Chloroplast membranes of the green alga Acetabularia mediterranea. II. Topography of the chloroplast membrane

The localization of the chlorophyll-protein complexes inside the thylakoid membrane of Acetabularia mediterranea was determined by fractionating the chloroplast membrane with EDTA and Triton X-100, by using pronase treatment, and by labeling the surface-exposed proteins with 125I. The effects of the various treatments were established by electrophoresis of the solubilized membrane fractions and electron microscopy. After EDTA and pronase treatment, the membrane structure was still intact. Only the two chlorophyll-protein complexes of 67,000 and 152,000 daltons and an additional polypeptides were found in the membrane before the EDTA and pronase treatment. The 125,000 dalton complex seems to be buried inside the lipid layer. The 23,000 dalton subunit of the 67,000 dalton complex is largely exposed to the surface of the EDTA-insoluble membrane and only the chlorophyll-binding subunit of 21,500 daltons is buried inside the lipid layer.     

Chloroplast membranes of the green alga Acetabularia mediterranea. I. Isolation of the photosystem II.

1. In the presence of Triton X-100, chloroplast membranes of the green alga Acetabularia mediterranea were disrupted into two subchloroplast fragments which differed in buoyant density. Each of these fractions had distinct and unique complements of polypeptides, indicating an almost complete separation of the two fragments. 2. One of the two subchloroplast fractions was enriched in chlorophyll b. It exhibited Photosystem II activity, was highly fluorescent and was composed of particles of approx. 50 A diameter. 3. The light-harvesting chlorophyll-protein complex of the Photosystem II-active fraction had a molecular weight of 67 000 and contained two different subunits of 23 000 and 21 500. The molecular ratio of these two subunits was 2:1.     

Rna transport from the nucleus to the cytoplasm in Acetabularia mediterranea]

The biosynthesis of nuclear RNA, its transport from the nucleus to the cytoplasm and distribution in the cytoplasm were studied in Acetabularia mediterranea under different light conditions. It was shown that the nuclear RNA incorportate 3H-uracil more rapidly in the darkness and the transport of labeled RNA from the nucleus slowed down after the transfer of plants in the cold medium in the darkness. To study the distribution of nuclear RNA in the cytoplasm, the 3H-uracil labeled nuclei were transplanted in the rhizoids of unlabeled plants, the dikaryons obtained were kept for different time in the light and in the darkness and the content of 3H-RNA was determined in different stem regions. It was shown that the transport of 3H-RNA in the cytoplasm is slowed down in the darkness and it is distributed by the basal-apical gradient. RNA is rapidly accumulated in the apical stem zone in the light and redistributed afterwards in the basal stem zones. The problem of relationship between the polarity and nuclear RNA distribution in Acetabularia is discussed.     

The Ionic Relations of Acetabularia mediterranea

The concentrations of K⁺, Na⁺, and Cl^– in the cytoplasmand the vacuole of Acetabularia mediterranea have been measured,as have the vacuolar concentrations of SO₄^–– andoxalate. The electrical potential difference between externalsolution, and vacuole and cytoplasm has been measured. The resultsindicate that Cl^– and SO₄^–– are probably transportedactively into the cell, and that active transport of Na⁺ isoutwards. The results for K⁺ are equivocal. The fluxes of K⁺,Na⁺, Cl^–, and S0₄^–– into the cell and theeffluxes of Na⁺ and Cl^– have been determined. The Cl^–fluxes are extremely large. In all cases the plasmalemma isthe rate-limiting membrane for ion movement. A technique isdescribed for the preparation of large, completely viable cellfragments containing only cytoplasm, with no vacuole.     

Regulation of UDPG pyrophosphorylase in Acetabularia mediterranea

The kinetic properties of UDPG pyrophosphorylase (glucosyl-1-phosphate uridyl transferase, EC 2.7.7.9) suggest that it may play a key role in the regulation of metabolism in Acetabularia mediterranea. The enzyme-catalyzed reaction is readily reversible in vitro, and has been assayed in both directions. The enzyme shows substrate inhibition by UDPG and UTP at substrate concentrations in excess of 2 mM. The kinetic behavior of the enzyme is consistent with the hypothesis that it catalyzes an ordered bisubstrate biproduct reaction in which G-1-P is the leading substrate, and UTP is the leading product. A plot of initial velocity vs. PPi concentration is sigmoid, indicating a cooperative homotropic effect. PGAL inhibits the reaction in the direction: UTP + G-1-P leads to UDPG + PPi It has no effect on the reverse reaction. The responses of the enzyme may serve to regulate the allocation of G-1-P between anabolic and catabolic pathways.     

Regulation of Nitrate Reductase in Acetabularia mediterranea

Nitrate reductase (NR) activity has been characterized on cell-freeextracts of the marine unicellular green alga, Acetabulariamediterranea. Reduced methyl viologen and bromophenol blue aswell as NAD(P)H were effective electron donors for the enzyme. NADH-NR activity increased during cell development reachingits maximum level when the cells approached their maximum length.A substantial increase was also seen in enucleated cells. Theenzyme activity was only present in the green parts of the celland became diminished as the stalks bleached in ageing cells.Under a 12 h light: 12 h darkness photoperiod, NADH-NR activityexhibited pronounced daily fluctuations reaching its maximumon the first hour of illumination (this increase was impairedby cycloheximide) and a minimum in the middle of the dark period. In N-starved algal cultures, NR was constitutively present atappreciable levels and new synthesis took place in the presenceof either NO₃ or low concentrations of NH⁺₄ . However, in thepresence of this cation the enzyme remained mostly in its inactiveform, and could be reactivated in vitro with ferrycyanide. Key words: Nitrate reductase, Acetabularia, daily rhythms, nitrate, ammonium     

The Membrane Potential of Acetabularia mediterranea

The cytoplasm of an Acetabularia cell is normally at a potential of about -170 mv relative to the external solution; the vacuole is also at this potential. Although there is strict flux equilibrium for all ions, the potential is more negative than the Nernst potentials of any of the permeating ions. Darkness, CCCP, low temperature, and reducing [Cl^-]_o by a factor of 25 all rapidly depolarize the membrane and inhibit Cl^- influx. Some of these treatments do not inhibit the effluxes of K⁺ and Na⁺. Increasing [K⁺]_o also depolarizes the membrane both under normal conditions and at low temperature; in the latter case the membrane is partially depolarized in normal seawater (low [K⁺]_o) and in high [K⁺]_o positive potentials of up to +15 mv are attained. It is concluded that the membrane potential is controlled by the electrogenic influx of Cl^-, and also, at least in some circumstances, by the diffusion of K⁺. In addition, it is suggested that electrogenic efflux of H⁺ may be important in transient nonequilibrium situations. An Appendix deals with the interpretation of simple nonsteady-state tracer kinetic data.     

Nucleic Acid Synthesis in the Chloroplasts of Acetabularia mediterranea

Radioautographic and radiochemical techniques were used to establish the presence of replicating DNA in the chloroplasts of Acetabularia mediterranea. These techniques also demonstrated that these chloroplasts synthesize RNA. It was found that label from thymine was also incorporated into DNA and RNA in these chloroplasts.

With the establishment of protein and nucleic acid synthesis in Acetabularia chloroplasts, it is clear that these chloroplasts carry out those metabolic processes which are most characteristic of autonomous cells.

     

Betacyanin biosynthesis in the isolated hypocotyls bei Acetabularia mediterranea

Summary Isolated hypocotyls synthesize betacyanin after light exposure in Amaranthus caudatus L. Pigment synthesizing capacity is reduced in the hypocotyls with increased incubation of seedlings in dark after 24h. External feeding of precursors of betacyanin L-tyrosine and DOPA enhances pigment synthesis in the isolated hypocotyls to equal that of intact hypocotyls. Cotyledons are probably the source of precursors while both cotyledons and hypocotyls are the sites of betacyanin synthesis. Betacyanin synthesizing capacity is progressively lost from the base of the hypocotyl and precursors could not induce pigment synthesis in these regions.     

Some pathways of biosynthesis and degradation of polyphosphates from green algae Acetabularia mediterranea]

The activity of ATP: polyphosphate phosphotransferase was detected in free-cellular extracts of Acetabularia mediterranea. The enzyme activity in cells originally deficient in phosphorus and subsequently transferred into the phosphate-containing medium increases 5-10-fold as compared to normal. Polyphosphate degradation in A. mediterranea is probably produced by polyphosphatase, which was also detected in the free-cellular extract. It was shown that the polyphosphatase activity has two pH optima, i.e. 4.5 and 7.5, and is considerably increased when the cells are transferred into the phosphate-free medium. It is assumed that high-molecular polyphosphates involved in A. Mediterranea metabolism are responsible for regulation of orthophosphate and ATP level in the cells by ATP: polyphosphate phosphotransferase and polyphosphatase.     

Isolation and characterization of different forms of thioredoxins from the green alga Acetabularia mediterranea: identification of an NADP/thioredoxin system in the extrachloroplastic fraction.

A procedure has been developed for the simultaneous purification to apparent homogeneity of chloroplast thioredoxins f and m, and nonchloroplast thioredoxin h, from the green alga Acetabularia mediterranea. In the chloroplast fraction, three thioredoxins were isolated: one f type thioredoxin (Mr 13.4 kDa) and two m type thioredoxin forms (Mr of 12.9 and 13.8 kDa). A Western blot analysis of crude and purified chloroplast thioredoxin preparations revealed that Acetabularia thioredoxin m was immunologically related to its higher-plant counterparts whereas thioredoxin f was not. In the nonchloroplast fraction, a single form of thioredoxin h (Mr 13.4 kDa) and its associated enzyme NADP-thioredoxin reductase (NTR) were evidenced. Acetabularia NTR was partially purified and shown to be an holoenzyme composed of two 33.0-kDa subunits as is the case for other plant and bacterial NTRs. Similarity was confirmed by immunological tests: the algal enzyme was recognized by antibodies to spinach and Escherichia coli NTRs. Acetabularia thioredoxin h seemed to be more distant from higher-plant type h thioredoxins as recognition by antibodies to thioredoxin h from spinach and wheat was weak. The algal thioredoxin h was also slightly active with spinach and E. coli NTRs. These results suggest that in green algae as in the green tissues of higher plants the NADP and chloroplast thioredoxin systems are present simultaneously, and might play an important regulatory role in their respective cellular compartments.     

Enhancer-controlled expression of the simian virus 40 T-antigen in the green alga Acetabularia.

Nuclei from Acetabularia mediterranea were isolated, microinjected with simian virus 40 (SV40) DNA and fused with cytoplasts from the same species. Various times after fusion of the injected nuclei the fusion products were screened for expression of the T-antigen by indirect immunofluorescence. One and two days after injection a bright fluorescence could be observed in the nuclei of Acetabularia. On the basis of this immunofluorescence we conclude that in Acetabularia cells the T-antigen is expressed and accumulated in the nucleus. Moreover, evidence is presented that the Acetabularia cell recognizes the SV40 enhancer sequence. The expression product of the SV40 DNA appears significantly earlier than the expression products of other foreign genes in Acetabularia. The results suggest that the well characterized SV40 can be used as a vector system for the introduction and expression of foreign genes in Acetabularia.