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.     

Comparative Study of Chlorophyll-Protein Complexes of Thylakoids of Bryopsis and Spinacia

properties, pigment compositions, Chl a/b ratios and apparent molecular weights of chlorophyll-protein complexes were compared between spinach and a marine green alga, Bryopsis corticulans. The results are as follows: 1. Ten chlorophyll-protein complexes were resolved from spinach thylakoid membranes solubilized by SDS in a final SDS/Chl weight ratio of 10:1, and subjected to SDS-PAGE with 11% resolution gel. CPIa 1–3 and CPI belonged to photosystem Ⅰ, and the rest to phorosystem Ⅱ. The maximum absorption of CPIa2, CPIas and CPI were all at 674nm, but that of CPIa1 at 670nm, and those of LHCII and D2 at 670 and 673nm, respectively. Chlorophyll ia PSⅡ was 63% of the total. In PSⅡ, most of chlorophyll was in LHCII which contained 86% of the chlorophyll in PSⅡ. In PSⅠ, chlorophyll in CPla was 72% of the total. Chlorophyll a was the main pigment in PSⅠ components which have Chl a/b ratio over 15. 2. Eight chlorophyll-protein complexes were isolated from B. corticulans with a SDS/Chi weight ratio of 8:1 and 8% resolution gel. The maximum absorption of CPIa, CPI, LHCII and D2 were respectively at 671nm, 673nm, 669nm and 664nm. PSⅡ contained 77% of the total chlorophyll. LHCII chlorophyll was 95% of the PSⅡ chlorophyll. CPI held 77% of PSⅠ chloro~ phyll. There was more chlorophyll b in Bryopsis complexes, especially in LHCI1 (Chl a/b< 0.8). The molecular weights of Bryopsis complexes were higher than those of the spinach complexes. Bryopsis LHCII contained siphoxanthin and siphothin, the marked pigments of Siphohales, as functional pigments. The above results revealed three points of difference between these two plants. Firstly, Chl a is the main pigment in spinach, whereas in Bryopsis the main pigments are Chl b and siphoxanthin. This is in accordance with the suggestion that plants may change their pigment composition to adapt light regime in the environment during evolution. Secondly, in Bryopsis, chlorophyll is concentrated in photosystem Ⅱ, but in spinach chlorophyll is shared evenly by two photosystems. Finally, CPI in Bryopsis contained the major part of chlorophyll in PSⅠ, yet in spinach CPIa is the superior.     

Influences of Blue and Red Light on the Photosynthetic Apparatus of Chlorella kessleri*

In white light of 33.2 μmol . m^?2 . s^?1 oxygen evolution of Chlorella kessleri is about 30 % higher after growth in blue light than after growth in red light of the same quantum fluence rate. When determined by the light-induced absorbance change at γ 820 nm, blue light-adapted cells possess about 60% more reaction centres per total chlorophyll in photosystem II. Correspondingly, the cells exhibit about 30% more Hill activity of PS II. Conversely, red light-adapted cells contain relatively more reaction centres and higher electron flow capacities of photosystem I. The distribution of total chlorophyll among the pigment-protein complexes, CPI, CPIa, CPa, and LHC II, corresponds to these data. There is more chlorophyll associated with the light-harvesting complex of PS II, LHC II, in cells under blue light conditions, but more chlorophyll bound to both complexes of PS I, CPI and CPIa, in cells under red light conditions. The respective ratios of chlorophyll a/chlorophyll b of all complexes are identical for blue and red light-adapted cells. This results in a higher relative amount of chlorophyll b in blue light-adapted cells. Total carotenoids per total chlorophyll are increased by 20% in red light-adapted cells. Their distribution among the pigment-protein complexes is unknown, however the ratios of lutein, neoxanthin and violaxanthin extractable from LHC II are different in blue (32.1:35.9:32.0) and in red (51.4:26.7:21.9) light-adaptod cells.     

The Chlorophyll-Protein Complexes Resolved from Ultrasonically Broken Thylakoid Memb-ranes of Blue-Green Algae

When the thylakoid membranes of blue-green algae were broken by ultrasonic vibrations and subjected to polyacrylamide gel electrophoresis at 4℃, six green zones were resolved. They were designated as CPIa, CPlb, CPI; CPal, CPa2, and FC. The absorption spectrum of CPI had a red maximum at 674 nm and a peak in the blue at 435 nm. It was identified as PS chlorophyll a-protein Complex, but was contaminated with minor PSⅡ which was implied by the appearance of fluorescence emission peak at 680 nm besides the main one at 725 nm at 77 K. The spectral properties of CPIa and CPlb were similar to that of CPl. The absorption spectra of CPa1 and CPa2 were similar, both having red maxima at 667 nm and peaks in the blue at 431.5 nm. Their fluorescence emission had the same peaks at 684 nm at 77 K indicating that they belonged to PSⅡ. It was recognized that CPal of 47 kD is the reaction center complex of photosystem Ⅱ and CPa2 of 40 kD is the internal antenna complex of photosystem Ⅱ. The spectral characteristics of the chlorophyll-protein complexes resolved by ultrasonic method were similar to those of the same complexes resolved by SDS solubilization, except the absorbance positions of CPa1 and CPa2 in the blue peak and the red one which shifted to blue about 3–5 nm. It was calculated that in thylakoid membranes of blue-green algae 40.93% chlorophyll was in PSⅠ, while 38.78% of chlorophyll in PSⅡ. The difference of chlorophyll contents between PSⅠ and PSⅡ was only 2.15%. Concerning the fact that minor PSⅡ compound remained in the part of PSⅠ zones, it might be concluded that the distribution of chlorophyll between PSⅠ and PSⅡ in blue-green algae was equal. This result was in agreement with the hypothesis that PSⅠ and PSⅡ operates in series in photosynthetic electron transport.     

Isolation of an intrinsic antenna chlorophyll a-protein from the photosystem I reaction center complex of the thermophilic cyanobacterium Synechococcus sp

A chlorophyll-protein was isolated from a Synechococcus P700-chlorophyll a-protein complex free from small subunits (CP1-e) by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis after treatment with 2% 2-mercaptoethanol and 2% SDS. In contrast to CP1-e which, when electrophoresed under denaturating conditions, showed two polypeptide bands of 62 and 60 kDa, the chlorophyll-protein contained only the 60-kDa polypeptide and hence is called CP60. The yield of CP60 was maximal with 1-2% SDS and 2-4% sulfhydryl reagents because the chlorophyll-protein was denatured at higher concentrations of the reagents. The absorption spectrum of CP60, which retained more than half of the chlorophyll alpha molecules originally associated with the 60-kDa subunit of the photosystem I reaction center complex, showed a red band maximum at 672 nm and a small absorption band around 700 nm at liquid nitrogen temperature. CP60 emitted a fluorescence band at 717 to 725 nm at 77 degrees K. The temperature dependence of the far red band of CP60 was essentially the same as that of CP1-e between 77 and 273 degrees K. No photoresponse of P700 was detected in CP60. The results suggest that the two polypeptides resolved by SDS-gel electrophoresis from CP1-e are apoproteins of two distinct chlorophyll-proteins and that CP60 represents a chlorophyll-bearing 60-kDa subunit functioning as an intrinsic antenna protein of the photosystem I reaction center complex. It will also be shown that the temperature dependence of the far red fluorescence band is not related to the photosystem I photochemistry.     

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.     

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)     

大麦突变种Chlorina-f 2类囊体膜的叶绿素蛋白复合体

当突变种大麦Chlorina-f 2的类囊体膜在SDS/叶绿素的重量比为10:1,叶绿素的浓度为0.5mg/ml的条件下增溶,并在SDS-聚丙烯酰胺凝胶电泳中进行分离时,共出现4条含叶绿素的带。按电泳迁移率的增加,这些带分别是CP Ⅰ,CPa 1,CPa 2和FC。光谱测定表明CP Ⅰ为混有少量光系统Ⅱ??成分的光系统Ⅰ反应中心复合体,CPa 2为光系统Ⅱ反应中心复合体,CPa 2为光系统Ⅱ内周天线复合体。属于光系统Ⅰ的CP Ⅰ的叶绿素含量占总叶绿素的45.6%,而属于光系统Ⅱ的CPa Ⅰ和CPa 2的叶绿素之和则占总叶绿素的43.2%。可见在缺b大麦中,两个都失缺其外周天线的光系统的叶绿素含量是基本相等的。这和光合作用中两个光反应相互串联的理论是完全一致的。     

Nuclear pea mutants deficient in chlorophyll b and the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II

Eight chlorophyll b deficient nuclear mutants of pea (Pisum sativum L.) have been characterized by low temperature fluorescence emission spectra of their leaves and by the ultrastructure, photochemical activities and polypeptide compositions of the thylakoid membranes. The room temperature fluorescence induction kinetics of leaves and isolated thylakoids have also been recorded. In addition, the effects of Mg²⁺ on the fluorescence kinetics of the membranes have been investigated. The mutants are all deficient in the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II. The low temperature fluorescence emission spectra of aurea-5106, xantha-5371 and –5820 show little or no fluorescence around 730 nm (photosystem I fluorescence), but possess maxima at 685 and 695 nm (photosystem II fluorescence). These three mutants have low photosystem II activities, but significant photosystem I activities. The long-wavelength fluorescence maximum is reduced for three other mutants. The Mg²⁺ effect on the variable component of the room temperature fluorescence (685 nm) induction kinetics is reduced in all mutants, and completely absent in aurea-5106 and xantha-5820. The thylakoid membranes of these 2 mutants are appressed pairwise in 2-disc grana of large diameter. Chlorotica-1-206A and–130A have significant long-wavelength maxima in the fluorescence spectra and show the largest Mg²⁺ enhancement of the variable part of the fluorescence kinetics. These two mutants have rather normally structured chloroplast membranes, though the stroma regions are reduced. The four remaining mutants are in several respects of an intermediate type.Abbreviations Chl chlorophyll - CPI Chi-protein complex I, F_o, F_v - F_m parameters of room temperature chlorophyll fluorescence induction kinetics - F685, F695 and F-1 components of low temperature chlorophyll emission with maximum at 685, 695 and ca 735 nm, respectively - PSI photosystem I - PSII photosystem II - LHCI and LHCII light-harvesting chlorophyll a/b complexes associated with PSI and PSII, respectively - SDS sodium dodecyl sulfate     

Identification of the photosystem I antenna polypeptides in barley. Isolation of three pigment-binding antenna complexes.

The light-harvesting antenna of barley photosystem I (LHCI) was isolated from native photosystem I (PSI) complexes and fractionated into three pigment-protein subcomplexes using two consecutive rounds of green gel electrophoresis. Each complex showed a characteristic polypeptide composition and low-temperature fluorescence emission spectrum; they were designated as LHCI-730, LHCI-680A and LHCI-680B. Their four apoproteins of 21, 22, 23 and 25 kDa were purified and NH2-terminal sequences were determined; in the case of the NH2-terminally blocked 25-kDa protein, an internal sequence was obtained after cleavage with endoproteinase Lys-C. This made possible an assignment of the four proteins to the four types (I-IV) of genes coding for chlorophyll a/b proteins of PSI (cab or lha genes). The LHCI-730 complex was isolated as a heterodimer composed of the 21-kDa (LHCI type IV) and the 22-kDa (LHCI type I) polypeptides. Each LHCI-680 complex had a single apoprotein. LHCI-680A consisted of the 25-kDa (LHCI type III) and LHCI-680B of the 23-kDa (LHCI type II) polypeptides. LHCI-680B was associated with the non-pigmented PSI-E subunit, indicating that this protein may function in the binding of this antenna to the reaction centre.     

The effect of the dark interval in intermittent light on thylakoid development: photosynthetic unit formation and light harvesting protein accumulation

Etiolated bean plants were grown in intermittent light with dark intervals of shorter or longer duration, to modulate the rate of chlorophyll accumulation, relative to that of the other thylakoid components formed. We thus produced conditions under which chlorophyll becomes more or less a limiting factor. We then tested whether LHC complexes can be incorporated in the thylakoid. It was found that an equal amount of chlorophyll, formed under the same total irradiation received, may be used for the stabilization of few and large-in-size PS units containing LHC components (short dark-interval intermittent light), or for the stabilization of many and small-in-size PS units with no LHC components (long dark-interval intermittent light). The size of the PS units diminishes as the dark-interval duration is increased, with no further change after 98 minutes. The PSII/cytf ratio remains constant throughout development in intermittent light and equal to that of mature chloroplasts (PSII/cytf = 1) except in the case of very long dark-interval regimes, where about half PSII units per cytf are present. The PSII/PSI ratio was found to be correlated with the PSII unit size (the larger the size, the lower the ratio). The number of PSI units operating on the same electron transfer chain varied depending on the size of the PSII unit (the larger the PSII unit size, the more the PSI units per chain). The results suggest that it is not the chlorophyll content per se which regulates the stabilization of LHC in developing thylakoids and consequently the size of the PS units, but rather the rate by which it is accumulated, relative to that of the other thylakoid components.Abbreviations Chl Chlorophyll - CL Continuous light - CPa the reaction center complex of PSII - CPI the reaction center complex of PSI - CPIa Chlorophyll protein complex containing the CPI and the light harvesting complex of PSI - fr w fresh weight - LDC Light dark cycles - LHC-I Light-harvesting complex of PSI - LHC-II Light harvesting complex of PSII - PS photosystem - PSI photosystem I - PSII photosystem II     

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)     

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)     

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.     

Organization and role of the long-wave chlorophylls in the photosystem I of the Cyanobacterium spirulina.

The data on the organization and function of the photosystem I pigment-protein complexes of the cyanobacterium Spirulina and the characteristics of pigment antenna of the photosystem I monomeric and trimeric core complexes are presented and discussed. We proved that the photosystem I complexes in the cyanobacterial membrane pre-exist mainly as trimers, though both types of complexes contribute to the photosynthetic electron transport. In contrast to monomers, the antenna of the photosystem I trimeric complexes of Spirulina contains the extreme long-wave chlorophyll form absorbing at 735 nm and emitting at 760 nm (77 K). The intensity of fluorescence at 760 nm depends strongly on the P700 redox state: it is maximum with the reduced P700 and strongly decreased with the oxidized P700 which is the most efficient quencher of fluorescence at 760 nm. The energy absorbed by the extreme long-wave chlorophyll form is active in the photooxidation of P700 in the trimeric complex. The data obtained indicate that the long-wave form of chlorophyll originates from interaction of the chlorophyll molecules localized on monomeric subunits forming the photosystem I trimer. Kinetic analysis of the P700 photooxidation and light-induced quenching of fluorescence at 760 nm (77 K) allows the suggestion that the excess energy absorbed by the antenna monomeric subunits within the trimer migrates via the extreme long-wave chlorophyll to the P700 cation radical and is quenched, which prevents the photodestruction of the pigment-protein complex.     

Characterization of Photosystem I Chlorophyll-Protein Complexes Reconstituted into Phosphatidylcholine Liposomes

Chlorophyll-protein complexes associated with photosystem Iwere isolated from native photosystem I particles (PS I-200)of spinach thylakoids by centrifugation in SDS-sucrose densitygradients. These complexes were designated CPIa (Chl/P700 ratioof 160), CPI' (CW/P700 ratio of 70), and LHCI (light-harvestingChl a/bprotein complex associated with photosystem I). CPI'was reconstituted with and without LHCI into phosphatidylcholineliposomes by a freeze-thaw technique. The first-order rate constantfor P700-photooxidation in proteoliposomes reconstituted withCPI' plus LHCI increased with an increase in the concentrationof phosphatidylcholine. When the concentration of phosphatidylcholinewas more than 20 times (by weight) that of chlorophyll in thecomplexes, the rate constant under lightlimiting conditionswas approximately double that of a mixture of two complexesnot reconstituted into liposomes. The fluorescence emissionspectrum (77 K) of the proteoliposomes reconstituted with CPI'plus LHCI displayed a longer wavelength band at 730–733nm which was very similar to the spectrum of CPIa and whichwas not displayed in the spectrum of a mixture of CPI' and LHCIwithout liposomes. The circular dichroism spectrum of a mixtureof CPI' and LHCI indicated that the intensity of both a positivepeak at 665 nm and a negative peak at 686 nm increased whena mixture of the two complexes was reconstituted into liposomes.These results suggest that some alteration of chlorophyll organizationoccurs in proteoliposomes reconstituted with both CPI' and LHCI,facilitating energy transfer from LHCI to the reaction centerof photosystem I. (Received July 18, 1986; Accepted March 12, 1987)     

Identification and analysis of the maize P700 chlorophyll a apoproteins PSI-A1 and PSI-A2 by high pressure liquid chromatography analysis and partial sequence determination

We recently described a pair of partially homologous maize chloroplast genes, one of which was shown to code for an apoprotein of the P700 chlorophyll a complex of photosystem I (Fish, L.E., Kück, U., and Bogorad, L. (1985) J. Biol. Chem. 260, 1413-1421). Two chlorophyll-free apoprotein bands from maize chlorophyll-protein complex I (CPI) can be resolved on lithium dodecyl sulfate (LDS)-urea polyacrylamide gels. Proteins in both bands react with antibodies prepared against CPI, but antibodies prepared against two synthetic peptides corresponding to predicted sequences of PSI-A1 react only with the upper band. The presence of products of the two genes, ps1A1 and ps1A2, in CPI was verified by analysis of cyanogen bromide (CNBr) fragments of the lower apoprotein band obtained from LDS-urea polyacrylamide gels by reverse-phase high pressure liquid chromatography. Amino-terminal sequencing of five CNBr fragments indicates that the lower band contains a product of the ps1A2 gene. The possibility of extensive processing was investigated because the apparent molecular masses of the maize CPI proteins are about 58-70 kDa on LDS-polyacrylamide gels rather than the predicted sizes of about 83 kDa. Antibodies against a synthetic peptide corresponding to a predicted sequence in PSI-A1 were used to determine that the amino-terminal end of PSI-A1 is intact beyond about position 52. The amino-terminal CNBr fragment of PSI-A2 was identified by sequencing, indicating that the amino-terminal end of PSI-A2 is not processed. The carboxyl-terminal CNBr fragment of PSI-A2 was also identified by sequencing. These results indicate that the PSI-A1 and PSI-A2 polypeptides are not extensively processed, although some processing at the carboxyl-terminal end has not been ruled out.     

Enrichment of a 50-kilodalton polypeptide in a photosystem II-phycobilisome particle from Porphyridium cruentum

A 50-kDa polypeptide was obtained from photosynthetically active phycobilisome-photosystem II preparations from the red alga Porphyridium cruentum after removal of phycobiliproteins. Removal of phycobiliproteins caused destabilization of the structure of the phycobilisome-photosystem II preparations and was accompanied by a decline in photosystem II activity (oxygen-evolution and dichlorophenol-indophenol (DPIP) reduction). The treatments in increasing relative effectiveness were: addition of EDTA (10 mM), lowering the pH (6.8----4.4), and lowering the ionic strength (to ca. 1 mM phosphate). The lowering of the ionic strength by dialysis resulted in a preparation highly enriched in a 50-kDa polypeptide (apparent molecular mass on SDS-PAGE). This preparation retained photosystem II activity as evidenced by the photoreduction of DPIP in the presence of diphenylcarbazide (222 mumol DPIP/mg chlorophyll/h). Also it had a 698-nm (77K) fluorescence emission maximum, as compared to a 668-nm emission in the unfractionated preparation, which indicates enrichment of the photosystem II reaction center. Comparing our results with those obtained from green plants and a cyanobacterium leads us to suggest that the reaction center II polypeptides are highly similar in all chlorophyll alpha-containing plants.     

Effect of DNAase on fluorescent properties of pigment-protein complexes, contained in photosystem II

It was found that chlorophyll fluorescence spectra and spectra of fluorescence excitation of pigment-protein complexes of photosystem II are affected by treatment with DNase. Pigment-protein complexes were isolated from pea thylakoid membranes. Spectra were measured at room temperature. It was shown that the treatment with DNase leads to a 30% increase in fluorescence yield at excitation in chlorophyll absorption bands in the fraction containing CP47, CP43, and CP29, and also in the fraction containing reaction center complexes with minor contaminations of light-harvesting complexes. Upon excitation at 260-300 nm and in the region of 500 nm, a diminishing of fluorescence yield takes place. These results suggest that pigments and/or pigment-protein complexes are bound to nucleic acids. This association, by influencing the pigment properties, can participate in the photoregulation of biochemical reactions through changes in the thermal dissipation of excited chlorophyll molecules.     

Relationship between biosynthesis of carotenoids and increasing complexity of photosystem I in mutant C-6D of Scenedesmus obtiquus

Dark-grown cells of the pigment mutant C-6D of Scenedesmus obliquus, strain D₃ (Gaffron 1939), contain only chlorophyll (Chl) a and carotenoid precursors. In these cells a functioning photosystem I (PSI) of basic structure was characterised by a high PSI activity and a low Chl/P700 ratio. The reaction-center complex of PSI (CPI) was shown to exist in the dark-grown cells. These findings demonstrate that the assembly of the core complex of PSI and its function are independent of the presence of carotenoids. Upon illumination, carotenoids, Ch1 b and additional Chl a were synthesized. Newly formed -carotene was shown by pigment analysis using high-performance liquid chromatography (HPLC) to be incorporated into CPI. Parallel to this process a shift of the long-wavelength fluorescence emission of PSI from 712–714 to 718–719 nm was observed. In the later stages of chloroplast differentiation, when xanthophylls and Chl b were synthesized, a higher-molecular-weight complex of PSI (CPIa) could be isolated. Pigment analysis demonstrated that CPIa contained xanthophylls and Chl b in addition to Chl a and -carotene. This indicates the formation of a light-harvesting antenna closely associated with PSI (LHCI). The addition of an LHCI to the reaction-center complex of PSI caused an increase in the absorption cross-section of PSI as shown by action spectroscopy and in-vivo fluorescence measurements. A model demonstrating the changes in the molecular organization of PSI during light-induced carotenoid biosynthesis in mutant C-6D of Scenedesmus obliquus is presented.Abbreviations Chl chlorophyll - CP chlorophyll-protein complex - LHC light-harvesting complex - HPLC high-performance liquid chromatography - PSI, II photosystem I, II - PAGE polyacrylamide gel electrophoresis This work was supported by the Deutsche Forschungsgemeinschaft and a scholarship of the Studienstiftung des deutschen Volkes to S. Römer. We thank Ms. K. Bölte for technical assistance and Mr. H. Becker for drafting the figures.