Modifying Plant Photosynthesis and Growth via Simultaneous Chloroplast Transformation of Rubisco Large and Small Subunits.

Engineering improved Rubisco for the enhancement of photosynthesis is challenged by the alternate locations of the chloroplast rbcL gene and nuclear RbcS genes. Here we develop an RNAi-RbcS tobacco (Nicotiana tabacum) master-line, tobRrΔS, for producing homogenous plant Rubisco by rbcL-rbcS operon chloroplast transformation. Four genotypes encoding alternative rbcS genes and adjoining 5'-intergenic sequences revealed that Rubisco production was highest (50% of the wild type) in the lines incorporating a rbcS gene whose codon use and 5' untranslated-region matched rbcL Additional tobacco genotypes produced here incorporated differing potato (Solanum tuberosum) rbcL-rbcS operons that either encoded one of three mesophyll small subunits (pS1, pS2, and pS3) or the potato trichome pST-subunit. The pS3-subunit caused impairment of potato Rubisco production by ∼15% relative to the lines producing pS1, pS2, or pST However, the ßA-ßB loop Asn-55-His and Lys-57-Ser substitutions in the pS3-subunit improved carboxylation rates by 13% and carboxylation efficiency (CE) by 17%, relative to potato Rubisco incorporating pS1 or pS2-subunits. Tobacco photosynthesis and growth were most impaired in lines producing potato Rubisco incorporating the pST-subunit, which reduced CE and CO2/O2 specificity 40% and 15%, respectively. Returning the rbcS gene to the plant plastome provides an effective bioengineering chassis for introduction and evaluation of novel homogeneous Rubisco complexes in a whole plant context.

The dependency of red Rubisco on its cognate activase for enhancing plant photosynthesis and growth.

Plant photosynthesis and growth are often limited by the activity of the CO2-fixing enzyme Rubisco. The broad kinetic diversity of Rubisco in nature is accompanied by differences in the composition and compatibility of the ancillary proteins needed for its folding, assembly, and metabolic regulation. Variations in the protein folding needs of catalytically efficient red algae Rubisco prevent their production in plants. Here, we show this impediment does not extend to Rubisco from Rhodobacter sphaeroides (RsRubisco)-a red-type Rubisco able to assemble in plant chloroplasts. In transplastomic tobRsLS lines expressing a codon optimized Rs-rbcLS operon, the messenger RNA (mRNA) abundance was ∼25% of rbcL transcript and RsRubisco ∼40% the Rubisco content in WT tobacco. To mitigate the low activation status of RsRubisco in tobRsLS (∼23% sites active under ambient CO2), the metabolic repair protein RsRca (Rs-activase) was introduced via nuclear transformation. RsRca production in the tobRsLS::X progeny matched endogenous tobacco Rca levels (∼1 µmol protomer·m2) and enhanced RsRubisco activation to 75% under elevated CO2 (1%, vol/vol) growth. Accordingly, the rate of photosynthesis and growth in the tobRsLS::X lines were improved >twofold relative to tobRsLS. Other tobacco lines producing RsRubisco containing alternate diatom and red algae S-subunits were nonviable as CO2-fixation rates (kcatc) were reduced >95% and CO2/O2 specificity impaired 30-50%. We show differences in hybrid and WT RsRubisco biogenesis in tobacco correlated with assembly in Escherichia coli advocating use of this bacterium to preevaluate the kinetic and chloroplast compatibility of engineered RsRubisco, an isoform amenable to directed evolution.

An improved Escherichia coli screen for Rubisco identifies a protein-protein interface that can enhance CO2-fixation kinetics.

An overarching goal of photosynthesis research is to identify how components of the process can be improved to benefit crop productivity, global food security, and renewable energy storage. Improving carbon fixation has mostly focused on enhancing the CO2 fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This grand challenge has mostly proved ineffective because of catalytic mechanism constraints and required chaperone complementarity that hinder Rubisco biogenesis in alternative hosts. Here we refashion Escherichia coli metabolism by expressing a phosphoribulokinase-neomycin phosphotransferase fusion protein to produce a high-fidelity, high-throughput Rubisco-directed evolution (RDE2) screen that negates false-positive selection. Successive evolution rounds using the plant-like Te-Rubisco from the cyanobacterium Thermosynechococcus elongatus BP1 identified two large subunit and six small subunit mutations that improved carboxylation rate, efficiency, and specificity. Structural analysis revealed the amino acids clustered in an unexplored subunit interface of the holoenzyme. To study its effect on plant growth, the Te-Rubisco was transformed into tobacco by chloroplast transformation. As previously seen for Synechocccus PCC6301 Rubisco, the specialized folding and assembly requirements of Te-Rubisco hinder its heterologous expression in leaf chloroplasts. Our findings suggest that the ongoing efforts to improve crop photosynthesis by integrating components of a cyanobacteria CO2-concentrating mechanism will necessitate co-introduction of the ancillary molecular components required for Rubisco biogenesis.

Seamless editing of the chloroplast genome in plants.

BACKGROUND: Gene editing technologies enable the precise insertion of favourable mutations and performance enhancing trait genes into chromosomes whilst excluding all excess DNA from modified genomes. The technology gives rise to a new class of biotech crops which is likely to have widespread applications in agriculture. Despite progress in the nucleus, the seamless insertions of point mutations and non-selectable foreign genes into the organelle genomes of crops have not been described. The chloroplast genome is an attractive target to improve photosynthesis and crop performance. Current chloroplast genome engineering technologies for introducing point mutations into native chloroplast genes leave DNA scars, such as the target sites for recombination enzymes. Seamless editing methods to modify chloroplast genes need to address reversal of site-directed point mutations by template mediated repair with the vast excess of wild type chloroplast genomes that are present early in the transformation process. RESULTS: Using tobacco, we developed an efficient two-step method to edit a chloroplast gene by replacing the wild type sequence with a transient intermediate. This was resolved to the final edited gene by recombination between imperfect direct repeats. Six out of 11 transplastomic plants isolated contained the desired intermediate and at the second step this was resolved to the edited chloroplast gene in five of six plants tested. Maintenance of a single base deletion mutation in an imperfect direct repeat of the native chloroplast rbcL gene showed the limited influence of biased repair back to the wild type sequence. The deletion caused a frameshift, which replaced the five C-terminal amino acids of the Rubisco large subunit with 16 alternative residues resulting in a ~30-fold reduction in its accumulation. We monitored the process in vivo by engineering an overlapping gusA gene downstream of the edited rbcL gene. Translational coupling between the overlapping rbcL and gusA genes resulted in relatively high GUS accumulation (~0.5 % of leaf protein). CONCLUSIONS: Editing chloroplast genomes using transient imperfect direct repeats provides an efficient method for introducing point mutations into chloroplast genes. Moreover, we describe the first synthetic operon allowing expression of a downstream overlapping gene by translational coupling in chloroplasts. Overlapping genes provide a new mechanism for co-ordinating the translation of foreign proteins in chloroplasts.

Horizontal gene transfer from genetically modified plants - Regulatory considerations.

Gene technology regulators receive applications seeking permission for the environmental release of genetically modified (GM) plants, many of which possess beneficial traits such as improved production, enhanced nutrition and resistance to drought, pests and diseases. The regulators must assess the risks to human and animal health and to the environment from releasing these GM plants. One such consideration, of many, is the likelihood and potential consequence of the introduced or modified DNA being transferred to other organisms, including people. While such gene transfer is most likely to occur to sexually compatible relatives (vertical gene transfer), horizontal gene transfer (HGT), which is the acquisition of genetic material that has not been inherited from a parent, is also a possibility considered during these assessments. Advances in HGT detection, aided by next generation sequencing, have demonstrated that HGT occurrence may have been previously underestimated. In this review, we provide updated evidence on the likelihood, factors and the barriers for the introduced or modified DNA in GM plants to be horizontally transferred into a variety of recipients. We present the legislation and frameworks the Australian Gene Technology Regulator adheres to with respect to the consideration of risks posed by HGT. Such a perspective may generally be applicable to regulators in other jurisdictions as well as to commercial and research organisations who develop GM plants.