Heavy Water Fractionation during Transpiration

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 18O measurements carried a record of hurricane activity. The motivation for our laboratory to study water isotopes was to enhance studies of transpiration efficiency (TE; the leaf contribution to water-use efficiency at the plant, crop, or ecosystem level). The instantaneous TE is A/[g w ν], where A is the rate of photosynthetic uptake of CO2, g 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 s, and boundary layer conductance, g b, in series), and ν is the leaf-to-air water vapor concentration difference. Carbon isotope composition of leaves can be measured to determine the carbon isotopic discrimination, Δ13C, during photosynthesis. In turn, Δ13C relates to A/g t, the ratio of CO2 assimilation rate to the total conductance to diffusion of CO2 from the atmosphere to the sites of carboxylation. The two measures are obviously related, and it was suggested that the oxygen isotope composition (δ 18O) of leaf material could be used to pick up differences in ν (Farquhar et al., 1994). Further, because g w affects δ 18O and A does not, it was hoped that measurements of Δ13C and δ 18O in organic matter could be combined to separate photosynthetic capacity effects on TE from g w effects (Yakir and Israeli, 1995; Saurer et al., 1997; Farquhar et al., 1998). The heavy isotopes of water, expressed in organic matter, have promise as a means of identifying genetic variation in g w. In an ecological and population biology context, the carbon, hydrogen, and oxygen isotope measurements could provide a practical surrogate 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. On earth, roughly 0.204% of oxygen is 18O and 0.037% is 17O; 0.015% of hydrogen is 2H. 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, 1H2 16O, 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 continues until the leaf water becomes sufficiently enriched that the exit of heavy and light molecules through the stomata matches that of the supply of water from the xylem. We note that, in steady state, R E must be close to the isotope ratio of source (soil) water, Rs. Thus, from Equations 5 and 6, the maximum isotope effect is the sum of the equilibrium and kinetic fractionation terms and is realized when atmospheric humidity is very low. The isotope effect is generally small when atmospheric humidity approaches 100%. 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 (Δ18O) 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 w or variation in evaporative demand, ν. Does this make sense? Enrichment of leaf water versus relative humidity in five C3 grass species. All grasses were grown in chambers where the isotope ratio of source water was held constant, leaf boundary layer development 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 a/w 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 Δe. There is a third effect that reinforces these. The enrichment of water in the leaf, ΔL, will usually be less than that at the sites of evaporation, Δe, 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 Péclet 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, ΔL, in a simple system would be given by ([1 − exp(−P)]/P) Δe. The Péclet number P is proportional to E, and so, as E increases, average leaf enrichment, ΔL, becomes increasingly depleted compared with Δe. It represents another reason for ΔL to decrease as g w increases. The Péclet 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 Δe. What is the experimental support for these mathematical ideas about a negative relationship between E and ΔL, when the source of variation is g w? At present, the data on direct effects of stomatal conductance on ΔL are fairly thin, and particularly so for the so-called Péclet 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 Péclet 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 Péclet effect. Cernusak et al. (2002, 2005) included the effect in simulating their field observations of ΔL, 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 ΔL less than Δe, and the dependence on changes in g w tended to be lost in the variation caused by changes in Δe. There are few direct measurements in the laboratory of the effect of changes in g w on ΔL. The main difficulty of such measurements is their intrinsically destructive nature, meaning one leaf per datum, together with what appears to be variable Péclet 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). Although the direct evidence that an increase in g 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‰ compared to the water. In the formation of Suc, the water undergoing exchange will be both that in the chloroplasts, probably close to Δe (Farquhar et al., 1993), and that in the cytosol, which is presumably less enriched. Barbour et al. (2000b) examined the oxygen isotopic composition of Suc 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‰, and, by subtracting that value from the enrichment of the Suc, they were able to obtain the composition of the effective substrate water. The latter was less enriched than Δe, and the difference increased with increasing E as would be expected from theory. Cernusak et al. (2003b) compared destructive measurements of ΔL with the enrichment of phloem Suc and also obtained a fractionation of 27‰. 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‰. 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 Suc. 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. The relationships of cotton leaf organic matter oxygen isotopic enrichment with stomatal conductance, g s (A); leaf temperature T l (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, 0, 10−5, 10−4, and 10−3 m), sprayed on the developing leaves. Data and figure are redrawn from Barbour and Farquhar (2000). The relevance of the Péclet effect to organic δ 18O 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 18O 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, ΔL, when the source of variation is evaporative demand, or decreasing ΔL, when the source of variation is g w. 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 well-watered 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 w all increased with year of release—the breeders had done their job well—and the canopy temperature and δ 18O 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 w in the field is in any case a dynamic variable, δ 18O 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. 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 w w i; Dongmann et al., 1974) and not to the net transpiration rate [g w(w i − w 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 w w a) than through the petiole [g w(w i − w a)]. Farquhar and Cernusak (2005) extended the model of Dongmann et al. (1974) to include the Péclet effect. They also included changes in leaf water content as these are thought to be important sometimes (Yakir, 1998). Cernusak et al. (2002, 2005) showed that inclusion of the Péclet effect was necessary for simulating the experimental results during the day, but less so at night when E was low. They found that the non-steady-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). 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. The detailed theory requires several elements. One needs to consider both mesophyll and veins (Allison et al., 1985), as well as the Péclet effect discussed already. The latter is essentially a radial effect, from xylem to stomata. But there is a longitudinal Péclet 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; Ogée et al., 2007). Recently Ogée 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 ΔM. The modeling of this aspect is sensitive to the nature of leaf tapering. 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). Helliker and (2007) and the changes in δ 18O of a that after falls as the They the of the one-way between the and the atmosphere and how material of this species may be used as a for δ 18O of water There are effects on atmospheric isotopic composition CO2 oxygen isotopic exchange with leaf water and soil water, and changes in the δ 18O of CO2 can be used to study spatial and temporal variation in the net exchange of CO2 in and its underlying by photosynthesis and (Farquhar et al., et al., It had been thought that CO2 from leaves would be in isotopic equilibrium with leaf water, a fractionation of about (Yakir et al., 1994). However, as with water vapor into and out of the leaf, it is the one-way that must be and it is that of CO2 from the by exchange with leaf water, can the isotopic of CO2 (Cernusak et al., what part of the leaf water and to what is not is released by the during photosynthesis. The isotopic composition of oxygen is an experimental of to that of the substrate water in the et al., 1993), and so an is required as to where the in the of isotopic enrichment from soil water to the sites of evaporation within leaves. in the oxygen isotope of atmospheric are on time in the effect et al., where atmospheric oxygen is enriched in 18O by compared with water. changes in the effect are in the study of in the of and et al., et al., 1994). well as the effect depends on fractionation during and on The processes to enrichment of water isotopes in leaves isotopes and the oxygen isotopes of CO2 in a as does the enrichment in is about of the enrichment of 18O In among and CO2 in the oxygen isotopes in a and with about of δ and δ 18O in atmospheric and, about of δ and δ 18O in atmospheric CO2 et al., 1995; et al., and et al., 2005). This a in that depends on the relative rates of photosynthetic production and If the latter rates are the rate of photosynthetic can be from the of the in et al., 1994). This was by et al. and et al. air in to the rate during the It of on or of the The most recent contribution to in this field fractionation of the three oxygen isotopes by et al., 2005). transpiration water vapor to the with an oxygen isotope composition to that of soil water. the evaporation of by leaves no fractionation when all the water has been However, soil evaporation and involve hydrogen and oxygen isotope fractionation of water molecules and and water at the of with a evaporative is enriched in the heavy compared with In it is important to be able to transpiration from soil The evaporation in the flux can be isotope mass such as those proposed by and and and et al. this was done for the by and who used their results to net primary for the from the data of and for C3 and studies with measurements of vapor pressure and carbon isotope composition (Farquhar and would have been of great in their of the isotopic composition of water vapor have been used to transpiration in et al., were to be in with the technique is to when of isotopic composition the are At an ecosystem may also be by making of isotopic composition of water vapor versus water vapor pressure a of a few et al., The at pressure is taken as the average composition of and then compared with soil water the composition of and the composition of water a calculated value based on the Craig-Gordon to et al., 2004). The underlying the applications to be our of leaf water isotopic composition, they involve different from those discussed water from a leaf after will be depleted (Farquhar and enriched water will also be the so that, on a time this of evaporation can be as not transpiration from a leaf at this time can be considered as not et al., The isotopic composition of water vapor a direct role in all discussed since it is a in the of leaf water isotopic enrichment et al., It is likely that detailed general models of the water will increasingly involve isotopic and leaf processes are here also at It is that plants play an important role in the it as well as being by will be better understood as we models and of the heavy isotopes of water. to the of the on Helliker for for in the and the for We an