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PubMed1999

Phytochrome types in Picea and Pinus. Expression patterns of PHYA-Related types.

Knowledge of the genes in gymnosperms encoding the apoproteins of the plant photoreceptor phytochrome is currently scanty as for gymnosperm nuclear protein coding sequences in general. Here we report two complete cDNA-derived sequences which code for two different types of gymnosperm phytochrome. One sequence stems from Norway spruce (Picea abies) and the other from Scots pine (Pinus sylvestris). More detailed studies have shown that both types of phytochrome gene are present in Norway spruce. From phylogenetic analyses, these types appear to branch off from progenitors that are also the common ancestors of the angiosperm PHYA/PHYC and PHYB/PHYD/PHYE lineages. Partial phytochrome sequences of other gymnosperms cluster with either the one type or the other of the gymnosperm phytochrome genes characterized here. Southern blot analysis of Picea DNA using probes derived from the full-length Picea gene indicated a family of at least five members. Whether they code for new types may be doubted since only two phylogenetic clusters were found. Studies using RNA-PCR of Picea RNA extracted from either light- or dark-grown seedlings indicated that the steady-state levels of the transcripts of two PHYA/C-related genes were hardly affected by light.

Cycadopsida↗

PubMed2001

Two S-adenosylmethionine synthetase-encoding genes differentially expressed during adventitious root development in Pinus contorta.

Two S-adenosylmethionine synthetase (SAMS) cDNAs, PcSAMS1 and PcSAMS2, have been identified in Pinus contorta. We found that the two genes are differentially expressed during root development. Thus, PcSAMS1 is preferentially expressed in roots and exhibits a specific expression pattern in the meristem at the onset of adventitious root development, whereas PcSAMS2 is expressed in roots as well as in shoots and is down-regulated during adventitious root formation. The expression of the two SAMS genes is different from the SAMS activity levels during adventitious root formation. We conclude that other SAMS genes that remain to be characterized may contribute to the observed SAMS activity, or that the activities of PcSAMS1 and PcSAMS2 are affected by post-transcriptional regulation. The deduced amino acid sequences of PcSAMS1 and PcSAMS2 are highly divergent, suggesting different functional roles. However, both carry the two perfectly conserved motifs that are common to all plant SAMS. At the protein level, PcSAMS2 shares about 90% identity to other isolated eukaryotic SAMS, while PcSAMS1 shares less than 50% identity with other plant SAMS. In a phylogenetic comparison, PcSAMS1 seems to have diverged significantly from all other SAMS genes. Nevertheless, PcSAMS1 was able to complement a Saccharomyces cerevisiae sam1 sam2 double mutant, indicating that it encodes a functional SAMS enzyme.

Amino Acid Sequence↗

PubMed2001

Elevated CO2 and ozone reduce nitrogen acquisition by Pinus halepensis from its mycorrhizal symbiont.

The effects of 700 µmol mol-1 CO2 and 200 nmol mol-1 ozone on photosynthesis in Pinus halepensis seedlings and on N translocation from its mycorrhizal symbiont, Paxillus involutus, were studied under nutrient-poor conditions. After 79 days of exposure, ozone reduced and elevated CO2 increased net assimilation rate. However, the effect was dependent on daily accumulated exposure. No statistically significant differences in total plant mass accumulation were observed, although ozone-treated plants tended to be smaller. Changes in atmospheric gas concentrations induced changes in allocation of resources: under elevated ozone, shoots showed high priority over roots and had significantly elevated N concentrations. As a result of different shoot N concentration and net carbon assimilation rates, photosynthetic N use efficiency was significantly increased under elevated CO2 and decreased under ozone. The differences in photosynthesis were mirrored in the growth of the fungus in symbiosis with the pine seedlings. However, exposure to CO2 and ozone both reduced the symbiosis-mediated N uptake. The results suggest an increased carbon cost of symbiosis-mediated N uptake under elevated CO2, while under ozone, plant N acquisition is preferentially shifted towards increased root uptake.

Journal Article↗

PubMed2001

Response to auxin changes during maturation-related loss of adventitious rooting competence in loblolly pine (Pinus taeda) stem cuttings.

Hypocotyl cuttings (from 20- and 50-day-old Pinus taeda L. seedlings) rooted readily within 30 days in response to exogenous auxin, while epicotyl cuttings (from 50-day-old seedlings) rarely formed roots within 60 days. Responses to auxin during adventitious rooting included the induction of cell reorganization and cell division, followed by the organization of the root meristem. Explants from the bases of both epicotyl and hypocotyl cuttings readily formed callus tissue in response to a variety of auxins, but did not organize root meristems. Auxin-induced cell division was observed in the cambial region within 4 days, and later spread to the outer cortex at the same rate in both tissues. Cells at locations that would normally form roots in foliated hypocotyl cuttings did not produce callus any differently than those in other parts of the cortex. Therefore, auxin-induced root meristem organization appeared to occur independently of auxin-induced cell reorganization/division. The observation that N-(1-naphthyl)phthalamic acid (NPA) promoted cellular reorganization and callus formation but delayed rooting implies the existence of an auxin signal transduction pathway that is specific to root meristem organization. Attempts to induce root formation in callus or explants without foliage were unsuccessful. Both the cotyledon and epicotyl foliage provided a light-dependent product other than auxin that promoted root meristem formation in hypocotyl cuttings.

Journal Article↗

PubMed2001

Novel cytokinins: The predominant forms in mature buds of Pinus radiata.

To elicit the roles of cytokinins in the regulation of maturation of Pinus radiata D. Don, the spectrum of endogenous cytokinins and their concentration in the mature buds were analysed using double-solvent extraction, column complex purification and separation, a novel immunoaffinity purification method, normal and reverse phase high-pressure liquid chromatography, enzymatic treatment, radioimmunoassay and electrospray MS/MS spectrometry. We have isolated two novel cytokinin glycosides whose proposed structures are isopentenyladenine-9-(glucopyranosyl riboside), dihydrozeatin-9-(glucopyranosyl riboside) and confirmed the presence of zeatin-9-(glucopyranosyl riboside). We have also found the presence of novel phosphorylated forms of these 3 cytokinin ribosyl-linked glycosides. Quantitative analyses revealed that the cytokinin ribosyl-linked glycosides predominate in P. radiata mature buds. Although cytokinin free base, riboside and nucleotide forms are also present, we could find no evidence of the traditional cytokinin O- or N-glucosides in the conifer buds. Thus, cytokinin metabolism in mature buds of P. radiata is very different from other species previously examined.

Journal Article↗

PubMed2001

Physiological response of Pinus halepensis needles under ozone and water stress conditions.

The aim of this study was to evaluate how physiological processes of potted Pinus halepensis plants, grown under controlled conditions, were affected by ozone (O3) and/or water stress, integrating the gas exchange and biochemical data with fluorescence OJIP polyphasic transient data. Plants submitted to only water stress (T1) and with ozone (T3) showed a strong decrease in stomatal conductance and gas exchange, coinciding with a reduction of maximum yield of photochemistry (varphipo) and very negative values of leaf water potential. Simultaneously, a great increase of both PSII antenna size, indicated by absorption per reaction centre, and electron transport per reaction centre were found. The reduction of photosynthesis in the O3-treated plants (T2) by a slowing down of the Calvin cycle was supported by the increase of related fluorescence parameters such as relative variable fluorescence, heat de-excitation constant, energy de-excitation by spillover, and the decrease of varphipo. We suggest an antagonistic effect between the two stresses to explain the delayed ozone-induced decrease of stomatal conductance values for T3 with respect to T1 plants, by an alteration of the physiological mechanisms of stomatal opening, which involve the increase of intra-cellular free-calcium induced by ABA under co-occurring water shortage. We emphasise the importance of considering the intensity of the individual stress factor in studies concerning the interaction of stresses.

Journal Article↗

PubMed2002

Requirement for far-red light to maintain secondary needle extension growth in northern but not southern populations of Pinus sylvestris (Scots pine).

Extension growth of secondary needles is under photoperiodic control in Pinus sylvestris. To test for the effects of far-red light on maintaining this extension growth, seedlings of six populations originating from latitudes between 57 degrees and 67 degrees N were raised for 11 weeks in continuous incandescent (metal halogen) light at 300 &mgr;mol m-2 s-1 and 20 degrees C and then transferred at the same temperature to a daily regime of 8 h incandescent light (230 &mgr;mol m-2 s-1) followed by a 16 h day extension with cool white fluorescent light (40 &mgr;mol m-2 s-1, R/FR ratio 7.5) or with incandescent lamps (20 &mgr;mol m-2 s-1, R/FR ratio 2.0). For the seedlings from the three populations north of 64 degrees, needle extension growth over 42 days in the FR-poor day extension treatment was lower by up to 40% than in the FR-rich day extension treatment, whereas for the seedlings from the three southern populations the needle extension growth was similar in both day extension treatments. The requirement for FR in day extensions is characteristic of 'light-dominant' photoperiodic control mechanisms. It appears that P. sylvestris changes from dark-dominant night timekeeping to light-dominant day timekeeping with increasing latitude, as with the photoperiodic control of budset in Picea abies.

Journal Article↗

PubMed2003

Casparian strips in needles of Pinus bungeana: isolation and chemical characterization.

By using cell wall degrading enzymes, Casparian strips were for the first time isolated from Pinus bungeana needle endodermis. They appeared as a fine network, similar to those isolated from roots. Fourier transform infrared spectroscopic analysis provided evidence that the Casparian strips were impregnated with lignin, suberin, cellulose and cell wall proteins.

Journal Article↗

PubMed2003

Regulation of phosphoenolpyruvate carboxylase in Pinus halepensis needles submitted to ozone and water stress.

Effects of ozone and/or drought stresses on phosphoenolpyruvate carboxylase (PEPc, EC 4.1.1.31) regulation in Pinus halepensis Mill. needles were assessed over 3 months in controlled conditions. Whereas moderated water stress applied to Aleppo pine had no effect on PEPc activity compared to the control, which was probably related to the high tolerance of this species to drought, ozone stress induced a dramatic increase of PEPc activity in pine needles. This stimulation of the anaplerotic pathway could provide substrates to repair processes, well known for being enhanced upon ozone exposure. The ozone-increased PEPc activity was related, to a certain extent, to an increase in protein and mRNA levels. The possible role of the stimulation of the phosphorylation status of the enzyme in the increased PEPc activity under ozone was also investigated. Following the demonstration of the existence of the phosphorylation site at the N terminal part of Aleppo pine PEPc, it was shown that, under ozone treatment, the light/dark PEPc activity ratio and the Ki (malate) for PEPc were increased. This strengthens the hypothesis of an ozone-related post-translational process, which could be part of an adaptation of the plants to prolonged stress. When ozone and water stress were applied in combination, the enhancement in PEPc activity was only related to changes in gene expression. This difference in PEPc regulation, compared to the effect of single stress, could be the consequence of the specific action of each stress on the enzyme. This study brings new insights into the regulation of PEPc in a C3 plant, Aleppo pine under these stresses. A different regulatory mechanism of PEPc is occurring according to the stress. The physiological implications of the increase in PEPc activity in response to ozone and/or water stress are discussed.

Journal Article↗

PubMed1994

Allozyme and chloroplast DNA variation in Italian and Greek populations of Pinus leucodermis.

Allozyme and chloroplast (cpDNA) variation was examined in five Italian and two Greek populations of Pinus leucodermis Ant. to estimate levels of genetic variability within and among populations and to establish the usefulness of allozyme and cpDNA markers in the taxonomic classification of these populations. Twenty-three isozyme gene loci were analysed, as well as restriction fragment length polymorphisms at two cpDNA spacer regions between tRNA genes. The level of genetic variability tended to be lower in the Greek populations but overall the observed levels of allozyme variation within and among populations were similar to those of other conifers. Identical cpDNA amplification and restriction patterns were observed among all individuals sampled from the seven populations. Taken together, the results of the allozyme and cpDNA analyses indicate that all seven populations belong to the same biological species.

Alleles↗

PubMed1974

Properties and substrate specificity of some reactions catalysed by a short-chain fatty acyl-coenzyme A synthetase from seeds of Pinus radiata.

1. Crude extracts of seeds of Pinus radiata catalysed acetate-, propionate-, n-butyrate- and n-valerate-dependent PP(i)-ATP exchange in the presence of MgCl(2), which was apparently due to a single enzyme. Propionate was the preferred substrate. Crude extracts did not catalyse medium-chain or long-chain fatty acid-dependent exchange. 2. Ungerminated dry seeds contained short-chain fatty acyl-CoA synthetase activity. The activity per seed was approximately constant for 11 days after imbibition and then declined. The enzyme was located only in the female gametophyte tissue. 3. The synthetase was purified 70-fold. 4. Some properties of the enzyme were studied by [(32)P]PP(i)-ATP exchange. K(m) values for acetate, propionate, n-butyrate and n-valerate were 4.7, 0.21, 0.33 and 2.1mm respectively. Competition experiments between acetate and propionate demonstrated that only one enzyme was involved and confirmed that the affinity of the enzyme for propionate was greater than that for acetate. CoA inhibited fatty acid-dependent PP(i)-ATP exchange. The enzyme catalysed fatty acid-dependent [(32)P]PP(i)-dATP exchange. 5. The enzyme also catalysed the fatty acyl-AMP-dependent synthesis of [(32)P]ATP from [(32)P]PP(i). Apparent K(m) (acetyl-AMP) and apparent K(m) (propionyl-AMP) were 57mum and 7.5mum respectively. The reaction was inhibited by AMP and CoA. 6. Purified enzyme catalysed the synthesis of acetyl-CoA and propionyl-CoA. Apparent K(m) (acetate) and apparent K(m) (propionate) were 16mm and 7.5mm respectively. The rate of formation of acetyl-CoA was enhanced by pyrophosphatase. 7. It was concluded that fatty acyl adenylates are intermediates in the formation of the corresponding fatty acyl-CoA.

Acetates↗

PubMed1974

The enzymology of short-chain fatty acyl-coenzyme A synthetase from seeds of Pinus radiata. Kinetic studies and a proposed reaction mechanism.

1. Short-chain fatty acyl-CoA synthetase from seeds of Pinus radiata was examined by acetate- and propionate-dependent PP(i)-ATP exchange. Reaction mixtures came to equilibrium almost instantly as judged by rates of exchange and analysis of an incubation mixture. 2. The activity of the enzyme was correlated with the concentration of MgP(2)O(7) (2-) but not with the concentration of Mg(2+), as judged by PP(i)-ATP exchange and fatty acyl AMP-dependent synthesis of ATP in the presence of PP(i). In PP(i)-ATP exchange assays, no clear relationship between activity and any single species of ATP was apparent. 3. High concentrations of fatty acid inhibited PP(i)-ATP exchange. PP(i)-dATP exchange was less than PP(i)-ATP exchange at low concentrations of fatty acid, but at higher concentrations PP(i)-dATP exchange exceeded PP(i)-ATP exchange. The rate of synthesis of fatty acyl-CoA in the presence of dATP was less than with ATP. 4. ATP and propionate inhibited the synthesis of ATP from propionyl-AMP and PP(i). The inhibition by ATP was competitive with respect to propionyl-AMP and non-competitive with respect to PP(i). The inhibition by propionate was non-competitive with respect to propionyl-AMP and PP(i). 5. AMP was a competitive inhibitor of propionyl-AMP-dependent synthesis of ATP and competitively inhibited propionate-dependent PP(i)-ATP exchange when ATP was the variable substrate. 6. It was concluded that the first partial reaction catalysed by the enzyme is ordered; ATP is the first substrate to react with the enzyme and PP(i) is probably the only product released.

Acetates↗

PubMed1978

Isolation, characterization and distribution of adenosine 3':5'-cyclic monophosphate from Pinus radiata.

Cyclic AMP was extracted in 0.1 M-HCl from tissues of Pinus radiata and purified by gel filtration on Sephadex G-10, and chromatography on Dowex AG1 (X2) and polyethyleneimine-cellulose in two separate solvent systems. Presumptive cyclic AMP from 10kg batches of pine needles was characterized by countercurrent distribution in the presence of cyclic [8-3H]AMP. Statistical analysis of the curves for radioactivity and mass (determined by the Gilman competitive-binding assay) showed that the fit of the curves was highly significant for seven degrees of freedom. The distribution of cyclic AMP within P. radiata and various other plant tissues was determined by the Gilman procedure. The results suggest that there is no relationship between variations in cyclic AMP concentrations and the known function of the tissue in which it was measured.

Chromatography↗

PubMed1995

Flavin-dependent alcohol oxidase from the yeast Pichia pinus. Spatial localization of the coenzyme FAD in the protein structure: hot-tritium bombardment and ESR experiments.

The spatial localization of the coenzyme FAD in the quaternary structure of the alcohol oxidase from the yeast Pichia pinus was studied by tritium planigraphy and ESR methods. In the present paper we measured the specific radioactivity of FAD labelled as a part of the alcohol oxidase complex. The specific-radioactivity ratio for two FAD portions (FMN and AMP) was calculated. ESR experiments show 4 A (0.4 nm) to be the depth of immersion of paramagnetic isoalloxazines into alcohol oxidase octamer molecules. It is suggested that FAD molecules are bound to the surface of the octamer, rather than to the subunit interfaces. The orientation of the prosthetic group FAD in the alcohol oxidase protein is discussed.

Adenosine Monophosphate↗

PubMed2000

Paternity and gene dispersal in limber pine (Pinus flexilis James).

This study provides empirical information on intrapopulation gene dispersal via pollen, the size of genetic 'neighbourhoods', and interpopulation gene flow in a long-lived conifer, limber pine (Pinus flexilis). We used allozyme loci for a paternity analysis of 518 seeds produced in an isolated population of limber pine located in north-eastern Colorado, U.S.A., separated by 2 km from the nearest conspecific trees and nearly 100 km from populations in the Rocky Mountains. We also used indirect techniques (FST analyses) to estimate gene flow rates among subdivisions of the study population and among five widely separated populations. Within the main study population limber pine exhibited a polymorphism level of 50%, observed heterozygosity of 0.159, and 2.36 alleles per polymorphic locus. Mountain populations were slightly more variable. The main study population showed significant differentiation in allozymes among neighbouring subpopulations. The mean FST was 0.031 and the gene flow rate among subpopulations was estimated as 7.8 migrants per generation. Among widely separated populations the mean FST was 0.035 and the gene flow rate was estimated as 6.9 migrants per generation. The paternity analysis indicated a best estimate of 6.5% pollen immigration (minimum 1.1%) from populations 2 km to 100+ km away. For 4% of the seeds examined, paternity could be ascribed to a single tree in the study population. Fractional paternity and likelihood methods were used to estimate pollen dispersal distances for the remainder of the seeds. Mean pollen dispersal distance was estimated at 140 m using the fractional method, similar to results from the other techniques. This compares with a mean distance of 172 m between potential mates. These results suggest near-panmictic pollen dispersal over this population, which covers about 15 ha. The observed allozyme differences and surprisingly low estimates of among-subpopulation gene flow are ascribed to a probable restriction of gene dispersal by seeds.

Alleles↗

PubMed2001

Genetic diversity within and among Pinus pinaster populations: comparison between AFLP and microsatellite markers.

Twenty-three populations of Pinus pinaster (13 Aquitaine populations and 10 Corsican populations) were analysed at three microsatellite loci and 122 AFLP loci. The aims of the study were: (i) to compare levels of within-population and among-population diversity assessed with both kinds of markers; (ii) to compare Aquitaine and Corsican provenances of P. pinaster; and (iii) to know if both markers gave the same information for conservation purposes. Classical population genetics statistics were estimated and the ranking of populations obtained using different markers and/or parameters were compared by computing Spearman's rank correlations. Even though microsatellites showed a higher within-population diversity, they showed the same level of differentiation as AFLP markers. Moreover, both markers also showed a higher genetic diversity in the Aquitaine provenance and a higher differentiation among Corsican populations. AFLPs and microsatellites gave different population diversity rankings. Consequently, the results do not support the potential population identification within each provenance for conservation purposes.

Alleles↗

PubMed1999

Community structure of ectomycorrhizal fungi in a Pinus muricata forest: minimal overlap between the mature forest and resistant propagule communities.

We have investigated colonization strategies by comparing the abundance and frequency of ectomycorrhizal fungal species on roots in a mature Pinus muricata forest with those present as resistant propagules colonizing potted seedlings grown in the same soil samples. Thirty-seven fungal species were distinguished by internal transcribed spacer (ITS) restriction fragment length polymorphisms (RFLPs); most were identified to species level by sporocarp RFLP matches or to genus/family level by using sequence databases for the mitochondrial and nuclear large-subunit rRNA genes. The below-ground fungal community found in the mature forest contrasted markedly with the resistant propagule community, as only four species were found in both communities. The dominant species in the mature forest were members of the Russulaceae, Thelephorales and Amanitaceae. In contrast, the resistant propagule community was dominated by Rhizopogon species and by species of the Ascomycota. Only one species, Tomentella sublilacina (Thelephorales), was common in both communities. The spatial distribution of mycorrhizae on mature roots and propagules in the soil differed among the dominant species. For example, T. sublilacina mycorrhizae exhibited a unique bias toward the organic horizons, Russula brevipes mycorrhizae were denser and more clumped than those of other species and Cenococcum propagules were localized, whereas R. subcaerulescens propagules were evenly distributed. We suggest that species differences in resource preferences and colonization strategies, such as those documented here, contribute to the maintenance of species richness in the ectomycorrhizal community.

Ascomycota↗

PubMed2000

Glacial refugia of limber pine (Pinus flexilis James) inferred from the population structure of mitochondrial DNA.

To make inferences about the glacial refugia that harboured the limber pine, Pinus flexilis James, we examined the range-wide population structure of mitochondrial DNA (mtDNA) with eight size variants in the second intron of nad1. The data consisted of haplotypes from 704 trees collected from 40 localities. The value of FST for these populations was 0.80, which is a much larger value than has been reported for allozymes and chloroplast DNA (cpDNA) in limber pine, and it suggests that the number of seeds moving among localities per generation is approximately 0.12. Gene flow of this magnitude would allow mutation and subsequent genetic drift to have a substantial impact on the population structure of mtDNA. The majority of the mtDNA haplotypes are restricted to minor portions of the geographical range. The data are consistent with mtDNA differentiation in seven glacial refugia, followed by dispersal out of those refugia.

Alberta↗