Protocol
Authors
Lawren Sack, Jessica PasquetKok, Megan Bartlett
OVERVIEW
This protocol explains how to make measurements of basic pressure volume parameters using simple equipment, with a pressurevolume analysis spreadsheet tool (PVAST) to facilitate the analysis.
BACKGROUND
This protocol explains two methods (“bench dry method”, “squeeze method”) to estimate the parameters of pressurevolume (PV) curves. The bench dry method is most traditional, and allows measurement of a set of 510 leaves at once, but requires a substantial time allocation (> 24 hours), whereas the squeeze method allows focused measurement of a single leaf in 23 hours.
PV parameters include osmotic potential at full turgor and at the turgor loss point, relative water content at turgor loss point, modulus of elasticity at full turgor, apoplastic fraction, and relative and absolute capacitances at full turgor and at zero turgor (Koide et al., 2000; Sack et al., 2003). Relative water content at turgor loss point, modulus of elasticity, and relative capacitance can and should be calculated in terms of both total and symplastic water content.
MATERIALS/EQUIPMENT
 Analytical balance (to 0.001 g)
 Razor blade
 Fan (bench dry method)
 Pressure chamber (Plant Moisture Stress, Model 1000, Albany, Oregon, USA)
 WhirlPak bag (WhirlPak, Nasco, Fort Atkinson, Wisconsin, USA)
UNITS, TERMS, DEFINITIONS
Leaf water potential (ψ_{leaf}) equals osmotic potential (ψ_{o}) plus turgor potential (ψ_{P}) (see Table 1 for symbol definitions).
The turgor loss point (π_{tlp}) is the point at which turgor potential is 0, and thus leaf water potential equals osmotic potential. Consequently, the inverse of leaf water potential declines linearly with relative water content below the turgor loss point (Tyree & Hammel 1972, Koide et al. 2000). Plotting the inverse of leaf water potential (1/ψ_{leaf}) against relative water content allows determination of the π_{tlp} as the point of transition between the linear and nonlinear portions of the pressurevolume relationship.
Osmotic potential at full turgor (π_{ft}) is estimated by extrapolating the linear section to calculate the ψ_{o} at 100% relative water content, or full hydration. The π_{ft} and π_{tlp} can also be assessed with an osmometer method (see Measuring osmotic potential and turgor loss point using a vapor pressure osmometer from Bartlett & Sack 2015), which is less time and laborintensive than generating the pressurevolume curve, but does not allow for estimation of the other pressurevolume curve parameters (Bartlett et al. 2012).
The apoplastic fraction (a_{f}), or the proportion of leaf water content contained in the apoplast (inside the vasculature and bound to the cell walls), is estimated by extrapolating the xintercept of this linear section, or the relative water content at 1/ψ_{leaf} = 0. These parameters are estimated from the total relative water content (RWC^{*}), which includes the apoplastic water and the water contained in living cells (the symplasm).
The rest of the pressurevolume curve parameters can be calculated in two versions—i.e., based on the total relative water content (RWC^{*}) or on the relative water content of the symplasm (RWC). Expressing these parameters in terms of symplastic relative water content captures the volume of water available for metabolic processes in the living cells, while calculating these parameters from total relative water content allows a_{f} to be evaluated as an independent parameter (Bartlett, Scoffoni & Sack 2012). The relative water content at the turgor loss point measures the leaf hydration at wilting, and is visually estimated from the same nonlinear to linear transition as the turgor loss point.
The bulk modulus of elasticity estimates cell wall rigidity averaged across the leaf, and is determined from the change in turgor potential over the change in total relative water content (ε^{*}) or symplastic relative water content (ε) between full hydration and turgor loss point. See Notes for additional details regarding its calculation.
The relative capacitance is calculated as the slope of relative water content versus leaf water potential, and can be determined between full turgor and turgor loss point, and also separately, below turgor loss point, for total or symplastic relative water content. Absolute capacitance per leaf area can be estimated as relative capacitance multiplied by the mass of water per leaf area, but only for total relative water content, as weighing the leaf cannot distinguish between symplastic and apoplastic water.
Table 1. Symbols and definitions for the measured variables used to construct the pressurevolume curve, and for the variables calculated from the curve.
Symbol
(units) 
Symbol
(total) 
Symbol
(symplastic) 
Definition 
Measured variables  
ψ_{leaf }(MPa)  Leaf water potential  
(%)  RWC^{*}  Total relative water content  
Calculated water status variables  
Ψ_{p }(MPa)  Turgor (or pressure) potential  
Ψ_{O }(MPa)  Solute (or osmotic) potential  
(%)  RWC  Relative water content in living cells (symplasm)  
Calculated pressurevolume curve parameters  
π_{ft} (MPa)  Osmotic potential at full turgor, calculated as the solute potential at 100% relative water content  
π_{tlp} (MPa)  Osmotic potential at the turgor loss point, visually estimated as the point where the relationship between RWC^{*} and 1/ψ_{leaf} is linear  
a_{f }(%)  Apoplastic fraction, or the proportion of the leaf water content outside the living cells, calculated as the RWC^{*} at which 1/ψ_{leaf} = 0  
(%)  RWC^{*}_{tlp}  RWC_{tlp}  Total or symplastic RWC at turgor loss point, visually estimated from π_{tlp} 
(MPa)  ε^{*}  ε  Modulus of elasticity at full turgor, calculated as the change in turgor potential over the change in total or symplastic RWC 
(MPa^{1})

C^{*}_{ft}  C_{ft}  Relative capacitance at full turgor, calculated as the change in total or symplastic RWC over the change in ψ_{leaf} between full hydration and π_{tlp} 
(MPa^{1})  C^{*}_{tlp}  C_{tlp}  Relative capacitance at zero turgor, calculated as the change in total or symplastic RWC over the change in ψ_{leaf} after reaching π_{tlp} 
C_{ft }^{a}_{ }
_{ }(g m^{2} MPa^{1}) 
Absolute capacitance per leaf area at full turgor, calculated as C^{*}_{ft} ´ area ´ saturated water content 
PROCEDURE
Bench dry method: Pressurevolume curve parameters are determined by progressively drying leaves on a laboratory bench with a fan (optional), and measuring leaf water potential and leaf mass at intervals (ψ_{leaf}):
 Let shoots rehydrate overnight (optional)
 In the morning, remove leaf from shoot by cutting at base of petiole with a razor blade
 Measure or trace leaf for leaf area determination (optional, for determination of absolute capacitance per leaf area)
 Put the leaf in a WhirlPak bag
 Measure leaf water potential (ψ_{leaf}) with a pressure chamber.
 Weigh the bagged leaf using an analytical balance.
 Take the leaf from the plastic bag and let dry on the bench (optional: use fan to dry leaf faster, with leaf taped to a line or held in a frame).
 Let leaf equilibrate for 10 min in a WhirlPak bag.
 Repeat steps 4 to 7, attempting to capture ψ_{leaf} intervals of 0.20.3 MPa, until you achieve ψ_{leaf} of 3.0 MPa
 Weigh the WhirlPak bag without the leaf, dry the leaf in an oven at > 70°C for at least 48 h before dry mass determination.
 Use attached “Pressure volume analysis spreadsheet tool” (PVAST) to analyze data, including testing and correcting for “plateau effects” due to leaf rehydration.
Squeeze method: Pressurevolume curve parameters are determined by squeezing water out of the leaf using a pressure chamber to achieve different leaf water potential and weighing.
 Let shoots rehydrate overnight (optional)
 In the morning, remove leaf from shoot by cutting at base of petiole with a razor blade
 Measure or trace leaf for leaf area determination (optional, for determination of absolute capacitance per leaf area)
 Put the leaf in a WhirlPak bag
 Measure first leaf water potential (ψ_{leaf1st}) with a pressure chamber.
 Weigh the bagged leaf using an analytical balance.
 Replace bagged leaf in pressurebomb and increase the pressure until you reach ψ_{leaf1st} and then increase pressure by 0.2 MPa and note this pressure. Hold the pressure at this level while dabbing the petiole until there is no more water coming out.
 Weigh the bagged leaf using an analytical balance.
 Repeat steps 78, until you achieve ψ_{leaf} of 3.0 MPa.
 Weigh the WhirlPak bag without the leaf, dry the leaf in an oven at > 70°C for at least 48 h before dry mass determination.
 Use attached “Pressure volume analysis spreadsheet tool” (PVAST) to analyze data, including testing and correcting for “plateau effects” due to leaf rehydration.
NOTES AND TROUBLESHOOTING TIPS
 This protocol follows most previous studies in rehydrating the leaves before dehydrating and measuring for PV curve determination. While there is some debate in the literature over whether the pressurevolume curve parameters might be dynamic with respect to leaf water status (Kubiske & Abrams 1991), which would lead to shifts in parameters with rehydration, this is not clearly established. The benefits of rehydrating before PV curve determination include (1) fully capturing the nonlinear portion of the pressurevolume curve prior to turgor loss point, (2) assessing all samples at a standard hydration, and (3) ability to compare PV parameters with previously published data.
 The approach to the estimation of ε as dψ_{P} / dRWC assumes at least an approximately linear decline of turgor with RWC between full turgor and turgor loss point, which is consistent with most empirical data. Some previous studies have suggested that ε declines with turgor. In fact, this idea depends on the specific formula used to determine ε. Technically in the early literature ε is defined as dψ_{P} / dRWC × mean RWC, where mean RWC is the mean of the points in the interval used for estimating ε; this formulation treats the dehydrating leaf as a deformed solid, with no loss of material, like a Young’s Using that definition, ε does decline strongly with declining RWC, even if dψ_{P} / dRWC is approximately constant. However, many studies have simply calculated ε as dψ_{P} / dRWC without the multiplication by mean RWC; this formulation treats the leaf as a deflating object, given that water is lost during dehydration (Jones 2014). The issue of different definitions of ε can be rendered moot, by simply calculating ε for leaves at full turgor, and assuming that dψ_{P} / dRWC is approximately constant. In this case, the “mean RWC” for the leaves at full turgor = 1, and ε is the same using either definition for its calculation.
 If the petiole is too fragile, you can put parafilm around it at the beginning of the measurement. Do not forget to weigh the parafilm at the end of the measurement.
 If piece of leaf breaks, keep it inside the plastic bag, note when this occurred and do not forget to put all the pieces in the oven. Then, deduct the dry mass of the piece from the mass values after the breakage occurred.
 Note that the PV_analysis spreadsheet tool fits lines by standard major axis (Model II regression) so that either variable can be predicted from the other and the parameter calculation is more robust.
LINKS TO RESOURCES AND SUPPLIERS
Measurement of leaf water potential; see tutorial videos on Plant Moisture Systems (PMS) web site Plant Moisture Stress, Model 1000, Albany, Oregon, USA
WhirlPak bag (WhirlPak, Nasco, Fort Atkinson, Wisconsin, USA)
LITERATURE REFERENCES
Bartlett M. K., Scoffoni C. & Sack L. (2012) The determinants of leaf turgor loss point and prediction of drought tolerance of species and biomes: a global metaanalysis. Ecology Letters, 15(5), 393405.
Bartlett M. K., Scoffoni C., Ardy R., Zhang Y. Sun S. Cao KF. & Sack, L. (2012) Rapid determination of comparative drought tolerance traits: using an osmometer to predict turgor loss point. Methods in Ecology and Evolution, 3(5), 880888.
Jones H.G. (2014) Plants and Microclimate, 3^{rd} ed. Cambridge University Press, Cambridge UK.
Koide R.T., Robichaux R.H., Morse S.R. & Smith C.M. (2000) Plant water status, hydraulic resistance and capacitance. In: Plant Physiological Ecology: Field Methods and Instrumentation (eds R.W. Pearcy, J.R. Ehleringer, H.A. Mooney, & P.W. Rundel), pp. 161183. Kluwer, Dordrecht, the Netherlands.
Kubiske, M.E. & Abrams, M. D. (1991) Rehydration effects on pressurevolume relationships in four temperate woody species: variability with site, time of season and drought conditions. Oecologia, 85(4), 53742.
Sack L., Cowan P.D., Jaikumar N. & Holbrook N.M. (2003) The ‘hydrology’ of leaves: coordination of structure and function in temperate woody species. Plant, Cell and Environment, 26, 13431356.
Tyree M.T. & Hammel H.T. (1972) Measurement of turgor pressure and water relations of plants by pressure bomb technique. Journal of Experimental Botany, 23, 267&.