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     

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.     

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.     

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.     

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.     

Somatic embryo maturation from long-term suspension cultures of white spruce (Picea glauca)

Summary The production of cotyledonary somatic embryos of white spruce from cultures grown long-term as suspensions was investigated. We report the effects of removal of 2,4-dichlorophenoxyacetic acid (2,4-D) from the maintenance medium (ordinarily containing both 2,4-D and benzyl adenine) before (±)-ABA-stimulated maturation. In particular the use of a 1-wk culture period without 2,4-D was found to improve the production of normal-looking cotyledonary somatic embryos. Using high performance liquid chromatography analyses of culture supernatants, it was determined that this affect was not related to altered ABA metabolism. Germination of cotyledonary somatic embryos from cultures pretreated by the 1-wk culture period without 2,4-D was improved compared with similar embryos from cultures that had not been pretreated.     

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.     

Cell structural changes in the needles of Norway spruce exposed to long-term ozone and drought

Effects of ozone and/or drought on Norway spruce needles werestudied using light microscopy and electron microscopy. Saplingswere exposed to ozone in open-top chambers during 1992–1995and also to drought in the late summers of 1993–1995.Samples from current and previous year needles were collectedfive times during 1995. Ozone increased the numbers of peroxisomesand mitochondria, which suggests that defence mechanisms againstoxidative stress were active. The results from peroxisomes suggestthat the oxidative stress was more pronounced in the upper sideof the needles, and those from mitochondria that defence wasmore active in the younger needle generation. Possibly due tothe good nitrogen status and the active defence, no ozone-specificchloroplast alterations were seen. At the end of the season,older needles from ozone treatments had smaller central vacuolescompared with other needles. Cytoplasmic vacuoles around thenucleus were increased by ozone in the beginning of the experiment,and did not increase towards the end of the season as in thecontrols. These results from vacuoles may indicate that ozoneaffected the osmotic properties of the cells. Decreased numberand underdevelopment of sclerenchyma cells and proliferationof tonoplast were related to nutrient imbalance, which was enhancedby drought. Larger vascular cylinders and more effective starchaccumulation before and after the drought periods compensatedfor the reduced water status. Numbers of peroxisomes and mitochondriawere increased in the drought-exposed needles before the onsetof drought treatments of the study year, i.e. these changeswere memory effects. Interactions between ozone and droughtwere few.     

Endo-β-mannanase activity during dormancy alleviation and germination of white spruce (Picea glauca) seeds

It is not known how embryos of seeds of the Pinaceae protrude from their enclosing tissues to complete germination. Prior to protrusion of the radicle there is an increase in endo-β-1,4-mannanase (EC 3.2.1.78) activity associated with weakening of the micropylar megagametophyte/nucellus from seeds of white spruce ( Picea glauca [Moench.] Voss). Mannanase activity is present as three isoforms (pI values 5.0, 4.8, 4.7) in both the embryo and surrounding structures (megagametophyte and nucellus) prior to and during imbibition. Activity of all the isoforms increases in the chalazal and micropylar megagametophyte during germination. Activity then declines after the testa splits, typically 1 day prior to radicle protrusion, due partially to its leaching from the seed into the surrounding water. Activity increases in the cotyledons and axis as the embryo commences elongation. Seeds from dormant seedlots exhibit a lower germination percentage, relative to seeds from nondormant seedlots, and the force necessary for the embryo to puncture the surrounding structures tends to be greater. Although similar mannanase activities are present in unimbibed seeds of dormant and nondormant seedlots, during germination, enzyme activity in seeds of dormant seedlots is lower. Moist chilling alleviates dormancy in the seeds of the Pinaceae and, during 3 weeks of this treatment, mannanase activity slowly increases. After 3 weeks of moist chilling and regardless of whether the seedlot was dormant or not prior to moist chilling, the force necessary to puncture the micropylar megagametophyte and nucellus is lower, and the speed of germination greater. Seeds from previously dormant seedlots also complete germination to a greater percentage, relative to unchilled seeds from dormant seedlots. Upon transfer to 25°C, mannanase activity in moist-chilled seeds decreases during germination of all seedlots regardless of their previous dormancy status.     

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     

Structure of the intergenic spacer region from the ribosomal RNA gene family of white spruce (Picea glauca)

Five genomic clones containing ribosomal DNA repeats from the gymnosperm white spruce (Picea glauca) have been isolated and characterized by restriction enzyme analysis. No nucleotide variation or length variation was detected within the region encoding the ribosomal RNAs. Four clones which contained the intergenic spacer (IGS) region from different rDNA repeats were further characterized to reveal the sub-repeat structure within the IGS. The sub-repeats were unusually long, ranging from 540 to 990 bp but in all other respects the structure of the IGS was very similar to the organization of the IGS from wheat, Drosophila and Xenopus.     

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.     

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.     

Sodium-calcium interactions in two wheat species differing in salinity tolerance

White spruce [ Picea glauca (Moench) Voss.] seedlings were used to study the changes in cell wall composition and elasticity in mature needles before and after the resumption of growth following winter dormancy. Dormant seedlings showed high cell wall elasticity that decreased after the resumption of shoot growth. Cell wall hemicellulose content increased 3 days after planting and decreased after the buds flushed. Non-cellulosic glucose and arabinose were the sugars showing the most pronounced changes related to shoot growth. Arabinose was the most abundant sugar residue in the pectin and hemicellulose fractions and it decreased until day 10 after planting. At the same time, the levels of glucose in pectin and hemicellulose increased. The results provide evidence for cell wall carbohydrate turnover in dormant and active seedlings before and after bud flushing.     

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.     

A set of microsatellite markers for use in Sitka spruce (Picea sitchensis) developed from Picea glauca ESTs

Few microsatellite markers have been specifically developed for Picea sitchensis. In January 2004 the appearance of over 10 000 sequences of expressed regions of DNA from P. glauca in GenBank presented an opportunity for the development of additional microsatellite markers in Sitka spruce. Mono‐ and dinucleotide repeat sequences were located in these sequences and primers were designed around these regions. Amplification was attempted in Sitka material from a broad geographical range and the level of polymorphism was assessed. Primers were also tested in progeny of a controlled cross. Nine polymorphic loci that demonstrated Mendelian inheritance in Sitka were discovered in this study.     

Cryopreservation of interior spruce (Picea glauca engelmanni complex) embryogenic cultures

Summary Embryogenic cultures of interior spruce derived from 12 full-sib families were subjected to cryopreservation, with a 97 % success rate for 357 genotypes. Analyses suggested that cryotolerance was not related to family ranking (height increment), embryogenic potential or culture dispersability in suspension, and long-term storage in or above liquid nitrogen did not affect regenerative potential. By contrast, differences in cryotolerance among cell lines appeared to be prevalent in certain families. Analysis with a DNA fingerprinting probe used for clonal identification demonstrated no evidence of somaclonal variation as a result of cryopreservation. The results of this work indicate the applicability of cryopreservation as a long-term storage strategy for spruce embryogenic cultures from a wide genetic background.Abbreviations ABA ± abscisic acid - BA N⁶-benzyladenine - BSA bovine serum albumin - CTAB cetyldimethylethyl-ammonium bromide - DMSO dimethylsulfoxide - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid disodium salt - IBA indole-3-butyric acid - LN liquid nitrogen - PEG polyethylene glycol - SLS N-lauroyl sarcosine - Tris tris[hydroxymethylamino] methane - 2,4-D 2,4-dichlorophenoxyacetic acid     

Possible effect from shear stress on maturation of somatic embryos of Norway spruce (Picea abies)

Somatic embryogenesis is the only method with the potential for industrial scale clonal propagation of conifers. Implementation of the method has so far been hampered by the extensive manual labor required for development of the somatic embryos into plants. The utilization of bioreactors is limited since the somatic embryos will not mature and germinate under liquid culture conditions. The negative effect on mature embryo yields from liquid culture conditions has been previously described. We have described the negative effects of shear stress on the development of early stage somatic embryos (proembryogenic masses; PEMs) at shear stresses of 0.086 and 0.14 N/m². In the present study, additional flow rates were studied to determine the effects of shear stress at lower rates resembling shear stress in a suspension culture flask. The results showed that shear stress at 0.009, 0.014, and 0.029 N/m² inhibited the PEM expansions comparing with the control group without shear stress. This study also provides validation for the cross‐correlation method previously developed to show the effect of shear stress on early stage embryo suspensor cell formation and polarization. Furthermore, shear stress was shown to positively affect the uptake of water into the cells. The results indicate that the plasmolyzing effect from macromolecules added to liquid culture medium to stimulate maturation of the embryos are affected by liquid culture conditions and thus can affect the conversion of PEMs into mature somatic embryos. Bioeng. 2011; 108:1089–1099. © 2010 Wiley Periodicals, Inc.