Upstream non-coding region which confers polar expression to Ri plasmid root inducing gene rolB

Summary Root differentiation could be elicited on carrot discs by transformation with the agropine Ri plasmid rolB gene cloned in the binary vector Bin19, provided two conditions were met. Firstly, an adequate auxin supply had to be provided. This was achieved by co-inoculation with a strain carrying only the auxin synthetic genes of the T_R-DNA. Most of the resulting roots were then shown to harbour only rolB and no aux genes. Secondly, an extended non-coding region (1200 bp) at the 5 end of rolB had to be included in the construction. A shorter (300 bp) 5 region, including TATA and CCAAT boxes, was not sufficient to trigger root differentiation. Both the extended (B1185) and reduced (B310) 5 regions of rolB were then cloned upstream of the -glucuronidase (GUS) reporter gene and infections carried out both on the apical and on the basal side of carrot discs. Strong expression of GUS, visualized histochemically as an intense blue colouring of transformed cells was observed with B1185-GUS constructions on the apical side of the discs. Only occasionally could coloured cells be observed on the basal side of the discs with B1185-GUS and on both apical and basal sides with B310-GUS constructions. Strong GUS expression was, on the contrary, achieved on cells of both auxin-rich (apical) and auxin-depleted (basal) sides of the discs with the strong constitutive viral promoter, CaMV35S. These results indicate the presence of an upstream regulatory region which confers polar expression to the rolB gene and suggest a role for auxin in its activation.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

The Effect of Salicylic Acid on Peroxidase Activity in Wheat Calli Cocultured with the Bunt Pathogen Tilletia caries

The effect of salicylic acid (SA) on peroxidase activity in wheat (Triticum aestivum L.) calli cocultured with the bunt pathogen Tilletia caries was studied. Fungal infection was shown to activate cytoplasmic peroxidase. SA suppressed total peroxidase activity but did not inhibit the peroxidase with pI 9.8. A novel chitin-specific peroxidase with pI 3.5 appeared after the SA treatment. The infection of SA-treated cells with Tilletia caries activated the isoenzymes with pI 3.5, 4.8, and 7.5 and stimulated their secretion into the culture medium. The ability of SA to control wheat peroxidase activity during pathogenesis is discussed. The important role of this control in plant defense responses to the bunt pathogen is emphasized.     

Evolution of a protein superfamily: Relationships between vertebrate lens crystallins and microorganism dormancy proteins

Summary A search of sequence databases shows that spherulin 3a, an encystment-specific protein ofPhysarum polycephalum, is probably structurally related to the - and -crystallins, vertebrate ocular lens proteins, and to Protein S, a sporulation-specific protein ofMyxococcus xanthus. The - and -crystallins have two similar domains thought to have arisen by two successive gene duplication and fusion events. Molecular modeling confirms that spherulin 3a has all the characteristics required to adopt the tertiary structure of a single -crystallin domain. The structure of spherulin 3a thus illustrates an earlier stage in the evolution of this protein superfamily. The relationship of - and -crystallins to spherulin 3a and Protein S suggests that the lens proteins were derived from an ancestor with a role in stressresponse, perhaps a response to osmotic stress.     

Identification of hybrids betweenQuercus petraea andQ. robur (Fagaceae): Results obtained with RAPD markers confirm allozyme studies based on theGot-2 locus

RAPDs were employed as genetic markers to detect interspecific hybridization between the closely related oak speciesQuercus robur andQ. petraea. Fourteen primers were used in order to check the genetic status (pure or hybrid) of individuals classified morphologically. Among the 147 PCR fragments obtained 11 appear to be species-specific. In the phenotypically intermediate individuals different combinations of these species-specific bands were obtained. The patterns in these putative hybrids were not additive, which may be either the result of repeated backcrossing and introgression between the two species or of heterozygosity within the parental species. The results of the RAPD study are consistent with morphological analyses and allozyme data obtained for theGot-2 locus. Thus the RAPD markers used in this study may provide a powerful genetic tool for the identification of hybrids and the discrimination between the two pure species.     

Semper's (zoanthid) larvae: pelagic life,parentage and other problems

Semper's larvae were obtained from <300 out of 1800 plankton tows taken in the world's oceans (1964–1993). Zoanthellae (larvae of Sphenopidae) occurred at 217 stations and zoanthinae (larvae of Zoanthidae) at 86, the two larval types showing distributions clearly delimited by a minimum sea temperature (22 °C for zoanthellae, 18 °C for zoanthinae; a statistically significant difference, P<0.001). Length of formalin-fixed zoanthellae was 2–8.6 mm and of zoanthinae 1.5–5.9 mm. Endodermal zooxanthellae were present in 9/24 zoanthinae but in no zoanthellae (of 19). Three larvae contained an endo-commensal/parasitic amphipod. Septa were externally visible in larger zoanthinae and were counted in transverse sections of other larvae, a majority of which (both kinds) had 12 septa, the normal maximum. The pattern was brachycnemic in 40/43 larvae and anomalous (but non-macrocnemic) in three. If macrocnemic genera reproduce by Semper's larvae, they should have been represented in such a large sample. The distribution of adult Epizoanthus was examined: many species are deep sea (recorded down to 5000 m) but shallow-water species are relatively plentiful in, for example, the Adriatic and North Seas. No Semper's larva has ever been recorded from either. Some Parazoanthus species also occur in shallow water, especially associated with western Atlantic reef sponges. If they produce Semper's larvae, these have never been found. It is probable that macrocnemic zoanthids settle from planulae that do not develop into recognizable zoanthellae or zoanthinae.     

β-D-Glycan synthases and the CesA gene family: lessons to be learned from the mixed-linkage (1→3),(1→4)β-D-glucan synthase

Cellulose synthase genes (CesAs) encode a broad range of processive glycosyltransferases that synthesize (14)-D-glycosyl units. The proteins predicted to be encoded by these genes contain up to eight membrane-spanning domains and four `U-motifs' with conserved aspartate residues and a QxxRW motif that are essential for substrate binding and catalysis. In higher plants, the domain structure includes two plant-specific regions, one that is relatively conserved and a second, so-called `hypervariable region' (HVR). Analysis of the phylogenetic relationships among members of the CesA multi-gene families from two grass species,Oryza sativa and Zea mays, with Arabidopsis thaliana and other dicotyledonous species reveals that the CesA genes cluster into several distinct sub-classes. Whereas some sub-classes are populated by CesAs from all species, two sub-classes are populated solely by CesAs from grass species. The sub-class identity is primarily defined by the HVR, and the sequence in this region does not vary substantially among members of the same sub-class. Hence, we suggest that the region is more aptly termed a `class-specific region' (CSR). Several motifs containing cysteine, basic, acidic and aromatic residues indicate that the CSR may function in substrate binding specificity and catalysis. Similar motifs are conserved in bacterial cellulose synthases, the Dictyostelium discoideum cellulose synthase, and other processive glycosyltransferases involved in the synthesis of non-cellulosic polymers with (14)-linked backbones, including chitin, heparan, and hyaluronan. These analyses re-open the question whether all the CesA genes encode cellulose synthases or whether some of the sub-class members may encode other non-cellulosic (14)-glycan synthases in plants. For example, the mixed-linkage (13)(14)-D-glucan synthase is found specifically in grasses and possesses many features more similar to those of cellulose synthase than to those of other -linked cross-linking glycans. In this respect, the enzymatic properties of the mixed-linkage -glucan synthases not only provide special insight into the mechanisms of (14)-glycan synthesis but may also uncover the genes that encode the synthases themselves.     

Gene-sized DNA molecules of the macronuclei in three species of hypotrichs: Size distributions and absence of nicks

Macronuclear DNAs from three related hypotrichous ciliated protozoans were compared by agarose gel electrophoresis. Each was shown to be composed of DNA duplexes that yielded a unique pattern of bands overlying a continuous distribution of DNA sizes ranging from 400 bp to 20,000 bp. By EM, the number average molecular sizes for doublestranded DNA were 2,200 bp for Oxytricha sp., 2,514 bp for Stylonychia pustulata and 1,836 bp for Euplotes aediculatus. Contrary to previous reports we present evidence that the macronuclear DNAs in each of these three organisms lack single-stranded interruptions.     

Hexagonally ordered “rosettes” of particles in the plasma membrane ofMicrasterias denticulata Bréb. and their significance for microfibril formation and orientation

Summary Cells ofMicrasterias denticulata Bréb. at the stage of secondary wall formation have been studied by freeze-etching. It was found that the plasma membrane exhibits oval areas in which arrays of membrane particles occur. These particles form rosettes which are arranged in a hexagonally ordered lattice with a center to center spacing of 25 nm. Nearly the same periodicities can be found between microfibrils. It is concluded that the rosettes probably together with the thickened area of the plasma membrane below them represent the apparatus for the production and orientation of microfibrils. The hypothesis suggesting the incorporation of membrane templates functional in microfibril formation, originally advanced byKiermayer andDobberstein (1973) has received further support.     

Characterization of the photosynthetic electron transport chain in normal and photobleachedAnabaena cylindrica by flash spectroscopy

Electron transport of normal and photobleachedAnabaena cylindrica was studied using spectral and kinetic analyses of absorbance transients induced by single turnover flashes. Between 500 and 600 nm two positive bands (540 and 566 nm) and two negative bands (515 and 554 nm) were found. Absorbance changes at 515 and 540 nm were partly characterized. None of these absorbance changes represent an electrochromic shift. Absorbance changes at 554 and 566 nm correspond to the oxidation of cytochromef and the reduction of cytochromeb ₅₆₃, respectively. We found a very slight 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) sensitivity of cytochromef in normal cells, while DCMU was completely ineffective for cytochromef reduction in photobleached cells. The absorbance change of cytochromeb ₅₆₃ increased, while the absorbance change of cytochromef was smaller than in normal cells. The increased O₂ evolution in photobleached cells and the negligible electron transport via cytochromef suggest the participation of other electron acceptor(s) in the electron-transport chain of photobleachedAnabaena cylindrica.     

Effects of salinity on metabolism and life history characteristics of Daphnia magna

In the Baltic Sea area, the cladoceran Daphnia magna is commonly found in brackish water rockpools and it has been suggested that salinity is one of the niche dimensions that affects the distribution of the species. The salinity tolerance of D. magna was studied both in physiological and life history experiments. The experimental salinities were freshwater, 4S and 8S. The highest respiration and ammonium excretion rates were measured in the freshwater treatment with decreasing respiration and ammonium excretion rates at higher salinities. The lowest O/N ratio (oxygen consumption to ammonium excretion), describing the metabolic status of an organism, was obtained at 8S, although the only significant differences were detected when comparing to 4S treatments. Individual growth rate, reproductive output and population growth rate were highest at 4S. At 8S growth and reproduction were reduced as compared to freshwater and 4S. The life history parameters in the performed experiments indicated higher fitness (expressed as r) as well as more favourable conditions for growth and reproduction at 4S, whereas the O/N ratio was more difficult to interpret and, in this case, gave a less clear picture of the salinity influence.     

Release of carboxylic anions and protons by tomato roots in response to ammonium nitrate ratio and pH in nutrient solution

The exudation of certain organic anions and protons by roots which may affect solubility of metals and P and uptake by plants, is affected by nitrogen form and pH. The objective of this work was to study exudation of carboxylates and H⁺/OH^– by tomato plants in response to NH₄/NO₃ ratio and pH in nutrient solution. Four NH₄/(NH₄+NO₃) ratios (R= 0, 0.33, 0.67 and 1) and constant vs. variable solution pH treatments were investigated. The sum of the exudation rates of all carboxylates tended to decline with increasing R, particularly tri- and dicarboxylates. The molar fraction of the exuded tri- and dicarboxylates, averaged over all treatments and plant ages, increased in the order tartarate 2%), malate (6%), succinate (15%), citrate (26%) and fumarate (46%). At R=1 the solution pH dropped from 5.2 to 3 and at R=0 increased to 8. The R corresponding to the pH stat of tomato plant was 0.3. For the constant solution pH treatment, the effect of solution pH on carboxylate exudation rate was small as compared to the effect of R. The exudation of citrate and H⁺ efflux which were initiated when NO₃ and NH₄ uptake rates per plant exceeded certain threshold values, increased with plant age.     

Ultrastructure and behaviour of the spindle pole body of the aphid-pathogenic fungusErynia neoaphidis

Summary A detailed account of the ultrastructure and behaviour of the spindle pole body (SPB) of the entomophthoraceous fungusErynia neoaphidis is presented for the first time.The SPB consists of extranuclear (ENC) and intranuclear (INC) components. The ENC is a saucepan-shaped structure which lies in a pocket of the nuclear envelope. It is composed of a forked, fibrillar handle and a shallow, cylindrical pan. The pan has a wall of two layers, both of which are thickened with a regular periodicity so that they appear to be beaded. It is postulated that the pan is formed from rough endoplasmic reticulum and that it synthesizes the amorphous, electron-dense material coating the ENC.The INC is a saucer-shaped, electron-dense plaque in which the ends of the spindle microtubules terminate. During metaphase, a clear zone separates the INC from the nuclear envelope and persists until telophase. The roles of the amorphous, electron-dense material and the clear zone as well as the method of SPB replication are discussed.     

Purification and characterization of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Four antifreeze proteins (AFPs) were purified from larvae of the beetle Dendroides canadensis. The AFPs are similar in amino acid compositions, having high contents of hydrophilic amino acids (45–55 mol%) and cysteine (16 mol% Cys). Approximately half of the Cys residues form disulfide bridges, and both the disulfide bridges and free sulfhydryls are essential for activity. The N-terminals of the AFPs are blocked. The pH optimum of the AFPs is 7.8, but major loss of activity occurred only at very high pH (12.0). The detergents SDS and Triton X-100 did not inactivate the AFPs. Circular dichroism spectra indicate the presence of both and secondary structures in the AFPs, in addition to a large random structure component.Abbreviations AFP antifreeze protein - CD circular dichroism - DTT dithiothreitol - HPLC high pressure liquid chromatography - PAGE polyacrylamide gel electrophoresis - PAS periodic acid Schiff - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid     

<Emphasis Type="Italic">In vitro</Emphasis> screening of sugarcane to evaluate smut susceptibility

Tissue-cultured plantlets of three sugarcane (Saccharum spp.) cultivars having a known field smut reaction were screened for susceptibility to Ustilago scitaminea H&P Sydow. Plantlets were inoculated with 0.5 l of a suspension of equally mixed quantities of plus and minus mating type sporidia of U. scitaminea at concentrations ranging from 1×10¹ to 1×10⁶ cells. Fungal sori (whips) were produced in cultivar N12 (intermediate) 6weeks following inoculation with 1×10⁵ mixed sporidia and thereafter in cultivar NCo310 (susceptible) but not in cultivar N19 (resistant). Sori bearing teliospores were produced up to 3months following inoculation and incubation at 26°C. No sori were produced at mixed sporidial concentrations lower than 1×10⁵cells. The in vitro soral production in cultivars N19, N12 and NCo310 was 0, 27.5 and 47.5% respectively. Plantlets inoculated with 1×105sporidia of only one mating-type did not produce sori in any of the three cultivars tested. Blind scoring of an unknown sugarcane cultivar by this method corresponded exactly with its field smut rating.     

Different effects of inorganic and triethyl lead on growth and ultrastructure of lily pollen tubes

Summary Pollen tubes ofLilium longiflorum growingin vitro were treated for 1 h with inorganic lead (Pb) and with triethyl lead (TriEL) and studied by light and electron microscopy. Pb was considerably more toxic in relation to inhibition of pollen tube growth (EC₅₀=6 M Pb) than was TriEL (EC₅₀=60 M TriEL). On the other hand, at almost the entire concentration range tested (25-500 M) TriEL caused aberrant tubes and tube swellings. Pb did not cause tube swellings, even at highly growth-impairing concentrations. Pb (60 M) predominantly affected the ultrastructure of the growing cell walls without impairing the distribution of the cell organelles in the tube tips. In contrast, 50 and 100 M TriEL did not visibly influence cell wall ultrastructure but it severely damaged dictyosomes; 100 M TriEL also disturbed the original order of cell organelles in the tube tips. Cortical microtubules were selectively and completely destructed by TriEL at concentrations (50 M) where no effect on polar organization of the tube tips occurred but they remained unimpaired by 60 M Pb, indicating selective and effective interaction of TriEL with these cell organelles.Abbreviations EC⁵⁰ effective lead concentration causing 50% inhibition of pollen tube growth - MTs microtubules - Pb inorganic lead - TriAL trialkyl lead - TriEL triethyl lead     

Immunoreactivity of the corpuscles of Stannius of the garpike,Lepisosteus osseus,to antisera against salmon and trout stanniocalcins

Stanniocalcin-immunoreactive cells were localized in the corpuscles of Stannius of a holostean fish, the garpike (Lepisosteus osseus), using antisera against salmon and trout stanniocalcins and the peroxidase-antiperoxidase and protein A-gold immunohistochemical methods. The stanniocalcin-immunoreactive cells were periodic acid-Schiff-positive, and antibody staining was abolished if the antiserum was preabsorbed with corpuscle homogenate. Immunocytochemistry revealed two reactive cell types in the glandular parenchyma, and immunoreactivity was confined to the secretory granules. Staining of the granules was also abolished when the antisera were blocked with crude corpuscle homogenate. When corpuscle extracts from garpike were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis, a single dense band was evident with a molecular weight of 68 kDa under non-reducing conditions, whereas three bands were observed (29, 31, and 34 kDa) under reducing conditions. Staining of all bands disappeared following preabsorption of the antiserum with salmon stanniocalcin, trout stanniocalcin, or garpike corpuscle extract. The results are compared with stanniocalcins from another extant holostean, the bowfin (Amia calva), and from more modern bony fishes, the teleosts.     

Observations on the fine structure of the cercaria of Notocotylus attenuatus and formation of the cyst wall of the metacercaria

Summary The dorsal tegument of the mature cercaria of Notocotylus attenuatus is a syncytial, cytoplasmic layer, containing two types of secretory granule which are identifiable ultrastructurally. The type 1 secretory bodies are electron lucid, whereas most type 2 granules have a banded appearance. The ventral tegument contains granules which are secreted from the type 3 cells; the type 3 granules are membrane bound, electron dense, and consist of both an amorphous and a finely striated zone. The type 4 cells mainly contain cigar-shaped granules consisting of an amorphous core surrounded by concentric striations. The granules exhibit structural variability in shape and content. The type 4 cells undergo a cellular migration to the tegument during encystment. The structure of the posterior-lateral glands and mode of secretion of the granules are described. Possible functions of microtubules are discussed for each cell type. Details of some secretory processes involved in the formation of the hemispherical cyst wall are described. The layers of the cyst wall may be related to the granular contents of the various parenchymal cells of the cercaria. The tegument of the metacercaria originates primarily from the cytoplasm of the type 1, type 2, type 3 and type 4 cells.