An optimized fast-repetition-rate fluorometry method reveals photochemical flux bottlenecks in C3 photosynthesis that limit carboxylation and growth.

Using the fast-repetition-rate fluorometry (FRRF) technique to monitor the variable yield of chlorophyll fluorescence emission (Fv = Fm - Fo), we develop a pulse sequence to measure flux bottlenecks that limit the transport of electrons and protons from their water oxidation source through the photosynthetic electron transport chain (PETC) into CO2 carboxylation and triose phosphate production within the Calvin-Benson-Bassham (CBB) cycle of living organisms. FRRF uses a single source of microsecond flashes to excite photochemistry and produce fluorescence, thus removing slower non-photochemical quenching contributions to total Fv (pFv = total Fv - NPQ). pFv is the fraction of total Fv that originates from net charge separation and recombination in photosystem II: P ↔ P∗ ↔ P+QA- ↔... Subsequent charge-transfer steps are monitored, revealing fluxes from water oxidation (reducing P+) into downstream carrier pools of the PETC (oxidizing QA-). As these pools fill in sequence, QA/QA- repopulates at characteristic lag times as measured by pFv dynamics. Three temporal phases of pFv are observable in various phototrophs performing C3 photosynthesis that reveal five flux bottlenecks between redox carriers in the PETC and CBB cycle. pFv dynamics reveal that the PETC and CBB cycles operate synchronously, governed by the law of mass action and microscopic reversibility, with CO2 accelerating upstream reactions in both linear and cyclic electron pathways. pFv fluxes reliably monitor species-dependent phenotypic changes in PETC/CBB bottlenecks that determine carboxylation rate and influence cellular growth rate. This versatile technique can be applied to monitor the effects of transgene or environmental changes on light-use efficiency in diverse photosynthetic organisms.