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

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.

High levels of anoxygenic photosynthesis revealed by dual-frequency Fourier photoacoustics in Ailanthus altissima leaves.

In contrast to oxygenic photosynthesis, true anoxygenic photosynthesis is not associated with O2 evolution originated from water photolysis but still converts light energy to that of the phosphoanhydride bonds of ATP. In a narrow sense, anoxygenic photosynthesis is mainly known as to be related to the purple and green sulfur bacteria, but in a broad sense, it also occurs in the vascular plants. The portion of photosynthetic water photolysis that is compensated by the processes of O2 uptake (respiration, photorespiration, Mehler cycle, etc.) may be referred to as 'quasi' anoxygenic photosynthesis. Photoacoustic method allows for the separate detection of photolytic O2 at frequencies of measuring light about 20-40Hz, whereas at 250-400Hz, it detects the photochemical energy storage. We have developed a fast-Fourier transform photoacoustic method enabling measurements of both these signals simultaneously in one sample. This method allows to calculate oxygenic coefficients, which reflect the part of photochemically stored light energy that is used for the water photolysis. We show that the true anoxygenic photosynthesis in Ailanthus altissima Mill. leaves reached very high levels under low light, under moderate light at the beginning of the 1-h period, and at the end of the 40-min period under saturating light.

Chlorophyll fluorescence kinetics and oxygen evolution in Chlorella vulgaris cells: Blue vs. red light.

Oxygen evolution and chlorophyll fluorescence kinetics in cells of the Chlorella vulgaris strain (Europolytest, Russia) were studied under low, moderate and high photosynthetic photon flux densities (PPFD 40, 130 and 350 µmol photons m-2 s-1) of the red and blue actinic light. A novel method of a pulse amplitude modulated (PAM) Fourier chlorophyll fluorometry was applied to obtain photoinduction curves simultaneously for the red and blue measuring light for one sample. It was found that the red light did not induce oxygen evolution at low and moderate PPFD, whereas at high PPFD it caused a declining oxygen release. There was only a trace fluorescence kinetics at the low PPFD, but noticeable fluorescence kinetics under the red light was observed at the low and moderate PPFD. Particularly, the moderate red illumination of Chlorella cells excited a high chlorophyll fluorescence kinetics along with the absence of oxygen evolution that suggests anoxygenic photosynthesis. In contrast, the blue light induced a significant oxygen evolution as well as fluorescence kinetics already at low PPFD which were both further increased with the PPFD increasing. In addition, a high value of the chromatic divergence of quantum yield of photosystem II was revealed between the red and blue measuring light under high PPFD of the red actinic light.

Anthocyanin-dependent anoxygenic photosynthesis in coloured flower petals?

Chlorophylless flower petals are known to be composed of non-photosynthetic tissues. Here, we show that the light energy storage that can be photoacoustically measured in flower petals of Petunia hybrida is approximately 10-12%. We found that the supposed chlorophylless photosynthesis is an anoxygenic, anthocyanin-dependent process occurring in blue flower petals (ADAPFP), accompanied by non-respiratory light-dependent oxygen uptake and a 1.5-fold photoinduced increase in ATP levels. Using a simple, adhesive tape stripping technique, we have obtained a backside image of an intact flower petal epidermis, revealing sword-shaped ingrowths connecting the cell wall and vacuole, which is of interest for the further study of possible vacuole-related photosynthesis. Approaches to the interpretations of ADAPFP are discussed, and we conclude that these results are not impossible in terms of the known photochemistry of anthocyanins.