Climate of origin shapes variations in wood anatomical properties of 17 Picea species.

BACKGROUND: Variations in hydraulic conductivity may arise from species-specific differences in the anatomical structure and function of the xylem, reflecting a spectrum of plant strategies along a slow-fast resource economy continuum. Spruce (Picea spp.), a widely distributed and highly adaptable tree species, is crucial in preventing soil erosion and enabling climate regulation. However, a comprehensive understanding of the variability in anatomical traits of stems and their underlying drivers in the Picea genus is currently lacking especially in a common garden. RESULTS: We assessed 19 stem economic properties and hydraulic characteristics of 17 Picea species grown in a common garden in Tianshui, Gansu Province, China. Significant interspecific differences in growth and anatomical characteristics were observed among the species. Specifically, xylem hydraulic conductivity (Ks) and hydraulic diameter exhibited a significant negative correlation with the thickness to span ratio (TSR), cell wall ratio, and tracheid density and a significant positive correlation with fiber length, and size of the radial tracheid. PCA revealed that the first two axes accounted for 64.40% of the variance, with PC1 reflecting the trade-off between hydraulic efficiency and mechanical support and PC2 representing the trade-off between high embolism resistance and strong pit flexibility. Regression analysis and structural equation modelling further confirmed that tracheid size positively influenced Ks, whereas the traits DWT, D_r, and TSR have influenced Ks indirectly. All traits failed to show significant phylogenetic associations. Pearson's correlation analysis demonstrated strong correlations between most traits and longitude, with the notable influence of the mean temperature during the driest quarter, annual precipitation, precipitation during the wettest quarter, and aridity index. CONCLUSIONS: Our results showed that xylem anatomical traits demonstrated considerable variability across phylogenies, consistent with the pattern of parallel sympatric radiation evolution and global diversity in spruce. By integrating the anatomical structure of the stem xylem as well as environmental factors of origin and evolutionary relationships, our findings provide novel insights into the ecological adaptations of the Picea genus.

Cutting tree rings into time slices: how intra-annual dynamics of wood formation help decipher the space-for-time conversion.

Tree-ring anatomy, microdensity and isotope records provide valuable intra-annual information. However, extracting signals at that scale is challenged by the complexity of xylogenesis, where two major processes - cell enlargement and wall thickening - occur at different times and rates. We characterized the space-for-time association in the tree rings of three conifer species by examining the duration, overlapping, inter-tree synchronicity and interannual stability during cell enlargement and wall thickening across regular tree-ring sectors (portions of equal tangential width). The number of cells and cell differentiation rates determined the duration of sector formation, which augmented more rapidly throughout the ring for wall thickening than for enlargement. Increasing the number of sectors above c. 15 had a limited effect on improving time resolution because consecutive sector formation overlapped greatly in time, especially in narrow rings and during wall thickening. Increasing the number of sectors also resulted in lower synchronicity and stability of intermediate-sector enlargement, whereas all sectors showed high synchronicity and stability during wall thickening. Increasing the number of sectors had a stronger effect on enhancing time-series resolution for enlargement- than for wall-thickening-related traits, which would nevertheless produce more reliable intra-annual chronologies as a result of the more similar calendars across trees and years in wall thickening.

Cavitation fatigue in conifers: a study on eight European species.

After drought-induced embolism and repair, tree xylem may be weakened against future drought events (cavitation fatigue). As there are few data on cavitation fatigue in conifers available, we quantified vulnerability curves (VCs) after embolism/repair cycles on eight European conifer species. We induced 50% and 100% loss of conductivity (LC) with a cavitron, and analyzed VCs. Embolism repair was obtained by vacuum infiltration. All species demonstrated complete embolism repair and a lack of any cavitation fatigue after 50% LC . After 100% LC, European larch (Larix decidua), stone pine (Pinus cembra), Norway spruce (Picea abies), and silver fir (Abies alba) remained unaffected, while mountain pine (Pinus mugo), yew (Taxus baccata), and common juniper (Juniperus communis) exhibited 0.4-0.9 MPa higher vulnerability to embolism. A small cavitation fatigue observed in Scots pine (Pinus sylvestris) was probably biased by incomplete embolism repair, as indicated by a correlation of vulnerability shifts and conductivity restoration. Our data demonstrate that cavitation fatigue in conifers is species-specific and depends on the intensity of preceding LC. The lack of fatigue effects after moderate LC, and relevant effects in only three species after high LC, indicate that conifers are relatively resistant against cavitation fatigue. This is remarkable considering the complex and delicate conifer pit architecture and may be important considering climate change projections.

Cryogenic sequenced layering for the 3D reconstruction of biological objects.

Three-dimensional (3D) visualization is applied throughout many specialities, prompting an important breakthrough in accessibility and modeling of data. Experimental rendering and computerized reconstruction of objects has influenced many scientific achievements, facilitating one of the greatest advancements in medical education since the first illustrated anatomy book changed specialist training forever. Modern medicine relies on detailed, high quality virtual models for educational, experimental and clinical purposes. Almost all current virtual visualization methods rely on object slicing producing serial sections, which can then be digitalized or analyzed manually. The tendency to computerize serial sections roots from convenience, accessibility, decent visualization quality and automation capabilities. Drawbacks of serial section imaging is tissue damage occurring within each consequent sectioning. To utilize the important aspects of real-life object reconstruction, and maintain integrity of biological structures, we suggest a novel method of low-temperature layering of objects for digitization and computerized virtual reconstruction. Here we show the process of consequent imaging of each novel layer of a biological object, which provides a computer with high quality data for virtual reconstruction and creation of a multidimensional real-life model. Our method prevents tissue deformation and biodegradation due to specific methods used in preparation of the biological object. The resulting images can be applied in surgical training, medical education and numerous scientific fields for realistic reconstruction of biological objects.

Seasonal movement of chloroplasts in mesophyll cells of two Picea species.

Cold acclimation in evergreen conifers of temperate zone is associated with seasonal structural changes of mesophyll cells. Photoprotective reactions include the movement of chloroplasts from summer position when they are located along the cell walls to winter arrangement with their aggregation in one part of the cell. Special spatial arrangement of mesophyll in Picea species with chloroplasts located along the two opposite cell walls causes the very specific pattern of chloroplast movement. To reveal the intracellular apparatus involved in the seasonal organelle position changes, 3D reconstruction of mesophyll cell structure was applied. Two Picea species, P. obovata and P. pungens, from two geographic regions were studied in a 3-year course. The involvement of small transparent vacuoles in the development of cytoplasmic strands penetrating through the central vacuole and connecting two opposite lateral sides of the cell was shown. The nucleus and cytoplasmic organelles including chloroplasts move inwards the strand forming the cytoplasmic conglomerate enclosed by the vacuole at the cell center. Two Picea species have distinct differences in spatial organization of winter mesophyll cells and in structural events leading to its formation. Analysis of Picea species from two geographic regions over 3 years of monitoring reveals dependence of seasonal organelle movement on the dynamics of temperature decline in autumn.

Phylogenomics disentangles the evolutionary history of spruces (Picea) in the Qinghai-Tibetan Plateau: Implications for the design of population genetic studies and species delimitation of conifers.

A laborious and difficult task in current tree of life reconstruction is to resolve evolutionary relationships of closely related congeneric species that originated from recent radiations. This is particularly difficult for forest species with long generation times and large effective population sizes such as conifers. The Qinghai-Tibetan Plateau (QTP) and adjacent areas are considered a species diversity center of Picea, harboring 11 species (including 5 varieties) of this genus, but evolutionary relationships of these species are far from being resolved due to recent radiations, morphological convergence, and frequent interspecific gene flow. In this study, we use these spruce species to test whether phylotranscriptomic analysis, combined with population genetic analysis, can disentangle their evolutionary relationships, and to explore whether reticulate evolution has occurred among them. Phylogenomic analyses indicate that all spruce species in the QTP and neighboring areas, except P. asperata and P. crassifolia, cluster together, and in particular, nearly all taxa (including varieties) reflect reciprocally monophyletic lineages, although the two species P. likiangensis and P. brachytyla are not monophyletic. We found that, compared to herbaceous plants, many more genes (a minimum of 600 OGs for Picea) are required to resolve interspecific relationships of conifers. Contrary to previous studies, our data do not support a hybrid origin of P. purpurea, but suggests a hybrid origin for P. brachytyla var. brachytyla and P. likiangensis var. rubescens. We emphasize that the species or species complex used for population genetic and phylogeographical studies should be monophyletic.

Testing of the narrow crowned Norway spruce ideotype (Picea abies f. pendula) and the hybrids with normal crown form (pyramidalis) in multisite comparative trials.

The study aims to analyse the stability of the narrow crowned Norway spruce (pendula form) compared to the normal spruce form (pyramidalis form) and the hybrids of the two forms, in 5 field trials (Comandau, Lepsa 1&2, Ilva Mica and Voineasa) located in the Romanian Carpathians. Trees height (Th), breast height diameter (Dbh), height growth of the last year, crown diameter (Cd), number of branches per whorl (Nbw) and dominant branch diameter (Dbd) traits were measured and survival rate (Sr) was determined, at 20 years old. Also, branches finesse (Bf), trees volume (Tv) and trees slenderness (Ts) were calculated. In order to compare the wood density (Cwd) there were collected cores. In all trials ANOVA revealed significant (p < 0.05) differences between the two forms of spruce and the hybrids (mainly between those that have a different crown form mother), especially for the stability and quality traits. Factorial ANOVA revealed a high influence (p < 0.001) of the locality and also a significant influence (p < 0.05) of the locality × spruce form interaction. The factor "form" was significant for some traits involved in Norway spruce stability (Ts, Cd, Nbw). The pendula trees present higher values for Sr, Dbh and Tv, and lower values for Ts, Cd, Nbw, Dbd and Bf, compared to pyramidalis spruce form, which showed a higher stability. Heritability was in generally low (h2 < 0.4), with exceptions of Ts which presents a medium rate of heritage. For the same trait, different heritability was registered in different environmental conditions. The Cwd was higher only with 2% for the pendula form in Lepsa trial, while in Comandau trial the pyramidalis registered a higher value (7%). In the new breeding programme, the selection strategy may be pursued with the pendula trees selection based on Ts and branches traits.

Couplings in cell differentiation kinetics mitigate air temperature influence on conifer wood anatomy.

Conifer trees possess a typical anatomical tree-ring structure characterized by a transition from large and thin-walled earlywood tracheids to narrow and thick-walled latewood tracheids. However, little is known on how this characteristic structure is maintained across contrasting environmental conditions, due to its crucial role to ensure sap ascent and mechanical support. In this study, we monitored weekly wood cell formation for up to 7 years in two temperate conifer species (i.e., Picea abies (L.) Karst and Larix decidua Mill.) across an 8°C thermal gradient from 800 to 2,200 m a.s.l. in central Europe to investigate the impact of air temperature on rate and duration of wood cell formation. Results indicated that towards colder sites, forming tracheids compensate a decreased rate of differentiation (cell enlarging and wall thickening) by an extended duration, except for the last cells of the latewood in the wall-thickening phase. This compensation allows conifer trees to mitigate the influence of air temperature on the final tree-ring structure, with important implications for the functioning and resilience of the xylem to varying environmental conditions. The disappearing compensation in the thickening latewood cells might also explain the higher climatic sensitivity usually found in maximum latewood density.

Xylem anatomical adjustments prioritize hydraulic efficiency over safety as Norway spruce trees grow taller.

As a tree grows taller, the increase in gravitational pressure and path length resistance results in lower water potentials at a given flow rate and higher carbon construction costs to transport a given amount of water to the leaves. We investigated how hydraulic safety and efficiency are coordinated under the constraints of higher cavitation risks and higher carbon construction costs with increasing tree height. We combined measurements of xylem tracheid anatomical traits with the vulnerability to drought-induced embolism and hydraulic conductivity of the apical shoots of 2- to 37-m tall Picea abies trees growing at two sites in the Dolomites (Italian Eastern Alps). We found that the theoretical hydraulic conductivity of the apical shoots increased with tree height at both sites (P < 0.001) as a result of an increase in either total tracheid number or mean hydraulic diameter. The xylem water potential inducing 50% loss of apical conductance significantly increased from small (-4.45 ± 0.20 MPa) to tall trees (-3.65 ± 0.03 MPa) (P = 0.007). The more conductive xylem at the treetop of taller trees allows the full compensation for the height-related hydraulic constraints and minimizes the additional carbon costs of transporting water over a longer path length. The corresponding increase in vulnerability to cavitation shows that hydraulic efficiency is prioritized over safety during height growth.

Cell geometry across the ring structure of Sitka spruce.

For wood to be used to its full potential as an engineering material, it is necessary to quantify links between its cell geometry and the properties it exhibits at bulk scale. Doing so will make it possible to predict timber properties crucial to engineering, such as mechanical strength and stiffness, and the resistance to fluid flow, and to inform strategies to improve those properties as required, as well as to measure the effects of interventions such as genetic manipulation and chemical modification. Strength, stiffness and permeability of timber all derive from the geometry of its cells, and yet current practice is to predict them based on properties, such as bulk density, that do not directly describe the cell structure. This work explores links between micro-computed tomography data for structural-size pieces of wood, which show the variation of porosity across the wood's ring structure, and high-resolution tomography showing the geometry of the cells, from which we measure cell length, lumen area, porosity, cell wall thickness and the number density of cells. High-resolution scans, while informative, are time-consuming and expensive to run on a large number of samples at the scale of building components. By scanning the same volume of timber at both low and high resolutions (high-resolution scans over a near-continuous volume of timber of approx. 20 mm3 at 15 µm3 per voxel), we are able to demonstrate correlations between the measurements at the two different resolutions, reveal the physical basis for these correlations, and demonstrate that the data from the low-resolution scan can be used to estimate the variation in (small-scale) cell geometry throughout a structural-size piece of wood.

Why are the seed cones of conifers so diverse at pollination?

Background and Aims: Form and function relationships in plant reproductive structures have long fascinated biologists. Although the intricate associations between specific pollinators and reproductive morphology have been widely explored among animal-pollinated plants, the evolutionary processes underlying the diverse morphologies of wind-pollinated plants remain less well understood. Here we study how this diversity may have arisen by focusing on two conifer species in the pine family that have divergent reproductive cone morphologies at pollination. Methods: Standard histology methods, artificial wind pollination assays and phylogenetic analyses were used in this study. Key Results: A detailed study of cone ontogeny in these species reveals that variation in the rate at which their cone scales mature means that pollination occurs at different stages in their development, and thus in association with different specific morphologies. Pollination experiments nevertheless indicate that both species effectively capture pollen. Conclusions: In wind-pollinated plants, morphological diversity may result from simple variation in development among lineages rather than selective pressures for any major differences in function or performance. This work also illustrates the broader importance of developmental context in understanding plant form and function relationships; because plant reproductive structures perform many different functions over their lifetime, subtle differences in development may dramatically alter the specific morphologies that they use to meet these demands.

Robustness of xylem properties in conifers: analyses of tracheid and pit dimensions along elevational transects.

In alpine regions, tree hydraulics are limited by low temperatures that restrict xylem growth and induce winter frost drought and freezing stress. While several studies have dealt with functional limitations, data on elevational changes in functionally relevant xylem anatomical parameters are still scarce. In wood cores of Pinus cembra L. and Picea abies (L.) Karst. trunks, harvested along five elevational transects, xylem anatomical parameters (tracheid hydraulic diameter dh, wall reinforcement (t/b)2), pit dimensions (pit aperture Da, pit membrane Dm and torus Dt diameters) and respective functional indices (torus overlap O, margo flexibility) were measured. In both species, tracheid diameters decreased and (t/b)2 increased with increasing elevation, while pit dimensions and functional indices remained rather constant (P. cembra: Dt 10.3 ± 0.2 µm, O 0.477 ± 0.005; P. abies: Dt 9.30 ± 0.18 µm, O 0.492 ± 0.005). However, dh increased with tree height following a power trajectory with an exponent of 0.21, and also pit dimensions increased with tree height (exponents: Dm 0.18; Dt 0.14; Da 0.11). Observed elevational trends in xylem structures were predominantly determined by changes in tree size. Tree height-related changes in anatomical traits showed a remarkable robustness, regardless of the distributional ranges of study species. Despite increasing stress intensities towards the timberline, no adjustment in hydraulic safety at the pit level was observed.

Early root growth and architecture of fast- and slow-growing Norway spruce (Picea abies) families differ-potential for functional adaptation.

The relationship between the growth rate of aboveground parts of trees and fine root development is largely unknown. We investigated the early root development of fast- and slow-growing Norway spruce (Picea abies (L.) H. Karst.) families at a developmental stage when the difference in size is not yet observed. Seedling root architecture data, describing root branching, were collected with the WinRHIZO™ image analysis system, and mixed models were used to determine possible differences between the two growth phenotypes. A new approach was used to investigate the spatial extent of root properties along the whole sample root from the base of 1-year-old seedlings to the most distal part of a root. The root architecture of seedlings representing fast-growing phenotypes showed ~30% higher numbers of root branches and tips, which resulted in larger root extensions and potentially a better ability to acquire nutrients. Seedlings of fast-growing phenotypes oriented and allocated root tips and biomass further away from the base of the seedling than those growing slowly, a possible advantage in nutrient-limited and heterogeneous boreal forest soils. We conclude that a higher long-term growth rate of the aboveground parts in Norway spruce may relate to greater allocation of resources to explorative roots that confers a competitive edge during early growth phases in forest ecosystems.

Complex bud architecture and cell-specific chemical patterns enable supercooling of Picea abies bud primordia.

Bud primordia of Picea abies, despite a frozen shoot, stay ice free down to -50 °C by a mechanism termed supercooling whose biophysical and biochemical requirements are poorly understood. Bud architecture was assessed by 3D-reconstruction, supercooling and freezing patterns by infrared video thermography, freeze dehydration and extraorgan freezing by water potential measurements, and cell-specific chemical patterns by Raman microscopy and mass spectrometry imaging. A bowl-like ice barrier tissue insulates primordia from entrance by intrinsic ice. Water repellent and densely packed bud scales prevent extrinsic ice penetration. At -18 °C, break-down of supercooling was triggered by intrinsic ice nucleators whereas the ice barrier remained active. Temperature-dependent freeze dehydration (-0.1 MPa K-1 ) caused accumulation of extraorgan ice masses that by rupture of the shoot, pith tissue are accommodated in large voids. The barrier tissue has exceptionally pectin-rich cell walls and intercellular spaces, and the cell lumina were lined or filled with proteins, especially near the primordium. Primordial cells close to the barrier accumulate di, tri and tetrasaccharides. Bud architecture efficiently prevents ice penetration, but ice nucleators become active inside the primordium below a temperature threshold. Biochemical patterns indicate a complex cellular interplay enabling supercooling and the necessity for cell-specific biochemical analysis.

Cell-type- and tissue-specific transcriptomes of the white spruce (Picea glauca) bark unmask fine-scale spatial patterns of constitutive and induced conifer defense.

Plant defenses often involve specialized cells and tissues. In conifers, specialized cells of the bark are important for defense against insects and pathogens. Using laser microdissection, we characterized the transcriptomes of cortical resin duct cells, phenolic cells and phloem of white spruce (Picea glauca) bark under constitutive and methyl jasmonate (MeJa)-induced conditions, and we compared these transcriptomes with the transcriptome of the bark tissue complex. Overall, ~3700 bark transcripts were differentially expressed in response to MeJa. Approximately 25% of transcripts were expressed in only one cell type, revealing cell specialization at the transcriptome level. MeJa caused cell-type-specific transcriptome responses and changed the overall patterns of cell-type-specific transcript accumulation. Comparison of transcriptomes of the conifer bark tissue complex and specialized cells resolved a masking effect inherent to transcriptome analysis of complex tissues, and showed the actual cell-type-specific transcriptome signatures. Characterization of cell-type-specific transcriptomes is critical to reveal the dynamic patterns of spatial and temporal display of constitutive and induced defense systems in a complex plant tissue or organ. This was demonstrated with the improved resolution of spatially restricted expression of sets of genes of secondary metabolism in the specialized cell types.

Monitoring of Freezing Dynamics in Trees: A Simple Phase Shift Causes Complexity.

During winter, trees have to cope with harsh conditions, including extreme freeze-thaw stress. This study focused on ice nucleation and propagation, related water shifts and xylem cavitation, as well as cell damage and was based on in situ monitoring of xylem (thermocouples) and surface temperatures (infrared imaging), ultrasonic emissions, and dendrometer analysis. Field experiments during late winter on Picea abies growing at the alpine timberline revealed three distinct freezing patterns: (1) from the top of the tree toward the base, (2) from thin branches toward the main stem's top and base, and (3) from the base toward the top. Infrared imaging showed freezing within branches from their base toward distal parts. Such complex freezing causes dynamic and heterogenous patterns in water potential and probably in cavitation. This study highlights the interaction between environmental conditions upon freezing and thawing and demonstrates the enormous complexity of freezing processes in trees. Diameter shrinkage, which indicated water fluxes within the stem, and acoustic emission analysis, which indicated cavitation events near the ice front upon freezing, were both related to minimum temperature and, upon thawing, related to vapor pressure deficit and soil temperature. These complex patterns, emphasizing the common mechanisms between frost and drought stress, shed new light on winter tree physiology.

How does climate influence xylem morphogenesis over the growing season? Insights from long-term intra-ring anatomy in Picea abies.

Background and Aims: During the growing season, the cambium of conifer trees produces successive rows of xylem cells, the tracheids, that sequentially pass through the phases of enlargement and secondary wall thickening before dying and becoming functional. Climate variability can strongly influence the kinetics of morphogenetic processes, eventually affecting tracheid shape and size. This study investigates xylem anatomical structure in the stem of Picea abies to retrospectively infer how, in the long term, climate affects the processes of cell enlargement and wall thickening. Methods: Tracheid anatomical traits related to the phases of enlargement (diameter) and wall thickening (wall thickness) were innovatively inspected at the intra-ring level on 87-year-long tree-ring series in Picea abies trees along a 900 m elevation gradient in the Italian Alps. Anatomical traits in ten successive tree-ring sectors were related to daily temperature and precipitation data using running correlations. Key Results: Close to the altitudinal tree limit, low early-summer temperature negatively affected cell enlargement. At lower elevation, water availability in early summer was positively related to cell diameter. The timing of these relationships shifted forward by about 20 (high elevation) to 40 (low elevation) d from the first to the last tracheids in the ring. Cell wall thickening was affected by climate in a different period in the season. In particular, wall thickness of late-formed tracheids was strongly positively related to August-September temperature at high elevation. Conclusions: Morphogenesis of tracheids sequentially formed in the growing season is influenced by climate conditions in successive periods. The distinct climate impacts on cell enlargement and wall thickening indicate that different morphogenetic mechanisms are responsible for different tracheid traits. Our approach of long-term and high-resolution analysis of xylem anatomy can support and extend short-term xylogenesis observations, and increase our understanding of climate control of tree growth and functioning under different environmental conditions.

Proteomic analysis of stress-related proteins and metabolic pathways in Picea asperata somatic embryos during partial desiccation.

Partial desiccation treatment (PDT) stimulates germination and enhances the conversion of conifer somatic embryos. To better understand the mechanisms underlying the responses of somatic embryos to PDT, we used proteomic and physiological analyses to investigate these responses during PDT in Picea asperata. Comparative proteomic analysis revealed that, during PDT, stress-related proteins were mainly involved in osmosis, endogenous hormones, antioxidative proteins, molecular chaperones and defence-related proteins. Compared with those in cotyledonary embryos before PDT, these stress-related proteins remained at high levels on days 7 (D7) and 14 (D14) of PDT. The proteins that differentially accumulated in the somatic embryos on D7 were mapped to stress and/or stimuli. They may also be involved in the glyoxylate cycle and the chitin metabolic process. The most significant difference in the differentially accumulated proteins occurred in the metabolic pathways of photosynthesis on D14. Furthermore, in accordance with the changes in stress-related proteins, analyses of changes in water content, abscisic acid, indoleacetic acid and H2 O2 levels in the embryos indicated that PDT is involved in water-deficit tolerance and affects endogenous hormones. Our results provide insight into the mechanisms responsible for the transition from morphologically mature to physiologically mature somatic embryos during the PDT process in P. asperata.

In Planta Localization of Stilbenes within Picea abies Phloem.

Phenolic stilbene glucosides (astringin, isorhapontin, and piceid) and their aglycons commonly accumulate in the phloem of Norway spruce (Picea abies). However, current knowledge about the localization and accumulation of stilbenes within plant tissues and cells remains limited. Here, we used an innovative combination of novel microanalytical techniques to evaluate stilbenes in a frozen-hydrated condition (i.e. in planta) and a freeze-dried condition across phloem tissues. Semiquantitative time-of-flight secondary ion-mass spectrometry imaging in planta revealed that stilbenes were localized in axial parenchyma cells. Quantitative gas chromatography analysis showed the highest stilbene content in the middle of collapsed phloem with decreases toward the outer phloem. The same trend was detected for soluble sugar and water contents. The specimen water content may affect stilbene composition; the glucoside-to-aglycon ratio decreased slightly with decreases in water content. Phloem chemistry was correlated with three-dimensional structures of phloem as analyzed by microtomography. The outer phloem was characterized by a high volume of empty parenchyma, reduced ray volume, and a large number of axial parenchyma with porous vacuolar contents. Increasing porosity from the inner to the outer phloem was related to decreasing compactness of stilbenes and possible secondary oxidation or polymerization. Our results indicate that aging-dependent changes in phloem may reduce cell functioning, which affects the capacity of the phloem to store water and sugar, and may reduce the defense potential of stilbenes in the axial parenchyma. Our results highlight the power of using a combination of techniques to evaluate tissue- and cell-level mechanisms involved in plant secondary metabolite formation and metabolism.

Gap expansion in old-growth subarctic forests: the climate-pathogen connection.

We tested the hypothesis considering old-growth subarctic woodlands, free of fire, insect and stand-scale blowdown disturbances, to be at equilibrium with the climate. To do so, we explored the status of Hudsonian woodlands based on the natality/mortality ratio. The gap history of the woodland was reconstructed based on mapping and dating of dead gap-spruces (Picea mariana). Among the 25 gaps studied, 763 dead trees and only 14 saplings were recorded. The center of some gaps remained treeless over the last 1000 yr, and gap area doubled over the last 100 yr. The status of the tree population is in a demographic disequilibrium caused by the small replacement of dead spruces in all of the gaps. Episodes of 'mass' mortality occurred during several decades corresponding to years of favorable tree-ring growth. The natural process of gap-filling appears to be ineffective under current conditions. Good tree-ring growth of dying trees suggests abundant precipitation during the mortality episodes, but precipitation appears to be involved indirectly in the mortality process. The main cause of the widespread tree mortality during the last centuries of gap expansion appears to be biotic in origin. The impact of pathogenic fungal disease linked to late-lying snow cover is proposed for the mortality events.