A New Multiphase Single Flash Method of Determining Fm' Is Important for Field-Grown Plants in Order to Accurately Calculate Φpsii and the Rate of Electron Transport (J) Using Chlorophyll Fluorescence Measurements.

Determination of maximal fluorescence (Fm’) is necessary to estimate the effective quantum efficiency of Photosystem II (Φ_PSII) from chlorophyll fluorescence measurements. Fm’ is commonly measured using a constant saturating, multi-turnover flash of light of 400-1200 ms duration (rectangular flash, RF) for reducing the Q_A-PQ pool.  In many conditions, especially in field plants, full reduction of the Q_A-PQ pool cannot be achieved using such a protocol. Here we present a ‘multiphase’ flash (MPF) method to estimate the true Fm’ in these conditions.  We tested the hypotheses (1) that the RF method largely underestimates Fm’ under field conditions, and (2) that the MPF method can be used to provide a rapid and reliable Fm’ measurement within a single, multiturnover flash.  The MPF approach was tested experimentally under both easy- and difficult-to-saturate conditions, and the method was also cross-checked using an alternative method: a flash train (FT) of variable Q.  Under high, light-adapted conditions, estimates obtained using RF were considerably lower than the MPF results.  In field-grown sunflower (Helianthus annuus) and soybean (Glycine max), which were adapted to full sunlight, the RF method underestimated Fm’ by an average of 13% and 8%, respectively (both significant at α0.01).  This difference resulted in calculated Φ_PSII measurements that were underestimated by 20% in sunflower, and 23% in soybean (both significant at α0.01).  There were no significant differences between the MPF and FT approaches.  Combined gas exchange and fluorescence light response curve measurements were proportional when the rate of electron transport (J) was estimated using the MPF approach, but significantly diverged at the highest light intensities when using the RF method.  Correctly determining Fm’ not only impacts the Φ_PSII and J estimations, but also affects the accuracy of the calculations of mesophyll conductance (g_m) and CO₂ concentration in the chloroplast (C_c).