Calibration matters: On the procedure of using the chlorophyll fluorescence method to estimate mesophyll conductance

Estimates of mesophyll conductance (gm), when calculated from chlorophyll fluorescence, are uncertain, especially when the photosystem II (PSII) operating efficiency is measured from the traditional single saturation pulse methodology. The multiphase flash method has recently been recommended to replace the single saturation pulse method, allowing a more reliable estimation of gm. Also, many researchers still directly use the PSII operating efficiency to derive linear electron transport rate J (that is required to estimate gm), without appropriate calibration using measurements under non-photorespiratory conditions. Here we demonstrate for tomato and rice that (i) using the multiphase flash method did not yield realistic estimates of gm if no calibration was conducted; and (ii) using the single saturation pulse method still gave reasonable estimates of gm when calibration based on the non-photorespiratory measurements was properly conducted. Therefore, conducting calibration based on data under non-photorespiratory conditions was indispensable for a reliable estimation of gm, regardless whether the multiphase flash or the single saturation pulse method was used for measuring the PSII operating efficiency. Other issues related to the procedure of using the chlorophyll fluorescence method to estimate gm were discussed.