Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121–158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F V/F M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights.

The photosynthetic responses of wheat (Triticum aestivum L.) leaves to different levels of drought stress were analyzed in potted plants cultivated in growth chamber under moderate light. Low-to-medium drought stress was induced by limiting irrigation, maintaining 20 % of soil water holding capacity for 14 days followed by 3 days without water supply to induce severe stress. Measurements of CO2 exchange and photosystem II (PSII) yield (by chlorophyll fluorescence) were followed by simultaneous measurements of yield of PSI (by P700 absorbance changes) and that of PSII. Drought stress gradually decreased PSII electron transport, but the capacity for nonphotochemical quenching increased more slowly until there was a large decrease in leaf relative water content (where the photosynthetic rate had decreased by half or more). We identified a substantial part of PSII electron transport, which was not used by carbon assimilation or by photorespiration, which clearly indicates activities of alternative electron sinks. Decreasing the fraction of light absorbed by PSII and increasing the fraction absorbed by PSI with increasing drought stress (rather than assuming equal absorption by the two photosystems) support a proposed function of PSI cyclic electron flow to generate a proton-motive force to activate nonphotochemical dissipation of energy, and it is consistent with the observed accumulation of oxidized P700 which causes a decrease in PSI electron acceptors. Our results support the roles of alternative electron sinks (either from PSII or PSI) and cyclic electron flow in photoprotection of PSII and PSI in drought stress conditions. In future studies on plant stress, analyses of the partitioning of absorbed energy between photosystems are needed for interpreting flux through linear electron flow, PSI cyclic electron flow, along with alternative electron sinks.

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a b s t r a c tIn conditions of long-lasting moderate drought stress, we have studied the photoprotective responses in leaves of wheat (Triticum aestivum L., cv. Katya) related to the photosynthetic electron and proton transport. The dark-interval relaxation kinetics of electrochromic bandshift (ECS) indicated a decrease of electric and an increase of osmotic component of the proton motive force in drought stressed leaves, but neither the total proton motive force (pmf) nor the thylakoid proton conductance (gH + ) were affected.We observed the enhanced protection against overreduction of PSI acceptor side in leaves of drought stressed plants. This was obviously achieved by the rapid buildup of transthylakoid pH gradient at relatively low light intensities, directly associated to the steep increase of NPQ and the down-regulation of linear electron transport. It was further accompanied by the steep increase of redox poise at PSII acceptor side and PSI donor side. The early responses related to thylakoid lumen acidification in drought-stressed leaves could be associated with the activity of an enhanced fraction of PSI not involved in linear electron flow, which may have led to enhanced cyclic electron pathway even in relatively low light intensities, as well as to the drought-induced decrease of IP-amplitude in fast chlorophyll fluorescence kinetics.

Polyphasic chlorophyll a fluorescence represents a promising tool for detection of plant tolerance to various environmental stresses. In pot vegetation experiments, plants of seven winter wheat varieties were screened for their drought tolerance. The drought stress was initiated in plants by withholding water at the beginning of anthesis. While water content was measured continuously as relative water content (RWC), fast chlorophyll a fluorescence kinetics was measured and analysed on dehydrating intact leaves by the JIP-test (analysis of O-J-I-P fluorescence transient). Maximum quantum efficiency of PS II photochemistry (F V /F M ) parameter was almost unaffected by dehydration until the severe water stress occurred. In contrast to this a continuous decrease of performance index (PI abs ) parameter (Strasser et al. 1995) was observed from the very beginning of dehydration following the decrease of RWC. Statistically significant differences were also found in the PI abs parameter among all tested varieties. The results show that PI abs may serve as an index of plant/variety vitality and/or sensitivity to water stress reflecting their different drought tolerance.