Comparison of Modeled and Observed Environmental Influences on the Stable Oxygen and Hydrogen Isotope Composition of Leaf Water in Phaseolus vulgaris L

In this paper we describe how a model of stable isotope fractionation processes, originally developed by H. Craig and L. I. Gordon ([1965] in E Tongiorgi, ed, Proceedings of a Conference on Stable Isotopes in Oceanographic Studies and Paleotemperature, Spoleto, Italy, pp 9-130) for evaporation of water from the ocean, can be applied to leaf transpiration. The original model was modified to account for turbulent conditions in the leaf boundary layer. Experiments were conducted to test the factors influencing the stable isotopic composition of leaf water under controlled environment conditions. At steady state, the observed leaf water isotopic composition was enriched above that of stem water with the extent of the enrichment dependent on the leaf-air vapor pressure difference (VPD) and the isotopic composition of atmospheric water vapor (AWV). The higher the VPD, the larger was the observed heavy isotope content of leaf water. At a constant VPD, leaf water was relatively depleted in heavy isotopes when exposed to AWV with a low heavy isotope composition, and leaf water was relatively enriched in heavy isotopes when exposed to AWV with a large heavy isotope composition. However, the observed heavy isotope composition of leaf water was always less than that predicted by the model. The extent of the discrepancy between the modeled and observed leaf water isotopic composition was a strong linear function of the leaf transpiration rate.     

Relative growth rate, biomass allocation pattern and water use efficiency of three wheat cultivars during early ontogeny as dependent on water availability

We have investigated the water use efficiency of whole plants and selected leaves and allocation patterns of three wheat cultivars (Mexipak, Nesser and Katya) to explore how variation in these traits can contribute to the ability to grow in dry environments. The cultivars exhibited considerable differences in biomass allocation and water use efficiency. Cultivars with higher growth rates of roots and higher proportions of biomass in roots (Nesser and Katya) also had higher leaf growth rates, higher proportions of their biomass as leaves and higher leaf area ratios. These same cultivars had lower rates of transpiration per unit leaf area or unit root weight and higher biomass production per unit water use. They also had higher ratios of photosynthesis to transpiration, and lower ratios of intercellular to external CO₂ partial pressure. The latter resulted from large differences in stomatal conductance associated with relatively small differences in rates of photosynthesis. There was little variation between cultivars in response to drought, and differences in allocation pattern and plant water use efficiency between cultivars as found under well-watered conditions persisted under dry conditions. At the end of the non-watered treatment, relative growth rates and transpiration rates decreased to similar values for all cultivars. High ratios of photosynthesis to transpiration, and accordingly high biomass production per unit of transpiration, is regarded as a favourable trait for dry environments, since more efficient use of water postpones the decrease in plant water status.     

Relative growth rate, biomass allocation pattern and water use efficiency of three wheat cultivars during early ontogeny as dependent on water availability

We have investigated the water use efficiency of whole plants and selected leaves and allocation patterns of three wheat cultivars (Mexipak, Nesser and Katya) to explore how variation in these traits can contribute to the ability to grow in dry environments. The cultivars exhibited considerable differences in biomass allocation and water use efficiency. Cultivars with higher growth rates of roots and higher proportions of biomass in roots (Nesser and Katya) also had higher leaf growth rates, higher proportions of their biomass as leaves and higher leaf area ratios. These same cultivars had lower rates of transpiration per unit leaf area or unit root weight and higher biomass production per unit water use. They also had higher ratios of photosynthesis to transpiration, and lower ratios of intercellular to external CO₂ partial pressure. The latter resulted from large differences in stomatal conductance associated with relatively small differences in rates of photosynthesis. There was little variation between cultivars in response to drought, and differences in allocation pattern and plant water use efficiency between cultivars as found under well-watered conditions persisted under dry conditions. At the end of the non-watered treatment, relative growth rates and transpiration rates decreased to similar values for all cultivars. High ratios of photosynthesis to transpiration, and accordingly high biomass production per unit of transpiration, is regarded as a favourable trait for dry environments, since more efficient use of water postpones the decrease in plant water status.     

Modelling non‐steady‐state isotope enrichment of leaf water in a gas‐exchange cuvette environment

The combined use of a gas‐exchange system and laser‐based isotope measurement is a tool of growing interest in plant ecophysiological studies, owing to its relevance for assessing isotopic variability in leaf water and/or transpiration under non‐steady‐state (NSS) conditions. However, the current Farquhar & Cernusak (F&C) NSS leaf water model, originally developed for open‐field scenarios, is unsuited for use in a gas‐exchange cuvette environment where isotope composition of water vapour (δ_v) is intrinsically linked to that of transpiration (δ_E). Here, we modified the F&C model to make it directly compatible with the δ_v–δ_E dynamic characteristic of a typical cuvette setting. The resultant new model suggests a role of ‘net‐flux’ (rather than ‘gross‐flux’ as suggested by the original F&C model)‐based leaf water turnover rate in controlling the time constant (τ) for the approach to steady sate. The validity of the new model was subsequently confirmed in a cuvette experiment involving cotton leaves, for which we demonstrated close agreement between τ values predicted from the model and those measured from NSS variations in isotope enrichment of transpiration. Hence, we recommend that our new model be incorporated into future isotope studies involving a cuvette condition where the transpiration flux directly influences δ_v. There is an increasing popularity among plant ecophysiologists to use a gas‐exchange system coupled to laser‐based isotope measurement for investigating non‐steady state (NSS) isotopic variability in leaf water (and/or transpiration); however, the current Farquhar & Cernusak (F&C) NSS leaf water model is unsuited for use in a gas‐exchange cuvette environment due to its implicit assumption of isotope composition of water vapor (δ_v) being constant and independent of that of transpiration (δ_E). In the present study, we modified the F&C model to make it compatible with the dynamic relationship between δ_v and δ_E as is typically associated with a cuvette setting. Using an experiment conducted on cotton leaves, we show that the modified NSS model performed well in predicting the time constant for the exponential approach of leaf water toward steady state under cuvette conditions. Such a result demonstrates the applicability of this new model to gas‐exchange cuvette conditions where the transpiration flux directly influences δ_v, and therefore suggests the need to incorporate this model into future isotope studies that employ a laser‐cuvette coupled system.     

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 ¹⁸O 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 influence of transpiration on the equilibration of leaf water with atmospheric water vapour

Abstract. The authors examine the isotopic composition of leaf water, at natural abundance levels, as influenced by transpiration rate. The isotopic composition of water of wheat leaves ( Triticum aestivum L. var. Aroona) was followed while their transpiration rate adjusted to 'steady-state' environmental conditions. Leaf diffusive resistance was modified by short-term salt treatment and by plant culture in either nutrient solution, free-draining sand, or vermiculite. Resultant changes in ¹⁸O and ²H in leaf water are described and fitted to the model of Leaney et al. (1985). The treatments with lower transpiration rates were found to have a greater fraction of their leaf water equilibrated with water vapour in the atmosphere. Comparable results were obtained with both ¹⁸O and ²H, with some differences being interpreted in terms of turbulence in the vapour diffusion path. The fraction of the leaf water equilibrated with the atmosphere varied between leaves of different ages. However, this may have been due to their different positions in the canopy.     

The contributions of apoplastic,symplastic and gas phase pathways for water transport outside the bundle sheath in leaves

Water movement from the xylem to stomata is poorly understood. There is still no consensus about whether apoplastic or symplastic pathways are more important, and recent work suggests vapour diffusion may also play a role. The objective of this study was to estimate the proportions of hydraulic conductance outside the bundle sheath contributed by apoplastic, symplastic and gas phase pathways, using a novel analytical framework based on measurable anatomical and biophysical parameters. The calculations presented here suggest that apoplastic pathways provide the majority of conductance outside the bundle sheath under most conditions, whereas symplastic pathways contribute only a small proportion. The contributions of apoplastic and gas phase pathways vary depending on several critical but poorly known or highly variable parameters namely, the effective Poiseuille radius for apoplastic bulk flow, the thickness of cell walls and vertical temperature gradients within the leaf. The gas phase conductance should increase strongly as the leaf centre becomes warmer than the epidermis – providing up to 44% of vertical water transport for a temperature gradient of 0.2 K. These results may help to explain how leaf water transport is influenced by light absorption, temperature and differences in leaf anatomy among species.     

Effects of water stress on oxygen, hydrogen and carbon isotope ratios in two species of cotton plants

Abstract. Two cotton species ( Gossypium hirsutum L. cv. SJ-2 and Gossypium barbadence cv. S-5) were grown under irrigated (wet) and non-irrigated (dry) conditions in the same field. Leaf water was enriched in ¹⁸O and deuterium in the dry treatment relative to the wet treatment for both species. Only in plants of S-5 was a similar enrichment observed in leaf cellulose. In both species, the isotopic composition of leaf cellulose must reflect the isotopic composition of the actual water pool involved in cellulose synthesis. Therefore, our observations indicate that one species (SJ-2) can maintain a relative isolation of this water pool from direct evapotranspirational effects. Such plant species will more faithfully record, in the isotopic composition of organic matter, the isotopic composition of ground water. In contrast, the isotopic composition of organic matter in plants such as S-5 could be used as an integrated signal reflecting humidity conditions during growth. Water use efficiency, based on seed-cotton yield and total water applied, correlated linearly with differences in carbon isotopic ratios between species in both the wet and dry treatments and between treatments in each species.     

A model of stomatal conductance to quantify the relationship between leaf transpiration, microclimate and soil water stress

A model of stomatal conductance was developed to relate plant transpiration rate to photosynthetic active radiation (PAR), vapour pressure deficit and soil water potential. Parameters of the model include sensitivity of osmotic potential of guard cells to photosynthetic active radiation, elastic modulus of guard cell structure, soil‐to‐leaf conductance and osmotic potential of guard cells at zero PAR. The model was applied to field observations on three functional types that include 11 species in subtropical southern China. Non‐linear statistical regression was used to obtain parameters of the model. The result indicated that the model was capable of predicting stomatal conductance of all the 11 species and three functional types under wide ranges of environmental conditions. Major conclusions included that coniferous trees and shrubs were more tolerant for and resistant to soil water stress than broad‐leaf trees due to their lower osmotic potential, lignified guard cell walls, and sunken and suspended guard cell structure under subsidiary epidermal cells. Mid‐day depression in transpiration and photosynthesis of pines may be explained by decreased stomatal conductance under a large vapour pressure deficit. Stomatal conductance of pine trees was more strongly affected by vapour pressure deficit than that of other species because of their small soil‐to‐leaf conductance, which is explainable in terms of xylem tracheids in conifer trees. Tracheids transport water by means of small pit‐pairs in their side walls, and are much less efficient than the end‐perforated vessel members in broad‐leaf xylem systems. These conclusions remain hypothetical until direct measurements of these parameters are available.     

Leaf water content and palisade cell size

The palisade cell sizes in leaves of Eucalyptus pauciflora were estimated in paradermal sections of cryo-fixed leaves imaged in the cryo-scanning electron microscope, as a quantity called the cell area fraction (CAF). Cell sizes were measured in detached leaves as a function of leaf water content, in intact leaves in the field during a day"s transpiration as a function of balance pressure of adjacent leaves, and on leaf disks equilibrated with air of relative humidities from 100 to 58%. Values of CAF ranged from 0.82 at saturation to approx. 0.3 in leaves dried to a relative water content (RWC) of 0.5, and in the field to approx. 0.58 at 15 bar (1.5 MPa) balance pressure. At a CAF of 0.58, the moisture content of the cell walls is in equilibrium with air at 90% relative humidity, which is the estimated relative humidity in the intercellular spaces. It is shown that at this moisture content, the cell walls could be exerting a pressure of approx. 50 bar on the cell contents.     

Stable isotope composition of water in desert plants

A survey of the stable isotope content of tissue waters of plants from the Negev desert was conducted. Large differences were observed in the extent of enrichment of the heavy isotopes in leaf water relative to local precipitation among different plants. This is apparently caused by the species-dependent stratagems adopted by the plants to cope with water stress, primarily by differences in the depth of water uptake in the soil and through the timing of stomatal openings during the daily cycle. Salt stressed plants showed extreme variability in the isotopic composition of leaf–water. The results show that plants with adaptation to arid conditions can avoid the transpiration regime, which would lead to the strong isotopic enrichment in their leaf water expected under arid conditions. This has implications for the use of stable isotopes in plants as indicators of either plant ecophysiology or paleoclimate. Responsible Editor: Hans Lambers. G. Goodfriend is deceased.     

Dew water isotopic ratios and their relationships to ecosystem water pools and fluxes in a cropland and a grassland in China

Dew formation has the potential to modulate the spatial and temporal variations of isotopic contents of atmospheric water vapor, oxygen and carbon dioxide. The goal of this paper is to improve our understanding of the isotopic interactions between dew water and ecosystem water pools and fluxes through two field experiments in a wheat/maize cropland and in a short steppe grassland in China. Measurements were made during 94 dew events of the D and ¹⁸O compositions of dew, atmospheric vapor, leaf, xylem and soil water, and the whole ecosystem water flux. Our results demonstrate that the equilibrium fractionation played a dominant role over the kinetic fractionation in controlling the dew water isotopic compositions. A significant correlation between the isotopic compositions of leaf water and dew water suggests a large role of top-down exchange with atmospheric vapor controlling the leaf water turnover at night. According to the isotopic labeling, dew water consisted of a downward flux of water vapor from above the canopy (98%) and upward fluxes originated from soil evaporation and transpiration of the leaves in the lower canopy (2%).     

Root cooling strongly affects diel leaf growth dynamics,water and carbohydrate relations in Ricinus communis

In laboratory and greenhouse experiments with potted plants, shoots and roots are exposed to temperature regimes throughout a 24 h (diel) cycle that can differ strongly from the regime under which these plants have evolved. In the field, roots are often exposed to lower temperatures than shoots. When the root‐zone temperature in Ricinus communis was decreased below a threshold value, leaf growth occurred preferentially at night and was strongly inhibited during the day. Overall, leaf expansion, shoot biomass growth, root elongation and ramification decreased rapidly, carbon fluxes from shoot to root were diminished and carbohydrate contents of both root and shoot increased. Further, transpiration rate was not affected, yet hydrostatic tensions in shoot xylem increased. When root temperature was increased again, xylem tension reduced, leaf growth recovered rapidly, carbon fluxes from shoot to root increased, and carbohydrate pools were depleted. We hypothesize that the decreased uptake of water in cool roots diminishes the growth potential of the entire plant – especially diurnally, when the growing leaf loses water via transpiration. As a consequence, leaf growth and metabolite concentrations can vary enormously, depending on root‐zone temperature and its heterogeneity inside pots.     

Temporal and spatial variations in the oxygen-18 content of leaf water in different plant species

Temporal variations in the δ¹⁸ oxygen (δ¹⁸O) content of water transpired by leaves during a simulated diurnal cycle fluctuated around the δ¹⁸O content of the source water. Reconstructed variations in the δ¹⁸O values of leaf water differed markedly from those predicted by conventional models. Even when transpiring leaves were maintained under constant conditions for at least 3 h, strict isotopic steady-state conditions of leaf water (equality of the ¹⁸O/¹⁶O ratios in the input and transpired water) were rarely attained in a variety of plant species (Citrus reticu-lata, Citrus paradisi, Gossypium hirsutum, Helianthus annuns, Musa musaceae and Nicotinia tabacum). Isotopic analysis of water transpired by leaves indicated that leaves approach the isotopic steady state in two stages. The first stage takes 10 to 35 min (with a rate of change of about 3–3%h^?1), while in the second stage further approach to the isotopic steady state is asymptotic (with a rate of change of about 0–4% h^?1), and under conditions of low transpiration leaves can last for many hours. Substantial spatial isotopic heterogeneity was maintained even when leaves were at or near isotopic steady state. An underlying pattern in this isotopic heterogeneity is often discerned with increasing ¹⁸O/¹⁶O ratios from base to tip, and from the centre to the edges of the leaves. It is also shown that tissue water along these spatial isotopic gradients, as well as the average leaf water, can have ¹⁸O/¹⁶O ratios both lower and higher than those predicted by the conventional Craig and Gordon model. We concluded, first, that at any given time during the diurnal cycle of relative humidity the attainment of an isotopic steady state in leaf water cannot be assumed a priori and, secondly, that the isotopic enrichment pattern of leaf water reflects gradual enrichment along the water-flow pathway (e.g. as in a string of pools), rather than a single-step enrichment from source water, as is normally assumed.     

Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress

Most of the symplastic water transport in plants occurs via aquaporins, but the extent to which aquaporins contribute to plant water status under favorable growth conditions and abiotic stress is not clear. To address this issue, we constitutively overexpressed the Arabidopsis plasma membrane aquaporin, PIP1b, in transgenic tobacco plants. Under favorable growth conditions, PIP1b overexpression significantly increased plant growth rate, transpiration rate, stomatal density, and photosynthetic efficiency. By contrast, PIP1b overexpression had no beneficial effect under salt stress, whereas during drought stress it had a negative effect, causing faster wilting. Our results suggest that symplastic water transport via plasma membrane aquaporins represents a limiting factor for plant growth and vigor under favorable conditions and that even fully irrigated plants face limited water transportation. By contrast, enhanced symplastic water transport via plasma membrane aquaporins may not have any beneficial effect under salt stress, and it has a deleterious effect during drought stress.     

Transpiration rate and intercellular diffusive resistance in the tobacco leaf

The increase in the measured transpiration rate in tobacco leaves due to the experimentally decreased humidity of the bulk air was found to be significantly lower than the theoretical value calculated from the change of water vapour concentration gradients. Boundary layer and stomatal diffusive resistances remained unchanged under experimental conditions with no change of net photosynthetic CO₂ uptake. This suggests an increase in intercellular diffusive resistance with an increase in water vapour concentration gradient which is the driving force of water vapour diffusive part of transpiration flux. The increase can be ascribed to the lengthening of intercellular diffusive pathway as steeper water vapour concentration gradient in intercellular spaces results in an increased evaporating surface of intercellular cells thus moving the effective plane of vaporization in leaf mesophyll further inwards. Due to different and independent changes of concentration gradients for water vapour and CO₂, different length of intercellular diffusive pathways for CO₂ and water vapour may be expected.     

Comparison of measured and modeled variations in piñon pine leaf water isotopic enrichment across a summer moisture gradient

Stable hydrogen and oxygen isotopic composition of bulk leaf water (δD_lw and δ¹⁸O_lw) in piñon pine (Pinus edulis and P. monophylla) and gas exchange parameters were measured under field conditions to examine the effects of seasonal moisture stress on leaf water isotopic enrichment. Study sites were located near the lower elevation limit for piñon in the southwestern USA. Leaf-level transpiration measurements were made four times daily in spring, summer and early autumn; simultaneously, leaf samples were collected for water extraction and stable isotope analysis. Diurnal variations in δD_lw and δ¹⁸O_lw values were small, especially when leaf water residence times (molar leaf water content divided by transpiration rate) were high. Stomatal conductance explained most of the variance (60%) in leaf water enrichment across the dataset. Observed leaf water enrichment was compared with predictions of steady-state and nonsteady-state models. Nonsteady-state predictions fit observations the best, although D enrichment was often lower than predicted by any model. Hydrogen isotope ratios of leaf water and cellulose nitrate were strongly correlated, demonstrating preservation of a leaf water signal in wood and leaf cellulose.     

Environmental effects on oxygen isotope enrichment of leaf water in cotton leaves

The oxygen isotope enrichment of bulk leaf water (Delta(b)) was measured in cotton (Gossypium hirsutum) leaves to test the Craig-Gordon and Farquhar-Gan models under different environmental conditions. Delta(b) increased with increasing leaf-to-air vapor pressure difference (VPd) as an overall result of the responses to the ratio of ambient to intercellular vapor pressures (e(a)/e(i)) and to stomatal conductance (g(s)). The oxygen isotope enrichment of lamina water relative to source water (Delta(1)), which increased with increasing VPd, was estimated by mass balance between less enriched water in primary veins and enriched water in the leaf. The Craig-Gordon model overestimated Delta(b) (and Delta(1)), as expected. Such discrepancies increased with increase in transpiration rate (E), supporting the Farquhar-Gan model, which gave reasonable predictions of Delta(b) and Delta(1) with an L of 7.9 mm, much less than the total radial effective length L(r) of 43 mm. The fitted values of L for Delta(1) of individual leaves showed little dependence on VPd and temperature, supporting the assumption that the Farquhar-Gan formulation is relevant and useful in describing leaf water isotopic enrichment.     

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 (Δ_e) and mean lamina leaf water enrichment (Δ_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 Δ_L for ¹⁸O and ²H in beech seedlings under well-watered and water-limited conditions. We applied evaporative enrichment models of increasing complexity to predict Δ_e and Δ_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 Δ_e and Δ_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.     

Inferring foliar water uptake using stable isotopes of water

A growing number of studies have described the direct absorption of water into leaves, a phenomenon known as foliar water uptake. The resultant increase in the amount of water in the leaf can be important for plant function. Exposing leaves to isotopically enriched or depleted water sources has become a common method for establishing whether or not a plant is capable of carrying out foliar water uptake. However, a careful inspection of our understanding of the fluxes of water isotopes between leaves and the atmosphere under high humidity conditions shows that there can clearly be isotopic exchange between the two pools even in the absence of a change in the mass of water in the leaf. We provide experimental evidence that while leaf water isotope ratios may change following exposure to a fog event using water with a depleted oxygen isotope ratio, leaf mass only changes when leaves are experiencing a water deficit that creates a driving gradient for the uptake of water by the leaf. Studies that rely on stable isotopes of water as a means of studying plant water use, particularly with respect to foliar water uptake, must consider the effects of these isotopic exchange processes.