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\title{Leaf cell wall properties and stomatal density influence oxygen isotope
enrichment of leaf water}



\author[1]{Patrick Ellsworth}%
\author[2]{Patricia Ellsworth}%
\author[3]{Rachel Mertz}%
\author[4]{Nuria Koteyeva}%
\author[2]{Asaph B. Cousins}%
\affil[1]{USDA-ARS Southeast Area}%
\affil[2]{Washington State University}%
\affil[3]{Inari Agriculture}%
\affil[4]{V. L. Komarov Botanical Institute of Russian Academy of Sciences}%


\vspace{-1em}



  
  \date{April 28, 2026}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Oxygen isotopic composition (\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW})
of leaf water can help improve our understanding of how anatomy
interacts with physiology to influence leaf water transport. Leaf water
isotope models of \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} such as the
P\selectlanguage{ngerman}éclet effect model have been developed to predict
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}, and it incorporates
transpiration rate (\emph{E}) and the mixing length between unenriched
xylem water and enriched mesophyll water, which can occur in the
mesophyll (\emph{L}\textsubscript{m}) or veins
(\emph{L}\textsubscript{v}). Here we used two cell wall composition
mutants grown under two light intensities and RH to evaluate the effect
of cell wall composition on \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}. In
maize (\emph{Zea mays}), the compromised ultrastructure of the suberin
lamellae in the bundle sheath of the ALIPHATIC SUBERIN FERULOYL
TRANSFERASE mutant (\emph{Zmasft}) reduced barriers to apoplastic water
movement, resulting in higher \emph{E} and \emph{L}\textsubscript{v}
and, consequently, lower \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}. In
cellulose synthase-like F6 (\emph{Cslf6}) mutants and wildtype of rice
(\emph{Oryza sativa}), the difference in
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} in plants grown under high and
low growth light intensity co-varied with their differences in stomatal
density. These results show that cell wall composition and stomatal
density influence \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} by altering
the P\selectlanguage{ngerman}éclet effect and that stable isotopes can facilitate the
development of a physiologically and anatomically explicit water
transport model.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy




\textbf{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 \emph{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 (\emph{E} ) and leaf
anatomical structures can influence water flow within the leaf and the
degree of fractionation, impacting oxygen isotopic composition in leaf
water {[}\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}{]}(Yakir 1998; Barbour
\& Farquhar 2000; Barbour \emph{et al.} 2000a; Barbour 2007; Cernusak
\emph{et al.} 2016; Holloway-Phillips \emph{et al.} 2016). Because of
these complex interactions of \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}
with leaf physiology and anatomy, it is presently difficult to draw
clear conclusions from the experimental data (Barbour \emph{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
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}(Cernusak \emph{et al.} 2016;
Barbour \emph{et al.} 2017a).

Multiple models have been developed to help explain how environment and
physiological processes influence
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}. For example, the two-pool
(Roden \& Ehleringer 1999), the string-of-lakes models (Gat \& Bowser
1991), and the P\selectlanguage{ngerman}éclet effect (Barbour\emph{et al.} 2000b; Barbour
\emph{et al.}2004) have been developed to help explain how
evaporatively-enriched water at the sites of evaporation and unenriched
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{sw} influence
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}. 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 \emph{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
(\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e}) are considered two discrete
water pools that mix to form \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}.
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
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} varies with \emph{E} (Farquhar
\& Lloyd 1993).

The P\selectlanguage{ngerman}éclet 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 \emph{et al.} 2000a; Barbour \emph{et al.} 2000b). These
opposing flows in the Péclet model are dependent on \emph{E} and the
effective mixing length over which unenriched water from the xylem mixes
with enriched water from the sites of evaporation (\emph{L} ).
\emph{L}can 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 {[}\emph{L}
\textsubscript{m}{]} or in the veins where enriched mesophyll water
mixes with unenriched xylem water {[}\emph{L} \textsubscript{v}{]}
(Holloway-Phillips \emph{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 (\emph{L} ) and flow or
conductivity (\emph{E} ), which, in turn, affect
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} beyond the effect that
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{SW} 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 \emph{et al.} 2000b; Helliker \& Ehleringer 2000; Barbour
\& Farquhar 2004; Loucos \emph{et al.} 2015; Song \emph{et al.} 2015;
Holloway-Phillips \emph{et al.} 2016).

However, the P\selectlanguage{ngerman}éclet 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 \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}.
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
(\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e}) on leaf water enrichment above
source water (\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}). In other words,
anatomical features that potentially influence the mixing of water pools
and the flow of water out of the leaf (\emph{E} ) or that impact
\emph{L}can be tested on how they alter
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}. 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
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} up the leaf blade and
subsequently \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}(Helliker \&
Ehleringer 2000; Farquhar \& Gan 2003). In dicots, greater vein density
had the opposite effect in that \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}
was lower because the proportion of unenriched xylem water (\emph{f}
\textsubscript{sw}) was greater (Holloway-Phillips \emph{et al.}2016).
Stomatal density and pore size also influence
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}, where greater stomatal
densities, even when \emph{E} remained the same, had higher
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} because \emph{L} decreased with
stomatal density (Sternberg \& Manganiello 2014; Larcher \emph{et al.}
2015; Liang \emph{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 affects\emph{E} and \emph{L}
(Barbour \emph{et al.} 2017b).

Cell wall composition mutations modify how leaf anatomy influences water
movement andphysiological properties that affect \emph{E} , \emph{L} ,
and, ultimately, \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}. 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 \emph{et al.}2015).\emph{Zmasft} double mutants, which
were created in maize (\emph{Zea mays} L.) by mutating two paralogously
duplicated, unlinked maize orthologues of Arabidopsis thaliana
\emph{ALIPHATIC SUBERIN FERULOYL TRANSFERASE} , have a compromised
suberin llamelae ultrastructure that increased cell wall elasticity and
apoplastic water diffusion (Mertz \emph{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, influencing\emph{L}
\textsubscript{m} and \emph{L} \textsubscript{v}. Cellulose
synthase-like F6 (\emph{CslF6} ) in rice (\emph{Oryza sativa} L.)
mediates the biosynthesis of mixed linkage glucan (MLG), a
polysaccharide important in cell wall composition (Vega-S\selectlanguage{ngerman}ánchez \emph{et
al.} 2012). Loss-of-function mutants do not synthesize MLG (Vega-Sánchez
\emph{et al.} 2012; Smith-Moritz \emph{et al.} 2015) and have higher
cell wall porosity, which has been show to affect the internal
conductance of CO\textsubscript{2} (\emph{g} \textsubscript{m})
(Ellsworth \emph{et al.} 2018). This increase in porosity affected the
movement/conductance of CO\textsubscript{2} and may also alter
apoplastic water movement as well, which can alter water flow to the
stomates (\emph{E} ) or the length and tortuosity of water path
(\emph{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
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}.

To determine the influence of cell wall properties on
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} 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 \emph{E}
and\emph{L} impact \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}. The data
presented here demonstrates that changes in both cell wall properties
and stomatal density influence
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}through the impact of \emph{E}
and \emph{L} on the P\selectlanguage{ngerman}éclet effect.

\textbf{Theory and calculation of isotopic factors}

Leaf water isotopic composition
(\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}) is a mixture of two water
pools that represent the sources of isotopic variation within the leaf 
source water in the xylem (\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{SW}) and
water from the sites of evaporation
(\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e}; Equation 1), where using a
mass\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\(\delta^{18}O_{\text{LW}}=\ \left(1-\alpha\right)\delta^{18}O_{\text{sw}}+\ \alpha*\ \delta^{18}O_{e}\) & Equation 1\tabularnewline
\bottomrule
\end{longtable}

balance equation \selectlanguage{greek}\emph{α} \selectlanguage{english}is the fraction of unenriched water in leaf
xylem. To remove the effect of source water on
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}, the oxygen isotopic
composition in leaf water can be expressed as enrichment above source
water (\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}),\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\({}^{18}O_{\text{LW}}=\ \frac{\delta^{18}O_{\text{LW}}-\ \delta^{18}O_{\text{sw}}}{1-\ \delta^{18}O_{\text{LW}}}\) & Equation 2\tabularnewline
\bottomrule
\end{longtable}

where \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} and
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{sw} are expressed as ratios
of\textsuperscript{18}O/\textsuperscript{16}O (Barbour 2007; Cernusak
\emph{et al.} 2016). Therefore, eqn. 1 can be expressed as enrichments
(Eqn. 3).\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\({}^{18}O_{\text{LW}}=\ \alpha*\ ^{18}O_{e}\) & Equation 3\tabularnewline
\bottomrule
\end{longtable}

Water at the sites of evaporation is assumed to follow the Craig-Gordon
model, so \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} can be derived from
equilibrium fractionation (\selectlanguage{greek}ε\selectlanguage{english}\textsuperscript{+}), the ambient to
intercellular air vapor mole fraction (\emph{e}
\textsubscript{a}/\emph{e} \textsubscript{i}), the kinetic fractionation
(\selectlanguage{greek}ε\selectlanguage{english}\textsubscript{k}), and air vapor enrichment
(\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{v}; Eqn. 4) (Craig \& Gordon 1965;
Dongmann \emph{et al.} 1974; Flanagan \emph{et al.} 1991).\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\({}^{18}O_{e}=\varepsilon^{*}+\ \varepsilon_{k}+\ \frac{e_{a}}{e_{i}}\left({}^{18}O_{v}-\varepsilon_{k}\right)\) & Equation 4\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{greek}ε\selectlanguage{english}\textsuperscript{+} is the fractionation associated with the movement
of water from the liquid phase into the vapor phase as part of the
boundary layer around the leaf surface and is inversely related to
T\textsubscript{L} (Eqn. 5).\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\(\varepsilon^{+}=2.644-3.206\left(\frac{10^{3}}{T_{L}}\right)+1.534\ \left(\frac{10^{6}}{T_{L}^{2}}\right)\) & Equation 5\tabularnewline
\bottomrule
\end{longtable}

The fractionation (\selectlanguage{greek}\emph{ε} \selectlanguage{english}\textsubscript{k}) associated with water
vapor leaving the boundary layer is inversely proportional to the
stomatal (\emph{g} \textsubscript{s}) and boundary layer (\emph{g}
\textsubscript{bl}) conductances (Eqn. 6).\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\(\varepsilon_{k}=\frac{\left(32\frac{1}{g_{s}}+21\frac{1}{g_{b}}\right)}{\left(\frac{1}{g_{s}}+\frac{1}{g_{b}}\right)}\) & Equation 6\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} is nearly always less enriched
than \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e}, meaning that leaf water is
not a well-mixed pool undergoing evaporative enrichment, so two models
were developed to explain \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} based
on its isotopic sources: the two-pool model (Eqn. 1) and the P\selectlanguage{ngerman}éclet
effect model (Roden \& Ehleringer 1999; Barbour \& Farquhar 2000;
Helliker \& Ehleringer 2000; Gan \emph{et al.} 2003; Barbour 2007). The
Péclet effect model, instead of the fraction of water contributed by the
sites of evaporation (\selectlanguage{greek}α \selectlanguage{english}in Eqn. 3) being fixed, it was modified to be
dependent on the P\selectlanguage{ngerman}éclet number (P; Eqn. 7). The Péclet number\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\({}^{18}O_{\text{LW}}=\ \frac{{}^{18}O_{e}\ \left(1-e^{-P}\right)}{P}\) & Equation 7\tabularnewline
\bottomrule
\end{longtable}

represents the advective water flow from the xylem toward the sites of
evaporation and back diffusion from the sites of evaporation into the
rest of the leaf including the xylem.

The P\selectlanguage{ngerman}éclet number is proportional to the transpiration rate (\emph{E} )
and the effective mixing length (\emph{L} ) that water takes to move
from the veins to the sites of evaporation, and inversely proportional
to the molar density of water (\emph{C} ; 5.56 x 10\textsuperscript{-4}
mol m\textsuperscript{-3}) and the diffusivity of
H\textsubscript{2}\textsuperscript{18}O in water (\emph{D} ; Eqn. 8)
(Farquhar \emph{et al.} 1998; Barbour \& Farquhar 2000; Barbour \emph{et
al.} 2000b; Gan \emph{et al.} 2003; Barbour \& Farquhar 2004; Barbour
2007). The diffusivity of H\textsubscript{2}\textsuperscript{18}O in
water calculated according to Cuntz \emph{et al.} (2007).\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\(P=\ \frac{\text{LE}}{\text{CD}}\) & Equation 8\tabularnewline
\bottomrule
\end{longtable}

\emph{L} is the product of two components related to leaf anatomy: one
that accounts for the tortuosity of water pathway from the xylem to the
stomatal pore (\emph{l} ) and the other refers to the cross-sectional
area perpendicular to water flow relative to the leaf surface area
(\emph{k} ) (Barbour \& Farquhar 2004; Cernusak \& Kahmen 2013; Song
\emph{et al.} 2013; Sternberg \& Manganiello 2014). The assumption that
\emph{L} is constant for species, across treatments, and across a
gradient of transpiration rates has not always been supported (Song
\emph{et al.} 2013; Roden \emph{et al.} 2015). The lack of understanding
of \emph{L} and the factors that affect it largely arise from the
difficulty of measuring \emph{L} .

The determining evidence of the presence of the P\selectlanguage{ngerman}éclet effect is if the
fractional contribution of source water (unenriched xylem water;\emph{f}
\textsubscript{sw}) increases with \emph{E} (Eqn. 9; Song \emph{et al.}
2015; Holloway-Phillips \emph{et al.} 2016). A Péclet effect has failed
to be observed in several\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\(f_{\text{sw}}=1-\frac{{}^{18}O_{\text{LW}}}{{}^{18}O_{e}}\) & Equation 9\tabularnewline
\bottomrule
\end{longtable}

studies and species (Roden \& Ehleringer 1999; Cernusak \emph{et al.}
2003; Loucos \emph{et al.} 2015; Song \emph{et al.} 2015;
Holloway-Phillips \emph{et al.} 2016). Adherence to the P\selectlanguage{ngerman}éclet effect
model appears to depend on the relative importance of enriched vein
water to bulk leaf water and the interaction between xylem and mesophyll
water pools (Song \emph{et al.} 2015; Holloway-Phillips \emph{et al.}
2016).

Leaf anatomy was shown to play a distinct role in
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}, where parallel venation in
grasses increased the magnitude of enrichment from the base of a grass
leaf blade to the tip (Helliker \& Ehleringer, 2000). Even though
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e}remains the same over the length
of the leaf blade, \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{SW} (xylem
water) becomes progressively more enriched up the blade, supplying
increasingly more enriched water to the sites of evaporation and
creating a gradient in \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}. Vein
density increased the influence of the enriched mesophyll water on the
xylem, increasing the magnitude of
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} along the blade. The
string-of-lakes model described this progressive enrichment up the leaf
blade as a series of interconnected pools where the source of one pool
is the preceding pool (Gat \& Bowser 1991; Helliker \& Ehleringer 2000).
The Farquhar-Gan model was created, so that separate P\selectlanguage{ngerman}éclet effects were
associated with xylem and mesophyll (lamina) water pools (Eqn. 10; Gan
\emph{et al.} 2003; Holloway-Phillips \emph{et al.} 2016).\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\(\Delta^{18}O_{\text{LW}}=\ \phi_{x}\left(\Delta^{18}O_{e}e^{{-P}_{r}}\right)+\left(1-\phi_{x}\right)\left(\Delta^{18}O_{e}\frac{1-e^{{-P}_{m}}}{P_{m}}\right)\) & Equation 10\tabularnewline
\bottomrule
\end{longtable}

Where \selectlanguage{greek}ϕ\selectlanguage{english}\textsubscript{x} is the fraction of leaf water in the xylem, and
P\textsubscript{r} and P\textsubscript{m} are the xylem and mesophyll
P\selectlanguage{ngerman}éclet numbers, respectively. This model allows for different Péclet
effects in each tissue type (Gan \emph{et al.} 2003; Holloway-Phillips
\emph{et al.} 2016). The influence of the xylem Péclet depends on
physiological traits such as \emph{E} that regulates flow velocity and
anatomical traits, such as vein density, fraction vein volume, stomatal
density and size, and specific water content (Sternberg \& Manganiello
2014; Larcher \emph{et al.} 2015; Song \emph{et al.} 2015;
Holloway-Phillips \emph{et al.} 2016; Liang \emph{et al.} 2018).
Potentially multiple other anatomical traits that influence the movement
of water along the transpiration pathway and in the leaf alter
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}. The Farquhar-Gan model can be
modified by excluding the mesophyll P\selectlanguage{ngerman}éclet if it is small or nonexistent
compared to xylem Péclet (Eqn. 11). Therefore, the Péclet effect\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\(\Delta^{18}O_{\text{LW}}=\ \phi_{x}\left(\Delta^{18}O_{e}e^{{-P}_{r}}\right)+\left(1-\phi_{x}\right)\Delta^{18}O_{e}\) & Equation 11\tabularnewline
\bottomrule
\end{longtable}

model can be adapted to the species depending on the leaf anatomy and
physiology influencing \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}.

\textbf{Materials and Methods}

\emph{Plant Material}

\emph{Experiment 1: Maize suberin mutants (Zmasft)}

Maize (\emph{Zea mays} L.) seeds were planted in 11.3 L pots and placed
in a controlled-environment growth chamber (Biochambers, GRC-36,
Winnipeg, MB, Canada). The photoperiod was 16 h with a 1-hour ramp for
light reaching 1000 \selectlanguage{greek}μ\selectlanguage{english}mol quanta
m\textsuperscript{-2}s\textsuperscript{-1} at canopy level and 1-hour
ramp for temperature to mimic dawn and dusk. Day/night temperatures were
set at 28\selectlanguage{ngerman}°C/20°C, respectively and relative humidity was kept at 70 \%.
The pots were distributed in two light treatments even before
germination: high light (HL) exposed to 1200 \selectlanguage{greek}μ\selectlanguage{english}mol quanta
m\textsuperscript{-2}s\textsuperscript{-1} at canopy level and low light
(LL) exposed to 300 \selectlanguage{greek}μ\selectlanguage{english}mol quanta m\textsuperscript{-2}
s\textsuperscript{-1} at canopy level. The plants were grown
simultaneously in the same growth chamber and the height of the pots was
adjusted to maintain the desired light treatments throughout the
experiment.

Five replicates of each genotype were grown in a soil mixture of
Sunshine Mix LC-1 (Sun Gro Horticulture). Plants were watered daily and
fertilized every four days, initially with 20-20-20 and later with
15-5-15 CalMag. Experiment was conducted from March to May 2015, at
Washington State University, Pullman, WA, USA.

\emph{Experiment 2: Rice mixed linkage glucan mutants (cslf6)}

Rice (\emph{Oryza sativa} L.), Nipponbare cv (wild type) and
mutants\emph{cslf6-1} and \emph{cslf6-2} , which vary in the position of
their transposon insertion within the \emph{CslF6} genomic sequence of
Nipponbare cv\textsubscript{}(Vega-S\selectlanguage{ngerman}ánchez \emph{et al.} 2012;
Ellsworth \emph{et al.} 2018) were germinated on wet filter paper in a
petri dish. Seedlings were transplanted in trays containing Sunshine Mix
LC-1 soil (Sun Gro Horticulture) and turface (ratio of 3:1 \emph{v/v} )
and then after a month, they were transplanted into 7.5 L pots and
placed in a controlled environment growth chamber (Biochambers, GRC-36,
Winnipeg, MB, Canada). The photoperiod was 14 h with a two-hour ramp,
reaching 500 \selectlanguage{greek}μ\selectlanguage{english}mol photon m\textsuperscript{-2} s\textsuperscript{-1} at
canopy level. Day/night temperatures were set at 26/22 \selectlanguage{ngerman}°C, respectively
with a one-hour ramp, and relative humidity was kept at 70 \%. One week
after transplanting, plants were distributed into two light treatments:
high light (HL) plants were submitted to a gradual increase in
irradiance over 5 days to 1000 \selectlanguage{greek}μ\selectlanguage{english}mol photon m\textsuperscript{-2}
s\textsuperscript{-1} at canopy level and low light (LL) plants were
placed directly under 300 \selectlanguage{greek}μ\selectlanguage{english}mol photon m\textsuperscript{-2}
s\textsuperscript{-1} at canopy level. Plants were grown simultaneously
in the same growth chamber, and pot heights were adjusted to maintain
the desired light treatments at canopy level.

Five replicates of each genotype were watered daily and fertilized every
four days with a nutrient solution containing Sprint 330 iron chelate
(1.86 g L\textsuperscript{-1}), magnesium sulfate (0.88 g
L\textsuperscript{-1}), Scotts-Peters Professional 10-30-20 compound
(3.96 g L\textsuperscript{-1}), and Scott-Peters Soluble Trace Element
Mix (10.0 mg L\textsuperscript{-1}; Scotts). The experiment was
conducted from March to May 2014, at Washington State University,
Pullman, WA, USA.

\emph{Anatomical analysis}

For measurements of partial volume of total vasculature, vascular
bundles (both including and excluding the bundle sheath), and xylem,
cross sections were made on leaf 5 at the point of maximum lamina width.
The freehand transverse sections were immersed in water and imaged under
UV light filter (with excitation wavelength range 350-520nm) using cell
wall autofluorescence to maximize the contrast between the bundle sheath
cell wall and the surrounding mesophyll cells. Images were captured
using Leitz Epi-Fluorscent Microscope with Leica DFC425C Camera at 100x
for the leaf blade and 250x for the midrib. Additional images were taken
at 40x magnification for the leaf blade and 25x for the midrib to
calculate cross sectional leaf area. The section area of all vascular
bundles (including and excluding the bundle sheath) was measured. For
xylem area, diameter of each xylem element was measured twice
perpendicular to each other, and mean diameter was used to calculate
xylem area as the area of a circle. All measurements were made in ImageJ
(Schneider 2012). The total vasculature was calculated as area of the
vascular bundles in the leaf blade plus the entire midrib. Partial
volume of each was calculated as the total cross-sectional area of each
respective anatomical feature relative to the total leaf cross-sectional
area (blade area + midrib area).

The surface of three fresh leaves per genotype per treatment was
analyzed under the low vacuum mode of FEI Scanning Electron Microscope
(SEM) Quanta 200F (FEI Company, Field Emission Instruments, Hillsboro,
OR, USA). The leaves were the same or similar to those used for gas
exchange measurements and anatomical analyses. Images of the adaxial and
abaxial epidermal surfaces were randomly captured across the entire leaf
blade avoiding the central vein (min. 10 images per leaf) to determine
the number of stomata per mm\textsuperscript{2} of leaf surface
(stomatal density) and axis length of stomatal pores.

\emph{Gas Exchange Measurements}

Gas exchange measurements were performed in Pullman, WA, USA, with mean
atmospheric pressure of 92.1 kPa. The mid portion of 3-5 uppermost,
fully expanded leaves were used for gas exchange measurements using a
LI-6400XT and a 2 x 3 cm leaf chamber with a red and blue light emitting
diode light source (P/N 6400-02B, Li-Cor, Lincoln, NE, USA). Spot
measurements were performed inside the growth chamber, and the light
intensity, temperature, and relative humidity during gas exchange
measurements were maintained similar to the growth chamber conditions
under each treatment. Leaf-to-air vapor pressure deficit was kept at 1
kPa, flow rate maintained at 300 \selectlanguage{greek}μ\selectlanguage{english}mol air s\textsuperscript{-1}, and
oxygen partial pressure of 19.3 kPa (21 \%).

\emph{Leaf trait measurements}

The fully expanded leaf selected for transpiration measurements (section
above) was afterwards collected, photographed for calculation of area
using ImageJ (Schneider 2012) and placed in weighed glass tubes. Fresh
weight was first recorded and its water extracted according to
Vendramini and Sternberg Lda (2007) for measurements of
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}. The dried leaf material was
weighed to calculate leaf water content (LWC) and leaf dry mass per area
(LMA). LWC was calculated as the difference between fresh and dry leaf
weight per area and LMA was calculated as dry leaf weight per leaf area.

Root crowns were collected and placed in a separate glass tube for water
extraction (Vendramini \& Sternberg Lda 2007) and oxygen isotope
analysis, so that source water was characterized for each plant to
calculate leaf water oxygen isotope enrichment
(\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}). Vapor from the growth
chamber was collected every 30 min during transpiration measurements.
The vapor was sealed in 5 L inert foil gas sampling bags (Supelco
Analytical \#30228-U; Bellefonte, PA) that restrict exchange of water
vapor or CO\textsubscript{2} exchange with ambient air, and after
collection the vapor samples were measured immediately (details below).

\emph{Oxygen isotope analysis}

Root crown and leaf water oxygen isotope ratios
(\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}) were analyzed by isotope
ratio mass spectrometer (IRMS). A 0.3\% CO\textsubscript{2}/He gas
mixture was flushed through the vials and equilibrated with water for 48
h on a ThermoFinnigan GasBench II (Bremen). The equilibrated
CO\textsubscript{2} gas was separated at 40 \selectlanguage{ngerman}°C with a 25 m x 0.32 mm ID
GC column (Varian, poraplot Q) and analyzed with a continuous flow
isotope ratio mass spectrometer (Delta PlusXP, Thermofinnigan, Bremen)
(Brenna \emph{et al.} 1997; Qi \emph{et al.} 2003) to derive the oxygen
isotope ratios of the water. Standards (SLAP and Puerto Rico water) were
analyzed alongside the samples. Final delta values were the mean of 5
sample peaks reported relative to Vienna Standard Mean Ocean Water
(V-SMOW).

Irrigation water and water vapor were measured by isotope ratio-cavity
ringdown spectroscopy using a water analyzer (model L1102-i, Picarro,
Sunnydale, CA, USA). Each sample was analyzed six consecutive times, and
the mean of the last three analyses was used. Three laboratory
standards, calibrated to the V-SMOW scale, were interspersed among the
samples and used to correct the sample \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O values to
the V-SMOW scale. Water vapor was introduced into the water analyzer
through the inlet valve and analyzed for at least 15 minutes, and the
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O of water vapor
(\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{v}) was calculated as the mean
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O over the last five minutes. The
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{v} was corrected to the V-SMOW
scale in the same way that irrigated water samples were corrected.
Further \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{v} was corrected for water
concentration dependency. The stable isotope composition of oxygen
(\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O) was reported in \selectlanguage{greek}δ \selectlanguage{english}notation (Eqn. 12),\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\(\delta=\ \left(\frac{R_{\text{sample}}}{R_{\text{standard}}}-1\right)\) & Equation 12\tabularnewline
\bottomrule
\end{longtable}

where R\textsubscript{sample} and R\textsubscript{standard} are the
isotopic ratios of oxygen (\textsuperscript{18}O/\textsuperscript{16}O)
of the sample and international standard, respectively. The precision of
the analysis for each parameter was 0.2 %0 for leaf and root crown water
and water vapor and 0.07 %0 for irrigation water.

For calculation of effective leaf water mixing length, some parameters
were measured, and others were calculated. Steady state condition (SS)
was assumed for all calculations. Water isotope enrichment at the sites
of evaporation (\selectlanguage{greek}Δ\selectlanguage{english}\textsubscript{e}) was calculated for each leaf (Craig
\& Gordon 1965; Dongmann \emph{et al.} 1974) relative to source water
(\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} -
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{sw}; Eqn 2).
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{v} is the atmospheric water vapor
enrichment above plant water source
(\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{v} -
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{sw}), which in this study was root
crown water. We used \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{v} from the
atmosphere, as only 6.5 \% of the leaf was inside the cuvette during gas
exchange measurements for about 3-5 min.\emph{e}
\textsubscript{a}/\emph{e} \textsubscript{i} is the ratio of vapor
pressure external to the leaf to internal vapor pressure, \selectlanguage{greek}ε\selectlanguage{english}* is the
temperature-dependent (\selectlanguage{ngerman}°Kelvin) equilibrium fractionation factor between
vapor and water inside the leaf (Eqn. 5; Majoube 1971), and
\selectlanguage{greek}ε\selectlanguage{english}\textsubscript{k} is the kinetic fractionation during diffusion of
vapor from the leaf to the atmosphere (Eqn. 6; Farquhar \emph{et al.}
1989).

\emph{Statistical Analysis}

All statistical analyses and graphing were performed in R software
(version 3.2.2) (R Core Team, 2015) . The number of replicates varied
depending on the measured parameters. Leaf anatomy study was performed
in 3-5 replicates. The remaining measurements were performed in 5
replicates. If the dependent factor was not normal or homogeneous, the
data were transformed before using a two-way ANOVA. Two-way ANOVA
followed by contrasts was selected to estimate mean differences (LL
plants subtracted from HL plants) for each genotype separately for
individual parameters. Genotypes were combined for the ANCOVA followed
by contrast analysis to determine if changes in leaf water oxygen
isotope ratios co-varied with changes in other physiological and
anatomical traits (in terms of mean differences of HL and LL growth
treatment). Contrast analysis was considered the appropriate statistical
approach to interpret mean differences according to personal
consultation provided by the statistical facility at WSU.

According to P\selectlanguage{ngerman}éclet effect theory, the extent to that the Péclet effect
is occurring varies with leaf water pool, so the relative importance of
each Péclet effect is dependent on its strength and water pool size
(Farquhar \& Gan 2003; Holloway-Phillips \emph{et al.} 2016). The two
Péclet models fitted to the \emph{f} \textsubscript{sw} versus\emph{E}
data were the xylem and the mesophyll and xylem Péclet models where the
unknowns were \emph{L} \textsubscript{v} and\selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x}
in the xylem P\selectlanguage{ngerman}éclet model and\emph{L} \textsubscript{m}, \emph{L}
\textsubscript{v}, and\selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x} for the mesophyll and
xylem P\selectlanguage{ngerman}éclet model. The knowns were \emph{f} \textsubscript{sw} (1 -
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}/
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e}) and \emph{E} . Model fitting
was done with the Levenberg-Marquardt Nonlinear Least-Squares Algorithm
in R using the minpack.lm package.

\textbf{Results}

\emph{Maize Zmasft double mutant versus wildtype}

The enrichment of water at the sites of evaporation above source water
(\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e}) was 4.1 times more responsive
to changes in RH than to changes in light intensity, meaning
that\emph{e} \textsubscript{a}/\emph{e} \textsubscript{i} is a more
influential variable in determining
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} than\emph{g}
\textsubscript{s}-driven changes in \selectlanguage{greek}ε\selectlanguage{english}\textsubscript{k}.
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} did not differ between genotypes
in either RH or light intensity, while maize \emph{Zmasft} double
mutants were less enriched in leaf water oxygen isotope enrichment
(\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}) than wildtype in every
treatment (14.24 \selectlanguage{ngerman}± 1.48 and 16.14 ± 1.41 \selectlanguage{english}%0, respectively) (Table 1; Fig.
1a, b). In all treatments, \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} was
consistently less enriched in the \emph{Zmasft} double mutants than
wildtype by 0.5 to 1 %0 (Table 1; Fig. 1c, d). Additionally,
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} values trended lower in
wildtype, contrary to what is expected if differences in
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} were driving the genotypic
differences in \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}. Nonetheless,
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} has a drastic effect on
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} in that
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} was 11.65 \selectlanguage{ngerman}± 0.65 \selectlanguage{english}%0 lower at 80
\% RH than at 50 \% RH, ranging from 9.44 to 22.80 \selectlanguage{english}%0. The response to
light intensity was much less in that
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} was 2.90 \selectlanguage{ngerman}± 0.50 \selectlanguage{english}%0 lower at 300
than at 1200 \selectlanguage{greek}μ\selectlanguage{english}mol photons m\textsuperscript{-2} s\textsuperscript{-1},
and there was no difference at 80 \% RH (Table 1, S1; Fig. 1).

The fractional contribution of source water (unenriched xylem
water;\emph{f} \textsubscript{sw}) was slightly greater in
\emph{Zmasft}mutants (0.22 \selectlanguage{ngerman}± 0.03) than wildtype (0.16 ± 0.03).
Alternatively, for both genotypes \emph{f} \textsubscript{sw} was
greater under 1200 compared to 300 \selectlanguage{greek}µ\selectlanguage{english}mol quanta m\textsuperscript{-2}
s\textsuperscript{-1}(0.27 \selectlanguage{ngerman}± 0.1 and 0.1 ± 0.02, respectively) (Table 1;
Fig. 2a) and RH increased \emph{f} \textsubscript{sw} under 300 but not
1200 \selectlanguage{greek}µ\selectlanguage{english}mol quanta m\textsuperscript{-2} s\textsuperscript{-1} (Table 1;
Fig. 2a). Transpiration rate (\emph{E} ; mol H\textsubscript{2}O
m\textsuperscript{-2} s\textsuperscript{-1}) was greater in
the\emph{Zmasft} double mutants than in wildtype across treatments (P
\textless{} 0.0001), and this difference was most apparent under high
light intensity. At high light intensity, \emph{E} was higher than at
low light intensity, and RH only affected \emph{E} at low light
intensity (Table 1; Fig. 2b). Similarly, stomatal conductance (\emph{g}
\textsubscript{s}; mol H\textsubscript{2}O m\textsuperscript{-2}
s\textsuperscript{-1}) was 44 \% greater in\emph{Zmasft} double mutant,
and this genotypic difference was most apparent at high light intensity
(Table 1).

\emph{Rice mixed linkage glucan mutants (cslf6-1 and cslf6-2) versus
wildtype}

\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} was greater in the wildtype
(24.52 \selectlanguage{ngerman}± 1.00 \selectlanguage{english}%0) and \emph{cslf6-2} (23.69 +- 0.44 \selectlanguage{english}%0) than
\emph{cslf6-1}(22.12 +- 0.30 \selectlanguage{english}%0) (Table 2: Fig. 3a) and was higher at high
light (24.78 +- 0.63 \selectlanguage{english}%0) than at low light (22.10 +- 0.20 \selectlanguage{english}%0), regardless of
genotype (Table 2). \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} was
significantly higher in \emph{cslf6-1} than wildtype and \emph{cslf6-2}
mutants, but higher \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} in
\emph{cslf6-1} was not due to higher
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e}, which was lower in
\emph{cslf6-1} than in wildtype and \emph{cslf6-2} (Table 2; Fig. 3b).
There was a higher proportion of enriched than unenriched water (lower
\emph{f} \textsubscript{sw}) in \emph{cslf6-1} plants (0.36 \selectlanguage{ngerman}± 0.02)
compared to wildtype (0.49 ± 0.02) and \emph{cslf6-2} (0.48 ± 0.02)
mutant (Table 2). In all genotypes, \emph{f\textsubscript{sw}} was
higher under high light than low light (0.48 ± 0.02 and 0.40 ± 0.02,
respectively; Table 2; Fig. 4a). \emph{E} and \emph{g}
\textsubscript{s}were higher in wildtype (42 and 40 \%, respectively)
and \emph{cslf6-2}(32 and 34 \%, respectively) than in \emph{cslf6-1}
under high light, but there was no difference between genotypes under
low light (Table 2).\emph{E} and \emph{g} \textsubscript{s}
significantly decreased under low light compared to high light in
wildtype and \emph{cslf6-2} but not\emph{cslf6-1} (Table 2; Fig. 4b).

\emph{Anatomical measurements in maize and rice}

The partial volume of the leaf represented by vasculature (midrib and
vascular bundles in the blade), veins (both including and excluding the
bundle sheath cells), and xylem were not significantly different across
genotypes in either maize or rice, except for the partial volume of
veins including the bundle sheath in rice (Table 1 and 2). In rice, the
partial volume of veins including the bundle sheath was slightly greater
in WT (0.22 ± 0.02) than \emph{cslf6-1} (0.18 ± 0.01) and
\emph{cslf6-2}(0.19 ± 0.01), but there was no difference in anatomy with
growth light intensity (Table 2). The partial volume of total
vasculature, veins (both including and excluding the bundle sheath), and
xylem in maize (0.50 ± 0.01, 0.25 ± 0.01, 0.11 ± 0.01, 0.03 ± 0.01,
respectively) and in rice (0.53 ± 0.04, 0.20 ± 0.03, 0.08 ± 0.01, 0.01 ±
0.02, respectively) showed that about half of the leaf was vasculature,
and only a small fraction of the vasculature was conducting tissue
(Table 2).

In maize and rice, stomatal density (Sd) differed with growth light
intensity where plants under high light had greater Sd (117.2 ± 1.5 and
737 ± 35, respectively) than those grown under low light (88.7 ± 1.4 and
528 ± 31, respectively) (Table 1 and 2). The Sd was slightly higher in
leaves of \emph{Zmasft} double mutants (105.7 ± 3.4) than wildtype (99.8
± 3.9), but there were no differences across rice genotypes. Sd on both
adaxial and abaxial sides of the leaf was significantly higher under
high light, but neither was significantly different across genotypes
(Table 1, S1). In maize, abaxial stomata subsidiary cells length was
greater in wildtype grown under low light intensity (34.5 \selectlanguage{greek}µ\selectlanguage{english}m \selectlanguage{ngerman}± 2.0) than
all other conditions (25.7 to 26.6 \selectlanguage{greek}µ\selectlanguage{english}m; Table 1, S1).

Changes in \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} between plants grown
in high and low light (HL-LL) co-varied with changes in\emph{L}
\textsubscript{v}, \emph{E} , Sd, Sd\textsubscript{abaxial}, and partial
volume of veins excluding the bundle sheath (P \textless{} 0.05) and
partial volume of the total vasculature and\emph{g} \textsubscript{s} (P
= 0.05; Table S2). The mean genotypic difference (HL-LL) in Sd and
\emph{E} formed significant linear relationships with
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} (Fig. 5a, b), while \emph{E}
and Sd also formed a linear relationship (Fig. 5c). In contrast, the
change in \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}between plants grown
at high and low light did not co-vary with changes in
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e}, mesophyll and bundle sheath
cell wall thickness (bordering the IAS), leaf thickness, mesophyll cell
layers, volume fraction of intercellular air spaces (IAS) per mesophyll
area, mesophyll surface area exposed to IAS per unit leaf area (\emph{S}
\textsubscript{mes}), surface area of chloroplasts exposed to IAS per
unit of leaf area (S\textsubscript{c}), and leaf dry mass per area
{[}Table S2, these data were previously published in Ellsworth \emph{et
al.} (2018){]}.

\emph{Mesophyll and xylem P} \selectlanguage{ngerman}é\emph{clet models in maize and rice}

All genotypes of maize and rice formed significant positive
relationships between \emph{f} \textsubscript{sw} and transpiration rate
(\emph{E} ), suggesting a Péclet effect in these genotypes (Fig. 6a,b).
When \emph{f} \textsubscript{sw} and \emph{E} were fitted to the
mesophyll and xylem Péclet model (Eqn. 10) to solve for the following
three variables: mesophyll \emph{L} (\emph{L} \textsubscript{m}),
xylem\emph{L} (\emph{L} \textsubscript{v}), and the proportion of xylem
water (\selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x}), the model in maize converged, but
the estimated value for \emph{L} \textsubscript{m} was essentially 0 (2
x 10\textsuperscript{-17}). In rice the model failed to converge on a
significant value for \emph{L} \textsubscript{m}. Considering
that\emph{L} \textsubscript{m} was estimated as \textasciitilde{}0 in
maize and did not converge on a significant value in rice, it was
assumed that the mesophyll Peclet was nonexistent, and the location of
the Peclet effect was in the xylem only (Eqn. 11) for maize and rice. In
maize, the xylem Peclet model failed to converge on significant values
of\emph{L} \textsubscript{v} and \selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x} when all
data were used. However, when only data from 80 \% RH were
examined,\emph{Zmasft} had a larger estimate of \emph{L}
\textsubscript{v} (186 \selectlanguage{ngerman}± 86 mm) than wildtype (113 ± 47 mm; Table 3). At
50 \% RH, the xylem Péclet model did not converge. In rice, \emph{L}
\textsubscript{v}calculated from the xylem Péclet only model was smaller
in\emph{cslf6-1} than wildtype and \emph{cslf6-2} . In each species,
estimates of \selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x} were similar across genotypes,
but \selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x} was twice as large in rice than maize
(Table 3).

\emph{L} \textsubscript{v} was estimated using the xylem Peclet model
when the partial volumes of total vasculature, veins including and
excluding BS were used as \selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x} in maize and only
when the partial volume of total vasculature was used as
\selectlanguage{greek}ϕ\selectlanguage{english}\textsubscript{x} in rice. When the partial volume of leaf vasculature
or vascular bundles was used as \selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x} in the xylem
P\selectlanguage{ngerman}éclet model, estimates of \emph{L} \textsubscript{v} were greater
for\emph{Zmasft} double mutants than WT, and this difference was most
apparent at low light intensity (Table 1, 3). In rice,\emph{L}
\textsubscript{v} was lower in \emph{cslf6-1} than WT and\emph{cslf6-2}
. These results were similar to modelled\emph{L} \textsubscript{v}
(solving for both \emph{L} \textsubscript{v}and \selectlanguage{greek}\emph{ϕ}
\selectlanguage{english}\textsubscript{x}), except that \emph{L} \textsubscript{v}was
considerably smaller when partial pressure of the total vasculature was
used as \selectlanguage{greek}ϕ\selectlanguage{english}\textsubscript{x} in maize. The partial volume of veins,
including the bundle sheath, was most similar to\emph{f}
\textsubscript{sw} (especially at high light intensity) and\selectlanguage{greek}\emph{ϕ}
\selectlanguage{english}\textsubscript{x} values estimated from the xylem P\selectlanguage{ngerman}éclet model in maize
(Table 3). Rice, having larger \emph{f} \textsubscript{sw}values than
maize, the partial volume of the total vasculature of rice was most
similar to \emph{f} \textsubscript{sw} and\selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x}.

A sensitivity analysis of the interaction between\emph{L}
\textsubscript{v} and \selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x} in the xylem P\selectlanguage{ngerman}éclet
model showed that \selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x} in rice was greater than in
maize and that \emph{L} \textsubscript{v} had a large effect on\selectlanguage{greek}\emph{ϕ}
\selectlanguage{english}\textsubscript{x} until it reached its minimum threshold in both maize
and rice at approximately 75 mm (Fig. 7). Genotypic differences were
only apparent at low light, where \emph{L} \textsubscript{v} and\selectlanguage{greek}\emph{ϕ}
\selectlanguage{english}\textsubscript{x} were greater in \emph{Zmasft} than in wildtype. In
rice, \emph{L} \textsubscript{v} and\selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x} were
lower in \emph{cslf6-1} than wildtype and\emph{cslf6-2} when \emph{L}
\textsubscript{v} was greater than approximately 50 mm. The results
between maize at 50 \% RH and rice were similar in that \selectlanguage{greek}\emph{ϕ}
\selectlanguage{english}\textsubscript{x} was greater at high light than low light across the
range of \emph{L} \textsubscript{v} (Fig. 7).

\textbf{Discussion}

Observations of \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} values in maize
and rice are consistent with the P\selectlanguage{ngerman}éclet effect in the veins. Unlike the
two-pool model where the \emph{f} \textsubscript{sw} is a fixed volume
and not dependent on physiological factors (Roden \& Ehleringer 1999;
Roden \emph{et al.} 2015; Song \emph{et al.} 2015; Hirl \emph{et al.}
2019), a positive relationship between \emph{f} \textsubscript{sw} and
transpiration rate (\emph{E} ) was observed in all genotypes of maize
and rice. According to the Farquhar and Gan model,
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} is the accumulation of the
P\selectlanguage{ngerman}éclet effect in each tissue relative to the fraction of leaf water in
the tissue, but several studies have found that the xylem water pool is
the principal, if not the only, location of the Péclet effect (Helliker
\& Ehleringer 2000; Gan \emph{et al.} 2002; Farquhar \& Gan 2003; Gan
\emph{et al.} 2003; Holloway-Phillips \emph{et al.} 2016). In support of
this observation, we found that \emph{L} \textsubscript{m}estimates in
the mesophyll and xylem Péclet model were essentially 0, indicating that
the mesophyll Péclet effect was likely nonexistent. However, observed
variation in \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}across genotypes,
species, and treatments was not related to the differences in the
partial volume of the vasculature, meaning that variation in \emph{f}
\textsubscript{sw} was influenced by anatomical and physiological
factors regulating water movement and the relative importance of leaf
water pools.

Anatomical differences between C\textsubscript{3} and
C\textsubscript{4}grasses such as suberization of the bundle sheath
regulate the movement of water out of the vasculature and through the
mesophyll, contributing to variation in leaf water isotopic composition.
For example, it is not clear which tissues should be included in the
xylem water pool (\selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x}) of the xylem P\selectlanguage{ngerman}éclet model,
and\selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x} could include only the vein elements or
include associated ground tissue such as the bundle sheath and the large
quantity of parenchyma in the midrib (Gan\emph{et al.} 2003). In
C\textsubscript{3} rice, the estimated\selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x} (and
\emph{f} \textsubscript{sw}) were relatively similar to the partial
volume of the entire vasculature, which indicates that likely conducting
and ground tissues associated with the vasculature together are
isotopically one pool of water. Previous studies have made similar
conclusions (Gan \emph{et al.} 2003; Song \emph{et al.} 2015;
Holloway-Phillips \emph{et al.} 2016). In contrast, the influence of
xylem water in C\textsubscript{4} maize may be more constrained than in
rice in that \selectlanguage{greek}\emph{ϕ} \selectlanguage{english}\textsubscript{x} may be confined to the vascular
bundles and may not necessarily include all ground tissue associated
with the veins. This difference between C\textsubscript{3} and
C\textsubscript{4} grass species may lie in that C\textsubscript{3}
plants have less suberized bundle sheaths, leading to greater outside
xylem conductivity to support higher \emph{E} and a greater isotopic
influence of xylem water on the mesophyll water pool (Sonawane et al. in
review; Kocacinar \& Sage 2003). Therefore, the lower
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} in C\textsubscript{3} grass
species than C\textsubscript{4} species may depend on more than
anatomical features such as interveinal distance (vein density) and
include the facility of water movement out of the xylem and outside
xylem conductivity.

\selectlanguage{ngerman}\emph{Suberin mutation and the Péclet effect}

Both \emph{E} and \emph{L} \textsubscript{v} were affected by the
compromised ultrastructure of the bundle sheath cells and increased
apoplastic permeability in Zm\emph{asft} maize double mutants,
especially at the interface of suberin lamellae and adjoining
polysaccharide cell walls (Mertz et al. In review). Suberin is
considered essential as a functional barrier to unrestricted diffusion
of water across cell walls (Botha \emph{et al.} 1982; Schönherr 1982;
Mertz \& Brutnell 2014). Consequently, more permissive water flow in the
leaf of the \emph{Zmasft} double mutants led to greater water flow out
of the xylem as evidenced by greater \emph{E} under high light and more
water channels in and out of the xylem under low light, increasing the
overall effective mixing length in the xylem (\emph{L}
\textsubscript{v}). The likely reason for mutation-related differences
in \emph{E} under high light and\emph{L} \textsubscript{v} under low
light is an interaction between water demand and the lack of water
channeling. Under high light \emph{E}was 2-3.3x and 2.6-3.8x greater in
wildtype and the double mutant relative to low light, respectively, so
the principal water channels moved the vast majority of water out of the
xylem, leading to similar measures of \emph{L} \textsubscript{v}. In
contrast, transpiration demand was low under low light, so greater
apoplastic permeability allowed water to move through more tortuous
channels, increasing the overall\emph{L} \textsubscript{v} in the
\emph{Zmasft} mutants. Therefore, channeling of water outside the xylem
through suberin lamellae influenced the Péclet effect in leaf water by
restricting water flow (lower \emph{E} ) and reducing water movement
across the suberin lamellae (smaller \emph{L} \textsubscript{v}). As has
been seen previously, suberin and suberin lamellae play a role in the
plant's ability to regulate transpiration (Schreiber 2010; Vishwanath
\emph{et al.} 2015), but interestingly these differences are detectable
with stable isotopes. Potentially, through the Péclet effect model
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} can be used to detect and study
anatomical differences and their physiological consequences on water
movement in leaves.

\selectlanguage{ngerman}\emph{Mixed linkage glucan (MLG) and the Péclet effect}

The genotypes varied in their physiological and isotopic response to
growth light intensity. The genotypes (\emph{cslf6-1} , \emph{cslf6-2}
), which are transposon insertion lines created in a Nipponbare
background, did not always have the same phenotype (Upadhyaya \emph{et
al.} 2006; Vega-Sánchez \emph{et al.} 2012). They were similar in most
anatomical features and biochemical properties (Vega-Sánchez \emph{et
al.} 2012; Ellsworth \emph{et al.} 2018). However, genotype
\emph{cslf6-1} had lower values than \emph{cslf6-2} in several
physiological traits such as \emph{g\textsubscript{s}} , \emph{E} , and
both \emph{A} \textsubscript{max} and \emph{J} \textsubscript{max} on a
per gram basis (Ellsworth \emph{et al.}2018) and \emph{L}
\textsubscript{v} in this study. Because all plants were grown in the
same physical space and under the same conditions such as temperature
and relative humidity, variation in these traits across genotypes can be
either attributed to pleiotropic effects on the cell wall or background
effects as seen by the differences between Nipponbare and the isogenic
WT siblings of these different lines (Vega-Sánchez \emph{et al.} 2012).

The genotypic response of \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} to
growth light intensity can be explained within the P\selectlanguage{ngerman}éclet effect theory
by the corresponding response in stomatal density (Sd). Multiple studies
have shown that Sd and stomatal size influence
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} by modifying \emph{E}
and\emph{L} , and that differences in Sd were sufficient to drive
species-level differences in
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}between mangrove and nearby
freshwater species and across\emph{Arabidopsis} stomatal density lines
(Rosado \emph{et al.} 2013; Sternberg \& Manganiello 2014; Larcher
\emph{et al.} 2015; Liang \emph{et al.} 2018). In this study, lower
\emph{L} \textsubscript{v} in \emph{cslf6-1} was likely due to an
interaction between a genotype-driven reduction in \emph{E} related to
Sd and an increased mesophyll and xylem water exchange due to lower
water flow, which reduced the mixing length between enriched mesophyll
water and unenriched water in the xylem (Farquhar \& Gan 2003; Gan
\emph{et al.} 2003). Therefore, changes in Sd regulated the relative
differences in \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}, but Sd alone
did not predict the magnitude of
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}(Rosado \emph{et al.} 2013;
Larcher \emph{et al.} 2015). Conversely and similar to Sternberg and
Manganiello (2014), higher \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} at
high light in wildtype and \emph{cslf6-2} did not result in higher
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} because increased advective
flow (\emph{E} ) and longer mixing length (\emph{L} \textsubscript{v})
between xylem and mesophyll reduced back diffusion and the impact of
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} on
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}. The fact that the partial
volume of the veins along with Sd, \emph{L} \textsubscript{v},
and\emph{E} co-varied with
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}suggests that
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} was significantly influenced by
the entire water transport path through the leaf and, consequently, may
trace anatomical, physical, and physiological factors affecting water
movement.

\emph{Conclusions}

Cell wall and other anatomical properties can affect
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} by manipulating \emph{E}through
altering water flow along the transpiration stream and\emph{L}
\textsubscript{v} through altering water channeling out of the xylem and
to the stomata. This influence is apparent in variation in
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} between C\textsubscript{3} and
C\textsubscript{4} grass species that can be attributed to differences
in vasculature that limit water flow out of the vasculature in
C\textsubscript{4} species and may be an indication of differing
hydraulic attributes. Studying the interactions of anatomical features
and physiological traits in water movement through and outside of the
xylem can shed more light on
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}because either physiology or
anatomy alone fails to capture the isotopic implications of water
movement. Consequently, stable isotopes can be used to study this
interaction. With the development of gene editing,
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} can potentially be used to
better understand gene-phenotype relationships of traits related to
water movement in the leaf. For example, when studying genes associated
with Sd, differences in water movement may not be discernable from
measures of Sd \emph{per se} , but nevertheless differences in Sd can
influence \selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} and indicate a subtle
response of Sd to genotype or environmental conditions. Further
integration between leaf water transport through and outside the xylem
with stable isotope theory will facilitate the development of a
physiologically and anatomically explicit water transport model.

\textbf{Acknowledgements}

Our research is supported by the Office of Biological and Environmental
Research in the DOE Office of Science (DE-SC0008769) and by the Russian
Science Foundation (16-16-00089) for N.K. (leaf anatomy study and
anatomical traits analysis). We are grateful to the Core Facility Center
``Cell and Molecular Technologies in Plant Science'' of Komarov
Botanical Institute and Franceschi Microscopy and Imaging Center of
Washington State University for use of their facilities. Seed\emph{Oryza
sativa} , Nipponbare cultivar (wild type) and mutants\emph{cslf6-1} and
\emph{cslf6-2} were kindly provided by Drs. Miguel E. Vega-S\selectlanguage{ngerman}ánchez and
Pamela C. Ronald, Joint BioEnergy Institute, Emeryville, California
94608 and UC Davis Genome Center and Plant Pathology, University of
California, Davis, California 95616.

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\textbf{Table 1} . Leaf water isotope parameters for \emph{Zea mays}
wildtype and Zm\emph{asft} double mutants (aliphatic suberin feruloyl
transferase), grown under 300 or 1200 photosynthetic active photon flux
density (PAR; \selectlanguage{greek}μ\selectlanguage{english}mol photons m\textsuperscript{-2} s\textsuperscript{-1})
and either 50 or 80 \% relative humidity (RH). Stomatal measurements
were on plants grown at 80 \% RH. Results of the main effects of a
three-way ANOVA are included, except the stomatal traits. Stomatal
traits were analyzed using a two-way ANOVA because they were only
measured in plants grown under 80 \% RH. Significance was shown as ns,
*, **, and *** for not significant, P \textless{} 0.05, P \textless{}
0.01, and P \textless{} 0.001, respectively. The only interactions where
P \textless{} 0.05 for at least one trait were RH x PAR and Genotype x
PAR (Table S1).\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllll@{}}
\toprule
& RH 50 \% & RH 50 \% & RH 50 \% & RH 50 \% & RH 80 \% & RH 80 \% & RH
80 \% & RH 80 \% & & &\tabularnewline
\midrule
\endhead
& Light intensity 300 & Light intensity 300 & Light intensity 1200 &
Light intensity 1200 & Light intensity 300 & Light intensity 300 & Light
intensity 1200 & Light intensity 1200 & & &\tabularnewline
& Genotype & Genotype & Genotype & Genotype & Genotype & Genotype &
Genotype & Genotype & ANOVA & ANOVA & ANOVA\tabularnewline
Trait & WT & \emph{Zmasft} & WT & \emph{Zmasft} & WT & \emph{Zmasft} &
WT & \emph{Zmasft} & RH & PAR & Gen\tabularnewline
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} & 23.2\selectlanguage{ngerman}±0.2 & 22.2\selectlanguage{ngerman}±0.5 &
20.2\selectlanguage{ngerman}±0.3 & 19.6\selectlanguage{ngerman}±0.2 & 9.7\selectlanguage{ngerman}±0.2 & 9.2\selectlanguage{ngerman}±0.4 & 10.4\selectlanguage{ngerman}±0.2 & 9.3\selectlanguage{ngerman}±0.3 & *** & ns
& **\tabularnewline
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} & 23.1\selectlanguage{ngerman}±0.1 & 23.7\selectlanguage{ngerman}±0.5 & 27.5\selectlanguage{ngerman}±0.1
& 27.9\selectlanguage{ngerman}±0.2 & 11.2\selectlanguage{ngerman}±0.2 & 11.4\selectlanguage{ngerman}±0.2 & 13.9\selectlanguage{ngerman}±0.4 & 13.0\selectlanguage{ngerman}±0.6 & *** & *** &
ns\tabularnewline
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{SW} & -13.8\selectlanguage{ngerman}±0.2 & -13.5\selectlanguage{ngerman}±0.1 &
-15.0\selectlanguage{ngerman}±0.1 & -15.2\selectlanguage{ngerman}±0.1 & -15.6\selectlanguage{ngerman}±0.1 & -14.8\selectlanguage{ngerman}±0.1 & -14.9\selectlanguage{ngerman}±0.2 & -15.6\selectlanguage{ngerman}±0.1 &
*** & *** & ns\tabularnewline
\emph{f}\textsubscript{sw} & 0.0\selectlanguage{ngerman}±0.01 & 0.06\selectlanguage{ngerman}±0.03 & 0.26\selectlanguage{ngerman}±0.01 &
0.30\selectlanguage{ngerman}±0.004 & 0.14\selectlanguage{ngerman}±0.01 & 0.19\selectlanguage{ngerman}±0.03 & 0.25\selectlanguage{ngerman}±0.03 & 0.28\selectlanguage{ngerman}±0.04 & *** & *** &
**\tabularnewline
\emph{E}\textsuperscript{+} & 1.20\selectlanguage{ngerman}±0.07 & 1.22\selectlanguage{ngerman}±0.03 & 2.37\selectlanguage{ngerman}±0.15 &
3.20\selectlanguage{ngerman}±0.21 & 0.69\selectlanguage{ngerman}±0.04 & 0.80\selectlanguage{ngerman}±0.03 & 2.26\selectlanguage{ngerman}±0.09 & 3.04\selectlanguage{ngerman}±0.11 & *** & *** &
***\tabularnewline
\emph{g}\textsubscript{s}\textsuperscript{+} & 0.059\selectlanguage{ngerman}±0.004 & 0.057\selectlanguage{ngerman}±0.003
& 0.107\selectlanguage{ngerman}±0.008 & 0.147\selectlanguage{ngerman}±0.011 & 0.061\selectlanguage{ngerman}±0.004 & 0.069\selectlanguage{ngerman}±0.002 & 0.210\selectlanguage{ngerman}±0.01 &
0.322\selectlanguage{ngerman}±0.008 & ** & *** & **\tabularnewline
\emph{L}\textsubscript{v} (Vasc)\textsuperscript{++} & 0\selectlanguage{ngerman}±3 & 15.1\selectlanguage{ngerman}±6.6 &
43\selectlanguage{ngerman}±3 & 40\selectlanguage{ngerman}±3 & 63\selectlanguage{ngerman}±5 & 87\selectlanguage{ngerman}±16 & 43\selectlanguage{ngerman}±7 & 39\selectlanguage{ngerman}±9 & *** & ns & *\tabularnewline
\emph{L}\textsubscript{v} (VB)\selectlanguage{ngerman}\textsuperscript{§ ¶} & 0.2\selectlanguage{ngerman}±5.5 & 32\selectlanguage{ngerman}±14 &
155\selectlanguage{ngerman}±NA & NA & 163\selectlanguage{ngerman}±18 & 298\selectlanguage{ngerman}±86 & 74\selectlanguage{ngerman}±NA & 75.3\selectlanguage{ngerman}±0.04 & ** & ns &
.\tabularnewline
Sd & NA & NA & NA & NA & 84.8\selectlanguage{ngerman}±2.8 & 93.6\selectlanguage{ngerman}±1.6 & 116.6\selectlanguage{ngerman}±1.9 & 117.8\selectlanguage{ngerman}±5.5 &
NA & *** & **\tabularnewline
S\textsubscript{adaxial} & NA & NA & NA & NA & 33.6\selectlanguage{ngerman}±2.0 & 39.1\selectlanguage{ngerman}±1.2 &
51.6\selectlanguage{ngerman}±2.3 & 52.3\selectlanguage{ngerman}±1.8 & NA & *** & ns\tabularnewline
S\textsubscript{abaxial} & NA & NA & NA & NA & 51.12\selectlanguage{ngerman}±1.3 & 54.5\selectlanguage{ngerman}±1.6 &
65.0\selectlanguage{ngerman}±3.0 & 65.5\selectlanguage{ngerman}±3.7 & NA & *** & ns\tabularnewline
S\textsubscript{abaxial} length & NA & NA & NA & NA &
33.3\selectlanguage{ngerman}±3.0\textsuperscript{a} & 25.7\selectlanguage{ngerman}±1.1\textsuperscript{b} &
26.0\selectlanguage{ngerman}±0.6\textsuperscript{b} & 26.6\selectlanguage{ngerman}±0.4\textsuperscript{b} & NA & ns &
*\tabularnewline
S\textsubscript{adaxial} length & NA & NA & NA & NA & 34.5\selectlanguage{ngerman}±2.8 &
27.2\selectlanguage{ngerman}±2.1 & 29.6\selectlanguage{ngerman}±1.0 & 29.2\selectlanguage{ngerman}±0.5 & NA & ns & ns\tabularnewline
\emph{g}\textsubscript{s}/Sd & NA & NA & NA & NA & 0.71\selectlanguage{ngerman}±0.04 & 0.69\selectlanguage{ngerman}±0.03
& 1.31\selectlanguage{ngerman}±0.16 & 2.00\selectlanguage{ngerman}±0.29 & NA & *** & ns\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{+}\emph{Zmasft} had significantly higher \emph{E}
and\emph{g} \textsubscript{s} than WT (P \textless{} 0.0001). There was
a significant interaction between RH and light intensity in that E was
higher at both low and high RH compared to low light intensity. At low
light intensity, \emph{E} was significantly lower at 50 \% RH than at 80
\% RH.

\textsuperscript{++}Partial volume of leaf total vasculature including
all midrib tissue was 0.50 and 0.49 for WT and \emph{Zmasft} double
mutants, respectively, and there was no significant difference between
genotypes (P = 0.72). There was a significant interaction between
genotype and light intensity (P \textless{} 0.05), where\emph{L}
\textsubscript{v} was higher in the \emph{Zmasft} double mutant only
under 300 \selectlanguage{greek}μ\selectlanguage{english}mol m\textsuperscript{-2} s\textsuperscript{-1}. There was a
significant interaction between RH and light intensity (P \textless{}
0.0001), where \emph{L} \textsubscript{v} was higher under 300 \selectlanguage{greek}μ\selectlanguage{english}mol
m\textsuperscript{-2} s\textsuperscript{-1} at 50 \% RH than at 80 \%
RH.

\selectlanguage{ngerman}\textsuperscript{§}Partial volume of vascular bundles in the leaf
(including the bundle sheath) was 0.25 and 0.26 for WT and
\emph{Zmasft}double mutants, respectively, and genotypes were not
significantly different (P = 0.70). Genotypes were marginally different
in\emph{L} \textsubscript{v} (P = 0.06). There is a significant
interaction between genotype and light intensity (P \textless{} 0.05),
where\emph{L} \textsubscript{v} under 300 \selectlanguage{greek}μ\selectlanguage{english}mol
m\textsuperscript{-2}s\textsuperscript{-1} was higher in the
\emph{Zmasft} double mutant.\emph{L} \textsubscript{v} in the xylem
P\selectlanguage{ngerman}éclet model could not be calculated when \selectlanguage{greek}ϕ\selectlanguage{english}\textsubscript{x} was
considered the partial volume of vascular bundle excluding the bundle
sheath or xylem in the leaf.

\selectlanguage{ngerman}\textsuperscript{¶}Light intensity x Genotype interaction was
significant (P \textless{} 0.05).

\textbf{Table 2} . Leaf water isotope parameters for \emph{O.
sativa}wild type (Nipponbare), \emph{cslf6-1} and \emph{cslf6-2} , grown
under 1000 and 300 photosynthetic active photon flux density (P, \selectlanguage{greek}μ\selectlanguage{english}mol
photons m\textsuperscript{-2} s\textsuperscript{-1}). \emph{f}
\textsubscript{sw}is the proportion of unenriched water in bulk leaf
water. Calculation of L\textsubscript{v} made by using the partial
volume of vascular tissue including the entire midrib as \selectlanguage{greek}\emph{θ}
\selectlanguage{english}\textsubscript{x}. A two-way ANOVA was performed to test the
significance: * \emph{P} \textless{} 0.05, ** P \textless{} 0.01, *** P
\textless{} 0.001. Tukey post hoc test represented when PxG interaction
was significant. Values with the same superscripted letters are not
significantly different. Growth light condition is represented by P
(PAR) and genotype by G.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllll@{}}
\toprule
& Growth light condition & Growth light condition & Growth light
condition & Growth light condition & Growth light condition & Growth
light condition & Growth light condition & & &\tabularnewline
\midrule
\endhead
& 1000 & 1000 & 1000 & 300 & 300 & 300 & \textbf{ANOVA} & \textbf{ANOVA}
& \textbf{ANOVA} & \textbf{ANOVA}\tabularnewline
& WT & \emph{cslf6-1} & \emph{cslf6-2} & WT & \emph{cslf6-1} &
\emph{cslf6-2} & \textbf{P} & \textbf{P} & \textbf{G} &
\textbf{PxG}\tabularnewline
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} (%0)\textsuperscript{+} &
27.02\selectlanguage{ngerman}±1.02 & 22.36\selectlanguage{ngerman}±0.57 & 24.97\selectlanguage{ngerman}±0.17 & 22.01\selectlanguage{ngerman}±0.54 & 21.88\selectlanguage{ngerman}±0.19 &
22.42\selectlanguage{ngerman}±0.21 & ** & ** & *** & ns\tabularnewline
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW} (%0) & 12.5\selectlanguage{ngerman}±0.4 & 13.5\selectlanguage{ngerman}±0.2 &
12.4\selectlanguage{ngerman}±0.4 & 12.1\selectlanguage{ngerman}±0.5 & 14.7\selectlanguage{ngerman}±0.2 & 12.7\selectlanguage{ngerman}±0.3 & ns & ns & *** &
ns\tabularnewline
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{SW} (%0) & -15.78\selectlanguage{ngerman}±0.1 & -15.36\selectlanguage{ngerman}±0.14
& -15.76\selectlanguage{ngerman}±0.15 & -15.04\selectlanguage{ngerman}±0.11 & -15.16\selectlanguage{ngerman}±0.19 & -14.87\selectlanguage{ngerman}±0.15 & *** & *** & ns
& ns\tabularnewline
\emph{f}\textsubscript{sw} & 0.53\selectlanguage{ngerman}±0.02 & 0.39\selectlanguage{ngerman}±0.02 & 0.5\selectlanguage{ngerman}±0.01 &
0.45\selectlanguage{ngerman}±0.03 & 0.33\selectlanguage{ngerman}±0.01 & 0.43\selectlanguage{ngerman}±0.01 & *** & *** & *** & ns\tabularnewline
\emph{E} (mmol H\textsubscript{2}O m\textsuperscript{-2}
s\textsuperscript{-1}) & 5.6\selectlanguage{ngerman}±0.4\textsuperscript{a} &
3.5\selectlanguage{ngerman}±0.3\textsuperscript{b} & 4.8\selectlanguage{ngerman}±0.1\textsuperscript{a} &
3.2\selectlanguage{ngerman}±0.1\textsuperscript{b} & 3\selectlanguage{ngerman}±0.1\textsuperscript{b} &
3.4\selectlanguage{ngerman}±0.1\textsuperscript{b} & *** & *** & *** & **\tabularnewline
\emph{g}\textsubscript{s} (mol H\textsubscript{2}O m\textsuperscript{-2}
s\textsuperscript{-1}) & 0.55\selectlanguage{ngerman}±0.06\textsuperscript{a} &
0.36\selectlanguage{ngerman}±0.04\textsuperscript{b} & 0.5\selectlanguage{ngerman}±0.02\textsuperscript{a} &
0.32\selectlanguage{ngerman}±0\textsuperscript{b} & 0.3\selectlanguage{ngerman}±0.01\textsuperscript{b} &
0.34\selectlanguage{ngerman}±0.01\textsuperscript{b} & *** & *** & ** & *\tabularnewline
\emph{g}\textsubscript{s}/Sd (nmols s\textsuperscript{-1}) & 0.74\selectlanguage{ngerman}±0.07 &
0.53\selectlanguage{ngerman}±0.02 & 0.65\selectlanguage{ngerman}±0.02 & 0.65\selectlanguage{ngerman}±0.04 & 0.52\selectlanguage{ngerman}±0.01 & 0.64\selectlanguage{ngerman}±0.04 & ns & ns & **
& ns\tabularnewline
\emph{C}\textsubscript{i}/\emph{C}\textsubscript{a} & 0.77\selectlanguage{ngerman}±0.02 &
0.88\selectlanguage{ngerman}±0.01 & 0.88\selectlanguage{ngerman}±0 & 0.81\selectlanguage{ngerman}±0.01 & 0.9\selectlanguage{ngerman}±0 & 0.89\selectlanguage{ngerman}±0 & *** & *** & *** &
ns\tabularnewline
\emph{L}\textsubscript{v} (Vasc)\textsuperscript{++} & 103\selectlanguage{ngerman}±9 & 62\selectlanguage{ngerman}±4 &
108\selectlanguage{ngerman}±1 & 81\selectlanguage{ngerman}±18 & 39\selectlanguage{ngerman}±2 & 64\selectlanguage{ngerman}±8 & ** & ** & ** & ns\tabularnewline
Prop. vasculature\selectlanguage{ngerman}\textsuperscript{§} & 0.52\selectlanguage{ngerman}±0.04 & 0.50\selectlanguage{ngerman}±0.04 & 0.48\selectlanguage{ngerman}±0.01
& 0.55\selectlanguage{ngerman}±0.04 & 0.57\selectlanguage{ngerman}±0.05 & 0.54\selectlanguage{ngerman}±0.02 & ns & ns & ns & ns\tabularnewline
Prop. veins + BS\selectlanguage{ngerman}\textsuperscript{¶} & 0.23\selectlanguage{ngerman}±0.01 & 0.18\selectlanguage{ngerman}±0.01 & 0.20\selectlanguage{ngerman}±0.01
& 0.21\selectlanguage{ngerman}±0.004 & 0.19\selectlanguage{ngerman}±0.01 & 0.18\selectlanguage{ngerman}±0.01 & ns & ns & * & ns\tabularnewline
Prop. veins - BS & 0.08\selectlanguage{ngerman}±0.002 & 0.07\selectlanguage{ngerman}±0.002 & 0.08\selectlanguage{ngerman}±0.005 & 0.09\selectlanguage{ngerman}±0.005 &
0.08\selectlanguage{ngerman}±0.004 & 0.08\selectlanguage{ngerman}±0.001 & ns & ns & ns & ns\tabularnewline
Prop. xylem & 0.011\selectlanguage{ngerman}±0.002 & 0.012\selectlanguage{ngerman}±0.001 & 0.015\selectlanguage{ngerman}±0.003 & 0.015\selectlanguage{ngerman}±0.001 &
0.013\selectlanguage{ngerman}±0.004 & 0.015\selectlanguage{ngerman}±0.003 & ns & ns & ns & ns\tabularnewline
Sd (no./mm\textsuperscript{2}) & 745\selectlanguage{ngerman}±42 & 735\selectlanguage{ngerman}±7 & 730\selectlanguage{ngerman}±57 & 503\selectlanguage{ngerman}±32 &
557\selectlanguage{ngerman}±12 & 524\selectlanguage{ngerman}±50 & *** & *** & ns & ns\tabularnewline
Sd\textsubscript{abaxial} (no./mm\textsuperscript{2}) & 387\selectlanguage{ngerman}±26 & 381\selectlanguage{ngerman}±5 &
401\selectlanguage{ngerman}±22 & 267\selectlanguage{ngerman}±15 & 284\selectlanguage{ngerman}±5 & 279\selectlanguage{ngerman}±20 & *** & *** & ns & ns\tabularnewline
Sd\textsubscript{adaxial} (no./mm\textsuperscript{2}) & 358\selectlanguage{ngerman}±24 & 354\selectlanguage{ngerman}±2 &
329\selectlanguage{ngerman}±23 & 236\selectlanguage{ngerman}±15 & 273\selectlanguage{ngerman}±10 & 245\selectlanguage{ngerman}±18 & *** & *** & ns & ns\tabularnewline
Pore length (abaxial) & 13.3\selectlanguage{ngerman}±0.4 & 11.9\selectlanguage{ngerman}±0.8 & 13.1\selectlanguage{ngerman}±0.4 & 14.5\selectlanguage{ngerman}±0.6 &
12.4\selectlanguage{ngerman}±0.2 & 13.3\selectlanguage{ngerman}±0.4 & ns & ns & ns & ns\tabularnewline
Pore length (adaxial) & 13.5\selectlanguage{ngerman}±0.7 & 12.2\selectlanguage{ngerman}±0.7 & 12.9\selectlanguage{ngerman}±0.4 & 15.2\selectlanguage{ngerman}±0.9 &
13.2\selectlanguage{ngerman}±0.3 & 13.3\selectlanguage{ngerman}±0.9 & ns & ns & ns & ns\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{+}\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e} was
calculated from \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LWpm}.

\textsuperscript{++}Calulation of L\textsubscript{v} made by using the
partial volume of vascular tissue including the entire midrib as\selectlanguage{greek}\emph{θ}
\selectlanguage{english}\textsubscript{x}.

\selectlanguage{ngerman}\textsuperscript{§}Partial volume of vascular tissue including the
entire midrib

\textsuperscript{¶}WT and \emph{cslf6-2} were significantly greater
than\emph{cslf6-1} , and 1000 \selectlanguage{greek}μ\selectlanguage{english}mol photons
m\textsuperscript{-2}s\textsuperscript{-1} was significantly greater
than 300 \selectlanguage{greek}μ\selectlanguage{english}mol photons m\textsuperscript{-2} s\textsuperscript{-1}.
Proportion of leaf volume found in the vessels and midrib was used for
proportion of xylem water (\selectlanguage{greek}\emph{θ} \selectlanguage{english}\textsubscript{x}) in the xylem
P\selectlanguage{ngerman}éclet model.

\par\null

\textbf{Table 3.} Xylem Péclet model (Eqn. 11) fitted to \emph{f}
\textsubscript{sw} (1 -
\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{LW}/\selectlanguage{greek}Δ\selectlanguage{english}\textsuperscript{18}O\textsubscript{e})
and \emph{E} data. Unknowns were \emph{L} \textsubscript{v} and\selectlanguage{greek}\emph{ϕ}
\selectlanguage{english}\textsubscript{x} in the xylem P\selectlanguage{ngerman}éclet model. Values are ± SE.\emph{L}
\textsubscript{v} was calculated again when \selectlanguage{greek}ϕ\selectlanguage{english}\textsubscript{x}was
considered the partial volume of either total vasculature, veins
including the bundle sheath (V + BS) or excluding the sheath (V - BS),
or xylem. Means within each species and each column followed by
different letters are significantly different.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllll@{}}
\toprule
\begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Mean \emph{f}\textsubscript{sw}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Mean \emph{E} (mmol m\textsuperscript{-2}s\textsuperscript{-1})\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Eqn. 11 \emph{L}\textsubscript{v} (mm)\textsuperscript{+}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Eqn. 11 \selectlanguage{greek}\emph{ϕ}\selectlanguage{english}\textsubscript{x}\textsuperscript{+}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Part. vol. total vasculature\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\emph{L}\textsubscript{v} (mm)\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Prop. V+BS\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\emph{L}\textsubscript{v} (mm)\textsuperscript{++}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Part. vol. V-BS\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
\emph{L}\textsubscript{v} (mm)\textsuperscript{++}\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Part. vol. xylem\selectlanguage{ngerman}\textsuperscript{§}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
All maize genotypes\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.19\selectlanguage{ngerman}±0.02\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1.80\selectlanguage{ngerman}±0.16\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
148\selectlanguage{ngerman}±40\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.29\selectlanguage{ngerman}±0.03\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.50\selectlanguage{ngerman}±0.01\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
43\selectlanguage{ngerman}±5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.26\selectlanguage{ngerman}±0.01\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
126\selectlanguage{ngerman}±24\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.11\selectlanguage{ngerman}±0.005\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
107\selectlanguage{ngerman}±26\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.03\selectlanguage{ngerman}±0.001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Wildtype\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.16\selectlanguage{ngerman}±0.03\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1.59\selectlanguage{ngerman}±0.17\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
113\selectlanguage{ngerman}±47\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.30\selectlanguage{ngerman}±0.06\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.50\selectlanguage{ngerman}±0.01\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
37\selectlanguage{ngerman}±6\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.25\selectlanguage{ngerman}±0.01\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
87\selectlanguage{ngerman}±19\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.11\selectlanguage{ngerman}±0.01\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
75\selectlanguage{ngerman}±41\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.03\selectlanguage{ngerman}±0.001\textsuperscript{a}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\emph{asft}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.22\selectlanguage{ngerman}±0.03\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2.04\selectlanguage{ngerman}±0.29\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
186\selectlanguage{ngerman}±86\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.29\selectlanguage{ngerman}±0.04\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.49\selectlanguage{ngerman}±0.01\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
50\selectlanguage{ngerman}±9\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.26\selectlanguage{ngerman}±0.01\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
174\selectlanguage{ngerman}±46\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.11\selectlanguage{ngerman}±0.005\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
155\selectlanguage{ngerman}±30\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.03\selectlanguage{ngerman}±0.004\textsuperscript{a}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
All rice genotypes\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.44\selectlanguage{ngerman}±0.01\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
3.93\selectlanguage{ngerman}±0.20\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
36.7\selectlanguage{ngerman}±7.6\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.68\selectlanguage{ngerman}±0.08\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.53\selectlanguage{ngerman}±0.02\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
53\selectlanguage{ngerman}±1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.20\selectlanguage{ngerman}±0.01\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-------\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.08\selectlanguage{ngerman}±0.002\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-------\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.013\selectlanguage{ngerman}±0.001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Wildtype\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.49\selectlanguage{ngerman}±0.02\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4.42\selectlanguage{ngerman}±0.45\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
57.4\selectlanguage{ngerman}±15.1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.59\selectlanguage{ngerman}±0.06\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.53\selectlanguage{ngerman}±0.02\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
87\selectlanguage{ngerman}±11\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.22\selectlanguage{ngerman}±0.01\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-------\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.084\selectlanguage{ngerman}±0.003\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-------\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.013\selectlanguage{ngerman}±0.001\textsuperscript{a}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\emph{cslf6-1}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.36\selectlanguage{ngerman}±0.02\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
3.27\selectlanguage{ngerman}±0.18\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
26.9\selectlanguage{ngerman}±16.5\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.75\selectlanguage{ngerman}±0.32\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.53\selectlanguage{ngerman}±0.03\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
50\selectlanguage{ngerman}±4\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.18\selectlanguage{ngerman}±0.01\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-------\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.077\selectlanguage{ngerman}±0.003\textsubscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-------\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.013\selectlanguage{ngerman}±0.002\textsuperscript{a}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\emph{cslf6-2}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.47\selectlanguage{ngerman}±0.02\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4.11\selectlanguage{ngerman}±0.24\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
52.2\selectlanguage{ngerman}±16.3\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.59\selectlanguage{ngerman}±0.07\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.51\selectlanguage{ngerman}±0.02\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
77\selectlanguage{ngerman}±7\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.19\selectlanguage{ngerman}±0.01\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-------\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.081\selectlanguage{ngerman}±0.002\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-------\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.013\selectlanguage{ngerman}±0.002\textsuperscript{a}\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\selectlanguage{english}\textsuperscript{+}These values were from fitting the model to the data
from 80 \% RH only.

\selectlanguage{english}\textsuperscript{++}The model failed to calculate\emph{L}
\textsubscript{v} for rice.

\textsuperscript{SS}The model failed to calculate\emph{L}
\textsubscript{v} using partial volume of xylem elements as\selectlanguage{greek}\emph{ϕ}
\selectlanguage{english}\textsubscript{x} in both maize and rice.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-1/Fig-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-2/Fig-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-3/Fig-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-4/Fig-4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-5/Fig-5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-6/Fig-6}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-7/Fig-7}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{CJK}\end{document}

