Uptake, translocation and metabolism of 14C-labelled formaldehyde in the leaves of Epipremnum aureum (Golden Potho) and Ficus benjamina (Weeping Fig) were investigated. Plants were exposed in light and dark to 14C-formaldehyde (500 μg m−3) in gas exposure chambers. The amount of 14C-incorporation into the soluble (water-extractable) and insoluble fractions of leaves, stem sections and roots was determined. The soluble 14C-activity was fractionated by ion exchange chromatography followed by thin-layer chromatography/autoradiography. Approximately 60–70% of the applied 14C-formaldehyde was recovered from the plants. In the light about five times more 14C-formaldehyde was assimilated than in the dark. The amount of 14C-label derived from 14C-formaldehyde, which was incorporated into acid-stable metabolites, was enhanced to an even larger extent in the light. The 14C-activity pattern closely resembled the general labelling spectrum of photosynthates, obtained after a 14CO2 exposure. A substantial amount of labelled material, mostly sucrose, was translocated into the stems and roots. Our results suggest that in the light 14C enters the Calvin cycle after an enzymatic two-step oxidation process of 14C-formaldehyde to 14CO2. The activities of the respective enzymes, formaldehyde dehydrogenase and formate dehydrogenase, were determined. Among 27 ‘leafy’ indoor decorative plants, a screening experiment revealed no outstanding species with regard to its capacity for metabolism of formaldehyde, and rate of uptake through stomata was too low to justify claims that plants contribute usefully to indoor air purification.

This is a review of genetic mapping with molecular markers aimed at the non-specialist who wishes to use, or at least grasp the concepts behind, this powerful analytical tool. Restriction fragment length polymorphisms (RFLPs) are defined and used to illustrate the different aspects of mapping. The principles of segregation, recombination and linkage are considered and related to the idea of a molecular marker map. A description of a typical mapping population and how it is analysed follows. Traits to be mapped are divided into those controlled by ‘major’ genes and those governed by quantitative trait loci (QTLs). Exploitation of the map for marker-assisted selection, gene cloning and synteny comparisons is discussed, as are some of the limitations to the usefulness of molecular marker maps. Finally other marker systems are introduced, namely minisatellites or variable number tandem repeats (VNTRs); randomly amplified polymorphic DNA (RAPDs); microsatellites or simple sequence repeats (SSRs); and amplified fragment length polymorphisms (AFLPs).

Cell walls were isolated and purified from Ri T-DNA-transformed carrot roots to determine whether arbuscular mycorrhizal (AM) fungi need a signal from the host root in order to form appressoria. Large cell-wall pieces were generated by the Parr nitrogen bomb technique. Purified cell walls were sterilized and inoculated with single spores of Gigaspora gigantea (Nicol. & Gerd.) Gerdemann & Trappe and Gigaspora margarita Becker & Hall using an in vitro culture technique. Appressoria only formed on epidermal cell walls isolated from host roots (Daucus carota L.) but did not form on epidermal cell walls isolated from non-host roots (Beta vulgaris L.). Although appressoria formed on host-cell walls, penetration hyphae did not fully develop. The results of this study indicate that appressorium formation by these AM fungi is a contact recognition event that does not require a signal secreted from the host root or the presence of intact host cells.

The involvement of the cytoskeleton in symbiotic interactions such as arbuscular mycorrhizas has received little attention. In this paper, we examine the organization of actin in tobacco mycorrhizal roots and compare actin and tubulin patterns within arbuscule-containing cells.

Our results show drastic reorganization of microfilaments and microtubules upon fungal infection and how those new cytoskeletal patterns relate to the host cytoplasm rearrangement and the intracellular fungal structures. Whereas in uninfected cells a network of cortical and perinuclear actin filaments was observed, in infected cells actin filaments closely follow the fungal branches and envelop the whole arbuscule in a dense coating network. Microtubules are less closely connected with the fungus surface. They run across the whole arbuscule mass, linking branches to each other and to the host cell cortex and nucleus.

These major differences between the two cytoskeletal components are used to advance some suggestions concerning their contribution to structural functions in the plant–fungus interactions during the mycorrhizal symbiosis.

The changes in pigment composition, photosynthesis and PSII photochemistry were investigated in cells of Spirulina platensis adapted to salt stress (<0.75 M NaCl). A decrease in the phycocyanine/chlorophyll and no significant change in the carotenoid/chlorophyll ratio were observed in salt-adapted cells. Salt stress inhibited the apparent quantum efficiency of photosynthesis and PSII activity while stimulating PSI activity and dark respiration significantly. Salt stress also resulted in a decrease in overall activity of the electron transport chain, which could not be restored by diphenylcarbazide, an artificial electron donor to the reaction centres of PSII. Measurements of the polyphasic fluorescence rise in fluorescence transients including phases O, J, I and P showed that salt stress had no effect on the fluorescence yield at phase O but decreased the fluorescence yield at phases J, I and P. Analyses of the JIP test developed from the polyphasic rise of fluorescence transients showed that salt stress led to a decrease in both the maximum quantum efficiency of PSII photochemistry and the maximum quantum efficiency of electron transport beyond the primary quinone electron acceptor. However, salt stress induced no significant changes in the probability of transporting an electron beyond QA, the trapping flux per PSII reaction centre, or the electron transport flux per PSII reaction centre. A theoretical analysis of fluorescence parameters indicated a decrease in the rate constant of excitation energy trapping by PSII reaction centres. In addition, salt stress induced an increase in the complementary area above the fluorescence induction curve in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting an increase in the proportion of closed PSII reaction centres in salt-adapted cells. Based on these results, it is suggested that modifications in PSII photochemistry in salt-adapted Spirulina cells maintained a high conversion efficiency of excitation energy, such that no significant change was observed in either the trapping flux or the electron transport flux per PSII reaction centre.

A model system was developed for the molecular analysis of plant–mycorrhizal fungus interactions using Hordeum vulgare cv. Galleon and Glomus intraradices Schenk and Smith. High levels of early-colonization stage mycorrhizas (20–35% of the total root length) were routinely obtained in the roots of 12–14-d-old barley seedlings. Detailed colonization studies showed that most colonization occurred in the first 5–10 lateral roots, and that colonization in primary roots and younger lateral roots was low. Differential screening of a cDNA library prepared from the highly colonized region of the root system and subsequent northern analyses identified four differentially expressed cDNA clones. Two clones, BMR6 and BMR78, detect RNA transcripts that accumulate to much higher levels in mycorrhizal roots than in non-mycorrhizal root tissues. DNA hybridization analysis confirmed that both of the clones were of plant rather than fungal origin. Hybridization and sequence analysis strongly suggests that BMR78 represents a partial cDNA of a low copy proton-ATPase gene. This finding is consistent with the extensive proliferation of the periarbuscular membrane and high ATPase activity associated with it. BM6 detects a low copy gene whose function is unknown.

We investigated interactions between the effects of elevated atmospheric carbon dioxide concentrations ([CO2]) and soil water availability on root biomass, root length and nutrient uptake by spring wheat (Triticum aestivum cv. Tonic). We grew plants at 350 and 700 µmol mol−1 CO2 and with frequent and infrequent watering (‘wet’ and ‘dry’ treatments, respectively). Water use per plant was 1·25 times greater at 350 than at 700 µmol CO2 mol−1, and 1·4 times greater in the ‘wet’ than in the ‘dry’ treatment. Root biomass increased with [CO2] and with watering frequency. Elevated [CO2] changed the vertical distribution of the roots, with a greater stimulation of root growth in the top layers of the soil. These data were confirmed by the video data of root lengths in the ‘dry’ treatment, which showed a delayed root development at depth under elevated [CO2]. The apparent amount of N mineralized appeared to be equal for all treatments. Nutrient uptake was affected by [CO2] and by watering frequency, and there were interactions between these treatments. These interactions were different for N, K and P, which appeared to be related to differences in nutrient availability and mobility in the soil. Moreover, these interactions changed with time as the root system became larger with [CO2] and with watering frequency, and as fluctuations in soil moisture contents increased. Elevated [CO2] affected nutrient uptake in contrasting ways. Potassium uptake appeared to be reduced by the smaller mass flow of water reaching the root surface. However, this might be countered with time by the greater root biomass at elevated [CO2], by the greater soil moisture contents at elevated [CO2], enabling faster diffusion, or both. Phosphorus uptake appeared to be increased by the greater root biomass at elevated [CO2]. We conclude that plant nutrient uptake at elevated [CO2] is affected by interactions with water availability, though differences between nutrients preclude generalizations of the response.

The male sterile mutant, ms35, of Arabidopsis thaliana was produced by X-irradiation of seeds. The mutant produces fertile pollen, but is male sterile because the anthers do not dehisce. Anther development in ms35 plants occurs as in wild-type Arabidopsis until shortly after microspores are released from meiotic tetrads. Thereafter, in the wild type, bands of lignified, cellulosic secondary wall thickenings are laid down around the cells of the anther endothecium. In contrast, wall thickenings are not formed in the endothecium of the ms35 mutant. Development of other lignified tissues, for example the vascular tissue of the stamen, occurs normally in ms35 plants. In mutant anthers, as pollen maturation is completed, the stomium is cleaved but the anther wall does not retract to release pollen. The block in anther dehiscence in ms35 plants is specifically correlated with the absence of endothecial wall thickenings. The ms35 mutation represents the first genetic evidence in support of the proposed role of the endothecium in anther dehiscence. The ms35 gene was mapped to the top arm of chromosome 3 (hy2-(4.17±2.31 cM)-ms35-(32.14±5.45 cM)-gl1).

This paper considers the extent to which differences in pollen tube growth rates can provide prezygotic reproductive isolation between Mimulus nasutus and its presumed progenitor, Mimulus guttatus. Mimulus nasutus is partially cleistogamous, but its larger chasmogamous flowers offer appreciable opportunity for outcrossing. Mimulus nasutus was found to have smaller pollen grains and shorter styles than M. guttatus. No differences were observed in pollen grain germination on conspecific and heterospecific stigmas. However, pollen tube growth rates of M. nasutus were found to be much slower than those of M. guttatus in the styles of that species. Consequently, any M. nasutus pollen transferred to an M. guttatus stigma was found to be competitively disadvantaged in an M. gutattus style. By contrast, no difference in pollen tube growth rate was detected between the species when growing in M. nasutus styles, possibly because M. nasutus styles are unable to support fast pollen tube growth. We tested the prediction from the pollen tube studies that a 50[ratio ]50 mix of M. guttatus and M. nasutus pollen would produce 50% hybrid seeds when M. nasutus was the maternal parent, and near to 0% hybrid seed when M. guttatus was the maternal parent. The results were found to support this prediction. We conclude that pollen–pistil interactions can effect strong reproductive isolation between these species, as M. guttatus pollen tubes have a competitive advantage over those of M. nasutus in an M. guttatus style, but not in an M. nasutus style.

Two cultivars of sorghum (CSH-1 and Ochuti) were grown in the presence and absence of the root hemiparasite Striga hermonthica in uniform conditions in the field in Kenya, Africa. S. hermonthica had a marked influence on growth and photosynthesis of ‘CSH-1’; however, ‘Ochuti’ showed a less severe response to infection and tolerance of the parasite. The variation in genotype response might be partly explained by later attachment of the parasite and a lower level of infection. Laboratory studies were used to determine the importance of both variables in determining host response to infection. Early infection by S. hermonthica had a more negative effect on the host than late infection. The level of parasite biomass supported by the host also influenced host productivity but the relationship was nonlinear. Low degrees of parasite infection had a proportionately much greater effect on host grain weight than at greater parasite loading. Early infection of ‘Ochuti’ in laboratory conditions resulted in lower stem dry weight than in uninfected plants but not in smaller total plant biomass or lower rates of photosynthesis. In conclusion, the time of parasite attachment affected host performance and might explain much of the variation in host sensitivity both within and between studies. The level of parasite infection affected host performance to a lesser extent. In addition, late attachment and low levels of infection might have implications for control management strategies.

A strain of non-pathogenic Fusarium oxysporum Schlecht. emend. Snyd. & Hans. has been selected for its capacity to reduce the incidence of Fusarium wilt of tomato. Among the possible modes of action of this strain, competition with the pathogen for the colonization of the root surface and tissues has been proposed. In order to study the pattern of root colonization, young Lycopersicon esculentum Miller (tomato) plants grown in a nutrient solution were inoculated by a suspension of F. oxysporum microconidia and processed at time-intervals for microscopic observations. The fungal strain was transformed with the Gus reporter gene to facilitate the observations. Within 24 h of inoculation the root surface was colonized by a dense network of hyphae, with the exception of the apex, which was colonized only after 48 h. A few hyphae were observed penetrating into the epidermis, leading to the internal colonization of the root cortex. This colonization was always discontinuous, since defence reactions of the plant limited the extension of the fungus. The barrier formed by thickenings and coilings of the cell walls and hypertrophied cells was most frequently observed in the external cortex and, sometimes, deeper in the internal cortex, close to the vessels which were never colonized. Typical defence reactions such as wall appositions, intercellular plugging and intracellular osmiophilic deposits, were frequently observed. This is the first report, based on microscopic observations, of the capacity of a non-pathogenic strain of F. oxysporum to colonize roots of tomato.

The potential role of sclerophylly (leaf hardness and rigidity) in the control of leaf dehydration and rehydration was investigated in two sclerophylls (Viburnum tinus and Ilex aquifolium) and two non-sclerophylls (Hedera helix ssp. helix and Sambucus nigra). After leaves were dehydrated in the pressure chamber, water transport from the apoplast (mainly consisting of xylem conduits and mechanical cells) to symplast was detected 15 min from pressure release, in terms of a spontaneous increase in leaf water potential (Ψ1). This Ψ1 increase was much larger in sclerophylls than in non-sclerophylls.

Positive pressures applied to leaves simulated the tensions developing in the leaf apoplast under water stress conditions, causing water to be expelled from leaf xylem conduits and mechanical cells and transferred to the leaf symplast, thus leading to symplast rehydration and to the consequent Ψ1 increase.

No correlation was found between the leaf modulus of elasticity at full turgor or the degree of sclerophylly (in terms of the ratio of leaf d. wt to surface area) and the characteristic rehydration time of the leaves, i.e. between the two main parameters expressing the rigidity of the leaf blade and the rate of leaf rehydration. However, when changes in Ψ1 were measured as a function of leaf water deficit (RWD), equal Ψ1s corresponded with larger RWDs during leaf rehydration than during leaf dehydration in the two non-sclerophylls. In particular, the two sclerophylls showed rehydration of their leaf apoplast and symplast completely and simultaneously. By contrast, the two non-sclerophylls showed a persisting water loss, localized, it is likely, in their xylem conduits and mechanical cells.

In other words, the two sclerophylls did not recover from water loss more rapidly than non-sclerophylls but they recovered from xylem cavitation more completely. The major elasticity of the cavitation strain shown by the two sclerophylls studied was interpreted as of advantage to plants subjected to diurnal large drops in Ψ1 followed by nocturnal recovery. This is the case in Mediterranean sclerophylls growing in areas characterized by high humidity of the air condensing on the soil at night. The same mechanism of cavitation recovery, however, would be useless in very xeric areas.

The hypothesis is advanced that sclerophylly of Mediterranean species may derive from similar anatomical structures developed in species formerly adapted to more humid environments which later migrated to more arid zones.

Seedlings of Quercus petraea (oak), Fraxinus excelsior (ash) and Pinus sylvestris (Scots pine) were grown at two CO2 concentrations with two O3 and two water supply treatments for 3 yr in a factorial experiment. Oak was the most responsive species to all three treatments; elevated CO2 and irrigation increased biomass by an average of 79% and 41%, respectively, whereas the ozone treatment resulted in a 30% reduction in growth. Significant treatment interactions in this species demonstrated that CO2 ameliorated and irrigation exacerbated the effects of ozone. For Scots pine and ash, irrigation and elevated CO2 increased growth by approx. 60% and 20%, respectively, whereas ozone had no detectable effect on ash and resulted in a 15% reduction in growth of Scots pine. Carbon partitioning to the shoot was enhanced by both the CO2 and H2O treatments in oak, while branching was also increased in this species in response to elevated O3, resulting in changes to the allometric relationships. CO2 enhanced leaf production in oak and Scots pine, and together with the promotion of shoot allocation, this indicates an increased susceptibility to windthrow. Biomass accumulation expressed as relative growth rate, suggested three different time-dependent growth responses to elevated CO2; the CO2 fertilization effect was maintained through the third year of growth in oak, had disappeared in Scots pine and a negative effect was evident in ash. Foliar nitrogen and chlorophyll concentrations indicated a CO2-induced nitrogen deficiency in oak and ash, but not in Scots pine. Chlorophyll degradation in response to ozone was observed in oak, an effect that was enhanced by irrigation and reduced by CO2, presumably through stomatal mediated changes in effective ozone dose. These results therefore suggest that elevated atmospheric CO2 concentrations will enhance growth of some UK forest tree species, although this might only be apparent during the juvenile phase. However, nitrogen deficiencies might limit this enhancement on some sites while changes in allocation and leaf area might promote susceptibility to windthrow. Elevated CO2 also provides some protection against ozone pollution, especially in combination with limited soil moisture availability. These interactions between CO2, ozone and water supply should be taken into account when predicting the effects of environmental change on tree growth and forest productivity.

We grew cuttings of two early (mid Oct.) and two late (early Nov.) leaf-fall Populus tremuloides Michx. genotypes (referred to as genotype pairs) for c. 150 d in open-top chambers to understand how twice-ambient (elevated) CO2 and soil N availability would affect growth and C allocation. For the study, we selected genotypes differing in leaf area duration to find out if late-season photosynthesis influenced C allocation to roots. Both elevated CO2 and high soil N availability significantly increased estimated whole-tree photosynthesis, but they did so in different ways. Elevated CO2 stimulated leaf-level photosynthesis rates, whereas high soil N availability resulted in greater total plant leaf area. The early leaf-fall genotype pair had significantly higher photosynthesis rates per unit leaf area than the late leaf-fall genotype pair and elevated CO2 enhanced this difference. The early leaf-fall genotype pair had less leaf area than the late leaf-fall genotype pair, and their rate of leaf area development decreased earlier in the season. Across both genotype pairs, high soil N availability significantly increased fine root length production and mortality by increasing both the amount of root length present, and by decreasing the life span of individual roots. Elevated CO2 resulted in significantly increased fine root production and mortality in high N but not low N soil and did not affect fine root life span. The early leaf-fall genotype pair had significantly greater fine root length production than the late leaf-fall genotype pair across all CO2 and N treatments. These differences in belowground C allocations are consistent with the hypothesis that belowground C and N cycling is strongly influenced by soil N availability and will increase under elevated atmospheric CO2. In addition, this study reinforces the need for better understanding of the variation in tree responses to elevated CO2, within and among species.

In the northeastern part of Belgium, the Centaurea jacea complex shows extensive morphological variation and is represented by a diploid (2n = 22) and a tetraploid (2n = 44) cytotype. Polysomic inheritance of allozyme markers in the tetraploids, suggesting autopolyploidy, is here demonstrated for the first time. In order to test whether the two cytotypes occupy distinct habitats and possess different gene pools, patterns of allozymic and morphological variation were investigated in relation to ploidy level and site characteristics in 26 populations from the Belgian Ardennes. The two cytotypes showed a parapatric distribution, the diploids occurring at higher elevations (mostly above 500 m) than the tetraploids (mostly below 500 m). Three mixed populations were found near the contact zone of the two cytotypes. Within the mixed populations no triploid plant and no evidence for gene flow between cytotypes were found, despite widely overlapping flowering periods. The two cytotypes can be distinguished on the basis of morphological traits and enzymatic gene pools. The congruence of morphological and allozymic variation with chromosome numbers suggests a secondary contact between the two cytotypes with limited gene flow between them. The origin and persistence of the parapatric distribution are discussed.

The ability of seeds to germinate and establish seedlings in a predictable manner under a range of conditions has a direct contribution to the economic success of commercial crops, and should therefore be considered in crop improvement. We measured traits associated with seed vigour and pre-emergence seedling growth in a segregating population of 105 doubled haploid Brassica oleracea lines. The germination traits measured were: mean germination times for unstressed germination; germination under water stress or germination after a heat treatment; and conductivity of seed leachate. The seedling growth traits measured were: seed weight; seedling growth rate; and seedling size at the end of the exponential growth phase. There were some correlations, notably among germination traits, and between seed weight and pre-emergence seedling growth. Heritability of the various traits was typically in the 10–15% range, with heritability of conductivity and mean germination time under water stress 25 and 24% respectively. Collectively the results indicate that germination and pre-emergence seedling growth are under separate genetic control. Quantitative trait loci analyses were carried out on all measurements and revealed significant loci on linkage groups O1, O3, O6, O7 and O9. We suggest that genes at these loci are important in determining predictable seed germination and seedling establishment in practice.

Mycorrhizas can produce negative crop responses when phosphorus availability is sufficient in agricultural soils because the fungi are of no benefit in nutrient acquisition yet continue to colonize roots and invoke parasitic costs. Benomyl fungicide was used to test this prediction in the field by limiting mycorrhizal colonization of 2-yr-old Valencia orange trees (Citrus sinensis (L.) Osbeck) on four rootstocks of varying mycorrhizal dependency in P-deficient soil fertilized with and without phosphate. No known fungal pathogens of citrus roots controlled by benomyl were present on the trees or in the field soil. Young trees with or without P fertilization and benomyl treatment remained sufficient in P ([ges ]0·10% leaf P) throughout the 27-month study. Root zone drenches of benomyl reduced mycorrhizal colonization and leaf P status of Valencia orange trees on the three slower-growing rootstocks, trifoliate orange (Poncirus trifoliata (L. ) Raf.), Swingle citrumelo (Citrus paradisi Macf.×P. trifoliata) and sour orange (Citrus aurantium L.), for the duration of three growing seasons. Benomyl affected root colonization and P status of trees on the faster-growing rootstock, Volkamer lemon (Citrus volkameriana Tan. and Pasq.), less than for trees on the slower-growing rootstocks and the effects were sustained for only two seasons. The shorter duration of benomyl effect for trees on Volkamer lemon rootstock compared with the slower-growing rootstocks was explained by the loss of inhibition of mycorrhizal activity when roots grew out of the drench zone and mycorrhizas were no longer in direct contact with the fungicide. Benomyl treatment increased growth rate of Valencia orange on the slow-growing rootstocks from 5 to 17% after three seasons, and from 2 to 9% on Volkamer lemon rootstock after two seasons compared with the non-benomyl treated trees. The benomyl effect was attributed to reduction of costs of root colonization over time, and consequently, a greater availability of carbon assimilate for shoot growth of trees. Since mycorrhizal fungi are ubiquitous in fertilized agricultural soils and obligate biotrophs on the roots of most crop species, these results indicate a need to further investigate whether negative growth responses of P-sufficient plants in the field occur because mycorrhizal fungi are no longer behaving as mutualists.

Leaching of nitrate through soil profiles is a major cause of soil acidification. Legume species differ in their ability to take up nitrate (NO3−) and in the degree to which soil NO3− impairs legume nodulation and N2 fixation. This pot experiment examined species variation in N2 fixation and acid production in grain legumes grown for 6 wk on a sandy soil with Ca(NO3)2 added at 0–56.7 mg N kg−1 soil. The eight legumes studied were Lupinus angustifolius (‘Gungurru’), Lupinus albus (‘Kiev mutant’), Lupinus luteus (‘Teo’), Cicer arietinum (Selection T1587), Pisum sativum (‘Dundale’ and ‘Wirrega’), Vicia faba (‘Fiord’) and Lathyrus sativus (Selection 453). While number of nodules was not significantly affected by treatment with NO3−, increasing supply of NO3− decreased nodule mass, with P. sativum ‘Wirrega’ being most sensitive and C. arientinum and V. faba least sensitive. The concentrations of nitrogen in plants generally decreased as the supply of NO3− increased. Increasing supply of NO3− decreased the percentage of N2 fixation, most significantly in L. sativus and least in L. albus. In the absence of added NO3−, the amounts of H+ produced per unit biomass (specific acid production) differed by as much as 2.2-fold among the species, with C. arientinum generating most H+, followed by L. angustifolius, L. luteus, L. albus, V. faba, L. sativus, P. sativum ‘Dundale’ and P. sativum ‘Wirrega’. This species variation was even greater at higher NO3− supply. In the presence of NO3− at up to 14.2 mg N kg−1, acid production by these legumes correlated well with uptake of excess cations but not with the concentration of nitrogen in plants. For all species, release of H+ declined with increasing supply of NO3−, decreasing most in L. sativus and least in C. arientinum. The results suggest that there might be scope for selecting grain legume species in order to minimize soil acidification by decreasing leaching of NO3−.

A lacustrine sequence from Lake Sedmo Rilsko (altitude 2095 m) in the northwest of Rila Mountain, Bulgaria, was analysed on the basis of 84 pollen spectra and three 14C accelerator mass spectrometry dates. The lower part of the sequence (413–530 cm) corresponds to the Late-glacial. Three phases, two stadial and one interstadial, are characterized by the dominance of mountain–steppe herb vegetation composed of Artemisia, Chenopodiaceae and Poaceae, with single trees of Pinus and shrub land of Juniperus and Ephedra. The identification of pollen grains of Abies, Quercus robur-type, Corylus, Acer, Fagus and other mesophilous trees suggests that they survived the harsh Late-glacial conditions in refuges below an altitude of 1000 m, where moisture was sufficient for their growth. In the early Holocene period, Betula forests at high altitudes and, below them, closed deciduous forests with Quercus, Tilia, Ulmus and Corylus, occurred from 11800 until approx. 6700 cal. BP. The formation of the coniferous belt dominated by Pinus sylvestris, Pinus peuce and Abies alba lasted from between 6700 and 5000 cal. BP. The forest dynamics in the Subboreal and the Subatlantic ended with the invasion of Fagus sylvatica and Picea abies after approx. 4500 and 3300 cal. BP, respectively. Indications of anthropogenic activities, expansion of agriculture in the lowland foothills, and livestock grazing in the mountain meadows and pastures, are clearly evident from the pollen diagram from 2400 cal. BP onwards.

The fructan-β-fructosidase activity (1-FEH; EC 3.2.1.80) that degrades inulin in tubers of Helianthus tuberosus L. appears to be developmentally regulated; it was low in growing tubers but increased during dormancy and sprouting. In spite of relatively high 1-FEH activity in vitro, fructose concentration was very low in developing and dormant tubers and increased markedly only during sprouting. A fructan-β-fructosidase from such sprouting tubers was purified 41-fold to a single protein band on one-dimensional sodium dodecylsulphate–polyacrylamide gels. The purification procedure included ammonium sulphate precipitation, lectin-affinity chromatography on concanavalin A, anion-exchange and cation-exchange chromatography. The enzyme had an apparent molecular mass of 75000 measured by size-exclusion chromatography, and 79000 measured by one-dimensional sodium dodecylsulphate-polyacrylamide gel electrophoresis. It exhibited a high substrate specificity, hydrolysing terminal β-(2-1)-fructosyl-fructose-linkages in linear and branched fructan oligomers; β-(2-6)-linkages were hardly hydrolysed. Hydrolysis of inulin oligomers followed normal saturation kinetics: Km values for 1,1-kestotetraose and 1,1,1-kestopentaose were 8·3 mM and 12 mM, respectively. Fructosyl residues were hydrolysed from inulin oligomers by a multi-chain mechanism. The fructan-β-fructosidase showed optimal enzyme activity at pH 5·2, and it retained its full activity after pre-incubation for 1 h at up to 40 °C. The release of fructose from 5 mM 1,1-kestotetraose was reduced by 25% when 1-FEH was assayed in the presence of 10 mM sucrose. It is proposed that the inhibition of 1-FEH activity by sucrose is a mechanism for controlling fructan degradation in planta.