Formation of Chlorophyll-Protein Complexes during Greening. 2. Redistribution of Chlorophyll among Apoproteins

The formation of Chl-protein complexes (CPs) in cucumber cotyledonsduring a dark period after a brief illumination was studied.SDS-PAGE analysis showed that the P700-Chl a-protein complex(CP1) and Chl a-protein complex of the PS II core (CPa) increased,with a concomitant decrease in the light-harvesting Chl a/6-proteincomplex of PS II (LHCII), during 24-h dark incubation of cotyledonsafter 6h of continuous illumination. In agreement with theseresults, curve analysis revealed that spectral components characteristicof CP1 and CPa increased while those of Chi b decreased duringthe dark incubation. Since Chl is not synthesized in the dark,Chl must be released from LHCII and re-incorporated into CP1and CPa. The amounts of apoproteins of CP1 and 43 kDa protein(one of the apoproteins of CPa) increased during the dark incubation,and the increase could be inhibited by chloramphenicol (CAP).CP1 did not increase in the dark when tissues were incubatedwith CAP which inhibited the synthesis of apoproteins of CP1,indicating that CP formation by Chl redistribution needs newlysynthesized apoproteins. The decrease in LHCII apoproteins duringdark incubation was inhibited by CAP probably because Chl wasnot removed from LHCII by apoproteins of CP1 and CPa, whosesynthesis was blocked by the presence of CAP. When intermittently-illuminatedcotyledons containing a little LHCII were incubated with CaCl₂in the dark, Chl b and LHCII apoproteins accumulated with thedisappearance of 43 kDa protein; Chl of 43 kDa protein may beutilized for LHCII formation. We concluded that Chl moleculesonce bound with their apoproteins are redistributed among theapoproteins. (Received October 17, 1990; Accepted December 6, 1990)     

Photoconversion of a Water-Soluble Chlorophyll Protein from Chenopodium Album: Resonance Raman and Fourier Transform Infrared Study of Protein and Pigment Structures

A water-soluble Chl a/b-protein complex, CP668, from Chenopodiumalbum converts to another form of protein complex, CP743, uponlight illumination. Structural changes of pigments and proteinsupon photoconversion were studied using resonance Raman (RR)and Fourier transform infrared (FTIR) spectroscopies. RR spectraof CP668 and CP743 and a light-induced FTIR difference spectrumshowed that the macrocyle C=C bands of Chl a in CP668 considerablychanged upon conversion to the pigment (not chemically identifiedyet) in CP743. The C=C band pattern of the RR spectrum of CP743was similar to that of bacteriochlorophyll a, suggesting thatthe conjugated system of the CP743 pigment resembles a bacteriochlorinring. Judging from the C=O frequencies, the 13¹-keto C=O groupsof Chl a and b in CP668 are free from hydrogen bonding, whereasthe 13²-ester C=O groups of both Chl a and b and the 7-formylC=O of Chl b in CP668 are hydrogen bonded. Upon conversion toCP743, interactions of the 13¹-keto and 13²-ester C=O groupswere basically unaffected, demonstrating no drastic changesaround these C=O groups. FTIR spectra in the amide I' regionof CP668 and CP743 in D₂O buffer showed a peak at 1,633 cm^–1,which represents a major component of ß-sheet conformation.Second-derivative spectra of the amide I' bands as well as alight-induced FTIR difference spectrum suggested that drasticchange in the protein conformation does not occur upon photoconversion. (Received November 1, 1998; Accepted December 24, 1998)     

Formation of Chlorophyll-Protein Complexes during Greening 1. Distribution of Newly Synthesized Chlorophyll among Apoproteins

The relationship between the accumulation of Chl and the apoproteinsof the light-harvesting Chl a/b-protein complex of PS II (LHCII)during the greening of cucumber cotyledons was studied. LHCIIapoproteins were not detected in etiolated cotyledons. Uponillumination, Chl a was formed as a result of photoconversionof protochlorophyllide (Pchlide) which had accumulated in thedark. During the lag period that preceded the accumulation ofChl, a small amount of LHCII apoproteins appeared. The amountof LHCII apoproteins increased with increases in levels of Chlb, though somewhat more rapidly during the first 10 h of greening.Treatment with benzyladenine (BA) or levulinic acid (LA) wasused to vary the supply of Chl a for apoproteins by promotingor inhibiting the synthesis of Chl a, respectively. LA decreasedbut BA increased the rate of accumulation of Chl b and LHCIIapoproteins. Only small amounts of Chl b and LHCII apoproteinswere formed under intermittent illumination. However, in thepresence of chloramphenicol (CAP), which inhibits the synthesisof plastome-coded proteins including apoproteins of the P700-Chla-protein complex (CP1) and a Chl a-protein complex of PS II(CPa), we observed the accumulation of Chl b and LHCII apoproteins,both of which are of nuclear origin. During incubation in thedark after intermittent exposure to light, CAP alone allowedneither destruction nor accumulation of Chl b and LHCII apoproteins,but it did enhance the effect of CaCl₂ in inducing both Chlb and these apoproteins. These results can be explained by assumingthat apoproteins of CP1 and CPa have a higher affinity for Chla than do LHCII apoproteins. When the availability of Chl ais limited, these apoproteins compete with one another for Chla, with the resultant preferential formation of CP1 and CPa.However, when the supply of Chl a becomes large enough for saturationof apoproteins of CP1 and CPa, some of the Chl a is incorporatedinto LHCII apoproteins either directly or after conversion toChl b. Thus, the formation of different Chl-protein complexes(CPs) is regulated by the relative rates of synthesis of Chla and apoproteins and by differential affinities of the apoproteinsfor Chl a. ⁴Present address: Kyowa Hakko Co., Ltd., 4041, Ami-machi, Inashiki,Ibaraki, 300-03 Japan (Received September 14, 1989; Accepted April 26, 1990)     

Organization and Stability of Polypeptides Associated with the Chlorophyll a-b Light-Harvesting Complex of Photosystem-II

In the oxygen-evolving photosystem-II (PSII) of higher plantchioroplasts and green algae, most of the light-harvesting functionis performed by the chlorophyll (Chl) a-b-protein complex (LHC-II).On the average, the LHC-II contains about 210 Chl (a+b) moleculesper PSII reaction center. The polypeptide composition, copynumber and organization of assembly in the LHC-II complex arenot fully understood at present. This work utilized the chlorinaf2 mutant of barley (lacking Chl b and having a LHC-II antennaof only 13 Chl a molecules) to determine the organization andstability of assembly of proteins in the LHC-II. High-resolutionSDS-PAGE and immunoblot analysis showed the presence of fourmain constitutive polypeptides in the wild-type LHC-II (termedhere subunits a, b, c and d) with molecular masses in the range30–25 kDa. Of those, only subunit d (a 25 kDa polypeptide)was found to occur at an equal copy number per PSII reactioncenter in both wild-type and in the Chl b-less chlorina f2 mutant.All other subunits were either absent or existed in much loweramounts in the mutant. Subunit d is a polypeptide constituentof the major Chl-protein subcomplex (CPII) of the LHC-II. Itis stably incorporated in the thylakoid membrane in the absenceof Chl b and probably binds the 13 Chl a molecules in the residualLHC-II antenna of the chlorina f2 mutant. We propose that, ofall LHC-II polypeptides, subunit d is most proximal to the PSIIcore and may serve as a linker in the process of excitationenergy transfer from the bulk LHC-II to the PSII reaction centerin chloroplasts. (Received February 25, 1992; Accepted May 12, 1992)     

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)     

Changes in the Levels of Chlorophyll and Light-Harvesting Chlorophyll a/b Protein of PS II in Rice Leaves Aged under Different Irradiances from Full Expansion through Senescence

Effects of irradiance on changes in the amounts of chlorophyll(Chl) and light-harvesting chlorophyll a/b protein of PS II(LHCII) were examined in senescing leaves of rice (Oryza sativaL.). Results of treatments at two irradiances (100% and 20%natural sunlight) were examined after the full expansion ofthe 13th leaf throughout the course of senescence. With 20%sunlight, the Chl content decreased only a little during leafsenescence, while with 100% sunlight it decreased appreciably.Similarly, the amount of LHCII protein during treatment with20% sunlight remained almost constant. However, the ratio ofChl a/b during the shade treatment decreased significantly andthe rate of decrease was greater than during the full-sunlighttreatment. The ratio of Chl a/b for Chl a and b bound to LHCIIwas about 1.2, irrespective of leaf age or irradiance treatment.When the amounts of Chl bound to LHCII were calculated fromthe total leaf content of Chl and the ratio of Chl a/b, assuminga ratio of Chl a/b bound to LHCII of 1.2, they were well correlatedwith the amounts of LHCII protein. Changes in the amounts of LHCII synthesized during the two irradiancetreatments were examined using an ¹⁵ tracer. Incorporation of¹⁵N into LHCII declined dramatically during both treatmentsfrom full expansion through senescence, suggesting that therewas little synthesis of LHCII protein during that time. In addition,the amount of LHCII synthesized during senescence was lowerduring the shade treatment than during the 100% sunlight treatment.These results indicate that the absence of an apparent changein levels of LHCII with shade treatment during senescence wascaused by the very low rate of turnover of LHCII protein. (Received June 17, 1992; Accepted September 28, 1992)     

Chlorophyll-Protein Complexes Associated with Photosystem I Isolated from the Green Alga, Bryopsis maxima

Six chlorophyll (Chl)-protein complexes associated with photosystemI (CPla), and the PS I reaction center complex (CPl) were isolatedfrom the thylakoid membranes of the green alga, Bryopsis maxima,by SDS-polyacrylamide gel electrophoresis. CPla had four polypeptides(22, 24, 25, 26 kDa) in addition to the 67 kDa polypeptide ofCPl. These complexes may thus possibly be a combination of CPland antenna complexes for PS I. Six CPla showed almost the sameoptical properties, with absorption maxima at 650 and 677 nmand contained carotene and a small amount of xanthophylls. TheChl a/b ratios of these CPla were about 2, while that of CPlwas 14. CPla showed a fluorescence emission maximum at 695 nm;its excitation spectrum had peaks at 438, 470 and 540 nm, correspondingto the absorption maxima of Chl a, Chl b, xanthophylls, respectively.An antenna complex free of CPl has been detected in some plantsbut was not found in the present alga. ¹Present address: Department of Botany, The University of Adelaide,Adelaide, S.A. 5001, Australia (Received April 17, 1986; Accepted June 26, 1986)     

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.     

Presence of the Photoactive Reaction-Center Chlorophyll of PSI (P700) in Dark-Grown Cells of a Chlorophyll-Deficient Mutant of Chlorella kessleri

Compositions of pigments and polypeptides of pale green membranesthat had been isolated from dark-grown cells of a chlorophyll-deficientmutant of Chlorella kessleri were investigated. They containedChl a in a level corresponding to about 1% of that present inthe thylakoid membranes isolated from autotrophically grownwild-type cells and a trace amount of chlorophyllide a, butneither Chl b nor carotenoids. The polypeptide profile of themutant membranes was similar to that of membranes isolated fromwild-type cells that were grown in the dark. Neither the chlorophyll-bindingsubunits of PSI nor the apoproteins of LHCP were detected bySDS-PAGE and immunoblot analysis. However, the light-minus-darkdifference spectrum of the mutant membranes revealed the presenceof the reaction-center chlorophyll of PSI (P700) at a molarratio of 190 chlorophyll (Chl a plus Chlide a) per P700. P700was more stable than Chl a and Chlide a in the light so thatprolonged illumination led to a decline in the Chl/P700 ratioto 24. The initial rate of P700 photooxidation in the mutantmembranes was comparable to that in CP1 isolated from the dark-grownwild-type cells. Under illumination with strong light, the initialrate was decreased in parallel to the decrease in Chl/P700 ratio.The results suggest that most of Chi present in the mutant membranescan transfer excitation energy to P700. (Received March 13, 1998; Accepted August 7, 1998)     

The chlorophyll-protein complexes of Acetabularia. A novel chlorophyll ab complex which forms oligomers

(1) Five minor chlorophyll-protein complexes were isolated from thylakoid membranes of the green alga Acetabularia by SDS-polyacrylamide gel electrophoresis, after SDS or octylglucoside solubilization. None of them were related to CP I (Photosystem I reaction center core) or CP II (chlorophyll $\text{a}\text{b}$ light-harvesting complex). (2) Two complexes (CPa-1 and CPa-2) contained only chlorophyll (Chl) a, with absorption maxima of 673 and 671 nm, and fluorescence emission maxima of 683 nm compared to 676 nm for CP II. The complexes had apparent molecular masses of 43–47 and 38–40 kDa, and contained a single polypeptide of 41 and 37 kDa, respectively. They each account for about 3% of the total chlorophyll. (3) Three complexes had identical spectra, with Chl $\text{a}\text{b}$ ratios of 3–4 compared to 2 for thylakoid membranes, and a pronounced shoulder around 485 nm indicating enrichment in carotenoids. One of them was the complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432) and the other two were slightly different oligomeric forms of CP 29. They could be formed from CP 29 during reelectrophoresis; but about half the complex was isolated originally in an oligomeric form. Together they account for at least 7% of the total chlorophyll. Their function is unknown.     

The 28 kDa apoprotein of CP 26 in PS II binds copper

Photosystem II (PS II) particles isolated from spinach in the presence of 10 M CuSO₄ contained 1.2 copper/300 Chl that was resistant to EDTA. When CuSO₄ was not added during the isolation, PS II particles contained variable amounts of copper resistant to EDTA (0.1–1.1 copper/300 Chl). No correlation was found between copper content and oxygen evolving capacity of the PS II particles. To identify the copper binding protein, we developed a fractionation procedure which included solubilisation of PS II particles followed by precipitation with polyethylene glycol. A 22-fold purification of copper with respect to protein was achieved for a 28 kDa protein. Partial amino acid sequence of a 13 kDa fragment, obtained after V8 (endo Glu-C) protease treatment, showed identity with CP 26 over a 14 amino acid stretch. EPR measurements on the purified protein suggest oxygen and/or nitrogen as ligands for copper but tend to exclude sulfur. We conclude that the 28 kDa apoprotein of CP 26 from spinach binds one copper per molecule of CP 26. A possible function for this copper protein in the xanthophyll cycle is discussed.Abbreviations CP 26 and CP 29 chlorophyll a/b protein complex 26 and 29 - LHC II light-harvesting chlorophyll a/b protein complex of Photosystem II - SB14 sulfobetaine 14 A preliminary report of these results was presented at the IX Int. Congress on Photosynthesis, Nagoya, Japan, 1992.     

Thylakoid Membrane Development and Differentiation: Assembly of the Chlorophyll a-b Light-Harvesting Complex and Evidence for the Origin of Mr=19, 17.5 and 13.4 kDa Proteins

Pea plants were grown under intermittent illumination (ImL)conditions. The low dosage of light given to ImL plastids limitedthe rate of chlorophyll (Chl) a and Chl b biosynthesis and,therefore, it retarded the rate of photosynthetic unit formationand thylakoid membrane development. Depending on the developmentalstage of the photosynthetic unit, ImL plastids had variableChl a/Chl b ratios (2.7 <Chl a/Chlb<20) and showed distinctintermediates in the assembly of the chlorophyll a–b light-harvestingcomplex (LHC) of photosystem-II (PSII). The results are consistentwith a step-wise increment in the PSII antenna size involvingthree distinct forms of the PSII unit: (i) a PSII-core formwith about 37 Chl a molecules; (ii) a PSIL_ß form containingthe PSII-core and the LHC-II-inner antenna with a total of about130 Chl (a + b) molecules, and (iii) the mature PSII_a form containingPSII_ß and the LHC-II-peripheral antenna with a totalof 210–300 Chl (a + b) molecules. The thylakoid membranecontained polypeptide subunits b, c and d (the Lhcb1, 2 and3 gene products, respectively) when only the LHC-II-inner waspresent. Polypeptide subunit a, (the apoprotein of the chlorophyll-proteinknown as CP29), along with increased amounts of b and c appearedlater in the development of thylakoids, concomitant with theassembly of the LHC-II-peripheral. The results suggest thatpolypeptide subunit d has priority of assembly over subunita. It is implied that, of all LHC-II constituent proteins, subunitd is most proximal to the PSII-core complex and that it servesas a linker in the transfer of excitation energy from the bulkLHC-II (subunits b and c) to the PSII-core. The work also addressesthe origin of low-molecular-weight proteins (M_r = 19, 17.5 and13.4 kDa) which co-isolate with intact developing plastids andwhose abundance decreases during plastid development. Aminoacid compositional and immunoblot analyses show a nuclear histoneorigin for these low-molecular-weight proteins and suggest co-isolationof histone-containing nuclear vesicles along with intact developingplastids. ¹Present address: Plant Physiology Research Group, The Universityof Calgary, Department of Biological Sciences, 2500 UniversityDrive N.W., Calgary, Alberta CANADA T2N 1N4.     

Energy transfer for low temperature fluorescence in PS II mutant thylakoids

The Chl-protein complexes of three maize (Zea mays L.) mutants and one barley (Hordeum vulgare L.) mutant were analyzed using low temperature Chl fluorescence emissions spectroscopy and LDS-polyacrylamide gel electrophoresis. The maize mutants hcf-3, hcf-19, and hcf-114 all exhibited a high Chl fluorescence (hcf) phenotype indicating a disruption of the energy transfer within the photosynthetic apparatus. The mutations in each of these maize mutants affects Photosystem II. The barley mutant analyzed was the well characterized Chl b-less mutant chlorina-f2, which did not exhibit the hcf phenotype. Chlorina-f2 was used because no complete Chl b-less mutant of maize is available. Analysis of hcf-3, hcf-19, and hcf-114 revealed that in the absence of CP43, LHC II can still transfer excitation energy to CP47. These results suggest that in mutant membranes LHC II can interact with CP47 as well as CP43. This functional interaction of LHC II with CP47 may only occur in the absence of CP43, however, it is possible that LHC II is positioned in the thylakoid membranes in a manner which allows association with both CP43 and CP47.Abbreviations hcf high chlorophyll fluorescence - LDS lithium dodecyl sulfate - LHC II light-harvesting complex of Photosystem II - LHC I light-harvesting complex of Photosystem I - CPIa chlorophyll-protein complex consisting of LHC I and the PS I core complex - CPI chlorophyll-protein complex consisting of the PS I core complex - CP47 47 kDa chlorophyll-protein of the Photosystem II core - CP43 43 kDa chlorophyll-protein of the Photosystem II core - CP29 29 kDa chlorophyll-protein of Photosystem II - CP26 26 kDa chlorophyll-protein of Photosystem II - CP24 24 kDa chlorophyll-protein of Photosystem II - fp free pigments     

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.     

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)     

Identity of the Photosystem II reaction center polypeptide

A Photosystem-II (PS-II)-enriched chloroplast submembrane fraction has been subjected to non-denaturing gel-electrophoresis. Two chlorophyll a (Chl a)-binding proteins associated with the core complex were isolated and spectrally characterized. The Chl protein with apparent apoprotein mass of 47 kDa (CP47) displayed a 695 nm fluorescence emission maximum (77 K) and light-induced absorption characteristics indicating the presence of the reaction center Chl, P-680, and its primary electron acceptor, pheophytin. A Chl protein of apparent apoprotein mass of 43 kDa (CP43) displayed a fluorescence emission maximum at 685 nm. We conclude that CP43 serves as an antenna Chl protein and the PS II reaction center is located in CP47.     

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.     

Effect of Sodium Dodecyl Sulfate on Structure and Spectroscopic Characteristics of Water-Soluble Chlorophyll Protein Complex Isolated from Stems of Lepidium virginicum

The relationship between structure and spectroscopic characteristicsof the watersoluble chlorophyll protein complex isolated fromstems of Lepidium virginicum (CP663S) was studied. Additionof 0.08% SDS induced a red shift of the 663 nm absorption maximum.At the same time, under excitation at 435 nm, the maximum offluorescence emission shifted from 672 nm to 675 nm and thefluorescence yield increased. When CP663S was excited at 480nm, the 660 nm emission band of chlorophyll b became more prominent.Fluorescence lifetime of emission from chlorophyll a increasedon addition of SDS. The energy transfer from chlorophyll b tochlorophyll a was decreased by the SDS addition, as judged bythe fluorescence spectra and lifetime measurement. Symmetricalpositive and negative peaks of the circular dichroism (CD) spectrumaround 669 nm, which indicate the interaction between chlorophylla molecules at short distances, disappeared after addition ofSDS. These SDS-induced changes of spectroscopic characteristicsoccurred in similar SDS concentration ranges and were reversible.SDS polyacrylamide gel electrophoresis cleaved CP663S into subunits.Chlorophyll molecules moved with protein moieties. Glutaraldehydetreatment suppressed the effects of SDS on absorption, fluorescenceand CD characteristics. We conclude that chlorophyll moleculesin CP663S are in the hydrophobic region of the protein and theinteraction between chlorophyll a molecules occurs at shortdistances. Changes of spectroscopic characteristics are a resultof cleavage of CP663S. ¹Present address: National Institute for Basic Biology, Okazaki444, Japan. (Received November 22, 1982; Accepted May 31, 1983)     

Appearance of Chlorophyll-Protein Complexes in Greening Barley Seedlings

The sequential appearance of chlorophyll-protein complexes (CP)in greening barley leaves was studied by an improved methodof SDS-polyacrylamide gel electrophoresis (PAGE). Solubilizedthylakoid membranes were purified using a sucrose step gradientand CPs were separated by PAGE with low concentrations of SDSin solubilizing and reservoir buffers. At 10 min after the onsetof illumination, a chlorophyll-protein complex (CPX) was detected.It was a labile CP, its chlorophyll (Chl) being easily releasedfrom the apoprotein during electrophoresis. The P700-chlorophylla/b-protein complex (CPl) appeared after 45–60 min ofillumination together with P700 activity. Light-harvesting chlorophylla/b-protein complex (LHCP) began to accumulate at 2.5 h withthe beginning of Chl b synthesis. In some cases a small amountof CPa could be detected after 6 h of greening. The time-differencespectrum between homogenates of leaves illuminated for 30 and60 min had an absorbance maximum at 677 nm, showing that a redshift indicative of CPl formation began soon after completionof the Shibata shift. The time-difference spectrum between 3.5-hand 4.0-h illuminated leaves resembled the absolute spectrumof fully greened leaves, indicating that at this stage, spectralcomponents were being synthesized at the same ratio at whichthey exist in fully greened tissues. Both absolute and time-differencespectral data supported the SDS-PAGE results. (Received February 27, 1985; Accepted May 8, 1985)     

Prolonged incubation with low concentrations of mercury alters energy transfer and chlorophyll (Chl) a protein complexes in Synechococcus 6301: changes in Chl a absorption and emission characteristics and loss of the F695 emission band

Synechococcus PCC 6301 cells grown in the presence of low sublethal levels of (about 2 m) mercury induced alterations in chlorophyll (Chl) a absorption without significant alterations in phycocyanin. Chl a fluorescence emission in Hg²⁺ -raised cells showed a large (about 18 nm) blue shift in the peak emission. No major spectral changes in phycobilisome (PBsome) emission characteristic were noticed, indicating major structural alterations in Chl-protein complexes by incubation with Hg²⁺ ions. Low temperature (77 K) emission spectra of cells grown in the presence of Hg²⁺ showed a loss of the characteristic Chl a emission band at 695 nm (F695), which is known to be linked to photosystem II photochemistry and to originate from the Chl a of core antenna polypeptide CP 47 of photosystem II. The SDS-PAGE polypeptide profile of thylakoids indicates a loss of a polypeptide(s) with a molecular mass between 40 and 60 k Da by Hg²⁺ incubation of cells. Our results suggest that prolonged incubation of Synechococcus 6301 cells with low concentrations of Hg²⁺ affects the Chl a spectral properties and the structure of Chl-protein complexes.