Leaf Water Isotopic Enrichment Research Articles

Covariation between oxygen and hydrogen stable isotopes declines along the path from xylem water to wood cellulose across an aridity gradient.

Oxygen and hydrogen isotopes of cellulose in plant biology are commonly used to infer environmental conditions, often from time series measurements of tree rings. However, the covariation (or the lack thereof) between δ18 O and δ2 H in plant cellulose is still poorly understood. We compared plant water, and leaf and branch cellulose from dominant tree species across an aridity gradient in Northern Australia, to examine how δ18 O and δ2 H relate to each other and to mean annual precipitation (MAP). We identified a decline in covariation from xylem to leaf water, and onwards from leaf to branch wood cellulose. Covariation in leaf water isotopic enrichment (Δ) was partially preserved in leaf cellulose but not branch wood cellulose. Furthermore, whilst δ2 H was well-correlated between leaf and branch, there was an offset in δ18 O between organs that increased with decreasing MAP. Our findings strongly suggest that postphotosynthetic isotope exchange with water is more apparent for oxygen isotopes, whereas variable kinetic and nonequilibrium isotope effects add complexity to interpreting metabolic-induced δ2 H patterns. Varying oxygen isotope exchange in wood and leaf cellulose must be accounted for when δ18 O is used to reconstruct climatic scenarios. Conversely, comparing δ2 H and δ18 O patterns may reveal environmentally induced shifts in metabolism.

\u03b42Hn-alkane and \u03b418Osugar biomarker proxies from leaves and topsoils of the Bale Mountains, Ethiopia, and implications for paleoclimate reconstructions

The hydrogen isotopic composition of leaf wax–derived n-alkane (δ2Hn-alkane) and oxygen isotopic composition of hemicellulose–derived sugar (δ18Osugar) biomarkers are valuable proxies for paleoclimate reconstructions. Here, we present a calibration study along the Bale Mountains in Ethiopia to evaluate how accurately and precisely the isotopic composition of precipitation is imprinted in these biomarkers. n-Alkanes and sugars were extracted from the leaf and topsoil samples and compound–specific δ2Hn-alkane and δ18Osugar values were measured using a gas chromatograph–thermal conversion–isotope ratio mass spectrometer (GC–TC–IRMS). The weighted mean δ2Hn-alkane and δ18Osugar values range from − 186 to − 89‰ and from + 27 to + 46‰, respectively. Degradation and root inputs did not appear to alter the isotopic composition of the biomarkers in the soil samples analyzed. Yet, the δ2Hn-alkane values show a statistically significant species dependence and δ18Osugar yielded the same species–dependent trends. The reconstructed leaf water of Erica arborea and Erica trimera is 2H– and 18O–enriched by + 55 ± 5 and + 9 ± 1‰, respectively, compared to precipitation. By contrast, Festuca abyssinica reveals the most negative δ2Hn-alkane and least positive δ18Osugar values. This can be attributed to “signal–dampening” caused by basal grass leaf growth. The intermediate values for Alchemilla haumannii and Helichrysum splendidum can be likely explained with plant physiological differences or microclimatic conditions affecting relative humidity (RH) and thus RH–dependent leaf water isotope enrichment. While the actual RH values range from 69 to 82% (x̄ = 80 ± 3.4%), the reconstructed RH values based on a recently suggested coupled δ2Hn-alkane –δ18Osugar (paleo–) hygrometer approach yielded a mean of 78 ± 21%. Our findings corroborate (i) that vegetation changes, particularly in terms of grass versus non–grassy vegetation, need to be considered in paleoclimate studies based on δ2Hn-alkane and δ18Osugar records and (ii) that the coupled δ2Hn-alkane –δ18Osugar (paleo–) hygrometer approach holds great potential for deriving additional paleoclimatic information compared to single isotope approaches.

Isotope signature of maize stem and leaf and investigation of transpiration and water transport

Stable isotope signature of plant water contains essential information on water transport pathway and plant transpiration, which has been shown to be a powerful tracer in plant physiological and ecological processes. However, stable isotopes fractionation in processes of plant water transport and the relationship between transpiration rate (E) and effective pathway length (L) and their possible mechanisms are still largely mysterious and confusing. Here, we tested stable isotope signature of maize stem and leaf based on anatomical measurements and modeling, and propose a deuterium deviation in leaf water (Δd) to understand variability leaf water isotope enrichment and transpiration. We found isotopes fractionation occurred in maize stems in arid area. Leaf transpiration rate was strongly affected by Δd. The data revealed L has a negative power relationship with E, with a single power function of L = 284.77E−1.02; and the proportional deviation of leaf 18O enrichment 1 − ΔL/ΔE is negatively correlated with E under low E (E < 2.0 mmol m−2 s−1) and, a positively relationship under high E (E > 2.0 mmol m−2 s−1). Suggesting that a pivotal role of effective path length in driving variations in leaf transpiration rate. The deuterium deviation Δd may have great potential to serve as a new diagnostic tool for understanding pathways of water transport in plant. Care should be taken when examining source-water and estimating roots water uptake using the stable isotope method in arid areas, and further study is needed to be carried out and confirm the conclusions across a range of environmental conditions and species.

Can hydraulic design explain patterns of leaf water isotopic enrichment in C3 plants?

H2 18 O enrichment develops when leaves transpire, but an accurate generalized mechanistic model has proven elusive. We hypothesized that leaf hydraulic architecture may affect the degree to which gradients in H2 18 O develop within leaves, influencing bulk leaf stable oxygen isotope enrichment (ΔL ) and the degree to which the Péclet effect is relevant in leaves. Leaf hydraulic design predicted the relevance of a Péclet effect to ΔL in 19 of the 21 species tested. Leaves with well-developed hydraulic connections between the vascular tissue and the epidermal cells through bundle sheath extensions and clear distinctions between palisade and spongy mesophyll layers (while the mesophyll is hydraulically disconnected) may have velocities of the transpiration stream such that gradients in H2 18 O develop and are expressed in the mesophyll. In contrast, in leaves where the vascular tissue is hydraulically disconnected from the epidermal layers, or where all mesophyll cells are well connected to the transpiration stream, velocities within the liquid transport pathways may be low enough that gradients in H2 18 O are very small. Prior knowledge of leaf hydraulic design allows informed selection of the appropriate ΔL modelling framework.

Seasonal and diurnal trends in progressive isotope enrichment along needles in two pine species.

The Craig-Gordon type (C-G) leaf water isotope enrichment models assume a homogeneous distribution of enriched water across the leaf surface, despite observations that Δ18 O can become increasingly enriched from leaf base to tip. Datasets of this 'progressive isotope enrichment' are limited, precluding a comprehensive understanding of (a) the magnitude and variability of progressive isotope enrichment, and (b) how progressive enrichment impacts the accuracy of C-G leaf water model predictions. Here, we present observations of progressive enrichment in two conifer species that capture seasonal and diurnal variability in environmental conditions. We further examine which leaf water isotope models best capture the influence of progressive enrichment on bulk needle water Δ18 O. Observed progressive enrichment was large and equal in magnitude across both species. The magnitude of this effect fluctuated seasonally in concert with vapour pressure deficit, but was static in the face of diurnal cycles in meteorological conditions. Despite large progressive enrichment, three variants of the C-G model reasonably successfully predicted bulk needle Δ18 O. Our results thus suggest that the presence of progressive enrichment does not impact the predictive success of C-G models, and instead yields new insight regarding the physiological and anatomical mechanisms that cause progressive isotope enrichment.

Using Water Stable Isotopes to Trace Water Sources of Three Typical Japanese Tree Species under Heavy Rainfall Conditions

Stable isotopes of xylem water (18O and D) have been successfully used to determine sources of soil water for plant transpiration, but mainly in drought-prone environments. The water uptake strategies of three representative tree species in Japan, namely cedar (Cryptomeria japonica), larch (Larix kaempferi) and beech (Fagus crenata), were investigated using δ18O and δD of water (precipitation, soil and xylem), together with wood α-cellulose δ13C and δ18O, along one growing season. The study was carried out in the research forest of Yamagata University (Shonai region), a high precipitation area in Japan, which exceeds 3000 mm per year. Precipitation water δ18O and δD increased along the summer growing season, but oxygen and hydrogen isotopic composition of soil water remained essentially unchanged. In general, xylem water isotopes of cedar and larch followed the local meteoric water line, but beech xylem water was decoupled from soil and precipitation values in July and August. For this tree species, the xylem water isotopic records were more depleted than cedar and larch xylem water isotopic values and the precipitation water isotopic records, indicating that beech used more water from soil layers during July-August than the other two species, which mainly used newly-fallen precipitation. Wood δ18O showed an opposite seasonal trend to the one found for xylem water, likely because of leaf water isotope enrichment, which was in turn controlled by seasonal transpiration rate. The higher δ13C values of cedar during summer suggested that this species had enhanced water-use efficiency during the growing season compared with the deciduous species larch and beech. Our results highlight different water use strategies among forest tree species even in areas where the annual water balance is far from limiting plant performance.

Seasonal variation of δ18O and δ2H in leaf water of Fagus sylvatica L. and related water compartments

The study aims to assess variability in leaf water isotopic enrichment occurring in the field under natural conditions. We focused on seasonal variation and difference between sun-exposed and shaded leaves. Isotopic composition (δ18O, δ2H) of leaf water was monitored in a beech tree (Fagus sylvatica L.) growing in the forest-meadow ecotone together with δ18O (2H) of water compartments which are in close relation to this signal, namely twig and soil water. The sampling was carried out in approximately two-week intervals during five consecutive vegetation seasons.The δ18O (2H) data showed a distinct seasonal pattern and a consistency in relative differences between the seasons and sample categories. Leaf water was the most isotopically enriched water compartment. The leaf water enrichment decreased toward the autumn reflecting the change in δ18O (2H) of source water and evaporative demands. The soil and twig water isotopic signal was depleted against current precipitation as it partly retained the isotopic signature from winter precipitation however the seasonal pattern of soil and twig water followed that of precipitation. No significant differences between sun-exposed and shaded samples were detected. Nevertheless, the observed strong seasonal pattern of isotope composition of leaf, twig and soil water should be taken into account when using leaf water enrichment for further calculations or modeling.

A multiple time scale modeling investigation of leaf water isotope enrichment in a temperate grassland ecosystem

Understanding the controls on temporal variation in plant leaf δ2H and δ18O values is important for understanding carbon–water dynamics of the biosphere and interpreting a wide range of proxies for past environments. Explaining the enrichment mechanisms under field conditions is challenging. To clarify the leaf water isotopic enrichment process at the ecosystem scale, four models with a range of complexities that were previously conducted at the leaf scale have been tested to simulate canopy foliage water in a multispecies grassland ecosystem. Although the exact importance of considering non-steady-state or/and isotopic diffusion in bulk leaf isotopic simulations has been reported in previous studies, our findings suggested that the steady-state assumption (SSA) is practically acceptable as a first-order approximation. The SSA two-pool model was the best option for reproducing seasonality of the bulk-leaf-water isotopic ratio for a grassland ecosystem. Relative humidity at canopy layer as the most controlling factor for canopy foliage water stable isotope composition because of its high sensitivity and variation. The results highlighted that canopy foliage water was a well-behaved property that was predictable for a multispecies grassland ecosystem at hourly or daily time-scales.

Hydrogen isotope fractionation in leaf waxes in the Alaskan Arctic tundra

Leaf wax hydrogen isotopes (δDwax) are increasingly utilized in terrestrial paleoclimate research. Applications of this proxy must be grounded by studies of the modern controls on δDwax, including the ecophysiological controls on isotope fractionation at both the plant and landscape scales. Several calibration studies suggest a considerably smaller apparent fractionation between source water and waxes (εapp) at high latitudes relative to temperate or tropical locations, with major implications for paleoclimatic interpretations of sedimentary δDwax. Here we investigate apparent fractionation in the Arctic by tracing the isotopic composition of leaf waxes from production in modern plants to deposition in lake sediments using isotopic observations of precipitation, soil and plant waters, living leaf waxes, and waxes in sediment traps in the Brooks Range foothills of northern Alaska. We also analyze a lake surface sediment transect to compare present-day vegetation assemblages to εapp at the watershed scale. Source water and εapp were determined for live specimens of Eriophorum vaginatum (cottongrass) and Betula nana (dwarf birch), two dominant tundra plants in the Brooks Range foothills. The δD of these plants’ xylem water closely tracks that of surface soil water, and reflects a summer-biased precipitation source. Leaf water is enriched by 23±15‰ relative to xylem water for E. vaginatum and by 41±19‰ for B. nana. Evapotranspiration modeling indicates that this leaf water enrichment is consistent with the evaporative enrichment expected under the climate conditions of northern Alaska, and that 24-h photosynthesis does not cause excessive leaf water isotope enrichment. The εapp determined for our study species average −89±14‰ and −106±16‰ for B. nana n-alkanes and n-acids, respectively, and −182±10‰ and −154±26‰ for E. vaginatum n-alkanes and n-acids, which are similar to the εapp of related species in temperate and tropical regions, indicating that apparent fractionation is similar in Arctic relative to other regions, and there is no reduced fractionation in the Arctic. Sediment trap data suggest that waxes are primarily transported into lakes from local (watershed-scale) sources by overland flow during the spring freshet, and so δDwax within lakes depends on watershed-scale differences in water isotope compositions and in plant ecophysiology. As such, the large difference between our study species suggests that the relative abundance of graminoids and shrubs is potentially an important control on δDwax in lake sediments. These inferences are supported by δDwax data from surface sediments of 24 lakes where εapp, relative to δDxylem, averages −128±13‰ and −130±8‰ for n-acids and n-alkanes, respectively, and co-varies with vegetation type across watersheds. These new determinations of plant source water seasonality and εapp for the Arctic will improve the δDwax paleoclimate proxy at high latitudes.

Leaf water 18 O and 2 H enrichment along vertical canopy profiles in a broadleaved and a conifer forest tree.

Distinguishing meteorological and plant-mediated drivers of leaf water isotopic enrichment is prerequisite for ecological interpretations of stable hydrogen and oxygen isotopes in plant tissue. We measured input and leaf water δ2 H and δ18 O as well as micrometeorological and leaf morpho-physiological variables along a vertical gradient in a mature angiosperm (European beech) and gymnosperm (Douglas fir) tree. We used these variables and different enrichment models to quantify the influence of Péclet and non-steady state effects and of the biophysical drivers on leaf water enrichment. The two-pool model accurately described the diurnal variation of leaf water enrichment. The estimated unenriched water fraction was linked to leaf dry matter content across the canopy heights. Non-steady state effects and reduced stomatal conductance caused a higher enrichment of Douglas fir compared to beech leaf water. A dynamic effect analyses revealed that the light-induced vertical gradients of stomatal conductance and leaf temperature outbalanced each other in their effects on evaporative enrichment. We conclude that neither vertical canopy gradients nor the Péclet effect is important for estimates and interpretation of isotopic leaf water enrichment in hypostomatous trees. Contrarily, species-specific non-steady state effects and leaf temperatures as well as the water vapour isotope composition need careful consideration.

Identifying drivers of leaf water and cellulose stable isotope enrichment in Eucalyptus in northern Australia.

Several previous studies have investigated the use of the stable hydrogen and oxygen isotope compositions in plant materials as indicators of palaeoclimate. However, accurate interpretation relies on a detailed understanding of both physiological and environmental drivers of the variations in isotopic enrichments that occur in leaf water and associated organic compounds. To progress this aim we measured δ18O and δ2H values in eucalypt leaf and stem water and δ18O values in leaf cellulose, along with the isotopic compositions of water vapour, across a north-eastern Australian aridity gradient. Here we compare observed leaf water enrichment, along with previously published enrichment data from a similar north Australian transect, to Craig-Gordon-modelled predictions of leaf water isotopic enrichment. Our investigation of model parameters shows that observed 18O enrichment across the aridity gradients is dominated by the relationship between atmospheric and internal leaf water vapour pressure while 2H enrichment is driven mainly by variation in the water vapour-source water isotopic disequilibrium. During exceptionally dry and hot conditions (RH<21%, T>37°C) we observed strong deviations from Craig-Gordon predicted isotope enrichments caused by partial stomatal closure. The atmospheric-leaf vapour pressure relationship is also a strong predictor of the observed leaf cellulose δ18O values across one aridity gradient. Our finding supports a wider applicability of leaf cellulose δ18O composition as a climate proxy for atmospheric humidity conditions during the leaf growing season than previously documented.

A multi-proxy approach to drought reconstruction

In palaeoclimate reconstructions, the combination of proxy records measured in different climate archives is challenging because of the uncertainties associated with each proxy, but it can also help reduce some of these uncertainties. Here, we present a novel approach to combine speleothem and tree ring proxies for a drought reconstruction of the last 640 years: a fluid inclusion δ18O record from a stalagmite from Villars Cave (southwest France) and a tree ring cellulose δ18O record of Quercus spp. from the nearby Angouleme area. The δ18O of the fluid inclusions is taken as an estimate of the δ18O of the trees’ source water. Then, the cellulose and source water δ18O are used to calculate the leaf water isotopic enrichment, as well as relative humidity, which is the dominant controlling factor of this enrichment. The reconstructed long-term trends in relative humidity differ from a previously published reconstruction of moisture variability based on the tree ring record alone. Further measurements will be necessary to support either reconstruction. Nevertheless, this investigation demonstrates the great potential for combining isotope proxies from speleothems and tree rings to reconstruct both the low- and high-frequency variability of drought.

Carbon and hydrogen isotopic effects of stomatal density in Arabidopsis thaliana

Stomata are key gateways mediating carbon uptake and water loss from plants. Varied stomatal densities in fossil leaves raise the possibility that isotope effects associated with the openness of exchange may have mediated plant wax biomarker isotopic proxies for paleovegetation and paleoclimate in the geological record. Here we use Arabidopsis thaliana, a widely used model organism, to provide the first controlled tests of stomatal density on carbon and hydrogen isotopic compositions of cuticular waxes. Laboratory grown wildtype and mutants with suppressed and overexpressed stomatal densities allow us to directly test the isotope effects of stomatal densities independent of most other environmental or biological variables. Hydrogen isotope (D/H) measurements of both plant waters and plant wax n-alkanes allow us to directly constrain the isotopic effects of leaf water isotopic enrichment via transpiration and biosynthetic fractionations, which together determine the net fractionation between irrigation water and n-alkane hydrogen isotopic composition. We also measure carbon isotopic fractionations of n-alkanes and bulk leaf tissue associated with different stomatal densities. We find offsets of +15‰ for δD and −3‰ for δ13C for the overexpressed mutant compared to the suppressed mutant. Since the range of stomatal densities expressed is comparable to that found in extant plants and the Cenozoic fossil record, the results allow us to consider the magnitude of isotope effects that may be incurred by these plant adaptive responses. This study highlights the potential of genetic mutants to isolate individual isotope effects and add to our fundamental understanding of how genetics and physiology influence plant biochemicals including plant wax biomarkers.

Effects of stomatal density and leaf water content on the ¹⁸O enrichment of leaf water.

Leaf water isotopic composition is imprinted in several biomarkers of interest and it is imperative that we understand the isotopic enrichment of leaf water. Here, we test the effect of stomatal density and leaf water content on the oxygen isotopic composition of leaf water in transgenic Arabidopsis plants expressing different stomatal densities, and several other species showing a range of stomatal density. We grew Arabidopsis plants hydroponically and collected other species in the field. Stomatal density and leaf water content were determined for each plant. We measured transpiration and extracted leaf water for isotopic determination. Using these measurements and the current leaf water isotope model, we calculated several of the parameters related to leaf water isotopic enrichment. High stomatal density promoted leaf water isotope enrichment. No conclusion, however, can be drawn regarding the effect of leaf water content on leaf water isotope enrichment. Factors such as transpiration might mask the effect of stomatal density on leaf water isotopic enrichment. We propose a method by which stomatal density can be incorporated in the current Peclet model of leaf water isotope enrichment. These findings have important applications in the use of plant-based metabolic proxies in paleoclimate studies.

Reconstructing relative humidity from plant delta18O and deltaD as deuterium deviations from the global meteoric water line.

Cellulose delta18O and deltaD can provide insights on climates and hydrological cycling in the distant past and how these factors differ spatially. However, most studies of plant cellulose have used only one isotope, most commonly delta18O, resulting in difficulties partitioning variation in delta18O of precipitation vs. evaporative conditions that affect leaf water isotopic enrichment. Moreover, observations of pronounced diurnal differences from conventional steady-state model predictions of leaf water isotopic fractionation have cast some doubt on single isotope modeling approaches for separating precipitation and evaporation drivers of cellulose delta18O or deltaD. We explore a dual isotope approach akin to the concept of deuterium-excess (d), to establish deuterium deviations from the global meteoric water line in leaf water (deltad(l)) as driven by relative humidity (RH). To demonstrate this concept, we survey studies of leaf water delta18O and deltaD in hardwood vs. conifer trees. We then apply the concept to cellulose delta18O and deltaD using a mechanistic model of cellulose delta18O and deltaD to reconstruct deuterium deviations from the global meteoric water line (deltad(c)) in Quercus macrocarpa, Q. robur, and Pseudotsuga menziesii. For each species, deltad(c) showed strong correlations with RH across sites. deltad(c) agreed well with steady-state predictions for Q. macrocarpa, while for Q. robur, the relationship with RH was steeper than expected. The slope of deltad(c) vs. RH of P. menziesii was also close to steady-state predictions, but deltad(c) were more enriched than predicted. This is in agreement with our leaf water survey showing conifer deltad(l) was more enriched than predicted. Our data reveal that applications of this method should be appropriate for reconstructing RH from cellulose delta18O and deltaD after accounting for differences between hardwoods and conifers. Hence, deltad(c) should be useful for understanding variability in RH associated with past climatic cycles, across regional climates, or across complex terrain where climate modeling is challenging. Furthermore, deltad(c) and inferred RH values should help in constraining variation in source water delta18O.

Stomatal pore size and density in mangrove leaves and artificial leaves: effects on leaf water isotopic enrichment during transpiration.

We tested the hypothesis that the previously observed low isotopic enrichment of mangrove leaf water is caused by larger stomatal pores and lower densities compared with freshwater plants. First, we measured and compared pore size and density in mangroves, transitional and freshwater species in South Florida. We pooled this data with other reports encompassing 14 mangrove species and 134 freshwater species and tested for differences in pore size and density between mangroves and freshwater plants. Second, we built artificial leaves having different pore size and density and determined whether there were isotopic differences in their water after transpiration. Both the local survey and pooled data showed that mangrove leaves have significantly larger stomatal pores with lower densities compared with freshwater plants. Isotope enrichment of water from artificial leaves having larger less dense pores was lower than those having smaller and denser pores. Stomatal pore size and density has an effect on leaf water isotopic enrichment amongst other factors. Pore size and density probably affects key components of the Peclet ratio such as the distance advective flow of water must travel to the evaporative surface and the cross-sectional area of advective flow. These components, in turn, affect leaf water isotopic enrichment. Results from the artificial leaf experiment also mimic a recent finding that effective path length scales to the inverse of transpiration in real leaves. The implications of these findings further our understanding of leaf water isotope ratios and are important in applications of stable isotopes in the study of paleoclimate and atmospheric processes.

Are leaf physiological traits related to leaf water isotopic enrichment in restinga woody species?

During plant-transpiration, water molecules having the lighter stable isotopes of oxygen and hydrogen evaporate and diffuse at a faster rate through the stomata than molecules having the heavier isotopes, which cause isotopic enrichment of leaf water. Although previous models have assumed that leaf water is well-mixed and isotopically uniform, non-uniform stomatal closure, promoting different enrichments between cells, and different pools of water within leaves, due to morpho-physiological traits, might lead to inaccuracies in isotopic models predicting leaf water enrichment. We evaluate the role of leaf morpho-physiological traits on leaf water isotopic enrichment in woody species occurring in a coastal vegetation of Brazil known as restinga. Hydrogen and oxygen stable isotope values of soil, plant stem and leaf water and leaf traits were measured in six species from restinga vegetation during a drought and a wet period. Leaf water isotopic enrichment relative to stem water was more homogeneous among species during the drought in contrast to the wet period suggesting convergent responses to deal to temporal heterogeneity in water availability. Average leaf water isotopic enrichment relative to stem water during the drought period was highly correlated with relative apoplastic water content. We discuss this observation in the context of current models of leaf water isotopic enrichment as a function of the Péclet effect. We suggest that future studies should include relative apoplastic water content in isotopic models.

Isotopic composition of transpiration and rates of change in leaf water isotopologue storage in response to environmental variables

During daylight hours, the isotope composition of leaf water generally approximates steady-state leaf water isotope enrichment model predictions. However, until very recently there was little direct confirmation that isotopic steady-state (ISS) transpiration in fact exists. Using isotope ratio infrared spectroscopy (IRIS) and leaf gas exchange systems we evaluated the isotope composition of transpiration and the rate of change in leaf water isotopologue storage (isostorage) when leaves were exposed to variable environments. In doing so, we developed a method for controlling the absolute humidity entering the gas exchange cuvette for a wide range of concentrations without changing the isotope composition of water vapour. The measurement system allowed estimation of (18)O enrichment both at the evaporation site and for bulk leaf water, in the steady state and the non-steady state. We show that non-steady-state effects dominate the transpiration isoflux even when leaves are at physiological steady state. Our results suggest that a variable environment likely prevents ISS transpiration from being achieved and that this effect may be exacerbated by lengthy leaf water turnover times due to high leaf water contents.

The Péclet effect on leaf water enrichment correlates with leaf hydraulic conductance and mesophyll conductance for CO2

Leaf water gets isotopically enriched through transpiration, and diffusion of enriched water through the leaf depends on transpiration flow and the effective path length (L). The aim of this work was to relate L with physiological variables likely to respond to similar processes. We studied the response to drought and vein severing of leaf lamina hydraulic conductance (K(lamina) ), mesophyll conductance for CO(2) (g(m) ) and leaf water isotope enrichment in Vitis vinifera L cv. Grenache. We hypothesized that restrictions in water pathways would reduce K(lamina) and increase L. As a secondary hypothesis, we proposed that, given the common pathways for water and CO(2) involved, a similar response should be found in g(m) . Our results showed that L was strongly related to mesophyll variables, such as K(lamina) or g(m) across experimental drought and vein-cutting treatments, showing stronger relationships than with variables included as input parameters for the models, such as transpiration. Our findings were further supported by a literature survey showing a close link between L and leaf hydraulic conductance (K(leaf) = 31.5 × L(-0.43) , r(2) = 0.60, n = 24). The strong correlation found between L, K(lamina) and g(m) supports the idea that water and CO(2) share an important part of their diffusion pathways through the mesophyll.

Effect of water availability on leaf water isotopic enrichment in beech seedlings shows limitations of current fractionation models

Current models of leaf water enrichment predict that the differences between isotopic enrichment of water at the site of evaporation (Delta(e)) and mean lamina leaf water enrichment (Delta(L)) depend on transpiration rates (E), modulated by the scaled effective length (L) of water isotope movement in the leaf. However, variations in leaf parameters in response to changing environmental conditions might cause changes in the water path and thus L. We measured the diel course of Delta(L) for (18)O and (2)H in beech seedlings under well-watered and water-limited conditions. We applied evaporative enrichment models of increasing complexity to predict Delta(e) and Delta(L), and estimated L from model fits. Water-limited plants showed moderate drought stress, with lower stomatal conductance, E and stem water potential than the control. Despite having double E, the divergence between Delta(e) and Delta(L) was lower in well-watered than in water-limited plants, and thus, L should have changed to counteract differences in E. Indeed, L was about threefold higher in water-limited plants, regardless of the models used. We conclude that L changes with plant water status far beyond the variations explained by water content and other measured variables, thus limiting the use of current evaporative models under changing environmental conditions.