Vasilij Goltsev

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.

Delayed fluorescence in photosynthesis

Photosynthesis is a very efficient photochemical process. Nevertheless, plants emit some of the absorbed energy as light quanta. This luminescence is emitted, predominantly, by excited chlorophyll a molecules in the light-harvesting antenna, associated with Photosystem II (PS II) reaction centers. The emission that occurs before the utilization of the excitation energy in the primary photochemical reaction is called prompt fluorescence. Light emission can also be observed from repopulated excited chlorophylls as a result of recombination of the charge pairs. In this case, some time-dependent redox reactions occur before the excitation of the chlorophyll. This delays the light emission and provides the name for this phenomenon—delayed fluorescence (DF), or delayed light emission (DLE). The DF intensity is a decreasing polyphasic function of the time after illumination, which reflects the kinetics of electron transport reactions both on the (electron) donor and the (electron) acceptor sides of PS II. Two main experimental approaches are used for DF measurements: (a) recording of the DF decay in the dark after a single turnover flash or after continuous light excitation and (b) recording of the DF intensity during light adaptation of the photosynthesizing samples (induction curves), following a period of darkness. In this paper we review historical data on DF research and recent advances in the understanding of the relation between the delayed fluorescence and specific reactions in PS II. An experimental method for simultaneous recording of the induction transients of prompt and delayed chlorophyll fluorescence and decay curves of DF in the millisecond time domain is discussed.

Simultaneous analysis of prompt and delayed chlorophyll a fluorescence in leaves during the induction period of dark to light adaptation.

An attempt is made to reveal the relation between the induction curves of delayed fluorescence (DF) registered at 0.35-5.5 ms and the prompt chlorophyll fluorescence (PF). A simple formulation was proposed to link the ratio of the transient values of delayed and variable fluorescence with the redox state of the primary electron acceptor of Photosystem II--QA, and the thylakoid membrane energization. The term luminescence potential (UL) was introduced, defined as the sum of the redox potential of QA and the transmembrane proton gradient. It was shown that UL is proportional to the ratio of DF to the variable part of PF. The theoretical model was verified and demonstrated by analysing induction courses of PF and millisecond DF, simultaneously registered from leaves of barley--wild-type and the chlorophyll b-less mutant chlorina f2. A definitive correlation between PF and DF was established. If the luminescence changes are strictly due to UL, the courses of DF and PF are reciprocal and the millisecond DF curve resembles the first derivative of the PFt function.

Temperature Effects on Pea Plants Probed by Simultaneous Measurements of the Kinetics of Prompt Fluorescence, Delayed Fluorescence and Modulated 820 nm Reflection

Simultaneous in vivo measurements of prompt fluorescence (PF), delayed fluorescence (DF) and 820-nm reflection (MR) were made to probe response of pea leaves to 40 s incubation at high temperatures (25–50°C). We interpret our observation to suggest that heat treatment provokes an inhibition of electron donation by the oxygen evolving complex. DF, in a time range from several microseconds to milliseconds, has been thought to reflect recombination, in the dark, between the reduced primary electron acceptor QA – and the oxidized donor (P680+) of photosystem II (PSII). The lower electron transport rate through PSII after 45 and 50°C incubation also changed DF induction. We observed a decrease in the amplitude of the DF curve and a change in its shape and in its decay. Acceleration of P700+ and PC+ re-reduction was induced by 45°C treatment but after 50°C its reduction was slower, indicating inhibition of photosystem I. We suggest that simultaneous PF, MR and DF might provide useful information on assessing the degree of plant tolerance to different environmental stresses.

Preservation of photosynthetic electron transport from senescence-induced inactivation in primary leaves after decapitation and defoliation of bean plants.

The comparative effects of decapitation and defoliation on the senescence-induced inactivation of photosynthetic activity in primary leaves of bean plants were investigated. Decapitation was performed during different phases of bean plant ontogenesis, immediately after the appearance of the 1st, 2nd, 3rd and 4th composite leaf. In addition, we examined a variant with primary leaves and stem with an apical bud, but without composite leaves, i.e. defoliated plants. Analyses of chlorophyll fluorescence, millisecond delayed fluorescence and absorption at 830nm in primary leaves were undertaken to investigate the alterations in photosystems II and I electron transport during the decapitation-induced delayed senescence in the non-detached leaves. Analysis of the OKJIP transients using the JIP-test (see [Strasser R, Srivastava A, Tsimilli-Michael M. Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou G, Govindjee, editors. Chlorophyll a fluorescence: a signature of photosynthesis. The Netherlands: Kluwer Academic Publishers, 2004; pp. 321-362]) showed an increase in several biophysical parameters of photosystem II in decapitated plants, specifically, the density of active reaction centers on a chlorophyll basis, the yields of trapping and electron transport, and the performance index. We also observed a decrease in the absorbed light energy per reaction center. Such a decrease in light absorption could be a result of the photosystem II down regulation that appeared as an increase in Q(B)-non-reducing photosystem II centers. The effect was identical when all leaves except the primary leaves were removed. The variant with a preserved apical bud, the defoliated plant, showed values similar to those of decapitated plants with primary leaves only. The changes in the induction curves of the delayed fluorescence also indicated an acceleration of electron transport beyond photosystem II in the decapitated and in defoliated plants. In these plants, the photosystem I-driven electron transport was accelerated, and the size of the plastoquinone pool was enhanced. It was established that decapitation can retard the senescence of primary leaves, can expand leaf life span and can cause activation of both photosystems I and II electron transport. The decapitation procedure shows similarities to the process of defoliation. The overcompensation effect that is developed after defoliation could initially be manifested as an acceleration of the linear photosynthetic electron flow in the rest of the leaves.

Prompt chlorophyll a fluorescence as a rapid tool for diagnostic changes in PSII structure inhibited by salt stress in Perennial ryegrass.

Perennial ryegrass (Lolium perenne L.) is one of the most popular grass species in Europe. It is commonly used for establishing the lawns in urban areas, where the salt stress is one of the major environmental conditions limiting its growth. The basic aim of this study was the detailed in vivo analysis of the changes in photosynthetic efficiency, induced by salt stress, of two lawn varieties of Perennial ryegrass and to find out the variety of better properties to create lawn on the soils contaminated with salt. Two lawn varieties of L. perenne L. were used: Nira and Roadrunner. The salinization was applied 8 weeks after sowing by adding NaCl in water solution (0, 0.15, and 0.30 M). The measurements were carried out 8 times: 0, 24, 48, 96, 144, 192, 240 and 288 h after salinization. Our results revealed that the disturbance of PSII function could easily be estimated by measuring chlorophyll a fluorescence and analyzing that signal by JIP-test. Our work allowed to identify various limiting parameters of photosynthetic efficiency of perennial ryegrass lawn varieties grown under salt stress conditions. This knowledge can allow for selection of plants with a higher potential photosynthetic efficiency (vitality) during salt stress conditions, that can be used successfully neighboring roads, where salt is applied.

Variable chlorophyll fluorescence and its use for assessing physiological condition of plant photosynthetic apparatus

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.

Photosynthetic performance during leaf expansion in Malus micromalus probed by chlorophyll a fluorescence and modulated 820nm reflection.

The simultaneous measurements of prompt chlorophyll a fluorescence, delayed chlorophyll a fluorescence and modulated 820nm reflection allow collection and correlation of complementary information for the three domains of the photosynthetic electron transport chain - the PSII electron donor side, electron transport between PSII and PSI, and the PSI electron acceptor side. In this study, we used this approach to investigate photochemical activity during Malus micromalus leaf expansion. The results showed that as leaves expanded, the antenna size per reaction center for the two systems became smaller, and the energetic connectivity of PSII units decreased gradually. Meanwhile, the light trapping efficiency of PSII, electron transfer capacity at the donor side of PSII, exchange capacity of PQs at the QB site and the reoxidation capacity of PQH2 were all increased as leaves expanded. However, the capacity of PQH2 reoxidation increased at a slower rate than the exchange capacity of PQs at the QB site. In general, during leaf development, the photochemical activity of both PSII and PSI increased, although the increase in PSII activity was faster relative to PSI. The results from the three independent signals corroborate each other.

Chapter 15 – The Use of Chlorophyll Fluorescence Kinetics Analysis to Study the Performance of Photosynthetic Machinery in Plants

Plants under natural conditions experience unfavorable growth conditions. These can cause a reduction of their photosynthetic rate or even damage the photosynthetic apparatus, which can lead to a serious decrease of plant productivity and yield. Photosynthesis is sensitive to environmental limitations, which means that photosynthetic measurement is an important tool for plant stress studies. Nevertheless, classic methods, such as the measurement of photosynthetic rates through gas exchange (CO2, H2O, and O2), are time-consuming and give incomplete information about photosynthetic function. Thus, the introduction of methods based on chlorophyll a fluorescence has led to a significant breakthrough in photosynthesis research. In this chapter we discuss the wide range of chlorophyll a fluorescence applications to understand the response of the photosynthetic apparatus to various stress conditions. We concentrate on demonstrating the so-called “OJIP test” as a good tool to explore the response of photosystem II (PSII). In addition, we discuss the issue of PSII heterogeneity under unfavorable growth conditions.