Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins, pigment system II particles and chlorophyll a in diethylether solution by the curve-fitting method.

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25 degrees C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol 49, 421--429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm. The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0--2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6--4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively. Absorption spectra at 25 degrees C and at --196 degrees C of the water-soluble chlorophyll proteins were compared by the curve-fitting methods. The component bands at --196 degrees C were blue-shifted by 0.8--4.1 nm and narrower in half widths as compared to those at 25 degrees C.     

Differential changes in degradation of chlorophyll-protein complexes of photosystem I and photosystem II during flag leaf senescence of rice.

The stability of chlorophyll-protein complexes of photosystem I (PSI) and photosystem II (PSII) was investigated by chlorophyll (Chl) fluorescence spectroscopy, absorption spectra and native green gel separation system during flag leaf senescence of two rice varieties (IIyou 129 and Shanyou 63) grown under outdoor conditions. During leaf senescence, photosynthetic CO(2) assimilation rate, carboxylase activity of Rubisco, chlorophyll and carotenoids contents, and the chlorophyll a/b ratio decreased significantly. The 77 K Chl fluorescence emission spectra of thylakoid membranes from mature leaves had two peaks at around 685 and 735 nm emitting mainly from PSII and PSI, respectively. The total Chl fluorescence yields of PSI and PSII decreased significantly with senescence progressing. However, the decrease in the Chl fluorescence yield of PSI was greater than in the yield of PSII, suggesting that the rate of degradation in chlorophyll-protein complexes of PSI was greater than in chlorophyll-protein complexes of PSII. The fluorescence yields for all chlorophyll-protein complexes decreased significantly with leaf senescence in two rice varieties but the extents of their decrease were significantly different. The greatest decrease in the Chl fluorescence yield was in PSI core, followed by LHCI, CP47, CP43, and LHCII. These results indicate that the rate of degradation for each chlorophyll-protein complex was different and the order for the stability of chlorophyll-protein complexes during leaf senescence was: LHCII>CP43>CP47>LHCI>PSI core, which was partly supported by the green gel electrophoresis of the chlorophyll-protein complexes.     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins,pigment System II particles and chlorophyll a in diethylether solution by the curve-fitting method

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25°C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol. 49, 421–429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm.The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0–2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6–4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively.Absorption spectra at 25°C and at ?196°C of the water-soluble chlorophyll proteins were compared by the curve-fitting method. The component bands at ?196°C were blue-shifted by 0.8–4.1 nm and narrower in half widths as compared to those at 25°C.     

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)     

Chlorophyll-Protein Complexes of Blue-Green Algae Isolated by a New Gel Electrophoresis System with High Resolving Power

At least 13 chlorophyll bands from the thylakoid membranes of blue-green algae could be clearly resolved by SDS-PAGE employing a new improved procedure. They were designated as CPIa, CPIb, CPIc, CPId, CPIe, CPIf, CPIg, CHIh, CPal, CPa2, CPa3, CPa4 and FC. 8 chlorophyll-protein complexes, CPIa-CPIh, had the same absorption spectrum at 676 nm in the red and 436 nm in the blue region. They belonged to the chlorophyll-protein complexes of PS Ⅰ. 4 chlorophyll-protein complexes, CPal-CPa4, had a red absorption peak at 670­672 nm and a blue one at 436 nm. Their fluorescence emission peak at 77K was at 685 nm. They were chlorophyll-protein complexes of PS Ⅱ.     

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

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

Spectroscopic properties of the CP43 core antenna protein of photosystem II

CP43 is a chlorophyll-protein complex that funnels excitation energy from the main light-harvesting system of photosystem II to the photochemical reaction center. We purified CP43 from spinach photosystem II membranes in the presence of the nonionic detergent n-dodecyl-beta,D-maltoside and recorded its spectroscopic properties at various temperatures between 4 and 293 K by a number of polarized absorption and fluorescence techniques, fluorescence line narrowing, and Stark spectroscopy. The results indicate two "red" states in the Q(y) absorption region of the chlorophylls. The first peaks at 682.5 nm at 4 K, has an extremely narrow bandwidth with a full width at half-maximum of approximately 2.7 nm (58 cm(-1)) at 4 K, and has the oscillator strength of a single chlorophyll. The second peaks at approximately 679 nm, has a much broader bandshape, is caused by several excitonically interacting chlorophylls, and is responsible for all 4 K absorption at wavelengths longer than 685 nm. The Stark spectrum of CP43 resembles the first derivative of the absorption spectrum and has an exceptionally small overall size, which we attribute to opposing orientations of the monomer dipole moments of the excitonically coupled pigments.     

Photosynthetic Membranes of Porphyridium cruentum: An Analysis of Chlorophyll-Protein Complexes and Heme-Binding Proteins

Three chlorophyll-protein complexes (CP I, CP III, CP IV) were electrophoretically separated from thylakoids of the eukaryotic red alga Porphyridium cruentum. CP I contained the primary photochemical reaction center of photosystem I as judged by its light-induced reversible absorbance change at 700 nanometers, by its fluorescence emission maximum at 720 nanometers (−196°C), and by the molecular weight of its apoprotein (68,000 daltons). CP III and CP IV appeared to belong with photosystem II as suggested by the absence of light-reversible absorbance at 700 nanometers, by their fluorescence maximum at 690 nanometers (−196°C), and by the presence of a chlorophyll-binding polypeptide with a molecular weight of about 52,000 daltons. CP IV when completely denatured had two additional polypeptides of about 40,000 and 48,000 daltons. All three chlorophyll-protein complexes contained carotenoids: the chlorophyll/carotenoid molar ratio of 15:1 for CP I, and 20:1 for CP III and CP IV. The thylakoid membranes of P. cruentum contained four cytochromes, detected by heme-dependent peroxidase activity, but there was no observed association with the electrophoretically separated chlorophyll-protein complexes.     

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.     

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

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

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.     

Properties of protein-chlorophyll complexes from pea (Pisum sativum L.) leaves. The organization of chlorophyll.

Chlorophyll-protein-detergent complexes were prepared from pea chloroplasts by using sodium dodecylbenzenesulphonate and polyacrylamide-gel electrophoresis. Circular-dichroism spectra showed that complex CPI has a dimeric arrangement of chlorophyll a, with additional weaker interactions. Ellipticities were determined for both complexes and for purified chlorophylls in solution, and it is argued that the circular dichroism of complex CPII is derived from chlorophyll-protein interaction rather than from interaction between chlorophylls a and b. The detergent could be removed from the complexes by using urea and gel filtration, leaving the chlorophyll-protein in solution, although in each case a diminished ellipticity indicated some loss of organization. Three-peaked circular-dichroism spectra of chloroplast fragments before and after addition of detergent were compared with a curve obtained by summing graphically the spectra of complexes CPI, CPII and the free-pigment fraction. There was good correspondence at 650 nm, and the longer-wavelength peaks agreed in form and magnitude, but with discrepancies in position. It was concluded that complexes CPI and CPII pre-exist in the original material, but that there is an environmental effect which is destroyed when the complexes are extracted.     

Isolation of chlorophyll-protein complexes and quantification of electron transport components in Synura petersenii and Tribonema aequale

The chlorophyll-protein complexes of the yellow alga Synura petersenii (Chrysophyceae) and the yellow-green alga Tribonema aequale (Xanthophyceae) were studied. The sodiumdodecylsulfate/sodiumdesoxycholate solubilized photosynthetic membranes of these species yielded three distinct pigment-protein complexes and a non-proteinuous zone of free pigments, when subjected to SDS polyacrylamid gel electrophoresis. The slowest migrating protein was identical to complex I (CP I), the P-700 chlorophyll a-protein, which possessed 60 chlorophyll a molecules per reaction center in Tribonema and 108 in Synura. The zone of intermediate mobility contained chlorophyll a and carotenoids. The absorption spectrum of this complex was very similar to the chlorophyll a-protein of photosystem II (CP a), which is known from green plants. The fastest migrating pigment protein zone was identified as a light-harvesting chlorophyll-protein complex. In Synura this protein was characterized by the content of chlorophyll c and of fucoxanthin. Therefore this complex will be named as LH Chl a/c-fucocanthin protein. In addition to the separation of the chlorophyll-protein complexes the cellular contents of P-700, cytochrome f (bound cytochrome) and cytochrome c-553 (soluble cytochrome) were measured. The stoichiometry of cytochrome f: cytochrome c-553:P-700 was found to be 1:4:2.4 in Tribonema and 1:6:3.4 in Synurá.Abbreviations CP a chlorophyll a-protein of photosystem II - CP I P-700 chlorophyll a-protein - FP free pigment - LH Chl a/c light-harvesting chlorophyll a/c-protein - PAGE polyacrylamidgelelectrophoresis - SDS Sodiumdodecylsulfate - SDOC sodium-desoxycholate     

Membrane Development in the Cyanobacterium, Anacystis nidulans, during Recovery from Iron Starvation

Deprivation of iron from the growth medium results in physiological as well as structural changes in the unicellular cyanobacterium Anacystis nidulans R2. Important among these changes are alterations in the composition and function of the photosynthetic membranes. Room-temperature absorption spectra of iron-starved cyanobacterial cells show a chlorophyll absorption peak at 672 nanometers, 7 nanometers blue-shifted from its normal position at 679 nanometers. Iron-starved cells have decreased amounts of chlorophyll and phycobilins. Their fluorescence spectra (77K) have one prominent chlorophyll emission peak at 684 nanometers as compared to three peaks at 687, 696, and 717 nanometers from normal cells. Chlorophyll-protein analysis of iron-deprived cells indicated the absence of high molecular weight bands. Addition of iron to iron-starved cells induced a restoration process in which new components were initially synthesized and integrated into preexisting membranes; at later times, new membranes were assembled and cell division commenced. Synthesis of chlorophyll and phycocyanins started almost immediately after the addition of iron. The absorption peak slowly returned to its normal wavelength within 24 to 28 hours. The fluorescence emission spectrum at 77K changed over a period of 14 to 24 hours during which the 696- and 717-nanometer peaks grew to their normal levels, and the 684 nanometer peak moved to 687 nanometers and its relative intensity decreased to its normal level. Analysis of chlorophyll-protein complexes on polyacrylamide gels showed that high molecular weight chlorophyll-protein bands were formed during this time, and that low molecular weight bands (related to photosystem II) disappeared. The origin of the fluorescence emission at 687 and 696 nanometers is discussed in relation to the specific chlorophyll-protein complexes formed during iron reconstitution.     

Isolation of chlorophyll-protein complexes and quantification of electron transport components in Synura petersenil and Tribonema aequale

The chlorophyll-protein complexes of the yellow alga Synura petersenii (Chrysophyceae) and the yellow-green alga Tribonema aequale (Xanthophyceae) were studied. The sodiumdodecylsulfate/sodiumdesoxycholate solubilized photosynthetic membranes of these species yielded three distinct pigment-protein complexes and a non-proteinous zone of free pigments, when subjected to SDS polyacrylamid gel electrophoresis. The slowest migrating protein was identical to complex I (CP I), the P-700 chlorophyll a-protein, which possessed 60 chlorophyll a molecules per reaction center in Tribonema and 108 in Synura. The zone of intermediate mobility contained chlorophyll a and carotenoids. The absorption spectrum of this complex was very similar to the chlorophyll a-protein of photosystem II (CP a), which is known from green plants. The fastest migrating pigment protein zone was identified as a light-harvesting chlorophyll-protein complex. In Synura this protein was characterized by the content of chlorophyll c and of fucoxanthin. Therefore this complex will be named as LH Chl a/c-fucocanthin protein. In addition to the separation of the chlorophyll-protein complexes the cellular contents of P-700, cytochrome f (bound cytochrome) and cytochrome c-553 (soluble cytochrome) were measured. The stoichiometry of cytochrome f: cytochrome c-553:P-700 was found to be 1:4:2.4 in Tribonema and 1:6:3.4 in Synurá.     

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

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

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.     

Chlorophyll-protein composition of the thylakoid membrane from Prochlorothrix hollandica, a prokaryote containing chlorophyll b

The chlorophyll-protein complexes of the thylakoid membrane from Prochlorothrix hollandica were identified following electrophoresis under nondenaturing conditions. Five complexes, CP1-CP5, were resolved and these green bands were analyzed by spectroscopic and immunological methods. CP1 contains the photosystem I (PSI) reaction center, as this complex quenched fluorescence at room temperature, and had a 77 K fluorescence emission peak at 717 nm. CP4 contains the major chlorophyll-a-binding proteins of the photosystem II (PSII) core, because this complex contained polypeptides which cross-reacted to antibodies raised against Chlamydomonas PSII proteins 5 and 6. Furthermore, fluorescence excitation studies at 77 K indicated that only a Chl a is bound to CP4. Complexes CP2, CP3 and CP5 contained functionally bound Chl a and b as judged by absorption spectroscopy at 20 degrees C and fluorescence excitation spectra at 77 K. CP2, CP3 and CP5 all contain polypeptides of 30-33 kDa which are immunologically distinct from the LHC-II complex of higher plant thylakoids.     

Chiroptical properties of chlorophyll-protein complexes separated on Deriphat/polyacrylamide gel.

Circular dichroism (c.d.) was measured for four chlorophyll-protein complexes, resolved from sodium dodecyl sulphate extracts of chloroplasts by electrophoresis in polyacrylamide gel containing Deriphat 160 (disodium N-dodecyl beta-imidopropionate), a zwitterionic detergent. The slowest-band (1) complex was found to be identical with the complex CP1 as found on electrophoresis in the presence of anion detergent, but it was in a much higher yield (30% of the chlorophyll a). In band-2 and -3 protein complexes a c.d. pattern described for the complex CP2 could be recognized. Another c.d. component of a split-exciton type with extrema at 680 (-) and 669 (+)nm, together with evidence of disorganized chlorophyll, was found in band-2, -3 and -4 complexes. When a barley (Hordeum vulgare) mutant lacking chlorophyll b was examined, only bands 1 and 4 were obtained, and the c.d. of the band-4 complex was much less affected by disorganized chlorophyll. C.D. spectra resembling that of this band-4 complex could be generated by subtracting the c.d. of complex CP1 from the c.d. of photochemically active mutant chloroplast fragments, or by subtracting the c.d. of complexes CP1 and CP2 from pea (Pisum sativum) chloroplast fragments. The Deriphat appears to have preserved at least to some extent a new type of chlorophyll a-protein complex.     

Chlorophyll-protein complex composition during chloroplast development: A species comparison

Barley, maize, pea, soybean, and wheat exhibited differences in chlorophyll a/b ratio and chlorophyll-protein (CP) complex composition during the initial stages of chloroplast development. During the first hours of greening, the chlorophyll a/b ratios of barley, pea, and wheat were high (a/b8) and these species contained only the CP complex of photosystem I as measured by mild sodium dodecyl sulfate polyacrylamide gel electrophoresis. A decrease in chlorophyll a/b ratio and the observation of the CP complexes associated with photosystem II and the light-harvesting apparatus occurred at later times in barley, pea, and wheat. In contrast, maize and soybean exhibited low chlorophyll a/b ratios (a/b<8) and contained the CP complexes of both photosytem I and the light-harvesting apparatus at early times during chloroplast development. The species differences were not apparent after 8 h of greening. In all species, the CP complexes were stabilized during the later stages of chloroplast development as indicated by a decrease in the percentage of chlorophyll released from the CP complexes during detergent extraction. The results demonstrate that CP complex synthesis and accumulation during chloroplast development may not be regulated in the same way in all higher plant species.Abbreviations Chl chlorophyll - CP chlorophyll-protein - CPI P700 chlorophyll-a protein complex of photosystem I - CPa electrophoretic band that contains the photosystem II reaction center complexes and a variable amount of the photosystem I light-harvesting complex - LHC the major light-harvesting complex associated with photosystem II - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Paper No. 10335 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601.