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PubMed1999

Monoterpene synthases of loblolly pine (Pinus taeda) produce pinene isomers and enantiomers.

The turpentine fraction of conifer oleoresin is a complex mixture of monoterpene olefins and plays important roles in defense and in the mediation of chemical communication between conifer hosts and insect predators. The stereochemistry of the turpentine monoterpenes is critical in these interactions, influencing host recognition, toxicity, and potency of derived pheromones, and the stereochemical composition of these compounds lends insight into their biogenetic origin, with implications for the numbers and types of enzymes responsible and their corresponding genes. Analysis of the oleoresin from several tissues of loblolly pine (Pinus taeda) showed the derived turpentine to consist mainly of (+)-(3R:5R)-alpha-pinene and (-)-(3S:5S)-beta-pinene. Cell-free extracts from xylem tissue yielded three monoterpene synthases which together account for the monoterpene isomer and enantiomer content of the turpentine of this tissue. The major products of these enzymes, produced from the universal precursor of monoterpenes, geranyl diphosphate, were shown to be (+)-alpha-pinene, (-)-alpha-pinene, and (-)-beta-pinene, respectively. In most properties (molecular mass of approximately 60 kDa, K(m) for geranyl diphosphate of 3 microM, requirement for monovalent and divalent cations), these enzymes resemble other monoterpene synthases from conifer species.

Animals↗

PubMed1999

Multi-site modulation of flux during monolignol formation in loblolly pine (Pinus taeda).

Loblolly pine (Pinus taeda L.) cell suspension cultures secrete monolignols when placed in 8% sucrose/20 mM KI solution, and these were used to identify phenylpropanoid pathway flux-modulating steps. When cells were provided with increasing amounts of either phenylalanine (Phe) or cinnamic acid, cellular concentrations of immediate downstream products (cinnamic and p-coumaric acids, respectively) increased, whereas caffeic and ferulic acid pool sizes were essentially unaffected. Increasing Phe concentrations resulted in increased amounts of p-coumaryl alcohol relative to coniferyl alcohol. However, exogenously supplied cinnamic, p-coumaric, caffeic, and ferulic acids resulted only in increases in their intercellular concentrations, but not that of downstream cinnamyl aldehydes and monolignols. Supplying p-coumaryl and coniferyl aldehydes up to 40, 000-320,000-fold above the detection limits resulted in rapid, quantitative conversion into the monolignols. Only at nonphysiological concentrations was transient accumulation of intracellular aldehydes observed. These results indicate that cinnamic and p-coumaric acid hydroxylations assume important regulatory positions in phenylpropanoid metabolism, whereas cinnamyl aldehyde reduction does not serve as a control point.

Aldehydes↗

PubMed2001

Variation in the nrDNA ITS of Pinus subsection Cembroides: implications for molecular systematic studies of pine species complexes.

The pinyon pines (Pinus subsection Cembroides), distributed in semiarid regions of the western United States and Mexico, include a mixture of relictual and more recently evolved taxa. To investigate relationships among the pinyons, we screened and partially sequenced 3000-bp clones of the nuclear ribosomal DNA internal transcribed spacer (ITS) region for 16 taxa from subsect. Cembroides and nine representatives from four other subsections of subgenus Strobus. Restriction digests of clones reveal within-individual heterogeneity, suggesting that concerted evolution is operating slowly on the ITS in pine species. Two ITS clones were identified as pseudogenes. Tandem subrepeats in the ITS1 form stem loops comparable to those in other genera of Pinaceae and may be promoting recombination between rDNA repeats, resulting in ITS1 chimeras. Within the pinyon clade, phylogenetic structure is present, but different clones from the same (or different) individuals of a species are polyphyletic, indicating that coalescence of ITS copies within individual genomes predates evolutionary divergence in the group. At the level of subsection and above, the ITS region corresponds well with morphological and cpDNA evidence. Except for P. nelsonii, the pinyons are monophyletic, with both subsect. Cembroides and P. nelsonii forming a clade with the foxtail and bristlecone pines (subsect. Balfourianae) of western North America.

Base Sequence↗

PubMed1993

Molecular characterization of a cDNA clone encoding glutamine synthetase from a gymnosperm, Pinus sylvestris.

A full-length cDNA clone (pGSP114) encoding glutamine synthetase was isolated from a lambda gt11 library of the gymnosperm Pinus sylvestris. Nucleotide sequence analysis showed that pGSP114 contains an open reading frame encoding a protein of 357 amino acid residues with a calculated molecular mass of 39.5 kDa. The derived amino acid sequence was more homologous to cytosolic (GS1) (78-82%) than to chloroplastic (GS2) (71-75%) glutamine synthetase in angiosperms. The lack of N-terminal presequence and C-terminal extension which define the primary structure of GS2, also supports that the isolated cDNA encodes cytosolic GS. Southern blot analysis of genomic DNA from P. sylvestris and P. pinaster suggests that GS may be encoded by a small gene family in pine. GS mRNA was more abundant in cotyledons and stems than in roots of both Scots and maritime pines. Western blot analysis in P. sylvestris seedlings showed that only one GS polypeptide, similar in size to GS1 in P. pinaster, could be detected in several different tissues. Our results suggest that cytosolic GS is mainly responsible for glutamine biosynthesis in pine seedlings.

Amino Acid Sequence↗

PubMed1990

Type I and type II genes for the chlorophyll a/b-binding protein in the gymnosperm Pinus sylvestris (Scots pine): cDNA cloning and sequence analysis.

A cDNA library was constructed from mRNA prepared from light-treated seedlings of Scots pine (Pinus sylvestris L.) and cDNAs for the chlorophyll a/b-binding protein LHC-II were identified using a pea gene as the heterologous probe. Three cDNA clones were sequenced. The deduced amino acid sequences of two of the genes corresponded to Type I and one to Type II LHC-II proteins which were ca. 90% homologous to their angiosperm counterparts. The transit peptides of the Scots pine preLHC-II showed features common to angiosperm transit peptides. The three cDNAs had a 70 to 75% preference for G + C in the third base position. CpG and GpC profiles and degenerate codon position bias suggested that two of the corresponding genes lie within CpG islands.

Amino Acid Sequence↗

PubMed1992

Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants.

Chalcone synthase (CHS) and stilbene synthase (STS) are closely related polyketide synthases which are key enzymes in the biosynthesis of flavonoids and stilbenes. Scots pine (Pinus sylvestris) is an interesting plant for a direct comparison of the enzymes. It not only contains the usual flavonoids, but also an unusual chalcone derivative (pinocembrin), and it synthesizes stilbenes of the pinosylvin type. We analysed a CHS and a STS by molecular cloning and functional expression in Escherichia coli. The CHS was active not only with 4-coumaroyl-CoA (to naringenin chalcone), but also with cinnamoyl-CoA (leading to pinocembrin). The STS was identified as dihydropinosylvin synthase, because it preferred dihydrocinnamoyl-CoA to cinnamoyl-CoA. The protein deviated in 47 positions from the CHS consensus. It had 73.2% identity with the CHS from P. sylvestris and only 65.3% with a STS from peanut (Arachis hypogaea). We also investigated the regulation of both enzyme types in P. sylvestris plantlets exposed to stress. CHS was present in non-stressed plantlets, and induction led to a transient increase with a peak after 16 h. STS type activities were regulated differently and were absent in non-stressed plantlets. Increases were observed after a lag period of at least 6 h, and highest activities were obtained after 30 h. The analysis of the reactions in the plant extracts and the substrate specificity of the cloned STS indicate that the plants contain at least two different types of STS: the cloned dihydropinosylvin synthase and a pinosylvin synthase which preferentially utilizes cinnamoyl-CoA as substrate.

Acyltransferases↗

PubMed1994

Molecular characterization of a novel, nuclear-encoded, NAD(+)-dependent glyceraldehyde-3-phosphate dehydrogenase in plastids of the gymnosperm Pinus sylvestris L.

Angiosperms and algae possess two distinct glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes, an NAD(+)-dependent tetramer involved in cytosolic glycolysis and an NADP(+)-dependent enzyme of the Calvin cycle in chloroplasts. We have found that the gymnosperm Pinus sylvestris possesses, in addition to these, a nuclear-encoded, plastid-specific, NAD(+)-dependent GAPDH, designated GapCp, which has not previously been described from any plant. Several independent full-size cDNAs for this enzyme were isolated which encode a functional transit peptide and mature subunit very similar to that of cytosolic GAPDH of angiosperms and algae. A molecular phylogeny reveals that chloroplast GapCp and cytosolic GapC arose through gene duplication early in chlorophyte evolution. The GapCp gene is expressed as highly as that for GapC in light-grown pine seedlings. These findings suggest that aspects of compartmentalized sugar phosphate metabolism may differ in angiosperms and gymnosperms and furthermore underscore the contributions of endosymbiotic gene transfer and gene duplication to the nuclear complement of genes for enzymes of plant primary metabolism.

Amino Acid Sequence↗

PubMed1996

Differential redox regulation by glutathione of glutathione reductase and CuZn-superoxide dismutase gene expression in Pinus sylvestris L. needles.

Glutathione reductase (GR; EC 1.6.4.2) and superoxide dismutase (SOD; EC 1.15.1.1) are two well-known enzymes involved in the scavenging of reactive oxygen intermediates. However, little is known about the regulation of Gor and Sod genes in plant cells. To obtain information about hypothetical redox regulatory mechanisms controlling Gor and Sod gene expression we artificially enhanced the levels of reduced and oxidized forms of glutathione (GSH and GSSG) in Pinus sylvestris L. needles. Scots pine shoots were placed for 12 h in beakers containing 5 mM GSH, 5 mM GSSG or water. Increased levels of both GSSG and GSH were observed in the GSSG-treated needles after 3 h. In contrast, only the GSH level was increased by the GSH treatment. Thus, the GSH/GSSG ratio increased up to 15-fold during the GSH treatment and decreased approximately two-fold during the GSSG treatment. The GR activity was significantly higher (60%) when GSSG was applied, without any apparent change in the amount and isoform population of GR or accumulation of Gor gene transcripts. This indicates that the GR activity increased per se in the GSSG treatment. The level of cytosolic CuZn-Sod transcripts was decreased significantly by the GSH treatment without any change in enzyme activity. The chloroplastic CuZn-Sod gene generally showed a more stable transcript level in the different treatments. However, a similarity between the cytosolic and chloroplastic levels of CuZn-Sod transcripts could be observed in different treatments. This suggests that the redox state of glutathione plays an important role in the in vivo regulation of CuZn-Sod gene expression in plants.

Amino Acid Sequence↗

PubMed1995

The genes encoding subunit 3 of NADH dehydrogenase and ribosomal protein S12 are co-transcribed and edited in Pinus sylvestris (L.) mitochondria.

The nucleotide sequence of the region encoding NADH dehydrogenase subunit 3 and ribosomal protein S12 from Pinus sylvestris (L.) mitochondrial DNA (mtDNA) has been determined. A sequence comparison of this region with six individual cDNA clones prepared by RT-PCR revealed 35 C-to-T differences, showing the occurrence of RNA editing. All but one of these alterations in mRNA sequence change codon identities to specify amino acid better conserved in evolution. Most of these modifications take place within the nad3 gene changing 20% of the amino-acid sequence, which is much more than in angiosperms. Of six cDNA clones investigated, four clones of nad3 were differentially edited, but the editing of the rps12 sequences was identical. As in angiosperms, the two genes are separated by a short sequence of 52 bp, which is not edited. Two transcripts of about 0.9 kb and 1.2 kb, each encoding both proteins, have been detected by Northern hybridisation. The hybridisation of nad3 and rps12 probes with pine mtDNA digested with different restriction enzymes indicates that both genes are present in a single copy in pine mtDNA. The analysis of PCR amplification products with gene-specific primers shows a conserved order of these genes in a wide range of gymnosperms.

Amino Acid Sequence↗

PubMed1987

Electrophoretic analysis of genetic linkage in Scots pine (Pinus sylvestris L.).

Eighty megagametophytes from each of 24 Scots pines (Pinus sylvestris L.) were subjected to horizontal starch gel electrophoresis. The trees were from crosses among widely separated provenances, and each was polymorphic for 8 to 14 loci. Evidence for linkage among 275 two-locus combinations was tested using chi-square analysis. Data from different trees were pooled to calculate map distances for the species. Nineteen of the twenty-nine loci tested were linked in one of six groups; the groups varied in size from two to seven loci. Similarities in linkage relationships among Scots pine, other pines, and other species within the Pinaceae support karyological research that suggests extensive conservation of the conifer genome.

Electrophoresis, Starch Gel↗

PubMed2002

Effect of white pine blister rust (Cronartium ribicola) and rust-resistance breeding on genetic variation in western white pine (Pinus monticola).

Western white pine (Pinus monticola) is an economically and ecologically important species from western North America that has declined over the past several decades mainly due to the introduction of blister rust (Cronartium ribicola) and reduced opportunities for regeneration. Amplified fragment length polymorphism (AFLP) was used to assess the genetic variation in northern Idaho populations of western white pine (including rust-resistant breeding stock) in relation to blister rust. A total of 176 individuals from four populations was analyzed using 163 AFLP loci. Within populations, an average 31.3% of the loci were polymorphic (P), and expected heterozygosity (H(e)) was 0.123. Genetic differentiation values (G(st)) showed that 9.4% of detected genetic variation was explained by differences among populations. The comparison between the rust-resistant breeding stock and a corresponding sample derived from multiple natural populations produced similar values of P (35% vs. 34.4%) and H(e) (0.134 vs. 0.131). No apparent signs of a genetic bottleneck caused by rust-resistance breeding were found. However, a comparison of two natural populations from local geographic areas showed that the population with low pressure from blister rust had higher polymorphism and heterozygosity than the population that had experienced high mortality due to blister rust: P (30.7% vs. 25.1%) and H(e) (0.125 vs. 0.100), respectively. In addition, the population from low blister-rust pressure had twice as many unique alleles as the blister rust-selected population. The genetic distance and Dice's similarity coefficients among the four populations indicated that the local population that survived high blister-rust pressure was genetically similar to the rust-resistant breeding stock.

Crosses, Genetic↗

PubMed2003

Genetic maps for Pinus elliottii var. elliottii and P. caribaea var. hondurensis using AFLP and microsatellite markers.

Genetic maps for individual Pinus elliottii var. elliottii and P. caribaea var. hondurensis trees were generated using a pseudo-testcross mapping strategy. A total of 329 amplified fragment length polymorphic (AFLP) and 12 microsatellite markers were found to segregate in a sample of 93 interspecfic F(1) progeny. The male P. caribaea var. hondurensis parent was more heterozygous than the female P. elliottii var. elliottii parent with 19% more markers segregating on the male side. Framework maps were constructed using a LOD 5 threshold for grouping and interval support threshold of LOD 2. The framework map length for the P. elliottii var. elliottii megagametophyte parent (1,170 cM Kosambi; 23 linkage groups) was notably smaller than the P. caribaea var. hondurensis pollen parent (1,658 cM Kosambi; 27 linkage groups). The difference in map lengths was assumed to be due to sex-related recombination variation, which has been previously reported for pines, as the difference in map lengths not be accounted for by the larger number of markers mapping to the P. caribaea var. hondurensis parent - 109 compared with 78 in P. elliottii var. elliottii parent. Based on estimated genome sizes for these species, the framework maps for P. elliottii var. elliottii and P. caribaea var. hondurensis covered 82% and 88% of their respective genomes. The pseudo-testcross strategy was extended to include AFLP and microsatellite markers in an intercross configuration. These comprehensive maps provided further genome coverage, 1,548 and 1,828 cM Kosambi for P. elliottii var. elliottii and P. caribaea var. hondurensis, respectively, and enabled homologous linkage groups to be identified in the two parental maps. Homologous linkage groups were identified for 11 out of 24 P. elliottii var. elliottii and 10 out of 25 P. caribaea var. hondurensis groups. A higher than expected level of segregation distortion was found for both AFLP and microsatellite markers. An explanation for this segregation distortion was not clear, but it may be at least in part due to genetic mechanisms for species isolation in this wide cross.

Genetic Markers↗

PubMed2003

QTL associations for density and diameter in Pinus radiata and the potential for marker-aided selection.

A large full-sib family of radiata pine ( Pinus radiata Donn. ex D. Don) was used for quantitative trait locus (QTL) detection and independent verification. QTL detection experiments were carried out for juvenile wood density (JWD) and stem diameter at breast height (DBH) using selective genotyping. Evenly spaced RFLP and microsatellite markers were selected from an existing linkage map. QTLs were verified in an independent set of progeny from the same family. Based on map location, at least eight QTL positions for JWD and two for DBH were detected and verified. The percent variance accounted for by the markers ranged from 0.78% to 3.58%, suggesting a genomic architecture of many genes with small effect. Two unrelated "bridging" families were chosen as candidates for marker-aided selection (MAS), and six microsatellite markers showing an association with JWD or DBH were tested in these families. Of these, four markers showed a consistent association with JWD in one or both of the bridging families. Results from this study provide a basis for MAS in P. radiata.

Analysis of Variance↗

PubMed2004

Detection and verification of quantitative trait loci for resistance to Dothistroma needle blight in Pinus radiata.

Six related radiata pine ( Pinus radiata) full-sib families were used to detect and independently verify quantitative trait loci (QTLs) for resistance to Dothistroma needle blight, caused by Dothistroma septospora. The detection families had from 26 to 30 individuals each, and had either a common maternal (31053) or paternal (31032) parent; one family (cross 4) consisted of progeny from both parents, 31053 x 31032. Approximately 200 additional progeny from cross 4 were clonally replicated and planted at two sites, with at least five to seven ramets of each individual per site. Marker segregation data were collected from a total of 250 RFLP and microsatellite markers, and single factor ANOVAs were conducted separately for each family and marker. A number of putative associations were observed, some across more than one family. Permutation tests were used to confirm expected probabilities of multiple associations based on chance alone. Seven markers representing at least four QTLs for resistance to Dothistroma were identified as being significant in more than one family; one of these was significant at P<0.05 in three families and highly significant at P<0.01 in a fourth. Further confirmation was obtained by testing those markers that were significant in more than one of the detection families (or highly significant in cross 4) in the clonally replicated progeny from cross 4. Four QTL positions were verified in the clonal populations, with a total percent variation accounted for of 12.5.

Analysis of Variance↗

PubMed2005

AT-rich sequences containing Arabidopsis-type telomere sequence and their chromosomal distribution in Pinus densiflora.

Japanese red pine Pinus densiflora has 2 n=24 chromosomes and after FISH-detection of Arabidopsis-type (A-type) telomere sequences, many telomere signals were observed on these chromosomes at interstitial and proximal regions in addition to the chromosome ends. These interstitial and proximal signal sites were observed as DAPI-positive bands, suggesting that the interstitial and proximal telomere signal sites are composed of AT-rich highly repetitive sequences. Four DNA clones (PAL810, PAL1114, PAL1539, PAL1742) localized at the interstitial telomere signals were selected from AluI-digested genomic DNA library using colony blot hybridization probed with A-type telomere sequences and characterized using FISH and Southern blot hybridization. The AT-contents of these selected four clones were 60.8-76.3%, and repeat units of the telomere sequence and degenerated telomere sequences were found in their nucleotide sequences. Except for two sites of PAL1114, FISH signals of the four clones co-localized with interstitial and proximal A-type telomere sequence signals. FISH signals a showed similar distribution pattern, but the patterns of signal intensity were different among the four clones. PAL810, PAL1539 and PAL 1742 showed similar FISH signal patterns, and the differences were only with respect to the signal intensity of some signal sites. PAL1114 had unique signals that appeared on chromosomes 7 and 10. Based on results of the Southern blot hybridization these four sequences are not arranged tandemly. Our results suggest that the interstitial A-type telomere sequence signal sites were composed of a mixture of several AT-rich repetitive sequences and that these repetitive sequences contained A-type telomere sequences or degenerated A-type telomere sequence repeats.

AT Rich Sequence↗

PubMed2005

Screening for putative PGPR to improve establishment of the symbiosis Lactarius deliciosus-Pinus sp.

A screening for plant growth promoting rhizobacteria (PGPR) was carried out in the mycorrhizosphere of wild populations of Pinus pinea and P. pinaster, and in the mycosphere of associated Lactarius deliciosus. A total of 720 bacteria were isolated, purified, and grouped by morphological criteria. Fifty percent of the isolates were selected and tested for aminocyclopropanecarboxylic acid (ACC) degradation, auxin and siderophore production, and phosphate solubilization. Thirty eight percent of the isolates showed at least one of the evaluated activities. Nutrient-related traits were associated with P. pinaster, whereas hormone production traits predominated in P. pinea. These activities were found mostly in Gram positive isolates. After PCR-RAPDs (random amplified polymorphic DNA) analysis, 10 groups appeared with 85% similiarity when considering all isolates, indicating the low diversity in the system. One strain of each group was identified by 16S rDNA sequencing. Our results suggest that P. pinaster selects for mycorrhizosphere bacteria that mobilize nutrients, whereas P. pinea selects for bacteria that have the capacity to increase root growth via production of plant growth regulators.

Classification↗

PubMed2003

Somatic embryogenesis in loblolly pine (Pinus taeda L.): improving culture initiation with abscisic acid and silver nitrate.

Loblolly pine ( Pinus taeda L.) culture initiation was improved by the addition of abscisic acid (ABA) (3.7 micro M), silver nitrate (20 micro M), and guanosine 3',5'-cyclic monophosphate, 8-bromo-, sodium salt (10 micro M) to the medium and by raising cytokinin levels in the presence of 50 mg/l activated carbon (AC). Basal medium contained modified 1/2-P6 salts, 50 mg/l AC, Cu and Zn added to compensate for adsorption by AC, 1.5% maltose, 2% myo-inositol, 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l alpha-naphthaleneacetic acid (NAA), 0.55 mg/l 6-benzylaminopurine (BA), 0.53 mg/l kinetin, and 2 g/l Gelrite. Across 32 open-pollinated families initiation ranged from 0 to 53.4%, with an average of 17.9%. Further optimization of cytokinins to 0.63 mg/l BA and 0.61 mg/l kinetin along with the removal of ABA maintained initiation at 18.2% across 19 families. Survival of 2001 new initiations was tracked for 4-6 months. Survival averaged 28.8%. A test of 68 new initiations tracked closely for 4 months demonstrated that at least 80% of the cultures lost did not grow after transfer to the multiplication media, suggesting that many new initiations abort during the initiation process.

Abscisic Acid↗

PubMed2004

Antioxidants enhance in vitro plant regeneration by inhibiting the accumulation of peroxidase in Virginia pine (Pinus virginiana Mill.).

Plant tissue necrosis and subsequent cell death are usually observed during in vitro regeneration in conifers, especially in plant regeneration via somatic organogenesis in pine species. Cell death is correlated with the elevated levels of peroxides. In this investigation, the effects of antioxidants on in vitro regeneration of Virginia pine (Pinus virginiana Mill.) were evaluated. Antioxidants, polyvinylpolypyrrolidone (PVPP) and 1,4-dithio-DL-threitol (DTT), were found to improve callus formation, shoot differentiation and growth, and shoot rooting by inhibiting tissue necrosis during the initiation of cultures and subculture of shoots. These treatments enabled the recovery and regeneration plants at high frequency through somatic organogenesis. Compared to the control, the frequencies of callus formation, shoot growth, and shoot rooting increased 15, 26, and 19%, respectively, by addition of 5 g/l PVPP and 2 g/l DTT. Higher peroxidase activity of tissue cultures during subculture from callus proliferation medium to shoot differentiation medium and to rooting medium was observed. The addition of antioxidants reduces and inhibits browning by reducing the accumulation of peroxidase.

Antioxidants↗