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PubMed1994

Diterpenoid resin acid biosynthesis in conifers: characterization of two cytochrome P450-dependent monooxygenases and an aldehyde dehydrogenase involved in abietic acid biosynthesis.

Abietic acid is a major component of the rosin fraction of oleoresin synthesized by grand fir (Abies grandis), lodgepole pine (Pinus contorta), and many other conifer species as a defensive secretion against insect and pathogen attack. The diterpenoid resin acid is derived from geranylgeranyl pyrophosphate via abietadiene, with subsequent oxidation of the C18-methyl of this olefin to abietadienol, abietadienal, and abietic acid. The pathway was confirmed by administering [1,2-14C]acetic acid to grand fir stems which incorporated the radiolabel into abietadiene, the corresponding alcohol and aldehyde, as well as abietic acid. Three different enzymatic activities, catalyzing the sequential oxidation of the olefin to abietic acid, were demonstrated in cell-free stem extracts of both grand fir and lodgepole pine. The first two oxidation steps were catalyzed by the microsomal fraction and required both oxygen and a reduced pyridine nucleotide (NADPH preferred). Both activities were strongly inhibited by CO (blue light reversible) and were differentially sensitive to several substituted N-heterocyclic inhibitors, suggesting that these two enzymes are distinct, microsomal cytochrome P450-dependent monooxygenases. A third enzymatic activity, catalyzing the oxidation of abietadienal to abietic acid, was located in the soluble protein fraction. This oxidation reaction employed NAD+ as cofactor, but did not require oxygen and was not inhibited by CO, indicating that this last step of abietic acid biosynthesis is catalyzed by an operationally soluble aldehyde dehydrogenase.

Abietanes↗

PubMed1994

Diterpenoid resin acid biosynthesis in conifers: enzymatic cyclization of geranylgeranyl pyrophosphate to abietadiene, the precursor of abietic acid.

Abietic acid is a major component of the rosin fraction of oleoresin synthesized by conifer species, such as grand fir (Abies grandis) and lodgepole pine (Pinus contorta), as a defensive secretion against insect and pathogen attack. This diterpenoid resin acid is derived from geranylgeranyl pyrophosphate by conversion to abietadiene and sequential oxidation of the C18-methyl group of the precursor olefin to a carboxyl function. Resin acid biosynthesis is constitutively expressed at high levels in lodgepole pine stem and is induced to these levels by stem wounding in grand fir. Soluble enzyme extracts of lodgepole pine stem and of mechanically wounded grand fir stem catalyzed the divalent metal ion-dependent cyclization of [1-3H]geranylgeranyl pyrophosphate to (-)-abieta-7(8),13(14)-diene. The wound-inducible enzyme was partially purified and identified as an 80-kDa protein with general characteristics typical of those of other terpenoid cyclases. Although the enzymatic cyclization sequence almost certainly involves the formation of copalyl pyrophosphate and a pimaradiene as stable intermediates, no evidence for the separation of the corresponding partial activities was obtained. A pathway involving the production of various pimaradiene and abietadiene isomers is proposed to account for the origin of several common resin acids.

Abietanes↗

PubMed1995

Interaction between influenza virus proteins and pine cone antitumor substance that inhibits the virus multiplication.

Fractions obtained from pine cone extract (PCE) of Pinus parviflora Sieb. et Zucc. have been shown to suppress the growth of influenza virus. The inhibitory effects of one of the fractions, Fraction VII, on the formation of RNA-viral protein complex and the viral RNA synthesis were investigated. The formation of M1-RNA or NP-RNA complex was inhibited when M1 or NP was preincubated with the PCE fraction. The in vitro viral RNA synthesis was inhibited by the PCE fraction, while this inhibitory effect was titrated out by the increasing concentration of M1 protein. These results suggest that the major target of the PCE fraction was M1 protein.

Antineoplastic Agents↗

PubMed1998

Active oxygen scavengers during cold acclimation of Scots pine seedlings in relation to freezing tolerance.

Freezing injury of plants may be caused by the deleterious reactions of active oxygen species, and free-radical scavenging systems may be important in the alleviation of freezing stress. To test the feasibility of this hypothesis, enzymes and metabolites that cooperatively scavenge O2 and H2O2 were analyzed in Scots pine (Pinus sylvestris L.) seedlings during a stepwise cold acclimation procedure. Elevated levels of enzymatic scavengers such as ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were found, along with increased freezing tolerance during cold acclimation, supporting the hypothesis. Induction of the scavenging systems during acclimation is discussed in relation to freezing tolerance.

Acclimatization↗

PubMed1998

Memory effects in the action of ozone on conifers.

Conifers are known to possess relative ozone tolerance in short-term experiments. A scenario for ozone damage of conifers is now derived from the first exposure experiments in which both the initial biochemical response phase and delayed visible symptom development were studied. A number of early biochemical ozone responses could be detected in Norway spruce (Picea abies [L.] Karst.) and Scots pine (Pinus sylvestris L.). The stress metabolite catechin persisted over several months. In the year following ozone treatment of spruce, decreases in pigment content and photosynthetic capacity, as well as development of visible symptoms (chlorosis, banding), were determined in the needle age classes previously exposed to an accumulated hourly ozone dose above 40 ppb (AOT40) of >/=60-80 ppm small middle doth. The visible symptoms developed during spring emergence of the new flush. In the case of Scots pine, an ozone dose (AOT40) of >/=30 ppm small middle doth caused the premature shedding of needles 9 months after treatment. The delayed symptoms of both spruce and pine occurred during known phases of endogenous stress. The symptoms appeared to reflect an ozone "memory" imprinted by the induced early stress reactions. Ambient AOT40 ozone doses in Central Europe are in the range 4 and 50 ppm small middle doth per growing season. Ozone is proposed to potentially damage conifers through memory effects ("abiotic" pathway) or through predisposition for pathogen attack ("biotic" pathway).

Catechin↗

PubMed2001

A study of metal distribution from lignite fuels using trees as biological monitors.

Concentrations of five metals (Cd, Cu, Mn, Fe, Zn) were determined in tree leaves collected from an area with large coal-fired plants in Ptolemais, Macedonia, Greece. The analyses were carried out with flame atomic absorption spectroscopy. Mean heavy metal content in the tree leaves is, in descending order, Fe>Mn>Zn>Cu>Cd. In Populus nigra and Salix babylonica the dense piled leaves and the widespread root system are the reasons for high heavy metal uptake. Conifer trees Pinus nigra and Juniperus arizona, which have a rough leaf surface, also had elevated concentrations. Leaves with a smooth surface were less contaminated. The presence of one metal within leaf cells may reduce the uptake or toxicity of other metals. Fruits accumulated less heavy metal than the corresponding leaves. The most damaged areas are those in the direct vicinity of the coal power plants, and the rate of damage caused in the environment is affected by the main wind direction. All sampling sites in Ptolemais basin are generally more or less polluted when compared with the control site.

Coal↗

PubMed1998

Evaluation of the Structure and Acid-Base Properties of Bulk Wood by FT-Raman Spectroscopy.

The structure of pine wood (Pinus silvestris L.) has been analyzed by FT-Raman spectroscopy, taking birch wood and the wood components cellulose, hemicellulose (xylan), and lignin as well as previously characterized wood resins as references. The acid-base properties of bulk pine wood were evaluated by comparing the spectra recorded before and after the treatment with various solvents. After the treatment with the probe liquids having only a Lifshitz-van der Waals (LW) component, it was found that the LW interactions in pine wood take place without changing the main structure. After treatment with Lewis acid-base active probe liquids, the spectra indicate that, e.g., the intense peak located at approximately 2936 cm-1 (CH2 stretch) seems to disappear, suggesting that this peak may be related to Lewis acidity. In addition, after treatment with a Lewis acid, it was found that the intense peak located at approximately 1657 cm-1 (C&dbond;C) is shifted, relating to Lewis basicity. With the ratio approximately 2936/ approximately 1657 cm-1 as a measure of the acid-base properties of bulk wood, a value of about 2.00 indicates that the bulk pine wood is largely acidic. The pH determined supports the evaluation made by FT-Raman spectroscopy. Copyright 1998 Academic Press.

Journal Article↗

PubMed1990

Agrobacterium-mediated DNA transfer in sugar pine.

DNA transfer using Agrobacterium tumefaciens has been demonstrated in sugar pine, Pinus lambertiana Dougl. Shoots derived from cytokinin-treated cotyledons formed galls after inoculation with A. tumefaciens strains containing the plasmid pTiBo542. A selectable marker, neomycin phosphotransferase II, conferring resistance to kanamycin, was transferred into sugar pine using a binary armed vector system. Callus proliferated from the galls grew without hormones and in some cases, kanamycin-resistant callus could be cultured. Southern blots provided evidence of physical transfer of T-DNA and the nptII gene. Expression of the nptII gene under control of the nos promoter was demonstrated by neomycin phosphotransferase assays. Several aspects of DNA transfer were similar to those previously observed in angiosperms transformed by A. tumefaciens. This is the first evidence for DNA transfer by Agrobacterium in this species and the first physical evidence for transfer in any pine. These results bring us closer to genetic engineering in this commercially important genus of forest trees.

DNA↗

PubMed1993

A view of plant dehydrins using antibodies specific to the carboxy terminal peptide.

Dehydrins are characterized by the consensus KIKEKLPG amino acid sequence found near the carboxy terminus, and usually repeated from one to many times within the protein. A synthetic peptide containing this consensus sequence was used to produce specific antibodies that recognize dehydrins in a wide range of plants. This range covered two families of monocots, viz. Gramineae (Hordeum vulgare L., Triticum aestivum L., Zea mays L., Oryza sativa L.) and Liliaceae (Allium sativa L.), and five families of dicots, Malvaceae (Gossypium hirsutum L.), Solanaceae (Lycopersicon esculentum L.), Brassicaceae (Raphanus sativus L.), Fabaceae (Vigna unguiculata L.), and Cucurbitaceae (Cucumis sativus L.). Two families of gymnosperms, Pinaceae (Pinus edulis Engelm.) and Ginkgoaceae (Ginkgo biloba L.), were also included. For several plants in which dehydrin cDNA and genomic clones have previously been characterized, it now appears that the dehydrin family of proteins is larger, and the regulation of dehydrin expression much more complex, than earlier studies have shown.

Amino Acid Sequence↗

PubMed1991

Homologues of the green algal gidA gene and the liverwort frxC gene are present on the chloroplast genomes of conifers.

Strong hybridization signals were obtained from total DNA of two conifers, lodgepole pine (Pinus contorta) and Norway spruce (Picea abies), in a Southern blot analysis using a probe derived from the chloroplast gidA gene of the green alga Chlamydomonas reinhardtii. The pine fragments detected by the probe were found to originate from the chloroplast genome and, as judged by the signal intensity, this was also true for the spruce fragments. Sequence analysis of the hybridizing pine chloroplast DNA region revealed an open reading frame potentially encoding a 459 amino acid polypeptide, highly homologous to that deduced from the algal gene and to ORF465 of liverwort chloroplast DNA. Upstream of the gidA sequence, we found a trnN(GUU) gene and an open reading frame of 291 codons which was 78% identical to the frxC gene of liverwort. Since ORF465 is located immediately downstream of trnN and frxC in liverwort, the genetic organization of this region is very similar in the two plants. In contrast, neither the gidA nor the frxC gene is present in the chloroplast DNA of tobacco or rice. It was recently reported that deletions in the gidA region of the chloroplast genome of Chlamydomonas reinhardtii abolish the light-independent pathway of chlorophyll synthesis which exists in many algae and lower plants. The presence of the gidA gene on the chloroplast genomes of conifers may therefore be of significance with respect to the ability of these plants to synthesize chlorophyll in the dark.

Algal Proteins↗

PubMed1992

Characterization of cDNAs encoding CuZn-superoxide dismutases in Scots pine.

A Scots pine (Pinus sylvestris L.) cDNA library was screened with two heterologous cDNA probes (P31 and T10) encoding cytosolic and chloroplastic superoxide dismutases (SOD) from tomato. Several positive clones for cytosolic and chloroplastic superoxide dismutases were isolated, subcloned, mapped and sequenced. One of the cDNA clones (PS3) had a full-length open reading frame of 465 bp corresponding to 154 amino acid residues and showed approximately 85% homology with the amino acid sequences of angiosperm cytosolic SOD counterparts. Another cDNA clone (PST13) was incomplete, but encoded a putative protein with 93% homology to pea and tomato chloroplastic superoxide dismutase. The derived amino acid sequence from both cDNA clones matched the corresponding N-terminal amino acid sequence of the purified mature SOD isozymes. Northern blot hybridizations showed that, cytosolic and chloroplastic CuZn-SOD are expressed at different levels in Scots pine organs. Sequence data and Southern blot hybridization confirm that CuZn-SODs in Scots pine belong to a multigene family. The results are discussed in relation to earlier observations of CuZn-SODs in plants.

Amino Acid Sequence↗

PubMed1996

Cloning of a cDNA for a chitinase homologue which lacks chitin-binding sites and is down-regulated by water stress and wounding.

A cDNA clone (pLP6) of a gene which is repressed under water deficit was isolated from a loblolly pine (Pinus taeda L.) cDNA library and characterized. The predicted polypeptide encoded by pLP6 bears strong resemblance to a number of Class I chitinases. However, LP6 lacks most of the amino-terminal and, consequently the signal peptide, cysteine-rich chitin-binding domain and glycine/proline-rich "hinge' region, diagnostic of Class I chitinases, are absent. Although the cDNA is similar in size to its mRNA, the long open reading frame encoding the LP6 protein commences halfway through the mRNA, implying a 5'-untranslated region of over 700 nucleotides. Subfragments from the 5' end of pLP6 hybridize to the same mRNA as do probes consisting of the entire cDNA. Reverse transcription(RT)-PCR experiments confirm that the cDNA derives from a single mRNA molecule. Analysis of the 5'-UTR revealed six upstream open reading frames and four inverted repeat structures. Expression of the pLP6 gene is repressed by water deficit stress and wounding. Possible functions and origin of this gene are discussed.

Amino Acid Sequence↗

PubMed1991

Structure comparison and evolutionary relations between elongation factors EF-Tu (EF-1 alpha) and SUP 2 proteins.

On the basis of high homology and structural similarity, three genes, SUP2 Saccharomyces cerevisiae, SUP2 Pichia pinus and GST1 Homo sapiens, might be considered as members of one family named SUP2. Comparison of the primary structure of SUP2 proteins and elongation factors EF-Tu(EF-1 alpha) from 19 different species was performed. It was found that SUP2 proteins bear more homology to eukaryotic elongation factor than to procaryotic EF-Tu, though the degree of sequence conservation in SUP2 proteins is smaller than in EF-1 alpha factors. The extensive phylogenetic analysis of SUP2 and EF-Tu(EF-1 alpha) genes was performed by means of 3 methods, 2 phenetic and one cladystic (maximal parsimony). The data support the close relation of SUP2 genes to other elongation factor genes.

Amino Acid Sequence↗

PubMed1991

The effect of corticosterone on standard metabolic rates of small passerine birds.

Resting metabolic rates of Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) and pine siskins (Carduelis pinus) were evaluated at thermoneutral temperatures before and after administration of corticosterone (B) at physiological doses. There was no effect of B on basal metabolic rate of either species, but nocturnal metabolic rate varied significantly less over the 3-h period of measurement in B-treated sparrows and siskins than in control birds. These results, coupled with observations of caged birds, suggest that corticosterone has no direct effect on avian resting metabolism but does reduce the responsiveness of birds to external stimuli and thus promotes nocturnal restfulness.

Animals↗

PubMed1991

Evolutionary conservation of the chlorophyll a/b-binding proteins: cDNAs encoding type I, II and III LHC I polypeptides from the gymnosperm Scots pine.

cDNAs encoding three different LHC I polypeptides (Type I, Type II and Type III) from the gymnosperm Scots pine (Pinus sylvestris L.) were isolated and sequences. Comparisons of the deduced amino acid sequences with the corresponding tomato sequences showed that all three proteins were highly conserved although less so than the LHC II proteins. The similarities between mature Scots pine and tomato Types I, II and III LHC I proteins were 80%, 87% and 85%, respectively. Two of the five His residues that are found in AXXXH sequences, which have been identified as putative chlorophyll ligands in the Type I and Type II proteins, were not conserved. The same two regions of high homology between the different LHC proteins, which have been identified in tomato, were also found in the Scots pine proteins. Within the conserved regions, the Type I and Type II proteins had the highest similarity; however, the Type II and Type III proteins also showed a similarity in the central region. The results suggest that all flowering plants (gymnosperms and angiosperms) probably have the same set of LHC polypeptides. A new nomenclature for the genes encoding LHC polypeptides (formerly cab genes) is proposed. The names lha and lhb are suggested for genes encoding LHC I and LHC II proteins, respectively, analogous to the nomenclature for the genes encoding other photosynthetic proteins.

Amino Acid Sequence↗

PubMed1992

Chloroplast DNA of black pine retains a residual inverted repeat lacking rRNA genes: nucleotide sequences of trnQ, trnK, psbA, trnI and trnH and the absence of rps16.

A physical map of black pine (Pinus thunbergii) chloroplast DNA (120 kb) was constructed and two separate portions of its nucleotide sequence were determined. One portion contains trnQ-UUG, ORF510, ORF83, trnK-UUU (ORF515 in the trnK intron), ORF22, psbA, trnI-CAU (on the opposing strand) and trnH-GUG, in that order. Sequence analysis of another portion revealed the presence of a 495 bp inverted repeat containing trnI-CAU and the 3' end of psbA but lacking rRNA genes. The position of trnI-CAU is unique because most chloroplast DNAs have no gene between psbA and trnH (trnI-CAU is usually located further downstream). Black pine chloroplast DNA lacks rps16, which has been found between trnQ and trnK in angiosperm chloroplast DNAs, but possesses ORF510 instead. This ORF is highly homologous to ORF513 found in the corresponding region of liverwort chloroplast DNA and ORF563 located downstream from trnT in Chlamydomonas moewusii chloroplast DNA. A possible pathway for the evolution of black pine chloroplast DNA is discussed.

Amino Acid Sequence↗

PubMed1994

A new gene encoding tRNA(Pro) (GGG) is present in the chloroplast genome of black pine: a compilation of 32 tRNA genes from black pine chloroplasts.

The chloroplast genome of black pine (Pinus thunbergii), a gymnosperm, contains 32 different tRNA genes, 30 of which correspond to those previously identified in tobacco and rice chloroplast genomes. Two additional genes encode tRNA(Pro) (GGG) and tRNA(Arg) (CCG); the former is newly identified while the latter is present in liverwort, Physcomitrella patens and Angiopteris lygodiifolia, chloroplast genomes. Moreover, a partial copy of the split tRNA(Gly) (UCC) gene and full copies of tRNA(His) (GUG), tRNA(Thr) (GGU) and tRNA(Ser) (GCU) genes are present in the large single-copy region of the genome, suggesting extensive rearrangements of the chloroplast genome during evolution. No tRNA genes whose tRNA products can recognize codons CUU/C (Leu) and GCU/C (Ala) have been found. We propose that the 32 tRNAs are sufficient to read all the 61 sense codons in the black pine system using the "two-out-of-three" and the "U:N wobble" mechanisms.

Base Sequence↗

PubMed1994

Chloroplastic genomes of Ginkgo biloba and Chlamydomonas moewusii contain a chlB gene encoding one subunit of a light-independent protochlorophyllide reductase.

We have cloned and sequenced a Chlamydomonas moewusii chloroplastic DNA fragment that includes a 563 amino-acid open reading frame (ORF563, chlB) presenting 89% amino-acid homology with ORF513 from Marchantia polymorpha. It is also homologous to ORF510 from Pinus thunbergii but includes two insertions absent in both M. polymorphia and P. thunbergii. The derived polypeptide is 54% similar to the product of bchB from Rhodobacter capsulatus, identified as one subunit of a light-independent NADH-protochlorophyllide reductase. We also isolated and sequenced an homologous chloroplastic gene from the gymnosperm Ginkgo biloba. Northern hybridizations performed on RNA isolated from synchronized Chlamydomonas eugametos cells showed higher expression between the tenth hour of light and the eighth hour of darkness, peaking during the first 2 h of darkness.

Amino Acid Sequence↗