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Chloroplast reverse genetics: new insights into the function of plastid genes
reviews
Chloroplast reverse genetics: new insights into the function of plastid genes ,eooov. ,oo,o,. The complete sequences of 12 plastid genomes have recently been determined. This has revealed that in addition to k n o w n genes involved in plastid gene expression and photosynthesis, there are numerous open reading frames encoding genes of u n k n o w n function. Several of these genes have been inactivated in Chlamydomonas reinhardtii and tobacco by biolistic chloroplast transformation. These studies - coupled with molecular, biochemical and biophysical analysis - have revealed the existence of novel subunits and factors required for the accumulation and optimal functioning of photosynthetic complexes. Other plastid genes have also been identified that are essential for cell survival, but w h o s e function remains to be determined. p
lastid genomes of algae and land plants exist as circular DNA molecules with sizes in the range 50-400 kbp. In the unicellular alga Chlamydomonas reinhardtii there are 80 copies of the genome, whereas in mesophyll cells of higher plants there are up to 10000 copies. Most of the genomes examined contain two large inverted repeats harbouring the ribosomal RNA (rRNA) genes, and these are separated by two single-copy regions 1. Although the organization of genes in the plastid genome is generally well conserved in land plants, extensive genome rearrangements have occurred in algae. The complete nucleotide sequences of plastid genomes are now available for six vascular plants (Epifagus virginiana, Marchantia polymorpha, tobacco, rice, Pinus thunbergii and maize) and six unicellular eukaryotes (Euglena gracilis, the green alga Chlorella ellipsoidea, and three nongreen algae, Cyanophora paradoxa, Porphyra purpurea and Odontella sinensis) 2. The sequences for plastids from land plants and green algae reveal approximately 120 genes, which are mostly conserved among these organisms and can be divided into three major groups. The first group includes sequences required for plastid gene expression, with approximately 50 genes encoding subunits of RNA polymerase, rRNAs, transfer RNAs (tRNAs), ribosomal proteins and additional factors (e.g. elongation factor EF-Tu and initiation factor). The second group comprises approximately 40 genes, encoding components of the photosynthetic apparatus - mostly subunits of the thylakoid-associated complexes photosystem I (PSI), photosystem II (PSII), the cytochrome b6/f complex, the ATP synthase and the large subunit of the stromal enzyme Rubisco. A third group includes open reading frames whose function is still largely unknown. Some of these are conserved between species, and are designated ycf ('hypothetical chloroplast open reading frame'); others appear to be species-specific. A greater variation in plastid gene content occurs in the nongreen algae. For example, the plastid genome of Porphyra purpurea contains twice as many genes as land plants 3. Although not all the additional genetic material has been characterized, it includes genes involved in photosynthesis that are encoded by the nucleus in plants, and genes for plastid biosynthesis functions (e.g. for the synthesis of fatty acids, amino acids, pigments and thiamine). Other genes encode proteins involved in transport. © 1997 Elsevier Science Ltd
Transformation and reverse genetics of chloroplasts A major breakthrough in plastid research occurred in 1988 when Boynton et al. 4 established a biolistic chloroplast transformation method for C. reinhardtii. Once the DNAcoated particles have penetrated into the chloroplast, the transforming DNA is released and becomes incorporated into the plastid genome by homologous recombination. Transformants can be selected in several ways: it is possible to use mutants with lesions in chloroplast genes and transform them with the corresponding wild-type copies by selecting for photoautotrophic growth; alternatively, mutant alleles of the 16S and 23S rRNA genes that confer resistance to antibiotics can be used to transform wild-type cells on drug-containing medium 5. The most versatile chloroplast selectable marker is the bacterial aadA ('aminoglycoside adenyl transferase') gene, which has been engineered into a chloroplast expression cassette and confers resistance to spectinomycin and streptomycin ~ (Fig. 1). Because of the homologous plastid recombination system, this cassette allows specific gene disruptions and sitedirected mutagenesis to be performed on any chloroplast gene. Chloroplast transformation has also been achieved with tobacco plants using similar approaches 7's. The chloroplast genome is present in multiple copies, and thus any directed gene manipulation through transformation requires repeated subcloning of the transformants until complete segregation of the mutant and wild-type gene copies has been achieved. Disruption of chloroplast genes whose function is not essential for cell viability leads to a homoplasmic state in which all plastid gene copies are inactivated. This can occur for genes involved in photosynthesis, because photosynthetic function is dispensable in C. reinhardtii when the cells are grown with acetate as the carbon source, or in tobacco when sucrose is added to the growth medium. Homoplasmic gene disruptions can also be achieved for other dispensable plastid genes that may not necessarily have a role in photosynthesis. In contrast, inactivation of an essential plastid gene invariably leads to a heteroplasmic state in which mutant and wild-type gene copies coexist as long as the selective pressure for the disrupted gene is maintained (Fig. 2). Table 1 summarizes chloroplast gene disruptions performed in C. reinhardtii and tobacco. In addition to gene disruptions, it is possible to perform site-directed mutagenesis of chloroplast genes. In this case, PII $1360-1385(97)01121-7
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reviews
Role of small subunits of photosynthetic complexes
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Identification of novel proteins involved in photosynthesis The presence of several conserved unidentified open reading frames in the chloroplast genomes of higher and lower plants, and green, red and brown algae, suggests that the encoded proteins play an important role. For example, the ycf8 open reading frame is cotranscribed with psbB, which encodes a PSII subunit, in C. reinhardtii and land plants TM. As this is the only other operon besides the ribosomal operon to be conserved in C. reinhardtii and plants, co-expression of the psbB and ycf8 genes appears to be important. The Ycf8 polypeptide consists of 31 amino acids with a central hydrophobic region that may act as a transmembrane domain. The protein has been identified as a novel PSII subunit (PsbT) based on its severely reduced levels in various PSII-deficient mutants and on its cofractionation with PSII (Ref. 14). Mutants with lesions in the ycf8 gene are able to
Fig. 1. Targeting of the chloroplast aadA ('aminoglycosideadenyl transferase') gene expression cassette to a specific site in the chloroplast genome. The expression cassette consists of the aadA coding sequence fused on its 5' side to a chloroplast promoter (P) and 5'-untranslated region (5'UTR), and on its 3' side to a chloroplast 3'-untranslated region (3'UTR). Upon bombardment of the cells with the transforming plasmid, in which the expression cassette is flanked by chloroplast DNA sequences corresponding to the targeting region (indicated by a thick line), the cassette is inserted into the chloroplast genome by homologous recombination. The AAD protein catalyses the adenylation of streptomycin/spectinomycin and thereby inactivates the drug.
the aadA cassette is inserted at a site that is not essential for chloroplast function and is close to the target gene. The construct is introduced into the chloroplast genome via biolistic transformation by selecting for spectinomycin resistance. This 'reverse genetics' approach is a powerful tool for analyzing the function of chloroplast-encoded subunits of photosynthetic complexes and for identifying new subunits of these complexes. 420
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Biochemical analysis of the thylakoid photosynthetic complexes has revealed that, in addition to the larger catalytic subunits, they are comprised of numerous small molecular mass polypeptides. The larger subunits have been extensively characterized, but little attention has been devoted to these smaller subunits. Several of the smaller subunits have been disrupted: inactivation of the gene encoding PsbF ('photosystem Ir - the fourth letter designates a specific subunit) completely abolishes PSII activity (T.S. Mor and I. Ohad, unpublished); in contrast, cells lacking PsbI (a component of the PSII reaction centre) are still capable of photoautotrophic growth in low light, but not in high light, even though the amount of PSII is reduced to 10-20% of wild-type levels 9. Disruption of the psbH gene 1°'11 or the psbK gene 12 in C. reinhardtii leads to a considerable reduction in the accumulation of PSII and prevents photoautotrophic growth. However, inactivation ofpsbK in cyanobacteria only has a mild effect, because these mutants still grow photoautotrophically1~. These observations suggest that the PsbK subunit is not essential for the photochemical activity of PSII, but is required for the structural integrity of the complex, at least in C. reinhardtii. Extragenic suppressors of the psbK disruptions have recently been isolated, and these might provide new insight into the mechanism underlying the stability and turnover of the PSII complex (Y. Takahashi, J. van Dillewijn and J-D. Rochaix, unpublished).
reviews grow photoautotrophically at the same rate as the wild type, but PSII function and cell growth are impaired under high light and conditions of diminished chloroplast protein synthesis induced by spectinomycin 14. Hence this small PSII subunit appears to have a role in maintaining high photosynthetic activity under adverse growth conditions. A similar approach has been used to elucidate the function ofycf7, which encodes a 43 amino acid polypeptide containing a putative transmembrane domain 1~. Mutants in which ycf7 has been disrupted have considerably impaired photoautotrophic growth: they only accumulate 25-50% of the cytochrome b6/f complex as compared with the wild type, and the rate of electron transfer within the complex is reduced ~5.The Ycf7 product is absent in mutants lacking the cytochrome b6/f complex and copurifies with the cytochrome b6/f complex, indicating that Ycf7 is an authentic subunit of this complex and is required for its stability, accumulation and optimal efficiency~5. Recent results suggest that YcfY may be involved in the dimerization of the cytochrome b6/f complex1~. This subunit, called PetL, had escaped biochemical detection in earlier studies. Two chloroplast open reading frames, ycf3 and ycf4, have recently been shown to be required for stable accumulation of the PSI complex in C. reinhardtii, because disruption of either gene leads to a complete loss of PSI (Ref. 17). However, although the ycf3 and ycf4 gene products are found in the thylakoid membrane, they are not associated with PSI. These subunits also accumulate to the same level as in the wild type in mutants lacking PSI. The ycf4 gene has also been disrupted in cyanobacteria TM,but although the amount of PSI is reduced in Synechocystis sp. strain PCC 6803, the complex is not fully destabilized as in C. reinhardtii. These proteins could be involved in the synthesis of one of the PSI reaction centre polypeptides, might be required for the stability of the complex or could act as assembly factors. Another interesting chloroplast open reading frame is ycflO. Disruption of this gene in C. reinhardtii leads to increased light sensitivity and to a significant reduction in inorganic carbon uptake, although it is not yet clear whether this is a direct or indirect effect (N. Rolland et al., unpublished). The ycfl0 product has been localized to the plastid envelope ~9, and mutants affected in a similar gene in Synechocystis appear to have a defect in CO2 uptake 2°. More recently, the primary defect of these mutants has been traced to their inability to extrude protons 21. Analysis of the chloroplast ycf5 gene of C. reinhardtii has revealed that it displays limited sequence similarity with the cycK/ccll gene involved in cytochrome c biogenesis in bacteria ~2. A mutant of C. reinhardtii carrying a frameshift mutation within ycf5 is unable to attach heme to the membrane-bound apocytochrome f and to the soluble cytochrome c6, which acts as electron donor to PSI in copper-deficient cells23. The phototrophic growth deficiency could be complemented by transformation with the wild-type copy of ycf5 (Ref. 22). Targeted inactivation of ycf5 leads to the loss of both chloroplast c-type cytochromes. Thus, ycf5 has been renamed ccsA ('c-type cytochrome synthesis'). Reverse genetics has also been used to identify chloroplast genes involved in light-independent chlorophyll synthesis. In contrast to higher plants, which are only capable of synthesizing chlorophyll in the light, C. reinhardtii (and many lower plants and gymnosperms) can form chlorophyll both in a light-dependent and light-independent manner.
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Fig. 2. Targeted inactivation of a chloroplast gene. One chloroplast is shown containing five circular DNA toolecules. The targeted wild-type gene is indicated by an open box. The corresponding inactivated gene copy carrying the aadA ('aminoglycoside adenyl transferase') cassette introduced into the chloroplast by biolistic transformation is represented by a filled box. In the heteroplasmic state, both wild-type and mutant copies are maintained, because the former are essential for cell survival and the latter are required for growth in the presence of spectinomycin. In the homoplasmic state, all copies are inactivated, because the gene is not essential for nonphotosynthetic growth in the presence of an appropriate carbon source.
Disruption of three genes (chlB, chlL and chiN) leads to the loss of chlorophyll in the dark and to the accumulation of protochlorophyllide 24. These genes are homologous to three genes of Rhodobacter capsulatus found to be altered in mutants defective in bacteriochlorophyll formation 24. Thus, they appear to define minimal components of a novel enzyme complex that catalyses light-independent protochlorophyllide reduction. In addition, at least seven nuclear loci involved in this pathway have been found in C. reinhardtii. It remains to be seen whether they encode additional factors required for this complex or whether their products are necessary for the proper expression of the three chloroplast-encoded subunits. November1997,Vol.2, No. 11
42 1
reviews The chloroplast genomes from higher plants also contain eleven genes with homology to mitochondrial genes encoding subunits of NADH dehydrogenase 1. A role for such an enzyme in the chloro-respiratory chain was first postulated for C. reinhardtii 25, but none of the ndh ('NADH dehydrogenase') genes could be identified in the chloroplast genome of this species. The chloro-respiratory chain is believed to share the plastoquinone pool with the photosynthetic electron transport chain, and under anaerobic conditions in the dark the plastoquinone pool is reduced, presumably at the expense of NADH. Some of the ndh genes of tobacco have recently been disrupted (P. Nixon and P. Maliga, unpublished). Under normal growth conditions the mutant plants are indistinguishable from the wild type, but appear to be affected in their ability to reduce the plastoquinone pool in the dark. These preliminary results are thus compatible with the existence of an NADH:plastoquinone-oxidoreductase in the chloro-respiratory chain.
Essential chloroplast genes Protein synthesis Among the numerous mutants of C. reinhardtii affected in chloroplast function, some have been found to be partially deficient in plastid protein synthesis 26. However, no mutant was found with this process fully inactivated, suggesting that plastid protein synthesis is essential for the survival of C. reinhardtii. One would therefore predict that attempts to disrupt plastid genes involved in organellar protein synthesis would lead to a heteroplasmic state in which both wild-type and mutant copies containing the aadA expression cassette coexist. The heteroplasmic state persists for as long as the selection for drug resistance is maintained and in the absence of this selection pressure the mutant copies are rapidly lost 6'2;. Whether chloroplast protein synthesis is essential in higher plants is less clear. Antibiotics that specifically inhibit plastid ribosomes cause chlorophyll bleaching, but not death of calli from Nicotiana in tissue culture. The bleached cells are capable of division when sucrose is provided as the carbon source, although at a reduced rate 28. Plastid protein synthesis is also not required for calli cultured from roots of haploid rice plants derived from pollen grains 29.
Transcription Most transcription in mature chloroplasts is catalyzed by an RNA polymerase that is composed of ~2 ~, ~' and ~" subunits. It is similar to the Escherichia coli RNA polymerase, except that the chloroplast ~' and ~" subunits correspond to the N- and C-terminal domains of the bacterial ~' subunit, respectively3°. This enzyme initiates transcription of plastid genes from sequences resembling E. coli ¢rT°-typepromoters, and attempts to disrupt the genes encoding the subunits have yielded different results in C. reinhardtii and tobacco. In the green alga, the 5' part of the rpoB ('RNA polymerase') gene (rpoB1) is separated from the 3' part, called rpoB2 (Ref. 31). Disruption of rpoB1, rpoB2 and rpoC2 resulted in all cases in a heteroplasmic state, indicating that these genes are essential for cell survivaP '45. Surprisingly, no transcript of any of the C. reinhardtii rpo genes could be detected by mRNA (northern) hybridization, suggesting that these genes are expressed at very low levels 31. Resequencing of the rpoC2-ORF472 region of C. reinhardtii has recently revealed that it encodes a single open reading frame of 3119 residues (S. Nuotio and S. Purton, 4-22
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unpublished). A comparison between this protein sequence and that of RpoC2 from higher plants reveals clear blocks of homology separated by sequences juxtaposed in frame with coding sequences of known identity. Similar sequences have been reported for the clpP ('catalytic subunit of the ATPdependent Clp protease') (Ref. 33), rps3 (Ref. 31) and ycflO (N. Rolland and J.D. Rochaix, unpublished) genes of C. reinhardtii. In contrast, a homoplasmic deletion of the rpoB gene was obtained in tobacco34. Although the mutant plants are deficient in pigments and photosynthetic activity, and have to be grown on sucrose-containing medium, the plants develop normally, but at a reduced rate 34. The plastids of the mutant are smaller than in the wild type and lack the arrays of stacked thylakoid membranes, so that they resemble proplastids in this respect. The levels of transcripts of plastid genes involved in photosynthesis are strongly reduced in the mutant, whereas the amount of transcript from the plastid genes of the protein-synthesizing system is near to wildtype levels. These results clearly imply the existence of a plastid-localized, nuclear-encoded RNA polymerase that is required for the maintenance of the nonphotosynthetic plastid functions necessary for plant growth and development. Evidence for a polymerase of this sort has also emerged from several other findings. The nonphotosynthetic parasitic plant E. virginiana contains only a small plastid genome of 67 kb (Ref. 35), and although this genome lacks all E. coli-like RNA polymerase genes as well as genes for the photosynthetic apparatus, it is nevertheless transcribed. The identity of the RNA polymerase has not yet been elucidated. Further evidence for a nuclear-encoded RNA polymerase arises from the albostrians mutant of barley, which is severely deficient in plastid ribosomes3~. Although expression of the plastid-encoded RNA polymerase genes is strongly reduced in this mutant, transcription of several plastid genes can still be detected 3~. Ribosome-deficient plastids isolated from heat-bleached rye seedlings also possess detectable transcriptional activitya2. Finally, distinct RNA polymerases have been isolated from chloroplasts and found to be associated with both multisubunit complexes and single subunit enzymes 37. The differences observed between the rpoB disruption in tobacco and C. reinhardtii may reflect the fact that higher plants can apparently tolerate proplastid-like p]astids, whereas a mature chloroplast (with or without chlorophyll) is needed for survival of green algae. Another possibility is that C. reinhardtii lacks the nuclear-encoded RNA polymerase. It has been proposed that this enzyme plays an important role during the differentiation of proplastids to plastids, before the E. coli-like RNA polymerase is required for transcription of the high levels of gene transcripts associated with photosynthetically active chloroplasts 38. Assuming that chloroplast protein synthesis is necessary for growth and survival, the question arises as to which of the chloroplast-encoded proteins is essential besides the components involved in the expression of the plastid genetic system. Of about 20 chloroplast gene disruptions in C. reinhardtii, only two (aside from those performed on the rpo and ribosomal protein genes) give rise to a heteroplasmic state (Table 1) even after extensive subcloning of the transformants. One of these genes, clpP, encodes the chloroplast homologue of the catalytic subunit of the ATP-dependent Clp protease in E. coli39. The exact role of the chloroplast enzyme in protein processing and degradation is not yet
reviews
Organism
Gone~
Homoplasmic?
Photo-autotrophic growth?
Phenotyl ~b
Chlamydomonas reinhardtii psaA psaB psaC psaJ
YeS Yes Yes Yes
No No No :- . Yes
PSIPSI PSF Wild type
tscA psbF
Yes Yes
No : : No
psbH psbI psbK psbN
Yes Yes Yes ~ ~ " Yes
NO So~e, N~ • Yes . . . . .
psbT (ycf8) petA petB petD petL (ycf7) ycfl (0RF1995)
Yes
ycf4 ycf5 ycflO
Yes . . . . Yes Yes Yes No Yes Yes Yes Yes
Yes NO No No . . . . . . . . Some-:Not determined No No No No
Light sensitive 'i '.: : b6f~ b6f:: h6f: . . . . . . . . . : ' - : . - - ...... : Possibly b6F . . . . . . . . . . Wild type . . . . . . . . . . . . . . . PSIPSI b6fUptake of inorganic carbon
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Yes
Yes
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Yes
Yes
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Yes
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rpoB1 rpoB2 rpOC2-ORF
No No No
Not de~rmined Not determined Not determined
Chloro synthesis ir~ the dark Chlorophyll synthesis in the dark Chlorophyll synthesis in the dark Wild type Wild !ype Wild type
rps3 (0RF712)
No
Not determined
Wild type
.......
clpP ORF58 ORFB
NO Yes No
Some Yes Not determined
Wild type Wild type Wild type
....
12 A.J, Cain et at. unpublished. 14 43 43 : 43 15: 40 17 17 22 N. Rolland et at,, unpublished. 24 24 24 45 45 6, 45; S. Nuotio and S, Purton. unpublished. J-D RoChaix, unpublished: 33 15 A. Watson aud S. ~ r t o n . unpublished.
Tobacco
rbcL rpoB sprA ndh
Yes Yes Yes Yes
No No .... Yes
dear, but the observation t h a t the heteroplasmic clpP n u t a n t grows poorly photoautotrophically suggests t h a t the )rotease m a y interact with components of the photosyn~hetic apparatus 33. The other essential gene identified by chloroplast gene ]isruption in C. reinhardtii is a large open reading frame of
Rubisco Inactive in photosynthesis Wild .type Not dete
44 34 46 P. Nixon and P . Maliga, unpublished.
1995 codons 4°. This potentially encodes a product with a hydrophobic N-terminal domain of 350 amino acids and five putative t r a n s m e m b r a n e helices. The remaining part of the protein is very basic. Homologues of this open reading frame have been found in Nephroselmis olivacea and Chlorella vulgaris. I n addition, the predicted protein November 1997, Vol. 2, No. 11
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reviews displays structural similarity, but only low sequence identity to the ycfl product of land plants. Intriguingly, because it varies extensively in sequence and size, this open reading frame appears to be subjected to few evolutionary constraints. Of the 42 genes of the plastid genome of the nonphotosynthetic plant E. virginiana, 38 can be assigned to the plastid genetic system 35. It is interesting that the remaining genes include ycfl, accD (whose product is involved in fatty acid synthesis), 0RF2216 and clpP. Apparently the plastid genome and translation apparatus of E. virginiana have been maintained to express genes that are also found in the chloroplast genome of tobacco1. However, in contrast, accD, ycfl and 0RF2216 could not be identified in the plastid genome of rice ~.
Conclusions and perspectives Molecular analysis has led to the identification of novel functions controlled by chloroplast-encoded genes. Some of these functions are involved with photosynthesis, and others are essential for cell viability. In the latter case, in addition to the components of the plastid protein-synthesizing apparatus, at least two genes, clpP and the ycfl-like ORF1995 of C. reinhardtii, are notable. Although the role of the ATP-dependent ClpP protease has been studied extensively in E. coli 39, its function in plastids remains to be explored. The function of the membrane-associated Ycfl protein is also completely unknown and although ycfl is not present in all plastid genomes examined, it constitutes an important topic for future investigations. Plastid transformation has not only been important for elucidating the function of genes, it has also greatly helped the analysis of the molecular mechanisms underlying chloroplast gene expression. There is little doubt that this technology will continue to be powerful for gaining new insights into chloroplast DNA replication, transcription, RNA processing (including splicing and editing) and translation. A particularly interesting problem is how these different processes are regulated by the environment. The nuclear control of plastid gene expression is well documented, but the influence of the plastid on nuclear gene activity has also been investigated 4~. The nature of the chloroplast signal involved in this process is unknown, but as the number of plastid open reading frames of unknown function is rapidly shrinking, it should soon be possible to determine whether any of them is involved in this signal transduction.
Acknowledgements The author would like to thank E. Boudreau, P. Maliga, P. Nixon, I. Ohad, S. Purton and M. Sugita for communicating unpublished results, and M. Goldschmidt-Clermont for helpful comments. Research in the author's lab was supported by a grant from the Swiss National Fund. References 1 Sugiura, M. (1996) Structure and rephcation of chloroplast DNA, in Frontiers in Molecular Biology: Molecular Biology of Photosynthesis (Anderson, B., Salter, A.H. and Barber, J., eds), pp. 58-74, IRL Press 2 Reardon, E.M. and Price, C.A. (1995) Plastid genomes of three non-green algae are sequenced, Plant Mol. Biol. Rep. 13, 320-326 3 Reith, M. and Munholland, J. (1995) Complete nucleotide sequence of the Phorphyra purpurea chloroplast genome, Plant MoI. Biol. Rep. 13, 333-342 424
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4 Boynton, J.E. et al. (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles, Science 240, 1534-1538 5 Boynton, J.E. and Gillham, N.W. (1992) Chloroplast transformation in Chlamydomonas, Methods Enzymol. 217, 510-536 6 Goldschmidt-Clermont, M. (1991) Transgenic expression of aminoglycoside adenyl transferase in the chloroplast: a selectable marker for site-directed transformation of Chlamydomonas, Nucleic Acids Res. 19, 4083-4089 7 Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) Stable transformation of plastids in higher plants, Proc. Natl. Acad. Sci. U. S. A. 87, 8526-8530 8 Svab, Z. and Maliga, P. (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene, Proc. Natl. Acad. Sci. U. S. A. 90, 913-917 9 Kfmstner, P. et al. (1995) A mutant strain of Chlamydomonas reinhardtii lacking the chloroplast photosystem II psbI gene grows photoautotrophically, J. Biol. Chem. 270, 9651-9654 10 Ruffle, S.V. et al. (1995) The construction and analysis of a disruption mutant of psbH in Chlamydomonas reinhardtii, in Photosynthesis: from Light to Biosphere (Vol. 3) (Mathis, P., ed.), pp. 663466, Khiwer 11 Summer, J.S. etal. (1997) Requirement for the H phosphoproteinin photosystem II of Chlamydomonas reinhardtii, Plant Physiol. 113, 1359-1368 12 Takahashi, Y. et al. (1994) Directed disruption of the Chlamydomonas chtoroplastpsbK gene destabilizes the photosystem II reaction center complex, Plant Mol. Biol. 24, 779-788 1,3 Ikeuchi, M. et al. (1991) Cloning of thepsbK gene from Synechocystis sp. PCC 6803 and characterization of photosystem II in mutants lacking PSII-K, J. Biol. Chem. 266, 11111-11115 14 Monod, C. et al. (1994) The chloroplast ycf8 open reading frame encodes a photosystem II polypeptide which maintains photosynthetic activity under adverse growth conditions, EMBO J. 13, 2747-2754 15 Takahashi, Y. et al. (1996) The chloroplast ycf7 open reading frame encodes a small hydrophobic subunit of the cytochrome b6/f complex and is important for photoautotrophic growth of Chlamydomonas reinhardtii, EMBO J. 15, 3498-3506 16 Breyton, C. et al. Dimer to monomer conversion of the cytochrome b6f complex: causes and consequences, J. Biol. Chem. (in press) 17 Boudreau, E. et al. The chloroplast ycf3 andycf4 open reading frames are required for the accumulation of the photosystem I complex, EMBO J. (in press) 18 Wilde, A. et al. (1995) Inactivation of a Synechocystis sp strain PCC 6803 gene with homology to conserved chloroplast open reading frame 184 increases the photosystem II-to-photosystem I ratio, Plant Cell 7, 649-658 19 Sasaki, Y. et al. (1993) Chloroplast envelope protein encoded by chloroplast genome, FEBS Lett. 316, 93-98 20 Katoh, A. et al. (1996) cemA homologue essential to CO2 transport in the cyanobacterium Synechocystis PCC 6803, Proc. Natl. Acad. Sci. U. S. A. 93, 4006-4010 21 Katoh, A. et al. (1996) Absence of light-induced proton extrusion in a cotA-less mutant ofSynechocystis sp. strain PCC 6803, J. Bacteriol. 178, 5452-5455 22 Xie, Z. and Merchant, S. (1996) The plastid-encoded ccsA gene is required for heme attachment to chloroplast c-type cytochromes, J. Biol. Chem. 271, 4632-4639 23 Howe, G., Mets, L. and Merchant, S. (1995) Biosynthesis of cytochrome f in Chlamydomonas reinhardtii: analysis of the pathway in gabaculinetreated cells and in the heine attachment mutant B36, Mol. Gen. Genet. 246, 156-165 24 Bauer, C.E., Bollivar, D.W. and Suzuki, J.Y. (1993) Genetic analysis of photopigment biosynthesis in eubacteria: a guiding light for algae and plants, J. Bacteriol. 175, 3919-3925 25 Bennoun, P. (1982) Evidence for a respiratory chain in the chloroplast, Proe. Natl. Acad. Sci. U. S. A. 79, 4352-4356
reviews 26 Gillham, N.W. (1994) Organelle Genes and Genomes, Oxford University Press 27 Fischer, N. et al. (1996). Selectable marker recycling in the chloroplast, Mol. Gen. Genet. 251, 373-380 28 Malign, P. (1994) Isolation and characterization of mutants in plant cell culture, Annu. Rev. Plant Physiol. 35, 519-542 29 Harada, T. et al. (1992) Pollen-derived rice calli that have large deletions in plastid DNA do not require protein synthesis in plastids for growth, Mol. Gen. Genet. 233, 145-150 39 Gruissem, W. and Tonkyn, J.C. (1993) Control mechanisms of plastid gene expression, Crit. Rev. Plant Sci. 12, 19-55 31 Fong, S.E. and Surzycki, S.J. (1992) Chloroplast DNA polymerase genes of ChIamydomonas reinhardtii exhibit an unusual structure and arrangement, Curr. Genet. 21, 485-487 32 Winter, U. and Feierabend, J. (1990) Multiple coordinate controls contribute to a balanced expression of ribuiose 1,5-bisphospate carboxylase/oxygenasesubunits in rye leaves, Eur. J. Biochem. 187, 445-453 33 Huang, C. et al. (1994) The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth, Mel. Gen. Genet. 244, 15t-159 34 Allison, L.A., Simon, L.D. and Maliga, P. (1996) Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants, EMBO J. 15, 2802-2809 35 Wolfe, K.H., Morden, C.W. and Palmer, J.D. (1992) Function and evolution of a minimal plastid genome from a nonphotosynthetic parasitic plant, Proc. Natl. Acad. Sci. U. S. A. 89, 10648-10652 36 Hess, W.R. et al. (1993) Chloroplast rps15 and the rpoB/CiC2 gene cluster are strongly transcribed in ribosome-deficientplastids: evidence for a functioning non-chloroplast-encodedRNA polymerase, EMBO J. 12, 563-571 37 Lerbs-Mache, S. (1993) The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single subunit enzyme or catalytic
core of multimeric enzyme complexes?Proc. Natl. Acad. Sci, U. S. A. 90, 5509-5513 38 Mullet, J.E. (1993) Dynamic regulation of chloroplast transcription, Plant Physiol. 103, 309-313 39 Shapiro, J.A. (1993) A role for the Clp protease in activating Mumediated DNA rearrangements, J. Bacteriol. 175, 2625-2631 49 Boudreau, E. et al. (1997) A large open reading frame (ORF 1995) in the chloroplast DNA of Chlamydomonas reinhardtii encodes an essential protein, Mol. Gen. Genet. 256, 649-653 41 Taylor, W.C. (1989) Regulatory interactions between nuclear and plastid genomes, Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 211-233 42 Takahashi, Y. et al. (1991) Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I, EMBO J. 10, 2033-2040 43 Kuras, R. and Wollman, F-A. (1994) The assembly of cytochromeb6/f complexes:an approach using genetic transformation of the green alga Chlamydomonas reinhardtii, EMBO J. 13, 1019-1027 44 Kanevski, I. and Maliga, P. (t994) Relocation of the plastid rbcL gene to the nucleus yields functional ribulose-l,5 bisphosphate carboxylase in tobacco chloroplasts, Proc. Natl. Acad. Sci. U. S. A. 91, 1969-1973 45 Rochaix,J-D. (1995) Chlamydomonas reinhardtii as the photosynthetic yeast, Annu. Rev. Genet. 29, 209-230 46 Sugita, M. et al. Targeted deletion ofsprA from the tobacco plastid genome indicates that the encoded small RNA is not essential for pre16S rRNA maturation in plastids, Mol. Gen. Genet. (in press)
J6anlDavid Rocha:ixis a t ihe Depts
M~ie:~iai BioiogYaad
Geae#a;:Swi~etiand
The biosynthesis of glucosinolates Barbara Ann Ha kier and Liangcheng Du Glucosinolates and their hydrolytic products have a wide range of effects that are of biological and economic importance. In particular, these compounds have been shown to mediate interactions between plants and pests, and to reduce the feeding quality of rapeseed meal. Significant progress has now been made in understanding the biochemistry and genetics of glucosinolate biosynthesis, and the first Arabidopsis genes involved should soon be isolated. Hence, modifying the level of glucosinolates in Brassica crops, both to study interactions with pests and to improve flavour and nutritional qualities, should soon be a realistic possibility. lucosinolates - formerly t e r m e d t h e m u s t a r d oil glucosides - a r e amino acid-derived secondary p l a n t products t h a t contain a s u l p h a t e a n d a thioglucose moiety (Fig. 1), and occur in t h e order C a p p a r a l e s a n d a few o t h e r u n r e l a t e d fainilies of dicotyledons 1. T h e y are grouped into aliphatic, aromatic and indolyl glucosinolates b a s e d on w h e t h e r t h e y a r e derived from aliphatic amino acids (often methionine), phenylalanine or tyrosine, or tryptophan, respectively [or chain-elongated homologues t h e r e o f (e.g. homop h e n y l a l a n i n e and dihomomethionine)]. This d i v e r s i t y is f u r t h e r e x t e n d e d by secondary side-chain modifications such as hydroxylations, methylations, oxidations a n d des a t u r a t i o n s 2. A p p r o x i m a t e l y 100 different glucosinolates
G
© 1997 Elsevier Science Ltd
have been identified 2, 23 of which occur in A r a b i d o p s i s 3. Glucosinolates h a v e been detected in all organs of the plant, and are located w i t h i n t h e vacuole of the cell.
Glucosinolate degradation and the occurrence of degradation products in crops
The glucosinolate-myrosinase system The hydrolysis of glucosinolates is c a t a l y s e d by endogenous ~-thioglucosidases, the myrosinases, localized in t h e 'myrosin' cells s c a t t e r e d t h r o u g h o u t most p l a n t tissues. W i t h i n t h e s e cells t h e e n z y m e is stored inside m y r o s i n g r a i n s 4. The g l u c o s i n o l a t e - m y r o s i n a s e s y s t e m is a preformed two-component s y s t e m t h a t is a c t i v a t e d upon t i s s u e
PIt S1360-1385(97)01128-X
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425
Chloroplast reverse genetics: new insights into the function of plastid genes ,eooov. ,oo,o,. The complete sequences of 12 plastid genomes have recently been determined. This has revealed that in addition to k n o w n genes involved in plastid gene expression and photosynthesis, there are numerous open reading frames encoding genes of u n k n o w n function. Several of these genes have been inactivated in Chlamydomonas reinhardtii and tobacco by biolistic chloroplast transformation. These studies - coupled with molecular, biochemical and biophysical analysis - have revealed the existence of novel subunits and factors required for the accumulation and optimal functioning of photosynthetic complexes. Other plastid genes have also been identified that are essential for cell survival, but w h o s e function remains to be determined. p
lastid genomes of algae and land plants exist as circular DNA molecules with sizes in the range 50-400 kbp. In the unicellular alga Chlamydomonas reinhardtii there are 80 copies of the genome, whereas in mesophyll cells of higher plants there are up to 10000 copies. Most of the genomes examined contain two large inverted repeats harbouring the ribosomal RNA (rRNA) genes, and these are separated by two single-copy regions 1. Although the organization of genes in the plastid genome is generally well conserved in land plants, extensive genome rearrangements have occurred in algae. The complete nucleotide sequences of plastid genomes are now available for six vascular plants (Epifagus virginiana, Marchantia polymorpha, tobacco, rice, Pinus thunbergii and maize) and six unicellular eukaryotes (Euglena gracilis, the green alga Chlorella ellipsoidea, and three nongreen algae, Cyanophora paradoxa, Porphyra purpurea and Odontella sinensis) 2. The sequences for plastids from land plants and green algae reveal approximately 120 genes, which are mostly conserved among these organisms and can be divided into three major groups. The first group includes sequences required for plastid gene expression, with approximately 50 genes encoding subunits of RNA polymerase, rRNAs, transfer RNAs (tRNAs), ribosomal proteins and additional factors (e.g. elongation factor EF-Tu and initiation factor). The second group comprises approximately 40 genes, encoding components of the photosynthetic apparatus - mostly subunits of the thylakoid-associated complexes photosystem I (PSI), photosystem II (PSII), the cytochrome b6/f complex, the ATP synthase and the large subunit of the stromal enzyme Rubisco. A third group includes open reading frames whose function is still largely unknown. Some of these are conserved between species, and are designated ycf ('hypothetical chloroplast open reading frame'); others appear to be species-specific. A greater variation in plastid gene content occurs in the nongreen algae. For example, the plastid genome of Porphyra purpurea contains twice as many genes as land plants 3. Although not all the additional genetic material has been characterized, it includes genes involved in photosynthesis that are encoded by the nucleus in plants, and genes for plastid biosynthesis functions (e.g. for the synthesis of fatty acids, amino acids, pigments and thiamine). Other genes encode proteins involved in transport. © 1997 Elsevier Science Ltd
Transformation and reverse genetics of chloroplasts A major breakthrough in plastid research occurred in 1988 when Boynton et al. 4 established a biolistic chloroplast transformation method for C. reinhardtii. Once the DNAcoated particles have penetrated into the chloroplast, the transforming DNA is released and becomes incorporated into the plastid genome by homologous recombination. Transformants can be selected in several ways: it is possible to use mutants with lesions in chloroplast genes and transform them with the corresponding wild-type copies by selecting for photoautotrophic growth; alternatively, mutant alleles of the 16S and 23S rRNA genes that confer resistance to antibiotics can be used to transform wild-type cells on drug-containing medium 5. The most versatile chloroplast selectable marker is the bacterial aadA ('aminoglycoside adenyl transferase') gene, which has been engineered into a chloroplast expression cassette and confers resistance to spectinomycin and streptomycin ~ (Fig. 1). Because of the homologous plastid recombination system, this cassette allows specific gene disruptions and sitedirected mutagenesis to be performed on any chloroplast gene. Chloroplast transformation has also been achieved with tobacco plants using similar approaches 7's. The chloroplast genome is present in multiple copies, and thus any directed gene manipulation through transformation requires repeated subcloning of the transformants until complete segregation of the mutant and wild-type gene copies has been achieved. Disruption of chloroplast genes whose function is not essential for cell viability leads to a homoplasmic state in which all plastid gene copies are inactivated. This can occur for genes involved in photosynthesis, because photosynthetic function is dispensable in C. reinhardtii when the cells are grown with acetate as the carbon source, or in tobacco when sucrose is added to the growth medium. Homoplasmic gene disruptions can also be achieved for other dispensable plastid genes that may not necessarily have a role in photosynthesis. In contrast, inactivation of an essential plastid gene invariably leads to a heteroplasmic state in which mutant and wild-type gene copies coexist as long as the selective pressure for the disrupted gene is maintained (Fig. 2). Table 1 summarizes chloroplast gene disruptions performed in C. reinhardtii and tobacco. In addition to gene disruptions, it is possible to perform site-directed mutagenesis of chloroplast genes. In this case, PII $1360-1385(97)01121-7
November1997,Vol. 2, No. 11
41 9
reviews
Role of small subunits of photosynthetic complexes
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Identification of novel proteins involved in photosynthesis The presence of several conserved unidentified open reading frames in the chloroplast genomes of higher and lower plants, and green, red and brown algae, suggests that the encoded proteins play an important role. For example, the ycf8 open reading frame is cotranscribed with psbB, which encodes a PSII subunit, in C. reinhardtii and land plants TM. As this is the only other operon besides the ribosomal operon to be conserved in C. reinhardtii and plants, co-expression of the psbB and ycf8 genes appears to be important. The Ycf8 polypeptide consists of 31 amino acids with a central hydrophobic region that may act as a transmembrane domain. The protein has been identified as a novel PSII subunit (PsbT) based on its severely reduced levels in various PSII-deficient mutants and on its cofractionation with PSII (Ref. 14). Mutants with lesions in the ycf8 gene are able to
Fig. 1. Targeting of the chloroplast aadA ('aminoglycosideadenyl transferase') gene expression cassette to a specific site in the chloroplast genome. The expression cassette consists of the aadA coding sequence fused on its 5' side to a chloroplast promoter (P) and 5'-untranslated region (5'UTR), and on its 3' side to a chloroplast 3'-untranslated region (3'UTR). Upon bombardment of the cells with the transforming plasmid, in which the expression cassette is flanked by chloroplast DNA sequences corresponding to the targeting region (indicated by a thick line), the cassette is inserted into the chloroplast genome by homologous recombination. The AAD protein catalyses the adenylation of streptomycin/spectinomycin and thereby inactivates the drug.
the aadA cassette is inserted at a site that is not essential for chloroplast function and is close to the target gene. The construct is introduced into the chloroplast genome via biolistic transformation by selecting for spectinomycin resistance. This 'reverse genetics' approach is a powerful tool for analyzing the function of chloroplast-encoded subunits of photosynthetic complexes and for identifying new subunits of these complexes. 420
November1997,Vo[.2, No. 11
Biochemical analysis of the thylakoid photosynthetic complexes has revealed that, in addition to the larger catalytic subunits, they are comprised of numerous small molecular mass polypeptides. The larger subunits have been extensively characterized, but little attention has been devoted to these smaller subunits. Several of the smaller subunits have been disrupted: inactivation of the gene encoding PsbF ('photosystem Ir - the fourth letter designates a specific subunit) completely abolishes PSII activity (T.S. Mor and I. Ohad, unpublished); in contrast, cells lacking PsbI (a component of the PSII reaction centre) are still capable of photoautotrophic growth in low light, but not in high light, even though the amount of PSII is reduced to 10-20% of wild-type levels 9. Disruption of the psbH gene 1°'11 or the psbK gene 12 in C. reinhardtii leads to a considerable reduction in the accumulation of PSII and prevents photoautotrophic growth. However, inactivation ofpsbK in cyanobacteria only has a mild effect, because these mutants still grow photoautotrophically1~. These observations suggest that the PsbK subunit is not essential for the photochemical activity of PSII, but is required for the structural integrity of the complex, at least in C. reinhardtii. Extragenic suppressors of the psbK disruptions have recently been isolated, and these might provide new insight into the mechanism underlying the stability and turnover of the PSII complex (Y. Takahashi, J. van Dillewijn and J-D. Rochaix, unpublished).
reviews grow photoautotrophically at the same rate as the wild type, but PSII function and cell growth are impaired under high light and conditions of diminished chloroplast protein synthesis induced by spectinomycin 14. Hence this small PSII subunit appears to have a role in maintaining high photosynthetic activity under adverse growth conditions. A similar approach has been used to elucidate the function ofycf7, which encodes a 43 amino acid polypeptide containing a putative transmembrane domain 1~. Mutants in which ycf7 has been disrupted have considerably impaired photoautotrophic growth: they only accumulate 25-50% of the cytochrome b6/f complex as compared with the wild type, and the rate of electron transfer within the complex is reduced ~5.The Ycf7 product is absent in mutants lacking the cytochrome b6/f complex and copurifies with the cytochrome b6/f complex, indicating that Ycf7 is an authentic subunit of this complex and is required for its stability, accumulation and optimal efficiency~5. Recent results suggest that YcfY may be involved in the dimerization of the cytochrome b6/f complex1~. This subunit, called PetL, had escaped biochemical detection in earlier studies. Two chloroplast open reading frames, ycf3 and ycf4, have recently been shown to be required for stable accumulation of the PSI complex in C. reinhardtii, because disruption of either gene leads to a complete loss of PSI (Ref. 17). However, although the ycf3 and ycf4 gene products are found in the thylakoid membrane, they are not associated with PSI. These subunits also accumulate to the same level as in the wild type in mutants lacking PSI. The ycf4 gene has also been disrupted in cyanobacteria TM,but although the amount of PSI is reduced in Synechocystis sp. strain PCC 6803, the complex is not fully destabilized as in C. reinhardtii. These proteins could be involved in the synthesis of one of the PSI reaction centre polypeptides, might be required for the stability of the complex or could act as assembly factors. Another interesting chloroplast open reading frame is ycflO. Disruption of this gene in C. reinhardtii leads to increased light sensitivity and to a significant reduction in inorganic carbon uptake, although it is not yet clear whether this is a direct or indirect effect (N. Rolland et al., unpublished). The ycfl0 product has been localized to the plastid envelope ~9, and mutants affected in a similar gene in Synechocystis appear to have a defect in CO2 uptake 2°. More recently, the primary defect of these mutants has been traced to their inability to extrude protons 21. Analysis of the chloroplast ycf5 gene of C. reinhardtii has revealed that it displays limited sequence similarity with the cycK/ccll gene involved in cytochrome c biogenesis in bacteria ~2. A mutant of C. reinhardtii carrying a frameshift mutation within ycf5 is unable to attach heme to the membrane-bound apocytochrome f and to the soluble cytochrome c6, which acts as electron donor to PSI in copper-deficient cells23. The phototrophic growth deficiency could be complemented by transformation with the wild-type copy of ycf5 (Ref. 22). Targeted inactivation of ycf5 leads to the loss of both chloroplast c-type cytochromes. Thus, ycf5 has been renamed ccsA ('c-type cytochrome synthesis'). Reverse genetics has also been used to identify chloroplast genes involved in light-independent chlorophyll synthesis. In contrast to higher plants, which are only capable of synthesizing chlorophyll in the light, C. reinhardtii (and many lower plants and gymnosperms) can form chlorophyll both in a light-dependent and light-independent manner.
aadA
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Fig. 2. Targeted inactivation of a chloroplast gene. One chloroplast is shown containing five circular DNA toolecules. The targeted wild-type gene is indicated by an open box. The corresponding inactivated gene copy carrying the aadA ('aminoglycoside adenyl transferase') cassette introduced into the chloroplast by biolistic transformation is represented by a filled box. In the heteroplasmic state, both wild-type and mutant copies are maintained, because the former are essential for cell survival and the latter are required for growth in the presence of spectinomycin. In the homoplasmic state, all copies are inactivated, because the gene is not essential for nonphotosynthetic growth in the presence of an appropriate carbon source.
Disruption of three genes (chlB, chlL and chiN) leads to the loss of chlorophyll in the dark and to the accumulation of protochlorophyllide 24. These genes are homologous to three genes of Rhodobacter capsulatus found to be altered in mutants defective in bacteriochlorophyll formation 24. Thus, they appear to define minimal components of a novel enzyme complex that catalyses light-independent protochlorophyllide reduction. In addition, at least seven nuclear loci involved in this pathway have been found in C. reinhardtii. It remains to be seen whether they encode additional factors required for this complex or whether their products are necessary for the proper expression of the three chloroplast-encoded subunits. November1997,Vol.2, No. 11
42 1
reviews The chloroplast genomes from higher plants also contain eleven genes with homology to mitochondrial genes encoding subunits of NADH dehydrogenase 1. A role for such an enzyme in the chloro-respiratory chain was first postulated for C. reinhardtii 25, but none of the ndh ('NADH dehydrogenase') genes could be identified in the chloroplast genome of this species. The chloro-respiratory chain is believed to share the plastoquinone pool with the photosynthetic electron transport chain, and under anaerobic conditions in the dark the plastoquinone pool is reduced, presumably at the expense of NADH. Some of the ndh genes of tobacco have recently been disrupted (P. Nixon and P. Maliga, unpublished). Under normal growth conditions the mutant plants are indistinguishable from the wild type, but appear to be affected in their ability to reduce the plastoquinone pool in the dark. These preliminary results are thus compatible with the existence of an NADH:plastoquinone-oxidoreductase in the chloro-respiratory chain.
Essential chloroplast genes Protein synthesis Among the numerous mutants of C. reinhardtii affected in chloroplast function, some have been found to be partially deficient in plastid protein synthesis 26. However, no mutant was found with this process fully inactivated, suggesting that plastid protein synthesis is essential for the survival of C. reinhardtii. One would therefore predict that attempts to disrupt plastid genes involved in organellar protein synthesis would lead to a heteroplasmic state in which both wild-type and mutant copies containing the aadA expression cassette coexist. The heteroplasmic state persists for as long as the selection for drug resistance is maintained and in the absence of this selection pressure the mutant copies are rapidly lost 6'2;. Whether chloroplast protein synthesis is essential in higher plants is less clear. Antibiotics that specifically inhibit plastid ribosomes cause chlorophyll bleaching, but not death of calli from Nicotiana in tissue culture. The bleached cells are capable of division when sucrose is provided as the carbon source, although at a reduced rate 28. Plastid protein synthesis is also not required for calli cultured from roots of haploid rice plants derived from pollen grains 29.
Transcription Most transcription in mature chloroplasts is catalyzed by an RNA polymerase that is composed of ~2 ~, ~' and ~" subunits. It is similar to the Escherichia coli RNA polymerase, except that the chloroplast ~' and ~" subunits correspond to the N- and C-terminal domains of the bacterial ~' subunit, respectively3°. This enzyme initiates transcription of plastid genes from sequences resembling E. coli ¢rT°-typepromoters, and attempts to disrupt the genes encoding the subunits have yielded different results in C. reinhardtii and tobacco. In the green alga, the 5' part of the rpoB ('RNA polymerase') gene (rpoB1) is separated from the 3' part, called rpoB2 (Ref. 31). Disruption of rpoB1, rpoB2 and rpoC2 resulted in all cases in a heteroplasmic state, indicating that these genes are essential for cell survivaP '45. Surprisingly, no transcript of any of the C. reinhardtii rpo genes could be detected by mRNA (northern) hybridization, suggesting that these genes are expressed at very low levels 31. Resequencing of the rpoC2-ORF472 region of C. reinhardtii has recently revealed that it encodes a single open reading frame of 3119 residues (S. Nuotio and S. Purton, 4-22
November1997, Vol. 2, No. 11
unpublished). A comparison between this protein sequence and that of RpoC2 from higher plants reveals clear blocks of homology separated by sequences juxtaposed in frame with coding sequences of known identity. Similar sequences have been reported for the clpP ('catalytic subunit of the ATPdependent Clp protease') (Ref. 33), rps3 (Ref. 31) and ycflO (N. Rolland and J.D. Rochaix, unpublished) genes of C. reinhardtii. In contrast, a homoplasmic deletion of the rpoB gene was obtained in tobacco34. Although the mutant plants are deficient in pigments and photosynthetic activity, and have to be grown on sucrose-containing medium, the plants develop normally, but at a reduced rate 34. The plastids of the mutant are smaller than in the wild type and lack the arrays of stacked thylakoid membranes, so that they resemble proplastids in this respect. The levels of transcripts of plastid genes involved in photosynthesis are strongly reduced in the mutant, whereas the amount of transcript from the plastid genes of the protein-synthesizing system is near to wildtype levels. These results clearly imply the existence of a plastid-localized, nuclear-encoded RNA polymerase that is required for the maintenance of the nonphotosynthetic plastid functions necessary for plant growth and development. Evidence for a polymerase of this sort has also emerged from several other findings. The nonphotosynthetic parasitic plant E. virginiana contains only a small plastid genome of 67 kb (Ref. 35), and although this genome lacks all E. coli-like RNA polymerase genes as well as genes for the photosynthetic apparatus, it is nevertheless transcribed. The identity of the RNA polymerase has not yet been elucidated. Further evidence for a nuclear-encoded RNA polymerase arises from the albostrians mutant of barley, which is severely deficient in plastid ribosomes3~. Although expression of the plastid-encoded RNA polymerase genes is strongly reduced in this mutant, transcription of several plastid genes can still be detected 3~. Ribosome-deficient plastids isolated from heat-bleached rye seedlings also possess detectable transcriptional activitya2. Finally, distinct RNA polymerases have been isolated from chloroplasts and found to be associated with both multisubunit complexes and single subunit enzymes 37. The differences observed between the rpoB disruption in tobacco and C. reinhardtii may reflect the fact that higher plants can apparently tolerate proplastid-like p]astids, whereas a mature chloroplast (with or without chlorophyll) is needed for survival of green algae. Another possibility is that C. reinhardtii lacks the nuclear-encoded RNA polymerase. It has been proposed that this enzyme plays an important role during the differentiation of proplastids to plastids, before the E. coli-like RNA polymerase is required for transcription of the high levels of gene transcripts associated with photosynthetically active chloroplasts 38. Assuming that chloroplast protein synthesis is necessary for growth and survival, the question arises as to which of the chloroplast-encoded proteins is essential besides the components involved in the expression of the plastid genetic system. Of about 20 chloroplast gene disruptions in C. reinhardtii, only two (aside from those performed on the rpo and ribosomal protein genes) give rise to a heteroplasmic state (Table 1) even after extensive subcloning of the transformants. One of these genes, clpP, encodes the chloroplast homologue of the catalytic subunit of the ATP-dependent Clp protease in E. coli39. The exact role of the chloroplast enzyme in protein processing and degradation is not yet
reviews
Organism
Gone~
Homoplasmic?
Photo-autotrophic growth?
Phenotyl ~b
Chlamydomonas reinhardtii psaA psaB psaC psaJ
YeS Yes Yes Yes
No No No :- . Yes
PSIPSI PSF Wild type
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Yes Yes
No : : No
psbH psbI psbK psbN
Yes Yes Yes ~ ~ " Yes
NO So~e, N~ • Yes . . . . .
psbT (ycf8) petA petB petD petL (ycf7) ycfl (0RF1995)
Yes
ycf4 ycf5 ycflO
Yes . . . . Yes Yes Yes No Yes Yes Yes Yes
Yes NO No No . . . . . . . . Some-:Not determined No No No No
Light sensitive 'i '.: : b6f~ b6f:: h6f: . . . . . . . . . : ' - : . - - ...... : Possibly b6F . . . . . . . . . . Wild type . . . . . . . . . . . . . . . PSIPSI b6fUptake of inorganic carbon
chlB
Yes
Yes
chlL
Yes
Yes
chlN
-- Yes
Yes
ycf3
- PSF~ . PSIF ....
. . . . .
27 27 42 N, Fischer, E., Boudreauand J-D. Rochaix, unpublished,
. . . . .
.
Refs
.
.
.........
6
.
T, Mor and I. Ohad. unpublished. 10, 11
PSIILight sensitive PSIF Light sensitiv6
9
rpoB1 rpoB2 rpOC2-ORF
No No No
Not de~rmined Not determined Not determined
Chloro synthesis ir~ the dark Chlorophyll synthesis in the dark Chlorophyll synthesis in the dark Wild type Wild !ype Wild type
rps3 (0RF712)
No
Not determined
Wild type
.......
clpP ORF58 ORFB
NO Yes No
Some Yes Not determined
Wild type Wild type Wild type
....
12 A.J, Cain et at. unpublished. 14 43 43 : 43 15: 40 17 17 22 N. Rolland et at,, unpublished. 24 24 24 45 45 6, 45; S. Nuotio and S, Purton. unpublished. J-D RoChaix, unpublished: 33 15 A. Watson aud S. ~ r t o n . unpublished.
Tobacco
rbcL rpoB sprA ndh
Yes Yes Yes Yes
No No .... Yes
dear, but the observation t h a t the heteroplasmic clpP n u t a n t grows poorly photoautotrophically suggests t h a t the )rotease m a y interact with components of the photosyn~hetic apparatus 33. The other essential gene identified by chloroplast gene ]isruption in C. reinhardtii is a large open reading frame of
Rubisco Inactive in photosynthesis Wild .type Not dete
44 34 46 P. Nixon and P . Maliga, unpublished.
1995 codons 4°. This potentially encodes a product with a hydrophobic N-terminal domain of 350 amino acids and five putative t r a n s m e m b r a n e helices. The remaining part of the protein is very basic. Homologues of this open reading frame have been found in Nephroselmis olivacea and Chlorella vulgaris. I n addition, the predicted protein November 1997, Vol. 2, No. 11
423
reviews displays structural similarity, but only low sequence identity to the ycfl product of land plants. Intriguingly, because it varies extensively in sequence and size, this open reading frame appears to be subjected to few evolutionary constraints. Of the 42 genes of the plastid genome of the nonphotosynthetic plant E. virginiana, 38 can be assigned to the plastid genetic system 35. It is interesting that the remaining genes include ycfl, accD (whose product is involved in fatty acid synthesis), 0RF2216 and clpP. Apparently the plastid genome and translation apparatus of E. virginiana have been maintained to express genes that are also found in the chloroplast genome of tobacco1. However, in contrast, accD, ycfl and 0RF2216 could not be identified in the plastid genome of rice ~.
Conclusions and perspectives Molecular analysis has led to the identification of novel functions controlled by chloroplast-encoded genes. Some of these functions are involved with photosynthesis, and others are essential for cell viability. In the latter case, in addition to the components of the plastid protein-synthesizing apparatus, at least two genes, clpP and the ycfl-like ORF1995 of C. reinhardtii, are notable. Although the role of the ATP-dependent ClpP protease has been studied extensively in E. coli 39, its function in plastids remains to be explored. The function of the membrane-associated Ycfl protein is also completely unknown and although ycfl is not present in all plastid genomes examined, it constitutes an important topic for future investigations. Plastid transformation has not only been important for elucidating the function of genes, it has also greatly helped the analysis of the molecular mechanisms underlying chloroplast gene expression. There is little doubt that this technology will continue to be powerful for gaining new insights into chloroplast DNA replication, transcription, RNA processing (including splicing and editing) and translation. A particularly interesting problem is how these different processes are regulated by the environment. The nuclear control of plastid gene expression is well documented, but the influence of the plastid on nuclear gene activity has also been investigated 4~. The nature of the chloroplast signal involved in this process is unknown, but as the number of plastid open reading frames of unknown function is rapidly shrinking, it should soon be possible to determine whether any of them is involved in this signal transduction.
Acknowledgements The author would like to thank E. Boudreau, P. Maliga, P. Nixon, I. Ohad, S. Purton and M. Sugita for communicating unpublished results, and M. Goldschmidt-Clermont for helpful comments. Research in the author's lab was supported by a grant from the Swiss National Fund. References 1 Sugiura, M. (1996) Structure and rephcation of chloroplast DNA, in Frontiers in Molecular Biology: Molecular Biology of Photosynthesis (Anderson, B., Salter, A.H. and Barber, J., eds), pp. 58-74, IRL Press 2 Reardon, E.M. and Price, C.A. (1995) Plastid genomes of three non-green algae are sequenced, Plant Mol. Biol. Rep. 13, 320-326 3 Reith, M. and Munholland, J. (1995) Complete nucleotide sequence of the Phorphyra purpurea chloroplast genome, Plant MoI. Biol. Rep. 13, 333-342 424
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4 Boynton, J.E. et al. (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles, Science 240, 1534-1538 5 Boynton, J.E. and Gillham, N.W. (1992) Chloroplast transformation in Chlamydomonas, Methods Enzymol. 217, 510-536 6 Goldschmidt-Clermont, M. (1991) Transgenic expression of aminoglycoside adenyl transferase in the chloroplast: a selectable marker for site-directed transformation of Chlamydomonas, Nucleic Acids Res. 19, 4083-4089 7 Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) Stable transformation of plastids in higher plants, Proc. Natl. Acad. Sci. U. S. A. 87, 8526-8530 8 Svab, Z. and Maliga, P. (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene, Proc. Natl. Acad. Sci. U. S. A. 90, 913-917 9 Kfmstner, P. et al. (1995) A mutant strain of Chlamydomonas reinhardtii lacking the chloroplast photosystem II psbI gene grows photoautotrophically, J. Biol. Chem. 270, 9651-9654 10 Ruffle, S.V. et al. (1995) The construction and analysis of a disruption mutant of psbH in Chlamydomonas reinhardtii, in Photosynthesis: from Light to Biosphere (Vol. 3) (Mathis, P., ed.), pp. 663466, Khiwer 11 Summer, J.S. etal. (1997) Requirement for the H phosphoproteinin photosystem II of Chlamydomonas reinhardtii, Plant Physiol. 113, 1359-1368 12 Takahashi, Y. et al. (1994) Directed disruption of the Chlamydomonas chtoroplastpsbK gene destabilizes the photosystem II reaction center complex, Plant Mol. Biol. 24, 779-788 1,3 Ikeuchi, M. et al. (1991) Cloning of thepsbK gene from Synechocystis sp. PCC 6803 and characterization of photosystem II in mutants lacking PSII-K, J. Biol. Chem. 266, 11111-11115 14 Monod, C. et al. (1994) The chloroplast ycf8 open reading frame encodes a photosystem II polypeptide which maintains photosynthetic activity under adverse growth conditions, EMBO J. 13, 2747-2754 15 Takahashi, Y. et al. (1996) The chloroplast ycf7 open reading frame encodes a small hydrophobic subunit of the cytochrome b6/f complex and is important for photoautotrophic growth of Chlamydomonas reinhardtii, EMBO J. 15, 3498-3506 16 Breyton, C. et al. Dimer to monomer conversion of the cytochrome b6f complex: causes and consequences, J. Biol. Chem. (in press) 17 Boudreau, E. et al. The chloroplast ycf3 andycf4 open reading frames are required for the accumulation of the photosystem I complex, EMBO J. (in press) 18 Wilde, A. et al. (1995) Inactivation of a Synechocystis sp strain PCC 6803 gene with homology to conserved chloroplast open reading frame 184 increases the photosystem II-to-photosystem I ratio, Plant Cell 7, 649-658 19 Sasaki, Y. et al. (1993) Chloroplast envelope protein encoded by chloroplast genome, FEBS Lett. 316, 93-98 20 Katoh, A. et al. (1996) cemA homologue essential to CO2 transport in the cyanobacterium Synechocystis PCC 6803, Proc. Natl. Acad. Sci. U. S. A. 93, 4006-4010 21 Katoh, A. et al. (1996) Absence of light-induced proton extrusion in a cotA-less mutant ofSynechocystis sp. strain PCC 6803, J. Bacteriol. 178, 5452-5455 22 Xie, Z. and Merchant, S. (1996) The plastid-encoded ccsA gene is required for heme attachment to chloroplast c-type cytochromes, J. Biol. Chem. 271, 4632-4639 23 Howe, G., Mets, L. and Merchant, S. (1995) Biosynthesis of cytochrome f in Chlamydomonas reinhardtii: analysis of the pathway in gabaculinetreated cells and in the heine attachment mutant B36, Mol. Gen. Genet. 246, 156-165 24 Bauer, C.E., Bollivar, D.W. and Suzuki, J.Y. (1993) Genetic analysis of photopigment biosynthesis in eubacteria: a guiding light for algae and plants, J. Bacteriol. 175, 3919-3925 25 Bennoun, P. (1982) Evidence for a respiratory chain in the chloroplast, Proe. Natl. Acad. Sci. U. S. A. 79, 4352-4356
reviews 26 Gillham, N.W. (1994) Organelle Genes and Genomes, Oxford University Press 27 Fischer, N. et al. (1996). Selectable marker recycling in the chloroplast, Mol. Gen. Genet. 251, 373-380 28 Malign, P. (1994) Isolation and characterization of mutants in plant cell culture, Annu. Rev. Plant Physiol. 35, 519-542 29 Harada, T. et al. (1992) Pollen-derived rice calli that have large deletions in plastid DNA do not require protein synthesis in plastids for growth, Mol. Gen. Genet. 233, 145-150 39 Gruissem, W. and Tonkyn, J.C. (1993) Control mechanisms of plastid gene expression, Crit. Rev. Plant Sci. 12, 19-55 31 Fong, S.E. and Surzycki, S.J. (1992) Chloroplast DNA polymerase genes of ChIamydomonas reinhardtii exhibit an unusual structure and arrangement, Curr. Genet. 21, 485-487 32 Winter, U. and Feierabend, J. (1990) Multiple coordinate controls contribute to a balanced expression of ribuiose 1,5-bisphospate carboxylase/oxygenasesubunits in rye leaves, Eur. J. Biochem. 187, 445-453 33 Huang, C. et al. (1994) The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth, Mel. Gen. Genet. 244, 15t-159 34 Allison, L.A., Simon, L.D. and Maliga, P. (1996) Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants, EMBO J. 15, 2802-2809 35 Wolfe, K.H., Morden, C.W. and Palmer, J.D. (1992) Function and evolution of a minimal plastid genome from a nonphotosynthetic parasitic plant, Proc. Natl. Acad. Sci. U. S. A. 89, 10648-10652 36 Hess, W.R. et al. (1993) Chloroplast rps15 and the rpoB/CiC2 gene cluster are strongly transcribed in ribosome-deficientplastids: evidence for a functioning non-chloroplast-encodedRNA polymerase, EMBO J. 12, 563-571 37 Lerbs-Mache, S. (1993) The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single subunit enzyme or catalytic
core of multimeric enzyme complexes?Proc. Natl. Acad. Sci, U. S. A. 90, 5509-5513 38 Mullet, J.E. (1993) Dynamic regulation of chloroplast transcription, Plant Physiol. 103, 309-313 39 Shapiro, J.A. (1993) A role for the Clp protease in activating Mumediated DNA rearrangements, J. Bacteriol. 175, 2625-2631 49 Boudreau, E. et al. (1997) A large open reading frame (ORF 1995) in the chloroplast DNA of Chlamydomonas reinhardtii encodes an essential protein, Mol. Gen. Genet. 256, 649-653 41 Taylor, W.C. (1989) Regulatory interactions between nuclear and plastid genomes, Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 211-233 42 Takahashi, Y. et al. (1991) Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I, EMBO J. 10, 2033-2040 43 Kuras, R. and Wollman, F-A. (1994) The assembly of cytochromeb6/f complexes:an approach using genetic transformation of the green alga Chlamydomonas reinhardtii, EMBO J. 13, 1019-1027 44 Kanevski, I. and Maliga, P. (t994) Relocation of the plastid rbcL gene to the nucleus yields functional ribulose-l,5 bisphosphate carboxylase in tobacco chloroplasts, Proc. Natl. Acad. Sci. U. S. A. 91, 1969-1973 45 Rochaix,J-D. (1995) Chlamydomonas reinhardtii as the photosynthetic yeast, Annu. Rev. Genet. 29, 209-230 46 Sugita, M. et al. Targeted deletion ofsprA from the tobacco plastid genome indicates that the encoded small RNA is not essential for pre16S rRNA maturation in plastids, Mol. Gen. Genet. (in press)
J6anlDavid Rocha:ixis a t ihe Depts
M~ie:~iai BioiogYaad
Geae#a;:Swi~etiand
The biosynthesis of glucosinolates Barbara Ann Ha kier and Liangcheng Du Glucosinolates and their hydrolytic products have a wide range of effects that are of biological and economic importance. In particular, these compounds have been shown to mediate interactions between plants and pests, and to reduce the feeding quality of rapeseed meal. Significant progress has now been made in understanding the biochemistry and genetics of glucosinolate biosynthesis, and the first Arabidopsis genes involved should soon be isolated. Hence, modifying the level of glucosinolates in Brassica crops, both to study interactions with pests and to improve flavour and nutritional qualities, should soon be a realistic possibility. lucosinolates - formerly t e r m e d t h e m u s t a r d oil glucosides - a r e amino acid-derived secondary p l a n t products t h a t contain a s u l p h a t e a n d a thioglucose moiety (Fig. 1), and occur in t h e order C a p p a r a l e s a n d a few o t h e r u n r e l a t e d fainilies of dicotyledons 1. T h e y are grouped into aliphatic, aromatic and indolyl glucosinolates b a s e d on w h e t h e r t h e y a r e derived from aliphatic amino acids (often methionine), phenylalanine or tyrosine, or tryptophan, respectively [or chain-elongated homologues t h e r e o f (e.g. homop h e n y l a l a n i n e and dihomomethionine)]. This d i v e r s i t y is f u r t h e r e x t e n d e d by secondary side-chain modifications such as hydroxylations, methylations, oxidations a n d des a t u r a t i o n s 2. A p p r o x i m a t e l y 100 different glucosinolates
G
© 1997 Elsevier Science Ltd
have been identified 2, 23 of which occur in A r a b i d o p s i s 3. Glucosinolates h a v e been detected in all organs of the plant, and are located w i t h i n t h e vacuole of the cell.
Glucosinolate degradation and the occurrence of degradation products in crops
The glucosinolate-myrosinase system The hydrolysis of glucosinolates is c a t a l y s e d by endogenous ~-thioglucosidases, the myrosinases, localized in t h e 'myrosin' cells s c a t t e r e d t h r o u g h o u t most p l a n t tissues. W i t h i n t h e s e cells t h e e n z y m e is stored inside m y r o s i n g r a i n s 4. The g l u c o s i n o l a t e - m y r o s i n a s e s y s t e m is a preformed two-component s y s t e m t h a t is a c t i v a t e d upon t i s s u e
PIt S1360-1385(97)01128-X
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