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.

Analysis of plant behavior under diverse environmental conditions would be impossible without the methods for adequate assessment of the processes occurring in plants. The photosynthetic apparatus and its reaction to stress factors provide a reliable source of information on plant condition. One of the most informative methods based on monitoring the plant biophysical characteristics consists in detection and analysis of chlorophyll a fluorescence. Fluorescence is mainly emitted by chlorophyll a from the antenna complexes of photosystem II (PSII). However, fluorescence depends not only on the processes in the pigment matrix or PSII reaction centers but also on the redox reactions at the PSII donor and acceptor sides and even in the entire electron transport chain. Presently, a large variety of fluorometers from various manufacturers are available. Although application of such fluorometers does not require specialized training, the correct interpretation of the results would need sufficient knowledge for converting the instrumental data into the information on the condition of analyzed plants. This review is intended for a wide range of specialists employing fluorescence techniques for monitoring the physiological plant condition. It describes in a comprehensible way the theoretical basis of light emission by chlorophyll molecules, the origin of variable fluorescence, as well as relations between the fluorescence parameters, the redox state of electron carriers, and the light reactions of photosynthesis. Approaches to processing and analyzing the fluorescence induction curves are considered in detail on the basis of energy flux theory in the photosynthetic apparatus developed by Prof. Reto J. Strasser and known as a “JIP-test.” The physical meaning and relation of each calculated parameter to certain photosynthetic characteristics are presented, and examples of using these parameters for the assessment of plant physiological condition are outlined.

In natural conditions, plants growth and development depends on environmental conditions, including the availability of micro- and macroelements in the soil. Nutrient status should thus be examined not by establishing the effects of single nutrient deficiencies on the physiological state of the plant but by combinations of them. Differences in the nutrient content significantly affect the photochemical process of photosynthesis therefore playing a crucial role in plants growth and development. In this work, an attempt was made to find a connection between element content in (i) different soils, (ii) plant leaves, grown on these soils and (iii) changes in selected chlorophyll a fluorescence parameters, in order to find a method for early detection of plant stress resulting from the combination of nutrient status in natural conditions. To achieve this goal, a mathematical procedure was used which combines principal component analysis (a tool for the reduction of data complexity), hierarchical k-means (a classification method) and a machine-learning method—super-organising maps. Differences in the mineral content of soil and plant leaves resulted in functional changes in the photosynthetic machinery that can be measured by chlorophyll a fluorescent signals. Five groups of patterns in the chlorophyll fluorescent parameters were established: the ‘no deficiency’, Fe-specific deficiency, slight, moderate and strong deficiency. Unfavourable development in groups with nutrient deficiency of any kind was reflected by a strong increase in F _o and ΔV/Δt ₀ and decline in φ _Po, φ _Eo δ _Ro and φ _Ro. The strong deficiency group showed the suboptimal development of the photosynthetic machinery, which affects both PSII and PSI. The nutrient-deficient groups also differed in antenna complex organisation. Thus, our work suggests that the chlorophyll fluorescent method combined with machine-learning methods can be highly informative and in some cases, it can replace much more expensive and time-consuming procedures such as chemometric analyses.

Perennial ryegrass (Lolium perenne L.) belongs to the common cultivated grass species in Central and Western Europe. Despite being considered to be susceptible to drought, it is frequently used for forming the turf in urban green areas. In such areas, the water deficit in soil is recognized as one of the most important environmental factors, which can limit plant growth. The basic aim of this work was to explore the mechanisms standing behind the changes in the photosynthetic apparatus performance of two perennial ryegrass turf varieties grown under drought stress using comprehensive in vivo chlorophyll fluorescence signal analyses and plant gas exchange measurements. Drought was applied after eight weeks of sowing by controlling the humidity of the roots ground medium at the levels of 30, 50, and 70% of the field water capacity. Measurements were carried out at four times: 0, 120, and 240 h after drought application and after recovery (refilling water to 70%). We found that the difference between the two tested varieties’ response resulted from a particular re-reduction of P700⁺ (reaction certer of PSI) that was caused by slower electron donation from P680. The difference in the rate of electron flow from Photosystem II (PSII) to PSI was also detected. The application of the combined tools (plants’ photosynthetic efficiency analysis and plant gas exchange measurements) allowed exploring and explaining the specific variety response to drought stress.

The aim of this study was to understand the acclimatization mechanisms of photosynthetic apparatus in Brachypodium pinnatum (L.) P. Beauv grass during its expansion. Twelve populations differentiated by age: young (30–50 years old), intermediate age (ca. 100 y) and old (>300 y) were studied. It was confirmed that the decrease of the number of genotypes as a result of environmental stress and competition were reflected in changes in chlorophyll fluorescence (ChlF) parameters. The old stands were dominated by a few genotypes which seem to be the best acclimatized to the self-shading/competition by lowering their photosynthetic performance during light-phase of photosynthesis. On the other hand, the 'high-speed' photosynthetic rate observed in the young populations can be seen as acclimatization to very adverse conditions. Our results clearly confirm that ChlF is a powerful method of inferring physiological mechanisms of the expansion of tor grass. The Principal Component and Redundancy Analyses, followed with k-means classification, allowed to find the differentiation of groups of distinct ChlF parameters and enabled us to relate them to changes in genotypic diversity of populations. We conclude that the plastic morphological and physiological response to changeable habitat light conditions with its optimum in half-shade refers to its forest-steppe origin.