Changes in ploidy affect vascular allometry and hydraulic function in Mangifera indica trees.

The enucleated vascular elements of the xylem and the phloem offer an excellent system to test the effect of ploidy on plant function because variation in vascular geometry has a direct influence on transport efficiency. However, evaluations of conduit sizes in polyploid plants have remained elusive, most remarkably in woody species. We used a combination of molecular, physiological and microscopy techniques to model the hydraulic resistance between source and sinks in tetraploid and diploid mango trees. Tetraploids exhibited larger chloroplasts, mesophyll cells and stomatal guard cells, resulting in higher leaf elastic modulus and lower dehydration rates, despite the high water potentials of both ploidies in the field. Both the xylem and the phloem displayed a scaling of conduits with ploidy, revealing attenuated hydraulic resistance in tetraploids. Conspicuous wall hygroscopic moieties in the cells involved in transpiration and transport indicate a role in volumetric adjustments as a result of turgor change in both ploidies. In autotetraploids, the enlargement of organelles, cells and tissues, which are critical for water and photoassimilate transport at long distances, point to major physiological novelties associated with whole-genome duplication.

Plant water uptake from soil through a vapor pathway.

Water uptake from the soil via a vapor pathway was tested. Viburnum suspensum L. plants were divided into: (1) irrigated, (2) drought with vapor and (3) drought without vapor treatments. Each plant was placed into a larger bucket containing deuterium-labeled water as a vapor source (vapor treatment) or no water (drought and irrigation treatments). We also tested whether uptake via a vapor pathway could mitigate drought effects. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) were measured daily until the first visible signs of stress. Soil water content, stem water potential (Ψ) and the stable hydrogen isotope ratio (δ2 H) of soil and plant xylem water were then measured in all treatments. We show that water is taken up by plants through the vapor phase in dry soils. The δ2 H values of the soil water in the vapor treatment were highly enriched compared to the background isotope ratios of the non-vapor exposed irrigated and drought treatments. Stem water δ2 H values for the vapor treatment were significantly greater than those for irrigation and drought treatments not exposed to isotopically enriched vapor. In this experiment, movement of water to the plant via the vapor phase did not mitigate drought effects. A, E, plant Ψ and gs significantly decreased in the drought and vapor treatments relative to the controls, with no significant differences between vapor and drought treatments.

Protein coating composition targets nanoparticles to leaf stomata and trichomes.

Plant nanobiotechnology has the potential to revolutionize agriculture. However, the lack of effective methods to deliver nanoparticles (NPs) to the precise locations in plants where they are needed impedes these technological innovations. Here, model gold nanoparticles (AuNP) were coated with citrate, bovine serum albumin (BSA) as a protein control, or LM6-M, an antibody with an affinity for functional groups unique to stomata on leaf surfaces to deliver the AuNPs to stomata. One-month-old Vicia faba leaves were exposed via drop deposition to aqueous suspensions of LM6-M-coated AuNPs and allowed to air dry. After rinsing, Au distribution on the leaf surface was investigated by enhanced dark-field microscopy and X-ray fluorescence mapping. While citrate-coated AuNPs randomly covered the plant leaves, LM6M-AuNPs strongly adhered to the stomata and remained on the leaf surface after rinsing, and BSA-AuNPs specifically targeted trichome hairs. To the authors' knowledge, this is the first report of active targeting of live leaf structures using NPs coated with molecular recognition molecules. This proof-of-concept study provides a strategy for future targeted nanopesticide delivery research.

Stomatal traits as a determinant of superior salinity tolerance in wild barley.

Wild barley Hordeum spontaneum (WB) is the progenitor of a cultivated barley Hordeum vulgare (CB). Understanding efficient mechanisms evolved by WB to cope with abiotic stresses may open prospects of transferring these promising traits to the high yielding CB genotypes. This study aimed to investigate the strategies that WB plants utilise in regard to the control of stomatal operation and ionic homeostasis to deal with salinity stress, one of the major threats to the global food security. Twenty-six genotypes of WB and CB were grown under glasshouse conditions and exposed to 300 mM NaCl salinity treatment for 5 weeks followed by their comprehensive physiological assessment. WB had higher relative biomass than CB when exposed to salinity stress. Under saline conditions, WB plants were able to keep constant stomatal density (SD) while SD significantly decreased in CB. The higher SD in WB also resulted in a higher stomatal conductance (gs) under saline conditions, with gs reduction being 51% and 72% in WB and CB, respectively. Furthermore, WB showed faster stomatal response to light, indicating their better ability to adapt to changing environmental conditions. Experiments with isolated epidermal strips indicated that CB genotypes have the higher stomatal aperture when incubated in 80 mM KCl solution, and its aperture declined when KCl was substituted by NaCl. On the contrary, WB genotype had the highest stomatal aperture being exposed to 80 mM NaCl suggesting that WB plants may use Na+ instead of K+ for stomata movements. Overall, our data suggest that CB employ a stress-escaping strategy by reducing stomata density, to conserve water, when grown under salinity conditions. WB, on a contrary, is capable of maintaining relatively constant stomata density, faster stomatal movement and higher gs under saline conditions.

Arabidopsis ALIX Regulates Stomatal Aperture and Turnover of Abscisic Acid Receptors.

The plant endosomal trafficking pathway controls the abundance of membrane-associated soluble proteins, as shown for abscisic acid (ABA) receptors of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) family. ABA receptor targeting for vacuolar degradation occurs through the late endosome route and depends on FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FYVE1) and VACUOLAR PROTEIN SORTING23A (VPS23A), components of the ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT-I (ESCRT-I) complexes. FYVE1 and VPS23A interact with ALG-2 INTERACTING PROTEIN-X (ALIX), an ESCRT-III-associated protein, although the functional relevance of such interactions and their consequences in cargo sorting are unknown. In this study we show that Arabidopsis (Arabidopsis thaliana) ALIX directly binds to ABA receptors in late endosomes, promoting their degradation. Impaired ALIX function leads to altered endosomal localization and increased accumulation of ABA receptors. In line with this activity, partial loss-of-function alix-1 mutants display ABA hypersensitivity during growth and stomatal closure, unveiling a role for the ESCRT machinery in the control of water loss through stomata. ABA-hypersensitive responses are suppressed in alix-1 plants impaired in PYR/PYL/RCAR activity, in accordance with ALIX affecting ABA responses primarily by controlling ABA receptor stability. ALIX-1 mutant protein displays reduced interaction with VPS23A and ABA receptors, providing a molecular basis for ABA hypersensitivity in alix-1 mutants. Our findings unveil a negative feedback mechanism triggered by ABA that acts via ALIX to control the accumulation of specific PYR/PYL/RCAR receptors.

Mesophyll porosity is modulated by the presence of functional stomata.

The formation of stomata and leaf mesophyll airspace must be coordinated to establish an efficient and robust network that facilitates gas exchange for photosynthesis, however the mechanism by which this coordinated development occurs remains unclear. Here, we combine microCT and gas exchange analyses with measures of stomatal size and patterning in a range of wild, domesticated and transgenic lines of wheat and Arabidopsis to show that mesophyll airspace formation is linked to stomatal function in both monocots and eudicots. Our results support the hypothesis that gas flux via stomatal pores influences the degree and spatial patterning of mesophyll airspace formation, and indicate that this relationship has been selected for during the evolution of modern wheat. We propose that the coordination of stomata and mesophyll airspace pattern underpins water use efficiency in crops, providing a target for future improvement.

The stomatal flexoskeleton: how the biomechanics of guard cell walls animate an elastic pressure vessel.

In plants, stomatal guard cells are one of the most dynamic cell types, rapidly changing their shape and size in response to environmental and intrinsic signals to control gas exchange at the plant surface. Quantitative and systematic knowledge of the biomechanical underpinnings of stomatal dynamics will enable strategies to optimize stomatal responsiveness and improve plant productivity by enhancing the efficiency of photosynthesis and water use. Recent developments in microscopy, mechanical measurements, and computational modeling have revealed new insights into the biomechanics of stomatal regulation and the genetic, biochemical, and structural origins of how plants achieve rapid and reliable stomatal function by tuning the mechanical properties of their guard cell walls. This review compares historical and recent experimental and modeling studies of the biomechanics of stomatal complexes, highlighting commonalities and contrasts between older and newer studies. Key gaps in our understanding of stomatal functionality are also presented, along with assessments of potential methods that could bridge those gaps.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings.

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

The malate-activated ALMT12 anion channel in the grass Brachypodium distachyon is co-activated by Ca2+/calmodulin.

In plants, strict regulation of stomatal pores is critical for modulation of CO2 fixation and transpiration. Under certain abiotic and biotic stressors, pore closure is initiated through anionic flux, with calcium (Ca2+) playing a central role. The aluminum-activated malate transporter 12 (ALMT12) is a malate-activated, voltage-dependent member of the aluminum-activated malate transporter family that has been implicated in anionic flux from guard cells controlling the stomatal aperture. Herein, we report the characterization of the regulatory mechanisms mediating channel activities of an ALMT from the grass Brachypodium distachyon (BdALMT12) that has the highest sequence identity to Arabidopsis thaliana ALMT12. Electrophysiological studies in a heterologous cell system confirmed that this channel is malate- and voltage-dependent. However, this was shown to be true only in the presence of Ca2+ Although a general kinase inhibitor increased the current density of BdALMT12, a calmodulin (CaM) inhibitor reduced the Ca2+-dependent channel activation. We investigated the physiological relevance of the CaM-based regulation in planta, where stomatal closure, induced by exogenous Ca2+ ionophore and malate, was shown to be inhibited by exogenous application of a CaM inhibitor. Subsequent analyses revealed that the double substitutions R335A/R338A and R335A/K342A, within a predicted BdALMT12 CaM-binding domain (CBD), also decreased the channels' ability to activate. Using isothermal titration calorimetry and CBD-mimetic peptides, as well as CaM-agarose affinity pulldown of full-length recombinant BdALMT12, we confirmed the physical interaction between the CBD and CaM. Together, these findings support a co-regulatory mechanism of BdALMT12 activation by malate, and Ca2+/CaM, emphasizing that a complex regulatory network modulates BdALMT12 activity.

Beyond soil water potential: An expanded view on isohydricity including land-atmosphere interactions and phenology.

Over the past decade, the concept of isohydry or anisohydry, which describes the link between soil water potential (ΨS ), leaf water potential (ΨL ), and stomatal conductance (gs ), has soared in popularity. However, its utility has recently been questioned, and a surprising lack of coordination between the dynamics of ΨL and gs across biomes has been reported. Here, we offer a more expanded view of the isohydricity concept that considers effects of vapour pressure deficit (VPD) and leaf area index (AL ) on the apparent sensitivities of ΨL and gs to drought. After validating the model with tree- and ecosystem-scale data, we find that within a site, isohydricity is a strong predictor of limitations to stomatal function, though variation in VPD and leaf area, among other factors, can challenge its diagnosis. Across sites, the theory predicts that the degree of isohydricity is a good predictor of the sensitivity of gs to declining soil water in the absence of confounding effects from other drivers. However, if VPD effects are significant, they alone are sufficient to decouple the dynamics of ΨL and gs entirely. We conclude with a set of practical recommendations for future applications of the isohydricity framework within and across sites.

Contrasting pectin polymers in guard cell walls of Arabidopsis and the hornwort Phaeoceros reflect physiological differences.

BACKGROUND AND AIMS: In seed plants, stomata regulate CO2 acquisition and water relations via transpiration, while minimizing water loss. Walls of guard cells are strong yet flexible because they open and close the pore by changing shape over the substomatal cavity. Pectins are necessary for wall flexibility and proper stomata functioning. This study investigates the differences in pectin composition in guard cells of two taxa that represent key lineages of plants with stomata: Arabidopsis, an angiosperm with diurnal stomatal activity, and Phaeoceros, a bryophyte that lacks active stomatal movement. METHODS: Using immunolocalization techniques in transmission electron microscopy, this study describes and compares the localization of pectin molecule epitopes essential to stomata function in guard cell walls of Arabidopsis and Phaeoceros. KEY RESULTS: In Arabidopsis, unesterified homogalacturonans very strongly localize throughout guard cell walls and are interspersed with arabinan pectins, while methyl-esterified homogalacturonans are restricted to the exterior of the wall, the ledges and the junction with adjacent epidermal cells. In contrast, arabinans are absent in Phaeoceros, and both unesterified and methyl-esterified homogalacturonans localize throughout guard cell walls. CONCLUSIONS: Arabinans and unesterified homogalacturonans are required for wall flexibility, which is consistent with active regulation of pore opening in Arabidopsis stomata. In contrast, the lack of arabinans and high levels of methyl-esterified homogalacturonans in guard cell walls of Phaeoceros are congruent with the inability of hornwort stomata to open and close with environmental change. Comparisons across groups demonstrate that variations in guard cell wall composition reflect different physiological activity of stomata in land plants.

Colored shade nets induced changes in growth, anatomy and essential oil of Pogostemon cablin.

The purpose of this investigation was to determine the influence of colored shade nets on the growth, anatomy and essential oil content, yield and chemical composition of Pogostemon cablin. The plants were cultivated under full sunlight, black, blue and red nets. The harvesting was performed 5 months after planting and it was followed by the analysis of plant growth parameters, leaf anatomy, essential oil content, yield and chemical composition. The plants grown under red net have produced more leaf, shoot, total dry weight and leaf area. Plants cultivated under colored nets showed differences in morphological features. Plants maintained under red net had a higher leaf blade thickness and polar and equatorial diameter of the stomata ratio. Additionally, higher yield of essential oil in the leaves was observed under red and blue colored shade net. The essential oil of the plants grown under red net showed the highest relative percentage of patchoulol (66.84%). Therefore, it is possible using colored shade nets to manipulate P. cablin growth, as well as its essential oil production with several chemical compositions. The analyses of principal components allowed observing that pogostol has negative correlation with α-guaiene and α-bulnesene. There was difference in total dry weight and patchoulol content when the patchouli is cultured under the red colored shade nets.

Direct measurement of intercellular CO2 concentration in a gas-exchange system resolves overestimation using the standard method.

Intercellular CO2 concentration of leaves (Ci) is a critical parameter in photosynthesis. Nevertheless, uncertainties in calculating Ci arise as stomata close. Here, by modifying the assimilation chamber of a commercial gas-exchange equipment to directly measure Ci, we demonstrate overestimation of calculated Ci (i.e. Ci(c)) without stimulating stomatal closure. Gas exchange was measured on one side of the leaf while measured Ci (Ci(m)) was acquired simultaneously on the other side of the leaf in hypostomatous passion fruit (Passiflora edulis Sims) and amphistomatous sunflower (Helianthus annuus L.) and common bean (Phaseolus vulgaris L.). The adaxial surface showed comparable Ci(c) and Ci(m) in sunflower, whereas in common bean, where the adaxial surface has a low stomatal density, Ci(c) markedly differed from Ci(m) when the stomata remained open. However, the latter discrepancy disappeared when measuring the leaf flipped upside down so that the gas exchange was measured (i.e. Ci was calculated) on the abaxial side, which has a much higher stomatal density. The passion fruit showed the largest discrepancy on the astomatous side, indicating that the cuticle has a large impact on the calculation. Direct measurement of Ci is recommended as a more accurate estimate than the calculation when stomatal gas transport is restricted. Occurrence of overestimation and prospects for direct measurement are discussed.

Modelling the water-plant cuticular polymer matrix membrane partitioning of diverse chemicals in multiple plant species using the support vector machine-based QSAR approach.

In this study, a support vector machine (SVM) based multi-species QSAR (quantitative structure-activity relationship) model was developed for predicting the water-plant cuticular polymer matrix membrane (MX) partition coefficient, KMXw of diverse chemicals using two simple molecular descriptors derived from the chemical structures and following the OECD guidelines. Accordingly, the Lycopersicon esculentum Mill. data were used to construct the QSAR model that was externally validated using three other plant species data. The diversity in chemical structures and end-points were verified using the Tanimoto similarity index and Kruskal-Wallis statistics. The predictive power of the developed QSAR model was tested through rigorous validation, deriving a wide series of statistical checks. The MLOGP was the most influential descriptor identified by the model. The model yielded a correlation (r2) of 0.966 and 0.965 in the training and test data arrays. The developed QSAR model also performed well in another three plant species (r2 > 0.955). The results suggest the appropriateness of the developed model to reliably predict the plant chemical interactions in multiple plant species and it can be a useful tool in screening the new chemical for environmental risk assessment.

Polyamines increase nitric oxide and reactive oxygen species in guard cells of Arabidopsis thaliana during stomatal closure.

A comprehensive study which was undertaken on the effect of three polyamines (PAs) on stomatal closure was examined in relation to nitric oxide (NO) and reactive oxygen species (ROS) levels in guard cells of Arabidopsis thaliana. Three PAs-putrescine (Put), spermidine (Spd), and spermine (Spm)-induced stomatal closure, while increasing the levels of NO as well as ROS in guard cells. The roles of NO and ROS were confirmed by the reversal of closure by cPTIO (NO scavenger) and catalase (ROS scavenger). The presence of L-NAME (NOS-like enzyme inhibitor) reversed PA-induced stomatal closure, suggesting that NOS-like enzyme played a significant role in NO production during stomatal closure. The reversal of stomatal closure by diphenylene iodonium (DPI, NADPH oxidase inhibitor) or 2-bromoethylamine (BEA, copper amine oxidase inhibitor) or 1,12 diaminododecane (DADD, polyamine oxidase inhibitor) was partial. In contrast, the presence of DPI along with BEA/DADD reversed completely the closure by PAs. We conclude that both NO and ROS are essential signaling components during Put-, Spd-, and Spm-induced stomatal closure. The PA-induced ROS production is mediated by both NADPH oxidase and amine oxidase. The rise in ROS appears to be upstream of NO. Ours is the first detailed study on the role of NO and its dependence on ROS during stomatal closure by three major PAs.

Permanently open stomata of aquatic angiosperms display modified cellulose crystallinity patterns.

Most floating aquatic plants have stomata on their upper leaf surfaces, and usually their stomata are permanently open. We previously identified 3 distinct crystallinity patterns in stomatal cell walls, with angiosperm kidney-shaped stomata having the highest crystallinity in the polar end walls as well as the adjacent polar regions of the guard cells. A numerical bio-mechanical model suggested that the high crystallinity areas are localized to regions where the highest stress is imposed. Here, stomatal cell wall crystallinity was examined in 4 floating plants from 2 different taxa: basal angiosperms from the ANITA grade and monocots. It appears that the non-functional stomata of floating plants display reduced crystallinity in the polar regions as compared with high crystallinity of the ventral (inner) walls. Thus their guard cells are both less flexible and less stress resistant. Our findings suggest that the pattern of cellulose crystallinity in stomata of floating plants from different families was altered as a consequence of similar evolutionary pressures.

Smart Hydrogel-Based Valves Inspired by the Stomata in Plants.

We report the design of hydrogels that can act as "smart" valves or membranes. Each hydrogel is engineered with a pore (about 1 cm long and <1 mm thick) that remains closed under ambient conditions but opens under specific conditions. Our design is inspired by the stomatal valves in plant leaves, which regulate the movement of water and gases in and out of the leaves. The design features two different gels, active and passive, which are attached concentrically to form a disc-shaped hybrid film. The pore is created in the central active gel, and the conditions for opening the pore can be tuned based on the chemistry of this gel. For example, if the active gel is made from N-isopropylacrylamide (NIPA), the actuation of the pore depends on the temperature of water relative to 32 °C, which is the lower-critical solution temperature (LCST) of NIPA. The concentric design of our hybrid provides directionality to the volumetric transition of the active gel, i.e., it ensures that the pore opens as the active gel shrinks. In turn, contact with hot water (T > 32 °C) opens the pore and allows the water to pass through the gel. Conversely, the pore remains closed when the water is cold (T < 32 °C). The gel thereby acts as a "smart" valve that is able to regulate the flow of solvent depending on its properties. We have extended the concept to other stimuli that can cause gel-swelling transitions including solvent composition, pH, and light. Additionally, when two different gel-based valves are arranged in series, the assembly acts as a logical "AND" gate, i.e., water flows through the valve-combination only if it simultaneously satisfies two distinct conditions (such as its pH being below a critical value and its temperature being above a critical value).

Exploring novel genetic sources of salinity tolerance in rice through molecular and physiological characterization.

BACKGROUND AND AIMS: Agricultural productivity is increasingly being affected by the build-up of salinity in soils and water worldwide. The genetic base of salt-tolerant rice donors being used in breeding is relatively narrow and needs broadening to breed varieties with wider adaptation to salt-affected areas. This study evaluated a large set of rice accessions of diverse origins to identify and characterize novel sources of salt tolerance. METHODS: Diversity analysis was performed on 107 germplasm accessions using a genome-wide set of 376 single-nucleotide polymorphism (SNP) markers, along with characterization of allelic diversity at the major quantitative trait locus Saltol Sixty-nine accessions were further evaluated for physiological traits likely associated with responses to salt stress during the seedling stage. KEY RESULTS: Three major clusters corresponding to the indica, aus and aromatic subgroups were identified. The largest group was indica, with the salt-tolerant Pokkali accessions in one sub-cluster, while a set of Bangladeshi landraces, including Akundi, Ashfal, Capsule, Chikirampatnai and Kutipatnai, were in a different sub-cluster. A distinct aus group close to indica contained the salt-tolerant landrace Kalarata, while a separate aromatic group closer to japonica rice contained a number of traditional, but salt-sensitive Bangladeshi landraces. These accessions have different alleles at the Saltol locus. Seven landraces - Akundi, Ashfal, Capsule, Chikirampatnai, Jatai Balam, Kalarata and Kutipatnai - accumulated less Na and relatively more K, maintaining a lower Na/K ratio in leaves. They effectively limit sodium transport to the shoot. CONCLUSIONS: New salt-tolerant landraces were identified that are genetically and physiologically distinct from known donors. These landraces can be used to develop better salt-tolerant varieties and could provide new sources of quantitative trait loci/alleles for salt tolerance for use in molecular breeding. The diversity observed within this set and in other donors suggests multiple mechanisms that can be combined for higher salt tolerance.

The effect of vapour pressure deficit on stomatal conductance, sap pH and leaf-specific hydraulic conductance in Eucalyptus globulus clones grown under two watering regimes.

BACKGROUND AND AIMS: Stomatal conductance has long been considered of key interest in the study of plant adaptation to water stress. The expected increase in extreme meteorological events under a climate change scenario may compromise survival in Eucalyptus globulus plantations established in south-western Spain. We investigated to what extent changes in stomatal conductance in response to high vapour pressure deficits and water shortage are mediated by hydraulic and chemical signals in greenhouse-grown E. globulus clones. METHODS: Rooted cuttings were grown in pots and submitted to two watering regimes. Stomatal conductance, shoot water potential, sap pH and hydraulic conductance were measured consecutively in each plant over 4 weeks under vapour pressure deficits ranging 0·42 to 2·25 kPa. Evapotranspiration, growth in leaf area and shoot biomass were also determined. KEY RESULTS: There was a significant effect of both clone and watering regime in stomatal conductance and leaf-specific hydraulic conductance, but not in sap pH. Sap pH decreased as water potential and stomatal conductance decreased under increasing vapour pressure deficit. There was no significant relationship between stomatal conductance and leaf-specific hydraulic conductance. Stomata closure precluded shoot water potential from falling below -1·8 MPa. The percentage loss of hydraulic conductance ranged from 40 to 85 %. The highest and lowest leaf-specific hydraulic conductances were measured in clones from the same half-sib families. Water shortage reduced growth and evapotranspiration, decreases in evapotranspiration ranging from 14 to 32 % in the five clones tested. CONCLUSIONS: Changes in sap pH seemed to be a response to changes in atmospheric conditions rather than soil water in the species. Stomata closed after a considerable amount of hydraulic conductance was lost, although intraspecific differences in leaf-specific hydraulic conductance suggest the possibility of selection for improved productivity under water-limiting conditions combined with high temperatures in the early stages of growth.

An epidemiological assessment of stomatal ozone flux-based critical levels for visible ozone injury in Southern European forests.

Southern forests are at the highest ozone (O3) risk in Europe where ground-level O3 is a pressing sanitary problem for ecosystem health. Exposure-based standards for protecting vegetation are not representative of actual field conditions. A biologically-sound stomatal flux-based standard has been proposed, although critical levels for protection still need to be validated. This innovative epidemiological assessment of forest responses to O3 was carried out in 54 plots in Southeastern France and Northwestern Italy in 2012 and 2013. Three O3 indices, namely the accumulated exposure AOT40, and the accumulated stomatal flux with and without an hourly threshold of uptake (POD1 and POD0) were compared. Stomatal O3 fluxes were modeled (DO3SE) and correlated to measured forest-response indicators, i.e. crown defoliation, crown discoloration and visible foliar O3 injury. Soil water content, a key variable affecting the severity of visible foliar O3 injury, was included in DO3SE. Based on flux-effect relationships, we developed species-specific flux-based critical levels (CLef) for forest protection against visible O3 injury. For O3 sensitive conifers, CLef of 19 mmol m(-2) for Pinus cembra (high O3 sensitivity) and 32 mmol m(-2) for Pinus halepensis (moderate O3 sensitivity) were calculated. For broadleaved species, we obtained a CLef of 25 mmol m(-2) for Fagus sylvatica (moderate O3 sensitivity) and of 19 mmol m(-2) for Fraxinus excelsior (high O3 sensitivity). We showed that an assessment based on PODY and on real plant symptoms is more appropriated than the concentration-based method. Indeed, POD0 was better correlated with visible foliar O3 injury than AOT40, whereas AOT40 was better correlated with crown discoloration and defoliation (aspecific indicators). To avoid an underestimation of the real O3 uptake, we recommend the use of POD0 calculated for hours with a non-null global radiation over the 24-h O3 accumulation window.