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Published July 7, 2022 | Submitted
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Carbon isotope fractionation by an ancestral rubisco suggests biological proxies for CO₂ through geologic time should be re-evaluated


The history of Earth's carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO₂ is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. We tested the intersection of environment and evolution by measuring both biomass (ϵₚ) and enzymatic (ϵRubisco) carbon isotope fractionations of a cyanobacterial strain (Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to ≫1 Ga. This strain, nicknamed ANC, grows in ambient pCO₂ and displays larger ϵₚ values than WT, despite having a much smaller ϵ_(Rubisco) (17.23 ± 0.61‰ vs. 25.18 ± 0.31‰, respectively). Measuring both enzymatic and biomass fractionation revealed a surprising result -- ANC ϵₚ exceeded ANC ϵRubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. However, these models were corrected by accounting for cyanobacterial physiology, notably the CO₂ concentrating mechanism (CCM). Our model suggested that additional fractionating processes like powered inorganic carbon uptake systems contribute to ϵₚ, and this effect is exacerbated in ANC. Understanding the evolution of rubisco and the CCM is therefore critical for interpreting the carbon isotope record. Large fluctuations in that record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO₂.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. We thank Newton Nguyen for valuable guidance in the MCMC model used to calculate doubling times from growth curve data. We thank Victoria Orphan and Alex Sessions for access to lab space and analytical instruments, as well as lab managers Stephanie A. Connon, Fenfang Wu, and Nami Kitchen for assistance. This research was supported by the David and Lucille Packard Foundation (12540178), Simons Foundation (554187), NASA Exobiology (00010652), and the Schwartz-Reisman Collaborative Science Program (12520057). R.Z.W. was supported by a National Science Foundation Graduate Research Fellowship. Work in the lab of D.F.S. was supported by the US Department of Energy (DE-SC00016240). Work in the lab of P.M.S. was supported by a Society in Science–Branco Weiss fellowship from ETH Zürich and a Packard Fellowship from the David Lucile Packard Foundation. We thank Danielle Jorgens and Reena Zalpuri at the University of California Berkeley Electron Microscope Laboratory for advice and assistance in electron microscopy sample preparation and data collection. The authors have declared no competing interest.

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Submitted - 2022.06.22.497258v2.full.pdf


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August 20, 2023
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