Physical properties of the cell wall of photoautotrophic suspension cells fromChenopodium rubrum L.

Photoautotrophic suspension cells ofChenopodium rubrum were used to determine Donnan potential, charge density and pore-radius distribution in the cell wall. Experiments were done either with turgescent cells or with isolated cell walls. Titration of a cell-wall-generated 9-aminoacridine fluorescence quench with salts of mono- and divalent cations was used to determine Donnan potential and charge density. The experiments and theory were adapted from measurements of membrane surface charges. A tenfold increase in ionic strength, which decreases the repellant forces between charges of the same sign, led to an approximately threefold increase in the measured charge density, thus resulting in a much smaller decrease of the Donnan potential than would be expected if the charge density remained fixed. This decreased influence of ionic strength on the Donnan potential, resulting from the elasticity of the cell wall, was also measurable but less pronounced when the wall of intact cells was stretched by turgor. The porosity of the cell wall was determined by longterm uptake of polyethylene glycols of different molecular weights, and by gel filtration of polyethylene glycols and dextrans as well as mono- and disaccharides using intact suspension cells as matrix. Both methods gave a mean pore diameter of about 4.5 nm and a maximum pore size of 5.5 nm. The resulting pores-size distribution was slightly broader with the latter method.Abbreviations 9-AA 9-aminoacridine - DMBr₂ decamethoniumbromide=N,N,N,N,N,N hexamethyldecane-1,10-diaminebromide - DW dry weight after lyophilization - EDTA ethylene diaminetetra acetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FW fresh weight - Mops 3-(N-morpholino)propanesulfonic acid - MW molecular weight - PEG polyethylene glycol     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O ₂ ^⋅ and H₂O₂ during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-β-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O₂ consumption, only slightly but substantially inhibited in a competitive manner the O ₂ ^⋅ -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive Superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria. These results are interpreted in terms of a possible lysosomal membrane permeability to O ₂ ^⋅ causing organelle impairment by a process that, though leading to enzyme-marker leakage, does not involve lipid peroxidation.     

Studies on systematic embryology inScilla (Hyacinthaceae)

Scilla persica and 5 species of the so-calledS. hohenackeri group, namely,S. furseorum, S. puschkinioides, S. vvedenskyi, S. hohenackeri, andS. greilhuberi, have been investigated embryologically with special reference to embryo sac and endosperm development.Polygonum-type embryo sac development was stated inS. puschkinioides andS. greilhuberi. 8-nucleate, normally structured embryo sacs, which could not be specified further due to sparse availability of the material, were stated inS. furseorum, S. vvedenskyi, andS. hohenackeri. InS. persica the embryo sac develops according to the bisporicAllium-type. In most species endosperm development was stated to be nuclear, exceptS. hohenackeri, where the type could not be specified. Other traits of possible taxonomic significance are the number of layers in the outer integument, which is mostly 4, or 5–6 inS. furseorum, and the occurrence of polyploid versus haploid and early degenerating antipodal nuclei, the latter occurring only inS. persica andS. furseorum. These embryological characters may be useful for assessing taxonomic relationship of the present species with other allied groups withinScilla, in particular, theS. siberica alliance,S. messeniaca, and theS. bifolia alliance. TheAllium-type embryo sac, which occurs inS. persica, is also characteristic for theS. siberica alliance, and may be a common derived character. Lack of antipodal polyploidization, as characteristic forS. persica andS. furseorum, occurs also in theS. siberica alliance, and is perhaps another common derived trait indicating phylogenetic relationship. Nuclear endosperm development is more frequent in spring-flowering squills than helobial development, which has previously been stated inS. messeniaca, some species of theS. siberica alliance, and inS. litardierei. While helobial endosperm may be primitive forHyacinthaceae in general, it may, by reversal, also occur as a derived character, at least in some species of theS. siberica alliance.     

“Self-tanning”—a new and important source of stoichiometric error in cytophotometric determination of nuclear DNA content in plants

A Feulgen-densitometric comparison of nuclear DNA contents (C-values) was performed in various plant species (a fern, four gymnosperms, 16 woody and herbaceous angiosperms) after two types of fixation, additive (neutral formaldehyde) and non-additive (methanol-acetic acid, 3:1, MAA). Nuclei from tissues containing a significant amount of polyphenols (of the hydrolysable and non-hydrolysable tannin type) always showed reduced stainability and distorted spectral absorbance curves after MAA-fixation, while after formaldehyde-fixation no evidence for distorted staining was found. No fixation-dependent differences in Feulgen-DNA contents were stated in nuclei from tissues having no polyphenols. Distorted Feulgen-staining is a consequence of cellular self-tanning during fixation. Tanning is impaired by formaldehyde which binds to tannins and inactivates them. The rationale for using formaldehyde as a fixative in Feulgen-cytophotometry can be mainly seen in its capability of eliminating the self-tanning error. Standardization in plant DNA cytophotometry, and recent reports on unorthodox nuclear DNA variation in conifers are critically discussed.The author dedicates this paper, with emotions of respect and gratitude, to emer. O. Prof. DrElisabeth Tschermak-Woess on the occasion of the 70th anniversary of her birthday. She guided his Ph.D. Thesis in the years 1968 to 1972.     

Characterization of heterochromatin in different species of the Scilla siberica group (Liliaceae) by in situ hybridization of satellite DNAs and fluorochrome banding

Satellite DNAs have been isolated from the monocotyledonous plants Scilla siberica, S. amoena, S. ingridae (all are highly GC-rich), and S. mischtschenkoana by using the Ag⁺ –Cs₂SO₄ density centrifugation technique. Hybridization in situ has been performed with ₃H-cRNA to these satellite DNAs in all four species. In each species, the endogenous satellite DNA is located mainly in intercalary and major heterochromatin bands associated with terminal regions and nucleolar organizer regions (NORs) but not in centromeric regions. Patterns observed after cross-species hybridization show a high degree of satellite DNA homology between S. siberica, S. amoena, and S. ingridae. By contrast, satellite DNA of S. mischtschenkoana consists largely of different, non homologous DNA sequences, with two exceptions: (i) the NORs of all four species contain similar satellite sequences, and (ii) a strong homology exists between the satellite DNA of S. mischtschenkoana and centromeric DNA of S. siberica but not with those of S. amoena and S. ingridae. — Heterochromatin has also been characterized by the AT-specific fluorochromes quinacrine (Q) and DAPI and the GC-specific agent chromomycin A₃ (CMA₃), in combination with two counterstaining techniques. While CMA₃-fluorescence is largely in agreement with data on base composition and location of the specific satellite DNAs, the results with Q and DAPI are conflicting. Prolonged fixation has been found to change the fluorescence character in certain instances, indicating that other factors than the base sequence of the DNA also play a role in fluorochrome staining of chromosomes. The results are discussed in relation to the taxonomy and phylogeny of the four species.     

Heterogeneity of heterochromatin in plants: Comparison of Hy- and C-bands inVicia faba

The comparison of Hy- and C-bands reveals three types of heterochromatin (Hy+, Hy−, and Hy 0) inVicia faba. By C-banding, the total of constitutive heterochromatin is identified. The Hy-banded heterochromatin is restricted to the M-chromosome: The nucleolus-associated heterochromatin appears as two darkly stained (Hy+)-bands bordering the secondary constriction, while reduced staining (Hy−)-bands are located near the centromere. The heterochromatin of the S-chromosomes is of the (Hy 0)-type, i.e., it does not respond to the Hy-banding procedure. A complete karyogram of the C-banded chromosome complement is presented and discussed. A comparison of C-bands and cold-sensitive segments clearly shows that negatively as well as positively reacting cold-induced segments are partly heterochromatic, partly euchromatic.     

Aneusomaty inOrobanche gracilis

InOrobanche gracilis Sm. (collected in Vienna, Lower Austria, and N. Italy) the somatic chromosome numbers were found to be inter-and intra-individually variable. Most plants yielded counts of around the tetraploid level (2n = 73−91), a few of around the hexaploid level (2n = 112−116). Only 36% of the plants were eusomatic, only 24% eutetraploid (2n = 76). Depending on the variable somatic chromosome number, gametophytic numbers are also variable. Univalents, multivalents, and anaphase anomalies were of regular occurrence during meiosis of plants with more than 2n = 76 chromosomes. This is the first report of variable polyploid chromosome numbers (4x = 76, 6x = 114) inOrobanche sect.Orobanche. Aneusomaty is due to a constitutional tendency to nondisjunction during mitosis. The supernumerary chromosomes are considered to represent A- (= normal) rather than B-chromosomes. A possible causality between the chromosomal and the morphological variation, and the adaptive significance of this phenomenon are discussed.     

Fluxes and compartmentation of 3-O-methyl-D-glucose in Riccia fluitans

In thalli of the aquatic liverwort, Riccia fluitans, 3-O-methyl-D-glucose (3-OMG) is not metabolized. Intracellular compartmentation, accumulation and transmembrane fluxes of 3-OMG have been determined by compartmental analysis. A novel set of equations has been derived to extend this method to non-steady-state conditions of constant but unequal unidirectional fluxes. Efflux kinetics with 3-OMG and L-glucose revealed two intracellular flux compartments, presumably cytoplasm and vacuole; an additional quickly exchanging compartment (half-time approx. 1 min) has been assigned to the apoplast. With 1 mM 3-OMG given externally, cytoplasmic 3-OMG concentration (c _c) attains a quasi-steady state of about 10 mM lasting for >100 h, whereas the presumed vacuolar concentration (c _v) rises steadily, but does not reach flux equilibrium even after two weeks (c _v=46 mM). After 24 h incubation with 0.03 mM 3-OMG, c _c=1 mM approx., and c _v=3 mM approx., thus indicating accumulation by active hexose transport at both the plasmalemma and tonoplast. External D-glucose, but no D-mannitol, competitively inhibits 3-OMG uptake (cis-inhibition) and stimulates 3-OMG efflux at the plasmalemma by a factor up to 2.5. This trans-stimulation saturates half-maximally at 1.5 mM D-glucose. It clearly indicates a hexose carrier in the plasmalemma with one substrate-binding site for D-glocose and 3-OMG, alternately exposed to the cytoplasmic and outside compartment. The extent of the measured trans-stimulation can only be explained, if in the transport cycle the translocation of the empty substrate-binding site across the plasmalemma is rate-limiting.     

The role of thiosulfate in sulfur metabolism of Rhodopseudomonas globiformis

Rhodopseudomonas globiformis is able to assimilate both sulfur moieties of thiosulfate. During growth on ³⁵S-labelled thiosulfate the amino acids cysteine, homocysteine and methionine were labelled. The bulk of thiosulfate, however, was oxidized to tetrathionate and accumulated in the medium. A thiosulfate: acceptor oxidoreductase was partially purified and characterized. The enzyme oxidized thiosulfate to tetrathionate in the presence of ferricyanide. A c-type cytochrome isolated from this organism was reduced by this enzyme.     

Evidence for intergeneric incompatibility in ferns

The archegonial mucilage ofAthyrium filix-femina andA. distentifolium paralyses spermatozoids ofDryopteris filix-mas (and in one caseD. inaequalis) before they penetrate the archegonial venter. The archegonial mucilage ofDryopteris filix-mas has a weak positive chemotactic influence on the spermatozoids of the twoAthyrium species. The spermatozoids ofDryopteris were never observed in the archegonia ofAthyrium. Incompatibility was not observed within and between the twoAthyrium species, withinDryopteris filix-mas or betweenAthyrium filix-femina and twoAsplenium species.Contribution No. 327.