Plant Physiol. (1999) 119: 133-142

Plastome Engineering of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Tobacco to Form a Sunflower Large Subunit and Tobacco Small Subunit Hybrid¹

Ivan Kanevski, Pal Maliga, Daniel F. Rhoades, and Steven Gutteridge^*

Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08855-0759 (I.K., P.M.); and Central Research and Development Department, Experimental Station, DuPont Company, Wilmington, Delaware 19880-0402 (D.F.R., S.G.)

	ABSTRACT

Targeted gene replacement in plastids was used to explore whether the rbcL gene that codes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, the key enzyme of photosynthetic CO₂ fixation, might be replaced with altered forms of the gene. Tobacco (Nicotiana tabacum) plants were transformed with plastid DNA that contained the rbcL gene from either sunflower (Helianthus annuus) or the cyanobacterium Synechococcus PCC6301, along with a selectable marker. Three stable lines of transformants were regenerated that had altered rbcL genes. Those containing the rbcL gene for cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase produced mRNA but no large subunit protein or enzyme activity. Those tobacco plants expressing the sunflower large subunit synthesized a catalytically active hybrid form of the enzyme composed of sunflower large subunits and tobacco small subunits. A third line expressed a chimeric sunflower/tobacco large subunit arising from homologous recombination within the rbcL gene that had properties similar to the hybrid enzyme. This study demonstrated the feasibility of using a binary system in which different forms of the rbcL gene are constructed in a bacterial host and then introduced into a vector for homologous recombination in transformed chloroplasts to produce an active, chimeric enzyme in vivo.

	INTRODUCTION

Rubisco is the key enzyme of photosynthetic CO₂ fixation, catalyzing two competing reactions that involve the carboxylation and oxygenation of ribulose-P₂, and initiating the primary steps of photosynthetic C reduction and photorespiration. The relative specificities of these two reactions is not a fixed constraint and varies by at least a factor of 10 in Rubisco enzymes from divergent species (Jordan and Ogren, 1981, 1983; Parry et al., 1989; Read and Tabita, 1992). Even enzymes from similar organisms exhibit significant differences in relative specificity. Because the ratio of these two reactions varies naturally or can be manipulated using in vivo and in vitro mutational techniques (for review, see Hartman and Harpel, 1994; Gutteridge and Gatenby, 1995), it has long been considered an attractive goal to change the properties of the enzyme to favor carboxylation.

In higher plants Rubisco is composed of two distinct subunits. The large subunit is encoded on the plastid genome and synthesized on plastid ribosomes in the stroma, where it aggregates as octameric cores with the aid of chaperonins (Gatenby and Ellis, 1990). Each core associates with eight small subunits that are encoded on the nuclear genome, synthesized on cytoplasmic ribosomes, and subsequently imported into the chloroplast (for review, see Gatenby and Ellis, 1990; Gutteridge and Gatenby, 1995). The elements required to catalyze the reactions of the enzyme, substrate binding and product formation, are located on the large subunit (Andrews, 1988; Cleland et al., 1998). Investigations of the isolated octameric core of cyanobacterial Rubisco showed that partitioning of the bisphosphate substrate between carboxylation and oxygenation is similar to the holoenzyme, indicating that this discrimination is also inherently determined by structural elements of the large subunit (Andrews and Lorimer, 1985; Gutteridge, 1990, 1991). These studies indicated that altering the relative specificity of Rubisco might best be achieved by modifying the large subunit rather than the small subunit (Read and Tabita, 1992). Furthermore, unlike the small subunit, which is coded for by a multigene family in plants, the large subunits are a homogeneous population encoded by the plastid genome.

There are now a number of examples of mutants of Rubisco generated in vivo and in vitro that have altered activities both favorably and less so. Nearly all examples have involved mutagenesis of the enzyme expressed in a heterologous bacterial host or in the green alga Chlamydomonas reinhardtii (for review, see Spreitzer, 1993; Hartman and Harpel, 1994; Gutteridge and Gatenby, 1995). A cyanobacterial operon from Synechococcus PCC6301 coding for both large and small subunits has been used the most because the enzyme readily assembles in Escherichia coli. Unlike the cyanobacterial enzyme, higher-plant Rubisco is not amenable to heterologous expression (Cloney et al., 1993; Gutteridge and Gatenby, 1995), and attempts at modifying the properties of higher-plant enzymes have been thwarted. However, chloroplast-transformation technology has now developed to the point where it is possible to manipulate the plastid genome in both C. reinhardtii (Boynton et al., 1988) and tobacco (Nicotiana tabacum) (Svab et al., 1990; Maliga, 1993; Svab and Maliga, 1993), including the rbcL (Rubisco large subunit) gene (Kanevski and Maliga, 1994; Zhu and Spreitzer, 1994, 1996; for review, see Rochaix, 1997). To determine if the Rubisco large subunit can be replaced with the subunit of other species, yet still fold into an active holoenzyme by assembling with the indigenous small subunits, tobacco plastids were transformed with vectors harboring the rbcL genes from other photosynthetic organisms. Two were chosen that represent enzymes having distinct activities compared with the tobacco enzyme. Our aim was to determine whether these characteristics are also transferred to a hybrid enzyme. The rbcL gene from the cyanobacterium Synechococcus PCC6301 (rbcL-C) generates Rubisco with lower relative specificity than the tobacco enzyme (Andrews and Lorimer, 1985), whereas the gene from sunflower (Helianthus annuus) (rbcL-S) was used to try to enhance the activity of the enzyme (Parry et al., 1989).

	MATERIALS AND METHODS

Plasmid Construction

In the 3-flanking region of the gene, a XbaI restriction site was introduced to allow much of the rbcL genes to be ligated in-frame with the 5 end of the tobacco gene (rbcL-T), yielding plasmids pSGPM1 and pSGPM2, which harbor the rbcL-S and rbcL-C genes, respectively. The 1.6-kb NcoI/XbaI fragment in plasmid pIK76 was replaced with a 1.6-kb NcoI/XbaI fragment from plasmids pSGPM1 and pSGPM2 to obtain plasmids pIK80 and pIK81, respectively. Plastid-transformation vectors pIK83 and pIK84 were obtained by cloning the spectinomycin-resistance gene (aadA) on a HindIII fragment into the HindIII site of plasmids pIK80 and pIK81, respectively. The chimeric aadA gene derives from plasmid pIK82 and is identical to aadA in plasmid pZS197 (Svab and Maliga, 1993) except that (a) the first five amino acids of the tobacco large subunit are translationally fused with the aadA-coding region; (b) the XbaI site between the aadA-coding region and the psbA 3-untranslated region was filled in by the Klenow fragment of DNA polymerase I; and (c) the Ecl136II site upstream of the promoter was converted into a HindIII site by linker-ligation (5-CAAGCTTG-3).

Transformation and Regeneration of Transgenic Plants

1

1

DNA and RNA Gel-Blot Analysis

Immunoblotting

DNA Sequencing

Rubisco Isolation and Activity

The specificities of carboxylation relative to oxygenation were determined using [1-¹⁴C]ribulose-P₂ (5 Ci mol¹) consumed by the enzyme in solutions containing set ratios of CO₂:O₂. The solutions were buffered with CO₂-free 0.1 M triethanolamine HCl, pH 8.2, containing MgCl₂. The buffer was then equilibrated with the appropriate ratio of CO₂:O₂ using a precision Wosthoff gas-mixing pump. The enzyme solution to be used in the assays was also equilibrated with the same gas prior to adding up to 32 µg of a 1.6 mg mL¹ solution to the reaction tube. The reaction was started by the addition of the labeled ribulose-P₂ (10 nmol), and the vials were immediately sealed with caps that contained a small amount of silicon grease to remove any dead space from above the solution.

To achieve good precision of the specificity results, enough of the buffer solution was made so that spinach Rubisco, which has well-determined specificity, could be assayed in parallel with the tobacco variants. The assay solutions (300 µL) also contained 0.2 mM vanadate to inhibit unwanted phosphatase activity that tended to deplete the ribulose-P₂ during the reactions and 5 µg of carbonic anhydrase to maintain the HCO₃-CO₂ equilibrium. The reactions were stopped by addition of acetic acid (10%) after at least 90% of the ribulose-P₂ had been consumed. The labeled products of carboxylation and oxygenation were collected as filtrates from small Dowex 50 H⁺ spin columns that also removed the proteins and then separated chromatographically, as described previously (Gutteridge et al., 1989b, 1993). The amount of 2-phosphoglycolate to 3-phosphoglycerate produced from the labeled ribulose-P₂ was determined by liquid-scintillation counting.

The standard Michaelis-Menten kinetic parameters of the enzyme were based on measurements of the carboxylase activity determined from the incorporation of ¹⁴CO₂ into an acid-stable product (Lorimer et al., 1976) at various ribulose-P₂ and CO₂ concentrations.

	RESULTS

Chimeric rbcL Genes

View larger version (27K): [in this window] [in a new window]   Figure 1. Targeted replacement of rbcL-T in the tobacco plastid genome with the rbcL-S gene. A, Plastid-targeting region in plasmid pIK83 (plastid DNA is underlined) and the cognate region of the plastid genome (Nt-ptDNA) and of the Nt-pIK83-1 line. atpB (Shinozaki et al., 1986) and accD (Sasaki et al., 1993) are plastid genes. aadA is the plastid-selectable spectinomycin-resistance gene. Recombination endpoints (1-4) discussed in the text are marked by vertical arrows. Horizontal arrows represent mRNAs detected by the rbcL (P1) and aadA (P2) probes. Restriction enzyme recognition sites: P, PvuII; R, EcoRV; Xh, XhoI. B, Wild-type plastid genome copies are absent in four plants regenerated independently (lanes 1-4) from the Nt-pIK83-1 line. Data for wild-type tobacco (T) are also shown. DNA blots of EcoRV-digested total cellular DNA (1 µg per lane) were hybridized with the rbcL (P1) and aadA (P2) probes. The rbcL probe hybridized to a 7.1-kb DNA fragment of the wild-type Nt-ptDNA and a 4.2-kb fragment of the transplastome (Fig. 1A). The 4.2-kb transgenic fragment also hybridized with the aadA probe. Note the lack of wild-type 7.1-kb DNA fragments in the plastid transformants.

View larger version (30K): [in this window] [in a new window]   Figure 2. Amino acid sequences of the large (A) and small (B) subunits of tobacco (Mazur and Chui, 1985; Shinozaki et al., 1986) and sunflower (B. Ranty and S. Gutteridge, unpublished data, accession no. AF097517; Waksman and Freyssinet, 1987) Rubisco showing the positions where there are amino acid differences. A significant feature of the sunflower large subunit is the C terminus extended by eight residues. (Accession numbers for the tobacco large and small subunits are P00876 and P26666, respectively. Those for the sunflower large and small subunits are P45738 and P08705, respectively.)

A selectable spectinomycin-resistance gene (aadA) was cloned between the chimeric rbcL gene and the accD gene in the plastid DNA fragment to yield plasmids pIK83 and pIK84, carrying the rbcL-S and rbcL-C genes, respectively. The map of the plastid DNA fragment in the pIK83 plasmid with the chimeric gene is shown in Figure 1A.

Plastid Transformation

Out of six transplastomic lines, two carried the rbcL-S and aadA genes and three carried the aadA gene alone. Because the regenerated plants within each group were indistinguishable, only one line from each group was studied further. A novel rbcL was also identified in only one line, Nt-pIK83-4, which had formed by another homologous recombination event. In this variant the rbcL-R gene has 233 nucleotides of the tobacco large subunit coding region fused with the sunflower gene. Given the significant (90.4%) DNA sequence conservation between the tobacco and sunflower genes, recombination at this particular site was a chance event, and indicates the feasibility of obtaining different chimeric rbcL genes by in vivo recombination during transformation. The product of rbcL-R in the Nt-pIK83-4 line is identical to the sunflower large subunit except for a conservative change of Glu to Asp at position 19 and a change of Glu to Gln at residue 30 (Fig. 2A).

Transformation with plasmid pIK84 yielded seven transplastomic lines. The plastid genome of two lines (Nt-pIK84-7 and Nt-pIK84-15) carried integrated copies of both rbcL-C and aadA, whereas in five lines only the aadA gene integrated. Since plants representing the independently transformed Nt-pIK84-7 and Nt-pIK84-15 lines were indistinguishable, data are shown for the Nt-pIK84-15 line only.

The rbcL-T/rbcL-S and rbcL-T/rbcL-C gene pairs can be distinguished by 9 and 12 restriction fragment-length polymorphic markers, respectively (not shown). The absence of wild-type plastid DNA copies in the regenerated plants was verified by DNA gel-blot analysis utilizing six restriction fragment-length polymorphic markers for each of the chimeric genes. An example for shoots regenerated from the Nt-pIK83-1 line is shown in Figure 3.

View larger version (52K): [in this window] [in a new window]   Figure 3. Tobacco plants with heterologous rbcL-S and rbcL-C genes. A, Leaves of wild-type (top) and of Nt-pIK83-1 (rbcL-S, left) plants are green. The leaves of Nt-pIK84-15 plants (rbcL-C, right) are yellow in sterile culture on 3% Suc RM medium. Illumination was for 16 h at 180 µmol m2 s1. B, Nt-pIK83-1 (rbcL-S) shoot grafted on a wild-type plant. Note the pale-green color of transgenic leaves in contrast to the green of the rootstock in the greenhouse.

The leaves of tobacco plants carrying rbcL-S (line Nt-pIK83-1) and the novel chimeric rbcL-R gene (line Nt-pIK83-4) were indistinguishable from wild-type leaves on a medium containing 3% Suc in sterile culture (Fig. 3A). In contrast, the leaves of the same plants were pale green when grown in the greenhouse as grafts (Fig. 3B). Seeds were obtained from shoots grafted onto a wild-type plant. The leaves of homoplasmic Nt-pIK84-15 tobacco plants with rbcL-C were yellow even in sterile culture on Suc (Fig. 3A), and could not be grown into fertile plants in the greenhouse even if grafted onto wild-type root stock.

Expression of the rbcL-C Gene

View larger version (50K): [in this window] [in a new window]   Figure 4. Steady-state level of rbcL mRNA in transgenic leaves. Total cellular RNA (1 µg per lane) was from wild-type tobacco (lane T, rbcL-T), Nt-pIK83-1 (lane S, rbcL-S), Nt-pIK83-4 (lane R, rbcL-R), Nt-pIK83-10 (lane T*, rbcL-T), and Nt-pIK84-15 (lane C, rbcL-C) plants. RNA gel blots were hybridized (Staub and Maliga, 1994) with the rbcL probe (Fig. 1A, P1) (A) and with the aadA coding region probe (Fig. 1A, P2) (B). The position of monocistronic rbcL (1.8 kb) and aadA (1.0 kb) transcripts and of the dicistronic rbcL-aadA transcript (3.0 kb) are shown in Figure 1A.

Western analysis was carried out to follow the accumulation of the Rubisco large and small subunits. No large subunit polypeptide could be detected with antibodies raised to either the spinach (Fig. 5) or cyanobacterial large subunit (not shown). Furthermore, no ribulose-P₂ dependent CO₂ fixation was detected with standard assays for Rubisco activity. Therefore, if the large subunit is synthesized, it must be rapidly degraded. Minor amounts of the tobacco small subunit were detectable in these leaves at about the same level found in tobacco leaves, which lack the rbcL gene (Kanevski and Maliga, 1994).

View larger version (40K): [in this window] [in a new window]   Figure 5. Immunoblot analysis of Rubisco accumulation in transgenic leaves. Total cellular protein (1 µg per lane) was from Nt-pIK83-1 (lane S, rbcL-S), Nt-pIK83-4 (lane R, rbcL-R), and Nt-pIK84-15 (lane C, rbcL-C) plants. For comparison, a dilution series (1:10, 1:5, 1:3, and 1:2) of a wild-type tobacco extract (T) is also shown. The blots were probed with antibodies to the spinach Rubisco large (L) and small (S) subunits (Kanevski and Maliga, 1994).

Expression of the rbcL-S Gene

Rubisco Properties

	DISCUSSION

We report here replacement of the 1.8-kb rbcL-coding region with cognate sequences from heterologous sources. In higher plants manipulation of the plastid genome thus far has involved insertion of chimeric genes in intergenic regions (Staub and Maliga, 1993; Svab and Maliga, 1993; Carrer and Maliga, 1995), introduction of point mutations (Bock et al., 1994; Bock and Maliga, 1995), and targeted gene deletions (Kanevski and Maliga, 1994; Sugita et al., 1997; Burrows et al., 1998). As to the practicality of coding region replacement, valuable information was obtained concerning separation of the selectable marker from the engineered coding region via short homologous sequences between the two (approximately 0.2 kb of the rbcL 3-untranslated region).

Incorporation of the selectable marker aadA alone was observed in three of six pIK83-transformed lines and two of seven pIK84-transformed lines. Thus, in 50% to 70% of the lines aadA integration occurred without incorporation of any of the heterologous rbcL sequences. Integration of aadA alone indicated that homologous recombination events via the approximately 0.2-kb rbcL 3-untranslated region adjacent to the selectable marker must have occurred. However, when both aadA and the engineered rbcL integrated, recombination in all but one clone occurred upstream of the coding region. Therefore, recombination events in the heterologous coding region were relatively rare (one in five clones). Minor sequence variations in the coding region of heterologous rbcL genes may suppress recombination via short stretches of homology.

Plastid transformation and targeting the rbcL gene for replacement is therefore feasible at an acceptable frequency. Even the gene from a photosynthetic bacterium with only 50% homology can be stably incorporated into the plastid genome. This allows a binary procedure to be used to construct plant large subunit chimeras and mutant variants. The first step involves a bacterial host such as E. coli as a means to construct variants of the rbcL gene from any source. This is followed by transformation of the plant system to express the chimeric genes and produce protein. The full gene from Synechococcus PCC6301 was transcribed into mRNA, but the mRNA was not translated into protein. At this stage, it is unknown whether there is incompatibility at the level of translation, or if the protein, once produced, is unable to assemble correctly using the indigenous folding machinery. Western analysis indicated that if protein is produced it is transient and does not accumulate enough to be detected by antibodies raised against the cyanobacterial large subunit. However, in those cases in which higher plant/cyanobacterial rbcL chimeras are of interest (Gutteridge et al., 1989a) and have proved intractable using E. coli expression and refolding, this system might provide an alternative approach, one that can supply the amounts of enzyme required for detailed structural analysis using crystallography.

Incorporation of a rbcL-S gene in the plastid genome of tobacco did result in protein that was able to assemble with tobacco small subunits to form an active hybrid Rubisco. The transformants produced about 30% of the wild-type level of Rubisco, and the hybrid enzyme had about 20% of the carboxylase activity at saturating substrate concentrations. This was not enough activity for the plants to withstand greenhouse growing conditions to reach maturity and supply seed unless grafted onto wild-type stock. Nevertheless, enough leaf material was obtained from the plants grown on Suc for an analysis of the properties of the chimeric protein. Naturally, the holoenzyme must be a hybrid of sunflower large subunits and one or more small subunits encoded by the rbcS-T gene family (see Fig. 5). Assuming that the hybrid retains a significant number of small subunits during purification, the lower affinities for the substrates may simply reflect some binding incompatibility at the interface between the large and small subunits.

Although large subunits contain the active site and are catalytically active, even some distance (15 Å) from the active site, small subunits influence both turnover and substrate affinities (Andrews, 1988; Gutteridge, 1991; Read and Tabita, 1992). Figure 6 shows the structural elements that form one of the interfaces between the large and small subunits of the tobacco hexadecamer. Of the 32 differences in primary structure between the tobacco and sunflower large subunits, several residues are within or directly adjacent to residues at the large/small subunit interfaces (Knight et al., 1990; Curmi et al., 1992). For example (Fig. 6), Lys-429, which resides in helix 8 of the C-terminal /-barrel domain of the tobacco large subunit, is a Gln in the sunflower enzyme. The contacts that would normally exist between this side chain and Glu-25 of the A helix of the small subunit may be less than ideal in the hybrid enzyme. This helix has multiple differences in primary structure (Waksman and Freysinnet, 1987), including a Ser at position 28 in tobacco that is occupied by Lys in the sunflower small subunit (Fig. 2B). Presumably, the loss of a basic residue that would be expected to readily form ionic interactions with Gln-429 and Glu-433 in the native sunflower large subunit may disrupt the association with the small subunits in the hybrid.

View larger version (40K): [in this window] [in a new window]   Figure 6. Amino acids at one of the interfaces of the large and small subunits. The region shown involves residues of helix 8 of the C-terminal domain of the large subunit (Knight et al., 1990) and the N-terminal segment of the small subunit. The side chains of amino acids that differ between the two species, or are adjacent to sequence changes that make contacts at the subunit interface (Fig. 2), are shown in this Molscript (Kraulis, 1991) rendition of tobacco Rubisco (Curmi et al., 1992). The position of the N-terminal end of helix 8 is critical for ribulose-P2 binding at the active site. L-subunit, Large subunit; S-subunit, small subunit.

Sequence differences between tobacco and sunflower small subunits total 28 (Fig. 2B), about 10 of which are in positions to influence large/small subunit interactions. These differences, compounded by the heterogeneous population of the native small subunits and the uncertainty about small subunit content of the purified hybrid, may all contribute to the reason for lower substrate affinities. For example, one role of the small subunit located between adjacent large subunit dimers is to stabilize the large subunit core, the integrity of which may be compromised in the hybrid, contributing to weaker substrate affinities (Andrews, 1988). Another factor that must be considered is whether a hybrid Rubisco variant is able to bind activating CO₂. Carbamylation was compromised in a hybrid version of the enzyme, in which Arabidopsis Rubisco large subunits were partnered with pea small subunits in Arabidopsis nuclear transformants (Getzoff et al., 1998). The sunflower-tobacco hybrid also showed some inability to bind activating CO₂ at all sites (Table I), but this clearly cannot explain the much larger drop in overall turnover.

Apart from the differences in primary structure, a striking feature of the sunflower large subunit is the C-terminal extension of eight charged residues compared with the tobacco subunit, a region of the protein that affects relative specificity (Gutteridge et al., 1993). The hybrid nature of the enzyme might be such that this extension is unable to interact correctly with other structural elements of the protein. Nevertheless, given the feasibility of plastid transformation, we now have a means of investigating these amino acid differences by introducing specific mutations into the large subunit and/or generating structural chimeras rationally or by chance recombination. The identification of photosynthetic organisms with higher relative specificities than either tobacco or sunflower (Read and Tabita, 1992) make this an exciting prospect. Because small subunits might, at the very least, influence the integrity of the complex quaternary structure of the holoenzyme, and therefore influence turnover, coexpression of the two subunits in the plastid might be attempted to circumvent any subunit heterogeneity. In terms of maintaining the vigor of regenerated transformants, an additional factor that influences Rubisco activity is activase (Portis, 1995; Andrews, 1996), which ensures that Rubisco does not become inactivated during turnover. The interaction between activase and a hybrid enzyme composed of sunflower large subunits may not be ideal (Wang et al., 1992). A combination of plastid transformation to alter tandemly incorporated large and small subunits and nuclear transformation to introduce modified activase would be an interesting system with which to study this elusive but essential interaction.

	FOOTNOTES

   Received August 10, 1998; accepted October 15, 1998.

	ABBREVIATIONS

Abbreviation: ribulose-P₂, ribulose 1,5-bisphosphate.

	ACKNOWLEDGMENTS

We thank Zora Svab for the aadA gene, Benoit Ranty for the rbcL-S gene, and Charles Herrmann for technical assistance.

	LITERATURE  CITED

Andrews TJ (1988) Catalysis by cyanobacterial ribulose-bisphosphate carboxylase large subunit in the complete absence of small subunits. J Biol Chem 263: 12213-12219 [Abstract/Free Full Text]

Andrews TJ (1996) The bait in the rubisco mousetrap. Nature Struct Biol 3: 3-7 [Medline]

Andrews TJ, Lorimer GH (1985) Catalytic properties of a hybrid cyanobacterial large subunits and higher plant small subunits of ribulose bisphosphate carboxylase-oxygenase. J Biol Chem 260: 4632-4636 [Abstract/Free Full Text]

Bachmann B, Lueke W, Hunsmann G (1990) Improvement of PCR amplified DNA sequencing with the aid of detergents. Nucleic Acids Res 18: 1309 [Free Full Text]

Bock R, Kossel H, Maliga P (1994) Introduction of a heterologous editing site into the tobacco plastid genome: lack of RNA editing leads to a mutant phenotype. EMBO J 13: 4623-4628 [Web of Science][Medline]

Bock R, Maliga P (1995) In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL editing. Mol Gen Genet 247: 439-443 [CrossRef][Web of Science][Medline]

Boynton JE (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240: 1534-1538 [Abstract/Free Full Text]

Burrows P, Sazanov L, A, Svab Z, Maliga P, Nixon PJ (1998) Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes. EMBO J 17: 868-876 [CrossRef][Web of Science][Medline]

Carrer H, Maliga P (1995) Targeted insertion of foreign genes into the tobacco plastid genome without physical linkage to the selectable marker gene. Biotechnology 13: 791-794 [CrossRef]

Cleland WW, Andrews TJ, Hartman FC, Gutteridge S, Lorimer GH (1998) Mechanism of rubisco: the carbamate as general base. Chem Rev 98: 549-561 [CrossRef][Web of Science][Medline]

Cloney LP, Bekkaoui DR, Hemmingsen SM (1993) Co-expression of plastid chaperonin genes and a synthetic Rubisco operon in Escherichia coli. Plant Mol Biol 23: 1285-1290 [CrossRef][Web of Science][Medline]

Curmi PMG, Cascio D, Sweet RM, Eisenberg D, Schreuder H (1992) Crystal structure of the unactivated form of ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco refined at 2.0-Å resolution. J Biol Chem 267: 16980-16989 [Abstract/Free Full Text]

Gatenby AA, Ellis RJ (1990) Chaperone function: the assembly of ribulose bisphosphate carboxylase-oxygenase. Annu Rev Cell Biol 6: 125-149 [CrossRef][Web of Science]

Getzoff TP, Zhu G, Bohnert HJ, Jensen RG (1998) Chimeric Arabidopsis thaliana Rubisco containing pea small subunit protein is compromised in carbamylation. Plant Physiol 116: 695-702 [Abstract/Free Full Text]

Gutteridge S (1990) Limitations of the primary events of CO₂ fixation in photosynthetic organisms: the structure and mechanism of rubisco. Biochem Biophys Acta 1015: 1-14

Gutteridge S (1991) The relative catalytic specificities of the large subunit core of Synechococcus ribulose bisphosphate carboxylase/oxygenase. J Biol Chem 266: 7359-7362 [Abstract/Free Full Text]

Gutteridge S, Gatenby AA (1995) Rubisco synthesis, assembly, mechanism and regulation. Plant Cell 7: 809-819 [CrossRef][Web of Science][Medline]

Gutteridge S, Pierce J, Lorimer GH (1989a) Details of the reactions catalysed by mutant forms of rubisco. Plant Physiol Biochem 26: 675-682

Gutteridge S, Reddy GS, Lorimer G (1989b) The synthesis and purification of 2-carboxyarabinitol 1-phosphate, a natural inhibitor of ribulose 1,5-bisphosphate carboxylase, investigated by ³¹P NMR. Biochem J 260: 711-716 [Web of Science][Medline]

Gutteridge S, Rhoades DF, Herrmann C (1993) Site-specific mutations in a loop region of the C-terminal domain of the large subunit of ribulose bisphosphate carboxylase/oxygenase that influence substrate partitioning. J Biol Chem 268: 7818-7824 [Abstract/Free Full Text]

Hartman FC, Harpel MR (1994) Structure, function, regulation and assembly of ribulose-1,5-bisphosphate carboxylase/oxygenase. Annu Rev Biochem 63: 157-234

Jordan DB, Ogren WL (1981) Species variation in the specificity of ribulose bisphosphate carboxylase/oxygenase. Nature 291: 513-515 [CrossRef]

Jordan D, B, Ogren W, L (1983) Species variation in kinetic properties of ribulose 1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys 227: 425-433 [CrossRef][Web of Science][Medline]

Kanevski I, Maliga P (1994) Relocation of the plastid rbcL gene to the nucleus yields functional ribulose-1,5-bisphosphate carboxylase in tobacco chloroplasts. Proc Natl Acad Sci USA 91: 1969-1973 [Abstract/Free Full Text]

Knight S, Andersson I, Branden C-I (1990) Crystallographic analysis of ribulose 1,5-bisphosphate carboxylase from spinach at 2.4 Å resolution. J Mol Biol 215: 113-160 [CrossRef][Web of Science][Medline]

Kraulis P (1991) Molscript: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallog 24: 946-950 [CrossRef]

Lorimer GH, Badger MR, Andrews TJ (1976) The activation of ribulose-1,5-bisphosphate carboxylase by carbon dioxide and magnesium ions: equilibria, kinetics, a suggested mechanism and physiological implications. Biochemistry 15: 529-538 [CrossRef][Medline]

Maliga P (1993) Towards plastid transformation in flowering plants. Trends Biotechnol 11: 101-106

Mazur B, Chui C-F (1985) Sequence of a genomic DNA clone for the small subunit of ribulose bisphosphate carboxylase-oxygenase from tobacco. Nucleic Acids Res 13: 2373-2386 [Abstract/Free Full Text]

Mettler IJ (1987) A simple and rapid method for minipreparation of DNA from tissue cultured cells. Plant Mol Biol Rep 5: 346-349 [CrossRef]

Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15: 493-497

Parry MAJ, Keys AJ, Gutteridge S (1989) Variation in the specificity factor of C3 higher plant Rubiscos determined by the total consumption of ribulose-P₂. J Exp Bot 40: 317-320 [Abstract/Free Full Text]

Portis A (1995) Regulation of rubisco by rubisco activase. J Exp Bot 46: 1285-1291 [Abstract]

Read BA, Tabita FR (1992) A hybrid ribulose bisphosphate carboxylase/oxygenase enzyme exhibiting a substantial increase in substrate specificity factor. Biochemistry 31: 5553-5560 [CrossRef][Medline]

Rochaix J-D (1997) Chloroplast reverse genetics: new insights into the function of plastid genes. Trends Plant Sci 2: 419-425 [CrossRef]

Sasaki Y, Hakamada K, Suama Y, Nagano Y, Furusawa I, Matsuno R (1993) Chloroplast-encoded protein as a subunit of acetyl-CoA carboxylase in pea plant. J Biol Chem 268: 25118-25123 [Abstract/Free Full Text]

Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Schinozaki K, and others (1986) The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J 5: 2043-2049 [Web of Science][Medline]

Shinozaki K, Sugiura M (1985) Genes for the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase constitute a single operon in a cyanobacterium Anacystis nidulans 6301. Mol Gen Genet 200: 27-32 [CrossRef]

Spreitzer RJ (1993) Genetic dissection of Rubisco structure and function. Annu Rev Plant Physiol Plant Mol Biol 44: 411-434 [CrossRef][Web of Science]

Staub J, Maliga P (1993) Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA. EMBO J 12: 601-606 [Web of Science][Medline]

Staub JM, Maliga P (1994) Translation of psbA mRNA is regulated by light via the 5-untranslated region in tobacco plastids. Plant J 6: 547-553 [CrossRef][Web of Science][Medline]

Sugita M, Svab Z, Maliga P, Sugiura M (1997) Targeted deletion of sprA from the tobacco plastid genome indicates that the encoded small RNA is not essential for pre-16S rRNA maturation in plastids. Mol Gen Genet 257: 23-27 [CrossRef][Web of Science][Medline]

Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87: 8526-8530 [Abstract/Free Full Text]

Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90: 913-917 [Abstract/Free Full Text]

Waksman G, Freyssinet G (1987) Nucleotide sequence of a cDNA encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase from sunflower (Helianthus annuus). Nucleic Acids Res 15: 1328 [Free Full Text]

Wang ZY, Snyder GW, Esau BD, Portis AR, Ogren WL (1992) Species-dependent variation in the interaction of substrate-bound ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase. Plant Physiol 100: 1858-1862 [Abstract/Free Full Text]

Zhu G, Spreitzer RJ (1994) Directed mutagenesis of chloroplast ribulose-bisphosphate carboxylase/oxygenase: substitution at large-subunit asparagine 123 and serine 379 decrease CO₂/O₂ specificity. J Biol Chem 269: 3952-3956 [Abstract/Free Full Text]

Zhu G, Spreitzer RJ (1996) Directed mutagenesis of chloroplast ribulose-bisphosphate carboxylase/oxygenase: loop-6 substitutions complement for structural stability but decrease catalytic efficiency. J Biol Chem 271: 18494-18498 [Abstract/Free Full Text]

Copyright Clearance Center:   0032-0889/99/119//10
 
© 1999 American Society of Plant Physiologists