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PubMed1994

Pinus sylvestris L. needles contain extracellular CuZn superoxide dismutase.

Four new isoforms of superoxide dismutase (SOD; superoxide: superoxide oxidoreductase, EC 1.15.1.1.) were identified in extracellular washing fluid from Scots pine (Pinus sylvestris L.) needles. The isoforms had an apparent molecular mass of 33 kDa. No neutral carbohydrates were present in the enzymes. The enzymatic activities were inhibited by 3 mM NaCN. One of the putative extracellular SOD isoforms was purified and NH2-terminal-sequenced. The sequence contained the domain KAVAVL. The domains VEG and V(K/S)G, present in chloroplastic and cytosolic CuZn SODs of plants, respectively, were not detected. The enzyme was composed of two subunits of 17.8 kDa each. The isoelectric point was determined to be 6.5. The results suggest the existence of an extracellular SOD in Scots pine.

Amino Acid Sequence↗

PubMed1994

Histological changes in Pinus sylvestris L. in the proximal-zone around the Chernobyl power plant.

In September 1990, samples of wood and bark were collected from Pinus sylvestris L. at three locations exposed to different levels of radioactive fallout from the 1986 accident at the Chemobyl nuclear power plant (NPP). Cross-sections of wood from the most exposed location showed a distinct change in histology in the annual ring of 1986, a consequence of the accident on 26 April. The width of annual rings decreased after the accident, and the relative width of latewood in annual rings increased transiently in 1986 and subsequently decreased in 1987. In 1987, an increase in the number of vertical resin ducts was observed, related to contamination at the location, and the number of radial rays decreased at the two locations of higher contamination. The radionuclide content in the bark was found to correlate with the degree of damage in the wood. There are several hypotheses about the contribution from various types of radioactive contamination, but the results indicate that both 'cloud gamma' and deposited radioactivity (beta and gamma) were of importance. The present work suggests that detailed studies of dose-effect relationships after exposure to different dose rates and radiation qualities may establish the usefulness of pine trees as in situ, time-recording differential dosimeters of ionizing radiation.

Gamma Rays↗

PubMed1994

Isolation and purification of mitochondrial Mn-superoxide dismutase from the gymnosperm Pinus sylvestris L.

Manganese superoxide dismutase (Mn-SOD; EC 1.15.1.1) was purified from germinating seeds of Scots pine (Pinus sylvestris L.) 3 days after the start of imbibition. The purification schedule included (NH4)2SO4 fractionation, anion-exchange and hydrophobic-interaction chromatographies and chromatofocusing. Purified Mn-SOD had an apparent specific activity of 4,130 McCord-Fridovich units (mg protein)-1. The molecular mass of the holoenzyme was estimated to be 91 kDa by size-exclusion chromatography, and a molecular mass of 23 kDa was determined by SDS-PAGE. However, isoelectric focusing demonstrated that the purified enzyme consisted of three similarly migrating isoforms, with isoelectric points of approximately 6.5. NH2-terminal amino acid sequencing of purified Mn-SOD revealed no differences among the three isoforms. The comparison of the first 32 NH2-terminal amino acids with sequences of NH2-terminal amino acids of Mn-SODs from angiosperms reflected the phylogenetic distances between Scots pine, which is a gymnosperm, and angiospermic species. Cell fractionation suggested the mitochondrial localization of Mn-SODs and no evidence for glyoxysomal localization was found. Mn-SOD activity was absent from dry seeds. It was detectable at a considerable level after imbibition for 24 h, and it was again absent from 3-week-old seedlings.

Amino Acid Sequence↗

PubMed1994

[Segregation disturbance in chronically irradiated populations of Pinus sylvestris L. growing in the area of the Chernobyl nuclear power plant accident].

Segregation of polymorphic diallelic loci, which encode isozyme synthesis in seed endosperms of Pinus Sylvestris L. trees exposed to various levels of radiation after Chernobyl' meltdown, was studied. It was shown that directional gametic selection, expressed as segregation distortion in heterozygotes, is observed in irradiated populations for a number of loci. The detected selection has one-locus character and distinctly influences the gene pool of the next generation.

Accidents↗

PubMed1993

Lignification in cell suspension cultures of Pinus taeda. In situ characterization of a gymnosperm lignin.

Pinus taeda suspension cultures grown in medium containing 2,4-dichlorophenoxyacetic acid showed only primary cell wall formation and essentially no lignification, as determined by histochemical, ultrastructural, chemical, and NMR spectroscopic analyses. However, these cultures maintained a functional phenylpropanoid pathway as demonstrated by formation of the lignans (-)-matairesinol and (-)-pinoresinol. Administration of [1-13C]Phe to these cultures, followed by solid-state carbon-13 NMR spectral analysis of their cell walls, demonstrated that the phenylalanine incorporated into the cell wall matrix was primarily as protein, rather than lignin. Successive transfer of the 2,4-dichlorophenoxyacetic acid-grown cultures to alpha-naphthaleneacetic acid-containing medium induced cell wall thickening concomitant with lignification. The presence of lignin was confirmed by histochemical, ultrastructural, chemical, biochemical, and NMR spectroscopic analyses. Specific labeling of the lignin polymer in situ with [1-13C]-, [2-13C]-, and [3-13C]Phe and analysis of the cell wall preparations by solid-state carbon-13 NMR spectroscopy permitted the first direct determination of the in situ bonding patterns in a gymnosperm lignin. Several dominant interunit linkages were observed, including beta-O-aryl, furanofuran, phenylcoumarin, and phenolic-linked monolignols, consistent with those predicted but hitherto not proven. Finally, milled wood lignin derivatives prepared from these 13C-specifically enriched lignin tissues gave a relatively high fidelity copy of the native lignin.

Cell Wall↗

PubMed1993

[Mutagenesis of enzyme loci, induced in megaspores of Pinus sylvestris L. by ionizing radiation from the accident at the Chernobyl Atomic Power Plant].

Mutagenesis of alleles of 20-allozyme loci was studied using the electrophoresis methods in polyacrylamide gel in seeds of Pinus sylvestris within the 30 km zone of the Chernobyl NNP and the limits of contamination levels by Cs-137 from 5 up to 600 Cu/km (the absorbed doses being 0.5 to 10 Gy). The frequency of emergence of corresponding mutations which change the synthesis of enzymes in endosperms within the zone constituted on the average 2.7-10 per gene as compared with 0.6-10 in control version. Linear dependence was observed between low doses absorbed and the frequency of gene mutations, while in case of high doses this decreased. Genetic effectivity was 0.001 mutations/locus/Gy. Various radiosensitivity levels were shown among the loci studied. Spontaneous level of mutations frequency in P. sylvestris populations was found to be within the limits of 0.5 to 1.5 Gy under conditions of the 30 km zone. The high rate of mutagenesis was revealed in seeds of the first two post-accident reproductions.

Accidents↗

PubMed1999

A DEF/GLO-like MADS-box gene from a gymnosperm: Pinus radiata contains an ortholog of angiosperm B class floral homeotic genes.

The specification of floral organ identity during development depends on the function of a limited number of homeotic genes grouped into three classes: A, B, and C. Pairs of paralogous B class genes, such as DEF and GLO in Antirrhinum, and AP3 and PI in Arabidopsis, are required for establishing petal and stamen identity. To gain a better understanding of the evolutionary origin of petals and stamens, we have looked for orthologs of B class genes in conifers. Here we report cDNA cloning of PrDGL (Pinus radiata DEF/GLO-like gene) from radiata pine. We provide phylogenetic evidence that PrDGL is closely related to both DEF- and GLO-like genes of angiosperms, and is thus among the first putative orthologs of floral homeotic B function genes ever reported from a gymnosperm. Expression of PrDGL is restricted to the pollen strobili (male cones) and was not detected in female cones. PrDGL expression was first detected in emergent male cone primordia and persisted through the early stages of pollen cone bud differentiation. Based on the results of our phylogeny reconstructions and expression studies, we suggest that PrDGL could play a role in distinguishing between male (where expression is on) and female reproductive structures (where expression is off) in radiata pine. We speculate that this could be the general function of DEF/GLO-like genes in gymnosperms that may have been recruited for the distinction between stamens and carpels, the male and female reproductive organs of flowering plants, during the evolution of angiosperms out of gymnosperm-like ancestors.

Amino Acid Sequence↗

PubMed1994

Isolation and preliminary characterization of Pichia pinus mutants insensitive to glucose repression.

A new method for the isolation of glucose repression-insensitive mutants in the methylotrophic yeast Pichia pinus was developed. The method is based on screening of small suspension samples derived from 2-deoxyglucose-resistant colonies for alcohol oxidase activity. Alcohol oxidase activity was evaluated by determination of formaldehyde excreted by cells. Mutants with glucose non-repressible alcohol oxidase and catalase synthesis were obtained. All mutants grew poorly on D-xylose compared to the wild type, whereas growth on L-arabinose was similar to the wild type. Changes in the glucose transport system were suggested to be responsible for altered growth characteristics and defective glucose repression.

Alcohol Oxidoreductases↗

PubMed2000

Enzymatic synthesis of methylbutenol from dimethylallyl diphosphate in needles of Pinus sabiniana.

Methylbutenol (2-methyl-3-buten-2-ol) is an abundant volatile organic compound released from Western U.S. pines. To understand the mechanism of methylbutenol formation, we developed a sensitive gas chromatographic assay for its detection and determined that needles of gray pine (Pinus sabiniana) contain an enzyme that catalyzes the synthesis of methylbutenol from dimethylallyl diphosphate (DMAPP). The methylbutenol synthase activity was partially purified; its pH optimum was 7-8, and, like other prenyl diphosphate utilizing enzymes, it was dependent on the presence of a divalent cation, preferably Mn2+. The enzyme also required K+ or NH4+ for activity. The Km values for DMAPP and Mn2+ were about 4.8 and 6 mM, respectively. Geranyl diphosphate was not a substrate for the enzyme, so it is distinct from linalool synthase, a plant enzyme that catalyzes an analogous reaction. The methylbutenol synthase reaction may be responsible for the majority of light-dependent methylbutenol production by many pine species in the Western United States.

Butanols↗

PubMed1993

Chloroplast DNA transgresses species boundaries and evolves at variable rates in the California closed-cone pines (Pinus radiata, P. muricata, and P. attenuata).

We studied phylogenetic relationships among populations and species in the California closed-cone pines (Pinus radiata D. Don, P. attenuata Lemm., and P. muricata D. Don) via chloroplast DNA restriction site analysis. Data on genetic polymorphism within and among 19 populations in the three species were collected using 9 to 20 restriction enzymes and 38 to 384 trees. Because only five clades and extremely low intraclade diversity were found, additional phylogenetic data were collected using a single representative per clade and two outgroup species, P. oocarpa Schiede and P. jeffreyi Loud. In total, 25 restriction enzymes were employed and approximately 2.7 kb surveyed (2.3% of genome). The five clades recognized were Monterey pine, knobcone pine, and the southern, intermediate, and northern races of bishop pine. On the basis of bootstrapping, both Wagner and Dollo parsimony analyses strongly separated the northern and intermediate races of bishop pine from the southern race; knobcone pine from Monterey and bishop pines; and the closed-cone pines from the two outgroups. Approximate divergence times were estimated for the lineages leading to knobcone pine and to the intermediate and northern populations of bishop pine. The position of Monterey pine relative to bishop pine within their monophyletic clade was unresolved. Surprisingly, Montery pine and the southern race of bishop pine were much more similar to one another than was the southern race of bishop pine to its conspecific intermediate and northern races. Both the Monterey and southern bishop pine lineages also evolved severalfold more slowly than did the knobcone pine and intermediate-northern bishop pine lineages.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Evolution↗

PubMed2000

Effect of bioflavonoids extracted from the bark of Pinus maritima on proinflammatory cytokine interleukin-1 production in lipopolysaccharide-stimulated RAW 264.7.

Currently, bioflavonoids have been known to have strong antioxidant capacities, and a variety of efforts have been made to identify the utilities of bioflavonoids in treating various diseases based on their antioxidant capacities. The effects of bioflavonoids extracted from the bark of Pinus maritima Pycnogenol (PYC) on free radical formation, activation of redox sensitive transcription factors, as well as interleukin-1 beta (IL-1 beta) production were investigated in murine macrophage cell lines. PYC exerted strong scavenging activities against reactive oxygen species generated either by H(2)O(2) or PMA in RAW 264.7 and IC-21 cells, respectively. In situ ELISA, immunoblot analysis, and competitive RT-PCR demonstrated that PYC pretreatment of LPS-stimulated RAW 264.7 cells dose-dependently reduced both the production of IL-1 beta and its mRNA levels. Furthermore, in the same cells, PYC blocked the activation of nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1), two major transcription factors centrally involved in IL-1 beta gene expression. When RAW 264.7 cells were stimulated with LPS, the inhibitor protein I kappa B largely disappeared from cytosolic fractions. However, pretreatment of the cells with PYC abolished the LPS-induced I kappa B degradation. These results suggest that PYC can inhibit the expression of the proinflammatory cytokine IL-1 by regulating redox-sensitive transcription factors. This study may support the possibility that bioflavonoids including PYC can be used as antiinflammatory and immunosuppressive drugs based on their radical scavenging activities.

Animals↗

PubMed1992

Structure of the pine (Pinus thunbergii) chlorophyll a/b-binding protein gene expressed in the absence of light.

A gene for chlorophyll a/b-binding protein (cab) of pine (Pinus thumbergii) was isolated and sequenced. The gene (cab-6) contains an intron at a position equivalent to the type II cab genes of angiosperms. Transcript mapping analyses show that the amount of the mRNA in the dark is about half of that in the light. The cab-6 gene is expressed in dark-grown seedlings at a very high level, differing from angiosperm cab genes which are induced by light. The cab-6 gene typifies the coniferous plant cab genes in light-independent gene expression.

Amino Acid Sequence↗

PubMed1994

Identification and expression of the chloroplast clpP gene in the conifer Pinus contorta.

The clpP gene from the conifer Pinus contorta was identified and isolated from a chloroplast genomic library by heterologous hybridisation to the second exon of the chloroplast clpP gene in tobacco. DNA sequencing of two overlapping clones revealed an uninterrupted 615 bp open-reading frame with 41 to 65% similarity to the clpP genes in five other chloroplast genomes and Escherichia coli. The 615 bp sequence in P. contorta contained perfectly matched motifs for the serine and histidine active sites of the ClpP protease in E. coli. The location of the clpP gene was determined using a physical map of the P. contorta chloroplast genome, and was found to lie within a 10 kb region between the psbE/F and rpoB genes. Sequencing of the regions adjacent to the clpP gene revealed the first exon of the rps12 gene located 135 bp downstream. The genomic position of the first exon of the rps12 gene in relation to the clpP gene is conserved for all other chloroplast clpP genes identified so far. Northern blot analysis showed that the clpP gene in both P. contorta and P. sylvestris was present in several transcript of different length, ranging from 0.8 to 2.4 kb. The two longer transcripts in P. contorta also included the first exon of the rps12 gene. Mapping of the 5' end of the clpP transcripts by primer extension, however, revealed a single transcription initiation site 53 bp upstream of the first ATG codon. Analysis of total RNA isolated from the two pine species grown in darkness or moderate light conditions (250 mumol photons m-2 s-1) showed no significance difference in the level of expression of the clpP gene. The results suggest that the clpP gene in conifers is part of an operon which includes the first exon of the rps12 and the entire rpl20 gene, and is expressed in a light-independent manner as a polycistronic precursor which later undergoes post-transcriptional processing to give the mature monocistronic clpP mRNA.

ATP-Dependent Proteases↗

PubMed1994

Oleosins in the gametophytes of Pinus and Brassica and their phylogenetic relationship with those in the sporophytes of various species.

Oleosins, which are structural proteins on the surface of intracellular oil bodies, have been found in the sporophytic seeds of angiosperms. Here, we report an oleosin from the female gametophyte of gymnosperm Pinus ponderosa Laws. seed and another oleosin from the male gametophyte of Brassica napus L. With the pine seed gametophyte, we identified two putative oleosins of 15 and 10 kDa, which are similar to the oleosins in angiosperm seeds in terms of their presence in the oil bodies in massive quantity. The complete sequence of the cDNA encoding the gametophytic 15-kDa oleosin was obtained, and it has a predicted amino-acid sequence similar to those of oleosins in angiosperm sporophytic seeds. A Brassica napus pollen cDNA sequence, which was reported earlier, would encode an amino-acid sequence somewhat similar to those of seed oleosins. We tested if the dissimilarity signifies a substantially different oleosin in the Brassica male gametophyte or an analytic error. By direct sequencing of a polymerase chain reaction (PCR)-amplified fragment of genomic DNA, we obtained evidence showing that this reported dissimilarity is likely to have arisen from a sequencing error. Our predicted sequence of the Brassica pollen oleosin has all the structural characteristics of seed oleosins. A phylogenic tree of 20 oleosins, including those from sporophytic and gametophytic tissues of angiosperm and gymnosperm, was constructed based on their amino-acid sequences. We discuss the evolution of oleosins, and conclude that oleosins are ancient proteins with multiple lineages whose root cannot be determined at this time.

Amino Acid Sequence↗

PubMed1993

Light-independent and light-dependent protochlorophyllide-reducing activities and two distinct NADPH-protochlorophyllide oxidoreductase polypeptides in mountain pine (Pinus mugo).

Lower plants and gymnosperms synthesize chlorophyll and develop photosynthetically competent chloroplasts even when grown in the dark. In cell-free extracts of pine (Pinus mugo, Turra, ssp. mugo) seedlings, light-independent and light-dependent protochlorophyllide-reducing activities are present. Two distinct NADPH-protochlorophyllide-oxidoreductase (POR) polypeptides can be detected immunologically with an antiserum raised against the POR of barley. The subcellular localization and amounts of the two POR polypeptides are differentially affected by light: one of them is predominantly present in prolamellar bodies of etiochloroplasts and its abundance rapidly declines once the pine seedlings are exposed to light; the other is found in thylakoid membranes and its amount does not change during illumination of dark-grown seedlings. Two types of cDNA sequences are identified that encode two distinct POR polypeptides in pine. The relevance of these POR polypeptides for the two chlorophyll biosynthetic pathways active in gymnosperms is discussed.

Amino Acid Sequence↗

PubMed1991

Duplication of the psbA gene in the chloroplast genome of two Pinus species.

The psbA gene, encoding the D1 protein of photosystem II, was found to be duplicated in the chloroplast genome of two pine species, Pinus contorta and P. banksiana. Analysis of cloned overlapping restriction fragments of P. contorta chloroplast DNA showed that the two psbA genes have the same orientation and are separated by approximately 3.3 kb. The nucleotide sequences of the coding and the upstream regions of the two psbA copies were found to be identical, whereas the downstream sequences diverged from a point 20 bp 3' of the stop codons. Downstream of the gene copy designated psbAII, a dyad symmetry which allows the formation of a strong mRNA hairpin structure, and a trnH gene were found. No such elements, which are characteristic of psbA downstream regions, were found 3' of psbAI. This suggests that psbAII is the ancestral gene copy in P. contorta. Upon comparison with psbA from other plants, the pine 353-codon sequence appeared almost as distant from the angiosperm as from the liverwort counterpart. As compared to tobacco, 14 substitutions in the predicted amino acid sequence were found, most of which were located in the terminal regions of the protein.

Amino Acid Sequence↗

PubMed1983

Isozyme variation and heterozygosity in Pinus halepensis L.

The genetic polymorphism of a population of Pinus halepensis was studied using 12 electrophoretically detectable enzyme systems encoded by 7 monomorphic and 10 polymorphic loci. An estimation of population parameters revealed a relatively high average heterozygosity (H = 0.179). The total variation present in the population was partitioned into a high amount of genetic variation within groups, with only 1.3-4.4% of the total variation resulting from genic differences among groups.

Electrophoresis, Starch Gel↗

PubMed1981

Biochemical genetics of some seed proteins of Pinus radiata.

In a high-salt soluble fraction of the total protein from single seeds of Pinus radiata, up to 45 polypeptides were resolved on SDS-polyacrylamide gels. At least one-fifth of these polypeptides showed variation between seeds. In the 27,000--29,000 dalton region, two polypeptides were inherited as codominant alleles at a single locus and were shown to assort independently of another seed protein locus and three allozyme loci. A survey of 120 individuals from the five known native populations of P. radiata in California detected only the 27K and 29K alleles at the locus. In all populations, the 29K allele predominated, and the two island populations were monomorphic for the 29K allele. The 27 and 29 kdalton polypeptides were shown to have very similar amino acid sequences, and the allelic difference at this locus is most probably in the gene sequence for the polypeptide.

Alleles↗