPl and RlSf2 contained nucleotides 1 to 340 of RISf2. The subclone 2-3, specific for RISf3 and RISf5, contained nucleotides 1 to 311 of RISP3. The subclone specific for RISP4 contained nucleotides f to 401 of the RISP4 gene. Hybridization was detected by antidigoxigenin-alkaline phosphatase conjugate using the Genius system (Boehringer Mannheim). Color was allowed to develop generally from 24 to 48 hr before photographs were taken.

RNA lsolation and Blot Analysis

Total RNAs were isolated from tobacco leaves, stems, rmts, and flowers as described previously by Chomczynski and Sacchi (1987). Fifteen micrograms of total RNA from each sample was resolved on a 1.3% formaldehyde agarose gel (Goldberg, 1980). Ethidium bromide (EtBr) was added directly to samples to a final concentration of 50 pg/mL before loading. After the dye had migrated into the gel, the top portion of the gel that contained the EtBr was excised to reduce the gel background. After electrophoresis, a photograph was taken to document the relative loading in each sample. The gel was used directly for transfer in 10 x SSC to a Nytran membrane without further treatment. RNA was covalently bound to the Nytran membrane by UV cross-linking. The cDNA clone 2.2 (RISP2) was labeled with digoxigenin-11-dUTP by PCR and used for hybridization. The conditions were the same as for DNA blot analysis, except that Lumi-Phos 530 (Boehringer Mannheim) was used as the substrate for chemiluminescent detection. The oligonuclew tide probes used for the gene-specific RNA blot analyseswere RP92M (5'-CATCTTCTCCTCTTTGGGTGAGCTCGTTAT-3: specific for RISf 7 and RISP2), RP92N (5'-CTTGAGA(3TTGlGATTTAGGGTTTCGClW-3: specific for RISP3 and RISP5), and three RISP4-specific oligonucleotide probes RP93M1 (5'-CGCACAATATCCACCTCGAATGTTTGCATG-3?, RP93M2 (5'-AGTAACAGATGCCAAACCCCAGCAACTTCC-3'), and RP93N (5'-TCCAATACCTTACGAGGCGCTATTATTCAC-3').

446 The Plant Cell

The oligonucleotide probes were labeled at the 5' end with y3'P- ATP using the T4 polynucleotide kinase (Sambrook et al., 1989). Hy- bridization conditions using oligonucleotide probes were the same as for the DNA blot analysis. The blot was washed twice at room temperature in 2 x SSC, O.lO/o SDS, twice at 55OC in 2 x SSC, 0.1% SDS, and exposed to Kodak XR5 film with an intensifying screen at -80°C. The 18s rDNA gene of Cucurbita maxima (Kelly et al., 1990) was used as a control to monitor RNA loading. Hybridization signals were quan- titated by using a densitometer (Soft Laser Scanning Densitometer Model SLR9D/lD; Biomed Instruments, Inc., Fullerton, CA).

Preparation of a Polyclonal Antiserum against RlSP

A 750-bp Bglll-EcoRI fragment from the RlSf7 was subcloned into the expression plasmid pET5a (Novagene, Madison, WI). The resulting plasmid, pET/RISP, was expressed in Escherichia coli HMS174 (Novagene) essentially as recommended by the supplier. A protein band with an expected size of 20 kD was readily detectable by Coomas- sie Brilliant Blue R250 staining and identified as the RlSP by immunostaining (data not shown) with an antiserum raised against the Neumspora RlSP (kindly provided by U. Harnisch, lnstitut für Bio- chemie, Universitat Düsseldorf, Germany). The antigen was resolved by SDSPAGE (Laemmli, 1970), transferred to and excised from nitrocel- IuIose membranes (Schleicher €i Schuell), dissolved in dimethyl sulfoxide, and used to immunize rabbits according to standard procedures (Harlow and Lane, 1988). Preimmune sera were collected before the first injection and used as controls for immunodetection experiments. The second booster injection was given 2 weeks after the first injection. The antiserum collected 10 days after the second booster injection was used in this experiment.

Cell Fractionation and Protein Blot Analysis

Mitochondria were isolated from tobacco, maize, wheat, and pea as described previously by Huang et al. (1990). Total cellular protein extracts were isolated by grinding an equal amount of leaf or flower tissues in an identical volume of 2 x Laemmli gel loading buffer (Laemmli, 1970). The homogenate was then immediately boiled for 5 min and spun in a microcentrifuge for 5 min. The supernatant was saved for protein analysis. An equal volume of total cellular extracts rather than an equal amount of proteins was used for immunoblot analysis to avoid overestimation of leaf proteins caused by large amounts of chloroplast proteins in leaves. Thirty micrograms of mitochondrial proteins or 15 pL of total cellular extracts was separated on a 12% SDS-polyacrylamide gel (Laemmli, 1970) and transferred to nitrocellulose membranes (Towbin et al., 1979). The blots were blocked in TBST(50 mM Tris-HCI, pH 8, 150 mM NaCI, 0.1% Triton X-100) containing 1% BSA for 2 hr. The anti-RISP antiserum was added to the blocking solution (1:4000 Ivhr]), and incubation was continued at room temperature from 2 hr to overnight. The goat anti-rabbit IgG (whole molecules) peroxidase conjugate (Sigma) was used as the secondary antibody (1:5000 [v/v]), and the ECL system (Amersham) was used for detection.

ACKNOWLEDGMENTS

We thank Drs. Bernard Trumpower and Jim Siedow for valuable dis- cussions and comments on the manuscript; Dr. Albert Siegel for the

18s rDNA clone; Dr. Marc Boutry for the anti-ATPase p antiserum; Drs. John C. Gray and F. N. Madueiio for the chloroplast RlSP cDNA clone; Dr. Earl Wernsman for providing plant materials; Caro1 Griffin, Jane Suddith, and Mary Clark for excellent technical assistance; Wilma Hu for preparing photographs; Suzanne Quick for editorial assistance; and membeffi of the Levings laboratory for critical reading of the manuscript. This work was supported, in part, by grants from the National Science Foundation to C.S.L. and from the R. J. Reynolds Tobacco Company to J.H. and D.F.M.

Received November 10, 1993; accepted January 24, 1994.

REFERENCES

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1987). Current Protocols in Mo- lecular Biology (New York: Wiley).

Aviv, H., and Leder, P. (1972). Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid- cellulose. Proc. Natl. Acad. Sci. USA 69, 1408-1412.

Beckmann, J.D., Ljungdahl, P.O., Lopez, J.L., and Rumpower, B.L. (1987). lsolation and characterization of the nuclear gene encoding the Rieske iron-sulfur protein (RIP1) from Sacchammyces cerevisiae. J. Biol. Chem. 262, 8901-8909.

Beckmann, J.D., Ljungdahl, P.O., and Trumpower, B.L. (1989). Muta- tional analysis of the mitochondrial Rieske iron-sulfur protein of Sacchammyces cerevisiae. 1. Construction of a RlPl deletion strain and isolation of temperature-sensitive mutants. J. Biol. Chem. 264,

Berry, E.A., Huang, L.-s., and DeRose, V.J. (1991). Ubiquinol- cytochrome c oxidoreductase of higher plants. lsolation and characterization of the bc, complex from potato tuber mitochondria. J. Biol. Chem. 266, 9064-9077.

Braun, H.-P., and Schmitz, U.K. (1992). Affinity purification of cytochrome c reductase from potato mitochondria. Eur. J. Biochem.

Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extrac- tion. Anal. Biochem. 162, 156-159.

Davidson, E., Ohnishl, T., Atta-Asafo-Adjel, E., and Daldal, F. (1992). Potential ligands to the (2Fe-2Sj Rieske cluster of the cytochrome bc, complex of Rhodobacter capsulatus probed by site-directed mutagenesis. Biochemistry 31, 3342-3351.

Dellaporta, S.L., Wood, J., and Hicks, J.B. (1983). A plant DNA minipreparation: Version 11. Plant MOI. Biol. Rep. 1, 19-21.

Dennls, D.T. (1987). The Biochemistry of Energy Utilization in Plants (New York: Chapman and Hall).

De Paepe, R., Forchioni, A., ChBtrit, P., and Vedel, F. (1993). Spe- cific mitochondrial proteins in pollen: Presence of an additional ATP synthase p subunit. Proc. Natl. Acad. Sci. USA 90, 5934-5938.

Gerstel, D.U. (1966). Evolutionary problems in some polyploid crop plants. Hereditas 2 (suppl.), 481-504.

Goldberg, D.A. (1980). lsolation and partia1 characterization of the Dmsopbila alcohol dehydrogenase gene. Proc. Natl. Acad. Sci. USA 77, 5794-5798.

3713-3722.

208, 761-767.

Differential Expression of the RlSP Gene Family 447

Graham, L.A., and Trumpower, B.L. (1991). Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. 111. Import, protease processing, and assembly into the cytochrome bcl complex of iron-sulfur protein lacking the iron-sulfur cluster. J. Biol. Chem. 266, 22485-22492.

Gray, J.C., Kung, S.D., Wlldman, S.G., and Sheen, S.J. (1974). Ori- gin of Nicotiana tabacum L. detected by polypeptide composition of Fraction I protein. Nature 252, 226-227.

Harlow, E., and Lane, D. (1988). Antibodies: A Laboratory Manual (Cold Spring Harbor, NY Cold Spring Harbor Laboratory).

Harnisch, U., Weiss, H., and Sebald, W. (1985). The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing. Eur. J. Biochem. 149, 95-99.

Hartl, F.-U., Pfanner, N., Nicholson, D.W., and Neupert, W. (1989). Mitochondrial protein import. Biochim. Biophys. Acta 988, 1-45.

Hatefi, Y., Haavik, A.G., and Griffiths, D.E. (1962). Studies on the electron transfer system. XLI. Reduced coenzyme Q (QH,)- cytochrome c reductase. J. Biol. Chem. 237, 1681-1685.

Heinrlch, P. (1987). Constructing nested deletions for use in DNA sequencing. In Current Protocols in Molecular Biology, Suppl. 16, F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds (New York: Wiley), pp. 7.2.1-7.2.20.

Huang, J., Hack, E., Thornburg, R.W., and Myers, A.M. (1990). A yeast mitochondrial leader peptide functions in vivo as a dual targeting signal for both chloroplasts and mitochondria. Plant Cell2,

Huang, J., Struck, F., Matzinger, D.F., and Levings, C.S., 111. (1991). Functional analysis in yeast of cDNA coding for the mitochondrial Rieske iron-sulfur protein of higher plants. Proc. Natl. Acad. Sci.

Kelly, R.J., Johnson, R.D., and Siegel, A. (1990). Heterogeneity and organization of the ribosomal RNA genes of Cucurbita maxima. Plant MOI. Biol. 14, 927-933.

Koltunow, A.M., Ruettner, J., Cox, K.H., Wallroth, M., and GoldbeFg, R.B. (1990). Different temporal and spatial gene expression patterns occur during anther development. Plant Cell 2, 1201-1224.

Laemmll, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

Lind, C., Halldbn, C., and Mbller, I.M. (1991). Protein synthesis in mitochondria purified from roots, leaves and flowers of sugar beet. Physiol. Plant. 83, 7-16.

Logemann, J., Schell, J., and Willmltzer, L. (1987). lmproved method for the isolation of RNAfrom plant tissues. Anal. Biochem. 163,1620.

Madueiio, F., Napier, J.A., Cejudo, F.J., and Gray, J.C. (1992). Im- port and processing of the precursor of the Rieske FeS protein of tobacco chloroplasts. Plant MOI. Biol. 20, 289-299.

Mitchell, P. (1976). Possible molecular mechanisms of the protonmo- tive function of cytochrome systems. J. Theor. Biol. 62, 327-367.

Monbger, F., Mandaron, P., Nlogret, M.-F., Freyssinet, G., and Mache, R. (1992). Expression of chloroplast and mitochondrial genes during microsporogenesis in maize. Plant Physiol. 99, 396-400.

Nakajima, T., Maeshima, M., and Asahl, T. (1984). The subunit composition of sweet potato cytochrome b-cl complex. Agricul. Biol. Chem. 48, 3019-3025.

Newton, K.J., and Walbot, V. (1985). Maize mitochondria synthesize organ-specific polypeptides. Proc. Natl. Acad. Sci. USA 82,

1249-1 260.

USA 88, 10716-10720.

6879-6883.

Nishikimi, M., Hosokawa, Y., Toda, H., Suzuki, H., and Orawa, T. (1989). Cloning and sequence analysis of a cDNA encoding the Rieske iron-sulfur protein of rat mitochondrial cytochrome bcl complex. Biochem. Biophys. Res. Commun. 159, 19-25.

Nishlkimi, M., Hosokawa, Y., Toda, H., Suzuki, H., and Ozawa, T. (1990). The primary structure of human Rieske iron-sulfur protein of mitochondrial cytochrome bcl complex deduced from cDNA analysis. Biochem. Int. 20, 155-160.

Pichersky, E., Bernatrky, R., Tanksley, S.D., and Cashmore, A.R. (1986). Evidence for selection as a mechanism in the concerted evo- lution of Lycopersicon esculentum (tomato) genes encoding the small subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase. Proc. Natl. Acad. Sci. USA 83, 3880-3884.

Rieske, J.S. (1967). Preparation and properties of a respiratory chain iron-protein. Methods Enzymol. 10, 357-362.

Rieske, J.S., Zaugg, W.S., and Hansen, R.E. (1964). Studieson the electron transfer system. LIX. Distribution of iron and of the component giving an electron paramagnetic resonance signal at g = 1.90 in subfractions of complex 111. J. Biol. Chem. 239, 3023-3030.

Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A., and Arnhelm, N. (1985). Enzymatic amplification of P-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350-1354.

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Clon- ing: A Laboratory Manual, 2nd ed (Cold Spring Harbor, NY Cold Spring Harbor Laboratory).

Sanger, F., Nicklen, S., and Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,

Siedow, J.N., Power, S., de Ia Rosa, F.F., and Palmer, G. (1978). The preparation and characterization of highly purified, enzymically active complex 111 from baker's yeast. J. Biol. Chem. 253,2392-2399.

Southern, E.M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. MOI. Biol. 98,503-517.

Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76,

Trumpower, B.L. (1981). Function of the iron-sulfur protein of the cytochrome b-cl segment in electron-transfer and energy-conserving reactions of the mitochondrial respiratory chain. Biochim. Biophys. Acta 639, 129-155.

Trumpower, B.L. (1990). Cytochrome bc, complexes of microorgan- isms. Microbiol. Rev. 54, 101-129.

Trumpower, B.L., and Edwards, C.A. (1979). Purification of a recon- stitutively active iron-sulfur protein (oxidation-factor) from succinate-cytochrome c reductase complex of bovine heart mitochondria. J. Biol. Chem. 254, 8697-8706.

Usui, S., Yu, L., and Yu, C.A. (1990). Cloning and sequencing of a cDNA encoding the Rieske iron-sulfur protein of bovine heart mitochondrial ubiquinolqtcchrome c reductase. Biochem. Biophys. Res. Commun. 167, 575-579.

Vlelra, J., and Messing, J. (1987). Production of single-stranded plasmid DNA. Methods Enzymol. 153, 3-11.

von Heijne, G. (1986). Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 5, 1335-1342.

5463-5467.

4350-4354.

448 The Plant Cell

Warmke, H.E., and Lee, S.-L.J. (1978). Pollen abortion in T cytoplas- mic male-sterile corn (Zea mays): A suggested mechanism. Science

Weiss, H., and Kolb, H.J. (1979). lsolation of mitochondrial succinate: ubiquinone reductase, cytochrome c reductase and cytochrome c

oxidase from Neurospora crassa using nonionic detergent. Eur. J. Biochem. 99, 139-149.

Yang, X., and Trumpower, B.L. (1986). Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans. J. Biol. Chem. 261, 12282-12289.

200, 561-563.

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