A comparison of the three isoforms of the light-harvesting complex II using transient absorption and time-resolved fluorescence measurements

In this article we report the characterization of the energy transfer process in the reconstituted isoforms of the plant light-harvesting complex II. Homotrimers of recombinant Lhcb1 and Lhcb2 and monomers of Lhcb3 were compared to native trimeric complexes. We used low-intensity femtosecond transient absorption (TA) and time-resolved fluorescence measurements at 77 K and at room temperature, respectively, to excite the complexes selectively in the chlorophyll b absorption band at 650 nm with 80 fs pulses and on the high-energy side of the chlorophyll a absorption band at 662 nm with 180 fs pulses. The subsequent kinetics was probed at 30–35 different wavelengths in the region from 635 to 700 nm. The rate constants for energy transfer were very similar, indicating that structurally the three isoforms are highly homologous and that probably none of them play a more significant role in light-harvesting and energy transfer. No signature has been found in the transient absorption measurements at 77 K for Lhcb3 which might suggest that this protein acts as a relative energy sink of the excitations in heterotrimers of Lhcb1/Lhcb2/Lhcb3. Minor differences in the amplitudes of some of the rate constants and in the absorption and fluorescence properties of some pigments were observed, which are ascribed to slight variations in the environment surrounding some of the chromophores depending on the isoform. The decay of the fluorescence was also similar for the three isoforms and multi-exponential, characterized by two major components in the ns regime and a minor one in the ps regime. In agreement with previous transient absorption measurements on native LHC II complexes, Chl b → Chl a energy transfer exhibited very fast channels but at the same time a slow component (ps). The Chls absorbing at around 660 nm exhibited both fast energy transfer which we ascribe to transfer from ‘red’ Chl b towards ‘red’ Chl a and slow transfer from ‘blue’ Chl a towards ‘red’ Chl a. The results are discussed in the context of the new available atomic models for LHC II.     

Light-harvesting chlorophyll a-b complex requirement for regulation of Photosystem II photochemistry by non-photochemical quenching

Recently, it has been suggested (Horton et al. 1992) that aggregation of the light-harvesting a-b complex (LHC II) in vitro reflects the processes which occur in vivo during fluorescence induction and related to the major non-photochemical quenching (qE). Therefore the requirement of this chlorophyll a-b containing protein complex to produce qN was investigated by comparison of two barley mutants either lacking (chlorina f2) or depressed (chlorina¹⁰⁴) in LHC II to the wild-type and pea leaves submitted to intermittent light (IL) and during their greening in continuous light. It was observed that qN was photoinduced in the absence of LHC II, i.e. in IL grown pea leaves and the barley mutants. Nevertheless, in these leaves qN had no (IL, peas) or little (barley mutants) inhibitory effect on the photochemical efficiency of Q_A reduction measured by flash dosage response curves of the chlorophyll fluorescence yield increase induced by a single turn-over flash During greening in continuous light of IL pea leaves, an inhibitory effect on Q_A photoreduction associated to qN developed as Photosystem II antenna size increased with LHC II synthesis. Utilizing data from the literature on connectivity between PS II units versus antenna size, the following hypothesis is put forward to explain the results summarized above. qN can occur in the core antenna or Reaction Center of a fraction of PS II units and these units will not exhibit variable fluorescence. Other PS II units are quenched indirectly through PS II-PS II exciton transfer which develops as the proportion of connected PS II units increases through LHC II synthesis.     

Heat Stress and Spermidine: Effect on Chlorophyll Fluorescence in Tomato Plants

Two tomato (Lycopersicon esculentum L.) cultivars: Robin (tolerant) and Roma (sensitive to heat stress) were studied. Chlorophyll fluorescence induction parameters (F_v/F_p, A_max, and Rfd) at 25 °C showed that the PS2 activity was similar for both cultivars. The parameters, measured at 38 °C, decreased in both cultivars, but more in cv. Roma. Exogenous application of 4 mM spermidine improved the plant heat-resistance in both cultivars, and especially in cv. Roma. Analysis of chlorophyll fluorescence changes during linear increase in temperature showed that cv. Robin plants have higher ability to hardening and higher resistance to thermal damage of the pigment-protein complexes structure and the activity of PS2 than cv. Roma.     

The High Efficiency of Photosystem I in the Green Alga Chlamydomonas reinhardtii Is Maintained after the Antenna Size Is Substantially Increased by the Association of Light-harvesting Complexes II

Photosystems (PS) I and II activities depend on their light-harvesting capacity and trapping efficiency, which vary in different environmental conditions. For optimal functioning, these activities need to be balanced. This is achieved by redistribution of excitation energy between the two photosystems via the association and disassociation of light-harvesting complexes (LHC) II, in a process known as state transitions. Here we study the effect of LHCII binding to PSI on its absorption properties and trapping efficiency by comparing time-resolved fluorescence kinetics of PSI-LHCI and PSI-LHCI-LHCII complexes of Chlamydomonas reinhardtii. PSI-LHCI-LHCII of C. reinhardtii is the largest PSI supercomplex isolated so far and contains seven Lhcbs, in addition to the PSI core and the nine Lhcas that compose PSI-LHCI, together binding ∼320 chlorophylls. The average decay time for PSI-LHCI-LHCII is ∼65 ps upon 400 nm excitation (15 ps slower than PSI-LHCI) and ∼78 ps upon 475 nm excitation (27 ps slower). The transfer of excitation energy from LHCII to PSI-LHCI occurs in ∼60 ps. This relatively slow transfer, as compared with that from LHCI to the PSI core, suggests loose connectivity between LHCII and PSI-LHCI. Despite the relatively slow transfer, the overall decay time of PSI-LHCI-LHCII remains fast enough to assure a 96% trapping efficiency, which is only 1.4% lower than that of PSI-LHCI, concomitant with an increase of the absorption cross section of 47%. This indicates that, at variance with PSII, the design of PSI allows for a large increase of its light-harvesting capacities.     

Central-Cis isomers of lutein found in the major light-harvesting complex of Photosystem II (LHC IIb) of higher plants

Lutein (,-carotene-3,3-diol) is the major carotenoid of the light-harvesting systems of higher plants. Lutein was isolated at 4°C and in complete darkness from the bulk light-harvesting complex of Photosystem II of spinach (LHC IIb) and from BBY particles. Separation using normal-phase HPLC (with 2D detection) in comparison to the authentic isomers (prepared by iodine-sensitised isomerization) showed the presence of a number of geometrical isomers of this xanthophyll in PS II, namely all-trans (the major component); 13-cis, 13-cis and 15-cis-lutein. Iodine-sensitised photo-isomerization of all-trans lutein produced six geometrical isomers of lutein as determined by HPLC. The configuration of five of these isomers was determined by ¹H-NMR to be all-trans, 9-cis, 9-cis, 13-cis and 13-cis. In addition, small amounts of another isomer have been tentatively identified to be 15-cis lutein on the basis of its electronic absorption spectrum. The possible functional significance of the presence of cis-isomers of this carotenoid in LHC IIb is discussed.     

Prolonged incubation with low concentrations of mercury alters energy transfer and chlorophyll (Chl) a protein complexes in Synechococcus 6301: changes in Chl a absorption and emission characteristics and loss of the F695 emission band

Synechococcus PCC 6301 cells grown in the presence of low sublethal levels of (about 2 m) mercury induced alterations in chlorophyll (Chl) a absorption without significant alterations in phycocyanin. Chl a fluorescence emission in Hg²⁺ -raised cells showed a large (about 18 nm) blue shift in the peak emission. No major spectral changes in phycobilisome (PBsome) emission characteristic were noticed, indicating major structural alterations in Chl-protein complexes by incubation with Hg²⁺ ions. Low temperature (77 K) emission spectra of cells grown in the presence of Hg²⁺ showed a loss of the characteristic Chl a emission band at 695 nm (F695), which is known to be linked to photosystem II photochemistry and to originate from the Chl a of core antenna polypeptide CP 47 of photosystem II. The SDS-PAGE polypeptide profile of thylakoids indicates a loss of a polypeptide(s) with a molecular mass between 40 and 60 k Da by Hg²⁺ incubation of cells. Our results suggest that prolonged incubation of Synechococcus 6301 cells with low concentrations of Hg²⁺ affects the Chl a spectral properties and the structure of Chl-protein complexes.     

Characterization of the Porphyridium cruentum Chl a-binding LHC by in vitro reconstitution: LHCaR1 binds 8 Chl a molecules and proportionately more carotenoids than CAB proteins

The Porphyridium cruentum light harvesting complex (LHC) binds Chl a, zeaxanthin and -carotene and comprises at least 6 polypeptides of a multigene family. We describe the first in vitro reconstitution of a red algal light-harvesting protein (LHCaR1) with Chl a/carotenoid extracts from P. cruentum. The reconstituted pigment complex (rLHCaR1) is spectrally similar to the native LHC I, with an absorption maximum at 670 nm, a 77 K fluorescence emission peak at 677 nm (ex. 440 nm), and similar circular dichroism spectra. Molar ratios of 4.0 zeaxanthin, 0.3 -carotene and 8.2 Chl a per polypeptide for rLHCaR1 are similar to those of the native LHC I complex (3.1 zeaxanthin, 0.5 -carotene, 8.5 Chl a). The binding of 8 Chl a molecules per apoprotein is consistent with 8 putative Chl-binding sites in the predicted transmembrane helices of LHCaR1. Two of the putative Chl a binding sites (helix 2) in LHCaR1 were assigned to Chl b in Chl a/b-binding (CAB) LHC II [Kühlbrandt et al. (1994) Nature 367: 614–21]. This suggests either that discrimination for binding of Chl a or Chl b is not very specific at these sites or that specificity of binding sites evolved separately in CAB proteins. LHCaR1 can be reconstituted with varying ratios of carotenoids, consistent with our previous observation that the carotenoid to Chl ratio is substantially higher in P. cruentum grown under high irradiance. Also notable is that zeaxanthin does not act as an accessory light-harvesting pigment, even though it is highly likely that it occupies the position assigned to lutein in the CAB LHCs.This revised version was published online in October 2005 with corrections to the Cover Date.     

Cloning and expression of a gene coding for the major light-harvesting chlorophyll a/b protein of photosystem II in the green alga <Emphasis Type="Italic">Dunaliella salina</Emphasis>

Genes encoding proteins of the major light-harvesting complex of photosystem II (LHCII) in higher plants are well studied. However, little is known about the corresponding genes in the green alga Dunaliella salina, although this knowledge might provide valuable information about the respective roles of each LHCII protein at the molecular level under extreme environmental conditions. Here, we describe an additional LhcII gene from D. salina. An LhcII cDNA cloned by screening a D. salina cDNA library contains an open reading frame encoding a protein of 261 amino acids with a calculated molecular mass of 27.8 kDa. The deduced amino acid sequence shows high homology with other LHCII proteins. Genomic DNA—obtained by PCR using a specific primer set corresponding to the 5′ and 3′ untranslated regions—was used to determine the intron-exon structure. Short-term changes in mRNA levels after a shift from low-light to high-light or dark conditions were analyzed by real-time quantitative PCR, and indicated that this gene expresses different mRNA levels under different light conditions.     

Trophic relationships between primary producers and fish yields in Lake Balaton

Relationships between chlorophyll a content of the water, the shoreline-length: water area ratio and the annual total fish yield as catch per unit effort (CUE: kg ha^–1 100 h^–1 as annual mean values) have been calculated by multivariable regression. The determination coefficient (r² = 0.913) showed a significant dependence of fish yield on morphometry of different lake areas. Accordingly, fish carrying capacity of the open water areas, calculated from chlorophyll a content and S/A, ranged from 12 to 34%, but that of the littoral zone between 66 and 88%. These findings have also been supported by echo-sounding records of the horizontal distribution of fish.Bream (Abramis brama L.) contributes the majority (70–80%) of fish stock and yield. Its food mainly consists of zooplankton and benthic invertebrates in ratios that are widely variable with season and depend on the age of fish. Average daily food consumption of individuals (age group 3 + and over) varies between 2 and 5 g. Bream consumes two- to three-times more food in the SW basin than in the NE one. This means that the present stocks inhabiting areas from NE to SW consume annually 13249–20085 t yr^–1 of food. According to estimated calorific values, the annual energy consumption of local populations along the longitudinal axis of the lake varies between 93 and 141 kJ m^–2 yr^–1. The efficiency of energy transfer from primary producers to fish is low and varies from 0.04 to 0.1%.     

Phytoplankton growth rates in the freshwater tidal reaches of the Schelde estuary (Belgium) estimated using a simple light-limited primary production model

During the course of 1996, phytoplankton was monitored in the turbid, freshwater tidal reaches of the Schelde estuary. Using a simple light-limited primary production model, phytoplankton growth rates were estimated to evaluate whether phytoplankton could attain net positive growth rates and whether growth rates were high enough for a bloom to develop. Two phytoplankton blooms were observed in the freshwater tidal reaches. The first bloom occurred in March and was mainly situated in the most upstream reaches of the freshwater tidal zone, suggesting that it was imported from the tributary river Schelde. The second bloom occurred in July and August. This summer bloom was situated more downstream in the freshwater tidal reaches and appeared to have developed within the estuary. A comparison between phytoplankton growth rates estimated using a simple primary production model and flushing rate of the water indicated that no net increase in phytoplankton biomass was possible in March while phytoplankton could theoretically increase its biomass by 20% per day during summer. Chlorophyllaconcentrations at all times decreased strongly at salinities between 5–10 psu. This decline was ascribed to a combination of salinity stress and light limitation. Phytoplankton biomass and estimated annual net production were much higher in the freshwater tidal zone compared to the brackish reaches of the estuary (salinity > 10 psu) despite mixing depth to euphotic depth ratios being similar. Possible reasons for this high production include high nutrient concentrations, low zooplankton grazing pressure and import of phytoplankton blooms from the tributary rivers.     

Differential action of Hg2+ and Cd2+ on the phycobilisomes and chlorophyll a fluorescence,and photosystem II dependent electron transport in the cyanobacterium Anabaena flos-aquae

The effect of equimolar concentrations of Hg²⁺ and Cd²⁺ on the whole cell absorption spectra, absorption spectra of the extracted phycocyanin (PC) and fluorescence emission spectra of phycobilisomes (PBS) was investigated in the cells of Anabaena flos-aquae. The PC component of the PBS was found to be extremely sensitive to the Hg²⁺ rather than the Cd²⁺ ions. Further, the results showed that Hg²⁺ and Cd²⁺ induced decrease in the rate of Hill activity (H₂O - DCPIP) was partially restored by the electron donor NH₂OH, not by the diphenyl carbazide. Similarly, chlorophyll a fluorescence emission in the presence of metals showed that addition of NH₂OH could effectively reverse the metal induced alterations in the fluorescence emission intensity. These results, together, suggested that Hg²⁺ and Cd²⁺ caused damage to the photosystems (PS) II reaction center. However, a relatively higher stimulation of the chlorophyll a emission at 695 nm with a red shift of 4.0 nm in the presence of Hg²⁺, and Cd²⁺ induced preferential decrease in the emission intensity at 676 nm as compared with the peak at 695 nm were indicative of the differential action of Hg²⁺ and Cd²⁺ on the PS II.     

The redox state of the plastoquinone pool controls the level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoacclimation

A cytochrome b ₆ f deficient mutant of Lemna perpusilla maintains a constant and lower level of the light-harvesting chl a/b-binding protein complex II (LHC II) as compared to the wild type plants at low-light intensities. Inhibition of the plastoquinone pool reduction increases the LHC II content of the mutant at both low- and high-light intensities but only at high-light intensity in the wild type plants. Proteolytic activity against LHC II appears during high-light photoacclimation of wild type plants. However, the acclimative protease is present in the mutant at both light intensities. These and additional results suggest that the plastoquinone redox state serves as the major signal-transducing component in the photoacclimation process affecting both, synthesis and degradation of LHC II and appearance of acclimative LHC II proteolysis. The plastoquinol pool cannot be oxidized by linear electron flow in the mutant plants which are locked in a ‘high light’ acclimation state. The cytochrome b ₆ f complex may be involved indirectly in the regulation of photoacclimation via 1) regulation of the plastoquinone redox state; 2) regulation of the redox-controlled thylakoid protein kinase allowing exposure of the dephosphorylated LHC II to acclimative proteolysis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Consequences of LHC II deficiency for photosynthetic regulation in chlorina mutants of barley

The light-harvesting chlorophyll a/b proteins associated with PS II (LHC II) are often considered to have a regulatory role in photosynthesis. The photosynthetic responses of four chlorina mutants of barley, which are deficient in LHC II to varying degrees, are examined to evaluate whether LHC II plays a regulatory role in photosynthesis. The efficiencies of light use for PS I and PS II photochemistry and for CO₂ assimilation in leaves of the mutants were monitored simultaneously over a wide range of photon flux densities of white light in the presence and absence of supplementary red light. It is demonstrated that the depletions of LHC II in these mutants results in a severe imbalance in the relative rates of excitation of PS I and PS II in favour of PS I, which cannot be alleviated by preferential excitation of PS II. Analyses of xanthophyll cycle pigments and fluorescence quenching in leaves of the mutants indicated that the major LHC II components are not required to facilitate the light-induced quenching associated with zeaxanthin formation. It is concluded that LHC II is important to balance the distribution of excitation energy between PS I and PS II populations over a wide range of photon flux densities. It appears that LHC II may also be important in determining the quantum efficiency of PS II photochemistry by reducing the rate of quenching of excitation energy in the PS II primary antennae.Abbreviations Fm, Fv maximal and variable fluorescence yields in a light adapted state - LHC II light harvesting chlorophyll a/b protein complex associated with PS II - q_p photochemical quenching - A₈₂₀ light-induced absorbance change at 820 nm - ø_PSI, ø_PSII relative quantum efficiencies of PS I and PS II photochemistry - øCO₂ quantum yield of CO₂ assimilation     

Energy transfer in the light-harvesting complex II of Dunaliella tertiolecta is unusually sensitive to Triton X-100

Triton X-100, a detergent commonly used to solubilize higher plant thylakoid membranes, was found to be deleterious to Dunaliella LHC II. It disrupted the transfer of excitation energy from chlorophyll b to chlorophyll a. Based on analysis of pigments and immunoassays of LHC II apoproteins from sucrose density gradient fractions, Triton X-100 caused aggregation of the complex, but apparently did not remove chlorophyll b from the apoprotein. Following solubilization with Triton X-100 only CPI could be resolved by electrophoresis. In contrast, solubilization of Dunaliella thylakoids with octyl--D-glucopyranoside preserved energy transfer from chlorophyll b to chlorophyll a. This detergent also effectively prevented aggregation on sucrose gradients and preserved CPI oligomers, as well as LHCP1 and LHCP3 on non-denaturing gels. Solubilization with Deriphat gave similar results. We propose that room temperature fluorescence excitation and emission spectroscopy be used in conjunction with other biophysical and biochemical probes to establish the effects of detergents on the integrity of light harvesting chlorophyll protein complexes. Methods used here may be applicable to other chlorophytes which prove refractory to protocols developed for higher plants.Abbreviations LHC II light harvesting chlorophyll protein complex associated with photosystem II - LHCP1 and LHCP3 monomeric and oligomeric forms of LHC II, respectively, observed on non-denaturing gels - LiDS lithium dodecylsulphate - PMSF phenylmethylsulfonyl fluoride     

饱和白光引起的光系统II捕光复合体(LHCII)从反应中心复合体脱离不同于弱红光引起的状态1向状态2的转换

我们观测了不同光照预处理对拟南芥、小麦和大豆叶片光合作用和低温(77K) 叶绿素荧光参数F685、F735和F685/F735的影响.野生型拟南芥叶片光合作用对饱和光到有限光转变的响应曲线是V型,而缺乏叶绿体蛋白激酶的突变体STN7的这一曲线为L型. 饱和白光可以引起拟南芥叶片F685/F735的明显降低,但是F735没有明显增高,而弱红光可以导致拟南芥叶片F685/F735的明显降低和F735的明显增高,表明弱红光可以引起状态1向状态2的转变,同时伴随从光系统II脱离的LHC II与光系统I的结合,而饱和白光只能引起LHC II从光系统II反应中心复合体脱离.并且,低温叶绿素荧光分析结果证明,饱和白光可以引起大豆叶片LHC II脱离,但是不能引起小麦叶片LHC II脱离,而弱红光可以引起小麦叶片的这种状态转换,却不能引起大豆叶片的这种状态转换.因此,饱和白光引起的野生型拟南芥和大豆叶片的LHC II脱离不是一个典型的状态转换现象.     

Evolutionary conservation of the chlorophyll a/b-binding proteins: cDNAs encoding type I, II and III LHC I polypeptides from the gymnosperm Scots pine

Summary cDNAs encoding three different LHC I polypeptides (Type I, Type II and Type III) from the gymnosperm Scots pine (Pinus sylvestris L.) were isolated and sequenced. Comparisons of the deduced amino acid sequences with the corresponding tomato sequences showed that all three proteins were highly conserved although less so than the LHC II proteins. The similarities between mature Scots pine and tomato Types I, II and III LHC I proteins were 80%, 87% and 85%, respectively. Two of the five His residues that are found in AXXXH sequences, which have been identified as putative chlorophyll ligands in the Type I and Type II proteins, were not conserved. The same two regions of high homology between the different LHC proteins, which have been identified in tomato, were also found in the Scots pine proteins. Within the conserved regions, the Type I and Type II proteins had the highest similarity; however, the Type II and Type III proteins also showed a similarity in the central region. The results suggest that all flowering plants (gymnosperms and angiosperms) probably have the same set of LHC polypeptides. A new nomenclature for the genes encoding LHC polypeptides (formerly cab genes) is proposed. The names lha and lhb are suggested for genes encoding LHC I and LHC II proteins, respectively, analogous to the nomenclature for the genes encoding other photosynthetic proteins.     

Enhanced photosystem 2 thermostability during leaf growth of Elm (Ulmus pumila) seedlings

We examined photosynthetic activities and thermostability of photosystem 2 (PS2) in leaves of elm (Ulmus pumila) seedlings from initiation to full expansion. During leaf development, net photosynthetic rate (P _N) increased gradually and reached the maximum when leaves were fully developed. In parallel with the increase of P _N, chlorophyll (Chl) content was significantly elevated. Chl a fluorescence measurements showed that the maximum quantum yield of PS2 (ϕ_PS2), the efficiency a trapped exciton, moved an electron into the electron transport chain further than Q_A ⁻ (Ψ_o), and the quantum yield of electron transport beyond Q_A (ϕ_Eo) increased gradually. These results were independently confirmed by our low irradiance experiments. When subjected to progressive heat stress, the young leaves exhibited considerably lower ϕ_PS2 and higher minimal fluorescence (F₀) than the mature leaves, revealing the highly sensitive nature of PS2 under heat in the newly initiating leaves. Further analysis showed that PS2 structure in the newly initiating leaves was strongly altered under heat, as evidenced by the increased fluorescence signals at the position of the K step. We therefore demonstrated an inhibition in the oxygen-evolving complex (OEC) in the young leaves. This resulted in decrease in amount of the functional PS2 reaction centres and relative increase in the PS2 reaction centres with inhibited electron transport at the acceptor side under heat. We suggest that the enhanced thermostability of PS2 during leaf development is associated with improved OEC stability.     

Phosphorus dynamics following restoration measures in the Loosdrecht Lakes (The Netherlands)

External phosphorus loads to three shallow lakes in the Netherlands were reduced by eliminating waste-water discharge and by dephosphorization of the supply water, with which water level is controlled. Concentrations of total-phosphorus and chlorophyll a were significantly reduced during 1980–1986 in L. Breukeleveen, but not in L. Vuntus and L. Loosdrecht. In 1983–1986 the phosphorus flow through several trophic levels was determined. Changes over these years were not significant. External input to the lakes still contributes substantially to the phosphorus input. Release from the sediments also contributed to the cycling of the phosphorus. Excretion by large crustacean zooplankters was important in phosphorus recycling, and delivered 20–30% of the daily phytoplankton phosphorus demand. A similar contribution is expected from fish. If one wants recovery of the lakes to be accelerated, additional measures are needed.