Disturbed excitation energy transfer in Arabidopsis thaliana mutants lacking minor antenna complexes of photosystem II

Minor light-harvesting complexes (Lhcs) CP24, CP26 and CP29 occupy a position in photosystem II (PSII) of plants between the major light-harvesting complexes LHCII and the PSII core subunits. Lack of minor Lhcs in vivo causes impairment of PSII organization, and negatively affects electron transport rates and photoprotection capacity. Here we used picosecond-fluorescence spectroscopy to study excitation-energy transfer (EET) in thylakoid membranes isolated from Arabidopsis thaliana wild-type plants and knockout lines depleted of either two (koCP26/24 and koCP29/24) or all minor Lhcs (NoM). In the absence of all minor Lhcs, the functional connection of LHCII to the PSII cores appears to be seriously impaired whereas the “disconnected” LHCII is substantially quenched. For both double knock-out mutants, excitation trapping in PSII is faster than in NoM thylakoids but slower than in WT thylakoids. In NoM thylakoids, the loss of all minor Lhcs is accompanied by an over-accumulation of LHCII, suggesting a compensating response to the reduced trapping efficiency in limiting light, which leads to a photosynthetic phenotype resembling that of low-light-acclimated plants. Finally, fluorescence kinetics and biochemical results show that the missing minor complexes are not replaced by other Lhcs, implying that they are unique among the antenna subunits and crucial for the functioning and macro-organization of PSII.     

Acclimation of Arabidopsis thaliana to the light environment: regulation of chloroplast composition

The regulation by light of the composition of the photosynthetic apparatus was investigated in Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. When grown in high- and low-irradiance white light, wild-type plants and photomorphogenic mutants showed large differences in their maximum photosynthetic rate and chlorophyll a/b ratios; such changes were abolished by growth in red light. Photosystem I (PSI) and PSII levels were measured in wild-type plants grown under a range of light environments; the results indicate that regulation of photosystem stoichiometry involves the specific detection of blue light. Supplementing red growth lights with low levels of blue light led to large increases in PSII content, while further increases in blue irradiance had the opposite effect; this latter response was abolished by the hy4 mutation, which affects certain events controlled by a blue-light receptor. Mutants defective in the phytochrome photoreceptors retained regulation of photosystem stoichiometry. We discuss the results in terms of two separate responses controlled by blue-light receptors: a blue-high-fluence response which controls photosystem stoichiometry; and a blue-low-fluence response necessary for activation of such control. Variation in the irradiance of the red growth light revealed that the blue-high-fluence response is attenuated by red light; this may be evidence that photosystem stoichiometry is controlled not only by photoreceptors, but also by photosynthetic metabolism.Abbreviations BHF blue-high-fluence - BLF blue-low-fluence - Chl chlorophyll - FR far-red light - LHCII light-harvesting complex of PSII - P_max maximum photosynthetic rate - R red light - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This work was supported by Natural Environment Research Council Grant No. GR3/7571A. We would like to thank H. Smith (Botany Department, University of Leicester) and E. Murchie (INRA, Versailles) for helpful discussions.     

Polypeptide composition,assembly and phosphorylation patterns of the photosystem II antenna system of Chlamydomonas reinhardtii

In recent years major progress has been made in describing the gene families that encode the polypeptides of the light-harvesting antenna system of photosystem II (PSII). At the same time, advances in the biochemical characterization of these antennae have been hampered by the high degree of similarity between the apoproteins. To help interpret the molecular results, we have re-examined the composition, the assembly and the phosphorylation patterns of the light-harvesting antenna of PSII (LHCII) in the green alga Chlamydomonas reinhardtii Dang, using a non-Tris SDS-PAGE system capable of resolving polypeptides that differ by as little as 200 daltons. Research to date has suggested that in C. reinhardtii the LHCII comprises just four polypeptides (p11, p13, p16 and p17), and CP29 and CP26 just one polypeptide each (p9 and p10, respectively), i.e. a total of six polypeptides. We report here that these antenna systems contain at least 15 polypeptides, 10 associated with LHCII, 3 with CP29, and 2 with CP26. All of these polypeptides have been positively identified by means of appropriate antibodies. We also demonstrate substantial heterogeneity to the pattern of in-vitro phosphorylation, with major differences found among members of closely spaced and immunologically related polypeptides. Most intriguing is the fact that the polypeptides that cross-react with the anti-type 2 LHCII antibodies of higher plants (p16, and to a lesser extent p11) are not phosphorylated, whereas in higher plants these are the most highly phosphorylated polypeptides. Also, unlike in higher plants, CP29 is heavily phosphorylated. Phosphorylation does not appear to have any effect on the mobility of polypeptides on fully denaturing SDS-PAGE gels. To learn more about the accumulation and organization of the light-harvesting polypeptides, we have also investigated a chlorophyll b-less mutant, cbn1-48. The LHCII is almost completely lost in this mutant, along with at least some LHCI. But the accumulation of CP29 and CP26 and their binding to PSII core complexes, is relatively unaffected. As expected, the loss of antenna polypeptides is accompanied by a reduction of the size of large reaction-center complexes. Following in-vitro phosphorylation the number of phosphorylated proteins is greatly increased in the mutant thylakoids compared to wildtype thylakoids. We present a model of the PSII antenna system to account for the new polypeptide complexity we have demonstrated.This work was supported by National Institute of Health grant GM22912 to L.A.S. We would like to thank Anastasios Melis for helpful discussions.     

The State of Photosystem I Complexes in the Central Regions of Pea Granal Thylakoids

Grana-core and grana-margin fragments were obtained from pea (Pisum sativum L.) thylakoids, and both fractions contained photosystem I (PSI) complexes. The yield of these fractions exhibited variations for the plants grown during various periods of the summer season. Low-temperature fluorescence spectra, excitation spectra of long-wave fluorescence, and P700 kinetic characteristics were recorded for these fractions. PSI complexes in central granal regions were associated with PSII and the light-harvesting complexes of PSII, which followed from the excitation spectra of long-wave fluorescence and the kinetic characteristics of P700 light oxidation and dark reduction. The characteristics of the margin regions were changed depending on the fraction yield. If the yield was low, marginal fragments contained mainly PSI complexes. When the yield increased, PSI associates with PSII appeared. A spatial distribution and state of PSI complexes in granal thylakoids are discussed as related to the size and composition of the light-harvesting antenna.     

Chlorophyll-proteins from maize seedlings grown under intermittent light conditions

We studied the organization of the antenna system of maize (Zea mays L.) seedlings grown under intermittent light conditions for 11 d. These plants had a higher chlorophyll-a/b ratio, a higher ratio of carotenoids to chlorophyll and a lower ratio of chlorophyll to protein than plants grown in continuous light. We found all chlorophyll-protein complexes of maize to be present. However, the minor chlorophyll a/b-proteins CP29 and CP26, and to a greater extent CP24 and the major light-harvesting complex II were reduced relative to the photosystem (PS) II core-complex. Also the chlorophyll a/b-antennae of PSI were reduced relative to the reaction-centre polypeptides. When isolated by flatbed isoelectrofocussing, the chlorophyll-a/b complexes of PSII showed a higher chlorophyll-a/b ratio and a lower ratio of chlorophyll to protein than the same complexes from continuous light; additionally, they bound more carotenoids per protein than the latter. Thus the altered organization of the photosynthetic apparatus of plants from intermittent light is caused by two different factors: (i) the altered stoichiometry of chlorophyll-binding proteins and (ii) a different ratio of pigment to protein within individual chlorophyll-proteins.Abbreviations Chl chlorophyll - CL continuous light - F fraction - HPLC high-performance liquid chromatography - IEF isoelectrofocussing - IL intermittent light - LHCII light-harvesting complex II - PAGE polyacrylamide-gel electrophoresis - Phe pheophytin - SDS sodium dodecyl sulfate This work was supported by the grant no. 4.7240.90 from the Italian Ministry of Agriculture and Forestry. We thank Drs. R. Barbato (Dipartimento di Biologia, Padua, Italy) and Olivier Vallon (Institut de Biologie Physico-Chimique, Paris, France) for their gifts of antibodies, Drs. R. Barbato and P. Dainese (Dipartimento di Biologia, Padua, Italy) for fruitful discussion and Prof. G. Gennari (Dipartimento di Chimica fisica, Padua, Italy) for his assistance in recording the excitation spectra. J.M. was supported by a Stipendium from the Deutsche Forschungsgemeinschaft, which is gratefully acknowledged.     

Light regulation of photosynthetic membrane structure,organization, and function

The light environment during plant growth determines the structural and functional properties of higher plant chloroplasts, thus revealing a dynamically regulated developmental system. Pisum sativum plants growing under intermittent illumination showed chloroplasts with fully functional photosystem (PS) II and PSI reaction centers that lacked the peripheral chlorophyll (Chi) a/b and Chl a light-harvesting complexes (LHC), respectively. The results suggest a light flux differential threshold regulation in the biosynthesis of the photosystem core and peripheral antenna complexes. Sun-adapted species and plants growing under far-red-depleted illumination showed grana stacks composed of few (3–5) thylakoids connected with long intergrana (stroma) thylakoids. They had a PSII/PSI reaction center ratio in the range 1.3–1.9. Shade-adapted species and plants growing under far-red-enrichcd illumination showed large grana stacks composed of several thylakoids, often extending across the entire chloroplast body, and short intergrana stroma thylakoids. They had a higher PSII/PSI reaction center ratio, in the range of 2.2–4.0. Thus, the relative extent of grana and stroma thylakoid formation corresponds with the relative amounts of PSII and PSI in the chloroplast, respectively. The structural and functional adaptation of the photosynthetic membrane system in response to the quality of illumination involves mainly a control on the rate of PSII and PSI complex biosynthesis.     

Structural organization of photosynthetic apparatus in agranal chloroplasts of maize

We investigated the organization of photosystem II (PSII) in agranal bundle sheath thylakoids from a C(4) plant maize. Using blue native/SDS-PAGE and single particle analysis, we show for the first time that PSII in the bundle sheath (BS) chloroplasts exists in a dimeric form and forms light-harvesting complex II (LHCII).PSII supercomplexes. We also demonstrate that a similar set of photosynthetic membrane complexes exists in mesophyll and agranal BS chloroplasts, including intact LHCI.PSI supercomplexes, PSI monomers, PSII core dimers, PSII monomers devoid of CP43, LHCII trimers, LHCII monomers, ATP synthase, and cytochrome b(6)f complex. Fluorescence functional measurements clearly indicate that BS chloroplasts contain PSII complexes that are capable of performing charge separation and are efficiently sensitized by the associated LHCII. We identified a fraction of LHCII present within BS thylakoids that is weakly energetically coupled to the PSII reaction center; however, the majority of BS LHCII is shown to be tightly connected to PSII. Overall, we demonstrate that organization of the photosynthetic apparatus in BS agranal chloroplasts of a model C(4) plant is clearly distinct from that of the stroma lamellae of the C(3) plants. In particular, supramolecular organization of the dimeric LHCII.PSII in the BS thylakoids strongly suggests that PSII in the BS agranal membranes may donate electrons to PSI. We propose that the residual PSII activity may supply electrons to poise cyclic electron flow around PSI and prevent PSI overoxidation, which is essential for the CO(2) fixation in BS cells, and hence, may optimize ATP production within this compartment.     

Alterations of the chlorophyll-protein pattern in chronically photoinhibited Chenopodium rubrum cells

When photoautotrophic Chenopodium rubrum L. culture cells were exposed to high photon flux densities for seven consecutive light periods a marked reduction in photochemical efficiency, chlorophyll (Chl) content and Chl a/b ratio occurred. These alterations were accompanied by distinct changes in the pigment and protein composition of the thylakoid membranes. In photosystem II (PSII) a reduction in the relative contents of proteins from the reaction center (D1 protein, D2 protein and Cyt b₅₅₉) and the inner antenna (CP43 and CP47) was observed. In agreement with the reduction in the Chl a/b ratio an increase in the relative content of the major light-harvesting complex of PSII (LHCII) could be demonstrated. The minor chlorophyll-proteins of PSII were only slightly affected but PSI (quantified as total complex) showed a reduction upon chronic photoinhibition. The changes in protein composition were accompanied by a drastic increase in the contents of lutein and the xanthophyll-cycle pigments and by a reduction in the β-carotene content. The effects on lutein and xanthophyll-cycle pigment content were most pronounced in stroma thylakoids. Here, an increase in LHCII (which harbours these pigments) was clearly detectable. Considering the pigment content of LHCII, the change in its apoprotein content was not large enough to explain the pigment changes.     

Deletion mutations in a long hydrophilic loop in the photosystem II chlorophyll-binding protein CP43 in the cyanobacterium Synechocystis sp. PCC 6803

In order to investigate the role and function of the hydrophilic region between transmembrane regions V and CI in the photosystem II core antenna protein CP43, we introduced eight different deletions in psbC of Synechocystis sp; PCC 6803 resulting in a loss of 7–11 codons in evolutionary conserved domains in this region. All deletions resulted in an obligate photoheterotrophic phenotype (requirement of glucose for cell growth) and the absence of any detectable oxygen evolution activity. The various deletion mutations showed a different impact on the amount of CP43 in the thylakoid, ranging from wild-type levels of (a now slightly smaller) CP43 to no detectable CP43 at all. All deletions led to a decrease in the amount of the D1 and D2 proteins in the thylakoids with a larger effect on D2 than on D1. CP47, the other major chlorophyll-binding protein, was present in reduced but significant amounts in the thylakoid. Herbicide binding (diuron) was lost in all but one mutant indicating the PSII components are not assembled into functionally intact complexes. Fluorescence-emission spectra confirmed this notion. This indicates that the large hydrophilic loop of CP43 plays an important role in photosystem II, and even though a shortened CP43 is present in thylakoids of most mutants, functional characteristics resemble that of a mutant with interrupted psbC.Abbreviations CP chlorophyll-binding protein - DCPIP 2,6-dichlorophenolindophenol - DPC diphenylcarbazide - ferricyanide K₃Fe(CN)₆ - HEPES N-(2-hydroxyelthyl)piperazine-N-(2-hydroxypropane sulfonic acid) - MES 2-(N-morpholino)-ethanesulfonic acid - PCC Pasteur Culture Collection - PCR polymerase chain reaction - PS photosystem - Q_A first quinone acceptor in PSII - Q_B second quinone acceptor in PSII - Z redox-active tyrosine (Y161) in D1 serving as electron carrier between the Mn cluster and P680     

Repressible chloroplast gene expression in Chlamydomonas: A new tool for the study of the photosynthetic apparatus

A repressible/inducible chloroplast gene expression system has been used to conditionally inhibit chloroplast protein synthesis in the unicellular alga Chlamydomonas reinhardtii. This system allows one to follow the fate of photosystem II and photosystem I and their antennae upon cessation of chloroplast translation. The main results are that the levels of the PSI core proteins decrease at a slower rate than those of PSII. Amongst the light-harvesting complexes, the decrease of CP26 proceeds at the same rate as for the PSII core proteins whereas it is significantly slower for CP29, and for the antenna complexes of PSI this rate is comprised between that of CP26 and CP29. In marked contrast, the components of trimeric LHCII, the major PSII antenna, persist for several days upon inhibition of chloroplast translation. This system offers new possibilities for investigating the biosynthesis and turnover of individual photosynthetic complexes in the thylakoid membranes. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Characteristics of Photosystem I Complexes in the End Membranes of Pea Granal Thylakoids

Two fractions of the light fragments enriched in the photosystem I (PSI) complexes were obtained from pea (Pisum sativum L.) thylakoids by digitonin treatment and subsequent differential centrifugation. The ratio of chlorophyll a to chlorophyll b, chlorophyll/P700 spectra of low-temperature fluorescence, and excitation spectra of long-wave fluorescence were measured. These characteristics were shown to be different due to variation in the size and composition of the light-harvesting antenna of PSI complexes present in the particles obtained. The larger antenna size of one of the fractions was related to the incorporation of the pool of light-harvesting complex II (LHCII). A comparison with the data available allowed us to identify these particles as fragments of intergranal thylakoids and end membranes of granal thylakoids. The suggestion that an increase in the PSI light-harvesting antenna in intergranal thylakoids is related to the attachment of phosphorylated LHCII is discussed.     

Effect of Light Quality on the Composition, Function, and Structure of Photosynthetic Thylakoid Membranes of Asplenium australasicum (Sm.) Hook

The effect of light quality on the composition, function and structure of the thylakoid membranes, as well as on the photosynthetic rates of intact fronds from Asplenium australasicum, a shade plant, grown in blue, white, or red light of equal intensity (50 microeinsteins per square meter per second) was investigated. When compared with those isolated from plants grown in white and blue light, thylakoids from plants grown in red light have higher chlorophyll a/chlorophyll b ratios and lower amounts of light-harvesting chlorophyll a/b-protein complexes than those grown in blue light. On a chlorophyll basis, there were higher levels of PSII reaction centers, cytochrome f and coupling factor activity in thylakoids from red light-grown ferns, but lower levels of PSI reaction centers and plastoquinone. The red light-grown ferns had a higher PSII/PSI reaction center ratio of 4.1 compared to 2.1 in blue light-grown ferns, and a larger apparent PSI unit size and a lower PSII unit size. The CO₂ assimilation rates in fronds from red light-grown ferns were lower on a unit area or fresh weight basis, but higher on a chlorophyll basis, reflecting the higher levels of electron carriers and electron transport in the thylakoids.

The structure of thylakoids isolated from plants grown under the three light treatments was similar, with no significant differences in the number of thylakoids per granal stack or the ratio of appressed membrane length/nonappressed membrane length. The large freeze-fracture particles had the same size in the red-, blue-, and white-grown ferns, but there were some differences in their density. Light quality is an important factor in the regulation of the composition and function of thylakoid membranes, but the effects depend upon the plant species.

     

Photosynthetic characteristics of detached barley leaves during greening in the presence of SAN 9785

The effects of the pyridazinone compound SAN 9785 on the photosynthetic competence of leaves, on the photochemical activity of isolated thylakoids and on the formation and spectral properties of chlorophyll-protein complexes were studied during a 72-h greening period of detached etiolated leaves of barley (Hordeum vulgare L. cv. Horpácsi kétsoros). It was established that i) the photosynthetic capacity of the leaves decreased considerably (by 80 and 90%, as determined by¹⁴CO₂ fixation and fast fluorescence induction measurements, respectively); ii) the photochemical activity of isolated thylakoids from water to potassium ferricyanide and from dichlorophenol indophenol/ascorbate to methylviologen exhibited only slight reductions when expressed on a chlorophyll basis compared with the control; iii) the slow fluorescence induction curves of the treated leaves demonstrated the presence of a peculiar fluorescence component interrupting the quenching of fluorescence at around 1 min illumination; iv) a shortage of the chlorophyll-protein complex of photosystem I (CPI) occurred with a higher content of the monomer of the light harvesting complex in the thylakoids of treated leaves; and v) the fluorescence spectrum of the CPI band present in treated leaves indicates the destruction of the structural integrity of this complex during isolation from the membrane.Abbreviations Chl chlorophyll - CPI, CPII chlorophyll-protein complexes of the reaction centres of PSI and PSII - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DPIP 2,6-dichlorophenol indophenol - DPIPH₂ chemically reduced form of DPIP - F _o fluorescence of constant yield - F _v fluorescence of variable yield - F _i ,F _m mitial and maximum yield of fluorescence - LHCP³ monomer of the light-harvesting complex - LHCP² and LHCP¹ oligomers of the light-harvesting complex LHCP³ - PSI, PSII photosystems I, II - SAN 9785 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone, also known as BASF 13-338 - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Alteration of thylakoid membrane structure in Brassica rapa ssp. oleifera during ageing in high and low light

Abstract Alterations in the composition and structure of thylakoids were studied in Brassica rapa ssp. oleifera grown under high and low irradiance (800 μmol m^?2 s^?1 and 80 μmol m^?2 s^?1). During ageing, both high and low light induced a decrease in total protein particle density and in the relative amount of 80–90 Å cytochrome b6/f and 90–100 Å ATP-synthetase. The density of PSII complexes in stacked (EFs) and unstacked (EFu) thylakoids also decreased. In high light, a shift was noted towards smaller PSII complexes in the EFs face with decreasing attached antenna complex CP29, but the relative amount of the antenna chlorophyll a-protein complexes of photosystem II (CPa) remained stable. In contrast, the proportion of peripheral LHCH on the PFs face and the density of PFs particles increased together with an increase in grana size. In low light, a shift occurred towards larger PSII complexes on the EFs face, along with a decrease in the proportion of CPa complexes and the PFs particle density (peripheral LHCH), though a marked increase was observed in the proportion of chlorophyll a/b-protein complexes in SDS-PAGE. The amount of photosystem I in green gel remained fairly stable, although the density of PFu particles (including PSI) increased in low and slightly diminished in high light. The results indicate that the organization of thylakoid components depends strongly on the light conditions and stage of development.     

Extensive remodeling of the photosynthetic apparatus alters energy transfer among photosynthetic complexes when cyanobacteria acclimate to far-red light

Some cyanobacteria remodel their photosynthetic apparatus by a process known as Far-Red Light Photoacclimation (FaRLiP). Specific subunits of the phycobilisome (PBS), photosystem I (PSI), and photosystem II (PSII) complexes produced in visible light are replaced by paralogous subunits encoded within a conserved FaRLiP gene cluster when cells are grown in far-red light (FRL; λ = 700–800 nm). FRL-PSII complexes from the FaRLiP cyanobacterium, Synechococcus sp. PCC 7335, were purified and shown to contain Chl a, Chl d, Chl f, and pheophytin a, while FRL-PSI complexes contained only Chl a and Chl f. The spectroscopic properties of purified photosynthetic complexes from Synechococcus sp. PCC 7335 were determined individually, and energy transfer kinetics among PBS, PSII, and PSI were analyzed by time-resolved fluorescence (TRF) spectroscopy. Direct energy transfer from PSII to PSI was observed in cells (and thylakoids) grown in red light (RL), and possible routes of energy transfer in both RL- and FRL-grown cells were inferred. Three structural arrangements for RL-PSI were observed by atomic force microscopy of thylakoid membranes, but only arrays of trimeric FRL-PSI were observed in thylakoids from FRL-grown cells. Cells grown in FRL synthesized the FRL-specific complexes but also continued to synthesize some PBS and PSII complexes identical to those produced in RL. Although the light-harvesting efficiency of photosynthetic complexes produced in FRL might be lower in white light than the complexes produced in cells acclimated to white light, the FRL-complexes provide cells with the flexibility to utilize both visible and FRL to support oxygenic photosynthesis.This article is part of a Special Issue entitled Light harvesting, edited by Dr. Roberta Croce.     

Alteration in chloroplast structure and thylakoid membrane composition due to in vivo heat treatment of rice seedlings: correlation with the functional changes

Exposure of 25 °C-grown, seven-day-old rice seedlings to mild heat stress of 40 °C for 24 h in dark did not cause any change in protein or pigment content of the thylakoids, but produced major disorganization of chloroplast ultrastructure. This heat induced disorganization of thylakoid structure/organization caused significant (65 percnt;) loss in PSII activity, slight loss in PSI activity, and brought about a decrease in relative quantum efficiency of PSII. The herbicide ¹⁴C atrazine binding assay revealed a decreased number of binding sites of the herbicide and altered the herbicide dissociation constant, suggesting that the heat induced disorganization of the thylakoids affects the acceptor side of PSII. Cation induced Chla fluorescence analyses at room temperature and low temperature indicated thatin vivo heat exposure of rice seedlings altered the extent of energy transfer in favor of PSI. Immunoblotting analysis of several PSII polypeptides such as D1/D2 reaction dimer and Cyt b₅₅₉ showed no major changes due to mild heat exposure except for the PSII core antenna polypeptide (CP43), which could reflect the reduction in PSII activity observed in light saturation studies. Similarly, haeme staining did not indicate any change in other cytochrome related polypeptides. Our results therefore clearly suggest thatin vivo exposure of rice seedlings to elevated (40 °C) temperature caused thylakoid structural disorganization, and this disorganization of some of the thylakoid complexes resulted in a loss in thylakoid photochemical function.     

Developmental Loss of Photosystem II Activity and Structure in a Chloroplast-Encoded Tobacco Mutant, Lutescens-1

Lutescens-1, a tobacco mutant with a maternally inherited dysfunction, displayed an unusual developmental phenotype. In vivo measurement of chlorophyll fluorescence revealed deterioration in photosystem II (PSII) function as leaves expanded. Analysis of thylakoid membrane proteins by polyacrylamide gel electrophoresis indicated the physical loss of nuclear- and chloroplast-encoded polypeptides comprising the PSII core complex concomitant with loss of activity. Freeze fracture electron micrographs of mutant thylakoids showed a reduced density, compared to wild type, of the EF_s particles which have been shown previously to be the structural entity containing PSII core complexes and associated pigment-proteins. The selective loss of PSII cores from thylakoids resulted in a higher ratio of antenna chlorophyll to reaction centers and an altered 77 K chlorophyll fluorescence emission spectra; these data are interpreted to indicate functional isolation of light-harvesting chlorophyll a/b complexes in the absence of PSII centers. Examination of PSII reaction centers (which were present at lower levels in mutant membranes) by monitoring the light-dependent phosphorylation of PSII polypeptides and flash-induced O₂ evolution patterns demonstrated that the PSII cores which were assembled in mutant thylakoids were functionally identical to those of wild type. We conclude that the lutescens-1 mutation affected the correct stoichiometry of PSII centers, in relation to other membrane constituents, by disrupting the proper assembly and maintenance of PSII complexes in lutescens-1 thylakoid membranes.     

Targeted interruption of the psaA and psaB genes encoding the reaction-centre proteins of photosystem I in the filamentous cyanobacterium Anabaena variabilis ATCC 29413

The two reaction-centre proteins of the photosystem I (PSI) complex are encoded by two adjacent genes named psaA and psaB. We have performed targeted mutagenesis to insertionally inactivate each of these genes in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. The resulting mutant strains, termed psaA:: Nm^R and psaB:: Nm^R, were blue because of a high ratio of phycobilin to chlorophyll and were unable to grow in light. These mutant cells also lacked chemically reducible P700 (the reaction-centre chlorophylls of PSI) and as a consequence did not exhibit any PSI-mediated photochemical activity. However, their photosystem II (PSII) complexes were fully active. The loss of the PsaA and PsaB proteins and their associated chlorophyll molecules resulted in a five- to sevenfold decrease in the chlorophyll/PSII ratio in the mutant cells relative to the wild-type cells. Interestingly, the psaS:: Nm^R and not the psaA:: Nm^R mutant strain retained a small fluorescence peak (77K) at 721 nm originating from chlorophyll molecule(s) presumably bound to a small amount of the PsaA protein present in the psaB mutant. These results demonstrate that this organism is suitable for the manipulation of PSI reaction-centre proteins.     

Response of the Photosynthetic Apparatus in Dunaliella salina (Green Algae) to Irradiance Stress

The response of the photosynthetic apparatus in the green alga Dunaliella salina, to irradiance stress was investigated. Cells were grown under physiological conditions at 500 millimoles per square meter per second (control) and under irradiance-stress conditions at 1700 millimoles per square meter per second incident intensity (high light, HL). In control cells, the light-harvesting antenna of photosystem I (PSI) contained 210 chlorophyll a/b molecules. It was reduced to 105 chlorophyll a/b in HL-grown cells. In control cells, the dominant form of photosystem II (PSII) was PSII_α(about 63% of the total PSII) containing >250 chlorophyll a/b molecules. The smaller antenna size PSII_β centers (about 37% of PSII) contained 135 ± 10 chlorophyll a/b molecules. In sharp contrast, the dominant form of PSII in HL-grown cells accounted for about 95% of all PSII centers and had an antenna size of only about 60 chlorophyll a molecules. This newly identified PSII unit is termed PSII_γ. The HL-grown cells showed a substantially elevated PSII/PSI stoichiometry ratio in their thylakoid membranes (PSII/PSI = 3.0/1.0) compared to that of control cells (PSII/PSI = 1.4/1.0). The steady state irradiance stress created a chronic photoinhibition condition in which D. salina thylakoids accumulate an excess of photochemically inactive PSII units. These PSII units contain both the reaction center proteins and the core chlorophyll-protein antenna complex but cannot perform a photochemical charge separation. The results are discussed in terms of regulatory mechanism(s) in the plant cell whose function is to alleviate the adverse effect of irradiance stress.     

Correlation between thylakoid protein phosphorylation and molecular organization of the photosynthetic apparatus in a dynamic system

In-vitro thylakoid protein phosphorylation has been studied in synchronized cells of Scenedesmus obliquus at the 8- and 16-h of the life cycle, stages which are characterized by the maximum and minimum photosynthetic activities, respectively. The stage of maximum photosynthetic activity (8-h) is characterized by the highest protein phosphorylation in vitro and in vivo, by the largest proportion of the heavy subfraction of thylakoids, and by maximum oligomerization of the light-harvesting chlorophyll a/b-protein complex, altogether creating the highest energy charge of the thylakoid membranes. Protein phosphorylation in vitro decreases the amount of the heavy subfraction and increases the amount of oligomerization of the antenna of photosystem I (PSI) (increase of chlorophyll b in the light fraction). Concomittantly, PSII units become smaller (longer time for the rise in fluorescence induction) and photosynthetic efficiency increases (decrease of fluorescence yield). In-vivo protein phosphorylation is controlled mainly endogenously during the 8-h of the life cycle but is exogenously modulated by light to optimize the photosynthetic activity by redistribution of pigment-protein complexes. In-vitro protein phosphorylation seems to restore partially the conditions prevalent in vivo and lost during the preparation of membranes. The effect is greater in 16-h cells which have less-stable membranes. The regulatory mechanism between membrane stabilization and oligomerization on the one hand and redistribution of the light-harvesting chlorophyll a/b-protein complex from PSII to PSI on the other hand remains unexplained. We have confirmed that the mechanism of protein phosphorylation is regulated via plastohydroquinone, but experiments with the plastohydroquinone analogue 2,3,5,6-tetramethyl-p-benzoquinone demonstrated that plastohydroquinone is not solely responsible for the differences in protein phosphorylation of 8- and 16-h thylakoids. The inhibitory effect of ADP and the distinct rates of kinase reaction indicate that the adenylate energy charge and changes in the organization of the photosynthetic apparatus also contribute to the observed differences in protein phosphorylation. Phosphorylation in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea indicated that the 32-kDa phosphoprotein and the herbicide-binding Q_B protein may be the same. These experiments also indicated that 3-(3,4-dichlorophenyl)-1,1-dimethylurea-binding reduces kinase activity directly and not only by inhibiting electron transport.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP light-harvesting chlorophyll a/b-protein complex - PSI, II photosystem I, II - TMQ 2,3,5,6-tetramethyl-p-benzoquinone Dedicated to Professor Dr. W. Nultsch on the occasion of his 60th brithday