Chlorophyll-binding Proteins Research Articles

A pigment-binding protein essential for regulation of photosynthetic light harvesting.

Photosynthetic light harvesting in plants is regulated in response to changes in incident light intensity. Absorption of light that exceeds a plant's capacity for fixation of CO2 results in thermal dissipation of excitation energy in the pigment antenna of photosystem II by a poorly understood mechanism. This regulatory process, termed nonphotochemical quenching, maintains the balance between dissipation and utilization of light energy to minimize generation of oxidizing molecules, thereby protecting the plant against photo-oxidative damage. To identify specific proteins that are involved in nonphotochemical quenching, we have isolated mutants of Arabidopsis thaliana that cannot dissipate excess absorbed light energy. Here we show that the gene encoding PsbS, an intrinsic chlorophyll-binding protein of photosystem II, is necessary for nonphotochemical quenching but not for efficient light harvesting and photosynthesis. These results indicate that PsbS may be the site for nonphotochemical quenching, a finding that has implications for the functional evolution of pigment-binding proteins.

Assembly of the D1 Precursor in Monomeric Photosystem II Reaction Center Precomplexes Precedes Chlorophyll a–Triggered Accumulation of Reaction Center II in Barley Etioplasts

Assembly of plastid-encoded chlorophyll binding proteins of photosystem II (PSII) was studied in etiolated barley seedlings and isolated etioplasts and either the absence or presence of de novo chlorophyll synthesis. De novo assembly of reaction center complexes in etioplasts was characterized by immunological analysis of protein complexes solubilized from inner etioplast membranes and separated in sucrose density gradients. Previously characterized membrane protein complexes from chloroplasts were utilized as molecular mass standards for sucrose density gradient separation analysis. In etiolated seedlings, induction of chlorophyll a synthesis resulted in the accumulation of D1 in a dimeric PSII reaction center (RCII) complex. In isolated etioplasts, de novo chlorophyll a synthesis directed accumulation of D1 precursor in a monomeric RCII precomplex that also included D2 and cytochrome b(559). Chlorophyll a synthesis that was chemically prolonged in darkness neither increased the yield of RCII monomers nor directed assembly of RCII dimers in etioplasts. We therefore conclude that in etioplasts, assembly of the D1 precursor in monomeric RCII precomplexes precedes chlorophyll a-triggered accumulation of reaction center monomers.

Identification of salt-regulated genes in the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 by subtractive RNA hybridization.

To identify genes transcribed preferentially under salt stress, a subtractive RNA hybridization procedure was applied to the cyanobacterium Synechocystis sp. PCC 6803. The screening of a genomic library led to the identification of several RNA species that were more abundant in salt-stressed cells than in control cells. Salt-dependent transcription of the identified genes was verified in Northern blot experiments. In addition to the previously characterized genes cpn60 (encoding GroEL; a molecular chaperone) and isiA (encoding a chlorophyll-binding protein), genes encoding a protein of unknown function (slr0082) and a putative RNA helicase (slr0083) were identified as salt-regulated genes in Synechocystis. Genes slr0082 and slr0083, located at sites adjacent to each other on the Synechocystis chromosome, were transcribed from separate promoters and showed the most significant induction 1-3 h after salt shock. The salt-regulated promoters of these two genes were mapped. Genes cpn60, slr0082, and slr0083 were also found to be induced by a cold shock. The possible role of the identified gene products for salt adaptation of Synechocystis is discussed.

Turnover of the aggregates and cross-linked products of the D1 protein generated by acceptor-side photoinhibition of photosystem II

It is known that the reaction-center binding protein D1 in photosystem (PS) II is degraded significantly during photoinhibition. The D1 protein also cross-links covalently or aggregates non-covalently with the nearby polypeptides in PS II complexes by illumination. In the present study, we detected the adducts between the D1 protein and the other reaction-center binding protein D2 (D1/D2), the α-subunit of cyt b 559 (D1/cyt b 559), and the antenna chlorophyll-binding protein CP43 (D1/CP43) by SDS/urea-polyacrylamide gel electrophoresis and Western blotting with specific antibodies. The adducts were observed by weak and strong illumination (light intensity: 50–5000 μE m −2 s −1) of PS II membranes, thylakoids and intact chloroplasts from spinach, under aerobic conditions. These results indicate that the cross-linking or aggregation of the D1 protein is a general phenomenon which occurs in vivo as well as in vitro with photodamaged D1 proteins. We found that the formation of the D1/D2, D1/cyt b 559 and D1/CP43 adducts is differently dependent on the light intensity; the D1/D2 heterodimers and D1/cyt b 559 were formed even by illumination with weak light, whereas generation of the D1/CP43 aggregates required strong illumination. We also detected that these D1 adducts were efficiently removed by the addition of stromal components, which may contain proteases, molecular chaperones and the associated proteins. By two-dimensional SDS/urea-polyacrylamide gel electrophoresis, we found that several stromal proteins, including a 15-kDa protein are effective in removing the D1/CP43 aggregates, and that their activity is resistant to SDS.

Revealing the structure of the oxygen-evolving core dimer of photosystem II by cryoelectron crystallography.

Here we present cryoelectron crystallographic analysis of an isolated dimeric oxygen-evolving complex of photosystem II (at a resolution of approximately 0.9 nm), revealing that the D1-D2 reaction center (RC) proteins are centrally located between the chlorophyll-binding proteins, CP43 and CP47. This conclusion supports the hypothesis that photosystems I and II have similar structural features and share a common evolutionary origin. Additional density connecting the two halves of the dimer, which was not observed in a recently described CP47-RC complex that did not include CP43, may be attributed to the small subunits that are involved in regulating secondary electron transfer, such as PsbH. These subunits are possibly also required for stabilization of the dimeric photosystem II complex. This complex, containing at least 29 transmembrane helices in its asymmetric unit, represents one of the largest membrane protein complexes studied at this resolution.

Carotenoid sequestration in plants: the role of carotenoid-associated proteins

In plants, carotenoid accumulation and sequestration take place within chloroplasts and chromoplasts. In the chloroplast, practically all carotenoids are associated with chlorophyll-binding proteins, whereas chromoplasts have developed a unique mechanism to sequester carotenoids within specific lipoprotein structures. Recent research into the existence of a group of homologous genes that encode carotenoid-associated proteins that aid in the generation of carotenoid-lipoprotein structures in chromoplasts, offers a new framework for elucidating the carotenoid sequestration mechanism.

Spectroscopic characterization of pigment binding proteins in normal-grown and iron-stressed thermophilic cyanobacteria Synecococcus sp.

The results of low temperature absorption, fluorescence and hole burning spectroscopy of pigment binding proteins in normal-grown and iron-stressed thermophilic Synecococcus sp. are reported. These experiments revealed that the growing of Synecococcus sp. under the iron-limited condition affects spectral characteristics and excited energy transfer (EET) in pigment proteins. The comparison of low temperature absorption spectra of normal-grown and iron-stressed thermophilic cyanobacteria well documents major changes in composition of the antenna systems and in composition of the core of photosystem II. The absorption of membrane chlorophyll in particular, is blue-shifted from 679 nm in normal cells to 673 nm which is caused by absorption of chlorophyll binding protein CP 34 in stressed sample. The presence of CP 34 in the photosystem II (PS 1 1 Abbreviations: APC, allophycocyanin; CC, cylindrical core; Chl, chlorophyll; LT, low temperature; PBS, phycobilisomes; PSB, phonon sideband; PC, phycocyanin; PR, peripheral rods; PS, photosystem; RC, reaction center, ZPH, zero phonon hole. II) has also been seen in the low temperature fluorescence spectra where the increased luminescence at 685 nm has been observed. This implies the decreased efficiency of photosynthesis in iron-limited sample. We studied the energy transfer in PS II by the means of fluorescence and absorption hole-burning spectroscopy which enabled to study the influence of iron-stressed condition on energy transfer and pigment–protein interaction (Debye–Waller factor). The stressed cells exhibit the broadened spectral hole width at 682 and 685.5 nm.

Isolation of pigment-binding early light-inducible proteins from pea.

The early light-inducible proteins (ELIPs) in chloroplasts possess a high sequence homology with the chlorophyll a/b-binding proteins but differ from those proteins by their substoichiometric and transient appearance. In the present study ELIPs of pea were isolated by a two-step purification strategy: perfusion chromatography in combination with preparative isoelectric focussing. Two heterogeneous populations of ELIPs were obtained after chromatographic separation of solubilized thylakoid membranes using a weak anion exchange column. One of these populations contained ELIPs in a free form providing the first isolation of these proteins. To prove whether the isolated and pure forms of ELIP bind pigments, spectroscopic and chromatographic analysis were performed. Absorption spectra and TLC revealed the presence of chlorophyll a and lutein. Measurements of steady-state fluorescence emission spectra at 77 K exhibited a major peak at 674 nm typical for chlorophyll a bound to the protein matrix. The action spectrum of the fluorescence emission measured at 674 nm showed several peaks originating mainly from chlorophyll a. It is proposed that ELIPs are transient chlorophyll-binding proteins not involved in light-harvesting but functioning as scavengers for chlorophyll molecules during turnover of pigment-binding proteins.

Multiple light-harvesting II polypeptides from maize mesophyll chloroplasts are distinct gene products

The major light-harvesting complex of photosystem II in higher plants is known as LHCII. It is composed of a number of chlorophyllbinding proteins sharing epitopes with each other. The number of apoproteins resolved by fully denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis varies in different species. In order to know if this heterogeneity is caused by the expression of a number of homologous genes or if it is the product of post-translational modifications, we have resolved the six major apoproteins of Zea mays LHCII. Each protein is purified to homogeneity, subjected to direct protein sequencing and the sequences compared with those deduced from lhcb genes in maize and other organisms. All of the six proteins are distinct gene products, since they show differences in their primary structure. Three apoproteins are identified as products of type I lhcb genes and one each as type II and type III gene products. A sixth protein does not fit the requirements for any of the lhcb genes so far cloned and is therefore probably the product of an Ihcb gene type not yet described. Our results clearly show that the major source of LHCII protein heterogeneity is the expression of many lhcb genes. Fractionation of maize LHCII by non-denaturing flat-bed isoelectric focusing resolves at least five major isoforms showing distinct differences in their polypeptide composition and also differing in their spectroscopic properties, thus suggesting that individual Lhcb gene products have distinct pigment-binding properties.

Distinct “Assisted” and “Spontaneous” Mechanisms for the Insertion of Polytopic Chlorophyll-binding Proteins into the Thylakoid Membrane

The biogenesis of several bacterial polytopic membrane proteins has been shown to require signal recognition particle (SRP) and protein transport machinery, and one such protein, the major light-harvesting chlorophyll-binding protein (LHCP) exhibits these requirements in chloroplasts. In this report we have used in vitro insertion assays to analyze four additional members of the chlorophyll-a/b-binding protein family. We show that two members, Lhca1 and Lhcb5, display an absolute requirement for stroma, nucleoside triphosphates, and protein transport apparatus, indicating an "assisted" pathway that probably resembles that of LHCP. Two other members, however, namely an early light-inducible protein 2 (Elip2) and photosystem II subunit S (PsbS), can insert efficiently in the complete absence of SRP, SecA activity, nucleoside triphosphates, or a functional Sec system. The data suggest a possibly spontaneous insertion mechanism that, to date, has been characterized only for simple single-span proteins. Of the membrane proteins whose insertion into thylakoids has been analyzed, five have now been shown to insert by a SRP/Sec-independent mechanism, suggesting that this is a mainstream form of targeting pathway. We also show that PsbS and Elip2 molecules are capable of following either "unassisted" or assisted pathways, and we discuss the implications for the mechanism and role of SRP in chloroplasts.

ADP-Dependent phosphorylation regulates association of a DNA-binding complex with the barley chloroplast psbD blue-light-responsive promoter.

The chloroplast gene psbD encodes D2, a chlorophyll-binding protein located in the photosystem II reaction center. Transcription of psbD in higher plants involves at least three promoters, one of which is regulated by blue light. The psbD blue-light-regulated promoter (BLRP) consists of a -10 promoter element and an activating complex, AGF, that binds immediately upstream of -35. A second sequence-specific DNA-binding complex, PGTF, binds upstream of AGF between -71 and -100 in the barley (Hordeum vulgare) psbD BLRP. In this study we report that ADP-dependent phosphorylation selectively inhibits the binding of PGTF to the barley psbD BLRP. ATP at high concentrations (1-5 mM) inhibits PGTF binding, but in the presence of phosphocreatine and phosphocreatine kinase, this capacity is lost, presumably due to scavenging of ADP. ADP inhibits PGTF binding at relatively low concentrations (0.1 mM), whereas other nucleotides are unable to mediate this response. ADP-mediated inhibition of PGTF binding is reduced in the presence of the protein kinase inhibitor K252a. This and other results suggest that ADP-dependent phosphorylation of PGTF (or some associated protein) inhibits binding of PGTF to the psbD BLRP and reduces transcription. ADP-dependent phosphorylation is expected to increase in darkness in parallel with the rise in ADP levels in chloroplasts. ADP-dependent phosphorylation in chloroplasts may, therefore, in coordination, inactivate enzymes involved in carbon assimilation, protein synthesis, and transcription during diurnal light/dark cycles.

Genetic deletion of proteins resembling Type IV pilins in Synechocystis sp. PCC 6803: their role in binding or transfer of newly synthesized chlorophyll.

Upon non-denaturing gel electrophoresis of Synechocystis sp. PCC 6803 thylakoid extracts, a Type IV pilin-like protein encoded by open reading frame sll1694 was found in chlorophyll-containing bands. The Synechocystis sp. PCC 6803 genome also encodes two similar open reading frames, sll1695 and slr1456. Even though transcripts of sll1694 and slr1456 could be detected, deletion of the three open reading frames in systems with normal chlorophyll content had no effect. However, Sll1694 was found to affect the rate of chlorophyll synthesis and of the assembly of chlorophyll-binding proteins. In the sll1694/sll1695 deletion mutant in a PS I-less/chlL- background, which is unable to synthesize chlorophyll in darkness, chlorophyll synthesis during the first hours of illumination after dark incubation was 30% slower than in the PS I-less/chlL- strain. Moreover, the biogenesis of chlorophyll-protein complexes with a 77K chlorophyll fluorescence emission maximum at 685 mm was delayed by several hours in this mutant whereas the rate of biogenesis of photosystem II was not significantly affected. Furthermore, results of non-denaturing gel electrophoresis indicated that a chlorophyll-binding complex formed during the early hours of chlorophyll synthesis was altered in stability and mobility upon deletion of the three open reading frames. We propose that the protein encoded by sll1694 is involved in, but is not absolutely required for, delivering chlorophyll to nascent photosystems and antennae.

A phylogenetic assessment of the eukaryotic light-harvesting antenna proteins, with implications for plastid evolution.

The light-harvesting complexes (LHCs) are a superfamily of chlorophyll-binding proteins present in all photosynthetic eukaryotes. The Lhc genes are nuclear-encoded, yet the pigment-protein complexes are localized to the thylakoid membrane and provide a marker to follow the evolutionary paths of plastids with different pigmentation. The LHCs are divided into the chlorophyll a/b-binding proteins of the green algae, euglenoids, and higher plants and the chlorophyll a/c-binding proteins of various algal taxa. This work examines the phylogenetic position of the LHCs from three additional taxa: the rhodophytes, the cryptophytes, and the chlorarachniophytes. Phylogenetic analysis of the LHC sequences provides strong statistical support for the clustering of the rhodophyte and cryptomonad LHC sequences within the chlorophyll a/c-binding protein lineage, which includes the fucoxanthin-chlorophyll proteins (FCP) of the heterokonts and the intrinsic peridinin-chlorophyll proteins (iPCP) of the dinoflagellates. These associations suggest that plastids from the heterokonts, haptophytes, cryptomonads, and the dinoflagellate, Amphidinium, evolved from a red algal-like ancestor. The Chlorarachnion LHC is part of the chlorophyll a/b-binding protein assemblage, consistent with pigmentation, providing further evidence that its plastid evolved from a green algal secondary endosymbiosis. The Chlorarachnion LHC sequences cluster with the green algal LHCs that are predominantly associated with photosystem II (LHCII). This suggests that the green algal endosymbiont that evolved into the Chlorarachnion plastid was acquired following the emergence of distinct LHCI and LHCII complexes.

Purification and Properties of a Novel Chloroplast Stromal Peptidase: PROCESSING OF POLYPHENOL OXIDASE AND OTHER IMPORTED PRECURSORS

Polyphenol oxidases (PPOs) are nuclear-encoded chloroplast proteins that are targeted to the thylakoid lumen by a bipartite presequence. The N-terminal part of this sequence is removed by a stromal processing peptidase (SPP), and the resulting intermediate is translocated across the thylakoid and processed to the mature protein. A 4800-fold-purified SPP processed a PPO precursor (pPPO) at a site identical to that occurring in organelle. The in vitro product of SPP action on pPPO was further processed and translocated by thylakoids. This SPP processed other precursors but was inactive toward those of light-harvesting chlorophyll binding proteins. The enzyme appeared to be a metalloendopeptidase, like previously reported SPPs. However, it differed in substrate specificity, apparent size, and, most significantly, cleavage site of pPPO. Whereas the processing sites of lumen proteins determined so far were relatively distant from the hydrophobic core of the thylakoid targeting domain, pPPO was cleaved immediately before this domain. Cleavage removed the twin arginine motif characteristic of thylakoid targeting domains of lumen proteins, which are translocated by the DeltapH-dependent pathway. The possible significance of these observations to PPO translocation mechanism is discussed. It is suggested that several SPPs may exist in chloroplasts with preferences for different subsets of precursors.

Specific requirements for cytochrome c-550 and the manganese-stabilizing protein in photoautotrophic strains of Synechocystis sp. PCC 6803 with mutations in the domain Gly-351 to Thr-436 of the chlorophyll-binding protein CP47.

The requirement of cytochrome c-550 (PSII-V) in photosystem II (PSII) has been assessed in Synechocystis sp. PCC 6803 containing mutations between Gly-351 and Thr-436 of the loop E domain of the chlorophyll a-binding protein CP47. Six photoautotrophic strains were utilized to compare the effect of removal of either the manganese-stabilizing protein (PSII-O) or PSII-V on PSII activity in vivo. These were a wild-type control; two strains with amino acid deletions, Delta(R384-V392) and Delta(G429-T436); and three carrying specific amino acid substitutions, G351L/T365Q, G351L/E364Q/T365Q, and G351L/E353Q/E355Q/T365Q. The removal of PSII-O prevented the assembly of PSII in Delta(G429-T436) but not in Delta(R384-V392). Neither Delta(G429-T436) nor Delta(R384-V392) could support photoautotrophic growth in the absence of PSII-V. In chloride-limiting conditions, the photoautotrophic growth of Delta(R384-V392) was severely impaired and that of Delta(G429-T436) totally inhibited, and no strains lacking PSII-V could grow in chloride-limiting or calcium-limiting media. Substitutions at Gly-351, Glu-353, Glu-355, and Thr-365 produced phenotypes that were similar to those of the control in the presence or absence of PSII-O and PSII-V, but removal of PSII-O from G351L/E364Q/T365Q produced a significant reduction of assembled PSII centers and an enhanced sensitivity to photoinactivation while removal of PSII-V prevented photoautotrophic growth. The additional mutants E364Q:DeltaPSII-V and E364G:DeltaPSII-V demonstrated that this inhibition was a consequence of the mutation at Glu-364. These results also show that the removal of PSII-V, in vivo, produces phenotypes in the CP47 mutants examined that are either similar or more severe than those resulting from the removal of PSII-O.

Disruption of chloroplast biogenesis and plant development upon down‐regulation of a chloroplast processing enzyme involved in the import pathway

Summary Typically, nuclear‐encoded chloroplast proteins are synthesized as precursors and require proteolytic processing upon import before their assembly into functional complexes within the organelle. A cDNA encoding a chloroplast processing enzyme (CPE), which was originally identified as a protease that cleaves the precursor for the major light‐harvesting chlorophyll binding protein (preLHCP), was introduced into the tobacco genome in an antisense orientation to investigate the role of the enzyme in vivo . The presence of the antisense‐CPE gene resulted in chlorotic leaves, and retarded shoot and root growth. The introduction of the antisense‐CPE gene disrupted the normal pattern of plastid division. Chloroplast numbers in cotyledon and first leaf cells were reduced 25% compared to the control plants. Chloroplasts contained fewer thylakoids and large starch grains, the latter an indication of a change in carbon flux. CPE levels and activity were significantly lower in stromal extracts in the transgenic plants. Interestingly, in vitro import of precursor proteins was defective. Most of the preLHCP remained on the exterior of the organelle, and only a small fraction of preRBCA was imported, suggesting that a change in CPE levels can influence translocation across the envelope. Our in vivo results support the conclusion that CPE plays a critical role during chloroplast biogenesis, and that the pleiotropic effects of CPE down‐regulation reflect its function as a general stromal processing peptidase as part of the import machinery. Furthermore, these findings indicate the importance of regulating the expression of components of the import machinery for normal plant development.

Isolation and Characterization of Monomeric and Dimeric CP47-Reaction Center Photosystem II Complexes

Using the detergents n-dodecyl beta-D-maltoside and heptyl thioglycopyranoside, a subcore complex of photosystem II (PSII) has been isolated that contains the chlorophyll-binding protein, CP47, and the reaction center components, D1, D2, and cytochrome b559. We have found, by using sucrose density centrifugation, that the resulting preparation consisted of a mixture of dimeric and monomeric forms of the CP47 reaction center (RC) complex, having molecular masses of 410 +/- 30 and 200 +/- 28 kDa, respectively, as estimated by size exclusion chromatography. The level of the dimer in the preparation is significantly higher than the monomeric form. Both the monomer and dimer contain the proteins CP47, D1, and D2 and the alpha- and beta-subunits of cytochrome b559. Analyses by mass spectrometry and N-terminal sequencing showed that both forms of the CP47-RC complex contain the products of the psbI, psbTc (chloroplast gene), and psbW with molecular masses of 4195.5, 3849.6, and 5927.4 Da, respectively. In contrast to the monomeric form, the CP47-RC dimer contained two extra proteins with low molecular weights, identified as the products of the psbL and psbK genes having molecular masses of 4365.5 and 4292.1, respectively. It was also found that the dimer contained slightly more molecules of chlorophyll a (21 +/- 2.5) than the monomer (18 +/- 1.5), a characteristic also observed in the room temperature absorption spectrum by comparing the ratio of absorption at 416 and 435 nm. Of particular note was the finding that the dimer, but not the monomer, contained plastoquinone-9 (estimated to be 1.5 +/- 0.3 molecules per RC). The results indicate that the CP47-RC monomer is derived from the dimeric form of the complex, and therefore the latter is likely to represent an in vivo conformation. The PsbTc as well as the PsbI and PsbW proteins are identified as being intimately associated with the D1 and D2 proteins, and in the case of the dimer, importance is placed on the PsbL and PsbK proteins in sustaining plastoquinone binding and maintenance of the dimeric organization. Assuming only one copy of the alpha- and beta-subunits of cytochrome b559, the monomeric and dimeric forms of the complex would be expected to contain 21 and 23 x 2 transmembrane helices, respectively.

Frost hardening and photosynthetic performance of Scots pine ( Pinus sylvestris L.) needles. I. Seasonal changes in the photosynthetic apparatus and its function

Photosynthetic CO2 uptake, the photochemical efficiency of photosystem II, the contents of chlorophyll and chlorophyll-binding proteins, and the degree of frost hardiness were determined in three-year-old Scots pine (Pinus sylvestris L.) trees growing in the open air but under controlled daylength. The following conditions were compared: 9-h light period (short day), 16-h light period (long day), and natural daylength. Irrespective of induction by short-day photoperiods or by subfreezing temperatures, frost hardening of the trees was accompanied by a long-lasting pronounced decrease in the photosynthetic rates of one-year-old needles. Under moderate winter conditions, trees adapted to a long-day photoperiod, assimilated CO2 with higher rates than the short-day-treated trees. In the absence of strong frost, photochemical efficiency was lower under short-day conditions than under a long-day photoperiod. Under the impact of strong frost, photochemical efficiency was strongly inhibited in both sets of plants. The reduction in photosynthetic performance during winter was accompanied by a pronounced decrease in the content of chlorophyll and of several chlorophyll-binding proteins [light-harvesting complex (LHC)IIb, LHC Ib, and a chlorophyll-binding protein with MW 43 kDa (CP 43)]. This observed seasonal decrease in photosynthetic pigments and in pigment-binding proteins was irrespective of the degree of frost hardiness and was apparantly under the control of the length of the daily photoperiod. Under a constant 9-h daily photoperiod the chlorophyll content of the needles was considerably lower than under long-day conditions. Transfer of the trees from short-day to long-day conditions resulted in a significantly increased chlorophyll content, whereas the chlorophyll content decreased when trees were transferred from a long-day to a short-day photoperiod. The observed changes in photosynthetic pigments and pigment-binding proteins in Scots pine needles are interpreted as a reduction in the number of photosynthetic units induced by shortening of the daily light period during autumn. This results in a reduction in the absorbing capacity during the frost-hardened state.

Frost hardening and photosynthetic performance of Scots pine ( Pinus sylvestris L .). II. Seasonal changes in the fluidity of thylakoid membranes

The fluidity of chloroplast thylakoid membranes of frost-tolerant and frost-sensitive needles of␣three- to four-year-old Scots pine (Pinus sylvestris L.) trees, of liposomes produced from the lipids of the thylakoids of these needles, and of liposomes containing varying amounts of light-harvesting complex (LHC) II protein was investigated by means of electron paramagnetic resonance (EPR) measurements using spin-labelled fatty acids as probes. Broadening of the EPR-resonance signals of 16-doxyl stearic acid in chloroplast membranes of frost-sensitive needles and changes in the amplitudes of the peaks were observed upon a decrease in temperature from +30 °C to −10 °C, indicating a drastic loss in rotational mobility. The lipid molecules of the thylakoid membranes of frost-tolerant needles exhibited greater mobility. Moderate frost resistance could be induced in Scots pine needles by short-day treatment (Vogg et al., 1997, Planta, this issue), and growth of the trees under short-day illumination (9 h) resulted in a higher mobility of the chloroplast membrane lipids than did growth under long-day conditions (16 h). The EPR spectrum of thylakoids from frost-tolerant needles at −10 °C was typical of a spin label in highly fluid surroundings. However, an additional peak in the low-field range appeared in the subzero temperature range for the chloroplast membranes of frost-sensitive needles, which represents spin-label molecules in a motionally restricted surrounding. The EPR spectra of thylakoids and of liposomes of thylakoid lipids from frost-hardy needles were identical at +30 °C and −10 °C. The corresponding spectra from frost-sensitive plants revealed an additional peak for the thylakoids, but not for the pure liposomes. Hence, the domains with restricted mobility could be attributed to protein-lipid interactions in the membranes. Broadening of the spectrum and the appearance of an additional peak was observed with liposomes of pure distearoyl phosphatidyl glycerol modified to contain increasing amounts of LHC II. These results are discussed with respect to a loss of chlorophyll and chlorophyll-binding proteins in thylakoids of Scots pine needles under winter conditions.

A low-density genetic map of onion reveals a role for tandem duplication in the evolution of an extremely large diploid genome

The bulb onion, Allium cepa L., is a diploid (2n=2x=16) plant with a huge nuclear genome. Previous genetic and cytogenetic analyses have not supported a polyploid origin for onion. We developed a low-density genetic map of morphological markers, randomly amplified polymorphic DNAs (RAPD), and restriction fragment length polymorphisms (RFLP) as a tool for onion improvement and to study the genome organization of onion. A mapping population of 58 F3 families was produced from a single F1 plant from the cross of two partially inbred lines (Brigham Yellow Globe 15-23 and Alisa Craig 43). Segregations were established for restoration of male fertility in sterile cytoplasm, complementary light-red bulb color, 14 RAPDs, 110 RFLPs revealed by 90 anonymous cDNA clones, and 2 RFLPs revealed by a cDNA clone of alliinase, the enzyme responsible for the characteristic Allium flavors. Duplicated RFLP loci were detected by 21% of the clones, of which 53% were unlinked (>30 cM), 5% loosely linked (10–30 cM), and 42% tightly linked (<10 cM). This duplication frequency is less than that reported for paleopolyploids but higher than for diploid species. We observed 40% dominant RFLPs, the highest yet reported among plants. Among duplicated RFLP loci, 19% segregated as two loci each with two codominant alleles, 52% segregated as one locus with codominant alleles and one locus with only a dominant fragment, and 29% segregated as two loci with only dominant fragments. We sequenced cDNAs detecting duplicated RFLPs; 63% showed homology to known gene families (e.g., chlorophyll binding proteins, ubiquitin, or RuBISCO), and 37% were unique clones showing significant homology to known genes of low-copy number or no homology to database sequences. Duplicated RFLPs showing linkage could be due to retroviral-like sequences in adjacent coding regions or intrachromosomal, as opposed to whole genome, duplications. Previous cytological analyses and this genetic map support intrachromosomal duplication as a mechanism contributing to the huge onion genome.