Estimating the redox state of the plastoquinone pool in algae and cyanobacteria via OJIP fluorescence: perspectives and limitations

Abstract The redox state of the plastoquinone pool (PQ-redox) acts as a central element in a variety of intracellular signal pathways. Several methods for determining PQ-redox have been established. Although some of these methods may be quantitative, such as those based on liquid chromatography, they are typically sensitive to sample preparation. Here, we critically evaluate the use of fast chlorophyll a fluorescence induction kinetics (the so-called OJIP transient) for semi-quantitative PQ-redox estimation in green algae (Chlorella vulgaris) and cyanobacteria (Synechocystis sp. PCC 6803). The method, based on the evaluation of relative fluorescence yield at the J-step of the OJIP transient (VJ, VJ’), has already been reported; however, thus far, it has been used mostly for studying dark-acclimated leaves, which limits its range of application. Here, we show that the OJIP transient can be used for semi-quantitative estimation of PQ-redox in algal and cyanobacterial cell cultures, in addition to plants. We further show that it can reflect PQ-redox in both dark-acclimated and light-acclimated samples. Our systematic comparison of Multi-Color PAM, AquaPen, and FL 6000 fluorometers demonstrates that accurate measurement of VJ and VJ’ parameters in suspension cultures requires low culture density and a high-intensity saturation pulse. We further show that with increasing light intensity to which the cells are exposed, the state of photosystem II (PSII) changes due to light-induced reduction of quinone A (QA-) and conformational changes, which in turn influence both the sensitivity and dynamic range of the VJ’ parameter towards PQ-redox estimation. A comparison of fluorescence transients in Chlorella and Synechocystis revealed high homeostatic control over PQ-redox in Synechocystis, maintained by terminal oxidases present at the thylakoid membrane. While we discuss certain limitations, our systematic assessment suggests that the OJIP method has great potential to become a routine tool for semi-quantitative PQ-redox estimation under a wide range of experimental conditions in green algae and cyanobacteria. Competing Interest Statement The authors have declared no competing interest. Footnotes This version of the manuscript incorporates extensive revisions in response to comments from the editor and both reviewers. The title and wording throughout the text were updated to better reflect that the fluorescence-based approach provides an estimation of PQ redox state and is semi-quantitative rather than fully quantitative. Novelty statements were revised and appropriate earlier work on the VJ parameter was cited. Major changes include substantial expansion and clarification of the Discussion section. We now compare our results directly with previously published quantitative PQ/PQH2 data for multiple treatments, including darkness, DCMU, actinic light, high light, glycolaldehyde, methyl viologen, and KCN. These comparisons show that VJ reliably tracks PQ redox shifts and that our observations are consistent with earlier quantitative studies. The methodological limitations of the fluorescence approach, including conditions where PSII closure prevents reliable interpretation, are now described in significantly greater detail. We provide a more precise methodological description, including clarification of measuring light modulation, timing of the OJIP transient, sample-and-hold behavior, double normalization, and identification of the J-step via polynomial fitting. Several inaccuracies and ambiguous statements were corrected, including those related to UV absorbance methods, Dual-PAM instrumentation, photon flux density terminology, and potential actinic effects of the measuring light. The comparison of fluorometers in Figure 1 and its methodological relevance were clarified. The revision clarifies why a direct comparison with HPLC-based PQ/PQH2 measurements was not feasible. Although HPLC instruments are widely available, quantitative PQ/PQH2 analysis requires a specialized fluorescence detector and highly challenging sample-handling conditions to prevent PQH2 oxidation. Despite attempts to reproduce published protocols with multiple extraction solvents and storage approaches, only oxidized PQ was detectable, preventing acquisition of reliable comparative datasets. Nevertheless, the existing fluorescence dataset proved sufficiently rich to demonstrate consistent PQ-redox dynamics across organisms and treatments. Clear increases or decreases in VJ were observed in agreement with known effects of CBB cycle inhibition, terminal oxidase inhibition, methyl viologen addition, and light-to-dark and dark-to-light transitions. Additional clarifications were added regarding organism-specific responses, including the strong KCN effects in Chlorella. The manuscript has been language-edited throughout, formatting has been corrected to journal standards, the Abstract was rewritten, and the reference list reformatted. Overall, the revised manuscript now provides a clearer, more rigorous, and well-supported evaluation of the VJ parameter as a semi-quantitative tool for monitoring PQ-redox dynamics in algae and cyanobacteria. https://github.com/Computational-Biology-Aachen/OJIP-PQredox List of abbreviations - AL - actinic light - CBB - Calvin-Benson-Bassham cycle - COX - cytochrome c oxidase - Cyt b6/f - Cytochrome b6/f complex - Cyd - cytochrome bd quinol oxidase - DCMU - 3 (3,40-dichlorophenyl)-1,1-dimethylurea - FO, FO’ - initial fluorescence yield at the beginning of the OJIP curve in fully dark-acclimated samples (≥ 20 min in darkness) and in light-acclimated or partially dark-acclimated samples, respectively - FI, FJ and FP - fluorescence yield at the J, I and P point of the OJIP curve in fully dark-acclimated cells (≥ 20 min in darkness), respectively - FI’, FJ’ and FP’ - fluorescence yield at the J, I and P point of the OJIP curve in light- or partially dark-aclimated cells, respectively - FM, FM’ - maximal fluorescence yield in fully dark-acclimated cells (≥ 20 min in darkness) and in light-acclimated or partially dark-acclimated samples, respectively - FNR - ferredoxin-NADP+-oxidoreductase - GA - glycolaldehyde - HL - high light - J, I, P - J, I and P points of the OJIP curve, respectively - ML - measuring light - MV - methyl viologen - NDH-1 - NAD(P)H dehydrogenase-like complex type 1 - NPQ - non-photochemical quenching - OEC - oxygen evolving complex of PSII - OCP - orange carotenoid protein - PBS - phycobilisomes - PC - plastocyanin - PFD - photon flux density - PQ - plastoquinone - PQH2 - plastoquinol - PQ-redox - redox state of the PQ/PQH2 pool - PETC - photosynthetic electron transport chain - PSI - Photosystem I - PSI-CEF - cyclic electron flow around PSI - PSII - Photosystem II - PTOX - plastid terminal oxidase - QA - quinone A - QB - quinone B - SDH - succinate dehydrogenase - SP - saturating pulse - TOs - terminal oxidases - VJ, VJ’ - relative fluorescence yield at the J point of the OJIP curve measured in fully dark-acclimated and in partially dark- or light-acclimated samples, respectively - Y(NA) - quantum yield of non-photochemical energy dissipation due to acceptor side limitation of PSI