Zeaxanthin Synthesis, Energy Dissipation, and Photoprotection of Photosystem II at Chilling Temperatures

When leaves of a mangrove, Rhizophora mangle, were exposed to an excess of light at chilling temperatures, synthesis of zeaxanthin through violaxanthin de-epoxidation as well as nonphotochemical fluorescence quenching were markedly reduced. The results suggest a protective role of energy dissipation against the adverse effects of high light and chilling temperatures: leaves of R. mangle that had been preilluminated in 2% O₂, 0% CO₂ at low photon flux density and showed a high level of zeaxanthin, and leaves that had been kept in the dark and contained no zeaxanthin, were both exposed to high light and chilling temperatures (5°C leaf temperature) in air and then held under control conditions in low light in air at 25°C. Measurements of chlorophyll a fluorescence at room temperature showed that the photochemical efficiency of PSII and the yield of maximum fluorescence of the preilluminated leaf recovered completely within 1 to 3 hours under the control conditions. In contrast, the fluorescence responses of the predarkened leaf in high light at 5°C did not recover at all. During a dark/light transient in 2% O₂, 0% CO₂ in low light at 5°C, nonphotochemical fluorescence quenching increased linearly with an increase in the zeaxanthin content in leaves of R. mangle. In soybean (Glycine max) leaves, which contained a background level of zeaxanthin in the dark, a similar treatment with excess light induced a level of nonphotochemical fluorescence quenching that was not paralleled by an increase in the zeaxanthin content.     

Measurement of chlorophyll fluorescence within leaves using a modified PAM Fluorometer with a fiber-optic microprobe

By using a fiber-optic microprobe in combination with a modified PAM Fluorometer, chlorophyll fluorescence yield was measured within leaves with spatial resolution of approximately 20 m. The new system employs a miniature photomultiplier for detection of the pulse-modulated fluorescence signal received by the 20 m fiber tip. The obtained signal/noise ratio qualifies for recordings of fluorescence induction kinetics (Kautsky effect), fluorescence quenching by the saturation pulse method and determination of quantum yield of energy conversion at Photosystem II at different sites within a leaf. Examples of the system performance and of practical applications are given. It is demonstrated that the fluorescence rise kinetics are distinctly faster when chloroplasts within the spongy mesophyll are illuminated as compared to palisade chloroplasts. Photoinhibition is shown to affect primarily the quantum yield of the palisade chloroplasts when excessive illumination is applied from the adaxial leaf side. The new system is envisaged to be used in combination with light measurements within leaves for an assessment of the specific contributions of different leaf regions to overall photosynthetic activity and for an integrative modelling of leaf photosynthesis.This paper is dedicated to Ulrich Heber on the occasion of his 65th birthday, with great respect for his outstanding achievements in photosynthesis research.     

Analysis of the Heat Stress Induced Quenching of Variable Chlorophyll a Fluorescence in Beet Spinach Leaves: Possible Accumulation of Oxidized Quencher P680+ under High Illumination

Effect of preheating of beet spinach leaves on chlorophyll a fluorescence yield was analyzed with the help of additional high intensity illumination pulses using a pulse modulated fluorometer. Preheating at mildly elevated temperature (35–45°C) causes a shift in the redox state of secondary donor of photosystem II, possibly due to uncoupling of phosphorylation because of thermal induced membrane disorganization and associated alkalinization of intra thylakoid space. Also, at these preheating temperatures, a rise in photosystem I catalyzed electron transfer has been shown to occur. These two effects induce rapid quenching of Chi a fluorescence, which drops even in the presence of actinic light, below the level of initial fluorescence (F_o′ monitored by the weak modulated probing light. Preheating of leaf segments induces an increase in fluorescence in the presence of dluron, which blocks electron flow between two photosystems, and thus this increases in fluorescence yield (F_o′ as monitored by weak modulated light, is not solely due to disorganization of light harvesting Chi-protein complex but also due to a shift in the redox equilibrium of the donor at the oxidizing side of photosystem II resulting in rapid reduction of Q_A the stable primary acceptor of photosystem II. In 50°C preheated DCMU treated samples, the fluorescence yield increases in weak modulated light and it approaches that of maximal steady state (F_max) level. At preheating temperature of 48°–50°C, the inactivation of enzymes in the reducing side of photosystem I, causes an impairment of the reoxidation of QA and under this condition, a strong illumination causes quenching of Chi a fluorescence. This quenching seems to arise because of accumulation of the P680⁺, the oxidized physiological donor of photosystem which is a quencher of Chi a fluorescence. This quenching depended on the pulse intensity and duration which saturates P680⁺ accumulation and is greatly manifested when water oxidation complex is damaged.     

Blue light-modulation of chlorophyll a fluorescence transients in guard cell chloroplasts

Chlorophyll a fluorescence transients from mesophyll and single guard cell pairs of Vicia faba were measured by microspectrofluorometry. In both chloroplast types, fluorescence induction (O to P) was similar under actinic blue and green light. In slow transients from mesophyll cell chloroplasts, blue and green light induced identical, typical rapid quenching from P to S, and the M peak. In contrast, the P to S transient from guard cell (GC) chloroplasts irradiated with blue light showed a much slower quenching rate, and the P to T transition showed no M peak. Actinic green light induced mesophyll-like transients in GC chloroplasts, including rapid quenching from P to S and the M peak. Detection of these transients in single pairs of GC and isolated protoplasts ruled out mesophyll contamination as a signal source. Green light induced a rapid quenching and the M peak in GC chloroplasts from several species. The effect of CO₂ concentration on the fluorescence transients was investigated in the presence of HCO₃⁻ at pH 6.8 and 10.0. In transients induced by green light in both chloroplast types, a pH increase concomitant with a reduction in CO₂ concentration caused an increase in the initial rate of quenching and the elimination of the M peak. Actinic blue light induced mesophyll-like transients from GC chloroplasts in the presence of 10 micromolar KCN, a concentration at which the blue light-induced stomatal opening is inhibited. Addition of 100 to 200 micromolar phosphate also caused large increases in fluorescence quenching rates and a M peak. These results indicate that blue light modulates photosynthetic activity in GC chloroplasts. This blue light effect is not observed in the absence of transduction events connected with the blue light response and in the presence of high phosphate concentrations.     

Investigation of the molecular mechanism of the blue-light-specific excitation energy quenching in the plant antenna complex LHCII

Excitation of the major photosynthetic antenna complex of plants, LHCII, with blue light (470 nm) provides an advantage to plants, as it gives rise to chlorophyll a fluorescence lifetimes shorter than with excitation with red light (635 nm). This difference is particularly pronounced in fluorescence emission wavelengths longer than 715 nm. Illumination of LHCII preparation with blue light additionally induces fluorescence quenching, which develops on a minute timescale. This effect is much less efficient when induced by red light, despite the equalized energy absorbed in both the spectral regions. Simultaneous analysis of the fluorescence and photoacoustic signals in LHCII demonstrated that the light-driven fluorescence quenching is not associated with an increase in heat emission. Instead, a reversible light-induced conformational transformation of the protein takes place, as demonstrated by the FTIR technique. These findings are discussed in terms of the blue-light-specific excitation energy quenching in LHCII, which may have photoprotective applications.     

Proton uptake and quenching of bacteriochlorophyll fluorescence in Rhodopseudomonas spheroides

The addition of the cyclic cofactor 2,3,5,6-tetramethyl-p-phenylenediamine (diaminodurene) to a suspension of chromatophores of Rhodopseudomonas spheroides causes a light-dependent quenching of bacteriochlorophyll fluorescence. This effect is similar to one observed in chloroplasts and related to proton uptake. It is distinct from the quenching operative through the redox state of the primary electron donor and acceptor, as shown by its sensitivity to uncouplers and ionophorous antibiotics. The quenching is dependent on light intensity and diaminodurene concentration, and has a pH optimum at 7.1 where up to 70% of the fluorescence could be quenched in the presence of 0.33 mM diaminodurene.     

Fast Fluorescence Quenching from Isolated Guard Cell Chloroplasts of Vicia faba Is Induced by Blue Light and Not by Red Light

Chlorophyll a fluorescence transients from isolated Vicia faba guard cell chloroplasts were used to probe the response of these organelles to light quality. Guard cell chloroplasts were isolated from protoplasts by passing them through a 10-μm nylon net. Intact chloroplasts were purified on a Percoll gradient. Chlorophyll a fluorescence transients induced by actinic red or blue light were measured with a fluorometer equipped with a measuring beam. Actinic red light induced a monophasic quenching, and transients induced by blue light showed biphasic kinetics having a slow and a fast component. The difference between the red and blue light-induced transients could be observed over a range of fluence rates tested (200-800 μmol m⁻² s⁻¹). The threshold fluence rate of blue light for the induction of the fast component of quenching was 200 μmol m⁻² s⁻¹, but in the presence of saturating red light, fluence rates as low as 25 μmol m⁻² s⁻¹ induced the fast quenching. These results indicate that guard cell chloroplasts have a specific response to blue light.     

Measurement of transmembrane potentials in Rhodospirillum rubrum chromatophores with an oxacarbocyanine dye

The fluorescent dye 3,3-dipentyloxacarbocyanine (OCC) can be used as a fluorescence probe to measure transmembrane potentials across Rhodospirillum ruburm chromatophore membranes. A reversible fluorescence increase is observed in the light which is sensitive to inhibitors, permeable ions and uncouplers.Partial interchangeability between the electrical potential and the proton concentration gradient has been demonstrated by measurement of the fluorescence increase with OCC and the fluorescence quenching with 9-aminoacridine.OCC fluorescence changes can be induced also in the dark by injection of permeable salts and by rapid pH changes presumably indicating diffusion potentials. Using salt-induced diffusion potentials for calibrating the light signals and with several assumptions, the light-induced potentials were estimated as 170 mV for the maximal signal and 90–110 mV at the steady state.OCC has been shown to apparently increase the electrical conductivity of the chromatophore membrane, a fact which may be relevant to the mechanism of action of this probe.A red shift in the OCC absorption spectrum occurs when mixed with chromatophores, with a difference spectrum maximum at 495 nm. The absorption changes at 495 nm taking place in the light are similar in kinetics to the fluorescence changes. The absorbance spectrum of OCC in organic solvents is red shifted and the extent of the shift depends on the hydrophobicity of the medium. The difference spectrum compared to water in sec-butyl $\text{acetate}\text{n-}\text{hexane}$ (3 : 1, v/v) with a dipole moment of 5 was nearly identical to that of chromatophore-associated dye.The uncoupling properties of OCC at high concentrations and some difficulties in calibration limit the usefulness of this probe for quantitative measurements of transmembrane potentials.     

NMR (1H) analysis of crude extracts detects light stress in Beta vulgaris and Spinacia oleracea leaves

In highlight stress conditions, the mechanism of non-photochemical quenching (NPQ) of chlorophyll fluorescence is triggered at the chloroplast level. This process allows thermal quenching of the excessive excitation energy and it is strictly related to the efficiency of the xanthophyll cycle. Nowadays, the utilization of the nuclear magnetic resonance (NMR) spectroscopy provides a powerful complementary way for the identification and quantitative analysis of plant metabolites either in vivo or in tissue extracts. Seeing that the oxidative damage caused by light stress in plants and the consequent involvement of pigments are widely studied, NMR spectroscopy can be utilized to compare crude leaf extract at different levels of light stress, allowing an analysis of these compounds. In this paper, the identification of possible relationships between light stress and ¹H NMR signal variations is discussed. The analysis of the ¹H NMR (1D) spectra of two agronomic species (Spinacia oleracea and Beta vulgaris) exposed to different light intensities is presented. In particular, change in carotenoids and xanthophylls signals are analyzed.     

Slow fluorescence quenching of Type A chloroplasts. Resolution into two components

The divalent-cation-specific ionophore A23187 is used to define two components of the slow fluorescence quenching of type a spinach chloroplasts: ionophore-reversible and ionophore-resistant quenching. Ionophore-reversible quenching predominates at relatively low light intensities and approaches saturation as light levels are increased. It is sensitive to uncouplers and to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and is dark reversible. At high light intensities the bulk (> 80%) of slow fluorescence quenching is ionophore-resistant. Ionophore-resistant quenching is stimulated by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at pH 7.6 and by both CCCP and methylamine at pH 9.0. It is insensitive to DCMU and is not reversed in subsequent darkness. Taken together, the two components account for all quenching observed in Type A chloroplasts.Ionophore-reversible quenching is identified with the Mg²⁺-mediated fluorescence quenching described by Krause (Biochim. Biophys. Acta (1974) 333, 301–313) and by Barber and Telfer (in Membrane Transport in Plants (Dainty, J., and Zimmermann, U., eds.), pp. 281–288, Springer-Verlag, Berlin, 1974). Ionophore-resistant quenching, a first-order process requiring high light, resembles the quenching reported by Jennings et al. (Biochim. Biophys. Acta (1976) 423, 264–274).The resolution of the fluorescence quenching phenomenon into two distinct components reconciles the apparently contradictory observations of these earlier investigations.     

Inhibition of zeaxanthin formation and of rapid changes in radiationless energy dissipation by dithiothreitol in spinach leaves and chloroplasts

Dithiothreitol, which completely inhibits the de-epoxidation of violaxanthin to zeaxanthin, was used to obtain evidence for a causal relationship between zeaxanthin and the dissipation of excess excitation energy in the photochemical apparatus in Spinicia oleracea L. In both leaves and chloroplasts, inhibition of zeaxanthin formation by dithiothreitol was accompanied by inhibition of a component of nonphotochemical fluorescence quenching. This component was characterized by a quenching of instantaneous fluorescence (F_o) and a linear relationship between the calculated rate constant for radiationless energy dissipation in the antenna chlorophyll and the zeaxanthin content. In leaves, this zeaxanthin-associated quenching, which relaxed within a few minutes upon darkening, was the major component of nonphotochemical fluorescence quenching determined in the light, i.e. it represented the `high-energy-state' quenching. In isolated chloroplasts, the zeaxanthin-associated quenching was a smaller component of total nonphotochemical quenching and there was a second, rapidly reversible high-energy-state component of fluorescence quenching which occurred in the absence of zeaxanthin and was not accompanied by F_o quenching. Leaves, but not chloroplasts, were capable of maintaining the electron acceptor, Q, of photosystem II in a low reduction state up to high degrees of excessive light and thus high degrees of nonphotochemical fluorescence quenching. When ascorbate, which serves as the reductant for violaxanthin de-epoxidation, was added to chloroplast suspensions, zeaxanthin formation at low photon flux densities was stimulated and the relationship between nonphotochemical fluorescence quenching and the reduction state in chloroplasts then became more similar to that found in leaves. We conclude that the inhibition of zeaxanthin-associated fluorescence quenching by dithiothreitol provides further evidence that there exists a close relationship between zeaxanthin and potentially photoprotective dissipation of excess excitation energy in the antenna chlorophyll.     

Ultrafast fluorescence study on the location and mechanism of non-photochemical quenching in diatoms

The diatom algae, responsible for at least a quarter of the global photosynthetic carbon assimilation in the oceans, are capable of switching on rapid and efficient photoprotection, which helps them cope with the large fluctuations of light intensity in the moving waters. The enhanced dissipation of excess excitation energy becomes visible as non-photochemical quenching (NPQ) of chlorophyll a fluorescence. Intact cells of the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum, which show different NPQ induction kinetics under high light illumination, were investigated by picosecond time-resolved fluorescence under dark and NPQ-inducing high light conditions. The fluorescence kinetics revealed that there are two independent sites responsible for NPQ. The first quenching site is located in an FCP antenna system that is functionally detached from both photosystems, while the second quenching site is located in the PSII-attached antenna. Notwithstanding their different npq induction and reversal kinetics, both diatoms showed identical NPQ via both mechanisms in the steady-state. Their fluorescence decays in the dark-adapted states were different, however. A detailed quenching model is proposed for NPQ in diatoms.     

State Transition,Is It a Photochemical or Non-photochemical Process in Leaf in Response to Irradiance?

The response of steady-state fluorescence (Fs) to irradiance in apple (Malus pumila Mill. cv. Tengmu No.1/Malus hupehensis Rehd.) leaf increased and decreased at light levels below and above 400 mmol.m-2.s-1 photosynthetic photon flux density (PPFD), respectively, while the light-adapted maximal fluorescence (Fm') and minimal fluorescence (Fo') decreased constantly with the increasing PPFD, and the closure of photosystem Ⅱ reaction center (PSⅡ RC) increased continuously, reflected by the chlorophyll fluorescence parameter of (Fs-Fo')/(Fm'-Fo'). These facts indicated that decrease of Fs above 400 mmol.m-2.s-1 PPFD was not caused by closure of PSⅡ RC, but was mainly resulted from the process of light transfer from light-harvesting complexⅡ (LHCⅡ) to PSⅡ RC. In the presence of N-ethylmaleimide (NEM), an inhibitor of photosynthetic state transition, Fs kept on increasing in apple leaf at light levels from 400 to 700 mmol.m-2.s-1, which was the photosynthetic saturation irradiance of apple leaves. In addition, Fs still increased at light levels over 700 mmol.m-2.s-1 in apple leaf pre-treated with dithiothreitol (DTT), an inhibitor of xanthophyll cycle. These changes showed that state transition and xanthophyll cycle caused a decrease of Fs in apple leaf at light levels below and above the photosynthetic saturation irradiance, respectively. When apple leaf was pre-treated with NEM, the PSⅡ apparent rate of photochemical reaction (P-rate) and photochemical quenching (qP) decreased significantly in the light range of 600-800 mmol.m-2.s-1, but the non-photochemical quenching (qN) existed a small increase at 600-800 mmol.m-2.s-1 and a decrease above 800 mmol.m-2.s-1. These phenomena suggested that state transition was mainly a photochemical and a non-photochemical process in apple leaf responding to light lower and higher than photosynthetic saturation irradiance, respectively.     

Structural Implications of Fluorescence Quenching in the Shaker K+ Channel

When attached to specific sites near the S4 segment of the nonconducting (W434F) Shaker potassium channel, the fluorescent probe tetramethylrhodamine maleimide undergoes voltage-dependent changes in intensity that correlate with the movement of the voltage sensor (Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Science. 271:213–216; Cha, A., and F. Bezanilla. 1997. Neuron. 19:1127–1140). The characteristics of this voltage-dependent fluorescence quenching are different in a conducting version of the channel with a different pore substitution (T449Y). Blocking the pore of the T449Y construct with either tetraethylammonium or agitoxin removes a fluorescence component that correlates with the voltage dependence but not the kinetics of ionic activation. This pore-mediated modulation of the fluorescence quenching near the S4 segment suggests that the fluorophore is affected by the state of the external pore. In addition, this modulation may reflect conformational changes associated with channel opening that are prevented by tetraethylammonium or agitoxin. Studies of pH titration, collisional quenchers, and anisotropy indicate that fluorophores attached to residues near the S4 segment are constrained by a nearby region of protein. The mechanism of fluorescence quenching near the S4 segment does not involve either reorientation of the fluorophore or a voltage-dependent excitation shift and is different from the quenching mechanism observed at a site near the S2 segment. Taken together, these results suggest that the extracellular portion of the S4 segment resides in an aqueous protein vestibule and is influenced by the state of the external pore.     

Application of spectrally resolved fluorescence induction to study light-induced nonphotochemical quenching in algae

The light-induced nonphotochemical quenching (NPQ) can safely dissipate excess of absorbed light to heat. Here we describe an application of spectrally resolved fluorescence induction (SRFI) method for studying spectral variability of NPQ. The approach allows detection of spectrally-resolved nonphotochemical quenching (NPQ_λ) representing NPQ dependency on fluorescence emission wavelength in the whole spectral range of fluorescence emission. The experimental approach is briefly described and NPQ_λ is studied for the cryptophyte alga Rhodomonas salina and for green alga Chlorella sp. We confirm presence of NPQ_λ only in membrane-bound antennae (chlorophyll a/c antennae) and not in phycobiliproteins in lumen in cryptophyte and show that NPQ_λ is inhibited in the whole spectral range by NPQ inhibitors in Chlorella sp. We discuss variability in the quenching in the particular spectral ranges and applicability of the NPQ_λ parameter to study quenching locus in vivo.     

Measurement of chlorophyll fluorescence within leaves using a fibreoptic microprobe

Abstract. The distribution of chlorophyll fluorescence was measured within leaves of Medicago saliva with a fibre optic microprobe. Leaves were irradiated with broad band blue light (1000 μmol m⁻²s⁻¹) and chlorophyll fluorescence was measured at 688 nm. The amount of fluorescence measured within the leaf depended upon the direction in which the probe was inserted. When the probe was advanced directly through the leaf from the shaded towards the irradiated surface, the maximum amount of detected fluorescence occurred near the boundary between the palisade and spongy mesophyll. When the probe was advanced through the leaf from the opposite direction maximum detected fluorescence was at the boundary between the epidermis and palisade. These results appear to be a consequence of the blue light gradient, which declined exponentially within the palisade but was counterbalanced by increasing chlorophyll content within the leaf. Modelling indicates that the measured distribution of chlorophyll fluorescence can be explained by relatively uniform emission of fluorescence throughout the palisade layer, indicating that the chloroplasts may be photosynthetically specialized to their light environment within the leaf.     

ΔpH-Dependent Photosystem II Fluorescence Quenching Induced by Saturating, Multiturnover Pulses in Red Algae

We have previously shown that in the red alga Rhodella violacea, exposure to continuous low intensities of light 2 (green light) or near-saturating intensities of white light induces a ΔpH-dependent PSII fluorescence quenching. In this article we further characterize this fluorescence quenching by using white, saturating, multiturnover pulses. Even though the pulses are necessary to induce the ΔpH and the quenching, the development of the latter occurred in darkness and required several tens of seconds. In darkness or in the light in the presence of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, the dissipation of the quenching was very slow (more than 15 min) due to a low consumption of the ΔpH, which corresponds to an inactive ATP synthase. In contrast, under far-red illumination or in the presence of 3-(3,4-dichlorophenyl)-1,1′-dimethylurea (only in light), the fluorescence quenching relaxed in a few seconds. The presence of N,N′-dicyclohexyl carbodiimide hindered this relaxation. We propose that the quenching relaxation is related to the consumption of ΔpH by ATP synthase, which remains active under conditions favoring pseudolinear and cyclic electron transfer.     

Characterization of an on-line commercial fluorescence probe: modeling of the probe signal

A biosensor model was developed for a commercial NADH fluorescence probe to describe the single-frequency excitation and emission fluorescence behavior of an aqueous mixture of fluorophores. This model is essential in correlating the measured signals to the concentrations of fluorescent compounds in a bioreactor. In addition to the concentrations of fluorescent components, the relevant parameters of the model are the absorbance at both the excitation and the emission frequencies by the solvent and other absorbing species, the background signals, the light path length of the bioreactor vessel, the fluorescence yield, and the lampdetector configuration. Due to inner-filter effects and other interferences, the probe signal is intrinsically nonlinear in both the fluorophore concentration and the path length. An important parameter in the model is the geometric constant, S, which accounts for variations in the monitoring efficiency throughout the sample because fluorescent light is emitted in all directions. Previous models, derived from an unrealistic assumption that fluorescent light is emitted only in one direction parallel to the probe axis, are shown to be seriously deficient. The validity of the model was verified experimentally for a single-component solution in which both the fluorophore concentration and path length were varied.     

A method for estimating lateral diffusion coefficients in membranes from steady-state fluorescence quenching studies.

The Stern-Volmer theory, in which the quantum yield ratio (Io/I) depends linearly on the quencher concentration, will typically be inapplicable to fluorescence quenching in membranes. Numerical analysis shows that diffusion-controlled quenching results in a nonlinear concentration dependence for diffusion coefficients less than or of the order of 10(-6) cm2 s-1 and probe fluorescence lifetimes in the region of 10-100 ns. Lateral diffusion coefficients in membranes are typically overestimated an order of magnitude or more by the Stern-Volmer theory. An alternative empirical method is presented, which represents nonlinear concentration curves by a single parameter linear approximation determined by a least-squares analysis. The fitting parameter, P, depends on the interaction distance, the membrane thickness, the maximum extent of quenching and, in the case of biexponential probe fluorescence decay, the fluorescence kinetic parameters. P is presented in tabular form for a useful range of these parameters. The method is used to estimate diffusion coefficients for plastoquinone and plastoquinol from pyrene fluorescence quenching in soya bean phosphatidylcholine liposomes. It is found that the diffusion coefficients are nearly equal and in the region of 1.3-3.5 X 10(-7) cm2 s-1 for interaction radii of 1.5-0.5 nm, respectively.     

On the origin of a slowly reversible fluorescence decay component in the Arabidopsis npq4 mutant

Over-excitation of photosynthetic apparatus causing photoinhibition is counteracted by non-photochemical quenching (NPQ) of chlorophyll fluorescence, dissipating excess absorbed energy into heat. The PsbS protein plays a key role in this process, thus making the PsbS-less npq4 mutant unable to carry out qE, the major and most rapid component of NPQ. It was proposed that npq4 does perform qE-type quenching, although at lower rate than WT Arabidopsis. Here, we investigated the kinetics of NPQ in PsbS-depleted mutants of Arabidopsis. We show that red light was less effective than white light in decreasing maximal fluorescence in npq4 mutants. Also, the kinetics of fluorescence dark recovery included a decay component, qM, exhibiting the same amplitude and half-life in both WT and npq4 mutants. This component was uncoupler-sensitive and unaffected by photosystem II repair or mitochondrial ATP synthesis inhibitors. Targeted reverse genetic analysis showed that traits affecting composition of the photosynthetic apparatus, carotenoid biosynthesis and state transitions did not affect qM. This was depleted in the npq4phot2 mutant which is impaired in chloroplast photorelocation, implying that fluorescence decay, previously described as a quenching component in npq4 is, in fact, the result of decreased photon absorption caused by chloroplast relocation rather than a change in the activity of quenching reactions.