Marian Brestic

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.

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.

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.

Dissection of Photosynthetic Electron Transport Process in Sweet Sorghum under Heat Stress

Plant photosynthesis and photosystem II (PSII) are susceptible to high temperature. However, photosynthetic electron transport process under heat stress remains unclear. To reveal this issue, chlorophyll a fluorescence and modulated 820 nm reflection were simultaneously detected in sweet sorghum. At 43°C, J step in the chlorophyll a fluorescence transient was significantly elevated, suggesting that electron transport beyond primary quinone of PSII (QA) (primary quinone electron acceptor of PSII) was inhibited. PSI (Photosystem I) photochemical capacity was not influenced even under severe heat stress at 48°C. Thus, PSI oxidation was prolonged and PSI re-reduction did not reach normal level. The inhibition of electron transport between PSII and PSI can reduce the possibility of PSI photoinhibition under heat stress. PSII function recovered entirely one day after heat stress at 43°C, implying that sweet sorghum has certain self-remediation capacity. When the temperature reached 48°C, the maximum quantum yield for primary photochemistry and the electron transport from PSII donor side were remarkably decreased, which greatly limited the electron flow to PSI, and PSI re-reduction suspended. The efficiency of an electron transferred from the intersystem electron carrier (plastoquinol, PQH2) to the end electron acceptors at the PSI acceptor side increased significantly at 48°C, and the reason was the greater inhibition of electron transport before PQH2. Thus, the fragment from QA to PQH2 is the most heat sensitive in the electron transport chain between PSII and PSI in sweet sorghum.

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.