A procedure to introduce point mutations into the Rubisco large subunit gene in wild-type plants

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast-encoded Rubisco large subunit rbcL is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild-type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modification of rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast-encoded genes.     

Status of the substrate binding sites of ribulose bisphosphate carboxylase as determined with 2-C-carboxyarabinitol 1,5-bisphosphate

The properties of the tight and specific binding of 2-C-carboxy-d-arabinitol 1,5-bisphosphate (CABP), which occurs only to reaction sites of ribulose 1,5-bisphosphate carboxylase (Rubisco) that are activated by CO₂ and Mg²⁺, were studied. With fully active purified spinach (Spinacia oleracea) Rubisco the rate of tight binding of [¹⁴C]CABP fit a multiple exponential rate equation with half of the sites binding with a rate constant of 40 per minute and the second half of the sites binding at 3.2 per minute. This suggests that after CABP binds to one site of a dimer of Rubisco large subunits, binding to the second site is considerably slower, indicating negative cooperativity as previously reported (S Johal, BE Partridge, R Chollet [1985] J Biol Chem 260: 9894-9904). The rate of CABP binding to partially activated Rubisco was complete within 2 to 5 minutes, with slower binding to inactive sites as they formed the carbamate and bound Mg²⁺. Addition of [¹⁴C]CABP and EDTA stopped binding of Mg²⁺ and allowed tight binding of the radiolabel only to sites which were CO₂/Mg²⁺-activated at that moment. This approach estimated the amount of CO₂/Mg²⁺-activated sites in the presence of inactive sites and carbamylated sites lacking Mg²⁺. The rate of CO₂ fixation was proportional to the CO₂/Mg²⁺-activated sites. During light-dependent CO₂ fixation with isolated spinach chloroplasts, the amount of carbamylation was proportional to Rubisco activity either initially upon lysis of the plastids or following total activation with Mg²⁺ and CO₂. Lysis of chloroplasts in media with [¹⁴C]CABP plus EDTA estimated those carbamylated sites having Mg²⁺. The loss of Rubisco activation during illumination was partially due to the lack of Mg²⁺ to stabilize the carbamylated sites.     

Construction of a tobacco master line to improve Rubisco engineering in chloroplasts

The inability to assemble Rubisco from any photosynthetic eukaryote within Escherichia coli has hampered structure-function studies of higher plant Rubisco. Precise genetic manipulation of the tobacco chloroplast genome (plastome) by homologous recombination has facilitated the successful production of transplastomic lines that have either mutated the Rubisco large subunit (L) gene, rbcL, or replaced it with foreign variants. Here the capacity of a new tobacco transplastomic line, (cm)trL, to augment future Rubisco engineering studies is demonstrated. Initially the rbcL was replaced with the selectable marker gene, aadA, and an artificial codon-modified (cm)rbcM gene that codes for the structurally novel Rubisco dimer (L(2), approximately 100 kDa) from Rhodosprillum rubrum. To obtain (cm)trL, the aadA was excised by transiently introducing a T-DNA encoding CRE recombinase biolistically. Selection using aadA enabled transplantation of mutated and wild-type tobacco Rubisco genes into the (cm)trL plastome with an efficiency that was 3- to 10-fold higher than comparable transformations into wild-type tobacco. Transformants producing the re-introduced form I tobacco Rubisco variants (hexadecamers comprising eight L and eight small subunits, approximately 520 kDa) were identified by non-denaturing PAGE with fully segregated homoplasmic lines (where no L(2) Rubisco was produced) obtained within 6-9 weeks after transformation which enabled their Rubisco kinetics to be quickly examined. Here the usefulness of (cm)trL in more readily examining the production, folding, and assembly capabilities of both mutated tobacco and foreign form I Rubisco subunits in tobacco plastids is discussed, and the feasibility of quickly assessing the kinetic properties of those that functionally assemble is demonstrated.     

Antisense RNA inhibition of Rubisco activase expression

Ribulose bisphosphate carboxylase (Rubisco) activase catalyzes the activation of Rubisco in vivo. Activase antisense DNA mutants of tobacco have been generated to explore the control that activase exerts on the photosynthetic process. These mutants have up to 90% reductions in activase protein levels as a consequence of an inhibition of activase mRNA accumulation. It is shown that photosynthesis, measured as the rate of CO₂ exchange (CER), is modestly decreased in plants exposed to high irradiances. The decreases in CER in the transgenic plants are accompanied by corresponding decreases in Rubisco activation, indicating that activase has a direct effect on photosynthetic rates in the antisense plants by influencing the activation state of Rubisco. It is concluded that in high light conditions, control of photosynthesis is largely shared between Rubisco and activase. Plant growth is also impaired in mutant plants that have severe reductions in activase. The inhibition of activase in the antisense plants does not have an impact on the accumulation of Rubisco large subunit or small subunit mRNAs or proteins. This indicates that the concerted expression of the genes for activase (Rca) and Rubisco (rbcL and rbcS) in response to light, developmental factors and circadian controls is not due to feedback regulation of rbcL or rbcS by the amount of activase protein.     

Decreased ribulose-1,5-bisphosphate carboxylase/oxygenase in transgenic tobacco transformed with 'antisense'rbcS.VIII. Impact on photosynthesis and growth in tobacco growing under extreme high irradiance and high temperature

Wild-type and antisense rbcS tobacco (Nicotiana tabacum) plants were grown in a glasshouse in midsummer in Portugal with an irradiance of 1500–2000 μmol m⁻²s⁻¹ and daytime temperatures of 30–35 °C. The Rubisco content of the transformants was lower by 35, 80 and over 90% than that of the wild-type. Gas exchange was measured over three separate days. There was a near-linear relation between Rubisco content and photosynthetic rate during the period of high irradiance, allowing a flux control coefficient of 0.83–0.89 to be estimated. The relation deviated slightly from linearity, because the internal CO₂ concentration (c;) was higher in the transformants than in the wild-type (190 and 275 μmol mol⁻¹ in plants with 35 and 80% less Rubisco, respectively, compared with 175 μmol mol⁻¹ for wild-type), compensating to some extent for the decreased Rubisco content. This increase in c_i occurred because the stomatal conductance (g) remained unaltered or was even higher in plants with decreased Rubisco, despite the lower rate of CO₂ assimilation. As a consequence, water use efficiency declined. The decreased rate of photosynthesis was not accompanied by a stoichiometric decrease in apparent growth rate. These results are discussed in relation to earlier studies of the plant set in growth cabinets. It is concluded that tobacco can adjust over a wide range of growth conditions to avoid a onesided limitation by Rubisco, but that in extreme environmental conditions this capacity to adapt is exhausted.     

The temperature response of CO2 assimilation, photochemical activities and Rubisco activation in Camelina sativa, a potential bioenergy crop with limited capacity for acclimation to heat stress

The temperature optimum of photosynthesis coincides with the average daytime temperature in a species’ native environment. Moderate heat stress occurs when temperatures exceed the optimum, inhibiting photosynthesis and decreasing productivity. In the present study, the temperature response of photosynthesis and the potential for heat acclimation was evaluated for Camelina sativa, a bioenergy crop. The temperature optimum of net CO₂ assimilation rate (A) under atmospheric conditions was 30–32?°C and was only slightly higher under non-photorespiratory conditions. The activation state of Rubisco was closely correlated with A at supra-optimal temperatures, exhibiting a parallel decrease with increasing leaf temperature. At both control and elevated temperatures, the modeled response of A to intercellular CO₂ concentration was consistent with Rubisco limiting A at ambient CO₂. Rubisco activation and photochemical activities were affected by moderate heat stress at lower temperatures in camelina than in the warm-adapted species cotton and tobacco. Growth under conditions that imposed a daily interval of moderate heat stress caused a 63?% reduction in camelina seed yield. Levels of cpn60 protein were elevated under the higher growth temperature, but acclimation of photosynthesis was minimal. Inactivation of Rubisco in camelina at temperatures above 35?°C was consistent with the temperature response of Rubisco activase activity and indicated that Rubisco activase was a prime target of inhibition by moderate heat stress in camelina. That photosynthesis exhibited no acclimation to moderate heat stress will likely impact the development of camelina and other cool season Brassicaceae as sources of bioenergy in a warmer world.     

The two genes for the small subunit of RuBP Carboxylase/oxygenase are closely linked in Chlamydomonas reinhardtii

Summary Ribulose bisphosphate carboxylase-oxygenase (Rubisco) is a key enzyme in the photosynthetic fixation of CO₂ by the chloroplast. The synthesis of the enzyme is an example of the cooperation between the chloroplast and the nucleocytoplasmic compartments, as it is assembled from subunits encoded in the two respective genomes. I have used a synthetic oligonucleotide probe to isolate the nuclear Rubisco small subunit genes (rbcS) directly from a genomic library of Chlamydomonas reinhardtii DNA. They constitute only a small family: there are two rbcS genes, and an additional related sequence, in the C. reinhardtii genome. All three are clustered within 11kb at a single locus, and should thus be particularly well suited for genetic manipulation. The pattern of expression of rbcS RNA is dependent on the growth conditions.     

Simple Determination of the CO(2)/O(2) Specificity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by the Specific Radioactivity of [C]Glycerate 3-Phosphate

A new method is presented for measurement of the CO₂/O₂ specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The [¹⁴C]3-phosphoglycerate (PGA) from the Rubisco carboxylase reaction and its dilution by the Rubisco oxygenase reaction was monitored by directly measuring the specific radioactivity of PGA. ¹⁴CO₂ fixation with Rubisco occurred under two reaction conditions: carboxylase with oxygenase with 40 micromolar CO₂ in O₂-saturated water and carboxylase only with 160 micromolar CO₂ under N₂. Detection of the specific radioactivity used the amount of PGA as obtained from the peak area, which was determined by pulsed amperometry following separation by high-performance anion exchange chromatography and the radioactive counts of the [¹⁴C]PGA in the same peak. The specificity factor of Rubisco from spinach (Spinacia oleracea L.) (93 ± 4), from the green alga Chlamydomonas reinhardtii (66 ± 1), and from the photosynthetic bacterium Rhodospirillum rubrum (13) were comparable with the published values measured by different methods.     

Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes

The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803rbc, were detected within 8 days when grown under 5% CO₂ in air. These transformants were unable to grow in air (0.03% CO₂). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO₂ in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803rbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.Abbreviations Cm^r chloramphenicol resistance - Km^r kanamycin resistance - HCR high CO₂ requirer - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SSC sodium chloride and sodium citrate - wt wild-type     

Effects of elevated CO2 and temperature on photosynthesis and Rubisco in rice and soybean

Rice (Oryza sativa L. cv. IR-72) and soybean (Glycine max L. Merr. cv. Bragg), which have been reported to differ in acclimation to elevated CO₂, were grown for a season in sunlight at ambient and twice-ambient [CO₂], and under daytime temperature regimes ranging from 28 to 40°C. The objectives of the study were to test whether CO₂ enrichment could compensate for adverse effects of high growth temperatures on photosynthesis, and whether these two C₃ species differed in this regard. Leaf photosynthetic assimilation rates (A) of both species, when measured at the growth [CO₂], were increased by CO₂ enrichment, but decreased by supraoptimal temperatures. However, CO₂ enrichment more than compensated for the temperature-induced decline in A. For soybean, this CO₂ enhancement of A increased in a linear manner by 32–95% with increasing growth temperatures from 28 to 40°C, whereas with rice the degree of enhancement was relatively constant at about 60%, from 32 to 38°C. Both elevated CO₂ and temperature exerted coarse control on the Rubisco protein content, but the two species differed in the degree of responsiveness. CO₂ enrichment and high growth temperatures reduced the Rubisco content of rice by 22 and 23%, respectively, but only by 8 and 17% for soybean. The maximum degree of Rubisco down-regulation appeared to be limited, as in rice the substantial individual effects of these two variables, when combined, were less than additive. Fine control of Rubisco activation was also influenced by both elevated [CO₂] and temperature. In rice, total activity and activation were reduced, but in soybean only activation was lowered. The apparent catalytic turnover rate (K_cat) of rice Rubisco was unaffected by these variables, but in soybean elevated [CO₂] and temperature increased the apparent K_cat by 8 and 22%, respectively. Post-sunset declines in Rubisco activities were accelerated by elevated [CO₂] in rice, but by high temperature in soybean, suggesting that [CO₂] and growth temperature influenced the metabolism of 2-carboxyarabinitol-1-phosphate, and that the effects might be species-specific. The greater capacity of soybean for CO₂ enhancement of A at supraoptimal temperatures was probably not due to changes in stomatal conductance, but may be partially attributed to less down-regulation of Rubisco by elevated [CO₂] in soybean than in rice. However, unidentified species differences in the temperature optimum for photosynthesis also appeared to be important. The responses of photosynthesis and Rubisco in rice and soybean suggest that among C₃ plants species-specific differences will be encountered as a result of future increases in global [CO₂] and air temperatures.     

Activation of interspecies-hybrid Rubisco enzymes to assess different models for the Rubisco–Rubisco activase interaction

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is prone to inactivation from non-productive binding of sugar-phosphates. Reactivation of Rubisco requires conformational remodeling by a specific chaperone, Rubisco activase. Rubisco activase from tobacco and other plants in the family Solanaceae is an inefficient activator of Rubisco from non-Solanaceae plants and from the green alga Chlamydomonas reinhardtii. To determine if the Rubisco small subunit plays a role in the interaction with Rubisco activase, a hybrid Rubisco (SSNT) composed of tobacco small subunits and Chlamydomonas large subunits was constructed. The SSNT hybrid, like other hybrid Rubiscos containing plant small subunits, supported photoautotrophic growth in Chlamydomonas, but growth in air was much slower than for cells containing wild-type Rubisco. The kinetic properties of the SSNT hybrid Rubisco were similar to the wild-type enzyme, indicating that the poor growth in air was probably caused by disruption of pyrenoid formation and the consequent impairment of the CO₂concentrating mechanism. Recombinant Rubisco activase from Arabidopsis activated the SSNT hybrid Rubisco and hybrid Rubiscos containing spinach and Arabidopsis small subunits at rates similar to the rates with wild-type Rubisco. However, none of the hybrid Rubiscos was activated by tobacco Rubisco activase. That replacement of Chlamydomonas small subunits with plant small subunits does not affect the species-specific interaction between Rubisco and Rubisco activase suggests that the association is not dominated by the small subunits that surround the Rubisco central solvent channel. Therefore, the geometry of a side-on binding mode is more consistent with the data than a top-on or ring-stacking binding mode.     

Decreased CO2 availability and inactivation of Rubisco limit photosynthesis in cotton plants under heat and drought stress in the field

Heat and drought stresses are often coincident and constitute major factors limiting global crop yields. A better understanding of plant responses to the combination of these stresses under production environments will facilitate efforts to improve yield and water use efficiencies in a climatically changing world. To evaluate photosynthetic performance under dry-hot conditions, four cotton (Gossypium barbadense L.) cultivars, Monseratt Sea Island (MS), Pima 32 (P32), Pima S-6 (S6) and Pima S-7 (S7), were studied under well-watered (WW) and water-limited (WL) conditions at a field site in central Arizona. Differences in canopy temperature and leaf relative water content under WL conditions indicated that, of the four cultivars, MS was the most drought-sensitive and S6 the most drought-tolerant. Net CO₂ assimilation rates (A) and stomatal conductances (gs) decreased and leaf temperatures increased in WL compared to WW plants of all cultivars, but MS exhibited the greatest changes. The response of A to the intercellular CO₂ concentration (A–C_i) showed that, along with stomatal closure, non-stomatal factors associated with heat stress also limited A under WL conditions, especially in MS. The activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) decreased in WL compared to WW plants, consistent with thermal inhibition of Rubisco activase activity. The extent of Rubisco deactivation could account for the metabolic limitation to photosynthesis in MS. Taken together, these data reveal the complex relationship between water availability and heat stress for field-grown cotton plants in a semi-arid environment. Both diffusive (drought-stress-induced) and biochemical (heat-stress-induced) limitations contributed to decreased photosynthetic performance under dry-hot conditions.     

Influence of ozone on transgenic tobacco plants expressing reduced quantities of Rubisco

RbcS-antisense transformed tobacco plants (Nicotiana tabacum cv. Petit Havana) expressing reduced quantities of Rubisco protein were used to examine the role of Rubisco quantity in determining ozone (O₃) sensitivity. Transformed and wild-type plants were exposed to O₃ in the greenhouse and in the field. Stomatal conductance, net photosynthesis and Rubisco protein quantity were measured at various times. Antisense-transformed genotypes responded to O₃ by exhibiting rapid, severe foliar necrosis. The wild-type plants responded more slowly, exhibiting limited injury. Decreases in stomatal conductance, net photosynthesis or Rubisco quantity in plants exposed to O₃ were not observed in asymptomatic leaves. Total biomass was lower for the transformed genotypes and decreased in both genotypes after exposure to O₃. Shoot–root ratio and specific leaf area were higher in the transformed genotypes and increased in both genotypes with exposure to O₃. Measurements of intercellular airspace demonstrated the presence of larger intercellular spaces in the transformed plants. The indirect effects of the rbcS antisense transformation, including morphological changes in the leaf, probably rendered the transformed plants more sensitive to the oxidant. The decreased quantity of Rubisco is not thought to be directly related to increased O₃ sensitivity in the transformed plants.