Interrogating plant Rubisco-Rubisco activase interactions
Date
2018
Authors
Milward, Sara Eve
Journal Title
Journal ISSN
Volume Title
Publisher
Canberra, ACT : The Australian National University
Abstract
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 & 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.
Description
Keywords
Rubisco, activase, CO2 assimilation, AAA+ protein, protein-protein interaction, chloroplast transformation
Citation
Collections
Source
Type
Thesis (PhD)
Book Title
Entity type
Access Statement
License Rights
Restricted until
Downloads
File
Description