Leaf hydraulic properties of Antarctic plants: effects of growth temperature and its coordination with photosynthesis.

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.

Disentangling leaf structural and material properties in relationship to their anatomical and chemical compositional traits in oaks (Quercus L.).

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.

Deciduous and evergreen oaks show contrasting adaptive responses in leaf mass per area across environments.

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.

Day length regulates seasonal patterns of stomatal conductance in Quercus species.

Vapour pressure deficit is a major driver of seasonal changes in transpiration, but photoperiod also modulates leaf responses. Climate warming might enhance transpiration by increasing atmospheric water demand and the length of the growing season, but photoperiod-sensitive species could show dampened responses. Here, we document that day length is a significant driver of the seasonal variation in stomatal conductance. We performed weekly gas exchange measurements across a common garden experiment with 12 oak species from contrasting geographical origins, and we observed that the influence of day length was of similar strength to that of vapour pressure deficit in driving the seasonal pattern. We then examined the generality of our findings by incorporating day-length regulation into well-known stomatal models. For both angiosperm and gymnosperm species, the models improved significantly when adding day-length dependences. Photoperiod control over stomatal conductance could play a large yet underexplored role on the plant and ecosystem water balances.

Hydraulic and photosynthetic limitations prevail over root non-structural carbohydrate reserves as drivers of resprouting in two Mediterranean oaks.

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.

In situ warming in the Antarctic: effects on growth and photosynthesis in Antarctic vascular plants.

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 CO2 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.

Cell-level anatomical characteristics explain high mesophyll conductance and photosynthetic capacity in sclerophyllous Mediterranean oaks.

Leaf mass per area (LMA) has been suggested to negatively affect the mesophyll conductance to CO2 (gm ), which is the most limiting factor for area-based photosynthesis (AN ) in many Mediterranean sclerophyll species. However, despite their high LMA, these species have similar AN to plants from other biomes. Variations in other leaf anatomical traits, such as mesophyll and chloroplast surface area exposed to intercellular air space (Sm /S and Sc /S), may offset the restrictions imposed by high LMA in gm and AN 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 AN 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 gm , the most limiting factor for carbon assimilation in these species. Species with higher AN showed increased Sc /S, which implies increased gm without changes in stomatal conductance. The occurrence of this anatomical adaptation at the cell level allowed evergreen oaks to reach AN values comparable to congeneric deciduous species despite their higher LMA.

Physico-chemical properties of plant cuticles and their functional and ecological significance.

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.

Ultrasonic Sensing of Plant Water Needs for Agriculture.

Fresh water is a key natural resource for food production, sanitation and industrial uses and has a high environmental value. The largest water use worldwide (~70%) corresponds to irrigation in agriculture, where use of water is becoming essential to maintain productivity. Efficient irrigation control largely depends on having access to reliable information about the actual plant water needs. Therefore, fast, portable and non-invasive sensing techniques able to measure water requirements directly on the plant are essential to face the huge challenge posed by the extensive water use in agriculture, the increasing water shortage and the impact of climate change. Non-contact resonant ultrasonic spectroscopy (NC-RUS) in the frequency range 0.1-1.2 MHz has revealed as an efficient and powerful non-destructive, non-invasive and in vivo sensing technique for leaves of different plant species. In particular, NC-RUS allows determining surface mass, thickness and elastic modulus of the leaves. Hence, valuable information can be obtained about water content and turgor pressure. This work analyzes and reviews the main requirements for sensors, electronics, signal processing and data analysis in order to develop a fast, portable, robust and non-invasive NC-RUS system to monitor variations in leaves water content or turgor pressure. A sensing prototype is proposed, described and, as application example, used to study two different species: Vitis vinifera and Coffea arabica, whose leaves present thickness resonances in two different frequency bands (400-900 kHz and 200-400 kHz, respectively), These species are representative of two different climates and are related to two high-added value agricultural products where efficient irrigation management can be critical. Moreover, the technique can also be applied to other species and similar results can be obtained.

Wettability, polarity, and water absorption of holm oak leaves: effect of leaf side and age.

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.

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.

Stomatal encryption by epicuticular waxes as a plastic trait modifying gas exchange in a Mediterranean evergreen species (Quercus coccifera L.).

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.

The reflectivity in the S-band and the broadband ultrasonic spectroscopy as new tools for the study of water relations in Vitis vinifera L.

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.

Contrasting stem water uptake and storage dynamics of water-saver and water-spender species during drought and recovery.

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.

Leaf anatomical properties in relation to differences in mesophyll conductance to CO(2) and photosynthesis in two related Mediterranean Abies species.

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.

Cell-level anatomy explains leaf age-dependent declines in mesophyll conductance and photosynthetic capacity in the evergreen Mediterranean oak Quercus ilex subsp. rotundifolia.

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.

Relationship between ultrasonic properties and structural changes in the mesophyll during leaf dehydration.

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.

Minimum Leaf Conductance (g min) Is Higher in the Treeline of Pinus uncinata Ram. in the Pyrenees: Michaelis' Hypothesis Revisited.

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.

Contrasting functional strategies following severe drought in two Mediterranean oaks with different leaf habit: Quercus faginea and Quercus ilex subsp. rotundifolia.

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.

Air-coupled broadband ultrasonic spectroscopy as a new non-invasive and non-contact method for the determination of leaf water status.

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.