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.

Chlorophyll fluorescence analysis is one of the most powerful and widely used techniques to study the effect of stresses on the photosynthetic process. From the first utilization, the F_v/F_m ratio has been largely used as a sensitive indicator of plant photosynthetic performance. Decreases of this index are indicative of the reduction of photosystem II (PSII) efficiency, namely photoinhibition. In the last 20 years, application of chlorophyll fluorescence has been largely improved, and many other informative parameters have been established to detect PSII photochemical efficiency and the partitioning of light energy to alternative dissipative mechanisms (qE, energy-dependent quenching; qZ, zeaxanthin-dependent quenching and qI, photoinhibitory quenching; qH, sustained photoprotective antenna quenching; qM, quenching dependent to chloroplast movement; qT, light harvesting complexes II–I state-transition) such as the recently developed “photoprotective power” of non-photochemical quenching (pNPQ). This review reports a brief description of the main chlorophyll fluorescence parameters and a wide analysis of the current bibliography on the use of different parameters which are useful to detect events of PSII photoinhibition. In addition, in view of the inherent differences in morpho-anatomical, physiological and biochemical features between C3 and C4 metabolism, possible differences in terms of photoinhibition between C3 and C4 plant species under stress conditions are proposed. The attempt is to highlight the limits of their comparison in terms of susceptibility to photoinhibition and to propose direction of future research which, assisted by chlorophyll fluorescence, should improve the knowledge of the different sensitivity of C3 and C4 to abiotic stressors.

Whether flavonoids play significant antioxidant roles in plants challenged by photooxidative stress of different origin has been largely debated over the last few decades. A critical review of the pertinent literature and our experimentation as well, based on a free-of-scale approach, support an important antioxidant function served by flavonoids in plants exposed to a wide range of environmental stressors, the significance of which increases with the severity of stress. On the other side, some questions need conclusive answers when the putative antioxidant functions of plant flavonoids are examined at the level of both the whole-cell and cellular organelles. This partly depends upon a conclusive, robust, and unbiased definition of “a plant antioxidant”, which is still missing, and the need of considering the subcellular re-organization that occurs in plant cells in response to severe stress conditions. This likely makes our deterministic-based approach unsuitable to unveil the relevance of flavonoids as antioxidants in extremely complex biological systems, such as a plant cell exposed to an ever-changing stressful environment. This still poses open questions about how to measure the occurred antioxidant action of flavonoids. Our reasoning also evidences the need of contemporarily evaluating the changes in key primary and secondary components of the antioxidant defense network imposed by stress events of increasing severity to properly estimate the relevance of the antioxidant functions of flavonoids in an in planta situation. In turn, this calls for an in-depth analysis of the sub-cellular distribution of primary and secondary antioxidants to solve this still intricate matter.