Leaf water potential measurements using the pressure chamber: Synthetic testing of assumptions towards best practices for precision and accuracy.

Leaf water potential (ψleaf ), typically measured using the pressure chamber, is the most important metric of plant water status, providing high theoretical value and information content for multiple applications in quantifying critical physiological processes including drought responses. Pressure chamber measurements of ψleaf (ψleafPC ) are most typical, yet, the practical complexity of the technique and of the underlying theory has led to ambiguous understanding of the conditions to optimize measurements. Consequently, specific techniques and precautions diversified across the global research community, raising questions of reliability and repeatability. Here, we surveyed specific methods of ψleafPC from multiple laboratories, and synthesized experiments testing common assumptions and practices in ψleafPC for diverse species: (i) the need for equilibration of previously transpiring leaves; (ii) leaf storage before measurement; (iii) the equilibration of ψleaf for leaves on bagged branches of a range of dehydration; (iv) the equilibration of ψleaf across the lamina for bagged leaves, and the accuracy of measuring leaves with artificially 'elongated petioles'; (v) the need in ψleaf measurements for bagging leaves and high humidity within the chamber; (vi) the need to avoid liquid water on leaf surfaces; (vii) the use of 'pulse' pressurization versus gradual pressurization; and (viii) variation among experimenters in ψleafPC determination. Based on our findings we provide a best practice protocol to maximise accuracy, and provide recommendations for ongoing species-specific tests of important assumptions in future studies.

Most stomatal closure in woody species under moderate drought can be explained by stomatal responses to leaf turgor.

Reduced stomatal conductance (gs ) during soil drought in angiosperms may result from effects of leaf turgor on stomata and/or factors that do not directly depend on leaf turgor, including root-derived abscisic acid (ABA) signals. To quantify the roles of leaf turgor-mediated and leaf turgor-independent mechanisms in gs decline during drought, we measured drought responses of gs and water relations in three woody species (almond, grapevine and olive) under a range of conditions designed to generate independent variation in leaf and root turgor, including diurnal variation in evaporative demand and changes in plant hydraulic conductance and leaf osmotic pressure. We then applied these data to a process-based gs model and used a novel method to partition observed declines in gs during drought into contributions from each parameter in the model. Soil drought reduced gs by 63-84% across species, and the model reproduced these changes well (r(2) = 0.91, P < 0.0001, n = 44) despite having only a single fitted parameter. Our analysis concluded that responses mediated by leaf turgor could explain over 87% of the observed decline in gs across species, adding to a growing body of evidence that challenges the root ABA-centric model of stomatal responses to drought.

Is stomatal conductance optimized over both time and space in plant crowns? A field test in grapevine (Vitis vinifera).

Crown carbon gain is maximized for a given total water loss if stomatal conductance (gs ) varies such that the marginal carbon product of water (∂A/∂E) remains invariant both over time and among leaves in a plant crown, provided the curvature of assimilation rate (A) versus transpiration rate (E) is negative. We tested this prediction across distinct crown positions in situ for the first time by parameterizing a biophysical model across 14 positions in four grapevine crowns (Vitis vinifera), computing optimal patterns of gs and E over a day and comparing these to the observed patterns. Observed water use was higher than optimal for leaves in the crown interior, but lower than optimal in most other positions. Crown carbon gain was 18% lower under measured gs than under optimal gs . Positive curvature occurred in 39.6% of cases due to low boundary layer conductance (gbw ), and optimal gs was zero in 11% of cases because ∂A/∂E was below the target value at all gs . Some conclusions changed if we assumed infinite gbw , but optimal and measured E still diverged systematically in time and space. We conclude that the theory's spatial dimension and assumption of positive curvature require further experimental testing.

Rapid hydraulic recovery in Eucalyptus pauciflora after drought: linkages between stem hydraulics and leaf gas exchange.

In woody plants, photosynthetic capacity is closely linked to rates at which the plant hydraulic system can supply water to the leaf surface. Drought-induced embolism can cause sharp declines in xylem hydraulic conductivity that coincide with stomatal closure and reduced photosynthesis. Recovery of photosynthetic capacity after drought is dependent on restored xylem function, although few data exist to elucidate this coordination. We examined the dynamics of leaf gas exchange and xylem function in Eucalyptus pauciflora seedlings exposed to a cycle of severe water stress and recovery after re-watering. Stomatal closure and leaf turgor loss occurred at water potentials that delayed the extensive spread of embolism through the stem xylem. Stem hydraulic conductance recovered to control levels within 6 h after re-watering despite a severe drought treatment, suggesting an active mechanism embolism repair. However, stomatal conductance did not recover after 10 d of re-watering, effecting tighter control of transpiration post drought. The dynamics of recovery suggest that a combination of hydraulic and non-hydraulic factors influenced stomatal behaviour post drought.

Integrative field scale phenotyping for investigating metabolic components of water stress within a vineyard.

BACKGROUND: There is currently a high requirement for field phenotyping methodologies/technologies to determine quantitative traits related to crop yield and plant stress responses under field conditions. METHODS: We employed an unmanned aerial vehicle equipped with a thermal camera as a high-throughput phenotyping platform to obtain canopy level data of the vines under three irrigation treatments. High-resolution imagery (< 2.5 cm/pixel) was employed to estimate the canopy conductance (gc ) via the leaf energy balance model. In parallel, physiological stress measurements at leaf and stem level as well as leaf sampling for primary and secondary metabolome analysis were performed. RESULTS: Aerial gc correlated significantly with leaf stomatal conductance (gs ) and stem sap flow, benchmarking the quality of our remote sensing technique. Metabolome profiles were subsequently linked with gc and gs via partial least square modelling. By this approach malate and flavonols, which have previously been implicated to play a role in stomatal function under controlled greenhouse conditions within model species, were demonstrated to also be relevant in field conditions. CONCLUSIONS: We propose an integrative methodology combining metabolomics, organ-level physiology and UAV-based remote sensing of the whole canopy responses to water stress within a vineyard. Finally, we discuss the general utility of this integrative methodology for broad field phenotyping.

Stomatal and mesophyll conductances to CO2 in different plant groups: underrated factors for predicting leaf photosynthesis responses to climate change?

The climate change conditions predicted for the end of the current century are expected to have an impact on the performance of plants under natural conditions. The variables which are foreseen to have a larger effect are increased CO2 concentration and temperature. Although it is generally considered CO2 assimilation rate could be increased by the increasing levels of CO2, it has been reported in previous studies that acclimation to high CO2 results in reductions of physiological parameters involved in photosynthesis, like the maximum carboxylation rate (Vc,max), stomatal conductance (gs) and mesophyll conductance to CO2 (gm). On the one hand, most of the previous modeling efforts have neglected the potential role played by the acclimation of gm to high CO2 and temperature. On the other hand, the effect of climate change on plant clades other than angiosperms, like ferns, has received little attention, and there are no studies evaluating the potential impact of increasing CO2 and temperature on these species. In this study we predicted responses of several representative species among angiosperms, gymnosperms and ferns to increasing CO2 and temperature. Our results show that species with lower photosynthetic capacity - such as some ferns and gymnosperms - would be proportionally more favored under these foreseen environmental conditions. The main reason for this difference is the lower diffusion limitation imposed by gs and gm in plants having high capacity for photosynthesis among the angiosperms, which reduces the positive effect of increasing CO2. However, this apparent advantage of low-diffusion species would be canceled if the two conductances - gs and gm - acclimate and are down regulated to high CO2, which is basically unknown, especially for gymnosperms and ferns. Hence, for a better understanding of different plant responses to future climate, studies are urged in which the actual photosynthetic response/acclimation to increased CO2 and temperature of ferns, gymnosperms and other under-evaluated plant groups is assessed.