Closing in on maximum yield of chlorophyll fluorescence using a single multiphase flash of sub-saturating intensity

Estimation of the maximum chlorophyll fluorescence yield under illumination, or Fm, by traditional single saturation pulse (SP) methodology is prone to underestimation error because of rapid turnover within photosystem (PS) II. However, measurements of fluorescence yield during several single pulses of variable intensity describes the irradiance dependence of apparent Fm, from which estimates of Fm at infinite irradiance can be derived. While such estimates have been shown to result in valid approximations of Fm, the need to apply several single pulses limits its applicability. We introduce a novel approach that determines the relationship between apparent Fm and variable irradiance within a single approximate to 1s multiphase flash (MPF). Through experiments and simulations, we demonstrate that the rate of variation in irradiance during an MPF is critical for achieving quasi-steady-state changes in the proportions of PSII acceptor side redox intermediates and the corresponding fluorescence yields, which are prerequisites for accurately estimating Fm at infinite irradiance. The MPF methodology is discussed in the context of improving the accuracy of various parameters derived from chlorophyll fluorescence measurements, such as photochemical and non-photochemical quenchings and efficiencies. The importance of using MPF methodology for interpreting chlorophyll fluorescence, in particular for integrating fluorescence and gas exchange measurements, is emphasized.