An improved method of cell fractionation allowed the extraction of soluble (sNR) and membrane-associated (mNR) forms of nitrate reductase (NR) from a dinoflagellate, even though in previous studies only mNR had been found in these algae. Both activities were assayed in cell-free extracts of Peridinium gatunense from Lake Kinneret, Israel, after disruption of the cells and differential centrifugation. In the cultures used, sNR showed much higher NO3−-reducing activity. Only a low proportion, 2.5–3% of NR activity, was found to be associated with mNR. Moreover, mNR comprised two forms as indicated by protein solubilization: a tightly membrane-bound and a more weakly attached NR. Ascorbate inhibited all NR activities, but that of mNR recovered after its removal. Polyvinyl pyrrolidone (PVP) and DTT also diminished sNR and mNR activities. For both enzymes, pH optima (7.65) and temperature optima (13–25°C) were similar, and agreed with those for optimum growth of P. gatunense both in culture and in the lake. The most efficient electron donor was NADH, though NADPH sustained low NR activities. Carboxylic anions such as succinate and malate did not support any reduction of NO3−, nor did they cause any stimulation of sNR or mNR activities. Both forms of NR showed a high affinity for their substrates: Km was c. 10 μM for NO3− and c. 5 μM for NADH. The high efficiency of NO3− assimilation by Peridinium seems to be limited mainly by energy under otherwise optimal nutritional conditions and, at low nitrate concentrations, the low Km may be one of the main reasons for the high competitivity of this alga in Lake Kinneret.

This study examines the microtubular cytoskeleton during megasporogenesis in the Nun orchid, Phaius tankervilliae. The subepidermal cell located at the terminal end of the nucellar filament differentiates first into an archesporial cell and then enlarges to become the megasporocyte. The megasporocyte undergoes the first meiotic division, giving rise to two dyad cells of unequal size. Immunostaining reveals that microtubules become more abundant as the megasporocyte increases in size. Microtubules congregate around the nucleus forming a distinct perinuclear array and many microtubules radiate directly from the nuclear envelope. In the megasporocyte, prominent microtubules are readily detected at the chalazal end of the cell cytoplasm. After meiosis I, the chalazal dyad cell expands in size at the expense of the micropylar dyad cell. At this stage, new microtubule organizing centres can be found at the corners of the cells. The appearance of these structures is stage-specific and they are not found at any other stages of megasporogenesis. The functional dyad cell undergoes the second meiotic division, resulting in the formation of two megaspores of unequal size. The chalazal megaspore enlarges and eventually gives rise to the embryo sac. As the functional megaspore expands, the microtubules again form a distinct perinuclear array with many microtubules radiating from the nuclear envelope. A defined cortical array of microtubules has not been found in P. tankervilliae during the course of megasporogenesis.

Gravitropic responses of oat coleoptiles were measured in different growth media; humid air, natural soil and artificial soil (glass beads). The oat coleoptiles in soil and glass beads were monitored by NMR imaging, while those in humid air were imaged in darkness with an infrared-sensitive charge-coupled device (CCD) camera. The present study shows for the first time that gravitropic experiments can be performed in artificial soil using NMR imaging as a convenient and suitable recording method. Not only was it possible to follow the gravitropic curvatures in natural soil, but the artificial soil allowed plant images of sufficient spatial and temporal resolution to be recorded. The advantages of using artificial soil in magnetic resonance imaging studies are that the iron content of glass beads is very low compared with natural soil, and that the artificial soil matrix can easily be standardized with regard to particle size distribution and nutrient content. Two types of glass beads were used, the diameter of the small and the large beads being 300–400 and 420–840 μm, respectively. The growth rate of the coleoptiles in soil and in big beads was roughly the same and only slightly lower than in humid air, whereas small beads reduced the growth rate by approx. 16%. The bending rate of the coleoptiles during the gravitropic response was reduced by c. 65% in soil and 75% in bead mixtures relative to bending in air. It should be noted, however, that the maximum curvature of the coleoptile tip was of the same order in all cases, about 35°. This value may represent the largest possible curvature of the organ. The potential of NMR imaging to study how plant organs penetrate the soil under the influence of gravitropism, mechanical impedance and thigmotropism is also discussed.

Multiple-marker techniques based on PCR are highly informative in genetic analysis. The bulk of many genomes is comprised of repeated sequences, and thus anonymous markers derived from genomic DNA are likely to correspond to repeated DNA sequences. Here we show that repeated-sequence-derived markers can provide information similar in quality to that of other PCR-based markers. Presumably this reflects the abundance of this repeat type in anonymous PCR markers such as RAPDs or AFLPs.

The role of cytosolic pH (pHc) in growing germ tubes of the filamentous fungus Magnaporthe grisea was analysed by confocal ratio imaging of the pH-sensitive fluorescent dye 5(6)-carboxyseminaphthorhodafluor-1 (SNARF-1). The cytosol of these cells was successfully loaded with the acetoxymethyl ester of the dye and the pHc was visualized and quantified during conidium germination, germ tube growth and appressorium induction by simultaneous dual-emission confocal ratio imaging. Calibrations of the free acid in vitro and calibrations in vivo produced results indicating a similar dynamic response in the pH range 6.0–8.0 for both methods. The pHc in growing germ tubes was consistently pH 7.2±0.1 during all developmental stages analysed. Only slight changes in pHc (<0.1 pH unit) were found in response to alkaline external pH (pH 8.0). No changes in pHc occurred in response to an acidic extracellular pH (pH 6.0) or to the presence of nutrients. There was no observation of either pronounced gradients or changes in pHc in growing germ tubes accompanying conidium germination, germ tube growth or early appressorium formation.

During the middle of the 1992 austral winter in the northern Windmill Islands, continental Antarctica, a highly unusual climatic event occurred in which the air temperature exceeded 0°C for some 60 h, at the end of which there was a significant rain shower before the ambient temperature returned to subzero conditions. This event caused most of the snow cover to melt and refreeze as clear ice. Lichens were thus rehydrated in the dark, in some places completely inundated, then frozen in ice. The effect that these conditions had on the distribution of K, Na, Mg and Ca within the thallus was estimated for two of the dominant macrolichen species, Umbilicaria decussata (Vill.) Zahlbr. and Usnea sphacelata R.Br, from three sites on a knoll on Clark Peninsula. One site acted as a natural control, owing to the very deep snow cover at the site, which protected lichens from the rewetting event. Despite persistent differences between species and the various sites, there was no overall effect of the climatic event on the membrane integrity of either lichen species. Only Usnea sphacelata from the most exposed site showed a significant leakage of K across the cell membrane, which indicated a loss of membrane integrity. Overall, both species were tolerant of the extreme conditions, although Umbilicaria decussata was the more tolerant.

Previous work on Lolium perenne showed that sucrose[ratio ]sucrose fructosyltransferase (SST) activity did not increase concomitantly with fructan synthesis in regrowing leaves or in mature leaf blades of plants that have been subjected to carbohydrate-accumulating conditions. This was contrary to the pattern of SST activity in roots and stubble. To obtain further insight into the fructan synthesizing activities and to explain this discrepancy, total fructosyltransferase activity (FT) was assayed by increasing the sucrose and the soluble enzyme concentrations and was compared to sedimentable phlein sucrase activity (PS) throughout the regrowth period following defoliation in leaves, stubble and roots. Before analysis on 2-month-old plants, PS activity was characterized in dark-grown coleoptiles, using [U-14C]sucrose. PS activity had a pH optimum of 6.0 and produced 1-kestose in addition to high molecular weight fructans with a mean DP of 9. In 2-month-old plants, sedimentable PS and FT soluble reactions contained an initial sucrose concentration of 160 mM and 400 mM and proteins equivalent to 1.4 and 2.1 g f. wt of tissue, respectively. In stubble and roots, the FT preparation catalysed the synthesis of large fructans, and the overall pattern resembled the native fructans when separated by anion exchange HPLC. In regrowing leaves, the FT preparation produced low-DP fructans relatively more than in vivo but synthesized the high-DP fructan characteristic of the tissue. Moreover, FT activity did not remain at a low level like SST activity but increased from day 5 after defoliation when fructans began to accumulate. PS activity formed very few low- DP fructans and 1-kestose was the main product. Trisaccharides generated by PS activity represented 2–5% of the total trisaccharide synthesis. High-DP fructans were detectable only when the products of the reaction were concentrated 100 times. Results are discussed with respect to the relative contribution of FT and PS activities for the synthesis of 1-kestose and fructans in roots, stubble and leaves of Lolium perenne.

This paper describes the effects of hypomethylation with 5-azacytidine (5azaC) and dihydroxypropyladenine (DHPA) on protonemata of the moss Funaria hygrometrica Hedw. Following treatment with 5azaC or DHPA, hypomethylation of the EcoR II (CCA(T)GG) and Pst I (CTGCAG) sites in ribosomal DNA (rDNA) was confirmed using restriction enzyme analysis and Southern hybridization. Hypomethylation of the genome had profound effects on protonemal differentiation. Whilst apical cell organization and cell dimensions and shape remained unchanged, there was a marked retardation in both cytoplasmic and nuclear differentiation. Developmental abnormalities included: late and erratic side branch formation, some loss of the distinction between chloronema and caulonema, formation of aberrant buds, and loss of the potential to form brood and tmema cells after 5azaC treatment which in general had more profound effects than DHPA. Cytologically, caulonema cells were less highly polarized or unpolarized, and tended to retain small round chloroplasts, whilst nuclei in the hypomethylated protonema endoreduplicated to lower levels and tended to remain more spherical. Increased nucleolar volumes and loss of intranucleolar rDNA heterochromatin following hypomethylation might be the result of increased transcriptional activity of ribosomal RNA (rRNA) genes and drug-induced DNA decondensation, respectively. Growing hypomethylated protonema at 25°C induced extremely atypical cells and development. This temperature sensitivity and aberrations in development overall can be attributed to changes in the normal patterns of gene expression brought about by hypomethylation of gene promoter or regulator regions.

Hydrogen sulphide emission in lichens as a response to low concentration SO2 fumigation was investigated. In an open flow-through system several lichen species were fumigated with 36 ppb SO2. Two species were also fumigated with higher concentrations (72, 119, 122 and 198 ppb SO2). Hydrogen sulphide emission was monitored concurrently by cryogenic trapping and analysis with gas chromatography. All tested species increased H2S emission significantly in response to fumigation with 36 ppb SO2. Parmelina tiliacea (L.) Hale and Cladina rangiferina (L.) Wigg. released significantly more H2S (0·098±0·015 and 0·073±0·013 pmol H2S g−1 d. wt s−1, respectively) than Parmelina quercina (Ach.) Hale, Ramalina menziesii Tayl. and Parmelia sulcata Tayl. (0·028±0·01, 0·025±0·014 and 0·023±0·013 pmol H2S g−1 d. wt s−1, respectively). Release of H2S in Hypogymnia physodes was enhanced by increasing SO2 concentrations up to 72 ppb SO2. No significant difference in H2S emission in the dark vs. in the light was found. Generally, no correlation was found between photosynthetic activity and H2S emission for the tested species. Uptake of SO2 was similar for all species, at 24·7±5·6 pmol SO2 g−1 d. wt s−1 in 36 ppb SO2 and increasing at greater SO2 concentrations. Therefore, H2S-S release represents only 0·11–0·74% of SO2-S uptake.

Several generations of soil microbiologists and plant pathologists have attempted to unravel the microbial composition, population dynamics and various life-sustaining processes in the soil. However, until recently, this basic resource for plant growth and nutrient recycling has to a large extent remained a black box. Studies have been made of the behavior of individual microorganisms or their population dynamics, but the relationship between the growth of individual organisms or parts of organisms (such as fungal hyphae) and that of populations and communities has been largely unknown. In particular, the spread of fungal colonies in soil has been an enigma, but now Bailey et al. (pp. 000–000 in this issue) have clearly demonstrated that spatial behavior of Rhizoctonia colonies can switch from finite to invasive expansion dependent on the non-linear relationship between the distance among substrate particles and the probability of colonization.

Why don't the gas spaces of submerged organs of wetland plants flood extensively when damaged? In addressing this intriguing question, Soukup et al. (pp. 71–75 in this issue) report on the role of rhizome diaphragms as barriers to flooding in Phragmites australis. This should prompt some reappraisal of the ways in which flooding resistance can be realized, even perhaps in undamaged organs.

Most emergent wetland macrophytes have an abundance of interconnected internal gas space, much of it in the form of large voids transversely partitioned at intervals by perforated cellular plates termed diaphragms. Functionally, it provides a low-resistance pathway for internal oxygen transport to support the respiratory needs of submerged and buried organs (Armstrong, 1979; Armstrong et al., 1988; Crawford, 1992) and facilitates carbon dioxide removal. However, it does more than this, since it enables oxygen to be released from the root to where it can support aerobic microbial activity in otherwise anaerobic sediments, and phytotoxin immobilization or destruction (Armstrong et al., 1992; Begg et al., 1994; Gilbert & Frenzel, 1998). This oxygen release is regarded by some as a valuable aid to effluent purification by constructed wetlands. Perhaps a less desirable property of this gas-space provision is its recently discovered role in enhancing the emissions of greenhouse gases such as methane from wetlands (Brix et al., 1992; Chanton & Whiting, 1996; Crutzen, 1991; Dacey & Klug, 1979).

Monstera acuminata is a plant with a problem – it attains a large body size, yet has no secondary growth. For many years, observers have noted the differences in architecture between monocots and woody dicots, particularly in terms of overall size and comparisons of tree-like forms. However, very little is known about the functional morphology of smaller-bodied and ecologically significant climbing and hemiepiphytic monocots, of which M. acuminata is one. In its wider context, the diversity of plant growth forms contributes to the complex structure of many terrestrial ecosystems, where herbs, trees, shrubs, vines, lianas, root-climbers, epiphytes and hemiepiphytes may be said to comprise the ‘skeleton’ of the ecosystem (Speck & Rowe, 1999). This is seen readily, for example, in the humid tropics, which arguably possess the highest taxonomic and structural diversity that has ever been formed by plants on earth. In this issue, López-Portillo et al. (pp. 289–299) investigate the development of plant architecture in Monstera, and demonstrate a range of novel hydraulic parameters.

Spontaneous mutation of the cyanobacterium Nostoc muscorum to a caesium resistant (Cs+-R) phenotype resulted in severe impairment of its nitrogenase activity, oxygenic photosynthesis, chlorophyll a content and osmotolerance. Among alkali cations only Cs+ or Rb+ restored these physiological processes to an almost normal level. Parent and mutant were similar with respect to Cs+ or Rb+ uptake and accumulation and also to regulation by NH4+. The ions Na+ or K+, at much higher concentration, significantly influenced Cs+ uptake and accumulation in both the mutant and the parent. These findings have important implications for cyanobacteria growing in Cs+-polluted habitats.

Uptake of the lipophilic cation, tetraphenylphosphonium (TPP+), which is often used as a membrane potential probe, was studied in the cyanobacterium Synechococcus R-2 (PCC 7942) to determine if the technique was valid for these cells. Lipophilic cation probes are notorious for giving inconsistent estimates of membrane potential (ΔΨ) using the Nernst equation (ETPP+=ΔΨ). It is necessary to correct for the large amounts of TPP+ bound to the surface of the cells. The rate of equilibration of intracellular TPP+ is very fast (t½≈1–3 min), hence the permeability of the plasmalemma to TPP+ (PTPP+≈10–50 nm s−1) is very high. There appears to be an active uptake of TPP+ with a pH optimum at about pHo 7.5. TPP+ does not readily uncouple Synechococcus cells. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and dinitrophenol depolarized ETPP+, but monensin made ETPP+ more negative. Accumulation of TPP+ is not affected by [K+] in the medium in a way consistent with it acting as a reporter of the membrane potential. Addition of K+, NH4+ and HCO3− altered the uptake rate of TPP+ in a way consistent with electrogenic uptake of these ions but the effects were smaller than those found using the 86Rb+/valinomycin and 201TI+ probes. A comparison has been made with ΔΨ determinations using other membrane potential probes. ETPP+ was often close to the value of the membrane potential obtained using other methods under control conditions but effects of experimental treatments are sometimes inconsistent with those found using other ΔΨ probes. The TPP+ method is therefore unreliable and could be seriously misleading.

We tested a hypothesis that elevated ozone was an eliciting or contributing factor in outbreaks of the ‘top dying’ (or ‘subtop dying’) syndrome in Norway spruce (Picea abies). Progeny were used from open-pollinated trees within a stand with the ‘top dying’ syndrome. The mother trees were classified in relation to the expression of the ‘top dying’ syndrome, and progeny from the healthiest and least healthy thirds of the population were exposed to high and low concentrations of ozone for three seasons. Elevated ozone did not affect height growth of the trees. It did not measurably affect net photosynthesis, stomatal conductance or instantaneous water use efficiency. Chlorophyll and carotenoid contents were also not significantly affected by ozone concentration. In the first year, instantaneous water use efficiency was lower in the progeny of the unhealthy mother trees than in the healthy mother trees. Furthermore, the unhealthy mother trees tended to produce longer annual shoots and showed more winter damage at the end of the experiment. None of these parameters were related to ozone concentration in the atmosphere. These results do not support a hypothesis that elevated ozone is a significant contributory factor or an eliciting factor in the development of the syndrome.