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 cslf6-1 (0.18 ± 0.01) and 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 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 µm ± 2.0) than
all other conditions (25.7 to 26.6 µm; Table 1, S1).
Changes in Δ18OLW between plants grown
in high and low light (HL-LL) co-varied with changes inL v, E , Sd, Sdabaxial, and
partial volume of veins excluding the bundle sheath (P < 0.05)
and partial volume of the total vasculature andg s (P = 0.05; Table S2). The mean genotypic
difference (HL-LL) in Sd and E formed significant linear
relationships with Δ18OLW (Fig. 5a,
b), while E and Sd also formed a linear relationship (Fig. 5c).
In contrast, the change in Δ18OLWbetween plants grown at high and low light did not co-vary with changes
in Δ18Oe, 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
(S mes), surface area of chloroplasts exposed to
IAS per unit of leaf area (Sc), and leaf dry mass per
area [Table S2, these data were previously published in
Ellsworth et al. (2018)].
Mesophyll and xylem P éclet models in maize and rice
All genotypes of maize and rice formed significant positive
relationships between f sw and transpiration rate
(E ), suggesting a Péclet effect in these genotypes (Fig. 6a,b).
When f sw and E were fitted to the
mesophyll and xylem Péclet model (Eqn. 10) to solve for the following
three variables: mesophyll L (L m), xylemL (L v), and the proportion of xylem water
(ϕ x), the model in maize converged, but the
estimated value for L m was essentially 0 (2 x
10-17). In rice the model failed to converge on a
significant value for L m. Considering thatL m was estimated as ~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 ofL v and ϕ x when all data
were used. However, when only data from 80 % RH were examined,Zmasft had a larger estimate of L v (186 ±
86 mm) than wildtype (113 ± 47 mm; Table 3). At 50 % RH, the xylem
Péclet model did not converge. In rice, L vcalculated from the xylem Péclet only model was smaller incslf6-1 than wildtype and cslf6-2 . In each species,
estimates of ϕ x were similar across genotypes,
but ϕ x was twice as large in rice than maize
(Table 3).
L v was estimated using the xylem Peclet model
when the partial volumes of total vasculature, veins including and
excluding BS were used as ϕ x in maize and only
when the partial volume of total vasculature was used as
ϕx in rice. When the partial volume of leaf vasculature
or vascular bundles was used as ϕ x in the xylem
Péclet model, estimates of L v were greater forZmasft double mutants than WT, and this difference was most
apparent at low light intensity (Table 1, 3). In rice,L v was lower in cslf6-1 than WT andcslf6-2 . These results were similar to modelledL v (solving for both L vand ϕ x), except that L vwas considerably smaller when partial pressure of the total vasculature
was used as ϕx in maize. The partial volume of veins,
including the bundle sheath, was most similar tof sw (especially at high light intensity) andϕ x values estimated from the xylem Péclet model
in maize (Table 3). Rice, having larger f swvalues than maize, the partial volume of the total vasculature of rice
was most similar to f sw andϕ x.
A sensitivity analysis of the interaction betweenL v and ϕ x in the xylem
Péclet model showed that ϕ x in rice was greater
than in maize and that L v had a large effect onϕ 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 L v andϕ x were greater in Zmasft than in
wildtype. In rice, L v andϕ x were lower in cslf6-1 than wildtype andcslf6-2 when L v was greater than
approximately 50 mm. The results between maize at 50 % RH and rice were
similar in that ϕ x was greater at high light than
low light across the range of L v (Fig. 7).