Differences in mineral and osmotic balances enhance zinc translocation in an aquaporin overexpressing poplar.

Zinc (Zn) is an essential micronutrient for plants, but it is toxic beyond a certain threshold. Populus alba (L.) 'Villafranca' clone is known for its good tolerance to high Zn concentration compared to other poplar species. A line of this species overexpressing the tonoplast intrinsic aquaporin AQUA1 gene has showed an improved tolerance to Zn excess in comparison to the wild-type (wt) line. The aims of this work were to: 1) verify if AQUA1 plants can uptake Zn more efficiently after a longer period of exposure; 2) evaluate if a higher Zn uptake in transgenic lines can have negative effects; 3) assess Zn competing elements (iron and manganese), soluble sugars, osmolytes, and potassium to investigate differences in water and osmotic homeostasis between lines. Under Zn excess, AQUA1 plants showed a twofold Zn translocation factor and a higher xylem sap Zn concentration than the wt plants. Transgenic plants preferentially allocated Zn in aerial biomass and this different behaviour matched with modified manganese and iron balances suggesting that the increased Zn uptake might be related to a decrease in iron transport in the transgenic line. Moreover, a higher instantaneous water use efficiency in control conditions and an increase in bark soluble sugars under Zn excess could allow a higher resistance of AQUA1 plants to the water and osmotic perturbations caused by Zn. Indeed, the Zn excess increased the xylem osmolyte content only in wt plants. Further investigations are required to understand the role of AQUA1 in osmotic regulation.

High preseason temperature variability drives convergence of xylem phenology in the Northern Hemisphere conifers.

Wood growth is key to understanding the feedback of forest ecosystems to the ongoing climate warming. An increase in spatial synchrony (i.e., coincident changes in distant populations) of spring phenology is one of the most prominent climate responses of forest trees. However, whether temperature variability contributes to an increase in the spatial synchrony of spring phenology and its underlying mechanisms remains largely unknown. Here, we analyzed an extensive dataset of xylem phenology observations of 20 conifer species from 75 sites over the Northern Hemisphere. Along the gradient of increase in temperature variability in the 75 sites, we observed a convergence in the onset of cell enlargement roughly toward the 5th of June, with a convergence in the onset of cell wall thickening toward the summer solstice. The increase in rainfall since the 5th of June is favorable for cell division and expansion, and as the most hours of sunlight are received around the summer solstice, it allows the optimization of carbon assimilation for cell wall thickening. Hence, the convergences can be considered as the result of matching xylem phenological activities to favorable conditions in regions with high temperature variability. Yet, forest trees relying on such consistent seasonal cues for xylem growth could constrain their ability to respond to climate warming, with consequences for the potential growing season length and, ultimately, forest productivity and survival in the future.

The admixture of Quercus sp. in Pinus sylvestris stands influences wood anatomical trait responses to climatic variability and drought events.

Introduction: Forests are threatened by increasingly severe and more frequent drought events worldwide. Mono-specific forests, developed as a consequence of widespread management practices established early last century, seem particularly susceptible to global warming and drought compared with mixed-species forests. Although, in several contexts, mixed-species forests display higher species diversity, higher productivity, and higher resilience, previous studies highlighted contrasting findings, with not only many positive but also neutral or negative effects on tree performance that could be related to tree species diversity. Processes underlying this relationship need to be investigated. Wood anatomical traits are informative proxies of tree functioning, and they can potentially provide novel long-term insights in this regard. However, wood anatomical traits are critically understudied in such a context. Here, we assess the role of tree admixture on Pinus sylvestris L. xylem traits such as mean hydraulic diameter, cell wall thickness, and anatomical wood density, and we test the variability of these traits in response to climatic parameters such as temperature, precipitation, and drought event frequency and intensity. Methods: Three monocultural plots of P. sylvestris and three mixed-stand plots of P. sylvestris and Quercus sp. were identified in Poland and Spain, representing Continental and Mediterranean climate types, respectively. In each plot, we analyzed xylem traits from three P. sylvestris trees, for a total of nine trees in monocultures and nine in mixed stands per study location. Results: The results highlighted that anatomical wood density was one of the most sensitive traits to detect tree responses to climatic conditions and drought under different climate and forest types. Inter-specific facilitation mechanisms were detected in the admixture between P. sylvestris and Quercus sp., especially during the early growing season and during stressful events such as spring droughts, although they had negligible effects in the late growing season. Discussion: Our findings suggest that the admixture between P. sylvestris and Quercus sp. increases the resilience of P. sylvestris to extreme droughts. In a global warming scenario, this admixture could represent a useful adaptive management option.

A critical thermal transition driving spring phenology of Northern Hemisphere conifers.

Despite growing interest in predicting plant phenological shifts, advanced spring phenology by global climate change remains debated. Evidence documenting either small or large advancement of spring phenology to rising temperature over the spatio-temporal scales implies a potential existence of a thermal threshold in the responses of forests to global warming. We collected a unique data set of xylem cell-wall-thickening onset dates in 20 coniferous species covering a broad mean annual temperature (MAT) gradient (-3.05 to 22.9°C) across the Northern Hemisphere (latitudes 23°-66° N). Along the MAT gradient, we identified a threshold temperature (using segmented regression) of 4.9 ± 1.1°C, above which the response of xylem phenology to rising temperatures significantly decline. This threshold separates the Northern Hemisphere conifers into cold and warm thermal niches, with MAT and spring forcing being the primary drivers for the onset dates (estimated by linear and Bayesian mixed-effect models), respectively. The identified thermal threshold should be integrated into the Earth-System-Models for a better understanding of spring phenology in response to global warming and an improved prediction of global climate-carbon feedbacks.

Xylem anatomy and hydraulic traits in Vitis grafted cuttings in view of their impact on the young grapevine decline.

Grapevine grafting is an essential practice in viticulture and over the years, various bench grafting techniques have been developed to mechanize the nursery process and to increase the yield in number of viable cuttings. Bench grafting is a fundamental nursery practice that can potentially affect the quality of propagation material also in young decline associated to grapevine trunk diseases and has been recently reported to influence leaf symptoms development associated with diseases of Esca complex. The study aimed to investigate how three bench grafting methods [i.e., (i) Omega graft as mechanical technique, (ii) Whip and Tongue graft as manual technique and (iii) Full Cleft graft as semi-mechanical technique] can influence these phenomena. Specifically, the different methods were compared for their effect on the anatomical development of the grafting point and the functionality of the xylem, also considering two factors: the grapevine cultivar (Cabernet Sauvignon, Glera and Teroldego) and the scion/rootstock diameter (thin and large). Observations by light microscopy on the anatomical evolution and measurements on the xylem morphology and hydraulic traits were correlated with the grafting methods and the investigated varieties. The anatomical observations revealed that the mechanical (Omega) and semi-mechanical (Full Cleft) grafting methods have a faster callusing response while the manual technique (Whip and Tongue) has a slower but greater vascularization of the differentiated callus. Significant differences between cultivars and/or grafting types were also detected in necrotic area on the grafted tissues. Statistical analysis of the grapevine vessels suggested differences in xylem parameters between cultivars, while grafting type had no significant effects. On the other hand, the grafting type significantly affected the intrinsic growth rate. The study confirms the potential incidence of lesions and dysfunctionalities correlated with the grafting method applied, which can potentially induce grafted vine declines in vineyards due to the necrotic area detected on the grafted tissues.

Root Exposure to 5-Aminolevulinic Acid (ALA) Affects Leaf Element Accumulation, Isoprene Emission, Phytohormonal Balance, and Photosynthesis of Salt-Stressed Arundo donax.

Arundo donax has been recognized as a promising crop for biomass production on marginal lands due to its superior productivity and stress tolerance. However, salt stress negatively impacts A. donax growth and photosynthesis. In this study, we tested whether the tolerance of A. donax to salinity stress can be enhanced by the addition of 5-aminolevulinic acid (ALA), a known promoter of plant growth and abiotic stress tolerance. Our results indicated that root exposure to ALA increased the ALA levels in leaves along the A. donax plant profile. ALA enhanced Na+ accumulation in the roots of salt-stressed plants and, at the same time, lowered Na+ concentration in leaves, while a reduced callose amount was found in the root tissue. ALA also improved the photosynthetic performance of salt-stressed apical leaves by stimulating stomatal opening and preventing an increase in the ratio between abscisic acid (ABA) and indol-3-acetic acid (IAA), without affecting leaf methanol emission and plant growth. Supply of ALA to the roots reduced isoprene fluxes from leaves of non-stressed plants, while it sustained isoprene fluxes along the profile of salt-stressed A. donax. Thus, ALA likely interacted with the methylerythritol 4-phosphate (MEP) pathway and modulate the synthesis of either ABA or isoprene under stressful conditions. Overall, our study highlights the effectiveness of ALA supply through soil fertirrigation in preserving the young apical developing leaves from the detrimental effects of salt stress, thus helping of A. donax to cope with salinity and favoring the recovery of the whole plant once the stress is removed.

Localized stem heating from the rest to growth phase induces latewood-like cell formation and slower stem radial growth in Norway spruce saplings.

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.

Tree Growth Conditions Are Demanded When Optimal, Are Unwanted When Limited, but When Are They Suboptimal?

The recent climate projections predict that the intensity and frequency of extreme events will increase as a result of overall increasing mean temperature and reduced precipitations in the temperate regions of the Northern Hemisphere. How these changes will influence the harshness of the environment and the performances of trees growing under natural conditions remains an open question. In this commentary article, we would like to look at the concept of suboptimal growth conditions, widening its application from the traditional in vitro manipulation to trees growing in open air, addressing the main limitations and strengths of the upscaling results from cell to tree. We believe that the traditional single dose-effect approach is not suitable to explain the complex interactions between genotype and environment, occurring in open field or forest stands, where the intensity and frequency of the events are uncontrolled and unpredictable. As forests provide a wide range of ecosystem services, new parameters should be considered in the definition of the response thresholds in addition to growth. Thus, within this Special Issue, we stimulate the discussion over the development of new approaches and technologies that are able to define suitable threshold responses of trees under suboptimal natural conditions, with the aim to furnish new insights on the acclimation and adaptation processes in woody species under global change.

Photoperiod and temperature as dominant environmental drivers triggering secondary growth resumption in Northern Hemisphere conifers.

Wood formation consumes around 15% of the anthropogenic CO2 emissions per year and plays a critical role in long-term sequestration of carbon on Earth. However, the exogenous factors driving wood formation onset and the underlying cellular mechanisms are still poorly understood and quantified, and this hampers an effective assessment of terrestrial forest productivity and carbon budget under global warming. Here, we used an extensive collection of unique datasets of weekly xylem tissue formation (wood formation) from 21 coniferous species across the Northern Hemisphere (latitudes 23 to 67°N) to present a quantitative demonstration that the onset of wood formation in Northern Hemisphere conifers is primarily driven by photoperiod and mean annual temperature (MAT), and only secondarily by spring forcing, winter chilling, and moisture availability. Photoperiod interacts with MAT and plays the dominant role in regulating the onset of secondary meristem growth, contrary to its as-yet-unquantified role in affecting the springtime phenology of primary meristems. The unique relationships between exogenous factors and wood formation could help to predict how forest ecosystems respond and adapt to climate warming and could provide a better understanding of the feedback occurring between vegetation and climate that is mediated by phenology. Our study quantifies the role of major environmental drivers for incorporation into state-of-the-art Earth system models (ESMs), thereby providing an improved assessment of long-term and high-resolution observations of biogeochemical cycles across terrestrial biomes.

Osmotic adjustments support growth of poplar cultured cells under high concentrations of carbohydrates.

KEY MESSAGE: Poplar callus maintained a specific difference in osmotic potential with respect to media when supplemented with different carbohydrate concentrations. This balance in osmotic potential guaranteed the growth capacity. Osmotic stress is caused by several abiotic factors such as drought, salinity, or freezing. However, the threshold of osmotic potential that allows the growth under stress conditions has not been thoroughly studied. In this study, different levels of osmotic stress in Populus alba (L.) callus have been induced with the addition of mannitol or sorbitol in the medium (from 0 to 500 mM). The key factor for preserving the growth was observed to be the restoration of a constant difference in osmotic potential between callus and medium for all the tested conditions. The osmotic adjustments were primarily achieved with the uptake of mannitol or sorbitol from the media considering their chemical properties instead of their biological functions. The decrease in water content (from - 1 to - 10% after 21 days) and mineral elements, such as potassium, calcium, and magnesium, together with the alterations in cell morphology, did not show negative effects on growth. The activity of sorbitol dehydrogenase was detected for the first time in poplar (+ 4.7 U l-1 in callus treated with sorbitol compared to control callus). This finding suggested the importance of choosing carefully the molecules used to exert osmotic stress for separating the dual function of carbohydrates in osmotic adjustments and cell metabolism.

Characterization of bio-oil and bio-char produced by low-temperature microwave-assisted pyrolysis of olive pruning residue using various absorbers.

Olive pruning residue is largely formed during cultivation, and is usually disposed through open-air combustion directly in the field, but this habit is a possible source of pollution. The pyrolytic conversion of olive pruning residue has been run in a new and very appealing way using microwave as a heating source and different microwave absorbers in a multimode batch reactor. In this way, olive residue is converted into interesting bio-chemical products with a short pyrolysis time, ranging from 15 to 36 min, and with a peak temperature ranging from 450 K to 705 K according to the different microwave absorber. Thus, a very efficient and selective system was realized, which was able to address the process towards the formation of a large amount of bio-char (up to 61.2%) or a high formation of bio-oil (56.2%) and gas (41.7%) with a very low formation of bio-char (2.1%). However, when carbon and iron were used as microwave absorbers, it was possible to obtain an intermediate amount of bio-char (26-30%) and bio-oil (40 wt%). Bio-oils were collected as dark-brown liquids with low viscosity and density. A bio-oil with a low water concentration was obtained using carbon or iron as the microwave absorber. The bio-oils formed in all experiments contained a very large amount of acetic acid, even when NaOH was the microwave absorber. Furthermore, a large amount of aromatics were present in the bio-oil obtained using carbon as the microwave absorber.

Daily osmotic adjustments in stem may be good predictors of water stress intensity in poplar.

Drought events impair the carbon and water balances in plants. Climate changes highlight the importance to understand the limits of woody species to reallocate carbon in different processes and the mechanisms driving the osmotic adjustments during the day under stress. In this frame, the aim of this work was to investigate the plant capability to shift energy among competing sinks and preserve the osmotic balance during the day under severe short periods of water deficit. The role of carbohydrates as osmolytes as well as energy sources was investigated in poplar plants. Results highlighted that during water deficit soluble sugars, derived both from the new synthetised carbon and starch degradation, were principally convoyed in the bark. This increase in carbohydrates allowed the maintenance of a water reserve used during the day to prevent a water decrease within the xylem. The decrease of xylem sap osmotic potential during the night, driven by an increase of K, Ca, and fructose (+0.46, 0.52, and 0.26 mg ml-1 in water limited plants after 8 days of withholding water, respectively), probably further attracted water into the xylem. This response mechanism increased at higher water deficit intensity. The little variations in carbohydrates and mineral elements within the leaves highlighted the main role of sinks rather than sources in the early response to water deficit.

Whey and molasses as inexpensive raw materials for parallel production of biohydrogen and polyesters via a two-stage bioprocess: New routes towards a circular bioeconomy.

Consecutive dark-fermentation and photo-fermentation stages were investigated for a profitable circular bio-economy. H2 photo-production versus poly(3-hydroxybutyrate) (P3HB) accumulation is a modern biotechnological approach to use agro-food industrial byproducts as no-cost rich-nutrient medium in eco-sustainable biological processes. Whey and molasses are very important byproducts rich in nutrients that lactic acid bacteria can convert, by dark-fermentation, in dark fermented effluents of whey (DFEW) and molasses (DFEM). These effluents are proper media for culturing purple non-sulfur bacteria, which are profitable producers of P3HB and H2. The results of the present study show that Lactobacillus sp. and Rhodopseudomonas sp. S16-VOGS3 are two representative genera for mitigation of environmental impact. The highest productivity of P3HB (4.445 mg/(L·h)) was achieved culturing Rhodopseudomonas sp. S16-VOGS3, when feeding the bacterium with 20% of DFEM; the highest H2 production rate of 4.46 mL/(L·h) was achieved when feeding the bacterium with 30% of DFEM.

Poly-3-hydroxybutyrate and H2 production by Rhodopseudomonas sp. S16-VOGS3 grown in a new generation photobioreactor under single or combined nutrient deficiency.

The main goal of this investigation was setting up a growth strategy to separate H2 evolution from P3HB synthesis in order to increase cumulative P3HB in Rhodopseudomonas cells. The accumulation of poly-3-hydroxybutyrate (P3HB) was investigated culturing Rhodopseudomonas sp. S16-VOGS3 with three carbon substrates either as acetate, butyrate or lactate and with two nitrogen sources either as ammonium or glutamate. The investigation was carried out under several stress conditions caused by single or double nutrient deficiency. The content of P3HB in cell dry weight (CDW) was 21.8% with lactate; 24.6% with acetate and 27.6% with butyrate under sulfur deficient conditions. The P3HB content increased significantly culturing Rhodopseudomonas sp. S16-VOGS3 with butyrate following three phases of growth: phase-1, nutrient sufficient conditions; phase-2, nitrogen-deficiency and phase-3, sulfur-deficient conditions. Under this last phase, the highest P3HB content was achieved (34.4% of CDW). A combined production of P3HB and molecular H2 was obtained when Rhodopseudomonas sp. S16-VOGS3 was cultured with either acetate or butyrate under nitrogen sufficiency (glutamate) or nitrogen deficiency.

Dynamic changes in ABA content in water-stressed Populus nigra: effects on carbon fixation and soluble carbohydrates.

BACKGROUND AND AIMS: Hydraulic and chemical signals operate in tandem to regulate systemic plant responses to drought. Transport of abscisic acid (ABA) through the xylem and phloem from the root to shoot has been suggested to serve as the main signal of water deficit. There is evidence that ABA and its ABA-glycosyl-ester (ABA-GE) are also formed in leaves and stems through the chloroplastic 2-C-methylerythritol-5-phosphate (MEP) pathway. This study aimed to evaluate how hormonal and hydraulic signals contribute to optimize stomatal (gs), mesophyll (gm) and leaf hydraulic (Kleaf) conductance under well-watered and water-stressed conditions in Populus nigra (black poplar) plants. In addition, we assessed possible relationships between ABA and soluble carbohydrates within the leaf and stem. METHODS: Plants were subjected to three water treatments: well-watered (WW), moderate stress (WS1) and severe stress (WS2). This experimental set-up enabled a time-course analysis of the response to water deficit at the physiological [leaf gas exchange, plant water relations, (Kleaf)], biochemical (ABA and its metabolite/catabolite quantification in xylem sap, leaves, wood, bark and roots) and molecular (gene expression of ABA biosynthesis) levels. KEY RESULTS: Our results showed strong coordination between gs, gm and Kleaf under water stress, which reduced transpiration and increased intrinsic water use efficiency (WUEint). Analysis of gene expression of 9-cis-epoxycarotenoid dioxygenase (NCED) and ABA content in different tissues showed a general up-regulation of the biosynthesis of this hormone and its finely-tuned catabolism in response to water stress. Significant linear relationships were found between soluble carbohydrates and ABA contents in both leaves and stems, suggesting a putative function for this hormone in carbohydrate mobilization under severe water stress. CONCLUSIONS: This study demonstrates the tight regulation of the photosynthetic machinery by levels of ABA in different plants organs on a daily basis in both well-watered and water stress conditions to optimize WUEint and coordinate whole plant acclimation responses to drought.

Can sugar metabolism in the cambial region explain the water deficit tolerance in poplar?

Drought dramatically affects wood production by adversely impacting cambial cells and their derivatives. Photosynthesis and assimilate transport are also affected by drought conditions. Two poplar genotypes, Populus deltoides 'Dvina' and Populus alba 'Marte', demonstrated contrasting growth performance and water-carbon balance strategies; a mechanistic understanding of the water deficit response was provided by these poplar species. 'Marte' was found to be more anisohydric than 'Dvina'. This characteristic was associated with the capacity to reallocate carbohydrates during water deficits. In contrast, 'Dvina' displayed more conservative water management; carbohydrates were preferably stored or used for cellulose production rather than to achieve an osmotic balance between the phloem and the xylem. Data confirmed that the more 'risk-taking' characteristic of 'Marte' allowed a rapid recovery following water deficit and was connected to a different carbohydrate metabolism.

Monitoring intra-annual dynamics of wood formation with microcores and dendrometers in Picea abies at two different altitudes.

Seasonal analyses of cambial cell production and day-by-day stem radial increment can help to elucidate how climate modulates wood formation in conifers. Intra-annual dynamics of wood formation were determined with microcores and dendrometers and related to climatic signals in Norway spruce (Picea abies (L.) Karst.). The seasonal dynamics of these processes were observed at two sites of different altitude, Savignano (650 m a.s.l.) and Lavazè (1800 m a.s.l.) in the Italian Alps. Seasonal dynamics of cambial activity were found to be site specific, indicating that the phenology of cambial cell production is highly variable and plastic with altitude. There was a site-specific trend in the number of cells in the wall thickening phase, with the maximum cell production in early July (DOY 186) at Savignano and in mid-July (DOY 200) at Lavazè. The formation of mature cells showed similar trends at the two sites, although different numbers of cells and timing of cell differentiation were visible in the model shapes; at the end of ring formation in 2010, the number of cells was four times higher at Savignano (106.5 cells) than at Lavazè (26.5 cells). At low altitudes, microcores and dendrometers described the radial growth patterns comparably, though the dendrometer function underlined the higher upper asymptote of maximum growth in comparison with the cell production function. In contrast, at high altitude, these functions exhibited different trends. The best model was obtained by fitting functions of the Gompertz model to the experimental data. By combining radial growth and cambial activity indices we defined a model system able to synchronize these processes. Processes of adaptation of the pattern of xylogenesis occurred, enabling P. abies to occupy sites with contrasting climatic conditions. The use of daily climatic variables in combination with plant functional traits obtained by sensors and/or destructive sampling could provide a suitable tool to better investigate the effect of disturbances on response strategies in trees and, consequently, contribute to improving our prediction of tree growth and species resilience based on climate scenarios.

Compensatory mechanisms mitigate the effect of warming and drought on wood formation.

Because of global warming, high-latitude ecosystems are expected to experience increases in temperature and drought events. Wood formation will have to adjust to these new climatic constraints to maintain tree mechanical stability and long-distance water transport. The aim of this study is to understand the dynamic processes involved in wood formation under warming and drought. Xylogenesis, gas exchange, water relations and wood anatomy of black spruce [Picea mariana (Mill.) B.S.P.] saplings were monitored during a greenhouse experiment where temperature was increased during daytime or night-time (+6 °C) combined with a drought period. The kinetics of tracheid development expressed as rate and duration of the xylogenesis sub-processes were quantified using generalized additive models. Drought and warming had a strong influence on cell production, but little effect on wood anatomy. The increase in cell production rate under warmer temperatures, and especially during the night-time warming at the end of the growing season, resulted in wider tree-rings. However, the strong compensation between rates and durations of cell differentiation processes mitigates warming and drought effects on tree-ring structure. Our results allowed quantification of how wood formation kinetics is regulated when water and heat stress increase, allowing trees to adapt to future environmental conditions.

How do drought and warming influence survival and wood traits of Picea mariana saplings?

Warming and drought will occur with increased frequency and intensity at high latitudes in the future. How heat and water stress can influence tree mortality is incompletely understood. The aim of this study was to evaluate how carbon resources, stem hydraulics, and wood anatomy and density determine the ability of black spruce saplings to survive daytime or night-time warming (+ 6 °C in comparison with control) in combination with a drought period. Plant water relations, the dynamics of non-structural carbohydrates and starch, mortality rate, and wood anatomy and density of saplings were monitored. Warming, in conjunction with 25 d of water deficit, increased sapling mortality (10% and 20% in night-time and daytime warming, respectively) compared with the control conditions (0.8%). Drought substantially decreased gas exchange, and also pre-dawn and mid-day leaf water potential to values close to -3MPa which probably induced xylem embolism (xylem air entry point, P12, being on average around -3MPa for this species). In addition, the recovery of gas exchange never reached the initial pre-stress levels, suggesting a possible loss of xylem hydraulic conductivity associated with cavitation. Consequently, mortality may be due to xylem hydraulic failure. Warmer temperatures limited the replenishment of starch reserves after their seasonal minimum. Lighter wood was formed during the drought period, reflecting a lower carbon allocation to cell wall formation, preventing the adaptation of the hydraulic system to drought. Saplings of black spruce experienced difficulty in adapting under climate change conditions, which might compromise their survival in the future.