Overcoming limitations in bioengineering Rubisco in higher plant chloroplasts

Abstract

Better structure-function studies of higher plant Rubisco are imperative in improving catalytic potential of the enzyme Rubisco. In this thesis, a novel system to study Rubisco using an RNAi tobacco genotype is designed to provide a homogenous environment of large and small Rubisco subunits for a more genuine assessment of recombinant Rubisco catalysis, regulation and assembly as well as its photosynthetic capacity in tobacco. The application of technology and strategies discussed in this thesis will demonstrate a great leap forward in Rubisco bioengineering and recombinant protein expression in plant chloroplasts. The RbcS RNAi of the cmtrLRNAi-S genotype is stable up to three generations, having selectable resistance against the Basta herbicide while boasting no accumulation of transcript mRNA from the tobacco RbcS multigene family. Access and ability to manipulate the Rubisco S-subunit in higher plants have been the final crux in bioengineering Rubisco and is now possible using the cmtrLRNAi-S master line. Additionally, application of the intron-containing hairpin loop construct in RNAi silencing and its effectiveness as shown in this thesis strongly validates the use of this technology to study other genomic and proteomic components of photosynthesis in higher plants. The unperturbed growth of cmtrLRNAi-S to maturity in soil (albeit requiring elevated CO2 environment) and therefore the development of fertile pollen enhances the prowess of the cmtrLRNAi-S line to include stable transfer of the RNAi-RbcS system into tobacco genotypes using cross-pollination. New genotypes generated using pollen from cmtrLRNAi-S to fertilise genotypes producing S-subunits in the chloroplast mirror similar RbcS silencing found in cmtrLRNAi-S thus resulting in populations of homogenous, chloroplast-made S-subunits in the absence of endogenous (cytosolic) S-subunits. In summary, a more accurate system for determining the innumerable factors and limitations in recombinant Rubisco expression and biogenesis in higher plants can be achieved using cmtrLRNAi-S. The recent advent of cmtrLRNAi-S to intrinsically manipulate the S-subunit encourages further possibilities for comprehensive studies to overcome limitations in bioengineering higher plant Rubisco. The curious nature of the S-subunit multigene family and its indispensable role in higher plant photosynthesis once perplexing now serve as tools to bring fresh perspectives on the S-subunit’s import into the chloroplast, processing events and interaction with its counterpart subunit. The capacity to experiment on a single RbcS species in the chloroplast by its expression in an rbcL-rbcS dicistronic operon presents opportunities for differentiating members of the RbcS multigene family as well as to study the importance of structure-function differences between intra- and interspecies variants of RbcS. This thesis details preliminary knowledge gleaned from the first examples of homogenous hybrid Rubisco populations expressing foreign S-subunit genes from red Rubisco (G. monilis), C3 (N. tabacum and H. annuus) and C4 (F. bidentis and S. bicolor) plants as well as various approaches in regulatory elements and sequences for optimising synthesis of recombinant Rubisco in host surrogate tobacco. The mention of cmtrLRNAi-S in preceding theses from the Whitney laboratory and its use in the Whitney laboratory for various other projects in parallel to work done in the thesis is proof of a pioneering method for stable bioengineering of S-subunit and subsequently L8S8 Rubisco in higher plants. Ultimately, this thesis showcases new strategies for improving the transition of transcript mRNA coding for foreign and recombinant Rubisco as well as other potential proteins of interest to comparable levels of translated product in higher plant chloroplasts.