Excitation Energy In Photosystem Research Articles

Implementation of theoretical non-photochemical quenching (NPQ(T)) to investigate NPQ of chickpea under drought stress with High-throughput Phenotyping

Non-photochemical quenching (NPQ) is a protective mechanism for dissipating excess energy generated during photosynthesis in the form of heat. The accelerated relaxation of the NPQ in fluctuating light can lead to an increase in the yield and dry matter productivity of crops. Since the measurement of NPQ is time-consuming and requires specific light conditions, theoretical NPQ (NPQ(T)) was introduced for rapid estimation, which could be suitable for High-throughput Phenotyping. We investigated the potential of NPQ(T) to be used for testing plant genetic resources of chickpea under drought stress with non-invasive High-throughput Phenotyping complemented with yield traits. Besides a high correlation between the hundred-seed-weight and the Estimated Biovolume, significant differences were observed between the two types of chickpea desi and kabuli for Estimated Biovolume and NPQ(T). Desi was able to maintain the Estimated Biovolume significantly better under drought stress. One reason could be the effective dissipation of excess excitation energy in photosystem II, which can be efficiently measured as NPQ(T). Screening of plant genetic resources for photosynthetic performance could take pre-breeding to a higher level and can be implemented in a variety of studies, such as here with drought stress or under fluctuating light in a High-throughput Phenotyping manner using NPQ(T).

Melatonin alleviates photoinhibition in cucumber seedlings by modulating partitioning of absorbed excitation energy in photosystem Ⅱ

The aim of this study was to evaluate the effects of melatonin on photoinhibition under chilling stress in cucumber seedlings and to inquire into any mechanisms of mitigation. Under chilling stress, the net photosynthetic rate declined dramatically but the decline was significantly mitigated by irrigation with a melatonin solution. Possible mechanisms for this mitigation are that melatonin accelerates xanthophyll de-epoxidation by upregulating the transcription of the violaxanthin de-epoxidase gene (CsVDE) and down-regulating that of the zeaxanthin cyclase gene (CsZE) during chilling. There was also a rise in non-photochemical quenching (NPQ) if seedlings were pretreated with melatonin before chilling. The efficient operation of the xanthophyll cycle helped consume excessive excitation energy in photosystem (PS) Ⅱ and so protected the photosynthetic system. Melatonin also modulated the partitioning of absorbed excitation energy in PS Ⅱ as evidenced by alleviation of the decrease in quantum yield of photochemical energy conversion in PS Ⅱ under chilling stress, by alleviation of the rise in quantum yield of non-regulated, non-photochemical energy loss in PS II and by increasing the regulated non-photochemical energy loss in PS II. This study presents a new understanding of the mechanisms through which melatonin mitigates photoinhibition by modulating the partitioning of absorption energy in PS Ⅱ based on the xanthophyll cycle.

Chlorophyll fluorescence parameters to assess utilization of excitation energy in photosystem II independently of changes in leaf absorption

Measurement of Pulse-Amplitude-Modulated (PAM) chlorophyll a fluorescence is widely used method for obtaining information on the functional state of photosystem II (PSII). Recently, it has been shown that some of long-established fluorescence parameters must be interpreted with caution, when the light-induced chloroplast movements occur. In our work we have analyzed the effect of chloroplast movements on these parameters. We have derived new parameters that are independent of the change in PSII absorption occurring during measurement. To verify whether there is a need for new parameters or the difference between the parameters commonly used and the newly derived ones is insignificant, we conducted an experiment with Arabidopsis thaliana wild type plants and its phot1 phot2 mutant defective in chloroplast movement. Plants were exposed to light of different qualities (450, 470, 550 or 660 nm) and quantities (100, 400 or 1200 μmol m−2 s−1) for up to 40 min. Since the blue light-induced chloroplast avoidance reaction is a photoprotective mechanism, we expected that phot1 phot2 mutant will compensate the lack of this mechanism by increasing non-photochemical quenching. However, using the light at both 450 and 470 nm, the calculation of commonly used parameter, ΦNPQ (quantum yield of regulated light-induced thermal energy dissipation in PSII) based on Hendrickson et al. [L. Hendrickson, R.T. Furbank, W.S. Chow, Photosynth. Res. 82 (2004) 73–81] showed the opposite. On the other hand, the results obtained using our newly proposed formulae to determine quantum yield of PSII thermal energy dissipation were in line with our assumption. Thus, the experimental data showed that some formulae of fluorescence parameters are dependent on the change in PSII absorption and need to be interpreted carefully. On the contrary, the formulae introduced by us can remove the effect of changes in PSII absorption that occur during measurement, without additional measurements, and give the real estimate of light-induced non-photochemical quenching.

Assessment of the impact of photosystem I chlorophyll fluorescence on the pulse-amplitude modulated quenching analysis in leaves of Arabidopsis thaliana.

In their natural environment, plants are exposed to varying light conditions, which can lead to a build-up of excitation energy in photosystem (PS) II. Non-photochemical quenching (NPQ) is the primary defence mechanism employed to dissipate this excess energy. Recently, we developed a fluorescence-quenching analysis procedure that enables the protective effectiveness of NPQ in intact Arabidopsis leaves to be determined. However, pulse-amplitude modulation measurements do not currently allow distinguishing between PSII and PSI fluorescence levels. Failure to account for PSI contribution is suggested to lead to inaccurate measurements of NPQ and, particularly, maximum PSII yield (F v/F m). Recently, Pfündel et al. (Photosynth Res 114:189-206, 2013) proposed a method that takes into account PSI contribution in the measurements of F o fluorescence level. However, when PSI contribution was assumed to be constant throughout the induction of NPQ, we observed lower values of the measured minimum fluorescence level ([Formula: see text]) than those calculated according to the formula of Oxborough and Baker (Photosynth Res 54:135-142 1997) ([Formula: see text]), regardless of the light intensity. Therefore, in this work, we propose a refined model to correct for the presence of PSI fluorescence, which takes into account the previously observed NPQ in PSI. This method efficiently resolves the discrepancies between measured and calculated F o' produced by assuming a constant PSI fluorescence contribution, whilst allowing for the correction of the maximum PSII yield.

Collective Calculation of Actual Values of Non-Photochemical Quenching from Their Apparent Values after Chloroplast Movement and Photoinhibition

Chlorophyll fluorescence parameters such as Fv/Fm, NPQ and ΦII (YII) are widely used to estimate the fitness and photosynthetic activity of plant leaves and non-photochemical dissipation of excessive excitation energy in photosystem II. The effect of chloroplast movement on these fluorescence parameters reduces the accuracy of estimations of the size of de-excitation processes, but there is no method to calculate correct parameters from altered (fluctuated) parameters. Chloroplast movement was recently identified as the “middle” kinetic component of NPQ. In this paper, we devised a complex but reasonable mathematical method to remove the effect of chloroplast movement on fluorescence parameters, based on our previously reported fluorescence theory. The fraction of “S fluctuation” (designated as σ) was estimated from fluorescence observations and used to calculate the non-fluctuated Fs and F′m. fluorescence yields. From the σ values, the fractional change of light absorbance by a leaf caused by chloroplast movement was estimated at 70% - 100%, which varied according to the experimental conditions and plant species. The effect of photoinhibition on fluorescence parameters was also examined in this paper. The photochemical and non-photochemical de-excitation sizes during photoinhibition (measured by the parameters qPI and qSlow) changed on a single regression line. Using this correlation, qPI and qSlow can be predicted from Fv/Fm, and the non-fluctuated Fm and Fo values can be estimated from the fluctuated F″m and F″o values.

Circumvention of Over‐Excitation of PSII by Maintaining Electron Transport Rate in Leaves of Four Cotton Genotypes Developed under Long‐Term Drought

We investigated the patterns of response to a long-term drought in the field in cotton cultivars (genotypes) with known differences in their drought tolerance. Four cotton genotypes with varying physiological and morphological traits, suited to different cropping conditions, were grown in the field and subjected to a long-term moderate drought. In general, cotton leaves developed under drought had significantly higher area-based leaf nitrogen content (N (area)) than those under well irrigation. Droughted plants showed a lower light-saturated net photosynthetic rate (A (sat)) with lower stomatal conductance (g (s)) and intercellular CO (2) concentration (C (i)) than irrigated ones. Based on the responses of A (sat) to g (s) and C (i), there was no decreasing trend in A (sat) at a given g (s) and C (i) in droughted leaves, suggesting that the decline in A (sat) in field-grown cotton plants under a long-term drought can be attributed mainly to stomatal closure, but not to nonstomatal limitations. There was little evidence of an increase in thermal energy dissipation as indicated by the lack of a decrease in the photochemical efficiency of open PSII (F (v)'/F (m)') in droughted plants. On the basis of electron transport (ETR) and photochemical quenching (q (P)), however, we found evidence indicating that droughted cotton plants can circumvent the risk of excessive excitation energy in photosystem (PS) II by maintaining higher electron transport rates associated with higher N (area), even while photosynthetic rates were reduced by stomatal closure.

Elevated CO2 and limited nitrogen nutrition can restrict excitation energy dissipation in photosystem II of Japanese white birch (Betula platyphylla var. japonica) leaves

Elevated atmospheric CO2 concentration [CO2] and different levels of nitrogen (N) nutrition can influence the amount of excess excitation energy in photosystem (PS) II and related photosynthetic properties. The interactive effect of two [CO2] levels (ambient: 360 µM M−1 and elevated: 720 µM M−1) and two N levels (high: 700 mg N plant−1 and low: 100 mg N plant−1) on these properties was examined in seedlings of Japanese white birch (Betula platyphylla var. japonica) using simultaneous measurements of gas exchange and chlorophyll fluorescence. Photosynthetic acclimation to elevated [CO2], as indicated by a decline in carboxylation efficiency (CE), was observed in plants grown at elevated [CO2] especially under low N. Elevated [CO2] resulted in a decrease in area‐based leaf N content (Narea) irrespective of N treatment. The adverse effect of elevated [CO2] and low N on CE may have been exacerbated by a greater accumulation of leaf sugar and starch contents in these plants leading to a lower electron transport rate (ETR). While these plants also showed higher non‐photochemical quenching (NqP) that could offset the reduction in energy dissipation through ETR to some extent, they still have a higher risk of photoinhibition from excessive excitation energy in PSII as indicated by a decrease in photochemical quenching (qP). However, chronic photoinhibition was not observed in plant grown at elevated [CO2] and low N because they showed no difference in Fv/Fm (the maximum photochemical efficiency of PSII) from those grown at ambient [CO2] and low N after an overnight dark adaptation. High levels of NqP in plants grown at elevated [CO2] and low N reflect a near saturation of thermal energy dissipation. This impaired capacity of photoprotection would render these plants more vulnerable to photoinhibition in the event of additional environmental stresses such as drought, low or high temperature.

Pathways for Energy Transfer in the Core Light-Harvesting Complexes CP43 and CP47 of Photosystem II

The pigment-protein complexes CP43 and CP47 transfer excitation energy from the peripheral antenna of photosystem II toward the photochemical reaction center. We measured the excitation dynamics of the chlorophylls in isolated CP43 and CP47 complexes at 77 K by time-resolved absorbance-difference and fluorescence spectroscopy. The spectral relaxation appeared to occur with rates of 0.2–0.4 ps and 2–3 ps in both complexes, whereas an additional relaxation of 17 ps was observed only in CP47. Using the 3.8-Å crystal structure of the photosystem II core complex from Synechococcus elongatus (A. Zouni, H.-T. Witt, J. Kern, P. Fromme, N. Krauss, W. Saenger, and P. Orth, 2001, Nature, 409:739–743), excitation energy transfer kinetics were calculated and a Monte Carlo simulation of the absorption spectra was performed. In both complexes, the rate of 0.2–0.4 ps can be ascribed to excitation energy transfer within a layer of chlorophylls near the stromal side of the membrane, and the slower 2–3-ps process to excitation energy transfer to the calculated lowest excitonic state. We conclude that excitation energy transfer within CP43 and CP47 is fast and does not contribute significantly to the well-known slow trapping of excitation energy in photosystem II.

Decay Kinetics and Quantum Yields of Fluorescence in Photosystem I from Synechococcus elongatus with P700 in the Reduced and Oxidized State: Are the Kinetics of Excited State Decay Trap-Limited or Transfer-Limited?

Transfer and trapping of excitation energy in photosystem I (PS I) trimers isolated from Synechococcus elongatus have been studied by an approach combining fluorescence induction experiments with picosecond time-resolved fluorescence measurements, both at room temperature (RT) and at low temperature (5 K). Special attention was paid to the influence of the oxidation state of the primary electron donor P700. A fluorescence induction effect has been observed, showing a ∼12% increase in fluorescence quantum yield upon P700 oxidation at RT, whereas at temperatures below 160 K oxidation of P700 leads to a decrease in fluorescence quantum yield (∼50% at 5 K). The fluorescence quantum yield for open PS I (with P700 reduced) at 5 K is increased by ∼20-fold and that for closed PS I (with P700 oxidized) is increased by ∼10-fold, as compared to RT. Picosecond fluorescence decay kinetics at RT reveal a difference in lifetime of the main decay component: 34 ± 1 ps for open PS I and 37 ± 1 ps for closed PS I. At 5 K the fluorescence yield is mainly associated with long-lived components (lifetimes of 401 ps and 1.5 ns in closed PS I and of 377 ps, 1.3 ns, and 4.1 ns in samples containing ∼50% open and 50% closed PS I). The spectra associated with energy transfer and the steady-state emission spectra suggest that the excitation energy is not completely thermally equilibrated over the core-antenna-RC complex before being trapped. Structure-based modeling indicates that the so-called red antenna pigments (A708 and A720, i.e., those with absorption maxima at 708 nm and 720 nm, respectively) play a decisive role in the observed fluorescence kinetics. The A720 are preferentially located at the periphery of the PS I core-antenna-RC complex; the A708 must essentially connect the A720 to the reaction center. The excited-state decay kinetics turn out to be neither purely trap limited nor purely transfer (to the trap) limited, but seem to be rather balanced.

Nonphotochemical quenching of excitation energy in photosystem II. A picosecond time-resolved study of the low yield of chlorophyll a fluorescence induced by single-turnover flash in isolated spinach thylakoids.

Chlorophyll a fluorescence emission is widely used as a noninvasive measure of a number of parameters related to photosynthetic efficiency in oxygenic photosynthetic organisms. The most important component for the estimation of photochemistry is the relative increase in fluorescence yield between dark-adapted samples which have a maximal capacity for photochemistry and a minimal fluorescence yield (F0) and light-saturated samples where photochemistry is saturated and fluorescence yield is maximal (Fm). However, when photosynthesis is saturated with a short (less than 50 micro(s)) flash of light, which induces only one photochemical turnover of photosystem II, the maximal fluorescence yield is significantly lower (Fsat) than when saturation is achieved with a millisecond duration multiturnover flash (Fm). To investigate the origins of the difference in fluorescence yield between these two conditions, our time-resolved fluorescence apparatus was modified to allow collection of picosecond time-resolved decay kinetics over a short time window immediately following a saturating single-turnover flash (Fsat) as well as after a multiturnover saturating pulse (Fm). Our data were analyzed with a global kinetic model based on an exciton radical pair equilibrium model for photosystem II. The difference between Fm and Fsat was modeled well by changing only the rate constant for quenching of excitation energy in the antenna of photosystem II. An antenna-based origin for the quenching was verified experimentally by the observation that addition of the antenna quencher 5-hydroxy-1,4-naphthoquinone to thylakoids under Fm conditions resulted in decay kinetics and modeled kinetic parameters very similar to those observed under Fsat conditions in the absence of added quinone. Our data strongly support the origin of low fluorescence yield at Fsat to be an antenna-based nonphotochemical quenching of excitation energy in photosystem II which has not usually been considered explicitly in calculations of photochemical and nonphotochemical quenching parameters. The implications of our data with respect to kinetic models for the excited-state dynamics of photosystem II and the practical applications of the fluorescence yield parameters Fm and Fsat to calculations of photochemical yield are discussed.

Xanthophyll cycle-dependent quenching of photosystem II chlorophyll a fluorescence: formation of a quenching complex with a short fluorescence lifetime.

Excess light triggers protective nonradiative dissipation of excitation energy in photosystem II through the formation of a trans-thylakoid pH gradient that in turn stimulates formation of zeaxanthin and antheraxanthin. These xanthophylls when combined with protonation of antenna pigment-protein complexes may increase nonradiative dissipation and, thus, quench chlorophyll a fluorescence. Here we measured, in parallel, the chlorophyll a fluorescence lifetime and intensity to understand the mechanism of this process. Increasing the xanthophyll concentration in the presence of a pH gradient (quenched conditions) decreases the fractional intensity of a fluorescence lifetime component centered at approximately 2 ns and increases a component at approximately 0.4 ns. Uncoupling the pH gradient (unquenched conditions) eliminates the 0.4-ns component. Changes in the xanthophyll concentration do not significantly affect the fluorescence lifetimes in either the quenched or unquenched sample conditions. However, there are differences in fluorescence lifetimes between the quenched and unquenched states that are due to pH-related, but nonxanthophyll-related, processes. Quenching of the maximal fluorescence intensity correlates with both the xanthophyll concentration and the fractional intensity of the 0.4-ns component. The unchanged fluorescence lifetimes and the proportional quenching of the maximal and dark-level fluorescence intensities indicate that the xanthophylls act on antenna, not reaction center processes. Further, the fluorescence quenching is interpreted as the combined effect of the pH gradient and xanthophyll concentration, resulting in the formation of a quenching complex with a short (approximately 0.4 ns) fluorescence lifetime.

Iron-Induced Changes in Light Harvesting and Photochemical Energy Conversion Processes in Eukaryotic Marine Algae

The role of iron in regulating light harvesting and photochemical energy conversion processes was examined in the marine unicellular chlorophyte Dunaliella tertiolecta and the marine diatom Phaeodactylum tricornutum. In both species, iron limitation led to a reduction in cellular chlorophyll concentrations, but an increase in the in vivo, chlorophyll-specific, optical absorption cross-sections. Moreover, the absorption cross-section of photosystem II, a measure of the photon target area of the traps, was higher in iron-limited cells and decreased rapidly following iron addition. Iron-limited cells exhibited reduced variable/maximum fluorescence ratios and a reduced fluorescence per unit absorption at all wave-lengths between 400 and 575 nm. Following iron addition, variable/maximum fluorescence ratios increased rapidly, reaching 90% of the maximum within 18 to 25 h. Thus, although more light was absorbed per unit of chlorophyll, iron limitation reduced the transfer efficiency of excitation energy in photosystem II. The half-time for the oxidation of primary electron acceptor of photosystem II, calculated from the kinetics of decay of variable maximum fluorescence, increased 2-fold under iron limitation. Quantitative analysis of western blots revealed that cytochrome f and subunit IV (the plastoquinone-docking protein) of the cytochrome b(6)/f complex were also significantly reduced by lack of iron; recovery from iron limitation was completely inhibited by either cycloheximide or chloramphenicol. The recovery of maximum photosynthetic energy conversion efficiency occurs in three stages: (a) a rapid (3-5 h) increase in electron transfer rates on the acceptor side of photosystem II correlated with de novo synthesis of the cytochrome b(6)/f complex; (b) an increase (10-15 h) in the quantum efficiency correlated with an increase in D1 accumulation; and (c) a slow (>18 h) increase in chlorophyll levels accompanied by an increase in the efficiency of energy transfer from the light-harvesting chlorophyll proteins to the reaction centers.

Trapping and annihilation in the antenna system of Photosystem I

The primary charge separation in Photosystem I of pea chloroplasts was measured as a photovoltage in the pico- and nanosecond time range by applying laser flashes at 532 nm of variable energy and different duration (12 ns and 30 ps, respectively). Contributions to the photovoltage from Photosystem II was eliminated by addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination. The dependence of the photovoltage amplitude on the excitation energy could be described by an exponential saturation law when the excitation flash had a duration of 12 ns. Nearly the same dependence was found when the excitation source was the train of a mode-locked laser (approx. ten 30-ps flashes spaced by 7 ns; highest energy of a single flash, 80 μJ / cm −2). Even with single 30-ps flashes the photovoltage was only slightly smaller than the one elicited by 12-ns flashes of the same energy. These findings demonstrate that trapping of excitation energy by the reaction center of Photosystem I is much more effective than losses by annihilation and other loss processes. The photovoltage yield was nearly independent of the fraction of closed traps, thus demonstrating that the absorption cross section of Photosystem I is not altered by the closing of its reaction centers. By recording the rise time of the photovoltage with our highest time resolution we found that the trapping rate of the excitation energy in Photosystem I depended on the energy of the 30-ps flashes: at low excitation energies (less than 10 14 photons / cm 2 per pulse) trapping occurred within 90 ± 15 ps and at high excitation energy (10 15 photons / cm 2 per pulse) trapping and charge stabilization occurred within the time resolution of the apparatus, i.e., up to 50 ps. The trapping rate at low energies is in agreement with the one determined by fluorescence decay kinetics. Up to 50 ns there was no further detectable electrogenic phase (neither forward nor backward reactions). This demonstrates that all the electrogenicity, produced by the charge separation, takes place in less than 50 ps.

Transfer and trapping of excitation energy in Photosystem II as studied by chlorophyll a2 fluorescence quenching by dinitrobenzene and carotenoid triplet. The matrix model

1. The curves representing the reciprocal fluorescence yield of chlorophyll a of Photosystem II (PS II) in Chlorella vulgaris as a function of the concentration of m-dinitrobenzene in the states P Q and P Q -, are found to be straight parallel lines; P is the primary donor and Q the primary acceptor of PS II. In the weakly trapping state P Q - the half-quenching of dinitrobenzene is about 0.2 mM, in vitro it is of the order of 10 mM. The fluorescence yield as a function of the concentration of a quencher is described for three models for the structure of pigment systems: the model of separate units, the model of limited energy transfer between the units, and the matrix model. If it is assumed that the rate constant of quenching by dinitrobenzene is high and thus the number of dinitrobenzene molecules per reaction center low, it can be concluded that the pigment system of PS II in C. vulgaris is a matrix of chlorophyll molecules in which the reaction centers are embedded. Theoretical and experimental evidence is consistent with such an assumption. For Cyanidium caldarium the zero fluorescence yield Ф 0 and its quenching by dinitrobenzene were found to be much smaller than the corresponding quantities for C. vulgaris. Nevertheless, our measurements on C. caldarium could be interpreted by the assumption that the essential properties (rate constants, dinitrobenzene quenching) of PS II are the same for these two species belonging to such widely different groups. 2. The measured dinitrobenzene concentrations required for half-quenching in vivo and other observations are explained by (non-rate-limiting) energy transfer between the chlorophyll a molecules of PS II and by the assumptions that dinitrobenzene is approximately distributed at random in the membrane and does not diffuse during excitation. 3. The fluorescence kinetics of C. vulgaris during a 350 ns laser flash of variable intensity could be simulated on a computer using the matrix model. From the observed fluorescence quenching by the carotenoid triplet (C T) and the measurement of the number of C T per reaction center via difference absorption spectroscopy, the rate constant for quenching of C T is calculated to be k T = 3.3 · 10 11 s −1 which is almost equal to the rate constant of trapping by an open reaction center (Duysens, L.N.M. (1979) CIBA Foundation Symposium 61 (New Series), pp. 323–340). 4. The fluorescence quenching by C T in non-treated spinach chloroplasts after a 500 ns laser flash (Breton, J., Geacintov, N.E. and Swenberg, C.E. (1979) Biochim. Biophys. Acta 548, 616–635) could be explained within the framework of the matrix model when the value for k T is used as given in point 3. 5. The observations mentioned under point 1 indicate that the fluorescence yield Ф 0 for centers in trapping state P Q is probably for a fraction exceeding 0.8 emitted by PS II.

Competition between the 735 nm fluorescence and the photochemistry of Photosystem I in chloroplasts at low temperature

Fluorescence emission spectra of chloroplasts, initially frozen to −196°C, were measured at various temperatures as the sample was allowed to warm. The 735 nm emission band attributed to fluorescence from Photosystem I was approx. 10-fold greater at −196°C than at −78°C. The initial rate of photooxidation of P-700 was also measured at −196°C and −78°C and was found to be approximately twice as large at the higher temperature. It is proposed that the 735 nm emission band is fluorescence from a long wavelength form of chlorophyll, C-705, which acts as a trap for excitation energy in the antenna chlorophyl system of Photosystem I. Furthermore, it is proposed that C-705 only forms on cooling to low temperatures and that the temperature dependence of the 735 nm emission is the temperature dependence for the formation of C-705. C-705 and P-700 compete to trap the excitation energy in Photosystem I. It is estimated from the data that at −78°C P-700 traps approx. 20 times more energy than C-705 while, at −196°C, the two traps are approximately equally effective. By analogy, the 695 nm fluorescence which also appears on cooling to −196°C is attributed to traps in Photosystem II which form only on cooling to temperatures near −196°C.