Oligomerization of profilins from birch, man and yeast. Profilin, a ligand for itself?

 Profilins are structurally well conserved low molecular weight (12–15 kDa) eukaryotic proteins which interact with a variety of physiological ligands: (1) cytoskeletal components, e.g., actin; (2) polyphosphoinositides, e.g., phosphatidylinositol-4,5-bisphosphate; (3) proline-rich proteins, e.g., formin homology proteins and vasodilatator-stimulated phosphoprotein. Profilins may thus link the microfilament system with signal transduction pathways. Plant profilins have recently been shown to be highly crossreactive allergens which bind to IgE antibodies of allergic patients and thus cause symptoms of type I allergy. We expressed and purified from Escherichia coli profilins from birch pollen (Betula verrucosa), humans (Homo sapiens) and yeast (Schizosaccharomyces pombe) and demonstrated that each of these profilins is able to form stable homo- and heteropolymers via disulphide bonds in vitro. Circular dichroism analysis of oxidized (polymeric) and reduced (monomeric) birch pollen profilin indicates that the two states have similar secondary structures. Using ¹²⁵I-labeled birch pollen, yeast and human profilin in overlay experiments, we showed that disulphide bond formation between profilins can be disrupted under reducing conditions, while reduced as well as oxidized profilin states bind to actin and profilin-specific antibodies. Exposure of profilin to oxidizing conditions, such as when pollen profilins are liberated on the surface of the mucosa of atopic patients, may lead to profilin polymerization and thus contribute to the sensitization capacity of profilin as an allergen. Received: 25 February 1998 / Revision accepted: 12 May 1998     

Activity of the CaMV 35S promoter in various parts of transgenic early flowering birch clones

Early flowering together with small size would be useful for various biotechnical or genetic studies on trees. We report here the selection and micropropagation of early flowering birch (Betula pendula) clones (BPM1–12) obtained from seeds of birches bred elsewhere for early flowering. Under conditions that accelerate flowering (a high CO₂ level, strong and continuous illumination), the first male inflorescences emerged in 3–5 months, the trees then being 20–80 cm high. Transgenic lines (CaMV 35S-GUS INT) were produced through Agrobacterium-mediated gene transfer from BPM2, BPM5 and JR1/4 (a normally flowering birch). β-Glucuronidase (GUS) activities in the different lines, assayed 1–1.5 years after transformation, varied greatly. During further in vitro culture for 10 months, the activities decreased to 0.3–7% of the original values. GUS activities were detected in all organs studied, including the developing male inflorescences; the highest activity was in the roots. Received: 28 April 1997 / Revision received: 5 September 1997 / Accepted: 30 November 1997     

Refined mapping of the gene for autosomal dominant retinitis pigmentosa (RP17) on chromosome 17q22

Linkage analysis was performed on a large Dutch family with autosomal dominant retinitis pigmentosa. Linkage was found to the RP17 locus on chromosome 17q22, which was previously described in two South African families by Bardien et al. (1995, 1997). Assuming that the disease phenotypes in these families are caused by the same gene, the RP17 critical region is refined to a 7.7-cM interval between markers D17S1607 and D17S948. Two positional candidate genes, the retina-specific amine oxidase (RAO) gene (AOC2) and the cone transducin γ gene (GNGT2), were excluded. Received: 7 September 1998 / Accepted: 23 November 1998     

Comparisons of the efficiency of some promoters in silver birch (Betula pendula)

The efficiency of several promoters (pin2 from potato, ubiquitin from sunflower, rolC from Agrobacterium rhizogenes, act1 from rice and CaMV 35S from cauliflower mosaic virus) fused to the uidA reporter gene was measured after biolistic bombardment of birch leaves (Betula pendula L.). The highest level of β-glucuronidase (GUS) activity was achieved with the pin2 promoter and the lowest activity with the CaMV 35S promoter. The activity of the potato wound-inducible promoter (pin2) was also tested in stably transformed birch. The promoter showed induced activity after mechanical wounding and feeding by leaf weevils. The systemic effect was confirmed by enhanced GUS activity in non-wounded leaves. The results of this study indicated that the potato wound-inducible promoter maintains its function in birch and would be a suitable promoter in studies of insect-birch interaction at the molecular level. Received: 17 October 1996 / Revision received: 7 February 1997 / Accepted: 1 March 1997     

Mast cell alpha-chymase reduces IgE recognition of birch pollen profilin by cleaving antibody-binding epitopes.

In sensitized individuals birch pollen induces an allergic response characterized by IgE-dependent mast cell degranulation of mediators, such as alpha-chymase and other serine proteases. In birch and other plant pollens, a major allergen is profilin. In mammals, profilin homologues are found in an intracellular form bound to cytoskeletal or cytosolic proteins or in a secreted form that may initiate signal transduction. IgE specific to birch profilin also binds human profilin I. This cross-reactivity between airborne and endogenous proteins may help to sustain allergy symptoms. The current work demonstrates that cultured mast cells constitutively secrete profilin I, which is susceptible to degranulation-dependent proteolysis. Coincubation of chymase-rich BR mastocytoma cells with Ala-Ala-Pro-Phe-chloromethylketone (a chymase inhibitor) blocks profilin cleavage, which does not occur in degranulated HMC-1 mast cells, which are rich in tryptase, but chymase deficient. These data implicate chymase as the serine protease cleaving secreted mast cell profilin. Sequencing of chymase-cleaved profilins reveals hydrolysis at Tyr(6)-Val(7) and Trp(35)-Ala(36) in birch profilin and at Trp(32)-Ala(33) in human profilin, with all sites lying within IgE-reactive epitopes. IgE immunoblotting studies with sera from birch pollen-allergic individuals demonstrate that cleavage by chymase attenuates binding of birch profilin to IgE. Thus, destruction of IgE-binding epitopes by exocytosed chymase may limit further mast cell activation by this class of common plant allergens, thereby limiting the allergic responses in sensitized individuals.     

Birch family and environmental conditions affect endophytic fungi in leaves

We investigated whether variation in foliar endophyte frequency among mountain birch trees from different maternal families was due in part to genetic differences among trees. The effect of different environmental conditions on the susceptibility of these mountain birch families to foliar endophytes was tested. The 3-year study was conducted in two tree gardens (altitudinal difference 180 m) with ten families of mountain birch. The frequency of the commonest endophytic fungus in mountain birch leaves, Fusicladium sp., was significantly affected by mountain birch family, with the ranking of families relative to Fusicladium sp. frequency being consistent from environment to environment. Variation in the frequency of Melanconium sp. was difficult to interpret because of significant family × garden × year interaction. Fusicladium sp. and Melanconium sp. endophytes were most frequent in different tree individuals, families and gardens. We conclude that mountain birch trees show heritable variation in their foliar endophyte frequency, and thus one of the conditions necessary for foliar endophytes of mountain birch trees to be able to affect the evolution of their host plant is fulfilled. However, the effect of mountain birch family on the frequency of endophytic fungi varies depending on the endophytic genera in question and partially also on environmental conditions. Received: 28 March 1998 / Accepted: 14 October 1998     

Effects of defoliation and symbiosis on polyamine levels in pine and birch

 We report the effect of ectomycorrhizal fungi (Suillus variegatus, Paxillus involutus) and defoliation on polyamine concentrations in pine (Pinus silvestris) and birch (Betula pendula) foliage and roots. Symbiotic root tips showed consistently higher concentrations of putrescine than non-symbiotic roots. Partial defoliation had no effect on the polyamine levels in mycorrhizal pine or birch roots. The foliage of mycorrhizal pine seedlings had lower putrescine concentrations and higher spermidine than foliage of non-mycorrhizal plants, and defoliation reversed this pattern. The response to partial defoliation differed in birch foliage: mycorrhizal status had no effect and all new growth after defoliation had higher spermidine levels than in non-defoliated birch. The potential role of polyamines in mycorrhizal symbiosis is discussed. Accepted: 26 February 1997     

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C. Received: 5 August 1997 / Received revision: 6 November 1997 / Accepted: 7 November 1997     

Effects of leguminous ground cover competition on red birch and soil nutrient status in the nursery

Legumes as ground cover are regularly planted to increase nitrogen economy of crops and to improve soil. In the present study various clover species were evaluated as vegetative ground cover in nursery field production of micropropagated red birch (Betula pubescensEhrh. f. rubraUlvinen f. nova) in two 2-year experiments. The clover species and cultivars, Trifolium pratenseL. ‘Bjursele’, T. repens L. ‘Jogeva’, T. repens L. ‘Sonja’, T. hybridum L. ‘Frida’, T. incarnatum L. ‘Opolska’, T. resupinatum L. and T. subterraneum L. were compared to grass sod Festuca rubra L. ‘Ensylva’ and to a coverless ground (control). The last one was kept weed free by hand hoeing. Birch (leaves, stems, branches and roots) and soil nutrient concentrations (N, P, K, Ca, Mg and Fe) were analysed and nutrient ratios in birch determined. The annual clovers, T. incarnatumL., T. resupinatumL. and T. subterraneumL., provided about the same nutrient status in birch as did the control. Perennial clovers and grass were strong competitors with trees. High levels of P and Mg in birch leaves relative to N concentration were typical for poorly growing seedlings. Neither annual nor perennial clovers did generally improve soil nutrient status. This revised version was published online in June 2006 with corrections to the Cover Date.     

Does lignin modification affect feeding preference or growth performance of insect herbivores in transgenic silver birch (<Emphasis Type="Italic">Betula pendula</Emphasis> Roth)?

Transgenic silver birch (Betula pendula Roth) lines were produced in order to modify lignin biosynthesis. These lines carry COMT (caffeate/5-hydroxyferulate O-methyltransferase) gene from Populus tremuloides driven by constitutive promoter 35S CaMV (cauliflower mosaic virus) or UbB1 (ubiquitin promoter from sunflower). The decreased syringyl/guaiacyl (S/G) ratio was found in stem and leaf lignin of 35S CaMV-PtCOMT transgenic silver birch lines when compared to non-transformed control or UbB1–PtCOMT lines. In controlled feeding experiments the leaves of transgenic birch lines as well as controls were fed to insect herbivores common in boreal environment, i.e., larvae of Aethalura punctulata, Cleora cinctaria and Trichopteryx carpinata (Lepidoptera: Geometridae) as well as the adults of birch leaf-feeding beetles Agelastica alni (Coleoptera: Chrysomelidae) and Phyllobius spp. (Coleoptera: Curculionidae). The feeding preferences of these herbivores differed in some cases among the tested birch lines, but these differences could not be directly associated to lignin modification. They could as well be explained by other characteristics of leaves, either natural or caused by transgene site effects. Growth performance of lepidopteran larvae fed on transgenic or control leaves did not differ significantly.     

Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain

The ability of Alcaligenes eutrophus to grow and produce polyhydroxyalkanoates (PHA) on plant oils was evaluated. When olive oil, corn oil, or palm oil was fed as a sole carbon source, the wild-type strain of A. eutrophus grew well and accumulated poly(3-hydroxybutyrate) homopolymer up to approximately 80% (w/w) of the cell dry weight during its stationary growth phase. In addition, a recombinant strain of A. eutrophus PHB⁻4 (a PHA-negative mutant), harboring a PHA synthase gene from Aeromonas caviae, was revealed to produce a random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate from these plant oils with a high cellular content (approximately 80% w/w). The mole fraction of 3-hydroxyhexanoate units was 4–5 mol% whatever the structure of the triglycerides fed. The polyesters produced by the A. eutrophus strains from olive oil were 200–400 kDa (the number-average molecular mass). The results demonstrate that renewable and inexpensive plant oils are excellent carbon sources for efficient production of PHA using A. eutrophus strains. Received: 3 September 1997 / Received revision: 10 November 1997 / Accepted: 16 November 1997     

Evaluation of shelf life and rock phosphate solubilization of Burkholderia sp. in nutrient-amended clay, rice bran and rock phosphate-based granular formulation

Five phosphate-solubilizing bacteria (PSB) used in this study were isolated based on their ability to solubilize tricalcium phosphate (TCP) in Pikovskaya’s medium. Among the tested bacterial strains Burkholderia sp. strain CBPB-HIM showed the highest solubilization (363 μg of soluble P ml⁻¹) activity at 48 h of incubation. Further, this strain has been selected to assess its shelf life in nutrient-amended and -unamended clay, rice bran and rock phosphate (RP) pellet-based granular formulation. The results showed that the maximum viability of bacterium was observed in clay and rice bran (1:1) + 10% RP pellets than clay-RP pellets, irrespective of tested storage temperatures. Further, clay and rice bran (1:1) + 10% RP pellets amended with 1% glucose supported the higher number of cells compared to glycerol-amended and nutrient-unamended pellets. In this carrier solubilization of Morocco rock phosphate (MRP) by Burkholderia sp. strain CBPB-HIM was also investigated. The maximum of water and bicarbonate extractable P (206 and 245 μg P g⁻¹ of pellet respectively) was recorded in clay and rice bran (1:1) + 10% RP pellets amended with 1% glucose and glycerol respectively on day 5 of incubation. Therefore, this study proved the possibility of developing granular inoculant technology combining clay, rice bran and RP as substrates with phosphate-solubilizing Burkholderia.     

Growth and nitrogen utilization in seedlings of mountain birch (Betula pubescens ssp. tortuosa ) as affected by ultraviolet radiation (UV-A and UV-B) under laboratory and outdoor conditions

 Growth patterns and nitrogen economy were studied in pot-grown seedlings of mountain birch subjected to different ultraviolet radiation under both laboratory and outdoor conditions at Abisko in northern Sweden. In the laboratory, nutrient supply, temperature, humidity, ultraviolet radiation-A (UV-A, 320–400 nm) and B (UV-B, 280–320 nm) were controlled, while photosynthetically active radiation (PAR, 400–700 nm) and photoperiod varied naturally. Under outdoor conditions nutrient supply was controlled, and the irradiation treatments were ambient and above-ambient UV-B using additional fluorescent lamps. Mountain birch nitrogen economy was affected by increased ultraviolet radiation, as reflected by a changed relationship between plant growth and plant nitrogen both in the laboratory and outdoors. In the laboratory enhanced UV-A decreased leaf area per unit plant biomass (leaf area ratio) but increased biomass productivity, both per unit leaf area (leaf area productivity) and per unit leaf nitrogen (leaf nitrogen productivity). Low levels of UV-B affected growth patterns and nitrogen economy in a similar way to enhanced UV-A. High levels of UV-B clearly decreased relative growth rate and nitrogen productivity, as leaf area ratio, leaf area productivity and leaf nitrogen productivity were all decreased. Under outdoor conditions above-ambient levels of UV-B did not alter growth or biomass allocation traits of the seedlings, whilst nitrogen productivity was increased. Mountain birch seedlings originating from different mother trees varied significantly in their responses to different ultraviolet radiation. Received: 10 April 1997 / Accepted: 19 September 1997     

Elevated CO2 ameliorates birch response to high temperature and frost stress: implications for modeling climate-induced geographic range shifts

Despite predictions that both atmospheric CO₂ concentrations and air temperature will rise together, very limited data are currently available to assess the possible interactive effects of these two global change factors on temperate forest tree species. Using yellow birch (Betula alleghaniensis) as a model species, we studied how elevated CO₂ (800 vs. 400 μl l⁻¹) influences seedling growth and physiological responses to a 5°C increase in summer air temperatures (31/26 vs. 26/21°C day/night), and how both elevated CO₂ and air temperature during the growing season influence seedling ability to survive freezing stress during the winter dormant season. Our results show that while increased temperature decreases seedling growth, temperature-induced growth reductions are significantly lower at elevated CO₂ concentrations (43% vs. 73%). The amelioration of high-temperature stress was related to CO₂-induced reductions in both whole-shoot dark respiration and transpiration. Our results also show that increased summer air temperature, and to a lesser degree CO₂ concentration, make dormant winter buds less susceptible to freezing stress. We show the relevance of these results to models used to predict how climate change will influence future forest species distribution and productivity, without considering the direct or interactive effects of CO_2. Received: 5 June 1997 / Accepted: 16 December 1997     

Thermostable β-xylosidase from Thermomonospora curvata

Thermomonospora curvata produced a thermostable β-xylosidase during growth on birch xylan. The enzyme, extracted by sonication of early stationary phase mycelia, was purified by isoelectric focusing and size exclusion HPLC. The isoelectric point was pH 4.8. The molecular weight was estimated to be 102 000 by size exclusion HPLC and 112 000 by SDS-PAGE. Maximal activity occurred at pH 6–7 and 60–68°C. K _m values for xylobiose and p-nitrophenyl-β -D-xylopyranoside were 4.0 M and 0.6 M respectively. The enzyme was sensitive to low levels of Hg²⁺ (50% inhibition at 0.2 μM), but was stimulated by Co²⁺ and Pb²⁺. Addition of the xylosidase to a xylanase reaction mixture increased the liberation of xylose equivalents from xylan and decreased the proportion of xylobiose in the hydrolysate. Received 14 April 1997/ Accepted in revised form 21 October 1997     

The birch tube-maker Acrobasis betulella in a fragmented habitat: the importance of patch isolation and edges

We examined the response of the birch tube-maker Acrobasis betulella Hulst (Lepidoptera: Pyralidae) to habitat patch isolation and edges. Density of A.␣betulella larva was higher on distant islands than on islands close to the shore. Following experimental removal of all larvae from the islands, adults were able to recolonize even the most distant islands, and larval density was again positively correlated with the degree of isolation. Larval density was not correlated with island size or the amount of birch present on the islands. Larvae on more distant islands did not have lower mortality than those in less isolated sites. Larvae were found more often on edges than in the interior of birch stands and developed faster on edges. This positive edge effect, coupled with the ability of the adults to disperse to even our most distant islands, suggests that A. betulella would be favored in an environment fragmented at the same scale as our island system. Received: 15 April 1996 / Accepted: 30 September 1996     

Carbon and nitrogen flow in silver birch and Norway spruce connected by a common mycorrhizal mycelium

 Spruce and birch seedlings were grown together in boxes filled with unsterile peat. Both seedlings were colonized by the ectomycorrhizal fungus Scleroderma citrinum. The two plants thus shared a common external mycelium. ¹⁵N-labelled ammonium was supplied exclusively to the fungus, while the birch or the spruce plant was continuously fed with ¹³C-labelled CO₂ for 72 h. The carbon and nitrogen transfer rates were strikingly different for birch and spruce seedlings. The mycorrhizal mycelium received carbohydrates mainly from the birch plant and the nitrogen transfer by the fungus to the plants was largely directed towards the birch. Carbon assimilates were also transferred in both directions between birch and spruce; however, there was no conclusive evidence for a net transfer of carbon between the plants. Accepted: 20 September 1996     

Integration of flowering dates in phenology and pollen counts in aerobiology: analysis of their spatial and temporal coherence in Germany (1992–1999)

We studied the possibility of integrating flowering dates in phenology and pollen counts in aerobiology in Germany. Data were analyzed for three pollen types (Betula, Poaceae, Artemisia) at 51 stations with pollen traps, and corresponding phenological flowering dates for 400 adjacent stations (< 25 km) for the years 1992–1993 and 1997–1999. The spatial and temporal coherence of these data sets was investigated by comparing start and peak of the pollen season with local minima and means of plant flowering. Our study revealed that start of birch pollen season occurred on average 5.7 days earlier than local birch flowering. For mugwort and grass, the pollen season started on average after local flowering was observed; mugwort pollen was found 4.8 days later and grass pollen season started almost on the same day (0.6 days later) as local flowering. Whereas the peak of the birch pollen season coincided with the mean flowering dates (0.4 days later), the pollen peaks of the other two species took place much later. On average, the peak of mugwort pollen occurred 15.4 days later than mean local flowering, the peak of grass pollen catches followed 22.6 days after local flowering. The study revealed a great temporal divergence between pollen and flowering dates with an irregular spatial pattern across Germany. Not all pollen catches could be explained by local vegetation flowering. Possible reasons include long-distance transport, pollen contributions of other than phenologically observed species and methodological constraints. The results suggest that further research is needed before using flowering dates in phenology to extrapolate pollen counts.     

Element content in leaves of birch (Betula verrucosa Ehrh.) in an air polluted area

 The content of natural (S, N, P, Ca, Mg, K, Mn, Zn, Cu) and extraneous elements (Pb, Cd, Al) in leaves of birch growing in the Krusne hory Mts. (Czech Republic), a territory affected by air pollution (SO₂), was evaluated with regard to the altitude and age of birch stands, and in relation to stand nutrition. The contents of S, Ca, Mg, Zn and Pb culminated at the highest points of the altitudinal transect (elevation 900–1000 m). In contrast, at high altitudes the content of Cd, Al and Mn decreased. The content of elements in leaves was mostly not dependent on tree age. In terms of nutrition and growth of birch, the content of Cu was insufficient, the content of Ca, Mg, N, P and K was optimum, and the content of S, Zn and primarily Mn was higher. The content of Pb and Al was at natural background levels, that of Cd higher. Received: 3 July 1997 / Accepted: 19 May 1998     

Airborne Poaceae pollen in Porto (Portugal) and allergenic profiles of several grass pollen types

This work aims to investigate the presence of airborne grass pollen and to identify antigenic and allergenic profiles from eight different grass species collected in the Porto region (Portugal). Poaceae airborne pollen, sampled using a Hirst-type volumetric trap during 2003–2007, was the second most abundant type, and high concentrations were found from April to August. Pollen proteins extracted from the eight grass species collected were separated by SDS-PAGE, being the allergenic profile investigated by immunoblotting using sera from atopic patients and maize profilin polyclonal antibody (ZmPRO3). Pollen extract profiles showed several bands ranging from 10 to 97 kDa. In immunoblotting studies, a low molecular weight protein (12–13 kDa) was recognized by profilin antibody. Also, in all pollen extracts except Zea mays, the IgE binding proteins of 12–13 kDa were detected in sera from the 25 patients with different sensitization profiles presenting high IgE values (>80 kU/l). This protein can be considered as a potential causal agent of the allergic respiratory diseases.