How to correctly determine the different chlorophyll fluorescence parameters and the chlorophyll fluorescence decrease ratio RFd of leaves with the PAM fluorometer

This contribution is a practical guide to the measurement of the different chlorophyll (Chl) fluorescence parameters and gives examples of their development under high-irradiance stress. From the Chl fluorescence induction kinetics upon irradiation of dark-adapted leaves, measured with the PAM fluorometer, various Chl fluorescence parameters, ratios, and quenching coefficients can be determined, which provide information on the functionality of the photosystem 2 (PS2) and the photosynthetic apparatus. These are the parameters F-v, F-m, F-0, F-m', F-v', NF, and Delta F, the Chl fluorescence ratios F-v/F-m, F-v/F-0, Delta F/F-m', as well as the photochemical (q(P)) and non-photochemical quenching coefficients (q(N), q(CN), and NPQ). q(N) consists of three components (q(N) = q(E) + q(T) + q(I)), the contribution of which can be determined via Chl fluorescence relaxation kinetics measured in the dark period after the induction kinetics. The above Chl fluorescence parameters and ratios, many of which are measured in the dark-adapted state of leaves, primarily provide information on the functionality of PS2. In fully developed green and dark-green leaves these Chl fluorescence parameters, measured at the upper adaxial leaf side, only reflect the Chl fluorescence of a small portion of the leaf chloroplasts of the green palisade parenchyma cells at the upper outer leaf half Thus, PAM fluorometer measurements have to be performed at both leaf sides to obtain information on all chloroplasts of the whole leaf Combined high irradiance (HI) and heat stress, applied at the upper leaf side, strongly reduced the quantum yield of the photochemical energy conversion at the upper leaf half to nearly zero, whereas the Chl fluorescence signals measured at the lower leaf side were not or only little affected. During this HL-stress treatment, q(N), q(CN), and NPQ increased in both leaf sides, but to a much higher extent at the lower compared to the upper leaf side. q(N) was the best indicator for non-photochemical quenching even during a stronger HL-stress, whereas q(CN) and NPQ decreased with progressive stress even though non-photochemical quenching still continued. It is strongly recommended to determine, in addition to the classical fluorescence parameters, via the PAM fluorometer also the Chl fluorescence decrease ratio R-Fd (F-d/F-S), which, when measured at saturation irradiance is directly correlated to the net CO2 assimilation rate (P-N) of leaves. This R-Fd-ratio can be determined from the Chl fluorescence induction kinetics measured with the PAM fluorometer using continuous saturating light (cSL) during 4-5 min. As the R-Fd-values are fast measurable indicators correlating with the photosynthetic activity of whole leaves, they should always be determined via the PAM fluorometer parallel to the other Chl fluorescence coefficients and ratios.