O Spatial Patterns of Vein Xylem Water, Leaf - Plant Physiology

18O Spatial Patterns of Vein Xylem Water, Leaf Water, andDry Matter in Cotton Leaves

Kim Suan Gan, Suan Chin Wong, Jean Wan Hong Yong1, and Graham Douglas Farquhar*

Environmental Biology Group, Research School of Biological Sciences, Australian National University,Canberra, Australian Capital Territory 2601, Australia

Three leaf water models (two-pool model, Peclet effect, and string-of-lakes) were assessed for their robustness in predictingleaf water enrichment and its spatial heterogeneity. This was achieved by studying the 18O spatial patterns of vein xylemwater, leaf water, and dry matter in cotton (Gossypium hirsutum) leaves grown at different humidities using new experi-mental approaches. Vein xylem water was collected from intact transpiring cotton leaves by pressurizing the roots in apressure chamber, whereas the isotopic content of leaf water was determined without extracting it from fresh leaves withthe aid of a purpose-designed leaf punch. Our results indicate that veins have a significant degree of lateral exchange withhighly enriched leaf water. Vein xylem water is thus slightly, but progressively enriched in the direction of water flow. Leafwater enrichment is dependent on the relative distances from major veins, with water from the marginal and intercostalregions more enriched and that next to veins and near the leaf base more depleted than the Craig-Gordon modeledenrichment of water at the sites of evaporation. The spatial pattern of leaf water enrichment varies with humidity, asexpected from the string-of-lakes model. This pattern is also reflected in leaf dry matter. All three models are realistic, butnone could fully account for all of the facets of leaf water enrichment. Our findings acknowledge the presence of capacitancein the ground tissues of vein ribs and highlight the essential need to incorporate Peclet effects into the string-of-lakes modelwhen applying it to leaves.

The isotopic composition of leaf water reflects localhumidity, and its imprints on plant cellulose andother fossil materials have been widely explored forpalaeoclimatic reconstruction. To date, isotopic val-ues of wood cellulose (Epstein et al., 1977; Yapp andEpstein, 1982; Edwards et al., 1985; Edwards andFritz, 1986; Roden et al., 2000), grassland phytoliths(Webb and Longstaffe, 2000), and deer bone (Cormieet al., 1994) have been shown to be related to leafwater isotopic composition. Leaf water isotopic sig-nature is not only imprinted on plant organic matterbut is also recorded in atmospheric CO2 and O2. TheCO2 interacts and undergoes isotopic exchange withleaf water, and O2 is released by the plant duringphotosynthesis. Changes in the oxygen isotope ratiosof CO2 and O2 can thus be used to study variations inthe net exchange of CO2 in terrestrial ecosystems(Farquhar et al., 1993) and in the balance of terrestrialand marine productivity (Bender et al., 1985; Benderet al., 1994). Because all of these applications criticallydepend on estimation of the leaf water oxygen isoto-pic ratio, a good understanding of the nature of leafwater enrichment is needed.

The isotopic composition of leaf water is most com-monly estimated from the model of a freely evapo-

rating water surface (Craig and Gordon, 1965) whereisotopic fractionation is driven by the lower vaporpressure and diffusivity of the heavier molecules.Although the Craig-Gordon model basically de-scribes water enrichment at the sites of evaporationcompared with locally transpired water, it cannotadequately account for other aspects of leaf waterenrichment, particularly the spatial variation of leafwater 18O and/or D contents (Luo and Sternberg,1992; Bariac et al., 1994; Wang and Yakir, 1995; Hel-liker and Ehleringer, 2000). Also, the Craig-Gordonmodel has often been found to overestimate the iso-topic enrichment of bulk leaf water (Allison et al.,1985; Bariac et al., 1989; Walker et al., 1989; Walkerand Brunel, 1990; Yakir et al., 1990; Flanagan et al.,1991a, 1991b, 1994; Wang et al., 1998). To explainsuch observations, several other models have beensuggested in conjunction with the Craig-Gordonmodel, namely the two-pool model (Leaney et al.,1985), the Peclet model (Farquhar and Lloyd, 1993),and the string-of-lakes model (Gat and Bowser,1991). The objective of this paper is to examine andassess the applicability of these various leaf watermodels by studying the 18O spatial patterns of veinxylem water, leaf mesophyll water, and dry matter incotton (Gossypium hirsutum) leaves at different hu-midity treatments. To accomplish this, new experi-mental approaches are employed in the direct collec-tion of vein xylem water from an intact transpiringleaf using a root pressure chamber and in the isotopicmeasurement of leaf water without extracting it fromthe leaves with the aid of a purpose-designed leaf

1 Present address: Natural Sciences Academic Group, NationalInstitute of Education, Nanyang Technological University,Singapore.

* Corresponding author; e-mail [email protected]; fax61–2– 6125– 4919.

Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.007419.

1008 Plant Physiology, October 2002, Vol. 130, pp. 1008–1021, www.plantphysiol.org © 2002 American Society of Plant Biologists www.plantphysiol.orgon December 31, 2018 - Published by Downloaded from Copyright © 2002 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

punch. Bleeding xylem water directly from the peti-ole and veins of intact plants should give a goodrepresentation of the transpiration stream enteringthe leaf mesophyll cells. This technique allows us tomap the isotopic gradient along the main water flowin leaf veins.

Applying the Craig-Gordon model to leaves(Dongmann et al., 1974; Farquhar et al., 1989), theisotopic enrichment of leaf water above source water(�lw) at the evaporative sites of intercellular airspaces would be equal to the Craig-Gordon predic-tion (�C) where

�C � �k � �* � (�v��k)ea

ei(1)

where �v is the isotopic composition of atmosphericwater vapor relative to source water. The water va-por pressures in the atmosphere and intercellularspaces are ea and ei, respectively, �* is the equilibriumfractionation factor arising from the lower vaporpressure of H2

18O molecules at liquid-vapor phaseequilibrium, and �k is the kinetic fractionation factorcaused by the lower diffusivity of heavy H2

18O mol-ecules. Equation 1 assumes that isotopic steady statehas been achieved, where the isotopic composition oftranspired water (�E) equals the value of source water(�S). In calculating the kinetic fractionation factor, thedifferent layers of boundary resistance developingfrom the leaf evaporative sites to the fully turbulentatmospheric air need to be considered. The boundarylayer is fully stagnant in the leaf substomatal airspaces (18O kinetic fractionation, 28.5‰), whereas alaminar flow is expected near the leaf surface (18Okinetic fractionation, 18.9‰). Taking into account theweighted effects of these different boundary layers,Farquhar et al. (1989) expressed the overall kineticfractionation as

�k(‰) �28.5rs � 18.9rb

rs � rb(2)

where rs and rb are the leaf stomatal and boundarylayer resistances to water vapor diffusion, respec-tively. An alternative method to calculate �k is de-scribed by Buhay et al. (1996), taking leaf size andmorphology into consideration.

To explain the observation that bulk leaf water isless enriched than that predicted by the Craig-Gordon model, Leaney et al. (1985) described leafwater as consisting of two pools: evaporatively en-riched leaf tissue water and isotopically unalteredvascular water (Fig. 1A). On the basis of this defini-tion, the isotopic composition of bulk leaf water (�lw,bulk) could simply be expressed as

�lw,bulk � �C � f � �s � (1�f ) (3)

where f is the fraction of leaf water subject to frac-tionation and �C is the isotopic composition pre-dicted from the Craig-Gordon model (Eq. 1) with�C � � C � �s. To reconcile the differences betweenthe observed and Craig-Gordon predicted isotopicratios, the fraction of vein water would have to be inthe range of 25% to 50% total leaf water (Leaney etal., 1985; Walker et al., 1989). After the removal of themid-vein from leaves of Betula occidentalis and Popu-lus angustifolia, the estimated unenriched water frac-tion in the remaining leaves was reduced to 10%(Roden and Ehleringer, 1999).

Farquhar and Lloyd (1993) modified the physicalmodel of a single-leaf water pool to one having acontinuum of isotopic composition (Fig. 1B). Theyproposed that advection of unenriched water via thetranspiration stream opposes the back-diffusion ofenriched water from evaporation sites (Peclet effect).A continuous isotopic gradient is thereby createdwithin the leaf. The enrichment at the leaf evapora-tive sites (�e) can be described by Equation 1 and isproposed to decay exponentially to the isotopic sig-nature of source water (�s) at the veins. An integra-

Figure 1. Box-diagram representation of the leafwater models. A, Two-pool model; B, Pecletmodel; C, string-of-lakes model; and D, Peclet-continuous evaporative enrichment model in-corporating ground tissue capacitance. In thelast model, the left compartment represents veinribs and is further divided into two components:the capacitance arising from rib ground tissueand vein xylem water. The right compartmentrepresents leaf lamina tissue water. The intensityof shading indicates the extent of leaf waterenrichment. Double-headed arrows represent aPeclet effect, where the advection of unenrichedwater from the transpiration stream opposes theback-diffusion of enriched water; a bigger ar-rowhead implies a larger contribution in thatdirection.

18O Spatial Patterns of Leaf Constituents

Plant Physiol. Vol. 130, 2002 1009 www.plantphysiol.orgon December 31, 2018 - Published by Downloaded from Copyright © 2002 American Society of Plant Biologists. All rights reserved.


tion of this isotopic variation would give a lowerisotopic enrichment of bulk leaf water above sourcewater (�lw, bulk),

�lw,bulk � �C

1 � e��

�(4)

where leaf Peclet number, � � EL/CD. In the latterterm, E is transpiration rate, L is the scaled effectivepath length of water between the xylem and sites ofevaporation in leaves, C is the molar concentration ofwater (55.5 � 103 mol m�3), and D is the diffusivityof H2

18O in water (2.66 � 10�9 m2 s�1). Equation 4also predicts an increasing disparity between mea-sured �lw, bulk and the Craig-Gordon predicted �Cwith increasing transpiration rate. The Peclet modelnot only accounts for the lower than expected enrich-ment of bulk leaf water but also predicts, in principle,the nonuniform isotopic distribution of leaf water atthe small scale between stomata and xylem. How-ever, the Peclet model based on Equation 4 is unableto describe variation of isotopic enrichment on alarger scale.

An explanation for the larger scale spatial variationof leaf water was provided by Yakir (1992), whosuggested that the isotopic gradient within a leafcould be a consequence of enrichment along the pathof evaporation (Fig. 1C). This is analogous to a stringof evaporating lakes along a river. In this string-of-lakes model, the outflow from one evaporating ele-ment enters into the next element in the series, lead-ing to a progressive enrichment of heavy isotopesalong the path of water flow. A formulation of thismodel is given by Gat and Bowser (1991):

�n � �n�1 �

�v��n�1 ��

h

1�F�

E(1�h)

h

(5)

where �v is the isotopic composition of atmosphericwater vapor, �n is that of liquid water entering thenth evaporating element, F� and E represent theinflux and evaporative efflux of the nth element, h isthe relative humidity (RH) and, � � �* � (1 � h)�k.

Direct and indirect leaf water measurements sup-porting the different leaf water models have beenreported: Craig-Gordon model (Roden andEhleringer, 1999), Peclet model (Walker et al., 1989;Flanagan et al., 1991b; Barbour et al., 2000), andstring-of-lakes model (Yakir, 1992; Wang and Yakir,1995; Helliker and Ehleringer, 2000, 2002). Given theimportance of leaf water modeling to studies ofplant-environment interactions, there is a pressingneed to reconcile these apparently disparate results.To assess best the applicability of the various leafwater models, we examined the 18O content of wateralong its pathway in the leaf, from the petiole to thevein network and then to the lamina tissue. The firststep in our study was to clarify the extent of enrich-

ment in vein xylem water, which is presumed to beunfractionated and to have the same isotopic compo-sition as soil water (Leaney et al., 1985). Next, for abetter representation of the lamina tissue water, weremoved uncertainties such as the inclusion of veinwater in leaf water measurements. This was achievedusing a purpose-designed leaf punch that cuts andseals a small leaf disc sample for direct pyrolysisduring isotopic measurements. The 18O content of theleaf dry matter, which provides an integrated recordof leaf water isotopic composition, was analyzed as acheck on the results from leaf water measurements.Last, distribution of stomatal and venation densitiesacross the leaf were studied to assess the possibleanatomical basis for spatial heterogeneity of leafwater.

RESULTS

�18O Patterns of Vein Xylem Water

Water expressed from the petiole (�petiole ��6.6‰ � 0.1‰ [� se], n � 28) was not significantlymore enriched than the tap water (�s � �6.8‰ �0.1‰, n � 28) used to water the plants. At lowhumidity (vapor-pressure deficit [VPD] of air, 2.4kPa), the oxygen isotopic composition of petiolewater (�petiole � �6.5‰ � 0.1‰, n � 13) was similarto its high-humidity (VPD of air, 1.0 kPa) counter-part (�petiole � �6.7‰ � 0.1‰, n � 15). In contrastto the common assumption that the water pool in leafveins was unaltered with respect to the source water,we noted an increasing 18O enrichment as xylemwater moves along a vein (Fig. 2). At low humidity,enrichment of xylem water above petiole water (�vw)around the mid-point of the primary veins was usu-ally lower (0.07‰–0.4‰) than that at high humidity(0.5‰–0.9‰; Fig. 2A). However, enrichment near thedistal tip of the primary veins was similar (1.6‰–1.7‰) at both levels of humidity. Compared withprimary veins, secondary veins (Fig. 2B) showed ahigher degree of enrichment ranging from 0.7‰ to3.6‰. As before, upstream xylem water showed lessenrichment (0.7‰–1.6‰) than downstream waternear the vein endings (1.5‰–3.6‰). It appears thatxylem water near the vein endings tends to be moreenriched at low humidity than at high humidity,whereas no consistent humidity-driven difference isobserved for the upstream xylem water of secondaryveins.

To compare the two humidity treatments, we werecareful in our choice of incision locations, ensuringthat the relative positions of various sap collectionpoints were retained. However, there is natural vari-ation of venation from leaf to leaf. We also noted thatthe isotopic signature of xylem water near the veinendings where veins begin to taper was highly vari-able (deduced from the larger se; data not shown).Rather than comparing results from two leaves ofdifferent treatments, as presented in Figure 2, com-

Gan et al.

1010 Plant Physiol. Vol. 130, 2002 www.plantphysiol.orgon December 31, 2018 - Published by Downloaded from Copyright © 2002 American Society of Plant Biologists. All rights reserved.


parison is made from the same leaf with the same sapcollection points by changing the ambient humidityduring the experiment. On the basis of leaf watervolume and transpiration rate, we estimated the leafwater turnover time to be approximately 9 min at lowhumidity and 16 min at high humidity. Hence, 1 hafter a humidity step change was assumed to besufficient in achieving a new isotopic steady state. Ineither a step change from low to high humidity orvice versa (Table I), xylem water was mostly moreenriched at low humidity, with several incisionpoints showing a humidity-driven change in enrich-ment as high as 2.7‰. When �vw is normalizedagainst �C (the enrichment expected for leaf wateraccording to Eq. 1), the difference between humiditytreatments is not as distinct, even though the low-humidity leaves generally show higher �vw/�C val-ues. The results also demonstrate that on approach-ing the vein endings, the xylem water was enrichedby 0.13 to 0.17 �C. The effect of humidity change ongas exchange parameters is summarized in Table I. Ingeneral, increasing humidity led to an increase instomatal conductance and was accompanied by adecrease in transpiration rate and modest changes inassimilation rates.

�18O Patterns of Leaf Water and Organic Matter

A typical �18O pattern of leaf mesophyll water fora cotton leaf is shown in Figure 3. Leaf water clearlyis not isotopically uniform spatially and varies by asmuch as 10‰ at low humidity. Closer examinationshows a trend to more enriched waters at the leafedge and intercostal regions, whereas discs at the leafbase and adjacent to major veins are often less en-

riched. This enrichment pattern is seen to be moredistinct when �lw (the observed enrichment of leafwater over petiole water) is plotted against �C for the6 d of sampling (Fig. 4). The isotopic compositions ofleaf water from marginal and intercostal regionsmostly fall above the 1:1 line, indicating enrichmentabove that predicted by the Craig-Gordon model.Those adjacent to veins and at the leaf base are gen-erally more depleted than expected from the model.

At high humidity, there is less spatial heterogene-ity of leaf water, where for certain days, enrichmentat the leaf edge could be small or the venous regioncould be equally or more enriched than that pre-dicted (Fig. 5A). The relatively more homogeneousisotopic pattern at high humidity is also apparent inthe leaf dry matter (Fig. 5, B and C) where there is nosignificant difference in �om (isotopic enrichment ofleaf dry matter over source water) from one leaf zoneto another. Leaves grown in low humidity generallyhave significantly higher �om in the margin and in-tercostal regions and lower values for the venous andbasal regions. This reflects the measured isotopicpattern of leaf water. Figure 6 shows that the adaxialstomatal densities of the four leaf zones were notsignificantly different from one another. However,the abaxial stomatal density of the intercostal regionwas slightly higher than that of the other three zones.

DISCUSSION18O Spatial Variation of Vein Xylem and Leaf Water

Water expressed from the petiole of an intact cottonplant was not significantly more enriched than thetap water used to water the plants. This is in agree-

Figure 2. Spatial distribution of vein xylem water isotopic composition (�vw, ‰) in cotton leaves at low (top line) and high(bottom line) humidities. Given alongside in brackets is �vw/�C expressed as a percentage, indicating the proportioncontributed by back-diffusion of enriched water from leaf evaporative sites. �C refers to the enrichment of leaf water abovesource water (expressed from the petiole) and is calculated according to the Craig-Gordon model (Eq. 1). Mean �petiole were�6.5‰ and �6.7‰ at low and high humidities, respectively. Values of �v and ei are taken from the experiments on leafwater sampling. The calculated �C are 19.0‰ (low humidity) and 14.0‰ (high humidity). Data of �vw represent the averageof three samples on different days. The mean vapor pressure deficits of air at low (RH 44%) and high (RH 75%) humiditieswere 2.4 and 1.0 kPa, respectively. The letters A through F indicate vein incision locations for data in Table I.




ment with previous observations (White et al., 1985;Bariac et al., 1989, 1994) that there is little fraction-ation associated with uptake of soil water by roots, orits subsequent movement up the stem. An exceptionwould be the fractionation of hydrogen isotopes inmangroves (Lin and Sternberg, 1993). The absence ofisotopic fractionation during water uptake by mostplants has previously been used to support the as-sumption that water in leaf veins would similarly beunenriched relative to the soil water (Leaney et al.,1985). Previous sampling of vein water by pressuriz-ing a detached leaf in a pressure chamber indicatedgood agreement between the isotopic composition ofirrigation water and that assigned to vein water (Ya-kir et al., 1989). In contrast, based on vacuum distil-lation of excised vein segments, �2H of vein waterhad been shown to increase along the main vein fromthe leaf base to the tip (Luo and Sternberg, 1992). Thedata of Figure 2 give direct evidence of xylem waterenrichment in the leaf veins of an intact transpiringleaf. One possible contribution to the enrichment ofvein water is the back-diffusion of enriched waterfrom evaporative sites on the leaf lamina. The reportof the lateral escape of tritiated water from the xylemvessels of tomato (Lycopersicon esculentum) internodesbeing driven by diffusion (van Bel, 1976) lends sup-

port to this hypothesis. The tritiated water moveddown a concentration gradient and was mostly ab-sorbed in the cells and cell walls around the xylemvessels. The idea of progressive enrichment of veinwater by tissue water was first mooted by Yakir(1992) and elaborated by Wang and Yakir (1995) andHelliker and Ehleringer (2000) to explain the spatialheterogeneity of leaf water. This phenomenon isstrongly supported by our observations that the iso-topic composition of xylem water in veins (�vw) isresponsive to changes in environmental conditionssuch as humidity (Table I). Water at the evaporativesites is more enriched at lower humidity and theback-diffusion of this highly enriched water will con-sequently increase the extent of vein water enrich-ment at low humidity. �vw could be expressed math-ematically as

�vw � �s � (1�d) � �C � d (6)

where d is the proportional contribution by back-diffusion of enriched water from leaf lamina. Interms of enrichment over source water, Equation 6can be re-expressed as

�vw

�C�

�vw��s

�C��s� d (7)

Table I. �vw (‰) and �vw/�C of vein xylem water collected from various incision points (refer to Fig. 2 for location) before and after a stepchange of humidity (H) for two different cotton leaves (first line for leaf 3, second line for leaf 4) of the same plant

Collection of xylem water was made from the same incision points 1 h after the step change in humidity. The vapor pressure deficits of airat low (RH 46%) and high (RH 80%) humidity were 2.3 and 0.8 kPa, respectively. �C refers to the enrichment of leaf water above source water(expressed from the petiole, �s � �6.8‰) that is calculated according to the Craig-Gordon model (Eq. 1). Values of �v and ei were taken fromthe experiments on leaf water sampling. For low3 high humidity experiment, �C � 18.7‰ (low humidity) and 13.8‰ (high humidity); high3low humidity experiment, �C � 19.3‰ (low humidity) and 14.1‰ (high humidity). Different plants were used for each humidity step changeexperiment. Gas exchange data were recorded for leaf 5 of both plants throughout the experiment. Parameters gs, E, and A refer to stomatalconductance to water vapor diffusion, transpiration rate, and assimilation rate, respectively.

�vw (‰) �vw/�C

Low 3 High humidity High 3 Low humidity Low 3 High humidity High 3 Low humidity

Low H High H Difference High H Low H Difference Low H High H Difference High H Low H Difference

IncisionA 1.4 1.3 �0.1 1.7 2.2 0.5 0.08 0.09 0.02 0.12 0.11 �0.01

2.2 1.2 �1.0 1.7 1.9 0.2 0.12 0.09 �0.03 0.12 0.10 �0.02

B 2.4 1.0 �1.4 1.9 2.6 0.7 0.13 0.07 �0.05 0.13 0.13 0.001.3 1.3 0.0 1.9 1.9 0.0 0.07 0.10 0.03 0.14 0.10 �0.04

C 4.2 1.6 �2.7 1.6 2.6 1.0 0.23 0.11 �0.11 0.11 0.13 0.023.5 2.3 �1.2 2.5 3.4 0.9 0.19 0.17 �0.02 0.18 0.18 0.00

D 3.4 1.1 �2.3 1.8 1.9 0.1 0.18 0.08 �0.10 0.13 0.10 �0.033.1 2.0 �1.1 1.9 2.6 0.7 0.16 0.14 �0.02 0.13 0.13 0.00

E 4.2 2.0 �2.2 1.7 3.7 2.0 0.23 0.14 �0.08 0.12 0.19 0.072.7 2.0 �0.7 2.2 4.4 2.2 0.14 0.15 0.00 0.15 0.23 0.07

F 4.6 2.8 �1.8 1.4 3.4 2.0 0.24 0.20 �0.04 0.10 0.18 0.084.4 3.2 �1.2 2.0 3.0 1.0 0.23 0.23 �0.01 0.14 0.16 0.01

Mean 3.1 1.8 �1.3 1.9 2.8 0.9 0.17 0.13 �0.04 0.13 0.14 0.01SE 0.3 0.2 0.2 0.1 0.2 0.2 0.02 0.01 0.01 0.01 0.01 0.01Gas-exchange parameters

gs (mol m�2 s�1) 0.47 0.75 0.3 0.86 0.37 �0.5E (mmol m�2 s�1) 11.2 7.5 �3.7 8.0 9.6 1.6A (�mol m�2 s�1) 24.0 24.8 0.8 25.1 21.8 �3.3

Gan et al.



The term �vw/�C thus reflects the proportion ofvein water contributed by back-diffusion of enrichedwater. Figure 2 shows that �vw/�C increases down-stream along the vein and in secondary veins com-pared with primary veins. This implies that back-diffusion is actively occurring throughout the veinlength regardless of the vein type. Thus, enrichedwater continues to accumulate within the veins in thedirection of water flow.

Isotopic enrichment of leaf water (�lw) from theleaf base to the tip has been observed in several cropplants by Wang and Yakir (1995). Our results indicatethat leaf water enrichment should be more depen-dent on the relative distances from major veins be-cause discs sampled adjacent to the veins are noted tohave smaller �lw (Fig. 3). In general, leaf water fromthe marginal and intercostal regions is more enrichedthan the Craig-Gordon prediction, whereas that fromsites adjacent to veins and at the leaf base is moredepleted than expected (Figs. 4 and 5). This pattern is

more apparent in the low-humidity leaves, an obser-vation reinforced by the leaf dry matter isotopic pat-tern, �om (Fig. 5C). Leaf dry matter has a significantstore of nonstructural carbohydrates (21% total dryweight; Wong, 1990) from current assimilation, andits isotopic composition has been shown to vary withthe diurnal changes of �lw (Cernusak et al., 2002). Inaddition, �18O of cellulose, the major component inleaf dry matter, has been shown to be strongly cor-related to that of the dry matter of cotton leaves(Barbour and Farquhar, 2000) and the leaf water ofgrasses (Helliker and Ehleringer, 2002). Assuming aconstant lignin to cellulose ratio across the leaf, �omcould be expected to reflect the spatial heterogeneityof leaf water isotopic content arising from non-transient influences.

Anatomical Basis of Spatial Variation

Measurements of stomatal density did not reveal aspatial variation consistent with the leaf water mea-surements. Microscopic examination of the leaf vas-culature also showed no apparent difference in thevenation density across the whole-leaf area. Theseleaf anatomical studies show that the differential en-richment of leaf water across the leaf blade could notsimply be a direct result of particular leaf zoneshaving more or fewer evaporative sites or of entrap-ping more xylem water in the disc samples.

Figure 4. Comparison of the measured �lw and Craig-Gordon pre-dicted �C of leaf water for the four lamina zones (margin [x], inter-costal [�], venous [E], and basal [�]) of cotton leaves at low (blacksymbols) and high (gray symbols) humidities. Three leaves sampledon different days are shown for each humidity treatment. The meanvapor pressure deficits of air at low (RH 35%) and high (RH 75%)humidities were 2.8 and 1.0 kPa, respectively. The line represents a1:1 relationship.

Figure 3. A typical �18O (‰) spatial variation of cotton leaf meso-phyll water for the four lamina regions (margin, intercostal, venous,and basal) at low humidity (RH 40%; VPD of air, 2.4 kPa). TheCraig-Gordon values of �C (‰) � 22.1 (margin), 23.6 (intercostal),23.9 (venous), and 24.4 (basal). Variation of �C is attributable to thegradual increase of leaf temperature with progressive sampling fromthe margin to the base. Leaf discs containing a visible fine vein areexcluded from this representation.




Accounting for the 18O Spatial Variation of Leaf Water:String-of-Lakes Model

On the basis of our observations of specific leafregions having �lw values above and below thosepredicted by the Craig-Gordon equation, we deducethat water flow in cotton leaves could probably be-have somewhat like a string of interconnected evap-orating lakes. The resemblance to this hydrologicalmodel was first suggested by Yakir (1992) for maize(Zea mays) and has been noted in a variety of dicot-yledon plants and grasses (Wang and Yakir, 1995;Helliker and Ehleringer, 2000, 2002). From Figure 3,areas next to the veins and near the base, showing anenrichment less than the Craig-Gordon prediction,could represent the first elements in the string of lakes

and be feeding partially enriched water to surround-ing cells and the neighboring veins (accounting forenrichment in vein xylem water). The enriched waterwould not only move along a series of cells but couldalso be propagated through the veins to more distalareas. This could account for the higher degree ofenrichment at the intercostal lamina regions and at theleaf margin (representing terminal water elements).

The first quantitative application to leaves of theGat-Bowser formulation was by Helliker andEhleringer (2000). This approach requires the leafblade to be divided into a discrete, finite number ofevaporating elements. The authors chose seven ele-ments to represent the whole evaporative processoccurring in grasses. The rationale behind this choicewas not discussed, and the number was presumablychosen to give the best fit to the observed �lw or waschosen out of convenience to match the number ofsegments into which the whole blade was divided.We found that based on Equation 5, isotopic enrich-ment at any given point along the water pathway issensitive to the total number of evaporating ele-ments, even though the average enrichment over thewhole leaf is independent of this number (Fig. 7). Asmaller number of evaporating elements tends tooverestimate the isotopic enrichment near the leafbase and underestimate it toward the leaf tip. TheGat-Bowser formulation would need to be modifiedfor use in leaves where evaporative sites are contin-uous and non-discrete.

Different Isotopic Enrichment Patterns atDifferent Humidities

The string-of-lakes model not only predicts an in-creasing isotopic enrichment but also dictates a dif-ferent pattern of isotopic enrichment along the waterpathway for different humidity treatments, as de-picted in Figure 7. At high humidity, the increase inisotopic enrichment along the path of water flow ismuch smaller than that at low humidity. Thus, we

Figure 6. Stomatal densities on the four leaf lamina regions of acotton leaf. Adaxial (white) and abaxial (gray) stomatal densities areexpressed as number of stomata per square millimeter of leaf surface.Error bars represent SEs (n � 3–4).

Figure 5. Spatial differences in the four lamina regions (margin,intercostal, venous, and basal) of cotton leaves. A, �lw � �C (samedata set as Fig. 4); B, oxygen isotope composition of leaf dry matter,�om, obtained from leaf punches; C, �om, obtained from trimming, atlow and high humidities. The mean vapor pressure deficits of air atlow (RH 35%) and high (RH 75%) humidities were 2.8 and 1.0 kPa,respectively. For A and B, each set of bars represents a leaf sample.For C, each set of bars represents six leaf samples. Error bars repre-sent SE (n � 2–17). High-humidity treatments are gray.

Gan et al.



would expect the isotopic composition of leaf waterat high humidity to be similar along most of thewater pathway. This expectation is largely confirmedin the spatial variation of �lw and �om (Fig. 5). Athigh humidity, isotopic enrichment of the leaf drymatter is rather homogenous over the whole-leafarea, unlike the definite isotopic pattern obtained atlow humidity.

Our results on the 18O spatial variation of veinxylem water also support the prediction of differentisotopic enrichment patterns (as well as magnitudes)at different humidities. Smaller �vw/�C of upstreamxylem water was observed in the leaf veins at lowhumidity, compared with the high-humidity leaves(Fig. 2). At the vein endings, �vw/�C at low humidityis mostly greater than that at high humidity, thoughthis difference is not always statistically significant(Table I). Although the string-of-lakes model servedwell in predicting different spatial patterns of leafwater enrichment at different humidity, it could not

adequately account for our observations of bulk leafwater enrichment.

Lower Enrichment of Bulk Leaf Water Arising fromCapacitance in Vein Ribs

When cotton leaf water is extracted in bulk byazeotropic distillation, �lw, bulk is noted to be lowerthan �C despite the removal of big primary veinsfrom the leaves (Fig. 8). By solving Equation 3, thefraction of leaf water subject to fractionation ( f ) is

given by�lw,bulk��s

�C��s�

�lw,bulk

�Cwith the assumption

that the fractionated water is enriched at �C. Thus, ifthe vein water is assumed to be unfractionated ac-cording to the two-pool model, its proportion in thebulk leaf water would be indicated by the term

1 ��lw,bulk

�C. However, if vein water is partially en-

riched such that �vw � 0, �s in Equation 3 will bereplaced by �vw and the proportion of vein water in

bulk leaf water will be given by1 �

�lw,bulk

�C

1 ��vw

�C

. Averag-

ing the low- and high-humidity treatments (Fig. 8),there is approximately 30% unenriched water presentin the bulk leaf water of whole leaves with veinsintact. Upon the removal of primary veins, the pro-

Figure 7. Gat-Bowser formulation of the string-of-lakes model. Top,Effect of the number of evaporating elements on the 18O enrichment,�, along the whole length of a leaf blade at low humidity. Bottom,Different 18O enrichment patterns, �, along the length of a blade fordifferent RH (h) values. All lines are plotted by assuming 50 evapo-rating elements, of which the steps have been smoothed for clarity.Inset, An expansion of the bottom portion of the main graph. Notethat � at very high humidity can be larger than that at low humiditynear the basal region. l/lm refers to the relative distance from the leafbase with lm the maximum distance from base to tip. Values of �* �8.3‰, �k � 22.9‰, and �v � �5.1‰ are used in Equation 5,assuming all elements have equal evaporation rates.

Figure 8. Discrepancy between the measured �lw, bulk of bulk leafwater and the Craig-Gordon predicted �C for leaves of cotton plants.

The term 1 ��lw,bulk

�Csignifies the unenriched fraction present in the

bulk leaf water (see “Discussion”). Comparison is made betweenlow- and high-humidity treatments. The vapor pressure deficits of airat low (RH 25%) and high (RH 75%) humidity were 3.2 and 1.0 kPa,respectively. Unshaded bars refer to the removal of primary veins.Error bars represent SEs (n � 4).




portion of unenriched water in the bulk leaf water isreduced to approximately 15%. This implies that thewater fraction associated with primary veins consti-tutes about 15%. In view of our earlier finding thatvein water can be enriched by as much as 0.19�C atthe vein endings (Fig. 2) and that the average veinwater enrichment is likely to be less than this value,the water fraction contributed by the primary veinsshould be higher, with the maximum possible esti-mated to be about 18.5%. In relatively close agree-ment with these observations, our independent as-sessment of this water fraction by gravimetricanalysis gave a value of 14.2% � 1.9% total leafwater. Such a high proportion of non-fractionatingwater has previously been noted (Leaney et al., 1985;Walker et al., 1989) but criticized as unlikely (Luoand Sternberg, 1992) given independent estimates ofvein water fraction to be �5%. Anatomical analysisof mesophyll and vessel areas in young barley (Hor-deum vulgare) leaves suggested 0.8% total tissue wa-ter was found in the lumen of vessels (Rayan andMatsuda, 1988). Using a pressure bomb to expresswater from a single leaf at increasing balancing pres-sure, Yakir et al. (1989) estimated the vein waterfraction to be 1% to 3% total leaf water in sunflower(Helianthus annuus) and ivy leaves, and water in thecell walls to be another 22% to 38%.

In our experiment, the removal of primary veinsnot only excluded water in the big xylem vessels butalso removed the total water present in the massiveground tissues of vein ribs. The layers of collen-chyma forming the vein ridge are closely packedwith negligible air spaces for evaporative enrichmentand would be expected to have a large store of rela-tively unenriched water. Thus, the ground tissues ofvein ribs could be perceived as a capacitance havinglittle interaction with enriched water at the leaf evap-orative sites, and its isotopic content is expected to besimilar to that of the vein xylem.

Lower Enrichment of Bulk Leaf Water Arising fromPeclet Effect

The two-pool model suggested by Leaney et al.(1985) seemed to be appropriate in accounting for theobserved lower enrichment of bulk leaf water. How-ever, the unenriched water fraction need not arisefrom the vascular pool alone but could be attributedto other factors. Our results show that the vein xylemwater is partly enriched, with the extent of enrich-ment depending on the ambient humidity. Isotopemixing by diffusion can clearly be rather efficient inleaves. Yet, the degree of enrichment in vein watercould not match that observed for the lamina tissuewater in the same vicinity (Figs. 2 and 3). Despite theshort distance between these two tissues, their en-richment difference can be as large as 20‰ or more.It is likely that the mesophyll tissues are not directlyfed by the big veins but by some higher order fine

veins that are relatively more enriched. Also, a Pecleteffect might be involved, with the transpiration fluxopposing the back-diffusion of enriched water in thelamina and along the veins. As a result, the isotopiccontent of bulk leaf water should be lower than thatpredicted by the Craig-Gordon model.

It is noteworthy that the discrepancy between �Cand �lw, bulk of whole leaves, after normalizationagainst �C, did not vary with the ambient humiditydespite a difference in leaf transpiration rates (lowhumidity, 11.5 mmol m�2 s�1; high humidity, 8.8mmol m�2 s�1; Fig. 8). Upon the removal of primary

veins, 1 ��lw,bulk

�Cis larger at high humidity. In con-

trast, the Peclet model (Eq. 4) predicts an increasingdiscrepancy between �lw and �C with increasing

transpiration rate, E. That is, a larger 1 ��lw,bulk

�Cis

expected at lower humidity. There have been conflict-ing reports on the existence of such a relationship.Wang et al. (1998) and Roden and Ehleringer (1999)observed no clear dependency of �C � �lw, bulk on E,with the former study based on a collection of 90 plantspecies grown under the same climatic conditions. Thepositive relationship between �C � �lw, bulk and E,illustrated by Wang and Yakir (2000) and Gillon andYakir (2000), appears to be in agreement with thePeclet model but may well collapse after normaliza-tion against �C. Our results show that the discrepancy(�C � �lw, bulk) at low humidity is 2.5‰ larger thanthat at high humidity. This positive correlation withtranspiration rate is wiped out upon normalizationagainst �C, as shown in Figure 8. Nonetheless, a pos-

itive trend of 1 ��lw,bulk

�Cwith E, consistent with the

Peclet model, has been reported (Walker et al., 1989;Flanagan et al., 1994; Barbour et al., 2000). All of thestudies mentioned made direct isotopic measurementsof extracted leaf water except for that of Barbour andco-workers (2000). The latter demonstrated a convinc-ing positive relationship between 1 � (�sw/�C) andtranspiration rate based on the isotopic composition ofphloem Suc in castor bean (Ricinus communis), where�sw refers to the deduced isotopic composition of leafwater with which the Suc exchanges. Our conflicting

result, a negative relationship between 1 ��lw,bulk

�Cand

transpiration rate upon the removal of primary veins,requires resolution. First, we cannot rule out the pos-sibility of higher leaf water enrichment by evaporativeloss from the cut edges during vein removal at lowhumidity. Second and perhaps more important, waterin the primary veins consistently has lower �vw/�C atlow humidity, the difference from that at high humid-ity being up to 6% (Fig. 2A). In accordance, the re-moval of primary veins at low humidity would takeaway a larger proportion of unenriched water, giving

Gan et al.



a smaller 1 ��lw,bulk

�Cvalue. Because vein water is an

intrinsic component of the leaf water enrichment sys-tem, we recommend that for comparative studies,bulk leaf water should be extracted from whole leaveswithout any vein removal. Nonetheless, we could not

identify a clear relationship between 1 ��lw,bulk

�Cand

transpiration rate based on the bulk leaf water extrac-tion from whole leaves of cotton.

Applying the String-of-Lakes Model to Leaves for �18OLeaf Water Prediction

Assuming all water pools have equal evaporationrates, the average isotopic content of water in theinterconnected pools of the string-of-lakes modelshould, according to Equation 5, be equivalent to theCraig-Gordon predicted �C. To test this assumptionand to check the applicability of the string-of-lakesmodel in estimating leaf water isotopic content, aweighted mean of measured �lw was computed fromthe leaf discs sampling. Using a distribution ratio of4.7:10.7:4.3:1 for regions of margin:intercostal:venous:basal, weighted means of both the measured andmodeled leaf water isotopic compositions were ob-tained (Table II). At low humidity, the weighted mean�lw was 9.2% higher than the weighted mean �C, and11.8% higher in the case of high humidity. Althoughwe expect the weighted mean of �lw from leaf punchesto be larger than �lw, bulk because of the exclusion ofveins in leaf discs sampling, the weighted mean of �lwbeing larger than the Craig-Gordon prediction clearlydiverged from the expectations of all leaf water mod-els. A higher than expected �lw has previously beenencountered (Flanagan et al., 1993; Helliker andEhleringer, 2000), and the latter group explained theirobservations by applying the string-of-lakes model,

with water pools having variable transpiration ratesacross the length of the leaf. In our experiment, thesampling of the leaf discs was biased given that theleaf punch could not sample within 1 mm of the majorveins without rupturing the veins. Thus, the low de-gree of enrichment expected in the immediate vicinityof the veins was omitted from our sampling. This maypartially account for the higher value of weightedmean �lw compared with �C. It presumably explainsthe discrepancy with our bulk leaf water measure-ments, which consistently show enrichment less thanthe Craig-Gordon prediction.

It has been suggested that most of the discrepanciesbetween modeled and measured leaf water isotopiccompositions could be resolved by careful estimationof the kinetic fractionation factor, �k (Buhay et al.,1996). As expected from Equation 2, �k and conse-quently �C are sensitive to the boundary layer con-ductance to water vapor diffusion (gb), especially atthe lower range of gb (Fig. 9). Increasing gb from 0.5to 2.5 mol m�2 s�1 leads to a calculated enrichment ofleaf water by 2‰, whereas a gb increase from 2.5 to5.0 mol m�2 s�1 results in only 0.5‰ enrichment. Thedegree of uncertainty imposed by �k estimation onleaf water isotopic composition will thus be greatlyreduced with a highly turbulent boundary layer. Inour study, boundary layer conductance in the green-house determined by a mass transfer method was0.52 mol m�2 s�1. In view of the sensitivity of �C togb in this region, the value of gb was verified byanother method based on heat transfer and windvelocity. A value of 0.45 mol m�2 s�1 was obtained.Because minimal difference was noted between thetwo methods, an average value of 0.49 mol m�2 s�1

was used in all �C calculations.For a more realistic application of the string-of-

lakes model to leaves, Wang and Yakir (1995) pro-

Table II. Comparison of the weighted mean of measured �lw and Craig-Gordon predicted �C ofcotton leaves in low and high humidities

The vapor pressure deficits of air at low (RH 35%) and high (RH 75%) humidity were 2.8 and 1.0 kPa,respectively. Weighted mean of �lw and �C were calculated from the same data set of Fig. 5A, using aweighting ratio of margin:intercostal:venous:basal � 4.7:10.7:4.3:1. Leaf sampling was carried out ondifferent days.

Time of SamplingWeighted Mean

�lw

Weighted Mean�C

�lw � �C (�lw/�C) � 1

Low humidity ‰1115–1500 25.7 23.3 2.4 0.1051000–1225 27.3 25.1 2.2 0.0880950–1140 25.9 23.9 2.0 0.082

Mean 26.3 24.1 2.2 0.092SE 0.5 0.5 0.1 0.007High humidity

1000–1240 18.3 16.9 1.4 0.0821315–1535 22.0 19.4 2.6 0.1351015–1330 18.4 16.2 2.2 0.138

Mean 19.6 17.5 2.1 0.118SE 1.2 1.0 0.4 0.018




posed that the process of back-diffusion along thestring of water elements (a phenomenon clearly sup-ported by the xylem water isotopic pattern) be incor-porated into the Gat-Bowser formulation. We envis-age back-diffusion of enriched water occurring intwo dimensions; along the leaf length in the basipetaldirection and diffusion from the evaporative sitesinto the vein network (radial dimension), which fur-ther supplies the enriched water to other series ofcells further afield. However, the advective transpi-ration flux should oppose this diffusion process (Pe-clet effect), leading to two-dimensional (2-D) gradi-ents of isotopic variation (Fig. 1D). Simulation of a2-D model of leaf water using a 2-D advection diffu-sion program was described by Yakir (1998). Hemodeled the leaf as a square domain having a spec-ified leaf thickness, with water entering from the sideand exiting by evaporation from the top, with no fluxassumed at other boundaries. The modeled isotopicgradients of leaf water have clear 2-D characteristicsthat collapse to uni-dimensional during the nightwhen evaporation stops. His findings distinctly ex-emplify the significant role of transpiration flux inmodulating the spatial variation of leaf water enrich-ment during the day.

Implications of Leaf Water Spatial Heterogeneity

Despite the spatial heterogeneity of leaf water, Bar-bour et al. (2000) have shown that the isotopic label ofsugars exported from castor bean leaves behaves aspredicted by the Peclet model. This observation ismade from gas exchange leaf chamber experimentsunder optimum conditions where assimilation ratesacross the leaf area are most likely uniform. How-ever, for nonuniform assimilation rates across theleaf, spatial heterogeneity of leaf water could pose achallenge to our present applications of leaf waterisotopic composition as indicators of plant environ-ment and terrestrial productivity (Yakir, 1998). If as-similation rates are higher, for example, near the leaftip where leaf water enrichment is higher than theCraig-Gordon prediction, the �18O of retrodiffusedCO2 would be greater and could be misinterpreted ashigher terrestrial productivity. Such uneven distribu-tion of assimilation rate would also have implicationsfor the interpretation of the mean isotopic signatureof sugars formed from the entire leaf as well as the18O content of O2 produced during terrestrial photo-synthesis and the interpretation of the Dole effect(the balance between terrestrial and marine produc-tivity based on the deviation from 23.5‰ in the �18Oof atmospheric O2 [Bender et al., 1985, 1994]). Non-uniform assimilation rates are most likely to occurwhen the amount of light incident on a leaf is incon-sistent across the leaf, leading to spatial variations ofphotosynthetic capacity and 13C discrimination, 13�(Meinzer and Saliendra, 1997). We expect dicotyle-doneous leaves, in their natural orientation, to re-ceive more uniform incident rays than long, flaggingblades of monocotyledoneous plants. This expecta-tion is supported by the uniform distribution of 13�across beech (Fagus spp.) leaves (Schleser, 1990) andincreasing 13� from the base to the tip of leaves ofsugarcane (Saccharum officinarum; Meinzer and Sa-liendra, 1997) and maize (Sasakawa et al., 1989). Theerror introduced by the spatial heterogeneity of leafwater in the applications mentioned will thus be agreater concern in grasslands where their growthhumidity is generally low and large isotopic gradi-ents are expected across the leaf, as illustrated inFigure 5.

CONCLUSIONS

The three leaf water models examined in this paper(two-pool model, Peclet model, and string-of-lakesmodel) are found to be valid in describing the fol-lowing features of leaf water enrichment. The over-estimation of the isotopic enrichment of bulk leafwater by the Craig-Gordon model is well addressedby the two-pool and Peclet models, whereas spatialheterogeneity of leaf water enrichment can be ex-pected from the Peclet and string-of-lakes models.But none of the models on its own could fully ac-count for all of the facets of leaf water enrichment.

Figure 9. Relationship of the modeled �C (solid line) and kineticfractionation �k (dashed line) with boundary layer conductance gb atlow and high humidities. Modeled �C and �k are calculated usingEquations 1 and 2, respectively. Units for gb and gs (stomatal con-ductance) are moles per square meter per second; ea/ei refers to theratio of the vapor pressures in the atmosphere and intercellularspaces.

Gan et al.



For example, although a gradient of isotopic enrich-ment is projected by both the Peclet and the string-of-lakes models, only the latter model correctly pre-dicts the different isotopic enrichment patterns atdifferent humidities. Yet, the latter model could notadequately account for the lower degree of enrich-ment in bulk leaf water, which the two-pool modeland the Peclet model could. Also, the observed par-tial enrichment of vein xylem water is within expec-tations of the Peclet model, whereas the other twoeither assume no enrichment of vein water (two-poolmodel) or an enrichment equivalent to that at theneighboring evaporative sites (string-of-lakes mod-el). On the other hand, we could not identify a clear

relationship between 1 ��lw,bulk

�Cand transpiration

rate, a correlation expected from the Peclet model.Our findings acknowledge the presence of capaci-

tance in the ground tissues of vein ribs that are likelyto have similar isotopic contents to the vein xylem.This paper also draws attention to the need for mod-ification of the Gat-Bowser formulation of the string-of-lakes model to accommodate continuous, non-discrete evaporating elements and for incorporatingPeclet effects along the longitudinal and radial di-mensions into the string-of-lakes model.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Seeds of cotton (Gossypium hirsutum L. var Deltapine 90) plants weresown in 4.5-L polyvinylchloride pots containing sterilized garden soil mix-

ture supplied with slow-release fertilizer (Osmote Plus, Scotts-Sierra,Maysville, OH). The plants were grown under full sunlight between latesummer and early autumn in two greenhouses at the same temperature(30 � 1°C day and 22 � 1°C night) but with different RHs (40% � 10% and75% � 10%) maintained day and night. Tap water was used to water theplants twice daily. All samplings and measurements were carried out on 35-to 55-d-old plants in the greenhouses on cloud-free days during the timeperiod of 11 am to 3 pm when plant photosynthesis was at its maximum andgas exchange was observed to be at steady state.

Gas-Exchange Measurements

Leaf gas-exchange measurements were taken during sunny days using aportable gas-exchange system (model LI-6400, LI-COR, Lincoln, NE)equipped with the standard leaf chamber and the CO2 injector system forthe control of CO2 concentration. Measurements were made at a photosyn-thetic photon flux density of 1,200 �mol m�2 s�1 from an LED light source.

Boundary Layer Conductance Measurements

The boundary layer conductance to water vapor diffusion (gb) in thegreenhouses was first measured using a mass transfer method (Jarvis, 1971).A water-saturated filter paper (No. 1, Whatman, Clifton, NJ) was exposed,and the rate of weight loss from evaporation was recorded. The temperatureof the paper leaf model was constantly monitored using an infrared ther-mometer (Mikron Instrument, Oakland, NJ) with a resolution of 0.1°C. Theboundary layer conductance obtained was 0.52 � 0.04 mol m�2 s�1. Forverification, gb was also determined using the heat transfer method. Thewind speed in the greenhouse, 0.34 � 0.10 m s�1, was measured using anultrasonic anemometer (Gill Instruments, Lymington, Hampshire, UK). Us-ing equations given by Ball et al. (1988) for calculating the boundary layerresistance to water vapor based on heat transfer in a forced convection, weobtained a gb value of 0.45 � 0.06 mol m�2 s�1.

Vein Xylem Water Sampling

For sap collection with minimal perturbation, xylem water of an intactleaf was sampled concurrently from primary and secondary veins using aroot pressure chamber (Yong et al., 2000). While the whole root system ofthe plant was enclosed and pressurized in the chamber, a light incision wasmade on the leaf vein with a sharp razor blade. The pneumatic pressure wasadjusted to give a xylem sap flow of about 0.5 �L s�1 exuding from the finerveins. A sample of 0.7 �L of xylem water was directly siphoned off with apipette (2-�L micro-pipetteman, Gilson Medical Electronics, Middleton, WI)from the water bead formed at the incision point immediately after wipingoff the earlier exudate. The collected sap was quickly dispensed into asmooth-walled tin cup (4.5 � 2 mm) and sealed under argon with a mod-ified Carlo Erba liquid encapsulator equipped with a pneumatic actuator. Tocollect more xylem water for storage, a 10-�L capillary was held steadyagainst the incision, and its ends were immediately sealed with sealing waxafter filling. Sampling order followed the general rule of first scoring thevein endings in the vicinity of the leaf margin and gradually movinginwards toward the leaf base where vein diameter progressively increases.This minimized water-flow disruptions to areas yet to be sampled. Thepetiole of the same leaf was eventually cut to collect petiole water after thecompletion of vein xylem water sampling.

To determine humidity effects on the isotopic composition of vein xylemwater, a step change of humidity was carried out in another experiment.Xylem water was collected from the same vein incision points before and 1 hafter the step change of humidity.

Leaf Water and Organic Matter Sampling

To analyze leaf mesophyll water of cotton leaves without the inclusion ofvein water, leaf discs (diameter 3 mm) were cut out from an intact leaf(avoiding primary, secondary, and fine veins) with an improved version(Fig. 10) of the purpose-designed leaf punch (Gan et al., 2002). In brief,pressing down the plunger of the leaf punch cuts a leaf disc and alsoactivates a jet of argon that guides the disc to fall directly into a preweighedsmooth-walled tin cup (9 � 3.5 mm). The argon jet also flushes out air and

Figure 10. Cut-away view of the leaf punch device. The punch ishollow with sharp edges to cut a leaf disc and to deliver argon fordirecting the disc into the tin cup as well as purging the cup. The cupcan be immediately sealed with the push of a button that pneumat-ically operates the pincer arms.




hence excludes nitrogen and oxygen from the cup. The filled cup is imme-diately sealed with the push of a button (pneumatic actuation) and can bedirectly pyrolyzed before analysis in an Isotope Ratio Mass Spectrometer.Sampling began from the leaf margin and worked inward toward the petioleto minimize water-flow disruptions to the leaf lamina yet to be sampled.Leaf temperature profiles were captured using an IR scanner with a sensi-tivity of 0.1°C (Thermovision 870, AGEMA, Danderyd, Sweden) after everytwo to three leaf punches. Regular monitoring of leaf temperature wasessential because we noted that leaf temperature in the sampling regionwould gradually climb by 1°C to 2°C after cutting several leaf discs. Afterpunching out leaf discs from one side of the midrib, leaf temperature wasagain recorded by the IR scanner before the other one-half was trimmed,avoiding primary veins. The trimmed leaf segments were immediatelyimmersed in toluene (80 mL) for bulk leaf water extraction by azeotropicdistillation using a specially designed funnel as described by Revesz andWoods (1990). The leaf-half containing the punch holes was pressed be-tween layers of paper and dried in a 70°C oven. Small dry leaf segments inthe vicinity of each punch hole were collected for isotopic analysis of leaforganic matter.

To map spatial isotopic distribution of leaf organic matter, whole leavesof cotton from leaf positions 5 and 7 were harvested from each of threeplants. The leaf blades were trimmed into four distinct zones before ovendrying. The four zones were (a) margin, for lamina next to the leaf edge; (b)intercostal, for lamina remote from the secondary and primary veins; (c)venous, for lamina adjacent to the primary veins; and (d) basal, for laminacollected at the leaf base within approximately 20 mm of the petiole.Thereafter, the dried leaf segments were ground for isotopic analysis of leaforganic matter. To determine the distribution ratio of the four lamina zones,two fresh leaves were photocopied on paper, and each leaf area was dividedinto the four zones mentioned. From weighing the paper, the relative ratioof the four zones (margin, intercostal, venous, and basal) was found to be4.7:10.7:4.3:1, respectively.

For bulk leaf water analysis, whole leaves were sampled, and leaf waterwas extracted by azeotropic distillation, with some replicates having theprimary veins removed. To determine the percentage of water present in theprimary veins, leaf fresh weight was first measured, followed by the re-moval of primary veins. The fresh weights of the remaining lamina and theprimary rib skeleton were noted before oven drying. The leaf water contentand water associated with the primary ribs were obtained from the differ-ence between the fresh and dry weights of the lamina and primary ribs.

Throughout the period of leaf water sampling, atmospheric water vaporwas collected using a dry ice-ethanol cold trap. Over 20 d of leaf watermeasurements, �v � �12.6‰ � 1.3‰ (sd).

Oxygen Isotope Analysis

Oxygen isotopic analyses of water and dried and fresh leaf samples wereall performed using the continuous-flow pyrolysis technique described byFarquhar et al. (1997) with slight modifications. To minimize memoryproblems, the reaction column was packed with glassy carbon grit (3,150–4,000 �m, HTW, Thierhaupten, Germany) followed by a top layer (0.015 mthick) of nickelized carbon (50% [w/w] Ni, Alpha Resources, Sydney). Thesame analytical precision of 0.2‰ was achieved after reactor modification.Elemental oxygen standards were beet Suc and ANU-HP water, with thelatter (�18O � �5.5‰) also serving as the isotopic internal standard forwater samples. A standard of beet Suc containing 3% (w/w) nitrogen wasused for correcting isotopic composition of dry leaf samples because thelatter were found to contain about 3% (w/w) nitrogen. Isotopic calibrationagreement between water and organic standards has previously been per-formed (Gan et al., 2002), and a slope of 1.0027 was obtained over an isotopicrange of 90‰. Preparation of liquid samples for pyrolysis was similar to thatfor leaf xylem water sampling. Dried leaf samples of 1.0 to 1.5 mg wereaccurately weighed into tin capsules that were then crimped manually.Smooth-walled tin cups containing fresh leaf samples were directly pyro-lyzed. The fresh weight of the leaf disc was obtained from the differencebetween the total weight of the tin cup with the leaf disc and the weight ofthe empty tin cup. Direct pyrolysis of fresh and dry leaf samples gives themeasured values of �18OF and �18OD, respectively. The 18O ratio of leafwater (�18Olw) from the fresh leaf sample can be calculated by isotopic massbalance,

�18OF � x�18Olw � (1�x)�18OD

where x refers to the proportion contributed by leaf water in the totaloxygen pool of the fresh leaf sample. The value of x was determined fromthe thermal conductivity detector output of the gas chromatograph in thesame acquisition as the 18O analysis, the calculations of which are detailedin Gan et al. (2002). Overall,

�18Olw � �18OD �

OF(1�1816

OD)

OF�OD[�18OF��18OD]

where OF and OD are, respectively, the oxygen elemental composition offresh and dry leaf samples as obtained from the thermal conductivitydetector output of the gas chromatograph.

Stomatal Density Distribution

Fresh cotton leaf segments of approximately 0.25 to 0.50 cm2 weretrimmed from various places on the leaf lamina and placed on carbon pastebefore being frozen on a metal support precooled in liquid nitrogen. Scan-ning electron microscope (S-2250N Hitachi, Tokyo) images were takenunder standard conditions of 25 kV, 16 mm working distance, and low inputof water vapor. To quantify the distribution of stomatal densities over thewhole-leaf surface, we targeted four locations: margin, intercostal, venous,and basal. For every location, images of three to four different areas weretaken for each abaxial and adaxial surface. Each image represented an areaof 0.343 mm2 (the field of view at a magnification of 200�). The number ofstomata was counted directly from the prints and expressed as number ofstomata per mm2 of leaf surface.

Analysis of Leaf Vasculature

Leaf pigments and cell contents were cleared by incubating the fresh leafin methanol at 60°C for 1 h, followed by immersion in warm lactic acid for5 to 10 min. The venation pattern and density were examined under a lightmicroscope.

ACKNOWLEDGMENTS

We thank Peter Groeneveld and Jim Neale for helping to design andconstruct the leaf punch, Josette Masle for assistance with the analysis of leafvasculature, and an anonymous reviewer for helpful comments. The tech-nical support rendered by the Electron Microscopy Unit (Australian Na-tional University) is much appreciated.

Received April 23, 2002; returned for revision May 30, 2002; accepted June14, 2002.

LITERATURE CITED

Allison GB, Gat JR, Leaney FWJ (1985) The relationship between deute-rium and oxygen-18 delta values in leaf water. Chem Geol 58: 145–156

Ball MC, Cowan IR, Farquhar GD (1988) Maintenance of leaf temperatureand the optimisation of carbon gain in relation to water loss in a tropicalmangrove forest. Aust J Plant Physiol 15: 263–276

Barbour MM, Farquhar GD (2000) Relative humidity- and ABA-inducedvariation in carbon and oxygen isotope ratios of cotton leaves. Plant CellEnviron 23: 473–485

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

Bariac T, Gonzalez-Dunia J, Tardieu F, Tessier D, Mariotti A (1994) Spatialvariation of the isotopic composition of water (18O, 2H) in organs ofaerophytic plants: 1. Assessment under laboratory conditions. Chem Geol115: 307–315

Bariac T, Rambal S, Jusserand C, Berger A (1989) Evaluating water fluxesof field-grown alfalfa from diurnal observations of natural isotope con-centrations, energy budget and ecophysiological parameters. Agric For-est Meteorol 48: 263–283

Bender M, Labeyrie LD, Raynaud D, Lorius C (1985) Isotopic compositionof atmospheric O2 in ice linked with deglaciation and global primaryproductivity. Nature 318: 349–352

Gan et al.



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

Buhay WM, Edwards TWD, Aravena R (1996) Evaluating kinetic fraction-ation factors used for ecologic and paleoclimatic reconstructions fromoxygen and hydrogen isotope ratios in plant water and cellulose.Geochim Cosmochim Acta 60: 2209–2218

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

Cormie AB, Luz B, Schwarcz HP (1994) Relationship between the hydrogenand oxygen isotopes of deer bone and their use in the estimation ofrelative humidity. Geochim Cosmochim Acta 58: 3439–3449

Craig H, Gordon LI (1965) Deuterium and oxygen-18 variations in the oceanand the marine atmosphere. In E Tongiorgi, eds, Proceedings of a Con-ference on Stable Isotopes in Oceanographic Studies and Paleotempera-tures. Consiglio Nazionale delle Ricerche, Laboratorie Geologia Nuclear,Pisa, Italy, pp 9–130

Dongmann G, Nurnberg HW, Forstel H, Wagener K (1974) On the enrich-ment of H2

18O in the leaves of transpiring plants. Radiat Environ Biophys11: 41–52

Edwards TWD, Aravena RO, Fritz P, Morgan AV (1985) Interpretingpaleoclimate from 18O and 2H in plant cellulose: comparison with evi-dence from fossil insects and relict permafrost in southwestern Ontario.Can J Earth Sci 22: 1720–1726

Edwards TWD, Fritz P (1986) Assessing meteoric water composition andrelative humidity from 18O and 2H in wood cellulose: paleoclimaticimplications for southern Ontario, Canada. Appl Geochem 1: 715–723

Epstein S, Thompson P, Yapp CJ (1977) Oxygen and hydrogen isotopicratios in plant cellulose. Science 198: 1209–1215

Farquhar GD, Henry BK, Styles JM (1997) A rapid on-line technique fordetermination of oxygen isotope composition of nitrogen-containing or-ganic matter and water. Rapid Commun Mass Spectrom 11: 1554–1560

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

Farquhar GD, Lloyd J (1993) Carbon and oxygen isotope effects in theexchange of carbon dioxide between terrestrial plants and the atmo-sphere. In JR Ehleringer, AE Hall, GD Farquhar, eds, Stable Isotopes andPlant Carbon-Water Relations. Academic Press, New York, pp 47–70

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

Flanagan LB, Bain JF, Ehleringer JR (1991a) Stable oxygen and hydrogenisotope composition of leaf water in C3 and C4 plant species under fieldconditions. Oecologia 88: 394–400

Flanagan LB, Comstock JP, Ehleringer JR (1991b) Comparison of modeledand observed environmental influences on the stable oxygen and hydro-gen isotope composition of leaf water in Phaseolus vulgaris L. PlantPhysiol 96: 588–596

Flanagan LB, Marshall JD, Ehleringer JR (1993) Photosynthetic gas ex-change and the stable isotope composition of leaf water: comparison of axylem-tapping mistletoe and its host. Plant Cell Environ 16: 623–631

Flanagan LB, Phillips SL, Ehleringer JR, Lloyd J, Farquhar GD (1994)Effect of changes in leaf water oxygen isotopic composition on discrim-ination against C18O16O during photosynthetic gas exchange. Aust JPlant Physiol 21: 221–234

Gan KS, Wong SC, Farquhar GD (2002) Oxygen isotope analysis of plantwater without extraction procedure. In PA de Groot, ed, Handbook ofStable Isotope Analytical Techniques. Elsevier Science Publishing, NewYork (in press)

Gat JR, Bowser C (1991) The heavy isotope enrichment of water in coupledevaporative systems. In HP Taylor, JR O’Neil, IR Kaplan, eds, StableIsotope Geochemistry: A Tribute to Samuel Epstein. The GeochemicalSociety, Lancaster Press, St. Louis, pp 159–168

Gillon JS, Yakir D (2000) Internal conductance to CO2 diffusion and C18OOdiscrimination in C3 leaves. Plant Physiol 123: 201–213

Helliker BR, Ehleringer JR (2000) Establishing a grassland signature inveins: 18O in the leaf water of C3 and C4 grasses. Proc Natl Acad Sci USA97: 7894–7898

Helliker BR, Ehleringer JR (2002) Differential 18O enrichment of leaf cel-lulose in C3 versus C4 grasses. Funct Plant Biol 29: 435–442

Jarvis PG (1971) The Estimation of Resistances to Carbon Dioxide Transfer.In Z Sestak, J Catsky, PG Jarvis, eds, Plant Photosynthetic Production:Manual of Methods. Junk, The Hague, The Netherlands, pp 566–631

Leaney F, Osmond C, Allison G, Ziegler H (1985) Hydrogen-isotope com-position of leaf water in C3 and C4 plants: its relationship to thehydrogen-isotope composition of dry matter. Planta 164: 215–220

Lin G, Sternberg L (1993) Hydrogen isotopic fractionation by plant rootsduring water uptake in coastal wetland plants. In JR Ehleringer, AE Hall,GD Farquhar, eds, Stable Isotopes and Plant Carbon-Water Relations.Academic Press, New York, pp 497–510

Luo YH, Sternberg L (1992) Spatial D/H heterogeneity of leaf water. PlantPhysiol 99: 348–350

Meinzer FC, Saliendra NZ (1997) Spatial patterns of carbon isotope dis-crimination and allocation of photosynthetic activity in sugarcane leaves.Aust J Plant Physiol 24: 769–775

Rayan A, Matsuda K (1988) The relation of anatomy to water movementand cellular response in young barley leaves. Plant Physiol 87: 853–858

Revesz K, Woods PH (1990) A method to extract soil water for stableisotope analysis. J Hydrol 115: 397–406

Roden JS, Ehleringer JR (1999) Observations of hydrogen and oxygenisotopes in leaf water confirm the Craig-Gordon model under wide-ranging environmental conditions. Plant Physiol 120: 1165–1173

Roden JS, Lin GG, Ehleringer JR (2000) A mechanistic model for interpre-tation of hydrogen and oxygen isotope ratios in tree-ring cellulose.Geochim Cosmochim Acta 64: 21–35

Sasakawa H, Sugiharto B, O’Leary MH, Sugiyama T (1989) �13C values inmaize leaf correlate with phosphoenolpyruvate carboxylase levels. PlantPhysiol 90: 582–585

Schleser GH (1990) Investigations of the �13C pattern in leaves of Fagussylvatica L. J Exp Bot 41: 565–572

van Bel AJ (1976) Different mass transfer rates of labeled sugars andtritiated water in xylem vessels and their dependency on metabolism.Plant Physiol 57: 911–914

Walker CD, Brunel J-P (1990) Examining evapotranspiration in a semi-aridregion using stable isotopes of hydrogen and oxygen. J Hydrol 118: 55–75

Walker CD, Leaney FW, Dighton JC, Allison GB (1989) The influence oftranspiration on the equilibration of leaf water with atmospheric watervapour. Plant Cell Environ 12: 221–234

Wang XF, Yakir D (1995) Temporal and spatial variations in the oxygen-18content of leaf water in different plant species. Plant Cell Environ 18:1377–1385

Wang XF, Yakir D (2000) Using stable isotopes of water in evapotranspira-tion studies. Hydrol Process 14: 1407–1421

Wang XF, Yakir D, Avishai M (1998) Non-climatic variations in the oxygenisotopic compositions of plants. Global Change Biol 4: 835–849

Webb EA, Longstaffe FJ (2000) The oxygen isotopic compositions of silicaphytoliths and plant water in grasses: implications for the study ofpaleoclimate. Geochim Cosmochim Acta 64: 767–780

White JWC, Cook ER, Lawrence JR, Broecker WS (1985) The D/H ratios ofsap in trees: implications for water sources and tree ring D/H ratios.Geochim Cosmochim Acta 49: 237–246

Wong SC (1990) Elevated atmospheric partial pressure of CO2 and plantgrowth: II. Non-structural carbohydrate content in cotton plants and itseffect on growth parameters. Photosynth Res 23: 171–180

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

Yakir D (1998) Oxygen-18 of leaf water: a crossroad for plant-associatedisotopic signals. In H Griffiths, eds, Stable Isotopes. BIOS Scientific,Oxford, pp 147–168

Yakir D, DeNiro MJ, Gat JR (1990) Natural deuterium and oxygen-18enrichment in leaf water of cotton plants grown under wet an dryconditions: evidence for water compartmentation and its dynamics. PlantCell Environ 13: 49–56

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

Yapp CJ, Epstein S (1982) A re-examination of cellulose carbon-boundhydrogen �D measurements and some factors affecting plant-water D/Hrelationships. Geochim Cosmochim Acta 46: 955–965

Yong JWH, Wong SC, Letham DS, Hocart CH, Farquhar GD (2000) Effectsof elevated [CO2] and nitrogen nutrition on cytokinins in the xylem sapand leaves of cotton. Plant Physiol 124: 767–779




Documents

O Spatial Patterns of Vein Xylem Water, Leaf - Plant Physiology