Interrogating plant Rubisco-Rubisco activase interactions
Creators: name: Milward, Sara Eve
Publisher: name: Canberra, ACT : The Australian National University
Year: 2018
Subjects: Rubisco
activase
CO2 assimilation
AAA+ protein
protein-protein interaction
chloroplast transformation

description: Atmospheric CO2 fixation is catalysed by the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Despite the critical role Rubisco plays in the biosphere, it is a slow catalyst that poorly discriminates between substrate CO2 and O2, and is often the rate-limiting step of photosynthesis. These deficiencies have made improving Rubisco function a major target in steps towards enhancing leaf photosynthesis rate and plant growth. In pursuing this goal, one strategy is to identify solutions for improving the kinetics of plant Rubisco and introduce these altered Rubisco isoforms into crops (Sharwood, 2017). Unfortunately efforts to improve the performance of plant Rubiscos have so far proven unsuccessful. Success appears to be hindered by Rubisco’s complex catalytic mechanism and the extensive array of accessory proteins needed to assemble the eight large (L) and eight small (S) subunits into a L8S8 complex and to maintain it in a functional form. The complex catalytic chemistry of Rubisco is prone to inhibition by various sugar-phosphate ligands. These include catalytic misfire products as well as its own substrate, ribulose-1,5-bisphosphate (RuBP), which forms an inactive Rubisco-RuBP (ER) complex when bound to non-carbamylated (i.e. non-activated) catalytic sites. Release of these inhibitors is mediated by the AAA+ (ATPases associated with a variety of cellular activities) protein Rubisco activase (Rca); a Rubisco-specific metabolic repair chaperone for which there are convergently evolved structural isoforms found in most photosynthetic organisms (Bhat et al., 2017a; Mueller-Cajar, 2017). Rca is now also a promising target for manipulating photosynthetic performance, especially under elevated temperature stress and fluctuating light (Carmo-Silva &amp; Salvucci, 2013; Kumar et al., 2009; Kurek et al., 2007; Scafaro et al., 2016; Yamori et al., 2012). Critical to future Rubisco and Rca engineering efforts in plants is a greater mechanistic understanding of how Rca interacts with Rubisco, and the extent to which the kinetics of Rca differ between plant species. Utilising chloroplast transformation in tobacco, this thesis investigated the H9 helix and N-terminal region of Rca, and the βC-βD loop and C terminus of the Rubisco large (L-) subunit (RbcL) to determine their role in the Rubisco-Rca interaction in plants using both in vivo and in vitro methods. Capitalising on the regulatory incompatibility between Rubisco and Rca enzymes from Solanaceae and non-Solanaceae species, this thesis examined how mutations in the tobacco (Solanaceae) and Flaveria (F. bidentis and F. floridana, non-Solanaceae) enzymes influenced their kinetic properties and the Rca activation potential for inhibited ER complexes. The ability to continuously monitor the rate of ER activation provided by a NADH-coupled spectrophotometric method made it preferential to the alternative two-step 14CO2-fixation assay method. Optimising the assay conditions, particularly the accurate determination of enzyme concentrations, was crucial for obtaining reproducible kinetic values. Normalising the measurements of ER activation rate relative to the Rca ATPase activity (kcatATP ) proved critical in providing a means to determine the effect the Rca and Rubisco mutations had on interactivity. Using these assay conditions, mutagenic analyses of the Rca H9 helix found residue 317 plays a dominant role in defining the Rubisco selectivity of NtRca, and is critical for functional FbRca. N-terminal domain swapping modifications to NtRca and FbRca revealed amino acids at the junction of the N-terminal extension and the AAA+ module (residues 50 to 85) significantly influenced both kcatATP and the potential to interact with their cognate Rubiscos, but had little influence on altering interaction with the non-native Rubisco. In vivo analysis of Rubisco-Rca interaction was undertaken using tobacco genotypes producing differing tobacco Rubisco or recombinant Flaveria Rubisco mutants generated by chloroplast transformation of the rbcL gene into the plastome. In vitro analysis using purified enzymes uniformly showed residues 89 and 94 of the βC-βD loop of the F. bidentis, F. floridana and tobacco RbcLs influence interaction with Rca. In contrast, in vivo leaf gas exchange measurements of photosynthetic induction in the tobacco genotypes producing mutant RbcL showed mutation of residues 89 and 94 had little to no influence on their capacity to be activated by the endogenous NtRca. Combinatorial in vitro analyses using the range of RbcL and Rca mutants generated in this study were undertaken to better understand their interaction mechanism. It was found the avidity of Rubisco-Rca interactions were primarily defined by residue 94 in RbcL and 317 in Rca, and less so by RbcL residue 89 and Rca residue 320. Mutations to the RbcL C terminus had little influence on plant Rubisco-Rca interactions except when coupled with mutations that disrupted the connection between the RbcL βC-βD loop and the Rca H9 helix. The cumulative effect of the RbcL βC-βD loop and C-terminal modifications on Rubisco-Rca interactivity of these mutants implies involvement of the RbcL C terminus in the activation mechanism of Rca. This thesis showcases a novel approach to study the mechanism of plant Rubisco-Rca interactions by generating mutant Rubisco variants through tobacco plastome transformation. The work highlights large variations in the kinetics of NtRca (Solanaceae) and FbRca (non-Solanaceae) and provides evidence for variations in their Rubisco activation mechanism. For instance, while a D317K substitution in NtRca significantly enhanced its ability to activate Flaveria Rubisco, the reciprocal K317D mutation in FbRca had little impact on improving its capacity to activate tobacco Rubisco. Improving our understanding of the mechanistic differences in plant Rubisco-Rca interactions is critical for future Rubisco engineering endeavours. It is especially relevant in the context of ongoing efforts to transplant more efficient Rubisco variants into leaf chloroplasts where co-transformation with an appropriate Rca may be needed. Potential transgenic strategies for co-engineering compatible Rubisco and Rca isoforms in planta are also discussed.