Resolution of the light-harvesting chlorophyll a/b-protein of vicia faba chloroplasts into two different chlorophyll-protein complexes.

Thylakoids of Vicia faba chloroplasts disaggregated by sodium dodecyl sulfate were separated by means of different electrophoretic systems. Under the conditions of a high resolving gel system the chlorophyll containing zone previously termed chlorophyll-protein complex II or light-harvesting chlorophyll a/b-protein was found to be inhomogeneous. It represents a mixture of two distinct chlorophyll-proteins characterized by different spectral properties and different apoproteins. One chlorophyll-protein exhibits a chlorophyll a/b ratio of 0.9 and is associated with polypetides of 24,000 and 23,000 daltons. The 24,000 dalton band is proved to bind chlorophyll and has a light-harvesting function. The function of the 23,000 dalton band is unknown. The second chlorophyll-protein has a chlorophyll a/b ratio of 2.1 and an additional absorption maximum in the position of 637 nm. It is associated with only one polypeptide which has an apparent molecular weight of 23,000. The two 23,000 dalton polypeptides occurring in both complexes are not identical.     

Partial characterization of a minor chlorophyll-protein found in primary thylakoids of intermittently illuminated maize

A mild solubilization with sodium dodecyl sulphate of intermittently illuminated maize (Zea mays L. Mvsc 429) thylakoids allows the separation of a minor chlorophyll-protein in the position of the light harvesting chlorophyll-protein monomer of green plants by polyacrylamide gel electrophoresis. It contains mainly chlorophyll a, its chlorophyll b content may come from the slightly contaminating light harvesting chlorophyll a/b-protein. It represents about 15% of the chlorophyll in protochloroplasts. The new chlorophyll-protein has an absorption maximum at 672 nm, and only one fluorescence emission peak at 680 nm. A 34 kD polypeptide is the most abundant one in the polypeptide pattern of the complex. The function of the new chlorophyll-protein is unknown at present. Its relationship to other chlorophyll-proteins is discussed.     

Reinvestigation of the chlorophyll distribution among the chlorophyll-proteins and chlorophyll-protein complexes of Hordeum vulgare L.

Solubilization of barley (Hordeum vulgare L.) thylakoid membranes with sodium dodecylsulphate plus sodium deoxycholate with or without Triton X-100 and subsequent fractionation in the polyacrylamide gel electrophoresis system described in this paper resulted: (1) in the resolution of the chlorophyll-proteins and chlorophyll-protein complexes commonly known as CP1a, CP1, LHCP¹, LHCP², CPa and LHCP³; (2) in the highly increased stability of CP1 and CP1a, as judged by their chlorophyll content, (3) at the expense of the free pigment concentration (4) which could be reduced to a negligible amount. Some 40% of the total chlorophyll contained in the mature higher plant thylakoid membrane is associated with CP1 and CP1 a and as already suggested before [19] no significant amount of free chlorophyll occurs in vivo.Abbreviations chl chlorophyll - CP1 P₇₀₀-chla-protein - CPa P680-chla-protein - DOC sodium deoxychlolate - FC free chlorophyll - LHCP light-harvesting chlorophyll a/b-protein - PAGE(S) polyacrylamide gel electrophoresis (system) - SDS sodium dodecylsulphate - TX-100 Triton X-100     

Adaptation of the thylakoid membranes of pea chloroplasts to light intensities. I. Study on the distribution of chlorophyll-protein complexes

The effect of light intensity (16 h white light and 8 h dark) during growth of pea plants at 20°C on the chlorophyll composition and on the relative distribution of chlorophyll amongst the various chlorophyll-protein of pea thylakoids was studied. The chl a/chl b ratios increased from 2.1 to 3.2 as light intensity during growth varied from 10 to 840 Em^-2 s^-1. This function can be described by two straight lines intersecting at a transition point of approximately 200 Em^-2 s^-1. Similar discontinuities in the responses were observed in the changes in the relative distribution of chlorophyll amongst the various chlorophyll-protein complexes. This demonstrates that the chl a/chl b ratio of the various thylakoids is a good indicator of changes in the relative distribution of chlorophyll. As the chl a/chl b ratio decreased, the amount of chlorophyll associated with photosystem I complexes decreased, that with photosystem II core reaction centre complex was halved, and that with the main chl a/b-proteins of the light-harvesting complex was markedly increased.Abbreviations chl chlorophyll - PS photosystem - SDS sodium dodecyl sulphate - Tricine N-tris (hydroxymethyl) methylglycine     

Resolution of the light-harvesting chlorophyll ab-protein of Vicia faba chloroplasts into two different chlorophyll-protein complexes

Thylakoids of Vicia faba chloroplasts disaggregated by sodium dodecyl sulfate were separated by means of different electrophoretic systems. Under the conditions of a high resolving gel system the chlorophyll containing zone previously termed chlorophyll-protein complex II or light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ was found to be inhomogeneous. It represents a mixture of two distinct chlorophyll-proteins characterized by different spectral properties and different apoproteins. One chlorophyll-protein exhibits a chlorophyll $\text{a}\text{b}$ ratio of 0.9 and is associated with polypeptides of 24 000 and 23 000 daltons. The 24 000 dalton band is proved to bind chlorophyll and has a light-harvesting function. The function of the 23 000 dalton band is unknown. The second chlorophyll-protein has a chlorophyll $\text{a}\text{b}$ ratio of 2.1 and an additional absorption maximum in the position of 637 nm. It is associated with only one polypeptide which has an apparent molecular weight of 23 000. The two 23 000 dalton polypeptides occurring in both complexes are not identical.     

Characterization of early events leading to the formation of chlorophyll-proteins of PSII in Euglena gracilis

Development of chlorophyll-proteins in photosystem II was studied with Euglena gracilis Z. during dark-light transition. Upon illumination of the dark-grown cells, protochlorophyllide was photoconverted to chlorophyll(ide) a with a low efficiency (14%). After a lag time of 1-2 h, chlorophylls, apoproteins of antenna chlorophyll-protein complex CP 43/47 and of light-harvesting chlorophyll-protein complex (LHCII) accumulated in the thylakoid membrane in a coordinated fashion. There was, however, a significant difference in the stability between the newly formed LHCII and CP 43/47 judging from non-denaturing lithium dodecyl sulfate-polyacrylamide gel electrophoresis. The possibility that efficiencies of incorporation and stabilization of chlorophylls in the apoproteins differ among the chlorophyll-proteins in the early stage of greening of Euglena is discussed.     

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.     

The nature of the light-harvesting complex as defined by sodium dodecyl sulfate polyacrylamide gel electrophoresis

Reelectrophoresis of the oligomer form (CP II1) of the chlorophyll $\text{a}\text{b}$ light-harvesting complex (LHC) from the green alga Acetabularia yields two green bands which run at the position typical of the monomer (CP II). The upper green band (CP II₁) is enriched in the 27 kDa polypeptide of the LHC, while the lower is enriched in the 26 kDa polypeptide. The fact that both bands have both chlorophyll (Chl) a and b, and in the same ratio, implies that the LHC is made up of two Chl $\text{a}\text{b}$ proteins. Neither of these bands can be attributed to the Chl $\text{a}\text{b}$ complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432). Resolution of CP II₁ and CP II₂ of spinach can be obtained if sucrose gradient fractions of an octylglucoside extract are subjected to SDS-polyacrylamide gel electrophoresis. CP II₁ and CP II₂ are interpreted as being fundamental subunits of the light-harvesting complex as it is defined on SDS-polyacrylamide gels.     

Sequential extraction of chlorophyll from chlorophyll-protein complexes in lyophilized pea thylakoids with solvents of different polarity

Lyophilized chloroplasts of Pisum sativum (pea) have been extracted with petroleum ether of different polarity (obtained by adding varying amounts of ethanol to the petroleum ether). Extracted thylakoids have then been solubilized by sodium dodecyl sulphate (SDS) and chlorophyll-protein complexes have been isolated by polyacrylamide gel electrophoresis (PAGE). Absorption- and low temperature fluorescence emission spectro-scopy have been used to characterize thylakoids and purified chlorophyll-protein complexes. Weakly polar solvents extracted mainly chlorophyll a. SDS-PAGE scan profiles of similarly extracted thylakoids contained no photosystem II chlorophyll a reaction center antennae (CP-a_n) and the amount of photosystem I chlorophyll a reaction center antennae (CP-a₁) was reduced as compared with an unextracted control. This was due partly to the extraction of chlorophyll a prior to SDS-PAGE, and partly to the increased solubilization of chlorophyll a by SDS as a result of β-carotene extraction. By increasing the polarity of the solvent CP-a₁ also disappeared in the scan profile, leaving only the light-harvesting chlorophyll a/b-protein complex (CP-a/b) and SDS complexed chlorophyll. From these results we conclude that the chlorophyll molecules in the reaction center antennae are relatively more hydrophobically associated than the molecules in the light-harvesting CP-a/b complex. The chlorophyll a of CP-a_u and the far red absorbing chlorophyll a fraction of CP-a₁ appear to be the most hydrophobically associated chlorophyll molecules.     

管藻目绿藻叶绿素蛋白复合物特性及比较研究

By mild PAGE method, 11, 11, 7 and 9 chlorophyll-protein complexes were isolated from two species of siphonous green algae (Codium fragile (Sur.) Hariot and Bryopsis corticulans Setch.), green alga (Ulothrix flacca (Dillw.) Thur.), and spinach (Spinacia oleracea Mill.), respectively. Apparent molecular weights, Chl a/b ratios, distribution of chlorophyll, absorption spectra, low temperature fluorescence spectra of these complexes were determined, and compared with one another. PSⅠ complexes of two siphonous green algae are larger in apparent molecular weight because of the attachment of relative highly aggregated LHCⅠ. Four isolated light-harvesting complexes of PSⅡ are all siphonaxanthin-Chl a/b-protein complexes, and they are not monomers and oligomers like those in higher plants. Especially, the absence of 730 nm fluorescence in PSⅠ complexes indicates a distinct structure and energy transfer pattern.     

Polypeptide composition and spectral properties of light-harvesting chlorophyll ab-protein complexes from intact and trypsin-treated chloroplast thylakoid membranes

The polypeptide composition and spectral properties of isolated light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complexes from intact and trypsin-treated thylakoid membranes of Hordeum vulgare and Vicia faba are compared. The LHCP complexes consist of four distinct polypeptides with molecular weights between 21 000 and 25 000 occurring in equal relative amounts in the whole polypeptide spectra of thylakoid membranes. It is shown indirectly that the two major polypeptides very probably belong to different chlorophyll-proteins. The loss of a small segment from both polypeptides during trypsin digestion of thylakoids does not substantially alter the spectral properties and cation-mediated aggregation of isolated LHCP complexes.     

Functional and structural organization of chlorophyll in the developing photosynthetic membranes of Euglena gracilis Z. V-separation and characterization of pigment-protein complexes of the differentiated thylakoids

Low temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis following mild solubilization of Euglena thylakoid components allowed to resolve, in addition to the main CP1, CPa and LHCP chlorophyll-protein complexes, the additional CP1a and LHCP green bands. A carotenoid enriched band CPc can be separated from CPa using high acrylamide concentration. Pigment and polypeptide composition of these complexes were analyzed by absorption and fluorescence measurements and two dimensional gel electrophoresis. Spectral properties of CP1 and CP1a indicate an heterogenous organization of chlorophyll and the presence of significant amount of chlorophyll b in these complexes. They both contain a major 68 kilodalton polypeptide associated with three minor low molecular weight polypeptides in CP1a. CPa and CPc exhibit a characteristic fluorescence emission at 687 nm and they each contain one polypeptide of 54 and 41 Kda respectively. LHCP and LHCP are less abundant than in higher plant thylakoids and they contain a lower proportion of chl b (chl a: chl b=3). They include two polypeptides of 26 and 29 Kda.Abbreviations chl chlorophyll - SDS Sodium Dodecyl Sulfate - EDTA Ethylene Diamine Tetraacetic Acid - DTT Dithiothreitol     

Characterization of a 31 kDa polypeptide that accumulates in the light-harvesting apparatus of maize leaves during chilling

A 31 kDa polypeptide that accumulates in the thylakoids when maize leaves are chilled to 5°C in the light is characterized using monoclonal antibodies and analyses of chlorophyll-protein complexes. This polypeptide reacted with a monoclonal antibody, MLH2, that was specific for the 28 kDa polypeptide of the light-harvesting complex (LHCII) of pea leaves. On chilling leaves the appearance of a chlorophyll-protein complex having an apparent molecular weight of 31 kDa coincided with the appearance of a 31 kDa polypeptide and a decrease in the 29 kDa chlorophyll-protein, CP29. Returning the leaves to 25°C for 1 h produced a loss of both the 31 kDa chlorophyll-protein and 31 kDa polypeptide from the thylakoids, and an increase in the amount of CP29. Breakdown of the 31 kDa polypeptide in vitro was Mg²⁺-dependent and inhibited by EDTA and transition metal ions. It is suggested that the 31 kDa polypeptide may be a precursor of the apoprotein of CP29 and can bind chlorophyll. The appearance of the 31 kDa polypeptide correlated with a marked change in the 77 K fluorescence emission spectra of isolated LHCII particles, which did not revert with the disappearance of the 31 kDa on returning the leaves to 25°C for 1 h. The physiological significance of this spectral perturbation is discussed.     

The light-harvesting system of Euglena gracilis during the cell cycle

The apoproteins of the light-harvesting chlorophyll-protein complexes LHCI and CP29 (apparent molecular weights of 27 kDa and 29 kDa, respectively) of Euglena gracilis were identified immunologically. Both complexes are present in the thylakoids of autotrophically cultured Euglena cells during the whole cell cycle. The relative amount of each apoprotein tends to increase towards the end of the cell cycle. The light-harvesting chlorophyll-protein complex of photosystem II, LHCII, of E. gracilis contains chlorophyll a, chlorophyll b, neoxanthin, diadinoxanthin and beta-carotene. Its chlorophyll a/b ratio is about 1.7 during the whole cell cycle. About 9 h after cell division the ratio of diadinoxanthin to chlorophyll a is doubled for a time of 3–4 h. The relevance of this increase during one developmental stage is discussed in relation to the insertion and-or assembly of newly synthesized LHCII.Abbreviations LHCP light-harvesting chlorophyll-protein complex - PS photosystem This research was partly supported by the Deutsche Forschungsge meinschaft.     

Chlorophyll-proteins of the photosystem II antenna system

The chlorophyll-protein complexes of purified maize photosystem II membranes were separated by a new mild gel electrophoresis system under conditions which maintained all of the major chlorophyll a/b-protein complex (LHCII) in the oligomeric form. This enabled the resolution of three chlorophyll a/b-proteins in the 26-31-kDa region which are normally obscured by monomeric LHCII. All chlorophyll a/b-proteins had unique polypeptide compositions and characteristic spectral properties. One of them (CP26) has not previously been described, and another (CP24) appeared to be identical to the connecting antenna of photosystem I (LHCI-680). Both CP24 and CP29 from maize had at least one epitope in common with the light-harvesting antennae of photosystem I, as shown by cross-reactivity with a monoclonal antibody raised against LHCI from barley thylakoids. A complex designated Chla.P2, which was capable of electron transport from diphenylcarbazide to 2,6-dichlorophenolindophenol, was isolated by nondenaturing gel electrophoresis. It lacked CP43, which therefore can be excluded as an essential component of the photosystem II reaction center core. Fractionation of octyl glucoside-solubilized photosystem II membranes in the presence and absence of Mg2+ enabled the isolation of the Chla . P2 complex and revealed the existence of a light-harvesting complex consisting of CP29, CP26, and CP24. This complex and the major light-harvesting system (LHCII) are postulated to transfer excitation energy independently to the photosystem II reaction center via CP43.     

Adaptation of the thylakoid membranes of pea chloroplasts to light intensities. II. Regulation of electron transport capacities,electron carriers,coupling factor (CF1) activity and rates of photosynthesis

The electron transport rates of photosystems II and I, amounts of electron carriers, coupling factor activity and photosynthetic rates were investigated in thylakoids isolated from pea plants grown under a wide range of light intensities (16 h light-8 h dark). The electron transport rates of PS II and PS I, as partial reactions or in whole chain, and coupling factor activity on a unit chlorophyll basis, all increased as the light intensity available for growth was altered from a very low intensity of 10 E m^-2s^-1 to a high intensity of 840 E m^-2s^-1. Similarly, there were increases in the amounts of atrazine binding sites, plastoquinine, cytochrome f and P700 per unit chlorophyll; significantly, the amounts of reaction centres of PS II and PS I were not equal at any light intensity. The rate of change of all parameters with respect to light intensity could be represented by two straight lines of different slopes which met at a transition point corresponding to approximately 200 E m^-2s^-1 during growth. These photoadaptations were similar to those observed for both the relative distribution of chlorophyll in chlorophyll-protein complexes and the chl a/chl b ratios [Leong and Anderson, 1984, Photosynthesis Research 5:117–128]. Since these thylakoid components and functions were affected in the same direction by light intensity during growth and all show linear relationships with chl a/chl b ratios, it indicates that they are closely regulated and markedly well co-ordinated. Plants compensate for the limited amount of low light intensities by drastically increasing the light-harvesting antenna unit size of photosystem II and to a lesser extent that of photosystem I. Changes in the composition of the thylakoid membranes exert a regulatory effect on the overall photosynthetic rate up to approximately 450 E m^-2s^-1.Abbreviations chl chlorophyll - cyt cytochrome - PQ plastoquinone - PS photosystem     

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.     

Characterization of a spinach photosystem II core preparation isolated by a simplified method

A photosystem II core complex from spinach exhibiting high rates of electron transport was obtained rapidly and in high yield by treatment of a Tris-extracted, O2-evolving photosystem II preparation with the detergent dodecyl-beta-D-maltoside. The core complex was essentially free of light-harvesting chlorophyll-protein and photosystem I polypeptides, and was highly enriched in the polypeptides associated with the photosystem II reaction center (45 and 49 kDa), cytochrome b559, and three polypeptides in the region 32-34 kDa. The photosystem II core complex contained two chlorophyll-proteins which had a slightly higher apparent molecular mass than CPa-1 and CPa-2. Additionally, a high-molecular-mass chlorophyll-protein complex termed CPa* was observed, which exhibited a low fluorescence yield when illuminated with ultraviolet light. This observation suggests that CPa* contains a functionally efficient quencher of chlorophyll fluorescence, possibly P680.     

Absorption and fluorescence spectra of chlorophyll-proteins isolated from Euglena gracilis

A spectroscopic study of chlorophyll-protein complexes isolated from Euglena gracilis membranes was carried out to gain information about the state of chlorophyll in vivo and energy transfer in photosynthesis. The membranes were dissociated by Triton X-100 and separated into fractions by sucrose gradient centrifugation and hydroxyapatite chromatography. Four different types of chlorophyll-protein complexes were distinguished from each other and from detergent-solubilized chlorophyll in these fractions by examination of their absorption, fluorescence excitation (400–500 nm) and emission spectra at low temperature. These types were: (1). A mixture of antenna chlorophyll a- and chlorophyll ab-proteins with an absorption maximum at 669 and emission at 682 nm; (2) a P-700-chlorophyll a-protein (chlorophyll: P-700 = 30 : 1), termed CPI with an absorption maximum at 676 nm and emission maxima at 698 and 718 nm; (3) a second chlorophyll a-protein (CPI-2) less enriched in P-700, with an absorption maximum at 676 nm and emission maxima at 680, 722 and 731 nm; (4) a third chlorophyll a-protein (CPa₁) with no P-700, absorption maxima at 670 and 683 nm, and an unusually sharp emission maximum at 687 nm. Treatment of CPa₁ with sodium dodecyl sulfate drastically altered its spectroscopic properties indicating that at least some chlorophyll-proteins isolated with this detergent are partially denatured. The results suggest that the complex absorption spectra of chlorophyll in vivo are caused by varying proportions of different chlorophyll-protein complexes, each with different groups of chlorophyll molecules bound to it and making up a unique entity in terms of electronic transitions.