Low CO₂ results in a rearrangement of carbon metabolism to support C₄ photosynthetic carbon assimilation in Thalassiosira pseudonana

The mechanisms of carbon concentration in marine diatoms are controversial. At low CO₂, decreases in O₂ evolution after inhibition of phosphoenolpyruvate carboxylases (PEPCs), and increases in PEPC transcript abundances, have been interpreted as evidence for a C₄ mechanism in Thalassiosira pseudonana, but the ascertainment of which proteins are responsible for the subsequent decarboxylation and PEP regeneration steps has been elusive. We evaluated the responses of T. pseudonana to steady-state differences in CO₂ availability, as well as to transient shifts to low CO₂, by integrated measurements of photosynthetic parameters, transcript abundances and quantitative proteomics. On shifts to low CO₂, two PEPC transcript abundances increased and then declined on timescales consistent with recoveries of F_v/F_m, non-photochemical quenching (NPQ) and maximum chlorophyll a-specific carbon fixation (P_max), but transcripts for archetypical decarboxylation enzymes phosphoenolpyruvate carboxykinase (PEPCK) and malic enzyme (ME) did not change. Of 3688 protein abundances measured, 39 were up-regulated under low CO₂, including both PEPCs and pyruvate carboxylase (PYC), whereas ME abundance did not change and PEPCK abundance declined. We propose a closed-loop biochemical model, whereby T. pseudonana produces and subsequently decarboxylates a C₄ acid via PEPC₂ and PYC, respectively, regenerates phosphoenolpyruvate (PEP) from pyruvate in a pyruvate phosphate dikinase-independent (but glycine decarboxylase (GDC)-dependent) manner, and recuperates photorespiratory CO₂ as oxaloacetate (OAA).