Marek Zivcak

Frequently asked questions about in vivo chlorophyll fluorescence: practical issues

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.

Chlorophyll a fluorescence as a tool to monitor physiological status of plants under abiotic stress conditions

Plants living under natural conditions are exposed to many adverse factors that interfere with the photosynthetic process, leading to declines in growth, development, and yield. The recent development of Chlorophyll a fluorescence (ChlF) represents a potentially valuable new approach to study the photochemical efficiency of leaves. Specifically, the analysis of fluorescence signals provides detailed information on the status and function of Photosystem II (PSII) reaction centers, light-harvesting antenna complexes, and both the donor and acceptor sides of PSII. Here, we review the results of fast ChlF analyses of photosynthetic responses to environmental stresses, and discuss the potential scientific and practical applications of this innovative methodology. The recent availability of portable devices has significantly expanded the potential utilization of ChlF techniques, especially for the purposes of crop phenotyping and monitoring.

Photosystem II thermostability in situ: environmentally induced acclimation and genotype-specific reactions in Triticum aestivum L.

Photosystem II (PSII) thermostability and acclimation effects on PSII photochemical efficiency were analyzed in thirty field grown winter wheat (Triticum aestivum L.) genotypes using prompt chlorophyll a fluorescence kinetics before and after dark heat treatment. A gradual increase in temperature caused the appearance of K-bands at 300 μs on the chlorophyll fluorescence induction curve, indicating the impairment of the PSII donor side (even by heat treatment at 38 °C). An increase in basal fluorescence, commonly used as a criterion of PSII thermostability, was observed beyond a temperature threshold of 44 °C. Moreover, an acclimation shift (increase of critical temperature) was observed at the 3.5 °C identified for K-band appearance, but only by 1.1 °C for a steep increase in F(0). The single temperature approach with regular weekly observations completed within two months using dark heat treatment at 40 °C demonstrated that the acclimation effect is not gradual, but occurs immediately and is associated with an increase of daily temperature maxima over 30 °C. The acclimated heat treated samples had less effect on the donor side of PSII, the higher fraction of active Q(A)(-) reducing reaction centers and causing a much lower decrease of connectivity among PSII units compared to non-acclimated samples. In the non-treated plants the reduction of antennae size, increase of PSII connectivity and changes in the acceptor side occurred as a result of heat acclimation. The enhancement of PSII thermostability persisted over several weeks regardless of weather conditions. The genotype comparison identified three groups that differed either in initial PSII thermostability or in acclimation capacity; these groupings were clearly associated with the origin of the genotypes.

Photosynthetic responses of sun- and shade-grown barley leaves to high light: is the lower PSII connectivity in shade leaves associated with protection against excess of light?

In this study, we have compared photosynthetic performance of barley leaves (Hordeum vulgare L.) grown under sun and shade light regimes during their entire growth period, under field conditions. Analyses were based on measurements of both slow and fast chlorophyll (Chl) a fluorescence kinetics, gas exchange, pigment composition; and of light incident on leaves during their growth. Both the shade and the sun barley leaves had similar Chl a/b and Chl/carotenoid ratios. The fluorescence induction analyses uncovered major functional differences between the sun and the shade leaves: lower connectivity among Photosystem II (PSII), decreased number of electron carriers, and limitations in electron transport between PSII and PSI in the shade leaves; but only low differences in the size of PSII antenna. We discuss the possible protective role of low connectivity between PSII units in shade leaves in keeping the excitation pressure at a lower, physiologically more acceptable level under high light conditions.

PSII Fluorescence Techniques for Measurement of Drought and High Temperature Stress Signal in Crop Plants: Protocols and Applications

Field crops are frequently exposed to drought and high temperature in the field. As the stress tolerance is the major target of many research and breeding programmes, the efficient and reliable tools and methods useful in screening of the heat and drought stress effects are required. The techniques based on measurement of chlorophyll fluorescence induction belong recently to fundamentals of plant stress research; however, in most cases the very basic tools are used and its potential is not utilised sufficiently. This proposed chapter tries to summarise the knowledge, starting from basic theory through parameters and useful experimental protocols and results up to special kinds of application of chlorophyll fluorescence techniques. In addition to generally used pulse-amplitude-modulated (PAM) method with saturation pulse analysis, the fast fluorescence kinetics, the fluorescence imaging, as well as simultaneous measurements of chlorophyll fluorescence with other parameters and their potential application in drought and heat-stress research are discussed.

Low PSI content limits the photoprotection of PSI and PSII in early growth stages of chlorophyll b -deficient wheat mutant lines

In vivo analyses of electron and proton transport-related processes as well as photoprotective responses were carried out at different stages of growth in chlorophyll b (Chl b)-deficient mutant lines (ANK-32A and ANK-32B) and wild type (WT) of wheat (Triticum aestivum L.). In addition to a high Chl a–b ratio, ANK mutants had a lower content of photo-oxidizable photosystem I (PSI, Pm), and several parameters indicated a low PSI/PSII ratio. Moreover, simultaneous measurements of Chl fluorescence and P700 indicated a shift of balance between redox poise of the PSII acceptor side and the PSII donor side, with preferential reduction of the plastoquinone pool, resulting in an over reduced PSI acceptor side (high ΦNA values). This was the probable reason for PSI inactivation observed in the ANK mutants, but not in WT. In later growth phases, we observed partial relief of “chlorina symptoms,” toward WT. Measurements of ΔA520 decay confirmed that, in early growth stages, the ANK mutants with low PSI content had a limited capacity to build up the transthylakoid proton gradient (ΔpH) needed to trigger non-photochemical quenching (NPQ) and to regulate the electron transport by cytochrome b6/f. Later, the increase in the PSI/PSII ratio enabled ANK mutants to reach full NPQ, but neither over reduction of the PSI acceptor side nor PSI photoinactivation due to imbalance between the activity of PSII and PSI was mitigated. Thus, our results support the crucial role of proper regulation of linear electron transport in the protection of PSI against photoinhibition. Moreover, the ANK mutants of wheat showing the dynamic developmental changes in the PSI/PSII ratio are presented here as very useful models for further studies.

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.

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.

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.

Impact of Metal and Metal Oxide Nanoparticles on Plant: A Critical Review

An increasing need of nanotechnology in various industries may cause a huge environment dispersion of nanoparticles in coming years. A concern about nanoparticles interaction with flora and fauna is raised due to a growing load of it in the environment. In recent years, several investigators have shown impact of nanoparticles on plant growth and their accumulation in food source. This review examines the research performed in the last decade to show how metal and metal oxide nanoparticles are influencing the plant metabolism. We addressed here, the impact of nanoparticle on plant in relation to its size, concentration, and exposure methodology. Based on the available reports, we proposed oxidative burst as a general mechanism through which the toxic effects of nanoparticles are spread in plants. This review summarizes the current understanding and the future possibilities of plant-nanoparticle research.