Journal of Experimental Botany, Vol. 51, No. 90001, pp. 375-382,
February 2000
© 2000 Oxford University Press
The ycf 9 (orf 62) gene in the plant chloroplast genome encodes a hydrophobic protein of stromal thylakoid membranes
1 Laboratory of Plant Physiology and Molecular Biology, Department of Biology, University of Turku, FIN-20014 Turku, Finland
2 Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
3 Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK
Received 26 March 1999; Accepted 7 October 1999
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Abstract |
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There are still some open reading frames, orfs, with unknown function in the higher plant chloroplast genome. Of these conserved orfs, designated as ycfs (hypothetical chloroplast open reading frames), one is ycf 9 (orf 62) in the transcription unit with the psbC and psbD genes. The aim of this work was to investigate the function of ycf 9 by insertional inactivation of the gene with a selectable marker cassette, consisting of the aadA coding region connected to the trc promoter and rrnB terminator. This cassette was inserted 19 bp downstream from the start of the coding region of the tobacco ycf 9 gene. Two DNA constructs with the aadA cassette in opposite orientations were precipitated on 1 µm gold particles and delivered into leaves of Nicotiana tabacum, cultivar Samsun, by the biolistic method. Spectinomycin-resistant plants regenerated following bombardment with only the construct containing the aadA gene in the opposite orientation as ycf 9. In spite of several subsequent regeneration cycles on spectinomycin, the transplastomic plants did not reach homoplasmicity. This suggests that the ycf 9 gene product is essential for chloroplast function. Using a polyclonal antibody raised against the inner part of the gene product, the polypeptide was localized in the stromal thylakoid membranes of chloroplasts.
Key words: Chloroplast genome, chloroplast transformation, ycf 9, orf 62, tobacco.
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The complete nucleotide sequences of 12 plastid genomes have so far been determined (for review, see Rochaix, 1997). Plastomes from green plants and algae typically contain about 120 genes, most of them being conserved between organisms. Plastome genes can be divided into three main groups (Sugiura, 1992). The first consists of genes for plastome expression, the second contains genes for photosynthesis, and the third contains orfs whose function is still unknown in plants. These conservative orfs have been called ycfs (hypothetical chloroplast open reading frames, Hallic and Bairoch, 1994).
One of the conserved chloroplast open reading frames is ycf 9, which encodes a highly conserved hydrophobic protein of 62 amino acid residues (ORF 62, see Fig. 4). ycf 9 is located downstream of the psbD and psbC genes, encoding integral components of photosystem II, and is part of the complex transcription unit including these genes (Berends et al., 1987; Yao et al., 1989; Chen et al., 1994; Wada et al., 1994). ycf 9 appears not to have its own independent promoter. Instead, ycf 9 is included in transcripts from several promoters located upstream of psbD and psbC in dicots (Yao et al., 1989), and upstream of psbK in monocots (cereals) where ycf 9 is part of a transcription unit including psbK and psbI, as well as psbD and psbC (Chen et al., 1994; Wada et al., 1994). The co-transcription of ycf 9 with genes encoding photosystem II polypeptides has led to speculation that ORF62 may be an additional photosystem II component (Hird et al., 1987).
Recent advances in the transformation of the tobacco chloroplast genome (Svab et al., 1990; Svab and Maliga, 1993;Kanevski and Maliga, 1994; Allison et al., 1996; Sugita et al., 1997) enable targeted chloroplast gene disruption in higher plants, providing the means to assess the functional role of the ycfs. This strategy has been established and shown to be useful when identifying several ycfs in Chlamydomonas (Monod et al., 1994; Takahashi et al., 1996; Boudreau et al., 1997; Xie and Merchant, 1996). More information of the role of plastid ndh (Burrows et al., 1998) and sprA (Sugita et al., 1997) genes has already accumulated from characterization of the corresponding tobacco chloroplast gene disruption mutants.
In this study, the aim was to determine the functional role of ycf 9 in tobacco. For this purpose, the coding sequence of ycf 9 was disrupted by using an aadA expression cassette, conferring spectinomycin resistance, through biolistic transformation of the tobacco plastome. The location of the ycf 9 gene product in chloroplasts was also examined using antibodies to a 17 amino acids synthetic peptide from within ORF 62.
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Plant material and growth conditions
Nicotiana tabacum, var. Samsun, was grown aseptically on 0.7% agar-solidified MS salts, pH 5.7, containing 3% sucrose at 25 °C under 160 µmol photons m-2 s-1 (16 h light, 8 h dark). Leaves of plants either generated by germination of sterilized seeds or by regeneration of stem pieces were used as targets for plastome transformation.
Plasmid construction
The chloroplast targeting vectors pPM1 and pPM2 (Fig. 1
), derivatives of pBCSK+, carry a 1910 bp Hind III-Spe I tobacco plastome fragment obtained from the plasmid pTB20 (a kind gift of Professor M Sugiura) of the tobacco chloroplast genomic library (Shinozaki et al., 1986) and ligated into the Hind III and Spe I sites of pBCSK+. This fragment was interrupted by the aadA cassette, ligated into the Mun I site in orf 62. The aadA cassette used in the disruption of orf 62 consists of the aadA coding region (810 bp NcoI-PstI fragment from pUC-atpX-AAD, Goldschmidt-Clermont, 1991), placed between the trc promoter and the rrnB terminator (Fig. 1) in pKK 233–2. The whole entity trc-aadA-rrnB was removed from the construct as a 1.66 kb Hind II fragment. The protruding ends resulting from the Mun I digestion were filled in with the Klenow fragment of DNA polymerase I before ligation of the aadA cassette as a Hind II fragment. In the vectors pPM1 and pPM2, the aadA cassette is surrounded by 1211 bp (upstream) and 699 bp (downstream) of plastome DNA sequences which target the cassette to orf 62 during the plastome transformation process.
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Tobacco chloroplast transformation
Chloroplast transformation was performed using biolistic technology (Svab and Maliga, 1993) with modifications. The transforming DNA was precipitated on 1 µm gold particles at 4 °C. Microprojectile bombardment was carried out under a partial vacuum of 27.5 in Hg with a Bio-Rad PDS 1000 helium gun, using 1100 psi rupture discs and a helium cylinder pressure of 1300 psi. The stopping screen and macrocarriers were placed in the vacuum chamber on the second level from the top and the sample-containing Petri dish was placed on the fifth level from top. In the transformations, mature leaves from 4–6-week-old plants were used. The leaves were placed abaxial side up on a Whatman filter paper on top of RMOP agar 2 h before shooting to decrease the leaf turgor pressure. After 48 h, the leaves were cut into sections of approximately 5 mm in diameter and placed on RMOP agar containing spectinomycin hydrochloride (500 µg ml-1). Transgenic shoots were selected on spectinomycin (according to Svab et al., 1990) and they were subcultured on MS medium for rooting. The regeneration cycle was repeated four times and the expanded mature leaves from rooted plants were used for the transgenicity analysis.
Isolation of DNA from plants and analysis by PCR
Total plant DNA was isolated from 100 mg leaf pieces using the DNeasy Plant Mini Kit (Qiagen). This leaf total DNA was used as a template in PCR by Dynazyme polymerase (Finnzymes) to estimate the proportion of transformed to non-transformed plastome copies. The following pair of oligonucleotide primers was used: 5'-TAT CCG TAT ATA GAT ATA TG-3' (annealing with plastome nucleotides 37340–37360) and 5'-TTT TAT TCA AGA AGT TTG AC-3' (annealing with nucleotides 37925–37905). The PCR programme was as follows: denaturing 94 °C 1 min, annealing 50 °C 1 min, and synthesis 72 °C 3 min. 28 cycles were performed and the products were analysed on a 1% agarose gel.
Isolation and subfractionation of thylakoid membranes
Leaves were briefly homogenized in 50 mM HEPES pH 7.5, 5 mM MgCl2, and 0.33 M sorbitol, filtered through Miracloth and centrifuged for 3 min at 6000 g to pellet the chloroplasts. The pellet was washed with 50 mM HEPES pH 7.5, and 5 mM MgCl2 and resuspended in 50 mM HEPES pH 7.5, 10 mM MgCl2 and 0.1 M sorbitol. Thylakoids were subfractionated into stroma and grana lamellae by using the digitonin method (Kyle et al., 1984) with slight modifications (Baena-Gonzalez et al., 1999).
Separation of thylakoid complexes by sucrose gradient centrifugation
Thylakoids were solubilized with 1.25% (w/v) n-dodecyl-ß-D-maltoside and loaded onto a 0.1–1.0 M linear sucrose gradient (according to Baena-Gonzalez et al., 1999). After centrifugation at 180 000 g for 26 h at 4 °C, the gradients were fractionated into 600 µl fractions from top to bottom. The molecular mass of the resulting protein complexes was estimated by protein standards ranging from 67 to 699 kDa. The assignment of protein complexes in the gradient was based on immunoblot analysis and earlier experiments (Zhang et al., 1999).
SDS-PAGE, immunoblotting and chlorophyll determination
Thylakoid membranes, thylakoid membrane subfractions and sucrose gradient fractions were solubilized and the proteins separated in 15% (w/v) polyacrylamide gels containing 6 M urea (according to Laemmli, 1970). After electrophoresis the polypeptides were transferred to a polyvinyledene difluoride (PVFD) membrane and detected with antibodies by using the chemiluminescence kit of New England Biolabs. Polyclonal antibodies were used against the PSI complex, CP 43 (kind gifts from Professor R Barbato), the D1 polypeptide and ORF 62. The antibody used to recognize CP 43 recognizes CP 47 as well. To raise the polyclonal antiserum against ORF 62, rabbits were injected with a 17-mer synthetic peptide corresponding to amino acids from 29 to 45 (Research Genetics). Chlorophyll was extracted in 80% (v/v) buffered acetone and quantified (according to Porra et al., 1989).
Sequence analysis
Sequences similar to tobacco ORF 62 were found in the Internet with the Blast program (National Center for Biotechnological Information). The alignment of the received sequences was performed with the program Clustal W using span length of nine amino acids, gap opening penalty 10 and gap extension penalty 0.05. Hydrophobicity analysis of the ORF 62 sequence was performed by Expasy Molecular Biology Server (Proteomics tools). Identity and similarity of tobacco ORF 62 with corresponding sequences from other species was calculated by the program GeneDoc.
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Generation of plasmid constructs pPM1 and pPM2 for insertional inactivation of ycf 9
The role of ycf 9 of the plastome of tobacco leaves was examined by insertional inactivation of the gene with two chimeric gene constructs pPM1 and pPM2 (Fig. 1). In these constructs, the bacterial aadA gene, connected to the trc promoter and the rrnB terminator, was inserted in the Mun I site 19 bp downstream of the start of the ycf 9 coding region. The aadA gene (Hollingshead and Vapnek, 1985) encodes spectinomycin and streptomycin resistance, and is a selectable marker for chloroplast transformation (Svab and Maliga, 1993). The resulting gene constructs pPM1 and pPM2, which carry the aadA cassette in opposite orientations, contain a 1910 bp Hind III-Spe I chloroplast DNA fragment that targets the insertion of the aadA cassette into ycf 9.
Spectinomycin-resistant plants produced by plastome transformation
Two hundred tobacco leaves were bombarded with each construct, and in total 11 green calli (A to K) were obtained, all arising from transformations with the reverse aadA orientation (pPM2, Fig. 1). The calli developed from separate leaf sections and are regarded as different clones. From each callus, several spectinomycin-resistant plants were regenerated. These plants resembled wild-type plants and showed no evidence of gross morphological change. The possibility that the spectinomycin resistance was due to spontaneous mutations in the plastid genome was examined by placing leaf pieces on streptomycin medium (Svab and Maliga, 1993). Only plants regenerated from the calli G and F were phenotypically resistant to streptomycin and remained green during streptomycin selection, while leaf pieces derived from the other calli bleached.
The transformation efficiency was very low compared to tobacco chloroplast transformation in other laboratories (Svab and Maliga, 1993; Daniell et al., 1998). In these laboratories, tungsten, rather than gold, microprojectiles have been used to carry the transforming DNA to the target when producing stable transformants. However, both gold and tungsten particles have been used to introduce DNA for transient expression of green fluorescent protein in various plastid types (Hibberd et al., 1998). The low transformation efficiency may also be due to the use of the tobacco variety Samsun, rather than Petit Havana which has been used extensively elsewhere. However, Samsun has been used previously for transient gene expression in chloroplasts following microprojectile bombardment (Hibberd et al., 1998) and no difference in stable chloroplast transformation rates has been observed between Petit Havana and Samsun (PJ Linley and JC Gray, unpublished results). The structure of the transforming DNA, such as the plastome sequences flanking the selectable marker, and the promoter strength of the selectable marker gene, may have an effect on the transformation efficiency. The use of the chimeric trc promoter from Escherichia coli has not previously been reported for driving expression of the aadA gene for selection of chloroplast transformants, although it has been used for transient expression of green fluorescent protein in various plastid types (Hibberd et al., 1998).
Proportion of transformed to non-transformed plastome copies in the transgenic lines
The proportion of transformed to non-transformed plastome copies was estimated with a PCR analysis. The pair of oligonucleotide primers used in the analysis should result in an amplification product of 585 bp from the wild-type plastome and a product of 2257 bp from correctly transformed plastomes. The analysis was performed after the 2nd, 3rd and 4th regeneration cycle of the lines F and G. After the 2nd cycle, the proportion of transformed plastomes was estimated to be between 15% and 25%, and after the 4th cycle it was 50% in line G and 25% in line F, showing that the transplastomic plants did not reach homoplasmicity (Fig. 2). For an unknown reason, when total DNA from the transplastomic plants was used as a template, an extra band was often seen among the PCR products, migrating between the specific 585 and 2257 bp product bands. When this band was excized from the gel, purified and used as a template in PCR with the same pair of oligonucleotide primers, no product was obtained (not shown).
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DNA; in the following lanes different templates; 2: clone G; 3: clone F; 4: WT; 5: pPM1; 6, 7: clones A and B (spontaneous spectinomycin-resistant mutants).Northern blot analysis of total RNA extracted from leaves of lines G and F showed also, in addition to the five transcripts hybridizing to a psbC probe present in the wild-type plants, the presence of two additional transcripts, of 5.2 kb and 1.1 kb (Mäenpää et al., 1998). The 5.2 kb transcript presumably results from insertion of the aadA cassette into the psbD-psbC-ycf 9 transcription unit. The 1.1 kb transcript may be a processing product due to the presence of the aadA cassette.
It was believed that if orf 62 plays a role in photosynthesis, it should be possible to inactivate it and reach homoplasmicity, provided the selection and regeneration were performed on sucrose, in the same way as, for example, the rbcL gene inactivation reached homoplasmicity (Kanevski and Maliga, 1994). On the other hand, homoplasmicity may not be reached if the gene has a necessary house-keeping function (Rochaix, 1997). If this view is valid, the lack of homoplasmicity in the present orf 62 inactivation, even after the 4th regeneration cycle, possibly indicates a non-photosynthetic, but fundamental role of orf 62 in plant metabolism.
Hydrophobicity and similarity of ORF 62 to other proteins
Hydrophobicity analysis of the amino acid sequence of tobacco ORF 62, performed using a span-length of 9 amino acids, demonstrated the highly hydrophobic character of the protein (Fig. 3), pointing to a membrane protein with two membrane-spanning regions. It is difficult to predict the orientation of the protein in the membrane, because the hydrophilic region between the two hydrophobic domains contains only two charged residues, single aspartate and lysine residues. The protein therefore does not show a difference in charge distribution across the membrane, and its orientation cannot be predicted by the positive-inside rule (Gavel et al., 1991).
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Proteins homologous to ORF 62 are encoded in the plastid genomes of all organisms examined, except for the parasite beechdrops (Epifagus virginiana) which has lost all genes associated with photosynthetic functions (Wolfe et al., 1992). The proteins contain 60–65 amino acid residues and the sequences are aligned in Fig. 4
. A gene encoding an ORF 62 homologue is also present in the photosynthetic cyanobacterium Synechocystis 6803, although the open reading frame potentially encodes a larger protein with an additional stretch of 45 amino acid residues at the N-terminus. All the sequences examined show a high degree of similarity to tobacco ORF 62, ranging from 37% identical residues in S. 6803 to 95% in pea (Table 1).
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Localization of ORF 62 in chloroplast thylakoid membranes
To localize the ycf 9 gene product in chloroplasts, a 17 amino acid residue peptide corresponding to residues 29–45 (SPDGWSSNKNVVFSGTS) was synthesized and used to raise a polyclonal antiserum in a rabbit. This peptide shows the most hydrophilic character and probably represents a solvent-exposed region of the protein linking the two putative membrane-spanning regions. Using this antiserum, a small polypeptide migrating just ahead of the 6.5 kDa marker was detected in thylakoid membranes of tobacco, pumpkin and spinach (Fig. 5). The electrophoretic mobility of this protein corresponds well to the size of 6.553 kDa calculated from the sequence of tobacco ORF 62. The pre-immunoserum did not give any response in the thylakoid samples (Fig. 5). ORF 62 was shown to be present in the stromal thylakoid fraction and completely absent from the granal thylakoid fraction (Fig. 6), suggesting that it was not a component of photosystem II. Immunoblot analysis of sucrose gradient fractions of dodecyl maltoside-solubilized thylakoid membranes, using antibodies to D1, CP43, photosystem I, and ORF 62, revealed that ORF 62 was present in the same fractions as the photosystem I complex (Fig. 7). ORF 62 did not comigrate with the photosystem II polypeptides, providing further evidence that it is not a component of photosystem II. The co-transcription of ycf9 with psbD and psbC is therefore another example of a chloroplast transcription unit encoding proteins of different functional complexes, such as the psbB-psbH-petB-petD operon encoding subunits of photosystem II and the cytochrome bf complex (Rock et al., 1987; Westhoff and Herrmann, 1988). The co-migration of ORF 62 with the photosystem I complex in the sucrose gradient may suggest an association of ORF 62 with photosystem I. However, ORF 62 seems not to be among the well-characterized subunits of the complex (Nechustai et al., 1996). It may have a role in the assembly of the photosystem I complex, as recently suggested for ycf 3 and ycf 4, on the basis of gene inactivation studies in Chlamydomonas (Boudreau et al., 1997). However, a role of ORF 62 exclusively in photosystem I assembly would not necessarily explain the inability to obtain homoplasmic ycf 9 inactivation mutants.
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It is not known whether ORF 62 is present in non-green plastids. In such case an essential role of ORF 62 could be related to the assembly of membrane complexes present in the internal membranes of all plastid types. However, the absence of the ycf 9 gene in the Epifagus virginiana plastome (Wolfe et al., 1992) may suggest a function specifically related to chloroplasts, unless the gene has been transferred to the nucleus of Epifagus virginiana.
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Acknowledgements |
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The Finnish group acknowledges support from the Academy of Finland, from The Royal Society, London, and from Robinson College, Cambridge, UK. MSK was supported by a Scholarship from the Cambridge Commonwealth Fund and by an Overseas Research Student Award. The plasmid pTB20 was the generous gift of Professor M Sugiura and the antibodies against PSI complex and CP 43 were generous gifts of Professor R Barbato.
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Footnotes |
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4 To whom correspondence should be addressed. Fax: +358 2 333 5549. E-mail:pirmae{at}utu.fi
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References |
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