Interactions between beech and oak seedlings can modify the effects of hotter droughts and the onset of hydraulic failure.

Mixing species with contrasting resource use strategies could reduce forest vulnerability to extreme events. Yet, how species diversity affects seedling hydraulic responses to heat and drought, including mortality risk, is largely unknown. Using open-top chambers, we assessed how, over several years, species interactions (monocultures vs mixtures) modulate heat and drought impacts on the hydraulic traits of juvenile European beech and pubescent oak. Using modeling, we estimated species interaction effects on timing to drought-induced mortality and the underlying mechanisms driving these impacts. We show that mixtures mitigate adverse heat and drought impacts for oak (less negative leaf water potential, higher stomatal conductance, and delayed stomatal closure) but enhance them for beech (lower water potential and stomatal conductance, narrower leaf safety margins, faster tree mortality). Potential underlying mechanisms include oak's larger canopy and higher transpiration, allowing for quicker exhaustion of soil water in mixtures. Our findings highlight that diversity has the potential to alter the effects of extreme events, which would ensure that some species persist even if others remain sensitive. Among the many processes driving diversity effects, differences in canopy size and transpiration associated with the stomatal regulation strategy seem the primary mechanisms driving mortality vulnerability in mixed seedling plantations.

Drone-based assessment of microsite-scale hydrological processes promoted by restoration actions in early post-mining ecological restoration stages.

A successful choice of post-mining restoration activities in dry climates may depend on relevant features related to topographic characteristics, hydrological processes and vegetation development, which will determine functional recovery in these ecosystems. The combination of different restoration techniques to reestablish vegetation, such as sowing and plantation, implies the interspersion of bare-soil areas with vegetated areas in early plant development stages, which may result in an associated mosaic of hydrologic functioning. In this study, we conducted a drone-based assessment to disentangle the role played by microsite-scale hydrological processes (i.e., planting hole slope, sink volume capacity, individual catchment area, Flow Length Index) promoted by restoration actions in soil protection and vegetation development on the hillside scale. Based on two contrasting restoration scenarios (Steep hillside and Smooth hillside), the different applied restoration treatments conditioned the microtopographic processes on the planting hole scale and, therefore, resource redistribution. The main results showed higher planting hole functionality on the smooth hillsides than on steep hillside, which resulted in greater water availability and bigger vegetation patches. By addressing the role of hydrological processes on the microsite scale, our study contributes substantially to prior knowledge on the relevant factors for ecosystem development and post-mining restoration success. It also demonstrates that high-resolution drone images can be a very useful tool for monitoring restoration actions, especially in large, inaccessible and unstable restored areas.

Identification of distinctive physiological and molecular responses to salt stress among tolerant and sensitive cultivars of broccoli (Brassica oleracea var. Italica).

BACKGROUND: Salt stress is one of the main constraints determining crop productivity, and therefore one of the main limitations for food production. The aim of this study was to characterize the salt stress response at the physiological and molecular level of different Broccoli (Brassica oleracea L. var. Italica Plenck) cultivars that were previously characterized in field and greenhouse trials as salt sensitive or salt tolerant. This study aimed to identify functional and molecular traits capable of predicting the ability of uncharacterized lines to cope with salt stress. For this purpose, this study measured different physiological parameters, hormones and metabolites under control and salt stress conditions. RESULTS: This study found significant differences among cultivars for stomatal conductance, transpiration, methionine, proline, threonine, abscisic acid, jasmonic acid and indolacetic acid. Salt tolerant cultivars were shown to accumulate less sodium and potassium in leaves and have a lower sodium to potassium ratio under salt stress. Analysis of primary metabolites indicated that salt tolerant cultivars have higher concentrations of several intermediates of the Krebs cycle and the substrates of some anaplerotic reactions. CONCLUSIONS: This study has found that the energetic status of the plant, the sodium extrusion and the proline content are the limiting factors for broccoli tolerance to salt stress. Our results establish physiological and molecular traits useful as distinctive markers to predict salt tolerance in Broccoli or to design novel biotechnological or breeding strategies for improving broccoli tolerance to salt stress.

Towards a statistically robust determination of minimum water potential and hydraulic risk in plants.

Minimum water potential (Ψmin ) is a key variable for characterizing dehydration tolerance and hydraulic safety margins (HSMs) in plants. Ψmin is usually estimated as the absolute minimum tissue Ψ experienced by a species, but this is problematic because sample extremes are affected by sample size and the underlying probability distribution. We compare alternative approaches to estimate Ψmin and assess the corresponding uncertainties and biases; propose statistically robust estimation methods based on extreme value theory (EVT); and assess the implications of our results for the characterization of hydraulic risk. Our results show that current estimates of Ψmin and HSMs are biased, as they are strongly affected by sample size. Because sampling effort is generally higher for species living in dry environments, the differences in current Ψmin estimates between these species and those living under milder conditions are partly artefactual. When this bias is corrected using EVT methods, resulting HSMs tend to increase substantially with resistance to embolism across species. Although data availability and representativeness remain the main challenges for proper determination of Ψmin , a closer look at Ψ distributions and the use of statistically robust methods to estimate Ψmin opens new ground for characterizing plant hydraulic risks.

Distinctive physiological and molecular responses to cold stress among cold-tolerant and cold-sensitive Pinus halepensis seed sources.

BACKGROUND: Forest species ranges are confined by environmental limitations such as cold stress. The natural range shifts of pine forests due to climate change and proactive-assisted population migration may each be constrained by the ability of pine species to tolerate low temperatures, especially in northern latitudes or in high altitudes. The aim of this study is to characterize the response of cold-tolerant versus cold-sensitive Pinus halepensis (P. halepensis) seedlings at the physiological and the molecular level under controlled cold conditions to identify distinctive features which allow us to explain the phenotypic difference. With this objective gas-exchange and water potential was determined and the photosynthetic pigments, soluble sugars, glutathione and free amino acids content were measured in seedlings of different provenances under control and cold stress conditions. RESULTS: Glucose and fructose content can be highlighted as a potential distinctive trait for cold-tolerant P. halepensis seedlings. At the amino acid level, there was a significant increase and accumulation of glutathione, proline, glutamic acid, histidine, arginine and tryptophan along with a significant decrease of glycine. CONCLUSION: Our results established that the main difference between cold-tolerant and cold-sensitive seedlings of P. halepensis is the ability to accumulate the antioxidant glutathione and osmolytes such as glucose and fructose, proline and arginine.

Reductions in tree performance during hotter droughts are mitigated by shifts in nitrogen cycling.

Climate warming should result in hotter droughts of unprecedented severity in this century. Such droughts have been linked with massive tree mortality, and data suggest that warming interacts with drought to aggravate plant performance. Yet how forests will respond to hotter droughts remains unclear, as does the suite of mechanisms trees use to deal with hot droughts. We used an ecosystem-scale manipulation of precipitation and temperature on piñon pine (Pinus edulis) and juniper (Juniperus monosperma) trees to investigate nitrogen (N) cycling-induced mitigation processes related to hotter droughts. We found that while negative impacts on plant carbon and water balance are manifest after prolonged drought, performance reductions were not amplified by warmer temperatures. Rather, increased temperatures for 5 years stimulated soil N cycling under piñon trees and modified tree N allocation for both species, resulting in mitigation of hotter drought impacts on tree water and carbon functions. These findings suggest that adjustments in N cycling are likely after multi-year warming conditions and that such changes may buffer reductions in tree performance during hotter droughts. The results highlight our incomplete understanding of trees' ability to acclimate to climate change, raising fundamental questions about the resistance potential of forests to long-term, compound climatic stresses.

Tree water dynamics in a drying and warming world.

Disentangling the relative impacts of precipitation reduction and vapour pressure deficit (VPD) on plant water dynamics and determining whether acclimation may influence these patterns in the future is an important challenge. Here, we report sap flux density (FD ), stomatal conductance (Gs ), hydraulic conductivity (KL ) and xylem anatomy in piñon pine (Pinus edulis) and juniper (Juniperus monosperma) trees subjected to five years of precipitation reduction, atmospheric warming (elevated VPD) and their combined effects. No acclimation occurred under precipitation reduction: lower Gs and FD were found for both species compared to ambient conditions. Warming reduced the sensibility of stomata to VPD for both species but resulted in the maintenance of Gs and FD to ambient levels only for piñon. For juniper, reduced soil moisture under warming negated benefits of stomatal adjustments and resulted in reduced FD , Gs and KL . Although reduced stomatal sensitivity to VPD also occurred under combined stresses, reductions in Gs , FD and KL took place to similar levels as under single stresses for both species. Our results show that stomatal conductance adjustments to high VPD could minimize but not entirely prevent additive effects of warming and drying on water use and carbon acquisition of trees in semi-arid regions.

Integrating knowledge exchange and the assessment of dryland management alternatives - A learning-centered participatory approach.

The adoption of sustainable land management strategies and practices that respond to current climate and human pressures requires both assessment tools that can lead to better informed decision-making and effective knowledge-exchange mechanisms that facilitate new learning and behavior change. We propose a learning-centered participatory approach that links land management assessment and knowledge exchange and integrates science-based data and stakeholder perspectives on both biophysical and socio-economic attributes. We outline a structured procedure for a transparent assessment of land management alternatives, tailored to dryland management, that is based on (1) principles of constructivism and social learning, (2) the participation of stakeholders throughout the whole assessment process, from design to implementation, and (3) the combination of site-specific indicators, identified by local stakeholders as relevant to their particular objectives and context conditions, and science-based indicators that represent ecosystem services of drylands worldwide. The proposed procedure follows a pattern of eliciting, challenging, and self-reviewing stakeholder perspectives that aims to facilitate learning. The difference between the initial baseline perspectives and the final self-reviewed stakeholder perspectives is used as a proxy of learning. We illustrate the potential of this methodology by its application to the assessment of land uses in a Mediterranean fire-prone area in East Spain. The approach may be applied to a variety of socio-ecological systems and decision-making and governance scales.

Towards understanding resprouting at the global scale.

Understanding and predicting plant response to disturbance is of paramount importance in our changing world. Resprouting ability is often considered a simple qualitative trait and used in many ecological studies. Our aim is to show some of the complexities of resprouting while highlighting cautions that need be taken in using resprouting ability to predict vegetation responses across disturbance types and biomes. There are marked differences in resprouting depending on the disturbance type, and fire is often the most severe disturbance because it includes both defoliation and lethal temperatures. In the Mediterranean biome, there are differences in functional strategies to cope with water deficit between resprouters (dehydration avoiders) and nonresprouters (dehydration tolerators); however, there is little research to unambiguously extrapolate these results to other biomes. Furthermore, predictions of vegetation responses to changes in disturbance regimes require consideration not only of resprouting, but also other relevant traits (e.g. seeding, bark thickness) and the different correlations among traits observed in different biomes; models lacking these details would behave poorly at the global scale. Overall, the lessons learned from a given disturbance regime and biome (e.g. crown-fire Mediterranean ecosystems) can guide research in other ecosystems but should not be extrapolated at the global scale.

Physiological differences explain the co-existence of different regeneration strategies in Mediterranean ecosystems.

• Fire and drought are selective driving forces in Mediterranean plants, and thus their ability to resprout or recruit after these disturbances is of paramount importance. The contrast in regeneration niche between resprouters and seeders, and each group's different root characteristics, suggest that they are subjected to different degrees of environmental stress and, consequently, to different evolutionary forces. • We compared leaf traits, xylem traits related to hydraulic efficiency and vulnerability to cavitation, and the physiological response to an imposed drought between seedlings of resprouters and seedlings of seeders. We used 12 species co-existing in Mediterranean basin ecosystems. • Major differences were found in the xylem architecture and leaf traits, and in the response to drought conditions. Seeders were more efficient at transporting water to leaves but formed, in turn, a safer xylem network. They also presented higher photosynthesis and transpiration rates, and earlier stomatal closure with drought, but good leaf dehydration tolerance. • Seeders and resprouters can be considered two syndromes whose different functional characteristics are related to water availability and drought responses. These characteristics, together with their differences in rooting habits, account for their distinct regeneration niches and, thus, their co-existence.

Morphological and physiological divergences within Quercus ilex support the existence of different ecotypes depending on climatic dryness.

BACKGROUND AND AIMS: Several studies show apparently contradictory findings about the functional convergence within the Mediterranean woody flora. In this context, this study evaluates the variability of functional traits within holm oak (Quercus ilex) to elucidate whether provenances corresponding to different morphotypes represent different ecotypes locally adapted to the prevaling stress levels. METHODS: Several morphological and physiological traits were measured at leaf and shoot levels in 9-year-old seedlings of seven Q. ilex provenances including all recognized morphotypes. Plants were grown in a common garden for 9 years under the same environmental conditions to avoid possible biases due to site-specific characteristics. KEY RESULTS: Leaf morphometry clearly separates holm oak provenances into 'ilex' (more elongated leaves with low vein density) and 'rotundifolia' (short and rounded leaves with high vein density) morphotypes. Moreover, these morphotypes represent two consistent and very contrasting functional types in response to dry climates, mainly in terms of leaf area, major vein density, leaf specific conductivity, resistance to drought-induced cavitation and turgor loss point. CONCLUSIONS: The 'ilex' and 'rotundifolia' morphotypes correspond to different ecotypes as inferred from their contrasting functional traits. To the best of our knowledge, this is the first time that the combined use of morphological and physiological traits has provided support for the concept of these two holm oak morphotypes being regarded as two different species.

Alleviation of Zn toxicity by low water availability.

Heavy metal contamination and drought are expected to increase in large areas worldwide. However, their combined effect on plant performance has been scantly analyzed. This study examines the effect of Zn supply at different water availabilities on morpho-physiological traits of Quercus suber L. in order to analyze the combined effects of both stresses. Seedlings were treated with four levels of zinc from 3 to 150 µM and exposed to low watering (LW) or high watering (HW) frequency in hydroponic culture, using a growth chamber. Under both watering regimes, Zn concentration in leaves and roots increased with Zn increment in nutrient solution. Nevertheless, at the highest Zn doses, Zn tissue concentrations were almost twice in HW than in LW seedlings. Functional traits as leaf photosynthetic rate and root hydraulic conductivity, and morphological traits as root length and root biomass decreased significantly in response to Zn supply. Auxin levels increased with Zn concentrations, suggesting the involvement of this phytohormone in the seedling response to this element. LW seedlings exposed to 150 µM Zn showed higher root length and root biomass than HW seedlings exposed to the same Zn dose. Our results suggest that low water availability could mitigate Zn toxicity by limiting internal accumulation. Morphological traits involved in the response to both stresses probably contributed to this response.

Root architecture and hydraulic conductance in nutrient deprived Pistacia lentiscus L. seedlings.

Plants respond to low nutrient availability by modifying root morphology and root system topology. Root responses to nitrogen (N) and phosphorus (P) limitation may affect plant capacity to withstand water stress. But studies on the effect of nutrient availability on plant ability to uptake and transport water are scarce. In this study, we assess the effect of nitrogen and phosphorus limitation on root morphology and root system topology in Pistacia lentiscus L seedlings, a common Mediterranean shrub, and relate these changes to hydraulic conductivity of the whole root system. Nitrogen and phosphorus deprivation had no effect on root biomass, but root systems were more branched in nutrient limited seedlings. Total root length was higher in seedlings subjected to phosphorus deprivation. Root hydraulic conductance decreased in nutrient-deprived seedlings, and was related to the number of root junctions but not to other architectural traits. Our study shows that changes in nutrient availability affect seedling water use by modifying root architecture. Changes in nutrient availability should be taken into account when evaluating seedling response to drought.

Aridity and cold temperatures drive divergent adjustments of European beech xylem anatomy, hydraulics and leaf physiological traits.

Understanding plant trait coordination and variance across climatic gradients is critical for assessing forests' adaptive potential to climate change. We measured 11 hydraulic, anatomical and leaf-level physiological traits in European beech (Fagus sylvatica L.) along a moisture and temperature gradient in the French Alps. We assessed how traits covaried, and how their population-level variances shifted along the gradient. The intrapopulation variances of vessel size and xylem-specific conductivity reduced in colder locations as narrow vessels were observed in response to low temperature. This decreased individual-level water transport capacity compared with the warmer and more xeric sites. Conversely, the maximum stomatal conductance and Huber value variances were constrained in the arid and warm locations, where trees showed restricted gas exchange and higher xylem-specific conductivity. The populations growing under drier and warmer conditions presented wide variance for the xylem anatomical and hydraulic traits. Our results suggest that short-term physiological acclimation to raising aridity and heat in southern beech populations may occur mainly at the leaf level. Furthermore, the wide variance of the xylem anatomical and hydraulic traits at these sites may be advantageous since more heterogeneous hydraulic conductivity could imply populations' greater tree-tree complementarity and resilience against climatic variability. Our study highlights that both intrapopulation trait variance and trait network analysis are key approaches for understanding species adaptation and the acclimation potential to a shifting environment.

Physiological and Molecular Characterization of the Differential Response of Broccoli (Brassica oleracea var. Italica) Cultivars Reveals Limiting Factors for Broccoli Tolerance to Drought Stress.

Broccoli is a cruciferous crop rich in health-promoting metabolites. Due to several factors, including anthropogenic global warming, aridity is increasing in many cultivation areas. There is a great demand to characterize the drought response of broccoli and use this knowledge to develop new cultivars able to maintain yield under water constraints. The aim of this study is to characterize the drought response at the physiological and molecular level of different broccoli (Brassica oleracea L. var. Italica Plenck) cultivars, previously characterized as drought-sensitive or drought-tolerant. This approach aims to identify different traits, which can constitute limiting factors for drought stress tolerance in broccoli. For this purpose, we have compared several physiological parameters and the complete profiles of amino acids, primary metabolites, hormones, and ions of drought-tolerant and drought-sensitive cultivars under stress and control conditions. We have found that drought-tolerant cultivars presented higher levels of methionine and abscisic acid and lower amounts of urea, quinic acid, and the gluconic acid lactone. Interestingly, we have also found that a drought treatment increases the levels of most essential amino acids in leaves and in florets. Our results have established physiological and molecular traits useful as distinctive markers to predict drought tolerance in broccoli or which could be reliably used for breeding new cultivars adapted to water scarcity. We have also found that a drought treatment increases the content of essential amino acids in broccoli.

Distinctive Traits for Drought and Salt Stress Tolerance in Melon (Cucumis melo L.).

Melon (Cucumis melo L.) is a crop with important agronomic interest worldwide. Because of the increase of drought and salinity in many cultivation areas as a result of anthropogenic global warming, the obtention of varieties tolerant to these conditions is a major objective for agronomical improvement. The identification of the limiting factors for stress tolerance could help to define the objectives and the traits which could be improved by classical breeding or other techniques. With this objective, we have characterized, at the physiological and biochemical levels, two different cultivars (sensitive or tolerant) of two different melon varieties (Galia and Piel de Sapo) under controlled drought or salt stress. We have performed physiological measurements, a complete amino acid profile and we have determined the sodium, potassium and hormone concentrations. This has allowed us to determine that the distinctive general trait for salt tolerance in melon are the levels of phenylalanine, histidine, proline and the Na+/K+ ratio, while the distinctive traits for drought tolerance are the hydric potential, isoleucine, glycine, phenylalanine, tryptophan, serine, and asparagine. These could be useful markers for breeding strategies or to predict which varieties are likely perform better under drought or salt stress. Our study has also allowed us to identify which metabolites and physiological traits are differentially regulated upon salt and drought stress between different varieties.

Does sample length influence the shape of xylem embolism vulnerability curves? A test with the Cavitron spinning technique.

The Cavitron spinning technique is used to construct xylem embolism vulnerability curves (VCs), but its reliability has been questioned for species with long vessels. This technique generates two types of VC: sigmoid 's'-shaped and exponential, levelling-off 'r'-shaped curves. We tested the hypothesis that 'r'-shaped VCs were anomalous and caused by the presence of vessels cut open during sample preparation. A Cavitron apparatus was used to construct VCs from samples of different lengths in species with contrasting vessel lengths. The results were compared with VCs obtained using other independent techniques. When vessel length exceeded sample length, VCs were 'r'-shaped and anomalous. Filling vessels cut open at both ends with air before measurement produced more typical 's'-shaped VCs. We also found that exposing segments of 11 woody species in a Cavitron at the pressure measured in planta before sampling considerably increased the degree of embolism above the native state level for species with long vessels. We concluded that open vessels were abnormally more vulnerable to cavitation than intact vessels. We recommend restricting this technique to species with short conduits. The relevance of our conclusions for other spinning techniques is discussed.

Drought Tolerance in Pinus halepensis Seed Sources As Identified by Distinctive Physiological and Molecular Markers.

Drought is one of the main constraints determining forest species growth, survival and productivity, and therefore one of the main limitations for reforestation or afforestation. The aim of this study is to characterize the drought response at the physiological and molecular level of different Pinus halepensis (common name Aleppo pine) seed sources, previously characterized in field trials as drought-sensitive or drought-tolerant. This approach aims to identify different traits capable of predicting the ability of formerly uncharacterized seedlings to cope with drought stress. Gas-exchange, water potential, photosynthetic pigments, soluble sugars, free amino acids, glutathione and proteomic analyses were carried out on control and drought-stressed seedlings in greenhouse conditions. Gas-exchange determinations were also assessed in field-planted seedlings in order to validate the greenhouse experimental conditions. Drought-tolerant seed sources presented higher values of photosynthetic rates, water use efficiency, photosynthetic pigments and soluble carbohydrates concentrations. We observed the same pattern of variation of photosynthesis rate and maximal efficiency of PSII in field. Interestingly drought-tolerant seed sources exhibited increased levels of glutathione, methionine and cysteine. The proteomic profile of drought tolerant seedlings identified two heat shock proteins and an enzyme related to methionine biosynthesis that were not present in drought sensitive seedlings, pointing to the synthesis of sulfur amino acids as a limiting factor for drought tolerance in Pinus halepensis. Our results established physiological and molecular traits useful as distinctive markers to predict drought tolerance in Pinus halepensis provenances that could be reliably used in reforestation programs in drought prone areas.