Christiaan Sybesma (August 31, 1928-January 31, 2018), an extraordinary biophysicist of our time.

We provide here a brief Tribute to Christiaan Sybesma (1928-2018), a highly respected biophysicist of our time. We remember him by giving a brief highlight of his life and a glimpse of his outstanding contributions in photosynthesis. He was a charming and highly respected scientist of our time.

A simple routine for quantitative analysis of light and dark kinetics of photochemical and non-photochemical quenching of chlorophyll fluorescence in intact leaves.

Paper describes principles and application of a novel routine that enables the quantitative analysis of the photochemical O-J phase of the variable fluorescence F v associated with the reversible photo-reduction of the secondary electron acceptor QA of photosystem II (PSII) in algae and intact leaves. The kinetic parameters that determine the variable fluorescence F (PP)(t) associated with the release of photochemical quenching are estimated from 10 µs time-resolved light-on and light-off responses of F v induced by two subsequent light pulses of 0.25 (default) and 1000 ms duration, respectively. Application of these pulses allows estimations of (i) the actual value of the rate constants k L and k AB of the light excitation (photoreduction of QA) and of the dark re-oxidation of photoreduced QA ([Formula: see text]), respectively, (ii) the actual maximal normalized variable fluorescence [nF v] associated with 100 % photoreduction of QA of open RCs, and (iii) the actual size ß of RCs in which the re-oxidation of [Formula: see text] is largely suppressed (QB-nonreducing RC with k AB ~ 0). The rate constants of the dark reversion of Fv associated with the release of photo-electrochemical quenching F (PE) and photo-electric stimulation F (CET) in the successive J-I and I-P parts of the thermal phase are in the range of (100 ms)(-1) and (1 s)(-1), respectively. The kinetics of fluorescence changes during and after the I-P phase are given special attention in relation to the hypothesis on the involvement of a Δµ H+-dependent effect during this phase and thereafter. Paper closes with author's personal view on the demands that should be fulfilled for chlorophyll fluorescence methods being a correct and unchallenged signature of photosynthesis in algae and plants.

On the polyphasic quenching kinetics of chlorophyll a fluorescence in algae after light pulses of variable length.

This study reports on kinetics of the fluorescence decay in a suspension of the alga Scenedesmus quadricauda after actinic illumination. These are monitored as the variable fluorescence signal in the dark following light pulses of variable intensity and duration. The decay reflects the restoration of chlorophyll fluorescence quenching of the photosystem II (PSII) antennas and shows a polyphasic pattern which suggests the involvement of different processes. The overall quenching curve after a fluorescence-saturating pulse (SP) of 250-ms duration, commonly used in pulse amplitude modulation applications as the tool for estimating the maximal fluorescence (F m), has been termed P-O, in which P and O have the same meaning as used in the OJIP induction curve in the light. Deconvolution of this signal shows at least three distinguishable exponential phases with reciprocal rate constants of the order of 10, 10(2), and 10(3) ms. The size of the long (>10(3) ms) and moderate (~10(2) ms) lasting components relative to the complete quenching signal after an SP increases with the duration of the actinic pulse concomitantly with an increase in the reciprocal rate constants of the fast (~10 ms) and moderate quenching phases. Fluorescence responses upon single turnover flashes of 30-µs duration (STFs) given at discrete times during the P-O quenching were used as tools for identifying the quencher involved in the P-O quenching phase preceding the STF excitation. Results are difficult to interpret in terms of a single-hit two-state trapping mechanism with distinguishable quenching properties of open and closed reaction centers only. They give support for an earlier hypothesis on a double-hit three-state trapping mechanism in which the so-called semi-closed reaction centers of PSII are considered. In these trapping-competent centers the single reduced acceptor pair [PheQ A](1-), depending on the size of photoelectrochemically induced pH effects on the Q B-binding site, functions as an efficient fluorescence quencher.

Chlorophyll a fluorescence induction (Kautsky curve) in a Venus flytrap (Dionaea muscipula) leaf after mechanical trigger hair irritation.

This paper describes experiments on transient changes in chlorophyll a fluorescence in traps of the carnivorous plant Venus flytrap (Dionaea muscipula) that occur in association with mechanical stimulation of trigger hairs and propagation of action potentials (APs). The experiments show the following reproducible effects of APs on the fluorescence induction (Kautsky-, or OJIPSMT curve) in a 100 s low intensity light pulse (i) no change in the OJ phase attributed to release of photochemical quenching, (ii) a small enhancement, if at all of increase in the thermal JIP phase, (iii) a two- to threefold deceleration of the fluorescence decline (quenching) during the PSMT phase in the 2-100 s time range, and (iv) a transient 15-50% increase in variable fluorescence within ~20 s under steady state light condition with, after ~80 s, a 10% undershoot that reverses in several tens of seconds to the original steady state. The results are discussed in terms of a hypothesis that the fluorescence decline during the SMT phase of the Kautsky induction curve, attributed to NPQ, is caused by the Δµ(H+)-driven increase in proton conductance of the CF(o) channel of the ATPase during its activation. A signal-transducing role of Ca(2+) is suggested.

Effects of far-red light on fluorescence induction in infiltrated pea leaves under diminished ΔpH and Δφ components of the proton motive force.

Chlorophyll fluorescence induction curves induced by an actinic pulse of red light follow different kinetics in dark-adapted plant leaves and leaves preilluminated with far-red light. This influence of far-red light was abolished in leaves infiltrated with valinomycin known to eliminate the electrical (Δφ) component of the proton-motive force and was strongly enhanced in leaves infiltrated with nigericin that abolishes the ΔpH component. The supposed influence of ionophores on different components of the proton motive force was supported by differential effects of these ionophores on the induction curves of the millisecond component of chlorophyll delayed fluorescence. Comparison of fluorescence induction curves with the kinetics of P700 oxidation in the absence and presence of ionophores suggests that valinomycin facilitates a build-up of a rate-limiting step for electron transport at the site of plastoquinone oxidation, whereas nigericin effectively removes limitations at this site. Far-red light was found to be a particularly effective modulator of electron flows in chloroplasts in the absence of ΔpH backpressure on operation of the electron-transport chain.

Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes.

BACKGROUND: The thylakoid system in plant chloroplasts is organized into two distinct domains: grana arranged in stacks of appressed membranes and non-appressed membranes consisting of stroma thylakoids and margins of granal stacks. It is argued that the reason for the development of appressed membranes in plants is that their photosynthetic apparatus need to cope with and survive ever-changing environmental conditions. It is not known however, why different plant species have different arrangements of grana within their chloroplasts. It is important to elucidate whether a different arrangement and distribution of appressed and non-appressed thylakoids in chloroplasts are linked with different qualitative and/or quantitative organization of chlorophyll-protein (CP) complexes in the thylakoid membranes and whether this arrangement influences the photosynthetic efficiency. RESULTS: Our results from TEM and in situ CLSM strongly indicate the existence of different arrangements of pea and bean thylakoid membranes. In pea, larger appressed thylakoids are regularly arranged within chloroplasts as uniformly distributed red fluorescent bodies, while irregular appressed thylakoid membranes within bean chloroplasts correspond to smaller and less distinguished fluorescent areas in CLSM images. 3D models of pea chloroplasts show a distinct spatial separation of stacked thylakoids from stromal spaces whereas spatial division of stroma and thylakoid areas in bean chloroplasts are more complex. Structural differences influenced the PSII photochemistry, however without significant changes in photosynthetic efficiency. Qualitative and quantitative analysis of chlorophyll-protein complexes as well as spectroscopic investigations indicated a similar proportion between PSI and PSII core complexes in pea and bean thylakoids, but higher abundance of LHCII antenna in pea ones. Furthermore, distinct differences in size and arrangements of LHCII-PSII and LHCI-PSI supercomplexes between species are suggested. CONCLUSIONS: Based on proteomic and spectroscopic investigations we postulate that the differences in the chloroplast structure between the analyzed species are a consequence of quantitative proportions between the individual CP complexes and its arrangement inside membranes. Such a structure of membranes induced the formation of large stacked domains in pea, or smaller heterogeneous regions in bean thylakoids. Presented 3D models of chloroplasts showed that stacked areas are noticeably irregular with variable thickness, merging with each other and not always parallel to each other.

The analysis of PS II photochemical activity using single and multi-turnover excitations.

Paper describes chlorophyll a fluorescence measurements in algal cells, and intact plant leaves and isolated chloroplasts. It focuses on amplitude and 10 µs-resolved kinetics of variable fluorescence responses upon excitation with fluorescence-saturating pulses (SP) and with 25 µs saturating single turnover flashes (STF) which are exposed before, during and after a 100 s actinic illumination (AL) of low and high intensity. In addition to the amply documented suppression of the maximal variable fluorescence from F(m) to F(m)('), the relative proportion of the distinguished O-J-, J-I- and I-P-phases of an SP-induced response is shown to be distinctly different in dark- and light-adapted leaves. The O-J-phase in the 0.01-1 ms time range is much less sensitive to light adaptation than the other phases in the 1-200 ms range. In algae and chloroplasts, the amplitude F(m)(STF) of the STF-induced response is hardly affected by a shift from the dark- to the light-activated steady state. The results support the hypothesis that the maximal variable fluorescence F(m) induced by a multiple-turnover, fluorescence-saturating pulse (SP), is associated with the release of photochemical and photoelectrochemical quenching. It is argued that the OJIPMT- or Kautsky induction curve of variable chlorophyll fluorescence in the 0-100 s time range is the reflection of the release of photochemical quenching supplemented with a temporary Photosystem I (PSI)-dependent photoelectric stimulation and transient release of photoelectrochemical quenching of radiative energy loss in the Photosystem II (PSII) antennas, rather than solely of a decrease in PSII photochemical activity as is usually concluded.

Adaptation of photosystem II to high and low light in wild-type and triazine-resistant Canola plants: analysis by a fluorescence induction algorithm.

Plants of wild-type and triazine-resistant Canola (Brassica napus L.) were exposed to very high light intensities and after 1 day placed on a laboratory table at low light to recover, to study the kinetics of variable fluorescence after light, and after dark-adaptation. This cycle was repeated several times. The fast OJIP fluorescence rise curve was measured immediately after light exposure and after recovery during 1 day in laboratory room light. A fluorescence induction algorithm has been used for resolution and analysis of these curves. This algorithm includes photochemical and photo-electrochemical quenching release components and a photo-electrical dependent IP-component. The analysis revealed a substantial suppression of the photo-electrochemical component (even complete in the resistant biotype), a partial suppression of the photochemical component and a decrease in the fluorescence parameter F (o) after high light. These effects were recovered after 1 day in the indoor light.

Kinetic analyses and mathematical modeling of primary photochemical and photoelectrochemical processes in plant photosystems.

In this paper the model and simulation of primary photochemical and photo-electrochemical reactions in dark-adapted intact plant leaves is presented. A descriptive algorithm has been derived from analyses of variable chlorophyll a fluorescence and P700 oxidation kinetics upon excitation with multi-turnover pulses (MTFs) of variable intensity and duration. These analyses have led to definition and formulation of rate equations that describe the sequence of primary linear electron transfer (LET) steps in photosystem II (PSII) and of cyclic electron transport (CET) in PSI. The model considers heterogeneity in PSII reaction centers (RCs) associated with the S-states of the OEC and incorporates in a dark-adapted state the presence of a 15-35% fraction of Q(B)-nonreducing RCs that probably is identical with the S0 fraction. The fluorescence induction algorithm (FIA) in the 10 µs-1s excitation time range considers a photochemical O-J-D, a photo-electrochemical J-I and an I-P phase reflecting the response of the variable fluorescence to the electric trans-thylakoid potential generated by the proton pump fuelled by CET in PSI. The photochemical phase incorporates the kinetics associated with the double reduction of the acceptor pair of pheophytin (Phe) and plastoquinone Q(A) [PheQ(A)] in Q(B) nonreducing RCs and the associated doubling of the variable fluorescence, in agreement with the three-state trapping model (TSTM) of PS II. The decline in fluorescence emission during the so called SMT in the 1-100s excitation time range, known as the Kautsky curve, is shown to be associated with a substantial decrease of CET-powered proton efflux from the stroma into the chloroplast lumen through the ATPsynthase of the photosynthetic machinery.

Photoelectrochemical control of the balance between cyclic- and linear electron transport in photosystem I. Algorithm for P700+ induction kinetics.

Redox transients of chlorophyll P700, monitored as absorbance changes DeltaA810, were measured during and after exclusive PSI excitation with far-red (FR) light in pea (Pisum sativum, cv. Premium) leaves under various pre-excitation conditions. Prolonged adaptation in the dark terminated by a short PSII+PSI- exciting light pulse guarantees pre-conditions in which the initial photochemical events in PSI RCs are carried out by cyclic electron transfer (CET). Pre-excitation with one or more 10s FR pulses creates conditions for induction of linear electron transport (LET). These converse conditions give rise to totally different, but reproducible responses of P700- oxidation. System analyses of these responses were made based on quantitative solutions of the rate equations dictated by the associated reaction scheme for each of the relevant conditions. These provide the mathematical elements of the P700 induction algorithm (PIA) with which the distinguishable components of the P700+ response can be resolved and interpreted. It enables amongst others the interpretation and understanding of the characteristic kinetic profile of the P700+ response in intact leaves upon 10s illumination with far-red light under the promotive condition for CET. The system analysis provides evidence that this unique kinetic pattern with a non-responsive delay followed by a steep S-shaped signal increase is caused by a photoelectrochemically controlled suppression of the electron transport from Fd to the PQ-reducing Qr site of the cytb6f complex in the cyclic pathway. The photoelectrochemical control is exerted by the PSI-powered proton pump associated with CET. It shows strong similarities with the photoelectrochemical control of LET at the acceptor side of PSII which is reflected by release of photoelectrochemical quenching of chlorophyll a fluorescence.

Kinetic models of photosystem II should accommodate the effect of donor side quenching on variable chlorophyll A fluorescence in the microseconds time range.

Quantitative data on laser flash-induced variable fluorescence in the 100 ns to 1 ms time range (Belyaeva et al. in Photosynth Res 98:105-119, 2008) confirming those of others (Steffen et al. in Biochemistry 40:173-180, 2001, Biochemistry 44:3123-3132, 2005; Belyaeva et al. in Biophysics 51(6):976-990, 2006), need a substantial correction with respect to magnitude of the normalized variable fluorescence associated with single turnover-induced charge separation in RCs of PS II. Their data are conclusive with the involvement of donor side quenching, the release of which occurs with a rate constant in the range of tens of ms(-1), and presumed to be associated with reduction of Y(+)(z) by the OEC.

Photochemical and photoelectrochemical quenching of chlorophyll fluorescence in photosystem II.

This paper deals with kinetics and properties of variable fluorescence in leaves and thylakoids upon excitation with low intensity multi-turnover actinic light pulses corresponding with an excitation rate of about 10 Hz. These show a relatively small and amply documented rise in the sub-s time range towards the plateau level F(pl) followed by a delayed and S-shaped rise towards a steady state level F(m) which is between three and four fold the initial dark fluorescence level F(o). Properties of this retarded slow rise are i) rate of dark recovery is (1-6 s)(-1), ii) suppression by low concentration of protonophores, iii) responsiveness to complementary single turnover flash excitation with transient amplitude towards a level F(m) which is between five and six fold the initial dark fluorescence level F(o) and iv) in harmony with and quantitatively interpretable in terms of a release of photoelectrochemical quenching controlled by the trans-thylakoid proton pump powered by the light-driven Q cycle. Data show evidence for a sizeable fluorescence increase upon release of (photo) electrochemical quenching, defined as qPE. Release of qPE occurs independent of photochemical quenching defined here as qPP even under conditions at which qPP = 1. The term photochemical quenching, hitherto symbolized by qP, will require a new definition, because it incorporates in its present form a sizeable photoelectrochemical component. The same is likely to be true for definition and use of qN as an indicator of non photochemical quenching.

Higher concentration of Q(B)-nonreducing photosystem II centers in triazine-resistant Chenopodium album plants as revealed by analysis of chlorophyll fluorescence kinetics.

Plants resistant to triazine-type herbicides are known to be altered in their photosystem II reaction center. Serine at site 264 in D1 protein is replaced by glycine. The measurements of chlorophyll a fluorescence excitations with a variable number of saturating flashes in Chenopodium album plants show characteristic differences between the resistant and the wild-type plants. These differences appear in response to the first flash as well as in the rise pattern of subsequent flashes of a 12.5 Hz flash train. The differences indicate a higher concentration of Q(B)-nonreducing reaction centers in the resistant biotype, and confirm earlier results on a slower rate of electron transport between the primary and secondary electron acceptors.

Algorithm for analysis of OJDIP fluorescence induction curves in terms of photo- and electrochemical events in photosystems of plant cells: derivation and application.

The algorithm for simulation of the OJDIP fluorescence induction curve in chloroplasts under variable conditions is presented. It is derived from analyzes of chlorophyll a fluorescence kinetics upon excitation with single- (STF), twin- (TTF) and repetitive STF excitations, and from the rate equations that describe the sequence of transfer steps associated with the reduction of the primary quinone acceptor Q(A) and the release of photochemical fluorescence quenching of photosystem II (PSII) in multi-turnover excitation (MTF). The fluorescence induction algorithm (FIA) considers a photochemical O-J-D, a photo-electrochemical J-I and an I-P component (phase) which probably is associated with a photo-electric interaction between PSI and PSII. The photochemical phase incorporates the kinetics associated with the double reduction of the acceptor pair [PheQ(A)] in Q(B)-nonreducing reaction centers (RCs) and the associated doubling of the variable fluorescence, in agreement with the three-state trapping model (TSTM) of PSII. Application of and results with the algorithm are illustrated for MTF-induced OJDIP curves, measured in dark-adapted, in STF pre-excited and in DCMU inhibited thylakoids.

Analysis of initial chlorophyll fluorescence induction kinetics in chloroplasts in terms of rate constants of donor side quenching release and electron trapping in photosystem II.

The fluorescence induction F(t) of dark-adapted chloroplasts has been studied in multi-turnover 1 s light flashes (MTFs). A theoretical expression for the initial fluorescence rise is derived from a set of rate equations that describes the sequence of transfer steps associated with the reduction of the primary quinone acceptor Q (A) and the release of photochemical fluorescence quenching of photosystem II (PSII). The initial F(t) rise in the hundreds of mus time range is shown to follow the theoretical function dictated by the rate constants of light excitation (k (L)) and release of donor side quenching (k ( si )). The bi-exponential function shows sigmoidicity when one of the two rate constants differs by less than one order of magnitude from the other. It is shown, in agreement with the theory, that the sigmoidicity of the fluorescence rise is variable with light intensity and mainly, if not exclusively, determined by the ratio between rate of light excitation and the rate constant of donor side quenching release.

Contrasting effect of dark-chilling on chloroplast structure and arrangement of chlorophyll-protein complexes in pea and tomato: plants with a different susceptibility to non-freezing temperature.

The effect of dark-chilling and subsequent photoactivation on chloroplast structure and arrangements of chlorophyll-protein complexes in thylakoid membranes was studied in chilling-tolerant (CT) pea and in chilling-sensitive (CS) tomato. Dark-chilling did not influence chlorophyll content and Chl a/b ratio in thylakoids of both species. A decline of Chl a fluorescence intensity and an increase of the ratio of fluorescence intensities of PSI and PSII at 120 K was observed after dark-chilling in thylakoids isolated from tomato, but not from pea leaves. Chilling of pea leaves induced an increase of the relative contribution of LHCII and PSII fluorescence. A substantial decrease of the LHCII/PSII fluorescence accompanied by an increase of that from LHCI/PSI was observed in thylakoids from chilled tomato leaves; both were attenuated by photoactivation. Chlorophyll fluorescence of bright grana discs in chloroplasts from dark-chilled leaves, detected by confocal laser scanning microscopy, was more condensed in pea but significantly dispersed in tomato, compared with control samples. The chloroplast images from transmission-electron microscopy revealed that dark-chilling induced an increase of the degree of grana stacking only in pea chloroplasts. Analyses of O-J-D-I-P fluorescence induction curves in leaves of CS tomato before and after recovery from chilling indicate changes in electron transport rates at acceptor- and donor side of PS II and an increase in antenna size. In CT pea leaves these effects were absent, except for a small but irreversible effect on PSII activity and antenna size. Thus, the differences in chloroplast structure between CS and CT plants, induced by dark-chilling are a consequence of different thylakoid supercomplexes rearrangements.

Time sequence of the damage to the acceptor and donor sides of photosystem II by UV-B radiation as evaluated by chlorophyll a fluorescence.

The effects of ultraviolet-B (UV-B) radiation on photosystem II (PS II) were studied in leaves of Chenopodium album. After the treatment with UV-B the damage was estimated using chlorophyll a fluorescence techniques. Measurements of modulated fluorescence using a pulse amplitude modulated fluorometer revealed that the efficiency of photosystem II decreased both with increasing time of UV-B radiation and with increasing intensity of the UV-B. Fluorescence induction rise curves were analyzed using a mechanistic model of energy trapping. It appears that the damage by UV-B radiation occurs first at the acceptor side of photosystem II, and only later at the donor side.

On the chlorophyll a fluorescence yield in chloroplasts upon excitation with twin turnover flashes (TTF) and high frequency flash trains.

Chlorophyll fluorescence is routinely taken as a quantifiable measure of the redox state of the primary quinone acceptor Q(A) of PSII. The variable fluorescence in thylakoids increases in a single turnover flash (STF) from its low dark level F (o) towards a maximum F (m) (STF) when Q(A) becomes reduced. We found, using twin single turnover flashes (TTFs) that the fluorescence increase induced by the first twin-partner is followed by a 20-30% increase when the second partner is applied within 20-100 micros after the first one. The amplitude of the twin response shows a period-of-four oscillation associated with the 4-step oxidation of water in the Kok cycle (S states) and originates from two different trapped states with a life time of 0.2-0.4 and 2-5 ms, respectively. The oscillation is supplemented with a binary oscillation associated with the two-electron gate mechanism at the PSII acceptor side. The F(t) response in high frequency flash trains (1-4 kHz) shows (i) in the first 3-4 flashes a transient overshoot 20-30% above the F (m) (STF) = 3*F (o) level reached in the 1st flash with a partial decline towards a dip D in the next 2-3 ms, independent of the flash frequency, and (ii) a frequency independent rise to F (m) = 5*F (o) in the 3-60 ms time range. The initial overshoot is interpreted to be due to electron trapping in the S(0) fraction with Q(B)-nonreducing centers and the dip to the subsequent recovery accompanying the reoxidation of the double reduced acceptor pair in these RCs after trapping. The rise after the overshoot is, in agreement with earlier findings, interpreted to indicate a photo-electrochemical control of the chlorophyll fluorescence yield of PSII. It is anticipated that the double exciton and electron trapping property of PSII is advantageous for the plant. It serves to alleviate the depression of electron transport in single reduced Q(B)-nonreducing RCs, associated with electrochemically coupled proton transport, by an increased electron trapping efficiency in these centers.

The chlorophyll a fluorescence induction pattern in chloroplasts upon repetitive single turnover excitations: accumulation and function of QB-nonreducing centers.

The increase of chlorophyll fluorescence yield in chloroplasts in a 12.5 Hz train of saturating single turnover flashes and the kinetics of fluorescence yield decay after the last flash have been analyzed. The approximate twofold increase in Fm relative to Fo, reached after 30-40 flashes, is associated with a proportional change in the slow (1-20 s) component of the multiphasic decay. This component reflects the accumulation of a sizeable fraction of QB-nonreducing centers. It is hypothesized that the generation of these centers occurs in association with proton transport across the thylakoid membrane. The data are quantitatively consistent with a model in which the fluorescence quenching of QB-nonreducing centers is reversibly released after second excitation and electron trapping on the acceptor side of Photosystem II.