A Synechococcus sp. PCC 7942 mutant with a higher tolerance towards bentazone

In this article we describe the partial characterization of a Synechococcus sp. PCC 7942 mutant Mu1 with an enhanced resistance towards the herbicide bentazone (3-isopropyl-1H-2,1,3-benzothiadiazine-4(3H)-one 2,2-dioxide). The mutant was derived from a random mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (NSG) and exhibited superior growth rates, pigment content and overall photosynthetic activities under regular growth conditions compared to wild type. Whereas Synechococcus PCC 7942 wild type showed significant photoinhibition, especially in the presence of lincomycin, Mu1 was much more robust. A comparative analysis of the content of several photosynthesis-associated proteins revealed that Mu1 had an increased expression of PsbO on mRNA and protein level and that PsbO is tightly bound to Photosystem II, relative to wild type. This result was substantiated by mass spectrometer measurements of photosynthetic water oxidation revealing a higher stability and integrity of the water oxidizing complex in Mu1 cells grown under regular or calcium deficient conditions. Therefore, our results give rise to the possibility that the overexpression of PsbO in mutant Mu1 confers resistance to reactive oxygen species (ROS) formed as a consequence of bentazone binding to the acceptor side of PS II. In addition, we observed a significantly higher tolerance towards bentazone in iron depleted wild type cells, conditions under which the IdiA protein becomes expressed in highly elevated amounts. As we have previously shown, IdiA preferentially protects the acceptor site of PS II against oxidative stress, especially under iron limitation. Thus, it is likely that IdiA due to its topology interferes with bentazone binding or protects PS II against ROS generated in the presence of bentazone. This revised version was published online in June 2006 with corrections to the Cover Date.     

A fungus-type beta-galactofuranan in the cultivated Trebouxia photobiont of the lichen Ramalina gracilis

A structural characterization of polysaccharides extracted from the aposymbiotically cultured photobiont of the lichen Ramalina gracilis was carried out in order to compare them with those previously found in the symbiotic thallus. The photobiont was isolated from thallus fragments, following the method of Yamamoto, and cultivated in a liquid nutrient medium. Freeze-dried cells were defatted, and the polysaccharides extracted successively with water and aq. 10% KOH, each at 100 degrees C. After purification, the soluble fractions provided a polysaccharide containing a (1-->5)-linked beta-galactofuranosyl backbone, substituted in a small proportion at O-6 by beta-Galf units. Amylose was also found, as insoluble material obtained on freeze-thawing of the alkaline extract. These polysaccharides have not been found in the symbiotic thallus of Ramalina gracilis, which contained only water-soluble (isolichenan) and insoluble glucans (nigeran and laminaran), and galactomannan. Surprisingly, the galactofuranan has similarities with those found in some fungal cell walls.     

Evaluation of mitochondrial respiratory function in small biopsies of liver

Mitochondrial respiratory function was studied in permeabilized pig liver biopsies. The cell membrane was permeabilized mechanically in tissue samples of 2-7 mg, for application of a standardized substrate/inhibitor titration protocol in high-resolution respirometry. Specific respirometric tests demonstrated complete plasma membrane permeabilization and accessibility of substrates to intact mitochondria. High respiratory adenylate control ratios and cytochrome c conservation in the tissue preparation were comparable or even better than in isolated mitochondria. Citrate synthase and cytochrome c oxidase activities remained at 85% of controls after up to 98 h storage of liver tissue at 0 degrees C in histidine-tryptophan-ketoglutarate solution. Multiple mitochondrial defects, however, were indicated after 48 h cold storage by the decline in respiratory capacity, which was lowered to a larger extent with complex I substrates compared to respiration with substrates for complex II or IV, measured in the absence of cytochrome c. After prolonged ischemia, the adenylate control ratio was significantly reduced, and cytochrome c depletion was detected by the stimulatory effect of cytochrome c. High-resolution respirometry allows the assessment of mitochondrial function in a few milligrams of permeabilized liver tissue, without isolation of mitochondria. This provides a basis for the analysis of mitochondrial function in human liver biopsies.     

Comparison of photosystem II complexes isolated from tobacco and two chlorophyll deficient tobacco mutants

A comparative study of photosystem II complexes isolated from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contains normal stacked thylakoid membranes, and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked grana or essentially unstacked thylakoids with occasional membrane doublings, has been carried out. The corresponding photosystem II complexes had an O₂ evolving activity ranging from 290 (for the wild type) to 1100 mol O₂ x mg chlorophyll^-1 x h^-1 (for the mutant Su/su var. Aurea). The reduced photosynthetic unit size was also obvious in the mangenese and cytochrome_b559 content. The photosystem II complex from the wild type contained 4 Mn and 1 cytochrome_b559 per 200 to 280 chlorophylls, while the corresponding value for the mutant Su/su var. Aurea was 4 Mn and 1 cytochrome_b559 per 35 to 60 chlorophylls. We have also examined the polypeptide composition and show that the photosystem II complex from the wild type consisted of polypeptides of 48, 42, 33, 32, 30, 28, 23, 21, 18, 16 and 10 kDa, while the mutant complex mainly contained the polypeptides of 48, 42, 33, 32, 30, 28 and 10 kDa. In the mutant photosystem II complex the light-harvesting chlorophyll protein (peptide of 28 kDa) was reduced by a factor of 5 to 6 as compared to the wild type. With respect to the peptide composition and the photosynthetic unit size, the Triton-solubilized photosystem II complex from the mutant Su/su var. Aurea was very similar to O₂ evolving photosystem II reaction center core complexes.Abbreviations PS photosystem - chl chlorophyll - LHCP light-harvesting chlorophyll a/b protein complex     

Spaltöffnungs-Dünnstschnitte im Elektronenmikroskop

Zusammenfassung Mit Hilfe des Sjöstrandschen Ultramikrotoms gelang die Anfertigung von Dünnstschnittserien durch die Spaltöffnung vonHelleborus. An Hand der Aufnahmen werden beschrieben: Außen- und Innencuticula, Membranbau, besonders intermicellare Hohlraumsysteme in den Cutinleisten und Antiklinen, Tüpfel bzw. Plasmodesmen in Schließzellen, Hautgelenk und Epidermisaußenwand.     

ALGAL CALCIFICATION IN SOME CODIACEAE (CHLOROPHYTA): ULTRASTRUCTURE AND LOCATION OF SKELETAL DEPOSITS1,2

The capitular filaments of Penicillus and Rhipocephalus consist of an inner tube containing the cytoplasm and an outer calcified sheath. The sheath originates at the cell wall and differentiates into several layers which form the outer filament wall. CaCO₃ is deposited between organic layers within the sheath and is not in direct contact with the seawater. Pores within the sheath, usually uncalcified, may facilitate exchange of gases and solutes. The cytoplasm is characterized by vacuolar inclusions of calcium oxalate needles 50–150 μm long. A closed cortical surface is lacking. Udotea cyathiformis Dec. and U. conglutinata (Ellis & Sol.) Lam. are similar to Penicillus and Rhipocephalus, in addition showing some CaCO₃ between filaments (ICS-calcification). Udotea flabellum (Ellis & Sol.) Lam. is different as the filaments are profusely branched giving rise to a fully developed cortical surface. Pores and vacuolar calcium oxalate inclusions are absent. CaCO₃ deposition occurs within cortical filaments in between layers of the filament wall and subcortically in intercellular spares (ICS). Cortex calcification shows primary and secondary deposits bearing some resemblance to sheath calcification and to coralline red algae. In Rhipocephalus phoenix (Ellis & Sol.) Kütz., Penicillus pyriformis A. &E. Gepp, U. cyathiformis and U. conglutinata CaCO₃ is precipitated intracellularly within the sheath, in contrast to Halimeda and Cymopolia where it is deposited extracellularly in between filaments. U. flabellum takes an intermediate position showing both intra- and intercellular calcification. The sheath compartment volume is between 12.5 and 7500 μm³and 5–3 orders of magnitude smaller than the ICS-compartment. Compartment size and location of CaCO₃may bear on calcification mechanisms. One condition for such a mechanism may be restricted exchange of solutes (CO₂, CO₃^2-, HCO₃^-, O₂, Co²⁺). Codiaceae; filament ultrastructure; Penicillus; Rhipocephalus; Udotea     

Effect of ammonium and ferricyanide on nitrate utilization by Chlorella vulgaris

It has been shown previously that added ammonium salts cause a cessation of nitrate utilization in some Chlorella species. It has also been shown that Chlorella vulgaris can form an inactivated nitrate reductase which is an HCN complex. In the present study, a comparison has been made of the rate of nitrate utilization and the rate of nitrate reductase inactivation in Chlorella vulgaris in response to the addition of ammonium salts and light-dark changes. The rate of formation of HCN-inactivated enzyme is too slow to account for the prompt inhibition of nitrate utilization caused by adding ammonium. In contrast, when nitrate utilization is inhibited by addition of ferricyanide to intact cells, the HCN-inactivated enzyme is promptly formed in vivo, and might account for the inhibition of nitrate utilization, though inhibition of nitrate uptake can not be excluded.     

Cyanide formation in preparations from Chlorella vulgaris Beijerinck: Effect of sonication and amygdalin addition

Summary A critical evaluation of a method for recovering HCN from cell extracts is presented. Since crude extracts often bind or metabolize HCN extensively, the HCN recovered by distillation at room temperature represents only the difference between production and consumption. Sonication leads to HCN release from the alga, Chlorella vulgaris Beijerinck. Illumination of extracts at high light intensity in oxygen, with added Mn²⁺ and peroxidase, also stimulates HCN production. In both processes, the HCN is probably formed by oxidation of nitrogenous precursors. Chlorella extracts cause formation of HCN from added amygdalin. No evidence was found, however, for the presence of cyanogenic glycosides in the algae.