Induction of Adventitious Buds on Buds of Norway Spruce (Picea abies) Grown in vitro

Resting vegetative buds of Norway spruce, Picea abies (L.) Karst., were induced to form adventitious bud primordia when cultured on medium -containing cytokinin. After transfer of the induced buds to medium lacking cytokinin, adventitious buds developed. The adventitious buds arose from meristems formed de novo in the needle primordia. No differences were found in the ability to form adventitious buds among buds collected from trees ranging from 5–50 years old.     

Root Respiration in White Spruce (Picea glauca [Moench] Voss) Seedlings in Relation to Morphology and Environment

Roots of white spruce seedlings (Picea glauca [Moench] Voss) undergo respiratory changes during the year that are related to changing metabolic requirements. An alternative pathway is always present, functions during most of the year, and operates maximally during periods of root and shoot growth. Although some differences in respiration and the apportioning of respiration can be correlated to root morphology, the environment and the stage of shoot development are also important controls. Differences in respiration related to root morphology are not manifest in mitochondrial structure, but overall rates were found to correlate with the number of mitochondria present. Root respiration in seedlings grown under root growth capacity conditions reflects root and shoot growth at that time rather than root growth capacity.     

Peroxidase Activity in Relation to Suberization and Respiration in White Spruce (Picea glauca [Moench] Voss) Seedling Roots

Peroxidase (EC 1.11.1.7) activity is associated with suberization during endodermal development and metacutization in roots of white spruce (Picea glauca [Moench] Voss) seedlings. Histochemical analysis indicates a relationship between suberization and peroxidase activity, but peroxidase is ubiquitous. Increased peroxidase activity results from the induction of four anodic peroxidase isozymes in addition to quantitative increases in two anodic peroxidase isozymes. Four of these polymerized eugenol. Cold temperatures induce formation of two anodic isozymes and result in suberization. The increased peroxidase activity associated with suberization is correlated to residual respiration. In an attempt to elucidate this relationship, the effect of respiratory inhibitors on respiration and peroxidase activity are compared.     

An embryogenic cell suspension culture of Picea glauca (White spruce)

A cell suspension culture of Picea glauca (White spruce) which continuously produces somatic embryos has been established. Embryogenic callus derived from cultured zygotic embryos was used to initiate the culture. Numerous embryos at various early stages of development were recognized; they exhibited a meristematic embryonic region and suspensor consisting of elongate, vacuolated cells. The culture also contained clumps of meristematic cells and large irregular — shaped cells. The culture could be readily re-established on solid medium.     

Nuclear Magnetic Resonance Studies of Poly(3-Hydroxybutyrate) and Polyphosphate Metabolism in Alcaligenes eutrophus

The metabolic pathways of poly(3-hydroxybutyrate) (PHB) and polyphosphate in the microorganism Alcaligenes eutrophus H16 were studied by ¹H, ¹³C, and ³¹P nuclear magnetic resonance (NMR) spectroscopy and by conventional analytical techniques. A. eutrophus cells accumulated two storage polymers of PHB and polyphosphate in the presence of carbon and phosphate sources under aerobic conditions after exhaustion of nitrogen sources. The solid-state cross-polarization/magic-angle spinning ¹³C NMR spectroscopy was used to study the biosynthetic pathways of PHB and other cellular biomass components from ¹³C-labeled acetate. The solid-state ¹³C NMR analysis of lyophilized intact cells grown on [1-¹³C]acetate indicated that the carbonyl carbon of acetate was selectively incorporated both into the carbonyl and methine carbons of PHB and into the carbonyl carbons of proteins. The ³¹P NMR analysis of A. eutrophus cells in suspension showed that the synthesis of intracellular polyphosphate was closely related to the synthesis of PHB. The roles of PHB and polyphosphate in the cells were studied under conditions of carbon, phosphorus, and nitrogen source starvation. Under both aerobic and anaerobic conditions PHB was degraded, whereas little polyphosphate was degraded. The rate of PHB degradation under anaerobic conditions was faster than that under aerobic conditions. Under anaerobic conditions, acetate and 3-hydroxybutyrate were produced as the major extracellular metabolites. The implications of this observation are discussed in connection with the regulation of PHB and polyphosphate metabolism in A. eutrophus.     

Cryopreservation and Plant Regeneration from Embryogenic Cultures of Larch (Larixxeurolepis) and Black Spruce (Picea mariana)

A method has been developed for the routine cryopreservationof embryogenic cultures of hybrid larch (Larixxeurolepis) andblack spruce (Picea mariana Mill.). The method involves growingthe cultures in the presence of sorbitol and then briefly exposingthem to DMSO followed by controlled cooling to –40°C.The cultures were then submerged and stored in liquid nitrogen.Growth of the embryogenic cultures was monitored for 14 d afterrapid thawing and plating on to media. The highest relativeincrease in the tissue fresh weight, after storage in liquidnitrogen, was observed when embryogenic cultures of both specieswere pregrown for 24 h in a medium with 0·4 M sorbitoland then treated with 10% DMSO. This pretreatment also ensuredthe shortest lag phase in resuming the growth. The post-thawcultures gave rise to mature somatic embryos which developedinto plants Key words: Larixxeurolepis, Picea mariana, cryopreservation, embryogenic tissue, plant regeneration     

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.     

Carbon-13 Nuclear Magnetic Resonance Analysis of Metabolism in Soybean Labeled by CO(2)

Fourier transform ¹³C nuclear magnetic resonance spectra have been obtained of intact, fresh soybean ovules (Glycine max L. cv. Dare) harvested from pods subtended by a trifoliolate exposed to ¹³CO₂ 1 to 3 days earlier. The high resolution spectra are interpreted in terms of the labeled sugars and lipids in the ovule. Comparison of the spectra taken over the 3-day period permits qualitative estimates of sugar metabolism and rates of lipid synthesis. The spectra also contain information about the distribution of labels within the lipid chains. This information leads to a method of estimating the extent to which glucose degradation in the synthesizing soybean ovule is involved in the reactions of the phosphogluconate pathway.     

Photosynthetic Characteristics of Eastern Dwarf Mistletoe (Arceuthobium pusillum Peck) and its Effects on the Needles of Host White Spruce (Picea glauca [Moench] Voss)

Abstract: We studied the biochemical composition and photosynthetic characteristics of the aerial parasite eastern dwarf mistletoe (Arceuthobium pusillum) and the effect of infection on the needles of host white spruce (Picea glauca) in a coastal forest stand in Maine, USA. Eastern dwarf mistletoe was capable of photosynthetic oxygen evolution; however, rates were low and were exceeded by respiratory oxygen consumption at all light intensities through full sunlight. Therefore, eastern dwarf mistletoe acts as a net sink for host photosynthate. Relative to those of uninfected trees, needles from infected branches of white spruce were significantly smaller in terms of length, fresh weight, maximum cross section and the diameter of the vascular cylinder. Needles of uninfected and infected trees did not differ in terms of fresh weight to dry weight ratio, nor in nitrogen, soluble sugar or starch content. Needles of infected trees possessed significantly less α-carotene and neoxanthin, but did not otherwise differ from uninfected needles in terms of chlorophyll and carotenoid composition. Since specific physiological roles for α-carotene and neoxanthin have not been described, the functional significance of the decreases in their content is not known. Photosynthetic capacities of needles from infected and uninfected white spruce did not differ significantly, as measured by oxygen evolution. These findings suggest that dwarf mistletoe infection does not substantially perturb host white spruce source-sink balance at the end of the growing season and that carbon exchange dynamics between the host and parasite are unlikely to fully explain the detrimental effects of infection on white spruce.     

Carbon-13 Nuclear Magnetic Resonance Study of Metabolism of Propionate by Escherichia coli

We have evaluated the use of [1,2-13C2]propionate for the analysis of propionic acid metabolism, based on the ability to distinguish between the methylcitrate and methylmalonate pathways. Studies using propionate-adapted Escherichia coli MG1655 cells were performed. Preservation of the 13C-13C-12C carbon skeleton in labeled alanine and alanine-containing peptides involved in cell wall recycling is indicative of the direct formation of pyruvate from propionate via the methylcitrate cycle, the enzymes of which have recently been demonstrated in E. coli. Additionally, formation of 13C-labeled formate from pyruvate by the action of pyruvate-formate lyase is also consistent with the labeling of pyruvate C-1. Carboxylation of the labeled pyruvate leads to formation of [1,2-13C2]oxaloacetate and to multiply labeled glutamate and succinate isotopomers, also consistent with the flux through the methylcitrate pathway, followed by the tricarboxylic acid (TCA) cycle. Additional labeling of TCA intermediates arises due to the formation of [1-13C]acetyl coenzyme A from the labeled pyruvate, formed via pyruvate-formate lyase. Labeling patterns in trehalose and glycine are also interpreted in terms of the above pathways. The information derived from the [1, 2-13C2]propionate label is contrasted with information which can be derived from singly or triply labeled propionate and shown to be more useful for distinguishing the different propionate utilization pathways via nuclear magnetic resonance analysis.     

A Nuclear Magnetic Resonance Study of Water in Cold-acclimating Cereals

Continuous wave nuclear magnetic resonance (NMR) studies indicated that the line width of the water absorption peak (Δv½) from crowns of winter and spring wheat (Triticum aestivum L.) increased during cold acclimation. There was a negative correlation between Δv½ and crown water content, and both of these parameters were correlated with the lowest survival temperature at which 50% or more of the crowns were not killed by freezing (LT₅₀). Regression analyses indicated that Δv½ and water content account for similar variability in LT₅₀. Slow dehydration of unacclimated winter wheat crowns by artificial means resulted in similarly correlated changes in water content and Δv½. Rapid dehydration of unacclimated crowns reduced water content but did not influence Δv½. The incubation of unacclimated winter wheat crowns in a sucrose medium reduced water content and increased Δv½. The increase in Δv½ appears to be dependent in part on a reduction in water content and an increase in solutes.     

Enhanced Maturation and Desiccation Tolerance of White Spruce [Picea glauca (Moench) Voss] Somatic Embryos: Effects of a Non-plasmolysing Water Stress and Abscisic Acid

A non-plasmolysing moisture stress effected by polyethyleneglycol (PEG) was beneficial when applied to maturing white spruce(Picea glauca) somatic embryos for the following reasons. Anosmotic treatment of 5.0–7.5% PEG stimulated a threefoldincrease in the maturation frequency. The osmotically treatedsomatic embryos displayed higher dry weights and lower moisturecontents than the controls, indicating a greater accumulationof storage reserves. Moisture contents of mature, osmotically-treated,hydrated somatic embryos were 40–45%, in contrast to 57%for the non-osmotically treated controls. Desiccation was achievedby placing the somatic embryos in a range of relative-humidityenvironments. No clear trend for the effect of PEG on survivalof desiccated somatic embryos was observed; mean survival valuesranged from 34 to 62% when somatic embryos from all osmotictreatments were desiccated for 14 d at 81% relative humidity.Following this desiccation treatment, somatic embryos from allosmotic concentrations had moisture contents of 26–31%,similar to the 32% recorded for unimbibed zygotic embryos. Afterimbibition, moisture contents for these zygotic and somaticembryos were in the order of 60%. Somatic embryos matured withPEG remained quiescent during desiccation due to their low initialmoisture contents, and gave rise to plantlets of normal appearance.Gradual desiccation of the somatic embryos directly followingmaturation with abscisic acid (ABA) was crucial to survivalduring desiccation. A plasmolysing water stress effected bysucrose at osmotic potentials similar to PEG was detrimentalto somatic embryo maturation, thereby emphasizing the importanceof the choice of osmoticum. Desiccation, maturation, osmotic potential, Picea glauca, polyethylene glycol, somatic embryo, water stress, white spruce     

Metabolism of [U-13C]Aspartate by Astroglial Cultures: Nuclear Magnetic Resonance Analysis of the Culture Media

In brain the amino acid L-aspartate serves roles as: (1) putative transmitter, (2) protein precursor, (3) donor of atoms for the biosynthesis of pyrimidine and purine bases, and (4) fuel for energy metabolism. Astrocytes dominate aspartate clearance in brain, and in culture they take up aspartate and quickly metabolize it. In brain, only astrocytes were shown to express the enzymes for de novo pyrimidine biosynthesis. To gain more details about the spectrum of metabolites generated from aspartate and subsequently released by cultured astrocytes a ¹³C-nuclear magnetic resonance analysis was performed of [U-¹³C]aspartate supplemented incubation media exposed to astroglial cultures. The results show that astrocytes readily metabolize aspartate and release into their culture media ¹³C-isotopomers of lactate, glutamine, citrate and alanine. Despite the presence in astroglial cells of two tandem enzymes of pyrimidine biosynthesis and their mRNAs, pyrimidine nucleotide-related heterocyclic compounds such as dihydroorotate and orotate could not be detected in the culture media.     

Interactions between Carbon and Nitrogen Metabolism in Fibrobacter succinogenes S85: a 1H and 13C Nuclear Magnetic Resonance and Enzymatic Study

The effect of the presence of ammonia on [1-¹³C]glucose metabolism in the rumen fibrolytic bacterium Fibrobacter succinogenes S85 was studied by ¹³C and ¹H nuclear magnetic resonance (NMR). Ammonia halved the level of glycogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-acetate ratio. The ¹³C enrichment of succinate and acetate was precisely quantified by ¹³C-filtered spin-echo difference ¹H-NMR spectroscopy. The presence of ammonia did not modify the ¹³C enrichment of succinate C-2 (without ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic dilution of metabolites due to utilization of endogenous glycogen was not affected. In contrast, the presence of ammonia markedly decreased the ¹³C enrichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of the succinate synthesis pathway. The reversal of glycolysis was unaffected by the presence of ammonia as shown by ¹³C-NMR analysis. Study of cell extracts showed that the main pathways of ammonia assimilation in F. succinogenes were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthetase activity was not detected. Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation in the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities were evidenced by spectrophotometry and ¹H NMR. When cells were incubated in vivo with [1-¹³C]glucose, only ¹³C-labeled aspartate, glutamate, alanine, and valine were detected. Their labelings were consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.     

Highly Informative Single-Copy Nuclear Microsatellite DNA Markers Developed Using an AFLP-SSR Approach in Black Spruce (Picea mariana) and Red Spruce (P. rubens)

Background

Microsatellites or simple sequence repeats (SSRs) are highly informative molecular markers for various biological studies in plants. In spruce (Picea) and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana) and red spruce (Picea rubens) using a simple but efficient method based on a combination of AFLP and microsatellite technologies.

Principal Findings

A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce and 30 red spruce individuals drawn from many populations. The number of alleles at a polymorphic locus ranged from 2 to 18, with a mean of 9.3 in black spruce, and from 3 to 15, with a mean of 6.2 alleles in red spruce. The polymorphic information content or expected heterozygosity ranged from 0.340 to 0.909 (mean = 0.67) in black spruce and from 0.161 to 0.851 (mean = 0.62) in red spruce. Ten SSR markers showing inter-parental polymorphism inherited in a single-locus Mendelian mode, with two cases of distorted segregation. Primer pairs for almost all polymorphic SSR loci resolved microsatellites of comparable size in Picea glauca, P. engelmannii, P. sitchensis, and P. abies.

Significance

The AFLP-based microsatellite-enriched library appears to be a rapid, cost-effective approach for isolating and developing single-locus informative genomic microsatellite markers in black spruce. The markers developed should be useful in black spruce, red spruce and other Picea species for various genetics, genomics, breeding, forensics, conservation studies and applications.     

Visualization of Freezing Behaviors in Leaf and Flower Buds of Full-Moon Maple by Nuclear Magnetic Resonance Microscopy

1H-Nuclear magnetic resonance (NMR) microscopy was used to study the freezing behavior of wintering buds of full-moon maple (Acer japonicum Thunb.). The images obtained predominantly reflected the density of mobile (i.e. non-ice) protons from unfrozen water. A comparison of NMR images taken at different subfreezing temperatures revealed which tissues produced high- and low-temperature exotherms in differential thermal analyses. In leaf and lower buds of A. japonicum, the scales and stem bark tissues were already frozen by -7[deg]C, but the primordial inflorescence and terminal primordial shoots remained supercooled at -14[deg]C, and the lateral primordial shoots were unfrozen even at -21[deg]C. The freezing of these supercooled tissues was associated with their loss of viability. The size of the supercooled primordial shoots and inflorescences was gradually reduced with decreasing temperature, indicating extraorgan freezing in these tissues. During this process the formation of dark regions beneath the primordia and subsequent gradual darkening in the basal part of supercooled primordia were visible. As the lateral shoot primordia were cooled, the unfrozen area was considerably reduced. Since the lateral primordia remained viable down to -40[deg]C, with no detectable low-temperature exotherms, they probably underwent type I extraorgan freezing. Deep supercooling in the xylem was clearly imaged. NMR microscopy is a powerful tool for noninvasively visualizing harmonized freezing behaviors in complex plant organs.     

Kinetic and Nuclear Magnetic Resonance Studies of Xylose Metabolism by Recombinant Zymomonas mobilis ZM4(pZB5)

The specific rates of growth, substrate utilization, and ethanol production as well as yields of biomass and ethanol production on xylose for the recombinant Zymomonas mobilis ZM4(pZB5) were shown to be much less than those on glucose or glucose-xylose mixtures. Typical fermentations with ZM4(pZB5) growing on glucose-xylose mixtures followed two-phase growth kinetics with the initial uptakes of glucose and xylose being followed by slower growth and metabolic uncoupling on xylose after glucose depletion. The reductions in rates and yields from xylose metabolism were considered in the present investigation and may be due to a number of factors, including the following: (i) the increased metabolic burden from maintenance of plasmid-related functions, (ii) the production of by-products identified as xylitol, acetate, lactate, acetoin, and dihydroxyacetone by ¹³C-nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography, (iii) growth inhibition due to xylitol by the putative inhibitory compound xylitol phosphate, and (iv) the less energized state of ZM4(pZB5). In vivo ³¹P-NMR studies have established that the levels of NTP and UDP sugars on xylose were less than those on glucose, and this energy limitation is likely to restrict the growth of the recombinant strain on xylose media.     

生物大分子质谱电离技术的突破及核磁共振三维结构测定方法的建立——2002年诺贝尔化学奖简介

2002年诺贝尔化学奖授予了质谱和核磁共振领域的三位科学家以表彰他们对生物大分子鉴定及结构分析方法做出的贡献．其中两位科学家J．B．Fenn和K．Tanaka分别发展了生物大分子质谱分析的软解吸电离方法;另一科学家K．Wüthrich则将核磁共振技术成功地应用于生物大分子如蛋白质的溶液三维结构测定．他们的研究成果已使质谱和核磁共振技术成为生物大分子强有力的研究手段,极大地促进了生物大分子的研究进程,必将对整个生命科学研究产生深远的影响．     

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.