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.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis Huds.

A study was made of linolenic acid-dependent oxidative chlorophyll bleaching (CHLOX) by thylakoid membranes from senescing leaf tissue of a normal cultivar (cv. Rossa) and a non-yellowing mutant genotype (Bf 993) of Festuca pratensis Huds. To overcome the problem of variation in levels of endogenous chlorophyll substrate in membranes from different sources, light-harvesting complex (LHC) was used to supplement thylakoid pigment. It was shown that CHLOX is associated with both Photosystem I and LHC-rich thylakoid subfractions but that purified LHC has negligible associated CHLOX activity and stimulates the rate of bleaching by isolated entire chloroplast membranes. Non-senescent tissue of Bf 993 and Rossa had essentially identical thylakoid CHLOX levels, which subsequently declined during senescence in darkness. The half-life of CHLOX from the mutant was three times greater than that of the normal genotype. In both cultivars, the amount of CHLOX assayed in thylakoids isolated at different times during senescence was more than adequate to support the corresponding in-vivo rate of pigment degradation as calculated from the half-life for chlorophyll. It was concluded that the non-yellowing mutation is not expressed through a lack of CHLOX activity. The role of linolenic acid metabolism in the regulation of thylakoid structure and function during senescence, and as a likely site of the non-yellowing lesion, are discussed.Abbreviations CHLOX linolenic acid-dependent oxidative chlorophyll bleaching activity - CHLPX chlorophyll peroxidase - CPI chlorophyll-protein complex I - LHC light-harvesting complex - LNA linolenic acid - PSI photosystem I - PSII photosystem II - S relative senescence rate - t _1/2 lialf time for degradation     

Light-induced fluorescence quenching in the light-harvesting chlorophyll a/b protein complex

Summary Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.Abbreviations chla chlorophyll a - chlb chlorophyll b - F₀ fluorescence yield with reaction centers open - F_m fluorescence yield with reaction centres closed - F_i fluorescence at the plateau level of the fast induction phase - LHC II light-harvesting chlorophyll a/b protein complex II - PS II photosystem II - PSI photosystem I - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Immunochemical quantification of cytochrome f in leaves of a non-yellowing senescence mutant of Festuca pratensis

A non-competitive enzyme-linked immunosorbent assay (ELISA) which enables detection of as little as 0.1 ng cytochrome f in leaf extracts has been developed. No evidence for specific or non-specific interference by proteins other than cytochrome f was found. The assay was applied to a comparative study of age-related changes in the cytochrome f content of leaves of Festuca pratensis Huds. cv. Rossa, and a non-yellowing mutant genotype (Bf993) having a lesion in the mechanism responsible for thylakoid membrane disassembly. Cytochrome f in senescent leaves of the latter genotype was found to be present at significantly higher levels than in the wild-type, implying an inability on the part of the mutant to degrade this protein. The results obtained by ELISA were confirmed by antibody probing of Western blots.Abbreviations Ab antibody - BSA bovine serum albumin - chl chlorophyll - cyt cytochrome - ELISA enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - LHCP-2 light-harvesting chlorophyll a/b binding protein - OEC oxygen-evolving complex - PAGE polyacrylamide gel electrophoresis - POase peroxidase - SDS sodium dodecyl sulphate - PS II Photosystem II - TBS Tris buffered saline - Tris tris (hydroxymethyl) aminomethane     

The consequences of chlorophyll deficiency for photosynthetic light use efficiency in a single nuclear gene mutation of cowpea

The light harvesting and photosynthetic characteristics of a chlorophyll-deficient mutant of cowpea (Vigna unguilata), resulting from a single nuclear gene mutation, are examined. The 40% reduction in total chlorophyll content per leaf area in the mutant is associated with a 55% reduction in pigment-proteins of the light harvesting complex associated with Photosystem II (LHC II), and to a lesser extent (35%) in the light harvesting complex associated with Photosystem I (LHC I). No significant differences were found in the Photosystem I (PS I) and Photosystem II (PS II) contents per leaf area of the mutant compared to the wildtype parent. The decreases in the PS I and PS II antennae sizes in the mutant were not accompanied by any major changes in quantum efficiencies of PS I and PS II in leaves at non-saturating light levels for CO₂ assimilation. Although the chlorophyll deficiency resulted in an 11% decrease in light absorption by mutant leaves, their maximum quantum yield and light saturated rate of CO₂ assimilation were similar to those of wildtype leaves. Consequently, the large and different decreases in the antennae of PS II and PS I in the mutant are not associated with any loss of light use efficiency in photosynthesis.Abbreviations LHC I, LHC II light harvesting chlorophyll a/b protein complexes associated with PS I and PS II - A₈₂₀ light-induced absorbance change at 820 nm - ø_{PS I}, ø_{PS II} relative quantum efficiencies of PS I and PS II photochemistry     

Evaluation of the role of State transitions in determining the efficiency of light utilisation for CO2 assimilation in leaves

Wheat leaves were exposed to light treatments that excite preferentially Photosystem I (PS I) or Photosystem II (PS II) and induce State 1 or State 2, respectively. Simultaneous measurements of CO₂ assimilation, chlorophyll fluorescence and absorbance at 820 nm were used to estimate the quantum efficiencies of CO₂ assimilation and PS II and PS I photochemistry during State transitions. State transitions were found to be associated with changes in the efficiency with which an absorbed photon is transferred to an open PS II reaction centre, but did not correlate with changes in the quantum efficiencies of PS II photochemistry or CO₂ assimilation. Studies of the phosphorylation status of the light harvesting chlorophyll protein complex associated with PS II (LHC II) in wheat leaves and using chlorina mutants of barley which are deficient in this complex demonstrate that the changes in the effective antennae size of Photosystem II occurring during State transitions require LHC II and correlate with the phosphorylation status of LHC II. However, such correlations were not found in maize leaves. It is concluded that State transitions in C₃ leaves are associated with phosphorylation-induced modifications of the PS II antennae, but these changes do not serve to optimise the use of light absorbed by the leaf for CO₂ assimilation.Abbreviations Fm, Fo, Fv maximal, minimal and variable fluorescence yields - 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 - _{PS I}, _{PS II} relative quantum efficiencies of PS I and PS II photochemistry - _{CO 2} quantum yield of CO₂ assimilation     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Proteins of photosystem II

The senescence of leaves is characterized by yellowing as chlorophyll pigments are degraded. Proteins of the chloroplasts also decline during this phase of development. There exists a non-yellowing mutant genotype of Festuca pratensis Huds. which does not suffer a loss of chlorophyll during senescence. The fate of chloroplast membrane proteins was studied in mutant and wild-type plants by immune blotting and immuno-electron microscopy. Intrinsic proteins of photosystem II, exemplified by the light-harvesting chlorophyll a/b-binding protein (LHCP-2) and D1, were shown to be unusually stable in the mutant during senescence, whereas the extrinsic 33-kilodalton protein of the oxygen-evolving complex was equally lable in both genotypes. An ultrastructural study revealed that while the intrinsic proteins remained in the internal membranes of the chloroplasts, they ceased to display the heterogenous lateral distribution within the lamellae which was characteristic of nonsenescent chloroplasts. These observations are discussed in the light of possible mechanisms of protein turnover in chloroplasts.Abbreviations kDa kilodalton - LHCP-2 light-harvesting chlorophyll a/b-binding protein - M_r relative molecular mass - PSII photosystem II - SDS sodium dodecyl sulphate     

A mechanism of nonphotochemical energy dissipation, independent from PsbS, revealed by a conformational change in the antenna protein CP26

The regulation of light harvesting in higher plant photosynthesis, defined as stress-dependent modulation of the ratio of energy transfer to the reaction centers versus heat dissipation, was studied by means of carotenoid biosynthesis mutants and recombinant light harvesting complexes (LHCs) with modified chromophore binding. The npq2 mutant of Arabidopsis thaliana, blocked in the biosynthesis of violaxanthin and thus accumulating zeaxanthin, was shown to have a lower fluorescence yield of chlorophyll in vivo and, correspondingly, a higher level of energy dissipation, with respect to the wild-type strain and npq1 mutant, the latter of which is incapable of zeaxanthin accumulation. Experiments on purified thylakoid membranes from all three mutants showed that the major source of the difference between the npq2 and wild-type preparations was a change in pigment to protein interactions, which can explain the lower chlorophyll fluorescence yield in the npq2 samples. Analysis of the xanthophyll binding LHC proteins showed that the Lhcb5 photosystem II subunit (also called CP26) undergoes a change in its pI upon binding of zeaxanthin. The same effect was observed in wild-type CP26 upon treatment that leads to the accumulation of zeaxanthin in the membrane and was interpreted as the consequence of a conformational change. This hypothesis was confirmed by the analysis of two recombinant proteins obtained by overexpression of the Lhcb5 apoprotein in Escherichia coli and reconstitution in vitro with either violaxanthin or zeaxanthin. The V and Z containing pigment-protein complexes obtained by this procedure showed different pIs and high and low fluorescence yields, respectively. These results confirm that LHC proteins exist in multiple conformations, an idea suggested by previous spectroscopic measurements (Moya et al., 2001), and imply that the switch between the different LHC protein conformations is activated by the binding of zeaxanthin to the allosteric site L2. The results suggest that the quenching process induced by the accumulation of zeaxanthin contributes to qI, a component of NPQ whose origin was previously poorly understood.     

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     

Role of Light-harvesting Complex 2 Dissociation in Protecting the Photosystem 2 Reaction Centres Against Photodamage in Soybean Leaves and Thylakoids

The protective role of light-harvesting complex 2 (LHC2) dissociation from photosystem 2 (PS2) complex was explored by the 5-p-fluorosulfonylbenzoyl adenosine (FSBA, an inhibitor of protein kinase) treatment at saturating irradiance (SI) in soybean leaves and thylakoids. The dissociation of some LHC2s from PS2 complex occurred after SI treatment, but FSBA treatment inhibited the dissociation as demonstrated by analysis of sucrose density gradient centrifugation of thylakoid preparation and low-temperature (77 K) chlorophyll (Chl) fluorescence. A significant increase in F₀ and decrease in F_v/F_m occurred after SI, and the two parameters could largely recover to the levels of dark-adapted leaves after subsequent 3 h in the dark, but they could not recover in the FSBA-treated leaves at SI. Neither the electron transport activity of PS2 nor the D1 protein amount in vivo had significant change after SI without FSBA, whereas FSBA treatment at SI could result in significant decreases in both the PS2 electron transport activity and the D1 protein amount. When thylakoids instead of leaves were used, the PS2 electron transport activity and the D1 protein amount declined more after SI with FSBA than without FSBA. The phosphorylation level of PS2 core proteins increased, while the phosphorylation level of LHC2 proteins was reduced after SI. Also, the phosphorylation of PS2 core proteins could be greatly inhibited by the FSBA treatment at SI. Hence in soybean leaf the LHC2 dissociation is an effective strategy protecting PS2 reaction centres against over-excitation and photodamage by reducing the amount of photons transferred to the centres under SI, and the phosphorylation of PS2 core proteins plays an important role in the dissociation.     

Prolonged high light treatment of plant cells results in changes of the amount,the localization and the electrophoretic behavior of several thylakoid membrane proteins

The effect of a 30 h high light treatment on the amount and the localization of thylakoid proteins was analysed in low light grown photoautotrophic cells of Marchantia polymorpha and Chenopodium rubrum. High light treatment resulted in a net loss of D1 protein which was accompanied by comparable losses of other proteins of the PS II core (reaction center with inner antenna). LHC II proteins were not reduced correspondingly, indicating that these complexes are less affected by prolonged high light. High light influenced the distribution of PS II components between the grana and the stroma region of the thylakoid membrane, probably by translocation of the respective PS II proteins. Additionally, modifications of several thylakoid proteins were detected in high light treated cells of C. rubrum. These effects are discussed in relation to photoinhibitory damage and repair processes.Abbreviations BCA bioinchonic acid - chl chlorophyll - CF₁ coupling factor - CYC cycloheximide - GT grana thylakoids - HL high light - LL low light - PAGE polyacrylamide gel electrophoresis - PFD photon flux density - PS I Photosystem I - PS II Photosystem II - RC reaction center - SDS sodium dodecylsulfate - ST stroma thylakoids - Thyl unfractionated thylakoids     

The roles of specific xanthophylls in light utilization

To evaluate the role of specific xanthophylls in light utilization, wild-type and xanthophyll mutant plants (npq1, npq2, lut2, lut2npq1 and lut2npq2) from Arabidopsis thaliana were grown under three different light regimes: 30 (low light, LL), 150 (medium light, ML) and 450 (high light, HL) μmol photons m⁻² s⁻¹. We studied the pigment content, growth rate, xanthophyll cycle activity, chlorophyll fluorescence parameters and the response to photoinhibition. All genotypes differed strongly in the growth rates and the resistance against photoinhibition. In particular, replacement of lutein (Lut) by violaxanthin (Vx) in the lut2npq1 mutant did not affect the growth at non-saturating light intensities (LL and ML), but led to a pronounced reduction of growth under HL conditions, indicating an important photoprotective role of Lut. This was further supported by a much higher sensitivity of all Lut-deficient plants to photoinhibition in comparison with the wild type. In contrast, replacement of Lut by zeaxanthin (Zx) in lut2npq2 led to a pronounced reduction of growth under all light regimes, most likely related to the permanent non-photochemical dissipation of excitation energy by Zx at Vx-binding sites and the destabilization of antenna proteins by binding of Zx to Lut-binding sites. The high susceptibility of lut2npq2 to photoinhibition in comparison with npq2 further indicated that the photoprotective function of Zx is abolished in the absence of Lut. Thus, it can be concluded from our work that neither Vx nor Zx is able to fulfil the essential photoprotective function at Lut-binding sites under in vivo conditions.     

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     

Assembly and composition of the chlorophyll a-b light-harvesting complex of barley (Hordeum vulgare L.): Immunochemical analysis of chlorophyll b-less and chlorophyll b-deficient mutants

The chlorina-f2 mutant of barley (Hordeum vulgare L.) contains no chlorophyll b in its light-harvesting antenna, whereas the chlorina-103 mutant contains approximately 10% of the chlorophyll b found in wild-type. The absolute chlorophyll antenna size for Photosystem-II in wild-type, chlorina-103 and chlorina-f2 mutant was 250, 58 and 50 chlorophyll molecules, respectively. The absolute chlorophyll antenna size for Photosystem-I in wild-type, chlorina-103 and chlorina-f2 mutant was 210, 137 and 150 chlorophyll molecules, respoectively. In spite of the smaller PS I antenna size in the chlorina mutants, immunochemical analysis showed the presence of polypeptide components of the LHC-I auxiliary antenna with molecular masses of 25, 19.5 and 19 kDa. The chlorophyll a-b-binding LHC-II auxiliary antenna of PS II contained five polypeptide subunits in wild-type barley, termed a, b, c, d and e, with molecular masses of 30, 28, 27, 24 and 21 kDa, respectively. The polypeptide composition of the LHC-II auxiliary antenna of PS II was found to be identical in the two mutants, with only the 24 kDa subunit d present at an equal copy number per PS II in each of the mutants and in the wild-type barley. This d subunit assembles stably in the thylakoid membrane even in the absence of chlorophyll b and exhibits flexibility in its complement of bound chlorophylls. We suggest that polypeptide subunit d binds most of the chlorophyll associated with the residual PS II antenna in the chlorina mutants and that is proximal to the PS II-core complex.Abbreviations CP chlorophyll-protein - LHC the chlorophyll a-b binding light-harvesting complex - LHC-II subunit a the Lhcb4/5 gene product - subunit b the Lhcb1 gene product - subunit c Lhcb2 the gene product - subunit d the Lhcb3 gene product - subunit e the Lhcb6 gene product - PMSF phenylmethane sulphonyl fluoride - RC reaction center - Q_A the primary quinone electron acceptor of Photosystem-II - P700 the reaction center of PS I     

Chlorophyll fluorescence changes at high temperatures induced by linear heating of greening barley leaves

A relative decrease of the high temperature part (above 60°C) of the chlorophyll fluorescence temperature curve during 3 h to 10 h greening period of barley (Hordeum vulgare L.) leaves was found to be concomitant to a decrease of Chl alb ratio and to a gradual increase of LHCP/core ratio found by electrophoresis and the ratio of granal to total length of thylakoid membranes. It is suggested that the high temperature part of the fluorescence temperature curve depends inversely on the relative amount of LHC II in thylakoid membranes.Abbreviations Chl a(b) chlorophyll a(b) - CPa chlorophyll a protein complex of PS II - CP1 P700 chlorophyll a protein complex of PS I - FP free pigments - FTC fluorescence temperature curve - F(T30) fluorescence intensity at 30°C - LHC II light harvesting complex II - LHCP light harvesting chlorophyll protein - LHCP3 (LHCPm) monomeric form of LHC II - LHCPo oligomeric form of LHC II complex - M1 first maximum of FTC - M2 second maximum (region) of FTC - PAA polyacrylamide - PAR photosynthetically active radiation - PS I(II) Photosystem I(II) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Cytochrome b 6 f complex: Dynamic molecular organization,function and acclimation

The cytochrome b ₆ f complex occupies a central position in photosynthetic electron transport and proton translocation by linking PS II to PS I in linear electron flow from water to NADP⁺, and around PS I for cyclic electron flow. Cytochrome b ₆ f complexes are uniquely located in three membrane domains: the appressed granal membranes, the non-appressed stroma thylakoids and end grana membranes, and also the non-appressed grana margins, in contrast to the marked lateral heterogeneity of the localization of all other thylakoid multiprotein complexes. In addition to its vital role in vectorial electron transfer and proton translocation across the membrane, cytochrome b ₆ f complex is also involved in the regulation of balanced light excitation energy distribution between the photosystems, since its redox state governs the activation of LHC II kinase (the kinase that phosphorylates the mobile peripheral fraction of the chlorophyll a/b-proteins of LHC II of PS II). Hence, cytochrome b ₆ f complex is the molecular link in the interactive co-regulation of light-harvesting and electron transfer.The importance of a highly dynamic, yet flexible organization of the thylakoid membranes of plants and green algae has been highlighted by the exciting discovery that a lateral reorganization of some cytochrome b ₆ f complexes occurs in the state transition mechanism both in vivo and in vitro (Vallon et al. 1991). The lateral redistribution of phosphorylated LHC II from stacked granal membrane regions is accompanied by a concomitant movement of some cytochrome b ₆ f complexes from the granal membranes out to the PS I-containing stroma thylakoids. Thus, the dynamic movement of cytochrome b ₆ f complex as a multiprotein complex is a molecular mechanism for short-term adaptation to changing light conditions. With the concept of different membrane domains for linear and cyclic electron flow gaining credence, it is thought that linear electron flow occurs in the granal compartments and cyclic electron flow is localised in the stroma thylakoids at non-limiting irradiances. It is postulated that dynamic lateral reversible redistribution of some cytochrome b ₆ f complexes are part of the molecular mechanism involved in the regulation of linear electron transfer (ATP and NADPH) and cyclic electron flow (ATP only). Finally, the molecular significance of the marked regulation of cytochrome b ₆ f complexes for long-term regulation and optimization of photosynthetic function under varying environmental conditions, particularly light acclimation, is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - PS Photosystem     

An amino-proximal hydrophobic domain in the major light-harvesting chlorophyll a/b-protein is essential for membrane integration and protein stability

The major light-harvesting chlorophyll a/b-protein (LHCP) of higher plant chloroplasts is a nuclearencoded, integral thylakoid membrane protein that binds photosynthetic pigments and occurs in situ in an oligomeric form. We have previously examined structural and functional domains of the mature apoprotein by use of mutant LHCPs and in vitro assays for uptake and insertion. Results presented here demonstrate the effects of several mutations in the amino terminal domain of the mature apoprotein. Deletion of amino acid residues 12–58 greatly affected import into chloroplasts, while deletion or alteration of the hydrophobic region E⁶⁵VIHARWAM⁷³ led to rapid degradation of the mutant LHCP. We suggest that this amino-proximal region is essential for the stability of the LHCP and its ability to integrate into the thylakoid membranes. A structural/functional relationship of this region to a previously examined hydrophobic carboxy-proximal domain [Kohorn and Tobin (1989), The Plant Cell 1, 159–166] is proposed.Abbreviations BSA bovine serum albumin faction V - ELIPs early light-inducible proteins - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - LHCP light-harvesting chlorophyll a/b-protein - LHC IIb light-harvesting complex associated with Photosystem II - pLHCP precursor to LHCP - Rubisco ribulose 1,5-biphosphate carboxylase-oxygenase - SDS-PAGE sodium dodecyl sulfate-poly-acrylamide gel electrophoresis     

Contrasting modes of regulation of PS II light utilization with changing irradiance in normal and psbS mutant leaves of Arabidopsis thaliana

Complementary techniques of chlorophyll a fluorescence, steady state CO₂ exchange, and O₂ release during a multiple turnover flash were applied to compare responses to irradiance for leaves of wild type and psbS mutants. The latter included variants in which the psbS gene was deleted (npq4-1) or possessed a single point mutation (npq4-9). Nonphotochemical quenching (NPQ) was reduced by up to 80 and 50%, respectively, in these lines at high irradiance. Analysis of changes in steady-state fluorescence yields and quantum yield of linear electron transport in the context of the reversible radical pair model of Photosystem II (PS II) indicated that NPQ occurs by nonradiative deactivation of chlorophyll singlet states in normal leaves. In contrast, application of the same criteria together with the observed irreversibility of NPQ and decline in density of functional PS II reaction centers following excessive illumination indicated a change in reaction center properties for the psbS deletion phenotype (Npq4-1^–). Specifically, PS II reaction centers in Npq4-1^– convert to a photochemically inactive, yet strongly quenching, form in intense light. The possibility of formation of a carotenoid or chlorophyll cation quencher in the reaction center is discussed. Results for the point mutant phenotype (Npq4-9^–) were intermediate to those of wild-type and Npq4-1^–. Furthermore, wild-type leaves exhibited a significant reversible increase in the PS II in vivo rate constant for photochemistry (k_P0) in saturating compared to limiting light. Changes in k_P0 could not be accounted for in terms of a classic phosphorylation-dependent (state transition) mechanism. Changes in k_P0 may arise from alternate pigment—protein conformations that alter the way excitons equilibrate among PS II chromophores. The lack of similar irradiance-dependent changes in k_P0 for the psbS mutants suggests a role for the PS II-S protein in the regulation of exciton distribution.This revised version was published online in October 2005 with corrections to the Cover Date.     

Photosystem II in a mutant of Chlamydomonas reinhardtii lacking the 23 kDa psbP protein shows increased sensitivity to photoinhibition in the absence of chloride

The psbP gene product, the so called 23 kDa extrinsic protein, is involved in water oxidation carried out by Photosystem II. However, the protein is not absolutely required for water oxidation. Here we have studied Photosystem II mediated electron transfer in a mutant of Chlamydomonas reinhardtii, the FUD 39 mutant, that lacks the psbP protein. When grown in dim light the Photosystem II content in thylakoid membranes of FUD 39 is approximately similar to that in the wild-type. The oxygen evolution is dependent on the presence of chloride as a cofactor, which activates the water oxidation with a dissociation constant of about 4 mM. In the mutant, the oxygen evolution is very sensitive to photoinhibition when assayed at low chloride concentrations while chloride protects against photoinhibition with a dissociation constant of about 5 mM. The photoinhibition is irreversible as oxygen evolution cannot be restored by the addition of chloride to inhibited samples. In addition the inhibition seems to be targeted primarily to the Mn-cluster in Photosystem II as the electron transfer through the remaining part of Photosystem II is photoinhibited with slower kinetics. Thus, this mutant provides an experimental system in which effects of photoinhibition induced by lesions at the donor side of Photosystem II can be studied in vivo.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DPC 2,2-diphenylcarbonic dihydrazide - HEPES 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - P₆₈₀ the primary electron donor to PS II - PpBQ phenyl-p-benzoquinone - PS II Photosystem II - Q_A the first quinone acceptor of PS II - Q_B the second quinone acceptor of PS II - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)aminomethane - Tyr_D accessory electron donor on the D₂-protein - Tyr_Z tyrosine residue, acting as electron carrier between P₆₈₀ and the water oxidizing system     

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.