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.

• Salinity represents an abiotic stress constraint affecting growth and productivity of plants • Better solutions is to improve the level of salt resistance using natural genetic variability within crop species • Phenomic methodology employing different non-invasive imaging systems for detecting quantitative and qualitative changes caused by salt stress at the whole plant and canopy level. Hyperspectral imaging techniques provide unique opportunities for fast and reliable evaluation of numerous characteristics associated both with various structural, biochemical and physiological traits • Salt-soil-plant interaction and sustainable coastal agriculture need powerful phenotyping tools Salinity represents an abiotic stress constraint affecting growth and productivity of plants in many regions of the world. One of the possible solutions is to improve the level of salt resistance using natural genetic variability within crop species. In the context of recent knowledge on salt stress effects and mechanisms of salt tolerance, this review present useful phenomic approach employing different non-invasive imaging systems for detection of quantitative and qualitative changes caused by salt stress at the plant and canopy level. The focus is put on hyperspectral imaging technique, which provides unique opportunities for fast and reliable estimate of numerous characteristics associated both with various structural, biochemical Science of the Total Environment 578 (2017) 90-99 ⁎ Corresponding authors at:

Nitrogen deficiency influences importantly the plant photosynthetic capacity and crop productivity. Here, we employed the rapid, non-invasive measurements of chlorophyll a fluorescence kinetics for calculation of the integrative fluorescence parameters related to the leaf photosynthetic performance. In pot experiments with winter wheat (Triticum aestivum L.) we cultivated plants during the whole growing period in the soil substrate supplied with four different doses of nitrogen. The leaf nitrogen and chlorophyll content as well as the plant dry mass were analyzed after chlorophyll fluorescence records in three growth stages. Our results indicate that the commonly used parameter F v /F m (the maximum quantum yield of photochemistry) was almost insensitive to nitrogen treatment. In contrary, the performance index (PI abs ) and total performance index (PI tot ) were much more responsive and significant differences among plants of different nitrogen treatments as well as between the youngest and third leaf from the top were observed. Parameter PI tot was shown to express only small diurnal changes, thus being more reliable and more useful for comparison of different samples in field conditions than more frequently used parameter PI abs .

The impact of the genotype‐specific leaf morphological and anatomical characteristics on the ability of wheat plants to preserve leaf water balance and cell membranes stability under drought stress was investigated. Seedlings of six modern semi‐dwarf (carriers of Rht, Reduced height genes) and six old tall bread wheat varieties were subjected to soil drought by withholding watering for 6 days. Morpho‐anatomical traits (leaf area, perimeter, thickness, stomata and trichome density) of daily watered (control) plants were characterized by light microscopy, scanning and image analyses. The leaf water status in both control and stressed plants was determined by measuring the relative water content (RWC). The leaf cell membranes stability in stressed plants was estimated by conductometric determination of the membranes injury index. On average, the modern semi‐dwarf varieties had less leaf area and leaf perimeter, and less dissection index, a parameter characterizing the leaf shape. Under drought stress, the modern genotypes maintained better water balance evidenced by significantly higher leaf RWC and better‐preserved the cell membranes stability supported by significantly lower Injury index. The correlations between morpho‐anatomical traits in control plants and drought tolerance‐related traits showed that the higher the leaf dissection index (i.e. more oblong leaves), the greater the water loss and the leaf membrane damages after desiccation were. The effect of shape of the evaporating surface on the water loss was modelled using wet filter paper. Similar to plant leaves, the evaporation and, respectively, water loss from paper pieces of more oblong shape (i.e. higher dissection index) was more intensive. The elucidation of the impact of the leaf shape on transpiration might contribute to better understanding of the mechanisms used by plants to maintain water reserves during drought stress and could be a basis for developing of simple and fast screening methods aiding the selection of drought tolerant genotypes.