Temperature governs the relative contributions of cuticle and stomata to leaf minimum conductance

Introduction

Climate change is increasing the frequency and severity of hot drought events in many parts of the world, with further increases forecast for the coming century (Intergovernmental Panel on Climate Change (IPCC), 2021). During periods of water stress, plants typically reduce their stomatal aperture, restricting both water loss and carbon substrate availability for photosynthesis (Cowan & Farquhar, 1977). However, even with stomata maximally closed, leaves still lose water at a rate described by the leaf minimum conductance to water vapour, g_min (mol m⁻² s⁻¹; Table 1) (Duursma et al., 2019). While g_min is typically more than an order of magnitude less than stomatal conductance (g_sw; mol m⁻² s⁻¹) during more favourable conditions (Slot et al., 2021), plants may lose substantial amounts of water even under maximal stomatal closure due to high evaporative demand (Vicente-Serrano et al., 2020). Improved understanding of g_min is necessary, as transpiration during hot drought events can have substantial effects on plant mortality and landscape-scale water balance (Park Williams et al., 2013; Rogers et al., 2017; Hammond & Adams, 2019).

Table 1. List of symbols.

Symbol	Definition	Units
A	Net assimilation rate	μmol m−2 s−1
al	One-sided (projected) leaf area	m2
ca	Ambient air CO2 concentration	μmol mol−1
ci	Leaf intercellular CO2 concentration	μmol mol−1
E	Transpiration rate	mol m−2 s−1
gbw	Leaf boundary layer conductance to water vapour	mol m−2 s−1
gcw	Leaf cuticular conductance to water vapour	mol m−2 s−1
gmin	Leaf minimum conductance to water vapour	mol m−2 s−1
gsw	Stomatal conductance to water vapour	mol m−2 s−1
gsw,min	Minimum stomatal conductance	mol m−2 s−1
$$	Initial value of $$	mol m−2 s−1
$$	Stomatal conductance in excess of gsw,min	mol m−2 s−1
gtw	Leaf total conductance to water vapour	mol m−2 s−1
h	Elevation	m
Jmax	Maximum rate of RuBP regeneration	μmol m−2 s−1
LDMC	Leaf dry matter content	mg g−1
LMA	Leaf mass per unit area	g m−2
lsp	Stomatal pore length	μm
mdry	Leaf dry mass	g
mwet	Leaf wet (fresh) mass	g
M	Leaf mass	g
M0	Initial leaf mass	g
Patm	Atmospheric pressure	kPa
SVP	Saturation vapour pressure	kPa
t	Time	s
Vcmax	Maximum rate of Rubisco carboxylation	μmol m−2 s−1
VPD	Vapour pressure deficit	kPa
wa	Water vapour concentration in ambient air	mmol mol−1
wi	Water vapour concentration in leaf intercellular air spaces	mmol mol−1
wgs	Guard cell width	μm
xcut	Cuticle thickness	μm
xepi	Epidermis thickness	μm
xl	Leaf thickness	μm
xpal	Palisade mesophyll thickness	μm
xspg	Spongy mesophyll thickness	μm
β	Conductance conversion factor	g m mol−1
κ	Ratio of adaxial to abaxial ci	unitless
μ	Molar mass of water	g mol−1
ρs	Stomatal density	m−2
τ	Stomatal closure time constant	s

Even under complete stomatal closure conditions, water may still escape the leaf via the cuticle (i.e. cuticular conductance, g_cw; mol m⁻² s⁻¹) or via small apertures in incompletely closed stomata (i.e. minimum stomatal conductance, g_sw,min; mol m⁻² s⁻¹; Fig. 1) (Duursma et al., 2019). Careful measurements partitioning g_min into stomatal and cuticular components have yielded widely conflicting conclusions about the relative contributions of these pathways, from all water lost via the cuticle (Slot et al., 2021), to highly variable species-specific ratios (6–44% water lost via the stomata; Machado et al., 2021), to a majority of water lost via the stomata in one species (Márquez et al., 2022).

Fig. 1

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Leaf water fluxes during times of high and low water availability. During times of water availability and photosynthetic activity, stomata open to allow gas exchange with the atmosphere (upper image). In this case, leaf total conductance to water vapour g_tw is the sum of the stomatal conductance g_sw and the cuticular conductance g_cw (ignoring the boundary layer). During times of low water availability or low photosynthetic activity, stomata close to restrict water loss (lower image). With stomata fully closed, g_tw is at its minimum and is referred to as leaf minimum conductance g_min, which is a sum of g_cw and a minimum stomatal contribution g_sw,min. All conductances are expressed in units of mmol m⁻² s⁻¹.

As droughts often coincide with high temperatures, it is vital to understand the temperature dependence of g_min, as well as that of its underlying component pathways. The short-term temperature response of g_min frequently exhibits a monotonically increasing relationship with temperature over the range of c. 25–50°C, often with a ‘biphasic’ relationship (e.g. Schuster et al., 2016; Bueno et al., 2019; Billon et al., 2020; Wang et al., 2024; n.b., in nearly every study which has investigated the temperature response of g_min or g_cw, vapour pressure deficit (VPD) covaries with temperature; see the Discussion section). However, invariant relationships, or even declining g_min with temperature, have also been observed (e.g. Bueno et al., 2019; Slot et al., 2021). The short-term temperature dependence of g_cw also generally shows monotonically increasing relationships with temperature (e.g. Schreiber, 2001; Riederer, 2006), but this is typically measured on enzymatically isolated cuticles or leaf discs, rarely in vivo (cf., Márquez et al., 2021). Crucially, the short-term temperature dependencies of both g_min and g_cw have never been measured concurrently in vivo; thus, little is known about how these different water pathways respond to temperature.

Understanding how the different pathways of water loss contribute to the observed temperature sensitivity of g_min is necessary for informing theory on alternative plant water use strategies (Blonder et al., 2023). Recently, researchers have reported stomatal ‘decoupling’ at high leaf temperatures (Aparecido et al., 2020; Krich et al., 2022; Marchin et al., 2023). Stomatal decoupling occurs when stomatal conductance increases, or fails to decrease, despite declining assimilation rates at high leaf temperatures, contradicting theory based on optimality principles (Cowan & Farquhar, 1977; Medlyn et al., 2011). It is not known whether this decoupling is an adaptive response of the plant intended to cool the leaves (Michaletz et al., 2016; Garen et al., 2023) or a passive ‘failure’ mechanism (Slot et al., 2021; Blonder et al., 2023). Improved understanding of the temperature response of g_min and its component processes will help in dissecting the mechanism underlying this high-temperature water use behaviour.

Furthermore, understanding the temperature response of g_cw is necessary for improving gas exchange measurements. Recently, Márquez et al. (2021) proposed a new model for leaf gas exchange that accounts for g_cw (the Marquez-Stuart-Williams-Farquhar or MSF model). Previous gas exchange models calculated leaf intercellular CO₂ concentration c_i based on the ratio of diffusivities of CO₂ and H₂O in air (c. 1.6), under the assumption that all transpiration occurs via the stomata (von Caemmerer & Farquhar, 1981). However, while CO₂ and H₂O both move readily through the stomata, the cuticle is nearly impermeable to CO₂ (Boyer, 2015). Given that some water vapour escapes via the cuticle, a model that attributes all transpiration to the stomata will overestimate c_i (Tominaga et al., 2018). Photosynthetic capacity metrics such as V_cmax (maximum rate of Rubisco carboxylation) and J_max (maximum rate of RuBP regeneration) are estimated using the relationship between assimilation rate A and c_i (i.e. A–c_i curves) (Farquhar et al., 1980; Sharkey et al., 2007), hence errors in c_i may affect estimates of V_cmax and J_max, with potential consequences for process-based modelling frameworks that employ these metrics (Stinziano et al., 2019; Hussain et al., 2024). However, the effects of g_cw temperature dependence on metrics of photosynthetic capacity have not previously been investigated. To quantifying the magnitude of error and improve estimates of photosynthetic capacity, it is necessary to know whether and when to account for g_cw and its temperature dependence.

Here, we address these questions by measuring the short-term temperature responses of g_min and g_cw. Our study has three goals as follows:

1. to describe the temperature dependencies of g_min and g_cw, and to describe how the pathways of leaf water loss vary with temperature;
2. to test whether g_min and g_cw depend on anatomical, structural, and morphological leaf traits; and
3. to test whether g_cw and its temperature dependence cause errors in measurements of photosynthetic capacity.

We demonstrate that the contributions of stomata and cuticle to g_min vary with temperature, with the cuticular water loss pathway dominating at higher temperatures. We find temperature-dependent relationships between leaf conductance and leaf traits, and we further show that photosynthetic capacity metrics depend on g_cw, particularly at low stomatal conductance.