Drought increases Norway spruce susceptibility to the Eurasian spruce bark beetle and its associated fungi.

Drought affects the complex interactions between Norway spruce, the bark beetle Ips typographus and associated microorganisms. We investigated the interplay of tree water status, defense and carbohydrate reserves with the incidence of bark beetle attack and infection of associated fungi in mature spruce trees. We installed roofs to induce a 2-yr moderate drought in a managed spruce stand to examine a maximum of 10 roof and 10 control trees for resin flow (RF), predawn twig water potentials, terpene, phenolic and carbohydrate bark concentrations, and bark beetle borings in field bioassays before and after inoculation with Endoconidiophora polonica and Grosmannia penicillata. Drought-stressed trees showed more attacks and significantly longer fungal lesions than controls, but maintained terpene resin defenses at predrought levels. Reduced RF and lower mono- and diterpene, but not phenolic concentrations were linked with increased host selection. Bark beetle attack and fungi stimulated chemical defenses, yet G. penicillata reduced phenolic and carbohydrate contents. Chemical defenses did not decrease under mild, prolonged drought in our simulated small-scale biotic infestations. However, during natural mass attacks, reductions in carbon fixation under drought, in combination with fungal consumption of carbohydrates, may deplete tree defenses and facilitate colonization by I. typographus.

Chemical inhibition of xylem cellular activity impedes the removal of drought-induced embolisms in poplar stems - new insights from micro-CT analysis.

In drought-stressed plants a coordinated cascade of chemical and transcriptional adjustments occurs at the same time as embolism formation. While these processes do not affect embolism formation during stress, they may prime stems for recovery during rehydration by modifying apoplast pH and increasing sugar concentration in the xylem sap. Here we show that in vivo treatments modifying apoplastic pH (stem infiltration with a pH buffer) or reducing stem metabolic activity (infiltration with sodium vanadate and sodium cyanide; plant exposure to carbon monoxide) can reduce sugar accumulation, thus disrupting or delaying the recovery process. Application of the vanadate treatment (NaVO3, an inhibitor of many ATPases) completely halted recovery from drought-induced embolism for up to 24 h after re-irrigation, while partial recovery was observed in vivo in control plants using X-ray microcomputed tomography. Our results suggest that stem hydraulic recovery in poplar is a biological, energy-dependent process that coincides with accumulation of sugars in the apoplast during stress. Recovery and damage are spatially coordinated, with embolism formation occurring from the inside out and refilling from the outside in. The outside-in pattern highlights the importance of xylem proximity to the sugars within the phloem to the embolism recovery process.

Container volume affects drought experiments in grapevines: Insights on xylem anatomy and time of dehydration.

Plant stress experiments are commonly performed with plants grown in containers to better control environmental conditions. Nevertheless, the container can constrain plant growth and development, and this confounding effect is generally ignored, particularly in studies on woody species. Here, we evaluate the effect of the container volume in drought experiments using grapevine as a model plant. Grapevines grown in small (7 L, S) or large (20 L, L) containers were subjected to drought stress and rewatering treatments. We monitored plant stomatal conductance (gs ), midday stem water potential (Ψs ), and photosynthetic rate (AN ) throughout the experiment. The effect of the container volume on the stem and petiole xylem anatomy, as well as on the total leaf area (LA), was assessed before drought imposition. The results showed that LA did not differ between plants in L or S containers, but S vines exhibited a higher theoretical hydraulic conductance at the petiole level. Under drought L and S similarly reduced gs and AN , but plants in S containers reached lower Ψs than those in L. Nevertheless, upon rewatering droughted plants in S containers exhibited a faster stomata re-opening than those in L, probably as a consequence of the differences in the stress degree experienced and the biochemical adjustment at the leaf level. Therefore, a suitable experimental design should consider the container volume used in relation to the desired traits to be studied for unbiased results.

Gas exchange, biomass and non-structural carbohydrates dynamics in vines under combined drought and biotic stress.

BACKGROUND: Intensity of drought stress and pest attacks is forecasted to increase in the near future posing a serious threat to natural and agricultural ecosystems. Knowledge on potential effects of a combined abiotic-biotic stress on whole-plant physiology is lacking. We monitored the water status and carbon metabolism of a vine rootstock with or without scion subjected to water shortening and/or infestation with the sucking insect phylloxera (Daktulosphaira vitifoliae Fitch). We measured non-structural carbohydrates and biomass of different plant organs to assess the stress-induced responses at the root, stem, and leaf level. Effects of watering on root infestation were also addressed. RESULTS: Higher root infestation was observed in drought-stressed plants compared to well-watered. The drought had a significant impact on most of the measured functional traits. Phylloxera further influenced vines water and carbon metabolism and enforced the sink strength of the roots by stimulating photosynthates translocation. The insect induced carbon depletion, reprogramed vine development, while preventing biomass compensation. A synergic effect of biotic-abiotic stress could be detected in several physiological and morphological traits. CONCLUSIONS: Our results indicate that events of water shortage favour insects' feeding damage and increase the abundance of root nodosities. Root phylloxera infestation imposes a considerable stress to the plants which might exacerbate the negative effects of drought.

Insights from in vivo micro-CT analysis: testing the hydraulic vulnerability segmentation in Acer pseudoplatanus and Fagus sylvatica seedlings.

The seedling stage is the most susceptible one during a tree's life. Water relations may be crucial for seedlings due to their small roots, limited water buffers and the effects of drought on water transport. Despite obvious relevance, studies on seedling xylem hydraulics are scarce as respective methodical approaches are limited. Micro-CT scans of intact Acer pseudoplatanus and Fagus sylvatica seedlings dehydrated to different water potentials (Ψ) allowed the simultaneous observation of gas-filled versus water-filled conduits and the calculation of percentage loss of conductivity (PLC) in stems, roots and leaves (petioles or main veins). Additionally, anatomical analyses were performed and stem PLC measured with hydraulic techniques. In A. pseudoplatanus, petioles showed a higher Ψ at 50% PLC (Ψ50 -1.13MPa) than stems (-2.51 MPa) and roots (-1.78 MPa). The main leaf veins of F. sylvatica had similar Ψ50 values (-2.26 MPa) to stems (-2.74 MPa) and roots (-2.75 MPa). In both species, no difference between root and stems was observed. Hydraulic measurements on stems closely matched the micro-CT based PLC calculations. Micro-CT analyses indicated a species-specific hydraulic architecture. Vulnerability segmentation, enabling a disconnection of the hydraulic pathway upon drought, was observed in A. pseudoplatanus but not in the especially shade-tolerant F. sylvatica. Hydraulic patterns could partly be related to xylem anatomical traits.

Prediction of hydraulic conductivity loss from relative water loss: new insights into water storage of tree stems and branches.

More frequently occurring, drought waves call for a deeper understanding of tree hydraulics and fast and easily applicable methods to measure drought stress. The aim of this study was to establish empirical relationships between the percent loss of hydraulic conductivity (PLC) and the relative water loss (RWL) in woody stem axes with different P50 , i.e. the water potential (Ψ) that causes 50% conductivity loss. Branches and saplings of temperate conifer (Picea abies, Larix decidua) and angiosperm species (Acer campestre, Fagus sylvatica, Populus x canescens, Populus tremula, Sorbus torminalis) and trunk wood of mature P. abies trees were analyzed. P50 was calculated from hydraulic measurements following bench top dehydration or air injection. RWL and PLC were fitted by linear, quadratic or cubic equations. Species- or age-specific RWLs at P50 varied between 10 and 25% and P88 , the Ψ that causes 88% conductivity loss, between 18 and 44%. P50 was predicted from the relationship between Ψ and the RWL. The predictive quality for P50 across species was almost 1:1 (r2 = 0.99). The approach presented allows thus reliable and fast prediction of PLC from RWL. Branches and saplings with high hydraulic vulnerability tended to have lower RWLs at P50 and at P88 . The results are discussed with regard to the different water storage capacities in sapwood and survival strategies under drought stress. Potential applications are screening trees for drought sensitivity and a fast interpretation of diurnal, seasonal or drought induced changes in xylem water content upon their impact on conductivity loss.

Relationships between water status and photosystem functionality in a chlorolichen and its isolated photobiont.

MAIN CONCLUSION: Drought tolerance was greater in the whole lichen than in its isolated photobiont. Cell turgor state has an influence on the functionality of photosynthetic process in lichens. Irreversible thermodynamics is widely used to describe the water relations of vascular plants. However, poikilohydrous organisms like lichens and aeroterrestrial microalgae have seldom been studied using this approach. Water relations of lichens are generally addressed without separate analysis of the mycobiont and photobiont, and only few studies have correlated changes in photosynthetic efficiency of dehydrating lichens to accurate measurements of their water potential components. We measured water potential isotherms and chlorophyll a fluorescence in the lichen Flavoparmelia caperata harvested in different seasons, as well as in its isolated photobiont, the green alga Trebouxia gelatinosa, either exposed to water stress cycles or fully hydrated. No significant seasonal trends were observed in lichen water relations parameters. Turgor loss point and osmotic potential of the whole thallus were significantly lower than those measured in the photobiont, while differences between the water stressed photobiont and controls were not significant. Dehydration-induced drop of F v/F m was correlated with turgor loss, revealing that the photosynthetic activity of lichens partly depends on their turgor level. We provided one of the first quantitative evidences of the influence that turgor status could exert on the functionality of photosynthetic processes in lichens.

The pitfalls of in vivo imaging techniques: evidence for cellular damage caused by synchrotron X-ray computed micro-tomography.

Synchrotron X-ray computed micro-tomography (microCT) has emerged as a promising noninvasive technique for in vivo monitoring of xylem function, including embolism build-up under drought and hydraulic recovery following re-irrigation. Yet, the possible harmful effects of ionizing radiation on plant tissues have never been quantified. We specifically investigated the eventual damage suffered by stem living cells of three different species exposed to repeated microCT scans. Stem samples exposed to one, two or three scans were used to measure cell membrane and RNA integrity, and compared to controls never exposed to X-rays. Samples exposed to microCT scans suffered serious alterations to cell membranes, as revealed by marked increase in relative electrolyte leakage, and also underwent severe damage to RNA integrity. The negative effects of X-rays were apparent in all species tested, but the magnitude of damage and the minimum number of scans inducing negative effects were species-specific. Our data show that multiple microCT scans lead to disruption of fundamental cellular functions and processes. Hence, microCT investigation of phenomena that depend on physiological activity of living cells may produce erroneous results and lead to incorrect conclusions.

X-ray microtomography observations of xylem embolism in stems of Laurus nobilis are consistent with hydraulic measurements of percentage loss of conductance.

Drought-induced xylem embolism is a serious threat to plant survival under future climate scenarios. Hence, accurate quantification of species-specific vulnerability to xylem embolism is a key to predict the impact of climate change on vegetation. Low-cost hydraulic measurements of embolism rate have been suggested to be prone to artefacts, thus requiring validation by direct visualization of the functional status of xylem conduits using nondestructive imaging techniques, such as X-ray microtomography (microCT). We measured the percentage loss of conductance (PLC) of excised stems of Laurus nobilis (laurel) dehydrated to different xylem pressures, and compared results with direct observation of gas-filled vs water-filled conduits at a synchrotron-based microCT facility using a phase contrast imaging modality. Theoretical PLC calculated on the basis of microCT observations in stems of laurel dehydrated to different xylem pressures overall were in agreement with hydraulic measurements, revealing that this species suffers a 50% loss of xylem hydraulic conductance at xylem pressures averaging -3.5 MPa. Our data support the validity of estimates of xylem vulnerability to embolism based on classical hydraulic techniques. We discuss possible causes of discrepancies between data gathered in this study and those of recent independent reports on laurel hydraulics.

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.

The contribution of vascular and extra-vascular water pathways to drought-induced decline of leaf hydraulic conductance.

Drought stress can impair leaf hydraulic conductance (Kleaf), but the relative contribution of changes in the efficiency of the vein xylem water pathway and in the mesophyll route outside the xylem in driving the decline of Kleaf is still debated. We report direct measurements of dehydration-induced changes in the hydraulic resistance (R=1/K) of whole leaf (Rleaf), as well as of the leaf xylem (Rx) and extra-vascular pathways (Rox) in four Angiosperm species. Rleaf, Rx, and Rox were measured using the vacuum chamber method (VCM). Rleaf values during progressive leaf dehydration were also validated with measurements performed using the rehydration kinetic method (RKM). We analysed correlations between changes in Rx or Rox and Rleaf, as well as between morpho-anatomical traits (including dehydration-induced leaf shrinkage), vulnerability to embolism, and leaf water relation parameters. Measurements revealed that the relative contribution of vascular and extra-vascular hydraulic properties in driving Kleaf decline during dehydration is species-specific. Whilst in two study species the progressive impairment of both vascular and extra-vascular pathways contributed to leaf hydraulic vulnerability, in the other two species the vascular pathway remained substantially unaltered during leaf dehydration, and Kleaf decline was apparently caused only by changes in the hydraulic properties of the extra-vascular compartment.

Drought-induced xylem cavitation and hydraulic deterioration: risk factors for urban trees under climate change?

Urban trees help towns to cope with climate warming by cooling both air and surfaces. The challenges imposed by the urban environment, with special reference to low water availability due to the presence of extensive pavements, result in high rates of mortality of street trees, that can be increased by climatic extremes. We investigated the water relations and xylem hydraulic safety/efficiency of Quercus ilex trees growing at urban sites with different percentages of surrounding impervious pavements. Seasonal changes of plant water potential and gas exchange, vulnerability to cavitation and embolism level, and morpho-anatomical traits were measured. We found patterns of increasing water stress and vulnerability to drought at increasing percentages of impervious pavement cover, with a consequent reduction in gas exchange rates, decreased safety margins toward embolism development, and increased vulnerability to cavitation, suggesting the occurrence of stress-induced hydraulic deterioration. The amount of impermeable surface and chronic exposure to water stress influence the site-specific risk of drought-induced dieback of urban trees under extreme drought. Besides providing directions for management of green spaces in towns, our data suggest that xylem hydraulics is key to a full understanding of the responses of urban trees to global change.

Shoot desiccation and hydraulic failure in temperate woody angiosperms during an extreme summer drought.

Plant water status and hydraulics were measured in six woody angiosperms growing in a karstic woodland, during an extreme summer drought. Our aim was to take advantage of an unusual climatic event to identify key traits related to species-specific drought damage. The damage suffered by different species was assessed in terms of percentage of individuals showing extensive crown desiccation. Stem water potential (Ψstem ) and percent loss of hydraulic conductivity (PLC) were measured in healthy and desiccated individuals. Vulnerability to cavitation was assessed in terms of stem water potential inducing 50% PLC (Ψ50 ). Stem density (ρstem ) was also measured. Species-specific percentage of desiccated individuals was correlated to Ψ50 and ρstem . Crown desiccation was more widespread in species with less negative Ψ50 and lower ρstem . Desiccated individuals had lower Ψstem and higher PLC than healthy ones, suggesting that hydraulic failure was an important mechanism driving shoot dieback. Drought-vulnerable species showed lower safety margins (Ψstem  - Ψ50 ) than resistant ones. The Ψ50 , safety margins and ρstem values emerge as convenient traits to be used for tentative predictions of differential species-specific impact of extreme drought events on a local scale. The possibility that carbohydrate depletion was also involved in induction of desiccation symptoms is discussed.

Analyzing anatomy over three dimensions unpacks the differences in mesophyll diffusive area between sun and shade Vitis vinifera leaves.

Leaves grown at different light intensities exhibit considerable differences in physiology, morphology and anatomy. Because plant leaves develop over three dimensions, analyses of the leaf structure should account for differences in lengths, surfaces, as well as volumes. In this manuscript, we set out to disentangle the mesophyll surface area available for diffusion per leaf area (S m,LA) into underlying one-, two- and three-dimensional components. This allowed us to estimate the contribution of each component to S m,LA, a whole-leaf trait known to link structure and function. We introduce the novel concept of a 'stomatal vaporshed,' i.e. the intercellular airspace unit most closely connected to a single stoma, and use it to describe the stomata-to-diffusive-surface pathway. To illustrate our new theoretical framework, we grew two cultivars of Vitis vinifera L. under high and low light, imaged 3D leaf anatomy using microcomputed tomography (microCT) and measured leaf gas exchange. Leaves grown under high light were less porous and thicker. Our analysis showed that these two traits and the lower S m per mesophyll cell volume (S m,Vcl) in sun leaves could almost completely explain the difference in S m,LA. Further, the studied cultivars exhibited different responses in carbon assimilation per photosynthesizing cell volume (A Vcl). While Cabernet Sauvignon maintained A Vcl constant between sun and shade leaves, it was lower in Blaufränkisch sun leaves. This difference may be related to genotype-specific strategies in building the stomata-to-diffusive-surface pathway.

Changes in abscisic acid content during and after drought are related to carbohydrate mobilization and hydraulic recovery in poplar stems.

Drought compromises plant's ability to replace transpired water vapor with water absorbed from the soil, leading to extensive xylem dysfunction and causing plant desiccation and death. Short-term plant responses to drought rely on stomatal closure, and on the plant's ability to recover hydraulic functioning after drought relief. We hypothesize a key role for abscisic acid (ABA) not only in the control of stomatal aperture, but also in hydraulic recovery. Young plants of Populus nigra L. were used to investigate possible relationships among ABA, non-structural carbohydrates (NSC) and xylem hydraulic function under drought and after re-watering. In Populus nigra L. plants subjected to drought, water transport efficiency and hydraulic recovery after re-watering were monitored by measuring the percentage loss of hydraulic conductivity (PLC) and stem specific hydraulic conductivity (Kstem). In the same plants ABA and NSC were quantified in wood and bark. Drought severely reduced stomatal conductance (gL) and markedly increased the PLC. Leaf and stem water potential, and stem hydraulic efficiency fully recovered within 24 h after re-watering, but gL values remained low. After re-watering, we found significant correlations between changes in ABA content and hexoses concentration both in wood and bark. Our findings suggest a role for ABA in the regulation of stem carbohydrate metabolism and starch mobilization upon drought relief, possibly promoting the restoration of xylem transport capacity.

A simplified framework for fast and reliable measurement of leaf turgor loss point.

Drought tolerance shapes the distribution of plant species, and it is mainly determined by the osmotic potential at full turgor (π0) and the water potential at turgor loss point (Ψtlp). We provide a simplified framework for π0 and Ψtlp measurements based on osmometer determination of π0 (π0_osm). Specifically, we ran regression models to i) improve the predictive power of the estimation of π0 from π0_osm and morpho-anatomical traits; ii) obtain the most accurate model to predict Ψtlp on the basis of the global relationship between π0 and Ψtlp. The inclusion of the leaf dry matter content (LDMC), an easy-to-measure trait, in the regression model improved the predictive power of the estimation of π0 from π0_osm. When π0_osm was used as a simple predictor of Ψtlp, discrepancies arose in comparison with global relationship between π0 and Ψtlp. Ψtlp values calculated as a function of the π0 derived from π0_osm and LDMC (π0_fit) were consistent with the global relationship between π0 and Ψtlp. The simplified framework provided here could encourage the inclusion of mechanistically sound drought tolerance traits in ecological studies.

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.

The potential of Mid-Infrared spectroscopy for prediction of wood density and vulnerability to embolism in woody angiosperms.

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.

Less safety for more efficiency: water relations and hydraulics of the invasive tree Ailanthus altissima (Mill.) Swingle compared with native Fraxinus ornus L.

Invasion of natural habitats by alien trees is a threat to forest conservation. Our understanding of fundamental ecophysiological mechanisms promoting plant invasions is still limited, and hydraulic and water relation traits have been only seldom included in studies comparing native and invasive trees. We compared several leaf and wood functional and mechanistic traits in co-occurring Ailanthus altissima (Mill.) Swingle (Aa) and Fraxinus ornus L. (Fo). Aa is one of the most invasive woody species in Europe and North America, currently outcompeting several native trees including Fo. We aimed at quantifying inter-specific differences in terms of: (i) performance in resource use and acquisition; (ii) hydraulic efficiency and safety; (iii) carbon costs associated to leaf and wood construction; and (iv) plasticity of functional and mechanistic traits in response to light availability. Traits related to leaf and wood construction and drought resistance significantly differed between the two species. Fo sustained higher structural costs than Aa, but was more resistant to drought. The lower resistance to drought stress of Aa was counterbalanced by higher water transport efficiency, but possibly required mechanisms of resilience to drought-induced hydraulic damage. Larger phenotypic plasticity of Aa in response to light availability could also promote the invasive potential of the species.

Grapevine water relations and rooting depth in karstic soils.

Environmental sustainability of viticulture is negatively affected by prolonged droughts. In limestone dominated regions, there is limited knowledge on grapevine water status and on methods for accurate evaluation of actual water demand, necessary to appropriately manage irrigation. During a dry vintage, we monitored plant and soil water relations in old and young vines of Istrian Malvasia on Karst red soil. The vineyard with young vines was additionally subdivided into two areas, based on their soil type, 1) karst silty-clay loam, and 2) mixture of crushed rocks and karst silty-clay loam (stony soil). Seasonal changes in exploited water resources were estimated via analysis of oxygen isotope composition (δ18O) of rainfall, deep soil water, and xylem sap. We hypothesized that plants are able to thrive during drought thanks to the water stored in deep soil layers, while they rely less on superficial soil horizons. Our results show that vines growing on karstic substrates have deep roots securing the use of stable water sources during summer, with consequent favourable plant water status. In fact, both young and mature vines approached the threshold of severe water stress, but never surpassed it, as midday leaf water potentials were >-1.3MPa in all study sites. Vines roots showed flexible water uptake, i.e. the ability to absorb water from deep or shallow soil horizons during drought and after late-summer thunderstorms, which was particularly evident in vines growing on the stony soil. In fact, precipitations of 20mm were enough for plant water status recovery, due to fast infiltration. On the other hand, at least 50mm of rainfall were necessary to induce water status recovery in more compact soil (karst silty-clay loam). Our findings provide new knowledge on the rooting depth and water needs of vines growing on shallow soils overlying fractured limestone bedrock.