The mechanisms underlying the wavelength dependence of the quantum yield for CO 2 fixation (a) 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 a 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 a for leaves grown under the shade light spectrum (i.e., PSI light), whereas wavelengths overexciting PSII produced a higher a 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 a 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 CO 2 fixation. This also proves that combining different wavelengths can enhance quantum yields substantially.

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.

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, 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 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.

The nature of the highly efficient energy transfer in photosynthetic light-harvesting complexes is a subject of intense research. Unfortunately, the low fluorescence efficiency and limited photostability hampers the study of individual light-harvesting complexes at ambient conditions. Here we demonstrate an over 500-fold fluorescence enhancement of light-harvesting complex 2 (LH2) at the single-molecule level by coupling to a gold nanoantenna. The resonant antenna produces an excitation enhancement of circa 100 times and a fluorescence lifetime shortening to ~\n20 ps. The radiative rate enhancement results in a 5.5-fold-improved fluorescence quantum efficiency. Exploiting the unique brightness, we have recorded the first photon antibunching of a single light-harvesting complex under ambient conditions, showing that the 27 bacteriochlorophylls coordinated by LH2 act as a non-classical single-photon emitter. The presented bright antenna-enhanced LH2 emission is a highly promising system to study energy transfer and the role of quantum coherence at the level of single 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.protein multifunctionality | red forms A lthough conformational changes are essential for the function of proteins, little is known about their complex structural dynamics. Protein motions can partially be described by dynamic disorder in the crystalline, glass-like, or liquid states of matter (1-3). A feasible conceptual framework has been developed to visualize these dynamics as transitions between hierarchically ordered minima in the high-dimensional energy landscape of the protein (4, 5). Such a landscape represents all possible energy states as a function of the protein's conformation. The local minima, which signify conformational substates (CSs), are divided by energy barriers into different tiers. The order of a tier is characterized by an average barrier height, a higher barrier of which corresponds to a lower rate of conformational transitions (6). Protein-embedded pigments often serve as effective probes of conformational changes. In particular, strong intrapigment interactions in pigment aggregates can considerably increase the sensitivity of the pigments to the local environment (7). As a result, transitions between CSs are reflected as changes in the pigment electronic states and can be observed as distinct shifts in their absorption and emission energies. In conventional spectroscopy on large ensembles of proteins, energy equilibration after an excitation perturbation is monitored as the average of a very large number of possible trajectories on the energy landscapes of many similar proteins. In contrast, in single-molecule spectroscopy (SMS), the energy landscape of a single protein can be probed. For various pigment-protein complexes, this technique has proven successful to identify conform...

We have studied thylakoid membranes of Arabidopsis thaliana acclimated to different light conditions and have related protein composition to excitation energy transfer and trapping kinetics in Photosystem II (PSII). In high light: the plants have reduced amounts of the antenna complexes LHCII and CP24, the overall trapping time of PSII is only ∼180 ps, and the quantum efficiency reaches a value of 91%. In low light: LHCII is upregulated, the PSII lifetime becomes ∼310 ps, and the efficiency decreases to 84%. This difference is largely caused by slower excitation energy migration to the reaction centers in low-light plants due to the LHCII trimers that are not part of the C2S2M2 supercomplex. This pool of "extra" LHCII normally transfers energy to both photosystems, whereas it transfers only to PSII upon far-red light treatment (state 1). It is shown that in high light the reduction of LHCII mainly concerns the LHCII-M trimers, while the pool of "extra" LHCII remains intact and state transitions continue to occur. The obtained values for the efficiency of PSII are compared with the values of Fv/Fm, a parameter that is widely used to indicate the PSII quantum efficiency, and the observed differences are discussed.