Many intricacies of leaf-level photosynthesis can be probed by combining infrared gas analysis with pulse-amplitude-modulation chlorophyll a fluorometry. A key fluorescence yield (ΦF) parameter required for estimating many of the phenomena associated with the light reactions of photosynthesis is referred to as the maximum ΦF, which is termed Fm′ when measured on a light-adapted leaf. While ubiquitously used to assess many aspects of photosynthesis, Fm′ is problematic because it is prone to being underestimated. This error can be propagated to parameters and phenomena that are based on estimation of Fm′. Theoretical and experimental observations have shown that ΦF increases hyperbolically in response to increasing irradiance, asymptotically approaching the maximum ΦF, or Fm′, at extreme irradiances. Importantly, depending upon the convexity of the hyperbolic response, ΦF exhibits a linear and inverse relationship with the reciprocal of irradiance, a relationship previously referred to as a reciprocal plot. Given the negative slope of the reciprocal plot, estimates of ΦF at infinite irradiance can be obtained, even over sub-saturating irradiances, by linear regression and extrapolation of the resultant reciprocal plot to the y-intercept. Here, we show how to obtain data from a dynamic multiphase flash of sub-saturating irradiance, occurring within the time span of ~1 s, to generate a reciprocal plot that subsequently provides an accurate estimate of ΦF at infinite irradiance, or Fm′.
Maximum fluorescence yield Multiphase flash Photosynthesis Photosystem II Pulse-amplitude-modulation chlorophyll a fluorescence Saturation flash
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