Modifying Plant Photosynthesis and Growth via Simultaneous Chloroplast Transformation of Rubisco Large and Small Subunits

Avila, Elena Martin; Lim, Yi-Leen; Birch, Rosemary; Dirk, Lynnette M.A.; Buck, Sally Anne G; Rhodes, Timothy; Sharwood, Robert E.; Kapralov, Maxim V.; Whitney, Spencer M.

doi:10.1105/tpc.20.00288

Cited by 88 publications

(84 citation statements)

References 80 publications

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“…S5). In these assays we also found that the Cterminal hexa-histidine tag added to the small subunit in our constructs did not significantly affect kinetics, in contrast to that reported for tobacco Rubisco expressed in planta (45). However, the N-terminally processed ΔN1 variant that resembles natively processed RbcL in plants (Table 1) was among the fastest enzymes using both methods (Fig.…”

Section: A Dissection Of the Rbcl N-terminal Binding Motifsupporting

confidence: 46%

“…Future studies aiming to evaluate the effect of amino acid substitutions on plant carboxylase function should therefore use the ΔN1 variant. Although a C-terminal hexahistidine tag on the small subunit did not appear to affect kinetics in our system (Table 2), we agree it to be prudent practise not to include affinity tags when preparing Rubiscos for kinetic analysis (45).…”

Section: Discussionmentioning

confidence: 55%

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Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco

Guo

Mueller‐Cajar

2020

Journal of Biological Chemistry

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The photosynthetic CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms dead-end inhibited complexes while binding multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. Rubisco can be rescued from this inhibited form by molecular chaperones belonging to the ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas). The mechanism of green-type Rca found in higher plants has proved elusive, in part because until recently higher plant Rubiscos could not be expressed recombinantly. Identifying the interaction sites between Rubisco and Rca is critical to formulate mechanistic hypotheses. Towards that end here we purify and characterize a suite of 33 Arabidopsis Rubisco mutants for their ability to be activated by Rca. Mutation of 17 surface-exposed large subunit residues did not yield variants that were perturbed in their interaction with Rca. In contrast, we find that Rca activity is highly sensitive to truncations and mutations in the conserved N-terminus of the Rubisco large subunit. Large subunits lacking residues 1-4 are functional Rubiscos, but cannot be activated. Both T5A and T7A substitutions result in functional carboxylases that are poorly activated by Rca, indicating the side chains of these residues form a critical interaction with the chaperone. Many other AAA+ proteins function by threading macromolecules through a central pore of a disc-shaped hexamer. Our results are consistent with a model where Rca transiently threads the Rubisco large subunit N-terminus through the axial pore of the AAA+ hexamer.

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Section: A Dissection Of the Rbcl N-terminal Binding Motifsupporting

confidence: 46%

Section: Discussionmentioning

confidence: 55%

Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco

Guo

Mueller‐Cajar

2020

Journal of Biological Chemistry

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“…This is because the rbcS gene is nuclear encoded, multi-copy (in most plant species), undergoes recombination, and is less constrained by molecular chaperones. Moreover, as RbcS has an indirect influence on RuBisCO catalytic properties (Read and Tabita, 1992b, 1992a; Spreitzer, Peddi and Satagopan, 2005; Martin-Avila et al , 2020) and activity (Andrews, 1988; Lee and Tabita, 1990; Lee, Berka and Tabita, 1991), it is possible that this subunit may have helped the RuBisCO holoenzyme to evolve more rapidly and escape some phylogenetic constraint. However, the exact contribution of the RbcS to variation in holoenzyme kinetics, or indeed whether the influence of this protein has already been optimised, remain still to be fully understood.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, disparate lineages of Form I II, III and IV RuBisCO exist which catalyse CO 2 fixation and lack the small subunit entirely (Tabita et al , 2008; Banda et al , 2020). However, despite not being involved in the catalytic chemistry directly, the small subunit does have an indirect effect on the catalytic properties (Read and Tabita, 1992b, 1992a; Spreitzer, Peddi and Satagopan, 2005; Ishikawa et al , 2011; Joshi et al , 2015; Fukayama et al , 2019; Martin-Avila et al , 2020) and activity (Andrews, 1988; Lee and Tabita, 1990; Lee, Berka and Tabita, 1991) of RuBisCO, and thus is important to the function of Form I RuBisCO.…”

Section: Introductionmentioning

confidence: 99%

Rubisco adaptation is more limited by phylogenetic constraint than by catalytic trade-off

Emms

Rhodes

et al. 2020

Preprint

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RuBisCO assimilates CO2 to form the sugars that fuel life on earth. Correlations between RuBisCO kinetic traits across species have led to the proposition that RuBisCO adaptation is constrained by catalytic trade-offs. However, these correlations were founded on the assumption that kinetic measurements in different species are independent. Here, we show that this assumption is incorrect in angiosperms by evaluating the dependence of variation in RuBisCO kinetic traits on the phylogenetic tree that relates the enzymes. We show that there is significant phylogenetic signal in all carboxylase kinetic traits in angiosperms, and significant phylogenetic signal in the Michaelis constant for O2 in species that conduct C3 photosynthesis. When accounting for this non-independence, we show that the catalytic trade-off between carboxylase turnover and the Michaelis constant for CO2 is weak (∼30 % dependency) and that the correlations between all other RuBisCO kinetic traits are either not-significant or marginal (<9 % dependency). Finally, we demonstrate that phylogenetic constraints limit RuBisCO adaptation to a greater extent than catalytic trade-offs. Thus, the biochemical landscape of RuBisCO adaptation in angiosperms is predominantly limited by phylogenetic constraint and a partial trade-off between carboxylase turnover and the Michaelis constant for CO2.

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“…Eight T 0 plants with varying leaf phenotypes were transferred to soil and confirmed to contain the SpCas9 transgene by PCR using primers specific for the SpCas9 expression cassette. Four of these had visibly smaller and paler leaves than the non-transformed tissue culture control (i.e., wild-type), which is typical of Rubisco-deficient mutants (Khumsupan et al, 2020;Martin-Avila et al, 2020) (Figure 2A). The remaining four plants had a mixed pale and wild-type leaf phenotype indicative of chimeric mutations.…”

Section: Stable and Chimeric Mutations Were Identified In The T 0 Genmentioning

confidence: 96%

CRISPR-Cas9-Mediated Mutagenesis of the Rubisco Small Subunit Family in Nicotiana tabacum

et al. 2020

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Engineering the small subunit of the key CO2-fixing enzyme Rubisco (SSU, encoded by rbcS) in plants currently poses a significant challenge, as many plants have polyploid genomes and SSUs are encoded by large multigene families. Here, we used CRISPR-Cas9-mediated genome editing approach to simultaneously knock-out multiple rbcS homologs in the model tetraploid crop tobacco (Nicotiana tabacum cv. Petit Havana). The three rbcS homologs rbcS_S1a, rbcS_S1b and rbcS_T1 account for at least 80% of total rbcS expression in tobacco. In this study, two multiplexing guide RNAs (gRNAs) were designed to target homologous regions in these three genes. We generated tobacco mutant lines with indel mutations in all three genes, including one line with a 670 bp deletion in rbcS-T1. The Rubisco content of three selected mutant lines in the T1 generation was reduced by ca. 93% and mutant plants accumulated only 10% of the total biomass of wild-type plants. As a second goal, we developed a proof-of-principle approach to simultaneously introduce a non-native rbcS gene while generating the triple SSU knockout by co-transformation into a wild-type tobacco background. Our results show that CRISPR-Cas9 is a viable tool for the targeted mutagenesis of rbcS families in polyploid species and will contribute to efforts aimed at improving photosynthetic efficiency through expression of superior non-native Rubisco enzymes in plants.

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Modifying Plant Photosynthesis and Growth via Simultaneous Chloroplast Transformation of Rubisco Large and Small Subunits

Cited by 88 publications

References 80 publications

Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco

Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco

Rubisco adaptation is more limited by phylogenetic constraint than by catalytic trade-off

CRISPR-Cas9-Mediated Mutagenesis of the Rubisco Small Subunit Family in Nicotiana tabacum

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