Lettuce flavonoids screening and phenotyping by chlorophyll fluorescence excitation ratio.

MAIN CONCLUSION: Environmentally induced variation and the genotypic differences in flavonoid and phenolic content in lettuce can be reliably detected using the appropriate parameters derived from the records of rapid non-invasive fluorescence technique. The chlorophyll fluorescence excitation ratio method was designed as a rapid and non-invasive tool to estimate the content of UV-absorbing phenolic compounds in plants. Using this technique, we have assessed the dynamics of accumulation of flavonoids related to developmental changes and environmental effects. Moreover, we have tested appropriateness of the method to identify the genotypic differences and fluctuations in total phenolics and flavonoid content in lettuce. Six green and two red genotypes of lettuce (Lactuca sativa L.) grown in pots were exposed to two different environments for 50 days: direct sunlight (UV-exposed) and greenhouse conditions (low UV). The indices based on the measurements of chlorophyll fluorescence after red, green and UV excitation indicated increase of the content of UV-absorbing compounds and anthocyanins in the epidermis of lettuce leaves. In similar, the biochemical analyses performed at the end of the experiment confirmed significantly higher total phenolic and flavonoid content in lettuce plants exposed to direct sun compared to greenhouse conditions and in red compared to green genotypes. As the correlation between the standard fluorescence indices and the biochemical records was negatively influenced by the presence of red genotypes, we proposed the use of a new parameter named Modified Flavonoid Index (MFI) taking into an account both absorbance changes due to flavonol and anthocyanin content, for which the correlation with flavonoid and phenolic content was relatively good. Thus, our results confirmed that the fluorescence excitation ratio method is useful for identifying the major differences in phenolic and flavonoid content in lettuce plants and it can be used for high-throughput pre-screening and phenotyping of leafy vegetables in research and breeding applications towards improvement of vegetable health effects.

Applying hyperspectral imaging to explore natural plant diversity towards improving salt stress tolerance.

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 and physiological traits. The method also provides possibilities to combine plant and canopy analyses with a direct determination of salinity in soil. The future perspectives in salt stress applications as well as some limits of the method are also identified.

Frequently asked questions about chlorophyll fluorescence, the sequel.

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.

Genotypically Identifying Wheat Mesophyll Conductance Regulation under Progressive Drought Stress.

Photosynthesis limitation by CO2 flow constraints from sub-stomatal cavities to carboxylation sites in chloroplasts under drought stress conditions is, at least in some plant species or crops not fully understood, yet. Leaf mesophyll conductance for CO2 (gm) may considerably affect both photosynthesis and water use efficiency (WUE) in plants under drought conditions. The aim of our study was to detect the responses of gm in leaves of four winter wheat (Triticum aestivum L.) genotypes from different origins under long-term progressive drought. Based on the measurement of gas-exchange parameters the variability of genotypic responses was analyzed at stomatal (stomata closure) and non-stomatal (diffusional and biochemical) limits of net CO2 assimilation rate (AN). In general, progressive drought caused an increasing leaf diffusion resistance against CO2 flow leading to the decrease of AN, gm and stomatal conductance (gs), respectively. Reduction of gm also led to inhibition of carboxylation efficiency (Vcmax). On the basis of achieved results a strong positive relationship between gm and gs was found out indicating a co-regulation and mutual independence of the relationship under the drought conditions. In severely stressed plants, the stomatal limitation of the CO2 assimilation rate was progressively increased, but to a less extent in comparison to gm, while a non-stomatal limitation became more dominant due to the prolonged drought. Mesophyll conductance (gm) seems to be a suitable mechanism and parameter for selection of improved diffusional properties and photosynthetic carbon assimilation in C3 plants, thus explaining their better photosynthetic performance at a whole plant level during periods of drought.

Effect of photosystem I inactivation on chlorophyll a fluorescence induction in wheat leaves: Does activity of photosystem I play any role in OJIP rise?

Interpretation of the fast chlorophyll a fluorescence induction is still a subject of continuing discussion. One of the contentious issues is the influence of photosystem I (PSI) activity on the kinetics of the thermal JIP-phase of OJIP rise. To demonstrate this influence, we realized a series of measurements in wheat leaves subjected to PSI photoinactivation by the sequence of red saturation pulses (15,000 µmol photons m(-2) s(-1) for 0.3 s, every 10 s) applied in darkness. Such a treatment led to a moderate decrease of maximum quantum efficiency of PSII (by ~8%), but a strong decrease of the number of oxidizable PSI (by ~55%), which considerably limited linear electron transport and CO2 assimilation. Surprisingly, the PSI photoinactivation had low effects on OJIP kinetics of variable fluorescence. In particular, the amplitude of variable fluorescence of IP-step (ΔVIP), which has been considered to be a measure of PSI content, was not decreased, despite the low content of photooxidizable PSI. On the other hand, the slower relaxation of chlorophyll fluorescence after saturation pulse as well as the results of the double-hit method suggest that PSI inactivation treatment led to an increase of the fraction of QB-nonreducing PSII reaction centers. Our results somewhat challenge the mainstream interpretations of JIP-thermal phase, and at least suggest that the IP amplitude cannot serve to estimate reliably the PSI content or the PSI to PSII ratio. Moreover, these results recommend the use of the novel method of PSI inactivation, which might help clarify some important issues needed for the correct understanding of the OJIP fluorescence rise.

Reduced glutamine synthetase activity plays a role in control of photosynthetic responses to high light in barley leaves.

The chloroplastic glutamine synthetase (GS, EC 6.3.1.2) activity was previously shown to be the limiting step of photorespiratory pathway. In our experiment, we examined the photosynthetic high-light responses of the GS2-mutant of barley (Hordeum vulgare L.) with reduced GS activity, in comparison to wild type (WT). The biophysical methods based on slow and fast chlorophyll fluorescence induction, P700 absorbance, and gas exchange measurements were employed. Despite the GS2 plants had high basal fluorescence (F0) and low maximum quantum yield (Fv/Fm), the CO2 assimilation rate, the PSII and PSI actual quantum yields were normal. On the other hand, in high light conditions the GS2 had much higher non-photochemical quenching (NPQ), caused both by enhanced capacity of energy-dependent quenching and disconnection of PSII antennae from reaction centers (RC). GS2 leaves also maintained the PSII redox poise (QA(-)/QA total) at very low level; probably this was reason why the observed photoinhibitory damage was not significantly above WT. The analysis of fast chlorophyll fluorescence induction uncovered in GS2 leaves substantially lower RC to antenna ratio (RC/ABS), low PSII/PSI ratio (confirmed by P700 records) as well as low PSII excitonic connectivity.

Photosynthetic proton and electron transport in wheat leaves under prolonged moderate drought stress.

In 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.

Photosynthetic electron transport and specific photoprotective responses in wheat leaves under drought stress.

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.

Photosynthesis is improved by exogenous calcium in heat-stressed tobacco plants.

Effects of exogenous calcium chloride (CaCl(2)) (20 mM) on photosynthetic gas exchange, photosystem II photochemistry, and the activities of antioxidant enzymes in tobacco plants under high temperature stress (43°C for 2 h) were investigated. Heat stress resulted in a decrease in net photosynthetic rate (P(n)), stomatal conductance as well as the apparent quantum yield (AQY) and carboxylation efficiency (CE) of photosynthesis. Heat stress also caused a decrease of the maximal photochemical efficiency of primary photochemistry (F(v)/F(m)). On the other hand, CaCl(2) application improved P(n), AQY, and CE as well as F(v)/F(m) under high temperature stress. Heat stress reduced the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD), whereas the activities of these enzymes either decreased less or increased in plants pretreated with CaCl(2); glutathione reductase (GR) activity increased under high temperature, and it increased more in plants pretreated with CaCl(2). There was an obvious accumulation of H(2)O(2) and O(2)(-) under high temperature, but CaCl(2) application decreased the contents of H(2)O(2) and O(2)(-) under heat stress conditions. Heat stress induced the level of heat shock protein 70 (HSP70), while CaCl(2) pretreatment enhanced it. These results suggested that photosynthesis was improved by CaCl(2) application in heat-stressed plants and such an improvement was associated with an improvement in stomatal conductance and the thermostability of oxygen-evolving complex (OEC), which might be due to less accumulation of reactive oxygen species.