Major Chlorophyll-protein Complexes Research Articles

Characterization of linear and quadratic electrochromic probes in Chlorella sorokiniana and Chlamydomonas reinhardtii

Field-indicating absorption changes have been measured in mutant strains of Chlorella sorokiniana and Chlamydomonas reinhardtii lacking one or several chlorophyll-protein complexes. Using mutants which lack PS II centers and most of the chlorophyll antenna, we could characterize two types of probe, with linear or quadratic response to the membrane potential. The probes with linear response present an electrochromic spectrum with maxima at 514 and 486 nm and a minimum at 472 nm; those which respond quadratically present a spectrum with maxima at 464 and 504 nm and a minimum at 479 nm. By measuring the relative contribution of these probes upon a weak actinic flash, the offset of the membrane potential may be estimated under various experimental conditions. In anaerobiosis in the dark, a large permanent membrane potential arises from the hydrolysis of ATP, mainly of mitochondria! origin. We have also analyzed the electrochromic absorption changes in other mutant strains lacking either PS II only,or PS I and the major fraction of light-harvesting complexes. The quadratic probes are present to a similar extent in every strain investigated, which suggests that they are not associated with any of the major chlorophyll-protein complexes. These probes are also conserved in higher plants. In contrast, the linear electrochromic changes are roughly proportional to the overall amount of chlorophyll, either associated with the photocenter or with the antenna.

Absolute absorption and relative fluorescence excitation spectra of the five major chlorophyll-protein complexes from spinach thylakoid membranes

The absolute absorption and relative fluorescence excitation spectra have been obtained for the five major chlorophyll-protein complexes from spinach chloroplasts, resolved by mild SDS-polyacrylamide gel electrophoresis. Electro-elution of the chlorophyll-protein complexes from the gels enabled their absolute absorption spectra to be determined. The mean molar extinction coefficient from 400 to 720 nm was independent of the type of protein complex and the chlorophyll a b ratio, averaging 2230 ± 40 m 2 per mol chlorophyll. The mean molar extinction coefficient was used to normalize the absorption spectra of the protein complexes while in the gel slices. The distribution of chlorophyll between the pigment complexes can be determined at 662 nm rather than from the mean of two scans at 650 nm and 675 nm. Recombining the absorption spectra from the pigment-protein complexes gave reasonable absolute agreement with the spectrum of spinach thylakoids. However, the solubilization led to (i) a shift in the red peak to shorter wavelengths by 4.5 nm, and (ii) a loss of carotenoids which also shifted the blue shoulder to shorter wavelengths. The absorption spectra for the two photosystems were predicted with phosphorylated (State 2) and nonphosphorylated (State 1) light-harvesting complexes. The 77 K fluorescence excitation spectra of the chlorophyll-protein complexes were measured in an attempt to assess the excitation spectra of PS I and PS II in terms of their chlorophyll-protein complexes. Due to the relative nature of fluorescence detection, the ratio of fluorescence excitation at 470 relative to that at 400 nm was used to compare the spectra. The discrepancies between the absorption and fluorescence spectra for the Photosystem I complexes meant that fluorescence emission was an insensitive method for assessing the degree of association of the light-harvesting chlorophyll a b- complex with each photosystem.

Proton-induced grana formation in chloroplasts. Distribution of chlorophyll-protein complexes and Photosystem II photochemistry

Lowering the pH of the incubation medium to pH 5.4 leads to grana formation morphologically similar to that induced by metal cations. The same phenomenon is observed in EDTA-washed chloroplasts, indicating that it is not due in part to electrostatic ‘masking’ by residual cations associated with the membranes. Digitonin fractionation studies have indicated that the distribution of the major chlorophyll-protein complexes between granal and stromal membrane regions is similar at pH 5.4 in the absence of Mg 2+, and at pH 7.4 in the presence of Mg 2+. Chlorophyll fluorescence induction studies have indicated that the primary photochemistry of Photosystem II (PS II) is stimulated by lowering the pH to 5.4, just as it is upon metal cation addition at higher pH values. The failure to observe such an increase at pH 5.4 by measuring electron transport to ferricyanide is attributed to a combination of an inhibition by this pH of electron transport at a site after Q reduction and an increase in the number of PS II centres detached from the plastoquinone pool. We conclude that the stacked configuration of chloroplast membranes leads to increased PS II primary photochemistry, which is most simply explained in terms of a redistribution of excitation energy towards PS II.

Synthesis of polypeptides of the chlorophyll—protein complexes in isolated chloroplasts of Euglena gracilis

A novel purification procedure has been described for obtaining intact Euglena grucilis chloroplasts in gradients of Percoll with a high and strictly lightdependent capacity for protein synthesis [ 11. We have now further tested such chloroplasts with respect to the synthesis of thylakoid membrane proteins in connection with a general project on the genetic information potential of chloroplast DNA [2]. Three major chlorophyll-protein complexes have been described in higher plants [3-81, Chiizmydumonas [9] and Cuulerpu cuctoides [lo] but none so far for Euglenu grucilis. Here we characterize three chlorophyll-protein complexes, namely CPr, CPa and LHCP (in the nomenclature of [3]), which we obtain upon dissociation of washed thylakoid membranes of EugZenu. Furthermore, we show that one major and three minor proteins associated with CPr and two major and three minor proteins associated with CPa are synthesized by isolated chloroplasts. Two major proteins of LHCP seem not to be synthesized within the chloroplast. The results are compared with data obtained by others with Chlumydomonus and with higher plants.

A chloroplast membrane lacking Photosystem II. Thylakoid stacking in the absence of the Photosystem II particle

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.

Thylakoid membrane fragments with different chlorophyll A, chlorophyll C and fucoxanthin compositions isolated from the brown seaweed Ecklonia radiata

Thylakoids of the brown seaweed Ecklonia radiata have been fragmented by incubation with Triton X-100. Membrane fragments with different pigment compositions have been separated by sucrose density gradient centrifugation and also by hydroxylapatite chromatography. A heavier, green fraction with a chlorophyll a/P700 ratio of 80 had similar spectral characteristics to the P700-chlorophyll a-protein complex of green plants. Three lighter fractions, rich in chlorophyll c, were also isolated. One of these, an orange-brown fraction was also rich in fucoxanthin; fluorescence spectroscopy shows that both the chlorophyll c and fucoxanthin effectively transfer their energy to chlorophyll a. This fraction is likely to have an analogous function to the major chlorophyll-protein complex of higher plants, the light-harvesting chlorophyll a/ b-protein complex.

Polypeptide Composition of Chlorophyll-Protein Complexes from Romaine Lettuce

The protein moiety of the two major chlorophyll-protein complexes associated with chloroplast membranes of outer, dark green leaves of a romaine lettuce shoot (Lactuca sativa L. var. Romana) has been analyzed by discontinuous sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complex II, also termed light-harvesting chlorophyll-protein complex, is shown to consist of a major polypeptide of 25 kilodaltons (kD) and two minor ones of 27.5 and 23 kD. The 25 kD subunit is the single largest polypeptide component of the chloroplast membranes, accounting for about 25% of their total protein. Complex I contains only high molecular weight subunits, the major one being at 67 kD, these subunits representing only a small percentage of the chloroplast membrane total protein.These data, suggesting an oligomeric nature for the apoprotein of these two chlorophyll-protein complexes, are difficult to reconcile with the estimated molecular weights of the native complexes and raise some intriguing questions as to the types of interactions among the components of these major lipoproteins of the photosynthetic membranes.

Comparisons of Photosynthetic Activity and Lamellar Characteristics of Virescent and Normal Green Peanut Leaves

Summary Several characteristics of the chloroplast lamellar system and the photosynthetic activity of virescent and wild type peanut leaves have been compared. The mutant leaves exhibited a 42 % reduction in chlorophyll, a higher chlorophyll a/b ratio, and an alteration in the proportions of the two major chlorophyll-protein complexes of the chloroplast. The mutant leaves showed a reduction in the number of photosystem I reaction center components (the P 700-chlorophyll a-protein) per unit leaf area, and consequently a stoichiometric reduction in the number of photosystem I electron transport assemblages (P 700, cytochromes f and b 6 ). In addition the mutant had a photosynthetic unit size which was 1.5 times larger than the wild type. These findings indicated that the chlorophyll deficient leaves had many fewer, but larger, photosynthetic units than the normal green leaves. As a consequence of these alterations it is suggested that the observed low photosynthetic rates on a leaf area basis (10 mg CO 2 /dm 2 -hr) as well as on a chlorophyll basis (8 mg CO 2 /mg Chl-hr) were likely due to a much reduced capacity of the mutant chloroplasts to produce sufficient quantities of ATP and NADPH to support high levels of carbon fixation. Furthermore, the alterations in chlorophyll-protein content and organization into photosynthetic units can explain the reduction in chlorophyll and higher chlorophyll a/b ratio of the virescent leaves.

Composition and activity of the photosynthetic apparatus in temperature-sensitive mutants of higher plants.

Six nuclear mutants of corn, six of soybean, and seven of cotton displayed low temperature-induced virescence when grown in controlled environments. For the group of plants studied, an increase in leaf chlorophyll a/b ratio was correlated with a temperature-sensitive biosynthetic sequence leading to a reduction in total chlorophyll content. These pigment alterations were reflected in the composition and quantity of the two major chlorophyll-protein complexes of chloroplast membranes. Changes in the amount of the major light-harvesting chlorophyll-protein complex was a prime consequence of the nuclear mutations. A decrease in the light-harvesting chlorophyll component of the light reaction centers of the leaf may account for the decrease in size of the photo-synthetic unit frequently noted in chlorophyll-deficient mutants. Variations in the concentration of the chlorophyll-protein complexes in the chloroplast lamellae may be causally related to variations in CO(2) compensation points of mutant soybean and cotton plants.