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Photosynthetic electron transport adjustments in overwintering Scots pine (Pinus sylvestris L.).

As shown before [C. Ottander et al. (1995) Planta 197:176-183], there is a severe inhibition of the photosystem (PS) II photochemical efficiency of Scots pine (Pinus sylvestris L.) during the winter. In contrast, the in vivo PSI photochemistry is less inhibited during winter as shown by in vivo measurements of deltaA820/A820 (P700+). There was also an enhanced cyclic electron transfer around PSI in winter-stressed needles as indicated by 4-fold faster reduction kinetics of P700+. The differential functional stability of PSII and PSI was accompanied by a 3.7-fold higher intersystem electron pool size, and a 5-fold increase in the stromal electron pool available for P700+ reduction. There was also a strong reduction of the QB band in the thermoluminescence glow curve and markedly slower Q-A re-oxidation in needles of winter pine, indicating an inhibition of electron transfer between QA and QB. The data presented indicate that the plastoquinone pool is largely reduced in winter pine, and that this reduced state is likely to be of metabolic rather than photochemical origin. The retention of PSI photochemistry, and the suggested metabolic reduction of the plastoquinone pool in winter stressed needles of Scots pine are discussed in terms of the need for enhanced photoprotection of the needles during the winter and the role of metabolically supplied energy for the recovery of photosynthesis from winter stress in evergreens.

Adaptation, Physiological↗

Biosynthesis of 2-methyl-3-buten-2-ol emitted from needles of Pinus ponderosa via the non-mevalonate DOXP/MEP pathway of isoprenoid formation.

The volatile hemiterpene 2-methyl-3-buten-2-ol (MBO) is emitted from the needles of several pine species from the Western United States and contributes to ozone formation in the atmosphere. It is synthesised enzymatically from dimethylallyl diphosphate (DMAPP). We show here that needles of Pinus ponderosa Laws. incorporated [1-2H1]-1-deoxy-D-xylulose (d-DOX) into the emitted MBO, but not D,L-[2-13C]mevalonic acid lactone. Furthermore, MBO emission was inhibited by fosmidomycin, a specific inhibitor of the second enzyme of the mevalonate-independent pathway of isopentenyl diphosphate and DMAPP formation, i.e. the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway. We thus prove that MBO emitted from needles of P. ponderosa is primarily formed via the DOXP/MEP pathway.

Hemiterpenes↗

Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens.

Embryos of 24 open-pollinated families of loblolly pine (Pinus teade L.) were used as explants to conduct in vitro regeneration. Then, Agrobacterium tumefaciens strain GV3101 harboring the plasmid pPCV6NFHygGUSINT was used to transform mature zygotic embryos of seven families of loblolly pine. The frequency of transformation varied among families infected with A. tumefaciens. The highest frequency (100%) of transient beta-glucuronidase (GUS)-expressing embryos was obtained from family 11-1029 with over 300 blue spots per embryo. Expression of the GUS reporter gene was observed in cotyledons, hypocotyls, and radicles of co-cultivated mature zygotic embryos, as well as in callus and shoots derived from co-cultivated mature zygotic embryos. Ninety transgenic plants were regenerated from hygromycin-resistant callus derived from families W03. 8-1082 and 11-1029. and 19 transgenic plantlets were established in soil. The presence of the GUS gene in the plant genome was confirmed by polymerase chain reaction. Southern blot, and plant DNA/T-DNA junction analysis. These results suggest that an efficient A. tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for future studies on transferring economically important genes to loblolly pine.

Agrobacterium tumefaciens↗

Interaction between Laccaria laccata and Trichoderma virens in co-culture and in the rhizosphere of Pinus sylvestris grown in vitro.

Interactions between the ectomycorrhizal fungus Laccaria laccata and the soil fungus Trichoderma virens in co-culture and in the rhizosphere of Pinus sylvestris seedlings growing in vitro were investigated by light and scanning electron microscopy. The growth of T. virens was inhibited in co-culture. Shortened, more branched and sometimes deformed or injured hyphae of T. virens were observed in the zone of inhibition. Two-month-old mycorrhizae of P. sylvestris/ L. laccata were inoculated with a conidial suspension of T. virens and examined at intervals of 7-24 h and 2, 3, and 6 days post-inoculation (p.i.). On non-mycorrhizal roots, conidia germination was high and long hyphae formed 3 days p.i. On mycorrhizal roots, short germ tubes were observed only sporadically. At 3 days p.i., the mantle hyphae of L. laccata grew towards the conidia and coiled around them. Extremely dense coils of hyphae were found around clusters of conidia. Deformation of conidia, breaks in conidial walls and their partial degradation were observed 6 days p.i.

Agaricales↗

Wood formation from the base to the crown in Pinus radiata: gradients of tracheid wall thickness, wood density, radial growth rate and gene expression.

Wood formation was investigated at five heights along the bole for two unrelated trees of Pinus radiata. Both trees showed clear gradients in wood properties from the base to the crown. Cambial cells at the base of the tree were dividing 3.3-fold slower than those at the crown, while the average thickness of cell walls in wood was highest at the base. Cell wall thickness showed an overall correlation coefficient of >0.7 with wood density in both genotypes. Microscopic examination of developing tracheids showed that 33% of cells had formed secondary cell walls at the base of the tree, reducing to 3% at the crown. In total, 455 genes differentially expressed in developing xylem tissue from either the base or the crown were identified using modified differential display. RT-PCR analysis of 156 genes confirmed differential expression for 77%. Of the genes tested, 73% showed gradients in transcript abundance either up or down the bole of the tree, although the steepness of the gradients differed between genes. Genes involved in cell division and expansion tended to be more highly expressed in the crown of the tree, and two putative cell-cycle repressor genes were expressed 2-fold higher at the base. Conversely, transcripts of genes involved in secondary wall thickening were more abundant at the base of the tree. These results suggest that differences in the rate of cambial cell division, differences in the rate and duration of tracheid wall thickening, and differences in gene expression underpin the gradients of wood properties found in pines.

Cell Wall↗

Gene silencing of cinnamyl alcohol dehydrogenase in Pinus radiata callus cultures.

Xylem-derived Pinus radiata cell cultures, which can be induced to differentiate tracheary elements (TEs), were transformed with an RNAi construct designed to silence cinnamyl alcohol dehydrogenase (CAD), an enzyme involved in the biosynthesis of monolignols. Quantitative enzymatic CAD measurements revealed reduced CAD activity levels in most transclones generated. TEs from transclones with approximately 20% residual CAD activity did not release elevated levels of vanillin, which was derived from coniferyl-aldehyde through a mild alkali treatment. However, the activation of the phenylpropanoid pathway in transclones with approximately 20% residual CAD activity through the application of non-physiological concentrations of sucrose and l-phenylalanine produced phenotypic changes. The accumulation of metabolites such as dihydroconiferyl-alcohol (DHCA), which also accumulates in the P. taeda CAD mutant cad-n1, was observed. These results indicate that a substantial reduction in CAD activity is necessary for this enzyme to become a rate-limiting step in lignin biosynthesis in conifers such as P. radiata and confirm that transformable P. radiata callus cultures can be useful to investigate the function of xylogenesis-related genes in conifers.

Alcohol Oxidoreductases↗

cDNA isolation, functional expression, and characterization of (+)-alpha-pinene synthase and (-)-alpha-pinene synthase from loblolly pine (Pinus taeda): stereocontrol in pinene biosynthesis.

The complex mixture of monoterpenes, sesquiterpenes, and diterpenes that comprises oleoresin provides the primary defense of conifers against bark beetles and their associated fungal pathogens. Monoterpene synthases produce the turpentine fraction of oleoresin, which allows mobilization of the diterpene resin acid component (rosin) and is also toxic toward invading insects; this is particularly the case for alpha-pinene, a prominent bicyclic monoterpene of pine turpentine. The stereochemistry of alpha-pinene is a critical determinant of host defense capability and has implications for host selection, insect pheromone biosynthesis, and tritrophic-level interactions. Pines produce both enantiomers of alpha-pinene, which appear to arise through antipodal reaction mechanisms by distinct enzymes. Using a cDNA library constructed with mRNA from flushing needles of loblolly pine (Pinus taeda), we employed a homology-based cloning strategy to isolate, and confirm by functional expression, the genes encoding (+)-(3R:5R)-alpha-pinene synthase, (-)-(3S:5S)-alpha-pinene synthase, and several other terpene synthases. The pinene synthases, which produce mirror-image products, share only 66% amino acid identity (72% similarity) but are similar in general properties to other monoterpene synthases of gymnosperms. The stereochemical control of monoterpene cyclization reactions, the evolution of "antipodal" enzymes, and the implications of turpentine composition in ecological interactions are discussed.

Amino Acid Sequence↗

Chloroplastic responses of ponderosa pine (Pinus ponderosa) seedlings to ozone exposure.

Integrity of chloroplast membranes is essential to photosynthesis. Loss of thylakoid membrane integrity has been proposed as a consequence of ozone (O(3)) exposure and therefore may be a mechanistic basis for decreased photosynthetic rates commonly associated with ozone exposure. To investigate this hypothesis, Pinus ponderosa seedlings were exposed to ambient air or ozone concentrations maintained at 0.15 or 0.30 microliter l(-1) for 10 h day(-1) for 51 days during their second growing season. Over the course of the study, foliage samples were periodically collected for thylakoid membrane, chlorophyll and protein analyses. Additionally, gas-exchange measurements were made in conjunction with foliage sampling to verify that observed chloroplastic responses were associated with ozone-induced changes in photosynthesis. Needles exposed to elevated ozone exhibited decreases in chlorophyll a and b content. The decreases were dependent on the duration and intensity of ozone exposure. When based on equal amounts of chlorophyll, ozone-exposed sample tissue exhibited an increase in total protein. When based on equal amounts of protein, ozone-exposed samples exhibited an increase in 37 kDa proteins, possibly consisting of breakdown products, and a possible decrease in 68 kDa proteins, Rubisco small subunit. There was also a change in the ratio of Photosystem I protein complexes CPI and CPII that may have contributed to decreased photosynthesis. Net photosynthetic rates were decreased in the high ozone treatment suggesting that observed structural and biochemical changes in the chloroplast were associated with alterations of the photosynthetic process.

Chlorophyll↗

Bioindication of the anthropogenic effects on micropopulations of Pinus sylvestris, L. in the vicinity of a plant for the storage and processing of radioactive waste and in the Chernobyl NPP zone.

Results of a comparative analysis of the frequency and spectrum of cytogenetic anomalies are presented for reproductive (seeds) and vegetative (needles) samples taken from Scotch pine (Pinus sylvestris, L.) micropopulations growing at sites with differing levels of radioactive contamination in the Chernobyl NPP 30 km zone, and at the location of a facility for the processing and storage of radioactive wastes (the 'Radon' LWPE, near the town of Sosnovy Bor in the Leningrad Region). The data obtained indicate the presence of genotoxic contaminants in the environment of the tree micropopulations. Chemical toxins make the main contribution to the environmental contamination in the Sosnovy Bor area as compared with the influence of ionising radiation in the Chernobyl 30 km zone. The higher radioresistance of seeds of Scotch pine growing on the area of the 'Radon' LWPE and in the centre of Sosnovy Bor town was revealed with acute gamma-radiation.

Air Pollution, Radioactive↗

Urban polluted forest soils induce elevated root peroxidase activity in Scots pine (Pinus sylvestris L.) seedlings.

Plant biomass. mycorrhizal status and root peroxidase activity were measured in ectomycorrhizal Scots pine (Pinus sylvestris L.) seedlings grown in urban polluted and native, non-polluted forest soils with added ammonium or potassium sulphates simulating N and S deposition of urban areas. Peroxidase activity in the fine roots of seedlings planted in polluted forest soils was higher than in those planted in non-polluted soils and correlated positively with the activities measured in an earlier study in the roots of mature Scots pines growing at the sites from where the soils were collected. Growth of seedlings and mycorrhizal status were not affected by the origin of soil. Exposing the seedlings to winter acclimation conditions for 6 weeks elevated peroxidase activity in the roots. The addition of ammonium or potassium sulphate to non-polluted soils did not induce elevated root peroxidase activity, although at the levels of 0.5 and 1.0 g of ammonium sulphate a slight increasing trend was observed. We suggest, that indirect biotic factors, i.e. changes in the community structure of soil fungi, early stages of recognition, and defence reactions of pine roots against saprophytic and pathogenic fungi may be participating in the elicitation of peroxidase (POD) activity, although the possible role of heavy metals cannot be excluded.

Adaptation, Physiological↗

Ozone impacts on loblolly pine (Pinus taeda L.) grown in a competitive environment.

One-year old, bare-root, loblolly pine (Pinus taeda L.) seedlings grown in an early successional plant community, were exposed to four ozone treatments, 12 h day(-1) (09:00-21:59 h CST), 7 day week(-1) in east-central Alabama for 2 years (1994-1995). Treatments included AA=ambient air (open plot; no chamber), CF = carbon-filtered air (approximately 0.5xAA), 1X = non-filtered air, and 2X = twice ambient (AA) air. Pine height and diameter were measured monthly for 2 years and total above-ground biomass determined at termination of the experiment. In addition, height, diameter and volume (d2h) growth was modeled for the 2-year study. Trees growing in the CF chambers exhibited the least growth for any variable measured. The predicted height growth rate (r) was the least for the CF- and 2X-treated pines when compared with the AA and 1X treatments. In addition, percent total vegetative cover was significantly negatively correlated with height. diameter and volume growth in 1994, but not 1995. Our findings regarding the trees growing in the CF treatments, are contrary to previous reports regarding ozone impacts to loblolly pine grown without competition from other vegetation.

Dose-Response Relationship, Drug↗

Effects of environmental biomass-producing factors on Cd uptake in two Swedish ecotypes of Pinus sylvestris.

A factorial design was used to study direct effects of external biomass-producing factors such as light, temperature and photoperiod on cadmium (Cd) uptake and indirect effects, via change in biomass production in two ecotypes of Scots pine (Pinus silvestris). The aim was to find out if the external factors affect the Cd uptake directly or via change in biomass production, and if the effect differs between ecotypes. Seedlings were grown under 10 combinations of external factors, i.e. temperature (15 and 20 degrees C), light intensity (50 and 200 micromol photons m(-2) S(-1)), photoperiod (18 h light/8 h darkness and continuous light) and external Cd concentration (totally 1.88 and 7.50 micromol). The treatment lasted for 18 days and Cd concentrations in roots and shoots were determined by AAS. The results showed that an increased biomass production increased the total Cd uptake but had a dilution effect on the Cd concentration, especially in the root tissues. The external factors tested did not have any direct effects on the Cd untake, only in the case of Cd translocation to the shoot did the higher temperature show a direct increase, but only in the southern ecotype. The two ecotypes reacted differently in Cd uptake and translocation to the external factors studied. The relative Cd uptake creased with increasing photoperiod in the northern but not in the southern ecotype. The southern ecotype decreased the Cd concentration in the shoot with increased light intensity caused by a dilution effect due to extensive shoot growth of this ecotype. The conclusion is that the uptake in pine seedlings is mainly regulated via biomass production, and not directly by light and temperature and that resulting plant Cd contents to a certain extent depend on plant origin.

Biomass↗

Biosorption of chromium(VI) from aqueous solution by cone biomass of Pinus sylvestris.

Biosorption of chromium(VI) on to cone biomass of Pinus sylvestris was studied with variation in the parameters of pH, initial metal ion concentration and agitation speed. The biosorption of Cr(VI) was increased when pH of the solution was decreased from 7.0 to 1.0. The maximum chromium biosorption occurred at 150 rpm agitation. An increase in chromium/biomass ratio caused a decrease in the biosorption efficiency. The adsorption constants were found from the Freundlich isotherm at 25 degrees C. The cone biomass, which is a readily available biosorbent, was found suitable for removing chromium from aqueous solution.

Adsorption↗

Physical-mechanical properties and chemical composition of Pinus taeda mature wood following a forest fire.

The objective of this study was to assess the effects of heat released during forest fires on wood properties of Pinus taeda L. trees submitted to different burning levels (increasing fire intensity, I-IV). Wood samples were collected from trees in each of the burning levels and also from trees not affected by fire (control). Specimens were then extracted to evaluate the physical and mechanical wood properties; chemical composition was evaluated only for burning level IV and control. The analysis of the results showed that fire effects over the physical-mechanical properties and chemical composition in all burning levels did not cause sufficient chemical degradation and strength reduction, which could be cause for rejection of those woods for normal use. In the case of structural use caution should be adopted for the wood from burning levels III and IV, which had their mechanical property values reduced.

Chemical Phenomena↗

Alteration of foliar flavonoid chemistry induced by enhanced UV-B radiation in field-grown Pinus ponderosa, Quercus rubra and Pseudotsuga menziesii.

Chromatographic analyses of foliage from several tree species illustrate the species-specific effects of UV-B radiation on both quantity and composition of foliar flavonoids. Pinus ponderosa, Quercus rubra and Pseudotsuga menziesii were field-grown under modulated ambient (1x) and enhanced (2x) biologically effective UV-B radiation. Foliage was harvested seasonally over a 3-year period, extracted, purified and the flavonoid fraction applied to a mu Bondapak/C(18) column HPLC system sampling at 254 nm. Total flavonoid concentrations in Quercus rubra foliage were more than twice (leaf area basis) that of the other species; Pseudotsuga menziesii foliage had intermediate levels and P. ponderosa had the lowest concentrations of total flavonoids. No statistically significant UV-B radiation-induced effects were found in total foliar flavonoid concentrations for any species; however, concentrations of specific compounds within each species exhibited significant treatment effects. Higher (but statistically insignificant) levels of flavonoids were induced by UV-B irradiation in 1- and 2-year-old P. ponderosa foliage. Total flavonoid concentrations in 2-year-old needles increased by 50% (1x ambient UV-B radiation) or 70% (2x ambient UV-B radiation) from that of 1-year-old tissue. Foliar flavonoids of Q. rubra under enhanced UV-B radiation tended to shift from early-eluting compounds to less polar flavonoids eluting later. There were no clear patterns of UV-B radiation effects on 1-year-old P. menziesii foliage. However, 2-year-old tissue had slightly higher foliar flavonoids under the 2x UV-B radiation treatment compared to ambient levels. Results suggest that enhanced UV-B radiation will alter foliar flavonoid composition and concentrations in forest tree species, which could impact tissue protection, and ultimately, competition, herbivory or litter decomposition.

Flavins↗

Agrobacterium-mediated transformation and assessment of factors influencing transgene expression in loblolly pine (Pinus taeda L.).

This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for beta-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.

Agrobacterium tumefaciens↗

Apparent homology of expressed genes from wood-forming tissues of loblolly pine (Pinus taeda L.) with Arabidopsis thaliana.

Pinus taeda L. (loblolly pine) and Arabidopsis thaliana differ greatly in form, ecological niche, evolutionary history, and genome size. Arabidopsis is a small, herbaceous, annual dicotyledon, whereas pines are large, long-lived, coniferous forest trees. Such diverse plants might be expected to differ in a large number of functional genes. We have obtained and analyzed 59,797 expressed sequence tags (ESTs) from wood-forming tissues of loblolly pine and compared them to the gene sequences inferred from the complete sequence of the Arabidopsis genome. Approximately 50% of pine ESTs have no apparent homologs in Arabidopsis or any other angiosperm in public databases. When evaluated by using contigs containing long, high-quality sequences, we find a higher level of apparent homology between the inferred genes of these two species. For those contigs 1,100 bp or longer, approximately 90% have an apparent Arabidopsis homolog (E value < 10-10). Pines and Arabidopsis last shared a common ancestor approximately 300 million years ago. Few genes would be expected to retain high sequence similarity for this time if they did not have essential functions. These observations suggest substantial conservation of gene sequence in seed plants.

3' Untranslated Regions↗

Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism.

Transcriptional profiling of the phenylpropanoid pathway in Pinus taeda cell suspension cultures was carried out using quantitative real time PCR analyses of all known genes involved in the biosynthesis of the two monolignols, p-coumaryl and coniferyl alcohols (lignin/lignan precursors). When the cells were transferred to a medium containing 8% sucrose and 20 mm potassium iodide, the monolignol/phenylpropanoid pathway was induced, and transcript levels for phenylalanine ammonia lyase, cinnamate 4-hydroxylase, p-coumarate 3-hydroxylase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase, and cinnamyl alcohol dehydrogenase were coordinately up-regulated. Provision of increasing levels of exogenously supplied Phe to saturating levels (40 mm) to the induction medium resulted in further up-regulation of their transcript levels in the P. taeda cell cultures; this in turn was accompanied by considerable increases in both p-coumaryl and coniferyl alcohol formation and excretion. By contrast, transcript levels for both cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase were only slightly up-regulated. These data, when considered together with metabolic profiling results and genetic manipulation of various plant species, reveal that carbon allocation to the pathway and its differential distribution into the two monolignols is controlled by Phe supply and differential modulation of cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase activities, respectively. The coordinated up-regulation of phenylalanine ammonia lyase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase in the presence of increasing concentrations of Phe also indicates that these steps are not truly rate-limiting, because they are modulated according to metabolic demand. Finally, the transcript profile of a putative acid/ester O-methyltransferase, proposed as an alternative catalyst for O-methylation leading to coniferyl alcohol, was not up-regulated under any of the conditions employed, suggesting that it is not, in fact, involved in monolignol biosynthesis.

Base Sequence↗