Hypothesis for the evolution of three-helix Chl a/b and Chl a/c light-harvesting antenna proteins from two-helix and four-helix ancestors

The nuclear-encoded Chl a/b and Chl a/c antenna proteins of photosynthetic eukaryotes are part of an extended family of proteins that also includes the early light-induced proteins (ELIPs) and the 22 kDa intrinsic protein of PS II (encoded by psbS gene). All members of this family have three transmembrane helices except for the psbS protein, which has four. The amino acid sequences of these proteins are compared and related to the three-dimensional structure of pea LHC II Type I (Kühlbrandt and Wang, Nature 350: 130–134, 1991). The similarity of psbS to the three-helix members of the family suggests that the latter arose from a four-helix ancestor that lost its C-terminal helix by deletion. Strong internal similarity between the two halves of the psbS protein suggests that it in turn arose as the result of the duplication of a gene encoding a two-helix protein. Since psbS is reported to be present in at least one cyanobacterium, the ancestral four-helix protein may have been present prior to the endosymbiotic event or events that gave rise to the photosynthetic eukaryotes. The Chl a/b and Chl a/c antenna proteins, and the immunologically-related proteins in the rhodophytes may have had a common ancestor which was present in the early photosynthetic eukaryotes, and predated their division into rhodophyte, chromophyte and chlorophyte lineages. The LHC I-LHC II divergence probably occurred before the separation of higher plants from chlorophyte algae and euglenophytes, and the different Types of LHC I and LHC II proteins arose prior to the separation of angiosperms and gymnosperms.Abbreviations CAB Chl a/b-binding - ELIP early light-induced protein - FCP fucoxanthin-Chl a/c protein - PCR polymerase chain reaction - TMH trans-membrane helix     

Excitation dynamics and relaxation in the major antenna of a marine green alga Bryopsis corticulans

The light-harvesting complexes II (LHCIIs) of spinach and Bryopsis corticulans as a green alga are similar in structure, but differ in carotenoid (Car) and chlorophyll (Chl) compositions. Carbonyl Cars siphonein (Spn) and siphonaxanthin (Spx) bind to B. corticulans LHCII likely in the sites as a pair of lutein (Lut) molecules bind to spinach LHCII in the central domain. To understand the light-harvesting and photoprotective properties of the algal LHCII, we compared its excitation dynamics and relaxation to those of spinach LHCII been well documented. It was found that B. corticulans LHCII exhibited a substantially longer chlorophyll (Chl) fluorescence lifetime (4.9 ns vs 4.1 ns) and a 60% increase of the fluorescence quantum yield. Photoexcitation populated ³Car* equally between Spn and Spx in B. corticulans LHCII, whereas predominantly at Lut620 in spinach LHCII. These results prove the functional differences of the LHCIIs with different Car pairs and Chl a/b ratios: B. corticulans LHCII shows the enhanced blue-green light absorption, the alleviated quenching of ¹Chl*, and the dual sites of quenching ³Chl*, which may facilitate its light-harvesting and photoprotection functions. Moreover, for both types of LHCIIs, the triplet excitation profiles revealed the involvement of extra ³Car* formation mechanisms besides the conventional Chl-to-Car triplet transfer, which are discussed in relation to the ultrafast processes of ¹Chl* quenching. Our experimental findings will be helpful in deepening the understanding of the light harvesting and photoprotection functions of B. corticulans living in the intertidal zone with dramatically changing light condition.     

Coordinate expression of light-harvesting chlorophyll a/b gene family of photosystem II and chlorophyll a oxygenase gene regulated by salt-induced phosphorylation in Dunaliella salina

As a stress factor, salt induces the phosphorylation of light-harvesting chlorophyll (Chl) a/b proteins (LHCII) in Dunaliella salina. In this study, we found that the salt-induced phosphorylation of LHCII was not affected by phosphatase, and that salt simultaneously regulated both the phosphorylation of LHCII and the expression of genes encoding light-harvesting Chl a/b proteins of photosystem II (lhcb) and the gene encoding Chl a oxygenase (cao) in dark-adapted D. salina. The mRNA accumulation patterns of lhcb and cao were similar, which further affected the size of LHCII and the ratio of Chl a to Chl b. Therefore, we inferred this simultaneous regulation is one of the mechanisms of D. salina to adapt to the high-salinity environment.     

Preferential accumulation of apoproteins of the light-harvesting chlorophyll a/b-protein complex in greening barley leaves treated with 5-aminolevulinic acid

In order to study the coordinate accumulation of chlorophyll (Chl) and apoproteins of Chl-protein complexes (CPs) during chloroplast development, we examined changes in the accumulation of the apoproteins in barley (Hordeum vulgare L.) leaves when the rate of Chl synthesis was altered by feeding 5-aminolevulinic acid (ALA), a precursor of Chl biosynthesis. Pretreatment with ALA increased the accumulation of Chl a and Chl b 1.5- and 2.3-fold, respectively, after 12 cycles of intermittent light (2 min light followed by 28 min darkness). Apoproteins of the light-harvesting Chl a/b-protein complex of photosystem II (LHCII) were increased 2.4-fold with ALA treatment. However, apoproteins of the P700-Chl a-protein complex (CP1) and the 43-kDa apoprotein of a Chl a-protein complex of photosystem II (CPa) were not increased by ALA application. With respect to CPs themselves, LHCII was increased when Chl synthesis was raised by ALA feeding, whereas CP1 exhibited no remarkable increase. These results indicate that LHCII serves a role in maintaining the stoichiometry of Chl to apoproteins by acting as a temporary pool for Chl molecules.Abbreviations ALA 5-aminolevulinic acid - Chl chlorophyll - CP chlorophyll-protein complex - CPa chlorophyll a-protein complex of PSII - CP1 P700-chlorophyll a-protein complex - LDS lithium dodecyl sulfate - LHCII light-harvesting chlorophyll a/b-protein complex of PSII This work was supported by the Grants-in-Aid for Scientific Research (04304004) from the Ministry of Education, Science and Culture, Japan.     

Expression of ELIPs and PS II-S protein in spinach during acclimative reduction of the Photosystem II antenna in response to increased light intensities

The PS II-S protein and the so-called early light-inducible proteins (ELIPs) are homologous to the chlorophyll a/b-binding (Cab) gene products functioning in light-harvesting. The functional significance of these two CAB homologues is not known although they have been considered to bind pigments and in the case of the PS II–S protein this has been experimentally supported. The role of these two proteins does not appear to be light-harvesting but instead they are suggested to play a role as quenchers of free chlorophyll molecules during biogenesis and/or degradation of pigment-binding proteins. Such a role would be essential to eliminate the toxic and damaging effects that can be induced by free chlorophyll in the light. To this end the expression and characteristics of the ELIPs and the PS II–S protein were investigated in spinach leaves acclimating from low to high light intensities. Under these conditions there is a reduction in the antenna size of Photosystem II due to proteolytic digestion of its major chlorophyll a/b-binding protein (LHC II). During this acclimative proteolysis, up to one third of LHC II can be degraded and consequently substantial amounts of chlorophyll molecules will lose their binding sites. Our results reveal that there is a close correlation between ELIP accumulation and the onset of the LHC II degradation as low light-grown spinach leaves are subjected to increased light intensities. In contrast, there was no change in the relative level of the PS II–S protein during the acclimation process. It is concluded that the role for the ELIPs may be related to binding of liberated chlorophyll molecules and quenching of the toxic effects during LHC II degradation. In addition it was shown that in spinach four different ELIP species can be expressed and that they show different accumulation patterns in response to increased light intensities.     

Photosynthetic apparatus organization and function in the wild type and a chlorophyll b-less mutant of Chlamydomonas reinhardtii. Dependence on carbon source

 The assembly, organization and function of the photosynthetic apparatus was investigated in the wild type and a chlorophyll (Chl) b-less mutant of the unicellular green alga Chlamydomonas reinhardtii, generated via DNA insertional mutagenesis. Comparative analyses were undertaken with cells grown photoheterotrophically (acetate), photomixotrophically (acetate and HCO⁻ ₃) or photoautotrophically (HCO⁻ ₃). It is shown that lack of Chl b diminished the photosystem-II (PSII) functional Chl antenna size from 320 Chl (a and b) to about 95 Chl a molecules. However, the functional Chl antenna size of PSI remained fairly constant at about 290 Chl molecules, independent of the presence of Chl b. Western blot and kinetic analyses suggested the presence of inner subunits of the Chl a-b light-harvesting complex of PSII (LHCII) and the entire complement of the Chl a-b light-harvesting complex of PSI (LHCI) in the mutant. It is concluded that Chl a can replace Chl b in the inner subunits of the LHCII and in the entire complement of the LHCI. Growth of cells on acetate as the sole carbon source imposes limitations in the photon-use efficiency and capacity of photosynthesis. These are manifested as a lower quantum yield and lower light-saturated rate of photosynthesis, and as lower variable to maximal (F_v/F_max) chlorophyll fluorescence yield ratios. This adverse effect probably originates because acetate shifts the oxidation-reduction state of the plastoquinone pool, and also because it causes a decrease in the amount and/or activity of Rubisco in the chloroplast. Such limitations are fully alleviated upon inclusion of an inorganic carbon source (e.g. bicarbonate) in the cell growth medium. Further, the work provides evidence to show that transformation of green algae can be used as a tool by which to generate mutants exhibiting a permanently truncated Chl antenna size and a higher (per Chl) photosynthetic productivity of the cells. Received: 10 November 1999 / Accepted: 22 December 1999     

Comparison of chlorophyll a spectra in wild-type and mutant barley chloroplasts grown under day or intermittent light

The absorption (640–710 nm) and fluorescence emission (670–710 nm) spectra (77 K) of wild-type and Chl b-less, mutant, barley chloroplasts grown under either day or intermittent light were analysed by a RESOL curve-fitting program. The usual four major forms of Chl a at 662, 670, 678 and 684 nm were evident in all of the absorption spectra and three major components at 686, 693 and 704 nm in the emission spectra. A broad Chl a component band at 651 nm most likely exists in all chlorophyll spectra in vivo. The results show that the mutant lacks not only Chl b, but also the Chl a molecules which are bound to the light-harvesting, Chl a/b, protein complex of normal plants. It also appears that the absorption spectrum of this antenna complex is not modified appreciably by its isolation from thylakoid membranes.Abbreviations Chl chlorophyll - DL daylight - ImL intermittent light - WT wildtype - LHC light-harvesting Chl a/b protein complex - S.E. standard error of the mean DBP-CIW No. 763.     

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.     

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     

Reconstitution of pigment-containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed inEscherichia coli

A gene for a light-harvesting chlorophyll (Chl) a/b-binding protein (LHCP) from pea (Pisum sativum L.) has been cloned in a bacterial expression vector. Bacteria (Escherichia coli) transformed with this construct produced up to 20% of their protein as pLHCP, a derivative of the authentic precursor protein coded for by the pea gene with three amino-terminal amino acids added and-or exchanged, or as a truncated LHCP carrying a short amino-terminal deletion into the mature protein sequence. Following the procedure of Plumley and Schmidt (1987, Proc. Natl. Acad. Sci. USA84, 146–150), all bacteria-produced LHCP derivatives can be reconstituted with acetone extracts from pea thylakoids or with isolated pigments to yield pigment-protein complexes that are stable during partially denaturing polyacrylamide-gel electrophoresis. The spectroscopic properties of these complexes closely resemble those of the light-harvesting complex associated with photosystem II (LHCII) isolated from pea thylakoids. The pigment requirement for the reconstitution is highly specific for the pigments found in native LHCII: Chl a and b as well as at least two out of three xanthophylls are necessary. Varying the Chl a:Chl b ratios in the reconstitution mixtures changes the yields of complex formed but not the Chl a:Chl b ratio in the complex. We conclude that LHCP-pigment assembly in vitro is highly specific and that the complexes formed are structurally similar to LHCII. The N-terminal region of the protein can be varied without affecting complex formation and therefore does not seem to be involved in pigment binding. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

The 26- and 14-kDa phosphoproteins associated with spinach chloroplast envelope membranes are distinct membrane-bound pools of the light-harvesting complex of photosystem II and of the small subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase

Two chloroplast envelope proteins from spinach (Spinacia oleracea L.) exhibiting relative molecular masses (M_rs) of 26 and 14 kDa are apparently phosphorylated by a unique Ca²⁺-dependent serine protein kinase. The activity of this enzyme shows the same sensitivity towards pH, Ca²⁺, Mg²⁺, H7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine] and ATP concentrations (Siegenthaler and Bovet 1993, Planta 190, 231–240). Autoradiographic analyses following two-dimensional-gel electrophoresis (isoelectric focusing and SDS-PAGE) associated with Western blotting experiments indicate that these two phosphoproteins appeared to be pools of the light-harvesting complex of photosystem II (LHCII) and of the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit, respectively. Immunoprecipitation of envelope-phosphorylated proteins, using immunoglobulins (IgG) directed to the apoprotein of LHCII and to the holoenzyme of Rubisco confirmed that LHCII and the Rubisco small subunit effectively incorporated ³²P from (-³²P)ATP in isolated envelope membranes. We propose that, in agreement with the fact that protein import is driven by ATP, the phosphorylation of LHCII and the Rubisco small subunit could take place after the processing of precursor proteins and could be an obligatory step for their internalization into chloroplasts.Abbreviations 2D two dimensional - IEF isoelectric focusing - IgG immunoglobulin G - LHCII light-harvesting chlorophyll a/b proteins of PSII - LHCII A apoprotein a of LHCII - LHCIIB apoprotein b of LHCII - LS Rubisco large subunit - Mops (3-[N-morpholino]propanesulfonic acid) - M_r relative molecular mass - PI isoelectric point - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SS Rubisco small subunit The authors are grateful to Delphine Herrmann and Xavier Denys for their technical assistance. They also greatly thank Prof. R. J. Ellis and Dr. L. Barnett (Warwick University, UK) and Dr. P. Schürmann (University of Neuchâtel, Switzerland) for providing them with antibodies directed to the pea and spinach Rubisco holoenzymes and Dr. M. Spangfort (Lund University, Sweden) for his gift of the antibody directed to the pea LHCII apoprotein. This study was supported by the Swiss National Science Foundation. This work was part of a doctoral program carried out by L.B. in the Laboratoire de Physiologie végétale, Université de Neuchâtel, Switzerland.     

Evidence for an association of the early light-inducible protein (ELIP) of pea with photosystem II

The precursor to the nuclear-coded 17 kDa early light-inducible protein (ELIP) of pea has been transported into isolated intact chloroplasts. The location of the mature protein in the thylakoid membranes was investigated after using cleavable crosslinkers such as DSP and SAND in conjunction with immuno-fractionation methods and by application of mild detergent fractionation. We show that ELIP is integrated into the membranes via the unstacked stroma thylakoids. After isolation of protein complexes by solubilization of membranes with Triton X-100 and sucrose density-gradient centrifugation the crosslinked ELIP comigrates with the PS II core complex. Using SAND we identified ELIP as a 41–51 kDa crosslinked product while with DSP four products of 80 kDa, 70 kDa, 50–42 kDa and 23–21 kDa were found. The immunoprecipitation data suggested that the D₁-protein of the PS II complex is one of the ELIP partners in crosslinked products.Abbreviations chl chlorophyll - D₁ herbicide-binding protein - DSP dithiobis-(succinimidylpropionate) - ELIP early light-inducible protein - LHC I and LHC II light-harvesting chlorophyll a/b complex associated with photosystem I or II - PAGE polyacrylamide gel electrophoresis - poly(A)-rich RNA polyadenyd mRNA - PS I and PS II photosystems I and II - SAND sulfosuccinimidyl 2-(m-azido-o-nitro-benzamido)-ethyl-1,3-dithiopropionate - Triton X-100 octylphenoxypolyethoxyethanol     

Novel aspects of chlorophyll a/b-binding proteins

The light-harvesting proteins (LHC) constitute a multigene family including, in higher plants, at least 12 members whose location, within the photosynthetic membrane, relative abundance and putative function appear to be very different. The major light-harvesting complex of photosystem II (LHCII) is the most abundant membrane protein in the biosphere and fulfil a constitutive light-harvesting function for photosystem II while the early light-induced proteins (ELIPs) are expressed in low amounts under stress conditions. Primary sequence analysis suggests that all these proteins share a common structure which was resolved at 3.7 Å resolution by electron crystallography in the case of the major LHCII complex: Three transmembrane helices connected by hydrophilic loops coordinate seven chlorophyll a and five chlorophyll b molecules by histidine, glutamine, asparagine lateral chains as well as by charge compensated ionic pairs of glutamic acid and arginine residues; moreover, at least two xantophyll molecules are located at the centre of the structure in close contact with seven porphyrins, tentatively identified as chlorophyll a. The antenna system is also involved in the regulation of excitation energy transfer to reaction centre II. This function has been attributed to three members of the protein family, namely CP29, CP26 and CP24 (also called minor chlorophyll proteins) which have been recently characterised and shown to bind most of the xantophyll cycle carotenoids, thus suggesting that the non-photochemical quenching mechanism is acting in these proteins. Further support to this assignment comes from the recent identification of protonation sites in CP29 and CP26 by covalent dicyclohexhylcarbodiimide binding suggesting that these respond to low lumenal pH. In addition, CP29 is reversibly phosphorylated under light and cold stress conditions, undergoing conformational change, supporting the hypothesis that these subunits, present in low amounts in photosystem II, have a major regulatory role in the light-harvesting function and are thus important in environmental stress resistance.     

Changes in the Levels of Chlorophyll and Light-Harvesting Chlorophyll a/b Protein of PS II in Rice Leaves Aged under Different Irradiances from Full Expansion through Senescence

Effects of irradiance on changes in the amounts of chlorophyll(Chl) and light-harvesting chlorophyll a/b protein of PS II(LHCII) were examined in senescing leaves of rice (Oryza sativaL.). Results of treatments at two irradiances (100% and 20%natural sunlight) were examined after the full expansion ofthe 13th leaf throughout the course of senescence. With 20%sunlight, the Chl content decreased only a little during leafsenescence, while with 100% sunlight it decreased appreciably.Similarly, the amount of LHCII protein during treatment with20% sunlight remained almost constant. However, the ratio ofChl a/b during the shade treatment decreased significantly andthe rate of decrease was greater than during the full-sunlighttreatment. The ratio of Chl a/b for Chl a and b bound to LHCIIwas about 1.2, irrespective of leaf age or irradiance treatment.When the amounts of Chl bound to LHCII were calculated fromthe total leaf content of Chl and the ratio of Chl a/b, assuminga ratio of Chl a/b bound to LHCII of 1.2, they were well correlatedwith the amounts of LHCII protein. Changes in the amounts of LHCII synthesized during the two irradiancetreatments were examined using an ¹⁵ tracer. Incorporation of¹⁵N into LHCII declined dramatically during both treatmentsfrom full expansion through senescence, suggesting that therewas little synthesis of LHCII protein during that time. In addition,the amount of LHCII synthesized during senescence was lowerduring the shade treatment than during the 100% sunlight treatment.These results indicate that the absence of an apparent changein levels of LHCII with shade treatment during senescence wascaused by the very low rate of turnover of LHCII protein. (Received June 17, 1992; Accepted September 28, 1992)     

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.     

Distribution of Chlorophyll-Protein Complexes during Chilling in the Light Compared with Heat-Induced Modifications

The effects of chilling in the light (4 days at 5°C and 100-200 micromoles of photons per square meter per second) on the distribution of chlorophyll (Chl) protein complexes between appressed and nonappressed thylakoid regions of pumpkin (Cucurbita pepo L.) chloroplasts were studied and compared with the changes occurring during in vitro heat treatment (5 minutes at 40°C) of isolated thylakoids. Both treatments induced an increase (18 and 65%, respectively) in the relative amount of the antenna Chl a protein complexes (CP47 + CP43) of photosystem II (PSII) in stroma lamellae vesicles. Freeze-fracture replicas of light-chilled material revealed an increase in the particle density on the exoplasmic fracture face of unstacked membrane regions. These two treatments differed markedly, however, in respect to comigration of the light-harvesting Chl a/b protein complex (LHCII) of PSII. The LHCII/PSII ratio in stroma lamellae vesicles remained fairly constant during chilling in the light, whereas it dropped during the heat treatment. Moreover, it was a minor light-harvesting Chl a/b protein complex of PSII, CP29, that increased most in stroma lamellae vesicles during light-chilling. Changes in the organization of LHCII during chilling were suggested by a shift to particles of smaller sizes on the protoplasmic fracture face of stacked membrane regions and a decrease in the amount of trans-Δ³-hexadecenoic acid in the phosphatidyldiacylglycerol fraction.     

Polypeptide sequence of the chlorophyll a/b/c-binding protein of the prasinophycean alga Mantoniella squamata

The primary structure of the Chla/b/c-binding protein from Mantoniella squamata is determined. This is the first report that protein sequencing reveals one modified amino acid resulting in a LHCP-specific TFA-cleavage site. The comparison of the sequence of Mantoniella with other Chla/b-and Chla/c-binding proteins shows that the modified amino acid is located in a region which is highly conserved in all these proteins. The alignment also reveals that the LHCP of Mantoniella is related to the Chla/b-binding proteins. Finally, possible Chl-binding regions are discussed.Abbreviations a.m.u. atomic mass unit - LHC light-harvesting complex - LHC II major LHC of Photosystem II - LHCP light-harvesting chlorophyll-binding protein - LSIMS liquid secondary ion mass spectrometry - TFA trifluoroacetic acid     

Acclimation of Arabidopsis thaliana to the light environment: Changes in composition of the photosynthetic apparatus

Acclimation to changes in the light environment was investigated in Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. Plants grown under four light regimes showed differences in their development, morphology, photosynthetic performance and in the composition of the photosynthetic apparatus. Plants grown under high light showed higher maximum rates of oxygen evolution and lower levels of light-harvesting complexes than their low light-grown counterparts; plants transferred to low light showed rapid changes in maximum photosynthetic rate and chlorophyll-a/b ratio as they became acclimated to the new environment. In contrast, plants grown under lights of differing spectral quality showed significant differences in the ratio of photosystem II to photosystem I. These changes are consistent with a model in which photosynthetic metabolism provides signals which regulate the composition of the thylakoid membrane.Abbreviations Aac1 gene encoding actin - Chl chlorophyll - F far-red-enriched light (R:FR = 0.72) - FR far-red light - H high light (400 mol · m^–2 · s^–1) - L low light (100 ml · m^–2 · s^–1) - LHCII light-harvesting complex of PSII - Lhcb genes encoding the proteins of LHCII - R red light - Rbcs genes encoding the small subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - W white light (R:FR = 1.40) 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 (University of Sheffield) for helpful discussions.     

Characterization of Cytoplasmic and Nuclear Mutations Affecting Chlorophyll and Chlorophyll-Binding Proteins during Senescence in Soybean

Soybean plants (Glycine max [L.] Merr. cv Clark) carrying nuclear and cytoplasmic “stay-green” mutations, which affect senescence, were examined. Normally, the levels of chlorophyll (Chl) a and b decline during seedfill and the Chl a/b ratio decreases during late pod development in cv Clark. Plants homozygous for both the d₁ and d₂ recessive alleles, at two different nuclear loci, respectively, retained most (64%) of their Chl a and b and exhibited no change in their Chl a/b ratio. Combination of G (a dominant nuclear allele in a third locus causing only the seed coat to stay green during senescence) with d₁d₂ further inhibited the loss of Chl in the leaf. Whereas the thylakoid proteins seem to be degraded in normal Clark leaves during late pod development, they were not substantially diminished in d₁d₂ and Gd₁d₂ leaves. In plants carrying a cytoplasmic mutation, cytG, Chl declined in parallel with normal cv Clark; however, the cytG leaves had a much higher level of Chl b, and somewhat more Chl a, remaining at abscission, enough to color the leaves green. In cytG, most thylakoid proteins were degraded, but the Chl a/b-binding polypeptides of the light-harvesting complex in photosystem II (LHCII), and their associated Chl a and b molecules, were not. Thus, the combination of d₁ and d₂ causes broad preservation of the thylakoid proteins, whereas cytG appears to selectively preserve LHCII. The cytG mutation may be useful in elucidating the sequence of events involved in the degradation of LHCII proteins and their associated pigments during senescence.