Mechanisms of FWU
The
presented results unequivocally show that open stomata contribute to FWU
and highlight the role of the cuticle in the process. In both P.
dulcis and P. communis , (‘fully’) open stomata by FC treatment
resulted in increased water absorption rates across the entire Ψ range
evaluated relative to a ‘cuticular only’ path (stomata ‘closed’) and to
partially open stomata (Figure 3). Indeed, open stomata reduced the time
for full Ψ recovery via FWU by up to half and enabled up to 3-4 times
higher fluxes and hydraulic conductance (i.e. Qmax and
Kmax, Table 1). Interestingly, the hydraulic resistance
(Rmin) of P. dulcis leaves with open stomata, was
the same as that estimated for the mesophyll or high-resistance leaf
compartment when water was supplied via the petiole (260 MPa
m2 s g-1; Guzmán-Delgado et al.
2018; Zwieniecki et al. 2007). It must be, however, mentioned that in
that study leaves were younger (collected in May) than in the present
study (collected in September), leading to differences in P.
dulcis FWU parameters (e.g. Rmin of intact leaves was
~660 vs ~1,000 MPa m2s g-1 in May and September, respectively). Such a
discrepancy might indicate that FWU is influenced by temporal changes in
leaf surface properties, as reported by Cavallaro et al. (2020). Even if
open stomata facilitate FWU, the rates are still orders of magnitude
lower than the hydraulic suction via the petiole, which suggests that a
simple flow of liquid water into the leaf via stomata does not occur.
To further asses the principles of water movement into a leaf from its
wet surface across open stomata, we can first calculate the flux if
liquid bridges covering the pore volume were formed. For simplicity we
assume that open stomata are cylinders of radius equal to half of the
maximum pore width recorded here (r = 1.27 µm for P. dulcis and r
= 1.04 µm for P. communis ) and length equal to the average pore
depth (l = 6.73 µm for P. dulcis and l = 10.00 µm for P.
communis ). Considering a pressure difference equal to the Ψ at
Qmax (ΔΨ = 1.11 MPa for P. dulcis and ΔΨ = 1.27
MPa for P. communis ) and using measured stomatal densities (ρ =
211.5 stomata mm-2 for P. dulcis and ρ = 179.8
stomata mm-2 for P. communis ), we can estimate
the flux of liquid water (Ql) through the porous
epidermis using the Hagen–Poiseuille equation (Ql =
[π r4 ΔΨ / 8 ηw l] ρ; where
ηw is water dynamic viscosity). The estimated liquid
flux would be 4.0x104 and 1.2x104 g
m-2 s-1 for P. dulcis andP. communis , respectively. Even with the highly conservative
assumption of path resistance equal to half of the leaf thickness l≈150
µm, the respective flow rate would be 1.8x103 and
7.7x102 g m-2 s-1,
yet the observed flux was in the range of
~2.5x10-3 g m-2s-1, i.e. about six orders of magnitude lower, thus
precluding the possibility of flood type water penetration via open
stomata. Hence, we have to consider another, more plausible scenario,
where hydrophobic ledges over guard cells (Figure 4a, b) prevent the
formation of liquid bridges through stomatal pores, but still allow for
the formation of a water film over the pore, as intuited from ESEM
analyses. In such a scenario, we can raise a question as to whether
vapor diffusion across open stomata can account for the observed
increase in flux over that of cuticular uptake only. Assuming the same
parametrization (stomatal density, ρ, pore radius, r, and pore length,
l) as for liquid flow, and using only the epidermis thickness as the
barrier for vapor flux we can estimate the vapor flux
(Qv) through stomata using the general diffusion
equation based on Fick’s laws (Qv = [D ΔC / l] π
r2 ρ, where D is the diffusion coefficient of water
vapor in air, and ΔC is the concentration gradient between saturated air
at the droplet surface and air at ~99% humidity in the
leaf air space). By adding the contribution of the cuticle
(Qmax for ABA leaves) to the calculated vapor flux
through stomata, we obtain fluxes of 2.02x10-3 and
1.01x10-3 g m-2s-1 for P. dulcis and P. communis ,
respectively, which are within the same order of magnitude as those
recorded (3.35x10-3 and 1.97x10-3 g
m-2 s-1). If the path length is
increased to 150 µm, the respective vapor fluxes are still
1.15x10-3 and 6.83x10-4 g
m-2 s-1 (65% difference). This
simple, conservative analysis provides strong support for the notion
that, in the presented case, the main mechanism of water entry though
stomata is vapor diffusion. There is, however, space for a marginal
amount of water entering the leaf as submicron suspended water droplets
(some fog droplets; Eichert et al. 2008), and as thin water films
created along the pore walls of few stomata (Burkhardt et al. 2012;
Eichert and Burkhardt 2001; Eichert et al. 2008). Additionally, even if
epidermal surfaces are not wet, dehydrated organs can rehydrate from
near saturated air (close to 100% RH, and higher than air RH of the
stressed tissue; ΔC>0) at the surface, as previously
predicted (Binks et al. 2020; Vesala et al. 2017) and experimentally
found (Guzmán-Delgado et al. 2017; Laur and Hacke 2014).
Using chemical treatments on plants may result in equivocal responses of
the physical processes under study if affected by the biological
activity influenced by the treatment. Both FC and ABA were shown to
modify membrane permeability, sometimes affecting leaf hydraulic
properties (Blatt and Clint 1989; Coupel-Ledru et al. 2017). While this
effect could not be avoided, our trial evaluating FWU via the adaxial
surface of FC, ABA and control leaves of P. dulcis suggests that
cuticular uptake was not affected by the treatments, so that the effect
of open stomata leading to increased FWU is related to a new water
pathway and not changes in membrane permeability. The lack of a
significant effect on FWU by chemically induced variations in membrane
hydraulic properties also suggests that FWU is dominated by the
resistance related to the physico-chemical properties of the cuticle and
by vapor diffusion, but not membrane facilitated liquid fluxes.
Thus, the epidermal surface acts
as a high-resistance pathway controlling the kinetics of leaf
rehydration via FWU (Fuenzalida et al. 2019; Guzmán-Delgado et al.
2018). Most importantly, the pattern of leaf rehydration is independent
from the absorption pathway, whether it is the cuticle or the cuticle
with open stomata, suggesting a similar internal path for water
redistribution.
Foliar water uptake (Kmax) increased significantly with
increasing gs (R2=0.9999, p< 0.001, Figure 1c Supporting Information), but the slope was
significantly greater in P. communis than in P. dulcis .
The absence of a common relationship across species was also found by
Limm et al. (2009), and may indicate that species-specific stomatal
features, leaf rheology and tortuosity of the mesophyll path when
stomata are open can exert further influence on the absorption process.
Indeed, while both species show traits that could be associated with
greater protection against excess water entry (and dehydration), such as
outer and inner cuticular ledges and substomatal cuticles, P.
communis – the species with lower FWU capacity (lower
Kmax) – has more prominent inner ledges and a cuticle
that extends farther into the substomatal cavity, even lining
parenchymatic cells (Figure 4). Furthermore, changes in the properties
of the chemically heterogeneous cuticle and other cell wall regions
underneath can affect the species’ capacity for FWU through stomata, and
when the cuticle is the dominant path (Boanares et al. 2018; Guzmán et
al. 2014a,b; Guzmán-Delgado et al. 2018). These changes may be driven by
the stages of cuticle hydration, starting with a partial wetting of the
cuticle from the outer towards the inner side, followed by epidermal
cell rehydration and then rewetting of the cuticle from its inner side
(towards the leaf surface), shortening the diffusion pathways and
creating liquid bridges (Guzmán-Delgado et al. 2018).