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Parenchyma is an important component of the secondary xylem. It has multiple functions and its fraction is known to vary substantially across angiosperm species. However, the physiological significance of this variation is not yet fully understood. Here, we examined how different types of parenchyma (ray parenchyma [RP], axial parenchyma [AP] and AP in direct contact with vessels [APV]) are coordinated with three essential xylem functions: water conduction, storage of non-structural carbohydrate (NSC) and mechanical support. Using branch sapwood of 15 co-occurring drought-adapted woody species from the subtropical Bonin Islands, Japan, we quantified 10 xylem anatomical traits and examined their linkages to hydraulic properties, storage of soluble sugars and starch and sapwood density. The fractions of APV and AP in the xylem transverse sections were positively correlated with the percentage loss of conductivity in the native condition, whereas that of RP was negatively correlated with the maximum conductivity across species. Axial and ray parenchyma fractions were positively associated with concentrations of starch and NSC. The fraction of parenchyma was independent of sapwood density, regardless of parenchyma type. We also identified a negative relationship between hydraulic conductivity and NSC storage and sapwood density, mirroring the negative relationship between the fractions of parenchyma and vessels. These results suggest that parenchyma fraction underlies species variation in xylem hydraulic and carbon use strategies, wherein xylem with a high fraction of AP may adopt an embolism repair strategy through an increased starch storage with low cavitation resistance.

In trees, wood formation needs carbon import from the photosynthetic source tissues. Sugar transporters play important roles in carbohydrate transport into wood-forming cells. Sugars will eventually be exported transporters (SWEETs) play essential roles in many physiological processes. However, the roles of this family in the growth and development of woody plants have not been systematically investigated. In this study, 27 SWEET genes were identified in the Populus trichocarpa genome. These SWEET genes were classified into four clades based on their phylogenetic relationships, gene structures, conserved motifs and chromosomal locations. Representative SWEET members from each clade were selected for further studies. The PagSWEETs of Populus alba × Populus glandulosa were localized to plasma membrane, vacuolar, endoplasmic reticulum or Golgi. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that PagSWEETs have distinct expression patterns in various tissues, and PagSWEET5, 7, 10b, 10c, 15b, 17a and 17c exhibited high expression levels in stems. PagSWEET7 is localized to the cytoplasmic membrane and specifically expressed in the phloem as detected by histochemical GUS ($\beta $ - glucuronidase) assays. Xylem production and xylem sugar content were greater in developing wood of PagSWEET7 overexpression than wild-type lines. Collectively, these results provide valuable information for further investigating functions of PagSWEET genes, and identify PagSWEET7 as a candidate gene for using biotechnology to modify the wood formation in poplar.

Wood density (WD) is often used as a proxy for hydraulic traits such as vulnerability to drought-induced xylem cavitation and maximum water transport capacity, with dense-wooded species generally being more resistant to drought-induced xylem cavitation, having lower rates of maximum water transport and lower sapwood capacitance than light-wooded species. However, relationships between WD and the hydraulic traits that they aim to predict have not been well established in tropical forests, where modeling is necessary to predict drought responses for a high diversity of unmeasured species. We evaluated WD and relationships with stem xylem vulnerability by measuring cavitation curves, sapwood water release curves and minimum seasonal water potential (Ψmin) on upper canopy branches of six tree species and three liana species from a single wet tropical forest site in Panama. The objective was to better understand coordination and trade-offs among hydraulic traits and the potential utility of these relationships for modeling purposes. We found that parameters from sapwood water release curves such as capacitance, saturated water content and sapwood turgor loss point (Ψtlp,x) were related to WD, whereas stem vulnerability curve parameters were not. However, the water potential corresponding to 50% loss of hydraulic conductivity (P50) was related to Ψtlp,x and sapwood osmotic potential at full turgor (πo,x). Furthermore, species with lower Ψmin showed lower P50, Ψtlp,x and πo,x suggesting greater drought resistance. Our results indicate that WD is a good easy-to-measure proxy for some traits related to drought resistance, but not others. The ability of hydraulic traits such as P50 and Ψtlp,x to predict mortality must be carefully examined if WD values are to be used to predict drought responses in species without detailed physiological measurements.

Insect defoliation impacts forest productivity worldwide, highlighting the relevance of plant–insect interactions. The larch budmoth (Zeiraphera griseana Hübner) is one of the most extensively studied defoliators, where numerous tree ring-based analyses on its host (Larix decidua Mill.) have aided in identifying outbreak dynamics over the past millennia. Yet, outbreaks have been widely absent after the early 1980s, and little is known about the in situ tree physiological responses and the allocation of carbon resources during and after defoliation. In summer 2018, we tracked an ongoing larch budmoth outbreak in a well-studied larch forest in the Swiss Alps. We performed biweekly monitoring on an affected and unaffected site using a unique combination of xylogenesis observations, measurements of non-structural carbohydrates, isotopic analysis of needle assimilates and ground-based and remote-sensed leaf trait observations. The budmoth induced a defoliation that lasted 40 days and could be detected by satellite observations. Soluble sugars significantly decreased in needles and stem phloem of the defoliated trees, while starch levels remained stable in the stem and root xylem compared to the control. Carbon and oxygen isotope ratios in needle assimilates indicated that neither photosynthetic assimilation rates nor stomatal conductance was different between sites before, during and after the outbreak. Defoliated trees ceased cell wall thickening 17 days earlier than unaffected trees, showing the earliest halt of ring formation recorded from 2007 untill 2013 and causing significant thinner cell walls, particularly in the latewood. No significant differences were found for cell enlargement rates and ring width. Our study revealed that an outbreak causes a downregulation of cell wall thickening first, while no starch is mobilized or leaf physiology is adjusted to compensate for the reduced carbon source due to defoliation. Our observations suggest that affected larch trees prioritize leaf recovery and carbon storage over wood biomass development.

Ice formation and propagation in the xylem of plants is a complex process. During freezing of xylem sap, gases dissolved in liquid sap are forced out of the ice lattice due to their low solubility in ice, and supersaturation of xylem sap as well as low water potential (Ѱ) are induced at the ice–liquid interface. Supersaturation of gases near the ice front may lead to bubble formation and potentially to cavitation and/or to burst of gases driven out from the branch. In this study, we investigated the origin and dynamics of freezing-related gas bursts and ultrasonic acoustic emissions (AEs), which are suggested to indicate cavitation. Picea abies (L.) H. Karst. and Salix caprea L. branch segments were exposed to frost cycles in a temperature test chamber, and CO2 efflux (indicating gas bursts) and AEs were recorded. On freezing, two-thirds of the observed gas bursts originated from the xylem and only one-third from the bark. Simultaneously with gas bursts, AEs were detected. Branch Ѱ affected both gas bursts and AEs, with high gas burst in saturated and dry samples but relevant AEs only in the latter. Repeated frost cycles led to decreasing gas burst volumes and AE activity. Experiments revealed that the expanding ice front in freezing xylem was responsible for observed gas bursts and AEs, and that branch Ѱ influenced both processes. Results also indicated that gas bursts and cavitation are independently induced by ice formation, though both may be relevant for bubble dynamics during freezing.

Seasonality in tree cambial activity and xylem formation encompass large variation in environmental conditions. Abiotic stressors such as warming or drought also modulate plant behavior at species and individual level. Despite xylem formation susceptibility to carbon (C) and water availability, it is still unknown which are the key physiological variables that regulate xylogenesis, and to what extent plant performance contributes to further explain the number of cells in the different phases of xylem development. Xylogenesis and physiological behavior was monitored in saplings of Pinus pinaster Aiton, a bimodal growth pattern species, distributed in different irrigation regimes. Xylogenesis and plant physiological behavior were compared between treatments and the relationship between climate, physiology and the number of cells in the cambium, enlargement and cell-wall thickening phases was evaluated. Xylogenesis regulation shifted from physiological to climatic control as cell differentiation advanced to mature tracheids. The number of cells in the cambium increased with assimilation rates and decreased with the water potential gradient through the plant. Enlargement was the most susceptible phase to plant relative water content, whereas no physiological variable contributed to explain the number of cells in the wall thickening phase, which declined as temperatures increased. All treatments showed a bimodal growth pattern with a second growth period starting when primary growth was completed and after plants had experienced the highest summer hydraulic losses. Our study demonstrates the importance of including physiological responses and not only climate to fully understand xylogenesis, with special attention to the enlargement phase. This is critical when studying species with a bimodal growth pattern because the second growth peak responds to internal shifts of C allocation and may strongly depend on plant hydraulic responses and not on a fine tuning of cambial activity with soil water availability.

The increased frequency and intensity of drought pose great threats to the survival of trees, especially in dioecious tree species with sexual differences in mortality and biased sex ratios. The sex-specific mechanisms underlying stem xylem anatomy and function and carbon metabolism in drought resistance and recovery were investigated in dioecious Populus cathayana Rehder. The sex-specific drought resistance and subsequent recovery were linked to the xylem anatomy and carbon metabolism. Females had a greater xylem vessel area per vessel, biomass and theoretically hydraulic efficiency under well-watered conditions. Conversely, males had a lower xylem lumen area, but greater vessel numbers, and a higher cell wall thickness, suggesting a theoretically conservative water-use strategy and drought resistance. The recovery of photosynthetic ability after drought in males was largely dependent on the recovery of xylem function and the regulation of the xylem carbohydrate metabolism. Additionally, the number of upregulated genes related to xylem cell wall biogenesis was greater in males relative to females under drought stress and subsequent rewatering, which facilitated drought resistance and xylem function restoration in males. These results suggested that sex-specific drought resistance and restoration were related to xylem anatomy and function, carbohydrate metabolism and cell turgor maintenance.

Resprouting plants are distributed in many vegetation communities worldwide. With increasing resprout age post-severe-disturbance, new stems grow rapidly at their early age, and decrease in their growth with gradually decreasing water status thereafter. However, there is little knowledge about how stem hydraulic strategies and anatomical traits vary post-disturbance. In this study, the stem water potential (Ψstem), maximum stem hydraulic conductivity (Kstem-max), water potential at 50% loss of hydraulic conductivity (Kstem P50) and anatomical traits of Caragana korshinkii resprouts were measured during a 1- to 13-year post-disturbance period. We found that the Kstem-max decreased with resprout age from 1-year-old resprouts (84.2 mol m−1 s−1 MPa−1) to 13-year-old resprouts (54.2 mol m−1 s−1 MPa−1) as a result of decreases in the aperture fraction (Fap) and the sum of aperture area on per unit intervessel wall area (Aap). The Kstem P50 of the resprouts decreased from 1-year-old resprouts (−1.8 MPa) to 13-year-old resprouts (−2.9 MPa) as a result of increases in vessel implosion resistance (t/b)2, wood density (WD), vessel grouping index (GI) and decreases in Fap and Aap. These shifts in hydraulic structure and function resulted in an age-based divergence in hydraulic strategies i.e., a change from an acquisitive strategy to a conservative strategy, with increasing resprout age post-disturbance.

Recent climate projections predict a more rapid increase of winter temperature than summer and global temperature averages in temperate and cold environments. As there is relatively little experimental knowledge on the effect of winter warming on cambium phenology and stem growth in species growing in cold environments, the setting of manipulative experiments is considered of primary importance, and they can help to decipher the effect of reduced winter chilling and increased forcing temperatures on cambium reactivation, growth and xylem traits. In this study, localized stem heating was applied to investigate the effect of warming from the rest to the growth phase on cambium phenology, intra-annual stem growth dynamics and ring wood features in Picea abies (L.) H.Karst. We hypothesized that reduced winter chilling induces a postponed cambium dormancy release and decrease of stem growth, while high temperature during cell wall lignification determines an enrichment of latewood-like cells. The heating device was designed to maintain a +5 °C temperature delta with respect to air temperature, thus allowing an authentic scenario of warming. Continuous stem heating from the rest (November) to the growing phase determined, at the beginning of radial growth, a reduction of the number of cell layers in the cambium, higher number of cell layers in the wall thickening phase and an asynchronous stem radial growth when comparing heated and ambient saplings. Nevertheless, heating did not induce changes in the number of produced cell layers at the end of the growing season. The analyses of two-photon fluorescence images showed that woody rings formed during heating were enriched with latewood-like cells. Our results showed that an increase of 5 °C of temperature applied to the stem from the rest to growth might not influence, as generally reported, onset of cambial activity, but it could affect xylem morphology of Norway spruce in mountain environments.

Coordination between sapwood-specific hydraulic conductivity (Ks) and stomatal conductance (gs) has been identified in previous studies; however, coordination between leaf hydraulic conductance (Kleaf) and gs, as well as between Kleaf and Ks is not always consistent. This suggests that there is a need to improve our understanding of the coordination among hydraulic and gas exchange traits. In this study, hydraulic traits (e.g., Ks and Kleaf) and gas exchange traits, including gs, transpiration (E) and net CO2 assimilation (Aarea), were measured across 33 co-occurring subtropical woody species. Kleaf was divided into two components: leaf hydraulic conductance inside the xylem (Kleaf-x) and outside the xylem (Kleaf-ox). We found that both Kleaf-x and Kleaf-ox were coordinated with gs and E, but the correlations between Kleaf-ox and gs (or E) were substantially weaker, and that Ks was coordinated with Kleaf-x, but not with Kleaf-ox. In addition, we found that Ks, Kleaf-x and Kleaf-ox together explained 63% of the variation in gs and 42% of the variation in Aarea across species, with Ks contributing the largest proportion of explanatory power, whereas Kleaf-ox contributed the least explanatory power. Our results demonstrate that the coordination between leaf water transport and gas exchange, as well as the hydraulic linkage between leaf and stem, were weakened by Kleaf-ox. This highlights the possibility that water transport efficiencies of stem and leaf xylem, rather than that of leaf tissues outside the xylem, are important determinants of stomatal conductance and photosynthetic capacity across species.

Hydraulic safety and efficiency have become the central concept of the interpretation of the structure and function of vessels and their interconnections. Plants form an appropriate xylem network structure to maintain a balance of hydraulic safety vs efficiency. The term ‘tracheid bridge’ is used to describe a possible pathway of water transport between neighboring vessels via tracheids, and this pathway could also provide increased safety against embolisms. However, the only physiological study of such a structure thus far has been in Hippophae rhamnoides Linn. To test the function of tracheid bridges, this research examined four species that have relatively long and solitary vessels, which are two of the criteria for efficient tracheid bridges. Tracheids contributed less than 2.2% of the total conductance of the vessels in these species, but in theory, tracheids could serve as very efficient transport connector pathways that may or may not make direct vessel-to-vessel contact via pit fields between adjacent vessels. In some species, tracheid bridges may represent the dominant pathway for water flow between vessels, whereas in other species, tracheid bridges may be sub-dominant or virtually nil. Broader searches of woody taxa are needed to reveal the functional importance of tracheid bridges in vascular plants.

Wood fibers form thick secondary cell wall (SCW) in xylem tissues to give mechanical support to trees. NAC SECONDARY WALL THICKENING PROMOTING FACTOR3/SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1 (NST3/SND1) and NST1 were identified as master regulators of SCW formation in xylem fiber cells in the model plant Arabidopsis thaliana. In Populus species, four NST/SND orthologs have been conserved and coordinately control SCW formation in wood fibers and phloem fibers. However, it remains to be elucidated whether SCW formation in other xylem cells, such as ray parenchyma cells and vessel elements, is regulated by NST/SND orthologs in poplar. We knocked out all NST/SND genes in hybrid aspen using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system and investigated the detailed histological appearance of stem tissues in the knockout mutants. Observation by light microscopy and transmission electron microscopy showed that SCW was severely suppressed in wood fibers, phloem fibers and xylem ray parenchyma cells in the knockout mutants. Although almost all wood fibers lacked SCW, some fiber cells formed thick cell walls. The irregularly cell wall-forming fibers retained primary wall and SCW, and were mainly located in the vicinity of vessel elements. Field emission-scanning electron microscope observation showed that there were no apparent differences in the structural features of pits such as the shape and size between irregularly SCW-forming wood fibers in the knockout mutants and normal wood fibers in wild-type. Cell wall components such as cellulose, hemicellulose and lignin were deposited in the cell wall of irregularly SCW-forming wood fibers in quadruple mutants. Our results indicate that four NST/SND orthologs are master switches for SCW formation in wood fibers, xylem ray parenchyma cells and phloem fibers in poplar, while SCW is still formed in limited wood fibers, which are located at the region adjacent to vessel elements in the knockout mutants.

Although conifers have significant ecological and economic value, information on transcriptional regulation of wood formation in conifers is still limited. Here, to gain insight into secondary cell wall (SCW) biosynthesis and tracheid formation in conifers, we performed wood tissue-specific transcriptome analyses of Pinus densiflora (Korean red pine) using RNA sequencing. In addition, to obtain full-length transcriptome information, PacBio single molecule real-time iso-sequencing was carried out using RNAs from 28 tissues of P. densiflora. Subsequent comparative tissue-specific transcriptome analysis successfully pinpointed critical genes encoding key proteins involved in biosynthesis of the major secondary wall components (cellulose, galactoglucomannan, xylan and lignin). Furthermore, we predicted a total of 62 NAC (NAM, ATAF1/2 and CUC2) family transcription factor members and identified seven PdeNAC genes preferentially expressed in developing xylem tissues in P. densiflora. Protoplast-based transcriptional activation analysis found that four PdeNAC genes, homologous to VND, NST and SND/ANAC075, upregulated GUS activity driven by an SCW-specific cellulose synthase promoter. Consistently, transient overexpression of the four PdeNACs induced xylem vessel cell-like SCW deposition in both tobacco (Nicotiana benthamiana) and Arabidopsis leaves. Taken together, our data provide a foundation for further research to unravel transcriptional regulation of wood formation in conifers, especially SCW formation and tracheid differentiation.

Research on wood phenology has mainly focused on reactivation of the cambium in spring. In this study we investigated if summer drought advances cessation of wood formation and if it has any influence on wood structure in late successional forest trees of the temperate zone. The end of xylogenesis was monitored between August and November in stands of European beech and pedunculate oak in Belgium for two consecutive years, 2017 and 2018, with the latter year having experienced an exceptional summer drought. Wood formation in oak was affected by the drought, with oak trees ceasing cambial activity and wood maturation about 3 weeks earlier in 2018 compared with 2017. Beech ceased wood formation before oak, but its wood phenology did not differ between years. Furthermore, between the 2 years, no significant difference was found in ring width, percentage of mature fibers in the late season, vessel size and density. In 2018, beech did show thinner fiber walls, whereas oak showed thicker walls. In this paper, we showed that summer drought can have an important impact on late season wood phenology xylem development. This will help to better understand forest ecosystems and improve forest models.

Interlocked grain occurs when the orientation of xylem fibres oscillates, alternating between left- and right-handed spirals in successive wood layers. The cellular mechanisms giving rise to interlocked grain, thought to involve the slow rotation of fusiform initials within the vascular cambium, remain unclear. We suggest that observations of wood structure at the cellular level, but over large areas, might reveal these mechanisms. We assayed timber from several commercially important tropical angiosperms from the genus Khaya (African mahogany) that exhibit interlocked grain using X-ray computed microtomography followed by orthogonal slicing and image processing in ImageJ. Reconstructed tangential longitudinal sections were processed with the ImageJ directionality plug-in to directly measure fibre orientation and showed grain deviations of more than 10° from vertical in both left- and right-handed directions. Grain changed at locally constant rates, separated by locations where the direction of grain change sharply reversed. Image thresholding and segmentation conducted on reconstructed cross sections allowed the identification of vessels and measurement of their location, with vessel orientations then calculated in Matlab and, independently, in recalculated tangential longitudinal sections with the directionality plug-in. Vessel orientations varied more than fibre orientations, and on average deviated further from vertical than fibres at the locations where the direction of grain change reversed. Moreover, the reversal location for vessels was shifted ~400 μm towards the pith compared with the fibres, despite both cell types arising from the same fusiform initials within the vascular cambium. We propose a simple model to explain these distinct grain patterns. Were an auxin signal to control both the reorientation of cambial initials, as well as coordinating the end-on-end differentiation and linkage of xylem vessel elements, then it would be possible for fibres and vessels to run at subtly different angles, and to show different grain reversal locations.

Cultivation of fast-growing tree species is often confined to marginal land. Mixed cropping with Robinia pseudoacacia, a legume tree species that forms a symbiosis with N2-fixing bacteria, has been proposed to be a measure to improve soil fertility and to achieve beneficial effects on the cocultivated tree species. The goal of our study was to examine the influence of a Robinia mixture on wood chemistry, anatomy and gene expression in poplar. We hypothesized that annual wood growth is stimulated in species mixtures due to the positive effects of Robinia on nitrogen availability and complementary resource use. Alternatively, we hypothesized that competition, especially for water, has negative effects on the wood growth of poplar. We used two commercial biomass clones, Hybride 275 (H275, Populus trichocarpa × Populus maximowiczii) and Max1 (Populus nigra × P. maximowiczii), which were planted at two locations with contrasting soil fertility in monoculture or mixed plots with Robinia to investigate the annual wood increment, wood nitrogen and δ13C, wood anatomy (length, cell wall thickness, lumina and frequencies of fibers and vessels) and transcriptional profiles in the developing xylem of 4-year-old stems. In a mixture with Robinia, the annual stem increment was reduced, nitrogen in wood was enhanced, δ13C in wood was decreased, vessel and fiber frequencies were increased and fiber lengths and fiber lumina were decreased. Transcriptional profiles showed stronger differences between the genotypes and sites than between mono and mixed cultivation. The transcriptional abundances of only one gene (the putative nitrate transporter, NRT1.2) and one gene ontology term (‘immune system process’) were significantly enriched in wood-forming tissues in response to the mixture, irrespective of the poplar genotype and growth location. Weighted gene coexpression network analyses extracted gene modules that linked wood nitrogen mainly to vessel traits and wood δ13C with fiber traits. Collectively, molecular and anatomical changes in poplar wood suggest beneficial effects on the water and N supply in response to the mixture with Robinia. These alterations may render poplars less drought-susceptible. However, these benefits are accompanied by a reduced wood increment, emphasizing that other critical factors, presumably light competition or allelopathic effects, overrule a potential growth stimulation.

Mixed forests of Quercus ilex L. and Pinus pinea L. are widely found throughout the Mediterranean Basin, being representative of two co-existing functional types: evergreen-sclerophyllous drought-resistant species and Mediterranean-adapted drought-avoidant conifers. Their contrasting physiological strategies to cope with water deficit influence all the processes regulating their growth such as wood formation, leading to peculiar tree-ring anatomical features such as intra-annual density fluctuations (IADFs). Intra-annual density fluctuations are abrupt changes in wood anatomical traits within a tree ring, appearing as latewood-like cells within earlywood or earlywood-like cells within latewood, and are frequently found in Mediterranean species as a response to seasonal climate changes. In this study, we characterized the anatomical traits and composition of carbon and oxygen stable isotopes in IADFs occurring in tree rings of Q. ilex and P. pinea trees co-existing at a same site in Southern Italy, in order to link their xylem hydraulic properties with the related physiological mechanisms. The relationships between IADF occurrence and seasonal mean temperature and total precipitation were investigated, with the aim of assessing whether they can be used as indicators of species-specific responses to intra-annual climate fluctuations. Results show that IADF period of formation is during autumn months for both species. The influence of climate on IADF occurrence was found to be an indicator of species-specific response to climate: an increased stomatal conductance associated to the formation of a wood safer against embolism was found in Q. ilex, while a tighter stomatal control associated to a more efficient wood with regard to hydraulic conductivity occurred in P. pinea. Moreover, the assessment of the influence of climate on IADF occurrence indicates that, with rising temperatures, Q. ilex would form fewer IADFs compared with P. pinea. Other study cases are desirable to assess the suggested forecasts and to link the plasticity of the species to form IADFs with their effective adaptive capability to compete for resources, and to explain how it may influence future population development.

Brassinosteroids (BRs) as steroid hormones play an important role in plant growth and development. However, little is known about how BRs affect secondary wall biosynthesis in woody plants. In this study, we cloned and characterized PtoDWF4, a homologus gene of Arabidopsis DWF4 encoding a cytochrome P450 protein, from Populus tomentosa. qRT-PCR analysis showed that PtoDWF4 was highly expressed in stems, especially in xylem. Overexpression of PtoDWF4 (PtoDWF4-OE) in poplar promoted growth rate and biomass yield, increased area and cell layers of xylem. Transgenic plants showed a significant increase in plant height and stem diameter compared with the wild type. In contrast, the CRISPR/Cas9-generated mutation of PtoDWF4 (PtoDWF4-KO) resulted in significantly decreased biomass production in transgenic plants. Further studies revealed that constitutive expression of PtoDWF4 up-regulated the expression of secondary cell wall (SCW) biosynthesis-related genes, whereas knock-out of PtoDWF4 down-regulated their expression. Quantitative analysis of cell wall components showed a significant increase in PtoDWF4-OE lines but a reduction in PtoDWF4-KO lines compared with wild-type plants. Taken together, our results indicate that PtoDWF4 plays a positive role in improving growth rate and elevating biomass production in poplar.

Knowing more precisely the cambial phenology and wood formation dynamics of trees can lead to a better understanding on how trees react to short-term changes in environmental conditions. Such an understanding could also shed light on the physiological foundation of climate-growth interactions at a regional scale. Although it has been documented that temperature is an important factor determining the cambial phenology in cold and humid climates, there is less agreement on the driver(s) that trigger the onset and end of wood formation in cold and arid climates. Here, the phenological traits of cambial activity and xylem formation were analyzed biweekly along an altitudinal transect ranging from 3580 to 3980 m above sea level, a transect that covers the distribution of Qilian juniper (Juniperus przewalskii Kom.) along a slope of the Tibetan Plateau. Cambial phenology and the duration and rate of wood formation were assessed from anatomical observations during the growing season of the developing xylem obtained from microcores collected from the stem of 10 trees total in 2012 (five at two altitudes each) and 25 trees (five at five altitudes each) in 2013. We found that the onset of wood formation was significantly correlated with altitude in 2013, with onset beginning 8.2 days earlier with every 100 m decrease in elevation. The change in onset with elevation corresponds to a change of 14.1 days °C-1 when adjusted for the monitored altitudinal lapse rate of -0.58 °C per 100 m. The duration of wood formation lasted from mid-May to mid-August, with the length of the 2013 growing season decreasing from 97 to 65 days from low to high elevation. Although the end of growing season appeared minimally related to altitude during both growing seasons, differences in end of wood production and wood formation between the two growing seasons were significant. It appears that summer drought conditions constricted the end of growing season across all elevations along our transect in 2013. Sensitivity analysis found xylem growth was positively correlated with rate and duration of wood production, with the former explaining most variability in growth. Our findings provide new data on the timing and duration of wood formation and help quantify the potential impacts of global warming on tree growth and productivity in cold and arid regions.

Wood formation is a complex developmental process under multi-level transcriptional control executed by a large set of transcription factors. However, only limited members have been characterized to be key regulators of lignin biosynthesis in poplar. Here we report the conserved and unique functions of PtoMYB170, a transcription factor identified from Populus tomentosa (Chinese white poplar), in lignin deposition and drought tolerance in comparison with its duplicate paralog PtoMYB216. PtoMYB170 is preferentially expressed in young leaves and xylem tissues. Overexpression of PtoMYB170 in transgenic poplar plants resulted in stronger lignification and more thickened secondary wall in xylem compared with wild-type plants, whereas the CRISPR/Cas9-generated mutation of PtoMYB170 weakened lignin deposition, thereby leading to a more flexible and collapsed xylem phenotype. Transient expression experiments demonstrated that PtoMYB170 specifically activated the expression of lignin biosynthetic genes, consistent with the function of PtoMYB216. However, GUS staining assays revealed that PtoMYB170 was specifically expressed in guard cells of transgenic Arabidopsis while PtoMYB216 was not. Heterologous expression of PtoMYB170 in Arabidopsis enhanced stomatal closure in the dark and resulted in drought tolerance of the transgenic plants through reduced water loss, indicating a diversified role from PtoMYB216. These results revealed the PtoMYB170-dependent positive transcriptional regulation on lignin deposition in poplar and its coordinated function in enhancing drought tolerance by promoting dark-induced stomatal closure.

In recent decades, anthropogenic activities have increased nitrogen (N) deposition in terrestrial ecosystems. This higher availability of N is expected to impact plant growth. However, the effects of N deposition on tree growth remain inconclusive due to the wide variability of experimental methods used. This study aimed to test the effect of short-term N addition on the intra-annual wood formation of Chinese red pine (Pinus massoniana Lamb.) in a warm-temperate forest of Central China. From 2013, solution containing 25 kg N ha-1 year-1 was applied monthly to the understory of experimental plots from April to December to double the current natural N deposition. Each week from March to December in 2014 and 2015, cambial activity and the timings and dynamics of xylem formation were monitored by collecting microcores from stems. Xylem formation lasted from March to November, producing an average of 19 and 33 cells for all studied trees in 2014 and 2015, respectively. No difference in xylem cell production was observed between control and N-treated trees. Moreover, N-treated trees had similar timings, rates and durations of xylem formation as control trees. These findings indicated that short-term N addition was unable to affect timings and dynamics of xylem formation in Chinese red pine of warm-temperate forest.

European mistletoe (Viscum album L.) has largely infested Central European forests and causes high mortality probability particularly in dry years. However, little information is available about the consequences of mistletoe infestation for metabolic processes in bark and wood of its host, despite their important roles in infestation defense. We analyzed the tissue hydration, carbohydrate composition, phytohormone profile, reactive oxygen species and anti-oxidant levels in bark and wood of Scots pines (Pinus sylvestris L.), as dependent on mistletoe infestation. As a consequence of mistletoe infestation, host bark and wood showed impaired hydration and reduced total carbon content. In the bark, soluble sugar and lignin contents increased, apparently at the expense of holo-cellulose. Hydrogen peroxide accumulation was accompanied by increased glutathione and decreased reduced ascorbic acid levels. Mistletoe infestation mediated alteration of the phytohormone profile in bark and wood of its host. Cytokinins, jasmonic acid and abscisic acid levels increased in both tissues, whereas salicylic acid and indole-3-acetic acid, which were only detected in the bark, declined. The present results show that mistletoe infestation affects both the host's anti-oxidative defense system and the phytohormone profile after establishment of the xylem tapping haustorium. The significance of these processes for the development of the woody mistletoe stem and the haustorium is discussed.

Gas inside wood plays an important role in plant functioning, but there has been no study examining the adaptive nature of gas inside wood across plants differing in biomechanical demands. Using a comparative approach, we measured gas volumetric content, xylem’s anatomical traits and wood density of 15 tree and 16 liana species, to test whether gas content varies between these plant types strongly differing in their biomechanical demands. We asked (i) whether trees and lianas differ in gas content and (ii) how anatomical traits and wood density are related to gas content. Lianas had significantly less gas content in their branches compared with tree species. In tree species, gas content scaled positively with fiber, vessel and xylem cross-sectional area and fiber and vessel diameter, and negatively with dry-mass density. When pooling trees and lianas together, fiber cross-sectional area was the strongest predictor of gas content, with higher xylem cross-sectional area of fiber associated with higher gas content. In addition, we showed, through a simple analytical model, that gas inside wood increases the minimum branch diameter needed to prevent rupture, and this effect was stronger on trees compared with lianas. Our results support the view that gas inside wood plays an important role in the evolution of biomechanical functioning in different plant forms. Gas inside wood may also play an important role in physiological activities such as water transport, storage, photosynthesis and respiration, but it is still unknown whether these roles are or are not secondary to the mechanical support.

Secondary cell wall (SCW) biosynthesis during wood formation in trees is controlled by a multilevel regulatory network that coordinates the expression of substantial genes. However, few transcription factors involved in the negative regulation of secondary wall biosynthesis have been characterized in tree species. In this study, we isolated an R2R3 MYB transcription factor MYB189 from Populus trichocarpa, which is expressed predominantly in secondary vascular tissues, especially in the xylem. A novel repression motif was identified in the C-terminal region of MYB189, which indicates this factor was a transcriptional repressor. Overexpression (OE) of MYB189 in Arabidopsis and poplar resulted in a significant reduction in the contents of lignin, cellulose and hemicelluloses. Vascular development in stems of MYB189 OE lines was markedly inhibited, leading to a dramatic decrease in SCW thickness of xylem fibers. Gene expression analyses showed that most of the structural genes involved in the biosynthesis of lignin, cellulose and xylans were significantly downregulated in MYB189-overexpressing poplars compared with the wild-type control. Chromatin immunoprecipitation-quantitative real-time polymerase chain reaction and transient expression assays revealed that MYB189 could directly bind to the promoters of secondary wall biosynthetic genes to repress their expression. Together, these data suggest that MYB189 acts as a repressor to regulate SCW biosynthesis in poplar.

Xylem resistance to embolism formation determines the species-specific drought tolerance and the survival prospects of plants under extreme climatic conditions. Fourier Transform-Infrared (FTIR) spectroscopy is a cost-effective and rapid analytical tool with potential beyond its current use in plant physiology. We tested the use of FTIR spectroscopy as a method for estimating wood density (WD) and xylem resistance to embolism formation (P50) in 24 angiosperm species. Higher WD was associated with more negative P50 (r2 = 0.41). Partial least squares regression was applied to establish models of FTIR spectra and the reference data. They showed a high predictive quality for WD (r2 = 0.73), whereas the prediction of P50 was weaker (r2 = 0.49). By including WD in the model as an additional factor influencing P50, its predictive power significantly increased (r2 = 0.59). The spectral range in the model elaboration has been also narrowed (bands of lignin, cellulose, hemicellulose), but this did not influence the model descriptors, suggesting that for P50 prediction broad spectral range is more informative than narrow band regions reflecting main wood constituents. In conclusion, FTIR spectroscopy associated with WD measurements has proven to be a promising alternative to traditional methods for screening of individual- or species-specific resistance to embolism in angiosperms.

The release of water from storage compartments to the transpiration stream is an important functional mechanism that provides the buffering of sudden fluctuations in water potential. The ability of tissues to release water per change in water potential, referred to as hydraulic capacitance, is assumed to be associated with the anatomy of storage tissues. However, information about how specific anatomical parameters determine capacitance is limited. In this study, we measured sapwood capacitance (C) in terminal branches and roots of five temperate tree species (Fagus sylvatica L., Picea abies L., Quercus robur L., Robinia pseudoacacia L., Tilia cordata Mill.). Capacitance was calculated separately for water released mainly from capillary (CI; open vessels, tracheids, fibres, intercellular spaces and cracks) and elastic storage compartments (CII; living parenchyma cells), corresponding to two distinct phases of the moisture release curve. We found that C was generally higher in roots than branches, with CI being 3-11 times higher than CII Sapwood density and the ratio of dead to living xylem cells were most closely correlated with C In addition, the magnitude of CI was strongly correlated with fibre/tracheid lumen area, whereas CII was highly dependent on the thickness of axial parenchyma cell walls. Our results indicate that water released from capillary compartments predominates over water released from elastic storage in both branches and roots, suggesting the limited importance of parenchyma cells for water storage in juvenile xylem of temperate tree species. Contrary to intact organs, water released from open conduits in our small wood samples significantly increased CI at relatively high water potentials. Linking anatomical parameters with the hydraulic capacitance of a tissue contributes to a better understanding of water release mechanisms and their implications for plant hydraulics.

Cellulose synthase A genes (CesAs) are responsible for cellulose biosynthesis in plant cell walls. In this study, functions of secondary wall cellulose synthases PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B were characterized during wood formation in Populus trichocarpa (Torr. & Gray). CesA RNAi knockdown transgenic plants exhibited stunted growth, narrow leaves, early necrosis, reduced stature, collapsed vessels, thinner fiber cell walls and extended fiber lumen diameters. In the RNAi knockdown transgenics, stems exhibited reduced mechanical strength, with reduced modulus of rupture (MOR) and modulus of elasticity (MOE). The reduced mechanical strength may be due to thinner fiber cell walls. Vessels in the xylem of the transgenics were collapsed, indicating that water transport in xylem may be affected and thus causing early necrosis in leaves. A dramatic decrease in cellulose content was observed in the RNAi knockdown transgenics. Compared with wildtype, the cellulose content was significantly decreased in the PtrCesA4, PtrCesA7 and PtrCesA8 RNAi knockdown transgenics. As a result, lignin and xylem contents were proportionally increased. The wood composition changes were confirmed by solid-state NMR, two-dimensional solution-state NMR and sum-frequency-generation vibration (SFG) analyses. Both solid-state nuclear magnetic resonance (NMR) and SFG analyses demonstrated that knockdown of PtrCesAs did not affect cellulose crystallinity index. Our results provided the evidence for the involvement of PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B in secondary cell wall formation in wood and demonstrated the pleiotropic effects of their perturbations on wood formation.

China's subtropical forests play a vital role in sequestering global carbon; therefore, it is critical to conduct a precise investigation of intra-annual wood formation in these ecosystems to clarify the mechanisms behind this. Two field experiments were established in Chinese subtropical forests to monitor weekly the intra-annual xylem formation of Pinus massoniana Lamb. from January to December 2015, using the recently developed micro-sampling approach. The effects of climate on wood formation were also assessed using linear or mixed models. Results indicate that there is an inactive period that might be semi-dormancy in subtropical pine ecosystems in January compared with the complete dormancy in temperate and boreal ecosystems and the fully active or short-term dormancy in tropical ecosystems. The duration of xylem formation of Chinese red pine in subtropical China in 2015 was 4-6 months longer than temperate and boreal forests. Moreover, trees were found to grow better during the dry season than the wet season, indicating that the Chinese red pine ecosystem is more strongly regulated by net energy than by environmental factors. Our findings indicate that China's subtropical pine forests may benefit from the expected longer dry seasons, possibly leading to better forest growth and improved carbon sequestration under continued climate warming.

Conifers have incurred high mortality during recent global-change-type drought(s) in the western USA. Mechanisms of drought-related tree mortality need to be resolved to support predictions of the impacts of future increases in aridity on vegetation. Hydraulic failure, carbon starvation and lethal biotic agents are three potentially interrelated mechanisms of tree mortality during drought. Our study compared a suite of measurements related to these mechanisms between 49 mature piñon pine (Pinus edulis Engelm.) trees that survived severe drought in 2002 (live trees) and 49 trees that died during the drought (dead trees) over three sites in Arizona and New Mexico. Results were consistent over all sites indicating common mortality mechanisms over a wide region rather than site-specific mechanisms. We found evidence for an interactive role of hydraulic failure, carbon starvation and biotic agents in tree death. For the decade prior to the mortality event, dead trees had twofold greater sapwood cavitation based on frequency of aspirated tracheid pits observed with scanning electron microscopy (SEM), smaller inter-tracheid pit diameter measured by SEM, greater diffusional constraints to photosynthesis based on higher wood δ(13)C, smaller xylem resin ducts, lower radial growth and more bark beetle (Coleoptera: Curculionidae) attacks than live trees. Results suggest that sapwood cavitation, low carbon assimilation and low resin defense predispose piñon pine trees to bark beetle attacks and mortality during severe drought. Our novel approach is an important step forward to yield new insights into how trees die via retrospective analysis.

Plants transport water under negative pressure and this makes their xylem vulnerable to cavitation. Among plant organs, root xylem is often highly vulnerable to cavitation due to water stress. The use of centrifuge methods to study organs, such as roots, that have long vessels are hypothesized to produce erroneous estimates of cavitation resistance due to the presence of open vessels through measured samples. The assumption that roots have long vessels may be premature since data for root vessel length are sparse; moreover, recent studies have not supported the existence of a long-vessel artifact for stems when a standard centrifuge technique was used. We examined resistance to cavitation estimated using a standard centrifuge technique and compared these values with native embolism measurements for roots of seven woody species grown in a common garden. For one species we also measured vulnerability using single-vessel air injection. We found excellent agreement between root native embolism and the levels of embolism measured using a centrifuge technique, and with air-seeding estimates from single-vessel injection. Estimates of cavitation resistance measured from centrifuge curves were biologically meaningful and were correlated with field minimum water potentials, vessel diameter (VD), maximum xylem-specific conductivity ( Ksmax) and vessel length. Roots did not have unusually long vessels compared with stems; moreover, root vessel length was not correlated to VD or to the vessel length of stems. These results suggest that root cavitation resistance can be accurately and efficiently measured using a standard centrifuge method and that roots are highly vulnerable to cavitation. The role of root cavitation resistance in determining drought tolerance of woody species deserves further study, particularly in the context of climate change.

A better understanding and prediction of the impact of changing climate on tree stem growth could greatly benefit from the combination of anatomical and ecophysiological knowledge, yet the majority of studies focus on one research field only. We propose an approach that combines the method of pinning (cambial wounding) to timestamp anatomical X-ray computed microtomography images with continuous measurements of sap flow and stem diameter variations. By pinning the cambium of well-watered and drought-treated young African tropical trees of the species Maesopsis eminii Engl. we could quantify wood formation during a specific period of time and relate it to tree physiology and prevailing microclimate. Integrating continuous plant measurements and high-frequency pinning proved very useful to visualize and quantify the effects on stem growth of drought in M. eminii. Wood formation completely stopped during drought, and was associated with a strong shrinkage in stem diameter. Next, an unexpected increase in stem diameter was observed during drought, probably caused by root pressure, but not accompanied by wood formation. Our proposed approach of combining continuous plant measurements with cambial pinning is very promising to relate ecophysiology to stem anatomy and to understand the mechanisms underlying tree stem growth and bridge the gaps between the two research fields.

Wood density can be considered as a measure of the internal wood structure, and it is usually used as a proxy measure of other mechanical and functional traits. Eucalyptus is one of the most important commercial forestry genera worldwide, but the relationship between wood density and vulnerability to cavitation in this genus has been little studied. The analysis is hampered by, among other things, its anatomical complexity, so it becomes necessary to address more complex techniques and analyses to elucidate the way in which the different anatomical elements are functionally integrated. In this study, vulnerability to cavitation in two races of Eucalyptus globulus Labill. with different wood density was evaluated through Path analysis, a multivariate method that allows evaluation of descriptive models of causal relationship between variables. A model relating anatomical variables with wood properties and functional parameters was proposed and tested. We found significant differences in wood basic density and vulnerability to cavitation between races. The main exogenous variables predicting vulnerability to cavitation were vessel hydraulic diameter and fibre wall fraction. Fibre wall fraction showed a direct impact on wood basic density and the slope of vulnerability curve, and an indirect and negative effect over the pressure imposing 50% of conductivity loss (P₅₀) through them. Hydraulic diameter showed a direct negative effect on P₅₀, but an indirect and positive influence over this variable through wood density on one hand, and through maximum hydraulic conductivity (kₛ ₘₐₓ) and slope on the other. Our results highlight the complexity of the relationship between xylem efficiency and safety in species with solitary vessels such as Eucalyptus spp., with no evident compromise at the intraspecific level., Instituto de Fisiología Vegetal

Planting provenances originating from southern to northern locations has been discussed as a strategy to speed up species migration and mitigate negative effects of climate change on forest stability and productivity. Especially for drought-susceptible species such as European beech (Fagus sylvatica L.), the introduction of drought-tolerant provenances from the south could be an option. Yet, beech has been found to respond plastically to environmental conditions, suggesting that the climate on the plantation site might be more important for tree growth than the genetic predisposition of potentially drought-adapted provenances. In this study, we compared the radial growth, wood-anatomical traits and leaf phenology of four beech provenances originating from southern (Bulgaria, France) and northern locations (Sweden, the Netherlands) and planted in a provenance trial in the Netherlands. The distribution of precipitation largely differs between the sites of origin. The northern provenances experience a maximum and the southern provenances experience a minimum of rainfall in summer. We compared tree productivity and the anatomy of the water-conducting system for the period from 2000 to 2010, including the drought year 2003. In addition, tree mortality and the timing of leaf unfolding in spring were analysed for the years 2001, 2007 and 2012. Comparison of these traits in the four beech provenances indicates the influence of genetic predisposition and local environmental factors on the performance of these provenances under moderate site conditions. Variation in radial growth was controlled by environment, although the growth level slightly differed due to genetic background. The Bulgarian provenance had an efficient water-conducting system which was moreover unaffected by the drought in 2003, pointing to a high ability of this provenance to cope well with dry conditions. In addition, the Bulgarian provenance showed up as most productive in terms of height and radial growth. Altogether, we conclude that the similarity in ring-width variation among provenances points to environmental control of this trait, whereas the differences encountered in wood-anatomical traits between the well-performing Bulgarian provenance and the other three provenances, as well as the consistent differences in flushing pattern over 3 years under various environmental conditions, support the hypothesis of genetic control of these features.

The hydraulic performance of woody species during drought is currently of high interest in the context of climate change. It is known that woody species have the capacity to mitigate water shortage by using internally stored water. Elastic shrinkage of living cells and also water release during cavitation contribute to the so-called 'hydraulic capacitance' (C) of the plant, which adds water to the transpiration stream and buffers fluctuations in water potential. Although sap-conducting conduits may ultimately serve as a water pool, cavitation will hamper the conduction of sap. Both hydraulic conductivity and C are thus inextricably linked and the interaction between both should be studied to better understand hydraulic functioning of woody species during drought. However, measurements of C are scarce and no distinction is usually made between C from elastic storage and C supplied by cavitation. In this paper, we propose a new method to assess both the decrease in hydraulic conductivity and the change in C during bench dehydration of a whole-branch segment using continuous measurements of acoustic emissions, radial diameter shrinkage and gravimetrical water loss. With this method we could establish proper vulnerability curves for grapevine (Vitis vinifera L. 'Johanniter') and quantify C during dehydration. Our results showed that loss in hydraulic conductivity during the cavitation phase was accompanied by 22-92% gain in hydraulic capacitance; therefore, a certain degree of cavitation may be tolerated in grapevine during periods of drought stress.

Wood represents one of the most important renewable commodities for humanity and plays a crucial role in terrestrial ecosystem carbon-cycling. Wood formation is the result of a multitude of events that require the concerted action of endogenous and exogenous factors under the influence of photoperiod, for instance genes and plant growth regulators. Beyond providing mechanical support and being responsible for the increase in stem radial diameter, woody tissues constitute the vascular system of trees and are capable of reacting to environmental stimuli, and as such are therefore quite plastic and responsive. Despite the ecological and economic importance of wood, not all aspects of its formation have been unveiled. Many gaps in our knowledge are still present, which hinder the maximal exploitation of this precious bioresource. This review aims at surveying the current knowledge of wood formation and the available molecular data addressing the relationship between wood production and environmental factors, which have crucial influences on the rhythmic regulation of cambial activity and exert profound effects on tree stem growth, wood yield and properties. We will here go beyond wood sensu stricto, i.e., secondary xylem, and extend our survey to other tissues, namely vascular cambium, phloem and fibres. The purpose is to provide the reader with an overview of the complexity of the topic and to highlight the importance of progressing in the future towards an integrated knowledge on the subject.

Despite its major economic importance and the vulnerability of Picea abies (L.) H. Karst. to climate change, how its radial growth at intra-annual resolution is influenced by weather conditions in forest stands with a high production capacity has scarcely been explored. Between 2009 and 2011, phenological variation in seasonal cambial cell production (CP) was analysed in adult P. abies trees from three contrasting sites, differing in altitude and latitude. The results indicate that the timing of cambial CP is a highly synchronic process within populations since in all cases the cambium simultaneously started and stopped producing xylem and phloem cells. Our results also demonstrate that the phenology of cambial CP is highly variable and plastic between years, depending on seasonal temperature and precipitation variation. Differences among sites, however, are only partially explained by different environmental (elevation and altitude) and climatic conditions, suggesting that local adaptation may also play a decisive role in the strategy of P. abies for adapting wood and phloem increments to function optimally under local conditions.

International audience; Secondary xylem (wood) is formed through an intricate biological process that results in a highly variable final product. Studies have focused on understanding the molecular events for wood formation in conifers. In this process environmental, ontogenic and genetic factors influence variation in wood characteristics, including anatomical, chemical and physical properties. The main objective of this study was to analyse the ageing (ontogenic) effect on protein accumulation in wood-forming tissues along a cambial age (CA) gradient, ranging from juvenile wood (JW) sampled at the top of the tree, to mature wood (MW) sampled at the bottom of the tree. A total of 62 proteins whose accumulation varied by at least 1.5-fold according to CA were selected and identified by ESI-MS/MS; 30 of these were more abundant in MW and 32 were more abundant in JW. Consistent with earlier findings, our results show that JW is a tissue characterized by a high energy demand with the accumulation of gene products involved in energy, protein fate and cellular transport, while proteins identified in MW (heat shock response, oxygen and radical detoxification, and the S-adenosyl methionine cycle) support the idea that this tissue undergoes extended cell-wall thickening and a delay of programmed cell death.

Low water potentials in xylem can result in damaging levels of cavitation, yet little is understood about which hydraulic traits have most influence in delaying the onset of hydraulic dysfunction during periods of drought. We examined three traits contributing to longer desiccation times in excised shoots of 11 species from two sites of contrasting aridity: (i) the amount of water released from plant tissues per decrease in xylem water potential (WΨ); (ii) the minimum xylem water potential preceding acute water stress (defined as P50L; water potential at 50% loss of leaf conductance); and (iii) the integrated transpiration rate between the points of full hydration and P50L (Wtime). The time required for species to reach P50L varied markedly, ranging from 1.3 h to nearly 3 days. WΨ, P50L and Wtime all contributed significantly to longer desiccation times, explaining 28, 22 and 50% of the variance in the time required to reach P50L. Interestingly, these three traits were nearly orthogonal to one another, suggesting that they do not represent alternative hydraulic strategies, but likely trade off with other ecological strategies not evaluated in this study. The majority of water lost during desiccation (60-91%) originated from leaves, suggesting an important role for leaf capacitance in small plants when xylem water potentials decrease below -2 MPa.

The analysis of xylem cell anatomical features in dated tree rings provides insights into xylem functional responses and past growth conditions at intra-annual resolution. So far, special focus has been given to the lumen of the water-conducting cells, whereas the equally relevant cell wall thickness (CWT) has been less investigated due to methodological limitations. Here we present a novel approach to measure tracheid CWT in high-resolution images of wood cross-sections that is implemented within the specialized image-analysis tool 'ROXAS'. Compared with the traditional manual line measurements along a selection of few radial files, this novel image-analysis tool can: (i) measure CWT of all tracheids in a tree-ring cross-section, thus increasing the number of individual tracheid measurements by a factor of ~10-20; (ii) measure the tangential and radial walls separately; and (iii) laterally integrate the measurements in a customizable way from only the thinnest central part of the cell walls up to the thickest part of the tracheids at the corners. Cell wall thickness measurements performed with our novel approach and the traditional manual approach showed comparable accuracy for several image resolutions, with an optimal accuracy-efficiency balance at 100× magnification. The configurable settings intended to underscore different cell wall properties indeed changed the absolute levels and intra- and inter-annual patterns of CWT. This versatility, together with the high data production capacity, allows to tailor the measurements of CWT to the specific goal of each study, which opens new research perspectives, e.g., for investigating structure-function relationships, tree stress responses and carbon allocation patterns, and for reconstructing climate based on intra- and inter-annual variability of anatomical wood density.