Targeted Metabolomics Reveals Light/Dark-Dependent Reallocation of Central Carbon and Nitrogen Metabolism in the Diatom Phaeodactylum tricornutum

Background/Objectives: Diel reprogramming of central metabolism is well documented in vascular plants; whether diatoms organize carbon and nitrogen metabolism in the same way is unknown for want of absolutely quantified metabolite time series covering a complete light/dark cycle. Methods: Wild-type Phaeodactylum tricornutum was grown under a 12 h light/12 h dark cycle and sampled at the 3rd, 6th, and 12th h of both phases (n = 3 per time point; 18 samples). Ninety metabolites were detected and absolutely quantified by targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS), and the 72 metabolites were retained for downstream analysis after excluding those with extensive missingness; time effects were tested by one-way analysis of variance (ANOVA) (p < 0.05). Results: Forty metabolites (55.6%) changed significantly over time and clustered into five diel patterns. The citrate branch of the tricarboxylic acid (TCA) cycle declined in the light and recovered in the dark, malate and fumarate peaked at early dark (D3), and α-ketoglutarate reached its minimum at D3. Photorespiratory intermediates were generally higher in the light, except glyoxylate (D3 peak). Glutamate-family pools bottomed out at the end of the light phase; ornithine fell monotonically, the largest drop among measured amino acids. Conclusions: Central carbon and nitrogen metabolism of P. tricornutum undergoes phased reallocation over the light/dark cycle. Pool dynamics suggest a diel, bidirectional exchange of carbon skeletons between the TCA cycle and amino acid metabolism via the α-ketoglutarate node, with early-dark four-carbon (C4) accumulation coinciding with amino acid catabolic replenishment (carbon origin awaits 13C tracing). Photorespiratory intermediates followed a light-high, dark-low pattern consistent with suppression, but not abolition, of photorespiration by the CO2-concentrating mechanism (CCM). This dataset provides a metabolite-level, absolutely quantified reference for diatom diel metabolism.