FGF-2 and S100beta immunoreactivities increase in reactive astrocytes, but not in microglia, in ascending dopamine pathways following a striatal 6-OHDA-induced partial lesion of the nigrostriatal system

Partial lesions were induced in rat midbrain dopamine ascending pathways by intrastriatal injection of 6-hydroxydopamine (6-OHDA), and after two weeks changes were observed in the immunoreactivities of S100beta, a calcium-binding protein, and basic fibroblast growth factor (FGF-2), which is neurotrophic. Semiquantitative microdensitometric image analysis revealed increased intensities of FGF-2 and S100beta immunostaining in putative glial profiles of the ipsilateral neostriatum, pars compacta (SNc) and reticulata (SNr) of the substantia nigra and ventral tegmental area (VTA). Double immunofluorescence and immunoperoxidase procedures, using antibodies against glial fibrillary acidic protein and OX-42, showed that these increased immunoreactivities were restricted to reactive astrocytes; they were not observed in reactive microglia. These results indicate that reactive astrocytes may exert paracrine trophic actions through S100beta and FGF-2 in the midbrain dopamine ascending pathways after striatal 6-OHDA treatment. Interactions between S100beta and FGF-2 may be relevant to neuronal maintenance and repair following dopamine injury.     

Nucleotide diversity at two phytochrome loci along a latitudinal cline in Pinus sylvestris

Forest tree species provide many examples of well-studied adaptive differentiation, where the search for the underlying genes might be possible. In earlier studies and in our common conditions in a greenhouse, northern populations set bud earlier than southern ones. A difference in latitude of origin of one degree corresponded to a change of 1.4 days in number of days to terminal bud set of seedlings. Earlier physiological and ecological genetics work in conifers and other plants have suggested that such variation could be governed by phytochromes. Nucleotide variation was examined at two phytochrome loci (PHYP and PHYO, homologues of the Arabidopsis thaliana PHYB and PHYA, respectively) in three populations: northern Finland, southern Finland and northern Spain. In our samples of 12-15 sequences (2980 and 1156 base pairs at the two loci) we found very low nonsynonymous variation; pi was 0.0003 and 0.0002 at PHYP and PHYO loci, respectively. There was no functional differentiation between populations at the photosensory domains of either locus. The overall silent variation was also low, only 0.0024 for the PHYP locus. The low estimates of silent variation are consistent with the estimated low synonymous substitution rates between Pinus sylvestris and Picea abies at the PHYO locus. Despite the low level of nucleotide variation, haplotypic diversity was relatively high (0.42 and 0.41 for fragments of 1156 nucleotides) at the two loci.     

Long-distance colonization,isolation by distance,and historical demography in a relictual Mexican pinyon pine (Pinus nelsonii Shaw) as revealed by paternally inherited genetic markers (cpSSRs)

Pinus nelsonii is a relictual pinyon pine distributed across a wide altitudinal range in semiarid zones in Mexico near the border between the States of Nuevo León and Tamaulipas. It also occurs in small patches in the State of San Luis Potosí. Pinus nelsonii is classified in the monotypic subsection Nelsoniae, separated from other pinyon pines (subsection Cembroides), because it possesses several distinctive characters including persistent fascicle sheaths, connate needles, and a distinctive wood anatomy. In the present study, chloroplast simple sequence repeats (cpSSRs) were used to estimate genetic variation in most known populations (nine) of P. nelsonii. The genetic variation (HT = 0.73; 27 haplotypes in 256 individuals) is moderate when compared to other pine species. Population differentiation ranged between low and moderate (FST = 0.13 and RST = 0.05), as did the Nei and Goldstein genetic distances between populations. However, this pattern varied depending on whether the infinite alleles or stepwise mutation model was used. In the former case a significant isolation by distance was found, but not in the latter. A significant association between geographical and genetic structure in one clade, through a nested clade analysis, was found, which suggested long-distance colonization between 125000 and 309000 years ago. We found weak evidence for a population expansion. A mismatch distribution suggests that P. nelsonii populations underwent an expansion 4.25 times their size between 59000 and 146000 years ago. On the other hand, the populations' star-like phylogeny and a slight parabolic relationship between coalescence times and lineage number also suggest weak population expansion. Overall, this species appears to have been in demographic stasis for a large proportion of the time detected by the markers used.     

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.     

Pollen pool heterogeneity in shortleaf pine, Pinus echinata Mill

Pollen is the dominant vector of gamete exchange for most temperate tree species. Because pollen movement influences the creation, maintenance and erosion of genetic structure in adult populations, it is important to understand what factors influence the process of pollen movement. Isolation by distance in pollen donor populations can create highly structured pollen polls by increased sampling of local fathers. Extrinsic factors, such as the intervening vegetative structure and local pollen donor densities, can also influence the genetic composition of local pollen pools. Using paternally inherited chloroplast microsatellite markers, we examined the structure and diversity of pollen pools in Pinus echinata Mill. in southern Missouri, USA. Our analysis is based on a multivariate AMOVA analysis of stands ( approximately 1 ha; six per region) nested within regions (approximately 800 ha; four each). Significant multilocus structure of the pollen pool within regions (phiSR = 0.095), but not among regions (phiRT = 0.010), indicates that pollen movement is relatively restricted. Furthermore, the significant correlation between pairwise genetic and physical distances (Mantel correlation; rho = 0.32) provided support for the isolation by distance hypothesis. Our results indicated that availability of pollen donors did not affect diversity of the pollen pool, measured by the number of unique multilocus genotypes at each stand. However, pollen pool diversity was negatively associated with vegetative structure, measured as total forest tree density. Our findings indicated that on-going pollen movement within continuous forest is relatively restricted as a result of both isolation by distance and vegetative structure.     

缙云山马尾松种群生物量生殖配置研究

 对缙云山亚热带常绿阔叶林不同演替阶段马尾松(Pinus massoniana)种群生殖器官的生物量配置进行了系统研究。结果显示从蕾期、花期到果熟期,马尾松种群生殖器官的生物量配置逐渐增加,但不同演替阶段的生殖配置格局各异。马尾松纯林各生殖阶段的生殖配置分别为1.31％,7.61％,23.25％;针阔混交林各生殖阶段的生殖配置分别为0.6％,3.29％,15.14％;林缘旷地各生殖阶段的生殖配置分别为0.76％,3.78％,18.44％。一年中缙云山马尾松种群的生殖配置动态变化呈现低一渐高一高一低的规律性。缙云山马尾松种群的生殖年龄大致可分为4个时期,即幼龄生殖期、过渡生殖期、稳定生殖期和衰退生殖期。种群密度和群落透光度与其生殖配置显著相关。     

In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls

A high-efficiency two-step culture procedure for direct somaticorganogenesis in loblolly pine (Pinus taeda L.) resulting inthe formation of multiple shoot structures induced on cotyledons andhypocotyls of mature zygotic embryos is described. Mature zygoticembryos of eight genotypes of loblolly pine were used as explants toinduce direct somatic organogenesis with this two-step culture method,involving the induction and the differentiation of direct adventitiousshoots. After mature zygotic embryos of eight genotypes of loblolly pinewere cultured on induction medium containing 2,4-dichlorophenoxyaceticacid (2,4-D) or -naphthaleneacetic acid (NAA), 6-benzyladenine(BA), and kinetin for 2–3 weeks, embryos were transferred todifferentiation medium. Adventitious shoot regeneration via directsomatic organogenesis with the frequency of 8.7–27.8% wasobtained from mature zygotic embryo cultures of the genotypes tested.The highest mean number of 32.6 adventitious shoots per mature zygoticembryo was produced from genotype La. The tissue culture protocol of invitro shoot regeneration via direct somatic embryogenesis was optimizedafter examining the periods of the induction culture, chillingtreatment, glutamine concentration, and basic medium levels. Rooting wasachieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid(IBA), 0.5 mg/l gibberellic acid (GA₃), and 1 mg/l6-benzyladenine (BA), and regenerated plantlets were established insoil. These results suggested that adventitious shoot regeneration viadirect somatic organogenesis could be useful for clonal micropropagationof some genotypes of loblolly pine and for establishing a transformationsystem of this coniferous species.     

Comparative population structure and genetic diversity of Arceuthobium americanum (Viscaceae) and its Pinus host species: insight into host-parasite evolution in parasitic angiosperms

In a recent study we revealed that the parasitic angiosperm Arceuthobium americanum is comprised of three distinct genetic races, each associated with a different host in regions of allopatry. In order to assess the role of host identity and geographical isolation on race formation in A. americanum, we compared the genetic population structure of this parasite with that of its three principal hosts, Pinus banksiana, Pinus contorta var. latifolia and Pinus contorta var. murrayana. Despite the fact that A. americanum was divided into three genetic races, hosts were divided into only two genetic groups: (i) Pinus banksiana and hybrids, and (ii) P. contorta var. latifolia and var. murrayana. These findings suggest that factors such as geographical isolation and adaptation to different environmental conditions are important for race formation in the absence of host-driven selection pressures. To assess factors impacting population structure at the fine-scale, genetic and geographical distance matrices of host and parasite were compared within A. americanum races. The lack of a relationship between genetic and geographical distance matrices suggests that isolation-by-distance plays a negligible role at this level. The effect of geographical isolation may have been diminished because of the influence of factors such as random seed dispersal by animal vectors or adaptation to nongeographically patterned environmental conditions. Host-parasite interactions might also have impacted the fine-scale structure of A. americanum because the parasite and host were found to have similar patterns of gene flow.     

Fire and site type effects on the long-term carbon and nitrogen balance in pristine Siberian Scots pine forests

Effects of fire and site type on carbon (C) and nitrogen (N) balances were determined by following the change of total and component C and N pools along four chronosequences of fire-prone Siberian Scots pine ecosystems. These differed in the mean return interval of surface fires (unburned – moderately burned, 40 years – heavily burned, 25 years) and site quality (lichen versus Vaccinium site type). Of the Vaccinium site type (higher site quality) only a moderately burned chronosequence was studied. A total of 22 even-aged stands were investigated with stand ages ranging from 2 to 383 years. The C balance was dominated by the opposing dynamics of coarse woody debris (CWD) and biomass and could be divided into three phases: (1) Young stands (up to 40 years)acted as a net source for C of 6-10 mol C m-2 year-1 because the previous generation CWD pool originating from stand-replacing crown fires decayed much faster than biomass increased. During this period the C pool in the unburned lichen type chronosequence decreased from 807 to 480 mol C m-2. (2) Middle aged stands (40-100 years) being in a stage of maximum biomass accumulation were a net sink of 8-10 mol C m-2 year-1. (3)Maturestands (100 to > 350 years) continued to sequester C at a lower rate (0.8-2.5mol C m-2 year-1). Differences in the rates of C sequestration during the two later phases could be explained by the complex interaction between surface fire regime and site type. Recurrent surface fires resulted in enhanced mortality and regularly redistributed C from the living to the CWD pool thereby lowering the rate of C sequestration. Site quality determined the potential to recover from disturbance by fire events. Differences in site type did not correlate with soil and total ecosystem N pool size. However, the N status of needles as well as the N pool of physiologically active tissue was highest in the stands of the Vaccinium type. The woody C pool (biomass + CWD) was sensitive to differences in surface fire regime and site type. It was lowest in the heavily burned lichen type chronosequence (297 ± 108 mol C m-2), intermediate in the unburned and moderately burned lichen type chronosequence (571 ± 179 mol C m-2) and highest in the moderately burned Vaccinium type chronosequence (810 ± 334 mol C m-2). In contrast, the total soil C pool (organic plus mineral layer down to a depth of 25 cm) was independent of stand age, surface fire regimeand site type and fluctuated around a value of 250 mol C m-2. The organic layer C pool oscillated in response to recurring surface fires and its C pool was dependent on time since fire increasing at a rate of about 1.5 mol C m-2 year-during the first 40 years and then reaching a plateau of 170 mol C m-2. The total ecosystem N pool was 7.4 ± 1.5 mol N m-2 on average of which only 25 % were stored in biomass or coarse woody debris. Total ecosystem N was independent of stand age, surface fire regime and site type. No correlation was found between total ecosystem C and N pools. Average total ecosystem C:N ratio was 114 ± 35 mol C mol N-1. A conceptual model illustrating how changes in the regime of stand-replacing crown fires and recurrent surface fires and changes in site quality interact in determining the long-term C balance in Siberian Scots pine forests is presented.