title: Bioengineering Monocot Crop Rubisco in Tobacco Chloroplasts
Creators: name: Yuan, Ding
Publisher: name: The Australian National University
Year: 2022

description: Prior tobacco rbcL plastome transformation outcomes identified phylogenetic linkages between foreign plant fLSu sequences and their capacity to form hybrid fL8tS8 Rubisco in tobacco chloroplasts (i.e. comprising endogenous tobacco tSSu). Closer analysis revealed six amino acids common to the plant LSu able to form hybrid Rubisco. This thesis describes three tobacco transformation approaches to compare the biogenesis of hybrid rice, wheat and maize Rubisco in tobacco comprising either the wild-type LSu (rL, wL, mL) or mutated versions that coded 6 amino acids from the tobacco LSu (rL6, wL6, mL6). The first transformation approach involved transient agrobacterium mediated tp-rbcL expression analyses (the tp coding the Rubisco SSu transit peptide) into the transplastomic Nicotiana benthamiana NbL2 line where tobacco Rubisco has been replaced with Rhodospirullum rubrum L2 Rubisco (chapter 3). Only the mutated rL6, wL6 and mL6 subunits were found to form hybrid fL8tS8 in the NbL2 leaves. Extending on this, the same monocot rbcL and rbcL6 genes were stably transformed into the tobacco plastome (chapter 4). Differences in the rates of photosynthesis and growth correlated with the higher amount of hybrid maize Rubisco made in the tob-mL6tS lines relative to the hybrid wheat Rubisco content in the tob-wL6tS genotype. By contrast, little or no hybrid rice Rubisco was made in the tob-rL6tS (or tob-rLtS) lines and so they could only be grown in tissue culture. A third transformation approach introduced synthetic rbcL-rbcS operons into the plastome of cmtrLRNAi-S, a new tobacco line where rbcS mRNA production has been RNAi-silenced. The biogenesis properties of each monocot Rubisco in tobacco was enhanced when co-transformed with either its cognate or a non-cognate monocot SSu (chapter 5). Differences in the monocot Rubisco biogenesis properties among the seven transplastomic genotypes generated indicated the rice and maize Rubisco subunits had greater structural compatibility that with the wheat enzyme. Moreover, interchanging the SSu composition of both enzymes had a pervasive impact on catalysis, with incorporating the maize SSu increasing the kcatc and KC of rice Rubisco, and vice-versa for maize Rubisco incorporating the rice SSu. By contrast, wheat Rubisco incorporating maize SSu showed no change in kcatc and only a minor increase in KC, emphasising caution around assuming a universal impact of C4-plant Rubisco SSu's on C3-plant Rubisco catalysis. This thesis showcases the discovery of amino acid differences in monocot Rubisco LSu that impair their compatibility with the protein folding machinery of tobacco chloroplasts. Additional transplastomic lines made to study differing mutational combinations of the maize L6Su showed only 3 amino acid substitutions were needed for maize Rubisco production in tobacco, albeit in amounts &gt;30-fold lower than wild-type tobacco (chapter 6). Limitations in monocot Rubisco production in tobacco, and the catalytic impact of the 6 LSu amino acid substitutions on maize, but not wheat or rice. Rubisco catalysis highlights some challenges around developing tobacco as a plastome transformation surrogate for translational structure-function testing of monocot Rubisco in a whole plant context. Breakthrough understanding into the complex multi-chaperone mediate LSu folding and assembly process in higher plants has the potential to aid such efforts through elucidating the sequence complementarity requirements between monocot LSu and its ancillary proteins - providing the genetic blueprints for targeted modification of the tobacco chloroplast protein folding machinery by gene editing.