18
O Spatial Patterns of Vein Xylem Water, Leaf Water, and
Dry 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, Péclet effect, and string-of-lakes) were assessed for their robustness in predicting
leaf water enrichment and its spatial heterogeneity. This was achieved by studying the 18O spatial patterns of vein xylem
water, leaf water, and dry matter in cotton (Gossypium hirsutum) leaves grown at different humidities using new experimental approaches. Vein xylem water was collected from intact transpiring cotton leaves by pressurizing the roots in a
pressure chamber, whereas the isotopic content of leaf water was determined without extracting it from fresh leaves with
the aid of a purpose-designed leaf punch. Our results indicate that veins have a significant degree of lateral exchange with
highly enriched leaf water. Vein xylem water is thus slightly, but progressively enriched in the direction of water flow. Leaf
water enrichment is dependent on the relative distances from major veins, with water from the marginal and intercostal
regions more enriched and that next to veins and near the leaf base more depleted than the Craig-Gordon modeled
enrichment of water at the sites of evaporation. The spatial pattern of leaf water enrichment varies with humidity, as
expected from the string-of-lakes model. This pattern is also reflected in leaf dry matter. All three models are realistic, but
none could fully account for all of the facets of leaf water enrichment. Our findings acknowledge the presence of capacitance
in the ground tissues of vein ribs and highlight the essential need to incorporate Péclet effects into the string-of-lakes model
when applying it to leaves.
The isotopic composition of leaf water reflects local
humidity, and its imprints on plant cellulose and
other fossil materials have been widely explored for
palaeoclimatic reconstruction. To date, isotopic values of wood cellulose (Epstein et al., 1977; Yapp and
Epstein, 1982; Edwards et al., 1985; Edwards and
Fritz, 1986; Roden et al., 2000), grassland phytoliths
(Webb and Longstaffe, 2000), and deer bone (Cormie
et al., 1994) have been shown to be related to leaf
water isotopic composition. Leaf water isotopic signature is not only imprinted on plant organic matter
but is also recorded in atmospheric CO2 and O2. The
CO2 interacts and undergoes isotopic exchange with
leaf water, and O2 is released by the plant during
photosynthesis. Changes in the oxygen isotope ratios
of CO2 and O2 can thus be used to study variations in
the net exchange of CO2 in terrestrial ecosystems
(Farquhar et al., 1993) and in the balance of terrestrial
and marine productivity (Bender et al., 1985; Bender
et al., 1994). Because all of these applications critically
depend on estimation of the leaf water oxygen isotopic ratio, a good understanding of the nature of leaf
water enrichment is needed.
The isotopic composition of leaf water is most commonly estimated from the model of a freely evapo1
Present address: Natural Sciences Academic Group, National
Institute of Education, Nanyang Technological University,
Singapore.
* Corresponding author; e-mail [email protected]; fax
61–2– 6125– 4919.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.007419.
1008
rating water surface (Craig and Gordon, 1965) where
isotopic fractionation is driven by the lower vapor
pressure and diffusivity of the heavier molecules.
Although the Craig-Gordon model basically describes water enrichment at the sites of evaporation
compared with locally transpired water, it cannot
adequately account for other aspects of leaf water
enrichment, particularly the spatial variation of leaf
water 18O and/or D contents (Luo and Sternberg,
1992; Bariac et al., 1994; Wang and Yakir, 1995; Helliker and Ehleringer, 2000). Also, the Craig-Gordon
model has often been found to overestimate the isotopic enrichment of bulk leaf water (Allison et al.,
1985; Bariac et al., 1989; Walker et al., 1989; Walker
and Brunel, 1990; Yakir et al., 1990; Flanagan et al.,
1991a, 1991b, 1994; Wang et al., 1998). To explain
such observations, several other models have been
suggested in conjunction with the Craig-Gordon
model, namely the two-pool model (Leaney et al.,
1985), the Péclet model (Farquhar and Lloyd, 1993),
and the string-of-lakes model (Gat and Bowser,
1991). The objective of this paper is to examine and
assess the applicability of these various leaf water
models by studying the 18O spatial patterns of vein
xylem water, leaf mesophyll water, and dry matter in
cotton (Gossypium hirsutum) leaves at different humidity treatments. To accomplish this, new experimental approaches are employed in the direct collection of vein xylem water from an intact transpiring
leaf using a root pressure chamber and in the isotopic
measurement of leaf water without extracting it from
the leaves with the aid of a purpose-designed leaf
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18
punch. Bleeding xylem water directly from the petiole and veins of intact plants should give a good
representation of the transpiration stream entering
the leaf mesophyll cells. This technique allows us to
map the isotopic gradient along the main water flow
in leaf veins.
Applying the Craig-Gordon model to leaves
(Dongmann et al., 1974; Farquhar et al., 1989), the
isotopic enrichment of leaf water above source water
(⌬lw) at the evaporative sites of intercellular air
spaces would be equal to the Craig-Gordon prediction (⌬C) where
⌬C ⫽ ␧k ⫹ ␧* ⫹ (⌬v⫺␧k)
ea
ei
28.5rs ⫹ 18.9rb
rs ⫹ rb
where rs and rb are the leaf stomatal and boundary
layer resistances to water vapor diffusion, respectively. An alternative method to calculate ⑀k is described by Buhay et al. (1996), taking leaf size and
morphology into consideration.
To explain the observation that bulk leaf water is
less enriched than that predicted by the CraigGordon model, Leaney et al. (1985) described leaf
water as consisting of two pools: evaporatively enriched leaf tissue water and isotopically unaltered
vascular water (Fig. 1A). On the basis of this definition, the isotopic composition of bulk leaf water (␦lw,
bulk) could simply be expressed as
(1)
where ⌬v is the isotopic composition of atmospheric
water vapor relative to source water. The water vapor pressures in the atmosphere and intercellular
spaces are ea and ei, respectively, ⑀* is the equilibrium
fractionation factor arising from the lower vapor
pressure of H218O molecules at liquid-vapor phase
equilibrium, and ⑀k is the kinetic fractionation factor
caused by the lower diffusivity of heavy H218O molecules. Equation 1 assumes that isotopic steady state
has been achieved, where the isotopic composition of
transpired water (␦E) equals the value of source water
(␦S). In calculating the kinetic fractionation factor, the
different layers of boundary resistance developing
from the leaf evaporative sites to the fully turbulent
atmospheric air need to be considered. The boundary
layer is fully stagnant in the leaf substomatal air
spaces (18O kinetic fractionation, 28.5‰), whereas a
laminar flow is expected near the leaf surface (18O
kinetic fractionation, 18.9‰). Taking into account the
weighted effects of these different boundary layers,
Farquhar et al. (1989) expressed the overall kinetic
fractionation as
␧k(‰) ⫽
O Spatial Patterns of Leaf Constituents
(2)
␦lw,bulk ⫽ ␦C 䡠 f ⫹ ␦s 䡠 (1⫺f )
(3)
where f is the fraction of leaf water subject to fractionation and ␦C is the isotopic composition predicted from the Craig-Gordon model (Eq. 1) with
⌬ C ⫽ ␦ C ⫺ ␦ s. To reconcile the differences between
the observed and Craig-Gordon predicted isotopic
ratios, the fraction of vein water would have to be in
the range of 25% to 50% total leaf water (Leaney et
al., 1985; Walker et al., 1989). After the removal of the
mid-vein from leaves of Betula occidentalis and Populus angustifolia, the estimated unenriched water fraction in the remaining leaves was reduced to 10%
(Roden and Ehleringer, 1999).
Farquhar and Lloyd (1993) modified the physical
model of a single-leaf water pool to one having a
continuum of isotopic composition (Fig. 1B). They
proposed that advection of unenriched water via the
transpiration stream opposes the back-diffusion of
enriched water from evaporation sites (Péclet effect).
A continuous isotopic gradient is thereby created
within the leaf. The enrichment at the leaf evaporative sites (␦e) can be described by Equation 1 and is
proposed to decay exponentially to the isotopic signature of source water (␦s) at the veins. An integraFigure 1. Box-diagram representation of the leaf
water models. A, Two-pool model; B, Péclet
model; C, string-of-lakes model; and D, Pécletcontinuous evaporative enrichment model incorporating ground tissue capacitance. In the
last model, the left compartment represents vein
ribs and is further divided into two components:
the capacitance arising from rib ground tissue
and vein xylem water. The right compartment
represents leaf lamina tissue water. The intensity
of shading indicates the extent of leaf water
enrichment. Double-headed arrows represent a
Péclet effect, where the advection of unenriched
water from the transpiration stream opposes the
back-diffusion of enriched water; a bigger arrowhead implies a larger contribution in that
direction.
Plant Physiol. Vol. 130, 2002
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1009
Gan et al.
tion of this isotopic variation would give a lower
isotopic enrichment of bulk leaf water above source
water (⌬lw, bulk),
⌬lw,bulk ⫽ ⌬C
1 ⫺ e⫺㜷
㜷
(4)
where leaf Péclet number, 㜷 ⫽ EL/CD. In the latter
term, E is transpiration rate, L is the scaled effective
path length of water between the xylem and sites of
evaporation in leaves, C is the molar concentration of
water (55.5 ⫻ 103 mol m⫺3), and D is the diffusivity
of H218O in water (2.66 ⫻ 10⫺9 m2 s⫺1). Equation 4
also predicts an increasing disparity between measured ⌬lw, bulk and the Craig-Gordon predicted ⌬C
with increasing transpiration rate. The Péclet model
not only accounts for the lower than expected enrichment of bulk leaf water but also predicts, in principle,
the nonuniform isotopic distribution of leaf water at
the small scale between stomata and xylem. However, the Péclet model based on Equation 4 is unable
to describe variation of isotopic enrichment on a
larger scale.
An explanation for the larger scale spatial variation
of leaf water was provided by Yakir (1992), who
suggested that the isotopic gradient within a leaf
could be a consequence of enrichment along the path
of evaporation (Fig. 1C). This is analogous to a string
of evaporating lakes along a river. In this string-oflakes model, the outflow from one evaporating element enters into the next element in the series, leading to a progressive enrichment of heavy isotopes
along the path of water flow. A formulation of this
model is given by Gat and Bowser (1991):
␧
h
␦n ⫽ ␦n⫺1 ⫹
F⫹(1⫺h)
1⫹
E h
␦v⫺␦n⫺1 ⫹
(5)
where ␦v is the isotopic composition of atmospheric
water vapor, ␦n is that of liquid water entering the
nth evaporating element, F⫹ and E represent the
influx and evaporative efflux of the nth element, h is
the relative humidity (RH) and, ⑀ ⫽ ⑀* ⫹ (1 ⫺ h)⑀k.
Direct and indirect leaf water measurements supporting the different leaf water models have been
reported: Craig-Gordon model (Roden and
Ehleringer, 1999), Péclet model (Walker et al., 1989;
Flanagan et al., 1991b; Barbour et al., 2000), and
string-of-lakes model (Yakir, 1992; Wang and Yakir,
1995; Helliker and Ehleringer, 2000, 2002). Given the
importance of leaf water modeling to studies of
plant-environment interactions, there is a pressing
need to reconcile these apparently disparate results.
To assess best the applicability of the various leaf
water models, we examined the 18O content of water
along its pathway in the leaf, from the petiole to the
vein network and then to the lamina tissue. The first
step in our study was to clarify the extent of enrich1010
ment in vein xylem water, which is presumed to be
unfractionated and to have the same isotopic composition as soil water (Leaney et al., 1985). Next, for a
better representation of the lamina tissue water, we
removed uncertainties such as the inclusion of vein
water in leaf water measurements. This was achieved
using a purpose-designed leaf punch that cuts and
seals a small leaf disc sample for direct pyrolysis
during isotopic measurements. The 18O content of the
leaf dry matter, which provides an integrated record
of leaf water isotopic composition, was analyzed as a
check on the results from leaf water measurements.
Last, distribution of stomatal and venation densities
across the leaf were studied to assess the possible
anatomical basis for spatial heterogeneity of leaf
water.
RESULTS
⌬18O Patterns of Vein Xylem Water
Water expressed from the petiole (␦petiole ⫽
⫺6.6‰ ⫾ 0.1‰ [⫾ se], n ⫽ 28) was not significantly
more enriched than the tap water (␦s ⫽ ⫺6.8‰ ⫾
0.1‰, n ⫽ 28) used to water the plants. At low
humidity (vapor-pressure deficit [VPD] of air, 2.4
kPa), the oxygen isotopic composition of petiole
water (␦petiole ⫽ ⫺6.5‰ ⫾ 0.1‰, n ⫽ 13) was similar
to its high-humidity (VPD of air, 1.0 kPa) counterpart (␦petiole ⫽ ⫺6.7‰ ⫾ 0.1‰, n ⫽ 15). In contrast
to the common assumption that the water pool in leaf
veins was unaltered with respect to the source water,
we noted an increasing 18O enrichment as xylem
water 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 usually lower (0.07‰–0.4‰) than that at high humidity
(0.5‰–0.9‰; Fig. 2A). However, enrichment near the
distal tip of the primary veins was similar (1.6‰–
1.7‰) at both levels of humidity. Compared with
primary veins, secondary veins (Fig. 2B) showed a
higher degree of enrichment ranging from 0.7‰ to
3.6‰. As before, upstream xylem water showed less
enrichment (0.7‰–1.6‰) than downstream water
near the vein endings (1.5‰–3.6‰). It appears that
xylem water near the vein endings tends to be more
enriched at low humidity than at high humidity,
whereas no consistent humidity-driven difference is
observed for the upstream xylem water of secondary
veins.
To compare the two humidity treatments, we were
careful in our choice of incision locations, ensuring
that the relative positions of various sap collection
points were retained. However, there is natural variation of venation from leaf to leaf. We also noted that
the isotopic signature of xylem water near the vein
endings where veins begin to taper was highly variable (deduced from the larger se; data not shown).
Rather than comparing results from two leaves of
different treatments, as presented in Figure 2, com-
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Plant Physiol. Vol. 130, 2002
18
O Spatial Patterns of Leaf Constituents
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 proportion
contributed by back-diffusion of enriched water from leaf evaporative sites. ⌬C refers to the enrichment of leaf water above
source 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 leaf
water sampling. The calculated ⌬C are 19.0‰ (low humidity) and 14.0‰ (high humidity). Data of ⌬vw represent the average
of three samples on different days. The mean vapor pressure deficits of air at low (RH 44%) and high (RH 75%) humidities
were 2.4 and 1.0 kPa, respectively. The letters A through F indicate vein incision locations for data in Table I.
parison is made from the same leaf with the same sap
collection points by changing the ambient humidity
during the experiment. On the basis of leaf water
volume and transpiration rate, we estimated the leaf
water turnover time to be approximately 9 min at low
humidity and 16 min at high humidity. Hence, 1 h
after a humidity step change was assumed to be
sufficient in achieving a new isotopic steady state. In
either a step change from low to high humidity or
vice versa (Table I), xylem water was mostly more
enriched at low humidity, with several incision
points showing a humidity-driven change in enrichment as high as 2.7‰. When ⌬vw is normalized
against ⌬C (the enrichment expected for leaf water
according to Eq. 1), the difference between humidity
treatments is not as distinct, even though the lowhumidity leaves generally show higher ⌬vw/⌬C values. The results also demonstrate that on approaching the vein endings, the xylem water was enriched
by 0.13 to 0.17 ⌬C. The effect of humidity change on
gas exchange parameters is summarized in Table I. In
general, increasing humidity led to an increase in
stomatal conductance and was accompanied by a
decrease in transpiration rate and modest changes in
assimilation rates.
⌬18O Patterns of Leaf Water and Organic Matter
A typical ⌬18O pattern of leaf mesophyll water for
a cotton leaf is shown in Figure 3. Leaf water clearly
is not isotopically uniform spatially and varies by as
much as 10‰ at low humidity. Closer examination
shows a trend to more enriched waters at the leaf
edge and intercostal regions, whereas discs at the leaf
base and adjacent to major veins are often less enPlant Physiol. Vol. 130, 2002
riched. This enrichment pattern is seen to be more
distinct when ⌬lw (the observed enrichment of leaf
water over petiole water) is plotted against ⌬C for the
6 d of sampling (Fig. 4). The isotopic compositions of
leaf water from marginal and intercostal regions
mostly fall above the 1:1 line, indicating enrichment
above that predicted by the Craig-Gordon model.
Those adjacent to veins and at the leaf base are generally more depleted than expected from the model.
At high humidity, there is less spatial heterogeneity of leaf water, where for certain days, enrichment
at the leaf edge could be small or the venous region
could be equally or more enriched than that predicted (Fig. 5A). The relatively more homogeneous
isotopic pattern at high humidity is also apparent in
the leaf dry matter (Fig. 5, B and C) where there is no
significant difference in ⌬om (isotopic enrichment of
leaf dry matter over source water) from one leaf zone
to another. Leaves grown in low humidity generally
have significantly higher ⌬om in the margin and intercostal regions and lower values for the venous and
basal regions. This reflects the measured isotopic
pattern of leaf water. Figure 6 shows that the adaxial
stomatal densities of the four leaf zones were not
significantly different from one another. However,
the abaxial stomatal density of the intercostal region
was slightly higher than that of the other three zones.
DISCUSSION
18
O Spatial Variation of Vein Xylem and Leaf Water
Water expressed from the petiole of an intact cotton
plant was not significantly more enriched than the
tap water used to water the plants. This is in agree-
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1011
Gan et al.
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 step
change 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 air
at 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 from
the experiments on leaf water sampling. For low 3 high humidity experiment, ⌬C ⫽ 18.7‰ (low humidity) and 13.8‰ (high humidity); high 3
low humidity experiment, ⌬C ⫽ 19.3‰ (low humidity) and 14.1‰ (high humidity). Different plants were used for each humidity step change
experiment. Gas exchange data were recorded for leaf 5 of both plants throughout the experiment. Parameters gs, E, and A refer to stomatal
conductance to water vapor diffusion, transpiration rate, and assimilation rate, respectively.
⌬vw (‰)
Low 3 High humidity
⌬vw/⌬C
High 3 Low humidity
Low H
High H
Difference
High H
Low H
1.4
2.2
1.3
1.2
⫺0.1
⫺1.0
1.7
1.7
2.2
1.9
B
2.4
1.3
1.0
1.3
⫺1.4
0.0
1.9
1.9
C
4.2
3.5
1.6
2.3
⫺2.7
⫺1.2
D
3.4
3.1
1.1
2.0
E
4.2
2.7
F
Mean
Incision
A
SE
Gas-exchange parameters
gs (mol m⫺2 s⫺1)
E (mmol m⫺2 s⫺1)
A (␮mol m⫺2 s⫺1)
1012
High 3 Low humidity
Low H
High H
Difference
High H
Low H
Difference
0.5
0.2
0.08
0.12
0.09
0.09
0.02
⫺0.03
0.12
0.12
0.11
0.10
⫺0.01
⫺0.02
2.6
1.9
0.7
0.0
0.13
0.07
0.07
0.10
⫺0.05
0.03
0.13
0.14
0.13
0.10
0.00
⫺0.04
1.6
2.5
2.6
3.4
1.0
0.9
0.23
0.19
0.11
0.17
⫺0.11
⫺0.02
0.11
0.18
0.13
0.18
0.02
0.00
⫺2.3
⫺1.1
1.8
1.9
1.9
2.6
0.1
0.7
0.18
0.16
0.08
0.14
⫺0.10
⫺0.02
0.13
0.13
0.10
0.13
⫺0.03
0.00
2.0
2.0
⫺2.2
⫺0.7
1.7
2.2
3.7
4.4
2.0
2.2
0.23
0.14
0.14
0.15
⫺0.08
0.00
0.12
0.15
0.19
0.23
0.07
0.07
4.6
4.4
2.8
3.2
⫺1.8
⫺1.2
1.4
2.0
3.4
3.0
2.0
1.0
0.24
0.23
0.20
0.23
⫺0.04
⫺0.01
0.10
0.14
0.18
0.16
0.08
0.01
3.1
0.3
1.8
0.2
⫺1.3
0.2
1.9
0.1
2.8
0.2
0.9
0.2
0.17
0.02
0.13
0.01
⫺0.04
0.01
0.13
0.01
0.14
0.01
0.01
0.01
0.47
11.2
24.0
0.75
7.5
24.8
0.3
⫺3.7
0.8
0.86
8.0
25.1
0.37
9.6
21.8
ment with previous observations (White et al., 1985;
Bariac et al., 1989, 1994) that there is little fractionation associated with uptake of soil water by roots, or
its subsequent movement up the stem. An exception
would be the fractionation of hydrogen isotopes in
mangroves (Lin and Sternberg, 1993). The absence of
isotopic fractionation during water uptake by most
plants has previously been used to support the assumption that water in leaf veins would similarly be
unenriched relative to the soil water (Leaney et al.,
1985). Previous sampling of vein water by pressurizing a detached leaf in a pressure chamber indicated
good agreement between the isotopic composition of
irrigation water and that assigned to vein water (Yakir et al., 1989). In contrast, based on vacuum distillation of excised vein segments, ␦2H of vein water
had been shown to increase along the main vein from
the leaf base to the tip (Luo and Sternberg, 1992). The
data of Figure 2 give direct evidence of xylem water
enrichment in the leaf veins of an intact transpiring
leaf. One possible contribution to the enrichment of
vein water is the back-diffusion of enriched water
from evaporative sites on the leaf lamina. The report
of the lateral escape of tritiated water from the xylem
vessels of tomato (Lycopersicon esculentum) internodes
being driven by diffusion (van Bel, 1976) lends sup-
Difference
Low 3 High humidity
⫺0.5
1.6
⫺3.3
port to this hypothesis. The tritiated water moved
down a concentration gradient and was mostly absorbed in the cells and cell walls around the xylem
vessels. The idea of progressive enrichment of vein
water by tissue water was first mooted by Yakir
(1992) and elaborated by Wang and Yakir (1995) and
Helliker and Ehleringer (2000) to explain the spatial
heterogeneity of leaf water. This phenomenon is
strongly supported by our observations that the isotopic composition of xylem water in veins (␦vw) is
responsive to changes in environmental conditions
such as humidity (Table I). Water at the evaporative
sites is more enriched at lower humidity and the
back-diffusion of this highly enriched water will consequently increase the extent of vein water enrichment at low humidity. ␦vw could be expressed mathematically as
␦vw ⫽ ␦s 䡠 (1⫺d) ⫹ ␦C 䡠 d
(6)
where d is the proportional contribution by backdiffusion of enriched water from leaf lamina. In
terms of enrichment over source water, Equation 6
can be re-expressed as
⌬vw ␦vw⫺␦s
⫽
⫽d
⌬C
␦ C⫺ ␦ s
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(7)
Plant Physiol. Vol. 130, 2002
18
O Spatial Patterns of Leaf Constituents
more apparent in the low-humidity leaves, an observation reinforced by the leaf dry matter isotopic pattern, ⌬om (Fig. 5C). Leaf dry matter has a significant
store of nonstructural carbohydrates (21% total dry
weight; Wong, 1990) from current assimilation, and
its isotopic composition has been shown to vary with
the diurnal changes of ⌬lw (Cernusak et al., 2002). In
addition, ⌬18O of cellulose, the major component in
leaf dry matter, has been shown to be strongly correlated to that of the dry matter of cotton leaves
(Barbour and Farquhar, 2000) and the leaf water of
grasses (Helliker and Ehleringer, 2002). Assuming a
constant lignin to cellulose ratio across the leaf, ⌬om
could be expected to reflect the spatial heterogeneity
of leaf water isotopic content arising from nontransient influences.
Anatomical Basis of Spatial Variation
Figure 3. A typical ⌬18O (‰) spatial variation of cotton leaf mesophyll water for the four lamina regions (margin, intercostal, venous,
and basal) at low humidity (RH 40%; VPD of air, 2.4 kPa). The
Craig-Gordon values of ⌬C (‰) ⫽ 22.1 (margin), 23.6 (intercostal),
23.9 (venous), and 24.4 (basal). Variation of ⌬C is attributable to the
gradual increase of leaf temperature with progressive sampling from
the margin to the base. Leaf discs containing a visible fine vein are
excluded from this representation.
The term ⌬vw/⌬C thus reflects the proportion of
vein water contributed by back-diffusion of enriched
water. Figure 2 shows that ⌬vw/⌬C increases downstream along the vein and in secondary veins compared with primary veins. This implies that backdiffusion is actively occurring throughout the vein
length regardless of the vein type. Thus, enriched
water continues to accumulate within the veins in the
direction of water flow.
Isotopic enrichment of leaf water (⌬lw) from the
leaf base to the tip has been observed in several crop
plants by Wang and Yakir (1995). Our results indicate
that leaf water enrichment should be more dependent on the relative distances from major veins because discs sampled adjacent to the veins are noted to
have smaller ⌬lw (Fig. 3). In general, leaf water from
the marginal and intercostal regions is more enriched
than the Craig-Gordon prediction, whereas that from
sites adjacent to veins and at the leaf base is more
depleted than expected (Figs. 4 and 5). This pattern is
Plant Physiol. Vol. 130, 2002
Measurements of stomatal density did not reveal a
spatial variation consistent with the leaf water measurements. Microscopic examination of the leaf vasculature also showed no apparent difference in the
venation density across the whole-leaf area. These
leaf anatomical studies show that the differential enrichment of leaf water across the leaf blade could not
simply be a direct result of particular leaf zones
having more or fewer evaporative sites or of entrapping more xylem water in the disc samples.
Figure 4. Comparison of the measured ⌬lw and Craig-Gordon predicted ⌬C of leaf water for the four lamina zones (margin [x], intercostal [⫹], venous [E], and basal [䡺]) of cotton leaves at low (black
symbols) and high (gray symbols) humidities. Three leaves sampled
on different days are shown for each humidity treatment. The mean
vapor pressure deficits of air at low (RH 35%) and high (RH 75%)
humidities were 2.8 and 1.0 kPa, respectively. The line represents a
1:1 relationship.
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1013
Gan et al.
and be feeding partially enriched water to surrounding cells and the neighboring veins (accounting for
enrichment in vein xylem water). The enriched water
would not only move along a series of cells but could
also be propagated through the veins to more distal
areas. This could account for the higher degree of
enrichment at the intercostal lamina regions and at the
leaf margin (representing terminal water elements).
The first quantitative application to leaves of the
Gat-Bowser formulation was by Helliker and
Ehleringer (2000). This approach requires the leaf
blade to be divided into a discrete, finite number of
evaporating elements. The authors chose seven elements to represent the whole evaporative process
occurring in grasses. The rationale behind this choice
was not discussed, and the number was presumably
chosen to give the best fit to the observed ␦lw or was
chosen out of convenience to match the number of
segments into which the whole blade was divided.
We found that based on Equation 5, isotopic enrichment at any given point along the water pathway is
sensitive to the total number of evaporating elements, even though the average enrichment over the
whole leaf is independent of this number (Fig. 7). A
smaller number of evaporating elements tends to
overestimate the isotopic enrichment near the leaf
base and underestimate it toward the leaf tip. The
Gat-Bowser formulation would need to be modified
for use in leaves where evaporative sites are continuous and non-discrete.
Different Isotopic Enrichment Patterns at
Different Humidities
Figure 5. Spatial differences in the four lamina regions (margin,
intercostal, venous, and basal) of cotton leaves. A, ⌬lw ⫺ ⌬C (same
data set as Fig. 4); B, oxygen isotope composition of leaf dry matter,
⌬om, obtained from leaf punches; C, ⌬om, obtained from trimming, at
low and high humidities. The mean vapor pressure deficits of air at
low (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 represent SE (n ⫽ 2–17). High-humidity treatments are gray.
The string-of-lakes model not only predicts an increasing isotopic enrichment but also dictates a different pattern of isotopic enrichment along the water
pathway for different humidity treatments, as depicted in Figure 7. At high humidity, the increase in
isotopic enrichment along the path of water flow is
much smaller than that at low humidity. Thus, we
Accounting for the 18O Spatial Variation of Leaf Water:
String-of-Lakes Model
On the basis of our observations of specific leaf
regions having ␦lw values above and below those
predicted by the Craig-Gordon equation, we deduce
that water flow in cotton leaves could probably behave somewhat like a string of interconnected evaporating lakes. The resemblance to this hydrological
model was first suggested by Yakir (1992) for maize
(Zea mays) and has been noted in a variety of dicotyledon 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 an
enrichment less than the Craig-Gordon prediction,
could represent the first elements in the string of lakes
1014
Figure 6. Stomatal densities on the four leaf lamina regions of a
cotton leaf. Adaxial (white) and abaxial (gray) stomatal densities are
expressed as number of stomata per square millimeter of leaf surface.
Error bars represent SEs (n ⫽ 3–4).
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Plant Physiol. Vol. 130, 2002
18
O Spatial Patterns of Leaf Constituents
adequately account for our observations of bulk leaf
water enrichment.
Lower Enrichment of Bulk Leaf Water Arising from
Capacitance in Vein Ribs
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 for
different RH (h) values. All lines are plotted by assuming 50 evaporating elements, of which the steps have been smoothed for clarity.
Inset, An expansion of the bottom portion of the main graph. Note
that ⌬ at very high humidity can be larger than that at low humidity
near the basal region. l/lm refers to the relative distance from the leaf
base 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.
would expect the isotopic composition of leaf water
at high humidity to be similar along most of the
water pathway. This expectation is largely confirmed
in the spatial variation of ⌬lw and ⌬om (Fig. 5). At
high humidity, isotopic enrichment of the leaf dry
matter is rather homogenous over the whole-leaf
area, unlike the definite isotopic pattern obtained at
low humidity.
Our results on the 18O spatial variation of vein
xylem water also support the prediction of different
isotopic enrichment patterns (as well as magnitudes)
at different humidities. Smaller ⌬vw/⌬C of upstream
xylem water was observed in the leaf veins at low
humidity, compared with the high-humidity leaves
(Fig. 2). At the vein endings, ⌬vw/⌬C at low humidity
is mostly greater than that at high humidity, though
this difference is not always statistically significant
(Table I). Although the string-of-lakes model served
well in predicting different spatial patterns of leaf
water enrichment at different humidity, it could not
Plant Physiol. Vol. 130, 2002
When cotton leaf water is extracted in bulk by
azeotropic distillation, ⌬lw, bulk is noted to be lower
than ⌬C despite the removal of big primary veins
from the leaves (Fig. 8). By solving Equation 3, the
fraction of leaf water subject to fractionation ( f ) is
␦lw,bulk⫺␦s ⌬lw,bulk
given by
⫽
with the assumption
␦ C⫺ ␦ s
⌬C
that the fractionated water is enriched at ⌬C. Thus, if
the vein water is assumed to be unfractionated according to the two-pool model, its proportion in the
bulk leaf water would be indicated by the term
⌬lw,bulk
1⫺
. However, if vein water is partially en⌬C
riched such that ⌬vw ⬎ 0, ␦s in Equation 3 will be
replaced by ␦vw and the proportion of vein water in
⌬lw,bulk
1⫺
⌬C
. Averagbulk leaf water will be given by
⌬vw
1⫺
⌬C
ing the low- and high-humidity treatments (Fig. 8),
there is approximately 30% unenriched water present
in the bulk leaf water of whole leaves with veins
intact. Upon the removal of primary veins, the pro-
Figure 8. Discrepancy between the measured ⌬lw, bulk of bulk leaf
water and the Craig-Gordon predicted ⌬C for leaves of cotton plants.
⌬lw,bulk
The term 1 ⫺
signifies the unenriched fraction present in the
⌬C
bulk leaf water (see “Discussion”). Comparison is made between
low- and high-humidity treatments. The vapor pressure deficits of air
at 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).
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1015
Gan et al.
portion of unenriched water in the bulk leaf water is
reduced to approximately 15%. This implies that the
water fraction associated with primary veins constitutes about 15%. In view of our earlier finding that
vein water can be enriched by as much as 0.19⌬C at
the vein endings (Fig. 2) and that the average vein
water enrichment is likely to be less than this value,
the water fraction contributed by the primary veins
should be higher, with the maximum possible estimated to be about 18.5%. In relatively close agreement with these observations, our independent assessment of this water fraction by gravimetric
analysis gave a value of 14.2% ⫾ 1.9% total leaf
water. Such a high proportion of non-fractionating
water has previously been noted (Leaney et al., 1985;
Walker et al., 1989) but criticized as unlikely (Luo
and Sternberg, 1992) given independent estimates of
vein water fraction to be ⱕ5%. Anatomical analysis
of mesophyll and vessel areas in young barley (Hordeum vulgare) leaves suggested 0.8% total tissue water was found in the lumen of vessels (Rayan and
Matsuda, 1988). Using a pressure bomb to express
water from a single leaf at increasing balancing pressure, Yakir et al. (1989) estimated the vein water
fraction to be 1% to 3% total leaf water in sunflower
(Helianthus annuus) and ivy leaves, and water in the
cell walls to be another 22% to 38%.
In our experiment, the removal of primary veins
not only excluded water in the big xylem vessels but
also removed the total water present in the massive
ground tissues of vein ribs. The layers of collenchyma forming the vein ridge are closely packed
with negligible air spaces for evaporative enrichment
and would be expected to have a large store of relatively unenriched water. Thus, the ground tissues of
vein ribs could be perceived as a capacitance having
little interaction with enriched water at the leaf evaporative sites, and its isotopic content is expected to be
similar to that of the vein xylem.
Lower Enrichment of Bulk Leaf Water Arising from
Péclet Effect
The two-pool model suggested by Leaney et al.
(1985) seemed to be appropriate in accounting for the
observed lower enrichment of bulk leaf water. However, the unenriched water fraction need not arise
from the vascular pool alone but could be attributed
to other factors. Our results show that the vein xylem
water is partly enriched, with the extent of enrichment depending on the ambient humidity. Isotope
mixing by diffusion can clearly be rather efficient in
leaves. Yet, the degree of enrichment in vein water
could not match that observed for the lamina tissue
water in the same vicinity (Figs. 2 and 3). Despite the
short distance between these two tissues, their enrichment difference can be as large as 20‰ or more.
It is likely that the mesophyll tissues are not directly
fed by the big veins but by some higher order fine
1016
veins that are relatively more enriched. Also, a Péclet
effect might be involved, with the transpiration flux
opposing the back-diffusion of enriched water in the
lamina and along the veins. As a result, the isotopic
content of bulk leaf water should be lower than that
predicted by the Craig-Gordon model.
It is noteworthy that the discrepancy between ⌬C
and ⌬lw, bulk of whole leaves, after normalization
against ⌬C, did not vary with the ambient humidity
despite a difference in leaf transpiration rates (low
humidity, 11.5 mmol m⫺2 s⫺1; high humidity, 8.8
mmol m⫺2 s⫺1; Fig. 8). Upon the removal of primary
⌬lw,bulk
veins, 1 ⫺
is larger at high humidity. In con⌬C
trast, the Péclet model (Eq. 4) predicts an increasing
discrepancy between ⌬lw and ⌬C with increasing
⌬lw,bulk
transpiration rate, E. That is, a larger 1 ⫺
is
⌬C
expected at lower humidity. There have been conflicting 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 plant
species grown under the same climatic conditions. The
positive relationship between ⌬C ⫺ ⌬lw, bulk and E,
illustrated by Wang and Yakir (2000) and Gillon and
Yakir (2000), appears to be in agreement with the
Péclet model but may well collapse after normalization against ⌬C. Our results show that the discrepancy
(⌬C ⫺ ⌬lw, bulk) at low humidity is 2.5‰ larger than
that at high humidity. This positive correlation with
transpiration rate is wiped out upon normalization
against ⌬C, as shown in Figure 8. Nonetheless, a pos⌬lw,bulk
itive trend of 1 ⫺
with E, consistent with the
⌬C
Péclet model, has been reported (Walker et al., 1989;
Flanagan et al., 1994; Barbour et al., 2000). All of the
studies mentioned made direct isotopic measurements
of extracted leaf water except for that of Barbour and
co-workers (2000). The latter demonstrated a convincing positive relationship between 1 ⫺ (⌬sw/⌬C) and
transpiration rate based on the isotopic composition of
phloem Suc in castor bean (Ricinus communis), where
⌬sw refers to the deduced isotopic composition of leaf
water with which the Suc exchanges. Our conflicting
⌬lw,bulk
result, a negative relationship between 1 ⫺
and
⌬C
transpiration rate upon the removal of primary veins,
requires resolution. First, we cannot rule out the possibility of higher leaf water enrichment by evaporative
loss from the cut edges during vein removal at low
humidity. Second and perhaps more important, water
in the primary veins consistently has lower ⌬vw/⌬C at
low humidity, the difference from that at high humidity being up to 6% (Fig. 2A). In accordance, the removal of primary veins at low humidity would take
away a larger proportion of unenriched water, giving
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Plant Physiol. Vol. 130, 2002
18
⌬lw,bulk
value. Because vein water is an
⌬C
intrinsic component of the leaf water enrichment system, we recommend that for comparative studies,
bulk leaf water should be extracted from whole leaves
without any vein removal. Nonetheless, we could not
⌬lw,bulk
identify a clear relationship between 1 ⫺
and
⌬C
transpiration rate based on the bulk leaf water extraction from whole leaves of cotton.
a smaller 1 ⫺
Applying the String-of-Lakes Model to Leaves for ⌬18O
Leaf Water Prediction
Assuming all water pools have equal evaporation
rates, the average isotopic content of water in the
interconnected pools of the string-of-lakes model
should, according to Equation 5, be equivalent to the
Craig-Gordon predicted ⌬C. To test this assumption
and to check the applicability of the string-of-lakes
model in estimating leaf water isotopic content, a
weighted mean of measured ⌬lw was computed from
the leaf discs sampling. Using a distribution ratio of
4.7:10.7:4.3:1 for regions of margin:intercostal:venous:
basal, weighted means of both the measured and
modeled leaf water isotopic compositions were obtained (Table II). At low humidity, the weighted mean
⌬lw was 9.2% higher than the weighted mean ⌬C, and
11.8% higher in the case of high humidity. Although
we expect the weighted mean of ⌬lw from leaf punches
to be larger than ⌬lw, bulk because of the exclusion of
veins in leaf discs sampling, the weighted mean of ⌬lw
being larger than the Craig-Gordon prediction clearly
diverged from the expectations of all leaf water models. A higher than expected ⌬lw has previously been
encountered (Flanagan et al., 1993; Helliker and
Ehleringer, 2000), and the latter group explained their
observations by applying the string-of-lakes model,
O Spatial Patterns of Leaf Constituents
with water pools having variable transpiration rates
across the length of the leaf. In our experiment, the
sampling of the leaf discs was biased given that the
leaf punch could not sample within 1 mm of the major
veins without rupturing the veins. Thus, the low degree of enrichment expected in the immediate vicinity
of the veins was omitted from our sampling. This may
partially account for the higher value of weighted
mean ⌬lw compared with ⌬C. It presumably explains
the discrepancy with our bulk leaf water measurements, which consistently show enrichment less than
the Craig-Gordon prediction.
It has been suggested that most of the discrepancies
between modeled and measured leaf water isotopic
compositions could be resolved by careful estimation
of the kinetic fractionation factor, ⑀k (Buhay et al.,
1996). As expected from Equation 2, ⑀k and consequently ⌬C are sensitive to the boundary layer conductance to water vapor diffusion (gb), especially at
the lower range of gb (Fig. 9). Increasing gb from 0.5
to 2.5 mol m⫺2 s⫺1 leads to a calculated enrichment of
leaf water by 2‰, whereas a gb increase from 2.5 to
5.0 mol m⫺2 s⫺1 results in only 0.5‰ enrichment. The
degree of uncertainty imposed by ⑀k estimation on
leaf water isotopic composition will thus be greatly
reduced with a highly turbulent boundary layer. In
our study, boundary layer conductance in the greenhouse determined by a mass transfer method was
0.52 mol m⫺2 s⫺1. In view of the sensitivity of ⌬C to
gb in this region, the value of gb was verified by
another method based on heat transfer and wind
velocity. A value of 0.45 mol m⫺2 s⫺1 was obtained.
Because minimal difference was noted between the
two 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-oflakes model to leaves, Wang and Yakir (1995) pro-
Table II. Comparison of the weighted mean of measured ⌬lw and Craig-Gordon predicted ⌬C of
cotton 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 a
weighting ratio of margin:intercostal:venous:basal ⫽ 4.7:10.7:4.3:1. Leaf sampling was carried out on
different days.
Time of Sampling
Low humidity
1115–1500
1000 –1225
0950 –1140
Mean
SE
High humidity
1000 –1240
1315–1535
1015–1330
Mean
SE
Plant Physiol. Vol. 130, 2002
Weighted Mean
⌬lw
Weighted Mean
⌬C
25.7
27.3
25.9
26.3
0.5
18.3
22.0
18.4
19.6
1.2
⌬lw ⫺ ⌬C
(⌬lw/⌬C) ⫺ 1
23.3
25.1
23.9
24.1
0.5
2.4
2.2
2.0
2.2
0.1
0.105
0.088
0.082
0.092
0.007
16.9
19.4
16.2
17.5
1.0
1.4
2.6
2.2
2.1
0.4
0.082
0.135
0.138
0.118
0.018
‰
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1017
Gan et al.
Implications of Leaf Water Spatial Heterogeneity
Figure 9. Relationship of the modeled ⌬C (solid line) and kinetic
fractionation ⑀k (dashed line) with boundary layer conductance gb at
low and high humidities. Modeled ⌬C and ⑀k are calculated using
Equations 1 and 2, respectively. Units for gb and gs (stomatal conductance) are moles per square meter per second; ea/ei refers to the
ratio of the vapor pressures in the atmosphere and intercellular
spaces.
posed that the process of back-diffusion along the
string of water elements (a phenomenon clearly supported by the xylem water isotopic pattern) be incorporated into the Gat-Bowser formulation. We envisage back-diffusion of enriched water occurring in
two dimensions; along the leaf length in the basipetal
direction and diffusion from the evaporative sites
into the vein network (radial dimension), which further supplies the enriched water to other series of
cells further afield. However, the advective transpiration flux should oppose this diffusion process (Péclet effect), leading to two-dimensional (2-D) gradients of isotopic variation (Fig. 1D). Simulation of a
2-D model of leaf water using a 2-D advection diffusion program was described by Yakir (1998). He
modeled the leaf as a square domain having a specified leaf thickness, with water entering from the side
and exiting by evaporation from the top, with no flux
assumed at other boundaries. The modeled isotopic
gradients of leaf water have clear 2-D characteristics
that collapse to uni-dimensional during the night
when evaporation stops. His findings distinctly exemplify the significant role of transpiration flux in
modulating the spatial variation of leaf water enrichment during the day.
1018
Despite the spatial heterogeneity of leaf water, Barbour et al. (2000) have shown that the isotopic label of
sugars exported from castor bean leaves behaves as
predicted by the Péclet model. This observation is
made from gas exchange leaf chamber experiments
under optimum conditions where assimilation rates
across the leaf area are most likely uniform. However, for nonuniform assimilation rates across the
leaf, spatial heterogeneity of leaf water could pose a
challenge to our present applications of leaf water
isotopic composition as indicators of plant environment and terrestrial productivity (Yakir, 1998). If assimilation rates are higher, for example, near the leaf
tip where leaf water enrichment is higher than the
Craig-Gordon prediction, the ⌬18O of retrodiffused
CO2 would be greater and could be misinterpreted as
higher terrestrial productivity. Such uneven distribution of assimilation rate would also have implications
for the interpretation of the mean isotopic signature
of sugars formed from the entire leaf as well as the
18
O content of O2 produced during terrestrial photosynthesis and the interpretation of the Dole effect
(the balance between terrestrial and marine productivity based on the deviation from 23.5‰ in the ␦18O
of atmospheric O2 [Bender et al., 1985, 1994]). Nonuniform assimilation rates are most likely to occur
when the amount of light incident on a leaf is inconsistent across the leaf, leading to spatial variations of
photosynthetic capacity and 13C discrimination, 13⌬
(Meinzer and Saliendra, 1997). We expect dicotyledoneous leaves, in their natural orientation, to receive more uniform incident rays than long, flagging
blades of monocotyledoneous plants. This expectation is supported by the uniform distribution of 13⌬
across beech (Fagus spp.) leaves (Schleser, 1990) and
increasing 13⌬ from the base to the tip of leaves of
sugarcane (Saccharum officinarum; Meinzer and Saliendra, 1997) and maize (Sasakawa et al., 1989). The
error introduced by the spatial heterogeneity of leaf
water in the applications mentioned will thus be a
greater concern in grasslands where their growth
humidity is generally low and large isotopic gradients are expected across the leaf, as illustrated in
Figure 5.
CONCLUSIONS
The three leaf water models examined in this paper
(two-pool model, Péclet model, and string-of-lakes
model) are found to be valid in describing the following features of leaf water enrichment. The overestimation of the isotopic enrichment of bulk leaf
water by the Craig-Gordon model is well addressed
by the two-pool and Péclet models, whereas spatial
heterogeneity of leaf water enrichment can be expected from the Péclet and string-of-lakes models.
But none of the models on its own could fully account for all of the facets of leaf water enrichment.
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Plant Physiol. Vol. 130, 2002
18
O Spatial Patterns of Leaf Constituents
ture supplied with slow-release fertilizer (Osmote Plus, Scotts-Sierra,
Maysville, OH). The plants were grown under full sunlight between late
summer 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% and
75% ⫾ 10%) maintained day and night. Tap water was used to water the
plants twice daily. All samplings and measurements were carried out on 35to 55-d-old plants in the greenhouses on cloud-free days during the time
period of 11 am to 3 pm when plant photosynthesis was at its maximum and
gas exchange was observed to be at steady state.
Gas-Exchange Measurements
Leaf gas-exchange measurements were taken during sunny days using a
portable gas-exchange system (model LI-6400, LI-COR, Lincoln, NE)
equipped with the standard leaf chamber and the CO2 injector system for
the control of CO2 concentration. Measurements were made at a photosynthetic photon flux density of 1,200 ␮mol m⫺2 s⫺1 from an LED light source.
Boundary Layer Conductance Measurements
Figure 10. Cut-away view of the leaf punch device. The punch is
hollow with sharp edges to cut a leaf disc and to deliver argon for
directing the disc into the tin cup as well as purging the cup. The cup
can be immediately sealed with the push of a button that pneumatically operates the pincer arms.
For example, although a gradient of isotopic enrichment is projected by both the Péclet and the stringof-lakes models, only the latter model correctly predicts the different isotopic enrichment patterns at
different humidities. Yet, the latter model could not
adequately account for the lower degree of enrichment in bulk leaf water, which the two-pool model
and the Péclet model could. Also, the observed partial enrichment of vein xylem water is within expectations of the Péclet model, whereas the other two
either assume no enrichment of vein water (two-pool
model) or an enrichment equivalent to that at the
neighboring evaporative sites (string-of-lakes model). On the other hand, we could not identify a clear
⌬lw,bulk
relationship between 1 ⫺
and transpiration
⌬C
rate, a correlation expected from the Péclet model.
Our findings acknowledge the presence of capacitance in the ground tissues of vein ribs that are likely
to have similar isotopic contents to the vein xylem.
This paper also draws attention to the need for modification of the Gat-Bowser formulation of the stringof-lakes model to accommodate continuous, nondiscrete evaporating elements and for incorporating
Péclet effects along the longitudinal and radial dimensions into the string-of-lakes model.
MATERIALS AND METHODS
Plant Material and Growth Conditions
Seeds of cotton (Gossypium hirsutum L. var Deltapine 90) plants were
sown in 4.5-L polyvinylchloride pots containing sterilized garden soil mix-
Plant Physiol. Vol. 130, 2002
The boundary layer conductance to water vapor diffusion (gb) in the
greenhouses 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 temperature
of the paper leaf model was constantly monitored using an infrared thermometer (Mikron Instrument, Oakland, NJ) with a resolution of 0.1°C. The
boundary layer conductance obtained was 0.52 ⫾ 0.04 mol m⫺2 s⫺1. For
verification, gb was also determined using the heat transfer method. The
wind speed in the greenhouse, 0.34 ⫾ 0.10 m s⫺1, was measured using an
ultrasonic anemometer (Gill Instruments, Lymington, Hampshire, UK). Using equations given by Ball et al. (1988) for calculating the boundary layer
resistance to water vapor based on heat transfer in a forced convection, we
obtained 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 intact
leaf was sampled concurrently from primary and secondary veins using a
root pressure chamber (Yong et al., 2000). While the whole root system of
the plant was enclosed and pressurized in the chamber, a light incision was
made on the leaf vein with a sharp razor blade. The pneumatic pressure was
adjusted to give a xylem sap flow of about 0.5 ␮L s⫺1 exuding from the finer
veins. A sample of 0.7 ␮L of xylem water was directly siphoned off with a
pipette (2-␮L micro-pipetteman, Gilson Medical Electronics, Middleton, WI)
from the water bead formed at the incision point immediately after wiping
off the earlier exudate. The collected sap was quickly dispensed into a
smooth-walled tin cup (4.5 ⫻ 2 mm) and sealed under argon with a modified Carlo Erba liquid encapsulator equipped with a pneumatic actuator. To
collect more xylem water for storage, a 10-␮L capillary was held steady
against the incision, and its ends were immediately sealed with sealing wax
after filling. Sampling order followed the general rule of first scoring the
vein endings in the vicinity of the leaf margin and gradually moving
inwards toward the leaf base where vein diameter progressively increases.
This minimized water-flow disruptions to areas yet to be sampled. The
petiole of the same leaf was eventually cut to collect petiole water after the
completion of vein xylem water sampling.
To determine humidity effects on the isotopic composition of vein xylem
water, a step change of humidity was carried out in another experiment.
Xylem water was collected from the same vein incision points before and 1 h
after the step change of humidity.
Leaf Water and Organic Matter Sampling
To analyze leaf mesophyll water of cotton leaves without the inclusion of
vein 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 also
activates a jet of argon that guides the disc to fall directly into a preweighed
smooth-walled tin cup (9 ⫻ 3.5 mm). The argon jet also flushes out air and
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1019
Gan et al.
hence excludes nitrogen and oxygen from the cup. The filled cup is immediately sealed with the push of a button (pneumatic actuation) and can be
directly pyrolyzed before analysis in an Isotope Ratio Mass Spectrometer.
Sampling began from the leaf margin and worked inward toward the petiole
to minimize water-flow disruptions to the leaf lamina yet to be sampled.
Leaf temperature profiles were captured using an IR scanner with a sensitivity of 0.1°C (Thermovision 870, AGEMA, Danderyd, Sweden) after every
two to three leaf punches. Regular monitoring of leaf temperature was
essential because we noted that leaf temperature in the sampling region
would gradually climb by 1°C to 2°C after cutting several leaf discs. After
punching out leaf discs from one side of the midrib, leaf temperature was
again recorded by the IR scanner before the other one-half was trimmed,
avoiding primary veins. The trimmed leaf segments were immediately
immersed in toluene (80 mL) for bulk leaf water extraction by azeotropic
distillation using a specially designed funnel as described by Revesz and
Woods (1990). The leaf-half containing the punch holes was pressed between layers of paper and dried in a 70°C oven. Small dry leaf segments in
the vicinity of each punch hole were collected for isotopic analysis of leaf
organic matter.
To map spatial isotopic distribution of leaf organic matter, whole leaves
of cotton from leaf positions 5 and 7 were harvested from each of three
plants. The leaf blades were trimmed into four distinct zones before oven
drying. 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 lamina
collected at the leaf base within approximately 20 mm of the petiole.
Thereafter, the dried leaf segments were ground for isotopic analysis of leaf
organic matter. To determine the distribution ratio of the four lamina zones,
two fresh leaves were photocopied on paper, and each leaf area was divided
into the four zones mentioned. From weighing the paper, the relative ratio
of the four zones (margin, intercostal, venous, and basal) was found to be
4.7:10.7:4.3:1, respectively.
For bulk leaf water analysis, whole leaves were sampled, and leaf water
was extracted by azeotropic distillation, with some replicates having the
primary veins removed. To determine the percentage of water present in the
primary veins, leaf fresh weight was first measured, followed by the removal of primary veins. The fresh weights of the remaining lamina and the
primary rib skeleton were noted before oven drying. The leaf water content
and water associated with the primary ribs were obtained from the difference between the fresh and dry weights of the lamina and primary ribs.
Throughout the period of leaf water sampling, atmospheric water vapor
was collected using a dry ice-ethanol cold trap. Over 20 d of leaf water
measurements, ␦v ⫽ ⫺12.6‰ ⫾ 1.3‰ (sd).
Oxygen Isotope Analysis
Oxygen isotopic analyses of water and dried and fresh leaf samples were
all performed using the continuous-flow pyrolysis technique described by
Farquhar et al. (1997) with slight modifications. To minimize memory
problems, the reaction column was packed with glassy carbon grit (3,150–
4,000 ␮m, HTW, Thierhaupten, Germany) followed by a top layer (0.015 m
thick) of nickelized carbon (50% [w/w] Ni, Alpha Resources, Sydney). The
same analytical precision of 0.2‰ was achieved after reactor modification.
Elemental oxygen standards were beet Suc and ANU-HP water, with the
latter (␦18O ⫽ ⫺5.5‰) also serving as the isotopic internal standard for
water samples. A standard of beet Suc containing 3% (w/w) nitrogen was
used for correcting isotopic composition of dry leaf samples because the
latter were found to contain about 3% (w/w) nitrogen. Isotopic calibration
agreement between water and organic standards has previously been performed (Gan et al., 2002), and a slope of 1.0027 was obtained over an isotopic
range of 90‰. Preparation of liquid samples for pyrolysis was similar to that
for leaf xylem water sampling. Dried leaf samples of 1.0 to 1.5 mg were
accurately weighed into tin capsules that were then crimped manually.
Smooth-walled tin cups containing fresh leaf samples were directly pyrolyzed. The fresh weight of the leaf disc was obtained from the difference
between the total weight of the tin cup with the leaf disc and the weight of
the empty tin cup. Direct pyrolysis of fresh and dry leaf samples gives the
measured values of ␦18OF and ␦18OD, respectively. The 18O ratio of leaf
water (␦18Olw) from the fresh leaf sample can be calculated by isotopic mass
balance,
␦18OF ⫽ x␦18Olw ⫹ (1⫺x)␦18OD
1020
where x refers to the proportion contributed by leaf water in the total
oxygen pool of the fresh leaf sample. The value of x was determined from
the thermal conductivity detector output of the gas chromatograph in the
same acquisition as the 18O analysis, the calculations of which are detailed
in Gan et al. (2002). Overall,
18
O )
16 D 18
[␦ OF⫺␦18OD]
OF⫺OD
OF(1⫺
␦ Olw ⫽ ␦ OD ⫹
18
18
where OF and OD are, respectively, the oxygen elemental composition of
fresh and dry leaf samples as obtained from the thermal conductivity
detector output of the gas chromatograph.
Stomatal Density Distribution
Fresh cotton leaf segments of approximately 0.25 to 0.50 cm2 were
trimmed from various places on the leaf lamina and placed on carbon paste
before being frozen on a metal support precooled in liquid nitrogen. Scanning electron microscope (S-2250N Hitachi, Tokyo) images were taken
under standard conditions of 25 kV, 16 mm working distance, and low input
of water vapor. To quantify the distribution of stomatal densities over the
whole-leaf surface, we targeted four locations: margin, intercostal, venous,
and basal. For every location, images of three to four different areas were
taken for each abaxial and adaxial surface. Each image represented an area
of 0.343 mm2 (the field of view at a magnification of 200⫻). The number of
stomata was counted directly from the prints and expressed as number of
stomata per mm2 of leaf surface.
Analysis of Leaf Vasculature
Leaf pigments and cell contents were cleared by incubating the fresh leaf
in methanol at 60°C for 1 h, followed by immersion in warm lactic acid for
5 to 10 min. The venation pattern and density were examined under a light
microscope.
ACKNOWLEDGMENTS
We thank Peter Groeneveld and Jim Neale for helping to design and
construct the leaf punch, Josette Masle for assistance with the analysis of leaf
vasculature, and an anonymous reviewer for helpful comments. The technical support rendered by the Electron Microscopy Unit (Australian National University) is much appreciated.
Received April 23, 2002; returned for revision May 30, 2002; accepted June
14, 2002.
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