The aim of this educational review is to provide practical information on the hardware, methodology, and the hands on application of chlorophyll (Chl) a fluorescence technology. We present the paper in a question and answer format like frequently asked questions. Although nearly all information on the application of Chl a fluorescence can be found in the literature, it is not always easily accessible. This paper is primarily aimed at scientists who have some experience with the application of Chl a fluorescence but are still in the process of discovering what it all means and how it can be used. Topics discussed are (among other things) the kind of information that can be obtained using different fluorescence techniques, the interpretation of Chl a fluorescence signals, specific applications of these techniques, and practical advice on different subjects, such as on the length of dark adaptation before measurement of the Chl a fluorescence transient. The paper also provides the physiological background for some of the applied procedures. It also serves as a source of reference for experienced scientists.

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.

SummaryVascular plants have evolved a long-term light acclimation strategy primarily relying on the regulation of the relative amounts of light-harvesting complex II (LHCII) and of the two photosystems, photosystem I (PSI) and photosystem II (PSII). We investigated whether such a model is also valid in Selaginella martensii, a species belonging to the early diverging group of lycophytes.Selaginella martensii plants were acclimated to three natural light regimes (extremely low light (L), medium light (M) and full sunlight (H)) and thylakoid organization was characterized combining ultrastructural, biochemical and functional methods.From L to H plants, thylakoid architecture was rearranged from (pseudo)lamellar to predominantly granal, the PSII : PSI ratio changed in favour of PSI, and the photochemical capacity increased. However, regulation of light harvesting did not occur through variations in the amount of free LHCII, but rather resulted from the flexibility of the association of free LHCII with PSII and PSI.In lycophytes, the free interspersed LHCII serves a fixed proportion of reaction centres, either PSII or PSI, and the regulation of PSI-LHCII(-PSII) megacomplexes is an integral part of long-term acclimation. Free LHCII ensures photoprotection of PSII, allows regulated use of PSI as an energy quencher, and can also quench endangered PSI.

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Evolution of vascular plants required compromise between photosynthesis and photodamage. We analyzed representative species from two divergent lineages of vascular plants, lycophytes and euphyllophytes, with respect to the response of their photosynthesis and light-harvesting properties to increasing light intensity. In the two analyzed lycophytes, Selaginella martensii and Lycopodium squarrosum, the medium phase of non-photochemical quenching relaxation increased under high light compared to euphyllophytes. This was thought to be associated with the occurrence of a further thylakoid phosphoprotein in both lycophytes, in addition to D2, CP43 and Lhcb1-2. This protein, which showed light intensity-dependent reversible phosphorylation, was identified in S. martensii as Lhcb6, a minor LHCII antenna subunit of PSII. Lhcb6 is known to have evolved in the context of land colonization. In S. martensii, Lhcb6 was detected as a component of the free LHCII assemblies, but also associated with PSI. Most of the light-induced changes affected the amount and phosphorylation of the LHCII assemblies, which possibly mediate PSI-PSII connectivity. We propose that Lhcb6 is involved in light energy management in lycophytes, participating in energy balance between PSI and PSII through a unique reversible phosphorylation, not yet observed in other land plants.