Petar H. Lambrev

Identification of a slowly inducible zeaxanthin-dependent component of non-photochemical quenching of chlorophyll fluorescence generated under steady-state conditions in Arabidopsis

The induction and relaxation of non-photochemical quenching (NPQ) under steady-state conditions, i.e. during up to 90min of illumination at saturating light intensities, was studied in Arabidopsis thaliana. Besides the well-characterized fast qE and the very slow qI component of NPQ, the analysis of the NPQ dynamics identified a zeaxanthin (Zx) dependent component which we term qZ. The formation (rise time 10-15min) and relaxation (lifetime 10-15min) of qZ correlated with the synthesis and epoxidation of Zx, respectively. Comparative analysis of different NPQ mutants from Arabidopsis showed that qZ was clearly not related to qE, qT or qI and thus represents a separate, Zx-dependent NPQ component.

Far-red fluorescence: A direct spectroscopic marker for LHCII oligomer formation in non-photochemical quenching

Time‐resolved fluorescence on oligomers of the main light‐harvesting complex from higher plants indicate that in vitro oligomerization leads to the formation of a weakly coupled inter‐trimer chlorophyll–chlorophyll (Chl) exciton state which converts in tens of ps into a state which is spectrally broad and has a strongly far‐red enhanced fluorescence spectrum. Both its lifetime and spectrum show striking similarity with a 400 ps fluorescence component appearing in intact leaves of Arabidopsis when non‐photochemical quenching (NPQ) is induced. The fluorescence components with high far‐red/red ratio are thus a characteristic marker for NPQ conditions in vivo. The far‐red emitting state is shown to be an emissive Chl–Chl charge transfer state which plays a crucial part in the quenching.

Singlet Energy Dissipation in the Photosystem II Light‐Harvesting Complex Does Not Involve Energy Transfer to Carotenoids

The energy dissipation mechanism in oligomers of the major light-harvesting complex II (LHC II) from Arabidopsis thaliana mutants npq1 and npq2, zeaxanthin-deficient and zeaxanthin-enriched, respectively, has been studied by femtosecond transient absorption. The kinetics obtained at different excitation intensities are compared and the implications of singlet-singlet annihilation are discussed. Under conditions where annihilation is absent, the two types of LHC II oligomers show distributive biexponential (bimodal) kinetics with lifetimes of approximately 5-20 ps and approximately 200-400 ps having transient spectra typical for chlorophyll excited states. The data can be described kinetically by a two-state compartment model involving only chlorophyll excited states. Evidence is provided that neither carotenoid excited nor carotenoid radical states are involved in the quenching mechanism at variance with earlier proposals. We propose instead that a chlorophyll-chlorophyll charge-transfer state is formed in LHC II oligomers which is an intermediate in the quenching process. The relevance to non-photochemical quenching in vivo is discussed.

On the relationship between non-photochemical quenching and photoprotection of Photosystem II.

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is thought to be an indicator of an essential regulation and photoprotection mechanism against high-light stress in photosynthetic organisms. NPQ is typically characterized by modulated pulse fluorometry and it is often assumed implicitly to be a good proxy for the actual physiological photoprotection capacity of the organism. Using the results of previously published ultrafast fluorescence measurements on intact leaves of w.t. and mutants of Arabidopsis (Holzwarth et al. 2009) we have developed exact relationships for the fluorescence quenching and the corresponding Photosystem II acceptor side photoprotection effects under NPQ conditions. The approach based on the exciton-radical pair equilibrium model assumes that photodamage results from triplet states generated in the reaction center. The derived relationships allow one to distinguish and determine the individual and combined quenching as well as photoprotection contributions of each of the multiple NPQ mechanisms. Our analysis shows inter alia that quenching and photoprotection are not linearly related and that antenna detachment, which can be identified with the so-called qE mechanism, contributes largely to the measured fluorescence quenching but does not correspond to the most efficient photoprotective response. Conditions are formulated which allow simultaneously the maximal photosynthetic electron flow as well as maximal acceptor side photoprotection. It is shown that maximal photoprotection can be achieved if NPQ is regulated in such a way that PSII reaction centers are open under given light conditions. The results are of fundamental importance for a proper interpretation of the physiological relevance of fluorescence-based NPQ data.

Kinetic and Spectral Resolution of Multiple Nonphotochemical Quenching Components in Arabidopsis Leaves

Using novel specially designed instrumentation, fluorescence emission spectra were recorded from Arabidopsis (Arabidopsis thaliana) leaves during the induction period of dark to high-light adaptation in order to follow the spectral changes associated with the formation of nonphotochemical quenching. In addition to an overall decrease of photosystem II fluorescence (quenching) across the entire spectrum, high light induced two specific relative changes in the spectra: (1) a decrease of the main emission band at 682 nm relative to the far-red (750–760 nm) part of the spectrum (Δ F682); and (2) an increase at 720 to 730 nm (Δ F720) relative to 750 to 760 nm. The kinetics of the two relative spectral changes and their dependence on various mutants revealed that they do not originate from the same process but rather from at least two independent processes. The Δ F720 change is specifically associated with the rapidly reversible energy-dependent quenching. Comparison of the wild-type Arabidopsis with mutants unable to produce or overexpressing the PsbS subunit of photosystem II showed that PsbS was a necessary component for Δ F720. The spectral change Δ F682 is induced both by energy-dependent quenching and by PsbS-independent mechanism(s). A third novel quenching process, independent from both PsbS and zeaxanthin, is activated by a high turnover rate of photosystem II. Its induction and relaxation occur on a time scale of a few minutes. Analysis of the spectral inhomogeneity of nonphotochemical quenching allows extraction of mechanistically valuable information from the fluorescence induction kinetics when registered in a spectrally resolved fashion.

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.

Low temperature tolerance of tobacco plants transformed to accumulate proline, fructans, or glycine betaine. Variable chlorophyll fluorescence evidence

Tobacco (Nicotiana tabacum L.) has been transformed to accumulate different compatible solutes (proline, fructans, or glycine betaine) in order to improve its tolerance to abiotic stress. Photosynthetic activity of wild Type (wt) and transformed tobacco plants before and after freezing stress was studied by measuring chlorophyll (Chl) fluorescence. The JIP test of Chl fluorescence induction was used to analyze in details the functional activity of photosystem 2. No significant differences were found among wild Type and transgenic plants after 12 h of freezing. Both plant Types maintained the same values of the measured parameters [FV/FM, PI(CSM), ABS/RC, TR0/RC, ET/RC] after recovery of stress. The studied Chl fluorescence parameters decreased only for the wild Type plants, stressed for 24 h at −2 °C. The strong inhibition of photosynthetic reactions in the wt plant after 24 h of freezing could not be restored. The evaluated parameters of transgenic plants did not change significantly after 24 h at −2 °C and successfully survived freezing stress.

Effect of phosphorylation on the thermal and light stability of the thylakoid membranes

Higher plant thylakoid membranes contain a protein kinase that phosphorylates certain threonine residues of light-harvesting complex II (LHCII), the main light-harvesting antenna complexes of photosystem II (PSII) and some other phosphoproteins (Allen, Biochim Biophys Acta 1098:275, 1992). While it has been established that phosphorylation induces a conformational change of LHCII and also brings about changes in the lateral organization of the thylakoid membrane, it is not clear how phosphorylation affects the dynamic architecture of the thylakoid membranes. In order to contribute to the elucidation of this complex question, we have investigated the effect of duroquinol-induced phosphorylation on the membrane ultrastructure and the thermal and light stability of the chiral macrodomains and of the trimeric organization of LHCII. As shown by small angle neutron scattering on thylakoid membranes, duroquinol treatment induced a moderate (~10%) increase in the repeat distance of stroma membranes, and phosphorylation caused an additional loss of the scattering intensity, which is probably associated with the partial unstacking of the granum membranes. Circular dichroism (CD) measurements also revealed only minor changes in the chiral macro-organization of the complexes and in the oligomerization state of LHCII. However, temperature dependences of characteristic CD bands showed that phosphorylation significantly decreased the thermal stability of the chiral macrodomains in phosphorylated compared to the non-phosphorylated samples (in leaves and isolated thylakoid membranes, from 48.3°C to 42.6°C and from 47.5°C to 44.3°C, respectively). As shown by non-denaturing PAGE of thylakoid membranes and CD spectroscopy on EDTA washed membranes, phosphorylation decreased by about 5°C, the trimer-to-monomer transition temperature of LHCII. It also enhanced the light-induced disassembly of the chiral macrodomains and the monomerization of the LHCII trimers at 25°C. These data strongly suggest that phosphorylation of the membranes considerably facilitates the heat- and light-inducible reorganizations in the thylakoid membranes and thus enhances the structural flexibility of the membrane architecture.

The negatively charged amino acids in the lumenal loop influence the pigment binding and conformation of the major light-harvesting chlorophyll a/b complex of photosystem II

The major chlorophyll (Chl) a/b complexes of photosystem II (LHCIIb), in addition to their primary light-harvesting function, play key roles in the organization of the granal ultrastructure of the thylakoid membranes and in various regulatory processes. These functions depend on the structural stability and flexibility of the complexes. The lumenal side of LHCIIb is exposed to broadly variable pH environments, due to the build-up and decay of the pH gradient during photosynthesis. Therefore, the negatively charged amino acids in the lumenal loop might be of paramount importance for adjusting the structure and functions of LHCIIb. In order to clarify the structural roles of these residues, we investigated the pigment stoichiometries, absorption, linear and circular dichroism spectra of the reconstituted LHCIIb complexes, in which the negatively charged amino acids in the lumenal loop were exchanged to neutral ones (E94G, E107V and D111V). The mutations influenced the pigment binding and the molecular architecture of the complexes. Exchanging E94 to G destabilized the 3(10) helix in the lumenal loop structure and led to an acquired pH sensitivity of the LHCIIb structure. We conclude that these amino acids are important not only for pigment binding in the complexes, but also in stabilizing the conformation of LHCIIb at different pHs.

The ultrastructure and flexibility of thylakoid membranes in leaves and isolated chloroplasts as revealed by small-angle neutron scattering ☆ ☆☆

We studied the periodicity of the multilamellar membrane system of granal chloroplasts in different isolated plant thylakoid membranes, using different suspension media, as well as on different detached leaves and isolated protoplasts-using small-angle neutron scattering. Freshly isolated thylakoid membranes suspended in isotonic or hypertonic media, containing sorbitol supplemented with cations, displayed Bragg peaks typically between 0.019 and 0.023Å(-1), corresponding to spatially and statistically averaged repeat distance values of about 275-330 Å⁻¹. Similar data obtained earlier led us in previous work to propose an origin from the periodicity of stroma thylakoid membranes. However, detached leaves, of eleven different species, infiltrated with or soaked in D2O in dim laboratory light or transpired with D2O prior to measurements, exhibited considerably smaller repeat distances, typically between 210 and 230 Å⁻¹, ruling out a stromal membrane origin. Similar values were obtained on isolated tobacco and spinach protoplasts. When NaCl was used as osmoticum, the Bragg peaks of isolated thylakoid membranes almost coincided with those in the same batch of leaves and the repeat distances were very close to the electron microscopically determined values in the grana. Although neutron scattering and electron microscopy yield somewhat different values, which is not fully understood, we can conclude that small-angle neutron scattering is a suitable technique to study the periodic organization of granal thylakoid membranes in intact leaves under physiological conditions and with a time resolution of minutes or shorter. We also show here, for the first time on leaves, that the periodicity of thylakoid membranes in situ responds dynamically to moderately strong illumination. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.