Isolation and Characterization of a Light-Harvesting Chlorophyll a/b Protein Complex Associated with Photosystem I

A chlorophyll a/b protein complex has been isolated from a resolved native photosystem I complex by mildly dissociating sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chlorophyll a/b protein contains a single polypeptide of molecular weight 20 kilodaltons, and has a chlorophyll a/b ratio of 3.5 to 4.0. The visible absorbance spectrum of the chlorophyll a/b protein complex showed a maximum at 667 nanometers in the red region and a 77 K fluorescence emission maximum at 681 nanometers. Alternatively, by treatment of the native photosystem I complex with lithium dodecyl sulfate and Triton, the chlorophyll a/b protein complex could be isolated by chromatography on Sephadex G-75. Immunological assays using antibodies to the P₇₀₀-chlorophyll a-protein and the photosystem II light-harvesting chlorophyll a/b protein show no cross-reaction between the photosystem I chlorophyll a/b protein and the other two chlorophyll-containing protein complexes.     

Photoregulation of the Light-Harvesting Chlorophyll Protein Complex Associated with Photosystem II in Dunaliella tertiolecta: Evidence that Apoprotein Abundance but Not Stability Requires Chlorophyll Synthesis

The marine chlorophyte Dunaliella tertiolecta Butcher responds to a one-step transition from a high growth irradiance level (700 micromoles quanta per square meter per second) to a low growth irradiance level (70 micromoles quanta per square meter per second) by increasing the total amount of light-harvesting chlorophyll (Chl) a/b binding protein associated with photosystem II (LHC II), and by modifying the relative abundance of individual LHC II apoproteins. When high light-adapted cells were incubated with gabaculine, which inhibits Chl synthesis, and transferred to low light, the LHC II apoproteins were still synthesized and the ³⁵S-labeled LHC II apoproteins remained stable after a 24 hour chase. These results suggest that Chl synthesis is not required for stability of the LHC II apoproteins in this alga. However, when the control cells are transferred from high light to low light, the amount of the four LHC II apoproteins per cell increases, whereas it does not in the presence of gabaculine. These results suggest that Chl synthesis is required for a photoadaptive increase in the cellular level of LHC II.     

Effects of 5-Aminolevulinic Acid on the Accumulation of Chlorophyll b and Apoproteins of the Light-Harvesting Chlorophyll a/b-Protein Complex of Photosystem II

The effects were examined of 5-aminolevulinic acid (ALA) onthe accumulation of Chl and apoproteins of light-harvestingChl a/b-protein complex of photosystem II (LHCII) in cucumbercotyledons under intermittent light. A supply of ALA preferentiallyincreased the accumulation of Chl a during intermittent illumination.However, when cotyledons were pretreated with a brief exposureto light or benzyladenine (BA), the stimulatory effect of ALAon the increase in the level of Chl b was greater than thatin the level of Chl a, resulting in decreased ratios of Chla/b. Time-course experiments with preilluminated cotyledonsrevealed that LHCII apoproteins accumulated rapidly within thefirst 30 min of intermittent illumination with a decline duringsubsequent incubation in darkness. A supply of ALA did not affectthe accumulation of LHCII apoproteins during the intermittentlight period, but it efficiently inhibited the decline in theirlevels during the subsequent darkness. After exposure to a singlepulse of light of BA-treated cotyledons, the prompt increasein levels of LHCII apoproteins was not accompanied by the formationof Ch b, which began to accumulate later. The pattern of changesin levels of LHCII apoproteins was quite similar to that inlevels of Chl a. These results suggest that LHCII apoproteinsare first stabilized by binding with Chl a and that an increasedsupply of Chl a and the accumulation of LHCII apoproteins areprerequisites for the formation of Chl b. ¹Present address: Department of Chemistry, Faculty of Scienceand Technology, Meijo University, Aichi, 468 Japan.     

Synthesis and Breakdown of the Apoproteins of Light-Harvesting Chlorophyll a/b Proteins in Chlorophyll b-deficient Mutants of Rice

Turnover, in the light, of apoproteins of light-harvesting chlorophylla/6-proteins for Photo-system I and II (LHC-I and LHC-II, respectively)was studied with the wild-type and three chlorophyll 6-deficientmutants of rice. (1) Synthesis of the 24 and 25 kDa apoproteinsof LHC-II and the 20 and 21 kDa apoproteins of LHC-I was examinedby incubating leaf segments with [³⁵S]-methionine. The threerice mutants, chlorina 2, which totally lacks chlorophyll b,and chlorina 11 and 14, which are partially deficient in chlorophyllb, synthesized the apoproteins as rapidly as did the wild typerice. (2) Pulse-chase experiments showed that breakdown of theapoproteins proceeded slowly, such that only a small proportionof the newly synthesized apoproteins was lost during the 48h of the chase in normal rice leaves. By contrast, large fractionsof the labelled apoproteins were rapidly degraded within thefirst several hours of the chase period in the chlorina mutants.The greater the deficiency in chlorophyll b of the mutant, thelarger were the rate and extent of the protein breakdown. Thisresult indicates that chlorophyll b is needed to stabilize theapoproteins of LHC-II and LHC-I. (3) However, even in chlorina2, there were small fractions of the apoproteins with lifetimesas long as those of apoproteins in the wild-type rice, suggestingthat the newly synthesized apoproteins are partially protectedby a factor(s) other than chlorophyll b. (4) The rate of turnoverof the apoproteins was significantly reduced in the dark andstrongly inhibited by prior treatment of leaf segments withchloramphenicol. (Received November 24, 1988; Accepted March 17, 1989)     

Resolution of Chlorophyll a/b-Protein Complexes by Polyacrylamide Gel Electrophoresis: Evidence for the Heterogeneity of Light-Harvesting Chlorophyll a/b-Protein Complexes

Laboratory for Plant Ecological Studies, Faculty of Science,Kyoto University, Kyoto 606, Japan P700-Chl a-protein complexes(CP1 and CP1*), Chl-protein complexes of PS II core (CPa-1 andCPa-2), light-harvesting Chi a/A-protein complexes (LHCPo andLHCPm) and CP29 of spinach thylakoids were resolved by SDS-polyacrylamide-gelelectrophoresis (PAGE) under non-denaturing conditions. TheLHCP oligomer purified by electrophoresis, had 29.5- and 27-kDapolypeptides. CP1, CP29 and two LHCPs (LHCP-1 and LHCP-2) ofspinach thylakoids were separated by a lithium dodecylsulfate(LDS) PAGE system with high resolution. The two LHCPs showedthe same absorption spectrum on the gel. When LHCP oligomerwas reelectrophoresed by this system it also gave LHCP-1, andLHCP-2. LHCP-1 had both 29.5- and 27- kDa polypeptides, butLHCP-2 had only 29.5 kDa polypeptide. Both polypeptides seemedto bind Chi. The heterogeneity of LHCP was also observed withbean thylakoids. (Received August 5, 1987; Accepted September 17, 1987)     

Light-Harvesting Chlorophyll a/b Complexes: Interdependent Pigment Synthesis and Protein Assembly

The biogenetic interdependence of light-harvesting chlorophyll (Chl) a/b proteins (LHCPs) and antenna pigments has been analyzed for two nuclear mutants of Chlamydomonas that have low levels of Chl b, neoxanthin, and loroxanthin. In mutant PA2.1, the apoprotein precursors (pLHCP II) of the major light-harvesting complex LHC II were synthesized at approximately wild-type rates, processed to their mature size, and rapidly degraded. Because the bulk of labile LHCP II in PA2.1 was soluble, a thylakoid integration factor apparently is defective in this strain. Chl a, Chl b, neoxanthin, and loroxanthin synthesis and accumulation were coordinately reduced in PA2.1, indicating that LHCP II play important regulatory or substrate roles in de novo synthesis of these pigments. Mutant GE2.27 is impaired principally in Chl b synthesis but nonetheless accumulated wild-type levels of all LHCPs. Topology studies of the GE2.27 LHCP II demonstrated that their insertion into thylakoids was incomplete even though they were not structurally altered. Thus, Chl b formation mediates conformational changes of LHCP II after thylakoid integration is initiated. GE2.27 also exhibited very low rates of neoxanthin synthesis and was unable to accumulate loroxanthin. Revertant GE2.27 strains with varying capacities for Chl b formation provided additional evidence that neoxanthin synthesis and accumulation are coupled with the final steps of LHCP II integration into thylakoids. We propose that biogenesis of LHC includes interdependent pigment synthesis/assembly events that occur during LHCP integration into the thylakoid membrane and that defects in these events account for the pleiotropic characteristics of many Chl b-deficient mutants.     

Thermostability and Photostability of Photosystem II in Leaves of the Chlorina-f2 Barley Mutant Deficient in Light-Harvesting Chlorophyll a/b Protein Complexes

The chlorophyll-b-less chlorina-f2 barley mutant is deficient in the major as well as some minor light-harvesting chlorophyll-protein complexes of photosystem II (LHCII). Although the LHCII deficiency had relatively minor repercussions on the leaf photosynthetic performances, the responses of photosystem II (PSII) to elevated temperatures and to bright light were markedly modified. The chlorina-f2 mutation noticeably reduced the thermostability of PSII, with thermal denaturation of PSII starting at about 35[deg]C and 38.5[deg]C in chlorina-f2 and in the wild type, respectively. The increased susceptibility of PSII to heat stress in chlorina-f2 leaves was due to the weakness of its electron donor side, with moderate heat stress causing detachment of the 33-kD extrinsic PSII protein from the oxygen-evolving complex. Prolonged dark adaptation of chlorina-f2 leaves was also observed to inhibit the PSII donor side. However, weak illumination slowly reversed the dark-induced inhibition of PSII in chlorina-f2 and cancelled the difference in PSII thermostability observed between chlorina-f2 and wild-type leaves. The mutant was more sensitive to photoinhibition than the wild type, with strong light stress impairing the PSII donor side in chlorina-f2 but not in the wild type. This difference was not observed in anaerobiosis or in the presence of 3-(3,4-dichlorophenyl)- 1,1-dimethylurea, diuron. The acceptor side of PSII was only slightly affected by the mutation and/or the aforementioned stress conditions. Taken together, our results indicate that LHCII stabilize the PSII complexes and maintain the water-oxidizing system in a functional state under varying environmental conditions.     

Conversion of Chlorophyll b to Chlorophyll a and the Assembly of Chlorophyll with Apoproteins by Isolated Chloroplasts

The photosynthetic apparatus is reorganized during acclimation to various light environments. During adaptation of plants grown under a low-light to high-light environment, the light-harvesting chlorophyll a/b-protein complexes decompose concomitantly with an increase in the core complex of photosystem II. To study the mechanisms for reorganization of photosystems, the assembly of chlorophyll with apoproteins was investigated using isolated chloroplasts. When [14C]chlorophyllide b was incubated with chloroplasts in the presence of phytyl pyrophosphate, it was esterified and some of the [14C]chlorophyll b was converted to [14C]chlorophyll a via 7-hydroxymethyl chlorophyll. [14C]Chlorophyll a and b were incorporated into chlorophyll-protein complexes. Light-harvesting chlorophyll a/b-protein complexes of PSII had a lower [14C]chlorophyll a to [14C]chlorophyll b ratio than P700-chlorophyll a-protein complexes, indicating the specific binding of chlorophyll to apoproteins in our systems. 7-Hydroxymethyl chlorophyll, an intermediate molecule from chlorophyll b to chlorophyll a, did not become assembled with any apoproteins. These results indicate that chlorophyll b is released from light-harvesting chlorophyll a/b-protein complexes of photosystem II and converted to chlorophyll a via 7-hydroxymethyl chlorophyll in the lipid bilayer and is then used for the formation of core complexes of photosystems. These mechanisms provide the fast, fine regulation of the photosynthetic apparatus during construction of photosystems.     

Purification and Characterization of Light-Harvesting Chlorophyll a/b-Protein Complexes of Photosystem II from the Green alga, Bryopsis maxima

Light-harvesting chlorophyll a/b-proteins of photosystem II(LHC II) were purified from thylakoid membranes of the greenalga, Bryopsis maxima. Extraction with digitonin did not solubilizechlorophylls (Chl) and carotenoids to any significant extent.Two forms of purified LHC II, P4 and P5, with respective apparentparticle sizes of 280 and 295 kDa, were obtained by sucrosedensity gradient centrifugation and column chromatography onDEAE-Toyopearl. P4 and P5 had similar spectral absorption at77 K with Chl a maxima at 674, 658 and 438 nm and Chl b maximaat 649 and 476 nm. Carotene was not present in P4 or P5. Fluorescenceexcitation spectra demonstrated that Chl b, siphonaxanthin andsiphonein can efficiently transfer absorbed light energy toChl a. P4 and P5 each contained two apoproteins of 28 and 32kDa, with similar but not identical amino acid compositions.P5 contained 6 molecules of Chl a, 8 of Chl b and 5 of xanthophyll(three molecules of siphonaxanthin and one each of siphoneinand neoxanthin) per polypeptide. (Received September 11, 1989; Accepted December 11, 1989)     

Immunological Quantification of Proteins Related to Light-Harvesting Chlorophyll a/b Protein Complexes of the Two Photosystems in Rice Mutants Totally and Partially Deficient in Chlorophyll b

Ten rice chlorina mutants of Type I, which totally lack chlorophyllb and hence are unable to synthesize light-harvesting chlorophylla/b protein complexes of photosystem II (LHC-II), containedmRNA for proteins related to LHC-II. Immunoblotting with anantiserum, which had been raised against the 24 and 25 kDa apoproteinsof LHC-II and found to cross-react with the 26 kDa protein ofLHC-II and the 20 and 21 kDa apoproteins of light-harvestingchlorophyll a/b protein complexes of photosystem I (LHC-I),revealed that all the five proteins related to LHC-Iand LHC-IIwere present in reduced amounts in the Type I mutants. ThreeType HA mutants, which have a chlorophyll a/b ratio of 10, weremore abundant in the apoproteins, while three Type IIB mutantswith the ratio of 15 were heterogeneous in terms of the apoproteincontent. All the chlorina mutants contained less P700 comparedwith the wild type rice, but were relatively more abundant inthe LHC-I proteins than the LHC-II proteins. The results showthat all the rice chlorina strains are mutants of chlorophyllb synthesis and the deficiency of chlorophyll b differentlyaffects accumulation of the apoproteins of LHC-I and LHC-II.To balance light absorption between the two photosystem, lossof LHC-II is partly counter-balanced by a decrease in the numberof PSI complexes in the mutants. (Received January 21, 1988; Accepted April 28, 1988)     

Isolation of Lamellar Aggregates of the Light-Harvesting Chlorophyll a/b Protein Complex of Photosystem II with Long-Range Chiral Order and Structural Flexibility

Isolation of LHCII, the light-harvesting chlorophyll a/b complex of photosystem II, based on the procedure described by Krupaet al.(1987,Plant Physiol.84, 19–24), was optimized for obtaining purified lamellar aggregates with long-range chiral order and structural flexibility (the capability of undergoing light-induced reversible structural changes). By varying the concentration of the detergent Triton X-100 for the solubilization of thylakoid membranes, we obtained four types of LHCII aggregates: (i) With low detergent concentration, ≤0.6% (v/v), the aggregates contained lipids in high amount. These preparations with Chl a/b ratios of about 1.4 contained minor antenna complexes with a fingerprint of an additional CD band at (+) 505 nm; they formed disordered lamellae and exhibited no or weak psi-type CD bands (psi, polymerization- or salt-induced), which did not possess the ability to undergo light-induced changes (ΔCD). (ii) At the optimal concentration, around 0.7 ± 0.1% (v/v), the detergent removed some lipids and most of the minor complexes, and the Chl a/b ratio dropped to 1.0–1.1. LHCII formed loosely stacked two-dimensional lamellae which exhibited psi-type CD bands and large light-induced reversible structural changes (ΔCD). (iii) At detergent concentration above the optimum, around 0.8–1% (v/v), the lipid content of LHCII decreased and minor complexes could not be detected. LHCII formed disordered aggregates and showed neither psi-type CD nor ΔCD. (iv) High concentrations (≥1.1% (v/v)) Triton X-100 led to very pure but largely delipidated samples assembled into tightly stacked three-dimensional lamellar structures with intense psi-type CD but no ΔCD.     

Phytochrome Regulation of Greening in Pisum: Chlorophyll Accumulation and Abundance of mRNA for the Light-Harvesting Chlorophyll a/b Binding Proteins

A brief pulse of red light eliminates or reduces the lag in chlorophyll accumulation that occurs when dark-grown pea seedlings are transferred to continuous white light. The red light pulse also induces the accumulation of specific mRNAs. We compared time courses, escape from reversal by far-red light, and fluence-response behavior for induction of mRNA for the light-harvesting chlorophyll a/b binding proteins (Cab mRNA) with those for induction of rapid chlorophyll accumulation in seedlings of Pisum sativum cv Alaska. In both cases the time courses of low fluence and very low fluence responses diverged from each other in a similar fashion: the low fluence responses continued to increase for at least 24 hours, while the very low fluence responses reached saturation by 8 to 16 hours. Both responses escaped from reversibility by far-red slowly, approaching the red control level after 16 hours. The fluence-response curve for the Cab mRNA increase, on the other hand, showed threshold and saturation at fluences 10-fold lower than threshold and saturation values for the greening response. Therefore, the level of Cab mRNA, as measured by the presence of sequences hybridizing to a cDNA probe, does not limit the rate of chlorophyll accumulation after transfer of pea seedlings to white light. The Cab mRNA level in the buds of seedlings grown under continuous red light remained high even when the red fluence rate was too low to allow significant greening. In this case also, abundance of Cab mRNA cannot be what limits chlorophyll accumulation.     

Regulation of Electron Transport Composition in Cyanobacterial Photosynthetic System: Stoichiometry among Photosystem I and II Complexes and Their Light-Harvesting Antennae and Cytochrome b6/fComplex

This study was done to confirm our previous observation withthe pattern of changes in electron transport composition inducedby an imbalance of the electron transport state. Contents ofphotosystem (PS) I and II complexes and their antennae and Cytb₆/f complex were determined for systems of cyanobacterium SynechocystisPCC 6714 of different PS I/PS II ratios. The results indicatedthat (1) the observed changes in the PS I/PS II ratio are not-dueto regulation of the activities of the respective PS's but tochanges in their contents, (2) the molar ratio between PS IIand Cyt b₆/f complexes was fairly constant when marked changesoccurred in the PS I content, and (3) the PS II and Cyt b₆/fcontents per cell remained fairly constant while the PS I contentchanged markedly. These findings agree with our previous observationwith autotrophic cells of Anacystis nidulans Tx 20 and supportour argument that in cyanobacterial and red algal electron transportsystems, the content of the terminalcomponent(s), such as PSI complex, is regulated in order to maintain a balance betweenthe electron influx by PS II action to the system and the effluxby PS I action from it. (Received June 3, 1987; Accepted September 20, 1987)     

Organization of Chlorophyll a in the Light-Harvesting Chlorophyll a/b Protein Complex as Shown by Circular Dichroism : Liquid Crystal-Like Domains

The development of thylakoid stacking, accumulation of the light-harvesting chlorophyll a/b protein complex (LHCP), and the changes of circular dichroism (CD) which reflect the organization of chlorophyll molecules in greening thylakoids of bean Phaseolus vulgaris cv Red Kidney leaves were investigated.

Chloroplasts formed under intermittent light contained large double sheets of membrane with extensive appression in addition to separate lamellae. Thylakoids of such chloroplasts were devoid of LHCP and exhibited a relatively small CD in the chlorophyll absorption region. Upon continuous illumination, the rearrangement of membranes to characteristic grana and the accumulation of the LHCP was accompanied by the gradual appearance of the very intense CD signal with peaks at 682 to 684 (+) and 665 to 672 nanometers (−). The magnitude of differential absorption was approximately 100 times larger than that of the chlorophyll a in solution. This suggests a superhelical liquid crystal-like organization for LHCP, a texture which can be altered by changes of the electric field in the photosynthetic membranes.

     

Chlorophyll b-Deficient Mutants of Rice: II. Antenna Chlorophyll a/b-Proteins of Photosystem I and II

Chlorophyll-protein complexes of the wild type and 16 strainsof chlorina mutants of rice were investigated by gel electrophoresis.An antenna chlorophyll a/b-protein of photosystem II (LHC-II)was present in reduced amounts in Type II chlorina mutants whichhave the chlorophyll a/b ratios of 10–15, and was totallyabsent from Type I chlorina mutants which lack chlorophyll b.Another antenna chlorophyll-protein of photosystem I (LHC-I)containing two polypeptides of 20 and 21 kDa was also presentin the Type II mutants but not in the Type I mutants. The polypeptideprofiles of the thylakoid membranes indicate that Type I mutantslack both the 20 and 21 kDa polypeptides, whereas the abundanceof the two polypeptides relative to the CPI apoprotein in theType II mutants is comparable with that in the wild type. Itis concluded that the 20 and 21 kDa polypeptides are both relatedto LHC-I and are normally synthesized and accumulated in theType II mutants. (Received June 6, 1985; Accepted August 6, 1985)     

Changes in Thermostability of Photosystem Ⅱ and Leaf Lipid Composition of Rice Mutant with Deficiency of Light-harvesting Chlorophyll a/b Protein Complexes

We studied the difference in thermostability of photosystem Ⅱ （PSII） and leaf lipid composition between a T-DNA insertion mutant rice （Oryza sativa L.） VG28 and its wild type Zhonghuau. Native green gel and SDS-PAGE electrophoreses revealed that the mutant VG28 lacked all light-harvesting chlorophyll a/b protein complexes. Both the mutant and wild type were sensitive to high temperatures, and the maximal efficiency of PSII photochemistry （FJ Fm） and oxygen-evolving activity of PSII in leaves significantly decreased with increasing temperature. However, the PSII activity of the mutant was markedly more sensitive to high temperatures than that of the wild type. Lipid composition analysis showed that the mutant had less phosphatidylglycerol and sulfoquinovosyl diacylglycerol compared with the wild type. Fatty acid analysis revealed that the mutant had an obvious decrease in the content of 16：1t and a marked increase in the content of 18：3 compared with the wild type. The effects of lipid composition and unsaturation of membrane lipids on the thermostability of PSII are discussed.     

Photo and Nutritional Regulation of the Light-Harvesting Chlorophyll a/b-Binding Protein of Photosystem II mRNA Levels in Euglena

In Euglena gracillis var bacillaris, light exposure increases the level of mRNA encoding the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCPII) approximately twofold. LHCPII mRNA levels increased in the dark upon either malate or ethanol addition. LHCPII mRNA is present but LHCPII is not synthesized in the bleached mutants W₃BUL and W₁₀BSmL, which lack protochlorophyll(ide) and most if not all of the chloroplast genome. Light exposure increased LHCPII mRNA levels in W₃BUL but not in W₁₀BSmL. Carbon availability and light acting through a nonchloroplast photoreceptor appear to regulate LHCPII mRNA levels. A chloroplast photoreceptor and/or a product produced by the chloroplast appear to regulate LHCPII mRNA translation.