Quantitative separation of spinach thylakoids into Photosystem II-enriched inside-out vesicles and Photosystem I-enriched right-side-out vesicles

Yeda press disruption of thylakoids in the presence of magnesium followed by aqueous polymer two-phase partitioning fractionated the total thylakoid membrane material into two distinctly different fractions. One fraction comprised approx. 60% of the material on a chlorophyll basis and contained inside-out vesicles while the other fraction (40%) contained right-side-out vesicles. The sidedness of the vesicles was determined from the direction of their light-induced proton translocation. The inside-out vesicles showed a pronounced Photosystem (PS) II enrichment as judged by their high PS II and low PS I activities. Moreover, they showed a high ratio between the PS II reaction centre chlorophyll-protein complex and the PS I reaction centre chlorophyll-protein complex (CP I). The chlorophyll $\text{a}\text{b}$ ratio was as low as 2.3 compared to 3.2 for the starting material. In contrast, the right-side-out vesicles showed a pronounced PS I enrichment. Their chlorophyll $\text{a}\text{b}$ ratio was 4.3–4.9. The tight stacking induced by Mg²⁺ allows a quantitative formation of inside-out vesicles from the appressed thylakoid regions while mainly non-appressed thylakoids turn right-side-out. The possibility of fractionating all of the thylakoid material into two sub-populations with markedly different composition with respect to PS I and PS II argues against a close physical association between the two photosystems and in favour of their spatial separation in the plane of the membrane. This fractionation procedure, which can be completed within 1 h and gives high yields of both PS II inside-out thylakoids and PS I right-side-out thylakoids, should be very useful for facilitating and improving studies on both the transverse and lateral organization of the thylakoid membrane.     

The reconstitution of a Photosystem II protein complex, P-700-chlorophyll a-protein complex and light-harvesting chlorophyll ab-protein

A Photosystem II reaction centre protein complex was extracted from spinach chloroplasts using digitonin. This complex showed (i) high rates of dichloroindophenol and ferricyanide reduction in the presence of suitable donors, (ii) low-temperature fluorescence at 685 nm with a variable shoulder at 695 nm which increased as the complex aggregated due to depletion of digitonin and (iii) four major polypeptides of 47, 39, 31 and 6 kDa on dissociating polyacrylamide gels. The Photosystem II protein complex, together woth the P-700-chlorophylla protein complex and light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHCP) also isolated using digitonin, were reconstituted with lipids from spinach chloroplasts to form proteoliposomes. The low-temperature (77 K) fluorescence properties of the various proteoliposomes were analysed. The $\text{F}{}_{685}\text{F}{}_{695}$ ratios of the Photosystem II reaction centre protein complex-liposomes decreased as the lipid to protein ratios were increased. The $\text{F}{}_{681}\text{F}{}_{697}$ ratios of LHCP-liposomes were found to behave similarly. Light excitation of chlorophyll b at 475 nm stimulated emission from both the Photosystem II protein complex (F₆₈₅ and F₆₉₅) and the P-700-chlorophyll a-protein complex (F₇₃₅) when LHCP was reconstituted with either of these complexes, demonstrating energy transfer between LHCP and PS I or II complexes in liposomes. No evidence was found for energy transfer from the PS II complex to the P-700-chlorophyll a-protein complex reconstituted in the same proteoliposome preparation. Proteoliposome preparations containing all three chlorophyll-protein complexes showed fluorescence emission at 685, 700 and 735 nm.     

The action of lipase on chloroplast membranes: II. Polypeptide patterns of bean galactolipase- and phospholipase A2-treated thylakoid membranes

Thylakoid membranes obtained from bean chloroplasts treated with bean galactolipase or phospholipase A₂ (from Crotalus terr. terr.) showed marked changes in their polypeptide patterns when separated on SDS-PAGE. The obtained results have been discussed with regard to the relationship between chloroplast lipids and polypeptides originating from chlorophyll-protein complexes of bean thylakoids. A coexistence between galactolipids and the peripheral antennae in PS I complex and LHCP³ as well as a conspicuous role of phospholipids in PSI and PSII centre chlorophyll-protein complexes has to be underlined.Abbreviations CP1 chlorophyll a-protein complex of PSI - CPa chlorophyll a-protein complex of PSII - D₁₀ digitonin subchloroplast particles enriched in PSII - D₁₄₄ digitonin subchloroplast particles enriched in PSI - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP^1–3 light harvesting chlorophyll a/b protein complexes - PAGE polyacrylamide gel electrophoresis - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulphate - TCA trichloroacetic acid - Tricine N-Tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethan     

The action of lipase on chloroplast membranes: II. Polypeptide patterns of bean galactolipase- and phospholipase A2-treated thylakoid membranes

Thylakoid membranes obtained from bean chloroplasts treated with bean galactolipase or phospholipase A₂ (from Crotalus terr. terr.) showed marked changes in their polypeptide patterns when separated on SDS-PAGE. The obtained results have been discussed with regard to the relationship between chloroplast lipids and polypeptides originating from chlorophyll-protein complexes of bean thylakoids. A coexistence between galactolipids and the peripheral antennae in PS I complex and LHCP³ as well as a conspicuous role of phospholipids in PSI and PSII centre chlorophyll-protein complexes has to be underlined.Abbreviations CP1 chlorophyll a-protein complex of PSI - CPa chlorophyll a-protein complex of PSII - D₁₀ digitonin subchloroplast particles enriched in PSII - D₁₄₄ digitonin subchloroplast particles enriched in PSI - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea - LHCP^1-3 light harvesting chlorophyll a/b protein complexes - PAGE polyacrylamide gel electrophoresis - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulphate - TCA trichloroacetic acid - Tricine N-Tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethan     

Effects of cations upon chloroplast membrane subunit interactions and excitation energy distribution

When isolated chloroplasts from mature pea (Pisum sativum) leaves were treated with digitonin under “low salt” conditions, the membranes were extensively solubilized into small subunits (as evidenced by analysis with small pore ultrafilters). From this solubilized preparation, a photochemically inactive chlorophyll · protein complex (chlorophyll $\text{a}\text{b}$ ratio, 1.3) was isolated. We suggest that the detergent-derived membrane fragment from mature membranes is a structural complex within the membrane which contains the light-harvesting chlorophyll $\text{a}\text{b}$ protein and which acts as a light-harvesting antenna primarily for Photosystem II.Cations dramatically alter the structural interaction of the light-harvesting complex with the photochemically active system II complex. This interaction has been measured by determining the amount of protein-bound chlorophyll b and Photosystem II activity which can be released into dispersed subunits by digitonin treatment of chloroplast lamellae. When cations are present to cause interaction between the Photosystem II complex and the light-harvesting pigment · protein, the combined complexes pellet as a “heavy” membranous fraction during differential centrifugation of detergent treated lamellae. In the absence of cations, the two complexes dissociate and can be isolated in a “light” submembrane preparation from which the light-harvesting complex can be purified by sucrose gradient centrifugation.Cation effects on excitation energy distribution between Photosystems I and II have been monitored by following Photosystem II fluorescence changes under chloroplast incubation conditions identical to those used for detergent treatment (with the exception of chlorophyll concentration differences and omission of detergents). The cation dependency of the pigment · protein complex and Photosystem II reaction center interactions measured by detergent fractionation, and regulation of excitation energy distribution as measured by fluorescence changes, were identical. We conclude that changes in substructural organization of intact membranes, involving cation induced changes in the interaction of intramembranous subunits, are the primary factors regulating the distribution of excitation energy between Photosystems II and I.     

Localization of photosynthetic electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays

The organization of the electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays was investigated. Grana-containing mesophyll chloroplasts (chlorophyll a to chlorophyll b ratio of about 3.0) possessed the full complement of the various electron transport components, comparable to chloroplasts from C₃ plants. Agranal bundle sheath chloroplasts ($\text{Chl}\text{a}\text{Chl}\text{b}\text{> 5.0}$) contained the full complement of photosystem (PS) I and of cytochrome (cyt) f but lacked a major portion of PS II and its associated Chl $\text{a}\text{b}$ light-harvesting complex (LHC), and most of the cyt b₅₅₉. The kinetic analysis of system I photoactivity revealed that the functional photosynthetic unit size of PS I was unchanged and identical in mesophyll and bundle sheath chloroplasts. The results suggest that PS I is contained in stroma-exposed thylakoids and that it does not receive excitation energy from the Chl $\text{a}\text{b}$ LHC present in the grana. A stoichiometric parity between PS I and cyt f in mesophyll and bundle sheath chloroplasts indicates that biosynthetic and functional properties of cyt f and P₇₀₀ are closely coordinated. Thus, it is likely that both cyt f and P₇₀₀ are located in the membrane of the intergrana thylakoids only. The kinetic analysis of PS II photoactivity revealed the absence of PS II_αfrom the bundle sheath chloroplasts and helped identify the small complement of system II in bundle sheath chloroplasts as PS II_β. The distribution of the main electron transport components in grana and stroma thylakoids is presented in a model of the higher plant chloroplast membrane system.     

Properties of thylakoids and thylakoid particles derived from structurally different chloroplasts

Structurally and functionally different tobacco chloroplasts were subjected to digitonin treatment and subsequent fractional centrifugation. The light-harvesting $\text{chlorophyll}\text{a}\text{chlorophyll}\text{b-}\text{protein}$ complex was found to be enriched in the most dense fraction regardless of the presence of grana in the original preparation. It is suggested that isolated thylakoid membranes and fragments thereof which contain sufficient light-harvesting protein may, under appropriate ionic conditions, form aggregates even when they originate from unstacked thylakoid systems. Comparative studies of fluorescence properties and polypeptide composition of the thylakoids suggest that the light-harvesting protein does not contribute significantly to the fluorescence spectrum of isolated chloroplasts as long as this protein is intimately associated with the Photosystem II (PS II) pigment-protein complex responsible for the 685 nm emission. While the PS II-deficient mutant chloroplasts of the variegated tobacco variety NC 95 lacked both the 685 nm fluorescence component and two or three PS II proteins, one of these proteins was found to be very prominent in our chlorophyll b-deficient mutant thylakoids which also displayed an intense 685 nm fluorescence peak. This correlation supports the contention that a 45 kdalton polypeptide is an apoprotein of pigments associated with the PS II reaction center.     

Relationship between chloroplast structure and O2 evolution rate of leaf discs in plants from different biotopes in South Finland

Abstract The chloroplast ultrastructure, especially the thylakoid organization, the polypeptide composition of the thylakoid membranes and photosynthetic O₂ evolution rate, chlorophyll (Chl) content and Chi a/b ratio were studied in leaves of nine plants growing in contrasting biotopes in the wild in South Finland. All the measurements were made at the beginning of the period of main growth on leaves approaching full expansion, when the CO₂-saturated O₂ evolution rate (measured at 20°C and 1500 μmol photons m^?2s^?1) was at a maximum, ranging from 19.2 to 6.9 μmol O₂ cm^?2 h^?1. Among the species, the Chi a/b ratio varied between 3.75 and 2.71. In the mesophyll chloroplasts, the ratio of the total length of appressed to non-appressed thylakoid membranes varied between 1.07 and 1.79, the number of partitions per granum varied between 2.8 and 12.0 and the grana area between 21 and 42% of the chloroplast area. There was a significant relationship between the rate of O₂ evolution of the leaf discs and the thylakoid organization in the mesophyll chloroplasts. The higher the O₂ evolution rate, the lower was the ratio of the total length of appressed to non-appressed thylakoid membranes and also the lower the grana area. Although the relationship of the photosynthetic rate with the Chi content and the Chi a/b ratio of the leaves was not as clear, a significant negative correlation existed between the Chi a/b ratio and the ratio of appressed to non-appressed thylakoid membranes, indicating lateral heterogeneity in the distribution of different Chl- protein complexes.     

Effect of light quality on chloroplast-membrane organization and function in pea

The effect of light quality during plant growth of chloroplast membrane organization and function in peas (Pisum sativum L. cv. Alaska) was investigated. In plants grown under photosystem (PS) I-enriched (far-red enriched) illumination both the PSII/PSI stoichiometry and the electrontransport capacity ratios were high, about 1.9. In plants grown under PSII-enriched (far-red depleted) illumination both the PSII/PSI stoichiometry and the electron-transport capacity ratios were significantly lower, about 1.3. In agreement, steady-state electron-transport measurements under synchronous illumination of PSII and PSI demonstrated an excess of PSII in plants grown under far-red-enriched light. Sodium dodecylsulfate polyacrylamide gel electrophoretic analysis of chlorophyll-containing complexes showed greater relative amounts of the PSII reaction center chlorophyll-protein complex in plants grown under farred-enriched light. Additional changes were observed in the ratio of light-harvesting chlorophyll a/b protein to PSII reaction center chlorophyll-protein under the two different light-quality regimes. The results demonstrate the dynamic nature of chloroplast structure and support the notion that light quality is an important factor in the regulation of chloroplast membrane organization and-function.Abbreviations and symbols Chl chlorophyll - CPa PSII reaction center chlorophyll protein complex - CPI PSI chlorophyll protein complex - FR-D light depleted in far-red sensitizing primarily PSII - FR-E light enriched in far-red sensitizing primarily PSI - LHCP PSII light-harvesting chlorophyll a/b protein complex - P ₇₀₀ primary electron donor of PSI - PSI, PSII photosystems I and II, respectively - Q primary electron acceptor of PSII     

Localization of the reaction side of plastocyanin from immunological and kinetic studies with inside-out thylakoid vesicles

(1) The effect of four active antisera against plastocyanin on Photosystem I-driven electron transport and phosphorylation was investigated in spinach chloroplasts. Partial inhibition of electron transport and stimulation of plastocyanin-dependent phosphorylation were sometimes observed after adding amounts of antibodies which were in large excess and not related to the plastocyanin content of the chloroplasts. This indicates effects of the antibodies on the membrane. (2) The antibodies against plastocyanin neither directly nor indirectly agglutinated unbroken chloroplast membranes. (3) The plastocyanin content of right-side-out and inside-out thylakoid vesicles isolated by aqueous polymer two-phase partition from chloroplasts disrupted by Yeda press treatment was determined by quantitative rocket electroimmunodiffusion. Right-side-out vesicles retained about 25%, inside-out vesicles none of the original amount of plastocyanin. (4) The effect of externally added plastocyanin on the reduction of P-700 was studied by monitoring the absorbance changes at 703 nm after a long flash. In inside-out vesicles P-700 was reduced by the added plastocyanin but not in right-side-out vesicles and class II chloroplasts. These results provide strong evidence for a function of plastocyanin at the internal side of the thylakoid membrane.     

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.     

Structural and functional organization of the photosystems in spinach chloroplasts. Antenna size,relative electron-transport capacity,and chlorophyll composition

The structural and functional organization of the spinach chloroplast photosystems (PS) I, II_α and II_β was investigated. Sensitive absorbance difference spectrophotometry in the ultraviolet (?A₃₂₀) and red (?A₇₀₀) regions of the spectrum provided information on the relative concentration of PS II and PS I reaction centers. The kinetic analysis of PS II and PS I photochemistry under continuous weak excitation provided information on the number (N) of chlorophyll (Chl) molecules transferring excitation energy to PS II_α, PS II_β and PS I. Spinach chloroplasts contained almost twice as many PS II reaction centers compared to PS I reaction centers. The number N_α of chlorophyll (Chl) molecules associated with PS II_α was 234, while N_β = 100 and N_{PS I} = 210. Thus, the functional photosynthetic unit size of PS II reaction centers was different from that of PS I reaction centers. The relative electron-transport capacity of PS II was significantly greater than that of PS I. Hence, under light-limiting green excitation when both Chl a and Chl b molecules are excited equally, the limiting factor in the overall electron-transfer reaction was the turnover of PS I. The Chl composition of PS I, PS II_α and PS II_β was analyzed on the basis of a core Chl a reaction center complex component and a Chl $\text{a}\text{b-}\text{LHC}$ component. There is a dissimilar Chl $\text{a}\text{b-}\text{LHC}$ composition in the three photosystems with 77% of total Chl b associated with PS II_α only. The results indicate that PS II_α, located in the membrane of the grana partition region, is poised to receive excitation from a wider spectral window than PS II_β and PS I.     

Some effects of 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone (San 9785) on the development of chloroplast thylakoid membranes in Hordeum vulgare L.

Chloroplast ultrastructural and photochemical features were examined in 6-d-old barley (Hordeum vulgare L. cv. Sundance) plants which had developed in the presence of 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone (San 9785). In spite of a substantial modification of the fatty-acid composition of thylakoid lipids there were no gross abnormalities in chloroplast morphology, and normal amounts of membrane and chlorophyll were present. Fluorescence kinetics at 77K demonstrated considerable energetic interaction of photosystem (PS)I and PSII chlorophylls within the altered lipid environment. An interference with electron transport was indicated from altered room-temperature fluorescence kinetics at 20°C. Subtle changes in the arrangements of chloroplast membranes were consistently evident and the overall effects of these changes was to increase the proportion of appressed to nonappressed membranes. This correlated with a lower chlorophyll a/b ratio, an increase in the amount of light-harvesting chlorophylls as determined by gel electrophoresis and fluorescence emission spectra, and an increase in excitation-energy transfer from PSII to PSI, as predicted from current ideas on the organisation of photosystems in appressed and non-appressed thylakoid membranes.Abbreviations CP1 P700-chlorophyll a protein - F_o, F_m, F_v minimal, maximal and variable fluorescence yield - LHCP light-harvesting chlorophyll-protein complex - PSI, PSII photosystem I, II - San 9785 4-chloro-5(dimethylamino)-2-phenyl-3(2H)-pyridazinone     

Preparation of O2-evolving Photosystem-II subchloroplasts from spinach

An O₂-evolving Photosystem II subchloroplast preparation was obtained from spinach chloroplasts, using low concentrations of digitonin and Triton X-100. The preparation showed an O₂ evolution activity equivalent to 20% of the uncoupled rate of fresh broken chloroplasts, but had no significant Photosystem-I-dependent O₂ uptake activity. The preparation showed a chlorophyll $\text{a}\text{b}$ ratio of 1.9 and a $\text{P-700}\text{chlorophyll}$ ratio of $\text{1}\text{2400}$. Absorption spectra at room temperature and fluorescence emission spectra of chlorophyll at 77 K suggested a significant decrease in Photosystem I antenna chlorophylls in the O₂-evolving Photosystem II preparation.     

Adenocarcinoma of the human exocrine pancreas: presence of secretin and caerulein receptors

We have measured the cytochrome compositions of subfractions derived from appressed and non-appressed thylakoids by centrifugation and aqueous two-phase partition. Cytochrome $\text{b}$-559 (HP) was not detectable in the fraction derived from non-appressed thylakoids. Cytochromes $\text{f}\text{,}\text{b}$-563 and $\text{b}$-559 (LP) were all evenly distributed throughout the thylakoid membrane. This distribution points to plastocyanin as a possible lateral shuttle of reducing equivalents between spatially separated photosystems.Cytochrome $\text{f}$ was accessible to externally added plastocyanin in the inside-out vesicles but not in vesicles of normal sidedness. This strongly supports a location at the inner side of the thylakoid membrane. Cytochrome $\text{b}$-563 was slowly reduced by dithionite in vesicles with both normal and inside-out orientation suggesting a location within the membrane interior.     

Enhanced thermal tolerance of photosynthesis and altered chloroplast ultrastructure in a mutant of Arabidopsis deficient in lipid desaturation

A mutant of Arabidopsis thaliana, deficient in activity of the chloroplast n-6 desaturase, accumulated high levels of C_16:1 and C_18:1 lipids and had correspondingly reduced levels of polyunsaturated lipids. The altered lipid composition of the mutant had pronounced effects on chloroplast ultrastructure, thylakoid membrane protein and chlorophyll content, electron transport rates, and the thermal stability of the photosynthetic membranes. The change in chloroplast ultrastructure was due to a 48% decrease in the amount of appressed membranes that was not compensated for by an increased amount of nonappressed membrane. This resulted in a net loss of 36% of the thylakoid membrane per chloroplast and a corresponding reduction in chlorophyll and protein content. Electrophoretic analysis of the chlorophyll-protein complexes further revealed a small decrease in the amount of light-harvesting complex. Relative levels of whole chain and protosystem II electron transport rates were also reduced in the mutant. In addition, the mutation resulted in enhanced thermal stability of photosynthetic electron transport. These observations suggest a central role of polyunsaturated lipids in determining chloroplast structure and maintaining normal photosynthetic function and demonstrate that lipid unsaturation directly affects the thermal stability of photosynthetic membranes.     

The effect of magnesium and phosphorylation of light-harvesting chlorophyll ab-protein on the yield of P-700-photooxidation in pea chloroplasts

The yield of P-700 photooxidation has been studied in isolated chloroplast membranes by measuring the extent of the flash-induced absorption increase at 820 nm (ΔA₈₂₀) in the microsecond time range. The extent of ΔA₈₂₀ induced by non-saturating laser flashes was increased by the following treatments. (1) Suspension of chloroplast membranes in Mg²⁺ free medium (plus 15 mM K⁺) which leads to unstacking of grana (as detected by a decrease in chlorophyll fluorescence). (2) Reduction of Q, the primary acceptor of Photosystem II, in the presence of 20 μM 3-(3,4 dichlorophenyl)-1,1-dimethylurea by a saturating xenon flash, fired 300 ms before the laser flash. (3) Phosphorylation of light harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complex, which occurs in the presence of ATP after activation of protein kinase in the dark with NADPH and ferredoxin. We conclude that the Mg²⁺ concentration, the redox state of Q and the protein-phosphorylation all can control the photochemical efficiency of P-700 photooxidation in isolated chloroplasts, and we discuss these results in relation to control of excitation energy distribution between the two photosystems. We also discuss the significance of these results in relation to the regulation of photosynthetic electron transport in vivo.     

Photosynthetic responses of Oryza sativa L. seedlings to cadmium stress: physiological,biochemical and ultrastructural analyses

In the present study, photosynthetic responses induced by cadmium stress in chlorophyll biosynthesis, photochemical activities, the stability of thylakoid membranes chlorophyll-protein complexes and the chloroplast ultrastructure of the cereal crop Oryza sativa L. were characterized. Cadmium inhibited the biosynthesis of chlorophyll by interfering with activity of δ-aminolevulinic acid dehydratase in the rice seedlings. For the photochemical activities analyses, the extent of the decrease in photosystem II activity was much greater than that in the PS I activity. The variations in the chlorophyll a fluorescence parameters also indicated that cadmium toxicity drastically affected the photochemistry of PS II. Biochemical analyses by BN-PAGE and protein immunoblot showed that cadmium toxicity considerably affected the stability of PS II-core, cytb ₆ /f, RuBisCO, PSI + LHCI and LHCII (Trimeric). We observed the rate of the thylakoid membranes protein degradation, was mainly at the level of RbcL, PsaA, Lhca1 and D1. In addition, the damages to chloroplast structure and thylakoid stacking analyzed by transmission electron microscopy were indicative of general disarray in the photosynthetic functions exerted by cadmium toxicity. These results are valuable for understanding the biological consequences of heavy metals contamination particularly in soils devoted to organic agriculture.     

Light-Harvesting Chlorophyll a/b Protein : Membrane Insertion, Proteolytic Processing, Assembly into LHC II, and Localization to Appressed Membranes Occurs in Chloroplast Lysates

The apoprotein of the light-harvesting chlorophyll a/b protein (LHCP) is a major integral thylakoid membrane protein that is normally complexed with chlorophyll and xanthophylls and serves as the antenna complex of photosystem II. LHCP is encoded in the nucleus and synthesized in the cytosol as a higher molecular weight precursor that is subsequently imported into chloroplasts and assembled into thylakoids. In a previous study it was established that the LHCP precursor can integrate into isolated thylakoid membranes. The present study demonstrates that under conditions designed to preserve thylakoid structure, the inserted LHCP precursor is processed to mature size, assembled into the LHC II chlorophyll-protein complex, and localized to the appressed thylakoid membranes. Under these conditions, light can partially replace exogenous ATP in the membrane integration process.     

Electron transfer between the two photosystems. I. Flash excitation under oxidizing conditions

The redox changes of cytochrome b-563 (cytochrome b), cytochrome f, plastocyanin and P-700 were measured on dark-adapted chloroplasts after illumination by a series of flashes in oxidizing conditions (0.1 mM ferricyanide). In these conditions, the plastoquinone pool is fully oxidized and the only available plastoquinol are those formed by Photosystem (PS) II reaction. According to the two-electron gate mechanism proposed by Bouges-Bocquet (Bouges-Bocquet, B. (1973) Biochim. Biophys. Acta 314, 250–256), plastoquinol is mainly formed after the second and the fourth flashes. After the second flash, the reoxidation of plastoquinol occurs by a concerted reaction which reduces most of the cytochrome b present and a fraction of PS I donors. Most of these electrons are stored on P-700, which implies a large equilibrium constant between the secondary PS I donors and P-700. One electron is stored on cytochrome b during a time ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 1}\text{s}$) much longer than the dark interval between flashes. After the fourth flash, a new plastoquinol molecule is formed, which induces the reduction of PS I donors with no corresponding further reduction of cytochrome b. The number of electrons transferred after the fourth flash is larger than that transferred after the second flash although the rate of transfer is lower. To interpret these data, we assume that the plastoquinol formed after the fourth flash is reoxidized by a second concerted reaction: one electron is directly transferred to PS I donors while the other cooperates with the electron stored on cytochrome b to reduce a plastoquinone molecule localized on a site close to the outer face of the membrane. This newly formed plastoquinol crosses the membrane and transfers a second electron to PS I donors. This interpretation resembles a model proposed by Velthuys (Velthuys, B.R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2765?2769) and which belongs to the modified Q-cycle class of models.