Introduction
The oxygen isotopic composition has been used to trace water transport
into and through plants such as the identification of sources water
taken up by a plant (Dawson & Ehleringer
1991; Ellsworth & Sternberg 2014) and
water movement through the leaf (Barbour &
Farquhar 2004; Barbour et al.2017a). However, the use of oxygen isotopic composition to trace water
transport is dependent on understanding the factors that influence water
movement and the physical and biological processes that fractionate
oxygen isotopes in water. For example, physiological factors such as
transpiration (E ) and leaf anatomical structures can influence
water flow within the leaf and the degree of fractionation, impacting
oxygen isotopic composition in leaf water
[δ18OLW](Yakir
1998; Barbour & Farquhar 2000;
Barbour et al. 2000a;
Barbour 2007;
Cernusak et al. 2016;
Holloway-Phillips et al. 2016).
Because of these complex interactions of
δ18OLW with leaf physiology and
anatomy, it is presently difficult to draw clear conclusions from the
experimental data (Barbour et al.2017a). Therefore, a better understanding of how water movement within
a leaf interacts with the physical and physiological processes must
first be understood to be able to fully exploit
δ18OLW(Cernusak et al. 2016;
Barbour et al. 2017a).
Multiple models have been developed to help explain how environment and
physiological processes influence
δ18OLW. For example, the two-pool
(Roden & Ehleringer 1999), the
string-of-lakes models (Gat & Bowser
1991), and the Péclet effect (Barbouret al. 2000b; Barbour et al.2004) have been developed to help explain how evaporatively-enriched
water at the sites of evaporation and unenriched
δ18Osw influence
δ18OLW. In all of these models,
unenriched water supplied through the xylem becomes enriched at the
sites of evaporation based on a modified Craig-Gordon model, where
enrichment is a function of relative humidity, leaf temperature, and
oxygen isotopic composition of vapor (See Theory section;
Craig & Gordon 1965;
Farquhar et al. 1998). The
two-pool model operates under the principles of mass balance where
unenriched water in the xylem and enriched water from the sites of
evaporation (δ18Oe) are considered two
discrete water pools that mix to form
δ18OLW. Alternatively, the
string-of-lakes model used in grasses considers that instead of two
discrete water pools there are multiple discrete pools in series up the
leaf blade, where the source water to each pool is the water from the
pool preceding it. This leads to a progressive enrichment of water from
the first pool at the base of the leaf blade to the last pool towards
the leaf tip. However, the two-pool and the string-of-lakes models do
not address cases when δ18OLW varies
with E (Farquhar & Lloyd 1993).
The Péclet effect model accounts for the continuous gradation from
unenriched water moving through the xylem to enriched water at the sites
of evaporation through opposing flows of advection and back-diffusion
(Barbour et al. 2000a;
Barbour et al. 2000b). These
opposing flows in the Péclet model are dependent on E and the
effective mixing length over which unenriched water from the xylem mixes
with enriched water from the sites of evaporation (L ). Lcan be unique to each tissue type where these two isotopically-different
water pools are mixing, such as in the mesophyll as water moves from the
xylem to the inner-stomatal cavity [L m] or in
the veins where enriched mesophyll water mixes with unenriched xylem
water [L v]
(Holloway-Phillips et al. 2016).
In this manner, isotopic mixing occurs radially from the xylem out to
the stomata and longitudinally in the veins where enriched water enters
the veins through back diffusion and is carried toward the leaf tip
(Farquhar & Gan 2003). Leaf traits such
as cell wall composition that can affect the extent that mixing occurs
by influencing the length and tortuosity od the path that water takes
through the xylem and mesophyll (L ) and flow or conductivity
(E ), which, in turn, affect
δ18OLW beyond the effect that
δ18OSW has. Each of these models have
found empirical support under specific conditions and for certain
species; however, none of them can universally explained the isotopic
behavior of leaf water (Roden &
Ehleringer 1999; Barbour & Farquhar
2000; Barbour et al. 2000b;
Helliker & Ehleringer 2000;
Barbour & Farquhar 2004;
Loucos et al. 2015;
Song et al. 2015;
Holloway-Phillips et al. 2016).
However, the Péclet effect model is unique in that it can be used to
test hypotheses on how leaf anatomical features affect the movement of
leaf water and influence δ18OLW.
Specifically, this model can be used to test how leaf traits influence
the advection of water from the xylem to the sites of evaporation and
back diffusion of water from the sites of evaporation into the mesophyll
and xylem, which determines the influence of the enrichment of water at
the sites of evaporation above source water
(Δ18Oe) on leaf water enrichment above
source water (Δ18OLW). In other words,
anatomical features that potentially influence the mixing of water pools
and the flow of water out of the leaf (E ) or that impact Lcan be tested on how they alter
Δ18OLW. For example, greater vein
density increased the oxygen isotopic enrichment along the grass leaf
blade because it increased mixing of enriched mesophyll water with xylem
water, leading to the progressive enrichment of xylem water and
Δ18Oe up the leaf blade and
subsequently Δ18OLW(Helliker & Ehleringer 2000;
Farquhar & Gan 2003). In dicots, greater
vein density had the opposite effect in that
Δ18OLW was lower because the
proportion of unenriched xylem water (f sw) was
greater (Holloway-Phillips et al.2016). Stomatal density and pore size also influence
Δ18OLW, where greater stomatal
densities, even when E remained the same, had higher
Δ18OLW because L decreased with
stomatal density (Sternberg & Manganiello
2014; Larcher et al. 2015;
Liang et al. 2018). These findings
have led to increased interest in anatomical features such as cell wall
composition that partly control the permeability of the cell wall and,
in turn, influence the relative importance of apoplastic water movement
from the xylem to the sites of evaporation, which ultimately affectsE and L (Barbour et
al. 2017b).
Cell wall composition mutations modify how leaf anatomy influences water
movement andphysiological properties that affect E , L ,
and, ultimately, Δ18OLW. For example,
suberin in the suberin lamellae provides a barrier to apoplastic water
movement and passive water loss from the vasculature
(Mertz & Brutnell 2014;
Vishwanath et al.2015).Zmasft double mutants, which were created in maize
(Zea mays L.) by mutating two paralogously duplicated, unlinked
maize orthologues of Arabidopsis thaliana ALIPHATIC SUBERIN
FERULOYL TRANSFERASE , have a compromised suberin llamelae
ultrastructure that increased cell wall elasticity and apoplastic water
diffusion (Mertz et al. In
review). Greater apoplastic diffusion and cell wall elasticity can
increase available water for transpiration, while less barriers to water
movement can increase the number of water channels and modify the
relative importance of each channel that water takes in and out of the
leaf vasculature and from the vasculature to the stomata, influencingL m and L v. Cellulose
synthase-like F6 (CslF6 ) in rice (Oryza sativa L.)
mediates the biosynthesis of mixed linkage glucan (MLG), a
polysaccharide important in cell wall composition
(Vega-Sánchez et al. 2012).
Loss-of-function mutants do not synthesize MLG
(Vega-Sánchez et al. 2012;
Smith-Moritz et al. 2015) and have
higher cell wall porosity, which has been show to affect the internal
conductance of CO2 (g m)
(Ellsworth et al. 2018). This
increase in porosity affected the movement/conductance of
CO2 and may also alter apoplastic water movement as
well, which can alter water flow to the stomates (E ) or the
length and tortuosity of water path (L ). Therefore, these mutants
provide a means to evaluate the effect of cell wall composition on leaf
physiology with respect to water transport through its impact on
Δ18OLW.
To determine the influence of cell wall properties on
Δ18OLW we tested how the loss of MLG
and suberin changed water movement from the xylem to the sites of
evaporation under environmental conditions (growth light and relative
humidity) that have been demonstrated to influence how E andL impact Δ18OLW. The data
presented here demonstrates that changes in both cell wall properties
and stomatal density influence Δ18OLWthrough the impact of E and L on the Péclet effect.