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First published online July 24, 2003; 10.1104/pp.103.026146 Plant Physiology 133:287-294 (2003) © 2003 American Society of Plant Biologists
Photosynthesis and Growth of Tobacco with a Substituted Bacterial Rubisco Mirror the Properties of the Introduced EnzymeMolecular Plant Physiology, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 2601, Australia
Complete replacement, by biolistic plastid transformation, of the hexadecameric ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of tobacco (Nicotiana tabacum) with the dimeric version from the bacterium, Rhodospirillum rubrum, resulted in fully autotrophic and reproductive tobacco plants that required high CO2 concentrations to grow (Whitney SM, Andrews TJ [2001  ] Proc Natl Acad Sci USA 98: 14738-14743). Growth and photosynthesis of these plants was compared with that of nontransformed tobacco and other controls where the rbcL gene for the large subunit of tobacco Rubisco was linked to the aadA selectable-marker gene, simulating the gene arrangement of the transformants with R. rubrum Rubisco. An arrangement of the rbcL and aadA genes that gave rise to an abundant monocistronic rbcL transcript and a one-fifth as abundant bicistronic rbcL-aadA transcript had Rubisco levels and photosynthetic properties similar to those of nontransformed tobacco. Direct linkage of the rbcL and aadA genes, resulting in exclusive production of a bicistronic mRNA transcript analogous to that of the transformants with R. rubrum Rubisco, reduced transcript abundance and tobacco Rubisco content. The analogous transcript with the R. rubrum rbcM gene substituted for rbcL was not only reduced in abundance, but was also translated less efficiently. The photosynthetic rates of the transformants and controls were measured at high CO2 concentrations, using a mass spectrometric method. The rates and their responses to atmospheric CO2 concentration mirrored the amounts and the kinetic properties of the Rubiscos present. The contents of total nitrogen, carbohydrates, and photosynthetic metabolites of the leaves were also consistent with the content and type of Rubisco.
All plants depend on the photosynthetic CO2-fixing enzyme, Rubisco, to supply them with combined carbon. Plants, algae, and many bacteria have the so-called Form I Rubisco, which has a complex quaternary structure composed of eight large (50-55 kD) subunits, which bear the active sites, and eight small (12-18 kD) subunits (Roy and Andrews, 2000 ; Spreitzer and Salvucci, 2002). Two tetramers of small subunits glue four dimers of large subunits together in a (L2)4(S4)2 arrangement (Andersson, 1996). However, some bacteria and dinoflagellates have Rubiscos without small subunits. In these Form II Rubiscos, the basic L2 dimer occurs singly, as in Rhodospirillum rubrum, or as higher oligomers (Tabita, 1999).
Recently, we replaced the Form I Rubisco of tobacco (Nicotiana tabacum) with the Form II dimer from R. rubrum by substituting the rbcL gene for the large subunit in the tobacco plastid genome with the rbcM gene from R. rubrum (Whitney and Andrews, 2001a
The tobacco-rubrum plants had only approximately one-third as much R. rubrum Rubisco as the nontransformed controls had tobacco Rubisco (Whitney and Andrews, 2001a
The requirement of R. rubrum Rubisco for high concentrations of CO2 conferred on the transformed plants a requirement for CO2 concentrations so high that CO2 assimilation was barely detectable with conventional gas-exchange equipment based on infrared analyzers (Whitney and Andrews, 2001a
Construction of Tobaccos With rbcL in Contexts Similar to That of rbcM in Tobacco-rubrum The antibiotic-resistance gene, aadA, required to select plastome transformants, was introduced to the tobacco plastome in two ways. In LEV1, a promoterless aadA gene was inserted immediately downstream of the rbcL terminator, giving rise to the possibility of two mRNA transcripts—a monocistronic one containing rbcL alone and a bicistronic one with rbcL and aadA. LEV3 was similar, except that the rbcL terminator was deleted so that only a bicistronic transcript was possible. LEV3 mimics the gene arrangement used in tobacco-rubrum (Fig. 1). Five and seven spectinomycin-resistant plantlets were obtained from 10 sets of leaves bombarded with the pLEV1 and pLEV3 transforming plasmids, respectively. After three rounds of regeneration on spectinomycin-containing medium, DNA blots showed that all of the plantlets were homoplasmic (Fig. 2). Two lines each of LEV1 (L1a and L1b) and LEV3 (L3a and L3b) transformants were grown to maturity in soil and their flowers were pollinated with wild-type pollen. In the CO2-enriched atmosphere required for growth of tobacco-rubrum plants, the LEV1 and LEV3 plants grew similarly to nontransformed tobacco and substantially faster than tobacco-rubrum plants. However, eventually, all of the plants reached a similar size and bore the same number of leaves (Fig. 3). The thickness of the leaves (0.4 ± 0.1 mm) and the density of stomata (1.0 ± 0.1 x 104 cm-2) were similar in all genotypes (data not shown).
The Rubisco content of leaves of LEV1 plants was similar to that of the nontransformed controls, but, in LEV3 and tobacco-rubrum plants, it was reduced to 50% and 20%, respectively (Fig. 4A). Total soluble leaf protein was similar in LEV1, LEV3, and nontransformed plants, but was reduced in tobacco-rubrum plants, with most of the reduction attributable to the missing Rubisco (Fig. 4A). Under the high-CO2 growth conditions, Rubisco's carbamylation status inversely reflected its abundance (Fig. 4B).
Leaves of LEV1 and nontransformed plants had similar amounts of monocistronic rbcL transcripts (Figs. 2 and 4C). In addition, the LEV1 plants had a bicistronic rbcL-aadA transcript that was approximately 20% as abundant. The single bicistronic rbcL- aadA transcript of LEV3 leaves and rbcM-aadA transcript in tobacco-rubrum leaves (Fig. 2) was present at approximately 45% and 70%, respectively, of the level of the rbcL transcript in nontransformed leaves (Fig. 4C). Although the bicistronic rbcL-aadA transcript in LEV3 plants was less abundant, it was translated with an efficiency similar to that of the monocistronic rbcL transcript in LEV1 and nontransformed plants (Fig. 4D). By contrast, the translational efficiency of the bicistronic rbcM-aadA transcript in tobacco-rubrum plants was approximately one-third of that of the rbcL transcripts. Thus, the large reduction of Rubisco content of the tobacco-rubrum plants is a product of a modest reduction in message content and a larger reduction in its translational efficiency.
RNA blotting showed that LEV1 and nontransformed plants produced similar amounts of mRNA from their nuclear rbcS gene family. LEV3 and tobacco-rubrum plants had 1.6- and 2.5-fold higher amounts, respectively (data not shown). Presumably, this is a response to the reduction in content, or total lack, of tobacco Rubisco.
Where it was possible to compare them, there was generally reasonable agreement between CO2-assimilation measurements made with the infrared-analyzer and mass-spectrometer systems (Fig. 5). The membrane-inlet cuvette of the mass spectrometer system is unstirred and there are likely to be large boundary layer resistances adjacent to the leaf disc. These, or calibration imprecision, may be the cause of the slightly lower maximal assimilation rates measured with this system. At both levels of illumination, the reduced Rubisco content of the LEV3 plants compared with nontransformed plants was manifest as a reduction in slope of the curves at low CO2 pressures. However, at the much higher CO2 pressures under which the plants were grown, little difference in assimilation rate was apparent, indicating that both types of plants were limited by light-supported D-ribulose-1,5-bisphosphate (ribulose-P2) regeneration under these conditions. As reported previously (Whitney and Andrews, 2001a
The reduced Rubisco content of the tobacco-rubrum plants caused only modest increases in the ribulose-P2 content of leaves sampled under the growth conditions—no larger than that seen in LEV3 leaves (Fig. 6). By contrast, the 3-phospho-D-glycerate (P-glycerate) content, which was similar in LEV1, LEV3, and nontransformed tobacco, was massively reduced in tobacco-rubrum leaves, leading to an approximately 10-fold increase in the P-glycerate: ribulose-P2 ratio (Fig. 6).
The photosynthetic impairment of the tobacco- rubrum plants was particularly obvious in their leaf dry matter. Although the fresh weight per unit area of the leaves was little diminished (<20%) compared with the other three genotypes, the dry-to-fresh weight ratio was reduced by approximately 60% (Fig. 7A). Most of this reduction was attributable to the near absence of starch and Glc from the tobacco- rubrum leaves (Fig. 7B). This massive reduction in storage carbohydrate caused an approximately 70% reduction in total leaf carbon (Fig. 7D), far outstripping more modest changes in total leaf nitrogen (Fig. 7E) and leading to a halving of the C:N ratio (Fig. 7F). Differences between the LEV1, LEV3, and nontransformed plants were small and consistent only in the case of total leaf N, which ranked approximately according to Rubisco content (Fig. 7E). Chlorophyll content was not reduced by reduction in or substitution of Rubisco (Fig. 7C), and the chlorophyll a:chlorophyll b ratio was 2.4 ± 0.1 in all cases (not shown), indicating that none of the genotypes suffered photoinhibition in the high-CO2 growth atmosphere.
Low Rubisco Content of Tobacco-rubrum Plants Is Caused Mostly by Impaired Translation
Previous 35S pulse-labeling experiments showed that little turnover occurred with R. rubrum Rubisco in tobacco-rubrum plants and tobacco Rubisco in the controls (Whitney and Andrews, 2001a
The unaltered translational efficiency of the bicistronic rbcL-aadA transcript in the LEV3 plants, compared with the monocistronic rbcL transcript in nontransformed or LEV1 plants (Fig. 4D), demonstrates that the tobacco chloroplast is unable to increase the translation rate of the bicistronic message to compensate for its lesser abundance. This contrasts with the situation in the Chlamydomonas reinhardtii chloroplast where reductions in transcript abundance have been shown to be sometimes compensated by increases in translational efficiency, making transcript abundance nonlimiting in the expression of some chloroplast-encoded proteins, including the large subunit of Rubisco (Hosler et al., 1989
Measurement of photosynthetic gas exchange with equipment based on a membrane-inlet mass spectrometer allowed measurements at much higher CO2 partial pressures than are accessible with conventional equipment based on sensitive infrared analyzers. These measurements confirmed earlier suspicions, based on measurements at low CO2 only (Whitney and Andrews, 2001a At high CO2, the 50% reduced tobacco Rubisco content of the LEV3 plants was still sufficient to support wild-type rates of CO2 assimilation that were limited solely by the rate of light-driven ribulose-P2 regeneration, regardless of the intensity of illumination (Fig. 5). This accords with the wild-type growth characteristics of these plants at 25 mbar CO2 pressure (Fig. 3).
By contrast, CO2 assimilation by the tobacco- rubrum plants clearly displayed the much higher Kcair (apparent Km for CO2 at air levels of O2) of the R. rubrum enzyme (Jordan and Ogren, 1981 The ribulose-P2 and P-glycerate contents of leaves sampled under 450 µmol m-2 s-1 illumination in the growth cabinet (an intensity midway between the two intensities used for gas-exchange measurements; Fig. 5) showed that photosynthesis in the tobacco- rubrum plants was limited by Rubisco activity under these conditions. Although the ribulose-P2 contents were not very different for all four genotypes, the severe reduction of P-glycerate content (and the large increase in ribulose-P2:P-glycerate ratio) testify to the Rubisco-activity limitation in the tobacco-rubrum leaves (Fig. 6). By contrast, the more balanced ribulose-P2 and P-glycerate contents (Fig. 6), coupled with diminished Rubisco carbamylation status (Fig. 4B), are consistent with ribulose-P2-regeneration limitation applying in LEV1, LEV3, and nontransformed plants under the same conditions.
Before the advent of technology for manipulating the higher-plant plastome, genetic manipulation of Rubisco in plants was restricted to targeting the RbcS gene for the nuclear-encoded small subunit. Its expression was reduced by antisense genes in tobacco (Rodermel et al., 1988
The absence of detectable small subunits in the tobacco-rubrum plants (Whitney and Andrews, 2001a
Although the tobacco-rubrum plants had sufficient Rubisco activity for their photosynthesis to encounter the ribulose-P2-regeneration limitation at low light intensity (Fig. 5), it would be interesting to investigate whether higher contents of R. rubrum Rubisco would be able to eliminate the growth impairment under the better illuminated conditions in the high- CO2 growth cabinet. The present experiments indicate that perhaps a doubling of the present content of bacterial Rubisco would be required to achieve this. This will probably necessitate improvement of the translatability of the rbcM mRNA. Experiments with a synthetic rbcM with a plastid-optimized codon preference are in progress.
Construction and Transformation of Plasmid pLEV3
The transforming plasmid pLEV3 is equivalent to pRubLev14 (Whitney and Andrews, 2001a
The plastid genome of tobacco cv Petit Havana [N,N] was transformed biolistically with plasmids pLEV1 and pLEV3, and spectinomycin-resistant plantlets were regenerated in tissue culture as described (Svab and Maliga, 1993
LEV1, LEV3, and tobacco-rubrum (tr1; Whitney and Andrews, 2001a
All physiological, anatomical, and biochemical measurements were made on samples taken 8 h into the 14-h photoperiod from the fifth leaf below the apical meristem of T2-generation tobacco-rubrum, LEV3 and LEV1 transformants, and nontransformed plants of similar physiological age. At this stage, the plants were 15.5 ± 0.8 cm tall, they bore 12.5 ± 0.5 leaves, and the fifth leaf was 14 ± 0.5 cm in diameter. Because the tobacco-rubrum plants grew slower than the others, the former were chronologically older at the time they reached this stage. The plants were grown at 450 µmol quanta m-2 s-1 in 2.5% (v/v) CO2 as described above.
Total DNA was extracted from leaves, electrophoresed, and blotted as described previously (Whitney et al., 1999
Total RNA was isolated from the fifth leaf of T2-generation transformants and nontransformed plants (see above) with Tri-reagent (Sigma, St. Louis) according to the manufacturer's instructions. RNA was electrophoretically separated through formaldehyde-denaturing agarose gels (Sambrook et al., 2000 The abundances of rbcL- or rbcM-containing mRNA transcripts were measured in blots of gels loaded with 10 µg of total leaf RNA, using the rbcL2 and aadA DNA probes. Two RNA samples were extracted from each leaf sampled. The abundances of the monocistronic rbcL mRNA in nontransformed and LEV1 plants, and of the bicistronic rbcL-aadA1 and rbcL-aadA3 mRNAs in LEV1 and LEV3 leaf samples were measured using the rbcL2 probe. Similarly, the abundances of the bicistronic rbcM-aadA3, rbcL-aadA1, and rbcL-aadA3 mRNAs in the tobacco-rubrum, LEV1, and LEV3 leaf samples were measured in duplicate blots probed with the aadA probe (Fig. 2B).
Total N and total C content of dried leaf material were measured using an elemental analyzer (model EA 1110; Carlo Erba Instruments, Milan). Leaf discs (0.5 cm2) were snap frozen in liquid N2 and were immediately embedded onto a grooved aluminum stud using equal volumes of colloidal graphite (Agar Scientific, Essex, UK) and tissue-TEK embedding compound 4583 (Mile Scientific, Naperville, IL). While still frozen, and at approximately 13 Pa of water vapor pressure, leaf thickness and abaxial stomatal density were measured using a scanning electron microscope (model S-2250N; Hitachi, Tokyo) at an acceleration voltage of 27 kV.
Soluble leaf protein and the content and carbamylation status of Rubisco were measured as described (Whitney and Andrews, 2001a
Photosynthetic gas exchange by leaves in air was measured after transfer to the laboratory using a flow-through photosynthesis system (model LI-6400; Li-Cor) as described (Whitney et al., 1999
We thank Stephanie McCaffery for assistance with the carbohydrate analyses and Heather Kane and Grant Pearce for reading the manuscript. Received May 6, 2003; returned for revision May 27, 2003; accepted May 27, 2003. * Corresponding author; e-mail john.andrews{at}anu.edu.au; fax 61-2-6125-5075.
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Rodermel S, Haley J, Jiang CZ, Tsai CH, Bogorad L (1996) A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA. Proc Natl Acad Sci USA 93: 3881-3885 Rodermel SR, Abbott MS, Bogorad L (1988) Nuclear-organelle interactions: nuclear antisense gene inhibits ribulose bisphosphate carboxylase enzyme levels in transformed tobacco plants. Cell 55: 673-681[CrossRef][Web of Science][Medline] Roy H, Andrews TJ (2000) Rubisco: assembly and mechanism. In RC Leegood, TD Sharkey, S von Caemmerer, eds, Photosynthesis: Physiology and Metabolism. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 53-83
Ruuska SA, Badger MR, Andrews TJ, von Caemmerer S (2000) Photosynthetic electron sinks in transgenic tobacco with reduced amounts of Rubisco: little evidence for significant Mehler reaction. J Exp Bot 51: 357-368 Sambrook J, Fritsch EF, Maniatis T (2000) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
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Whitney SM, Andrews TJ (2001a) Plastome-encoded bacterial ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) supports photosynthesis and growth in tobacco. Proc Natl Acad Sci USA 98: 14738-14743
Whitney SM, Andrews TJ (2001b) The gene for the ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit relocated to the plastid genome of tobacco directs the synthesis of small subunits that assemble into Rubisco. Plant Cell 13: 193-205 Whitney SM, Baldet P, Hudson GS, Andrews TJ (2001) Form I Rubiscos from non-green algae are expressed abundantly but not assembled in tobacco chloroplasts. Plant J 26: 535-547[CrossRef][Web of Science][Medline]
Whitney SM, von Caemmerer S, Hudson GS, Andrews TJ (1999) Directed mutation of the Rubisco large subunit of tobacco influences photorespiration and growth. Plant Physiol 121: 579-588 This article has been cited by other articles:
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ABSTRACT
RESULTS
, n = 14), LEV3 (L3, 
, n = 9), and LEV1 (L1, 
, n = 9) transformants and nontransformed plants (nt, 
, n = 6). C, Shoot dry weight measured 37 d (L1, L3, and nt) and 55 d (tr) after cotyledon emergence.
, 5.2 µmol Rubisco sites m-2; 
, 6.2 µmol Rubisco sites m-2) and LEV3 (L3a; 
, 6.9 µmol Rubisco sites m-2; L3b; 
, 6.9 µmol Rubisco sites m-2) plants of similar physiological ages (24.6 ± 1.7 cm in height, bearing 15 leaves) grown at 450 µmol m-2 s-1 were measured at 25°C at the different light levels shown. The Li-Cor measurements for tobacco- rubrum plants reported previously (Whitney and Andrews, 2001a
