Net O2 exchange rates under dark and light conditions across different stem compartments.

Woody plants display some photosynthetic activity in stems, but the biological role of stem photosynthesis and the specific contributions of bark and wood to carbon uptake and oxygen evolution remain poorly understood. We aimed to elucidate the functional characteristics of chloroplasts in stems of different ages in Fraxinus ornus. Our investigation employed diverse experimental approaches, including microsensor technology to assess oxygen production rates in whole stem, bark, and wood separately. Additionally, we utilized fluorescence lifetime imaging microscopy (FLIM) to characterize the relative abundance of photosystems I and II (PSI : PSII chlorophyll ratio) in bark and wood. Our findings revealed light-induced increases in O2 production in whole stem, bark, and wood. We present the radial profile of O2 production in F. ornus stems, demonstrating the capability of stem chloroplasts to perform light-dependent electron transport. Younger stems exhibited higher light-induced O2 production and dark respiration rates than older ones. While bark emerged as the primary contributor to net O2 production under light conditions, our data underscored that wood chloroplasts are also photosynthetically active. The FLIM analysis unveiled a lower PSI abundance in wood than in bark, suggesting stem chloroplasts are not only active but also acclimate to the spectral composition of light reaching inner compartments.

Restricted O2 consumption in pea roots induced by hexanoic acid is linked to depletion of Krebs cycle substrates.

Plant roots are exposed to hypoxia in waterlogged soils, and they are further challenged by specific phytotoxins produced by microorganisms in such conditions. One such toxin is hexanoic acid (HxA), which, at toxic levels, causes a strong decline in root O2 consumption. However, the mechanism underlying this process is still unknown. We treated pea (Pisum sativum L.) roots with 20 mM HxA at pH 5.0 and 6.0 for a short time (1 h) and measured leakage of key electrolytes such as metal cations, malate, citrate and nonstructural carbohydrates (NSC). After treatment, mitochondria were isolated to assess their functionality evaluated as electrical potential and O2 consumption rate. HxA treatment resulted in root tissue extrusion of K+ , malate, citrate and NSC, but only the leakage of the organic acids and NSC increased at pH 5.0, concomitantly with the inhibition of O2 consumption. The activity of mitochondria isolated from treated roots was almost unaffected, showing just a slight decrease in oxygen consumption after treatment at pH 5.0. Similar results were obtained by treating the pea roots with another organic acid with a short carbon chain, that is, butyric acid. Based on these results, we propose a model in which HxA, in its undissociated form prevalent at acidic pH, stimulates the efflux of citrate, malate and NSC, which would, in turn, cause starvation of mitochondrial respiratory substrates of the Krebs cycle and a consequent decline in O2 consumption. Cation extrusion would be a compensatory mechanism in order to restore plasma membrane potential.

Contrasting Responses of Two Grapevine Cultivars to Drought: The Role of Non-structural Carbohydrates in Xylem Hydraulic Recovery.

Xylem embolism is one of the possible outcomes of decreasing xylem pressure when plants face drought. Recent studies have proposed a role for non-structural carbohydrates (NSCs) in osmotic pressure generation, required for refilling embolized conduits. Potted cuttings of grapevine Grenache and Barbera, selected for their adaptation to different climatic conditions, were subjected to a drought stress followed by re-irrigation. Stem embolism rate and its recovery were monitored in vivo by X-ray micro-computed tomography (micro-CT). The same plants were further analyzed for xylem conduit dimension and NSC content. Both cultivars significantly decreased Ψpd in response to drought and recovered from xylem embolism after re-irrigation. However, although the mean vessel diameter was similar between the cultivars, Barbera was more prone to embolism. Surprisingly, vessel diameter was apparently reduced during recovery in this cultivar. Hydraulic recovery was linked to sugar content in both cultivars, showing a positive relationship between soluble NSCs and the degree of xylem embolism. However, when starch and sucrose concentrations were considered separately, the relationships showed cultivar-specific and contrasting trends. We showed that the two cultivars adopted different NSC-use strategies in response to drought, suggesting two possible scenarios driving conduit refilling. In Grenache, sucrose accumulation seems to be directly linked to embolism formation and possibly sustains refilling. In Barbera, maltose/maltodextrins could be involved in a conduit recovery strategy via the formation of cell-wall hydrogels, likely responsible for the reduction of conduit lumen detected by micro-CT.

Flooding and Soil Properties Control Plant Intra- and Interspecific Interactions in Salt Marshes.

The stress gradient hypothesis (SGH) states that plant-plant interactions shift from competition to facilitation in increasing stress conditions. In salt marshes, edaphic properties can weaken the application of the SGH by amplifying the intensity of flooding and controlling plant zonation. We identified facilitative and competitive interactions along flooding gradients and tested the role of edaphic properties in exacerbating stress and shaping plant-plant interactions. Morphological traits of two target halophytes (Limonium narbonense and Sarcocornia fruticosa), flooding intensity, soil texture and soil organic C were recorded. The relative plant fitness index was assessed for the two species based on the relative growth in plurispecific rather than monospecific plant communities. Plant fitness increased with increasing stress supporting the SGH. L. narbonense showed larger fitness in plurispecific stands whereas S. fruticosa performed better in conspecific stands. Significant intra- or interspecific interactions were observed along the stress gradient defined by the combination of flooding and clay content in soil. When considering the limited soil organic C as stressor, soil properties were more important than flooding in defining plant-plant interactions. We highlight the need for future improvements of the SGH approach by including edaphic stressors in the model and their possible interactions with the main abiotic drivers of zonation.

Transient Effects of Snow Cover Duration on Primary Growth and Leaf Traits in a Tundra Shrub.

With the recent climate warming, tundra ecotones are facing a progressive acceleration of spring snowpack melting and extension of the growing season, with evident consequences to vegetation. Along with summer temperature, winter precipitation has been recently recognised as a crucial factor for tundra shrub growth and physiology. However, gaps of knowledge still exist on long-living plant responses to different snowpack duration, especially on how intra-specific and year-to-year variability together with multiple functional trait adjustments could influence the long-term responses. To fill this gap, we conducted a 3 years snow manipulation experiment above the Alpine treeline on the typical tundra species Juniperus communis, the conifer with the widest distributional range in the north emisphere. We tested shoot elongation, leaf area, stomatal density, leaf dry weight and leaf non-structural carbohydrate content of plants subjected to anticipated, natural and postponed snowpack duration. Anticipated snowpack melting enhanced new shoot elongation and increased stomatal density. However, plants under prolonged snow cover seemed to compensate for the shorter growing period, likely increasing carbon allocation to growth. In fact, these latter showed larger needles and low starch content at the beginning of the growing season. Variability between treatments slightly decreased over time, suggesting a progressive acclimation of juniper to new conditions. In the context of future warming scenarios, our results support the hypothesis of shrub biomass increase within the tundra biome. Yet, the picture is still far from being complete and further research should focus on transient and fading effects of changing conditions in the long term.

Shade-induced reduction of stem nonstructural carbohydrates increases xylem vulnerability to embolism and impedes hydraulic recovery in Populus nigra.

Nonstructural carbohydrates (NSCs) have been suggested to affect xylem transport under fluctuating water availability, but conclusive evidence is still lacking. We tested the effect of shade-induced NSC depletion on xylem vulnerability to embolism and hydraulic recovery on Populus nigra saplings. Vulnerability was assessed in light-exposed (L) and shaded (S) plants with the hydraulic method, and in vivo with the optical method and X-ray micro-computed tomography. Plants were stressed to 80% loss of hydraulic conductance (PLC) and re-irrigated to check for possible recovery. We measured PLC, bark and wood NSC content, as well as xylem sap pH, surface tension (γsap ) and sugar concentration, before, during and after drought. Shading induced depletion of stem NSC (mainly starch) reserves. All methods converged in indicating higher xylem vulnerability in S than in L plants. This difference was not explained by xylem vessel and pit anatomy or by γsap . Shading impeded sap acidification and sugar accumulation during drought in S plants and prevented hydraulic recovery, which was observed in L plants. Our results highlight the importance of stem NSCs to sustain xylem hydraulic functioning during drought and suggest that light and/or adequate stem NSC thresholds are required to trigger xylem sap chemical changes involved in embolism recovery.

Relationships between population traits, nonstructural carbohydrates, and elevation in alpine stands of Vaccinium myrtillus.

PREMISE: Despite great attention given to the relationship between plant growth and carbon balance in alpine tree species, little is known about shrubs at the treeline. We hypothesized that the pattern of main nonstructural carbohydrates (NSCs) across elevations depends on the interplay between phenotypic trait plasticity, plant-plant interaction, and elevation. METHODS: We studied the pattern of NSCs (i.e., glucose, fructose, sucrose, and starch) in alpine stands of Vaccinium myrtillus (above treeline) across an elevational gradient. In the same plots, we measured key growth traits (i.e., anatomical stem features) and shrub cover, evaluating putative relationships with NSCs. RESULTS: Glucose content was positively related with altitude, but negatively related with shrub cover. Sucrose decreased at high altitude and in older populations and increased with higher percentage of vascular tissue. Starch content increased at middle and high elevations and in stands with high shrub cover. Moreover, starch content was negatively related with the number of xylem rings and the percentage of phloem tissue, but positively correlated with the percentage of xylem tissue. CONCLUSIONS: We found that the increase in carbon reserves across elevations was uncoupled from plant growth, supporting the growth limitation hypothesis, which postulates NSCs accumulate at high elevation as a consequence of low temperature. Moreover, the response of NSC content to the environmental stress caused by elevation was buffered by phenotypic plasticity of plant traits, suggesting that, under climate warming conditions, shrub expansion due to enhanced plant growth would be pronounced in old but sparse stands.

Analysis of Non-Structural Carbohydrates and Xylem Anatomy of Leaf Petioles Offers New Insights in the Drought Response of Two Grapevine Cultivars.

In grapevine, the anatomy of xylem conduits and the non-structural carbohydrates (NSCs) content of the associated living parenchyma are expected to influence water transport under water limitation. In fact, both NSC and xylem features play a role in plant recovery from drought stress. We evaluated these traits in petioles of Cabernet Sauvignon (CS) and Syrah (SY) cultivars during water stress (WS) and recovery. In CS, the stress response was associated to NSC consumption, supporting the hypothesis that starch mobilization is related to an increased supply of maltose and sucrose, putatively involved in drought stress responses at the xylem level. In contrast, in SY, the WS-induced increase in the latter soluble NSCs was maintained even 2 days after re-watering, suggesting a different pattern of utilization of NSC resources. Interestingly, the anatomical analysis revealed that conduits are constitutively wider in SY in well-watered (WW) plants, and that water stress led to the production of narrower conduits only in this cultivar.

The Possible Role of Non-Structural Carbohydrates in the Regulation of Tree Hydraulics.

The xylem is a complex system that includes a network of dead conduits ensuring long-distance water transport in plants. Under ongoing climate changes, xylem embolism is a major and recurrent cause of drought-induced tree mortality. Non-structural carbohydrates (NSC) play key roles in plant responses to drought and frost stress, and several studies putatively suggest their involvement in the regulation of xylem water transport. However, a clear picture on the roles of NSCs in plant hydraulics has not been drawn to date. We summarize the current knowledge on the involvement of NSCs during embolism formation and subsequent hydraulic recovery. Under drought, sugars are generally accumulated in xylem parenchyma and in xylem sap. At drought-relief, xylem functionality is putatively restored in an osmotically driven process involving wood parenchyma, xylem sap and phloem compartments. By analyzing the published data on stem hydraulics and NSC contents under drought/frost stress and subsequent stress relief, we found that embolism build-up positively correlated to stem NSC depletion, and that the magnitude of post-stress hydraulic recovery positively correlated to consumption of soluble sugars. These findings suggest a close relationship between hydraulics and carbohydrate dynamics. We call for more experiments on hydraulic and NSC dynamics in controlled and field conditions.

Non-structural carbohydrate and hydraulic dynamics during drought and recovery in Fraxinus ornus and Ostrya carpinifolia saplings.

The maintenance of hydraulic function during and after a drought event is crucial for tree survival, but the importance of non-structural carbohydrates (NSCs) in the recovery phase is still debated. We tested whether higher NSC availability facilitates post-drought hydraulic recovery, by applying a short-term drought (Sdr) and a long-term drought combined with shading (Ldr+sh) in Fraxinus ornus and Ostrya carpinifolia. Plants were then re-irrigated and recovery was checked 24 h later, by measuring water potential, stem percentage loss of hydraulic conductance (PLC) and NSC content. The relative magnitude of hydraulic and carbon constraints was also assessed in desiccated plants. During drought, PLC increased only in F. ornus, while it was maintained almost constant in O. carpinifolia due to tighter stomatal control of xylem pressure (i.e. more isohydric). In F. ornus, only Sdr plants maintained high NSC contents at the end of drought and, when re-irrigated, recovered PLC to control values. Whereas hydraulic failure was ubiquitous, only F. ornus depleted NSC reserves at mortality. Our results suggest that preserving higher NSC content at the end of a drought can be important for the hydraulic resilience of trees.

Drought-induced dieback of Pinus nigra: a tale of hydraulic failure and carbon starvation.

Ongoing climate change is apparently increasing tree mortality rates, and understanding mechanisms of drought-induced tree decline can improve mortality projections. Differential drought impact on conspecific individuals within a population has been reported, but no clear mechanistic explanation for this pattern has emerged. Following a severe drought (summer 2012), we monitored over a 3-year period healthy (H) and declining (D) Pinus nigra trees co-occurring in a karstic woodland to highlight eventual individual-specific physiological differences underlying differential canopy dieback. We investigated differences in water and carbon metabolism, and xylem anatomy as a function of crown health status, as well as eventual genotypic basis of contrasting drought responses. H and D trees exploited the same water pools and relied on similar hydraulic strategies to cope with drought stress. Genetic analyses did not highlight differences between groups in terms of geographical provenance. Hydraulic and anatomical analyses showed conflicting results. The hydraulic tracheid diameter and theoretical hydraulic conductivity were similar, but D trees were characterized by lower water transport efficiency, greater vulnerability to xylem conduit implosion and reduced carbohydrate stores. Our results suggest that extreme drought events can have different impacts on conspecific individuals, with differential vulnerability to xylem embolism likely playing a major role in setting the fate of trees under climate change.

Hydraulic recovery from xylem embolism in excised branches of twelve woody species: Relationships with parenchyma cells and non-structural carbohydrates.

Embolism repair ability has been documented in numerous species. Although the actual mechanism driving this phenomenon is still debated, experimental findings suggest that non-structural carbohydrates (NSC) stored in wood parenchyma would provide the osmotic forces to drive the refilling of embolized conduits. We selected 12 broadleaved species differing in vulnerability to xylem embolism (P50) and amount of wood parenchyma in order to check direct evidence about the possible link(s) between parenchyma cells abundance, NSC availability and species-specific capacity to reverse xylem embolism. Branches were dehydrated until ∼50% loss of hydraulic conductivity was recorded (PLC ∼50%). Hydraulic recovery (ΔPLC) and NSC content was, then, assessed after 1h of rehydration. Species showed a different ability to recover their hydraulic conductivity from PLC∼50%. Removing the bark in the species showing hydraulic recovery inhibited the embolism reversal. Strong correlations between the ΔPLC and: a) the amount of parenchyma cells (mainly driven by the pith area), b) the consumption of soluble NSC have been recorded. Our results support the hypothesis that refilling of embolized vessels is mediated by the mobilization of soluble NSC and it is mainly recorded in species with a higher percentage of parenchyma cells that may be important in the hydraulic recovery mechanism as a source of carbohydrates and/or as a source of water.

Priming xylem for stress recovery depends on coordinated activity of sugar metabolic pathways and changes in xylem sap pH.

Some plant species are capable of significant reduction of xylem embolism during recovery from drought despite stem water potential remains negative. However, the functional biology underlying this process is elusive. We subjected poplar trees to drought stress followed by a period of recovery. Water potential, hydraulic conductivity, gas exchange, xylem sap pH, and carbohydrate content in sap and woody stems were monitored in combination with an analysis of carbohydrate metabolism, enzyme activity, and expression of genes involved in sugar metabolic and transport pathways. Drought resulted in an alteration of differential partitioning between starch and soluble sugars. Upon stress, an increase in the starch degradation rate and the overexpression of sugar symporter genes promoted the efflux of disaccharides (mostly maltose and sucrose) to the apoplast. In turn, the efflux activity of the sugar-proton cotransporters caused a drop in xylem pH. The newly acidic environment induced the activity of apoplastic invertases leading to the accumulation of monosaccharides in the apoplast, thus providing the main osmoticum necessary for recovery. During drought and recovery, a complex network of coordinated molecular and biochemical signals was activated at the interface between xylem and parenchyma cells that appeared to prime the xylem for hydraulic recovery.

Properties of the Permeability Transition of Pea Stem Mitochondria.

In striking analogy with Saccharomyces cerevisiae, etiolated pea stem mitochondria did not show appreciable Ca2+ uptake. Only treatment with the ionophore ETH129 (which allows electrophoretic Ca2+ equilibration) caused Ca2+ uptake followed by increased inner membrane permeability, membrane depolarization and Ca2+ release. Like the permeability transition (PT) of mammals, yeast and Drosophila, the PT of pea stem mitochondria was stimulated by diamide and phenylarsine oxide and inhibited by Mg-ADP and Mg-ATP, suggesting a common underlying mechanism; yet, the plant PT also displayed distinctive features: (i) as in mammals it was desensitized by cyclosporin A, which does not affect the PT of yeast and Drosophila; (ii) similarly to S. cerevisiae and Drosophila it was inhibited by Pi, which stimulates the PT of mammals; (iii) like in mammals and Drosophila it was sensitized by benzodiazepine 423, which is ineffective in S. cerevisiae; (iv) like what observed in Drosophila it did not mediate swelling and cytochrome c release, which is instead seen in mammals and S. cerevisiae. We find that cyclophilin D, the mitochondrial receptor for cyclosporin A, is present in pea stem mitochondria. These results indicate that the plant PT has unique features and suggest that, as in Drosophila, it may provide pea stem mitochondria with a Ca2+ release channel.

Shrub growth and plant diversity along an elevation gradient: Evidence of indirect effects of climate on alpine ecosystems.

Enhanced shrub growth and expansion are widespread responses to climate warming in many arctic and alpine ecosystems. Warmer temperatures and shrub expansion could cause major changes in plant community structure, affecting both species composition and diversity. To improve our understanding of the ongoing changes in plant communities in alpine tundra, we studied interrelations among climate, shrub growth, shrub cover and plant diversity, using an elevation gradient as a proxy for climate conditions. Specifically, we analyzed growth of bilberry (Vaccinium myrtillus L.) and its associated plant communities along an elevation gradient of ca. 600 vertical meters in the eastern European Alps. We assessed the ramet age, ring width and shoot length of V. myrtillus, and the shrub cover and plant diversity of the community. At higher elevation, ramets of V. myrtillus were younger, with shorter shoots and narrower growth rings. Shoot length was positively related to shrub cover, but shrub cover did not show a direct relationship with elevation. A greater shrub cover had a negative effect on species richness, also affecting species composition (beta-diversity), but these variables were not influenced by elevation. Our findings suggest that changes in plant diversity are driven directly by shrub cover and only indirectly by climate, here represented by changes in elevation.

Effects of prolonged drought on stem non-structural carbohydrates content and post-drought hydraulic recovery in Laurus nobilis L.: The possible link between carbon starvation and hydraulic failure.

Drought-induced tree decline is a complex event, and recent hypotheses suggest that hydraulic failure and carbon starvation are co-responsible for this process. We tested the possible role of non-structural carbohydrates (NSC) content on post-drought hydraulic recovery, to verify the hypothesis that embolism reversal represents a mechanistic link between carbon starvation and stem hydraulics. Measurements were performed in laurel plants subjected to similar water stress levels either over short or long term, to induce comparable embolism levels. Plants subjected to mild and prolonged water shortage (S) showed reduced growth, adjustment of turgor loss point driven by changes in both osmotic potential at full turgor and bulk modulus of elasticity, a lower content of soluble NSC and a higher content of starch with respect to control (C) plants. Moreover, S plants showed a lower ability to recover from xylem embolism than C plants, even after irrigation. Our data suggest that plant carbon status might indirectly influence plant performance during and after drought via effects on xylem hydraulic functioning, supporting the view of a possible mechanistic link between the two processes.

Contrasting oxygen dynamics in Limonium narbonense and Sarcocornia fruticosa during partial and complete submergence.

Terrestrial saltmarsh plants inhabiting flood-prone habitats undergo recurrent and prolonged flooding driven by tidal regimes. In this study, the role of internal plant aeration in contrasting hypoxic/anoxic conditions during submergence was investigated in the two halophytes Limonium narbonense Mill. and Sarcocornia fruticosa (L.) A.J. Scott. Monitoring of tissue O2 dynamics was performed in shoots and roots using microelectrodes under drained conditions, waterlogging, partial and complete submergence, in light or darkness. For both species, submergence in darkness resulted in significant declines in tissue O2 status and when in light, in rapid O2 increases first in shoot tissues and subsequently in roots. During partial submergence, S. fruticosa benefitted from snorkelling and efficiently transported O2 to roots, whereas the O2 concentration in roots of L. narbonense declined by more than 90%. Significantly thinner leaves and articles were recorded under high degree of flooding stress and both species showed considerably high tissue porosity. The presence of aerenchyma seemed to support internal aeration in S. fruticosa whereas O2 diffusion in L. narbonense seemed impeded, despite the higher porosity (up to 50%). Thus, the results obtained for L. narbonense, being well adapted to flooding, suggests that processes other than internal aeration could be involved in better flooding tolerance e.g. fermentative processes, and that traits resulting in flooding tolerance in plants are not yet fully understood.

Flavonoids and darkness lower PCD in senescing Vitis vinifera suspension cell cultures.

BACKGROUND: Senescence is a key developmental process occurring during the life cycle of plants that can be induced also by environmental conditions, such as starvation and/or darkness. During senescence, strict control of genes regulates ordered degradation and dismantling events, the most remarkable of which are genetically programmed cell death (PCD) and, in most cases, an upregulation of flavonoid biosynthesis in the presence of light. Flavonoids are secondary metabolites that play multiple essential roles in development, reproduction and defence of plants, partly due to their well-known antioxidant properties, which could affect also the same cell death machinery. To understand further the effect of endogenously-produced flavonoids and their interplay with different environment (light or dark) conditions, two portions (red and green) of a senescing grapevine callus were used to obtain suspension cell cultures. Red Suspension cell Cultures (RSC) and Green Suspension cell Cultures (GSC) were finally grown under either dark or light conditions for 6 days. RESULTS: Darkness enhanced cell death (mainly necrosis) in suspension cell culture, when compared to those grown under light condition. Furthermore, RSC with high flavonoid content showed a higher viability compared to GSC and were more protected toward PCD, in accordance to their high content in flavonoids, which might quench ROS, thus limiting the relative signalling cascade. Conversely, PCD was mainly occurring in GSC and further increased by light, as it was shown by cytochrome c release and TUNEL assays. CONCLUSIONS: Endogenous flavonoids were shown to be good candidates for exploiting an efficient protection against oxidative stress and PCD induction. Light seemed to be an important environmental factor able to induce PCD, especially in GSC, which lacking of flavonoids were not capable of preventing oxidative damage and signalling leading to senescence.

Species-specific reversal of stem xylem embolism after a prolonged drought correlates to endpoint concentration of soluble sugars.

Recent reports on tree mortality associated with anomalous drought and heat have raised interest into processes underlying tree resistance/resilience to water stress. Hydraulic failure and carbon starvation have been proposed as main causes of tree decline, with recent theories treating water and carbon metabolism as interconnected processes. We subjected young plants of two native (Quercus pubescens [Qp] and Prunus mahaleb [Pm]) and two invasive (Robinia pseudoacacia [Rp] and Ailanthus altissima [Aa]) woody angiosperms to a prolonged drought leading to stomatal closure and xylem embolism, to induce carbon starvation and hydraulic failure. At the end of the treatment, plants were measured for embolism rates and NSC content, and re-irrigated to monitor recovery of xylem hydraulics. Data highlight different hydraulic strategies in native vs invasive species under water stress, and provide physiological explanations for species-specific impacts of recent severe droughts. Drought-sensitive species (Qp and Rp) suffered high embolism rates and were unable to completely refill xylem conduits upon restoration of water availability. Species that better survived recent droughts were able to limit embolism build-up (Pm) or efficiently restored hydraulic functionality after irrigation (Aa). Species-specific capacity to reverse xylem embolism correlated to stem-level concentration of soluble carbohydrates, but not to starch content.

Rooting depth, water relations and non-structural carbohydrate dynamics in three woody angiosperms differentially affected by an extreme summer drought.

In 2012, an extreme summer drought induced species-specific die-back in woody species in Northeastern Italy. Quercus pubescens and Ostrya carpinifolia were heavily impacted, while Prunus mahaleb was largely unaffected. By comparing seasonal changes in isotopic composition of xylem sap, rainfall and deep soil samples, we show that P. mahaleb has a deeper root system than the other two species. This morphological trait allowed P mahaleb to maintain higher water potential (Ψ), gas exchange rates and non-structural carbohydrates content (NSC) throughout the summer, when compared with the other species. More favourable water and carbon states allowed relatively stable maintenance of stem hydraulic conductivity (k) throughout the growing season. In contrast, in Quercus pubescens and Ostrya carpinifolia, decreasing Ψ and NSC were associated with significant hydraulic failure, with spring-to-summer k loss averaging 60%. Our data support the hypothesis that drought-induced tree decline is a complex phenomenon that cannot be modelled on the basis of single predictors of tree status like hydraulic efficiency, vulnerability and carbohydrate content. Our data highlight the role of rooting depth in seasonal progression of water status, gas exchange and NSC, with possible consequences for energy-demanding mechanisms involved in the maintenance of vascular integrity.