Chlorophyll-protein complexes from higher plants: A procedure for improved stability and fractionation

An improved procedure for the electrophoretic fractionation of higher plant chlorophyllprotein complexes is described. Compared with currently used systems, it greatly reduces the amount of chlorophyll that is found unassociated with protein after electrophoresis and resolves four chlorophyll-protein complexes. The slowest migrating band has a red adsorption maximum at 674 nm or greater, contains chlorophyll a but not chlorophyll b, and has a molecular weight equivalency of 110,000. These properties are similar to the previously described CPI or P700-chlorophyll a-protein complex. The amount of the total chlorophyll in this material is increased by two to three fold over that present in the equivalent complex fractionated by previous procedures. The other three chlorophyll-protein complexes contain both chlorophylls a and b, and have molecular weight equivalencies of 80,000, 60,000, and 46,000. None of these complexes seems to correspond directly to the previously characterized light-harvesting chlorophyll $\text{a}\text{b}$-protein complex.     

Blue light photoreactivation of nitrate reductase from green algae and higher plants

The inactive form of NADH-nitrate reductase from spinach and $\text{Chlorella fusca}$ is fully reactivated in short periods of time when the enzyme-complex is illuminated with white or blue light but not with red light. Flavin nucleotides greatly accelerate the photoreactivation process. The results suggest that blue light might act as a modulating agent in the assimilation of nitrate in green algae and higher plants.     

Occurrence and distribution of filipin-sterol complexes in chloroplast envelope membranes of algae and higher plants as visualized by freeze-fracture

Summary The occurrence and planar distribution of 3--hydroxysterols in chloroplast envelope membranes of different algae and higher plants has been studied with the freeze-fracture technique using the polyene antibiotic filipin as cytochemical marker. The inner chloroplast envelope membrane in all organisms studied is devoid of filipin-sterol complexes. The outer chloroplast envelope membranes of isolated higher plant chloroplasts (spinach, pea) and of chloroplasts of the mossPolytrichum piliferum are lacking filipin-sterol complexes, thus indicating a very low concentration of 3--hydroxysterols in chloroplast envelope membranes of higher plants. In contrast filipin-sterol complexes are abundant in the outer chloroplast envelope membrane of the flagellatesChlamydomonas reinhardii, Cryptomonas erosa, andEuglena gracilis. The chloroplast-ER surrounding the plastid ofCryptomonas erosa also exhibits filipin-sterol complexes. Functional and phylogenetic aspects of these observations are discussed.Medizinische Cytobiologie, Westfälische Wilhelms-Universität, Westring 3, D-4400 Münster, Federal Republic of Germany.     

Codon usage in higher plants, green algae, and cyanobacteria

Codon usage is the selective and nonrandom use of synonymous codons by an organism to encode the amino acids in the genes for its proteins. During the last few years, a large number of plant genes have been cloned and sequenced, which now permits a meaningful comparison of codon usage in higher plants, algae, and cyanobacteria. For the nuclear and organellar genes of these organisms, a small set of preferred codons are used for encoding proteins. Codon usage is different for each genome type with the variation mainly occurring in choices between codons ending in cytidine (C) or guanosine (G) versus those ending in adenosine (A) or uridine (U). For organellar genomes, chloroplastic and mitochrondrial proteins are encoded mainly with codons ending in A or U. In most cyanobacteria and the nuclei of green algae, proteins are encoded preferentially with codons ending in C or G. Although only a few nuclear genes of higher plants have been sequenced, a clear distinction between Magnoliopsida (dicot) and Liliopsida (monocot) codon usage is evident. Dicot genes use a set of 44 preferred codons with a slight preference for codons ending in A or U. Monocot codon usage is more restricted with an average of 38 codons preferred, which are predominantly those ending in C or G. But two classes of genes can be recognized in monocots. One set of monocot genes uses codons similar to those in dicots, while the other genes are highly biased toward codons ending in C or G with a pattern similar to nuclear genes of green algae. Codon usage is discussed in relation to evolution of plants and prospects for intergenic transfer of particular genes.     

The chloroplast protein translocation complexes of Chlamydomonas reinhardtii: a bioinformatic comparison of Toc and Tic components in plants, green algae and red algae

The recently completed genome of Chlamydomonas reinhardtii was surveyed for components of the chloroplast protein translocation complexes. Putative components were identified using reciprocal BlastP searches with the protein sequences of Arabidopsis thaliana as queries. As a comparison, we also surveyed the new genomes of the bryophyte Physcomitrella patens, two prasinophyte green algae (Ostreococcus lucimarinus and Ostreococcus tauri), the red alga Cyanidioschizon merolae, and several cyanobacteria. Overall, we found that the components of the import pathway are remarkably well conserved, particularly among the Viridiplantae lineages. Specifically, C. reinhardtii contained almost all the components found in A. thaliana, with two exceptions. Missing from C. reinhardtii are the C-terminal ferredoxin-NADPH-reductase (FNR) binding domain of Tic62 and a full-length, TPR-bearing Toc64. Further, the N-terminal domain of C. reinhardtii Toc34 is highly acidic, whereas the analogous region in C. reinhardtii Toc159 is not. This reversal of the vascular plant model may explain the similarity of C. reinhardtii chloroplast transit peptides to mitochondrial-targeting peptides. Other findings from our genome survey include the absence of Tic22 in both Ostreococcus genomes; the presence of only one Toc75 homolog in C. merolae; and, finally, a distinctive propensity for gene duplication in P. patens.     

Resolution of mitochondrial and chloroplast membrane protein complexes from green leaves of potato on blue-native polyacrylamide gels

Purification of mitochondria and mitochondrial protein complexes from green tissues is often severely impaired by the presence of chloroplasts and their proteins. Here we present a method which allows analysis of respiratory protein complexes from potato leaves. The procedure includes the preparation of an organellar fraction specifically enriched in mitochondria and the separation of organellar protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). For the first time mitochondrial and chloroplast protein complexes have been resolved simultaneously in a native gel. BN-PAGE allowed the separation of eleven bands, including the mitochondrial NADH-dehydrogenase, the bc1 complex and the mitochondrial F1-ATP synthase as well as the chloroplast F1-ATP synthase, the cytochrome b6f complex, the two photosystems and the light harvesting complex. The resolution of the protein complexes in the first dimension was good enough to allow identification of all subunits of individual complexes in the second dimension under denaturing conditions. Thus, BN-PAGE offers an opportunity to analyze mitochondrial and chloroplast protein complexes from a single preparation from very small amounts of tissue. The implications of our findings, for studies on protein expression and turnover in different tissues and developmental stages, are discussed.     

Cross-reconstitution of various extrinsic proteins and photosystem II complexes from cyanobacteria, red algae and higher plants

Photosystem II (PSII) contains different extrinsic proteins required for oxygen evolution among different organisms. Cyanobacterial PSII contains the 33 kDa, 12 kDa proteins and cytochrome (cyt) c-550; red algal PSII contains a 20 kDa protein in addition to the three homologous cyanobacterial proteins; whereas higher plant PSII contains the 33 kDa, 23 kDa and 17 kDa proteins. In order to understand the binding and functional properties of these proteins, we performed cross-reconstitution experiments with combinations of PSII and extrinsic proteins from three different sources: higher plant (spinach), red alga (Cyanidium caldarium) and cyanobacterium (Synechococcus vulcanus). Among all of the extrinsic proteins, the 33 kDa protein is common to all of the organisms and is totally exchangeable in binding to PSII from any of the three organisms. Oxygen evolution of higher plant and red algal PSII was restored to a more or less similar level by binding of any one of the three 33 kDa proteins, whereas oxygen evolution of cyanobacterial PSII was restored to a larger extent with its own 33 kDa protein than with the 33 kDa protein from other sources. In addition to the 33 kDa protein, the red algal 20 kDa, 12 kDa proteins and cyt c-550 were able to bind to cyanobacterial and higher plant PSII, leading to a partial restoration of oxygen evolution in both organisms. The cyanobacterial 12 kDa protein and cyt c-550 partially bound to the red algal PSII, but this binding did not restore oxygen evolution. The higher plant 23 kDa and 17 kDa proteins bound to the cyanobacterial and red algal PSII only through non-specific interactions. Thus, only the red algal extrinsic proteins are partially functional in both the cyanobacterial and higher plant PSII, which implies a possible intermediate position of the red algal PSII during its evolution from cyanobacteria to higher plants.     

The chloroplast pigments of some green and yellow-green algae

Pigment analysis carried out by thin-layer chromatography confirms that Chlorocloster engadinensis Vischer, C. solani George and Nephrodiella brevis Vischer are all green algae (Chlorophyceae) and not yellow-green algae (Xanthophyceae) as has been suggested. The pigments of Coccomyxa elongata Jaag, C. simplex (Pringsheim) Mainx and Pyrobotrys stellata Korshikov are also typical of green algae. The pigments of Pleurochloris commutata Pascher, P. magna Boye Petersen, Polyedriella helvetica Vischer et Pascher, Mischococcus sphaerocephalus Vischer and Tribonema aequale Pascher, are different to those of the green algae in accordance with their being classified as yellow-green algae. However, a detailed comparison of the pigments of Pleurochloris and Tribonema suggests that whilst both contain chlorophyll, α, β-carotene and esterified vaucheriaxanthin, the major xanthophyll in Pleurochloris corresponds to violaxanthin and not to antheraxanthin as in Tribonema.     

The molecular size and conformation of the chloroplast DNA from higher plants.

Covalently closed circular choloroplast DNA (ctDNA) molecules have been isolated from pea, bean, spinach, lettuce, corn and oat plants by ethidium bromide/cesium choloride density-gradient entrifugation. As much as 30-40% of the total ctDNA could be isolated as closed circular DNA molecules and up to 80% of the total ctDNA was found in the form of circular molecules. The size of pea, spinach, lettuce, corn and oat ctDNA relative to an internal standard (phiX174 replicative form II monomer DNA) was determined by electron microscopy. The ctDNAs showed significant differences in their sizes, and their molecular weights ranged from 85.4 - 10(6) for corn ctDNA to 96.7 - 10(6) for lettuce ctDNA. Each of these ctDNAs contained 3-4% of the circular molecules as circular dimers and 1-2% of the circular molecules as catenated dimes. The molecular complexity of these ctDNAs was studied by renaturation kinetics using T4 DNA as a standard. The molecular weights of the unique sequences of the ctDNAs ranged from 83.7 - 10(6) for oat ctDNA to 93.1 - 10(6) for lettuce ctDNA, which are in excellent agreement with the sizes of the circular ctDNA molecules...     

Some effects of 2537 A on green algae and chloroplast preparations

1. The kinetics of the inactivation of photosynthesis by 2537 A in Chlorella pyrenoidosa and Scenedesmus D(1) indicate that, while the destruction process is largely a first order effect, higher order effects also occur, which become evident at low exposures. In agreement with previous observations, endogenous respiration is insensitive to exposures which inactivate photosynthesis. 2. In Scenedesmus D(1) a solid dose of ultraviolet has no more effect on the photosynthetic apparatus than a dose of equal total duration interrupted by periods of photosynthesis. Nor is any difference noted if the cells are in a different buffer, e.g. 0.05 M KH(2)PO(4), or carbonate-bicarbonate buffer 9. 3. In C. pyrenoidosa, a solid dose and an interrupted dose cause equal effects on photosynthesis when neutral phosphate buffer is used. If the ultraviolet exposure schedules are identical, equal effects are also noted in cells suspended in buffer 9, and in 0.05 M phosphate (pH 6.2). Solid exposures are, however, much more effective than interrupted exposures, when buffer 9 is used. 4. Oxygen evolution (Hill reaction), photosynthesis, and photoreduction in Scenedesmus D(1) are equally sensitive to a given dose of ultraviolet. The mechanism responsible for adaptation to hydrogen metabolism is not more sensitive to ultraviolet than is the photosynthetic mechanism. The O(2)/H(2)/CO(2) reaction in darkness is less sensitive to ultraviolet than any of the above reactions. 5. Glucose oxidation by C. pyrenoidosa, and colony formation in Scenedesmus D(1) are far more sensitive to a given dose of ultraviolet than photosynthesis in these organisms. 6. The photosynthetic apparatus of C. pyrenoidosa is more sensitive to ultraviolet than that of Scenedesmus D(1). 7. The Hill reaction in chloroplast fragments is also inactivated by 2537 A by a first order process. Exposures which inactivate this reaction completely have no effect on polyphenol oxidase, cytochrome oxidase, or catalase in the same chloroplast preparation. 8. After irradiation, the survival of photosynthesis in Scenedesmus D(1) and of the Hill reaction in chloroplast fragments are independent of the light intensity used to measure these processes. 9. No significant changes occur in the ultraviolet absorption of chloroplasts after an exposure to 2537 A, which completely inactivates the Hill reaction.     

Respiration from C3 plant green manure added to a C4 plant carbon dominated soil

Application of tree leaves (C₃ plants) on maize (Zea mays L.) (C₄ plant) fields is an agroforestry management technology to restore or maintain soil fertility. The rate at which the tree leaves decompose is crucial for the nutrient supply to the crop. We studied the in situ decomposition of Sesbania sesban (L.) Merr. leaves or C₃ sugar for 4 – 8 days after application to a maize field in Kenya. By using the difference of around 10‰ in natural abundance of ¹³C between the endogenous soil C (mainly C₄) and the applied C (C₃), we could calculate the contributions of the two C sources to soil respiration. The δ¹³C value of the basal respiration was from –15.9 to –16.7‰. The microbial response to the additions of leaves and sugar to this tropical soil was immediate. Application of sesbania leaves gave an initial peak in respiration rates that lasted from one to less than 6 days, after which it levelled off and remained about 2 – 3 times higher (230–270 mg C m^-2 h^-1) than the control respiration rates throughout the rest of the experiment (5 – 8 days). In the sugar treatment, there was no initial peak in respiration rate. The respiration rate was 170 mg C m^-2 h^-1 after 4 days. At the end of the experiments, after 4–8 days, as much as 14–17% of the added C had been respired and about 60% of the total respiration was from the added sesbania leaves or C₃ sugar. This non-destructive method allows repeated measurements of the actual rate of C mineralisation and facilitates decomposition studies with high temporal resolution in the field. This revised version was published online in August 2006 with corrections to the Cover Date.     

Light-induced pH changes in leaves of C4 plants

Light-induced changes in the fluorescence of the pH-indicating dyes pyranine or 5-(and 6-)carboxy-2, 7-dichlorofluorescein (CDCF) which had been fed to leaves were examined to monitor cellular pH changes. After short-term feeding of pyranine (pK 7.3) to leaves of Amaranthus caudatus L., a NAD-malic-enzyme-type C₄ plant, vascular bundles and surrounding cells became fluorescent. Fluorescence emission from mesophyll cells required longer feeding times. In CO₂-free air, pyranine fluorescence increased much more on illumination after mesophyll cells had become fluorescent than when only the vascular bundles and the bundle sheath of Amaranthus leaves had been stained. After short feeding times and in the absence of actinic illumination, CO₂ decreased pyranine fluorescence very slowly in Amaranthus and rapidly in C₃ leaves. After prolonged feeding times, the extent of the light-dependent increase in pyranine fluorescence was several times greater in different C₄ plants than in C₃ species. The kinetics of the fluorescence changes were also remarkably different in C₃ and C₄ plants. Carbon dioxide (500 l · l^–1) suppressed the light-induced increase in pyranine fluorescence more in C₄ than in C₃ leaves. Light-dependent changes in light scattering, which are indicative of chloroplast energization, and in 410-nm transmission, which indicate chloroplast movement, differed kinetically from those of the changes in pyranine fluorescence. Available evidence indicated that light-dependent changes in pyranine fluorescence did not originate from the apoplast of leaf cells. Microscopic observation led to the conclusion that, after prolonged feeding times or prolonged incubation, changes in pyranine fluorescence emitted from C₄ leaves reflect pH changes mainly in the cytosol of mesophyll cells. A transient acidification reaction indicated by quenching of pyranine fluorescence in the dark-light transient and not observed in C₃ species is attributed to the carboxylation of phosphoenolpyruvate. After short feeding times and in the absence of actinic illumination, CO₂ (250 l l^–1) decreased pyranine fluorescence very slowly in Amaranthus and more rapidly in C₃ leaves. After prolonged feeding times, both the rate and the extent of CO₂-dependent quenching of pyranine fluorescence increased, but the increase was insufficient to indicate the presence of highly active carbonic anhydrase in the compartment from which pyranine fluorescence was emitted. In contrast to pyranine, CDCF (pK 4.8) did not increase but rather decreased its fluorescence on illumination of an Amaranthus leaf, indicating acidification of an acidic compartment, most probably the vacuole of green leaf cells. The pattern of the acidification reaction was similar in C₄ and C₃ leaves. The remarkably large extent of the light-dependent increase in pyranine fluorescence from leaves of C₄ species and its slow kinetics are proposed to be caused by an alkalization of the cytosol which in the absence of CO₂ is larger in the mesophyll than in the bundle sheath. It gives rise to deprotonation of dye originally located in the mesophyll and, in addition, of dye which diffuses from the bundle sheath into the mesophyll following a pH gradient. Implications of slow diffusional transport of pyranine and CO₂ between mesophyll and bundle-sheath cells and the fast metabolite transport required in C₄ photosynthesis are discussed.Abbreviations CDCF 5-(and 6-)carboxy-2,7-dichlorofluorescein - DHAP dihydroxyacetone phosphate - PGA 3-phosphoglycerate This work was supported by the Sonderforschungsbereiche 176 and 251 of the University of Würzburg and by the Gottfried-Wilhelm-Leibniz Program of the Deutsche Forschungsgemeinschaft. A.S.R. was the recipient of a fellowship of the Alexander-von-Humboldt Foundation. We are grateful to Mrs. S. Neimanis for cooperation.     

Localization of superoxide dismutase in leaves of C3 and C4 plants

Chloroplasts, mitochondria and cytoplasm, isolated from pea,wheat, maize and sorghum mesophyll protoplasts, contain distinctforms of superoxide dismutase (SOD). In all species evaluated,chloroplasts exhibited a single cyanide-sensitive SOD. Thischloroplastic enzyme was the most anionic SOD observed in wholeleaf and protoplast extracts and constitutes 50–80% ofthe total soluble SOD. Pea and wheat protoplasts had only onecytoplasmic SOD, a cyanide-sensitive form of intermediate mobility;maize and sorghum had two such cytoplasmic enzymes. A singlecyanide-insensitive SOD was present in extracts from both C₃and C₄ tissues and was associated with mitochondria. Although bundle sheath cells of sorghum and maize are knownto be deficient in Photosystem II, there was no apparent differencein SOD between mesophyll and bundle sheath cells. Mesophyllprotoplasts and bundle sheath strands from these C₄ plants containedthe same forms of SOD. Levels of soluble SOD were similar, ona chlorophyll basis, in the two cell types as was distributionof activity among the various forms of the enzyme. (Received May 19, 1980; )