Photosynthesis improvement in crop plants through gene replacement

Food security is of increasing concern due to rising population along with decreasing arable land and water supplies, alongside the effects of climate change. It is predicted that by 2050, a 50% increase in food production will be required to prevent severe food shortages. One strategy for increasing food production is the improvement of photosynthesis, in particular the enzyme ribulose- 1,5-biphosphate carboxylase/oxygenase (Rubisco), the focus of this project. Rubisco catalyses the first step of the Calvin-Benson-Bassham cycle, fixing atmospheric CO2, ultimately producing sugars and starches. Both slow and non-specific, Rubisco catalysis is notoriously inefficient in higher plants. Plant Rubisco has two subunits, the large subunit expressed from the chloroplast genome and the small subunit expressed from multiple genes in the nuclear genome then transported to the chloroplast for holoenzyme assembly. Early research in tobacco regarding the replacement of the gene for the large subunit, rbcL, with other Rubisco variants led to the generation of chimeric rbcL or Rubisco hybrids. This issue was overcome via the development of a tobacco-rubrum master-line, which has the native rbcL replaced with a bacterially derived form 2 rbcL, derived from Rhodospirillum rubrum. The tobacco-rubrum master-line has stimulated research in the area, and has been utilised for further rbcL replacement experiments with a number of Rubisco variants. However, all of the previous research has been in tobacco. With the imperative being increasing crop yield in order to address food security, the primary aim of this thesis was the extension of rbcL replacement to food crop species. This thesis describes the first report of successful replacement of the gene for the large subunit, rbcL, with a gene for a bacterial L2 Rubisco, in Solanum tuberosum (potato). Like tobacco, potato belongs to the Solanaceae family and has established plastid transformation protocols. The transformation efficiency was similar to that previously reported. Homoplasmy was achieved using extended callus culture eliminating the need for further rounds of regeneration. Transplastomic potato-rubrum plant photosynthesis was supported by form 2 Rubisco only, however, due to the poor catalytic capability of R. rubrum Rubisco, the soil grown transplastomic plants were autotrophic only under elevated [CO2]. Further experiments for removal of the selectable marker gene via transient expression of Crerecombinase are currently underway. The next stage was to extend rbcL replacement to a food crop species other than Solanaceae, namely Brassica napus (canola), belonging to the Brassicaceae family, selected as there had been some reported success of biolistics for plastid transformation of B. napus, achieving heteroplasmic plants. Here, two different transformation protocols, biolistics and PEG-mediated uptake to protoplasts were utilised with biolistics generating green spectinomycin resistant callus that became overgrown with bleached non transformed tissue when transferred to shoot induction medium. Recent research has revealed that Brassicaceae response to the aadA selection system is different to that observed for Solanaceae, due to the presence acc2, expressed from the nuclear genome and coding for ACCase, allowing for cell division in the presence of spectinomycin. The development of acc2 knockdown lines alongside root callus tissue culture and regeneration system increased transformation efficiency in Arabidopsis thaliana. The development of a similar system for B. napus would allow for the generation of a canola-rubrum master-line that could then be utilised for further Rubisco replacement experiments. To understand the assembly of higher plant Rubisco, this project explored Rubisco chaperone proteins in particular, Bundle Sheath Defective II (BSDII). Here, BSDII overexpression from the nuclear genome in both potato and canola was attempted. The results indicated that BSDII was most likely silenced in potato. Of more interest to the overall project would be co-expression of foreign chaperone proteins alongside complementary Rubisco genes. The overall project aim of extension of photosynthesis improvement to one or more food crop species was achieved with replacement of rbcL in S. tuberosum, thus demonstrating the feasibility of extending this important research to other food crop species. Following the removal of the selectable marker gene, the potato-rubrum transplastomic plants generated will provide an important research tool to the wider plant biotechnology community for further Rubisco gene replacement experiments. The improvement of Rubisco in higher plant species will most likely require a combination of approaches, the majority of which require the introduction of Rubisco variants thus there is an increasingly urgent need for extension of plastid transformation protocols to important food crop species