Chlorophyll fluorometry in evaluating photosynthetic performance: key limitations, possibilities, perspectives and alternatives

Abstract

Non-destructive methods for the assessment of photosynthetic parameters of plants are widely applied to evaluate rapidly the photosynthetic performance, plant health, and shifts in plant productivity induced by environmental and cultivation conditions. Most of these methods are based on measurements of chlorophyll fluorescence kinetics, particularly on pulse modulation (PAM) fluorometry. In this paper, fluorescence methods are critically discussed in regard to some their possibilities and limitations inherent to vascular plants and microalgae. Attention is paid to the potential errors related to the underestimation of thylakoidal cyclic electron transport and anoxygenic photosynthesis. PAM-methods are also observed considering the color-addressed measurements. Photoacoustic methods are discussed as an alternative and supplement to fluorometry. Novel Fourier modifications of PAM-fluorometry and photoacoustics are noted as tools allowing simultaneous application of a dual or multi frequency measuring light for one sample.

Keywords: Anoxygenic photosynthesis; Fourier PAM; Fourier photoacoustics; Multicolor PAM; Photosystem II; Quantum yield.

Fig. 1

Imaging of the ratio of fluorescence decrease rate values (R_fd) distributed over the areas of a light-green young leaf (a) and dark-green old leaf (b) of Morus alba L. The reference colors are given using a lookup table (LUT). Fluorescence was monitored at 740 nm through an interference filter and excited by blue light, λ = 465 nm. The corresponding images of the same original leaves under white light are shown below (c, d). The imaging was performed using the method of Lysenko (2012). Unpublished data

Fig. 2

Principle of the application of fast Fourier transform (FFT) to the PAM fluorometry and photoacoustics. a mixed fluorescence or PA-signal in the time domain simultaneously excited in one sample by two sources of measuring light with frequencies f1 and f2; b single FFT-spectrum (in the frequency domain) calculated every 0.7 s from a number of amplitude readings (data massive; for example, 131,072 readings in 0.7 s). Respectively, the program saves the peak values of the amplitudes at f1 and f2 frequencies (see panel b) every 0.7 s in a log file (*.xlsx), from which the photoinduction curves may easily be obtained. When the bicolor Fourier modification of PAM-fluorometry is applied, f1 and f2 may be, for example, 340 (blue light) and 360 Hz (red light). When the Fourier photoacoustics is applied, f1 and f2 may be 20–30 and 250–300 Hz, where 20–30 Hz corresponds mainly to the photobaric signal, and 250–350 Hz—to the photothermal signal (see the text)