Nitrogen and phosphorus cycling in relation to stand age of Eucalyptus regnans F. Muell

Lupins, canola, ryegrass and wheat fertilized with Na₂ ³⁵SO₄ and either ¹⁵NH₄Cl or K¹⁵NO₃(N:S=10:1), were grown in the field in unconfined microplots, and the sources of N and S (fertilizer, soil, atmosphere, seed) in plant tops during crop development were estimated. Modelled estimates of the proportion of lupin N derived from the atmosphere, which were obtained independently of reference plants, were used to calculate the proportion of lupin N derived from the soil. Total uptake of N and S and uptake of labelled N and S increased during crop development. Total uptake of S by canola was higher than lupins, but labelled S uptake by lupins exceeded uptake by canola. The form of N applied had no effect on uptake of labelled and unlabelled forms of N or S. Ratios of labelled to unlabelled S and ratios of labelled to unlabelled N derived from soil sources decreased during growth, and were less for S than for N for each crop at each sampling time. Although ratios of labelled to unlabelled soil-derived N were similar between crops at 155, 176 and 190 days after sowing, ratios of labelled to unlabelled S for lupins were higher than for the reference crops and declined during this period. The ratios of labelled to unlabelled S in lupins and the reference plants therefore bore no relationship either to ratios of labelled to unlabelled soil-derived N in the plants, or to total S uptake by the plants. Therefore the hypothesis that equal ratios of labelled N to unlabelled soil-derived N in legumes (Rleg) and reference plants (Rref) would be indicated by equal ratios of labelled to unlabelled S was not supported by the data. The results therefore show that the accuracy of reference plant-derived values of Rleg cannot be evaluated by labelling with ³⁵S.     

A simple method for the determination of affinity and binding site concentration in receptor binding studies

In ligand binding studies, it is often difficult to apply kinetic analyses because of an uncertainty in experimental data obtained at high ligand concentrations. Under such circumstances, K_d value (an index of the affinity) and the binding site concentration may be estimated more accurately from the binding of a fixed concentration of labelled ligand observed in the presence of various concentrations of the non-labelled ligand, if the fraction of both labelled and non-labelled ligand bound is small. When there is no cooperative effect of the ligand binding, the K_d value may be calculated by subtracting the concentration of the labelled drug from the concentration of the non-labelled drug to cause a 50% reduction of the saturable binding of the labelled drug. From above values, the binding site concentration may be calculated. The proposed method is capable of examining the cooperativity of the ligand binding, the labelled drug concentration and the specific radioactivity of the labelled drug and does not require large amounts of the labelled drug.     

DNA barcoding of fresh seafood in Australian markets reveals misleading labelling and sale of endangered species

Flake and shark samples were purchased from outlets in several coastal Australian regions and genetically barcoded using the cytochrome oxidase subunit 1 (CO1) gene to investigate labelling reliability and species-specific sources of ambiguously labelled fillets. Of the 41 shark fillet samples obtained, 23 yielded high-quality CO1 sequences, out of which 57% (n = 13) were labelled ambiguously (misleading) and 35% (n = 8) incorrectly. In contrast, barramundi fillets, which are widely available and sought after in Australian markets, were shown to be accurately labelled. Species identified from shark samples, including the shortfin mako (n = 3) and the scalloped hammerhead (n = 1), are assessed by the IUCN as endangered and critically endangered, respectively, with several others classified as vulnerable and near threatened.     

Gibberellin-photoaffinity labelling of two polypeptides in plant plasma membranes

Two polypeptides of M _r 68 kDa and 18 kDa were gibberellin (GA)-photoaffinity labelled in vitro in plasma membrane preparations from oat ( Avena sativa L.) aleurone and from leaves and stems of wild-type and GA-sensitivity mutants of different species. Labelling of these polypeptides could be competed by biologically active, but not by inactive, GAs, indicating the likely biological significance of these interactions. On 2-dimensional gels the radiolabelled polypeptides were each resolved as one intensely labelled low abundance spot with a slightly lower pI form adjacent to it. There was a strong pH dependency for both labelling events, which correlated well with pH values at which GA are known to be most biologically active. A semi-dwarf GA-sensitivity mutant of sweet pea ( Lathyrus odoratus L.), lb , showed reduced photoaffinity labelling of both polypeptides compared with the wild type, Lb . In the GA-insensitive Arabidopsis thaliana mutant, gai , the level of labelling was the same as in wild type, GAI . This is the first report of GA-binding proteins in plant plasma membranes. Some preliminary sequence data are given for one of the labelled polypeptides. We discuss these mutants and consider their possible roles in GA perception or action.     

A comparative study of DNA synthesis in cotyledon protoplast cultures of Brassica napus,B. campestris and B. oleracea using an immunocytochemical technique

The present study was designed (1) to observe the characterization of 5-bromo-2′-dexoyuridine (BrdU) incorporation into cultured Brassica cotyledon protoplasts and (2) to investigate the genetic differences in the levels of nuclear DNA synthesis (expressed by the percentage of nuclei labelled with BrdU) in cotyledon protoplast cultures from 12 cultivars of three Brassica species (Brassica napus, B. campestris and B. oleracea) at an early stage using immunocytochemistry. Nuclei labelled with BrdU were different from those showing only staining with 4′-6′-diamidino-2-phenylindole (DAPI) under fluorescence and light microscopy. Two to 5% of nuclei were labelled with BrdU after 1 h of culture, indicating that nuclear DNA synthesis occurred at a very early stage of culture. The percentage of nuclei labelled with BrdU increased with time over the length of the culture period. The mean percentage of nuclei labelled with BrdU in the 12 cultivars was about 25% at 24 h after culture initiation. The curve of the increase in percentage of nuclei labelled with BrdU exhibited an S-shape from 1 to 24 h. However, cultivar differences in percentages of nuclei labelled with BrdU were very significant over the time course of 1-24 h from initial culture, with cultivars Eureka (B. napus), Global (B. napus), Narc 82 (B. napus), Bunyip (B. campestris) and Sugar Loaf (B. oleracea) having a consistently higher percentage of nuclei labelled with BrdU than the other cultivars. Species differences were also significant, with cultivars of B. napus showing much higher percentages than the tested cultivars of B. campestris and B. oleracea. The results indicate that the differences in nuclear DNA synthesis in Brassica cotyledon protoplast cultures were most likely at both intra- and interspecies levels.     

Clearance Rates of Crustacean Macrofiltrators: The Nature of in situ Rate Depressions in a Fertilized Oligotrophic Lake in the Kuokkel Area,Northern Sweden

Lake seston labelled by ¹⁴CO₂, ¹⁴C-acetate and ³²-PO₄ in parallel was used in zooplankton feeding experiments and checked for size distribution of the labelled particulates. The in situ experiments were made to find out if depressed Eudiaptomus and Holopedium clearance rates, in an artificially fertilized lake where seston was dominated by μ-algae, were due to low retention of such algae. Addition of a labelled reference alga (Chlamydomonas sp.) was used to find out if high food concentrations (above the Incipient Limiting Concentration) or toxins caused the depressions. Clearance rates mostly ranked highest for CO₂-labelled seston followed by ³²P and ¹⁴C-acetate labelled seston, reflecting approximately the size distributions of the labelled food. Clearance rates for the added reference alga were high and similar in the fertilized lake and in an oligotrophic reference lake. It was concluded that the detected “rate depression” could be explained purely by poor retention of small particles (passive size selection) by the crustaceans studied, but that active food selection offers optional explanations to the experimental results.     

Functions of digestive diverticula in marine invertebrates

Summary The movement of labelled glucose along the digestive tract of two ascidian species, Sidnyum argus and Dendrodoa grossularia is compared. In Dendrodoa grossularia, glucose absorption takes place in all parts of the digestive tract except the oesophagus. Labelled glucose passes through the digestive tract in less than 12 h, and it accumulates in the pyloric ampullae in less than 3 days. In this species, the pyloric gland has more capacity to stock glucidic compounds than other tissues. Under normal feeding conditions, after five days, there is no transfer of labelled glucose to other tissues. Whatever the substances given to Sidnyum argus, its pyloric gland never accumulates labelled products.     

Replication in Drosophila chromosomes

Prolongation of larval life in Drosophila melanogaster, by growing wild type larvae at lower temperature, or in animals carrying the X-linked mutation giant is known to result in a greater proportion of nuclei in salivary glands showing the highest level of polyteny. We have examined by autoradiography the patterns of ³H-thymidine incorporation during 10 min or 1 min pulses in salivary gland polytene chromosomes of older giant larvae and of wild type late third instar larvae of D. melanogaster grown since hatching either at 24 ° C or at 10 ° C. The various patterns of labelling and their relative frequencies are generally similar in glands from the warm-(24 ° C) or cold (10 ° C)-reared wild type larvae, except the interband (IB) labelling patterns which are very frequent in the later group but rare in the former. The IB type labelled nuclei in cold-reared wild type larvae show labelling ranging from only a few puffs/interbands labelled to nearly all puffs/interbands labelled. In warm-reared wild type larvae, very low labelled IB patterns are not seen. In older giant larvae, the ³H-thymidine labelling patterns are in most respects similar to those seen in cold-reared wild type larvae. In 1 min pulsed preparations from all larvae, the IB patterns are relatively more frequent than in corresponding 10 min pulsed preparations. No nuclei with the continuous (2C or 3C) type of labelling pattern, with all bands and interbands/puffs labelled, were seen in 1 min pulsed preparations from cold-reared wild type or in giant larvae, and only a few nuclei in 1 min pulsed preparations from warm-reared wild type larvae exhibited the 2C labelling pattern. Analysis of silver grain density on specific late replicating sites in late discontinuous (1D) type labelled nuclei suggests that the rate of DNA synthesis per chromosomal site is not different at the two developmental temperatures. It is suggested that correlated with the prolongation of larval life under cold-rearing conditions or in giant larvae, the polytene replication cycles are also prolonged. It is further suggested that the polytene S-period in these larvae is longer due to a considerable asynchrony in the initiation and termination of replication of different sites during a replication cycle.     

Lectin binding and uptake and glycoprotein characterization of isolated porcine aortic endothelial and smooth muscle cells

Endothelial and smooth muscle cells were isolated from porcine aorta and kept in short-term culture. To determine the terminal carbohydate composition of the plasma membranes from both cell populations, the cells were incubated with a panel of fluorescein-labelled lectins. Both cell populations shared a number of terminal carbohydrates, but the N-galactosamine specific lectin Wistaria floribunda agglutinin labelled only the endothelial cells. A lectin which selectively labelled smooth muscle cells was not found. Western blot analysis of isolated endothelial cell membrane glycoproteins indicated that most membrane glycoproteins are labelled by Wistaria floribunda agglutinin.     

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas

Summary Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): l-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis-to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.     

Immunocytochemical localization of pheromone-binding protein in moth antennae

Summary Odorant-binding proteins are supposed to play an important role in stimulus transport and/or inactivation in olfactory sense organs. In an attempt to precisely localize pheromone-binding protein in the antenna of moths, post-embedding immunocytochemistry was performed using an antiserum against purified pheromone-binding protein of Antheraea polyphemus. In immunoblots of antennal homogenates, the antiserum reacted exclusively with pheromone-binding protein of A. polyphemus, and cross-reacted with homologous proteins of Bombyx mori and Autographa gamma. On sections of antennae of male A. polyphemus and B. mori, exclusively the pheromone-sensitive sensilla trichodea are labelled; in A. gamma, label is restricted to a subpopulation of morphologically similar sensilla trichodea, which indicates that not all pheromone-sensitive sensilla contain the same type of pheromone-binding protein and accounts for a higher specificity of pheromone-binding protein than hitherto assumed. Within the sensilla trichodea, the extracellular sensillum lymph of the hair lumen and of the sensillum-lymph cavities is heavily labelled. Intracellular label is mainly found in the trichogen and tormogen cells: in endoplasmic reticulum, Golgi apparatus, and a variety of dense granules. Endocytotic pits and vesicles, multivesicular bodies and lysosome-like structures are also labelled and can be observed not only in these cells, but also in the thcogen cell and in the receptor cells. Cell membranes are not labelled except the border between thecogen cell and receptor cell and the autojunction of the thecogen cell. The intracellular distribution of label indicates that pheromone-binding protein is synthesized in the tormogen and trichogen cell along typical pathways of protein secretion, whereas its turnover and decomposition does not appear to be restricted to these cells but may also occur in the thecogen and receptor cells. The immunocytochemical findings are discussed with respect to current concepts of the function of pheromone-binding protein.     

Stable isotope labelling method for studies of saccharide metabolism in Agardhiella subulata

Stable isotopes are preferable in many ways to radioactive isotopes for metabolic studies designed to elucidate biosynthetic pathways. We have developed the methodology to utilize ¹³C-labelled compounds in tracer studies of saccharide metabolism in the red algae. Cultures of Agardhiella subulata were pulse-chase labelled with ¹³C0₂ and ¹²C0₂. Gas chromatography/mass spectrometry (GC-MS) and ¹³C-NMR provided for positive identification of labelled carbohydrate metabolites. In addition, GC-MS can be used to profile the monosaccharide composition of algal species and combined GC-MS and ¹³C-NMR can disclose which carbon(s) is (are) labelled and the extent of labelling. In ¹³C0₂ incubated plants, the label is clearly detected in floridoside and floridean starch. After chasing the labelled alga with ¹²CO₂ for three days or storing the pulse-chase labelled alga in darkness for 6 days, labels disappeared from both floridoside and starch and the contents of these two carbohydrates became very low. More detailed biochemical analysis is being continued to identify labelled cell wall polysaccharides and/or their precursors.     

Spin label saturation transfer ESR studies of protein-lipid interactions in Photosystem II-enriched membranes

Saturation transfer ESR has been used to study the dynamic behaviour of lipids in the appressed regions of thylakoid membranes from pea seedlings. Four different phospho- and galacto-lipid spin labels (phosphatidylcholine labelled at the 12 or 14 C-atom positions of the sn-2 chain, phosphatidylglycerol labelled at the 14-position of the sn-2 chain, and monogalactosyldiacylglycerol labelled at the 12-position of the sn-2 chain) were used to probe the lipid environment in photosystem II-enriched membranes prepared by detergent extraction. The ESR spectra show that the majority of the lipid in these preparations is strongly motionally restricted. Values for the effective rotational correlation times of the labelled chains were deduced from the lineheight ratios and integrals of thhe saturation transfer ESR spectra. The effective rotational correlation times were found to be in the 10⁵ range, indicating a very low lipid chain mobility which correlates with the low lipid content of these preparations. Comparison of the effective rotational correlation times deduced from the different diagnostic regions of the spectrum revealed little anisotropy in the chain mobility, indicating that the dominant motional mode was trans-gauche isomerization. The effective rotational correlation times deduced from the spectral integrals were similar to those deduced from the lineheight ratios, consistent with the absence of any appreciable fluid lipid component in these preparations. The results also indicate some selectivity of interaction between the lipid species, with phosphatidylcholine exhibiting appreciably slower motion than either phosphatidylglycerol or monogalactosyldiacylglycerol.     

Evidence for the priming role of the central retinula cell in ommatidium differentiation of Ephestia kuehniella

Summary In the developing compound eye of Ephestia kuehniella, within the advancing front of differentiation, regular cell clusters arise which consist of a central cell and two flanking cells. The central cell is destined to become the basal retinula cell later in development. Its crucial role in ommatidium formation is confirmed by ³H-thymidine labelling. Eye anlagen labelled early in the pupal stage incorporate thymidine within two distinct zones along the front of differentiation. After the ommatidia are completely differentiated, both zones contain labelled nuclei of all cell types which participate in ommatidia formation. Within the posterior zone, however, the basal retinula cells are always unlabelled, whereas in the anterior they show labelled nuclei. From this observation it must be concluded that the basal retinula cell first terminates proliferation (either alone or together with a few other cells) to become differentiated as the central retinula cell. These results agree with those found in Drosophila and indicate that the ordered stepwise addition of cells to a central founder cell is a widespread principle of ommatidia formation in insects.     

A simple technique for producing 13C or 14C-labelled fronds of Lemna gibba and their use in soil incubation investigations

The duckweed Lemna gibba required light and a suitable energy source such as sucrose, glucose or fructose, for maximum growth in culture. The requirement for light was relatively unimportant and the plants grew well in a photon flux density of only 52 μmol m^-2s^-1 PAR. The uptake and incorporation of uniformly labelled ¹⁴C-glucose into fronds was related only to the concentration of the sugar. When incubated with soil, labelled L. gibba behaved in a manner similar to that of labelled ryegrass roots which had been produced by a more elaborate technique using a ¹⁴CO₂ labelled atmosphere. During incubation with soil for 224 days the L. gibba material (specific activity 6133 Bq mg^-1 d. wt) lost 64% of its radioactivity as ¹⁴CO₂ and ryegrass (specific activity 6634 Bq mg^-1 d. wt) lost 49%. Alkaline extracted humic and fulvic acids from soil had specific activities for the L. gibba incubation of 3409 and 407 Bq mg^-1 solid and for ryegrass roots of 4609 and 546 Bq mg^-1 solid respectively. The production of ¹³C or ¹⁴C-labelled L. gibba can be undertaken using only simple equipment producing material the specific radioactivity of which can be controlled by adjusting the activity of the sugar energy source.     

Immunocytochemical detection of lignin-related epitopes in cell walls in bryophytes and the charalean alga Nitella

Lignins are complex phenolic heteropolymers present in xylem and sclerenchyma cell walls in tracheophytes. The occurrence of lignin-like polymers in bryophytes is controversial. In this study two polyclonal antibodies against homoguaiacyl (G) and guaiacyl/syringyl (GS) synthetic lignin-like polymers that selectively labelled lignified cell walls in tracheophytes also bound to cell walls in bryophytes, the GS antibody usually giving a stronger labelling than the G antibody. In contrast to tracheophytes, the antibody binding in liverworts and mosses was not tissue-specific. In the hornworts Megaceros flagellaris and M. fuegiensis the pseudoelaters and spores were labelled more intensely than the other cell types with the GS antibody. The cell walls in Nitella were labelled with both antibodies but no binding was observed in Coleochaete. The results suggest that the ability to incorporate G or GS moieties in cell walls is a plesiomorphy (primitive character) of the land plant clade.     

Lipopolysaccharide mobility in leaf tissue of Arabidopsis thaliana

Bacterial lipopolysaccharides (LPS) are triggers of defence responses in plants, and induce local as well as systemic acquired resistance. Arabidopsis thaliana plants pretreated with LPS show an increased resistance to the virulent bacterial plant pathogen Pseudomonas syringae pv. tomato DC3000. To investigate the mobilization and transport of LPS in Arabidopsis leaves, fluorescently labelled LPS (Alexa Fluor® 488 conjugate) from Salmonella minnesota was used. Leaves were pressure infiltrated with fluorescein‐labelled LPS and fluorescence microscopy was used to follow the movement and localization of LPS as a function of time. The observation of leaves 1 h after supplementation with fluorescein‐labelled LPS revealed a fluorescent signal in the intercellular space. Capillary zone electrophoresis was used for the detection and analysis of the labelled LPS in directly treated leaves and systemic leaves. In addition, gel electrophoresis was used to confirm LPS mobilization. The results indicated that LPS mobilization/translocation occurs through the xylem from local, treated leaves to systemic, untreated leaves. Consequently, care should be taken when ascribing the observed biochemical responses and induced resistance from LPS perception as being uniquely local or systemic, as these responses might overlap because of the mobility of LPS in the plant vascular system.     

Nitrogen assimilation in field-grown winter wheat: Direct measurements of nitrate reduction in roots using15N

Summary Nitrate fertiliser labelled with¹⁵N was applied to a field grown crop of winter wheat. Uptake and assimilation of fertiliser nitrate was studied by monitoring the appearance of labelled nitrate and labelled amino acids in the xylem sap. Shortly after applying¹⁵N-nitrate to the soil about 30 per cent of recently absorbed¹⁵N was in the reduced form, indicating that roots of cereal crops can make a substantial contribution in reducing nitrate. Seasonal changes in crop growth andin vivo NRA are also described.     

An in planta induced gene of Phytophthora infestans codes for ubiquitin

An in planta induced gene of Phytophthora infestans (the causal organism of potato late blight) was selected from a genomic library by differential hybridization using labelled cDNA derived from poly(A)+ RNA of P. infestans grown in vitro and labelled cDNA made from potato-P. infestans interaction poly(A)+ RNA as probes. Sequence analysis showed that the gene codes for ubiquitin, a highly conserved protein which plays an important role in several cellular processes. The structure of the polyubiquitin gene (designated ubi3 R) is consistent with the structure of other known polyubiquitin genes. It consists of three repeats in a head-to-tail arrangement without intervening sequences, each encoding a ubiquitin unit of 76 amino acids. The last ubiquitin unit is followed by an extra asparagine residue at the carboxy-terminal end. Northern and Southern blot analyses revealed that the polyubiquitin gene is a member of a multigene family, all genes of which show induced expression in planta.     

Cell cycle and DNA content of mitotic cells in brain ganglia ofdrosophila larvae

The programmes of replication of hetero- and euchromatin regions, mitotic cell cycle and the DNA content in metaphases in brain ganglia from late third instar larvae ofDrosophila melanogaster (wild type and a tumour bearing mutant, 1(2)gl, strain) and ofDrosophila nasuta were examined by autoradiography of [³H]thymidine labelled (continuous or pulse) cells and by cytophotometry, respectively. Brain ganglia labelled continuously with [³H]thymidine for 24 hin vitro showed a significantly high proportion of cells with incorporation of radioactivity restricted to heterochromatin only. Pulse labelling of brain ganglia from larvae ofDrosophila melanogaster andDrosophila nasuta followed by chase for different time intervals showed that (i) the frequency of labelled metaphases was more than 50% within 15 to 30 min of chase and remained higher than 50% in nearly all the chase samples till 24 h, (ii) euchromatin labelled metaphases appeared with a low frequency within 1 to 4 h chase period but the heterochromatin labelled metaphases continued to be more common in the later chase samples also, (iii) single chromatid labelled second cycle metaphases were seen within 1 to 4 h after the pulse, but their frequency did not increase in the later samples. Cytophotometry of feulgen-DNA and Hoechst 33258 stained metaphases in late third instar larval brain ganglia revealed a greater variation in the DNA content of individual metaphases, although the means were close to the expected 4 C content. It appears that in relation to the known asymmetric cell divisions of neuroblast and other neural cells, the mitotically active cells in brain ganglia comprise a heterogenous population with widely varying lengths of the different phases of cell cycle; some of them may not cycle regularly and may possibly have a discontinuous S-phase.