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Foliar application of GA3 during terminal long-shoot bud development stimulates shoot apical meristem activity in Pinus sylvestris seedlings.

The effect of exogenous gibberellin (GA3) on shoot apical meristem activity in conifer vegetative buds was investigated by spraying 0 or 0.1% GA3 on the foliage of first-year Scots pine (Pinus sylvestris L.) seedlings twice weekly for 9 weeks during development of the terminal long-shoot bud. Exogenous GA3 promoted mitotic activity in the apical zone, thereby increasing both the rate and duration of cataphyll formation and giving rise to a higher and wider apical meristem. The increase in number of cataphylls increased the number of axillary meristems, which developed as short-shoot buds.

Cell Differentiation↗

Correlating gene expression to physiological parameters and environmental conditions during cold acclimation of Pinus sylvestris, identification of molecular markers using cDNA microarrays.

Scots pine (Pinus sylvestris L.) seedlings were grown under different conditions (three field locations, two seasons and two climate room regimes), and then analyzed for freezing tolerance of shoots and roots and for transcript abundance in apical buds based on a cDNA microarray containing about 1500 expressed sequence tags (ESTs) from buds of cold-treated Scots pine seedlings. In a climate room providing long daily photoperiods and high temperatures, seedlings did not develop freezing tolerance, whereas seedlings in a climate room set to provide declining temperatures and day lengths developed moderate freezing tolerance. Control seedlings grown outside under field conditions developed full freezing tolerance. Differences in physiological behavior of the different seedling groups, combined with molecular analysis, allowed identification of a large group of genes, expression of which changed during the development of freezing tolerance. Transcript abundance of several of these genes was highly correlated with freezing tolerance in seedlings differing in provenance, field location or age, making them excellent candidate marker genes for molecular tests for freezing tolerance.

Acclimatization↗

Phosphorus and nitrogen limitations to photosynthesis in Rocky Mountain bristlecone pine (Pinus aristata) in Colorado.

We examined Pinus aristata Engelm. stands in four locations in Colorado: Almagre Mountain, Black Mountain, Goliath Peak and Quartzville. All stands are located at 3200-3700 m and face south-southeast. We measured maximum mass-based assimilation rates (A(max)) and nitrogen (N) and phosphorus (P) foliar concentrations on six foliar age classes, from which instantaneous photosynthetic N- and P-use efficiencies (PNUE and PPUE, respectively) and P:N ratios were estimated. Leaf mass per area (LMA) was also determined for each foliar age class from each site. Foliar age, P and N concentrations, and the P:N ratio explained the most variation in A(max) when data from all sites were combined. Leaf mass per area did not vary with foliar age class. Both P and N limit A(max), although P appears to be more limiting. The critical P:N ratio is approximately 0.12. Results for Black Mountain differed from the other sites, as A(max) was not correlated with age and was negatively correlated with LMA and P. Current findings showed no evidence of N saturation at the Front Range sites (Almagre Mountain and Goliath Peak); however, because P is a limiting nutrient, increased anthropogenic N availability at sites in the Front Range may cause adverse effects on photosynthesis, and perhaps growth, in the future.

Altitude↗

Utilization of polar metabolite profiling in the comparison of juvenile wood and compression wood in loblolly pine (Pinus taeda).

Juvenile wood (JW) of conifers is often associated with compression wood (CW), with which it is sometimes believed to be identical. To determine whether JW and CW can be distinguished metabolically, we compared gas chromatographic profiles of 25 polar metabolites from rooted cuttings of a single loblolly pine (Pinus taeda L.) clone raised in controlled environment chambers and subject to three treatments: (1) grown erect with minimal wind sway (control); (2) swayed by wind from oscillating fans; and (3) with 30-cm growth increments successively bent at an angle of 45 degrees to the vertical. Profiles were compared by principal component analysis. Substantial increases in abundances of coniferin and p-glucocoumaryl alcohol separated immature JW-forming xylem tissues of the control trees from the CW-forming xylem of the bent and swayed trees.

Carbohydrates↗

Anatomical-based defense responses of Scots pine (Pinus sylvestris) stems to two fungal pathogens.

We investigated the cellular responses of stem tissues of mature Scots pine (Pinus sylvestris L.) trees to inoculations with two fungal pathogens. The bark beetle vectored fungus, Leptographium wingfieldii Morelet, induced longer lesions in the bark, stronger swelling of polyphenolic parenchyma cells, more polyphenol accumulation and increased ray parenchyma activity compared with the root rot fungus, Heterobasidion annosum (Fr.) Bref., or mechanical wounding. Axial resin ducts in the xylem are a general feature of the preformed defenses of Scots pine, but there was no clear induction of additional traumatic axial resin ducts in response to wounding or fungal infection. The anatomical responses of Scots pine to pathogen infection were localized to the infection site and were attenuated away from bark lesions. The responses observed in Scots pine were compared with published studies on Norway spruce (Picea abies (L.) Karst.) for which anatomically based defense responses have been well characterized.

Ascomycota↗

Lignification in cell cultures of Pinus radiata: activities of enzymes and lignin topochemistry.

Enzymatic and topochemical aspects of lignification were studied in a Pinus radiata D. Don cell culture system that was induced to differentiate tracheary elements and sclereids with lignified secondary cell walls. The activities of the lignin-related enzymes phenylalanine ammonia lyase (PAL; EC 4.3.1.5) and cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) increased concomitantly with cell differentiation, indicating that the increase in enzyme activity was related to lignification of the cell walls and was not induced by stress. This result also indicates that PAL and CAD are suitable markers for tracheary element differentiation in coniferous gymnosperms. To further characterize lignification in this cell culture system, cellular UV-microspectrophotometry and thioacidolysis were employed. Typical UV-absorption spectra of lignin were obtained from the secondary cell walls of the tracheary elements and sclereids and from the compound middle lamella connecting differentiated cells, and the presence of lignin was confirmed by thioacidolysis. Certain aspects of lignin topochemistry in the cell walls of the tracheary elements were similar to cell walls of P. radiata wood, such as the high lignin concentration in the compound middle lamella connecting adjacent cells and the lower lignin concentration in the secondary cell walls. Therefore, the P. radiata cell culture system appears to be well suited to study the formation of lignified secondary cell walls in coniferous gymnosperms.

Alcohol Oxidoreductases↗

Restoration thinning and influence of tree size and leaf area to sapwood area ratio on water relations of Pinus ponderosa.

Ponderosa pine (Pinus ponderosa Dougl. ex P. Laws) forest stand density has increased significantly over the last century (Covington et al. 1997). To understand the effect of increased intraspecific competition, tree size (height and diameter at breast height (DBH)) and leaf area to sapwood area ratio (A(L):A(S)) on water relations, we compared hydraulic conductance from soil to leaf (kl) and transpiration per unit leaf area (Q(L)) of ponderosa pine trees in an unthinned plot to trees in a thinned plot in the first and second years after thinning in a dense Arizona forest. We calculated kl and Q(L) based on whole- tree sap flux measured with heat dissipation sensors. Thinning increased tree predawn water potential within two weeks of treatment. Effects of thinning on kl and Q(L) depended on DBH, A(L):A(S) and drought severity. During severe drought in the first growing season after thinning, kl and Q(L) of trees with low A(L):A(S) (160-250 mm DBH; 9-11 m height) were lower in the thinned plot than the unthinned plot, suggesting a reduction in stomatal conductance (g(s)) or reduced sapwood specific conductivity (K(S)), or both, in response to thinning. In contrast kl and Q(L) were similar in the thinned plot and unthinned plot for trees with high A(L):A(S) (260-360 mm DBH; 13-16 m height). During non-drought periods, kl and Q(L) were greater in the thinned plot than in the unthinned plot for all but the largest trees. Contrary to previous studies of ponderosa pine, A(L):A(S) was positively correlated with tree height and DBH. Furthermore, kl and Q(L) showed a weak negative correlation with tree height and a strong negative correlation with A(S) and thus A(L):A(S) in both the thinned and unthinned plots, suggesting that trees with high A(L):A(S) had lower g(s). Our results highlight the important influence of stand competitive environment on tree-size-related variation in A(L):A(S) and the roles of A(L):A(S) and drought on whole-tree water relations in response to thinning.

Arizona↗

Changes in freezing tolerance, plasma membrane H+-ATPase activity and fatty acid composition in Pinus resinosa needles during cold acclimation and de-acclimation.

It has previously been suggested that plasma membrane ATPase (PM H+-ATPase, EC 3.6.1.3.) is a site of incipient freezing injury because activity increases following cold acclimation and there are published data indicating that activity of PM H+-ATPase is modulated by changes in lipids associated with the enzyme. To test and extend these findings in a tree species, we analyzed PM H+-ATPase activity and the fatty acid (FA) composition of glycerolipids in purified plasma membranes (PMs) prepared by the two-phase partition method from current-year needles of adult red pine (Pinus resinosa Ait.) trees. Freezing tolerance of the needles decreased from -56 degrees C in March to -9 degrees C in May, and increased from -15 degrees C in September to -148 degrees C in January. Specific activity of vanadate-sensitive PM H+-ATPase increased more than two-fold following cold acclimation, despite a concurrent increase in protein concentration. During de-acclimation, decreases in PM H+-ATPase activity and freezing tolerance were accompanied by decreases in the proportions of oleic (18:1) and linoleic (18:2) acids and increases in the proportions of palmitic (16:0) and linolenic (18:3) acids in total glycerolipids extracted from the plasma membrane fraction. This pattern of changes in PM H+-ATPase activity and the 18:1, 18:2 and 18:3 fatty acids was reversed during cold acclimation. In the PM fractions, changes in FA unsaturation, expressed as the double bond index (1 x 18:1 + 2 x 18:2 + 3 x 18:3), were closely correlated with changes in H+-ATPase specific activity (r2 = 0.995). Changes in freezing tolerance were well correlated with DBI (r2 = 0.877) and ATPase specific activity (r2 = 0.833) in the PM fraction. Total ATPase activity in microsomal fractions also closely followed changes in freezing tolerance (r2 = 0.969). We conclude that, as in herbaceous plants, simultaneous seasonal changes in PM H+-ATPase activity and fatty acid composition occur during cold acclimation and de-acclimation in an extremely winter hardy tree species under natural conditions, lending support to the hypothesis that FA-regulated PM H+-ATPase activity is involved in the cellular response underlying cold acclimation and de-acclimation.

Acclimatization↗

Ectomycorrhizal fungi affect the physiological responses of Picea glauca and Pinus banksiana seedlings exposed to an NaCl gradient.

We tested the effects of ectomycorrhizal (ECM) inoculation on greenhouse-grown white spruce (Picea glauca (Moench) Voss) and jack pine (Pinus banksiana L.) seedlings to be used for revegetation of salt-affected tailing sands resulting from the exploitation of oil sand in northeastern Alberta, Canada. White spruce and jack pine seedlings were inoculated with three ECM fungi selected for their in vitro tolerance to excess Na+ and Cl-: Hebeloma crustuliniforme (Bull) Quel. UAMH 5247, Laccaria bicolor Maire (Orton) UAMH 8232 and a Suillus tomentosus (Kauff.) Sing., Snell and Dick isolate from a salt-affected site. The physiological responses of the seedlings to a gradient of NaCl concentration (0, 50, 100 and 200 mM) were assessed over four weeks by: (1) Na+ accumulation and allocation; (2) chlorophyll a fluorescence; (3) growth, (4) water content; and (5) organic osmolyte accumulation. Jack pine seedlings were more sensitive than white spruce seedlings to increasing Na+ and Cl- concentrations. Both species showed decreasing biomass accumulation, and increasing concentrations of organic osmotica and Na with increasing NaCl concentration. White spruce seedlings inoculated with the S. tomentosus isolate had the best growth response at all NaCl concentrations tested. Although jack pine seedlings inoculated with the L. bicolor or S. tomentosus isolate exhibited the highest growth in the 50 and 100 mM NaCl treatments, both fungi increased the photochemical stress and dehydration of their hosts in the 200 mM NaCl treatment. At the latter concentration, jack pine seedlings inoculated with H. crustuliniforme showed the greatest tolerance to salt stress. Although the different fungi altered the physiological response of the host in different ways, inoculation with salt-stress-tolerant ECM fungi increased growth and reduced the negative effects of excess NaCl. Use of controlled mycorrhization may increase survival of coniferous seedlings used for revegetation of salt-affected sites.

Dose-Response Relationship, Drug↗

Stomatal constraints may affect emission of oxygenated monoterpenoids from the foliage of Pinus pinea.

Dependence of monoterpenoid emission and fractional composition on stomatal conductance (G(V)) was studied in Mediterranean conifer Pinus pinea, which primarily emits limonene and trans-beta-ocimene but also large fractions of oxygenated monoterpenoids linalool and 1,8-cineole. Strong decreases in G(V) attributable to diurnal water stress were accompanied by a significant reduction in total monoterpenoid emission rate in midday. However, various monoterpenoids responded differently to the reduction in G(V), with the emission rates of limonene and trans-beta-ocimene being unaffected but those of linalool and 1,8-cineole closely following diurnal variability in G(V). A dynamic emission model indicated that stomatal sensitivity of emissions was associated with monoterpenoid Henry's law constant (H, gas/liquid phase partition coefficient). Monoterpenoids with a large H such as trans-beta-ocimene sustain higher intercellular partial pressure for a certain liquid phase concentration, and stomatal closure is balanced by a nearly immediate increase in monoterpene diffusion gradient from intercellular air-space to ambient air. The partial pressure rises also in compounds with a low H, but more than 1,000-fold higher liquid phase concentrations of linalool and 1,8-cineole are necessary to increase intercellular partial pressure high enough to balance stomatal closure. The system response is accordingly slower, and the emission rates may be transiently suppressed by low G(V). Simulations further suggested that linalool and 1,8-cineole synthesis rates also decreased with decreasing G(V), possibly as the result of selective inhibition of various monoterpene synthases by stomata. We conclude that physicochemical characteristics of volatiles not only affect total emission but also alter the fractional composition of emitted monoterpenoids.

Acyclic Monoterpenes↗

Evaluation of major genetic loci contributing to inbreeding depression for survival and early growth in a selfed family of Pinus taeda.

The magnitude of fitness effects at genetic loci causing inbreeding depression at various life stages has been an important question in plant evolution. We used genetic mapping in a selfed family of loblolly pine (Pinus taeda L.) to gain insights on inbreeding depression for early growth and viability. Two quantitative trait loci (QTLs) were identified that explain much of the phenotypic variation in height growth through age 3 and may account for more than 13% inbreeding depression in this family. One of these QTLs maps to the location of cad-nl, a lignin biosynthesis mutation. Both QTLs show evidence of overdominance, although evidence for true versus pseudo-overdominance is inconclusive. Evidence of directional dominance for height growth was noted throughout the genome, suggesting that additional loci may contribute to inbreeding depression. A chlorophyll-deficiency mutation, spf did not appear to be associated with growth effects, but had significant effects on survival through age 3. Previously identified embryonic viability loci had little or no overall effect on germination, survival, or growth. Our results challenge, at least in part, the prevailing hypothesis that inbreeding depression for growth is due to alleles of small effect. However, our data support predictions that loci affecting inbreeding depression are largely stage specific.

Biological Evolution↗

Evolution of genome size in pines (Pinus) and its life-history correlates: supertree analyses.

Genome size has been suggested to be a fundamental biological attribute in determining life-history traits in many groups of organisms. We examined the relationships between pine genome sizes and pine phylogeny, environmental factors (latitude, elevation, annual rainfall), and biological traits (latitudinal and elevational ranges, seed mass, minimum generation time, interval between large seed crops, seed dispersal mode, relative growth rate, measures of potential and actual invasiveness, and level of rarity). Genome sizes were determined for 60 pine taxa and then combined with published values to make a dataset encompassing 85 species, or 70% of species in the genus. Supertrees were constructed using 20 published source phylogenies. Ancestral genome size was estimated as 32 pg. Genome size has apparently remained stable or increased over evolutionary time in subgenus Strobus, while it has decreased in most subsections in subgenus Pinus. We analyzed relationships between genome size and life-history variables using cross-species correlations and phylogenetically independent contrasts derived from supertree constructions. The generally assumed positive relation between genome size and minimum generation time could not be confirmed in phylogenetically controlled analyses. We found that the strongest correlation was between genome size and seed mass. Because the growth quantities specific leaf area and leaf area ratio (and to a lesser extent relative growth rate) are strongly negatively related to seed mass, they were also negatively correlated with genome size. Northern latitudinal limit was negatively correlated with genome size. Invasiveness, particularly of wind-dispersed species, was negatively associated with both genome size and seed mass. Seed mass and its relationships with seed number, dispersal mode, and growth rate contribute greatly to the differences in life-history strategies of pines. Many life-history patterns are therefore indirectly, but consistently, associated with genome size.

Analysis of Variance↗

Genetic variation in piñon pine, Pinus edulis, associated with summer precipitation.

Three previous reports of microgeographical variation of glycerate dehydrogenase (Gly) frequencies in piñon, Pinus edulis, established the hypothesis that Gly frequencies contribute to adaptation to heterogeneous environments, specifically to variation in soil moisture. In each of these studies, the frequency of the Gly-3 allele or of Gly-33 homozygotes was higher on dry sites than on nearby moist sites. Here we attempt to extend these observations by testing the hypothesis that Gly frequencies respond to soil moisture variation on a range-wide scale. Gly frequencies were surveyed in 11 natural populations, and the frequency of the Gly-3 allele varied from 0.27 to 0.65 among the sample sites. Elevation varied from 1650 to 3100 m, and summer precipitation, defined as precipitation from April to August, varied from 13.7 to 26.4 cm. The soil types at the collection sites were schist, quaternary volcanic or a mixture of shale and sandstone. Logistic regression revealed that Gly frequencies did not respond to either elevation or soil type, but were related to summer precipitation (P < 0.01). The correlation between summer precipitation and the frequency of the Gly-3 allele was r = -0.92 (P < 0.001). Thus, the patterns of differentiation on microgeographical scales are consistent with greater differentiation on a range-wide scale.

Carbohydrate Dehydrogenases↗

Increased selfing and correlated paternity in a small population of a predominantly outcrossing conifer, Pinus sylvestris.

Outcrossing rate, the rates of ovule and seed abortion, and levels of correlated paternity were estimated in a small population of Pinus sylvestris, a predominantly outcrossing conifer, and were compared with estimates from two widely dispersed woodlands of the same species, showing a range of densities. On average, seed trees of the small population showed an eight-fold higher selfing rate (25 vs. 3%) and a 100-fold greater incidence of correlated paternity (19.6 vs. 0.2%) than did trees from the large populations. No evidence was found of pollen limitation within the remnant stand, as suggested by ovule abortion rates. Investigation of the mating patterns in the small population, based on the unambiguous genealogy of 778 open-pollinated seeds, showed a large departure from random mating. Only 8% of the possible mating pairs within the stand were observed. Correlated paternity rate within a maternal sibship was negatively associated (rs = -0.398, P < 0.050) with the distance to the nearest neighbour, and shared paternity among maternal sibships was negatively correlated (rs = -0.704, P < 0.001) with the distance between seed trees. Numerical simulations, based on the estimated individual pollen dispersal kernel, suggest that restricted dispersal might have been the key factor affecting mating patterns in the small population and, together with low population density, may account for the observed mating system variation between the small and the large populations. The results of this study show that a severe size reduction may substantially affect the mating system of a wind-pollinated, typically outcrossed plant species.

Computer Simulation↗

Inferring colonization history from analyses of spatial genetic structure within populations of Pinus strobus and Quercus rubra.

Many factors interact to determine genetic structure within populations including adult density, the mating system, colonization history, natural selection, and the mechanism and spatial patterns of gene dispersal. We examined spatial genetic structure within colonizing populations of Quercus rubra seedlings and Pinus strobus juveniles and adults in an aspen-white pine forest in northern Michigan, USA. A 20-year spatially explicit demographic study of the forest enables us to interpret the results in light of recent colonization of the site for both species. We assayed 217 Q. rubra seedlings and 171 P. strobus individuals at 11 polymorphic loci using nine allozyme systems. Plant genotypes and locations were used in an analysis of spatial genetic structure. Q. rubra and P. strobus showed similar observed levels of heterozygosity, but Q. rubra seedlings have less heterozygosity than expected. Q. rubra seedlings show spatial genetic clumping of individuals on a scale to 25 m and levels of genetic relatedness expected from the clumped dispersion of half-siblings. In contrast, P. strobus has low levels of genetic relatedness at the smallest distance class and positive spatial genetic structure at scales < 10 m within the plot. The low density of adult Q. rubra outside the study plot and limited, spatially clumped rodent dispersal of acorns is likely responsible for the observed pattern of spatial genetic structure and the observed heterozygote deficit (i.e. a Wahlund effect). We attribute weaker patterns observed in P. strobus to the longer dispersal distance of seeds and the historical overlap of seed shadows from adults outside of the plot coupled with the overlap of seed shadows from younger, more recently established reproductive adults. The study demonstrates the utility of long-term demographic data in interpreting mechanisms responsible for generating contemporary patterns of genetic structure within populations.

Genetic Variation↗

Contrasting phylogeographical patterns between mainland and island taxa of the Pinus luchuensis complex.

Species whose geographical distribution encompasses both mainland and island populations provide an ideal system for examining isolation and genetic divergence. In this study, paternally transmitted chloroplast DNA (cpDNA) and maternally transmitted mitochondrial DNA (mtDNA) were used to estimate population structure and phylogeography of Pinus luchuensis, a species found in eastern China (ssp. hwangshanensis), Taiwan (ssp. taiwanensis), and the Ryukyu Archipelago (ssp. luchuensis). Gene genealogies of both mtDNA and cpDNA reveal two major lineages. Molecular dating indicates that these lineages diverged before the colonization of P. luchuensis subspecies in Taiwan and the Ryukyu Archipelago. Both mtDNA and cpDNA show a lack of correspondence between molecular phylogeny and subspecies designation. Phylogeographical analysis suggests that paraphyly of the subspecies is the result of recent divergence rather than secondary contacts. In spite of the short divergence history of P. luchuensis on islands, the island populations show the same degree of genetic divergence as mainland populations. Low levels of genetic diversity in the mainland ssp. hwangshanensis suggest demographic bottlenecks. In contrast, the high heterogeneity of genetic composition for island populations is likely to be associated with a history of multiple colonization from the mainland. The spatial apportionment of organelle DNA polymorphisms is consistent with a pattern of stepwise colonization on island populations.

China↗

Analysis of spatial genetic structure in an expanding Pinus halepensis population reveals development of fine-scale genetic clustering over time.

We analysed the change of spatial genetic structure (SGS) of reproductive individuals over time in an expanding Pinus halepensis population. To our knowledge, this is the first empirical study to analyse the temporal component of SGS by following the dynamics of successive cohorts of the same population over time, rather than analysing different age cohorts at a single time. SGS is influenced by various factors including restricted gene dispersal, microenvironmental selection, mating patterns and the spatial pattern of reproductive individuals. Several factors that affect SGS are expected to vary over time and as adult density increases. Using air photo analysis, tree-ring dating and molecular marker analysis we reconstructed the spread of reproductive individuals over 30 years beginning from five initial individuals. In the early stages, genotypes were distributed randomly in space. Over time and with increasing density, fine-scale (< 20 m) SGS developed and the magnitude of genetic clustering increased. The SGS was strongly affected by the initial spatial distribution and genetic variation of the founding individuals. The development of SGS may be explained by fine-scale environmental heterogeneity and possibly microenvironmental selection. Inbreeding and variation in reproductive success may have enhanced SGS magnitude over time.

Biomarkers↗

Effective gene dispersal and female reproductive success in Mediterranean maritime pine (Pinus pinaster Aiton).

Understanding population-scale processes that affect allele frequency changes across generations is a long-standing interest in genetic, ecological and evolutionary research. In particular, individual differences in female reproductive success and the spatial scale of gene flow considerably affect evolutionary change and patterns of local selection. In this study, a recently developed maximum-likelihood (ML) method based on established offspring, the Seedling Neighbourhood Model, was applied and exponentially shaped dispersal kernels were fitted to both genetic and ecological data in a widespread Mediterranean pine, Pinus pinaster Aiton. The distribution of female reproductive success in P. pinaster was very skewed (about 10% of trees mothered 50% of offspring) and significant positive female selection gradients for diameter (gamma = 0.7293) and cone crop (gamma = 0.4524) were found. The selective advantage of offspring mothered by bigger trees could be due to better-quality seeds. These seeds may show more resilience to severe summer droughts and microsite variation related to water and nutrient availability. Both approaches, ecological and of parentage, consistently showed a long-distance dispersal component in saplings that was not found in dispersal kernels based on seed shadows, highlighting the importance of Janzen-Connell effects and microenvironmental variation for survival at early stages of establishment in this Mediterranean key forest tree.

Gene Flow↗