An open-source machine-learning approach for obtaining high-quality quantitative wood anatomy data from E. grandis and P. radiata xylem.

Quantitative wood anatomy is a subfield in dendrochronology that requires effective open-source image analysis tools. In this research, the bioimage analysis software QuPath (v0.4.4) is introduced as a candidate for accurately quantifying the cellular properties of the xylem in an automated manner. Additionally, the potential of QuPath to detect the transition of early- to latewood tracheids over the growing season was evaluated to assess a potential application in dendroecological studies. Various algorithms in QuPath were optimized to quantify different xylem cell types in Eucalyptus grandis and the transition of early- to latewood tracheids in Pinus radiata. These algorithms were coded into cell detection scripts for automatic quantification of stem microsections and compared to a manually curated method to assess the accuracy of the cell detections. The automatic cell detection approach, using QuPath, has been validated to be reproducible with an acceptable error when assessing fibers, vessels, early- and latewood tracheids. However, further optimization for parenchyma is still required. This proposed method developed in QuPath provides a scalable and accurate approach for quantifying anatomical features in stem microsections. With minor amendments to the detection and classification algorithms, this strategy is likely to be viable in other plant species.

Impact of intra-annual wood density fluctuation on tree hydraulic function: insights from a continuous monitoring approach.

Climate change significantly impacts global forests, leading to tree decline and dieback. To cope with climate change, trees develop several functional traits, such as intra-annual density fluctuations (IADFs) in tree rings. The formation of these traits facilitates trees to optimize resource allocation, allowing them to withstand periods of stress and eventually recover when the conditions become favourable again. This study focuses on a Pinus pinaster Aiton forest in a warm, drought-prone Mediterranean area, comparing two growing seasons with different weather patterns. The innovative continuous monitoring approach used in this study combines high-resolution monitoring of sap flow (SF), analysis of xylogenesis and quantitative wood anatomy. Our results revealed the high plasticity of P. pinaster in water use and wood formation, shedding light on the link between IADFs and tree conductance. Indeed, the capacity to form large cells in autumn (as IADFs) improves the total xylem hydraulic conductivity of this species. For the first time, a continuous SF measurement system captured the dynamics of bimodal SF during the 2022 growing season in conjunction with the bimodal growth pattern observed through xylogenesis monitoring. These results highlight the intricate interplay between environmental conditions, water use, wood formation and tree physiology, providing valuable insights into the acclimation mechanisms employed by P. pinaster to cope with weather fluctuations.

Formation of xylem tissues and secondary cell walls is diminished by severe and consecutive insect defoliation.

PREMISE: Insect defoliation of trees causes unusual changes to wood anatomy and slows radial growth that decreases tree value; however, the characteristics of these anatomical changes in hardwoods remain unclear. The aim of this study was to characterize the anatomy and histochemistry of the wood in trunks of Betula maximowicziana trees after severe insect defoliation. METHODS: Secondary xylem tissues were sampled from trunks that had been defoliated by Caligula japonica at Naie and Furano in central Hokkaido during 2006-2012, then cross-dated and examined microscopically and stained histochemically to characterize anatomical and chemical changes in the cells. RESULTS: White rings with thin-walled wood fibers and greatly reduced annual ring width in the subsequent year were observed in samples from both sites. From these results, the year that the white rings formed was determined, and severe defoliation was confirmed to trigger white ring formation. The characteristics may prove useful to detect the formation year of white rings. Scanning electron microscopy and histochemical analyses of the white rings indicated that the thickness of the S2 layer in the wall of wood fiber cells decreased, but xylan and lignin were still deposited in the cell walls of wood fibers. However, the walls of the fibers rethickened after the defoliation. CONCLUSIONS: Our results suggest that B. maximowicziana responds to a temporary lack of carbon inputs due to insect defoliation by regulating the thickness of the S2 layer of the cell wall of wood fibers. For B. maximowicziana, insect defoliation late in the growing season has serious deleterious effects on wood formation and radial growth.

Toward a better understanding of angiosperm xylogenesis: a new method for a cellular approach.

The kinetics of wood formation in angiosperms are largely unknown because their complex xylem anatomy precludes using the radial position of vessels and fibers to infer their time of differentiation. We analyzed xylogenesis in ring-porous ash (Fraxinus angustifolia) and diffuse-porous beech (Fagus sylvatica) over 1 yr and proposed a novel procedure to assess the period of vessel and fiber enlargement using a referential radial file (RRF). Our approach captured the dynamics of wood formation and provided a robust estimation of the kinetics of vessel and fiber enlargement. In beech, fibers and vessels had a similar duration of enlargement, decreasing from 14 to 5 d between April and July. In ash, wide vessels formed in April enlarged at a rate of 27 × 103 µm2 d-1 , requiring half the time of contemporary fibers (6 vs 12 d), and less time than the narrower vessels (14 d) formed in May. These findings reveal distinct cell-type-dependent mechanisms for differentiation in diffuse-porous and ring-porous trees, enhancing our understanding of angiosperm wood cell kinetics. Our approach presents an effective method for investigating angiosperm wood formation and provides a more accurate representation of vessel and fiber morphogenesis in wood formation models.

Addressing controversies in the xylem embolism resistance-vessel diameter relationship.

Although xylem embolism is a key process during drought-induced tree mortality, its relationship to wood anatomy remains debated. While the functional link between bordered pits and embolism resistance is known, there is no direct, mechanistic explanation for the traditional assumption that wider vessels are more vulnerable than narrow ones. We used data from 20 temperate broad-leaved tree species to study the inter- and intraspecific relationship of water potential at 50% loss of conductivity (P50 ) with hydraulically weighted vessel diameter (Dh ) and tested its link to pit membrane thickness (TPM ) and specific conductivity (Ks ) on species level. Embolism-resistant species had thick pit membranes and narrow vessels. While Dh was weakly associated with TPM , the P50 -Dh relationship remained highly significant after accounting for TPM . The interspecific pattern between P50 and Dh was mirrored by a link between P50 and Ks , but there was no evidence for an intraspecific relationship. Our results provide robust evidence for an interspecific P50 -Dh relationship across our species. As a potential cause for the inconsistencies in published P50 -Dh relationships, our analysis suggests differences in the range of trait values covered, and the level of data aggregation (species, tree or sample level) studied.

A safe breeding ground: genetically improved maritime pine for growth and stem form has more efficient but not more vulnerable xylem.

Through repeated cycles of selection and recombination, tree breeding programs deliver genetically improved varieties for a range of target characteristics such as biomass production, stem form, resistance to biotic stresses, wood properties, etc. However, in the context of increased drought and heat waves, it is not yet known whether growth performance will impede drought resistance. To address this question, we compared the hydraulic properties, such as hydraulic efficiency and hydraulic safety, in four varieties over successive varieties of genetically improved maritime pines (i.e., Pinus pinaster Aït.) for growth and stem form. We measured 22 functional traits related to hydraulic efficiency, hydraulic safety, xylem anatomy and wood density. We found that improved varieties presented higher hydraulic conductivity with larger tracheid lumen size and tracheid lumen fraction, and smaller wall thickness reinforcement and tracheid density, but not at the cost of reduced embolism resistance. The reported absence of trade-off between hydraulic conductivity and embolism resistance is a strong asset to improve biomass productivity, through increased hydraulic efficiency, without impacting drought resistance, and should enable new maritime pine varieties to cope with a drier climate. Our study is one of the first to reveal the hydraulic mechanisms over successive varieties of genetic improvement for tree growth. It provides guidelines for sustainable forest management through breeding for other forest tree species.

Tree-ring anatomy of Pinus cembra trees opens new avenues for climate reconstructions in the European Alps.

Tree rings form the backbone of high-resolution palaeoclimatology and represent one of the most frequently used proxy to reconstruct climate variability of the Common Era. In the European Alps, reconstructions were often based on tree-ring width (TRW) and maximum latewood density (MXD) series, with a focus on European larch. By contrast, only a very limited number of dendroclimatic studies exists for long-lived, multi-centennial Pinus cembra, despite the widespread occurrence of the species at treeline sites across the European Alps. This lack of reconstructions can be ascribed to the difficulties encountered in past studies in extracting a robust climate signal from TRW and MXD chronologies. In this study, we tested various wood anatomical parameters from P. cembra as proxies for the reconstruction of past air temperatures. To this end, we measured anatomical cell parameters and TRW of old-growth trees from the God da Tamangur forest stand, known for being the highest pure, and continuous P. cembra forest in Europe. We demonstrate that several wood anatomical parameters allow robust reconstruction of past temperature variability at annual to multidecadal timescales. Best results are obtained with maximum latewood radial cell wall thickness (CWTrad) measured at 40 µm radial band width. Over the 1920-2017 period, the CWTrad chronology explains 62 % and >80 % of interannual and decadal variability of air temperatures during a time window corresponding roughly with the growing season. These values exceed those found in past work on P. cembra and even exceed the values reported for MXD chronologies built with L. decidua and hitherto considered the gold standard for dendroclimatic reconstructions in the European Alps. The wood anatomical analysis of P. cembra records therefore unveils a dormant potential and opens new avenues for a species that has been considered unsuitable for climate reconstructions so far.

The vessel wall thickness-vessel diameter relationship across woody angiosperms.

PREMISE: Comparative anatomy is necessary to identify the extremes of combinations of functionally relevant structural traits, to ensure that physiological data cover xylem anatomical diversity adequately, and thus achieve a global understanding of xylem structure-function relations. A key trait relationship is that between xylem vessel diameter and wall thickness of both the single vessel and the double vessel+adjacent imperforate tracheary element (ITE). METHODS: We compiled a comparative data set with 1093 samples, 858 species, 350 genera, 86 families, and 33 orders. We used broken linear regression and an algorithm to explore changes in parameter values from linear regressions using subsets of the data set to identify a threshold, at 90-µm vessel diameter, in the wall thickness-diameter relationship. RESULTS: Below 90 µm diameter for vessels, virtually any wall thickness could be associated with virtually any diameter. Below this threshold, selection is free to favor a very wide array of combinations, such as very thick walls and narrow vessels in ITE-free herbs, or very thin-walled, wide vessels in evergreen dryland pioneers. Above 90 µm, there was a moderate positive relationship. CONCLUSIONS: Our analysis shows that the space of vessel wall thickness-diameter combinations is very wide, with selection apparently eliminating individuals with vessel walls "too thin" for their diameter. Most importantly, our survey revealed poorly studied plant hydraulic syndromes (functionally significant trait combinations). These data suggest that the full span of trait combinations, and thus the minimal set of hydraulic syndromes requiring study to span woody plant functional diversity adequately, remains to be documented.

The 2018 hot drought pushed conifer wood formation to the limit of its plasticity: Consequences for woody biomass production and tree ring structure.

Hot droughts are expected to increase in Europe and disturb forest ecosystem functioning. Wood formation of trees has the potential to adapt to those events by compensatory mechanisms between the rates and durations of tracheid differentiation to form the typical pattern of vital wood anatomical structures. We monitored xylogenesis and measured wood anatomy of mature silver fir (Abies alba Mill.) and Scots pine (Pinus sylvestris L.) trees along an elevational gradient in the Black Forest during the hot drought year of 2018. We assessed the kinetics of tracheid differentiation and the final tracheid dimensions and quantified the relationship between rates and durations of cell differentiation over the growing season. Cell differentiation kinetics were decoupled, and temperature and water availability signals were imprinted in the tree ring structure. The sudden decline in woody biomass production provided evidence for a disruption in carbon sequestration processes due to heat and drought stress. Growth processes of Scots pine (pioneer species) were mainly affected by the spring drought, whereas silver fir (climax species) growth processes were more disturbed by the summer drought. Our study provides novel insights on the plasticity of wood formation and carbon allocation in temperate conifer tree species in response to extreme climatic events.

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.

X-ray computed tomography for non-invasive dendrochronology reveals a concealed double panelling on a painting from Rubens' studio.

Dating the wood from historical art objects is a crucial step to ascertain their production time, and support or refute attribution to an artist or a workshop. Dendrochronology is commonly used for this purpose but requires access to the tree-ring pattern in the wood, which can be hindered by preparatory layers, polychromy, wax, or integrated frames. Here we implemented non-invasive dendrochronology based on X-ray computed tomography (CT) to examine a painting on panel attributed to Rubens' studio and its presumed dating around 1636 CE. The CT images achieved a resolution of 37.3 micron and revealed a double panelling, which was concealed by oak strips covering all four edges. The back (visible) board is made of deciduous oak (Quercus subg. Quercus), the most common type of wood used in 17th-century Netherlandish workshops, and was dated terminus post quem after 1557 CE. However, the front (original) board used for the painting has been identified through examination of the wood anatomy as a tropical wood, probably Swietenia sp., a species seldom used in Netherlandish paintings, and remains undated. Its very presence attests the global character of 17th-century trade, and demonstrates the use of exotic species in Flemish studios. The date of the oak board refutes previous results and suggests that this board was trimmed to meet the size of the tropical one, having been glued to it for conservation purposes or with deceiving intentions to pretend that the painting was made on an oak panel. These revelations have opened new lines of art historical inquiry and highlight the potential of X-ray CT as a powerful tool for non-invasive study of historical art objects to retrieve their full history.

Derived woodiness and annual habit evolved in African umbellifers as alternative solutions for coping with drought.

BACKGROUND: One of the major trends in angiosperm evolution was the shift from woody to herbaceous habit. However, reversals known as derived woodiness have also been reported in numerous, distantly related clades. Among theories evoked to explain the factors promoting the evolution of derived woodiness are moderate climate theory and cavitation theory. The first assumes that woody habit evolves in response to mild climate allowing for prolonged life span, which in turn leads to bigger and woodier bodies. The second sees woodiness as a result of natural selection for higher cavitation resistance in seasonally dry environments. Here, we compare climatic niches of woody and herbaceous, mostly southern African, umbellifers from the Lefebvrea clade to assess whether woody taxa in fact occur in markedly drier habitats. We also calibrate their phylogeny to estimate when derived woodiness evolved. Finally, we describe the wood anatomy of selected woody and herbaceous taxa to see if life forms are linked to any particular wood traits. RESULTS: The evolution of derived woodiness in chamaephytes and phanerophytes as well as the shifts to short-lived annual therophytes in the Lefebvrea clade took place at roughly the same time: in the Late Miocene during a trend of global climate aridification. Climatic niches of woody and herbaceous genera from the Cape Floristic Region overlap. There are only two genera with distinctly different climatic preferences: they are herbaceous and occur outside of the Cape Floristic Region. Therefore, studied herbs have an overall climatic niche wider than their woody cousins. Woody and herbaceous species do not differ in qualitative wood anatomy, which is more affected by stem architecture and, probably, reproductive strategy than by habit. CONCLUSIONS: Palaeodrought was likely a stimulus for the evolution of derived woodiness in the Lefebvrea clade, supporting the cavitation theory. The concurrent evolution of short-lived annuals withering before summer exemplifies an alternative solution to the same problem of drought-induced cavitation. Changes of the life form were most likely neither spurred nor precluded by any qualitative wood traits, which in turn are more affected by internode length and probably also reproductive strategy.

Prior secondary cell wall formation is required for gelatinous layer deposition and posture control in gravi-stimulated aspen.

Cell walls, especially secondary cell walls (SCWs), maintain cell shape and reinforce wood, but their structure and shape can be altered in response to gravity. In hardwood trees, tension wood is formed along the upper side of a bending stem and contains wood fiber cells that have a gelatinous layer (G-layer) inside the SCW. In a previous study, we generated nst/snd quadruple-knockout aspens (Populus tremula × Populus tremuloides), in which SCW formation was impaired in 99% of the wood fiber cells. In the present study, we produced nst/snd triple-knockout aspens, in which a large number of wood fibers had thinner SCWs than the wild type (WT) and some had no SCW. Because SCW layers are always formed prior to G-layer deposition, the nst/snd mutants raise interesting questions of whether the mutants can form G-layers without SCW and whether they can control their postures in response to changes in gravitational direction. The nst/snd mutants and the WT plants showed growth eccentricity and vessel frequency reduction when grown on an incline, but the triple mutants recovered their upright growth only slightly, and the quadruple mutants were unable to maintain their postures. The mutants clearly showed that the G-layers were formed in SCW-containing wood fibers but not in those lacking the SCW. Our results indicate that SCWs are essential for G-layer formation and posture control. Furthermore, each wood fiber cell may be able to recognize its cell wall developmental stage to initiate the formation of the G-layer as a response to gravistimulation.

Efflux and assimilation of xylem-transported CO2 in stems and leaves of tree species with different wood anatomy.

Determining the fate of CO2 respired in woody tissues is necessary to understand plant respiratory physiology and to evaluate CO2 recycling mechanisms. An aqueous 13 C-enriched CO2 solution was infused into the stem of 3-4 m tall trees to estimate efflux and assimilation of xylem-transported CO2 via cavity ring-down laser spectroscopy and isotope ratio mass spectrometry, respectively. Different tree locations (lower stem, upper stem and leafy shoots) and tissues (xylem, bark and leaves) were monitored in species with tracheid, diffuse- and ring-porous wood anatomy (cedar, maple and oak, respectively). Radial xylem CO2 diffusivity and xylem [CO2 ] were lower in cedar relative to maple and oak trees, thereby limiting label diffusion. Part of the labeled 13 CO2 was assimilated in cedar (8.7%) and oak (20.6%) trees, mostly in xylem and bark tissues of the stem, while limited solution uptake in maple trees hindered the detection of label assimilation. Little label reached foliar tissues, suggesting substantial label loss along the stem-branch transition following reductions in the radial diffusive pathway. Differences in respiration rates and radial xylem CO2 diffusivity (lower in conifer relative to angiosperm species) might reconcile discrepancies in efflux and assimilation of xylem-transported CO2 so far observed between taxonomic clades.

Linking wood anatomy with growth vigour and susceptibility to alternate bearing in composite apple and pear trees.

Excess vegetative growth and irregular fruit-bearing are often undesirable in horticultural practice. However, the biological mechanisms underlying these traits in fruit trees are not fully understood. Here, we tested if growth vigour and susceptibility of apple and pear trees to alternate fruit-bearing are associated with vascular anatomy. We examined anatomical traits related to water transport and nutrient storage in young woody shoots and roots of 15 different scion/rootstock cultivars of apple and pear trees. In addition, soil and leaf water potentials were measured across a drought period. We found a positive correlation between the mean vessel diameter of roots and the annual shoot length. Vigorously growing trees also maintained less negative midday leaf water potential during drought. Furthermore, we observed a close negative correlation between the proportions of total parenchyma in the shoots and the alternate bearing index. Based on anatomical proxies, our results suggest that xylem transport efficiency of rootstocks is linked to growth vigour of both apple and pear trees, while limited carbohydrate storage capacity of scions may be associated with increased susceptibility to alternate bearing. These findings can be useful for the breeding of new cultivars of commercially important fruit trees.

Wood day capacitance is related to water content, wood density, and anatomy across 30 temperate tree species.

Water released from wood during transpiration (capacitance) can meaningfully affect daily water use and drought response. To provide context for better understanding of capacitance mechanisms, we investigated links between capacitance and wood anatomy. On twigs of 30 temperate angiosperm tree species, we measured day capacitance (between predawn and midday), water content, wood density, and anatomical traits, that is, vessel dimensions, tissue fractions, and vessel-tissue contact fractions (fraction of vessel circumference in contact with other tissues). Across all species, wood density (WD) and predawn lumen volumetric water content (VWCL-pd ) together were the strongest predictors of day capacitance (r2adj = .44). Vessel-tissue contact fractions explained an additional ~10% of the variation in day capacitance. Regression models were not improved by including tissue lumen fractions. Among diffuse-porous species, VWCL-pd and vessel-ray contact fraction together were the best predictors of day capacitance, whereas among semi/ring-porous species, VWCL-pd , WD and vessel-fibre contact fraction were the best predictors. At predawn, wood was less than fully saturated for all species (lumen relative water content = 0.52 ± 0.17). Our findings imply that day capacitance depends on the amount of stored water, tissue connectivity and the bulk wood properties arising from WD (e.g., elasticity), rather than the fraction of any particular tissue.

CELLULOSE SYNTHASE INTERACTING 1 is required for wood mechanics and leaf morphology in aspen.

Cellulose microfibrils synthesized by CELLULOSE SYNTHASE COMPLEXES (CSCs) are the main load-bearing polymers in wood. CELLULOSE SYNTHASE INTERACTING1 (CSI1) connects CSCs with cortical microtubules, which align with cellulose microfibrils. Mechanical properties of wood are dependent on cellulose microfibril alignment and structure in the cell walls, but the molecular mechanism(s) defining these features is unknown. Herein, we investigated the role of CSI1 in hybrid aspen (Populus tremula × Populus tremuloides) by characterizing transgenic lines with significantly reduced CSI1 transcript abundance. Reduction in leaves (50-80%) caused leaf twisting and misshaped pavement cells, while reduction (70-90%) in developing xylem led to impaired mechanical wood properties evident as a decrease in the elastic modulus and rupture. X-ray diffraction measurements indicate that microfibril angle was not impacted by the altered CSI1 abundance in developing wood fibres. Instead, the augmented wood phenotype of the transgenic trees was associated with a reduced cellulose degree of polymerization. These findings establish a function for CSI1 in wood mechanics and in defining leaf cell shape. Furthermore, the results imply that the microfibril angle in wood is defined by CSI1 independent mechanism(s).

Wood Architecture and Composition Are Deeply Remodeled in Frost Sensitive Eucalyptus Overexpressing CBF/DREB1 Transcription Factors.

Eucalypts are the most planted trees worldwide, but most of them are frost sensitive. Overexpressing transcription factors for CRT-repeat binding factors (CBFs) in transgenic Eucalyptus confer cold resistance both in leaves and stems. While wood plays crucial roles in trees and is affected by environmental cues, its potential role in adaptation to cold stress has been neglected. Here, we addressed this question by investigating the changes occurring in wood in response to the overexpression of two CBFs, taking advantage of available transgenic Eucalyptus lines. We performed histological, biochemical, and transcriptomic analyses on xylem samples. CBF ectopic expression led to a reduction of both primary and secondary growth, and triggered changes in xylem architecture with smaller and more frequent vessels and fibers exhibiting reduced lumens. In addition, lignin content and syringyl/guaiacyl (S/G) ratio increased. Consistently, many genes of the phenylpropanoid and lignin branch pathway were upregulated. Most of the features of xylem remodeling induced by CBF overexpression are reminiscent of those observed after long exposure of Eucalyptus trees to chilling temperatures. Altogether, these results suggest that CBF plays a central role in the cross-talk between response to cold and wood formation and that the remodeling of wood is part of the adaptive strategies to face cold stress.

Fluorescence Scheimpflug LiDAR developed for the three-dimension profiling of plants.

This work proposes a novel fluorescence Scheimpflug LiDAR (SLiDAR) technique based on the Scheimpflug principle for three-dimension (3D) plant profile measurements. A 405 nm laser diode was employed as the excitation light source to generate a light sheet. Both the elastic and inelastic/fluorescence signals from a target object (e.g., plants) can be simultaneously measured by the fluorescence SLiDAR system employing a color image sensor with blue, green and red detection channels. The 3D profile can be obtained from the elastic signal recorded by blue pixels through elevation scanning measurements, while the fluorescence intensity of the target object is mainly acquired by red and green pixels. The normalized fluorescence intensity of the red channel, related to the chlorophyll distribution of the plant, can be utilized for the classification of leaves, branches and trunks. The promising results demonstrated in this work have shown a great potential of employing the fluorescence SLiDAR technique for 3D fluorescence profiling of plants in agriculture and forestry applications.