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Drought effects on fine-root and ectomycorrhizal-root biomass in managed Pinus oaxacana Mirov stands in Oaxaca, Mexico.

The effects of a severe drought on fine-root and ectomycorrhizal biomass were investigated in a forest ecosystem dominated by Pinus oaxacana located in Oaxaca, Mexico. Root cores were collected during both the wet and dry seasons of 1998 and 1999 from three sites subjected to different forest management treatments in 1990 and assessed for total fine-root biomass and ectomycorrhizal-root biomass. Additionally, a bioassay experiment with P. oaxacana seedlings was conducted to assess the ectomycorrhizal inoculum potential of the soil for each of the three stands. Results indicated that biomasses of both fine roots and ectomycorrhizal roots were reduced by almost 60% in the drought year compared to the nondrought year. There were no significant differences in ectomycorrhizal and fine-root biomass between the wet and dry seasons. Further, the proportion of total root biomass consisting of ectomycorrhizal roots did not vary between years or seasons. These results suggest that both total fine-root biomass and ectomycorrhizal-root biomass are strongly affected by severe drought in these high-elevation tropical pine forests, and that these responses outweigh seasonal effects. Forest management practices in these tropical pine forests should consider the effects of drought on the capacity of P. oaxacana to maintain sufficient levels of ectomycorrhizae especially when there is a potential for synergistic interactions between multiple disturbances that may lead to more severe stress in the host plant and subsequent reductions in ectomycorrhizal colonization.

Biomass↗

Zinc-tolerant Suillus bovinus improves growth of Zn-exposed Pinus sylvestris seedlings.

Scots pine (Pinus sylvestris L.) seedlings inoculated or not (NM) by a Zn-sensitive or a Zn-tolerant isolate of the ectomycorrhizal fungus Suillus bovinus (L. Fr.) Roussel were exposed to 0.1 or 150 muM Zn(2+) for 9 months. We hypothesized that inoculation with a Zn-tolerant S. bovinus isolate should result in added Zn resistance of the host plant. Plant and fungal growth as well as nutrient profiles and photosynthetic pigments in pine needles were quantified. In NM plants and in plants colonized by the Zn-sensitive isolate, plant growth, N, P, Mg and Fe assimilation were strongly inhibited under Zn stress and concurred with significantly reduced chlorophyll concentrations. In contrast, plants colonized by the Zn-tolerant isolate grew much better and remained physiologically healthier when exposed to elevated Zn. These results provide further evidence for the important role metal-adapted mycorrhizal fungi play as an effective biological barrier against metal toxicity in trees.

Basidiomycota↗

Paternal cytoplasmic transmission in Chinese pine (Pinus tabulaeformis).

In this paper, the stages of normal sexual reproduction between pollen tube penetration of the archegonium and early embryo formation in Pinus tabulaeformis are described, emphasizing the transmission of parental cytoplasm, especially the DNA-containing organelles--plastids and mitochondria. The pollen tube growing in the nucellus contained an irregular tube nucleus followed by a pair of sperm cells. The tube cytoplasm contained abundant organelles, including starch-containing plastids and mitochondria. The two sperm cells differed in their volume of cytoplasm. The leading sperm, with more cytoplasm, contained abundant plastids and mitochondria, while the trailing one, with a thin layer of cytoplasm, had very few organelles. The mature egg cell contained a great number of mitochondria, whereas it lacked normal plastids. At fertilization, the pollen tube penetrated into the egg cell at the micropylar end and released all of its contents, including the two sperms. One of the sperm nuclei fused with the egg nucleus, whereas the other one was retained by the receptive vacuole. Very few plastids and mitochondria of male origin were observed around the fusing sperm and egg nuclei, while the retained sperm nucleus was surrounded by a large amount of male cytoplasm. The discharged tube cytoplasm occupied a large micropylar area in the egg cell. In the free nuclear proembryo, organelles of maternal and paternal origins intermingled in the neocytoplasm around the free nuclei. Most of the mitochondria had the same features as those of the egg cell, but some appeared to be from sperm cells and tube cytoplasm. Plastids were obviously of male origin, with an appearance similar to those of the sperm or tube cells. After cellularization of the proembryo, maternal mitochondria became more abundant than the paternal ones and the plastids enlarged and began to accumulate starch. The results reveal the cytological mechanism for paternal inheritance of plastids and biparental inheritance of mitochondria in Chinese pine.

Cytoplasm↗

Characterization of two laccases of loblolly pine (Pinus taeda) expressed in tobacco BY-2 cells.

We previously showed that eight laccase genes (Lac 1-Lac 8) are preferentially expressed in differentiating xylem and are associated with lignification in loblolly pine (Pinus taeda) [Sato et al. (2001) J Plant Res 114:147-155]. In this study we generated transgenic tobacco suspension cell cultures that express the pine Lac 1 and Lac 2 proteins, and characterized the abilities of these proteins to oxidize monolignols. Lac 1 and Lac 2 enzymatic activities were detected only in the cell walls of transgenic tobacco cells, and could be extracted with high salt. The optimum pH for laccase activity with coniferyl alcohol as substrate was 5.0 for Lac 1 and between 5.0 and 6.0 for Lac 2. The activities of Lac 1 and Lac 2 increased as the concentration of CuSO(4) in the reaction mixtures increased in the range from 1 to 100 microM. Both enzymes were able to oxidize coniferyl alcohol and to produce dimers of coniferyl alcohol. These results are consistent with the hypothesis that Lac 1 and Lac 2 are involved in lignification in differentiating xylem of loblolly pine.

Cell Line, Transformed↗

Seasonal changes in needle water content and needle ABA concentration of Japanese red pine, Pinus densiflora, in declining forests on Mt. Gokurakuji, Hiroshima prefecture, Japan.

To evaluate the effects of air pollution on the decline of Pinus densiflora forests, various research has been conducted around Mt. Gokurakuji (34 degrees 23'N, 132 degrees 19'E, 693 m a.s.l.) north of the Seto Inland Sea, west Japan. To investigate the mechanisms responsible for decreases in photosynthesis (Pn) and stomatal conductance (gl), delta13C of needles and seasonal changes in the water content (WC) and abscisic acid concentration (ABA) of needles were measured in various stands. The delta13C values were less negative in declining stands and younger needles. ABA and WC were not correlated with each other. WC decreased consistently with needle age while the ABA showed a minimum in August and a smaller content in older needles. Monthly precipitation and the daily maximum vapor pressure were not correlated with ABA and WC. In declining stands, WC and ABA tended to be higher and lower, respectively, than in nondeclining stands. These results suggest that the trees in declining stands received less water stress than those in nondeclining stands and the differences in gl and delta13C are not caused by the difference in water stress. The possibilities of the effects of air pollution and the infection of pine-wood nematode on the physiological decline on the pine needles are discussed.

Abscisic Acid↗

Analysis of telomere length and telomerase activity in tree species of various life-spans, and with age in the bristlecone pine Pinus longaeva.

Normal somatic cells have a finite replicative capacity. With each cell division, telomeres (the physical ends of linear chromosomes) progressively shorten until they reach a critical length, at which point the cells enter replicative senescence. Some cells maintain telomere length by the action of the telomerase enzyme. The bristlecone pine, Pinus longaeva, is the oldest known living eukaryotic organism, with the oldest on record turning 4770 years old in 2005. To determine what changes occur, if any, in telomere length and telomerase activity with age, and what roles, if any, telomere length and telomerase activity may play in contributing to the increased life-span and longevity of P. longaeva with age, as well as in other tree species of various life-spans, we undertook a detailed investigation of telomere length and telomerase activity in such trees. The results from this study support the hypothesis that both increased telomere length and telomerase activity may directly/indirectly contribute to the increased life-span and longevity evident in long-lived pine trees (2000-5000 year life-spans) compared to medium-lived (400-500 year life-span) and short-lived (100-200 year life-span) pine trees, as well as in P. longaeva with age.

Aging↗

Detection of low genetic variation in a critically endangered Chinese pine, Pinus squamata, using RAPD and ISSR markers.

With only 32 individuals in the northeastern corner of Yunnan Province, China, Pinus squamata is one of the most endangered conifers in the world. Using two classes of molecular markers, RAPD and ISSR, its very low genetic variation was revealed. Shannon's index of phenotypic diversity (I) was 0.030, the mean effective number of alleles per locus (Ae) was 1.032, the percentage of polymorphic loci (P) was 6.45, and the expected heterozygosity (He) was 0.019 at the species level based on RAPD markers. The results of ISSR were consistent with those detected by RAPD but somewhat higher (I = 0.048, Ae = 1.042, P = 12.3, He = 0.029). The genetic variation of the subpopulation on the southwest-facing slope was much higher than that of the subpopulation on the northeast-facing slope, which may be attributed to the more diverse environment on the southwest-facing slope. The genetic differentiation between the two subpopulations was very low. The between-subpopulation variabilities, phiST, calculated from RAPD and ISSR data were 0.011 and 0.024. Because of the lack of fossil records and geological historical data, it was difficult to explain the extremely low genetic diversity of the species. We postulate that this ancient pine might have experienced strong bottlenecks during its long evolutionary history, which caused the loss of genetic variation. Genetic drift and inbreeding in post-bottlenecked small populations may be the major forces that contribute to low genetic diversity. Human activities such as logging may have accelerated the loss of genetic diversity in P. squamata.

Conservation of Natural Resources↗

Stable transformation of embryogenic tissues of Pinus nigra Arn. using a biolistic method.

The stable transformation of embryogenic tissues of Pinus nigra Arn., cell line E104, has been achieved using a biolistic approach. The introduced DNA consisted of the uidA reporter gene under the control of the double CaMV 35S promoter and the nptII selection gene controlled by the single CaMV 35S promoter. Three days after bombardment, putative transformed tissues were selected for continued proliferation on a medium containing 20 mg geneticin l(-1). Resistant embryogenic tissue recovery required 10-12 weeks. The integration of the nptII and uidA genes was confirmed by both histochemical/fluorimetric GUS assays and PCR amplification of the inserts in the five geneticin resistant sub-lines of line E104. The activity of the uidA reporter gene in transgenic, embryogenic tissue lines was stable and could be detected after one year of culture. Somatic embryo maturation was, however, poor and no plantlet regeneration could be obtained.

Biolistics↗

Toxic metals in the Austrian pine (pinus nigra) bark in the Thrace region, Turkey.

Austrian pine (Pinus nigra) bark was collected in thirty-seven localities in the Thrace region, Turkey, for the determination of airborne toxic metal distribution. Five toxic metals, As, Cd, Cu, Pb, and Zn, were monitored using atomic absorption spectrometry. Distribution maps of toxic elements in the region were plotted according to Geographic Information System technology. In general, the concentrations of all investigated elements were not high in the region apart from the vicinity of big cities, Tekirdag, Istanbul and, close to Bulgarian border. However, the highest concentrations were not at pollution level. They have a similar distribution pattern throughout the region.

Air Pollutants↗

Influence of fungal infection and wounding on contents and enantiomeric compositions of monoterpenes in phloem of Pinus sylvestris.

To identify chemical resistant markers induced by fungal or mechanical injury, young trees of Scots pine (Pinus sylvestris) were subjected to inoculations of blue stain fungi associated with the pine shoot beetles Tomicus piniperda and T. minor. Among the 20 trees selected for chemical analyses, 16 were divided into four groups: one as control and three were pretreated by wounding only, or by inoculation with either the blue stain fungus Leptographium wingfieldii or Ophiostoma canum. Four wk after pretreatment, all 16 pretreated trees were mass-inoculated with L. wingfieldii. The absolute and relative amounts, as well as the enantiomeric compositions of monoterpene hydrocarbons in the phloem, were determined via a small sample of the phloem before and after the pretreatment and mass inoculation, by using two-dimensional gas chromatography (2D GC) and GC-mass spectrometry (MS). After mass inoculation, the absolute amounts of most of the monoterpenes decreased in the phloem sampled >20 cm from the fungal infection, and were higher in the phloem sampled within the infected reaction zone. The relative amounts of both (-)-beta-pinene and (-)-limonene increased in phloem samples taken >20 cm above the fungal inoculation in the preinoculated trees compared with phloem sampled from the remaining four control trees. The enantiomeric compositions of beta-pinene and limonene changed, after fungal growth, at defined distances from the inoculation site: the proportion of the (-)-enantiomers was highest in the phloem sampled >20 cm from the fungal inoculation. Four wk after pretreatment, monoterpene production in the phloem at the site of inoculation was more enhanced by L. wingfieldii than by O. canum. However, the different virulence levels of the fungi did not affect the enantiomeric composition of the monoterpenes. The biosynthesis of monoterpene enantiomers is discussed in relation to induced pathogen resistance.

Animals↗

Overexpression of the pepper transcription factor CaPF1 in transgenic Virginia pine (Pinus Virginiana Mill.) confers multiple stress tolerance and enhances organ growth.

Transcription factors play an important role in regulating gene expression in response to stress and pathogen tolerance. We describe here that overexpression of an ERF/AP2 pepper transcription factor (CaPF1) in transgenic Virginia pine (Pinus virginiana Mill.) confers tolerance to heavy metals Cadmium, Copper, and Zinc, to heat, and to pathogens Bacillus thuringiensis and Staphylococcus epidermidis, as by the survival rate of transgenic plants and the number of decreasing pathogen cells in transgenic tissues. Measurement of antioxidant enzymes ascorbate peroxidase (APOX), glutathione reductase (GR), and superoxide dismutase (SOD) activities demonstrated that the level of the enzyme activities was higher in transgenic Virginia pine plants overexpressing the CaPF1 gene, which may protect cells from the oxidative damage caused by stresses, compared to the controls. Constitutive overexpression of CaPF1 gene enhanced organ growth by increasing organ size and cell numbers in transgenic Virginia pine plants over those in control plants.

Antioxidants↗

Cellular modelling of secondary radial growth in conifer trees: application to Pinus radiata (D. Don).

The radial growth of conifer trees proceeds from the dynamics of a merismatic tissue called vascular cambium or cambium. Cambium is a thin layer of active proliferating cells. The purpose of this paper was to model the main characteristics of cambial activity and its consecutive radial growth. Cell growth is under the control of the auxin hormone indole-3-acetic. The model is composed of a discrete part, which accounts for cellular proliferation, and a continuous part involving the transport of auxin. Cambium is modeled in a two-dimensional cross-section by a cellular automaton that describes the set of all its constitutive cells. Proliferation is defined as growth and division of cambial cells under neighbouring constraints, which can eliminate some cells from the cambium. The cell-growth rate is determined from auxin concentration, calculated with the continuous model. We studied the integration of each elementary cambial cell activity into the global coherent movement of macroscopic morphogenesis. Cases of normal and abnormal growth of Pinus radiata (D. Don) are modelled. Abnormal growth includes deformed trees where gravity influences auxin transport, producing heterogeneous radial growth. Cross-sectional microscopic views are also provided to validate the model's hypothesis and results.

Biological Transport, Active↗

Effects of delta5 polyunsaturated fatty acids of maritime pine (Pinus pinaster) seed oil on the fatty acid profile of the developing brain of rats.

Conifer (pine) seeds are a potential source of dietary oils, but their safety and nutritional properties are not well established. Conifer seed oils differ from common edible vegetable oils in having a series of unusual polyunsaturated fatty acids (PUFA) with a polymethylene-interrupted (PMI) double bond system and a double bond at the delta5 position. A rat study was conducted to assess whether delta5 PMI-PUFA of conifer seeds could alter the levels of n-6 and n-3 long-chain polyunsaturated fatty acids (LC-PUFA) in mothers' milk and the developing brain of fetuses and pups. Feeding maritime pine (Pinus pinaster) seed oil (MPO) diet with a delta5 PMI-PUFA content of 1.4 g/100 g throughout pregnancy and lactation resulted in a large incorporation of delta5 PMI-PUFA in mothers' milk (5.1 +/- 0.5% of total fatty acids). The fetus (17 d old) and pup (22 d) brains, however, accumulated very little (0.6 and 0.4% of total fatty acids, respectively) delta5 PMI-PUFA. Mother's milk and pup's brain of the MPO group contained normal levels of 20:4n-6, 22:4n-6, and 20:5n-3 compared to a reference group of rats fed a fat blend of sunflower, high-oleic sunflower, and canola oils. The level of 22:6n-3, however, was slightly but significantly (P < 0.05) higher in milk and pup brain of the MPO group. These results show that delta5 PMI-PUFA of MPO exert no negative effect on the levels of n-6 and n-3 LC-PUFA in rat brain during its early development.

Animals↗

Stilbene synthase from Scots pine (Pinus sylvestris).

Stilbene synthases are named according to their substrate preferences. By this definition, enzymes preferring cinnamoyl-CoA are pinosylvin synthases, and proteins with a preference for phenylpropionyl-CoA are dihydropinosylvin synthases. We investigated the assignment of a stilbene synthase cloned from Scots pine (Pinus sylvestris) as dihydropinosylvin synthase and the proposal of an additional pinosylvin synthase [1992, Plant Mol. Biol. 18, 489-503]. The results show that the previous interpretation was misled by several unexpected factors. Firstly, we found that the substrate preference and the activity of the plant-specific protein expressed in E. coli was influenced by bacterial factors. This was reduced by improvement of the expression system, and the subsequent kinetic analysis revealed that cinnamoyl-CoA rather than phenylpropionyl-CoA is the preferred substrate of the cloned stilbene synthase. Secondly, mixing experiments showed that extracts from P. sylvestris contain factor(s) which selectively influenced the substrate preference, i.e. the activity was reduced with phenylpropionyl-CoA, but not with cinnamoyl-CoA. This explained the apparent differences between plant extracts and the cloned enzyme expressed in E. coli. Taken together, the results indicate that the cloned enzyme is a pinosylvin synthase, and there is no evidence for a second stilbene synthase. This study cautions that factors in the natural and in new hosts may complicate the functional identification of cloned sequences.

Acyl Coenzyme A↗

High-level expression of Pinus sylvestris glutamine synthetase in Escherichia coli. Production of polyclonal antibodies against the recombinant protein and expression studies in pine seedlings.

In a previous work we reported the molecular characterization of a glutamine synthetase (GS; EC 6.3.1.2.) complementary DNA from a woody plant (Cantón et al. (1993) Plant Mol. Biol. 22, 819-828). The isolated cDNA (pGSP114) encoding a Scots pine (Pinus sylvestris) cytosolic subunit, has been subcloned into the expression vector pET3c to overproduce the GS polypeptide in Escherichia coli cells. The recombinant GS protein showed the same molecular size as a native Scots pine GS subunit. Antibodies against the pET3c-GSP114 encoded protein were raised in rabbits by injecting purified preparations and specificity was determined by immunoprecipitation of GS activity present in pine crude extracts. In spite of the antibodies were able to recognize both cytosolic and chloroplastic GS in tomato plants, they were unable to immunodetect chloroplastic GS in green cotyledons of pine seedlings and cytosolic GS was the unique recognized polypeptide. Unlike to that found in other plant species, cytosolic GS was strongly expressed in green tissues as determined by protein and Northern analysis. Our results suggest a key role for cytosolic GS in photosynthetic tissues of conifers.

Animals↗

Evidence of chemical reactions between di- and poly-glycidyl ether resins and tannins isolated from Pinus radiata D. Don bark.

This work evaluates the feasibility of reacting tannins isolated from Pinus radiata D. Don bark with epoxide resins of the diglycidyl and polyglycidyl ether type. To this end, gel times of aqueous tannin dispersions (40% w/w) with every one of nine selected resins (5% w/w), at previously established pH values (initial equal to 3.3, 4, 7 and 10), have been determined. Products of these reactions were analyzed by FT-IR spectroscopy, and the results were compared with those obtained from tannin-p-formaldehyde and (+)-catechin-p-formaldehyde systems, at the same pH values. Their mechanical properties were evaluated, by dynamic mechanical analysis, at two pH values (3.3 and 10). In general, it was concluded that tannin-epoxide resin systems behave similarly to tannin-paraformaldehyde systems, especially at basic pH values.

Catechin↗

Response surface optimization of the removal of nickel from aqueous solution by cone biomass of Pinus sylvestris.

Response surface methodology was applied to optimize the removal efficiency of Ni(II). Pinus sylvestris ovulate cones were used in this study. A 2(3) full-factorial central composite design was employed for experimental design and analysis of the results. The initial Ni(II) concentration (10-30 mg/l), pH (2.5-6.5) and biomass concentration (5-25 g/l) were the critical components of the removal optimized. The optimum pH, m (biomass concentration) and C0 (initial Ni(II) concentration) were found to be 6.17, 18.8 g/l and 11.175 mg/l, respectively. Under these conditions, removal efficiency of Ni(II) was 99.91%.

Adsorption↗

Kenaf (Hibiscus cannabinus L.) core and rice hulls as components of container media for growing Pinus halepensis M. seedlings.

The feasibility of replacing peat or perlite with rice hulls and ground kenaf core to grow a Mediterranean species (Pinus halepensis M.), was investigated. Treatments involved the following growing media: 70 peat:30 perlite as a control, 70 peat:30 rice hulls, 50 peat:50 rice hulls, kenaf (100%) and 60 kenaf:20 peat:20 rice hulls. The seedlings' quality and the physical and chemical properties of the substrates were evaluated. After this, the seedlings were planted in the field and their field performance was recorded. The results showed that the growth medium 70 peat:30 rice hulls can successfully be used for the production of P. halepensis, since the seedlings' nursery and field performance were similar to, or even better than those obtained with the control medium. The seedlings produced in the growth media containing kenaf did not attain a suitable planting size and showed poorer field performance than control seedlings.

Biomass↗