黄化油菜突变体Cr3529子叶类囊体膜光谱性质研究

以发育10d的黄化油菜突变体为材料,分析了突变体油菜子叶类囊体膜的色素含量、室温吸收光谱、叶绿素荧光发射和激发光谱以及蛋白内源荧光光谱的变化。数据显示：与野生型相比,突变体油菜子叶类囊体膜的光合色素Chl α和Chl b含量均减少．但Chl α／b比值升高;突变体油菜子叶类囊体膜叶绿素捕光能力和受激发能力均下降,且较依赖于Chl α捕光并将光能激发传递给PSⅡ反应中心;突变体油菜子叶类囊体膜的蛋白内源荧光也明显异于野生型。进一步表明突变体油菜子叶类囊体膜蛋白组成发生了改变。     

Changes of Thylakoid Membrane Stacks and Chl a/b Ratio of Chloroplast from Sacred Lotus (Nelumbo nucifera) Seeds During Their Germination Under Light

Changes of chloroplast thylakoid membrane stacks and Chl a/b ratio in the plumule of sacred lotus (Nelumbo nucifera Gaertn) seeds during their germination under light were as follows: Before germination there were giant grana and very low Chi a/b ratio (0.9) in the chloroplasts. Two days after germination, the thylakoid membranes of the giant grana gradually loosened and even destacked (disintegrated), the Chl a/b ratio was 1.06. Four clays after germination, the newly formed grana thylakoid membranes were 3–5 times shorter than those of the supergrana thylakoid membranes before germination and less grana stacks were seen; the Chl a/b ratio was 1.42. Six days after germination, the stacked thylakoi membranes became more orderly arranged. In addition the grana increased in number, the stroma thylakoid membranes were scarce, the Chl a/b ratio was 2.16. Eiglt days after germination, the thylakoid membranes in each granum decreased, but the total number of grana increased only slightly. In the meantime, some large starch grains and more stroma thylakoid membranes appeared; the Chl a/b ratio was 2.77. Ten days after germination normal thylakoid membrane structure was formed both in grana and stroma lamellae. They were arranged orderly as in the chloroplasts of other higher plants; the Chl a/b ratio was 2.80. The following conclusions could be drawn from the above mentioned results: 1) There was a negative correlation between the degree of stacking of the grana thylakoid membranes and the Chl a/b ratio. This statement further proved that the membranes stacking might mainly be induced by LHCII. 2) Development of the grana thylakoid membranes within chloroplasts from sacred lotus plumule followed that of the stroma thylakoid membranes, and the tendency of changes of their Chl 2/b ratio being from the lowest to the highest and then to normal were quite different from those of other higher plants. The chloroplasts iri the latter plants contain long parallel stacks of nonappressed primary thylakoids at second step, and the changes of their ratio of Chl a/b tend to be from the highest to the lowest and then to normal. There are indications that sacred lotus plumule might employ a distinctive developing pathway. This provides an important basis for Nelumbo to possess an unique position in phylogeny of Angiospermae.     

Function of chlorophyll d in reaction centers of photosystems I and II of the oxygenic photosynthesis of Acaryochloris marina

Reaction center chlorophylls (Chls) in photosystems II and I were studied in the isolated thylakoid membranes of a cyanobacterium, Acaryochloris marina, which contains Chls d and a as the major and minor pigments, respectively. The membranes contained PS I and II complexes at a 1.8:1 molar ratio on the basis of the spin densities on the tyrosine D radical and the photo-oxidized PS I primary donor (P740+). In the presence of ferricyanide, laser excitation induced bleach at 725 nm that recovered with time constants of 25 micros and 1.2 ms. The signal, designated P725, was suppressed by PS II inhibitors DCMU and hydroxylamine. The P725 spectrum was tentatively assigned to the absorption changes of the special pair Chl d, the accessory Chl d, and the acceptor pheophytin a in PS II. The addition of ascorbate induced the additional signal with a slow decay time constant of 4.5 ms. This signal showed a broad bleach at 740 nm and shift-type absorption changes at around 707 and 685 nm, which were assigned to the absorption changes of PS I special pair of Chl d (P740), the accessory Chl d, and the primary acceptor Chl a (A0), respectively. Mechanisms and the evolution of the Chl-d based reaction centers using far-red light are discussed together with the amino acid sequences of PS II D1 and D2 proteins.     

Photosystem 2-activity and thylakoid membrane polypeptides of <Emphasis Type="Italic">in vitro</Emphasis> cultured chrysanthemum as affected by NaCl

Long-term (30 d) effects of 100, 200, 300, and 400 mM NaCl on photosystem 2 (PS 2)-mediated electron transport activity and content of D1 protein in the thylakoid membranes of chrysanthemum (Dendranthema grandiflorum) cultured in vitro at low irradiance 20 μmol(photon) m⁻² s⁻¹ were investigated. 100 mM NaCl increased contents of chlorophylls (Chl) a and b, carotenoids (Car; xanthophylls + carotenes), and the ratio of Chl a/b, and Car/Chl a+b. However, further increase in NaCl concentration led to the significant reduction in the contents of Chl a, and Chl b, and increase in the ratio of Chl a/b and Car/Chl a+b. NaCl treatment decreased the PS 2-mediated electron transport activity and contents of various thylakoid membrane polypeptides including D1 protein.     

FUNCTIONAL BINDING OF DIATOM PIGMENTS TO A RHODOPHYTE LIGHT HARVESTING POLYPEPTIDE

A close relationship of light harvesting polypeptides (LHC) of rhodophytes, chromophytes and chlorophytes is inferred from the amino acid sequence similarity in three transmembrane helices, and from the conservation of 8 putative chlorophyll (Chl)-binding sites (Durnford et al. 1999, J. Mol. Evol. 48:59). Differences in Chl and carotenoid pigments have been a major classification feature. Thus, it was of interest to ascertain whether pigments from a diatom ( Thallasiosira fluviatilis ) could be functionally inserted into a red algal ( Porphyridium cruentum ) polypeptide. A recombinant polypeptide, LHCaR1, was reconstituted with pigment extracts from the diatom (Chls a and c , fucoxanthin, diadinoxanthin and β-carotene). The pigments were found attached to protein upon separation on sucrose gradients, and on non-denaturing gels. Absorption and fluorescence excitation spectra revealed individual peaks corresponding to the absorption maxima of Chl a at 438/672 nm; Chl c at 463/638 nm; and fucoxanthin at 493/540 nm. Fluorescence emission and CD spectra showed functional binding and suitable orientation for energy transfer from Chl c and carotenoids to Chl a. The LHCaR1 successfully folded in the presence of the heterologous pigments and bound 7 Chl a , 1 Chl c , 8 fucoxanthin, and 1.9 diadinoxanthin per polypeptide. By comparison, this polypeptide with P. cruentum pigments binds 8 Chl a , and 4 zeaxanthins, thus revealing its capability of functionally binding 8 Chls with variations in carotenoid numbers. Such a trait may have favored the diversification of a large family of LHCs and the successful radiation of photosynthetic eukaryotes into different light environments.     

低温胁迫期间水稻光合膜色素与蛋白水平的变化

对４℃和１１℃两种低温胁迫过程中水稻类囊体膜色素与蛋白组成的变化进行了比较研究。结果表明：４℃低温不仅使类囊体膜中的光合色素（叶绿素、类胡萝卜素）含量降低,而且还引起膜蛋白组成的深刻变化,表现在大部分原有膜蛋白组分的含量在低温下明显降低,同时在低温处理的第３天诱导出一条３２．５ＫＤ的新蛋白带。与４℃处理相比,１１℃低温处理只引起了光合色素含量的降低,而对类囊体膜蛋白组成的影响不大,另外发现,两种低     

Low growth temperature-induced changes to pigment composition and photosynthesis in Zea mays genotypes differing in chilling sensitivity

The effects on pigment composition and photosynthesis of low temperature during growth were examined in the third leaf of three chilling-tolerant and three chilling-sensitive genotypes of Zea mays L. The plants were grown under a controlled environment at 24 or 14 °C at a photon flux density (PFD) of 200 or 600 μ mol m^–2 s^–1. At 24 °C, the two classes of genotypes showed little differences in their photosynthetic activity and their composition of pigments. At 14 °C, photosynthetic activity was considerably reduced but the chilling-tolerant genotypes displayed higher photosynthetic rates than the chilling-sensitive ones. Plants grown at 14 °C showed a reduced chlorophyll (Chl) a + b content and a reduced Chl a / b ratio but an increased ratio of total carotenoids to Chl a + b . These changes in pigment composition in plants grown at low temperature were generally more pronounced in the chilling-sensitive genotypes than in the tolerant ones, particularly at high PFD. Furthermore, at 14 °C, all the genotypes showed increased ratios of lutein, neoxanthin and xanthophyll-cycle carotenoids to Chl a + b but a reduced ratio of β -carotene to Chl a + b , especially at high PFD. At 14 °C, the chilling-tolerant genotypes, when compared with the sensitive ones, were characterized by higher contents of β -carotene and neoxanthin, a lower content of xanthophyll-cycle carotenoids, a lower ratio of xanthophylls to β -carotene, and less of their xanthophyll-cycle carotenoid pool in the form of zeaxanthin. These differences between the two classes of genotypes were more pronounced at high PFD than at low PFD. The results are discussed in terms of the relationship that may exist in maize between pigment composition and the capacity to form an efficient photosynthetic apparatus at low growth temperature.     

Spectral signatures of five hydroxymethyl chlorophyll a derivatives chemically derived from chlorophyll b or chlorophyll f

Photosynthesis Research - Chlorophylls (Chls) are pigments involved in light capture and light reactions in photosynthesis. Chl a, Chl b, Chl d, and Chl f are characterized by unique absorbance...     

Exchange of pigment-binding amino acids in light-harvesting chlorophyll a/b protein

Four amino acids in the major light-harvesting chlorophyll (Chl) a/b complex (LHCII) that are thought to coordinate Chl molecules have been exchanged with amino acids that presumably cannot bind Chl. Amino acids H68, Q131, Q197, and H212 are positioned in helixes B, C, A, and D, respectively, and, according to the LHCII crystal structure [Kühlbrandt, W., et al. (1994) Nature 367, 614-621], coordinate the Chl molecules named a(5), b(6), a(3), and b(3). Moreover, a double mutant was analyzed carrying exchanges at positions E65 and H68, presumably affecting Chls a(4) and a(5). All mutant proteins could be reconstituted in vitro with pigments, although the thermal stability of the resulting mutant versions of recombinant LHCII varied significantly. All complexes reconstituted with the mutant proteins contained fewer chlorophyll molecules per two lutein molecules than complexes reconstituted with the wild-type protein. However, the chlorophyll-binding amino acids could not be unambiguously assigned to binding either chlorophyll a or b, as in most cases more than one chlorophyll molecule was lost due to the mutation. The changes in Chl stoichiometries suggest that in LHCII some chlorophyll positions can be filled with either Chl a or b. Only some of the point mutations in LHCII affected the ability of the apoprotein to assemble into trimeric LHCII upon insertion into isolated thylakoid membranes. Among these were exchanges of H68 with either F or L, suggesting that the stability of the LHCII trimer significantly depends on this amino acid or the Chl molecule named a(5) that is attached to it and is located close to the center of the trimeric complex. The ion pair bridge between E65 and R185 in LHCII does not appear to be essential for the proper folding of the protein.     

Photoinhibition in Chilling Stressed Leguminosae: Comparison of Vicia Faba and Pisum Sativum

The concentrations of photosynthetic pigments decreased in both chilling stressed species but the ratios of chlorophyll (Chl) a/b and total carotenoids (Car)/Chls were depressed only in faba bean. The contents of + carotene and lutein+lutein-5,6-epoxide remained unaffected in both species, but the de-epoxidation state involving the components of xanthophyll cycle increased in pea. Under chilling stress the photosynthetic electron transport associated with photosystem 2, PS2 (with and without the water oxidising complex) decreased in both plant species, the inhibition being higher in faba bean. The intrachloroplast quinone pool also decreased in both stressed species, yet an opposite trend was found for cytochrome b _559LP. Under stress an increasing peroxidation of thylakoid acyl lipids was detected in pea, but higher protein/Chl ratio was detected in faba bean. Thus the acceptor side of PS2 is mostly affected in both chilling stressed species, but faba bean is more sensitive.     

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)     

Mutant trimers of light-harvesting complex II exhibit altered pigment content and spectroscopic features

Mutants of plant light-harvesting complex II (LHC-II) were produced by refolding the complex in vitro from bacterially expressed apoprotein and purified pigments by a method which yields native-like LHC-II in a single step. Amino acid residues known from the structure of the complex [Kühlbrandt, W., et al. (1994) Nature 367, 614-621] to bind chlorophyll (Chl) were replaced with nonbinding residues by site-directed mutagenesis. Recombinant monomeric and trimeric pigment-protein complexes were separated by density gradient centrifugation, and their pigment composition was determined. Six out of nine mutants formed trimers with Chl a:Chl b ratios and Chl contents which suggested they were lacking one Chl a or b per polypeptide. In this way, the identities of Chls a1, a2, a3, b5, and b6 were confirmed as Chl a or b, respectively, whereas Chl b3 in the structure was found to be a Chl a. Absorption and fluorescence emission spectra of the mutant lacking Chl a2 indicated a central role for this Chl in energy transfer to the reaction center.     

Recombinant water-soluble chlorophyll protein from Brassica oleracea var. Botrys binds various chlorophyll derivatives

A gene coding for water-soluble chlorophyll-binding protein (WSCP) from Brassica oleracea var. Botrys has been used to express the protein, extended by a hexahistidyl tag, in Escherichia coli. The protein has been refolded in vitro to study its pigment binding behavior. Recombinant WSCP was found to bind two chlorophylls (Chls) per tetrameric protein complex but no carotenoids in accordance with previous observations with the native protein [Satoh, H., Nakayama, K., Okada, M. (1998) J. Biol. Chem. 273, 30568-30575]. WSCP binds Chl a, Chl b, bacteriochlorophyll a, and the Zn derivative of Chl a but not pheophytin a, indicating that the central metal ion in Chl is essential for binding. WSCP also binds chlorophyllides a and b and even the more distant Chl precursor Mg-protoporphyrin IX; however, these pigments fail to induce oligomerization of the protein. We conclude that the phytol group in bound Chl plays a role in the formation of tetrameric WSCP complexes. If WSCP in fact binds Chl or its derivative(s) in vivo, the lack of carotenoids in pigmented WSCP raises the question of how photooxidation, mediated by triplet-excited Chl and singlet oxygen, is prohibited. We show by spin-trap electron-paramagnetic resonance that the light-induced singlet-oxygen formation of WSCP-bound Chl is lower by a factor of about 4 than that of unbound Chl. This as-yet-unknown mechanism of WSCP to protect its bound Chl against photooxidation supports the notion that WSCP may function as a transient carrier of Chl or its derivatives.     

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.     

Excitation energy transfer pathways in Lhca4

EET in reconstituted Lhca4, a peripheral light-harvesting complex from Photosystem I of Arabidopsis thaliana, containing 10 chlorophylls and 2 carotenoids, was studied at room temperature by femtosecond transient absorption spectroscopy. Two spectral forms of Lut were observed in the sites L1 and L2, characterized by significantly different interactions with nearby chlorophyll a molecules. A favorable interpretation of these differences is that the efficiency of EET to Chls is about two times lower from the "blue" Lut in the site L1 than from the "red" Lut in the site L2 due to fast IC in the former case. A major part of the energy absorbed by the "red" Lut, approximately 60%-70%, is transferred to Chls on a sub-100-fs timescale from the state S(2) but, in addition, minor EET from the hot S(1) state within 400-500 fs is also observed. EET from the S(1) state to chlorophylls occurs also within 2-3 ps and is ascribed to Vio and/or "blue" Lut. EET from Chl b to Chl a is biphasic and characterized by time constants of approximately 300 fs and 3.0 ps. These rates are ascribed to EET from Chl b spectral forms absorbing at approximately 644 nm and approximately 650 nm, respectively. About 25% of the excited Chls a decays very fast-within approximately 15 ps. This decay is proposed to be related to the presence of the interacting Chls A5 and B5 located next to the carotenoid in the site L2 and may imply some photoprotective role for Lhca4 in the photosystem I super-complex.     

Structural modeling of the Lhca4 Subunit of LHCI-730 peripheral antenna in photosystem I based on similarity with LHCII

Peripheral chlorophyll a/b binding antenna of photosystem I (LHCI) from green algae and higher plants binds specific low energy absorbing chlorophylls (red pigments) that give rise to a unique red-shifted emission. A three-dimensional structural model of the Lhca4 polypeptide from the LHCI from higher plants was constructed on the basis of comparative sequence analysis, secondary structure prediction, and homology modeling using LHCII as a template. The obtained model of Lhca4 helps to visualize protein ligands to nine chlorophylls (Chls) and three potential His residues to extra Chls. Central domain of the Lhca4 comprising the first (A) and the third (C) transmembrane (TM) helices that binds 6 Chl molecules and two carotenoids is conserved structurally, whereas the interface between the first and the second TM helices and the outer surface of the second TM helix differ significantly among the LHCI and LHCII polypeptides. The model of Lhca4 predicts a histidine residue in the second TM helix, a potential binding site for extra Chl in close proximity to Chls a5 and b5 (labeling by Kühlbrandt). The interpigment interactions in the formed pigment cluster are suggested to cause a red spectral shift in absorption and emission. Modeling of the LHCI-730 heterodimer based on the model structures of Lhca1 and Lhca4 allowed us to suggest potential sites of pigment-pigment interactions that might be formed upon heterodimerization or docking of the LHCI dimers to the surface of PSI.     

Comparative Characterization of the Pigment Complex in Emergent, Floating, and Submerged Leaves of Hydrophytes

The content of chlorophylls (Chls) and carotenoids was studied in the leaves of 42 species of boreal aquatic plants with different degree of submergence (emergent, floating, and submerged) and isopalisade, dorsoventral, and homogenous types of mesophyll structure. Hydrophytes were shown to have a low Chl content (1–2 mg/g fr wt) and low Chls/carotenoids ratio (2.3–3.5) as compared to terrestrial plants. The pigment content per dry wt unit and unit leaf area was dependent on the type of mesophyll structure. It was a consequence of the changes in the parameters of leaf mesophyll structure characterizing the density of photosynthetic elements. In a sequence emergent floating submerged forms, the content of Chls and carotenoids decreased, and the photosynthetic capacity decreased due to a reduction in the chloroplast number per unit leaf area. Adaptation of submerged leaves to low illumination and slow CO₂ diffusion changed the functional properties of chloroplasts. An increase in the pigment content in the chloroplasts of submerged leaves (7 × 10^–9 mg Chl, 2 × 10^–9 mg carotenoids) as compared to emergent and floating leaves was accompanied by a decline in the photosynthetic capacity per Chl comprising 1.6 mg CO₂/(mg Chl h) versus 3.9 and 3.8 mg CO₂/(mg Chl h) in emergent and floating leaves, respectively.     

Origin of the chlorophyll b formyl oxygen in Chlorella vulgaris.

Chlorophyll (Chl) b is an accessory light-harvesting pigment of plants and chlorophyte algae. Chl b differs from Chl a in that the 3-methyl group on ring B of chl a is replaced by a 3-formyl group on Chl b. The present study determined the biosynthetic origin of the Chl b formyl oxygen in in vivo labeling experiments. A mutant strain of the unicellular chlorophyte Chlorella vulgaris, which can not synthesize Chls when cultured in the dark but rapidly greens when transferred to the light, was grown in the dark for several generations to deplete Chls, and then the cells were transferred to the light and allowed to form Chls in a controlled atmosphere containing 18O2. Chl a and Chl b were purified from the cells and analyzed by high-resolution mass spectroscopy. Analysis of the mass spectra indicated that over 76% of the Chl a molecules had incorporated an atom of 18O. For Chl b, 58% of the molecules had incorporated an atom of 18O at one position and 34% of the molecules had incorporated an atom of 18O at a second position. These results demonstrate that the isocyclic ring keto oxygen of both Chl a and Chl b, as well as the formyl oxygen of Chl b, is derived from O2.     

Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom, Chaetoceros gracilis

Diatoms occupy a key position as a primary producer in the global aquatic ecosystem. We developed methods to isolate highly intact thylakoid membranes and the photosystem I (PS I) complex from a marine centric diatom, Chaetoceros gracilis. The PS I reaction center (RC) was purified as a super complex with light-harvesting fucoxanthin-chlorophyll (Chl)-binding proteins (FCP). The super complex contained 224 Chl a, 22 Chl c, and 55 fucoxanthin molecules per RC. The apparent molecular mass of the purified FCP-PS I super complex (approximately 1000 kDa) indicated that the super complex was composed of a monomer of the PS I RC complex and about 25 copies of FCP. The complex contained menaquinone-4 as the secondary electron acceptor A1 instead of phylloquinone. Time-resolved fluorescence emission spectra at 77 K indicated that fast (16 ps) energy transfer from a Chl a band at 685 nm on FCP to Chls on the PS I RC complex occurs. The ratio of fucoxanthin to Chl a on the PS I-bound FCP was lower than that of weakly bound FCP, suggesting that PS I-bound FCP specifically functions as the mediator of energy transfer between weakly bound FCPs and the PS I RC.