Intraspecific variability of leaf form and function across habitat types.

Trait-based ecology has already revealed main independent axes of trait variation defining trait spaces that summarize plant adaptive strategies, but often ignoring intraspecific trait variability (ITV). By using empirical ITV-level data for two independent dimensions of leaf form and function and 167 species across five habitat types (coastal dunes, forests, grasslands, heathlands, wetlands) in the Italian peninsula, we found that ITV: (i) rotated the axes of trait variation that define the trait space; (ii) increased the variance explained by these axes and (iii) affected the functional structure of the target trait space. However, the magnitude of these effects was rather small and depended on the trait and habitat type. Our results reinforce the idea that ITV is context-dependent, calling for careful extrapolations of ITV patterns across traits and spatial scales. Importantly, our study provides a framework that can be used to start integrating ITV into trait space analyses.

Detecting and Quantifying Xylem Embolism by Synchrotron-Based X-Ray Micro-CT.

The vulnerability to xylem embolism is a key trait underlying species-specific drought tolerance of plants, and hence is critical for screening climate-resilient crops and understanding vegetation responses to drought and heat waves. Yet, accurate determination of embolism in plant's xylem is challenging, because most traditional hydraulic techniques are destructive and prone to artefacts. Hence, direct and in vivo synchrotron-based X-ray micro-CT observation of xylem conduits has emerged as a key reference technique for accurate quantification of vulnerability to xylem embolism. Micro-CT is nowadays a fundamental tool for studies of plant hydraulic architecture, and this chapter describes the fundamentals of acquisition and processing of micro-CT images of plant xylem.

Best Procedures for Leaf and Stem Water Potential Measurements in Grapevine: Cultivar and Water Status Matter.

The pressure chamber is the most used tool for plant water status monitoring. However, species/cultivar and seasonal effects on protocols for reliable water potential determination have not been properly tested. In four grapevine cultivars and two times of the season (early season, Es; late season, Ls, under moderate drought), we assessed the maximum sample storage time before leaf water potential (Ψleaf) measurements and the minimum equilibration time for stem water potential (Ψstem) determination, taking 24 h leaf cover as control. In 'Pinot gris', Ψleaf already decreased after 1 h leaf storage in both campaigns, dropping by 0.4/0.5 MPa after 3 h, while in 'Refosk', it decreased by 0.1 MPa after 1 and 2 h in Es and Ls, respectively. In 'Merlot' and 'Merlot Kanthus', even 3 h storage did not affect Ψleaf. In Es, the minimum Ψstem equilibration was 1 h for 'Refosk' and 10 min for 'Pinot gris' and 'Merlot'. In Ls, 'Merlot Kanthus' required more than 2 h equilibration, while 1 h to 10 min was sufficient for the other cultivars. The observed cultivar and seasonal differences indicate that the proposed tests should be routinely performed prior to experiments to define ad hoc procedures for water status determination.

The optical method based on gas injection overestimates leaf vulnerability to xylem embolism in three woody species.

Plant hydraulic traits related to leaf drought tolerance, like the water potential at turgor loss point (TLP) and the water potential inducing 50% loss of hydraulic conductance (P50), are extremely useful to predict the potential impacts of drought on plants. While novel techniques have allowed the inclusion of TLP in studies targeting a large group of species, fast and reliable protocols to measure leaf P50 are still lacking. Recently, the optical method coupled with the gas injection (GI) technique has been proposed as a possibility to speed up the P50 estimation. Here, we present a comparison of leaf optical vulnerability curves (OVcs) measured in three woody species, namely Acer campestre (Ac), Ostrya carpinifolia (Oc) and Populus nigra (Pn), based on bench dehydration (BD) or GI of detached branches. For Pn, we also compared optical data with direct micro-computed tomography (micro-CT) imaging in both intact saplings and cut shoots subjected to BD. Based on the BD procedure, Ac, Oc and Pn had P50 values of -2.87, -2.47 and -2.11 MPa, respectively, while the GI procedure overestimated the leaf vulnerability (-2.68, -2.04 and -1.54 MPa for Ac, Oc and Pn, respectively). The overestimation was higher for Oc and Pn than for Ac, likely reflecting the species-specific vessel lengths. According to micro-CT observations performed on Pn, the leaf midrib showed none or very few embolized conduits at -1.2 MPa, consistent with the OVcs obtained with the BD procedure but at odds with that derived on the basis of GI. Overall, our data suggest that coupling the optical method with GI might not be a reliable technique to quantify leaf hydraulic vulnerability since it could be affected by the 'open-vessel' artifact. Accurate detection of xylem embolism in the leaf vein network should be based on BD, preferably of intact up-rooted plants.

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.

Critical water contents at leaf, stem and root level leading to irreversible drought-induced damage in two woody and one herbaceous species.

Plant water content is a simple and promising parameter for monitoring drought-driven plant mortality risk. However, critical water content thresholds leading to cell damage and plant failure are still unknown. Moreover, it is unclear whether whole-plant or a specific organ water content is the most reliable indicator of mortality risk. We assessed differences in dehydration thresholds in leaf, stem and root samples, hampering the organ-specific rehydration capacity and increasing the mortality risk. We also tested eventual differences between a fast experimental dehydration of uprooted plants, compared to long-term water stress induced by withholding irrigation in potted plants. We investigated three species with different growth forms and leaf habits i.e., Helianthus annuus (herbaceous), Populus nigra (deciduous tree) and Quercus ilex (evergreen tree). Results obtained by the two dehydration treatments largely overlapped, thus validating bench dehydration as a fast but reliable method to assess species-specific critical water content thresholds. Regardless of the organ considered, a relative water content value of 60% induced significant cell membrane damage and loss of rehydration capacity, thus leading to irreversible plant failure and death.

Projections of leaf turgor loss point shifts under future climate change scenarios.

Predicting the consequences of climate change is of utmost importance to mitigate impacts on vulnerable ecosystems; plant hydraulic traits are particularly useful proxies for predicting functional disruptions potentially occurring in the near future. This study assessed the current and future regional patterns of leaf water potential at turgor loss point (Ψtlp ) by measuring and projecting the Ψtlp of 166 vascular plant species (159 angiosperms and 7 gymnosperms) across a large climatic range spanning from alpine to Mediterranean areas in NE Italy. For angiosperms, random forest models predicted a consistent shift toward more negative values in low-elevation areas, whereas for gymnosperms the pattern was more variable, particularly in the alpine sector (i.e., Alps and Prealps). Simulations were also developed to evaluate the number of threatened species under two Ψtlp plasticity scenarios (low vs. high plasticity), and it was found that in the worst-case scenario approximately 72% of the angiosperm species and 68% of gymnosperms within a location were at risk to exceed their physiological plasticity. The different responses to climate change by specific clades might produce reassembly in natural communities, undermining the resilience of natural ecosystems to climate change.

No Evidence for Light-Induced Embolism Repair in Cut Stems of Drought-Resistant Mediterranean Species under Soaking.

(1) Recent studies suggested that stem photosynthesis could favor bark water uptake and embolism recovery when stem segments are soaked in water under light conditions, but evidence for this phenomenon in drought-resistant Mediterranean species with photosynthetic stems is missing. (2) Embolism recovery upon immersion in water for 2 h-4 h under light was assessed (i) via a classical hydraulic method in leafless Fraxinus ornus and Olea europaea branch segments stressed to xylem water potentials (Yxyl) inducing ca. 50% loss of hydraulic conductivity (PLC) and (ii) via X-ray micro-CT imaging of the stem segments of drought-stressed potted F. ornus saplings. Hydraulic recovery was also assessed in vivo in intact drought-stressed F. ornus saplings upon soil re-irrigation. (3) Intact F. ornus plants recovered hydraulic function through root water uptake. Conversely, the soaked stem segments of both species did not refill embolized conduits, although Yxyl recovered to pre-stress levels (between -0.5 MPa and -0.2 MPa). (4) We hypothesize that xylem embolism recovery through bark water uptake, even in light conditions, may not be a common phenomenon in woody plants and/or that wounds caused by cutting short stem segments might inhibit the refilling process upon soaking.

Too dry to survive: Leaf hydraulic failure in two Salvia species can be predicted on the basis of water content.

Global warming is exposing plants to increased risks of drought-driven mortality. Recent advances suggest that hydraulic failure is a key process leading to plant death, and the identification of simple and reliable proxies of species-specific risk of irreversible hydraulic damage is urgently required. We assessed the predictive power of leaf water content and shrinkage for monitoring leaf hydraulic failure in two Mediterranean native species, Salvia ceratophylloides (Sc) and S. officinalis (So). The study species showed significant differences in relative water content (RWC) thresholds inducing loss of rehydration capacity, as well as leaf hydraulic conductance (KL) impairment. Sc turned out to be more resistant to drought than So. However, Sc and So showed different leaf saturated water content values, so that different RWC values actually corresponded to similar absolute leaf water content. Our findings suggest that absolute leaf water content and leaf water potential, but not RWC, are reliable parameters for predicting the risk of leaf hydraulic impairment of two Salvia species, and their potential risk of irreversible damage under severe drought. Moreover, the lack of any KL decline until the turgor loss point in Sc, coupled to consistent leaf shrinkage, rejects the hypothesis to use leaf shrinkage as a proxy to predict KL vulnerability, at least in species with high leaf capacitance. Robust linear correlations between KL decline and electrolyte leakage measurements suggested a role of membrane damage in driving leaf hydraulic collapse.

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.

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.

The extra-vascular water pathway regulates dynamic leaf hydraulic decline and recovery in Populus nigra.

Leaf hydraulic conductance (Kleaf ) is highly dynamic and typically responds to changes in water status and irradiance. However, the relative contribution of vascular (Kx ) and extra-vascular (Kox ) water pathways to Kleaf changes in response to water potential decline and recovery in function of light conditions remains poorly investigated. We investigated the dynamic responses of leaf hydraulics in Populus nigra L. by measuring Kleaf , Kx , and Kox changes under drought and upon recovery. Measurements were done at both low and high irradiance (LI and HI, respectively). Kleaf increased and became more vulnerable to dehydration under HI conditions than LI, due to marked changes of Kox . After re-watering, Kleaf recovered in parallel with Kox recovery, but Kleaf response to irradiance remained inhibited. Strong correlations between Kleaf and drought-induced membrane damage demonstrated the relevance of the cell-to-cell water pathway in driving the dynamic responses of Kleaf under drought and recovery. Our findings highlight the importance of coordination between water and light availability in modulating the overall Kleaf response to environmental conditions.

Water 'on the rocks': a summer drink for thirsty trees?

Drought-induced tree mortality frequently occurs in patches with different spatial and temporal distributions, which is only partly explained by inter- and intraspecific variation in drought tolerance. We investigated whether bedrock properties, with special reference to rock water storage capacity, affects tree water status and drought response in a rock-dominated landscape. We measured primary porosity and available water content of breccia (B) and dolostone (D) rocks. Saplings of Fraxinus ornus were grown in pots filled with soil or soil mixed with B and D rocks, and subjected to an experimental drought. Finally, we measured seasonal changes in water status of trees in field sites overlying B or D bedrock. B rocks were more porous and stored more available water than D rocks. Potted saplings grown with D rocks had less biomass and suffered more severe water stress than those with B rocks. Trees in sites with B bedrock had more favourable water status than those on D bedrock which also suffered drought-induced canopy dieback. Bedrock represents an important water source for plants under drought. Different bedrock features translate into contrasting below-ground water availability, leading to landscape-level heterogeneity of the impact of drought on tree water status and dieback.

A Leaf Selfie: Using a Smartphone to Quantify Leaf Vulnerability to Hydraulic Dysfunction.

Accurate predictions of species distribution under current and future climate conditions require modeling efforts based on clear mechanistic relationships between climate variables and plant physiological functions. Vulnerability of leaves to xylem embolism is a key mechanistic trait that might be included in these modeling efforts. Here, we propose a simple set-up to measure leaf vulnerability to embolism on the basis of the optical method using a smartphone, a light source, and a notebook. Our data show that this proposed set-up can adequately quantify the vulnerability to xylem embolism of leaf major veins in Populus nigra and Ostrya carpinifolia, producing values consistent with those obtained in temperate tree species with other methods, allowing virtually any laboratory to quantify species-specific drought tolerance on the basis of a sound mechanistic trait.

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.

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.

Vulnerability to xylem embolism correlates to wood parenchyma fraction in angiosperms but not in gymnosperms.

Understanding which structural and functional traits are linked to species' vulnerability to embolism formation (P50) may provide fundamental knowledge on plant strategies to maintain an efficient water transport. We measured P50, wood density (WD), mean conduit area, conduit density, percentage areas occupied by vessels, parenchyma cells (PATOT) and fibers (FA) on branches of angiosperm and gymnosperm species. Moreover, we compiled a dataset of published hydraulic and anatomical data to be compared with our results. Species more vulnerable to embolism had lower WD. In angiosperms, the variability in WD was better explained by PATOT and FA, which were highly correlated. Angiosperms with a higher P50 (less negative) had a higher amount of PATOT and total amount of nonstructural carbohydrates. Instead, in gymnosperms, P50 vs PATOT was not significant. The correlation between PATOT and P50 might have a biological meaning and also suggests that the causality of the commonly observed relationship of WD vs P50 is indirect and dependent on the parenchyma fraction. Our study suggests that angiosperms have a potential active embolism reversal capacity in which parenchyma has an important role, while in gymnosperms this might not be the case.

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.