Light- and CO2-dependent carbon isotope fractionation in the β-cyanobacterium Synechococcus sp. PCC 7002

Sarah Hurley, Boswell Wing, Henry C Holm , Ana Gonzalez-Nayeck, Benjamin Acosta, Claire Jasper, Chloe Huntzinger, Nicholas Hill, Margaret Habib, Jeffrey Cameron

Carbon isotope fractionation during photosynthetic carbon fixation by phytoplankton (εₚ) underpins proxies for reconstructing past atmospheric CO₂ concentrations. The interpretation of εₚ is commonly based on steady-state flux-balance models that link isotopic fractionation to the balance between cellular carbon demand and supply, with Rubisco carboxylation providing the primary fractionating step. Accumulating evidence shows that εₚ is also sensitive to other environmental factors, including irradiance, complicating mechanistic interpretation and proxy applications. Previous measurements of cyanobacterial εₚ have also been difficult to reconcile with the steady-state flux-balance framework because they span a narrow range and reach maximum values below the Rubisco kinetic isotope effect expected under conditions of high diffusive CO₂ supply. Here, we combined previously published high-irradiance measurements with new low-irradiance experiments to test how irradiance, CO₂ concentration, and carbon-concentrating mechanism (CCM) function affect εₚ in the β-cyanobacterium Synechococcus sp. PCC 7002. We compared a wild-type strain with a Δccm mutant lacking a functional CCM across multiple CO₂ conditions. In the wild type, εₚ varied by approximately 14‰ across treatments and responded strongly to both CO₂ and irradiance. Under high irradiance, εₚ increased with increasing CO₂, consistent with conventional steady-state flux-balance predictions, whereas under low irradiance, εₚ decreased with increasing CO₂, reversing the expected response. The largest εₚ changes occurred when the availability of the factor constraining growth (e.g., carbon availability or light) changed and followed the direction predicted by the flux-balance framework. However, εₚ also increased with irradiance at elevated CO₂ despite substantially faster growth. In contrast, the Δccm mutant exhibited a narrow εₚ range of approximately 1–2‰ across the three measured conditions. Maximum εₚ values were similar in the wild-type and Δccm strains at approximately 22‰ and were consistent with kinetic isotope effects measured for β-cyanobacterial Rubiscos. Our results indicate that, in Synechococcus sp. PCC 7002, the response of εₚ to CO₂ depends on irradiance and that loss of CCM function substantially reduces the sensitivity of εₚ to both variables. More broadly, the flux-balance framework captured the direction of the largest εₚ responses but did not explain the full response across irradiance and CO₂ conditions. Since irradiance changed the direction of the εₚ response to CO₂, changes in the light environment of organic-carbon production could affect the interpretation of εₚ-based pCO₂ proxy records.