Stable transformation of cultured cells of the liverwortMarchantia polymorpha by particle bombardment

Suspension-cultured cells (A-18 line) of the liverwortMarchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene (hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×10⁶ cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines.     

高维非自治系统的周期解

本文通过建立并利用齐次线性方程解的估计公式,获得了周期系统（1）的周期解的存在性、唯一性定理,对周期系统（2）给出了一个平稳振荡定理,最后给出了实例。     

Temporal and spatial variations in the oxygen-18 content of leaf water in different plant species

Temporal variations in the δ¹⁸ oxygen (δ¹⁸O) content of water transpired by leaves during a simulated diurnal cycle fluctuated around the δ¹⁸O content of the source water. Reconstructed variations in the δ¹⁸O values of leaf water differed markedly from those predicted by conventional models. Even when transpiring leaves were maintained under constant conditions for at least 3 h, strict isotopic steady-state conditions of leaf water (equality of the ¹⁸O/¹⁶O ratios in the input and transpired water) were rarely attained in a variety of plant species (Citrus reticu-lata, Citrus paradisi, Gossypium hirsutum, Helianthus annuns, Musa musaceae and Nicotinia tabacum). Isotopic analysis of water transpired by leaves indicated that leaves approach the isotopic steady state in two stages. The first stage takes 10 to 35 min (with a rate of change of about 3–3%h^?1), while in the second stage further approach to the isotopic steady state is asymptotic (with a rate of change of about 0–4% h^?1), and under conditions of low transpiration leaves can last for many hours. Substantial spatial isotopic heterogeneity was maintained even when leaves were at or near isotopic steady state. An underlying pattern in this isotopic heterogeneity is often discerned with increasing ¹⁸O/¹⁶O ratios from base to tip, and from the centre to the edges of the leaves. It is also shown that tissue water along these spatial isotopic gradients, as well as the average leaf water, can have ¹⁸O/¹⁶O ratios both lower and higher than those predicted by the conventional Craig and Gordon model. We concluded, first, that at any given time during the diurnal cycle of relative humidity the attainment of an isotopic steady state in leaf water cannot be assumed a priori and, secondly, that the isotopic enrichment pattern of leaf water reflects gradual enrichment along the water-flow pathway (e.g. as in a string of pools), rather than a single-step enrichment from source water, as is normally assumed.     

Monitoring uptake and contents of Mg,Ca and K in Norway spruce as influenced by pH and Al,using microprobe analysis and stable isotope labelling

In a model system using intact spruce trees (Picea abies [L.] Karst.) we followed the path of magnesium, calcium and potassium during uptake into the root and during long-range transport into the shoot, by multiple stable isotope labelling. The roots of two- and three-year-old spruce trees originating from soil culture were removed from the soil and, in part or in toto, exposed to labelling solutions containing the stable isotopes ²⁵Mg or ²⁶Mg, ⁴¹K and ⁴²Ca or ⁴⁴Ca. Optical-emission-spectroscopy (ICP-OES) of plant fractions and labelling solutions was combined with the quantitative analysis of stable isotope ratios in sections of shock frozen, cryosubstituted material using the laser-microprobe-mass-analyser (LAMMA). This combination allowed us to distinguish, both in bulk samples and on the cellular level between (i) the fraction of elements originally present in the plant before the start of the labelling, (ii) the material taken up from the labelling solution into the plant and (iii) any material released by the plant into the labelling solution.In single-root labelling experiments, roots of three-year-old spruce trees, grown in nursery soil, were exposed to various pH conditions. The exchange of Mg and Ca with the labelling solution was nearly 100% in the cell walls of the mycorrhized finest roots. This exchange was only slightly affected by a step down to pH 3.5. The absolute Mg and Ca content in the cell walls was moderately reduced by incubation at pH 3.5 and strongly reduced in the presence of Al at this pH. After a pH 3.5 and 2 mM Al treatment we found Al in the xylem cell walls and the cortex cell lumina at elevated concentrations. To analyse the combined effect of high Al and high proton concentrations on the long-range transport, we used a split-root system. The root mass of an intact two-year-old spruce tree, grown in mineral soil, was divided into even parts and both halves incubated in solutions with two sets of different stable isotopes of Mg and Ca (side A: no Al, ²⁵Mg and ⁴²Ca; side B: +Al, ²⁶Mg and ⁴⁴Ca) and ⁴¹K on both sides. We observed a large uptake of Mg, Ca and K into the plant and a pronounced release. The net uptake of all three elements was lower from the Al-doted solution. In cross-sections of the apical shoot we found after seven-day labelling period about 60–70% of the Mg and Ca and 30% of the K content in the xylem cell walls originating from both labelling solutions. The clear majority of the Mg and Ca label originated from the Al-doted side.     

Root water uptake of field-growing plants indicated by measurements of natural-abundance deuterium

Measurements of stable-isotope ratios of water extracted from stems and, in some studies, soils are increasingly being used to study the integrated root function of field-growing plants. This study explored if additional measurements on water extracted from roots could indicate the activity of roots in different areas of the soil profile and their influence on canopy water sources, so providing advantages over more common sampling strategies. Studies were conducted on trees and shrubs located in diverse habitats: a saline, semi-arid floodplain, a subhumid mountain-range front and a cold desert. At each site, roots, soil immediately surrounding the roots, and plant stems were sampled. Roots were taken from different depths in the soil, to approximately 2 m at one site. Overall, 80% of roots sampled had H isotope ratios different from the surrounding soil. The differences up to 37, were significant (p<0.05) at two of the sites. Thus water in most of the roots sampled did not come entirely, if at all, from the surrounding soil, illustrating movement and possible mixing of water within the root system. This condition was not simply related to the availability of water surrounding the soil, which was also measured. There were also differences in root and stem H isotope ratios (up to 17) in 67% of samples, although the difference was only significant in shallow samples from the floodplain. The general similarity in stem and root ²H values indicates that most roots sampled were involved in the main supply of water to the canopy. Patterns of root function varied between the individual sites. Trees were primarily using groundwater at the floodplain and mountain front sites, as the surface soils had mean matric potentials of-1800 kPa. At the mountain front site, the surface roots were transporting groundwater to the canopy in isolation form the surrounding soil. In contrast, surface roots at the floodplain were taking up water from the surrounding soil, although this water was not a significant source in the trees' overall water supply. This activity of surface roots would not have been evident from the ²H data without the root samples. At the cold desert the roots in moist surface soil provided the main source of water for the shrubs. There too the root data indicated different water uptake patterns than otherwise would have been assumed. The root data showed that groundwater could not have been a water source, a conclusion which had been reached in a previous study. Thus measurements of stable isotope ratios in root water may be a valuable tool in assessing water uptake patterns and root function.     

Uptake of 24Mg by excised pine roots: A preliminary study

Uptake of ²⁴Mg by excised roots of Pinus sylvestris L. during up to 4 h long incubations in 99.9 atom % ²⁴Mg (50 M) was measured by ICP-MS. A rapid initial uptake phase (30 min) was followed by a slower uptake. This was interpreted as a shift from a phase dominated by saturable ion exchange (free space uptake), to a non-saturable phase, during which the rate of uptake was 0.077±0.0.012 mol Mg g^–1 (d.wt.) h^–1. The metabolic uncoupler DNP (2,4-dinitrophenol) at 50 M decreased the Mg uptake rate by 35% only, but the effect of DNP was significant (p<0.01). Several problems related to a high variability in the experimental material were encountered, and further refinement of this approach in studies of plant Mg uptake is suggested.     

SOURCES OF INORGANIC CARBON FOR PHOTOSYNTHESIS BY THREE SPECIES OF MARINE DIATOM1

The utilization of inorganic carbon by three species of marine diatom, Skeletonema costatum (Grev.) Cleve. Ditylum brightwellii (West) Grun., and Chaetoceros calcitrans Paulsen was investigated using an inorganic carbon isotopic disequilibnum technique and inorganic carbon dose-response curves. Stable carbon isotope data of the diatoms are also presented. Observed rates of photosynthetic oxygen evolution were greater than could be accounted for by the theoretical rate of CO₂ supply from the uncatalyzed dehydration of HCO₃^? in the external medium, suggesting use of HCO₃^? as an inorganic carbon source. Data from the isotopic disequilibrium experiment demonstrate the use of both HCO₃^? and CO₂ for photosynthesis. Carbon isotope discrimination values support the use of HCO₃^? by the diatoms.     

Establishment and maintenance of the breeding territory in the polygynous great reed warbler

Settlement of male great reed warblers in the breeding ground was highly asynchronous at Lake Biwa in Japan. It took over 1 month from the appearance of the first male to a saturation in number of males. In resource-defense-polygyny, males are expected to try to defend as large an area as possible in the optimal habitat. In fact, a small number of the earliest settling males divided up the breeding ground almost completely as territories among themselves and these were later reduced in size with the addition of later settlers. The reduction was not due to a seasonal decline of aggressiveness on the part of the owners but to a higher level of intrusion pressure by later arriving males. The neighbor-neighbor relationship, once established, was rather stable. Home range overlap was small and territorial contacts were few between neighbors. Territorial boundaries seem not to shift despite the addition of new males as long as the neighbors were the same. The stable relationships between neighbors are expected to help the earlier resident to maintain his large and good territories throughout the breeding season and hence polygyny is favored in this species.     

Efficient membrane targeting of the chick nicotinic acetylcholine receptor α-subunit in stable insect cell lines

We have synthesised the -subunit of the chick nicotinic acetylcholine receptor (nAChR) in stable, continuous insect (Spodoptera frugiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovius immediate early gene promoter. Transformed cells were obtained by co-transfection of the insect cells with pIEK1.nAChR, encoding the -subunit cDNA, and pIEK1.neo, encoding the neomycin resistance gene. G-418-resistant clones were selected and expanded into continuous cell lines synthesising the chick nAChR -subunit. Using fluorescence microscopy and ligand binding studies we were able to demonstrate efficient membrane targeting of the receptor subunit in the insect cell plasma membrane. Stable insect cell lines may thus have significant advantages over transient baculovirus vectors for the synthesis and characterisation of heterologous receptor proteins.Abbreviations AcNPV Autographa californica nuclear polyhedrosis virus - BTX -bungarotoxin - BSA bovine serum albumin - FITC Fluoroscein isothiocyanate - G418 geneticin-418 - hpi hours post-infection - ie-1 immediate early 1 gene - nAChR nicotinic acetylcholine receptor alpha subunit - Sf Spodoptera frugiperda - tPA tissue plasminogen activator