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PubMed1993

cDNA sequence and expression of an intron-containing histone H2A gene from Norway spruce, Picea abies.

We have isolated a cDNA clone corresponding to a histone H2A gene from Norway spruce, Picea abies (L.) Karst. The clone was isolated on the basis of the preferential expression of the corresponding gene during germination. The identification of the clone was based on the high degree of nucleotide sequence identity (60-65%) to a range of eukaryotic histone H2A genes and the presence of a 9 amino acids long sequence identical to the conserved 'H2A box' in the deduced amino acid sequence. Like other plant histone genes, the spruce histone H2A gene encodes a polyadenylated transcript. Further, the spruce gene contains an intervening sequence of 891 bp in the coding region. The presence of introns is typical of a distinct class of replication-independent histone genes in other eukaryotes. However, the sequence of the spruce gene and its high expression in mitotically active tissues such as the apical meristem, strongly suggests that it belongs to the class of replication-dependent histone genes. This is the first documentation of an intervening sequence in this class of histone genes and the finding implies that introns were present in the ancestral histone H2A gene before the divergence of the two classes of histone genes.

Amino Acid Sequence↗
PubMed1990

T-DNA presence and opine production in tumors of Picea abies (L.) Karst induced by Agrobacterium tumefaciens A281.

The hypervirulent Agrobacterium tumefaciens strain A281 formed frequent tumors (31%) on Picea abies (Norway spruce), an economically important tree species in Swedish forests. Three-month-old seedlings were inoculated and tumors were established that grew hormone-independently in culture. Tumors contained agropine and mannopine/mannopinic acid as determined by acid pH paper electrophoresis. In addition, DNA hybridization studies showed that the DNA from these tumor lines contained sequences homologous to Ti plasmid T-DNA, whereas wild-type spruce seedling DNA did not. These results suggest that Agrobacterium vectors can be used for gene transfer into this important forest species.

DNA Probes↗
PubMed1996

Molecular characterization of a phosphoenolpyruvate carboxylase in the gymnosperm Picea abies (Norway spruce).

Phosphoenolpyruvate carboxylase (PEPC) genes and cDNA sequences have so far been isolated from a broad range of angiosperm but not from gymnosperm species. We constructed a cDNA library from seedlings of Norway spruce (Picea abies) and identified cDNAs coding for PEPC. A full-length PEPC cDNA was sequenced. It consists of 3522 nucleotides and has an open reading frame (ORF) that encodes a polypeptide (963 amino acids) with a molecular mass of 109551. The deduced amino acid sequence revealed a higher similarity to the C3-form PEPC of angiosperm species (86-88%) than to the CAM and C4 forms (76-84%). The putative motif (Lys/Arg-X-X-Ser) for serine kinase, which is conserved in all angiosperm PEPCs analysed so far, is also present in this gymnosperm sequence. Southern blot analysis of spruce genomic DNA under low-stringency conditions using the PEPC cDNA as a hybridization probe showed a complex hybridization pattern, indicating the presence of additional PEPC-related sequences in the genome of the spruce. In contrast, the probe hybridized to only a few bands under high-stringency conditions. Whereas this PEPC gene is highly expressed in roots of seedlings, a low-level expression can be detected in cotyledons and adult needles. A molecular phyiogeny of plant PEPC including the spruce PEPC sequence revealed that the spruce PEPC sequence is clustered with monocot and dicot C3- form PEPCs including the only dicot C4 form characterized so far.

Amino Acid Sequence↗
PubMed1993

Molecular cloning, sequence analysis and elicitor-/ozone-induced accumulation of cinnamyl alcohol dehydrogenase from Norway spruce (Picea abies L.).

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. We have previously isolated pure CAD enzyme from Norway spruce (Picea abies L.) cell culture. Here we report on partial protein sequences of the 42 kDa CAD polypeptide. A cDNA encoding CAD was isolated from the spruce cell culture. The open reading frame of a full-length cDNA coded for a 357 amino acid polypeptide with a calculated M(r) of 38,777 Da. The identity of the deduced polypeptide was verified by comparison with amino acid sequences of tryptic peptides from the purified enzyme. Southern blot analysis showed the presence of only one gene for CAD. Sequence comparison with CAD from tobacco and with a N-terminal protein sequence from loblolly pine CAD showed an identity of 69.7% and 91.5%, respectively. Treatment of spruce cell cultures with elicitor, as well as of seedlings with ozone both markedly increased the CAD mRNA level.

Alcohol Oxidoreductases↗
PubMed1993

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.

DNA, Ribosomal↗
PubMed1991

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 gymnosperm Picea glauca (white spruce). Promoter expression was assayed in three different tissues capable of in vitro regeneration, zygotic embryos, seedlings and embryogenic callus. The promoters tested include the light-inducible Arabidopsis 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-beta-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 beta-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.

Cloning, Molecular↗
PubMed1996

Expression of abundant mRNAs during somatic embryogenesis of white spruce [Picea glauca (Moench) Voss].

Embryogenic tissues of white spruce [Picea glauca (Moench) Voss] remain in an early developmental stage while cultured on 2,4-dichlorophenoxyacetic acid and N6-benzyladenine, but develop to cotyledonary embryos when these phytohormones are replaced by abscisic acid. Twenty-eight cDNAs were isolated from cotyledonary embryos by differential screening against immature embryo and non-embryonic tissues. Temporal expression patterns of these cDNAs during ABA-stimulated somatic embryo development were observed. This showed that clones could be allocated to various groups, including embryo-abundant, embryo-maturation-induced, and those whose expression was modulated during embryo development, germination or in non-embryogenic tissues. Expression corresponding to these cDNA clones showed that there were various responses to exogenous ABA or polyethylene glycol during a period of 48 h. Analyses of DNA and predicted amino acid sequence revealed that 12 of 28 cDNA clones had no known homologues, while others were predicted to encode different late-embryogenesis-abundant proteins, early methionine-labelled proteins, storage proteins, heat-shock proteins, glycine-rich cell wall protein, metallothionein-like protein and some other metabolic enzymes.

Base Sequence↗
PubMed1996

Characterization of three heat-shock-protein genes and their developmental regulation during somatic embryogenesis in white spruce [Picea glauca (Moench) Voss].

Three cDNAs (PgEMB22, 27 and 29) predicted to encode low-molecular-weight (LMW) heat-shock proteins (HSPs) were cloned and characterized from white spruce [Picea glauca (Moench) Voss] somatic embryo tissues by differentially screening a cotyledonary embryo cDNA library. Clone PgEMB22 is predicted to encode a putative mitochondria-localized LMW HSP, and PgEMB27 and 29 are predicted to encode different cytoplasmic class II LMW HSPs, although they share 84.7% identity within DNA coding regions and 83.0% identity for predicted proteins. They are developmentally regulated during somatic embryo development and subsequent embryo germination, in addition they show strong response to heat-shock stress. Transcripts of the two kinds of hsp genes could be detected in embryogenic tissues before induction of embryo maturation, but subsequently increased, being most abundant at late embryo stages. Gene expression levels were very low or not detectable in germinated plantlets or needle tissues from older plants. Abscisic acid and polyethylene glycol, stimulators for spruce embryo maturation, could also induce the hsp genes.

Abscisic Acid↗
PubMed1981

Aminopeptidases in seeds of picea abies (L.) Karst.: characterization of leucine aminopeptidase by molecular properties and inhibitors.

By either acrylamide or starch gel electrophoresis of Norway spruce (Picea abies) seed extracts, two prominent isoenzyme bands were obtained after staining for leucine aminopeptidase (LAP). These bands were proved to correspond to each other by reelectrophoresis in both gel media. Single endosperm studies with acrylamide gels showed clearly that, in addition to LAP, two bands are expressed after staining for alanine aminopeptidase (AAP) as well. Both the LAP and the AAP activities appeared together as a single peak between catalase and ferritin after gel chromatograhy on Sepharose. Isoelectric focusing in sucrose gradients proved the two LAP activities to have identical isoelectric points revealed that LAP, but not AAP, is detectable by standard starch gel electrophoretic procedures. The two LAP bands refer to approximate molecular weights of 71,000 and 131,000, respectively. Disaggregation studies did not conclusively determine whether these two bands represent different enzymes or not. only inhibitors succeeded in producing a definite differentiation by selective inhibition of one of the two bands. It is concluded that in both gel media the isoenzyme bands reflect the activity of two distinct leucine aminopeptidases.

Aminopeptidases↗
PubMed1997

Endochitinase and beta-1,3-glucanase genes are developmentally regulated during somatic embryogenesis in Picea glauca.

Two cDNAs isolated from white spruce [Picea glauca (Moench) Voss] somatic embryos, are predicted to encode a basic class IV chitinase and a beta-1,3-glucanase, respectively corresponding to genes PgChi-1 and PgGlu-1. Each represents a multigene family in spruce. Transcripts homologous to PgChi-1 or PgGlu-1 genes were highly abundant in embryogenic tissues and gradually decreased after tissues were placed on abscisic acid-containing maturation medium, with lowest abundance in globular embryos. Transcripts related to PgGlu-1 became highly abundant again in early cotyledonary embryos but decreased thereafter, whereas transcripts related to PgChi-1 were also highly abundant in late cotyledonary embryos and plantlets in vitro; transcripts were either low (PgChi-1) or were not detectable (PgGlu-1) in needles. Wounding, drying and flooding stresses enhanced PgChi-1- and PgGlu-1-related gene expression. Fungal cell wall suspension enhanced PgGlu-1-related transcript accumulation, but reduced PgChi-1-related transcript abundance within 24 h. PgChi-1 and PgGlu-1 and their homologues may have roles in plant defense, and possibly developmental roles during spruce somatic embryo maturation.

Amino Acid Sequence↗
PubMed2002

Pollen competition and seed-siring success in Picea abies.

The aim of the present work was to study pollen-tube competition in Picea abies. Controlled crossings were performed with pollen mixtures including pairs of pollen lots with fast and slowly elongating pollen-tubes. Paternity analysis using isozyme markers was performed on the progenies in order to study whether the in vitro pollen-germination vigour corresponds to the proportion of seeds sired by the pollen donor. Paternal success was found to be unequal, 15 out of 23 crossings producing progeny that differed significantly from the hypothetical ratio of 1:1. The paternal contribution in the majority of the crossings was as expected: the pollen parent with more-vigorous in vitro germination sired more seeds than the less-vigorous pollen. In the case of two pollen mixtures, however, the seed-siring success summed over the maternal trees was the opposite to the expected value. Despite these aberrations, the results support the hypothesis that pollen-tube competition is one of the factors contributing to male fitness in P. abies.However, when all the other factors affecting pollination and seed set under natural conditions are taken into account, it is clear that the seed-siring success of a particular paternal genotype cannot be predicted reliably by measuring only the in vitro pollen vigour.

Journal Article↗
PubMed2002

Megagametophyte-derived linkage maps of white spruce ( Picea glauca) based on RAPD, SCAR and ESTP markers.

We have constructed linkage maps for two parents of white spruce [ Picea glauca (Moench) Voss]. Haploid megagametophytes from 92 and 96 seeds of parents M2 and 80132, respectively, were analysed with RAPD, SCAR and ESTP markers. Fragments segregating in a 1:1 Mendelian ratio were classified and mapped using MAPMAKER, GMENDEL and JOINMAP. For M2, the analysis with JOINMAP resulted in 165 loci (152 RAPDs, 3 SCARs and 10 ESTPs) mapping to 23 linkage groups and covering 2,059.4 cM(Kosambi function, K). For 80132, the analysis resulted in 144 loci (137 RAPDs, 1 SCAR and 7 ESTPs) mapping to 19 linkage groups and covering 2,007.7 cM(K). The maps covered 87 and 73% of the entire genome of parents M2 and 80132, respectively. Similar results were obtained with MAPMAKER and GMENDEL. A comparison was made between the two individual maps and 16 loci were shared between the two maps.

Journal Article↗
PubMed1997

Characterization of cDNAs representing five abscisic acid-responsive genes associated with somatic embryogenesis in Picea glauca, and their responses their responses to abscisic acid stereostructure.

Five abscisic acid (ABA)-responsive cDNAs that were identified by differential screening of a white spruce [Picea glauca (Moench) Voss] cotyledonary somatic embryo cDNA library are characterized. Three of the cDNAs (PgEMB12, 14, 15) are predicted to encode homologues of different late-embryogenesis-abundant (LEA) proteins, a further two (PgEMB 5 and 23) share little similarity to any known DNA or protein sequences. When suspension cultures were fed 1.5 x 10(5) M S-(+)-ABA and assessed for transcript abundance over 48 h, gene expression corresponding to each lea-like gene was inducible and was evident during the 48-h period. Conversely, transcripts were at a very low abundance when suspensions were fed R-(-)-ABA. When suspensions were treated with a range of S-(+)-ABA and R-(-)-ABA concentrations (10(-7)-10(-4) M), expression was induced at S-(+)-ABA concentrations of 10(-5) M or above. Expression corresponding to PgEMB5 and PgEMB23 indicates less specificity for ABA stereostructure, with similar expression patterns being observed for either enantiomer during the 48 h after feeding. The two corresponding genes also appear more responsive to ABA concentration than the predicted lea genes, within the range examined (10(-7)-10(-4) M).

Abscisic Acid↗
PubMed2005

Chemical response of Picea glehnii seed-epiphytic Penicillium species to Pythium vexans under in vitro competitive conditions for mycelial growth.

The potential protection of Picea glehnii seedlings from damping-off by seed-epiphytic Penicillium species was investigated. We studied the chemical response of seed-epiphytic Penicillium species (Pen. cyaneum, Pen. damascenum, and Pen. implicatum) to Pythium vexans, a damping-off fungus, in vitro. Penicillium species were cultured singly or cocultured with Pyt. vexans for 14 or 18 d, and mycelial growth, pH of culture filtrate, antifungal activity of the culture filtrate against Pyt. vexans, and the amount of antifungal compound produced by each Penicillium species, were examined. The filtrate of both the single culture of Penicillium and the coculture of Penicillium and Pyt. vexans showed antifungal activity against Pyt. vexans. In a coculture with Pyt. vexans, Pen. cyaneum produced an antifungal compound (patulin) as in the single culture. Pen. damascenum cocultured with Pyt. vexans produced an antifungal compound (citrinin), as it did in the single culture and in larger amounts on day 10. Pen. implicatum produced two antifungal compounds, frequentin and palitantin, and the ratio of frequentin (with higher antifungal activity than palitantin) to palitantin was higher in the coculture with Pyt. vexans than in the single culture. Our results indicate that these Penicillium species have the ability to produce antifungal compounds and to keep anti-fungal activity under competitive condition with Pyt. vexans. The chemical response of these Penicillium species to Pyt. vexans may contribute to protect P. glehnii seedlings from damage by Pyt. vexans.

Antifungal Agents↗
PubMed1995

Phenolic extractives from root bark of Picea abies.

Five new compounds, viz. 3,4-dimethoxyphenyl 2-O-(3-O-methyl-alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside, 4'-hydroxyphenacyl beta-D-glucopyranoside, (2R,3R)-2,3-dihydro-7-hydroxy-2-(4'- hydroxy-3'-methoxyphenyl)- 3-hydroxymethyl-5-benzofuranpropanol 4'-O-(3-O-methyl-alpha-L-rhamnopyranoside), 1-(4'-hydroxy-3'-methoxyphenyl)-2-[4"-(3-hydroxypropyl)-2"-methoxyphe noxy]-1,3-propanediol 4'-O-beta-D-xylopyranoside and 3'-O-methylcatechin 7-O-beta-D-glucopyranoside, together with several known compounds were isolated from the root bark of Picea abies. The structures were elucidated on the basis of chemical and spectroscopic evidence.

Carbohydrate Conformation↗
PubMed1990

Monoterpene patterns of different tissues and plant parts of Norway spruce (Picea abies L. Karst.).

Norway spruce saplings (Picea abies L. Karst.) were exposed for five years to controlled concentrations of ozone and/or sulphur dioxide in open-top chambers. The monoterpenes of needles, twigs (bark and wood), bark and buds harvested from these trees were qualitatively and quantitatively analysed by combined gas chromatography-mass spectrometry. Different tissues and plant parts, showed notable differences in their percentage distribution. However, comparison of the monoterpene patterns of corresponding plant material obtained from trees which had been exposed to different fumigation regimes showed no significant effects of these pollutants.

Journal Article↗
PubMed1990

Effects of low-level long-term ozone fumigation and acid mist on photosynthesis and stomata of clonal Norway spruce (Picea abies (L.) Karst.).

Four clones of 3-year-old Norway spruce (Picea abies (L.) Karst.), grown on two soils, were from July 1986 to September 1987 exposed to ozone fumigation (50 microg m(-3) as a control, 100 microg m(-3) plus peaks between 130 and 360 microg m(-3) as treatment) and acid mist of pH 3.0 (versus mist pH 5.6 in the control). Climatic conditions, identical for both control and treatment, followed a diurnal and seasonal pattern characteristic of medium high altitudes of the Bavarian Forest, an area affected by the new-type forest decline. Gas-exchange measurements were carried out on the plants from December 1986 until the end of the 14-month's exposure using a series of climate-controlled minicuvettes. ANOVA of the four clones investigated towards the end of the experiment gave hints of a treatment-related depression of the photosynthetic capacity of the previous year's needles (age-class 1986). Within this age-class only one of the clones (11) showed a significant treatment effect, indicating an age-class dependence and a genetic influence of the treatment-related depression of the photosynthetic capacity. The current year's flush was not impaired through the ozone and acid mist exposure. Analysis also revealed clear effects of soil, clone and needle age on photosynthetic parameters.

Journal Article↗
PubMed1990

Biochemical response of Norway spruce (Picea abies (L.) Karst.) towards 14-month exposure to ozone and acid mist: part I--Effects on polyphenol and monoterpene metabolism.

Three-year-old clonal Picea abies (L.) Karst. plants, grown either on a sandy (No. 1) or on a calcareous (No. 2) soil, were treated with ozone (100 microg m(-3) and peaks of up to 360 microg m(-3)) and acid mist (pH 3.0) over two vegetation periods. Needles of the current (1987) and previous (1986) year were analysed at the end of the experiment for biosynthetic enzymes and in vivo activity of the phenylpropanoid pathway, for products of polyphenol metabolism (clones 11 and 14), and for mono- and sesquiterpenes (clones 14 and 16). 1. Polyphenol metabolism. The activity of cinnamyl alcohol dehydrogenase, an enzyme of the lignin pathway, was increased by the treatment by up to 83% in the needles of both age classes. Chalcone synthase was measured in mature tree material for the first time. This enzyme, as well as phenylalanine ammonia-lyase activities and the content of catechin, astringin, isorhapontin, picein and p-hydroxyacetophenone, exhibited no significant treatment-dependent differences. However, soil and age-class dependent differences occurred. Pulse-labelling experiments with l[U-(14)C]phenylalanine and [2-(14)C]acetate were carried out at four different stages of shoot development and showed label incorporation into (+)-catechin and proanthocyanidins. There was no effect of the ozone and acid mist treatment. 2. Monoterpenes. The content of needle terpenes was generally diminished by the ozone and acid mist treatment. Other factors tested, such as clone, needle age and soil, exerted a highly significant influence on the content of most of the needle monoterpenes.

Journal Article↗