Protocol

 

Authors

Lawren Sack, Jessica Pasquet-Kok, Megan Bartlett

OVERVIEW

This protocol explains how to make measurements of basic pressure volume parameters using simple equipment, with a pressure-volume analysis spreadsheet tool (PVAST) to facilitate the analysis.

BACKGROUND

This protocol explains two methods (“bench dry method”, “squeeze method”) to estimate the parameters of pressure-volume (PV) curves. The bench dry method is most traditional, and allows measurement of a set of 5-10 leaves at once, but requires a substantial time allocation (> 24 hours), whereas the squeeze method allows focused measurement of a single leaf in 2-3 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)
• Whirl-Pak bag (Whirl-Pak, 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 pressure-volume 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 labor-intensive than generating the pressure-volume curve, but does not allow for estimation of the other pressure-volume 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 x-intercept 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 pressure-volume 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 pressure-volume curve, and for the variables calculated from the curve.

Symbol (units)	Symbol (total)	Symbol (symplastic)	Definition
Measured variables
ψleaf (MPa)			Leaf water potential
(%)	RWC*		Total relative water content
Calculated water status variables
Ψp (MPa)			Turgor (or pressure) potential
ΨO (MPa)			Solute (or osmotic) potential
(%)		RWC	Relative water content in living cells (symplasm)
Calculated pressure-volume curve parameters
πft (MPa)			Osmotic potential at full turgor, calculated as the solute potential at 100% relative water content
πtlp (MPa)			Osmotic potential at the turgor loss point, visually estimated as the point where the relationship between RWC* and -1/ψleaf is linear
af (%)			Apoplastic fraction, or the proportion of the leaf water content outside the living cells, calculated as the RWC* at which  -1/ψleaf = 0
(%)	RWC*tlp	RWCtlp	Total or symplastic RWC at turgor loss point, visually estimated from πtlp
(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	Cft	Relative capacitance at full turgor, calculated as the change in total or symplastic RWC over the change in ψleaf between full hydration and πtlp
(MPa-1)	C*tlp	Ctlp	Relative capacitance at zero turgor, calculated as the change in total or symplastic RWC over the change in ψleaf after reaching πtlp
Cft 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: Pressure-volume 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):

1. Let shoots rehydrate overnight (optional)
2. In the morning, remove leaf from shoot by cutting at base of petiole with a razor blade
3. Measure or trace leaf for leaf area determination (optional, for determination of absolute capacitance per leaf area)
4. Put the leaf in a Whirl-Pak bag
5. Measure leaf water potential (ψ_leaf) with a pressure chamber.
6. Weigh the bagged leaf using an analytical balance.
7. 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).
8. Let leaf equilibrate for 10 min in a Whirl-Pak bag.
9. Repeat steps 4 to 7, attempting to capture ψ_leaf intervals of 0.2-0.3 MPa, until you achieve ψ_leaf of -3.0 MPa
10. Weigh the Whirl-Pak bag without the leaf, dry the leaf in an oven at > 70°C for at least 48 h before dry mass determination.
11. Use attached “Pressure volume analysis spreadsheet tool” (PVAST) to analyze data, including testing and correcting for “plateau effects” due to leaf rehydration.

Squeeze method: Pressure-volume curve parameters are determined by squeezing water out of the leaf using a pressure chamber to achieve different leaf water potential and weighing.

1. Let shoots rehydrate overnight (optional)
2. In the morning, remove leaf from shoot by cutting at base of petiole with a razor blade
3. Measure or trace leaf for leaf area determination (optional, for determination of absolute capacitance per leaf area)
4. Put the leaf in a Whirl-Pak bag
5. Measure first leaf water potential (ψ_leaf1st) with a pressure chamber.
6. Weigh the bagged leaf using an analytical balance.
7. Replace bagged leaf in pressure-bomb 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.
8. Weigh the bagged leaf using an analytical balance.
9. Repeat steps 7-8, until you achieve ψ_leaf of -3.0 MPa.
10. Weigh the Whirl-Pak bag without the leaf, dry the leaf in an oven at > 70°C for at least 48 h before dry mass determination.
11. 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 pressure-volume 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 pressure-volume 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

Whirl-Pak bag (Whirl-Pak, 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 meta-analysis. Ecology Letters, 15(5), 393-405.

Bartlett M. K., Scoffoni C., Ardy R., Zhang Y. Sun S. Cao K-F. & 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), 880-888.

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. 161-183. Kluwer, Dordrecht, the Netherlands.

Kubiske, M.E. & Abrams, M. D. (1991) Rehydration effects on pressure-volume relationships in four temperate woody species: variability with site, time of season and drought conditions. Oecologia, 85(4), 537-42.

Sack L., Cowan P.D., Jaikumar N. & Holbrook N.M. (2003) The ‘hydrology’ of leaves: co-ordination of structure and function in temperate woody species. Plant, Cell and Environment, 26, 1343-1356.

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-&.