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)     

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)     

Stoichiometries of Photosystem I and Photosystem II in Rice Mutants Differently Deficient in Chlorophyll b

Stoichiometries of photosystem I (PSI) and photosystem II (PSII)reaction centers in a cultivar of rice, Norin No. 8, and threechlorophyll b-deficient mutants derived from the cultivar wereinvestigated. Quantitation of PSI by photooxidation of P-700and chromatographic assay of vitamin K₁ showed that, on thebasis of chlorophyll, the mutants have higher concentrationsof PSI than the wildtype rice. Greater increases were observedin the PSII contents measured by photoreduction of Q_A, bindingof a radioactive herbicide and atomic absorption spectroscopyof Mn. Consequently, the PSII to PSI ratio increased from 1.1–1.3in the wild-type rice to 1.8 in chlorina 2, which contains noChl b, and to 2.0–3.3 in chlorina 11 and chlorina 14,which have chlorophyll a/b ratios of 9 and 13, respectively.Measurement of oxygen evolution with saturating single-turnoverflashes revealed that, whereas at most 20% of PSII centers areinactive in oxygen evolution in the wildtype rice, the non-functionalPSII centers amount to about 50% in the three mutant strains.The fluorescence induction kinetics was also analyzed to estimateproportions of the inactive PSII in the mutants. The data obtainedsuggest that plants have an ability to adjust the stoichiometryof the two photosystems and the functional organization of PSIIin response to the genetically induced deficiency of chlorophyllb. (Received July 29, 1994; Accepted February 7, 1996)     

Chlorophyll b-Deficient Mutants of Rice: I. Absorption and Fluorescence Spectra and Chlorophyll a/b Ratios

Absorption spectra and their fourth derivatives of chloroplastsisolated from 16 different rice chlorina mutants were determinedat liquid nitrogen temperature. The results suggest that Chlb is absent from 10 mutants labelled chlorina-1 to -10, while6 mutants named chlorina-11 to -16 contain low levels of Chlb. Low temperature fluorescence emission spectra indicate thata light-harvesting Chl a/b protein of photosystem I is presentin chlorina-11 to -16 but not in chlorina-1 to -10. Reinvestigationof Chl a/b ratios by a sensitive fluorescence method shows thatthe 16 mutants are divided into three groups different in thedegree of Chl b-deficiency; chlorina-1 to -10 totally lack Chlb (Type I), chlorina-11 to -13 have Chl a/b ratio of about 10(Type II-A) and chlorina-14 to -16 have the ratio of about 15(Type II-B). (Received June 6, 1985; Accepted August 6, 1985)     

Antenna Sizes of Photosystem I and Photosystem II in Chlorophyll b-Deficient Mutants of Rice. Evidence for an Antenna Function of Photosystem II Centers That are Inactive in Electron Transport

Light-harvesting capacities of photosystem I (PSI) and photosystemII (PSII) in a wild-type and three chlorophyll b-deficient mutantstrains of rice were determined by measuring the initial slopeof light-response curve of PSI and PSII electron transport andkinetics of light-induced redox changes of P-700 and Q_A, respectively.The light-harvesting capacity of PSI determined by the two methodswas only moderately reduced by chlorophyll b-deficiency. Analysisof the fluorescence induction that monitors time course of Q_Aphotoreduction showed that both relative abundance and antennasize of PSII_a decrease with increasing deficiency of chlorophyllb and there is only PSII_rß in chlorina 2 which totallylacks chlorophyll b. The numbers of antenna chlorophyll moleculesassociated with the mutant PSII centers were, therefore, threeto five times smaller than that of PSII_a in the wild type rice.Rates of PSII electron transport determined on the basis ofPSII centers in the three mutants were 60–70% of thatin the normal plant at all photon flux densities examined, indicatingthat substantial portions of the mutant PSII centers are inactivein electron transport. The initial slopes of light-responsecurves of PSII electron transport revealed that the functionalantenna sizes of the active populations of PSII centers in themutants correspond to about half that of PSII in the wild typerice. Thus, the numbers of chlorophyll molecules that serveas antenna of the oxygen-evolving PSII centers in the mutantsare significantly larger than those that are actually associatedwith each PSII center. It is proposed that the inactive PSIIserves as an antenna of the active PSII in the three chlorophyllb-deficient mutants of rice. In spite of the reduced antennasize of PSII, therefore, the total light-harvesting capacityof PSII approximately matches that of PSI in the mutants. (Received July 29, 1994; Accepted February 7, 1996)     

Supramolecular Assembly and Function of Subunits Associated with the Chlorophyll a-b Light-Harvesting Complex II (LHC II) in Soybean Chloroplasts

The chlorophyll (Chl) a-b light harvesting complex II (LHC II)contains more than 80% of the light-harvesting pigments of photosystemII (PS II) in chloroplasts. The supramolecular assembly andfunction of this auxiliary antenna system was investigated inChi b-deficient and Chi b-less mutant chloroplasts from soybeanand barley plants, and in their wild-type counterparts. Fourdistinct LHC II polypeptides were resolved by SDS-PAGE (subunitsa, b, c and d), having apparent molecular masses of 29, 28,27.2 and 26.8 kDa, respectively. The analysis of LHC II subunitcomposition in different developmental stages of the PS II unitin soybean (3>Chla/Chlbb>6), indicated the associationof specific subunits with the LHC H-inner and LHC II-peripheralin the chloroplast. The amount of subunit a in PS II was constantover a broad range of Chl a/Chl b ratios, suggesting that thissubunit is closely associated with the PS II-core complex. Subunitd also appeared to be constant over a wide range of Chl a/Chlb ratios, suggesting close association with the LHC II-inner.The PS II content in subunits b and c increased with the PSII antenna development in soybean but the ratio of b/c remainedconstant in all developmental stages and equal to 2 :1. Subunita was present in the Chl b-less chlorina f2 mutant of barleygrown under continuous illumination but was absent under intermittentillumination. The results suggest that each subunit binds 13-15Chl molecules. A working hypothesis is presented on the PS IIantenna development and LHC II subunit composition in soybeanchloroplasts. (Received October 11, 1988; Accepted January 19, 1989)     

Diurnal fluctuation in the composition of chlorophyll a/b light harvesting antenna of photosystem II in young wheat leaves

We investigated the diurnal fluctuation in the composition of the light harvesting chlorophyll a/b antenna of photosystem II in young wheat (Triticum aestivum) leaves grown under periodic day/night irradiation. By means of gel electrophoresis of the polypeptides of thylakoid membranes, we determined the amount of 25 kDa and 27 kDa polypeptides, which are the main components of the peripheral and inner antenna subpopulations, respectively. Our data show a preferential fluctuation in the amount of the 25 kDa protein relative to the 27 kDa polypeptide, in parallel to the fluctuation in the amount of chlorophyll a/b antenna of photosystem II, which suggests that the peripheral antenna plays a role in the diurnal adjustment of the antenna size.     

The detection,isolation and characterization of a light-harvesting complex which is specifically associated with Photosystem I

10% of the chlorophyll associated with a ‘native’ Photosystem (PS) I complex (110 chlorophylls/P-700) is chlorophyll (Chl) b. The Chl b is associated with a specific PS I antenna complex which we designate as LHC-I (i.e., a light-harvesting complex serving PS I). When the native PS I complex is degraded to the core complex by LHC-I extraction, there is a parallel loss of Chl b, fluorescence at 735 nm, together with 647 and 686 nm circular dichroism spectral properties, as well as a group of polypeptides of 24-19 kDa. In this paper we present a method by which the LHC-I complex can be dissociated from the native PS I. The isolated LHC-I contains significant amounts of Chl b (Chl $\text{a}\text{b ? 3.7}$). The long-wavelength fluorescence at 730 nm and circular dichroism signal at 686 nm observed in native PS I are maintained in this isolated complex. This isolated fraction also contains the low molecular weight polypeptides lost in the preparation of PS I core complex. We conclude that we have isolated the PS I antenna in an intact state and discuss its in vivo function.     

Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II

A collection of 17 monoclonal antibodies elicited against the light-harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC-II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b.     

Effect of Chloramphenicol on Etiochloroplasts of Wild and Chlorophyll b-Less Barley Genotypes

Greening of etiolated seedlings of wild and Chl b-less barley(Hordeum vulgare L.) genotypes in the presence of D-threochloramphenicol(CAP) led to macrogranal arrangements accompanying the inhibitionof Chl synthesis and an enhancement of the total protein contentin differentiated etiochloroplasts. In treated mutant plastids,protein/Chl ratio reached up to 100. No light-dependent O₂ evolution was detected in CAP-treatedplastids which had deficiency in polypeptides belonging to thephotosystem II (PSII) centres. On the other hand, plastids displayeda high photosystem I (PSI) activity despite the absence of the92 kDa polypeptide linked to the PSI centre. The accumulationof polypeptides ranging from 16 to 20 kDa suggest that theycould originate from primary complexes consisting of few Chlmolecules, but they were sufficient to allow the activity ofthe reaction centres. No accumulation of the 25–27 kDapolypeptides linked to the PSII antenna was detected. The increase in the proportion of trans-₃hexadecenoic acid (16:1tr) in phosphatidylglycerol (PG) of etiochloroplasts from bothtypes after CAP treatment could indicate an alteration of theregulation process of 16:1 tr biosynthesis occurring in plastids.The formation of macrograna could optimize the energy transferin altered thylakoid membranes. The accumulation of PG-16:1tr molecules could be related to the formation of active primarycomplexes in thylakoid when Chl synthesis is altered. (Received March 30, 1988; Accepted June 1, 1988)     

Correspondence of Apoproteins of Light-Harvesting Chlorophyll a/b Complexes Associated with Photosystem I to cab Genes: Evidence for a Novel Type IV Apoprotein

Apoproteins of spinach and pea light-harvesting chlorophylla/b complexes associated with photosystem I (LHCI) were identifiedby their chlorophyll fluorescence spectra and protein sequences.Spinach LHCI holocomplex consisted of four apoproteins of 25kDa, 23 kDa, 21 kDa and 20.5 kDa. LHCI subcomplex isolated bysucrose density gradient centrifugation fluoresced at 680 nmwith a shoulder around 700–710 nm at 77 K. It containedthe 23 kDa protein of which the N-terminal sequence correspondedto Type II gene of LHCI. Another LHCI subcomplex isolated bygel electrophoresis emitted at 679 nm and contained the 25 kDaprotein, of which the N-terminus was blocked. Its internal sequenceswere determined after protease treatment and found to be homologousto Type III gene of LHCI. An oligomeric subcomplex of LHCI isolatedby gel electrophoresis emitted at 726 nm and consisted of the21 kDa and 20.5 kDa apoproteins. N-terminal sequence of the20.5 kDa component corresponded to the Type I gene of LHCI.The 21 kDa component did not have any clear homologue, but itsN-terminal sequence was weakly but significantly homologousto all LHC components particularly to Type I LHCI among others.It was, thus, concluded that the 21 kDa protein is the fourthtype of LHCI apoprotein. Similar sequence homology was foundfor pea LHCI apoproteins. (Received September 10, 1990; Accepted November 22, 1990)     

Antibodies to the photosystem I chlorophyll a + b antenna cross-react with polypeptides of CP29 and LHCII

A chlorophyll (a + b)--protein complex associated with photosystem I (PSI) was isolated from a larger PSI complex (CPIa) produced by electrophoresis of barley thylakoids solubilized with 300 mM octyl glucoside. It had an apparent Mr of 35,000-43,000 on 7.5% and 10% acrylamide gels respectively, and a chlorophyll a/b ratio of 2.5 +/- 1.5. Denaturation released four polypeptides migrating between 21-24 kDa. They were well separated from the polypeptides of the two photosystem II chlorophyll a + b antenna complexes: LHCII (25-27 kDa) and CP29 (28-29 kDa). In order to study the PSI antenna complex, antibodies were raised against highly purified CPIa. The antigen appeared to be pure when electrophoresed, blotted and reacted with its antiserum, i.e. anti-CPIa detected only the 64-66-kDa CPI apoprotein and the four 21-24 kDa antenna polypeptides. However, when blotted against the whole spectrum of thylakoid proteins, it cross-reacted with both LHCII and CP29 apoproteins. Removal of anti-CPI activity from the anti-CPIa did not affect these cross-reactions, showing that they were not due to antibodies directed against CPI. To show that the same antibody population was reacting with both the photosystem I and photosystem II antenna polypeptides, anti-CPIa was adsorbed onto highly purified CPIa on nitrocellulose. The bound antibody was eluted and used again in a Western blot against whole thylakoid proteins. This selected antibody population showed the same relative strength of reaction with photosystem I and photosystem II antenna polypeptides as the original antibody population had. Similar observations have been made with antibodies to the two photosystem II antenna complexes. We therefore conclude that there are antigenic determinants in common among the chlorophyll a + b binding polypeptides, and predict that there could be amino acid sequence similarities.     

Assembly and composition of the chlorophyll a-b light-harvesting complex of barley (Hordeum vulgare L.): Immunochemical analysis of chlorophyll b-less and chlorophyll b-deficient mutants

The chlorina-f2 mutant of barley (Hordeum vulgare L.) contains no chlorophyll b in its light-harvesting antenna, whereas the chlorina-103 mutant contains approximately 10% of the chlorophyll b found in wild-type. The absolute chlorophyll antenna size for Photosystem-II in wild-type, chlorina-103 and chlorina-f2 mutant was 250, 58 and 50 chlorophyll molecules, respectively. The absolute chlorophyll antenna size for Photosystem-I in wild-type, chlorina-103 and chlorina-f2 mutant was 210, 137 and 150 chlorophyll molecules, respoectively. In spite of the smaller PS I antenna size in the chlorina mutants, immunochemical analysis showed the presence of polypeptide components of the LHC-I auxiliary antenna with molecular masses of 25, 19.5 and 19 kDa. The chlorophyll a-b-binding LHC-II auxiliary antenna of PS II contained five polypeptide subunits in wild-type barley, termed a, b, c, d and e, with molecular masses of 30, 28, 27, 24 and 21 kDa, respectively. The polypeptide composition of the LHC-II auxiliary antenna of PS II was found to be identical in the two mutants, with only the 24 kDa subunit d present at an equal copy number per PS II in each of the mutants and in the wild-type barley. This d subunit assembles stably in the thylakoid membrane even in the absence of chlorophyll b and exhibits flexibility in its complement of bound chlorophylls. We suggest that polypeptide subunit d binds most of the chlorophyll associated with the residual PS II antenna in the chlorina mutants and that is proximal to the PS II-core complex.Abbreviations CP chlorophyll-protein - LHC the chlorophyll a-b binding light-harvesting complex - LHC-II subunit a the Lhcb4/5 gene product - subunit b the Lhcb1 gene product - subunit c Lhcb2 the gene product - subunit d the Lhcb3 gene product - subunit e the Lhcb6 gene product - PMSF phenylmethane sulphonyl fluoride - RC reaction center - Q_A the primary quinone electron acceptor of Photosystem-II - P700 the reaction center of PS I     

Comparison of photosystem II complexes isolated from tobacco and two chlorophyll deficient tobacco mutants

A comparative study of photosystem II complexes isolated from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contains normal stacked thylakoid membranes, and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked grana or essentially unstacked thylakoids with occasional membrane doublings, has been carried out. The corresponding photosystem II complexes had an O₂ evolving activity ranging from 290 (for the wild type) to 1100 mol O₂ x mg chlorophyll^-1 x h^-1 (for the mutant Su/su var. Aurea). The reduced photosynthetic unit size was also obvious in the mangenese and cytochrome_b559 content. The photosystem II complex from the wild type contained 4 Mn and 1 cytochrome_b559 per 200 to 280 chlorophylls, while the corresponding value for the mutant Su/su var. Aurea was 4 Mn and 1 cytochrome_b559 per 35 to 60 chlorophylls. We have also examined the polypeptide composition and show that the photosystem II complex from the wild type consisted of polypeptides of 48, 42, 33, 32, 30, 28, 23, 21, 18, 16 and 10 kDa, while the mutant complex mainly contained the polypeptides of 48, 42, 33, 32, 30, 28 and 10 kDa. In the mutant photosystem II complex the light-harvesting chlorophyll protein (peptide of 28 kDa) was reduced by a factor of 5 to 6 as compared to the wild type. With respect to the peptide composition and the photosynthetic unit size, the Triton-solubilized photosystem II complex from the mutant Su/su var. Aurea was very similar to O₂ evolving photosystem II reaction center core complexes.Abbreviations PS photosystem - chl chlorophyll - LHCP light-harvesting chlorophyll a/b protein complex     

Alteration of Components of Chlorophyll-Protein Complexes and Distribution of Excitation Energy Between the Two Photosystems in Two New Rice Chlorophyll b-less mutants

Two new yellow rice chlorophyll (Chl) b-less (lack) mutants VG28-1 and VG30-5 differ from the other known Chl b-less mutants with larger amounts of soluble protein and ribulose-1,5-bisphosphate carboxylase/oxygenase small sub-unit and smaller amounts of Chl a. We investigated the altered features of Chl-protein complexes and excitation energy distribution in these two mutants, as compared with wild type (WT) rice cv. Zhonghua 11 by using native mild green gel electrophoresis and SDS-PAGE, and 77 K Chl fluorescence in the presence of Mg²⁺. WT rice revealed five pigment-protein bands and fourteen polypeptides in thylakoid membranes. Two Chl b-less mutants showed only CPI and CPa pigment bands, and contained no 25 and 26 kDa polypeptides, reduced amounts of the 21 kDa polypeptide, but increased quantities of 32, 33, 56, 66, and 19 kDa polypeptides. The enhanced absorption of CPI and CPa and the higher Chl fluorescence emission ratio of F685/F720 were also observed in these mutants. This suggested that the reduction or loss of the antenna LHC1 and LHC2 was compensated by an increment in core component and the capacity to harvest photon energy of photosystem (PS) 1 and PS2, as well as in the fraction of excitation energy distributed to PS2 in the two mutants. 77 K Chl fluorescence spectra of thylakoid membranes showed that the PS1 fluorescence emission was shifted from 730 nm in WT rice to 720 nm in the mutants. The regulation of Mg²⁺ to excitation energy distribution between the two photosystems was complicated. 10 mM Mg²⁺ did not affect noticeably the F685/F730 emission ratio of WT thylakoid membranes, but increased the ratio of F685/F720 in the two mutants due to a reduced emission at 685 nm as compared to that at 720 nm.     

The origin of the long-wavelength fluorescence emission band (77 degrees K) from photosystem I

Isolated photosystem I (PSI)-110 particles, prepared using a minimal concentration of Triton X-100 [J. E. Mullet, J. J. Burke, and C. J. Arntzen (1980) Plant Physiol. 65, 814-822] and further subjected to short-term solubilization with sodium dodecyl sulfate (SDS), were resolved into four pigment-containing bands on polyacrylamide gel electrophoresis (PAGE). We have identified these in order of increasing electrophoretic mobility as being (a) CPIa, (b) CPI, (c) the light-harvesting complex of photosystem I (LHC-I), and (d) a free pigment-zone. LHC-I had an absorption maximum in the red at 668-669 nm and a shoulder at 650 nm, which was resolved by its first-derivative spectrum to indicate the presence of chlorophyll b. LHC-I exhibited a 77 degrees K fluorescence emission maximum at 729-730 nm. The 77 degrees K fluorescence emission maxima of CPIa and CPI, excised from the gel, were at 729 and 722 nm, respectively. The LHC-I band, excised from the gel and rerun on dissociating SDS-PAGE, was resolved into two polypeptide doublets of 24-22.5 and 21-20.5 kDa. The CPIa band under similar conditions was resolved into polypeptides of 68, 24, 22.5, 21, 20.5, 19, 15, and 14 kDa; on the contrary, CPI contained only the 68-kDa polypeptide. When intact thylakoids were subjected to "nondenaturing" SDS-PAGE, LHC-I comigrated with an oligomeric form (dimer) of the light-harvesting chlorophyll a/b pigment-protein that preferentially serves photosystem II (LHCP-II). When this combined LHC-I/LHCP-II pigment-protein band was prepared by SDS-PAGE from isolated stroma lamellae, it exhibited a long-wavelength fluorescence band near 730 nm at 77 degrees K. When a similar preparation was obtained from sucrose density gradients containing SDS [J. Argyroudi-Akoyunoglou and H. Thomou (1981) FEBS Lett. 135, 171-181], it was found to be enriched in a 21-kDa polypeptide. The data suggest that the 21-kDa polypeptide of LHC-I is the chlorophyll-containing polypeptide responsible for the long-wavelength fluorescence of LHC-I; other polypeptides in the complex (20.5, 22.5, and 24 kDa) presumably bind chlorophyll and also serve an antennae function.     

Further investigations on structural and catalytic properties of O2 evolving preparations from tobacco and two chlorophyll deficient tobacco mutants

Previous investigations (Specht, S., Pistorius, E.K. and Schmid, G.H.: Photosynthesis Res. 13, 47–56, 1987) of Photosystem II membranes from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contain normally stacked thylakoid membranes and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked or essentially unstacked thylakoids with occasional membrane doublings, have been extended by using monospecific antisera raised against the three extrinsic polypeptides of 33,21 and 16 kDa. The results show that all three peptides are synthesized as well in wild type tobacco as in the two mutants to about the same level and that they are present in thylakoid membranes of all three plants. However, in the mutants the 16 and 21 kDa peptides (but not the 33 kDa peptide) are easily lost during solubilization of Photosystem II membranes. In the absence of the 16 and 21 kDa peptide Photosystem II membranes from the mutants have a higher O₂ evolving activity without addition of CaCl₂ than the wild type Photosystem II membranes. On the other hand, after removal of the 33 kDa peptide no significant differences in the binding of Mn could be detected among the three plants. The results also show that reaction center complexes from wild type tobacco and the mutant Su/su are almost identical to the Triton-solubilized Photosystem II membranes from the mutant Su/su var. Aurea.Abbreviations PS photosystem - chl chlorophyll - LHCP light harvesting chlorophyll a/b protein complex - WT wild type - OEE1, OEE2 and OEE3 oxygen evolution enhancing complex of 29–36 kDa, 21–24 kDa and 16–18 kDa, respectively     

The effect of outer antenna complexes on the photochemical trapping rate in barley thylakoid Photosystem II

We have investigated the previous suggestions in the literature that the outer antenna of Photosystem II of barley does not influence the effective photosystem primary photochemical trapping rate. It is shown by steady state fluorescence measurements at the F₀ fluorescence level of wild type and the chlorina f2 mutant, using the chlorophyll b fluorescence as a marker, that the outer antenna is thermally equilibrated with the core pigments, at room temperature, under conditions of photochemical trapping. This is in contrast with the conclusions of the earlier studies in which it was suggested that energy was transferred rapidly and irreversibly from the outer antenna to the Photosystem II core. Furthermore, the effective trapping time, determined by single photon counting, time-resolved measurements, was shown to increase from 0.17±0.017 ns in the chlorina Photosystem II core to a value within the range 0.42±0.036-0.47±0.044 ns for the wild-type Photosystem II with the outer antenna system. This 2.5-2.8-fold increase in the effective trapping time is, however, significantly less than that expected for a thermalised system. The data can be explained in terms of the outer antenna increasing the primary charge separation rate by about 50%.     

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)     

Evidence from Crosslinking for a Close Association of the Extrinsic 33 kDa Protein with the 9.4 kDa Subunit of Cytochrome b 559 and the 4.8 kDa Product of the psb I Gene in Oxygen-Evolving Photosystem II Complexes from Spinach

Oxygen-evolving photosystem II complexes from spinach, whichlack light-harvesting chlorophyll a/b proteins, were treatedwith a bifunctional crosslinking reagent, hexamethylene-diisocyanate.Identification of crosslinked proteins with antisera raisedagainst various constituent proteins of the oxygen-evolvingPS II complex showed that the extrinsic 33 kDa protein is locatedless than 11 Å from the 9.4 kDa subunit of cytochromeb 559 and the 4.8 kDa product of psb I gene. (Received October 14, 1991; Accepted February 6, 1992)