Diel Measurement Timing, Dark Acclimation and Sample Handling Alter Interpretation of OJIP Chlorophyll a Fluorescence...

OJIP chlorophyll a fluorescence is widely used for the rapid assessment of photosynthetic performance, but field workflows may alter the physiological state of the plants probed before the saturating pulse. We compared eight dark-acclimation and sample-handling workflows in field-grown Sida hermaphrodita and Miscanthus × giganteus: daytime clip acclimation, evening and night measurements with or without clip acclimation, flashlight-assisted night measurements, and detached-shoot measurements after laboratory green-light exposure. The same marked leaves were measured sequentially, and the analysis focused on primary OJIP fluorescence levels, phase-specific O-J, J-I and I-P kinetics, area above the transient, FV/FM and FK/FJ. Workflow choice altered OJIP trajectories in both species. Evening and night workflows generally increased the area above the transient relative to the Day-clip method, indicating that short daytime clip acclimation and prolonged natural darkness did not produce equivalent reference states. Night-flashlight and Detached-green workflows introduced further deviations, including altered FO, changed? O-J behavior and I-P rise kinetics. The magnitude and direction of workflow effects were species-specific. These results show that dark-acclimation timing, pre-measurement light exposure and detached-shoot handling are not interchangeable technical details in field OJIP measurements. Workflows should therefore be selected according to the biological question, kept constant within comparisons, and reported with timing, dark-acclimation duration, light exposure and handling conditions.