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As the single link between leaves and the rest of the plant, petioles must develop conductive tissues according to the water influx and sugar outflow of the leaf lamina. A scaling relationship between leaf area and anatomical traits of xylem and phloem is expected to improve the efficiency of these tissues. However, the different constraints compromising the functionality of both tissues (e.g., risk of cavitation) must not be disregarded. Additionally, deciduous and evergreen plants may have different strategies to produce and package their petiole conduits to cope with environmental restrictions. We explored in 33 oak species the relationships between petiole anatomical traits, leaf area, stomatal conductance, and photosynthesis rate. Results showed allometric scaling between anatomical structure of xylem and phloem with leaf area. We also found correlations between photosynthesis rate, stomatal conductance, and anatomical traits in the petiole. The main novelty is how oaks present a different strategy depending on the leaf habit. Deciduous species tend to increase their diameters to achieve greater leaf-specific conductivity. By contrast, evergreen oaks develop larger xylem conductive areas for a given leaf area than deciduous ones. This trade-off between safety-efficiency in petioles has never been attributed to the leaf habit of the species., (© 2024 John Wiley & Sons Ltd.)

One of the well-documented effects of regional warming in Antarctica is the impact on flora. Warmer conditions modify several leaf anatomical traits of Antarctic vascular plants, increasing photosynthesis and growth. Given that CO2 and water vapor partially share their diffusion pathways through the leaf, changes in leaf anatomy could also affect the hydraulic traits of Antarctic plants. We evaluated the effects of growth temperature on several anatomical and hydraulic parameters of Antarctic plants and assessed the trait co-variation between these parameters and photosynthetic performance. Warmer conditions promoted an increase in leaf and whole plant hydraulic conductivity, correlating with adjustments in carbon assimilation. These adjustments were consistent with changes in leaf vasculature, where Antarctic species displayed different strategies. At higher temperature, Colobanthus quitensis decreased the number of leaf xylem vessels, but increased their diameter. In contrast, in Deschampsia antarctica the diameter did not change, but the number of vessels increased. Despite this contrasting behavior, some traits such as a small leaf diameter of vessels and a high cell wall rigidity were maintained in both species, suggesting a water-conservation response associated with the ability of Antarctic plants to cope with harsh environments., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Drought is projected to occur more frequently and intensely in the coming decades, and the extent to which it will affect forest functioning will depend on species-specific responses to water stress. Aiming to understand the hydraulic traits and water dynamics behind water-saver and water-spender strategies in response to drought and recovery, we conducted a pot experiment with two species with contrasting physiological strategies, Scots pine (Pinus sylvestris L.) and Portuguese oak (Quercus faginea L.). We applied two cycles of soil drying and recovery and irrigated with isotopically different water to track fast changes in soil and stem water pools, while continuously measuring physiological status and xylem water content from twigs. Our results provide evidence for a tight link between the leaf-level response and the water uptake and storage patterns in the stem. The water-saver strategy of pines prevented stem dehydration by rapidly closing stomata which limited their water uptake during the early stages of drought and recovery. Conversely, oaks showed a less conservative strategy, maintaining transpiration and physiological activity under dry soil conditions, and consequently becoming more dehydrated at the stem level. We interpreted this dehydration as the release of water from elastic storage tissues as no major loss of hydraulic conductance occurred for this species. After soil rewetting, pines recovered pre-drought leaf water potential rapidly, but it took longer to replace the water from conductive tissues (slower labeling speed). In contrast, water-spender oaks were able to quickly replace xylem water during recovery (fast labeling speed), but it took longer to refill stem storage tissues, and hence to recover pre-drought leaf water potential. These different patterns in sap flow rates, speed and duration of the labeling reflected a combination of water-use and storage traits, linked to the leaf-level strategies in response to drought and recovery., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background and Aims: The existence of sclerophyllous plants has been considered an adaptive strategy against different environmental stresses. Given that it literally means 'hard-leaved', it is essential to quantify the leaf mechanical properties to understand sclerophylly. However, the relative importance of each leaf trait for mechanical properties is not yet well established., Methods: Genus Quercus is an excellent system to shed light on this because it minimizes phylogenetic variation while having a wide variation in sclerophylly. We measured leaf anatomical traits and cell wall composition, analysing their relationship with leaf mass per area and leaf mechanical properties in a set of 25 oak species., Key Results: The upper epidermis outer wall makes a strong and direct contribution to the leaf mechanical strength. Moreover, cellulose plays a crucial role in increasing leaf strength and toughness. The principal component analysis plot based on leaf trait values clearly separates Quercus species into two groups corresponding to evergreen and deciduous species., Conclusions: Sclerophyllous Quercus species are tougher and stronger owing to their thicker epidermis outer wall and/or higher cellulose concentration. Furthermore, section Ilex species share common traits, although they occupy different climates. In addition, evergreen species living in mediterranean-type climates share common leaf traits irrespective of their different phylogenetic origin., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Leaves of Mediterranean evergreen tree species experience a reduction in net CO2 assimilation (AN) and mesophyll conductance to CO2 (gm) during aging and senescence, which would be influenced by changes in leaf anatomical traits at cell level. Anatomical modifications can be accompanied by the dismantling of photosynthetic apparatus associated to leaf senescence, manifested through changes at the biochemical level (i.e., lower nitrogen investment in photosynthetic machinery). However, the role of changes in leaf anatomy at cell level and nitrogen content in gm and AN decline experienced by old non-senescent leaves of evergreen trees with long leaf lifespan is far from being elucidated. We evaluated age-dependent changes in morphological, anatomical, chemical and photosynthetic traits in Quercus ilex subsp. rotundifolia Lam., an evergreen oak with high leaf longevity. All photosynthetic traits decreased with increasing leaf age. The relative change in cell wall thickness (Tcw) was less than in chloroplast surface area exposed to intercellular air space (Sc/S), and Sc/S was a key anatomical trait explaining variations in gm and AN among different age classes. The reduction of Sc/S was related to ultrastructural changes in chloroplasts associated to leaf aging, with a concomitant reduction in cytoplasmic nitrogen. Changes in leaf anatomy and biochemistry were responsible for the age-dependent modifications in gm and AN. These findings revealed a gradual physiological deterioration related to the dismantling of the photosynthetic apparatus in older leaves of Q. ilex subsp. rotundifolia., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The growth of past, present, and future forests was, is and will be affected by climate variability. This multifaceted relationship has been assessed in several regional studies, but spatially resolved, large-scale analyses are largely missing so far. Here we estimate recent changes in growth of 5800 beech trees (Fagus sylvatica L.) from 324 sites, representing the full geographic and climatic range of species. Future growth trends were predicted considering state-of-the-art climate scenarios. The validated models indicate growth declines across large region of the distribution in recent decades, and project severe future growth declines ranging from -20% to more than -50% by 2090, depending on the region and climate change scenario (i.e. CMIP6 SSP1-2.6 and SSP5-8.5). Forecasted forest productivity losses are most striking towards the southern distribution limit of Fagus sylvatica, in regions where persisting atmospheric high-pressure systems are expected to increase drought severity. The projected 21 st century growth changes across Europe indicate serious ecological and economic consequences that require immediate forest adaptation., (© 2022. The Author(s).)

The search for a universal explanation of the altitudinal limit determined by the alpine treeline has given rise to different hypotheses. In this study, we revisited Michaelis' hypothesis which proposed that an inadequate "ripening" of the cuticle caused a greater transpiration rate during winter in the treeline. However, few studies with different explanations have investigated the role of passive mechanisms of needles for protecting against water loss during winter in conifers at the treeline. To shed light on this, the cuticular transpiration barrier was studied in the transition from subalpine Pinus uncinata forests to alpine tundra at the upper limit of the species in the Pyrenees. This upper limit of P. uncinata was selected here as an example of the ecotones formed by conifers in the temperate mountains of the northern hemisphere. Our study showed that minimum leaf conductance in needles from upper limit specimens was higher than those measured in specimens living in the lower levels of the sub-alpine forest and also displayed lower cuticle thickness values, which should reinforce the seminal hypothesis by Michaelis. Our study showed clear evidence that supports the inadequate development of needle cuticles as one of the factors that lead to increased transpirational water losses during winter and, consequently, a higher risk of suffering frost drought., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bueno, Alonso-Forn, Peguero-Pina, de Souza, Ferrio, Sancho-Knapik and Gil-Pelegrín.)

Increases in leaf mass per area (LMA) are commonly observed in response to environmental stresses and are achieved through increases in leaf thickness and/or leaf density. Here, we investigated how the two underlying components of LMA differ in relation to species native climates and phylogeny, across deciduous and evergreen species. Using a phylogenetic approach, we quantified anatomical, compositional and climatic variables from 40 deciduous and 45 evergreen Quercus species from across the Northern Hemisphere growing in a common garden. Deciduous species from shorter growing seasons tended to have leaves with lower LMA and leaf thickness than those from longer growing seasons, while the opposite pattern was found for evergreens. For both habits, LMA and thickness increased in arid environments. However, this shift was associated with increased leaf density in evergreens but reduced density in deciduous species. Deciduous and evergreen oaks showed fundamental leaf morphological differences that revealed a diverse adaptive response. While LMA in deciduous species may have diversified in tight coordination with thickness mainly modulated by aridity, diversification of LMA within evergreens appears to be dependent on the infrageneric group, with diversification in leaf thickness modulated by both aridity and cold, while diversification in leaf density is only modulated by aridity., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Foundation.)

Nowadays, evergreen sclerophyllous and winter-deciduous malacophyllous oaks with different paleogeographical origins coexist under Mediterranean-type climates, such as the mixed forests of the evergreen Quercus ilex subsp. rotundifolia Lam. and the winter-deciduous Quercus faginea Lam. Both Mediterranean oaks constitute two examples of contrasting leaf habit, so it could be expected that they would have different functional strategies to cope with summer drought. In this study, we analysed photosynthetic, photochemical and hydraulic traits of different organs for Q. faginea and Q. ilex subsp. rotundifolia under well-watered conditions and subjected to very severe drought. The coordinated response between photosynthetic and hydraulic traits explained the higher photosynthetic capacity of Q. faginea under well-watered conditions, which compensated its shorter leaf life span at the expense of higher water consumption. The progressive imposition of water stress evidenced that both types of Mediterranean oaks displayed different functional strategies to cope with water limitations. Specifically, the decrease in mesophyll conductance associated with edaphic drought seems to be the main factor explaining the differences found in the dynamics of net CO2 assimilation throughout the drought period. The sharp decline in photosynthetic traits of Q. faginea was coupled with a strong decrease in shoot hydraulic conductance in response to drought. This fact probably avoided extensive xylem embolism in the stems (i.e., 'vulnerability segmentation'), which enabled new leaf development after drought period in Q. faginea. By contrast, leaves of Q. ilex subsp. rotundifolia showed effective photoprotective mechanisms and high resistance to drought-induced cavitation, which would be related with the longer leaf life span of the evergreen Mediterranean oaks. The co-occurrence of both types of Mediterranean oaks could be related to edaphic conditions that ensure the maintenance of soil water potential above critical values for Q. faginea, which can be severely affected by soil degradation and climate change., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

The absorption of water and solutes by plant leaves has been recognised since more than two centuries. Given the polar nature of water and solutes, the mechanisms of foliar uptake have been proposed to be similar for water and electrolytes, including nutrient solutions. Research efforts since the 19th century focussed on characterising the properties of cuticles and applying foliar sprays to crop plants as a tool for improving crop nutrition. This was accompanied by the development of hundreds of studies aimed at characterising the chemical and structural nature of plant cuticles from different species and the mechanisms of cuticular and, to a lower extent, stomatal penetration of water and solutes. The processes involved are complex and will be affected by multiple environmental, physico-chemical and physiological factors which are only partially clear to date. During the last decades, the body of evidence that water transport across leaf surfaces of native species may contribute to water balances (absorption and loss) at an ecosystem level has grown. Given the potential importance of foliar water absorption for many plant species and ecosystems as shown in recent studies, the aim of this review is to first integrate current knowledge on plant surface composition, structure, wettability and physico-chemical interactions with surface-deposited matter. The different mechanisms of foliar absorption of water and electrolytes and experimental procedures for tracing the uptake process are discussed before posing several outstanding questions which should be tackled in future studies., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Predicted increases in atmospheric concentration of carbon dioxide (CO 2 ) coupled with increased temperatures and drought are expected to strongly influence the development of most of the plant species in the world, especially in areas with high risk of desertification like the Mediterranean basin. Helianthemum almeriense is an ecologically important Mediterranean shrub with an added interest because it serves as the host for the Terfezia claveryi mycorrhizal fungus, which is a desert truffle with increasingly commercial interest. Although both plant and fungi are known to be well adapted to dry conditions, it is still uncertain how the increase in atmospheric CO 2 will influence them. In this article we have addressed the physiological responses of H. almeriense × T. claveryi mycorrhizal plants to increases in atmospheric CO 2 coupled with drought and high vapor pressure deficit. This work reports one of the few estimations of mesophyll conductance in a drought deciduous Mediterranean shrub and evaluates its role in photosynthesis limitation. High atmospheric CO 2 concentrations help desert truffle mycorrhizal plants to cope with the adverse effects of progressive drought during Mediterranean springs by improving carbon net assimilation, intrinsic water use efficiency and dispersal of the species through increased flowering events., (© 2020 Scandinavian Plant Physiology Society.)

Resprouting is an ancestral trait in angiosperms that confers resilience after perturbations. As climate change increases stress, resprouting vigor is declining in many forest regions, but the underlying mechanism is poorly understood. Resprouting in woody plants is thought to be primarily limited by the availability of non-structural carbohydrate reserves (NSC), but hydraulic limitations could also be important. We conducted a multifactorial experiment with two levels of light (ambient, 2-3% of ambient) and three levels of water stress (0, 50 and 80 percent losses of hydraulic conductivity, PLC) on two Mediterranean oaks (Quercus ilex and Q. faginea) under a rain-out shelter (n = 360). The proportion of resprouting individuals after canopy clipping declined markedly as PLC increased for both species. NSC concentrations affected the response of Q. ilex, the species with higher leaf construction costs, and its effect depended on the PLC. The growth of resprouting individuals was largely dependent on photosynthetic rates for both species, while stored NSC availability and hydraulic limitations played minor and non-significant roles, respectively. Contrary to conventional wisdom, our results indicate that resprouting in oaks may be primarily driven by complex interactions between hydraulics and carbon sources, whereas stored NSC play a significant but secondary role., (© 2020 John Wiley & Sons Ltd.)

Plants prevent uncontrolled water loss by synthesizing, depositing and maintaining a hydrophobic layer over their primary aerial organs-the plant cuticle. Quercus coccifera L. can plastically respond to environmental conditions at the cuticular level. When exposed to hot summer conditions with high vapour-pressure deficit (VPD) and intense solar radiation (Mediterranean atmospheric conditions; MED), this plant species accumulates leaf cuticular waxes even over the stomata, thereby decreasing transpirational water loss. However, under mild summer conditions with moderate VPD and regular solar radiation (temperate atmospheric conditions; TEM), this effect is sharply reduced. Despite the ecophysiological importance of the cuticular waxes of Q. coccifera, the wax composition and its contribution to avoiding uncontrolled dehydration remain unknown. Thus, we determined several leaf traits for plants exposed to both MED and TEM conditions. Further, we qualitatively and quantitatively investigated the cuticular lipid composition by gas chromatography. Finally, we measured the minimum leaf conductance (gmin) as an indicator of the efficacy of the cuticular transpiration barrier. The MED leaves were smaller, stiffer and contained a higher load of cuticular lipids than TEM leaves. The amounts of leaf cutin and cuticular waxes of MED plants were 1.4 times and 2.6 times higher than that found for TEM plants, respectively. In detail, MED plants produced higher amounts of all compound classes of cuticular waxes, except for the equivalence of alkanoic acids. Although MED leaves contained higher cutin and cuticular wax loads, the gmin was not different between the two habitats. Our findings suggest that the qualitative accumulation of equivalent cuticular waxes might compensate for the higher wax amount of MED plants, thereby contributing equally to the efficacy of the cuticular transpirational barrier of Q. coccifera. In conclusion, we showed that atmospheric conditions profoundly affect the cuticular lipid composition of Q. coccifera leaves, but do not alter its transpiration barrier properties., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The wide band and air-coupled ultrasonic resonant spectroscopy together with a modified Simulated Annealing metaheuristic algorithm and a 1D layered acoustic-model are used to resolve the structure of plant leaves. In particular, this paper focuses on the extraction of the surface density of the different layers of tissue in leaves having a relatively simple structure. There are three main reasons to select the surface density as the focus of this study: (i) it is a parameter directly extracted by the proposed technique and it requires no further processing, (ii) it is relevant in order to study the dynamic of the water within the different tissues of the leaves and also to study the differential development of the different tissues, and (iii) unlike other parameters provided by this technique (like resonant frequency, impedance, ultrasonic elastic modulus, or ultrasonic damping), this parameter can be easier to understand as it is a direct measure of mass per unit surface. The selection of leaves with a simple structure is justified by the convenience of avoiding an unnecessary complication of the data extraction step. In this work, the technique was applied to determine the surface density of the palisade and spongy parenchyma layers of tissue of Ligustrum lucidum , Vitis vinifera , and Viburnum tinus leaves. The first species was used to study the variation of the surface density at full turgor with the thickness of the leaf, while the two other species were used to study the variation of the surface densities with the variation in the leaf relative water content. Consistency of the results with other conventional measurements (like overall surface density, and cross-section optical and cryo-SEM images) is discussed. The results obtained reveal the potential of this technique; moreover, the technique presents the additional advantage that can be applied in-vivo as it is completely non-invasive, non-destructive, fast, and equipment required is portable., (Copyright © 2020 Alvarez-Arenas, Sancho-Knapik, Peguero-Pina and Gil-Pelegrín.)

Plants prevent uncontrolled water loss by synthesizing, depositing and maintaining a hydrophobic layer over their primary aerial organs-the plant cuticle. Quercus coccifera L. can plastically respond to environmental conditions at the cuticular level. When exposed to hot summer conditions with high vapour-pressure deficit (VPD) and intense solar radiation (Mediterranean atmospheric conditions; MED), this plant species accumulates leaf cuticular waxes even over the stomata, thereby decreasing transpirational water loss. However, under mild summer conditions with moderate VPD and regular solar radiation (temperate atmospheric conditions; TEM), this effect is sharply reduced. Despite the ecophysiological importance of the cuticular waxes of Q. coccifera, the wax composition and its contribution to avoiding uncontrolled dehydration remain unknown. Thus, we determined several leaf traits for plants exposed to both MED and TEM conditions. Further, we qualitatively and quantitatively investigated the cuticular lipid composition by gas chromatography. Finally, we measured the minimum leaf conductance (gmin) as an indicator of the efficacy of the cuticular transpiration barrier. The MED leaves were smaller, stiffer and contained a higher load of cuticular lipids than TEM leaves. The amounts of leaf cutin and cuticular waxes of MED plants were 1.4 times and 2.6 times higher than that found for TEM plants, respectively. In detail, MED plants produced higher amounts of all compound classes of cuticular waxes, except for the equivalence of alkanoic acids. Although MED leaves contained higher cutin and cuticular wax loads, the gmin was not different between the two habitats. Our findings suggest that the qualitative accumulation of equivalent cuticular waxes might compensate for the higher wax amount of MED plants, thereby contributing equally to the efficacy of the cuticular transpirational barrier of Q. coccifera. In conclusion, we showed that atmospheric conditions profoundly affect the cuticular lipid composition of Q. coccifera leaves, but do not alter its transpiration barrier properties., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Non-contact resonant ultrasound spectroscopy (NC-RUS) has been proven as a reliable technique for the dynamic determination of leaf water status. It has been already tested in more than 50 plant species. In parallel, relative water content (RWC) is highly used in the ecophysiological field to describe the degree of water saturation in plant leaves. Obtaining RWC implies a cumbersome and destructive process that can introduce artefacts and cannot be determined instantaneously., Results: Here, we present a method for the estimation of RWC in plant leaves from non-contact resonant ultrasound spectroscopy (NC-RUS) data. This technique enables to collect transmission coefficient in a [0.15-1.6] MHz frequency range from plant leaves in a non-invasive, non-destructive and rapid way. Two different approaches for the proposed method are evaluated: convolutional neural networks (CNN) and random forest (RF). While CNN takes the entire ultrasonic spectra acquired from the leaves, RF only uses four relevant parameters resulted from the transmission coefficient data. Both methods were tested successfully in Viburnum tinus leaf samples with Pearson's correlations between 0.92 and 0.84., Conclusions: This study showed that the combination of NC-RUS technique with deep learning algorithms is a robust tool for the instantaneous, accurate and non-destructive determination of RWC in plant leaves., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

The Antarctic Peninsula has experienced a rapid warming in the last decades. Although recent climatic evidence supports a new tendency towards stabilization of temperatures, the impacts on the biosphere, and specifically on Antarctic plant species, remain unclear. We evaluated the in situ warming effects on photosynthesis, including the underlying diffusive, biochemical and anatomical determinants, and the relative growth of two Antarctic vascular species, Colobanthus quitensis and Deschampsia antarctica, using open top chambers (OTCs) and gas exchange measurements in the field. In C. quitensis, the photosynthetic response to warming relied on specific adjustments in the anatomical determinants of the leaf CO 2 transfer, which enhanced mesophyll conductance and photosynthetic assimilation, thereby promoting higher leaf carbon gain and plant growth. These changes were accompanied by alterations in the leaf chemical composition. By contrast, D. antarctica showed no response to warming, with a lack of significant differences between plants grown inside OTCs and plants grown in the open field. Overall, the present results are the first reporting a contrasting effect of in situ warming on photosynthesis and its underlying determinants, of the two unique Antarctic vascular plant species, which could have direct consequences on their ecological success under future climate conditions., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

[This corrects the article DOI: 10.1371/journal.pone.0183970.].

Most aerial plant surfaces are covered with a lipid-rich cuticle, which is a barrier for the bidirectional transport of substances between the plant and the surrounding environment. This review article provides an overview of the significance of the leaf cuticle as a barrier for the deposition and absorption of water and electrolytes. After providing insights into the physico-chemical properties of plant surfaces, the mechanisms of foliar absorption are revised with special emphasis on solutes. Due to the limited information and relative importance of the leaf cuticle of herbaceous and deciduous cultivated plants, an overview of the studies developed with Alpine conifers and treeline species is provided. The significance of foliar water uptake as a phenomenon of ecophysiological relevance in many areas of the world is also highlighted. Given the observed variability in structure and composition among, for example, plant species and organs, it is concluded that it is currently not possible to establish general permeability and wettability models that are valid for predicting liquid-surface interactions and the subsequent transport of water and electrolytes across plant surfaces., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Submediterranean forests are considered an ecotone between Mediterranean and Eurosiberian ecosystems, and are very sensitive to global change. A decline of Scots pine (Pinus sylvestris L.) and a related expansion of oak species (Quercus spp.) have been reported in the Spanish Pre-Pyrenees. Although this has been associated with increasing drought stress, the underlying mechanisms are not fully understood, and suitable monitoring protocols are lacking. The aim of this study is to bring insight into the physiological mechanisms anticipating selective decline of the pines, with particular focus on carbon and water relations. For this purpose, we performed a sampling campaign covering two growing seasons in a mixed stand of P. sylvestris and Quercus subpyrenaica E.H del Villar. We sampled seasonally twig xylem and soil for water isotope composition (δ18O and δ2H), leaves for carbon isotope composition (δ13C) and stems to quantify non-structural carbohydrates (NSC) concentration, and measured water potential and leaf gas exchange. The first summer drought was severe for both species, reaching low predawn water potential (-2.2 MPa), very low stomatal conductance (12 ± 1.0 mmol m-2 s-1) and near-zero or even negative net photosynthesis, particularly in P. sylvestris (-0.6 ± 0.34 μmol m-2 s-1 in oaks, -1.3 ± 0.16 μmol m-2 s-1 in pines). Hence, the tighter stomatal control and more isohydric strategy of P. sylvestris resulted in larger limitations on carbon assimilation, and this was also reflected in carbon storage, showing twofold larger total NSC concentration in oaks than in pines (7.8 ± 2.4% and 4.0 ± 1.3%, respectively). We observed a faster recovery of predawn water potential after summer drought in Q. subpyrenaica than in P. sylvestris (-0.8 MPa and -1.1 MPa, respectively). As supported by the isotopic data, this was probably associated with a deeper and more reliable water supply in Q. subpyrenaica. In line with these short-term observations, we found a more pronounced negative effect of steadily increasing drought stress on long-term growth in pines compared with oaks. All these observations confer evidence of early warning of P. sylvestris decline and indicate the adaptive advantage of Q. subpyrenaica in the area., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Phylogenetic analysis by maximum likelihood (PAML) has become the standard approach to study positive selection at the molecular level, but other methods may provide complementary ways to identify amino acid replacements associated with particular conditions. Here, we compare results of the decision tree (DT) model method with ones of PAML using the key photosynthetic enzyme RuBisCO as a model system to study molecular adaptation to particular ecological conditions in oaks (Quercus). We sequenced the chloroplast rbcL gene encoding RuBisCO large subunit in 158 Quercus species, covering about a third of the global genus diversity. It has been hypothesized that RuBisCO has evolved differentially depending on the environmental conditions and leaf traits governing internal gas diffusion patterns. Here, we show, using PAML, that amino acid replacements at the residue positions 95, 145, 251, 262 and 328 of the RuBisCO large subunit have been the subject of positive selection along particular Quercus lineages associated with the leaf traits and climate characteristics. In parallel, the DT model identified amino acid replacements at sites 95, 219, 262 and 328 being associated with the leaf traits and climate characteristics, exhibiting partial overlap with the results obtained using PAML.

Leaf dry mass per unit area (LMA) has been suggested to negatively affect the mesophyll conductance to CO2 (gm), the most limiting factor for photosynthesis per unit leaf area (AN) in many evergreens. Several anatomical traits (i.e., greater leaf thickness and thicker cell walls) constraining gm could explain the negative scaling of gm and AN with LMA across species. However, the Mediterranean sclerophyll Quercus ilex L. shows a major within-species variation in functional traits (greater LMA associated with higher nitrogen content and AN) that might contrast the worldwide trends. The objective of this study was to elucidate the existence of variations in other leaf anatomical parameters determining gm and/or biochemical traits improving the capacity of carboxylation (Vc,max) that could modulate the relationship of AN with LMA across this species. The results revealed that gm was the most limiting factor for AN in all the studied Q. ilex provenances from Spain and Italy. The within-species differences in gm can be partly attributed to the variation in several leaf anatomical traits, mainly cell-wall thickness (Tcw), chloroplast thickness (Tchl) and chloroplast exposed surface area facing intercellular air spaces (Sc/S). A positive scaling of gm and AN with Vc,max was also found, associated with an increased nitrogen content per area. A strong correlation of maximum photosynthetic electron transport (Jmax) with AN further indicated a coordination between the carboxylase activity and the electron transport chain. In conclusion, we have confirmed the strong ecotypic variation in the photosynthetic performance of individual provenances of Q. ilex. Thus, the within-species increases found in AN for Q. ilex with increasing foliage robustness can be explained by a synergistic effect among anatomical (at the subcellular and cellular level) and biochemical traits, which markedly improved gm and Vc,max., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Particular physiological traits allow the vascular plants Deschampsia antarctica Desv. and Colobanthus quitensis (Kunth) Bartl. to inhabit Antarctica. The photosynthetic performance of these species was evaluated in situ, focusing on diffusive and biochemical constraints to CO2 assimilation. Leaf gas exchange, Chl a fluorescence, leaf ultrastructure, and Rubisco catalytic properties were examined in plants growing on King George and Lagotellerie islands. In spite of the species- and population-specific effects of the measurement temperature on the main photosynthetic parameters, CO2 assimilation was highly limited by CO2 diffusion. In particular, the mesophyll conductance (gm)-estimated from both gas exchange and leaf chlorophyll fluorescence and modeled from leaf anatomy-was remarkably low, restricting CO2 diffusion and imposing the strongest constraint to CO2 acquisition. Rubisco presented a high specificity for CO2 as determined in vitro, suggesting a tight co-ordination between CO2 diffusion and leaf biochemistry that may be critical ultimately to optimize carbon balance in these species. Interestingly, both anatomical and biochemical traits resembled those described in plants from arid environments, providing a new insight into plant functional acclimation to extreme conditions. Understanding what actually limits photosynthesis in these species is important to anticipate their responses to the ongoing and predicted rapid warming in the Antarctic Peninsula., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Leaf mass per area (LMA) has been suggested to negatively affect the mesophyll conductance to CO 2 (g m ), which is the most limiting factor for area-based photosynthesis (A N ) in many Mediterranean sclerophyll species. However, despite their high LMA, these species have similar A N to plants from other biomes. Variations in other leaf anatomical traits, such as mesophyll and chloroplast surface area exposed to intercellular air space (S m /S and S c /S), may offset the restrictions imposed by high LMA in g m and A N in these species. Seven sclerophyllous Mediterranean oaks from Europe/North Africa and North America with contrasting LMA were compared in terms of morphological, anatomical and photosynthetic traits. Mediterranean oaks showed specific differences in A N that go beyond the common morphological leaf traits reported for these species (reduced leaf area and thick leaves). These variations resulted mainly from the differences in g m , the most limiting factor for carbon assimilation in these species. Species with higher A N showed increased S c /S, which implies increased g m without changes in stomatal conductance. The occurrence of this anatomical adaptation at the cell level allowed evergreen oaks to reach A N values comparable to congeneric deciduous species despite their higher LMA., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

The main objective of this study was to apply the air-coupled broad-band ultrasonic spectroscopy in attached transpiring leaves of Vitis vinifera L. to monitor changes in leaf water potential (Ψ) through the measurements of the standardized value of the resonant frequency associated with the maximum transmitance ( f/f o ). With this purpose, the response of grapevine to a drought stress period was investigated in terms of leaf water status, ultrasounds, gas exchange and sugar accumulation. Two strong correlations were obtained between f/f o and Ψ measured at predawn (pd) and at midday (md) with different slopes. This fact implied the existence of two values of Ψ for a given value of f/f o , which was taken as a sign that the ultrasonic technique was not directly related to the overall Ψ, but only to one of its components: the turgor pressure ( P ). The difference in Ψ at constant f/f o (δ) was found to be dependent on net CO 2 assimilation ( A ) and might be used as a rough estimator of photosynthetic activity. It was then, the other main component of Ψ, osmotic potential (π), the one that may have lowered the values of md Ψ with respect to pd Ψ by the accumulation of sugars associated to net CO 2 assimilation. This phenomenon suggests the existence of a diurnal osmotic adjustment in this species associated to sugars production in well-watered plants.

Wood formation in European beech (Fagus sylvatica L.) and Scots pine (Pinus sylvestris L.) was intra-annually monitored to examine plastic responses of the xylem phenology according to altitude in one of the southernmost areas of their distribution range, i.e., in the Moncayo Natural Park, Spain. The monitoring was done from 2011 to 2013 at 1180 and 1580 m a.s.l., corresponding to the lower and upper limits of European beech forest in this region. Microcores containing phloem, cambium and xylem were collected biweekly from twenty-four trees from the beginning of March to the end of November to assess the different phases of wood formation. The samples were prepared for light microscopy to observe the following phenological phases: onset and end of cell production, onset and end of secondary wall formation in xylem cells and onset of cell maturation. The temporal dynamics of wood formation widely differed among years, altitudes and tree species. For Fagus sylvatica, the onset of cambial activity varied between the first week of May and the third week of June. Cambial activity then slowed down and stopped in summer, resulting in a length of growing season of 48-75 days. In contrast, the growing season for P. sylvestris started earlier and cambium remained active in autumn, leading to a period of activity varying from 139-170 days. The intra-annual wood-formation pattern is site and species-specific. Comparison with other studies shows a clear latitudinal trend in the duration of wood formation, positive for Fagus sylvatica and negative for P. sylvestris.

Leaves growing in the forest understory usually present a decreased mesophyll conductance (gm) and photosynthetic capacity. The role of leaf anatomy in determining the variability in gm among species is known, but there is a lack of information on how the acclimation of gm to shade conditions is driven by changes in leaf anatomy. Within this context, we demonstrated that Abies pinsapo Boiss. experienced profound modifications in needle anatomy to drastic changes in light availability that ultimately led to differential photosynthetic performance between trees grown in the open field and in the forest understory. In contrast to A. pinsapo, its congeneric Abies alba Mill. did not show differences either in needle anatomy or in photosynthetic parameters between trees grown in the open field and in the forest understory. The increased gm values found in trees of A. pinsapo grown in the open field can be explained by occurrence of stomata at both needle sides (amphistomatous needles), increased chloroplast surface area exposed to intercellular airspace, decreased cell wall thickness and, especially, decreased chloroplast thickness. To the best of our knowledge, the role of such drastic changes in ultrastructural needle anatomy in explaining the response of gm to the light environment has not been demonstrated in field conditions., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

'White oaks'--one of the main groups of the genus Quercus L.--are represented in western Eurasia by the 'roburoid oaks', a deciduous and closely related genetic group that should have an Arcto-Tertiary origin under temperate-nemoral climates. Nowadays, roburoid oak species such as Quercus robur L. are still present in these temperate climates in Europe, but others are also present in southern Europe under Mediterranean-type climates, such as Quercus faginea Lam. We hypothesize the existence of a coordinated functional response at the whole-shoot scale in Q. faginea under Mediterranean conditions to adapt to more xeric habitats. The results reveal a clear morphological and physiological segregation between Q. robur and Q. faginea, which constitute two very contrasting functional types in response to climate dryness. The most outstanding divergence between the two species is the reduction in transpiring area in Q. faginea, which is the main trait imposed by the water deficit in Mediterranean-type climates. The reduction in leaf area ratio in Q. faginea should have a negative effect on carbon gain that is partially counteracted by a higher inherent photosynthetic ability of Q. faginea when compared with Q. robur, as a consequence of higher mesophyll conductance, higher maximum velocity of carboxylation and much higher stomatal conductance (gs). The extremely high gs of Q. faginea counteracts the expected reduction in gs imposed by the stomatal sensitivity to vapor pressure deficit, allowing this species to diminish water losses maintaining high net CO2 assimilation values along the vegetative period under nonlimiting soil water potential values. In conclusion, the present study demonstrates that Q. faginea can be regarded as an example of adaptation of a deciduous oak to Mediterranean-type climates., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The accumulation of epicuticular waxes over stomata in Quercus coccifera L. contributes to a severe reduction in maximum stomatal conductance (g s,max) under Mediterranean (MED) conditions. However, this phenomenon was not observed in this species under temperate (TEM) conditions, which could lead to differences in the ability to assimilate CO2 between the sites. We hypothesise that the overall importance of such a reduction in gs,max on photosynthesis is modulated by other factors affecting carbon gain, mainly mesophyll conductance to CO2 (g m), through a plastic response to changes in environmental conditions (i.e., vapour pressure deficit, VPD, and mean daily quantum flux density, Q int). The results reveal that leaves grown at the TEM site did not show an increased ability for net CO2 assimilation (A N), mainly due to an equal gm at both sites. This fact is explained by a trade-off between an increased conductance of the gas phase (g ias) and a reduced conductance of the liquid phase (g liq) at the TEM site compared with the MED site. In spite of the reduction in gs,max at the MED site, transpiration (E) did not diminish during midsummer to the levels of the TEM site due to a higher VPD found at the MED site, yielding a higher water use efficiency (AN/E) at the TEM site. Moreover, photosynthetic nitrogen use efficiency was also higher at the TEM site, indicating these leaves can reach similar values of AN with lower nitrogen investment that those at the MED site. These results suggest that Q. coccifera does not always use the main resources (water and nutrients) at leaf level as efficiently as possible. Moreover, the different patterns of resource use (in particular N), together with the functional plasticity, cannot overcome the morpho-functional constraints that limit photosynthetic activity, even under potentially favourable conditions., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Plant species living in the understory increase carbon (C) allocation toward leaf production for maximizing light capture at the expense of roots and stems, with negative consequences for the whole-plant hydraulic conductance. Moreover, under some conditions, the high atmospheric evaporative demand occurring in Mediterranean areas may be not well buffered by the canopy, which might be the case for relict conifer Abies pinsapo Boiss. growing in the forest understory. We hypothesized that acclimation to combined understory shade and high atmospheric dryness can be achieved through the adjustment of water losses to cope with the restriction in water transport. The results reveal high structural plasticity in A. pinsapo that allows light harvesting of this species to maximize light capture in the forest understory, and maintain a positive C balance under low light conditions. However, growth in the understory resulted in reduced leaf-specific conductivity, up to approximately four to five times, implying decreased plant capacity to supply water to the leaves. In order to cope with the high atmospheric evaporative demand in the understory, there is an adjustment of the stomatal conductance to the hydraulic conductivity by means of a reduction in the stomatal density in understory individuals, which is due to the almost complete lack of stomata in the adaxial side of the needles. To the extent of our knowledge, such a drastic phenotypic response found in a conifer when growing under shaded conditions had not been previously reported., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Plant trichomes play important protective functions and may have a major influence on leaf surface wettability. With the aim of gaining insight into trichome structure, composition, and function in relation to water-plant surface interactions, we analyzed the adaxial and abaxial leaf surface of holm oak (Quercus ilex) as a model. By measuring the leaf water potential 24 h after the deposition of water drops onto abaxial and adaxial surfaces, evidence for water penetration through the upper leaf side was gained in young and mature leaves. The structure and chemical composition of the abaxial (always present) and adaxial (occurring only in young leaves) trichomes were analyzed by various microscopic and analytical procedures. The adaxial surfaces were wettable and had a high degree of water drop adhesion in contrast to the highly unwettable and water-repellent abaxial holm oak leaf sides. The surface free energy and solubility parameter decreased with leaf age, with higher values determined for the adaxial sides. All holm oak leaf trichomes were covered with a cuticle. The abaxial trichomes were composed of 8% soluble waxes, 49% cutin, and 43% polysaccharides. For the adaxial side, it is concluded that trichomes and the scars after trichome shedding contribute to water uptake, while the abaxial leaf side is highly hydrophobic due to its high degree of pubescence and different trichome structure, composition, and density. Results are interpreted in terms of water-plant surface interactions, plant surface physical chemistry, and plant ecophysiology., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

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., (© The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Responses to drought stress by water withholding have been studied in 1 year old Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) seedlings from seven provenances from Andalusia (southern Spain). Several physiological parameters, including predawn xylem water potentials and relative water content in soil, roots, and leaves as well as maximum quantum efficiency and yield of PSII were evaluated for 28 days in both irrigated and nonirrigated seedlings. The leaf proteome map of the two provenances that show the extreme responses (Seville, GSE, is the most susceptible, while Almerı́a, SSA, is the least susceptible) was obtained. Statistically significant variable spots among provenances and treatments were subjected to MALDI-TOF/TOF-MS/MS analysis for protein identification. In response to drought stress, ~12.4% of the reproducible spots varied significantly depending on the treatment and the population. These variable proteins were mainly chloroplastic and belonged to the metabolism and defense/stress functional categories. The 2-DE protein profile of nonirrigated seedlings was similar in both provenances. Physiological and proteomics data were generally in good agreement. The general trend was a decrease in protein abundance upon water withholding in both provenances, mainly in those involved in ATP synthesis and photosynthesis. This decrease, moreover, was most marked in the most susceptible population compared with the less susceptible one.

The large water requirements of Vitis vinifera L. together with an increase in temperature and drought events imply the need for irrigation in the driest areas of its distribution range. Generous watering may reduce grape quality so irrigation should be precisely regulated through the development of new methods of accurate irrigation scheduling based on plant 'stress sensing'. Two new methods, the reflectivity in the S-band and the broadband ultrasonic spectroscopy, can be used as non-invasive and reproducible techniques for the study of plant water relations in V. vinifera. On one hand, the measurement of reflectance at frequencies around 2.4 GHz gives an excellent accuracy when the changes in the existing area (S) between two reflectance curves are correlated with the relative water content (RWC). On the other hand, an improvement of the broadband ultrasonic spectroscopy based on the enlargement of the analysis frequency window provides, apart from the determination of the turgor loss point (TLP), additional information about the leaves without additional computational cost or additional leaf information requirements. Before TLP, the frequency associated with the maximum transmittance (f/f(o)), the macroscopic elastic constant of the leaf in the Z direction (c(33)) and, specially, the variation of the attenuation coefficient with the frequency (n), were highly correlated with changes in RWC. Once turgor is lost, a shift in the parameters directly related to the attenuation of the signal was also observed. The use of both techniques allows for a more convincing knowledge of the water status in V. vinifera., (© 2012 Scandinavian Plant Physiology Society.)

The turgor loss point (TLP), which is considered a threshold for many physiological processes, may be useful in plant-breeding programs or for the selection of reforestation species. Obtaining TLP through the standard pressure-volume (p-v) curve method in a large set of species is highly time-consuming and somewhat subjective. To solve this problem, we present an objective and a less time-consuming technique based on the leaf resonance able to calculate the relative water content (RWC) at TLP (RWCTLP). This method uses air-coupled broadband ultrasonic spectroscopy to obtain the sigmoidal relation between RWC and the standardized resonant frequency (f/fo). For the 13 species measured, the inflexion point of the RWC-f/fo relationship ( ) was not statistically different from the value of RWC at the TLP obtained with the p-v curves (RWCTLP p-v).

Under excess light, the efficient PSII light-harvesting antenna is switched into a photoprotected state in which potentially harmful absorbed energy is thermally dissipated. Changes occur rapidly and reversibly, enhanced by de-epoxidation of violaxanthin (V) to zeaxanthin (Z). This process is usually measured as non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence. Using instrumentation for instantaneous leaf freezing, NPQ, spectral reflectance, and interconversions within the xanthophyll cycle with time resolution of seconds were recorded from Quercus coccifera leaves during low light (LL) to high light (HL) transitions, followed by relaxation at LL. During the first 30 s of both the LL to HL and HL to LL transitions, no activity of the xanthophyll cycle was detected, whereas 70-75% of the NPQ was formed and relaxed, respectively, by that time, the latter being traits of a rapidly reversible photoprotective energy dissipation. Three different Z pools were identified, which play different roles in energy dissipation and photoprotection. In conclusion, ΔpH was crucial to NPQ formation and relaxation in Q. coccifera during light transitions. Only a minor fraction of Z was associated to quenching, whereas the largest Z pool was not related to thermal dissipation. The latter is proposed to participate in photoprotection acting as antioxidant.

The adaptive benefit of stomatal crypts remains a matter of controversy. This work studies the effect on gas exchange of cuticular rims that overarch the stomatal pore in the Mediterranean species Quercus coccifera L. growing under Mediterranean (lower relative humidities and high summer temperatures) or oceanic conditions (higher daily relative humidities and mild temperatures). After microscopic assessment of the leaf surfaces and stomatal architecture, the impact of the cuticular 'cup' on gas exchange was evaluated by employing three-dimensional finite element models. Here, we provide evidence for a high plasticity of the Q. coccifera cuticular cup, with much larger vents under oceanic conditions compared to small vents under Mediterranean conditions. This structure adds a substantial fixed resistance thereby strongly decreasing gas exchange under Mediterranean conditions. The cuticular cup, which also increases leaf internal humidity, might buffer the rapid changes in vapour pressure deficit (VPD) often observed under Mediterranean conditions. Since water loss of guard and adjacent epidermal cells regulates stomatal aperture, we suggest that this structure allows an efficient regulation of stomatal conductance and optimum use of resources under high VPD. This study provides evidence that plasticity of stomatal architecture can be an important structural component of hydraulic adaptation to different climate conditions., (© 2012 Blackwell Publishing Ltd.)

Abies alba and Abies pinsapo are closely related species with the same ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (rbcL) but contrasting hydraulic traits and mesophyll structure occurring in the Iberian Peninsula under contrasting conditions. As photosynthesis and hydraulic capacities often co-scale, we hypothesize that these species differ in mesophyll conductance to CO(2) (g(m) ). g(m) and key anatomical traits were measured in both species. Drought-adapted population of A. pinsapo has higher photosynthesis than the more mesic population of A. alba, in agreement with its higher hydraulic capacity. However, A. alba exhibits the largest stomatal conductance (g(s) ), and so water use efficiency (WUE) is much higher in A. pinsapo. The differences in photosynthesis were explained by differences in g(m) , indicating a correlation between hydraulic capacity and g(m) . We report a case where g(m) is the main factor limiting photosynthesis in one species (A. alba) when compared with the other one (A. pinsapo). The results also highlight the discrepancy between g(m) estimates based on anatomical measurements and those based on gas exchange methods, probably due to the very large resistance exerted by cell walls and the stroma in both species. Thus, the cell wall and chloroplast properties in relation to CO(2) diffusion constitute a near-future research priority., (© 2012 Blackwell Publishing Ltd.)

We studied the intraspecific variability of maritime pine in a set of morphological and physiological traits: soil-to-leaf hydraulic conductance, intrinsic water-use efficiency (WUE, estimated by carbon isotope composition, δ(13)C), root morphology, xylem anatomy, growth and carbon allocation patterns. The data were collected from Pinus pinaster Aiton seedlings (25 half-sib families from five populations) grown in a greenhouse and subjected to water and water-stress treatments. The aims were to relate this variability to differences in water availability at the geographic location of the populations, and to study the potential trade-offs among traits. The drought-stressed seedlings demonstrated a decrease in hydraulic conductance and root surface area and increased WUE and root tip number. The relationships among the growth, morphological, anatomical and physiological traits changed with the scale of study: within the species, among/within populations. The populations showed a highly significant relationship between the percentage reduction in whole-plant hydraulic conductance and WUE. The differences among the populations in root morphology, whole-plant conductance, carbon allocation, plant growth and WUE were significant and consistent with dryness of the site of seed origin. The xeric populations exhibited lower growth and a conservative water use, as opposed to the fast-growing, less water-use-efficient populations from mesic habitats. The xeric and mesic populations, Tamrabta and San Cipriano, respectively, showed the most contrasting traits and were clustered in opposite directions along the main axis in the canonical discriminant analysis under both the control and drought treatments. The results suggest the possibility of selecting the Arenas population, which presents a combination of traits that confer increased growth and drought resistance.

Abies alba and Abies pinsapo are two closely related fir species that occur in the Iberian Peninsula under very different environmental conditions. Abies alba proliferates in the humid European mountains, including the Spanish Pyrenees. In contrast, A. pinsapo is a relict species that occurs in some restricted areas of the Mediterranean mountain ranges in Spain and Morocco, which experience intense summer drought periods. To cope with the high atmospheric evaporative demand during summer, A. pinsapo may either have a high resistance to xylem cavitation or develop a very efficient conducting system to reduce the soil-to-leaf water potential gradient. To investigate such hypotheses, we measured (i) the xylem vulnerability to cavitation for different populations, and (ii) several anatomical and hydraulic parameters indicating xylem sufficiency for -supplying water to the shoot in two contrasting populations of both species. Our results show that the resistance to cavitation was not different between species or populations. However, hydraulic conductivity (K(h)), specific hydraulic conductivity (K(s)), leaf-specific conductivity (LSC) and whole-shoot hydraulic conductance (K(shoot)) were higher in A. pinsapo, indicating a higher efficiency of water transport, which should contribute to maintaining its xylem tension below the threshold for rapidly increasing cavitation. The higher K(s) in A. pinsapo was largely a result of its wider tracheids, suggesting that this species may be much more vulnerable to freeze-thaw-induced cavitation than A. alba. This is consistent with the absence of A. pinsapo in northern mountain ranges with cooler winters. These physiological differences could partly explain the niche segregation and the geographical separation of these two firs.

Studies of variability in Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.), the dominant tree species in the typical Mediterranean forest, have been carried out by using electrophoresis-based proteomic analysis of acorns. Ten populations distributed throughout the Andalusia region have been surveyed. Acorns were sampled from individual trees and proteins extracted from seed flour by using the TCA-acetone precipitation protocol. Extracts were subjected to SDS-PAGE and 2-DE for protein separation, gel images captured, spot or bands quantified, and subjected to statistical analysis (ANOVA, SOM and clustering). Variable bands or spots among populations were subjected to MALDI-TOF/TOF and LC-MS/MS for identification. The protein yield of the used protocol varied among populations, and it was in the 2.92-5.92 mg/g dry weight range. A total of 23 bands were resolved by SDS-PAGE in the 3-35 kDa Mr range, with 8 and 12, out of the total, showing respectively qualitative and quantitative statistically significant differences among populations. Data allowed grouping populations, with groups being correlated according to geographical location and climate conditions, to northern and southern, as well as the discrimination of both mesic and xeric groups. Acorn flour extracts from the most distant populations were analyzed by 2-DE, and 56 differential spots were proposed as markers of variability. Identified proteins were classified into two principal categories; storage and stress/defense protein. Besides providing the first reference map of mature acorn seeds, the use of SDS-PAGE and proteomics in characterizing natural biodiversity in forest trees will be discussed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The broad-band ultrasonic spectroscopy technique allows the determination of changes in the relative water content (RWC) of leaves with contrasting structural features. Specifically, the standardized frequency associated with the maximum transmittance (f/f(o)) is strongly related to the RWC. This relationship is characterized by the existence of two phases separated by an inflexion point (associated with the turgor loss point). To obtain a better understanding of the strong relationship found between RWC and f/f(o), this work has studied the structural changes experienced by Quercus muehlenbergii leaves during dehydration in terms of ultrasounds measurements, cell wall elasticity, leaf thickness, leaf density, and leaf structure. The results suggest that the decrease found in f/f(o) before the turgor loss point can be attributed to the occurrence of changes in the estimation of the macroscopic effective elastic constant of the leaf (c(33)), mainly associated with changes in the bulk modulus of elasticity of the cell wall (ε). These changes are overriding or compensating for the thickness decreases recorded during this phase. On the other hand, the high degree of cell shrinkage and stretching found in the mesophyll cells during the second phase seem to explain the changes in the acoustic properties of the leaf beyond the turgor loss point. The formation of large intercellular spaces, which increased the irregularity in the acoustic pathway, may explain the increase of the attenuation coefficient of ultrasounds once the turgor loss point threshold is exceeded. The direct measurement of c(33) from ultrasonic measurements would allow a better knowledge of the overall biomechanical properties of the leaf further than those derived from the P-V analysis.

The implementation of non-destructive methods for the study of water changes within plant tissues and/or organs has been a target for some time in plant physiology. Recent advances in air-coupled ultrasonic spectroscopy have enabled ultrasonic waves to be applied to the on-line and real-time assessment of the water content of different materials. In this study, this technique has been applied as a non-destructive, non-invasive, non-contact, and repeatable method for the determination of water status in Populusxeuramericana and Prunus laurocerasus leaves. Frequency spectra of the transmittance of ultrasounds through plant leaves reveal the presence of at least one resonance. At this resonant frequency, transmittance is at its maximum. This work demonstrates that changes in leaf relative water content (RWC) and water potential (Psi) for both species can be accurately monitored by the corresponding changes in resonant frequency. The differential response found between both species may be due to the contrasting leaf structural features and the differences found in the parameters derived from the P-V curves. The turgor loss point has been precisely defined by this new technique, as it is derived from the lack of significant differences between the relative water content at the turgor loss point (RWC(TLP)) obtained from P-V curves and ultrasonic measurements. The measurement of the turgor gradient between two different points of a naturally transpiring leaf is easily carried out with the method introduced here. Therefore, such a procedure can be an accurate tool for the study of all processes where changes in leaf water status are involved.

There is increasing evidence suggesting that iron (Fe) deficiency induces not only leaf chlorosis and a decline of photosynthesis, but also structural changes in leaf morphology, which might affect the functionality of leaves. In this study, we investigated the effects of Fe deficiency on the water relations of peach (Prunus persica (L.) Batsch.) leaves and the responses of previously chlorotic leaves to Fe resupply via the root or the leaf. Iron deficiency induced a decline of maximum potential photosystem II (PSII) efficiency (F(V)/F(M)), of rates of net photosynthesis and transpiration and of water use efficiency. Iron chlorosis was associated with a reduction of leaf xylem vessel size and of leaf hydraulic conductance. In the course of the day, water potentials in chlorotic leaves remained higher (less negative) than in green leaves. In chlorotic leaves, normal stomatal functioning was disturbed, as evidenced by the lack of opening upon withdrawal of external CO(2) and stomatal closure after sudden illumination of previously darkened leaves. We conclude that the Fe deficiency induced limitations of xylem conductivity elicited a water saving strategy, which poses an additional challenge to plant growth on high pH, calcareous soils. Fertilisation with Fe improved photosynthetic performance but the proper xylem structure and water relations of leaves were not fully restored, indicating that Fe must be available at the first stages of leaf growth and development.

The ability of three Mediterranean oaks (Quercus coccifera L., Quercus ilex ssp. ballota (Desf.) Samp and Quercus suber L.) to cope with intense drought was investigated. Water stress reduced stomatal conductance and photosynthesis in these species. Drought-mediated changes in photosynthetic-related parameters allowed the characterisation of the specific photo-protective mechanisms. Specifically, Q. suber downregulated photosynthetic electron transport rates (ETR) closing PSII reaction centres (i.e. decreasing photochemical quenching) and through an antheraxanthin (A) + zeaxanthin (Z)-mediated diminished intrinsic PSII efficiency (Φ exc. ). These changes were lower in Q. coccifera and Q. ilex ssp. ballota, which decreased further ETR photo-inactivating PSII centres (evidenced by their low predawn F v /F m ratios at high water stress). The predawn F v /F m ratio decreased in Q. coccifera largely due to F m decreases, whereas in Q. ilex ssp. ballota F v /F m decreases were due to F 0 increases, below -4 MPa. These F v /F m decreases were well correlated with increases in the A + Z photo-protective pigments. An analysis of dark respiration and photorespiration as alternative electron sinks under intense drought stress also revealed interspecific differences. The largest imbalance between electrons generated and consumed increased potentially oxidative damage in Q. suber. Subsequently, only Q. suber showed loss of chlorophyll, which is one of the main targets of oxidative damage. Data suggest that Q. coccifera and Q. ilex ssp. ballota seem more able than Q. suber to withstand highly xeric conditions. Therefore, our results question the consideration of Mediterranean evergreen oaks as a homogeneous physiological group.

The existence of major vertical gradients within the leaf is often overlooked in studies of photosynthesis. These gradients, which involve light heterogeneity, cell composition, and CO(2) concentration across the mesophyll, can generate differences in the maximum potential PSII efficiency (F (V)/F (M) or F (V)/F (P)) of the different cell layers. Evidence is presented for a step gradient of F (V)/F (P) ratios across the mesophyll, from the adaxial (palisade parenchyma, optimal efficiencies) to the abaxial (spongy parenchyma, sub-optimal efficiencies) side of Quercus coccifera leaves. For this purpose, light sources with different wavelengths that penetrate more or less deep within the leaf were employed, and measurements from the adaxial and abaxial sides were performed. To our knowledge, this is the first report where a low photosynthetic performance in the abaxial side of leaves is accompanied by impaired F (V)/F (P) ratios. This low photosynthetic efficiency of the abaxial side could be related to the occurrence of bundle sheath extensions, which facilitates the penetration of high light intensities deep within the mesophyll. Also, leaf morphology (twisted in shape) and orientation (with a marked angle from the horizontal plane) imply direct sunlight illumination of the abaxial side. The existence of cell layers within leaves with different photosynthetic efficiencies makes appropriate the evaluation of how light penetrates within the mesophyll when using Chl fluorescence or gas exchange techniques that use different wavelengths for excitation and/or for driving photosynthesis.

Quercus coccifera L. is a Mediterranean sclerophyllous shrub with a high capacity to resist intense drought stress. Therefore, it could be used in the study of physiological changes suffered by plants at very low water potentials. A remote sensing sensor was used to measure continuously the physiological reflectance index (PRI; defined as the changes in reflectance at 531 nm with respect to those at 570 nm; PRI = [(R531 - R570)/(R531 + R570)] at canopy level and under field conditions in an artificial carpet of seedlings of Q. coccifera during a drought cycle. Correlations between leaf level-measured chlorophyll fluorescence parameters as well as the de-epoxidation state of the xanthophyll cycle [(A + Z)/(V + A + Z)] and canopy level-measured PRI were reasonably good (R (2) = 0.57-0.63, P < 0.01), and quite interesting for water stress remote sensing purposes. The instrument's temporal resolution allowed us to follow the rapid response of PRI to changing photosynthetic active radiation, and to resolve, in response to cloud-induced changes in light intensity, a fast and a slow PRI component. We report the disappearance of the rapid one under conditions of intense drought in response to a sudden increase in light intensity. The underlying photoprotection mechanisms that Q. coccifera shows in response to intense drought stress periods seem to be related to the existence of a low intrathylakoid lumenal pH at the end of the drought cycle. Under intense drought, these mechanisms allow this species to avoid oxidative damage, which was evidenced by the maintenance of an unaltered photosynthetic pigment composition and constant photosystem II efficiency in the mornings. It is concluded that, contrary to early reports, PRI is a sensible, indirect, non-destructive water stress indicator, even in plants experiencing intense drought.