Transitory pulse-like treatment with IAA solution more effectively induces xylogenesis in callus culture than permanent presence of the auxin

Differentiation of Acerpseudoplatanus L. cells into tracheary elements (TE) and an increase in the number of TE inHaplopappus gracilis (Nutt.) A. Gray calli were observed after pulse-treatment of the cultures grown in glass tubes with indole-3-acetic acid (IAA) solutions. The effect was enhanced if the treatment was repeated in three subsequent days. The manner of IAA application which caused a wave-like pattern of IAA flow through the callus culture appeared to be more important than the IAA concentration. Induction of differentiation of A.pseudoplatanus cells and an increase in the number of TE inH. gracilis callus did not occur when the calli were grown on agar media supplemented with increased concentrations of IAA.     

Effects of the physiological state of five yeast species on H+-ATPase-related processes

Effects of starvation and glucose preincubation on membrane potential, ATPase-mediated acidification and glutamic acid transport were studied in yeast speciesSaccharomyces cerevisiae, Schizosaccharomyces pombe, Dipodascus magnusii, Lodderomyces elongisporus andRhodotorula gracilis. Themembrane potential was highest after preincubation with glucose in all species butL. elongisporus andR. gracilis. In all cases the membranes were depolarized in the presence of 20 mmol/L KCl and hyperpolarized with 50 μmol/L diethylstilbestrol (DES). Theextracellular acidification caused by addition of glucose was highest after preincubation with glucose in all cases except inR. gracilis where there was none. In all cases except inR. gracilis addition of KCl caused a marked increase in the acidification rate. Addition of DES with glucose caused a large decrease in rate inS. cerevisiae but had much less effect on the other species.Transport of glutamic acid was clearly increased after pretreatment with glucose inS. cerevisiae, S. pombe andD. magnusii (mainly due to enhanced synthesis of the carrier) but actually decreased inR. gracilis andL. elongisporus. Addition of DES had an inhibitory effect in all species but much more pronounced inS. cerevisiae andS. pombe than in others. In general, both the acidification and the transport of glutamate were enhanced after preincubation with glucose but much more so in the semianaerobic species, such asS. cerevisiae, than in the strict aerobes (R. gracilis) where the effect was occasionally negative. There was no relationship between the ATPase-mediated acidification and the membrane potential.     

Genetic variability of Bouteloua gracilis populations differing in forage production at the southernmost part of the North American Graminetum

Bouteloua gracilis (blue grama grass) native populations have been shown to be highly variable, however the genetic basis of this variability has not been well established. Determining the extent of genetic variability within and among plant populations have important repercussions for the management and conservation of species, and in particular for those subjected to intensive use such as forage plants. Using RAPD, this study was undertaken to investigate the genetic variability of four B. gracilis native populations developed in three grasslands and one shrubland at the southernmost part of the North American Graminetum in México. Significant differences in grass aboveground production were found among the study sites, while considerable genetic variation within each of the four blue grama populations evaluated was detected. The molecular analysis, based on 55 individuals, revealed a total of 108 scorable repeatable bands, with 99 of them being polymorphic (overall polymorphism= 91.7%). Within every population each individual was genetically distinct and no population-specific bands (fixed marker differences) were identified. Pair-wise Φ _ST comparisons indicated that the four blue grama populations examined were significantly different in their genetic constitution (P<0.001). AMOVA revealed that most of the genetic variation detected in Bouteloua gracilis was explained by intra- (88.53%), rather than by inter-population (11.47%) differences. UPGMA based on the Φ _ST values indicated that the blue grama population collected from the shrubland displayed the RAPD profiles that most differed among the study sites. Possible causes of these results could reside on intensive grazing reducing, and proper management conserving, the forage production and genetic diversity of blue grama native populations. Our results are consistent with previous studies analyzing population genetic variation in outcrossing grasses and, in particular, with ecological and cytological evidence for a high genetic variability in native populations of B. gracilis. The implications of our findings and prospective studies to be undertaken using molecular tools in the study of blue grama biology and ecology are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of a ferrate-containing preparation on diverse metabolic processes in yeast

A plant-sap-derived preparation containing bi-and tervalent ferrate anions was tested on growth, respiration on glucose, and membrane transport of 6-deoxy-d-glucose (6-dGlc) and 2-aminoisobutyric acid (Aib) in several yeast species,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Lodderomyces elongisporus, Rhodotorula gracilis, andDipodascus magnusii. Growth was enhanced by as much as 65%, respiration was not affected significantly except for a decrease inR. gracilis, transport of 6-dGlc was not affected while that of Aib was increased by up to 45% inR. gracilis and up to 27% inL. elongisporus.     

Asymmetric somatic cell hybridization in plants

Summary An investigation into the possible application of UV radiation as a pretreatment for the donor cells in asymmetric plant cell hybridization protocols has been carried out. A comparison was made between the effects of UV doses in the range 700–4200 J/m² and those of ⁶⁰Co gamma radiation over the range 0.15–1 kGy on Beta vulgaris suspension cell protoplasts. The investigation had two aspects. Firstly, alterations to cell physiology (cell wall resynthesis, viability, division and colony formation) in irradiated protoplasts were examined during a 4-week culture period. Results have indicated that a dose of 700 J/m² UV is necessary to prevent further cell division and colony formation in these cells. A dose of 0.15 kGy gamma radiation generally prevented colony formation, although some early cell division did occur (as was also observed even after 0.45 kGy had been applied). Membrane integrity, as measured after 6 days, using fluorescein diacetate staining, was not affected by either treatment within the dose ranges applied. Secondly, denaturing (alkaline) gel electrophoresis, in association with a pulsed field gel DNA preparation technique, was used to determine the degree of in vivo DNA damage following the radiation treatments. After UV radiation, considerable fragmentation of the DNA was observed, the extent of which was dose-dependent. Gamma radiation, however, appeared to result in fewer DNA lesions, with only the 1 kGy treatment revealing a pattern significantly altered from that of the control. These results augur well for the potential use of UV radiation in asymmetric fusion experiments.     

Lipid content and utilization of lipids in planktonic copepods in Lake Pääjärvi,southern Finland

The variation of the lipid content of a planktonic copepod species, Eudiaptomus gracilis G. O. Sars, was studied in an oligotrophic lake, Pääjärvi, southern Finland, during 1982. The variation was caused by changes in the food resources and by the use of lipids for reproduction. The lipids are partly present in the form of droplets, which provide an energy reserve for some of food shortage, or may be used in reproduction. The utilization of lipid droplets for nutrition was studied by culturing specimens of two species, Eudiaptomus gracilis and Thermocyclops oithonoides G. O. Sars, in the absence of food. It was found that about half of the energy required for the 30-hour experimental period was obtained from the lipid droplets. However, not all the animal groups could use their lipid droplets and they had to use other reserves to satisfy their energy requirement. Lipid droplets seemed to have only a minor effect on the sinking rate of Eudiaptomus gracilis.     

Overexpression, Purification, and Generation of a Thermostable Variant ofZymomonas mobilisFructokinase

The gene encoding fructokinase (EC 2.7.1.4) fromZymomonas mobilishas been expressed at high level inEscherichia coliby modifying the ribosome binding site using the polymerase chain reaction. A simple two-step purification from extracts of the recombinant cells results in highly purified enzyme suitable for use in fructose determination. Using the polymerase chain reaction in mutagenic conditions, a variant of fructokinase was isolated which was more thermostable than the wild type, taking the 30 min half-life from 70.1 to 72.4°C. The purified thermostable variant had the same specific activity as the wild type. Sequencing of the variant indicated that only one amino acid was changed, with Ser 69 becoming Ala. Searches of the mutant libraries for variants that were (a) active with glucose or (b) had reduced inhibition by glucose were unsuccessful.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. VII. CELLULAR VARIATION

Variation in long-continued cultures of Haplopappus gracilis and Daucus carota has been investigated. A strain of carrot tissue was isolated that grew with a compact habit, in contrast to the highly friable habit of the parent strain. Its dividing cells were arranged quite differently than in the parent strain. Earlier work had shown that Haplopappus cultures could be reversibly altered in their pigmentation and form, by changing the culture medium. This was confirmed, and it was further shown that pronounced changes in nitrogenous compounds also occurred in response to factors in the medium. However, strains of Haplopappus were isolated which differed persistently from the parent strain, even when they were maintained under the same conditions. The variant strains, grown in the same medium, showed differences in their content of nitrogenous compounds. Stock cultures also changed spontaneously with time with respect to their content of nitrogenous substances. Acriflavine, at low concentration, inhibited the growth and formation of colonics by cells plated on nutrient agar, but, by prolonged exposure to sublethal amounts of the drug, resistant strains were isolated. Certain of the spontaneous variant strains were found to differ from each other and from the parent strain in their chromosome complements in ways that are described and to which the observed changes in morphology and metabolism of the cultures may be attributed. The variations that may occur in the free cells in culture are contrasted with the greater uniformity of the cells as they exist in the plant body.     

Effects of different 2,4-D concentrations on the cytogenetic behaviour of plant cells culturedin vitro

Cytogenetic effects of different concentrations of 2,4-D on suspension cultures ofVicia hajastana Grossh. andHaplopappus gracilis (Nutt.) Gray were studied using a simple, chemically defined medium. InVicia, the frequency of dicentric chromosomes showed a negative association with the 2,4-D concentration. In both the species, the frequencies of anaphase bridges were negatively associated, while those of the diploid cells showed a positive association with the 2,4-D concentration in the culture medium. A decrease in the frequency of diploid cells was observed in the cultures transferred to a lower concentration of 2,4-D. This decrease was transient inVicia, while inH. gracilis a new equilibrium at a lower level was established. Both endoreduplication and selection appear to be involved in the achievement of the lower equilibrium inH. gracilis, although selection appears to be the major factor.     

Characterization of a Variant ofAutographa californicaNuclear Polyhedrosis Virus with a Nonfunctional ORF 603

A new genotypic variant ofAutographa californicanuclear polyhedrosis virus (AcMNPV), the V8 variant, was originally identified by an additionalHindIII site in theHindIII–F fragment. Insect bioassays of this variant displayed a decreased time of mortality compared with the L1 variant of AcMNPV inSpodoptera frugiperdalarvae but not inTrichoplusia nilarvae. A 1.8-kb region containing the 3′ end of ORF 5,lef-2,ORF 603, and the 5′ end of the polyhedrin gene (polh) of both L1 and V8 was sequenced. V8 exhibited extensive sequence variation in the region between the 3′ end oflef-2and the 5′ end ofpolh; V8 had six amino acid substitutions in thelef-2gene product and a nonfunctional ORF 603. A site-specific frameshift mutation in ORF 603 of the L1 variant was constructed to determine the effect of ORF 603 inS. frugiperdalarvae. Truncation of ORF 603 was found to decrease the time of mortality inS. frugiperdalarvae. The insect-selective toxin gene,tox34, was inserted into the V8 variant by direct cloning. The efficacy of this recombinant as a biopesticide was equivalent to similar L1 recombinants.     

Nutritional changes induce xylogenesis in callus ofHaplopappus gracilis (NUTT.) A. Gray

Xylogenesis was induced in cultures of calli ofHaplopappus gracilis (NUTT.) A. Gray by metabolic shocks brought about by changes in nutrition. Xylogenesis was observed to occur in response to three changes in callus nutrition, caused by the following modifications of Eriksson’s nutrient medium and culture conditions: i) omission of sucrose in the medium for 3 to 6 days and then transfer of the calli onto complete Eriksson’s medium; ii) replacement of sucrose by 0.36*mol l_-1 xylose for the whole culture period; iii) omission of nitrogen sources(i.e. glycine and NH₄NO₃ omission and replacement of KNO₃ with equimolar KCI) for the whole culture period. The induction of xylogenesis in response to nutritional stress inH. gracilis can be used as a suitable model system for research concerning plant cell differentiation.     

Cytological Characterization of a Giant Strain of Euglena gracilis Obtained from Dark-starved Cultures

Euglena gracilis green cells were dark-starved for four months. After this period almost the entire population died, while a few giant, viable cells appeared in the culture. The giantism was maintained after repeated subcultures in growth medium in light or dark conditions. However, the phenomenon was not permanent, and the morphological characteristics of the wild-type Euglena were gradually restored. In giant cells nuclei enlarged greatly, DNA content increased and the Golgi apparatus greatly proliferated. Chloroplasts and mitochondria increased in number and size and often presented structural modifications when compared with normal Euglena. Importantly, in the giant cells that were maintained in darkness in resting or growth conditions chloroplasts persisted as structured organelles which appeared red-fluorescent under UV illumination. Whether giantism is a phenotypic or a genotypic change is still debated. In our case, the evolution of this phenomenon, chiefly the enhanced DNA content, suggests that teratism is a multiploid mutation with the possibility of a return to the normoploid condition. Constitutive chloroplasts are present in most algae, except for a few species, among which is Euglena gracilis. The persistence of differentiated plastids in darkness in giant Euglena is considered to be a return to an ancestral condition and may, therefore, be phylogenetically important.     

Antibiotics and apochlorosis

Hydroxylamine, an inhibitor of deoxyribonucleic acids (DNA), ribonucleic acids (RNA) and proteosynthesis interferes with the bleaching effect of streptomycin on growing cells ofEuglena gracilis. The addition of hydroxylamine to a green autotrophic culture ofEuglena gracilis inhibits, depigmentation of the culture by streptomycin. Otherwise, streptomycin alone, without, hydroxylamine, is a powerful bleaching agent and when added to a growing culture ofEuglena gracilis, transforms the green, autotrophic cells to permanently colourless, heterotrophic cells. Phenethyl alcohol, an inhibitor of RNA synthesis, and chloramphenicol, an inhibitor of proteosynthesis, do not block the bleaching effect of streptomycin. It can be concluded from these results that the bleaching effect of streptomycin is related to its interference in the plastid DNA.     

CRYPTIC DIVERSITY IN PHYTOPLANKTON CULTURES IS REVEALED USING A SIMPLE PLATING TECHNIQUE1

A simple technique has been developed to probe the occurrence of cryptic genetic diversity in populations of laboratory‐maintained phytoplankton cultures. This agarose‐based method allows the investigator to plate a broad range of fragile algae, including representatives of the Raphidophyceae, Synurophyceae, Prymnesiophyceae, and Dinophyceae. Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara was selected as a model system for our genetic diversity study. Further optimization of the plating technique for this alga demonstrated that colony formation was independent of the physiological state of the parent culture and yet dependent on incident light intensity. The density at which cells were plated affected colony formation and the rate of growth, with intermediate densities (～10³ cells per plate) performing best. High‐metal stress was used as the selective screen for assessing genetic variability within a single H. akashiwo culture. To this end, “clonal” lines (quotation marks used to indicate that within‐clone diversity was expected) were generated from individual plated colonies, and their tolerance was measured by plating on selective medium. Results of these experiments suggest the following: (i) “clonal” lines generated from a single H. akashiwo culture displayed a significant variation in stress tolerance; (ii) “clonal” lines chosen for their ability to grow on selective plates retained this tolerance in the absence of stress, indicating that the observed variation is heritable; and (iii) genetic variation is continually generated in growing cultures. Our results are consistent with a conceptual model, presented here, in which stress tolerance among the individuals in a culture has a genetic basis that varies over a continuous spectrum.     

Morphological and molecular variation in Mitchella undulata,with special reference to the systematic treatment of the dwarf form from Yakushima

Morphological and molecular variation in Mitchella undulata Siebold et Zucc. was examined to evaluate the genetic basis for recognizing the dwarf variety, M. undulata var. minor Masamune. Considerable variation in leaf size in M. undulata, but no obvious morphological discontinuities, were found between the normal and dwarf varieties. Instead, a weak cline running from the Pacific Ocean to the Sea of Japan was found. Anatomical observations of leaf blades revealed that the large variation in leaf size can be attributed to variation in the number of leaf cells and not to differences in cell size. A molecular analysis based on sequences of rDNA internal transcribed spacer regions indicated that there were two major genotypes in M. undulata with minor variation in haplotypes resulting from additional substitutions or putative recombination. The dwarf form from Yakushima was neither genetically uniform nor apparently differentiated from other populations. From these results, we conclude that the dwarf form of M. undulata should be treated at the rank of forma.     

Phenotypic and genetic differences between opaque and translucent colonies of Vibrio alginolyticus

Many pathogens undergo phase variation between rugose and smooth colony morphology or between opaque and translucent colony morphology, which is mainly due to the variation in the surface polysaccharides. In this study, Vibrio alginolyticus ZJ-51 displayed phase variation between opaque, rugose colonies (Op) and translucent, smooth colonies (Tr). Unlike the vibrios reported previously, Tr cells of ZJ-51 enhanced biofilm formation and motility, but they did not differ from Op cells in the quantity of surface polysaccharides produced. Real time PCR was used to analyze the expression of the genes involved in polysaccharide biosynthesis, flagellar synthesis, and the AI-2 quorum-sensing system. The results revealed that the K-antigen capsule gene cluster (which consists of homologs to the cpsA-K in Vibrio parahaemolyticus) and O-antigen polysaccharide gene cluster (which contains homologs to the wza-wzb-wzc) were significantly more transcribed in Tr cells. The AI-2 quorum-sensing genes showed enhanced expression in the Tr variant which also exhibited greater expression of genes associated with polar flagellar biosynthesis. These results suggest that colony phase variation might affect the virulence and survival ability in the stressful environment inhabited by V. alginolyticus.     

Chloroplast Genome Evolution in the Euglenaceae

Over the last few years multiple studies have been published outlining chloroplast genomes that represent many of the photosynthetic euglenid genera. However, these genomes were scattered throughout the euglenophyceaean phylogenetic tree, and focused on comparisons with Euglena gracilis. Here, we present a study exclusively on taxa within the Euglenaceae. Six new chloroplast genomes were characterized, those of Cryptoglena skujai, E. gracilis var. bacillaris, Euglena viridis, Euglenaria anabaena, Monomorphina parapyrum, and Trachelomonas volvocina, and added to six previously published chloroplast genomes to determine if trends existed within the family. With this study: at least one genome has now been characterized for each genus, the genomes of different strains from two taxa were characterized to explore intraspecific variability, and a second taxon has been characterized for the genus Monomorphina to examine intrageneric variability. Overall results showed a large amount of variability among the genomes, though a few trends could be identified both within Euglenaceae and within Euglenophyta. In addition, the intraspecific analysis indicated that the similarity of a genome sequence between strains was taxon dependent, and the intrageneric analysis indicated that the majority of the evolutionary changes within the Euglenaceae occurred intergenerically.     

GEOGRAPHICAL CORRELATION OF MORPHOLOGICAL VARIATION IN SILVETIA COMPRESSA (FUCACEAE,FUCALES, PHAEOPHYCEAE)

Silvetia compressa ( J. Agardh) Serrão et al. is a common member of the upper intertidal fucoid community on the Pacific coast of America from Humboldt County, California, to Punta Baja, Baja California, Mexico. A relatively narrow range of morphological variability is exhibited by most mainland populations, regardless of latitude, but some mainland populations and all insular populations participate in a complex pattern that we have attempted to analyze. A few populations on the Monterey Peninsula in which the fronds are atypically delicate were described by Setchell & Gardner as f. gracilis, to which was assigned a population from Santa Catalina Island. After comparing populations from various parts of the range of the species, including all of the Channel Islands, we conclude that two subspecies may be recognized. In subsp. compressa, which includes f. gracilis as a growth form and occurs chiefly on the mainland, the frond is robust with long tapered receptacles. In the variant subspecies, which is chiefly insular but also occurs on the coast of northern Baja California, the typical frond has slender axes as in f. gracilis, but is more densely branched and has short ellipsoidal receptacles. Comparison of nucleotide sequences from the ITS regions of rDNA revealed an identical pattern for subsp. compressa from Baja California and central California, including populations assignable to f. gracilis. By contrast, the pattern for the variant subspecies differed by 2 bp (0.3%) from that of subsp. compressa.     

Field measurements of photosynthesis,water-use efficiency,and growth inAgropyron smithii (C3) andBouteloua gracilis (C4) in the Colorado shortgrass steppe

Summary Field measurements of gas exchange and growth were conducted on a C₃ grass,Agropyron smithii, and a C₄ grass,Bouteloua gracilis, in order to further establish the adaptive significance of the C₄ pathway under natural conditions. Maximum rates of leaf area expansion in tillers and maximum seasonal photosynthesis rates of both species occurred during the cool, early summer month of June. The occurrence of maximum growth and photosynthesis inB. gracilis during this cool period was apparently related to its occupation of warm microenvironments next to the ground surface. As temperatures increased during the midsummer, photosynthesis rates decreased to 47% and 55% of the seasonal maximum inB. gracilis andA. smithii, respectively. Water-use efficiencies in both species were similar or slightly higher forB. gracilis during June, the period of maximum growth. By mid-July, however, leaves of the C₃ grass,A. smithii, exhibited water-use efficiencies approximately half as high asB. gracilis. These differences in water-use efficiency were the result of differences in stomatal conductance, rather than differences in daily CO₂ uptake rates which were similar in both species. The results demonstrate that in certain environments there are no offset periods of growth and maximum photosynthesis during the growing season in these C₃ and C₄ species. The greater amounts of daily water use inA. smithii during the midsummer might contribute to its much greater abundance in lowland sites in the shortgrass steppe. The C₄ grass,B. gracilis, occurs in dry upland sites in addition to the more mesic lowland sites.     

Newly established cell lines fromDrosophila larval CNS express neural specific characteristics

Summary From the central nervous system ofDrosophila melanogaster 3rd instar larvae, eight continuous cell lines have been established (named ML-DmBG1 to 8). Using ML-DmBG2, single colony isolation was carried out and six colonial clones were obtained. All reacted to the antibody to horseradish peroxidase, which is a neuronal marker in insects. Acetylcholine, a known neurotransmitter inDrosophila, was detected in three of the colonial clones by high performance liquid chromatography. Therefore, it is concluded that the established colonial clones are neural cells originating in the larval central nervous system. Among them, some variation was observed with respect to morphology, acetylcholine content, and reactivity to anti-HRP. The variation may reflect the heterogeneity of cells composing the central nervous system.