Photosynthesis Research 27: 1-14, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.

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Molecular biology of the C 3 photosynthetic carbon reduction cycle Christine A. Raines ~, Julie C. Lloyd 1 & Tristan A. Dyer 2

1Biology Department, University of Essex, Wivenhoe Park, Colchester C04 3SQ, UK (for correspondence and~or reprints); 2Cambridge Laboratory, John lnnes Centre for Plant Science Research, Colney Lane, Norwich, NR4 7UJ, UK Received 22 July 1990; accepted in revised form 30 September 1990

Key words: Calvin cycle, chloroplast metabolism, plant nuclear genes Abstract In recent years the enzymes of the C 3 photosynthetic carbon reduction (PCR) cycle have been studied using the techniques of molecular biology. In this review we discuss the primary protein sequences and structural predictions that have been made for a number of these enzymes, which, with the input of crystallographic analysis, gives the opportunity to understand the mechanisms of enzyme activity. The genome organisation and gene structure of the PCR enzymes is another area which has recently expanded, and we discuss the regulation of the genes encoding these enzymes and the complex interaction of various factors which influence their expression.

Introduction The reactions of the C 3 photosynthetic carbon reduction (PCR) cycle were elucidated by Calvin and colleagues in the 1950s. A wealth of information now exists on the functioning of this pathway, and on the enzymes which catalyse the individual reactions (Buchanan 1980, Robinson and Walker 1981, Leegood et al. 1985, Woodrow and Berry 1988). The PCR cycle involves eleven different enzymes and, with the exception of ribulose-l,5-bisphosphate carboxylase which catalyses the first step in this pathway, little was known until recently about the molecular biology of this cycle. The investigation of the PCR enzymes using molecular techniques has begun to advance our knowledge of the relationships between the structure and function of these enzymes and also the factors which control the development and maintenance of this pathway. These aspects will be the focus of this review and we have tried to give a comprehensive coverage of all the enzymes. Of necessity, the section on Rubisco is brief in comparison to the literature

but where possible we have given pertinent and review references.

The PCR proteins The protein sequences of a number of the PCR enzymes have been elucidated either by direct protein sequencing or by deducing the amino acid sequence from nucleotide sequence data. It is possible from primary structural information to learn more about the enzymes and how they function. Firstly, conserved regions which may have functional importance can be pinpointed by making sequence comparisons of the same enzyme from diverse sources. Structural predictions can then be used to give a more detailed picture of the features which determine enzyme activity. In addition, using cloned copies of D N A encoding these enzymes, the large amounts of protein needed to form crystals for the resolution of 3D structure by X-ray crystallography can be obtained.

Rubisco ( Rubulose- l,5-bisphosphate carboxylase/oxygenase - EC 4.1.1.39) Rubisco is by far the best studied plant enzyme and its properties have been extensively reviewed (Andrews and Lorimer 1987, Gutteridge 1990). Rubisco is a bifunctional enzyme with opposing carboxylase and oxygenase activities. The ratio between these competing reactions determines the efficiency of CO 2 fixation and the importance of this in determining plant productivity has made it a target for genetic manipulation (Ellis and Gatenby 1984). This work has, in turn, revealed the complex biology of this hexadecameric protein which involves not only the interaction of nuclear and chloroplast genomes, but also requires the synthesis of two Rubiscoassociated proteins; the plastid chaperonin and Rubisco activase. In higher plants and cyanobacteria, Rubisco has two sizes of subunit, referred to as the large (Lsu) and small (Ssu) subunits (Table 1). These have molecular weights of about 52- and 15-kDa, respectively, and are in a LsS 8 configuration giving the holoenzyme a molecular weight of about 536 kDa. The 3D structure of the Rubisco enzyme has now been resolved (Andersson et al. 1989, Knight et al. 1989). The L 8 core of the enzyme is essentially a tetramer of interlocking dimers capped at both ends by small subunits, each of which is in contact with two large subunits of adjacent pairs. The Lsu has two domains; the main structural component, an alpha/ beta barrel, is at the carboxyl-terminus and this region interacts with the amino-terminal domain of the adjacent Lsu forming the active site between the large-subunit dimer (Andersson et al. 1989, Knight et al. 1989). The Ssu is essential for full catalytic activity of higher plant Rubisco, although the mechanism of action is incompletely understood (Andrews 1988). The genes encoding the Lsu and Ssu were the first chloroplast (Bedbrook et al. 1979) and plant nuclear genes (Bedbrook et al. 1980) to be sequenced. Since that time a large number of both Ssu (at least 33 from more than 10 species) (for review see Dean et al. 1989) and Lsu genes have been sequenced. The sequence of the Lsu is highly conserved, indeed the residues which bind the substrate are invariant in all Rubiscos studied

to date, and it has been used extensively in phylogenetic and evolutionary studies (Ritland and Clegg 1987). This is not the case with the Ssu where up to 20% divergence is possible (Manzara and Gruissem 1988, Dean et al. 1989). Despite the significant sequence conservation between Lsu from various sources, the holoenzymes differ appreciably in their carboxylation/ oxygenation ratios (Andrews and Lorimer 1987), raising the possibility that the CO2/O 2 specificity of the holoenzyme is conferred by the Ssu. However, although Ssu has been shown to induce active site conformational changes (Scheidner et al. 1990), experimental data indicate that the ratio of carboxylation to oxygenation is determined by the Lsu alone (Andrews and Lorimer 1985, Incharoensakdi et al. 1986). Random mutagenesis of Chlamydomonas has shown that it is possible to influence the CO2/O 2 specificity of Rubisco by substituting residues in the Lsu that are not essential for catalysis (Chen et al. 1988, Chen and Spreitzer 1989). Using a combination of site-directed mutagenesis and chemical modification, a 5-fold lowering of the substrate specificity of L z Rubisco from Rhodospirillum rubrum has been achieved (Smith et al. 1990). In addition, the roles of individual amino acid residues within the catalytic domain of the Lsu of Rhodospirillum rubrum has been investigated using site-directed mutagenesis (Mural et al. 1990). This approach has depended largely on the ability to express the mutagenised proteins in E. coli, and for this reason has mainly been successful with prokaryotic enzymes. The synthesis and assembly of the higher plant enzyme is very complex, requiring additional proteins for assembly and activation, and it is not yet possible to produce an active LsS 8 protein from higher plants in E. coli.

Rubisco-associated proteins The assembly of the Rubisco complex is believed to be mediated by a protein termed the plastid chaperonin (Hemmingsen et al. 1988, Goloubinoff et al. 1989, Roy 1989, Hemmingsen 1990, Ellis 1990) which binds to both Lsu and Ssu prior to assembly (Lubben et al. 1989). This protein belongs to an important group of proteins, the molecular chaperones, that mediate the correct

3 Table 1. Summary of the structural data on the C 3 PCR enzymes and Rubisco-associated proteins. Sources are referenced in the appropriate sections in the text Enzyme

Monomer mol. wt

Holoenzyme structure

Number of aminoacids

Coding genome ~

Cytosolic isoenzyme

Transit Mature Ribulose-l,5-bisphosphate carboxylase Phosphoglycerate kinase Glyceraldehyde phosphate dehydrogenase Triose phosphate isomerase Aldolase Fructose-l,6-bisphosphatase Transketolase Sedoheptulose-1,7-bisphosphatase Ribulose phosphate epimerase Ribose phosphate isomerase Ribulose-5-phosphate kinase Plastid chaperonin

Lsu 52000 Ssu 15000 42992 A 36000 B 41000 27000 38000 39778 37-38000 35-38000 23000 b 26000 39200 61000 60000 41000 45000

Rubisco activase

Hetero 16-mer (L8S8) Monomer Homotetramer (A4) and Heterotetramer (A2B2) Homodimer Homotetramer Homotetramer Homotetramer Homodimer Homodimer b Homodimer Homodimer

None 46-57 72 >56 >53 Not Not 51 Not Not Not Not 53

Hetero 14-mer (a7/37)

9

Heterohexamer (or Homohexamer)

475-482 122-128 408 336 385 known known 358 known known known known 351

543 Not known 58 378 58 415

C N N N N N N N Nc N Nc Nc N

No No Yes Yes Yes Yes Yes Yes Yes No Yes Yes No

N N N

a C = chloroplast, N = nucleus; This data refers to the mammalian enzyme; c Likely coding compartment, but not yet determined experimentally.

folding and assembly of a number of other proteins. It is likely that the plastid chaperonin plays an important role in many protein assembly processes during chloroplast biogenesis (Ellis 1990). The plastid chaperonin is an oligomeric structure having a molecular weight of >700kDa. Its structure is heteromeric comprising subunits of two sizes: alpha (61kDa) and beta (60kDa) (Hemmingsen et al. 1988, Hemmingsen 1990). The enzyme Rubisco activase has been found in all higher plants so far analysed and is essential for activation of the Rubisco enzyme. The primary structures of the nuclear encoded spinach and Arabidopsis activases have been elucidated and this protein comprises two sizes of subunit, one of 41 and the other 45kDa (Werneke et al. 1988, Werneke et al. 1989). The sequence data indicate that these two polypeptides arise from the alternative splicing of a common precursor mRNA (Werneke et al. 1989) and they are functionally indistinguishable (Werneke et al. 1988).

3-Phosphoglycerate kinase (EC 2.7.2.3) Plants

have

two

phosphoglycerate

kinase

(PGKase) isozymes, one in the chloroplast and the other in the cytosol. These enzymes are antigenically related, but can be distinguished on the basis of isoelectric focusing properties and also with regard to substrate and Mg 2+ affinity (Anderson and Advani 1970, Kopke-Secundo et al. 1990, McMorrow and Bradbeer 1990). We have recently isolated and sequenced cDNA clones containing the entire coding region of both the chloroplastic and cytosolic versions of wheat PGKase (Longstaff et al. 1989). The chloroplast enzyme sequence contains 480 codons, the first 72 of which, code for a transit peptide and the remaining 408 for the mature protein (Table 1). A high level of homology (82%) exists between the chloroplastic and cytosolic isozymes and both of these enzymes share a 45-60% sequence homology with PGKase enzymes of other organisms (Longstaff et al. 1989, Watson and Littlechild 1990). The PGKase enzymes of yeast and horse have been shown, by X-ray crystallography, to be monomeric enzymes with a bilobal structure joned by a flexible hinge region (Banks et al. 1979, Watson et al. 1982). Modelling of the chloroplast enzyme using its derived amino acid sequence reveals a similar

bilobal structure (McMorrow et al. unpublished observation). The substrates are thought to bind in the cleft between the two lobes and the cleft then closes allowing the reaction to take place in an anhydrous environment.

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) In higher plants there are two chloroplast GAPDH subunits, GapA (36 kDa) and GapB (41 kDa) (Table 1). The functional enzyme is a tetramer with either a n m 4 or A2B 2 subunit structure (for review see Cerff 1982). Sequence analysis of tobacco cDNA clones encoding the GapA and GapB subunits has revealed that they are highly homologous with each other at both the nucleotide (71%) and amino acid (77%) levels (Shih et al. 1986). It has been suggested that the extra residues of the GapB subunit forms a 'tail' which may anchor the enzyme to the inner surface of the chloroplast envelope (Brinkmann et al. 1989). The sequence of the cytosolic (GapC) isozyme of the GAPDH enzyme (which is a homotetramer) is available from a number of plant species and comparison of this subunit protein sequence with that of the chloroplast (GapA) shows that these enzymes share only 45% homology (Shih et al. 1986, Martin and Cerff 1986, Brinkmann et al. 1987). The subunits of the chloroplast enzyme have amino acid sequences with pronounced prokaryotic features. In this respect they are substantially different from the cytosolic enzyme which resembles its homologues in other eukaryotes more than it does the chloroplast enzyme or those of prokaryotes. This has led to the proposal that the chloroplastic and cytosolic GAPDH isozymes have evolved from different lineages and strongly supports the endosymbiont theory of chloroplast origin (Martin and Cerff 1986, Shih et al. 1986, Gray 1989). The 3D structure of GAPDH from both a eukaryote and a prokaryote have been studied in some detail and were found to be very similar (Skarzynski et al. 1987). It seems that the initial binding of the NAD co-enzyme triggers a number of sequential structural changes and a model has been proposed to explain how these changes

enable the enzyme to perform its function (Skarzynski and Wonacott 1988).

Triose phosphate isomerase (EC 5.3.1.1) The chloroplastic triose phosphate isomerase (TPI) is a homodimeric enzyme with a subunit molecular weight of about 27kDa (Pichersky and Gottlieb 1984) (Table 1). This enzyme is clearly distinguishable by isoelectric focussing and peptide digest mapping from the cytosolic version, despite their having antigenic properties in common (Pichersky and Gottlieb 1984, Kurzok and Feierabend 1984). The sequence of the cytosolic enzyme is known and shares a high percentage (about 80%) of homology with TPI from a number of eukaryotic and procaryotic sources (Marchionni and Gilbert 1986). Comparison of the plant cytosolic isozyme sequence with the limited N-terminal amino acid sequence available for the chloroplast enzyme (Pichersky and Gottlieb 1984) shows some similarity between them. The 3-D structure of TPI is particularly noteworthy in that it was in this enzyme that the /3/a-barrel motif (common to many enzymes, and reviewed by Farber and Petsko 1990) was first identified (Banner et al. 1975, Muirhead 1983). Subsequent studies have shown that the substrate binds in a pocket which is then closed off by a flexible loop which shields the substrate from attack by water. This allows isomerization rather than hydrolysis of the substrate to take place. Although each active site pocket is mainly formed by residues from one subunit, residues from the other subunit help exclude water from the active-site domain and ensure that the appropriate hydrogen bonds are formed with the substrate. Thus, monomers only have very low activity (Brown and Kollman 1987).

Aldolase (EC 4.1.2.13) Two reactions of the PCR cycle involve aldolase activity and both steps seem to be catalysed by the same enzyme (Table 1). Electrophoretic analysis of this enzyme indicates that it has a 38-kDa subunit which forms tetramers to produce the active enzyme complex. The chloroplastic and cytosolic isozymes pos-

sess similar but not identical catalytic and structural properties (Schnarrenberger and Kruger 1986), and antisera raised against the spinach chloroplast and cytosolic enzymes cross-react with the complementary enzyme (Marsh et al. 1989). The only sequence data available on the chloroplast enzyme are 29 amino acid residues in the N-terminal region of the protein. This region is conserved between the chloroplast isozyme and the maize cytosolic and other eukaryotic aldolases (Marsh et al. 1989). There is a full length cDNA sequence for the maize cytosolic enzyme which has an overall level of similarity of about 55% with its eukaryotic homologues (Kelly and Tolan 1986). Surprisingly, given this relatively low level of homology, active heterotetramers can be formed between plant cytosolic and mammalian subunits (Heil and Lebherz 1978). Crystallographic studies of rabbit adolase show that, like Rubisco and triose phosphate isomerase, each subunit has a /3/or-barrel structure made up of alternating a-helixes and /3-sheets (Sygusch et al. 1987). The C-terminal region covers the active site pocket which is in the barrel and regulates access to it. In those aldolases for which the sequence is known, active site residues are absolutely conserved, but homologies in the C-terminal region are minimal. This suggests that it could be this region which is responsible in part for the variable substrate specificities which different aldolases exhibit.

Fructose- 1,6-bisphosphatase (EC 3.1.3.11) All FBPases appear to be homotetramers with molecular weights of about 160kDa (Table 1). Recently, the coding sequence for the wheat chloroplast subunit has been isolated and characterised (Raines et al. 1988) and shows that the molecular weight of the 358 residue monomer is 39 778. Alignment of FBPase amino acid sequences from both eukaryotic and prokaryotic sources with the wheat chloroplast sequence shows that FBPase enzymes are highly conserved. Overall, 45% of the residues are identical and another 20% are conservative substitutions (Raines et al. 1988). However, in both wheat and spinach, the chloroplast enzyme is unique in having an insertion of at least 12 extra

residues which appear to be involved in the regulation of its activity by light via the ferredoxin/thioredoxin system (Raines et al. 1988, Marcus et al. 1988). Recently, this hypothesis has been reinforced by the publication of spinach cytosolic protein sequence data which, whilst displaying 81% identity with the chloroplast isozyme, lacks this putative light-regulatory region (Ladror et al. 1990). The 3D structure of the pig kidney enzyme shows that the four subunits are arranged in a planar fashion with the four putative substrate binding sites located at the interfaces between adjoining subunits (Ke et al. 1989a, 1989b). Molecular modelling of the chloroplast sequence based on this structure indicates that the residues that may be involved in light regulation are exposed on the surface of the enzyme, some distance away from the active site (T.A. Dyer, unpublished results).

Transketolase (EC 2.2.1.1) Transketolase is a very poorly characterised enzyme, especially in plants. The chloroplast and cytosolic forms seem to have very similar properties (Feierabend and Gringel 1983). The main distinguishing feature of the chloroplast isozyme is that, unlike the cytosolic form, it is magnesium-activated (Murphy and Walker 1982). With respect to its size, there seems to be general agreement that in eukaryotes transketolase has a mass of about 150 kDa. However, while the enzyme from human blood (Takeuchi et al. 1986, Mocali and Paoletti 1989), rat and yeast (reviewed by Kochetov 1987) seemed to be a dimer of two identical subunits, the enzyme from plants was suggested to be homotetrameric with subunits of about 38 kDa (Table 1). Perhaps the reason for this disparity will soon be resolved as a recent analysis of the yeast transaldolase gene predicts that each subunit has a mass of 37 038 Da and so are very similar in size to those in plants. Recently, some preliminary crystallographic data for the wheat enzyme has been published (Schneider et al. 1989).

Sedoheptulose- 1,7-bisphosphatase Sedoheptulose-l,7-bisphosphatase (SBPase) has

6 no cytosolic counterpart and is found only in chloroplasts and photosynthetic prokaryotes. In higher plants the enzyme has been purified from maize and spinach and is apparently a homodimer with a subunit molecular weight of between 35-38kDa (Nishizawa and Buchanan 1981, Cadet et al. 1987) (Table 1). Of particular interest is its relationship to chloroplastic FBPase, as their substrates are so similar. They are both activated by reduced thioredoxin (Nishizawa and Buchanan 1981) and they are immunologically related, albeit rather poorly (Cadet and Meunier 1988). At this stage the main structural difference between the two enzymes appears to be that SBPase is a homodimer whereas FBPase is homotetrameric. Also, their amino acid compositions differ appreciably (Cadet and Meunier 1988). A putative coding sequence for the enzyme has been isolated and is being characterised (C.A. Raines, J.C. Lloyd, T.A. Dyer, unpublished results).

homodimeric enzyme has been determined using cDNA clones isolated from wheat (Raines et al. 1989) and spinach (Roesler and Ogren 1988, Milanez and Mural 1988); the subunit molecular weight is calculated to be 39.2kDa (Table 1). The wheat and spinach amino acid sequences are identical in 86% of positions and there is particularly high conservation of structure near the amino terminus, in which the two are identical in over 50 amino acids of the mature protein. This region is thought to be involved in both the light regulation of the catalytic activity of the enzyme and in ATP-binding. Two cysteine residues, Cys16 and Cys-53, have been shown to be involved in thioredoxin activation of this enzyme (Krieger et al. 1987, Porter et al. 1988). Affinity labelling studies (Krieger and Miziorko 1986) suggest that Cys-16 may also be part of the ATP-binding domain but not essential for catalysis. Further, such studies have implicated another residue (Lys-68), which lies outside the conserved region, in ATP binding (Miziorko et al. 1990).

D-ribulose 5-phosphate 3-epimerase (EC 5.1.3.1) Of all the enzymes of the PCR cycle, probably the least is known about this epimerase and the only molecular data available are from mammalian sources. The ribulose phosphate epimerase enzymes from calf (Wood 1979) and human (Karmali et al. 1983) are homodimers with a subunit molecular weight of about 23 kDa (Table

1). Ribose 5-phosphate isomerase (EC 5.3.1.6) Ribose-5-phosphate isomerase has been purified from spinach and tobacco, and the enzyme has two identical subunits of molecular weight 26 kDa (Rutner 1970, Kawashima and Tanabe 1976, Babadzhanova and Bakaeva 1987). There is, apparently, reversible dissociation between the subunits and it has been suggested that it might have a regulatory role in photosynthesis.

Ribulose-5-phosphate kinase ( phosphoribulokinase EC 2.7.1.19) Phosphoribulokinase is unique to the PCR cycle and is only found in photosynthetic organisms. The complete 351 amino acid sequence of this

PCR isozymes Most of the chloroplast PCR enzymes (see Table 1) have cytosolic counterparts which function in the glycolytic pathway of both plants and other organisms. In higher plants, both the cytosolic and chloroplastic enzymes are encoded in the nuclear genome and, in all the cases studied so far, they are products of separate genes (Shih et al. 1986, Martin and Cerff 1986, Chao et al. 1989). Comparisons of the nuclear encoded chloroplastic sequences with those of their cytosolic counterparts have contributed to the debate on the evolutionary origins of chloroplasts (Gray 1989). The chloroplast GAPDH sequences are more similar to those of prokaryotes than those of eukaryotes or of the plant cytosolic isoenzyme, supporting the endosymbiont theory (Martin and Cerff 1986, Shih et al. 1986, Brinkmann et al. 1987). There is now some evidence to suggest that this will also be the case for the FBPase isozymes (Raines et al. 1988, Ladror et al. 1990). The situation for PGKase seems more complex and it has been proposed that recombination has occurred between genes, possibly in quite recent times, so that now the chloroplast

proteins display both eukaryotic and prokaryotic features (Longstaff et al. 1989). Evidence from studies on aldolase and triose phosphate isomerase enzymes also suggests that recombination between the chloroplastic and cytosolic isozymes may have occurred. This proposal is tentative since little sequence data are available for the chloroplast versions of these enzymes, and it is largely based on biochemical analysis.

Transit sequences

Chloroplast proteins encoded in the nuclear genome are synthesised as precursor proteins with amino terminal extensions termed transit peptides which facilitate transport to the chloroplast (Highfield and Ellis 1978). Transit peptides have also been shown to be capable of directing foreign proteins to the chloroplast (Lubben et al. 1988), implying that the transit sequence must contain in its structure the information necessary for protein transport. Comparisons of a limited number of transit peptide sequences (from Rubisco Ssu, lightharvesting chlorophyll a/b binding protein and ferredoxin) led Karlin-Neumann and Tobin (1986) to propose a 'framework of homology' and some of these characteristics are shared by other PCR enzymes (Raines et al. 1989). However, these features are not evident in either the plastocyanin (Smeekens et al. 1985) or nitrite reductase (Back et al. 1988) presequences. The level of variation in primary structure between the wheat PCR transit peptides is quite striking, yet they are from the same plant species, are all transported to the stroma and function in the same biochemical pathway. Comparison of the PRKase transit sequences from wheat and spinach (Raines et al. 1989) and the G A P D H from maize and tobacco indicate that a higher level of homology is found between the transit peptides of the same protein from different species than between different proteins from the same species of plant; this was also shown for other chloroplast proteins (for review see Keegstra et al. 1989). Although no consensus transit peptide sequence has been found, many of these peptides have features in common: they are rich in serine,

threonine, valine and alanine; usually contain few acidic amino acids and have a net positive charge. As more sequence data emerges it seems unlikely that important functional features of transit sequences will be elucidated from studies of the linear amino acid sequence alone. Secondary structure analysis has not yet revealed the specific characteristics involved, but indications are that factors such as amphiphilicity, similar to that found in mitochondria, may have an important role to play for chloroplast transit peptides (Keegstra et al. 1989).

PCR gene organisation and characterisation

The genes encoding Rubisco Ssu were amongst the first higher plant genes to be isolated and a wealth of data has now accumulated concerning the Ssu gene families in a range of dicot and monocot species, including tomato, potato, pea, Petunia, Arabidopsis, Lemna and wheat (reviewed by Dean et al. 1989). The entire complement of Ssu genes has been isolated and fully sequenced only from tomato (Sugita et al. 1987) and Petunia (Dean et al. 1987) where there are 5 and 8 copies, respectively, per haploid genome. In both species the genes are organised into three loci, two of which contain a single copy with the remaining copies clustered at the third locus. Some conservation of intron positions has been observed amongst the Ssu genes studied. Dicot species have two introns at such sites and some copies have one additional intron. The limited number of monocot Ssu genes sequenced have only one intron, and this may be positioned at either of the two sites conserved in dicots, depending on the species. In contrast to the Ssu genes, there is only limited information on the genetic organisation of the other PCR enzymes. A recent study on the PCR genes of hexaploid wheat (Chao et al. 1989) used probes for all the enzymes for which cloned copies were available. Chloroplastic FBPase, PRKase and chloroplastic PGKase were all found to be present as single copy genes per haploid genome. Two copies of the chloroplastic G A P D H were detected, probably representing the A and B subunits of the chloroplast isozymes. The multigene family arrangement of the

Ssu genes therefore does not extend to all other members of the PCR cycle. This study included determining the chromosomal location of the wheat PCR gene copies (including Ssu), which were found to be distributed throughout the genome (see Chao et al. 1989). The only other published PCR gene which has been isolated and characterised is for maize chloroplast GAPDH (Quigley et al. 1988). This gene contains three introns, two of which are within the transit peptide coding region. The third, in the mature protein sequence, is at a position which is also conserved in the nematode GAPDH gene. The possible evolutionary implications of this have been considered (Quigley et al. 1988). In our laboratory, cloned copies of the wheat genes encoding chloroplastic FBPase and PRKase have been isolated and sequenced (Lloyd et al. in preparation). These genes contain three and four introns, respectively, all of which lie within the coding regions of the mature proteins.

PCR gene regulation The study of the expression of genes encoding enzymes of the PCR cycle has focused almost entirely on the Rubisco enzyme complex. The biogenesis of Rubisco is complicated by the fact that the subunits of this enzyme are encoded in separate compartments. Co-ordination of their synthesis may therefore involve communication between the nucleus and chloroplast (Taylor 1989); the possibility that Ssu transcript or protein levels influence the accumulation of holoenzyme is being investigated using antisense constructions in transgenic tobacco plants (Rodermel et al. 1988). Extensive studies of the nuclear encoded Ssu genes have revealed that complex regulatory mechanisms are involved in the synthesis of this component. The expression of Ssu is influenced by light, developmental stage, tissue type and to a lesser extent endogenous circadian rhythms (Kuhlemeier et al. 1987, Nagy et al. 1988a, 1988b, Giuliano et al. 1988a). The light response of the Ssu genes is mediated at the level of transcription (Gallagher and Ellis 1982) and involves the photoreceptor phytochrome in immature plants and both a blue light receptor

and phytochrome in mature tissue (Gallagher et al. 1985, Fluhr and Chua 1986). Individual members of the Ssu multigene families have also been shown to be differentially regulated in a number of species (for recent reviews see Manzara and Gruissem 1988, Dean et al. 1989). The Rubisco Lsu is encoded in the chloroplast genome and, although both light and developmental factors are involved in regulating the synthesis of this protein, unlike nuclear-encoded proteins, post-transcriptional control plays a major role (Mullet 1988, Gruissem 1989). It has been proposed that the differential accumulation of chloroplast mRNAs during development is controlled in part by mRNA stability and that translation of Lsu is light regulated (Berry et al. 1985, 1988). Until recently, little was known about the control of synthesis of other enzymes in the PCR cycle. Shih and Goodman (1988) have shown that the synthesis of chloroplastic GAPDH mRNA is light regulated at the transcriptional level, and found a correlation between the kinetics of GAPDH and Ssu mRNA synthesis. We have shown that the steady-state transcript levels of FBPase (Raines et al. 1988), PRKase (Raines et al. 1989) and PGKase (Longstaff et al. 1989) are regulated in a tissue dependent, light-induced and developmentally regulated manner. These data do not distinguish between rates of mRNA synthesis or stabilisation of transcripts, but, given the information on Ssu and GAPDH, it seems likely that significant control is exerted at the level of transcription. One question we are keen to address is what is the mechanism by which the nucleus controls the expression of a set of genes encoding enzymes involved in a biochemical pathway. Figure 1 (a, b and c) shows the steady state mRNA levels of a number of PCR enzymes in wheat. It is clear from these data that there is some co-ordination of mRNA synthesis of these components. Figure la shows that there is a lag phase in the accumulation of PCR enzyme mRNAs in etiolated tissue when the plants are transferred into the light. This delay is reduced when plants which developed in the light are given a period of darkness (40h) and then re-illuminated (Fig. lb). This difference may reflect the time required for chloroplast development to reach a certain stage.

Fig. I. RNA blot analysis of PCR enzymes in wheat. (a) 5 day old etiolated wheat leaves were illuminated for increasing periods of time. (b) 5 day old green leaves were put into the dark for 40 h and then re-illuminated over the time course indicated. (c) Leaves from 5 day old plants were cut into 6 sections of 2 cm each (1-6) numbered from the base as depicted. Sections 1 and 2 were repeated in an independent experiment and are shown on the left. Ten/xg of mRNA were loaded onto each track. Blots were probed as described (Raines et al. 1988) with a cDNA insert encoding chloroplastic fructose-l,6-bisphosphatase (FBP), ribulose-5-phosphate kinase (PRK), Rubisco small subunit (ssu) and both the chloroplastic (chl) and cytosolic (cyt) phosphoglycerate kinase (PGK).

10 Evidence supporting this proposal comes from the analysis of the levels of mRNAs in segments along the axis of the wheat leaf (Fig. lc). It is becoming increasingly clear that the developmental stage of the leaf has a significant role in controlling the light-regulated expression of nuclear photosynthetic genes, through competence to respond to phytochrome (Fourcroy et al. 1989) and also in relation to chloroplast development (Stockhaus et al. 1989, Sugita and Gruissem 1987). Therefore, although all but one of the proteins of the PCR cycle are encoded in the nuclear genome, it is important to consider the possible involvement of a chloroplastic factor in the biogenesis of this pathway (Taylor 1989). Whilst post-transcriptional mechanisms are likely to contribute to the control of the levels of the PCR proteins, it has become clear that transcription plays a major role in determining the expression of nuclear genes. For detailed analysis of these processes, cloned copies of the genes of interest are required and for this reason most of the information has been obtained for Rubisco Ssu. DNA sequence comparisons made to identify conserved motifs in upstream sequences and functional studies of deletions made in these regions have together yielded a great deal of information. Manzara and Gruissem (1988) have compared the upstream regions of all the tomato Rubisco Ssu genes with one another and also with all other published Ssu sequences, in an attempt to correlate the presence of conserved sequences with expression patterns. Deletion experiments, dependent on the introduction of manipulated sequences into transgenic plants to study their expression, reveal the 5' upstream sequences of Ssu genes to contain many different regulatory elements (reviewed by Jenkins 1988). Recent work has focused on identifying DNA binding sites of trans-acting factors (reviewed by Gilmartin et al. 1990) and for the best studied stimulus, light, a very complex picture is emerging. At least three DNA-binding proteins, GT-1 (Green et al. 1988), GBF (Giuliano et al. 1988b) and AT-1 (Datta and Cashmore 1989), have been found to interact with Ssu 5' upstream sequences. Clearly, the attention given to these topics should in the near future greatly increase our understanding of the molecular mechanisms of the control of Ssu transcription. As more

sequences become available from PCR genes other than Ssu it may be possible to define the components which confer the coordinate pattern of regulation observed.

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