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

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


| INTRODUCTION
The use of the pressure chamber to study plant water relations and the hydraulics of water transport dates from the beginnings of experimental biology in the work of Stephen Hales (1727). Formerly known as the 'pressure bomb'-still referred to this way colloquiallythe glass version of the instrument was abandoned due to explosions (Dixon, 1914), and a steel chamber version was re-invented by Scholander et al. (1965). Today's versions are the most common instruments used to measure leaf water potential (ψ leaf ), with pressure-release valves for safety, and digital pressure transducers for high precision. In the typical procedure, a leaf is excised from a plant and inserted in the instrument chamber lid with the cut surface of the petiole protruding out through a gasket. The lid is replaced, holding the leaf blade within the sealed chamber, and the chamber is pressurized until the petiole cut surface is wetted by the rising meniscus of xylem sap, indicating that the 'balancing pressure' applied has compensated for the negative pressure on the water column within the equilibrated leaf. The measurement assumes the Cohesion Tension theory for the ascent of sap (Angeles et al., 2004;Steudle, 2001), i.e., that the tension in the leaf xylem at the time that it is attached to the stem can be measured by pressurizing the equilibrated leaf and observing the balancing pressure. At that moment, assuming the xylem sap solute potential is negligible there is an equilibrium between the xylem tension, and the water potentials of the apoplast and the symplast, thus with bulk ψ leaf (Tyree & Zimmermann, 2002).
The pressure chamber has a range of critical applications, e.g., collection of xylem sap for determination of different components, or quantifying leaf water potential (ψ leafPC ; Table S1). To ensure its reliability, ψ leafPC has been compared with other techniques. Stem hygrometers (Dixon & Tyree, 1984), leaf psychrometers (Boyer & Knipling, 1965;Kursar et al., 2009;Tyree et al., , 2003, and the xylem pressure probe (Balling & Zimmermann, 1990;Melcher et al., 1998) enable estimates of stem xylem water potential (ψ stem ) and ψ leaf . These techniques can be less destructive than pressure chamber measurements, not necessarily requiring leaf excision, but can be more intensive, time-consuming, and less practical in the field due to their sensitivity to thermal gradients. Yet these approaches have corroborated the accuracy of ψ leafPC as have experiments based on transducers (Wei et al., 2000), centrifugation (Holbrook et al., 1995), air-injecting branches (Sperry et al., 1996), and comparisons with stem diameter variation (Cochard et al., 2001).
Several seminal reviews and studies on ψ leafPC have been published, and the approach has thus been used to study a huge diversity of plant species. However, the published literature does not provide clarity on many specific practices related to preparing, storing, and manipulating samples (Table S2), and there exists no consensus or standard protocol. Current published studies typically provide little information about the specific ψ leafPC measurement methods, except in few cases in which modifications are pivotal for a specific purpose, e.g., measuring plant-scale ψ (Knipfer et al., 2020), or to validate a methodology for a specific context, e.g., applying appropriate leaf storage conditions to maintain ψ leaf through time or determining the equilibration time for measuring ψ stem (Li et al., 2021;Wu et al., 2020). Coverage of ψ leafPC in trait measurement handbooks and online protocols (Boyer, 2011;Pérez-Harguindeguy et al., 2013) offers important guidance for beginning users but still include scant details and recommendations on critical aspects, and even include some advice at odds with common assumptions, e.g., suggestions to measure leaves as soon as possible after sampling without equilibration (Pérez-Harguindeguy et al., 2013). The current lack of standardized procedures reduces confidence in comparisons among the great variety of studies basing key conclusions on ψ leafPC .
Here, we surveyed specific methods of ψ leafPC from multiple laboratories, and synthesized experiments testing eight common assumptions and practices in ψ leafPC (Tables 1 and 2). We compiled tests from seven independent laboratories around the world on a wide range of species, aiming at the collaborative development of best practices recommendations to improve the reliability of ψ leafPC and to serve as updated guidelines for beginning and expert users.

| MATERIALS AND METHODS
We structured this study by, first, identifying the conditions required in theory for an accurate measurement, which rely on eight key assumptions-those related to sampling conditions or sample manipulations before measurement, and those related to measurement technique. Second, based on the discovery that multiple research groups had been conducting tests of given assumptions, we compiled strategies for establishing the required conditions and specific tests to achieve them (all experiments for each assumption are summarized in Tables 1 and 2). This study represents tests of different assumptions by multiple groups on different species, with conclusions that diverge in some cases. Our primary aim was to identify practices that would optimise the method in practical use, and provide a common practice protocol and guidance for testing given assumptions on specific species when needed.
The experiments were conducted on 25 species overall, diverse in geographical location, phylogeny, leaf morphology and ecological specialization. Experiments were performed independently by seven research teams situated in USA, such as Los Angeles (team UCLA), and West Lafayette (team PURDUE); in Australia, such as Sydney (team SYDNEY), and Hobart (team UTAS); and in Europe, such as Innsbruck (Austria, team INNSBRUCK), Seville (Spain, team IRNAS), and Clermont-Ferrand (France, team PIAF-INRAE). Details and descriptions of specific methods applied by each team are presented in Table S3.
2.1 | Methodology applied for each experiment to test assumptions towards best practices 2.1.1 | Assumptions related to sampling conditions or sample manipulations before measurement 2.1.1.1 | ASSUMPTION 1. Need for leaf equilibration before measurement According to this assumption, after excision of a leaf from the plant (and especially a transpiring leaf), bulk ψ leaf may change until equilibration throughout the leaf is reached, and thus, leaves should be equilibrated before measurement.
The IRNAS team tested whether leaf transpiration rate before leaf excision would affect the equilibration of bulk ψ leaf in Olea europaea leaves. Six current-year shoots from a single tree were selected and four mature leaves from two consecutive internodes per shoot (all with similar light exposure) were excised with a razor blade at 4:00 Greenwich Mean Time (GMT) (predawn), 8:00 GMT (midmorning) and 12:00 GMT (midday) (GMT). Each individual leaf was excised and rapidly enclosed in a small, unsealed plastic bag wrapped in soaked paper towel within a larger, sealed zip-lock plastic bag, previously exhaled into. This prevented direct contact of the leaf with liquid water while also creating a humid environment. Bags were stored in a portable icebox with ice packs not touching the bagged leaves. One leaf per shoot was measured within 2-3 min after excision, which was the time needed to reach the pressure chamber and perform the measurement and treated as the control (t 0 ), and a second leaf was measured at t 0 + 30 min. The remaining two leaves were used to test Assumption 2.
The SYDNEY team tested the equilibration of transpiring Eucalyptus camaldulensis leaves under two different conditions. At mid-day, five transpiring leaves were collected close together from a single shoot from each of six individual saplings, such that leaves on each shoot were similar in ψ leaf (leaf pairs measured on shoots of three other plants varied by less than 0.08 MPa). Leaves were enclosed in a plastic bag after excision and stored in an icebox with ice packs not touching the bagged leaves to avoid abrupt changes in leaf temperature that might result in condensation. In the first storage condition, bags in which the leaves were enclosed contained a moistened paper towel ('moist' condition); in the second condition, no paper towel was used but a researcher exhaled into the bag before quickly sealing it ('exhalation' condition). ψ leaf was measured for one leaf from each plant 1 min after excision as the control (t 0 ), and for leaves at t 0 + 10 min and t 0 + 1 h; the remaining two leaves per plant were used to test Assumption 2.
2.1.1.2 | ASSUMPTION 2. Leaves measured for ψ leaf may be stored before measurement According to this assumption, when leaves have reached equilibrium, ψ leaf will be maintained for several days provided the right storage protocol is used.
The IRNAS team tested the effect of storage of equilibrated leaves for the two remaining leaves from each of the six O. europaea shoots described for Assumption 1 collected at different times of the day, i.e., at different transpiration rates, and stored as explained above. Equilibrated leaves measured at t 0 + 30 min were treated as the control, and compared with leaves measured at t 0 + 4 h, and t 0 + 1.5 day.
The SYDNEY team tested the two remaining leaves from each of the six E. camaldulensis saplings described for Assumption 1 under two different storage conditions ('moist' and 'exhalation' conditions).
Equilibrated leaves measured at t 0 + 1 h were treated as the control, and compared with leaves measured at t 0 + 4 h, and t 0 + 1 day.
The UTAS team also tested the effects of partially dehydrated leaves of gymnosperm, fern and lycophyte species. Three to five 1a.
The ψ of O. europaea leaves, collected at midday (12:00 GMT), were 0.31 ± 0.10 MPa lower after 30 min of equilibration than the values measured immediately after excision (p < 0.01; Figure 1a; see Table S4 for all statistical results). The decline in ψ leaf during equilibration increased with the hydration status of leaves at excision. Only leaves that were already relatively dehydrated at excision (ψ leaf below ca. −1.4 MPa), showed little further decline during equilibration. The effect of equilibration was not significant for olive leaves sampled either at predawn (04:00 GMT) or in the early morning (08:00 GMT). These leaves were probably already equilibrated.
1b. Testing transpiring leaves stored under different conditions. Transpiring E. camaldulensis leaves stored in bags with moist paper towel ("moist" condition) or in bags with only air exhaled by a researcher ("exhalation" condition) for 10 min and 1 h.
1b.The ψ of E. camaldulensis leaves collected at midday and immediately measured did not differ from the values after equilibration for 10 min and 1 h ( Figure 1b). Exhalation treatment may allow further dehydration. were compared to unbagged and bagged leaves (at least 1 h), i.e., minimally and nontranspiring leaves, respectively.

3b.
No significant differences were found between ψ stem and ψ leaf in either minimally transpiring (un-bagged) leaves or leaves in which transpiration was further reduced by bagging (upper panels of Figure 3b). However, in P. lusitanica, the relationship between ψ stem and ψ leaf of previously bagged leaves had a significantly lower slope than the 1:1 line (p < 0.001; lower panels of Figure 3b).  (Richter, 1997;Figure 2.7 in Boyer, 1995).

5.
In three of five species, ψ of leaves fully exposed to the dry chamber was significantly lower than leaves that were enclosed in bags and measured in a humidified chamber ( Figure 5).

7.
In two of three species, approaching balance pressure by continuously increasing the pressure at a medium-low rate led to a small but significant underestimation of ψ leaf compared to

8.
Variation in ψ leaf measurements from a given equilibrated branch of C.
betulus was higher among investigators, in terms of coefficient of variation (CV), in newly trained non-experienced researchers relative to more experienced researchers. However, no significant differences were found in the mean among all investigators (p = 0.084) ( Figure 8).
(Continues) LEAF WATER POTENTIAL MEASUREMENTS | 2045 plants or branches transpired until ψ leaf approximated that corresponding to stomatal closure, determined previously in independent studies, and then were placed under non-transpiring conditions. One leaf per plant or branch was sampled and measured as the control (t 0 ). Then, samples were double bagged with a moist paper towel within a second bag and stored under laboratory conditions. For gymnosperms, samples were remeasured, following appropriate precautions, i.e., limiting water loss through the cut surface by avoiding driving 'gushing' of water due to overpressurization, and releasing the pressure very slowly (Wenkert et al., 1978) at t 0 + 6 h, 12 h and 1 day, and for the lycophyte and the ferns at t 0 + 1 day to According to this assumption, covered and non-transpiring leaves or shoots will equilibrate their ψ leaf with the branch or plant to which they are connected. This assumption is typically made when constructing hydraulic vulnerability curves, as branches are progressively bench dried and leaves or shoots are sampled for measurement of ψ leafPC , and stem or leaf hydraulic conductance is also measured, assuming equilibrium is maintained.
The INNSBRUCK team tested this assumption in 20 Pinus cembra branches. After laboratory processing (Table S3), subsamples (twigs, short twigs, or needles) were measured over a range of ψ of 0 to −3 MPa. ψ of three end twigs (length ca. 10 cm, with numerous short twigs) per branch, were considered the control samples, and compared with those of three short twigs (including 5 needles), and three single needles per branch. Measurements on single needles were made only to ca. −2 MPa, below which sealing into the lid of the pressure chamber was difficult and gas leaking complicated the determination of the endpoint.
The PIAF-INRAE team tested whether leaves measured for ψ leaf on dehydrating seedlings were equilibrated with the stem in which ψ stem was monitored using a stem psychrometer. This assumption is particularly important in the measurement of optical vulnerability curves (Brodribb et al., 2016 For the results discussed in terms of percentage (%) difference of tested leaves relative to control leaves, these were calculated as (Δψ leaf /-ψ leaf of control leaves) × 100. We fitted linear regressions to the relationships between Δψ leaf and control ψ leaf using SigmaPlot (version 12.0, Systat Software, Inc.). We plotted in figures only the regressions with p < 0.05 and when statistical analyses showed significant differences overall between tested and control leaves (see below); when overall significant differences were found but regressions were not significant, a horizontal line at the average of Δψ leaf was plotted instead. We report the mean effect sizes for Δψ leaf (MPa) ± standard errors.
We used linear mixed models with Tukey's post hoc comparisons to test the differences between test and control leaves overall for each Assumption at p < 0.05. We used the "lme()" function from the "nlme" package (Pinheiro et al., 2017) and the "glht()" function from the "multcomp" package (Hothorn et al., 2008) for post hoc multiple pair comparisons. We used random factor structure to account for experimental design (e.g., leaf identity within branch); when random factors were not necessary, the function "gls()" was used instead. The ψ leaf data were transformed when needed

| RESULTS
The ψ leaf remained stable when tested after 6 h, 12 h, and 1 day for gymnosperms, and when tested for 1, 2, 3 and 4 days for ferns and a lycophyte ( Figure 2c).
For two of three other species tested, storage under nontranspiring conditions ('bench' and 'fridge') was feasible for 1 day, 3 days and 7 days, and declines in ψ leaf were observed when leaves were stored for 14 days (Figure 2d). M. grandiflora leaves showed declines in ψ leaf during storage for 1 day and longer on the bench, and 3 days and longer in the refrigerator, suggesting that for this species, storage for more than one day in the refrigerator would lead to loss of precision in ψ leaf measurement ( Figure 2d).

| Test of Assumption 3. Leaves approximately equilibrate in ψ within non-transpiring single branches and seedlings
In branches of P. cembra, equilibrated at ψ leaf <−0.5 MPa, values were virtually the same across twigs, short twigs and needles, despite variation reaching ±100% when ψ leaf approached values very close to zero (Figure 3a). In branches equilibrated to lower water potentials, variation among different sampled organs was ±20% between twigs and short twigs or needles ( Figure 3a).
In dehydrated seedlings of L. ovalifolium and P. lusitanica, leaves equilibrated to similar ψ leaf , and similar to ψ stem . However, in strongly dehydrated branches of P. lusitanica, i.e., below TLP, and 50% loss of leaf xylem function, individual ψ stem values were higher than ψ leaf values for both bagged and un-bagged leaves ( Figure 3b).

| Test of Assumption 4. For most species tested, homogeneous equilibration within the leaf enables lamina to be excised without altering ψ leaf measurement
For four of five species tested, partial excision of lamina to artificially create 'elongated petioles' caused variation in ψ leaf , but no differences on average from an intact leaf. For M. grandiflora, partial excision of lamina led to ψ leaf values lower on average by 0.10 ± 0.03 MPa (p < 0.001) (Figure 4).

(a) (b)
F I G U R E 1 Assumption 1, i.e., need for leaf equilibration before measurement. Controls represent leaf water potential (ψ leaf ) measured at t 0 , i.e., measured soon after leaf excision, and Δψ leaf is the difference in ψ leaf between control and tested leaves (with a positive value indicating a less negative value for treatment than control) measured after different equilibration times (denoted as t 0 + time). Each point represents a pair of matched leaves, i.e., the difference in ψ leaf between tested and control leaves. 3.5 | Test of Assumption 5. Accurate measurements require bagging leaves and maintaining high humidity in the pressure chamber In three of five species, measured ψ leaf was significantly lower when leaves were measured un-bagged and without humidity inside the chamber. The effect was higher for leaves mildly dehydrated, i.e., at ψ leaf values closer to zero ( Figure 5).
3.6 | Test of Assumption 6. When measuring ψ leaf , air enters the leaf epidermis and pressurizes the intercellular spaces, and thus leaves should be measured with no liquid water on their surfaces For the three species tested, measuring leaves immersed under water increased ψ leaf (Figure 6).

| Test of Assumption 7.
Step pressurization is most accurate, with gradual pressurization acceptable for strongly dehydrated leaves For two of three species tested, ψ leaf measured by continuously applying pressure was significantly lower, though this effect was only pronounced for mildly dehydrated leaves, i.e., for ψ leaf values > −1 MPa (Figure 7).
3.8 | Test of Assumption 8. ψ leaf Measurements are reliable across multiple experimenters, given protocols are standardized within research teams F I G U R E 2 Assumption 2, i.e., leaves measured for leaf water potential (ψ leaf ) may be stored before measurement. Controls represent ψ leaf from equilibrated leaves and Δψ leaf is the difference in ψ leaf between control and tested leaves (with a positive value indicating a less negative value for treatment than control) measured after different storage times (denoted as t 0 + time). Each point represents a pair of matched leaves, i.e., the difference in ψ leaf between tested and control leaves. Different species or plant types (when appearing on the same plot) are depicted with different symbol types. Regression lines are plotted when statistical analyses showed significant differences between tested and control leaves and regressions were p < 0.05, and horizontal lines are plotted when significant differences were found but regressions were not significant.  F I G U R E 3 Assumption 3, i.e., homogeneous equilibration among leaves and with their bearing stems. (a) Branches of P. cembra collected in the field, dehydrated on the bench, and equilibrated for 1.5 h at different levels of ψ before measuring twigs, short twigs, and needles. Control samples were twigs and tested samples were short twigs or needles. Differences between control and tested samples (Δψ) over control ψ is presented in terms of absolute values (left panel) and % of difference respect to control ψ (right panel). Each point represents a pair of matched leaves, i.e., the difference in ψ leaf between tested and control leaves. (b) L. ovalifolium and P. lusitanica seedlings dehydrated on the bench under laboratory conditions. Stem water potential (ψ stem ) was measured with a stem psychrometer, and ψ leaf was measured on control leaves, previously bagged (i.e., equilibrated with ψ stem ) and on un-bagged leaves with a pressure chamber. Upper panels show differences (Δψ leaf ) between control and un-bagged leaves or ψ stem over control ψ leaf . Lower panels show the relationships between ψ stem and equilibrated, bagged ψ leaf . Each point represents one replicate.  (Bernardi, 2000;Karlic & Richter, 1979;Ritchie & Hinckley, 1975;Turner, 1981Turner, , 1988. One of the possible causes of this lack of consensus is difficulty in defining and understanding what the pressure chamber measures. According to early interpretations (Scholander et al., 1965), which are still the most widely accepted (Sperry et al., 1996;Wei et al., 1999), for the F I G U R E 4 Assumption 4, i.e., homogeneous equilibration within the leaf, tested on E. erythrocorys ( ), H. arbutifolia ( ), M. grandiflora ( ), E. camaldulensis ( ), and Z. diploperennis ( ). Controls represent ψ leaf measured with no modifications on the leaves, and Δψ leaf is the difference in ψ leaf between leaves with elongated petioles and control leaves (with a positive value indicating a less negative value for treatment than control). The horizontal line indicates that a significant difference was found between tested and control leaves but the regression was not significant. Each point represents a pair of matched leaves, i.e., the difference in ψ leaf between tested and control leaves [Color figure can be viewed at wileyonlinelibrary.com] F I G U R E 5 Assumption 5, i.e., accurate measurements require high humidity and bagged leaf in the chamber, tested on E. erythrocorys ( ), H. arbutifolia ( ), M. grandiflora ( ) C. cunninghamiana ( ), and E. obliqua ( ). Controls represent ψ leaf measured with moisture and bagged inside the chamber, and Δψ leaf is the difference in ψ leaf between leaves without bag and any moisture inside the chamber and control leaves (with a positive value indicating a less negative value for treatment than control). Δψ leaf over control ψ leaf is presented in terms of absolute values (left panel) and % of difference respect to control ψ leaf (right panel). The horizontal line indicates that a significant difference was found between tested and control leaves but the regression was not significant. Each point represents a pair of matched leaves, i.e., the difference in ψ leaf between tested and control leaves [Color figure can be viewed at wileyonlinelibrary.com] F I G U R E 6 Assumption 6, i.e., gas enters the leaf, tested on E. erythrocorys ( ), H. arbutifolia ( ), and M. grandiflora ( ). Controls represent ψ leaf measured while immersed, and Δψ leaf is the difference in ψ leaf between leaves measured immersed in water inside the chamber and control leaves (with a positive value indicating a less negative value for treatment than control). Regression lines are plotted when statistical analyses showed significant differences between tested and control leaves and regressions were p < 0.05, and horizontal lines are plotted when significant differences were found but regressions were not significant. Each point represents a pair of matched leaves, i.e., the difference in ψ leaf between tested and control leaves. is close to zero (Boyer, 1969). Yet, how this equilibrium water potential should correspond to that of the transpiring leaf before excision has not been made clear.
Modelling approaches may help to resolve the underlying physical and physiological mechanisms. Thus, a spatially explicit model which coupled water and heat transport outside the xylem of leaves and mapped the distribution of ψ across leaf tissues in relation to anatomy and environmental factors (Buckley et al., 2015(Buckley et al., , 2017 showed that in transpiring leaves, large ψ gradients may occur between the xylem and the most distal epidermal tissues. Indeed, such gradients have also been measured with xylem and cell pressure probe experiments (Shackel & Brinckmann, 1985;Shackel, 1987;Zwieniecki et al. 2002). When a transpiring leaf is excised, causing the retreating water in the xylem, the pressure gradients will collapse if it is stored in a bag to avoid evaporation, as its ψ will equilibrate (and change) over time across the leaf tissues. Higher ψ tissues, likely closer to the xylem, will tend to lose water to more dehydrated, lowerψ tissues in the mesophyll and/or epidermis.
The degree that the ψ immediately measured for an excised leaf would differ from that of a subsequently equilibrated leaf would depend on transpiration rate, and on the distribution and biophysical properties of leaf tissue of the species (Sack & Holbrook, 2006;Zwieniecki et al., 2007). For transpiring leaves with large pressure gradients from the xylem to the mesophyll, an instantly measured ψ leaf value would likely be biased upward by the higher ψ of the xylem and bundle sheath tissues, especially if these leaves had substantial water in these tissues, and during equilibration, given very high elastic modulus of the xylem cells, ψ leaf would decline closer to that of the mesophyll cells. The excision of transpiring leaves will also cause rapid dehydration (Turner & Long, 1980), so leaves must be enclosed under nontranspiring conditions within 1-2 s after excision. Given that transpiration is halted when the leaf is sampled and bagged, the water potential values within the leaf would collapse to an equilibrium value that would represent the volume weighted mean of the leaf tissues (Tyree & Zimmermann, 2002), and thus closer to the bulk ψ leaf during transpiration than the immediate measure. Our empirical results supported the necessity for equilibration of leaves that were sampled during transpiration under appropriate storage conditions (see below), with a need to test the required equilibration time, which could be as short as 10 min for certain species, or potentially longer for others.
F I G U R E 7 Assumption 7, i.e. gradual versus step pressurization, tested on E. erythrocorys ( ), H. arbutifolia ( ), and M. grandiflora ( ). Controls represent ψ leaf measured with step pressurization or 'pulsing', and Δψ leaf is the difference in ψ leaf between control leaves and leaves measured by continuously increasing the pressure at a constant rate (with a positive value indicating a less negative value for treatment than control). Δψ leaf over control ψ leaf is presented in terms of absolute values (left panel) and % of difference respect to control ψ leaf (right panel). The horizontal line indicates that a significant difference was found between tested and control leaves but the regression was not significant. Each point represents a pair of matched leaves, i.e., the difference in ψ leaf between tested and control leaves [Color figure can be viewed at wileyonlinelibrary.com] LEAF WATER POTENTIAL MEASUREMENTS | 2053 4.2 | Storing equilibrated leaves under fully humid conditions in which there is no transpiration maintains stable ψ leaf ψ leaf is expected to be stable over time when leaves are not transpiring, i.e., ψ gradients within the leaf are equilibrated, and leaves are stored optimally before measurement. Our results suggest that this was the case for all species, though the conditions for long-term maintenance of ψ leaf need to meet important criteria, and in some cases may be context dependent.
Indeed, E. camaldulensis leaves stored in bags that were only exhaled into showed a decrease in ψ leaf over time, probably due to leaf dehydration. To minimise transpiration, the leaf needs to be stored in high relative humidity to reduce the driving force for water loss, and darkness, and potentially high CO 2 to induce stomatal closure may be required, as well as constant temperature to prevent condensation on the leaf surface (Boyer, 1995). Our experiments showed that storage with simple procedures preserved ψ leaf of all species for up to 1 day and for most species, up to 3 days. Notably, a high humidity around the leaf as provided by wet paper towel is needed as part of the storage, and we did not find that ψ leaf increased due to uptake of water vapor under these conditions, excepting a very slight but statistically non-significant trend for E. camaldulensis. In fact, this leaf rehydrating behaviour can occur for leaves kept in fog chambers (Guzmán-Delgado et al., 2018) or in contact with water (Fernández et al., 2017).
Nevertheless, we do not recommend long periods of storage (e.g., 1-2 weeks) as degradation of the leaf tissues, e.g., caused by biochemical and osmotic processes such as lysis of carbohydrates or depolymerisation of starch, may generate a variation in ψ leaf values, as observed in our results not only for these long storage times but also for some leaves stored for shorter times (1.5 days in O. europaea). For one species tested, M. grandiflora, ψ leaf declined after 1 day stored under non-transpiring conditions on the laboratory bench, indicating this species dehydrated more easily than the others under the same conditions. This might be due to rapid osmotic adjustment, and/or tissue damage after excision, or just due to dehydration, especially if leaf surface trichomes play an important role as water storage sites and dehydrate even in humid air (Nguyen et al., 2017). Data from a greater diversity of species are needed to confirm the general feasibility of storing leaves for hours or days before ψ leaf measurement (see Appendix). We recommend conducting of tests for each species in designing measurement campaigns, to enable the convenience of sampling leaves in the field, storing and measuring them later in the laboratory. 4.3 | Influence on ψ leaf values by modifications of the samples or measurement procedures

| Equilibrating leaves on branches or seedlings
Transpiration causes gradients in ψ across organs in intact plants (Klepper et al., 1973). When this transpiration is prevented by covering the foliage, an equilibration time under non-transpiring conditions is needed for these gradients to dissipate, and for their ψ to equalize. Reported equilibration times for branches vary widely in the literature, from 10 min to 1.5 h or longer (Hochberg, 2020;Levin, 2019;Li et al., 2021;Rockwell et al., 2011). The tests compiled here used the most common times reported in the literature (1-1.5 h) to ensure equilibration and found these to be sufficient for ψ leaf of leaves on single branches at different levels of dehydration to equilibrate, with a small difference in ψ between needles and twigs in P. cembra likely due to a technical issue in measuring conifers. The release of resin from ducts may mask the emergence of water from the cut end in conifers, especially at ψ >−0.5 MPa. Yet, this difference may not be important for well-practiced operators, with repeatability of ±0.03-0.05 MPa.
Similarly, the ψ stem monitored with psychrometers in seedlings represented the ψ of all parts of the plant at equilibrium, given that stomata were virtually closed under the low irradiance laboratory conditions, except under strongly dehydrated samples. Notably, the ψ stem of P. lusitanica diverged from ψ leaf when ψ was close to TLP and 50% loss of leaf xylem functionality (P50); at this stage, embolism may lead to hydraulic disconnection between different tissues or organs in some species that would result in water losses at different rates, and a slower or incomplete equilibration across the plant (Brodribb et al., 2017). These discrepancies indicate that equilibration issues for branches dehydrated beyond cavitation thresholds would necessitate selecting ψ stem measurements as most appropriate according to the experimental design (Ritchie & Hinckley, 1971).

| Elongating the petioles
Many researchers measuring small leaves have relied on pretreatments such as cutting a portion of leaf lamina to elongate the petiole (e.g., Richter, 1997). For ψ leaf measurements in grasses, Boyer (1995) recommended to cut one side of the leaf lamina of wide grass leaves perpendicularly, almost to the midrib, and to tear the leaf toward the apex from the cut, before measuring that sample. Our results mostly supported this approach. However, variation was usually high among such treated leaves, and apparently greater for leaves dehydrated to ψ leaf >−1.5 MPa; ψ leaf was also significantly lower in one of four species subjected to this modification. This species was M. grandiflora, corroborating the ability of their leaves to dehydrate easily which agrees with results from Assumption 2 for this species. Thus, whether removing a portion of the leaf lamina affects ψ leaf must be determined beforehand for the species under study (Appendix). Notably, another modification, which we did not test in our study, i.e., recutting the petiole, has always been discouraged in seminal papers (Richter et al., 1972;Scholander et al., 1965;Turner, 1988). Cutting large portions of petioles is obviously not recommended (Hochberg, 2020), but for some species the effect on ψ leaf has seemed to be negligible (Levin, 2019), and more data testing this modification are required (Appendix).
4.3.3 | The need to measure leaves bagged and with high humidity inside the chamber For decades, seminal works have recommended maintaining high humidity inside the pressure chamber during measurements (Turner, 1981(Turner, , 1988, even modifying it so that gas enters the chamber by bubbling through water (Boyer, 1967). Given the initial steepness of pressure-volume curves (Bartlett et al., 2012), water loss from an exposed excised leaf might cause a dramatic decline in ψ leaf .
However, there have been scarce empirical data showing the effect of measuring leaves without humidifying the chamber (Cheung et al., 1975;Leach et al., 1982). We found that chamber conditions can strongly influence ψ leaf and thus that care must be taken to provide adequate humidity without inadvertently rehydrating samples (Appendix; Turner, 1988;Tyree et al., 1978), especially for leaves measured at moderate and high ψ leaf .

| Measuring with gradual or step pressurization
Most pressure chamber users apply a constant and slow rate of pressurization during measurement of balance pressure (personal communications from researchers of several laboratories). Several experiments testing the influence of different rates of increase in pressure have been reported, with mixed results (Naor & Peres, 2001;Ritchie & Hinckley, 1975), concluding that the maximum rate applied should be determined for the species under study (Ritchie & Hinckley, 1975). We did not compare different rates of pressurization but a constant rate (0.02 MPa s −1 ) (Turner, 1981(Turner, , 1988) versus a pulsed rate of increase, i.e., small increments followed by waiting periods. As the highest decrease from 'gradual' compared to 'pulsing' values was observed at ψ leaf >−1 MPa, we recommend the 'pulsing' methodology for accuracy of the endpoint identification when ψ leaf is expected to be high, although a low and continuous rate of pressurization would be acceptable when ψ leaf is expected to be lower and to enable measurement of a larger number of samples under time constraints (Appendix). We consider a 'pulsing' methodology to be more accurate because a specific time is allocated for pressure to equilibrate within the leaf. Therefore, pulsing or slow pressurization is needed to avoid overestimation (Naor & Peres, 2001), but this careful approach comes at the cost of rapid measurement. assumptions not yet considered in our study (Appendix), not to only improve our understanding of the best means for reliable, accurate and precise measurements, but also to yield further insights on underlying processes affecting the species' water relations.

SUPPORTING INFORMATION
Additional supporting information can be found online in the Supporting Information section at the end of this article. The aim of this Appendix is to present best practices for pressure chamber ψ leaf measurements derived from our knowledge of typical practices, the tests performed in the present study, and consultation with other experts. We also highlight when additional tests would be useful to design protocols for measuring given species.

Collecting samples
When the target leaf has been selected, caution must be taken in cutting the petiole.
The petiole must be long enough to fit the gasket of the pressure chamber (see Section 3 for recommendations measuring shortpetiole species), and the cleaner and more perpendicular the cut, the better for its visualization. Always use sharp razor blades.
Since the leaf may quickly dehydrate after excision, it should be immediately (within 1-2 s after excision) and appropriately stored.
Based on our tests of assumptions 1 and 2, we advise minimising leaf dehydration. Immediately before, or immediately after excising a leaf, (i) insert it, with its petiole inside, in an individual small zip lock plastic bag previously exhaled into (and thus containing high humidity and CO 2 to halt transpiration), (ii) squeeze the air out of the bag, (iii) seal the bag if convenient and insert it into another bag containing soaked paper towel and thus providing a saturated atmosphere around the bagged leaf, and (iv) store the double-bagged leaf in a cooling box containing ice packs, while using a barrier (e.g., a cardboard box) to prevent contact between samples and ice packs, thus avoiding abrupt changes of temperature.

Equilibration and storage of samples before measurements
Store leaves in double bags as explained above. We recommend measuring leaves once equilibrium has been reached and avoid long storage periods (>1 day) that may lead to loss of accuracy. For transpiring leaves, the minimum equilibration time should be tested beforehand; 10 to 30 min was sufficient for our tested species when excised during transpiration. Allowable storage time before measurement appeared to differ among species.
1. Test for determining the storage time required for leaves to equilibrate. Collect ca. 10 leaves from the same branch and under the same conditions, store them as stated above, and measure three leaves immediately (this will also validate that the initial ψ leaf is the same) and, then, every 5-10 min.
Once a plateau is reached, the leaves may be considered as

Manipulation of samples before measurements
The time that leaves remain on the bench when manipulated must be minimised and if leaves are subjected to treatments, the exposed leaf lamina should be covered with a plastic bag with wet paper towel inside to avoid dehydration. If the leaves have standing moisture on their surfaces (e.g., predawn samples), we recommend wiping them with a dry paper towel before measurement since it could rehydrate when measured (see tests of Assumption 6).
Although recutting the petiole has been discouraged in seminal works (Richter et al., 1972;Scholander et al., 1965;Turner, 1988) due to a possible effect of increasing ψ leaf , in some species, recutting of small amounts of the petiole may be essential to see the xylem and may not affect ψ leaf , e.g., vine leaves (Levin, 2019). We recommend that, if recutting the petiole is necessary, removing long portions of petioles should be avoided as this shortens the distance that the water column would travel (Hochberg, 2020 An important recommendation for measuring twigs of conifer species, is to remove the bark and peel away the 3-5 mm of resinous outer layers to better identify the endpoint.

Measurement technique
Create high humidity conditions inside the chamber with abundant damp paper towel and enclose the leaf in a bag. These conditions are particularly important for mildly or moderately dehydrated leaves, i.e., with high ψ leaf (see test of Assumption 5).
Caution must be taken using bags inside the chamber to avoid a safety risk if these block the safety valve making it difficult to fully depressurise the chamber after use. We suggest that the bag be arranged so as not to interfere with the safety valve, which is usually at the bottom of the chamber. Additionally, care must be taken such that the top of the plastic bag does not interfere with proper sealing of the chamber lid.
We suggest mounting a dissecting microscope above the pressure chamber, with fibre-optic lights, to clearly observe the cut petiole surface and to best resolve the balance pressure.
A common question among pressure chamber users is how to decide on the measurement endpoint: what proportion of the petiole surface conductive area must be wetted? This may depend on the plant type and species, and thus, identifying first the xylem through which sap will appear is essential. We recommend noting the pressure at the first sign of water at the cut end, and additionally, the pressure when the entire xylem area is wetted. These values may be compared, and, if not too different, an average may be used. We also suggest considering that the endpoint has been exceeded when water is 'bubbling'. These technicalities should be discussed among researchers; according to our test of assumption 8, variation among researchers in the approach should be minimised for what would be considered the endpoint.
If many samples need to be measured, and leaves to be measured are of low ψ leaf , it is acceptable to use a continuous medium-slow rate of pressurization (0.02-0.03 MPa s −1 ). However, if only a few samples need to be measured and, in particular, if they are at high ψ leaf , pulsing the pressure increase is recommended (see test of Assumption 7), i.e., increasing the pressure by low increments (e.g., 0.05-0.10 MPa), and holding the pressure for 5-10 s (15-30 s when approaching the value) to test for balance pressure. If the approximate value of ψ leaf is known and it is low enough (≤−1 MPa), higher rates of pressurization can be applied at the beginning to build the chamber pressure; then, after holding the pressure for 5-10 s, the pressurization rate should be slowed to a slow pressurization rate, 0.007-0.01 MPa s −1 for the last 30% of the range until the balance pressure is reached. Although this last procedure was not exactly tested in our experiments (we evaluated a continuous increase in pressure vs. a pulsed increase, both at the same rate of pressurization), the differences between the two techniques evaluated tended to occur at high ψ leaf . Thus, using a slow pressurization rate for the last 30% of the ψ leaf range should allow precise identification of the endpoint, and also more rapid measurements for leaves at very low ψ leaf .
To validate the measurement, in case the endpoint was not clear, or to check whether the balance pressure was overestimated (as would occur if water was gushing out or 'bubbling' vigorously at the xylem cut surface), after the balancing pressure is achieved, i.e., when water reaches the cut surface, slowly decrease the pressure by ca. 0.5 MPa or until the water recedes, and then increase the pressure again very slowly in small pulses, holding the pressure for 15-30 s between pulses until a new balance pressure is reached. Recognize that the new water potential measurement may be slightly lower than the original measurement due to loss of water from the petiole.

5.
Test of the effect of re-measuring the same leaf on ψ leaf .
When the same leaf is re-measured, it is assumed that the first measurement does not change the water content of the leaf, and so its ψ leaf . To test this assumption, collect a branch and equilibrate it under non-transpiring conditions for 1-2 h by covering each leaf individually with a Ziploc plastic bag containing a wet paper towel inside and the entire branch in a bigger, dark plastic bag with more wet paper towel inside. For the measurements, collect a leaf, weight it bagged on a precision balance, with the petiole inside the bag and the bag sealed, and measure it immediately taking the precautions specified by Wenkert et al. (1978), i.e., minimising the water loss through the cut surface by very slowly incrementing the pressure when approaching the balance pressure value, and depressurising very slowly to avoid possible damage to mesophyll cells.
After removing the leaf from the chamber, weigh it again immediately after inserting the petiole inside the bag.
Measure it a second time, repeating the same procedure, to check whether mass and ψ leaf have changed.
The determination of the balance pressure may be more difficult when embolism has occurred within the leaf xylem vessels, as an airwater mixture is often observed at the cut surface, though ψ leaf determination might still be possible. In these cases, we recommend continuing to increase the pressure until the xylem cut surface is entirely covered, but if this does not occur, the pressure at the first sign of water remains the most acceptable value.

Some extra recommendations on measurement technique
Caution is necessary when plant material contains resin or mucilage ducts or laticifers, so measurements should be performed in test material beforehand to train on the visual distinction between resin, mucilage or latex and water. With practice, the different fluids can be clearly distinguished visually by their viscosity and reflectance.
Caution must be taken during the closure of the chamber to not squeeze some parts of the leaf. The sealing force (compression of the gasket) should be increased with increasing pressure, always taking care not to over-squeeze the petiole. A good recommendation for low ψ leaf might be to wrap parafilm or dental epoxy around the base of the petiole to enhance the seal and prevent overtightening the lid and snapping the petiole. Another recommendation for herbaceous species, which are usually more difficult to measure, is not to force the gasket much to avoid smashing the petiole, even if there is some leakage. Small leaks are not problematic, indeed better than crushing the tissue, as long as the chamber can be pressurized. Using softer gaskets may also be very helpful. These ways to cope with the challenges of herbaceous leaves are based on our own experience, and we encourage researchers starting work on herbaceous species to evaluate different assumptions to optimize the use of the pressure chamber.
At the end of the measurement, samples should be checked for possible damage that may have led to incorrect values.
LEAF WATER POTENTIAL MEASUREMENTS