Resolving the contributions of photosystem II, photosystem I, and phycobilisomes to the in vivo fluorescence yield in the red alga Cyanidioschyzon merolae.

Analysis of in vivo fluorescence yield measured in the red and far-red regions is widely used to estimate photosynthetic parameters related to photosystem II (PSII). Besides PSII, photosystem I (PSI) and phycobilisomes (PBS), the latter present in cyanobacteria, red algae, and glaucophytes, also emit fluorescence. Real-time fluorescence responses of PSI and PBS have not been fully characterized, as practical methods to resolve fluorescence contributions from PSII, PSI, and PBS are limited. To resolve the contributions of PSII, PSI, and PBS to the in vivo fluorescence yield, an analytical workflow is proposed based on fluorescence induction curves and variable fluorescence kinetics measured across four detection wavebands. The fluorescence induction curves and variable fluorescence kinetics were measured using blue and amber lights, which preferentially excited the photosystems (PSII and PSI) and PBS, respectively, in the unicellular red alga Cyanidioschyzon merolae. Both PSI and PBS contributed small but measurable portions to the fluorescence induction curves, enabling estimation of the relative fluorescence amplitudes across detection wavebands through constrained non-negative matrix factorization (NMF). The relative fluorescence amplitudes were used to estimate the relative fluorescence yields of PSII, PSI, and PBS, as well as the PSII-related parameter ΦPSII through least-squares fitting of variable fluorescence data. The resulting kinetics obtained from the optimal NMF models revealed multiple processes associated with nonphotochemical quenching of PSII and provided insights into the regulation of excitonic energy transfer among PSII, PSI, and PBS. The analytical workflow serves as an effective approach to uncover PSI and PBS fluorescence yield kinetics across diverse photosynthetic organisms.