Heavy Water Fractionation During Transpiration1

By Farquhar, Graham D; Cernusak, Lucas A; Barnes, Belinda

MOTIVATION

A small proportion of water molecules contain the heavier isotopes of hydrogen and oxygen. There is a tendency for these heavier molecules of water to accumulate in leaves during transpiration. This has several interesting repercussions, including effects on the isotopic composition of organic matter, and of atmospheric water vapor, carbon dioxide, and oxygen. In turn, these effects aid temporal reconstruction of climate and spatial and temporal reconstruction of primary production in various ways. A recent, novel report by Miller et al. (2006) showed that tree-ring ^sup 18^O measurements carried a record of hurricane activity.

The motivation for our laboratory to study water isotopes was to enhance studies of transpiration effi-ciency (TE; the leaf contribution to water-use efficiency at the plant, crop, or ecosystem level). The instanta-neous TE is A/[g^sub w^v], where A is the rate of photosyn-thetic uptake of CO^sub 2^, g^sub w^ is the conductance to diffusion of water vapor to the atmosphere from the sites of evaporation within the leaf (made up of the stomatal conductance, g^sub s^, and boundary layer conductance, g^sub b^, in series), and v is the leaf-to-air water vapor concen-tration difference. Carbon isotope composition of leaves can be measured to determine the carbon iso-topic discrimination, Δ^sup 13^C, during photosynthesis. In turn, Δ^sup 13^C relates to A/g^sub t^, the ratio of CO^sub 2^ assimilation rate to the total conductance to diffusion of CO^sub 2^ from the atmosphere to the sites of carboxylation. The two measures are obviously related, and it was suggested that the oxygen isotope composition (δ^sup 18^O) of leaf material could be used to pick up differences in v (Farquhar et al., 1994).

Further, because g^sub w^ affects δ^sup 18^O and A does not, it was hoped that measurements of Δ^sup 13^C and δ^sub 18^O in organic matter could be combined to separate photo- synthetic capacity effects on TE from g^sub w^ effects (Yakir and Israeli, 1995; Saurer et al, 1997; Farquhar et al., 1998). The heavy isotopes of water, expressed in or-ganic matter, have promise as a means of identifying genetic variation in g^sub w^. In an ecological and population biology context, the carbon, hydrogen, and oxygen isotope measurements could provide a practical sur-rogate for measures of the marginal water cost of carbon gain, ∂E/ ∂A, with E the rate of transpiration (Cowan and Farquhar, 1977), as one might expect different stomatal and photosynthesis strategies to be associated with environments with differing rainfall statistics (Cowan, 1982).

In this Update on heavy water fractionation, we focus on how transpiration “leads” to enrichment and ask whether enrichment is a measure of transpiration. We then sketch some applications.

THE BASICS OF WHY LEAVES BECOME ENRICHED

On earth roughly 0.204% of oxygen is ^sup 18^O and 0.037% is ^sup 17^O; 0.015% of hydrogen is ^sup 2^H. There are usually more of these heavy isotopes in leaf water than in the soil/xylem water because (1) the vapor pressure of heavy water is less than that of the most common isotopolog, ^sup 1^H^sub 2^^sup 16^O, and (2) the binary diffusivity with air of heavy water vapor is less than that of light water. So, when water evaporates from the leaf, heavier molecules tend to be left behind. This process con-tinues until the leaf water becomes sufficiently en-riched that the exit of heavy and light molecules through the stomata matches that of the supply of water from the xylem.

Thus, from Equations 5 and 6, the maximum isotope effect is the sum of the equilibrium and kinetic frac-tionation terms and is realized when atmospheric humidity is very low. The isotope effect is generally small when atmospheric humidity approaches 100%.

AS E INCREASES, ISOTOPIC ENRICHMENT SHOULD ALSO INCREASE. TRUE OR FALSE?

Heavy water enrichment increases with increase in E. This was the recent conclusion of Sheshshayee et al. (2005) in a paper titled “Oxygen isotope enrichment (Δ^sup 18^O) as a measure of time averaged transpiration rate.” The authors report a relationship that holds regardless of whether the variation in E is caused by variation in g^sub w^ or variation in evaporative demand, v. Does this make sense?

If the source of variation is evaporative demand, it does, as can be seen by reducing relative humidity h in the equations above. Supporting evidence comes from many sources, and a nice example is from Helliker and Ehleringer (2002a, 2002b; Fig. 1).

Figure 1. Enrichment of leaf water versus relative humidity in five C^sub 3^ grass species. All grasses were grown in chambers where the isotope ratio of source water was held constant, leaf boundary layer develop-ment was minimized by high wind speeds, and only relative humidity varied across treatments. Note that relative humidity was calculated based on leaf temperature as opposed to ambient temperature similar to the convention of Equation 5, where h = w^sub a^/w^sub i^. Black and white circles are data from Helliker and Ehleringer (2002a) and Helliker and Ehleringer (2002b), respectively.

So as far as stomatal-induced changes in transpiration rate are concerned, we have two effects that should lead to a negative relationship between E and isotopic enrichment, these being of opposite sign to the positive effect of evaporative demand on both E and Δ^sub e^. There is a third effect that reinforces these. The enrichment of water in the leaf, Δ^sub L^, will usually be less than that at the sites of evaporation, Δ^sub e^, because mass flow from the xylem of unenriched water will oppose the diffusion of enriched water from the sites of evaporation back toward the xylem. Farquhar and Lloyd (1993) called this the Pclet effect, after a French mathematician, and formalized it as follows: Enrichment falls off from the value at the sites of evaporation, as exp(-P), where P is the dimensionless number vl/D, with v = velocity, l = distance from sites, and D the diffusivity of heavy water in water. Velocity is proportional to transpiration rate. Average enrichment, Δ^sub L^, in a simple system would be given by ([1 - exp(-P)]/P) Δ^sub e^. The Pclet number P is proportional to E, and so, as E increases, average leaf enrichment, Δ^sub L^, becomes increasingly depleted compared with Δ^sub e^. It represents another reason for Δ^sub L^ to decrease as g^sub w^ increases. The Pclet effect should also apply when E is increasing because of a reduction in h, but the calculated negative effect on average leaf water enrichment is less than the direct effect of increasing Δ^sub e^.

What is the experimental support for these mathematical ideas about a negative relationship between E and Δ^sub L^, when the source of variation is g^sub w^? At present, the data on direct effects of stomatal conductance on Δ^sub L^ are fairly thin, and particularly so for the so-called Pclet effect. It is a common, but not universal, observation that leaf water is less enriched than would be predicted from the modified Craig-Gordon equation. However, the evidence that the Pclet effect explains the shortfall is as yet largely indirect. Barbour et al. (2004) reanalyzed the data of Roden and Ehleringer (1999b) and found that the predictions of leaf water enrichment were improved by including the Pclet effect. Cernusak et al. (2002, 2005) included the effect in simulating their field observations of Δ^sub L^, and it also improved the fit greatly compared to the simple use of the Craig-Gordon model. This was so for both hydrogen and oxygen isotopes. However, this improvement is largely one of making Δ^sub L^ less than Δ^sub e^, and the dependence on changes in g^sub w^ tended to be lost in the variation caused by changes in Δ^sub e^. There are few direct measurements in the laboratory of the effect of changes in g^sub w^ on Δ^sub L^. The main difficulty of such measurements is their intrinsically destructive nature, meaning one leaf per datum, together with what appears to be variable Pclet lengths, l, between individual leaves. If aquaporins play a role in determining the effective length (Barbour and Farquhar, 2004) and if they are dynamic (Flexas et al., 2006), this might explain some of the variability. One cannot subsample leaves and hope to obtain representative values of average leaf water enrichment, as enrichment is often heterogeneous (Yakir et al., 1989; Bariac et al., 1994).

OXYGEN ISOTOPE ENRICHMENT OF PLANT ORGANIC MATTER

Although the direct evidence that an increase in g^sub w^ should reduce leaf water enrichment is lacking, there is indirect evidence via the effects on isotopic composition of organic matter. The factors controlling oxygen isotopic composition of organic matter are better understood than those controlling the hydrogen isotope composition, and for the former the major step appears to be the exchange of oxygen atoms between water and carbonyl oxygens in triose phosphates via a gem-diol intermediate. The analogous equilibration between acetone and water was studied by Sternberg and DeNiro (1983), who found that the oxygen in the organic matter ended up enriched by about 28[per thousand] compared to the water. In the formation of Suc, the water undergoing exchange will be both that in the chloroplasts, probably close \to Δ^sub e^ (Farquhar et al., 1993), and that in the cytosol, which is presumably less enriched. Barbour et al. (2000b) examined the oxygen isotopic composition of Sue bled from the petioles of castor bean (Ricinus communis) leaves undergoing gas-exchange measurements. The technique has the advantage that repeated measurements can be made of the same leaf under differing conditions. The results were consistent with a fractionation of 27[per thousand], and, by subtracting that value from the enrichment of the Sue, they were able to obtain the composition of the effective substrate water. The latter was less enriched than Δ^sub e^, and the difference increased with increasing E as would be expected from theory. Cernusak et al. (2003b) compared destructive measurements of Δ^sub L^ with the enrichment of phloem Sue and also obtained a fractionation of 27[per thousand]. However, the underlying processes, including their spatial distribution, are complex and may involve processes other than carbonyl exchange (Schmidt et al., 2001) Thus, at this stage it is unknown how reliable, or constant, is the fractionation taken here as 27[per thousand]. Suc molecules are broken down to Glc and rejoined to make cellulose. This exposes some carbonyl oxygens again to water, and, since that water is often less enriched than in source leaf cells, the enrichment in cellulose is likely to be less than that in the feeding Sue. The proportion exposed is at minimum 20%, but more if there is futile recycling of hexose phosphates through triose phosphates. The net proportion appears to be around 40% (Cernusak et al., 2005). Despite these complexities, clear leaf water signals can be discerned in the oxygen isotope composition of cellulose and, indeed, of general organic matter.

An example of g^sub w^ affecting cellulose enrichment was provided by cotton (Gossypium hirsutum) plants fed various concentrations of abscisic acid (ABA; Barbour and Farquhar, 2000). See Figure 2. As concentration increased, stomata closed (diminishing the stomatal conductance, g^sub s^, as well as g^sub w^) and the cellulose became more enriched. That is, a reduction of transpiration was associated with an increase in oxygen isotopic composition. Reassuringly, the reverse association was seen in plants where E was reduced by higher humidity. So increasing E via effects on evaporative demand were consistent with increased heavy water accumulation in the photosynthesizing leaves, while increasing E via effects on g^sub w^ were consistent with decreased accumulation. Nevertheless, contrary to this, Sheshshayee et al. (2005) observed a positive relationship between g^sub w^ and oxygen isotopic composition in plants fed ABA. However, recently Thompson et al. (A. Thompson, J. Andrews, B.J. Mulholland, J.M.T. MCKee, W. Howard, H.W. Hilton, J.S. Horridge, G.D. Farquhar, and I.B. Taylor, unpublished data) examined two tomato (Lycopersicon esculentum) lines, sp12 and sp5, that overexpress a gene encoding 9-cis- epoxycarotenoid dioxygenase and are involved in abscisic synthesis. The increases in ABA content were associated with a decrease in g^sub w^ and with an increase in organic δ^sup 18^O, thus conforming to theoretical expectations. Further, unpublished data of J. Masle and G. Farquhar show that plants growing in compacted soils have lower g^sub w^ and are enriched in δ^sup 18^O compared to plants growing in loose soil.

The relevance of the Pclet effect to organic δ^sup 18^O in trees is also supported by reanalysis of the cellulose data of Roden and Ehleringer (1999a, 2000) by Barbour et al. (2004). Cernusak et al. (2003a) showed that among Eucalyptus globulus trees at three adjacent field sites with differing soil water contents, variation in phloem sugar δ^sup 18^O was negatively associated with variation in transpiration rates among the trees.

Thus, apart from the puzzling and, therefore, interesting results of Sheshshayee et al. (2005), the conclusion is that increasing E can be associated with either increasing enrichment of heavy water, Δ^sub L^, when the source of variation is evaporative demand, or decreasing Δ^sub L^, when the source of variation is g^sub w^.

Figure 2. The relationships of cotton leaf organic matter oxygen isotopic enrichment with stomatal conductance, g^sub s^ (A); leaf temperature T^sub 1^ (B); and E for plants grown at 43% relative humidity (white squares) and 76% relative humidity (black circles; C). The source of variation was the concentration of the hormone, ABA (from left to right within each section, O, 10^sup -5^, 10^sup – 4^, and 10^sup -3^ M), sprayed on the developing leaves. Data and figure are redrawn from Barbour and Farquhar (2000).

^sup 18^O, STOMATAL CONDUCTANCE, AND BREEDING FOR IMPROVED GRAIN YIELD IN IRRIGATED WHEAT

Carbon isotope discrimination has been used as a selection criterion in wheat (Triticum aestivum) breeding, and as a result water-use efficient wheat varieties have been released commercially (Condon et al., 2002; Rebetzke et al., 2002) for dryland agriculture. In wellwatered conditions, yield potential is sought, and that appears to be associated with increases in both photosynthetic capacity and stomatal conductance and decrease in canopy temperature. CIMMYT grew eight short spring wheat species that they had released between 1962 and 1988 during three seasons in northwest Mexico. It was found that yield, A, and g^sub w^ all increased with year of release-the breeders had done their job well- and the canopy temperature and δ^sub 18^O of the leaf cellulose decreased with year of release (Barbour et al., 2000a), again as theory would suggest.

It is often easier to collect leaves in the field for subsequent mass spectrometric analysis than to directly measure stomatal conductance with a porometer. And since g^sub w^ in the field is in any case a dynamic variable, δ^sub 18^O of the leaf organic matter is an attractive measure when genetic differences in average conductance are sought. The measure is most effective when h is low, as was the case with the measurements in Mexico.

NON-STEADY STATE

At night time one would expect the leaf to lose its enrichment in heavy water because evaporative demand goes down. From what we have seen, it would be incorrect to think that enrichment should decline because closing stomata reduce E. In fact, night-time stomatal conductance is required for the leaf to lose its enrichment. The time constant for changes in isotopic composition of leaf water relates to the one-way flux out of the leaf (g^sub w^W^sub i^; Dongmann et al., 1974) and not to the net transpiration rate [g^sub w^(w^sub i^ - w^sub a^)]. This led Farquhar and Cernusak (2005) to observe that at relative humidities, h, greater than 50%, more water enters the leaf from the air (g^sub w^w^sub a^) than through the petiole [g^sub w(w^sub i^ - w^sub a^)].

Cernusak et al. (2002, 2005) showed that inclusion of the Pclet effect was necessary for simulating the experimental results during the day, but less so at night when E was low. They found that the nonsteady-state treatment was vital for reasonable simulation of observations at night, but less so during the day when open stomata allowed the leaf enrichment to follow changes in evaporative demand. The interest here in night-time stomatal opening is in whether it allows the leaf to lose enrichment in heavy water. This interest intersects with that in the isotopic exchange of oxygen between leaf water and CO2 in the atmosphere. If the stomata are open at night, CO2 can enter the leaf, exchange oxygen isotopically with leaf water, and diffuse back out to the atmosphere without any photosynthesis being involved (Cernusak et al., 2004).

SPATIAL HETEROGENEITY IN ENRICHMENT OF HEAVY WATER IN LEAVES

One of the frustrating features of heavy water enrichment, its variation within a single leaf, has turned out to be interesting and illuminating. Some of the variation is probably systematic with the ends of leaves being enriched compared to the bases. Some may be random and perhaps associated with dynamic stomatal heterogeneity (Peak et al., 2004). Yakir et al. (1989) proposed that there may be different metabolic pools of water within the leaf. This could conceivably come about because of hydraulic isolation of some parts of the tissue. One would think that internal vapor exchange would ensure that no part of the leaf is really isolated from the rest. Nevertheless, with the current interest in aquaporins and their possible dynamic nature, reversible isolation within cells could be imagined.

Progressive enrichment occurs along the leaf. It is seen in both monocots and dicots, but is easier to study in monocots. It occurs because when water first enters the leaf at the base, light water is preferentially transpired, leaving the heavy water behind. This heavy water tends to get swept further along the leaf. The process continues until a point is reached where the exchange no longer causes any enrichment. At this point the enrichment of the mesophyll will be maximal, Δ^sub M^, and equal to the enrichment of the locally transpired water, Δ^sub E^. We adapt this condition from the Craig-Gordon enrichment (Eq. 5):

Δ^sub e^, = Δ^sub c^ [asymptotically =] ε^sup +^ + (Δ^sub v^ – ε^sub k^)h,

and it then follows (Farquhar and Gan, 2003) that Δ^sub M^ [asymptotically =] Δ^sub C^/h. So the greatest enrichment along a leaf is the normal Craig-Gordon value divided by the relative humidity. Of course, the base of the leaf must be depleted so that the whole leaf average enrichment at the sites of evaporation is still given by the CraigGordon average. The origin of these concepts was a string-of-lakes model of Gat and Bowser (1991), which Yakir (1992) suggested could be applied to a line of evaporating cells. This was done by Helliker and Ehleringer (2000) and app\lied to observations on monocot leaves. They used a finite number of “lakes,” and the concept was extended to an infinite number of elements by Farquhar and Gan (2003) and later applied to maize (Zea mays) leaves by Gan et al. (2003).

The detailed theory requires several elements. One needs to consider both mesophyll and veins (Allison et al., 1985), as well as the Pclet effect discussed already. The latter is essentially a radial effect, from xylem to stomata. But there is a longitudinal Pclet effect also, and here the value of P will be large, as the advection along a vein will be great compared to back diffusion. The full theory is complex and depends on the pattern of transpiration and on any taper of the leaf and of xylem elements (Farquhar and Gan, 2003; Barnes et al., 2004; Oge et al, 2007). Recently Oge et al. (2007) have used an iterative model to extend progressive enrichment into non-steady-state conditions. The detailed experimental testing of these models requires multiple measurements in space and time. So far the uncertainty has mostly related to why the leaf tip is less enriched than Δ^sub M^. The modeling of this aspect is sensitive to the nature of leaf tapering.

APPLICATIONS

Thus far we have considered the application in terms of interpretation of the isotopic composition of organic matter. Ecophysiological applications include resource utilization by mistletoes (Amyema miquelii, Amyema preisii; Cernusak et al., 2004) and interpretation of effects of pollution (Saurer et al., 2001). Recently, Helliker and Griffiths (2007) modeled and validated the changes in δ^sup 18^O of Tillandsia usneoides (Spanish moss, a rootless epiphytic crassulacean acid metabolism plant) that occur after rain falls as the plant dries out. They emphasize the importance of the one-way fluxes between the plant and the atmosphere and show how herbarium material of this species may be used as a proxy for δ^sub 18^O of water vapor.

There are effects on atmospheric isotopic composition also. Atmospheric CO2 undergoes oxygen isotopic exchange with leaf water and soil water, and changes in the δ^sub 18^O of CO2 can be used to study spatial and temporal variation in the net exchange of CO2 in terrestrial ecosystems, and its underlying causes, dominated by photosynthesis and respiration (Farquhar et al., 1993; Cuntz et al., 2003). It had earlier been thought that CO2 respired from leaves would be in isotopic equilibrium with leaf water, minus a diffusional fractionation factor of about 8[per thousand] (Yakir et al., 1994). However, as with water vapor fluxes into and out of the leaf, it is the one-way fluxes that must be considered, and it is now recognized that invasion of CO2 from the atmosphere, followed by exchange with leaf water, can enrich the isotopic signature of respired CO2 (Cernusak et al, 2004), although just what part of the leaf water exchanges, and to what degree, is not fully understood.

O2 is released by the plant during photosynthesis. The isotopic composition of evolved oxygen is identical (within an experimental error of <0.01[per thousand]) to that of the substrate water in the chloroplast thylakoids (Guy et al., 1993), and so an understanding is required as to where the chloroplasts sit in the catena of isotopic enrichment from soil water to the sites of evaporation within leaves. Changes in the oxygen isotope ratios of atmospheric O2 are revealed on long time scales in the Dole effect (Dole et al., 1954), where atmospheric oxygen is currently enriched in ^sup 18^O by 23.5[per thousand] compared with mean ocean water. Recorded changes in the Dole effect are applied in the study of variations in the balance of terrestrial and marine productivity (Bender et al., 1985; Bender et al., 1994). As well as oxygenesis, the Dole effect depends on fractionation during respiration and weakly on stratospheric phenomena.

The processes leading to enrichment of water isotopes in leaves fractionate O2 isotopes and the oxygen isotopes of CO2 in a mass- dependent way, as does respiration. That is, the enrichment in ^sup 17^O over ^sup 16^O is about half of the enrichment of O ^sup 18^over ^sup 16^O. In contrast, photochemical reactions among O3, O2, and CO2 in the stratosphere fractionate oxygen isotopes in a non- mass-dependent way and with about equal lowering of δ^sup 17^O and δ^sup 18^O in atmospheric O2, and, similarly, about equal enhancement of δ^sup 17^O and δ^sup 18^O in atmospheric CO2 (Thiemens et al., 1995; Luz et al., 1999; Luz and Barkan, 2000; Hoag et al., 2005). This causes a ^sup 17^O anomaly in O2 that depends on the relative rates of photosynthetic O2 production and stratospheric photochemical reactions. If the latter rates are known, the overall global rate of photosynthetic oxygenesis can be estimated from the magnitude of the ^sup 17^O anomaly in O2 (Bender et al., 1994). This approach was applied by Luz et al. (1999) and Blunier et al. (2002), using air trapped in polar ice cores, to estimate the global rate during the last glacial period. It relies, of course, on assumptions concerning constancy or otherwise of the stratospheric reaction rates. The most recent contribution to plant physiology in this field describes fractionation of the three stable oxygen isotopes by photosynthesizing organisms (Helman et al., 2005).

Leaf transpiration contributes water vapor to the atmosphere, with an oxygen isotope composition equal to that of soil water. Similarly, the evaporation of rain intercepted by leaves involves no fractionation when all the water has been evaporated. However, soil evaporation and sublimation (snow) involve hydrogen and oxygen isotope fractionation of water molecules (Moser and Stichler, 1975; Gibson, 2001), and water at the mouth of rivers with a notable evaporative component is enriched in the heavy isotopes, compared with catchment precipitation. In hydrology it is important to be able to distinguish transpiration from soil evaporation. The evaporation component in the evapotranspiration (ET) flux can be estimated using isotope mass balance equations, such as those proposed by Gat and Bowser (1991), Gat and Matsui (1991), and Gibson et al. (1993, 1996). Recently, this was done for the Mississippi watershed by Lee and Veizer (2003), who used their results to predict net primary productivity for the region. Their calculations incorporated “water-use efficiencies” derived from the data of Shantz and Piemeisel (1927) for C3 and C4 species; however, modern studies with measurements of vapor pressure deficits and carbon isotope composition (Farquhar and Richards, 1984) would have been of great benefit in their approach.

Measurements of the isotopic composition of water vapor have been used to estimate transpiration fluxes in rice (Oryza sativa) crops (Brunel et al., 1992). Such estimates were shown to be in good agreement with one-dimensional aerodynamic energy budget calculations, although the technique is restricted to stable conditions when vertical gradients of isotopic composition near the ground are relatively high.

At an ecosystem scale, ET may also be partitioned by making Keeling plots of isotopic composition of water vapor versus water vapor partial pressure over a period of a few hours (Yepez et al., 2003). The intercept at infinite partial pressure is taken as the average composition of ET and then compared with soil water (representing the composition of transpired water) and the composition of evaporated water (using a calculated value based on the Craig-Gordon equation applied to soils; Williams et al., 2004). The assumptions underlying the above applications need to be tested against our understanding of leaf water isotopic composition, although they involve different timescales from those discussed earlier. For example, initially, intercepted water evaporated from a leaf surface after rain will be depleted (Farquhar and Cernusak, 2005), while remaining enriched water will also be evaporated over the following hours, so that, on a daily time scale, this particular form of evaporation can be regarded as not discriminating. Similarly, transpiration from a leaf itself at this time scale can be considered as not discriminating (Harwood et al., 1999).

The isotopic composition of water vapor plays a direct role in nearly all topics discussed above since it is a key component in the calculation of leaf water isotopic enrichment (Lee et al., 2006). It is likely that detailed general circulation models of the water cycle will increasingly involve isotopic considerations, and leaf processes are relevant here also (see IPILPS program at http:// ipilps.ansto.gov.au).

It is now accepted that plants play an important role in the world’s climate, affecting it as well as being affected by it. These plant-atmosphere interactions will be better understood as we probe them using models and measurement of the heavy isotopes of water.

ACKNOWLEDGMENTS

G.D.F. wishes to thank the organizers of the Snowbird Conference on Transpiration, Brent Helliker for providing Figure 1, Michelle Selman for assistance in preparing the manuscript, and the Australian Research Council for support. We thank an anonymous reviewer.

Received November 17, 2006; accepted November 28, 2006; published January 8, 2007.

1 This work was supported by the Australian Research Council (Discovery support to G.D.F.).

LITERATURE CITED

Allison G, Gat J, Leaney F (1985) The relationship between deuterium and oxygen-18 delta values in leaf water. Chem Geol 58: 145-156

Barbour MM, Farquhar GD (2000) Relative humidity- and ABA- induced variation in carbon and oxygen isotope ratios of cotton leaves. Plant Cell Environ 23: 473-485

Barbour MM, Farquhar GD (2004) Do pathways of water movement and leaf anatomical dimensions allow development of gradients in (H2O)O- 18 between veins and the sites of evaporation within leaves? Plant Cell Environ 27: 107-121

Barbour M\M, Fischer RA, Sayre KD, Farquhar GD (2000a) Oxygen isotope ratio of leaf and grain material correlates with stomatal conductance and grain yield in irrigated wheat. Aust J Plant Physiol 27: 625-637

Barbour MM, Roden JS, Farquhar GD, Ehleringer JR (2004) Expressing leaf water and cellulose oxygen isotope ratios as enrichment above source water reveals evidence of a Peclet effect. Oecologia 138: 426-435

Barbour MM, Schurr U, Henry BK, Wong SC, Farquhar GD (2000b) Variation in the oxygen isotope ratio of phloem sap sucrose from castor bean. Evidence in support of the Pclet effect. Plant Physiol 123:671-679

Bariac T, Gonzalezdunia J, Tardieu F, Tessier D, Mariotti A (1994) Spatial variation of the isotopic composition of water (O- 18, H-2) in organs of aerophytic plants. 1. Assessment under laboratory conditions. Chem Geol 115: 307-315

Barnes B, Farquhar G, Gan K (2004) Modelling the isotope enrichment of leaf water. J Math Biol 48: 672-702

Bender M, Labeyrie LD, Raynaud D, Lorius C (1985) Isotopic composition of atmospheric O2 in ice linked with deglaciation and global primary productivity. Nature 318: 349-352

Bender M, Sowers T, Labeyrie L (1994) The Dole effect and its variations during the last 130,000 years as measured in the Vostok ice core. Global Biogeochem Cycles 8: 363-376

Blunier T, Barnett B, Bender M, Hendricks MB (2002) Biological oxygen productivity during the last 60,000 years from triple oxygen isotope measurements. Global Biogeochem Cycles 16: 1029-1043

Brunei J, Simpson H, Herczeg R, Whitehead R, Walker G (1992) Stable isotope composition of water vapour as an indicator of transpiration fluxes from rice crops. Water Resour Res 28: 1407- 1416

Cappa CD, Hendricks MB, DePaolo DJ, Cohen RC (2003) Isotopic fractionation of water during evaporation. J Geophys Res 108: 4525- 4534

Cernusak LA, Arthur DJ, Pate JS, Farquhar GD (2003a) Water relations link carbon and oxygen isotope discrimination to phloem sap sugar concentration in Eucalyptus globuhts. Plant Physiol 131: 1544-1554

Cernusak LA, Farquhar GD, Pate JS (2005) Environmental and physiological controls over oxygen and carbon isotope composition of Tasmanian blue gum, Eucalyptus globulus. Tree Physiol 25: 129-146

Cernusak LA, Farquhar GD, Wong SC, Stuart-Williams H (2004) Measurement and interpretation of the oxygen isotope composition of carbon dioxide respired by leaves in the dark. Plant Physiol 136: 3350-3363

Cernusak LA, Pate JS, Farquhar GD (2002) Diurnal variation in the stable isotope composition of water and dry matter in fruiting Lupinus angustifolius under field conditions. Plant Cell Environ 25: 893-907

Cernusak LA, Wong SC, Farquhar GD (2003b) Oxygen isotope composition of phloem sap in relation to leaf water in Ricinus communis. Funct Plant Biol 30: 1059-1070

Condon AG, Richards RA, Rebetzke GJ, Farquhar GD (2002) Improving intrinsic water-use efficiency and crop yield. Crop Sci 42: 122-131

Cowan I (1982) Regulation of water use in relation to carbon gain in higher plant. In O Lange, P Nobel, C Osmond, H Ziegler, eds, Physiological Plant Ecology II: Water Relations and Carbon Assimilation. SpringerVerlag, Berlin, pp 589-613

Cowan IR, Farquhar GD (1977) Stomatal function in relation to leaf metabolism and environment. Symp Soc Exp Biol 31: 471-505

Craig H, Gordon LI (1965) Deuterium and oxygen-18 variations in the ocean and the marine atmosphere. In E Tongiorgi, ed, Proceedings of a Conference on Stable Isotopes in Oceanographic Studies and Palaeotemperatures. Lischi and Figli, Pisa, Italy, pp 9-130

Cuntz M, Ciais P, Hoffmann G, Knorr W (2003) A comprehensive global three-dimensional model of δ^sup 18^O in atmospheric CO2. 1: Validation of surface processes. J Geophys Res 108: 4527

Dole M, Lane G, Rudd D, Zaukelies DA (1954) Isotopic composition of atmospheric oxygen and nitrogen. Geochim Cosmochim Acta 6: 65-78

Dongmann G, Nornberg HW, Forstel H, Wagener K (1974) On the enrichment of H2^sup 18^O in the leaves of transpiring plants. Radial Environ Biophys 11: 41-52

Farquhar G, Barbour MM, Henry BK (1998) Interpretation of oxygen isotope composition of leaf material. In H Griffiths, ed, Stable Isotopes: Integration of Biological, Ecological and Geochemical Processes. BIOS Scientific Publishers, Oxford, pp 27-62

Farquhar G, Condon A, Masle J (1994) On the use of carbon and oxygen isotope composition and mineral ash content in breeding for improved rice production under favourable, irrigated conditions. In K Cassman, ed, Breaking the Yield Barrier. International Rice Research Institute, Manila, The Philippines, pp 95-101

Farquhar G, Lloyd J (1993) Carbon and oxygen isotope effects in the exchange of carbon dioxide between terrestrial plants and the atmosphere. In Ehleringer JR, Hall AE, Farquhar GD eds, Stable Isotopes and Plant Carbon-Water Relations. Academic Press, San Diego, pp 47-70

Farquhar GD, Cernusak LA (2005) On the isotopic composition of leaf water in the non-steady state. Funct Plant Biol 32: 293-303

Farquhar GD, Gan KS (2003) On the progressive enrichment of the oxygen isotopic composition of water along a leaf (reprinted from Plant, Cell and Environment, Vol 26, pp 801-819, 2003). Plant Cell Environ 26: 1579-1597

Farquhar GD, Hubick KT, Condon AG, Richards RA (1989) Carbon isotope fractionation and plant water-use efficiency. In PW Rundel, JR Ehleringer, KA Nagy, eds, Stable Isotopes in Ecological Research, Springer-Verlag, New York, pp 21-40

Farquhar GD, Lloyd J, Taylor JA, Flanagan LB, Syvertsen JP, Hubick KT, Wong SC, Ehleringer JR (1993) Vegetation effects on the isotope composition of oxygen in atmospheric CO2. Nature 363: 439- 443

Farquhar GD, Richards RA (1984) Isotopic composition of plant carbon correlates with water-use efficiency of wheat genotypes. Aust J Plant Physiol 11: 539-552

Flanagan LB, Comstock JP, Ehleringer JR (1991) Comparison of modeled and observed environmental influences on the stable oxygen and hydrogen isotope composition of leaf water in Phaseolus vulgaris L. Plant Physiol 96: 588-596

Flexas J, Ribas-Carbo M, Hanson DT, Bota J, Otto B, Cifre J, McDowell N, Medrano H, Kaldenhoff R (2006) Tobacco aquaporin NtAQP1 is involved in mesophyll conductance to CO2 in vivo. Plant J 48: 427- 439

Gan K, Wong S, Yong J, Farquhar G, Barbour MM, Henry BK (2003) Evaluation of models of leaf water ^sup 18^O enrichment using measurements of spatial patterns of vein xylem, leaf water and dry matter in maize leaves. Plant Cell Environ 26: 1479-1495

Gat J, Bowser C (1991) The heavy isotope enrichment of water in coupled evaporative systems. In H Taylor, J O’Neil, I Kaplan, eds, Stable Isotope Geochemistry: A Tribute to Samuel Epstein. The Geochemical Society, St. Louis, pp 159-168

Gat JR, Matsui E (1991) Atmospheric water-balance in the Amazon basin: an isotopic evapotranspiration model. J Geophys Res 96: 13179- 13188

Gibson JJ (2001) Forest-tundra water balance signals traced by isotopic enrichment in lakes. J Hydrol 251: 1-13

Gibson JJ, Edwards TWD, Bursey GG, Prowse TD (1993) Estimating evaporation using stable isotopes: quantitative results and sensitivity analysis for two catchments in northern Canada. Nordic Hydrol 24: 79-94

Gibson JJ, Prowse TD, Edwards TWD (1996) Evaporation from a small lake in the continental Arctic using multiple methods. Nordic Hydrol 27: 1-24

Guy R, Fogel ML, Berry J (1993) Photosynthetic fractionation of the stable isotopes of oxygen and carbon. Plant Physiol 101: 37-47

Harwood K, Gillon J, Roberts A, Griffiths H (1999) Determinants of isotopic coupling of CO2 and water vapour within a Qiiercus pelraeit forest canopy. Oecologia 119:109-119

Harwood KG, Gillon JS, Griffiths H, Broadmeadow MSJ (1998) Diurnal variation of Δ^sup 13^(CO2), ΔC^sup 18^O^sup 16^O and evaporative site enrichment of δH^sub 2^^sup 18^O in Piper aduncum under field conditions in Trinidad. Plant Cell Environ 21: 269-283

Helliker BR, Ehleringer J (2000) Establishing a grassland signature in veins: ^sup 18^O in the leaf water of C^sub 3^ and C^sub 4^ grasses. Proc Natl Acad Sci USA 97: 7894-7898

Helliker BR, Ehleringer JR (2002a) Differential O^sup 18^ enrichment of leaf cellulose in C^sub 3^ versus C^sub 4^ grasses. Funct Plant Biol 29: 435-442

Helliker BR, Ehleringer JR (2002b) Grass blades as tree rings: environmentally induced changes in the oxygen isotope ratio of cellulose along the length of grass blades. New Phytol 155: 417-424

Helliker BR, Griffiths H (2007) Towards a plant-based proxy for the isotope ratio of atmospheric water vapor. Glob Change Biol (in press)

Helman Y, Barkan E, Eisenstadt D, Luz B, Kaplan A (2005) Fractionation of the three stable oxygen isotopes by oxygen- producing and oxygen-consuming reactions in photosynthetic organisms. Plant Physiol 138: 2292-2298

Hoag K, Still C, Fung I, Boering KA (2005) Triple oxygen isotope composition of tropospheric carbon dioxide as a tracer of terrestrial gross carbon fluxes. Geophys Res Lett 32: 2802

Lee DH, Veizer J (2003) Water and carbon cycles in the Mississippi River basin: potential implications for the Northern Hemisphere residual terrestrial sink. Global Biogeochem Cycles 17:1037

Lee X, Smith R, Williams J (2006) Water vapour O^sup 18^/O^sup 16^ isotope ratio in surface air in New England, USA. Tellus B Chem Phys Meteorol 58: 293-304

Luz B, Barkan E (2000) Assessment of oceanic productivity with the triple-isotope composition of dissolved oxygen. Science 288: 2028-2031

Luz B, Barkan E, Bender ML, Thiemens MH, Boering KA (1999) Triple- isotope composition of atmospheric oxygen as a tracer of biosphere productivity. Nature 400: 547-550

Merlivat L (1978) Molecular diffusivities of H^sub 2^sup 16^O, HD^sup 16^O, and H^sub 2^^sup 18^O in gases. J Chem Phys 69: 2864- 2871

Miller DLMC, Grissino-Mayer HD, Mock CJ, UhIe ME, Sharp Z (2006) Tree-ring isotope records of tropical cyclone activity. Proc Natl Acad Sci USA 103: 14294-14297

Moser H, Stichler W (1975) Deuterium and oxygen-18 contents as an inde\x of the properties of snow covers. In Snow Mechanics: Proceedings of the Grindelwald Symposium. Publication No. 114. IASH Publishing, WaIlingford, UK, pp 122-135

Oge J, Cuntz M, Peylin P, Bariac T (2007) Non-steady-state, non- uniform transpiration rate and leaf anatomy effects on the progressive stable isotope enrichment of leaf water along monocot leaves. Plant Cell Environ (in press)

Peak D, West JD, Messinger SM, Mott KA (2004) Evidence for complex, collective dynamics and emergent, distributed computation in plants. Proc Natl Acad Sci USA 101: 918-922

Rebetzke GJ, Condon AG, Richards RA, FarquharGD (2002) Selection for reduced carbon isotope discrimination increases aerial biomass and grain yield of rainfed bread wheat. Crop Sci 42: 739-745

Roden JS, Ehleringer JR (1999a) Hydrogen and oxygen isotope ratios of tree-ring cellulose for riparian trees grown long-term under hydroponically controlled environments. Oecologia 121: 467- 477

Roden JS, Ehleringer JR (1999b) Observations of hydrogen and oxygen isotopes in leaf water confirm the Craig-Gordon model under wideranging environmental conditions. Plant Physiol 120:1165-1173

Roden JS, Ehleringer JR (2000) Hydrogen and oxygen isotope ratios of tree ring cellulose for field-grown riparian trees. Oecologia 123: 481-489

Saurer M, Aellen K, Siegwolf R (1997) Correlating δC^sup 13^ and δO^sup 18^ in cellulose of trees. Plant Cell Environ 20: 1543-1550

Saurer M, Siegwolf R, Scheidegger Y (2001) Canopy gradients in δ^sup 18^O of organic matter as ecophysiological tool. Isotopes Environ Health Stud 37: 13-24

Schmidt HL, Werner RA, Rossmann A (2001) O-18 pattern and biosynthesis of natural plant products. Phytochemistry 58: 9-32

Shantz HL, Piemeisel LN (1927) The water requirement of plants at Akron, CoIo. J Agric Res 34: 1093-1190

Sheshshayee MS, Bindumadhava H, Ramesh R, Prasad TG, Lakshminarayana MR, Udayakumar M (2005) Oxygen isotope enrichment (Delta O-18) as a measure of time-averaged transpiration rate. J Exp Bot 56: 3033-3039

Sternberg LdS, DeNiro MJ (1983) Biogeochemical implications of the isotopic equilibrium fractionation factor between the oxygen atoms of acetone and water. Geochim Cosmochim Acta 47: 2271-2274

Thiemens M, Jackson T, Brenninkmeijer C (1995) Observation of a mass-independent oxygen isotopic composition in terrestrial stratospheric CO2, the link to ozone chemistry, and the possible occurrence in the martian atmosphere. Geophys Res Lett 22: 255-257

Williams D, Cable W, Hultine K, Hoedjes J, Yepez E, Simonneaux V, Er-Raki S, Boulet G, de Bruin H, Chehbouni A, et al (2004) Evapotranspiration components determined by stable isotope, sap flow and eddy covariance techniques. Agric For Meteorol 125: 241-258

Yakir D (1992) Water compartmentation in plant tissue: isotopic evidence. In GN Somero, CB Osmond, L Bolis, eds, Water and Life. SpringerVerlag, Berlin, pp 205-222

Yakir D (1998) Oxygen-18 of leaf water: a crossroad for plant associated isotopic signals. In H Griffiths, ed, Stable Isotopes: Integration of Biological, Ecological, and Geochemical Processes. BIOS Scientific Publishers, Oxford, pp 147-188

Yakir D, Berry JA, Giles L, Osmond CB (1994) Isotopic heterogeneity of water in transpiring leaves: identification of the component that controls the δ^sup 18^O of atmospheric O2 and CO2. Plant Cell Environ 17: 73-80

Yakir D, DeNiro MJ, Rundel PW (1989) Isotopic inhomogeneity of leaf water: evidence and implications for the use of isotopic signals transduced by plants. Geochim Cosmochim Acta 53: 2769-2773

Yakir D, Israeli Y (1995) Reduced solar irradiance effects on net primary productivity (NPP) and the δ^sup 13^C and δ^sup 18^O values in plantations of Mtisa sp. Musaceae. Geochim Cosmochim Acta 59: 2149-2151

Yepez E, Williams D, Scott R, Lin G (2003) Partitioning overstory and understory evapotranspiration in a semi-arid savanna ecosystem from the isotopic composition of water vapor. Agric For Meteorol 119: 53-68

Graham D. Farquhar*, Lucas A. Cernusak2, and Belinda Barnes3

Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 2600, Australia

2 Present address: Smithsonian Tropical Research Institute, P.O. Box 0843-03092, Balboa, Ancon, Republic of Panama.

3 Present address: National Centre for Epidemiology and Popu- lation Health, Australian National University, Canberra, Australian Capital Territory 2600, Australia.

* Corresponding author; e-mail graham.farquhar@anu.edu.au; fax 61- 2-6125-4919.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Graham D. Farquhar (graham.farquhar@anu.edu.au).

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