Christine Scoffoni

Caetano Albuquerque

Hervé Cochard

Thomas N Buckley

Leila R Fletcher

Marissa A Caringella

Megan Bartlett

Craig R Brodersen

Steven Jansen

Andrew J Mcelrone

Marissa A Caringella

Lawren C Sack

H Albuquerque

T N Cochard

L R Buckley

M Fletcher

M K Caringella

A J Bartlett

The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana

Keywords: aquaporins, drought, leaf water relations, plant modelling, pv-curves

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Plant growth requires a copious water supply because the rate of CO2 uptake for photosynthesis depends on stomatal conductance, which results in transpiratory water loss. Because stomata close in dehydrating leaves, photosynthesis and growth depend on the efficiency of water replacement to the mesophyll. Thus, in the past two decades, many studies focusing on diverse species have shown the centrality of the plant hydraulic system in determining leaf-scale gas exchange and plant productivity [START_REF] Sack | Leaf hydraulics[END_REF]Brodribb et al., 2007;[START_REF] Scoffoni | Author and Title Google Scholar: Author Only Title Only Author and Title[END_REF]. Our aim was to test hypotheses for the dynamics of hydraulic traits and their influence on gas exchange during dehydration using the model species Arabidopsis. Establishing a framework for testing the influence of hydraulic traits in Arabidopsis can help address recent debates and open avenues for discovery of genetic associations in natural and mutant genotypes under moist conditions and during soil and/or atmospheric drought.

The leaf accounts for a large proportion of plant hydraulic resistance [START_REF] Sack | Leaf hydraulics[END_REF]. Thus, theoretical and empirical studies have shown strong correlations of stomatal conductance (gs) and photosynthetic rate (Amax) with leaf hydraulic conductance (Kleaf; determined as the flow rate divided by water potential driving force, in units mmol m -2 s -1 MPa -1 ) across species under well-watered conditions (Nardini and Salleo, 2003;Brodribb and Holbrook, 2004;[START_REF] Sack | Leaf hydraulics[END_REF][START_REF] Scoffoni | Author and Title Google Scholar: Author Only Title Only Author and Title[END_REF], and within given species during dehydration (Brodribb andHolbrook, 2006, 2007;Bartlett et al., 2016;Scoffoni and Sack, 2017). A high Kleaf enabling higher gs and Amax could be achieved through a high vein length per area, larger and/or more numerous xylem conduits (and/or xylem pits), and more conductive mesophyll and bundle sheath anatomy and biochemistry (Brodribb et al., 2007;[START_REF] Choat | Structure and function of bordered pits: new discoveries and impacts on whole-plant hydraulic function[END_REF][START_REF] Caringella | Leaf hydraulic conductance varies with vein anatomy across Arabidopsis thaliana wild-type and leaf vein mutants[END_REF]Scoffoni et al., 2015;[START_REF] Scoffoni | Author and Title Google Scholar: Author Only Title Only Author and Title[END_REF][START_REF] Stewart | Arabidopsis thaliana Ei-5: Minor Vein Architecture Adjustment Compensates for Low Vein Density in Support of Photosynthesis[END_REF]). Yet, the linkages of Kleaf and gas exchange as leaves dehydrate to turgor loss point are still under debate. Early studies suggested that Kleaf decline drives stomatal closure under high vapor pressure deficits at mid-day (Brodribb and Holbrook, 2003a;Bucci et al., 2003) and during drought (Salleo et al., 2001;Brodribb and Holbrook, 2003b;Nardini and Salleo, 2003). Several recent studies suggested that in some species, Kleaf might not decline until embolism forms in the leaf vein xylem (Brodribb et al., 2016a;Brodribb et al., 2016b;[START_REF] Skelton | Casting light on xylem vulnerability in an herbaceous species reveals a lack of segmentation[END_REF], which for many species does not occur until past the point of stomatal closure and bulk leaf turgor loss (Brodribb et al., 2016b;[START_REF] Hochberg | Stomatal closure, basal leaf embolism, and shedding protect the hydraulic integrity of grape stems[END_REF]Scoffoni et al., 2017a).

Similarly, xylem wall collapse may drive Kleaf declines in pine needles and minor veins of an oak species, but only below turgor loss point (Cochard et al., 2004a;Zhang et al., 2016). Avoiding Kleaf decline during transpiration when leaves are hydrated above turgor loss point has been suggested as adaptive, maintaining leaf water potential and open stomata, though at the risk of sustaining water potentials that would induce xylem cavitation under high vapor pressure deficits (Brodribb and Holbrook, 2006). Numerous studies in the last decade have shown that species differ in whether Kleaf declines at milder, similar or more severe leaf water potentials than at stomatal closure, and that Kleaf decline depends mechanistically on processes in multiple tissues-the venation, bundle sheath, and mesophyll pathways of liquid and vapor transport (reviewed by Scoffoni and Sack, 2017). Indeed, a meta-analysis of the literature found that on average, across species (and methods for Kleaf determination), Kleaf declined by 30-80% before turgor loss point (Scoffoni and Sack, 2017). Recent work focusing on partitioning leaf xylem and outside-xylem resistances during dehydration suggested the outside-xylem hydraulic conductance (Kox) as the primary driver of Kleaf decline [START_REF] Trifilo | The contribution of vascular and extra-vascular water pathways to droughtinduced decline of leaf hydraulic conductance[END_REF]Scoffoni et al., 2017a;Scoffoni and Sack, 2017), which could be triggered by the loss of cell connectivity, cell shrinkage, and/or changes in membrane aquaporin activity (Laur and Hacke, 2014b;[START_REF] Scoffoni | Leaf shrinkage with dehydration: coordination with hydraulic vulnerability and drought tolerance[END_REF]Scoffoni et al., 2017a), potentially mediated by effects of ABA in the bundle sheath (Pantin et al., 2013). A recent study in rice has attributed to Kleaf decline a strong causal role in driving stomatal closure during dehydration [START_REF] Wang | Leaf Hydraulic Vulnerability Triggers the Decline in Stomatal and Mesophyll Conductance during drought in Rice (Oryza sativa)[END_REF].

Debate has also focused on the light response of Kleaf. Previous studies have found many species to exhibit a rapid enhancement of Kleaf in response to increased irradiance [START_REF] Sack | The hydraulic conductance of the angiosperm leaf lamina: a comparison of three measurement methods[END_REF]Nardini et al., 2005b;[START_REF] Tyree | The dependence of leaf hydraulic conductance on irradiance during HPFM measurements: any role for stomatal response?[END_REF][START_REF] Cochard | Putative role of aquaporins in variable hydraulic conductance of leaves in response to light[END_REF][START_REF] Scoffoni | The rapid light response of leaf hydraulic conductance: new evidence from two experimental methods[END_REF][START_REF] Voicu | Light response of hydraulic conductance in bur oak (Quercus macrocarpa) leaves[END_REF][START_REF] Guyot | Combined impacts of irradiance and dehydration on leaf hydraulic conductance: insights into vulnerability and stomatal control[END_REF][START_REF] Xiong | Differential coordination of stomatal conductance, mesophyll conductance, and leaf hydraulic conductance in response to changing light across species[END_REF], but not all [START_REF] Sack | The hydraulic conductance of the angiosperm leaf lamina: a comparison of three measurement methods[END_REF][START_REF] Gasco | Resistance to water flow through leaves of Coffea arabica is dominated by extra-vascular tissues[END_REF][START_REF] Tyree | The dependence of leaf hydraulic conductance on irradiance during HPFM measurements: any role for stomatal response?[END_REF][START_REF] Scoffoni | The rapid light response of leaf hydraulic conductance: new evidence from two experimental methods[END_REF][START_REF] Xiong | Differential coordination of stomatal conductance, mesophyll conductance, and leaf hydraulic conductance in response to changing light across species[END_REF]. Activation of PIP2,1 and PIP2,2 aquaporins under high irradiance at high water potential has been shown to also enhance Kleaf in some [START_REF] Cochard | Putative role of aquaporins in variable hydraulic conductance of leaves in response to light[END_REF], though not all species [START_REF] Voicu | Aquaporin gene expression and apoplastic water flow in bur oak (Quercus macrocarpa) leaves in relation to the light response of leaf hydraulic conductance[END_REF]. A higher Kleaf under high light could potentially help buffer rapid changes in VPD and prevent stomata from closing (Cairns [START_REF] Murphy | Differential leaf expansion can enable hydraulic acclimation to sun and shade[END_REF]Scoffoni et al., 2015). In Arabidopsis, one study estimated hydraulic conductance by pushing water into entire rosettes suspended underwater in a dark pressure chamber, and found it was higher for leaves acclimated to dark rather than high irradiance (Prado et al., 2013), though no study has investigated this response at the leaf level.

Results

Leaf hydraulics and gas exchange and their responses to leaf dehydration and irradiance in Arabidopsis

Arabidopsis Col-0 exhibited high maximum leaf hydraulic conductance (Kleaf), stomatal conductance (gs), minimum epidermal conductance (gmin), as well as light-saturated photosynthetic rate (Aarea) (Figure 1, Figure 2, Table 2). The partitioning of hydraulic resistances in the leaf indicated a similar distribution of resistances in the xylem and outside-xylem pathways (45.6 vs.

54.4% respectively; Table 2).

Arabidopsis showed a strong vulnerability to dehydration in Kleaf and gas exchange (Figure 1). Notably, the range of water potential measured on intact plants diurnally, and on detached leaves during bench dehydration was similar (Figure 1, Figure 2). Kleaf responded non-linearly to dehydration, with steep declines before 50% loss of its initial Kleaf by -0.17 MPa (Kleaf P50), and gradually slowing down its response to further dehydration (Table 2, Figure 1). Both gs and Aarea responded linearly to declining Ψleaf (Figure 2), reaching 50% loss of initial rates by -0.37 and -0.38 MPa respectively, and 95% loss at similar Ψleaf values of -0.71MPa (Table 2). At turgor loss point, Kleaf had declined by ca. 88%, and stomata were nearly fully closed (Table 2, Figure 1).

Leaves acclimated to high irradiance had significantly higher Kleaf values than leaves acclimated to low irradiance, with a 60% enhancement of Kleaf from low to high irradiance in wellhydrated leaves of Col-0. (Figure 1; t-test done on residuals of Kleaf, i.e., difference of observed values relative to those predicted from the best fit function through all data combined: (𝐾 leaf = 8.33 + 83.7 × exp (-(9.47 × Ψ leaf ))). Residuals for Kleaf were 7.4 mmol m -2 s -1 MPa -1 higher under high irradiance across the entire vulnerability curve (p = 0.01), 7.9 mmol m -2 s -1 MPa -1 higher www.plantphysiol.org on October 26, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.

Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743 considering only leaves above turgor loss point (p = 0.01), and 13.9 mmol m -2 s -1 MPa -1 higher considering only leaves at hydration above -0.2MPa (p = 0.04). However, leaves acclimated to high and low irradiance were similar in their Kleaf P50 (-0.17 vs. -0.16 MPa respectively; Figure 1).

Diurnal responses of gas exchange

Photosynthetic and stomatal responses were measured over the course of two days, from 0900 to 1800. Our results showed that the diurnal pattern of whole-plant hydraulic conductance and gas exchange reflected the dynamics of Ψleaf, as evidenced by the strong trends of Kplant, gs and Amax versus Ψleaf (r 2 = 0.45-0.81; p < 0.02; Figure 2). Of all the potential environmental drivers, vapor pressure deficit (VPD) most strongly correlated with gs dynamics diurnally (r 2 = 0.18; p = 0.002; Supplemental Figure S1).

Independent effects analysis of potential drivers of diurnal dynamics in gs, including environmental factors and Ψleaf showed that Ψleaf was the most important statistically, contributing 77% towards the diurnal variation (Supplemental Figure S2). The VPD contributed 11%, and temperature, PAR and time of day each contributed only 4% to the observed variation (Supplemental Figure S2).

Testing for vein xylem embolism and collapse during leaf dehydration using micro-computed tomography

We scanned leaves using in vivo micro-computed tomography for dehydrated plants to visualize potential xylem embolism. In 14/18 leaves attached to plants that spanned the observed range of Ψleaf (-0.05 to -0.87 MPa), no gas embolism was observed in major or minor veins (Figure 3). In 4/18 scans, we observed 1-2 embolized conduits in the midrib and/or secondary veins; notably, these leaves were not the most dehydrated (Ψleaf = -0.13 to -0.45 MPa; Figure 4; Table 3) but were within the same range as other leaves that did not exhibit embolism. In all three leaves that showed embolized midrib conduits, the embolism spanned the entire length of the scanned section, and we were unable to measure the total vessel length (Figure 4; Table 3). For two leaves, the embolized midrib conduit extended into a secondary vein. In the fourth leaf, an isolated embolised conduit in the secondary vein was observed (Figure 4, Table 3). All embolized conduits were of average diameter ( range of water potentials investigated. The resolution of the micro-CT scans was not sufficient to determine whether conduit collapse occured in higher-order veins.

Modelling the impact of embolism and collapse on Kx

Spatially explicit modelling of the leaf xylem (Table 1) showed that the very low level of observed xylem conduit embolism would reduce leaf xylem hydraulic conductance (Kx) by 1.2 to 4.7%

(Table 3). Because resolution was not sufficient to determine whether conduit collapse occured in higher-order veins, we simulated the potential impact of such collapse if it had occurred. These simulations showed that if higher-order veins were to collapse to the same % of conduit diameter as recently reported for minor veins of Quercus rubra (Zhang et al., 2016), this would decrease Kx by 3-7.5% (Table 3). Under a more extreme scenario in which collapse of tertiary and minor veins caused a 50% decline in their conductivity, Kx would be reduced by 12-17% (Table 3), which would decrease Kleaf by 7-9%.

Modelling the putative causes of Kox decline

Spatially explicit modelling of the outside-xylem pathways using MOFLO 2.0 (Table 1) suggested that the main factor accounting for the decline in Kox observed at -0.5 MPa was most likely reduction of cell membrane permeability in combination with an apoplastic barrier at the bundle sheath (Figure 5). Under high irradiance, an 80% reduction of cell membrane permeability would cause a 68.4% decrease in Kox; adding an 80% reduction in cell connectivity would further decrease Kox by 0.2 % (Figure 5). When performing these simulations with no apoplastic barrier at the bundle sheath, the impact of an 80% reduction of cell membrane permeability caused only a 24.5% decrease in Kox (Figure 5). Simulating the impact of changes of temperature gradients due to light absorption changed the percent loss of Kox by 1-3% across simulations (Supplemental Table S1).

Finally, simulating the impact of cell shrinkage from full turgor to -0.5 MPa resulted in an increase in Kox by 7 to 15%, due to the increase in vein density caused by leaf shrinkage and the consequent decrease in outside-xylem water flow pathlengths (Figure 5).

Partitioning the contribution of Kleaf vulnerability to gs decline

In a transpiring leaf, a low Ψleaf would result from low water potentials proximally to the leaf (i.e., in the soil or roots; which is greater, given Kleaf vulnerability. Thus, given that gs declines with Ψleaf, Kleaf vulnerability will amplify the reduction of gs at a given soil water potential and vapor pressure deficit. Using a partitioning analysis, we applied the observed parameters of gs and Kleaf decline in Arabidopsis to compute the marginal % contribution of Kleaf vulnerability to the decline of gs (Table 1). Our results

showed that Kleaf vulnerability contributes strongly to gs decline in transpiring leaves early in dehydration, due to amplification of Ψleaf decline; when gs declines by 30%, 70% of this response is due to Kleaf vulnerability rather than low water potential proximal to the leaf (Figure 6). The contribution of Kleaf vulnerability to gs decline remains >40% until gs declined by 50%, and becomes less important as stomata approach full closure. When gs has declined by 95%, the contribution of Kleaf vulnerability to gs decline is < 1%.

Using the SurEau whole-plant physiology model to estimate the influence of Kleaf decline on gas exchange, productivity and survival

We tested the importance of Kleaf vulnerability prior to turgor loss point in reducing gs and photosynthesis on plant carbon balance and survival in simulations using SurEau [START_REF] Martin-Stpaul | Plant resistance to drought depends on timely stomatal closure[END_REF] (Figure 7A-D; Table 1). In simulations, the experimentally observed Kleaf vulnerability caused an up to -0.36 MPa lower Ψleaf at midday under well-hydrated conditions (yellow and red lines), compared to constant Kleaf simulations (light and dark blue lines) (Figure 7B). This lower Ψleaf in turn reduced gs and cumulative CO2 assimilation (An, tot) by up to 62 and 17% respectively under well-hydrated conditions (Figure 7A-B), but cumulative water-use efficiency (calculated as An, tot/total transpiration) increased by 28% (inset in Figure 7B). Given finite soil water supply in these simulations, this higher water-use efficiency led to up to 24% higher An, tot over the entire course of the simulated drought. Indeed, because Kleaf vulnerability results in lower gs during the early stage of the drought, the soil water potential (approximated as nightime Ψleaf in Figure 7B) is maintained at higher levels as drought ensues, leading to the maintenance of higher gs during later drought (Figure 7A). Additionally, because Ψleaf does not drop as fast during the course of the drought, these simulations showed that given Kleaf vulnerability, the onset of leaf xylem embolism occurs later during drought such that plants survive up to 6 days longer under drying soil (Figure 7D). These simulations resulted in similar findings whether or not Kroot was set as vulnerable or constant, highlighting Kleaf vulnerability as a main driver of improved water-use efficiency, An, tot and survival during soil drying.

Drought tolerance in Arabidopsis

Arabidopsis Col-0 exhibits low leaf mass per area, a high degree of area shrinkage during dehydration, high minimum epidermal conductance (gmin), high osmotic potential at full turgor and turgor loss point, low modulus of elasticity and relative water content at turgor loss point (Table 1).

Discussion

Our

Drivers of leaf hydraulic conductance decline during dehydration

Our results suggest that changes in outside-xylem pathways are the main drivers of the response of Kleaf to dehydration in Arabidopsis. MicroCT imaging showed that embolism was rare in major vein xylem conduits and nonexistent in minor veins. Only one or two embolized conduits (representing on average 6-11% of the conduits in the midrib) were found in 4/18 samples, with no trend of embolism with increasing water stress prior to turgor loss point. This low vulnerability to embolism in leaves parallels findings for Arabidopsis inflorescence stems, which have P50 values lower than -2.5 MPa [START_REF] Tixier | Arabidopsis thaliana as a model species for xylem hydraulics: does size matter?[END_REF]. The few rare observed leaf vein xylem emboli likely arose from methodological artifacts. In the 3/4 samples in which embolized conduits were observed, the embolized conduit spanned the entire section. One possibility is that air may have entered the conduit when plants were removed from the soil for dehydration, if air entered conduits from damaged roots, and conduits were continuous into the major veins of scanned leaves.

Similarly, air may have entered when the two leaves were excised from the plant for initial water potential measurement, if conduits spanned from these leaves to others in the rosette including the scanned leaves. Alternatively, these few embolisms could be the result of defects in the development of these conduits (Pickard, 1981;[START_REF] Tyree | Biophysical perspectives of xylem evolution: is there a tradeoff of hydraulic efficiency for vulnerability to dysfunction[END_REF]. Indeed, we found a single isolated embolism event occured in a secondary vein of one of our samples. Such isolated embolism events have been reported in leaf veins of other angiosperm species (Scoffoni et al., 2017b) and stem xylem (Brodersen et al., 2013;[START_REF] Choat | Synchrotron X-ray microtomography of xylem embolism in Sequoia sempervirens saplings during cycles of drought and recovery[END_REF][START_REF] Choat | Non-invasive measurement of vulnerability to drought induced embolism by X-ray Microtomography[END_REF].

MicroCT imaging did not reveal any conduit collapse in the midrib or secondary veins across the range of observed water potentials. Since the resolution of the microCT imaging did not permit assessment of xylem conduit collapse in higher-order veins, we tested the potential effect of collapse of minor veins on Kleaf using a spatially explicit model of the leaf vein system. These simulations suggested that if xylem conduit collapse in the tertiary and minor veins were to occur within the range of water potentials in which Kleaf declined, this collapse would have a quantitatively small effect, i.e., causing <10% decline in Kleaf at -0.5MPa. This finding was consistent with previous model results showing that extreme collapse of minor veins would cause Kleaf to decline only by up to 4% for four diverse species (Scoffoni et al., 2017b). Previous studies found collapse of xylem conduits in pine needles and the minor veins of oak leaves, but only past turgor loss point, and suggested this could act as a circuit breaker to protect the stem xylem from embolism formation (Cochard et al., 2004a;Zhang et al., 2016). An early decline in outside-xylem pathways would act in a similar way, hastening stomatal closure, before xylem collapse would occur (Scoffoni et al., 2017a; and see following sections). Past turgor loss point, the Arabidopsis leaf undergoes drastic shrinkage in area and thickness, and it is likely that xylem in the midrib and higher-order veins would collapse, especially as the Arabidopsis xylem cell walls are helicoidal, and mostly consisting of thick primary walls (Figure 8). Future studies are needed to investigate the collapse of xylem and its influence on the rehydration capacity of strongly dehydrated leaves.

Response of leaf hydraulic conductance to dehydration and coordination with gas-exchange

In Arabidopsis, we did not observe any embolism in leaf xylem conduits prior to, or even moderately past the point of stomatal closure and turgor loss point. This finding is consistent with recent work on tomato and grapevine showing stomata closed before any embolism were observed using an optical visualization technique [START_REF] Hochberg | Stomatal closure, basal leaf embolism, and shedding protect the hydraulic integrity of grape stems[END_REF][START_REF] Skelton | Casting light on xylem vulnerability in an herbaceous species reveals a lack of segmentation[END_REF]. Here, we confirm this finding for the first time using microCT on Arabidopsis. This finding is also consistent [START_REF] Charra-Vaskou | Hydraulic efficiency and safety of vascular and non-vascular components in Pinus pinaster leaves[END_REF][START_REF] Delzon | Recent advances in tree hydraulics highlight the ecological significance of the hydraulic safety margin[END_REF]Bouche et al., 2016;Brodribb et al., 2016b;Scoffoni et al., 2017a;Scoffoni et al., 2017b). A recent study on sunflower showed that xylem embolism occurs after turgor loss point even in plants that acclimate to drought:

plants grown under water-limited conditions adjusted osmotically and had a more negative turgor loss point (-0.3 MPa shift), and leaf xylem P50 also shifted to a more negative value (-0.6 MPa shift) [START_REF] Cardoso | Coordinated plasticity maintains hydraulic safety in sunflower leaves[END_REF]. (McAdam and Brodribb, 2016;[START_REF] Sussmilch | Up-regulation of NCED3 and ABA biosynthesis occur within minutes of a decrease in leaf turgor but AHK1 is not required[END_REF]Sack et al., 2018).

Indeed, ABA signaling may contribute to stomatal closure both directly at the guard cells and also by contributing to Kleaf decline in dehydrating leaves by reducing cell membrane permeability in the bundle sheath and mesophyll via changes in aquaporin expression [START_REF] Shatil-Cohen | Bundle-sheath cell regulation of xylem-mesophyll water transport via aquaporins under drought stress: a target of xylem-borne ABA?[END_REF]Pantin et al., 2013). Indeed, our MOFLO 2.0 simulations showed that Kox decline was best explained by reduced cell membrane permeability and to a lesser extent, cell connectivity. In Arabidopsis, stress-induced changes in cell membrane permeability, mediated by aquaporins, can have a strong impact on root hydraulic conductance [START_REF] Javot | The role of aquaporins in root water uptake[END_REF]. Alternatively, some studies have suggested that photosynthetic rate and carbon sink activities could regulate stomata and plant hydraulics (Nikinmaa et al., 2013;[START_REF] Körner | Paradigm shift in plant growth control[END_REF]Rockwell et al., 2018). Indeed, this is well known in cropping systems such as grapevine, where the presence of strong sinks such as fruits have been shown to stimulate photosynthesis [START_REF] Hofäcker | Investigations on the photosynthesis of vines influence of defoliation, topping, girdling and removal of grapes[END_REF]Petrie et al., 2000). Recent studies have found that excess cellular sugar concentrations under high irradiance, and/or during dehydration could trigger stomata closure (Nikinmaa et al., 2013;Rockwell et al., 2018). Excess sucrose may be transported to the guard cells by the transpiration stream, and the subsequent increase in osmolytes at the guard cell apoplast could induce stomatal closure in some species, especially during periods of high photosynthetic rates [START_REF] Lu | Sucrose: a solute that accumulates in the guard-cell apoplast and guard-cell symplast of open stomata[END_REF][START_REF] Lu | A New Mechanism for the Regulation of Stomatal Aperture Size in Intact Leaves (Accumulation of Mesophyll-Derived Sucrose in the Guard-Cell Wall of Vicia faba)[END_REF]Kang et al., 2007a;Kang et al., 2007b). Indeed, the increase in sucrose concentration at the guard cells could act as more than a simple osmolyte, as it can depolarize the guard cell plasma membrane, activating potassium channels [START_REF] Jarzyniak | Membrane transporters and drought resistance -a complex issue[END_REF], and an increase in the level of sugar-sensing enzymes in the guard cells can accelerate stomatal closure by stimulating ABA production [START_REF] Kelly | Hexokinase mediates stomatal closure[END_REF][START_REF] Van Houtte | Overexpression of the Trehalase Gene AtTRE1 Leads to Increased Drought Stress Tolerance in Arabidopsis and Is Involved in Abscisic Acid-Induced Stomatal Closure[END_REF][START_REF] Li | Glucose-and mannose-induced stomatal closure is mediated by ROS production, Ca2+ and water channel in Vicia faba[END_REF][START_REF] Li | Glucose triggers stomatal closure mediated by basal signaling through HXK1 and PYR/RCAR receptors in Arabidopsis[END_REF]Medeiros et al., 2018). Additionally, excess sucrose concentrations can decrease Kox and thus Kleaf, potentially via deactivation of aquaporins [START_REF] Kelly | Sugar and hexokinase suppress expression of PIP aquaporins and reduce leaf hydraulics that preserves leaf water potential[END_REF].

In conclusion, Kleaf, gs and Aarea show a coordinated decline during leaf dehydration in Arabidopsis, with a potentially strong direct effect of declining Kleaf in inducing stomatal closure via a decrease in water potential. The decline in Kleaf and stomatal conductance may be jointly driven by accumulation of sugar and/or ABA accumulation in dehydrating leaves, or Kleaf declines may contribute to this accumulation. Future studies are needed to decipher the exact sequence of events leading stomata to close. (set to the average value measured at Ψleaf of-0.1 to -0.2 MPa, i.e., the range at which gs was at its maximum). This dynamic Kox with water potential caused an up to 60% decline in gs but only an up to 12% decline in CO2 assimilation, resulting in a higher water-use efficiency and greater overall assimilation when considered over the entire period of soil drying. This benefit for low Kox raises the question of why plants should invest in a high Kox (or Kleaf) in maximally hydrated leaves.

Putative role of

Indeed, high Kleaf values at Ψleaf above -1.0MPa have at times been neglected when constructing vulnerability curves (Blackman et al., 2012;Blackman et al., 2014) under the presumption that leaves simply do not operate at such high water potentials in planta. However, a high Kox (and thus Kleaf) in well-hydrated leaves which declines during dehydration prior to turgor loss point would offer advantages; it would enable high gs and greater CO2 assimilation under well-watered conditions. This would be particularly beneficial for a short-lived species such as Arabidopsis, which is required to grow rapidly when water availability is high. Previous studies have found that maximum Kox (and Kleaf) was high and declined rapidly with water potential in herbs [START_REF] Scoffoni | Decline of leaf hydraulic conductance with dehydration: relationship to leaf size and venation architecture[END_REF]Nolf et al., 2016;Scoffoni et al., 2017a) than long-lived drought tolerant chaparral trees (Scoffoni et al., 2017a). Ephemeral species such as Arabidopsis, or desert plants with short-lived leaves, would especially benefit from high CO2 assimilation rates, and thus high Kleaf after a rainfall event, and "gear down" by reducing Kleaf and thus transpiration rates when water becomes scarce, to improve their water-use efficiency and survive longer under soil drying [START_REF] Grubb | A reassessment of the strategies of plants which cope with shortages of resources[END_REF]. In these simulations, root hydraulic vulnerability had a small effect on water-use efficiency in Arabidopsis.

The much greater effect of leaf over root is due to the very high proportion of hydraulic resistance in the leaf (85.7%), due to the lack of stem in the vegetative phase of this rosette species. The hydraulic vulnerability of roots and their influence on the control of gas exchange is still under debate given experimental challenges. Debate is ongoing over whether root xylem is highly vulnerable [START_REF] Hacke | Drought-induced xylem dysfunction in petioles, branches, and roots of Populus balsamifera L and Alnus glutinosa (L) Gaertn[END_REF][START_REF] Hacke | Drought experience and cavitation resistance in six shrubs from the Great Basin, Utah[END_REF], or resistant [START_REF] Rodriguez-Dominguez | Mapping xylem failure in disparate organs of whole plants reveals extreme resistance in olive roots[END_REF] to xylem embolism. However, just as in leaves (Scoffoni et al., 2017a), the root extraxylem flow pathways might be more vulnerable. In grapevine, lacunae formation in fine root cortical cells may cause a strong decline in Kroot under drying-soil conditions, which would help decouple the plant from drying soil and preserve its vascular system from embolism [START_REF] Cuneo | Mechanical failure of fine root cortical cells initiates plant hydraulic decline during drought[END_REF]. Notably, plant competition for soil water is not simulated in the SurEau model. As such, we assume that plants have evolved to efficiently utilize soil water, and not overspend it [START_REF] Cowan | Water use and optimization of carbon assimilation[END_REF]Buckley et al., 2017b).

The light response of leaf hydraulic conductance in Arabidopsis

Maximum Kleaf for well-hydrated leaves often increases in response to light; this response has been found for 15 of 30 species tested, in species of 23 plant families [START_REF] Sack | The hydraulic conductance of the angiosperm leaf lamina: a comparison of three measurement methods[END_REF][START_REF] Gasco | Resistance to water flow through leaves of Coffea arabica is dominated by extra-vascular tissues[END_REF][START_REF] Tyree | The dependence of leaf hydraulic conductance on irradiance during HPFM measurements: any role for stomatal response?[END_REF][START_REF] Cochard | Putative role of aquaporins in variable hydraulic conductance of leaves in response to light[END_REF][START_REF] Scoffoni | The rapid light response of leaf hydraulic conductance: new evidence from two experimental methods[END_REF][START_REF] Voicu | Light response of hydraulic conductance in bur oak (Quercus macrocarpa) leaves[END_REF][START_REF] Guyot | Combined impacts of irradiance and dehydration on leaf hydraulic conductance: insights into vulnerability and stomatal control[END_REF][START_REF] Xiong | Differential coordination of stomatal conductance, mesophyll conductance, and leaf hydraulic conductance in response to changing light across species[END_REF]. Further, in some species, the light enhancement of Kleaf is reduced in dehydrated leaves, or, equivalently for those species, Kleaf declines with dehydration more steeply under high irradiance [START_REF] Guyot | Combined impacts of irradiance and dehydration on leaf hydraulic conductance: insights into vulnerability and stomatal control[END_REF]. In Arabidopsis, a previous study suggested that the hydraulic conductance of entire rosettes of Arabidopsis had increased when acclimated to low rather than high irradiance (Prado et al., 2013). In our experiments using the evaporative flux method, we found significantly higher Kleaf values throughout the range of water potentials tested for leaves acclimated to high irradiance, with a 60% enhancement of Kleaf from low to high irradiance in well-hydrated leaves of Col-0. Discrepancies between these results may have arisen due to methodological differences, given that in the study of Prado et al. (2013), hydraulic conductance was measured by pushing water inward through the stomata of entire rosettes suspended under water in darkness within a pressure chamber.

Notably, the light enhancement in Kleaf found in Arabidopsis did not result in a shift in P50.

This finding indicates a proportional shift to lower values under low irradiance, throughout the range of water potentials, contrary to findings for four woody species in which leaves acclimated to high irradiance were more vulnerable to Kleaf decline with dehydration [START_REF] Guyot | Combined impacts of irradiance and dehydration on leaf hydraulic conductance: insights into vulnerability and stomatal control[END_REF]). The light enhancement of Kleaf would provide a greater hydraulic supply to meet the demand of leaves acclimated to high irradiance, i.e., given strong and rapid dynamics of air temperature and humidity and wind, and thus higher vapor pressure deficit and leaf boundary layer conductance.

Further, given strong transient dehydration during transpiration under these conditions, the higher Kleaf would contribute to rapid mesophyll rehydration at high water potential and thus enable the recovery of gs and photosynthetic rate. The light enhancement of Kleaf could be caused by stronger temperature gradients throughout the leaf under high light and/or changes in aquaporin expression [START_REF] Cochard | Putative role of aquaporins in variable hydraulic conductance of leaves in response to light[END_REF]. Our simulations of Kox under low and high light using MOFLO 2.0 indicated that Kleaf would be minimally enhanced by temperature gradients in the leaf caused by light absorption, pointing to a role for aquaporins instead. This is consistent with the molecular evidence that aquaporin expression is sensitive to light [START_REF] Cochard | Putative role of aquaporins in variable hydraulic conductance of leaves in response to light[END_REF]Ben Baaziz et al., 2012) that mutltiple aquaporin isoforms are involved in a range of responses such as Kleaf decline during drought and Kleaf light enhancement [START_REF] Cochard | Putative role of aquaporins in variable hydraulic conductance of leaves in response to light[END_REF][START_REF] Pou | A putative role for TIP and PIP aquaporins in dynamics of leaf hydraulic and stomatal conductances in grapevine under water stress and re-watering[END_REF]Laur and Hacke, 2014b, a). Furthermore, aquaporins may also be involved in cell rehydration [START_REF] Marco | VvPIP2;4N aquaporin involvement in controlling leaf hydraulic capacitance and resistance in grapevine[END_REF]. Finally, aquaporins have also been suggested to play a role in a rapid enhancement of Kleaf when Arabidopsis is suddenly exposed to low relative humidity, compensating for the increased evapotranspiration, and allowing stomata to remain open [START_REF] Levin | Low air humidity increases leaf-specific hydraulic conductance of Arabidopsis thaliana (L.) Heynh (Brassicaceae)[END_REF].

Contribution of hydraulic traits to Arabidopsis thaliana whole-plant physiology

Arabidopsis Col-0 has high values of Kleaf, gs and Aarea relative to previously published values of diverse angiosperm species [START_REF] Flexas | Leaf mesophyll conductance and leaf hydraulic conductance: an introduction to their measurement and coordination[END_REF]Scoffoni and Sack, 2017), and displays strong sensitivity in Kleaf and gas exchange to dehydration. This physiological behavior is consistent with Arabidopsis' ruderal ecology, establishing and producing flowers and seeds in open or disturbed habitats in spring/early summer [START_REF] Koornneef | Naturally occurring genetic variation in Arabidopsis thaliana[END_REF]. The high values of Kleaf were driven by an especially high Kox (106 mmol m -2 s -1 MPa -1 ). This high Kox is not untypical in herbs; in Salvia canariensis, maximum Kox reached 231 mmol m -2 s -1 MPa -1 (Scoffoni et al., 2017a). Notably, a high Kx,Kox and Kleaf are often achieved with allocation to substantial vein length per area (VLA), which increases flow paths in parallel within the xylem and reduces flow distance outside the xylem [START_REF] Sack | Leaf venation: structure, function, development, evolution, ecology and applications in past, present and future[END_REF]; Arabidopsis possesses a relatively low VLA but flow distance is strongly reduced by its very thin leaf, which would also reduce Kox (Brodribb et al., 2007;[START_REF] Buckley | How Does Leaf Anatomy Influence Water Transport outside the Xylem?[END_REF]. Furthemore, a high aquaporin activity, and/or cell wall permeability especially at the bundle sheath could potentially influence Kox; across several Arabidopsis mutants, maximum Kleaf was associated with an anatomical index of bundle sheath conductivity [START_REF] Caringella | Leaf hydraulic conductance varies with vein anatomy across Arabidopsis thaliana wild-type and leaf vein mutants[END_REF]. The high Kx value could potentially arise from xylem structure, i.e., the numbers and sizes of xylem cells within minor veins [START_REF] Caringella | Leaf hydraulic conductance varies with vein anatomy across Arabidopsis thaliana wild-type and leaf vein mutants[END_REF][START_REF] Stewart | Arabidopsis thaliana Ei-5: Minor Vein Architecture Adjustment Compensates for Low Vein Density in Support of Photosynthesis[END_REF], in combination with high conductance between xylem conduits. Indeed, our TEM imaging showed very little secondary lignification of xylem conduits throughout the midrib and other vein orders (Figure 8), such that the bulk of midrib conduit walls are effectively one large pit membrane (i.e., primary unlignified wall) with water potentially leaking throughout the surface, a structure that would strongly reduce pit wall resistance and thus total xylem resistance [START_REF] Choat | Structure and function of bordered pits: new discoveries and impacts on whole-plant hydraulic function[END_REF].

Arabidopsis Col-0 also exhibits strong drought sensitivity, with its very low leaf mass per area [START_REF] Wright | The worldwide leaf economics spectrum[END_REF] to that of the water potential at stomatal closure (gs95) and at 88% loss of Kleaf, around -0.7MPa.

This detailed characterization of Arabidopsis Col-0 hydraulics traits, and their dynamics during leaf dehydration and implications for whole-plant responses highlights useful avenues for high throughput phenotyping, and the elucidation of genetic mechanisms controlling these key traits, which would be loci for manipulation of gas exchange and drought tolerance.

Material and methods

Plant material and growth conditions

Measurements were performed on Arabidopsis thaliana Col-0 (ecotype Colombia), hereafter refered to as Arabidopsis, grown continuously from December 2015 through November 2016. We grew Arabidopsis thaliana in a climate-controlled greenhouse at the University of California, Los

Angeles. Seeds were sown in lawns in pots (3.13" width x 4.88"length x 2.31" deep) in soil

(1:1:2:1:1 mixture of washed plaster sand, loam, peat moss, perlite, vermiculite), and coldacclimated at 4°C for three days in a chamber, then brought to the temperature-controlled greenhouse (minimum, mean, and maximum values for temperature, 19.3°C, 22.5°C, and 33.2°C;

for humidity, 24%, 63%, and 92%; for irradiance (from 0900 to 1600), 11.2, 169, 1369 µmol photons m -2 s -1 ). We recognize that many researchers often grow Arabidopsis in growth chambers under <300 µmol photons m -2 s -1 irradiance, and future work should consider the variation in leaf physiology, morphology and anatomy driven by this lower irradiance. We chose to grow Arabidopsis in a greenhouse setting where plants are exposed to light fluctuations, with temporary high light peaks, as is experienced in the field. Indeed, this has been shown to impact plant growth (Poorter et al., 2016). Further, growing plants under such high irradiance means that these were not light-limited, and thus, our findings can be compared with those for other species grown without light limitation, as is typical in studies of plant hydraulic physiology.

When plants had true leaves after approximately one week, they were thinned to one individual per pot. Plants were watered regularly to keep soil moist. After approx. 6 weeks, at which points plants had >10-20 leaves, mature and healthy leaves were chosen for gas exchange, hydraulic and x-ray micro-computed tomography measurements.

Leaf hydraulic conductance

Pots were transported to the laboratory, watered and enclosed overnight in plastic bags filled with wet paper towels to ensure a saturated atmosphere. To obtain a vulnerability curve spanning a range of leaf water potential (Ψleaf), well-hydrated and dehydrated leaves were measured. To obtain Kleaf values at high Ψleaf, mature and healthy leaves were directly cut at their base under water and their petioles placed in a petri dish containing ultra-pure water (0.22 μm Thornton 200 CR;

Millipore) prior to being connected to the evaporative flux system described below. To obtain Kleaf values at low Ψleaf, individuals were removed from the soil and dehydrated on the bench for 0.25-2 hours, after which they were placed in bags which had previously been exhaled into, within a second bag filled with wet paper towels, to ensure high vapor and CO2 concentration, to reduce stomatal opening and facilitate equilibration for 30 min. Two leaves were then measured for initial Ψleaf, using a pressure chamber (Plant Moisture Stress Model 1000; PMS Instrument Co, Albany, OR, USA), with a grass fitting in the compression lid; for a few leaves with round petioles, silicon adaptors were used [START_REF] Shatil-Cohen | Bundle-sheath cell regulation of xylem-mesophyll water transport via aquaporins under drought stress: a target of xylem-borne ABA?[END_REF]. On average, the two leaves measured for initial Ψleaf differed by 0.051 MPa ± 0.008 standard error. A third mature and healthy leaf from the dehydrated individual was measured for Kleaf. After the leaf petiole was cut under water, it was gently wrapped with parafilm and connected via tubing to a water source on a balance (±10 μg, models XS205 and AB265; Mettler Toledo, Columbus, OH, USA), which logged the flow rate into the leaf every 5 s to a computer. The leaf was placed over a fan and under a light source (>1000 μmol m -2 s -1 ; model 73828, 1000 W UV filter; SearsRoebuck, Hoffman Estates, IL, USA).

A water bath was placed between the leaf and the light to avoid overheating the leaf, which was kept between 23 and 28°C as measured using a thermocouple (Cole-Parmer). After a minimum of 30 min to ensure light acclimation [START_REF] Scoffoni | The rapid light response of leaf hydraulic conductance: new evidence from two experimental methods[END_REF] and once the flow had stabilized with no upward or downward trend, the average steady state flow rate for the last 5 min was recorded and leaf temperature was measured (Cole-Parmer). The leaf was rapidly removed from the system, its petiole dabbed dry, and placed in a bag which had previously been exhaled into. [START_REF] Weast | Handbook of chemistry and physics[END_REF][START_REF] Yang | Hydraulic resistance in Acer saccharum shoots and its influence on leaf water potential and transpiration[END_REF]; this correction also approximately applies for the temperature dependence of vapor phase transport across this range of temperature [START_REF] Buckley | How Does Leaf Anatomy Influence Water Transport outside the Xylem?[END_REF].

Leaf hydraulic vulnerability curves were obtained as the plot of Kleaf vs. the most negative Ψleaf experienced by the leaf (either the initial or final).

Kleaf vulnerability curves were measured under very low laboratory irradiance (light source off; <3 μmol photons m -2 s -1 ) and high irradiance (>1000 μmol m -2 s -1 ). Measurements in very low and high irradiance were performed on the same day using leaves taken from the same individuals when possible, i.e.,when two leaves from the same individual were mature and healthy.

Notably, the aim of this experiment was to test for a rapid light enhancement of Kleaf for highlight-grown individuals. Future studies are needed to investigate the plasticity in Kleaf and other physiological and morphological traits for Arabidopsis across different light growth regimes, as found to be important in a study of species of Hawaian lobeliads (Scoffoni et al., 2015).

Leaf xylem hydraulic conductance

Leaf xylem hydraulic conductance was measured for six leaves (taken from six different individuals) using the vacuum pump method [START_REF] Kolb | A method for measuring xylem hydraulic conductance and embolism in entire root and shoot systems[END_REF]Nardini et al., 2001;[START_REF] Sack | Hydraulic analysis of water flow through leaves of sugar maple and red oak[END_REF][START_REF] Scoffoni | Are leaves "freewheelin'"? Testing for a Wheeler-type effect in leaf xylem hydraulic decline[END_REF][START_REF] Trifilo | The contribution of vascular and extra-vascular water pathways to droughtinduced decline of leaf hydraulic conductance[END_REF]. Briefly, individuals were rehydrated in the laboratory overnight, and kept in dark plastic bags filled with wet paper towels to ensure high humidity. The next morning, a leaf was cut off the plant under ultra-pure water and placed in a petri dish over a white light transilluminator table (Model TW, UVP, Upland, CA, USA) to allow visualization of the fourth-order veins (=minor veins). Using a fresh scalpel, 8-15 cuts per cm 2 of leaf area were made to the lamina, severing minor veins, to ensure that outside-xylem pathways would be bypassed [START_REF] Sack | Hydraulic analysis of water flow through leaves of sugar maple and red oak[END_REF]Nardini and Salleo, 2005;Nardini et al., 2005b;[START_REF] Sack | Leaf hydraulic architecture correlates with regeneration irradiance in tropical rainforest trees[END_REF]. Great care was taken to avoid cutting major veins; if they were cut by accident, the leaf was discarded. Once the cuts were made, the leaf petiole was wrapped with parafilm and inserted through a small rubber stopper that had been perforated using a cork borer. The small rubber stopper was then connected to a tube fitting connected to silicone tubing (ColeParmer, Vernon Hills, IL, USA). The rubber stopper allowed a good seal around the petiole without crushing. We obtained a vacuum tight seal by tightening the tubing around the rubber stopper with zipties and We applied five increasing levels of partial vacuum, resulting in absolute pressures between 0.06 and 0.02 MPa, and recorded the flow rate of water entering the leaf from a water source on a balance (±10 μg, models XS205 and AB265; Mettler Toledo, Columbus, OH, USA).

The average flow rate of the last 5 min of stability for a given pressure was recorded, along with the temperature. The flow rate was normalized to 25°C correct for the temperature response of the viscosity of water [START_REF] Weast | Handbook of chemistry and physics[END_REF][START_REF] Yang | Hydraulic resistance in Acer saccharum shoots and its influence on leaf water potential and transpiration[END_REF]. Leaf xylem hydraulic conductance (Kx)

was calculated as the slope of the flow rate vs. pressure, and normalised by leaf area, measured at the end of the experiment with a flatbed scanner. The percent hydraulic resistance in the leaf xylem (%Rx) and outside-xylem (%Rox) were calculated as:

(1)

%𝑅 x = 1 𝐾 x ⁄ 1 𝐾 leaf ⁄ × 100 (2)
%𝑅 ox = 100 -%𝑅 x

Diurnal measurements of stomatal conductance, photosynthetic rate and whole plant hydraulic conductance as a function of leaf water potential

Diurnal measurements of light-saturated photosynthetic rate (Amax) and stomatal conductance (gs)

were performed in the greenhouse on 40 individuals on 10-11 November 2016 from 0900 to 1800 using a portable gas exchange system (LI-6400; LI-Cor, Lincoln, NE, USA). The chamber CO2 was set at 400 ppm and irradiance at 1000 μmol m -2 s -1 photosynthetically active radiation, and leaf to air vapor pressure deficit was maintained between 0.4 and 0.6 kPa. Measurements were taken after the leaf had equilibrated in the chamber for 10 min; Amax and gs were logged 5 times at 10-sec intervals, and these five measurements were averaged. We checked that 10 min was sufficient equilibration time; n = 7 leaves were kept in the chamber for an additional five minutes; no significant differences were found between values taken at 10 vs. those taken at 15 min (paired t-test; p = 0.08). To verify gs measurements, additional measurements were taken using a porometer on the abaxial side of the leaf (Delta-T Devices; Cambridge, UK) on 10-11 November 2016 from 0900 to 1800. As expected, the gs values obtained from the LICOR and porometer were within the same range of values and thus were pooled together during the analyses.

At the end of the measurement, the leaf was excised with a razor blade and immediately placed in a sealable bag (Whirl-Pak, Nasco, Fort Atkinson, WI, USA), which had been previously exhaled in, and the bagged leaves were placed in a second bag filled with wet paper towels. After at least 30-min equilibration, Ψleaf was measured using a pressure chamber as described above.

Whole-plant hydraulic conductance (Kplant; mmol m -2 s -1 MPa -1 ) was estimated under the assumption that soil water potential was fully saturated throughout the day (thus, Ψsoil = 0 MPa).

Though we did not directly measure Ψsoil, plants were well-watered and soil was always moist.

Thus, Kplant was determined from the stomatal conductance obtained from the porometer data described above (measurements performed under ambient light irradiance), ambient VPD at the time of measurement, and leaf water potential:

(3)

𝐾 plant = 𝑔 s ×𝑉𝑃𝐷 Ψ soil -Ψ leaf

X-ray micro-computed tomography

To visualize leaf vein xylem embolism and tissue shrinkage, we used X-ray micro-computed tomography (microCT) at the synchrotron at the Advanced Light Source (ALS) in Berkeley, California (Beamline 8.3.2). We imaged the xylem within the midrib and lamina tissues in 18 leaves of a range of leaf water potential from 9 individuals in February 2016 at 1.27-μm resolution.

Three additional individuals were further scanned in November 2016 at a higher resolution of 0.638 μm to check for potential collapse in xylem conduits of the midrib. Arabidopsis individuals grown as described above were transported as carry-on in a plane to ALS. Individuals were fully rehydrated at the start of the experiment, and whole plants were removed from the soil and dehydrated on the bench for different times to obtain a range of water potentials, and equilibrated in double-sealed plastic bags for 30 min, after which two leaves were excised to obtain initial leaf water potential. Two of the leaves remaining attached to the plant were juxtaposed within a styrofoam holder and 0.653-0.869 mm length scans were made of their midrib and surrounding lamina at the center of each leaf. A small piece of copper wire was attached at the center of the leaves to help center the samples for scanning. Kapton tape (DuPont, Wilmington, DE, USA) was used to tape the leaves and the copper wire to the styrofoam holder to minimize sample movement during the scan. The styrofoam with the sample enclosed was placed in a Plexiglass cylinder, attached to a custom-built aluminium sample holder mounted on an air-bearing stage, and wet paper towels were placed above the sample in the Plexiglass cylinder to minimize evaporation during the measurement. At the end of the measurement, final leaf water potential was recorded and leaf areas were measured. No significant differences in water potential before and after the measurement were observed (paired t-test; p = 0.70; n = 8). Scans were made at 20-23 keV in the synchrotron X-ray beam, and rotated 180° with the instrument to enable visualization of the full 3D internal structure of the leaf. Scans took 5-10 min to complete depending on the scan area selected. Three-dimensional volume renderings were made using the AVIZO 8.1.1 software (VSG Inc., Burlington, MA, USA), and used to count the number of embolized conduits in the entire sample and different vein orders. For the four samples that showed embolism, we measured the length of the embolized conduit and the widths of both conduit axes at three locations along the sample length. We also visualized for each section the water-filled conduits within the midrib and secondary veins to observe any potential deformation or collapse.

Using ImageJ software (version 1.46r; National Institutes of Health), we measured lamina tissue and cell dimensions on three cross-sectional images randomly selected in the middle of each sample. For each image, we measured thickness of the lamina and of each tissue, i.e., the abaxial and adaxial epidermises including the cuticle, and the palisade and spongy mesophyll, at three locations within the sample. We also measured the area, perimeter and diameters as well as the % intercellular airspace of palisade and spongy mesophyll cells.

Drought tolerance traits

The leaf turgor loss point, osmotic potential at full turgor, relative water content at turgor loss point and modulus of elasticity were calculated from a pressure-volume curve constructed using 29 leaves from 20 individuals previously rehydrated overnight (Supplemental Figure S3). Initial leaf mass was obtained for each single leaf before dehydration to a range of Ψleaf. Leaf water potentials

were measured with a pressure chamber after 30 min of equilibration in bags with high humidity, after which the leaf mass was measured again, along with leaf area, before it was placed in a drying oven at 70°C and measured for dry mass after 72 hours. Pressure-volume curve parameters were obtained following standard protocols (Sack and PrometheusWiki, 2010).

We measured the minimum epidermal conductance (=cuticular plus residual stomatal conductance; gmin) on nine mature leaves from nine individuals in June 2016 by following a standard protocol (Sack et al., 2010). Individual leavess were rehydrated covered in plastic in the laboratory the night before measurements. The next day, nine leaves excised, their cut petioles were sealed with wax, and their fresh mass and leaf area were measured, before dehydration for an hour taped to a fishing line above a fan, to ensure stomatal closure. Leaves were then repeatedly taken off the fan and bagged and measured for mass every 20 min. After eight measurements were obtained for a given leaf, its area was measured again. The gmin was calculated as the slope of mass over time divided by the average mole fraction vapor pressure deficit (VPD) during the measurement and normalized by the average of the initial and final leaf area given shrinkage with dehydration during measurement. VPD was calculated from the temperature and relative humidity measurements obtained from a weather station (HOBO Micro Station with Smart Sensors, Onset, Bourne, MA, USA). Finally, each individual leaf was dried in an oven at 70°C for three days, and dry mass and area were obtained to calculate leaf dry mass per hydrated area (LMA; in g m -2 ) and the percent area shrinkage in the dried leaf relative to the hydrated leaf (PLAdry; %).

Leaf anatomy

Data for leaf venation and leaf cross-sectional anatomy of Col-0 to aid with interpretation of microCT images were obtained from a previous study [START_REF] Caringella | Leaf hydraulic conductance varies with vein anatomy across Arabidopsis thaliana wild-type and leaf vein mutants[END_REF].

To visualize xylem conduit walls, transmitted electron microscopy (TEM) was performed on three leaves from three Col-0 individuals in Germany. made using a JEOL 1400 TEM (JEOL, Tokyo, Japan) at an accelerating voltage of 120 kV. Images were taken with a digital camera (Soft Imagign System, Münster, Germany).

Modelling of hydraulic function across scales from tissues to whole plant

We applied a framework of four models across scales to compute the mechanisms underlying Kleaf decline inside and outside the xylem, the causal role of Kleaf decline in driving stomatal closure, and the implications for gas exchange under simulated drought regimes (Table 1).

We first estimated the causal importance of mechanistic drivers of Kleaf decline using spatially explicit models of the leaf veins (K_LEAF; Cochard et al., 2004b;Scoffoni et al., 2017b) and outside-xylem pathways (MOFLO 2.0; Buckley et al., 2017a). Using K_LEAF, we tested whether xylem embolism and/or conduit collapse could explain the observed decline in Kleaf. We first tested for the impact of the embolisms observed with microCT imaging in the midrib and secondary veins on the xylem hydraulic conductance (Kx) and ultimately Kleaf (see Supplemental

Methods for more information on model parameterization; Supplemental Table S2). We tested the potential effect of the collapse of tertiary and minor vein conduits on Kx under two scenarios: (1) a "realistic" impact of conduit collapse on conduit conductivity (13% decline in tertiary and minor vein conductivity, similar to that observed in Quercus rubra at turgor loss point by Zhang et al., 2016), and (2) a more severe conduit collapse scenario which would induce 50% decline in tertiary and minor vein conduit conductivity (see Supplemental Methods). Using MOFLO 2.0, we investigated the potential drivers of decline in outside-xylem hydraulic conductance with dehydration. We simulated the impact of an 80% decline in cell membrane permeability, and/or decline in cell-to-cell liquid phase hydraulic connectivity given the anatomical changes due to cell shrinkage at -0.5 MPa under different scenarios: (1) with or without an apoplastic barrier to liquidphase water transport across the bundle sheath, (2) under either no light or with an irradiance of 600 μmol m -2 s -1 photosynthetically active radiation to clarify a potential role of transdermal temperature gradients (Supplemental Methods; Supplemental Table S2).

We then quantified the direct influence of Kleaf decline on gs decline with dehydration, using a partitioning approach. We first considered the empirical maximum likelihood functions relating S3).

Statistics

We selected functions for the responses of Kplant, Kleaf, gs and Amax to Ψleaf using a maximum likelihood framework [START_REF] Burnham | Model selection and multimodel inference[END_REF]Sack et al., 2006). For the gs and Amax curve fitting, extremely low values at the beginning or end of the day when stomata were shut in wellhydrated leaves (Ψleaf > -0.01 MPa) were discarded, and likely represented the effects of the mechanical advantage of epidermal cells preventing stomatal opening in turgid leaves [START_REF] Guyot | Combined impacts of irradiance and dehydration on leaf hydraulic conductance: insights into vulnerability and stomatal control[END_REF]; these points represented 3/63 and 2/26 of the points for gs and Amax respectively. We selected the maximum likelihood model using the optim function in R 3.4.1 (http://www.rproject.org;). We fitted four types of functions to the curves, as previously used in the literature [START_REF] Scoffoni | Dynamics of leaf hydraulic conductance with water status: quantification and analysis of species differences under steady-state[END_REF], where Y= Kleaf, Aarea or gs, and Ψleaf is leaf done in the literature (i.e., [START_REF] Sack | The 'hydrology' of leaves: co-ordination of structure and function in temperate woody species[END_REF]Nardini et al., 2005a;Brodribb and Jordan, 2008;[START_REF] Scoffoni | The rapid light response of leaf hydraulic conductance: new evidence from two experimental methods[END_REF]Scoffoni et al., 2015). Because of the two dimensionalities of these sections, embolism in the midrib and secondary vein might appear disconnected (C, E). Note that while the embolism was present in only one conduit per cross-sectional image, multiple conduits spanned the length of the midrib and secondary vein (as can be observed in K, where two conduits can be seen connected to one another). Most likely, a first conduit in the midrib embolized, and all the conduits directly connected to that one upstream embolized after. In one sample, an embolized conduit was observed isolated in the secondary vein (L-N), while in another sample, an embolized conduit was observed spanning the midrib length (O-Q). Table 1 and Methods). The Kox was first computed based on the decline of observed cell size and air space alone (grey bars), which resulted in an increase in Kox (negative percent loss of Kox;

mainly due to shortening of pathways from the veins to stomata). We then modelled Kox decline according to three scenarios (though always including the effect of tissue dimensional changes):

an 80% decline at -0.5MPa in (1) cell connectivity (red bars), ( 2 conductance (Kleaf) decline to that of stomatal conductance (gs) with dehydration (Table 1). during the progression of a simulated soil drought (SurEau Model, see Table 1 and Methods).

Four scenarios were modelled: (1) both leaf hydraulic conductance (Kleaf) and root hydraulic conductance (Kroot) were vulnerable to dehydration prior to turgor loss point (yellow lines), (2)

Kleaf was vulnerable, but not Kroot (red lines), 3) Kroot was vulnerable, but not Kleaf (light blue lines), or (4) neither Kleaf nor Kroot was vulnerable (dark blue lines). The inset in (C) shows cumulative water-use efficiency (WUE; calculated as cumulative CO2 assimilation (A) over total transpiration rate (E)) over time. Scenarios including a vulnerable Kleaf showed leaves that showed highest water-use efficiency, cumulative CO2 assimilation and survived longer under drought conditions. (Cochard et al., 2004;Scoffoni et al., 2017) Model the influence of xylem embolism and potential conduit collapse on K x and K leaf Leaf size, number of secondary veins and theoretical conductivities from the different veins orders at (1) at full turgor, and after accounting (2) for the decline caused by observed embolism in midrib and/or secondary veins, and (3) for the decline potentially caused by collapsed xylem conduits of tertiary and higher order veins (under a "realistic" collapsed scenario as observed in an oak species (Zhang et al., 2016) which caused 13% PLC, and a more severe scenario-causing 50% PLC)

Leaf xylem hydraulic conductance

Neither embolism nor xylem conduit collapse caused a decline in K x substantial enough to explain the observed decline in K leaf .

MOFLO 2.0 (Buckley et al., 2017) Model the influence of changes in outside-xylem pathways on K ox and K leaf Cell shrinkage and % intercellular airspace at -0.5MPa obtained from microCT, stomatal conductance (abaxial and adaxial), vapor pressure deficit and bulk leaf temperature. Simulations were performed under no light or 600 μmol m -2 s -1 photosynthetically active radiation, with or without an apoplastic barrier at the bundle sheath, and with or without an 80% decline in cell membrane permeability and/or cell connectivity.

Leaf outside-xylem hydraulic conductance

Reduction of cell membrane permeability in the context of an apoplastic barrier would account for most of the K leaf decline observed at -0.5MPa. Temperature gradients through the leaf due to irradiance had little impact on K ox . Marginal contribution of K decline (refined from Rodriguez-Dominguez et al., 2016) Quantify the influence of K leaf decline on g s decline Parameters from the maximum likelihood function of g s and K leaf vs. Ψ leaf , VPD set at a constant value (1.5 kPa), and a computed range of % g s decline (0 to 100% decline in gs with Ψ leaf ). embolized conduits are depicted in yellow). The embolized conduit in the midrib and secondary vein can be seen in cross-sections (B, D, G, I) and longitudinal sections of the microCT scan (C, E, H, J). The arrows point to the embolized conduit (appearing as black in the microCT image).

Because of the two dimensionalities of these sections, embolism in the midrib and secondary vein might appear disconnected (C, E). Note that while the embolism was present in only one conduit per cross-sectional image, multiple conduits spanned the length of the midrib and secondary vein (as can be observed in K, where two conduits can be seen connected to one another). Most likely, a first conduit in the midrib embolized, and all the conduits directly connected to that one upstream embolized after. In one sample, an embolized conduit was observed isolated in the secondary vein (L-N), while in another sample, an embolized conduit was observed spanning the midrib length (O-Q). 1 and Methods). The K ox was first computed based on the decline of observed cell size and air space alone (grey bars), which resulted in an increase in K ox (negative percent loss of K ox ; mainly due to shortening of pathways from the veins to stomata). We then modelled K ox decline according to three scenarios (though always including the effect of tissue dimensional changes): an 80% decline at -0. during the progression of a simulated soil drought (SurEau Model, see Table 1 and Methods).

Four scenarios were modelled: (1) both leaf hydraulic conductance (K leaf ) and root hydraulic conductance (K root ) were vulnerable to dehydration prior to turgor loss point (yellow lines), (2) K leaf was vulnerable, but not K root (red lines), 3) K root was vulnerable, but not K leaf (light blue lines), or (4) neither K leaf nor K root was vulnerable (dark blue lines). The inset in (C) shows cumulative water-use efficiency (WUE; calculated as cumulative CO 2 assimilation (A) over total transpiration rate (E)) over time. Scenarios including a vulnerable K leaf showed leaves that showed highest water-use efficiency, cumulative CO 2 assimilation and survived longer under drought conditions.

  water potential: linear (Y = a Ψleaf + y0), two-parameter sigmoidal (Y = a / (1+ e ( -(Ψleaf -x0)/b) )), logistic (Y = a / (1+ (Ψleaf / x0) b )), and exponential (Y = y0 + a×e -(b ×Ψleaf) ). We estimated the maximum Y value by extrapolating to Ψleaf = 0 and, as indices of decline with dehydration, the Ψleaf at which maximum Y values decreased by 50% and 95%. Because the best-fit function for the Kleaf vulnerability curve was exponential and the Y value at Ψleaf = 0 was extrapolated to a very high unrealistic value, we also estimated the maximum Kleaf by averaging all points above -0.1 MPa (Kmax), as has been typically

Figure captions Figure 1 .

 captions1 Figure captions Figure 1. Decline of leaf hydraulic conductance (Kleaf) measured under high (>1000 μmol photons m -2 s -1 ) or low (< 3 μmol photons m -2 s -1 ) irradiance. The maximum likelihood function is shown for Kleaf vulnerability acclimated under high light (𝐾 leaf = 6.83 + 81.4 exp(-7.56 × |Ψ leaf |)), and low light (𝐾 leaf = 8.98 + 84.2 exp(-13.2 × |Ψ leaf |)). The dashed line respresents the water potential at 50% loss of Kleaf (similar in both treatments).

Figure 2 .

 2 Figure 2. Plant hydraulic and gas exchange response to dehydration in Arabidopsis. Decline of the whole plant hydraulic conductance (Kplant; A), stomatal conductance (gs; B) and lightsaturated photosynthetic rate (Aarea; C) with dehydration. Each point represents a different measured leaf. Kplant was obtained from the porometer data by dividing transpiration by leaf

Figure 3 .

 3 Figure 3. Lack of embolism observed in midrib conduits of Arabidopsis thaliana (Col-0) across levels of dehydration as revealed by in vivo images of leaf midribs subjected to progressive dehydration using micro-computed tomography (A-C). Water-filled cells appear in light grey in microCT. If air-filled (i.e., embolized) conduits were present, they would appear as black in the xylem portion of the midrib. There was no embolism, as shown in these images by the red arrows pointing at the entirely light grey midrib xylem. The leaf water potential (Ψleaf) has been provided for each image. The inset in (A) represents a leaf midrib cross-section imaged under light microscopy, with the red arrow pointing to the xylem tissue (dark blue conduits).

Figure 4 .

 4 Figure 4. Rare embolisms were observed in a few individual leaves. In two samples, an embolized conduit was observed in the midrib; it continued into a secondary vein (A, F; the embolized conduits are depicted in yellow). The embolized conduit in the midrib and secondary vein can be seen in cross-sections (B, D, G, I) and longitudinal sections of the microCT scan (C, E, H, J). The arrows point to the embolized conduit (appearing as black in the microCT image).

Figure 5 .

 5 Figure 5. Results from simulations using a spatially explicit model of leaf outside-xylem water to test for potential drivers of the decline in Kox in dehydrating leaves transport (MOFLO 2.0, see

  ) cell membrane permeability (blue bars), and (3) cell wall thickness (black bars). All simulations were run with (Ap; darker color) or without an apoplastic barrier (No Ap; lighter color) at the bundle sheath cells. The yellow star on the x-axis represents the % observed Kleaf decline at -0.5MPa (measured with the evaporative flux method, see Methods).

Figure 6 .

 6 Figure 6. Model simulations mapping the contribution of the decline of leaf hydraulic

Figure 7 .

 7 Figure 7. Daily simulated patterns of stomatal conductance (A), leaf water potential (B), cumulative CO2 assimilation (C) and the percent loss of leaf xylem hydraulic conductance (D)

Figure 8 .

 8 Figure 8. Transmitted-electron microscopy of Arabidopsis thaliana (Col-0) midrib crosssections. In A, the entire xylem portion of the midrib can be seen. Black arrows point to the lack of secondary lignified wall around xylem conduits. These long primary wall sections can be observed in more detail in B. The white arrow points to a lignified portion of the secondary

Figure 1 .Figure 2 .

 12 Figure 1. Decline of leaf hydraulic conductance (K leaf ) measured under high (>1000 μmol photons m -2 s -1 ) or low (< 3 μmol photons m -2 s -1 ) irradiance. The maximum likelihood function is shown for K leaf vulnerability acclimated under high light (𝐾 leaf = 6.83 + 81.4 exp(-7.56 × |Ψ leaf |)), and low light (𝐾 leaf = 8.98 + 84.2 exp(-13.2 × |Ψ leaf |)). The dashed line respresents the water potential at 50% loss of K leaf (similar in both treatments).

Figure 3 .Figure 4 .

 34 Figure 3. Lack of embolism observed in midrib conduits of Arabidopsis thaliana (Col-0) across levels of dehydration as revealed by in vivo images of leaf midribs subjected to progressive dehydration using micro-computed tomography (A-C). Water-filled cells appear in light grey in microCT. If air-filled (i.e., embolized) conduits were present, they would appear as black in the xylem portion of the midrib. There was no embolism, as shown in these images by the red arrows pointing at the entirely light grey midrib xylem. The leaf water potential (Ψ leaf ) has been provided for each image. The inset in (A) represents a leaf midrib cross-section imaged under light microscopy, with the red arrow pointing to the xylem tissue (dark blue conduits).

Figure 5 .

 5 Figure5. Results from simulations using a spatially explicit model of leaf outside-xylem water to test for potential drivers of the decline in K ox in dehydrating leaves transport (MOFLO 2.0, see Table1and Methods). The K ox was first computed based on the decline of observed cell size and air space alone (grey bars), which resulted in an increase in K ox (negative percent loss of K ox ; mainly due to shortening of pathways from the veins to stomata). We then modelled K ox decline according to three scenarios (though always including the effect of tissue dimensional changes): an 80% decline at -0.5MPa in (1) cell connectivity (red bars), (2) cell membrane permeability (blue bars), and (3) cell wall thickness (black bars). All simulations were run with (Ap; darker color) or without an apoplastic barrier (No Ap; lighter color) at the bundle sheath cells. The yellow star on the x-axis represents the % observed K leaf decline at -0.5MPa (measured with the evaporative flux method, see Methods).

Figure 6 .Figure 7 .

 67 Figure5. Results from simulations using a spatially explicit model of leaf outside-xylem water to test for potential drivers of the decline in K ox in dehydrating leaves transport (MOFLO 2.0, see Table1and Methods). The K ox was first computed based on the decline of observed cell size and air space alone (grey bars), which resulted in an increase in K ox (negative percent loss of K ox ; mainly due to shortening of pathways from the veins to stomata). We then modelled K ox decline according to three scenarios (though always including the effect of tissue dimensional changes): an 80% decline at -0.5MPa in (1) cell connectivity (red bars), (2) cell membrane permeability (blue bars), and (3) cell wall thickness (black bars). All simulations were run with (Ap; darker color) or without an apoplastic barrier (No Ap; lighter color) at the bundle sheath cells. The yellow star on the x-axis represents the % observed K leaf decline at -0.5MPa (measured with the evaporative flux method, see Methods).

Figure 8 .

 8 Figure8. Transmitted-electron microscopy of Arabidopsis thaliana (Col-0) midrib crosssections. In A, the entire xylem portion of the midrib can be seen. Black arrows point to the lack of secondary lignified wall around xylem conduits. These long primary wall sections can be observed in more detail in B. The white arrow points to a lignified portion of the secondary xylem wall. We hypothesize that the xylem resistance through these deeply helicoidal xylem conduits is greatly reduced, as unlignified primary cells effectively work as one large pit membrane.

Table 3

 3

; midrib conduit diameters measured under light microscopy ranged from 2.79 to 10.3 μm). No collapsed conduits were observed in midrib and secondary vein conduits at the www.plantphysiol.org on October 26, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743 7

  results demonstrate a potential strong role for outside-xylem pathways in the decline of Kleaf

	with leaf dehydration, contributing to stomatal closure and the reduction of photosynthetic rate in
	Arabidopsis thaliana (Col-0). Strong declines in Kleaf were associated with declines in Kplant, gs and
	Aarea, at water potentials where no significant embolism was observed using microCT. The absence
	of leaf xylem embolism before stomatal closure and hydraulic decline point to changes in outside-
	xylem pathways as the cause of observed Kleaf decline and imply no functional role of xylem
	dysfunction in this species' response of gas exchange to leaf dehydration. Modelling showed that
	Kleaf vulnerability has a strong causal role in determining stomatal closure, and further, that Kleaf
	vulnerability would improve plant carbon balance and survival during drought.

  , high minimum epidermal conductance (gmin), very high osmotic potential at full turgor, low modulus of elasticity and relative water content at turgor loss point, and a turgor loss point that is among the highest values reported across angiosperm species(Bartlett et al., 2012), similar

, a very high degree of area shrinkage during dehydration (58% shrinkage www.plantphysiol.org on October 26, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743 Scoffoni et al. Kleaf and gas exchange coordination in Arabidopsis 16 16 when dry)

  The bagged leaf was placed into a second bag filled with wet paper towels, and left to equilibrate for 30 min, after which final Ψleaf, was measured. Leaf area was manually traced onto paper, scanned, and measured potential of the water fed to the petiole [0 MPa] minus measured leaf), normalized by leaf area and corrected for the dependence of water viscosity on temperature (to a reference value of 25°C;

using ImageJ software (version 1.46r; National Institutes of Health). Leaf hydraulic conductance (Kleaf) was calculated as the flow rate divided by the leaf water potential driving force (the water www.plantphysiol.org on October 26, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743

  the petiole to the exposed end of the rubber stopper using super glue (Loctite 409 glue; Henkel Corp., Los Angeles, CA, USA) with accelerator (Loctite 712 accelerator). Leaves were placed inside a vacuum flask with a thermocouple (Cole-Parmer) connected by a fourway valve to

	a vacuum pump (Gast) and a high-precision pressure gauge (±0.002 MPa; J4605 Marsh/Bellofram;
	Marshall Instruments Inc., Anaheim, CA, USA).

Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743 sealing

  As Ψleaf declines during leaf dehydration, the resulting declines in gs and Kleaf lead to changes in their ratio, gs/Kleaf. If Kleaf declines more rapidly than gs with Ψleaf, such that the ratio gs/Kleaf increases, the decline in Ψleaf will be amplified, and consequently so will the decline in gs itself. of soil and atmospheric drought. We simulated transpiration, stomatal conductance, cumulative photosynthetic rate, cumulative water-use efficiency, water potential and the percent loss of xylem hydraulic conductance (PLC) daily and during the course of soil drying until plant death (i.e., PLC = 100%). We performed these simulations following four different scenarios: (1) Kleaf and Kroot were both vulnerable to dehydration prior to turgor loss point (using the function of Kleaf vs. Ψleaf measured with the EFM, and the vulnerability of Kroot obtained from that of Kleaf and Kplant, assuming no stem resistance in Arabidopsis; Supplemental FigureS4);(2) Kleaf was vulnerable but not Kroot (Kroot was kept constant until xylem embolism occurred in the root); (3) Kroot was vulnerable but not Kleaf (Kleaf was kept constant until xylem embolism occurred in the leaf); and (4) neither Kleaf nor Kroot were vulnerable to dehydration (i.e., both Kleaf and Kroot were kept at constant values until xylem embolism occurred) (Supplemental Methods; Supplemental Table

	Scoffoni et al.								Kleaf and gas exchange coordination in Arabidopsis 24
	(5)	𝐾 leaf = 6.83 + 81.4 exp(-7.56 × |Ψ leaf |)
	Ψleaf is in turn a function of gs, Kleaf, soil water potential (Ψsoil) and the water vapor mole fraction
	gradient (Δw):
	(6) Ψ leaf = Ψ soil -Δw	𝑔 s 𝐾 leaf
				𝜕𝑔 s		𝜕𝐾 leaf
	(7)	𝐹 =	𝜕𝐾 leaf 𝜕𝑔 s 𝜕Ψ leaf				,
				𝜕Ψ leaf
	where the partial derivative in the numerator is the sensitivity of gs to Ψleaf with Ψsoil and Δw held
	constant (1.5 kPa). That partial derivative is given by
	(8)	𝜕𝑔 s 𝜕𝐾 leaf	=	𝜕𝑔 s 𝜕Ψ leaf	𝜕Ψ leaf 𝜕𝐾 leaf	=	𝜕𝑔 s 𝜕Ψ leaf	[-∆𝑤 ( 𝐾 leaf 1	𝜕𝑔 s 𝜕𝐾 leaf	-	𝑔 s 𝐾 leaf	2 )]
	Solving for gs/Kleaf gives
	(9)	𝜕𝑔 s 𝜕𝐾 leaf	=	𝜕𝑔 s 𝜕Ψ leaf 1+ 𝜕𝑔 s 𝐾 leaf 𝑔 s ∆𝑤 2 𝜕Ψ leaf ∆𝑤 𝐾 leaf	=	𝜕𝑔 s 𝜕Ψ leaf 𝜕𝑔 s 𝜕Ψ leaf + 𝐾 leaf 𝑔 s 𝐾 leaf ∆𝑤
	Combining 7 and 9 gives F as
			𝜕𝐾 leaf	𝑔 s
	Kleaf and gs to leaf water potential: 𝜕Ψ leaf 𝐾 leaf (10) 𝐹 = 𝜕𝑔 s 𝜕Ψ leaf 𝐾 leaf + ∆𝑤
	(4)	𝑔 s = -451 × |Ψ leaf | + 339
											24

www.plantphysiol.org on October 26, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743 Therefore, Kleaf decline with dehydration would contribute to stomatal closure. The fraction of gs decline with Ψleaf that can be attributed to Kleaf decline, F, is Finally, using a simplified discrete-time soil-plant hydraulic model (SurEau; Martin-StPaul et al., 2017), we estimated the influence of Kleaf decline on stomatal closure under varying www.plantphysiol.org on October 26, 2018 -Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved. Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743 simulations

Table 1 .

 1 Modelling framework across scales to determine the underlying mechanisms linking Kleaf decline to gas exchange. Symbols: Aarea= leaf photosynthetic rate; gmin= minimum epidermal conductance; gmax= maximum stomatal conductance; gs= stomatal conductance; Kleaf= leaf hydraulic conductance; Kx= leaf xylem hydraulic conductance; Kox= leaf outside-xylem hydraulic conductance; PLC= percent loss of hydraulic conductance; VPD= vapor pressure deficit; Ψleaf= leaf water potential.

	Model	Purpose	Input

Table 2 .

 2 Parameters from the maximum likelihood function of K leaf vs. Ψ leaf , parameters from the function of K root vs. water potential, g min , g max , Farqhar's model inputs, PAR, air temperature, air humidity, time of day, transpiration under well-hydrated conditions, soil volume. Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743 Mean ± standard error for the physiological and anatomical traits measured for Arabidopsis thaliana (col-0). Symbols: K max : maximum leaf hydraulic conductance; K x : leaf xylem hydraulic conductance; %R ox : percent resistance outside the leaf xylem; g s : stomatal conductance; A max : maximum light saturated photosynthetic rate; P 50 , P 88 , and P 95 : leaf water potential at 50, 88 ns 95% decline in a given trait. American Society of Plant Biologists. All rights reserved. Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743

	Contribution of	K leaf decline explains most of the
	K leaf decline to g s	changes in g s during mild to
	decline with	moderate dehydration.
	dehydration.

Table 3 .

 3 Observations of embolized conduits and dimensions from microCT and their simulated impact on leaf xylem hydraulic conductance using the spatially explicit K_LEAF model. Because we did not have the resolution to determine whether conduit collapse occurs in tertiary and minor veins, two simulations were performed based on minor vein conduit collapse observed in Quercus rubra (see Methods). Note that two leaves were imaged at each water potential (22 leaves total); embolism at the four water potentials below were only found in one of the two leaves tested at that water potential. American Society of Plant Biologists. All rights reserved. Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743

	Leaf		Number of	Length of	Diameter of	Simulated percent loss of xylem hydraulic conducance
	water	embolized conduits	embolized	embolized conduit		(%)
	potential				conduit (μm)	conduit (μm)
	(MPa)
		midrib 2° 3°+ midrib	midrib	2°	Embolism	Embolism +	Embolism + "severe"
								only	"realistic" 3°+ vein	3°+ vein collapse
									collapse (13% PLC)	(50%PLC)
	-0.06	0	0	0	-	-	-	0	-	-
	-0.08	0	0	0	-	-	-	0	-	-
	-0.13	1	1	0	>898	7.3	8.7	7.13	9.99	19.1
	-0.16	0	0	0	-	-	-	0	-	-
	-0.18	2	2	0	258; >600	6.2; 5.3 5.2; 3.5 3.96	6.70	16
	-0.45	0	1	0	-	-	5.2	0.96	3.12	9.1
	-0.48	1	0	0	>850	5.9	-	1.16	3.96	13
	-0.50	0	0	0	-	-	-	0	-	-
	-0.69	0	0	0	-	-	-	0	-	-
	-0.79	0	0	0	-	-	-	0	-	-
	-0.87	0	0	0	-	-	-	0	2.82	12
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	Yang SD,
		39

Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R.,

Caringella

, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743 Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743 Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743 Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743 Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743 Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743

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(2016) Reversible leaf xylem collapse: a potential "circuit breaker" against cavitation. Plant Physiology 172: 2261-2274 Comment citer ce document : Scoffoni, C., Albuquerque, C., Cochard, H., Buckley, T. N., Fletcher, L. R., Caringella, M. A., Bartlett, M., Brodersen, C. R., Jansen, S., McElrone, A. J., Sack, L. (2018). The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana. Plant Physiology, 178 (4), 1584-1601. , DOI : 10.1104/pp.18.00743 1

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Acknowledgements

We thank Weimin Dang, Dula Parkinson, and Jessica Smith for technical assistance, and the University of California, Los Angeles, Plant Growth Facility and the Advanced Light Source in Berkeley, California (Beamline 8.3.2). This work was supported by the U.S. National Science Foundation (award nos. 1457279 and 1557906), a Humboldt Research Postdoctoral Fellowship, a CAPES/Brazil Fellowship, and the International Wheat Yield Partnership. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract no. DE-AC02-05CH11231.

To test for an effect of light on Kleaf, we selected the best-fit function for the response of Kleaf to Ψleaf, combining data for laboratory irradiance and high irradiance treatments, using a maximum-likelihood framework as explained above. We then calculated the residual variation for each leaf, subtracting the measured Kleaf (and irradiance) from the predicted Kleaf at the given Ψleaf, based on the best fit. We then performed a t-test on the residuals obtained for the high vs. low irradiance leaves across the entire range of Ψleaf, as well as just for points above turgor loss point, and for well-hydrated leaves (above -0.2MPa).

To determine the contribution of each correlated predictor variables (Time, PAR, Temperature, VPD, Ψleaf) to the observed variation in gs diurnally, we applied independent effects analysis (Murray and Conner, 2009)