Emilie Wientjes

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.

Photosynthetic Quantum Yield Dynamics: From Photosystems to Leaves

The quantum yield for CO2 fixation is wavelength dependent due to (1) light absorption by nonphotosynthetic pigments, (2) inefficient energy transfer, and (3) the excitation balance between the two photosystems. The growth-light spectrum alters the excitation balance by altering the photosystem composition, as shown both in vivo and in vitro. Enhancement effects can increase the quantum yield. The mechanisms underlying the wavelength dependence of the quantum yield for CO2 fixation (α) and its acclimation to the growth-light spectrum are quantitatively addressed, combining in vivo physiological and in vitro molecular methods. Cucumber (Cucumis sativus) was grown under an artificial sunlight spectrum, shade light spectrum, and blue light, and the quantum yield for photosystem I (PSI) and photosystem II (PSII) electron transport and α were simultaneously measured in vivo at 20 different wavelengths. The wavelength dependence of the photosystem excitation balance was calculated from both these in vivo data and in vitro from the photosystem composition and spectroscopic properties. Measuring wavelengths overexciting PSI produced a higher α for leaves grown under the shade light spectrum (i.e., PSI light), whereas wavelengths overexciting PSII produced a higher α for the sun and blue leaves. The shade spectrum produced the lowest PSI:PSII ratio. The photosystem excitation balance calculated from both in vivo and in vitro data was substantially similar and was shown to determine α at those wavelengths where absorption by carotenoids and nonphotosynthetic pigments is insignificant (i.e., >580 nm). We show quantitatively that leaves acclimate their photosystem composition to their growth light spectrum and how this changes the wavelength dependence of the photosystem excitation balance and quantum yield for CO2 fixation. This also proves that combining different wavelengths can enhance quantum yields substantially.

High-light vs. low-light: Effect of light acclimation on photosystem II composition and organization in Arabidopsis thaliana

The structural response of photosystem II (PSII) and its light-harvesting proteins (LHCII) in Arabidopis thaliana after long-term acclimation to either high or low light intensity was characterized. Biochemical and structural analysis of isolated thylakoid membranes by electron microscopy indicates a distinctly different response at the level of PSII and LHCII upon plant acclimation. In high light acclimated plants, the C(2)S(2)M(2) supercomplex, which is the dominating form of PSII in Arabidopsis, is a major target of structural re-arrangement due to the down-regulation of Lhcb3 and Lhcb6 antenna proteins. The PSII ability to form semi-crystalline arrays in the grana membrane is strongly reduced compared to plants grown under optimal light conditions. This is due to the structural heterogeneity of PSII supercomplexes rather than to the action of PsbS protein as its level was unexpectedly reduced in high light acclimated plants. In low light acclimated plants, the architecture of the C(2)S(2)M(2) supercomplex and its ability to form semi-crystalline arrays remained unaffected but the density of PSII in grana membranes is reduced due to the synthesis of additional LHCII proteins. However, the C(2)S(2)M(2) supercomplexes in semi-crystalline arrays are more densely packed, which can be important for efficient energy transfer between PSII under light limiting conditions.

LHCII is an antenna of both photosystems after long-term acclimation

LHCII, the most abundant membrane protein on earth, is the major light-harvesting complex of plants. It is generally accepted that LHCII is associated with Photosystem II and only as a short-term response to overexcitation of PSII a subset moves to Photosystem I, triggered by its phosphorylation (state1 to state2 transition). However, here we show that in most natural light conditions LHCII serves as an antenna of both Photosystem I and Photosystem II and it is quantitatively demonstrated that this is required to achieve excitation balance between the two photosystems. This allows for acclimation to different light intensities simply by regulating the expression of LHCII genes only. It is demonstrated that indeed the amount of LHCII that is bound to both photosystems decreases when growth light intensity increases and vice versa. Finally, time-resolved fluorescence measurements on the photosynthetic thylakoid membranes show that LHCII is even a more efficient light harvester when associated with Photosystem I than with Photosystem II.

The Role of Lhca Complexes in the Supramolecular Organization of Higher Plant Photosystem I

In this work, Photosystem I supercomplexes have been purified from Lhca-deficient lines of Arabidopsis thaliana using a mild detergent treatment that does not induce loss of outer antennas. The complexes have been studied by integrating biochemical analysis with electron microscopy. This allows the direct correlation of changes in protein content with changes in supramolecular structure of Photosystem I to get information about the position of the individual Lhca subunits, the association of the antenna to the core, and the influence of the individual subunits on the stability of the system. Photosystem I complexes with only two or three antenna complexes were purified, showing that the binding of Lhca1/4 and Lhca2/3 dimers to the core is not interdependent, although weak binding of Lhca2/3 to the core is stabilized by the presence of Lhca4. Moreover, Lhca2 and Lhca4 can be associated with the core in the absence of their “dimeric partners.” The structure of Photosystem I is very rigid, and the absence of one antenna complex leaves a “hole” in the structure that cannot be filled by other Lhcas, clearly indicating that the docking sites for the individual subunits are highly specific. There is, however, an exception to the rule: Lhca5 can substitute for Lhca4, yielding highly stable PSI supercomplexes with a supramolecular organization identical to the WT.

Conformational switching explains the intrinsic multifunctionality of plant light-harvesting complexes

The light-harvesting complexes of photosystem I and II (Lhcas and Lhcbs) of plants display a high structural homology and similar pigment content and organization. Yet, the spectroscopic properties of these complexes, and accordingly their functionality, differ substantially. This difference is primarily due to the charge-transfer (CT) character of a chlorophyll dimer in all Lhcas, which mixes with the excitonic states of these complexes, whereas this CT character is generally absent in Lhcbs. By means of single-molecule spectroscopy near room temperature, we demonstrate that the presence or absence of such a CT state in Lhcas and Lhcbs can occasionally be reversed; i.e., these complexes are able to interconvert conformationally to quasi-stable spectral states that resemble the Lhcs of the other photosystem. The high structural similarity of all the Lhca and Lhcb proteins suggests that the stable conformational states that give rise to the mixed CT-excitonic state are similar for all these proteins, and similarly for the conformations that involve no CT state. This indicates that the specific functions related to Lhca and Lhcb complexes are realized by different stable conformations of a single generic protein structure. We propose that this functionality is modulated and controlled by the protein environment.

The light-harvesting complexes of higher-plant Photosystem I: Lhca1/4 and Lhca2/3 form two red-emitting heterodimers.

The outer antenna of higher-plant PSI (Photosystem I) is composed of four complexes [Lhc (light-harvesting complex) a1-Lhca4] belonging to the light-harvesting protein family. Difficulties in their purification have so far prevented the determination of their properties and most of the knowledge about Lhcas has been obtained from the study of the in vitro reconstituted antennas. In the present study we were able to purify the native complexes, showing that Lhca2/3 and Lhca1/4 form two functional heterodimers. Both dimers show red-fluorescence emission with maxima around 730 nm, as in the intact PSI complex. This indicates that the dimers are in their native state and that LHCI-680, which was previously assumed to be part of the PSI antenna, does not represent the native state of the system. The data show that the light-harvesting properties of the two dimers are functionally identical, concerning absorption, long-wavelength emission and fluorescence quantum yield, whereas they differ in their high-light response. Implications of the present study for the understanding of the energy transfer process in PSI are discussed. Finally, the comparison of the properties of the native dimers with those of the reconstituted complexes demonstrates that all of the major properties of the Lhcas are reproduced in the in vitro systems.

During State 1 to State 2 Transition in Arabidopsis thaliana, the Photosystem II Supercomplex Gets Phosphorylated but Does Not Disassemble

Background: State transition balances the excitation pressure between the two photosystems of plants. Results: The organization of photosystem II supercomplexes and megacomplexes is the same in state 1 and state 2. Conclusion: Phosphorylation is not sufficient to induce the disassembly of the supercomplexes. Significance: This work helps to understand how plants optimize light harvesting under ever changing light conditions. Plants are exposed to continuous changes in light quality and quantity that challenge the performance of the photosynthetic apparatus and have evolved a series of mechanisms to face this challenge. In this work, we have studied state transitions, the process that redistributes the excitation pressure between photosystems I and II (PSI/PSII) by the reversible association of LHCII, the major antenna complex of higher plants, with either one of them upon phosphorylation/dephosphorylation. By combining biochemical analysis and electron microscopy, we have studied the effect of state transitions on the composition and organization of photosystem II in Arabidopsis thaliana. Two LHCII trimers (called trimers M and S) are part of the PSII supercomplex, whereas up to two more are loosely associated with PSII in state 1 in higher plants (called “extra” trimers). Here, we show that the LHCII from the extra pool migrates to PSI in state 2, thus leaving the PSII supercomplex and the semicrystalline PSII arrays intact. In state 2, not only is the mobile LHCII phosphorylated, but also the LHCII in the PSII supercomplexes. This demonstrates that PSII phosphorylation is not sufficient for disconnecting LHCII trimers S and M from PSII and for their migration to PSI.

Molecular Basis of Light Harvesting and Photoprotection in CP24 UNIQUE FEATURES OF THE MOST RECENT ANTENNA COMPLEX

CP24 is a minor antenna complex of Photosystem II, which is specific for land plants. It has been proposed that this complex is involved in the process of excess energy dissipation, which protects plants from photodamage in high light conditions. Here, we have investigated the functional architecture of the complex, integrating mutation analysis with time-resolved spectroscopy. A comprehensive picture is obtained about the nature, the spectroscopic properties, and the role in the quenching in solution of the pigments in the individual binding sites. The lowest energy absorption band in the chlorophyll a region corresponds to chlorophylls 611/612, and it is not the site of quenching in CP24. Chlorophylls 613 and 614, which are present in the major light-harvesting complex of Photosystem appear to be absent in CP24. In contrast to all other light-harvesting complexes, CP24 is stable when the L1 carotenoid binding site is empty and upon mutations in the third helix, whereas mutations in the first helix strongly affect the folding/stability of the pigment-protein complex. The absence of lutein in L1 site does not have any effect on the quenching, whereas substitution of violaxanthin in the L2 site with lutein or zeaxanthin results in a complex with enhanced quenched fluorescence. Triplet-minus-singlet measurements indicate that zeaxanthin and lutein in site L2 are located closer to chlorophylls than violaxanthin, thus suggesting that they can act as direct quenchers via a strong interaction with a neighboring chlorophyll. The results provide the molecular basis for the zeaxanthin-dependent quenching in isolated CP24.