ABA consumption in norway spruce (Picea abies) and white spruce (Picea glauca) somatic embryo cultures

Summary We investigated abscisic acid (ABA) metabolism among Norway and white spruce somatic embryo cultures which exhibited differences in maturation response when placed on racemic abscisic acid [(±)-ABA]. Differences in metabolic rate among the spruce genotypes could affect the ABA pool available for the maturation process, and might therefore be responsible for the differences in maturation response. The production of cotyledonary (stage 3) somatic embryos in cultures (genotypes) of Norway spruce (PA86:26A and PA88:25B) and of white spruce (WS1F cryoD and WS46) was compared. In each species pair one of the two genotypes failed to show stage 3 embryo development (respectively, PA88:25B and WS46). The investigation of ABA metabolism of each species pair showed that no substantial differences in ABA consumption or in the production of metabolites occurred. In each case ABA was metabolized to phaseic acid and dihydrophaseic acid over the 42-day culture period, metabolites were recoverable from the agar-solidified medium, and the sum of residual ABA and metabolites were equivalent to the ABA initially supplied. The results indicate that the process of ABA metabolism occurs essentially independently of somatic embryo maturation. NRCC no. 37345.     

Influence of excess nitrogen deposition on a white spruce (Picea glauca) stand in southern Alaska

Excess N delivered to forest ecosystems has been shown to alterinternal ecosystem biogeochemical cycles, contribute to forestdecline, and negatively affect the health of receiving waters.In the vicinity of the Nikiski Industrial Complex, Kenai Peninsula,Alaska, there has been recent concern about the influence ofNH3 emissions that have occurred for over twodecades on local soils and vegetation. The study site representedan opportunity to examine the influence of elevated N depositionon a northern coniferous ecosystem in an area with a low backgroundof N deposition. Overstory vegetation in the area is dominated bywhite spruce (Picea glauca Moench. Voss) and paper birch (Betulapapyrifera Marsh.). Mortality of both species has occurred adjacent(<2 km) to the industrial complex. Average annual Ndeposition rates ranged from 0.7 to 21.0 kg ha-1 y-1in the area, with the highest rates closest to the complex. Sulfatedeposition at the site was low. Due to the high NH3deposition, precipitation near the complex was less acidic thanprecipitation in general; bulk precipitation pH ranged from 5.51to 7.06. Within 1.80 km of the facility there was an increase inKCl- and resin- extractable soil NH4+ andNO3- in the O horizon, and a decrease in soil pHcompared to soils further from the facility. Spruce near thefacility had chlorotic foliage and thinning crowns, higherconcentrations of N, but lower foliar Ca and Mg. Foliar Mglevels approached deficiency levels, but foliar Ca was wellabove reported deficiency levels at all sites. Both Mg:N andCa:N ratios, however, suggest nutrient imbalances in the highN deposition zone. Canopy death and fertilization by N appearto have encouraged growth of the native bluejoint grass. Thepresence of elevated NO3- in O horizon soilextracts, elevated NO3- in resin bags placed betweenthe O and E horizons, and nutritional imbalances in the foliagesuggest that N saturation may be occurring in soils adjacent to thefacility.     

Responses of white spruce (Picea glauca) to experimental warming at a subarctic alpine treeline

From 2001 to 2004 we experimentally warmed 40 large, naturally established, white spruce [Picea glauca (Moench) Voss] seedlings at alpine treeline in southwest Yukon, Canada, using passive open‐top chambers (OTCs) distributed equally between opposing north and south‐facing slopes. Our goal was to test the hypothesis that an increase in temperature consistent with global climate warming would elicit a positive growth response. OTCs increased growing season air temperatures by 1.8°C and annual growing degree‐days by one‐third. In response, warmed seedlings grew significantly taller and had higher photosynthetic rates compared with control seedlings. On the south aspect, soil temperatures averaged 1.0°C warmer and the snow‐free period was nearly 1 month longer. These seedlings grew longer branches and wider annual rings than seedlings on the north aspect, but had reduced Photosystem‐II efficiency and experienced higher winter needle mortality. The presence of OTCs tended to reduce winter dieback over the course of the experiment. These results indicate that climate warming will enhance vertical growth rates of young conifers, with implications for future changes to the structure and elevation of treeline contingent upon exposure‐related differences. Our results suggest that the growth of seedlings on north‐facing slopes is limited by low soil temperature in the presence of permafrost, while growth on south‐facing slopes appears limited by winter desiccation and cold‐induced photoinhibition.     

Somaclonal variation in cryopreserved embryogenic clones of white spruce [Picea glauca (Moench) Voss.]

 Trees were regenerated from six white spruce embryogenic clones after cryopreservation for 3 and 4 years, respectively. Genetic stability was evaluated using randomly amplified polymorphic DNA (RAPD) fingerprints. Somaclonal variation was detected in some in vitro embryogenic cultures 2 and 12 months after they were re-established following cryopreservation but not in the corresponding regenerated trees. These results suggest that trees regenerated from cryopreserved cultures in subsequent years are primarily genetically stable in the genomic regions tested and that variation observed due to the in vitro culture process infrequently affects trees regenerated from normally maturing and germinating somatic embryos. However, trees regenerated from somatic embryos that matured or germinated abnormally in in vitro culture exhibited altered RAPD fragment patterns. Received: 20 July 1998 / Revision received: 15 October 1998 / Accepted: 14 December 1998     

Somatic embryogenesis and plant regeneration from suspension cultures of Picea glauca (White spruce)

Hakman, I. and von Arnold, S. 1988. Somatic embryogenesis and plant regeneration from suspension cultures of Picea glauca (White spruce). - Physiol. Plant. 72: 579–587.
Plantlets were regenerated from long-term embryogenic cultures of Picea glauca (Moench) Voss. (White spruce). Embryogenic calli, initiated from immature zygotic embryos and maintained by monthly subculture for 16 months, were used to establish suspension cultures. Small somatic embryos were continuously produced in liquid culture medium containing auxin and cytokinin and the cultures showed a sustained regeneration capacity for >6 months. Somatic embryos propagated in the suspension cultures developed further into embryos bearing cotyledons, about 1 month after transfer to solidified medium containing abscisic acid. Electron microscopic examination revealed that storage nutrients, lipids, proteins and carbohydrates, accumulated in the somatic embryos during this treatment with abscisic acid (ABA). Upon subculture to medium lacking plant growth regulators such embryos could develop into small green plantlets.     

Phylogeography of white spruce (Picea glauca) in eastern North America reveals contrasting ecological trajectories

Aim The main objective of this study was to investigate the origin of white spruce (Picea glauca) range discontinuities in eastern North America. A first analysis aimed at uncovering the glacial origin and post-glacial migration route of white spruce in the eastern part of the range. A second analysis aimed at evaluating whether disjunct white spruce populations in central Québec result from a recent expansion or are remnant stands following a northern range contraction. Location The first analysis covered the North American transcontinental boreal distribution of white spruce, and the second focused on the eastern part of its range. Methods A total of 589 P. glauca individuals from 51 populations, 24 of which were from a previous published source, were analysed for variation in three chloroplast DNA regions (trnT/L, trnL/F and ndhK/C). At the continental scale, haplotypic diversity, zones of genetic discontinuities, clustering analysis and analysis of molecular variance (AMOVA) were employed. In the eastern part of the range, haplotypic diversity was assessed with a permutation procedure, demographic history with mismatch analyses, and genetic structure with AMOVA. Results At the continental scale, various lines of evidence (genetic-diversity hotspots, endemic haplotypes, zones of genetic discontinuities, and pattern of molecular variation) show that different lineages migrated from three glacial refugia (Beringian, Mississippian, east Appalachian). Strong genetic discontinuities, low diversity and lack of evidence for demographic expansion were found among stands from isolated high hills in central Québec. Main conclusions White spruce in the eastern part of its range has an east Appalachian origin, a lineage genetically distinct from populations west of the Great Lakes. Previous studies have shown that the northward migration of white spruce is still ongoing as the northernmost populations expand into the tundra. In contrast, mixed white spruce and balsam fir (Abies balsamea) stands from isolated high hills in central Québec have already reached their Holocene maxima and are currently declining. This range contraction is probably a result of increased wildfire disturbance, which gradually creates a pattern of regional white spruce isolation and replacement by fire-prone black spruce (Picea mariana) forest.     

Cytochrome and alternative pathway respiration in white spruce (Picea glauca) roots. Effects of growth and measurement temperature

Interactions between growth temperature and measurement temperature were examined for their effects on white spruce [ Picea glauca (Moench) Voss] root respiration. Total dark respiration rates increased with measurement temperature and were unaffected by growth temperature. Partitioning of respiratory electron flow between the cytochrome and alternative pathways was also unaffected by growth temperature. The proportion of respiration mediated by the alternative pathway was constant at measurement temperatures between 4°C and 18°C, but was increased at higher temperatures. Changes in alternative pathway activity were paralleled by changes in capacity, and the alternative pathway was almost fully engaged at all temperatures. Roots grown at low temperature displayed higher carbohydrate levels than roots grown at higher temperatures, but respiration rate was unaffected. Spruce root respiration did not appear to acclimate to growth temperature, and the alternative pathway was not preferentially engaged at low temperature.     

Expression of inducible angiosperm promoters in a gymnosperm,Picea glauca (white spruce)

Electrical discharge particle acceleration was used to test the transient expression of numerous inducible angiosperm promoters in a gymnospermPicea glauca (white spruce). Promoter expression was assayed in three different tissues capable ofin vitro regeneration, zygotic embryos, seedlings and embryogenic callus. The promoters tested include the light-inducibleArabidopsis and soybean ribulose-1,5-bisphosphate small subunit promoters and a maize phosphoenolpyruvate carboxylase promoter; a soybean heat-shock-inducible promoter, a soybean auxin inducible promoter and a maize alcohol dehydrogenase promoter. Promoters were cloned into a promoter-less expression vector to form a promoter--glucuronidase-nopaline synthase 3 fusion. A similar construct was made using the cauliflower mosaic virus 35S (CaMV 35S) promoter as a control. All promoters were expressed in white spruce embryos, yet at levels lower than CaMV 35S. In addition, in the embryos the heat-shock and the alcohol dehydrogenase promoters showed inducible expression when given the proper induction stimulus. In seedlings, expression of all promoters was lower than in the embryos and expression was only inducible with the heat-shock promoter in the cotyledons. Of the tissues tested, the expression level of all promoters was lowest in embryogenic callus. Interestingly, the expression of the -glucuronidase gene in embryogenic callus was restricted to the proembryonal head cells regardless of the promoter used. These results clearly demonstrate the use of particle bombardment to test the transient expression of heterologous promoters in organized tissue and the expression of angiosperm promoters in a gymnosperm.     

Stable carbon isotopes as indicators of increased water use efficiency and productivity in white spruce (Picea glauca (Moench) Voss) seedlings

The relationship among water use efficiency (WUE), productivity and carbon isotopic composition (δ¹³C) in white spruce (Picea glauca (Moench) Voss) seedlings was investigated. Sixteen hundred seedlings representing 10 controlled crosses were planted in the field in individual buried sand-filled cylinders. The soil water content in the cylinders was measured using time domain reflectometry over two growing seasons and seedling water use determined by water balance. Two watering treatments were imposed: irrigation and dry land. There was significant (1.6–2.0%c) genetic variation in needle δ¹³C. Ranking of crosses in terms of δ¹³C was generally maintained over watering treatments and there was not a significant genetic versus environmental interaction. There was a positive correlation between δ¹³C and both intrinsic and long-term WUE (more positive δ¹³C with increased WUE) and between δ¹³C and productivity, suggesting a correlation due to variation in photosynthetic capacity. Root to shoot ratios did not increase in water-stressed plants, indicating that responses to drought were primarily at the level of gas exchange, rather than through morphological changes. Our results indicate that it should be possible to use δ¹³C as a surrogate for WUE and to select white spruce genotypes for high WUE without compromising yield.     

Effects of triadimefon and osmotic stress on plasma membrane composition and ATPase activity in white spruce (Picea glauca) needles

White spruce [ Picea glauca (Moench) Voss.] seedlings were grown in solution culture and treated with 20 mg I^-1 triadimefon [1-(chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)-2-butanol] for 4 weeks and then subjected to osmotic stress with polyethylene glycol 3350. Water potentials and electrolyte leakage were measured in control and triadimefon-treated seedlings before and after the plants were subjected to osmotic stress. The plasma membranes were isolated from needles to study their lipid composition and the activity of plasma-membrane bound ATPase. Triadimefon treatment reduced water potentials and increased leakage of electrolytes from seedlings. However, when the seedlings were exposed to osmotic stress, triadimefon-treated plants maintained higher water potentials and leaked less electrolytes compared with the control plants. Both triadimefon and osmotic stress treatments inhibited the activity of plasma membrane-bound ATPase and altered the composition of free sterols in the plasma membranes. Triadimefon-treated plants contained traces of campesterol, which was not present in control. Osmotic stress drastically reduced phospholipid:protein and sterol:protein ratios and increased sterol:phospholipid ratios in the plasma membranes     

Actin distribution in somatic embryos and embryogenic protoplasts of white spruce (Picea glauca)

Summary Examination of unfixed immature somatic embryos of white spruce (Picea glauca) with fluorescent rhodamine-labeled phalloidin revealed an extensive network of fine actin microfilaments (MFs) in the embryonal region which were not detected in specimens fixed with formaldehyde. Transition cells linking the embryonal region and suspensor cells contained fine MFs as well as bundles of MFs. The large, highly vacuolated suspensor cells were characterized by actin MF cables only. Treatment of embryos with cytochalasin B (CB) removed the fine MFs from the embryonal region and transition cells, but many MF cables in suspensor cells were resistant. Full recovery from CB treatment was observed in most somatic embryos. Embryogenic protoplasts capable of regenerating to somatic embryos in culture were released from only the embryonal region of somatic embryos. Both uninucleate and multinucleate embryogenic protoplasts retained the extensive network of fine actin MFs. In contrast, protoplasts derived from vacuolated suspensor cells and vacuolated free-floating cells contained thick MF bundles and were not embryogenic. Distinct MF cages enclosed nuclei in multinucleate protoplasts and may be responsible for preventing nuclear fusion. Microspectrophotometric analyses showed that the DNA contents of embryonal cells in the embryo and embryogenic protoplasts were similar and characteristic of rapidly dividing cell populations. However, transition and suspensor cells which released nonembryogenic protoplasts appeared to be arrested in G₁, and suspensor cells showed signs of DNA degradation.     

Early effects of triadimefon on water relations, sterol composition and plasma membrane ATPase in white spruce (Picea glauca) seedlings

Seedlings of white spruce ( Picea glauca [Moench] Voss.) were treated with triadimefon solution applied to the soil, and their early responses studied from 12 h to 7 days after treatment. Transpiration rates declined and respiration rates increased immediately after the commencement of triadimefon treatment. Photosynthetic rates declined less than transpiration rates, resulting in an increase in water use efficiency, whereas root and shoot water potentials remained unchanged during the first 5 days of triadimefon treatment. Triadimefon decreased root hydraulic conductivity and inhibited the activity of the plasma membrane ATPase. In addition, triadimefon-treated roots drastically increased the ratios between free sterols and sterol esters and decreased the ratios between sterol esters and acylated sterol glycosides.     

Microtubule organization and cell division in embryogenie protoplast cultures of white spruce (Picea glauca)

Summary Immunofluorescence methods were developed for examining the distribution of microtubules in freshly isolated and cultured protoplasts and regenerated somatic embryos of white spruce (Picea glauca). Freshly isolated protoplasts consisted of both uniand multinucleate types. Uninucleate protoplasts established parallel cortical microtubules during cell wall formation and cell shaping, divided within 24 h and developed into somatic embryos in culture. Dividing cells were characterized by preprophase bands (PPBs) of microtubules, atypical spindle microtubules focused at the poles and a typical phragmoplast at telophase. Multinucleate protoplasts also established parallel arrays of cortical microtubules during cell wall formation. In addition their nuclei divided synchronously within 4 days, then cell walls formed between the daughter nuclei. Individual multinucleate protoplast-derived colonies subsequently gave rise to elongate suspensor cells thereby forming embryo-like structures by 7 days.     

The effects of nitrogen stress on the stable carbon isotope composition, productivity and water use efficiency of white spruce (Picea glauca (Moench) Voss) seedlings

A, assimilation rate
a, fractionation against ¹³C for CO₂ diffusion through air
b, net fractionation against ¹³C during CO₂ fixation
C_a, ambient CO₂ concentration
C_c, CO₂ concentration at the chloroplast
C_i, intercellular CO₂ concentration
D, vapour pressure deficit
E_n, needle transpiration rate
E_p, whole plant water use
g_w, leaf internal transfer conductance to CO₂
g_s, stomatal conductance to water vapour
L, projected leaf area
NUE, nitrogen use efficiency
PEP, phosphoenolpyruvate
Rubisco, ribulose-1,5-biphosphate carboxylase
TDR, time domain reflectometry
WUE, water use efficiency
Δ, carbon isotope discrimination
δ¹³C, carbon isotope abundance parameter
δ¹³C_a, carbon isotopic composition of atmospheric CO₂
θ, volumetric soil water content

The effect of nitrogen stress on needle δ¹³C, water-use efficiency (WUE) and biomass production in irrigated and dry land white spruce (Picea glauca (Moench) Voss) seedlings was investigated. Sixteen hundred seedlings, representing 10 controlled crosses, were planted in the field in individual buried sand-filled cylinders. Two nitrogen treatments were imposed, nitrogen stressed and fertilized. The ranking of δ¹³C of the crosses was maintained across all combinations of water and nitrogen treatments and there was not a significant genetic versus environmental interaction. The positive relationships between needle δ¹³C, WUE and dry matter production demonstrate that it should be possible to use δ¹³C as a surrogate for WUE, and to select for increased WUE without compromising yield, even in nitrogen deficient environments. Nitrogen stressed seedlings had the lowest needle δ¹³C in both irrigated and dry land conditions. There was a positive correlation between needle nitrogen content and δ¹³C that was likely associated with increased photosynthetic capacity. There was some indication that decreased nitrogen supply led to increased stomatal conductance and hence lower WUE. There was a negative correlation between intrinsic water use efficiency and photosynthetic nitrogen use efficiency (NUE). This suggests that white spruce seedlings have the ability to maximize NUE when water becomes limited. There was significant genetic variation in NUE that was maintained across treatments. Our results suggest that in white spruce, there is no detectable effect of anaplerotic carbon fixation and that it is more appropriate to use a value of 29‰ (‘Rubisco only’) for the net discrimination against ¹³C during CO₂ fixation. This leads to excellent correspondence between values of C_i/C_a derived from gas exchange measurements or from δ¹³C.     

Effects of plant leachates from four boreal understorey species on soil N mineralization, and white spruce (Picea glauca) germination and seedling growth

BACKGROUND AND AIMS: Natural regeneration of white spruce (Picea glauca) after disturbance has been reported to be very poor. Here a study was made to determine whether C compounds released from understorey species growing together with white spruce could be involved in this regeneration failure, either by (1) changing soil nutrient dynamics, (2) inhibiting germination, and/or (3) delaying seedling growth. METHODS: Foliage leachates were obtained from two shrubs (Ledum palustre and Empetrum hermaphroditum) and one bryophyte (Sphagnum sp.) with high phenolic compound concentrations that have been reported to depress growth of conifers in boreal forests, and, as a comparison, one bryophyte (Hylocomium splendens) with negligible phenolic compounds. Mineral soil from a white spruce forest was amended with plant leachates to examine the effect of each species on net N mineralization. Additionally, white spruce seeds and seedlings were watered with plant leachates to determine their effects on germination and growth. KEY RESULTS: Leachates from the shrubs L. palustre and E. hermaphroditum contained high phenolic compound concentrations and dissolved organic carbon (DOC), while no detectable levels of C compounds were released from the bryophytes Sphagnum sp. or H. splendens. A decrease in net N mineralization was determined in soils amended with L. palustre or E. hermaphroditum leachates, and this effect was inversely proportional to the phenolic concentrations, DOC and leachate C/N ratio. The total percentage of white spruce germination and the growth of white spruce seedlings were similar among treatments. CONCLUSIONS: These results suggest that the shrubs L. palustre and E. hermaphroditum could negatively affect the performance of white spruce due to a decrease in soil N availability, but not by direct effects on plant physiology.     

Recent advance of white spruce (Picea glauca) in the coastal tundra of the eastern shore of Hudson Bay (Québec, Canada)

Aim The species‐specific response of tree‐line species to climatic forcing is a crucial topic in modelling climate‐driven ecosystem dynamics. In northern Québec, Canada, black spruce (Picea mariana) is the dominant species at the tree line, but white spruce (Picea glauca) also occurs along the maritime coast of Hudson Bay, and is expanding along the coast and on lands that have recently emerged because of isostatic uplift. Here we outline the present distribution, structure, dynamics and recent spread of white spruce from the tree line up to its northernmost position in the shrub tundra along the Hudson Bay coast. We aimed to obtain a minimum date of the arrival of the species in the area and to evaluate its dynamics relative to recent climate changes. Location White spruce populations and individuals were sampled along a latitudinal transect from the tree line to the northernmost individual in the shrub tundra along the Hudson Bay coast and in the Nastapoka archipelago in northern Québec and Nunavut, Canada (56°06′–56°32′ N). Methods White spruce populations were mapped, and the position, dimension, growth form and origin (seed or layering) of every individual recorded. Tree‐ring analyses of living and dead trees allowed an estimation of the population structure, past recruitment, growth trends and growth rate of the species. A macrofossil analysis was performed of the organic horizon of the northernmost white spruce stands and individuals. Radiocarbon dates of white spruce remains and organic matter were obtained. The rate of isostatic uplift was assessed by radiocarbon dating of drifted wood fragments. Results The first recorded establishment of white spruce was almost synchronous at all sites and occurred around ad 1660. Spruce recruitment was rather continuous at the tree line, while it showed a gap in the northern shrub tundra during the first decades of the 19th century. A vigorous, recent establishment of seedlings was observed in the shrub tundra; only wind‐exposed, low krummholz (stunted individuals) did not show any sexual regeneration. A period of suppressed growth occurred from the 1810s to the 1850s in most sites. A growth increase was evident from the second half of the 19th century and peaked in the 1880s and the 20th century. A shift from stunted to tree growth form has occurred since the mid‐19th century. No sample associated with white spruce remains gave a date older than 300 ¹⁴C years bp [calibrated age (cal.) ad 1430–1690]. Main conclusions White spruce probably arrived recently in the coastal tundra of Hudson Bay due to a delayed post‐glacial spread. The arrival of the species probably occurred during the Little Ice Age. The established individuals survived by layering during unfavourable periods, but acted as nuclei for sexual recruitment almost continuously, except in the northernmost and most exposed sites. Warmer periods were marked by strong seedling recruitment and a shift to tree growth form. Unlike white spruce, black spruce showed no evidence of an ongoing change in growth form and sexual recruitment. Ecological requirements and recent history of tree‐line species should be taken into account in order to understand the present dynamics of high‐latitude ecosystems.     

Frequency of somaclonal variation in plants of black spruce (Picea mariana,Pinaceae) and white spruce (P. glauca,Pinaceae) derived from somatic embryogenesis and identification of some factors involved in genetic instability

Plants of black spruce (Picea mariana, N = 7047 individuals) and white spruce (P. glauca, N = 3995 individuals) were regenerated from a total of 87 clones over a 5-yr period by somatic embryogenesis to study factors that might be associated with the appearance of variant phenotypes. Morphological evaluation of the plants showed several types of variation. These variations were grouped into nine types: dwarfism (type A), reduced height with various form anomalies (types B, C, and D), needle fasciation (type E), abnormality in tree architecture (type F), variegata phenotype (type G), and plants with an overall regular morphology but smaller than normal plants (type H). Plagiotropic plants were also observed (type I). Each plant from types A to H (except type C where no plants survived more than 6 mo) had retained its phenotype over 4–5 yr of growth. Some of the variant types could be related to chromosomic instability: chromosome counts showed aneuploid cells for type-A and type-D plants. The type I (plagiotropism) was not related to genetic instability but rather to physiological disorders. In total, spruce variants of types A–H were obtained at relatively low frequencies, i.e., 1.0% (39/3995) for white spruce and 1.6% (110/7047) for black spruce. Statistical analyses, conducted with family, clone, and time in maintenance as variables, showed that clone was the most important source of genetic instability followed by time in maintenance.     

Stress responses in alfalfa (Medicago sativa L.). 8. Cis-elements and trans-acting factors for the quantitative expression of a bean chalcone synthase gene promoter in electroporated alfalfa protoplasts

A chimeric gene consisting of a bean (Phaseolus vulgaris L.) chalcone synthase (CHS) promoter fused to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed, and further induced by fungal elicitor, when electroporated into alfalfa (Medicago sativa L.) suspension cell protoplasts. Functional analysis of 5 deletions of the CHS promoter-CAT construct in these protoplasts indicated that the region between –326 and –130 contained both activator and silencer elements. Co-electroporation experiments confirmed that these cis-acting elements were binding sites for functionally active trans factors. In vitro DNase I footprinting revealed four potential binding sites for alfalfa suspension cell nuclear proteins between positions –326 and –130 of the CHS promoter. These sites mapped to regions shown to contain functional cis-acting elements on the basis of the deletion analysis. Three of these sites mapped to previously identified binding sites for bean nuclear proteins. Competition gel retardation analysis using oligonucleotide probes containing binding site sequences revealed sequence-specific binding of alfalfa nuclear proteins to an AT-rich element and a putative GT-1 factor consensus binding sequence. Our results define cis elements and their cognate trans factors functionally active in determining the quantitative expression of a defense response gene in a heterologous transient expression system.Abbreviations CAT chloramphenicol acetyltransferase - CHS chalcone synthase (EC 2.3.1.74) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Interactions between extraradical ectomycorrhizal mycelia, microbes associated with the mycelia and growth rate of Norway spruce (Picea abies) clones

Despite their ecological relevance, field studies of the extraradical mycelia of ectomycorrhizal (ECM) fungi are rare. Here we examined in situ interactions between ECM mycelia and host vigour. Ectomycorrhizal mycelia were harvested with in-growth mesh bags buried under Norway spruce (Picea abies) clones planted in 1994 in a randomized block design. Mycelial biomass was determined and fungal species were identified by denaturing gradient gel electrophoresis (DGGE) and sequencing of the internal transcribed spacer 1 (ITS1) region. Microbial community structure in the mycelium was investigated by phospholipid fatty acid (PLFA) profiling. Compared to slow-growing spruce clones, fast-growing clones tended to support denser mycelia where the relative proportions of Atheliaceae fungi and PLFAs indicative of Gram-positive bacteria were higher. Ascomycetes and PLFAs representative of Gram-negative bacteria were more common with slow-growing clones. In general, the ECM mycelial community was similar to the ECM root-tip community. Growth rate of the hosts, the ECM mycelial community and the microbes associated with the mycelium were related, suggesting multitrophic interactions between trees, fungi and bacteria.