Dynamics of stomatal water relations following leaf excision

Authors

  • JULIA E. POWLES,

    1. Environmental Biology Group and Cooperative Research Centre for Greenhouse Accounting, Research School of Biological Sciences, Institute of Advanced Studies, The Australian National University, Canberra ACT 2601 Australia and
    2. School of Botany and Zoology, Faculty of Science, The Australian National University, Canberra ACT 2601 Australia
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  • THOMAS N. BUCKLEY,

    Corresponding author
    1. Environmental Biology Group and Cooperative Research Centre for Greenhouse Accounting, Research School of Biological Sciences, Institute of Advanced Studies, The Australian National University, Canberra ACT 2601 Australia and
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    • *

      Present address: Biology Department, Utah State University, Logan, UT 84322-5305, USA.

  • ADRIENNE B. NICOTRA,

    1. School of Botany and Zoology, Faculty of Science, The Australian National University, Canberra ACT 2601 Australia
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  • GRAHAM D. FARQUHAR

    1. Environmental Biology Group and Cooperative Research Centre for Greenhouse Accounting, Research School of Biological Sciences, Institute of Advanced Studies, The Australian National University, Canberra ACT 2601 Australia and
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Tom Buckley. Fax: +1 435 797 1575; e-mail: tom_buckley@alumni.jmu.edu

ABSTRACT

We examined the stomatal response to leaf excision in an evergreen woody shrub, Photinia × fraseri, using a novel combination of gas exchange, traditional water relations and modelling. Plants were kept outdoors in mild winter conditions (average daily temperature range: −1 to 12 °C) before being transferred to a glasshouse (temperature range: 20–30 °C) and allowed to acclimate for different periods before experiments. ‘Glasshouse plants’ were acclimated for at least 9 d, and ‘outdoor plants’ were acclimated for fewer than 3 d before laboratory gas exchange experiments. The transient stomatal opening response to leaf excision was roughly twice as long in outdoor plants as in glasshouse plants. To elucidate the reason for this difference, we inferred variables of stomatal water relations (epidermal and guard cell turgor pressures and guard cell osmotic pressure: Pe, Pg and πg, respectively) from stomatal conductance (gs) and bulk leaf water potential (ψl), using a hydromechanical model of gs. ψl was calculated from cumulative post-excision transpirational water loss using empirical relationships between ψl and relative water content obtained on similar leaves. Inferred Pg and Pe both declined immediately after leaf excision. Inferred πg also declined after a lag period. The kinetics of πg adjustment after the lag were similar in outdoors and glasshouse plants, but the lag period was much longer in outdoor plants. This suggests that the longer transient opening response in outdoor plants resulted from slower induction, not slower execution, of guard cell osmoregulation. We discuss the implications of our results for the mechanism of short-term stomatal responses to hydraulic perturbations, for dynamic modelling of gs and for leaf water status regulation.

INTRODUCTION

The control of gas exchange by leaf stomata has broad implications for the response of terrestrial vegetation to changes in environmental conditions, including global climate change (Hetherington & Woodward 2003). It is desirable to produce robust models of stomatal behaviour, ideally based on conserved physico-chemical mechanisms operating in and around stomatal guard cells. Great progress has been made in recent years in elucidating the signal transduction pathways by which guard cells respond to changes in light intensity, CO2 concentration and a variety of compounds such as abscisic acid (Assmann & Shimazaki 1999; McAinsh et al. 2000; Assmann & Wang 2001; Hetherington 2001; Schroeder, Kwak & Allen 2001; Zeiger et al. 2002; Dodd 2003; Hetherington & Woodward 2003; Vavasseur & Raghavendra 2005). Stomata also respond to short-term changes in hydraulic variables such as humidity (Cowan & Farquhar 1977; Mott & Parkhurst 1991; Cowan 1994; Monteith 1995; Oren et al. 1999), xylem hydraulic conductance (Saliendra, Sperry & Comstock 1995; Cochard et al. 2002; Brodribb & Holbrook 2003; Brodribb & Holbrook 2004) and soil water status (Raschke 1970; Fuchs & Livingston 1996; Comstock & Mencuccini 1998). However, there is still no consensus regarding the identity of the proximal effector(s) involved in stomatal responses to hydraulic perturbations, nor regarding the biophysical mechanisms by which those effectors induce changes in stomatal conductance (gs; see Table 1 for a list of symbols and units) (Buckley & Mott 2002b; Meinzer 2002; Franks 2004; Buckley 2005).

Table 1.  Symbols used in this paper
VariableSymbolUnits
Stomatal conductance (at excision, at peak of transient response)gs (gx, gp)mol m−2 s−1
Transpiration rateEmol m−2 s−1
Turgor pressure: epidermal guard cellPe, PgMPa
Osmotic pressure: guard cell, epidermal, saturated bulk leafπg, πe, πsMPa
Bulk leaf water potential (at excision, pre-dawn)ψl (ψx, ψpd)MPa
Turgor-conductance scaling factorχmol m−2 s−1 MPa−1
Net epidermal mechanical advantageM
Leaf relative water contentRWC
Leaf water content (at saturation, at excision)Q (Qs, Qx)mol
Fresh leaf weight (at saturation)FW (FWs)g
Dry leaf weightDWg
Pressure-chamber balance pressure readingPbMPa

Some observations suggest that gs is regulated by negative feedback from leaf water status. It is clear, for example, that gs tends to respond to variations in hydraulic supply and demand in a way that reduces the consequent change in bulk leaf water potential (ψl): gs declines to a new steady-state value when atmospheric humidity, soil water status or xylem hydraulic conductance are reduced. However, additional assumptions are required to make the ψl– gs feedback hypothesis consistent with what is known about stomatal hydromechanics. Stomatal aperture is determined not only by guard cell turgor pressure (Pg), which increases aperture, but also by epidermal turgor pressure (Pe), which reduces aperture. The effect of Pe is greater, so aperture increases if Pg and Pe decline by similar amounts (Sharpe, Wu & Spence 1987; Franks, Cowan & Farquhar 1998). The passive effect of water status on gs therefore produces positive, not negative, feedback. To produce negative feedback, Pg must be made more sensitive than Pe to hydraulic perturbations.

Two alternative hypotheses purport to explain how this happens. One holds that water lost directly from guard cells is replaced by flow through a large hydraulic resistance from epidermal cells to guard cells, which increases the passive sensitivity of Pg to changes in evaporative demand (Farquhar 1978; Maier-Maercker 1983; Dewar 1995; Dewar 2002; Eamus & Shanahan 2002). However, such a resistance would not cause Pg and Pe to differ in their sensitivities to perturbations upstream of the epidermis, such as changes in xylem hydraulic conductance or soil pressurization. The other hypothesis holds that epidermal water status is sensed by guard cells (via an unknown signal transduction process), which modulate their osmotic pressure (πg) in order to make steady-state πg proportional to epidermal water status (Darwin 1898; Darwin & Pertz 1911; Stålfelt 1929; Meidner 1986; Buckley, Mott & Farquhar 2003). Tracking of water status by πg would amplify the response of Pg to any change in hydraulic supply and demand in the epidermis.

It is not straightforward how best to evaluate these hypotheses and to work out their subtleties, because most of the variables of stomatal water relations are difficult to measure directly and to control independently by experiment. However, some insight may be gained by studying the transient ‘wrong-way’ response (WWR) that typically precedes, and is in the opposite direction to the steady-state response to hydraulic perturbations. WWRs often occur when any part of the soil—plant–atmosphere hydraulic flow continuum is perturbed, including changes in atmospheric humidity (Cowan & Farquhar 1977; Kappen, Andresen & Losch 1987; Grantz 1990), atmospheric pressure around the root system (Comstock & Mencuccini 1998) and transpiration rate (E) elsewhere in the same leaf or plant (Mott, Denne & Powell 1997; Buckley & Mott 2000), and in response to leaf excision (Darwin 1898; Iwanoff 1928; Rufelt 1963; Raschke 1970). The WWR is useful for studying the kinetics of guard cell osmoregulation because differences in the kinetic behaviours of water status and πg temporarily decouple gs from πg.

In the present study, we examined the stomatal response to leaf excision in an evergreen woody shrub, Photinia × fraseri. Preliminary work revealed a fortuitous discovery: that the duration of the WWR of gs to leaf excision was greater in plants that were kept outdoors in mild winter conditions than in plants that were kept in a glasshouse. The objective of this study was to determine the cause of these differences by characterizing the response kinetics of various stomatal water relations variables following leaf excision in these two groups of plants. To achieve this objective, we modified the protocol of Brodribb & Holbrook (2004), who estimated ψl over time after leaf excision, by applying pressure–volume curves to measurements of cumulative post-excision water loss (assessed by repeated weighing). In our version of the protocol, both water loss and gs were measured concurrently and with high temporal resolution (15 s) during the excision response, by enclosing leaves in a gas exchange chamber. The inference of ψl from water loss was validated by measuring ψl directly on leaves removed from the chamber at various times after excision. We applied time courses of gs and ψl to a simple model for gs to infer the dynamics of Pg, Pe and πg during the excision response.

MATERIALS AND METHODS

Plant material

All experiments used the woody evergreen shrub P. × fraseri (common names Photinia‘red tip’, ‘red robin’, or ‘superhedge’, a hybrid cross between P. glabra and P. serrulata, in the family Rosaceae). This species was chosen primarily because of its sturdy and long petioles, which facilitated the excision protocol and allowed for repeated pressure-chamber measurements. (Vicia faba L. seemed a logical choice initially, because parameters of stomatal hydromechanics have been estimated for that species, but its soft petioles proved not to be conducive to repeated pressure-chamber measurements.) P. × fraseri has alternate, finely serrated leaves and is characterized by flushes of new crimson foliage which turn deep green within a few weeks. Mature leaves are glossy, robust and hypostomatous. Eighteen-month-old plants were bought from a local nursery, kept in a semishaded outdoors location (7–17 °C day; −7 to 7 °C night) on the campus of the Australian National University, Canberra, between June and August 2004, and watered daily. Some plants were subsequently transferred into a glasshouse [daytime: 30 °C and 70% relative humidity (RH); night: 20 °C and 90% RH]. Plants used for experiments were grouped according to how long they had acclimated in the glasshouse before measurement: ‘outdoor plants’ were kept in the glasshouse three or fewer days before the experiments, and ‘glasshouse plants’ were kept in the glasshouse for at least 9 d before the experiments.

Leaf gas exchange

The experiments were performed using a laboratory-based open-flow gas exchange system described previously (e.g. Boyer, Wong & Farquhar 1997; Barbour et al. 2000). A single leaf was enclosed in a 22 cm × 18 cm chamber with a glass lid; air was stirred by a tangential fan to give a boundary layer conductance to water vapour of 5 mol m−2 s−1, and leaf temperature was held at 24 °C by circulating water from a water bath through a jacket under the chamber. Leaf temperature was measured with two copper–constantan thermocouples pressed against the lower surface of the leaf. Irradiance was provided by a metal-halide lamp. Compressed air was passed through soda lime columns to remove CO2, bubbled through a humidifier to saturate it with water vapour, passed through a temperature-controlled condenser to regulate inlet humidity, mixed with 12% CO2 in air using mass flow controllers, and passed through the chamber at a flow rate of 4 L min−1, monitored by a mass flowmeter (Brooks, Hatfield, PA, USA). Inlet CO2 concentration was kept near ambient (≈ 0.37 mg g−1). The CO2 partial pressures of incoming and outgoing air were measured with an infra-red gas analyser (IRGA; LI-6251; Li-Cor, Lincoln, NE, USA) operated in absolute mode and calibrated daily.

Previous experiments using this system measured vapour pressures of the incoming and outgoing air with an IRGA and alternated between measurements of incoming and outgoing air every 140 s. To permit data collection at shorter intervals, we measured vapour pressure with a Vaisala integrated RH and air temperature sensor (Humitter 50Y; Vaisala, Helinski, Finland) and switched from the alternating mode described above to ‘continuous’ mode (recording only the outgoing stream, but every 15 s) 30 min before excision. Subsequent calculations assumed the composition of incoming gas was constant; we checked for drift in the incoming stream by periodically switching back to the alternating mode. CO2 assimilation rate, transpiration rate (E) and gs were calculated from expressions given by von Caemmerer & Farquhar (1981).

Sample plants were brought to the laboratory on the afternoon prior to an experiment and kept well watered overnight. In the morning, a fully expanded, mature leaf from the 5th or 6th rank below the apex was sealed in the gas exchange chamber. Leaf-to-air vapour pressure difference (VPD) was set at 1–2 kPa (held constant during each experiment), and irradiance was increased in steps to 1000 µE m−2 s−1 between 0800 and 1000 h using neutral density filters. When gas exchange had reached steady-state, the petiole was excised close to the chamber, and the cut end was covered with parafilm. Gas exchange was measured continuously until stomatal closure occurred or until the leaf was removed to measure water potential (to validate the water potential inference method discussed below).

Magnitude and duration of transient opening response

To quantify aspects of the initial transient opening response of stomata to leaf excision, we first reduced high-frequency noise in the gs signal, using Gaussian smoothing in a moving 180 s window and a decay constant of 0.1 s−2, and then estimated the following parameters from the smoothed data: steady-state gs before excision (gx); gs at the peak of the transient response (gp); relative and absolute magnitude of the transient response [gp − gx and (gp– gx)/gx]; and duration of the transient response (time that gs > gx after excision). These parameters are illustrated in Fig. 1a .

Figure 1.

Measured time courses of stomatal conductance to water vapour (gs) after leaf excision, which occurred at time zero. (a) Two sample traces of absolute stomatal conductance: 25 June (an outdoor plant) and 01 July (a glasshouse plant). (b) All traces for outdoor plants, expressed relative to stomatal conductance at excision (gx). (c) All traces for glasshouse plants, expressed relative to gs at excision. In (b) and (c), the time courses from 25 June and 01 July are shown in bold.

Theory

We inferred the dynamics of stomatal water relations variables after leaf excision, using a theoretical framework based on several assumptions. First, ψl is an empirical function of leaf relative water content (RWC):

ψl = f(RWC).(1)

The function f is often taken as a composite of two lines, one of which applies above the point of turgor loss and the other below, and whose slopes are termed ‘capacitances’. In this study, we were less concerned with estimating capacitance values than with maximizing the empirical accuracy of the function f, so we used polynomial regressions instead. RWC is the ratio of leaf water content (Q) to saturated leaf water content (Qs). When a leaf is excised in air, Q changes at a rate equal to E: dQ/dt = –E(t), so Q(t) equals the water content at excision (Qx) minus the integral of E since excision:

image(2)

Equations 1 and 2 permit ψl to be estimated from E, which is measured by gas exchange. A third assumption was used to infer πg from gs: gs was assumed to be a linear combination of Pg and Pe, floored at zero (e.g. Sharpe et al. 1987):

gs = max{χ[Pg − (M + 1)Pe], 0},(3)

where M is the net mechanical advantage of the epidermis (M + 1 is conventionally denoted m and simply called the mechanical advantage), χ is a turgor-conductance scaling factor, and Pg and Pe are related to πg, ψl and πe by standard expressions of water relations:

Pe = max(ψl + πe, 0),(4)
Pg = max(ψl + πg, 0).(5)

Equation 4 assumes that the capacitor tissues represented by ψl are in close hydraulic contact with the epidermis (so that ψe is in quasi-static equilibrium with ψl). Similarly, Eqn 5 assumes that guard and epidermal cells are in quasi-static equilibrium with one another.

Inference of stomatal water relations

To infer the dynamics of πg during the excision response, we applied Eqns 1–5 to gas exchange measurements of E and gs as follows. Firstly, ψl was estimated from E using Eqns 1–2. Secondly, Pe was inferred from ψl using Eqn 4, in conjunction with a value for πe estimated from the pressure–volume analysis (discussed below). Thirdly, Pg was estimated from gs using Eqn 3, in conjunction with inferred values of Pe, an estimate for the quantity χM (discussed below), and a guess for the value of M (0.5, also discussed below). Finally, πg was calculated from Eqn 5 using estimated values of Pg and ψl.

The procedure described above requires estimates for several parameters: χ, M, πe, Qs, Qx, the polynomial coefficients in Eqn 1 and the value of ψl at leaf excision (ψx). Estimation of Qs, Qx, the polynomial coefficients, and ψx is discussed below under pressure–volume analysis. We assumed that πe is similar to bulk leaf osmotic pressure at saturation (πe ≈ πs). One constraint on the remaining parameters, χ and M, may be found by combining Eqns 3–5 to give

gs = χ[ − l + πg − (M + 1)πe],(6)

where Pe and Pg are understood to be non-negative. Immediately after excision, a decline in ψl and an increase in gs occur in concert before πg begins to change significantly. Hence, the product χM may be estimated from the initial slope of a phase plot of gs versus ψl after excision:

(∂gs/∂ψl)i = − χM,(7)

where the subscript ‘i’ denotes ‘initial’. We supplied the final constraint by making an arbitrary guess for the value of M. Most estimates of M in the literature are between 0.2 and 1.1, averaging around 0.6 (Meidner & Edwards 1975; Cooke et al. 1976; Edwards, Meidner & Sheriff 1976; Meidner & Bannister 1979; Buckley et al. 2003). Within this range, large M leads to large inferred Pg values (e.g. for M = 1.0, Pg inferred from our data is as large as 12.2 MPa). We therefore used a low value within this range (M = 0.5) for the simulations presented in Table 2 and Figs 2 and 3, and we quantified the sensitivity of our results to uncertainty in the value of M by repeating the simulations at different M-values between 0.3 and 1.0 (Table 3) .

Table 2.  Comparison of physiological measurements, properties of the wrong-way stomatal response to leaf excision (WWR), and inferred kinetic properties of guard cell osmotic pressure (πg) adjustment following leaf excision (assuming the net mechanical advantage, M, equals 0.5), for outdoor plants and glasshouse plants
VariableSymbolUnitsOutdoor plantsGlasshouse plantsP-value (notes)
  1. Notes: (a) two-tailed t-test assuming equal variances; (b) two-tailed t-test assuming unequal variances; (c) n = 8 and 12 for outdoor and glasshouse plants, respectively; (d) n = 7 and 8 for outdoor and glasshouse plants, respectively; (e) n = 4 for both outdoor and glasshouse plants.

Bulk leaf osmotic pressure at saturationπsMPa  1.00 ± 0.12  1.47 ± 0.122.4·10−7 (a,c)
Stomatal conductance at excisiongxmol m−2 s−1 0.092 ± 0.022 0.161 ± 0.0410.0016 (b,d)
Pre-dawn bulk leaf water potentialψpdMPa−0.231 ± 0.037−0.196 ± 0.0290.093 (b,c)
Duration of ‘wrong-way response’ (WWR)min 10.7 ± 1.4  5.4 ± 1.22.7·10−6 (a,d)
Relative size of WWR%   19 ± 10  7.9 ± 6.50.033 (b,d)
Absolute size of WWRmol m−2 s−1 0.017 ± 0.008 0.011 ± 0.0070.21 (a,d)
Time to 25% of πg decreaset25min 15.7 ± 3.2  8.2 ± 2.30.012 (b,e)
Time from 25 to 75% of πg decreaset(25−75)min  8.1 ± 2.4  8.7 ± 2.40.74 (a,e)
Time to 75% of πg decreaset75min 23.8 ± 2.8 16.7 ± 3.90.032 (b,e)
Figure 2.

Sample inferred time courses of water relations parameters (bulk leaf water potential, ψl; epidermal turgor pressure, Pe; guard cell turgor pressure, Pg; and guard cell osmotic pressure, πg) after leaf excision, for an outdoor plant (a, 25 June) and a glasshouse plant (b, 01 July). The vertical dotted line indicates the time of excision, and the horizontal dotted lines indicate the x-axis (y = zero).

Figure 3.

Inferred time courses of guard cell osmotic pressure (πg) expressed relative to the values of πg at the time of excision, with results compiled from all experiments on (a) outdoor plants and (b) glasshouse plants. The time courses from 25 June and 01 July are shown in bold for comparison with Figs 1 and 2.

Kinetic properties of πg adjustment were calculated from inferred πg time courses after Gaussian smoothing with a 220 s window and a decay constant of 0.01 s−2. Initial and final values of πg were calculated as the average of smoothed inferred values for 5 min before leaf excision and for 5 min prior to the end of the experiment, respectively.

Validation

The ψl inference method was validated against direct psychrometer measurements of ψl in eight experiments (three outdoor and five glasshouse plants), each terminated at a different time after excision. In each case, three leaf disks were taken from the sample leaf and equilibrated in separate psychrometer chambers for 16 h before measurement. (Psychrometer measurements are described below.) The method for inferring stomatal water relations parameters (Pe, Pg and πg) contains two major assumptions that we could not directly validate: that ψl and πs are representative of epidermal water potential and osmotic pressure, respectively, and that guard and epidermal cells are in quasi-static hydraulic equilibrium. These assumptions are evaluated in the Discussion.

Water relations parameters estimated by pressure–volume analysis

Parameters for pressure–volume curves (Eqn 1) and values of πs were estimated from the relationship between ψl and RWC as follows. Branches or leaves were excised underwater and rehydrated for up to 24 h to establish a water potential close to zero. A fully hydrated leaf was weighed to determine saturated fresh weight (FWs) and then placed in a pressure chamber (EMS, Santa Barbara, CA, USA) to estimate leaf balance pressure (Pb). Successive pairwise measurements of FW and Pb were acquired as the leaf slowly desiccated on the laboratory bench, until Pb exceeded the measuring range of the pressure chamber (−4.0 MPa). Leaves were then dried completely (verified by repeated weighing) to determine dry weight (DW). Absolute water content (Q) was calculated as Q = (FW − DW)/Mw (where Mw is the molar mass of water) and RWC was calculated as Q/Qs, where Qs is saturated water content [(FWs – DW)/Mw].

We compared the pressure chamber and psychrometer estimates of ψl by performing paired measurements on identical leaf samples across a range of RWC values. We found that Pb was linearly and reproducibly related to ψl (measured by the psychrometer), but with a slope far from unity: ψl = 0.6136(–Pb) + 0.0794 MPa (r2 = 0.9646, n = 17). The reason for the difference between –Pb and ψl is unclear. While some past comparisons of the two methods in the range of 0 to −2 MPa have found good agreement (Boyer 1967; Blum, Sullivan & Eastin 1973; Bennett, Cortes & Lorens 1986), others reported marked deviations (Kaufmann 1968; Barrs et al. 1970; Wilson et al. 1979; Turner, Spurway & Schulze 1984), and the validity of Pb as an estimate of ψl has been challenged on theoretical grounds (Canny & Roderick 2005; Roderick & Canny 2005). We feel that the psychrometer represents the more direct of the two measurements of water potential per se, so we converted all values of –Pb to ψl using the regression equation above. Pairwise measurements of RWC and ψl were grouped separately for outdoor plants and glasshouse plants, and polynomial functions (2nd- and 3rd-order, respectively) were fitted to each data set.

Bulk osmotic pressure at saturation (πs) was estimated for each sample leaf using the ‘osmotic line’ method of classical water relations (e.g. Tyree & Hammel 1972). There is no turgor pressure at low water potentials, so osmotic pressure (π) equals –ψl. However, π = nRT/V, where R is the gas constant, T is temperature, n is leaf osmotic content and V is the volume of water in the leaf, and V = VsRWC, where Vs is the leaf water volume at saturation. It follows that in the absence of turgor, –ψl = π = πs·(1/RWC). Thus, πs can be estimated from the slope of a line (the osmotic line) relating –ψl to 1/RWC at low ψl. To identify which data points should be included in the osmotic line for each leaf, we fitted lines to data subsets extending from the lowest measured ψl value to successively larger ψl values, and calculated πs as the average slope of all lines having r2 > 0.90. These πs estimates were validated against direct psychrometric measurements of πs on five leaves that had been frozen in liquid nitrogen to eliminate turgor; inferred values and direct measurements were not statistically different (Welch two-sample t-test assuming unequal variances, d.f. = 4, P = 0.94).

Water content at the time of excision (Qx) was calculated by adding the total water loss after of excision (determined from gas exchange) to the water content at the end of the experiment (determined from FW and DW). Sample leaves were rehydrated with the intent of calculating Qs; however, we found that complete rehydration was not possible for excised leaves that had been permitted to transpire for more than a few minutes. Thus, Qs was calculated on the basis of an assumed initial water potential at the time of excision (ψx), estimated for eight leaves that had been enclosed in the gas exchange chamber but removed at the time of excision. For three of these leaves, ψx was measured directly by thermocouple psychrometry (see below); the other five leaves were rehydrated (which was possible because these leaves were not significantly dehydrated) and ψx was estimated from Eqn 1. The average ψx was −0.42 ± 0.17 MPa. Initial relative water content was calculated from ψx and Eqn 1, permitting calculation of Qs from Qx.

Thermocouple psychrometry

Water potential measurements were made on leaf samples taken with a hole-punch, using a Wescor HR-33T Dew Point Microvoltmeter equipped with a C-52 Sample Chamber (Wescor, Logan, UT, USA), calibrated with a series of KCl solutions with osmotic pressures from 0.0 to 5.0 MPa. All measurements were made in hygrometric (automatic dew-point temperature depression) mode.

RESULTS

Water relations and gas exchange properties of outdoor and glasshouse plants

The measured relationships between RWC and ψl are shown in Fig. 4 for outdoor and glasshouse plants. The polynomial functions that best fitted these data were: ψl = 48.5·RWC2 − 74.8·RWC + 26.4 (r2 = 0.960, n = 58) for outdoor plants, and ψl = 83.5·RWC3 − 177.02·RWC2 + 125.08·RWC − 31.656 (r2 = 0.976, n = 177) for glasshouse plants.

Figure 4.

Relationship between leaf relative water content (RWC) and water potential (ψl) measured as described under pressure–volume analysis in the main text. Broken line and open symbols: glasshouse plants (acclimated in a glasshouse for ≥ 9 d prior to measurements). Solid line and closed symbols: outdoor plants (acclimated in a glasshouse for ≤ 3 d prior to measurements).

Bulk leaf osmotic pressure at saturation (πs, Table 2) was significantly lower in outdoor plants than in glasshouse plants (details given in Table 2). Outdoor plants also had lower steady-state stomatal conductance before leaf excision (gx, Table 2) than glasshouse plants. Pre-dawn water potential (ψpd, Table 2) was slightly but not significantly more negative in outdoor plants than in glasshouse plants.

Observed dynamics of gs following leaf excision

Stomatal conductance (gs) followed a similar qualitative trend after leaf excision in all experiments: gs initially increased, then decreased more substantially, and finally approached a minimum value close to zero after 2–4 h (Fig. 1). In some cases, the final approach towards zero was preceded by a single brief oscillation. Both the duration and the relative magnitude of the initial WWR of gs following leaf excision differed between outdoor and glasshouse plants, as is evident from inspection of the traces in Fig. 1 and from the compiled results of formal quantification of the WWR (Table 2). The WWR was longer and of larger relative magnitude in outdoor plants than in glasshouse plants, but the absolute magnitude of the WWR did not differ significantly between the two groups of plants (Table 2).

Inference of water potential from gas exchange

Bulk leaf water potential (ψl) was inferred from cumulative transpiration after leaf excision using Eqns 1 and 2, as described in Materials and methods. These inferences were validated against direct measurements of ψl on eight leaves removed from the chamber at a range of times following leaf excision in other experiments (of these eight leaves, three were from outdoor plants and five were from glasshouse plants). Inferred and measured ψl values were linearly related with a slope near unity [(inferred ψl) = 0.97 × (measured ψl) + 0.03 MPa; n = 8, r2 = 0.91; Fig. 5]. Inferred time courses of ψl are discussed below and presented in Fig. 2.

Figure 5.

Comparison of measured and inferred values of bulk leaf water potential (ψl) at the end of five experiments that were terminated early to validate the ψl inference technique. Measured values were acquired with a thermocouple psychrometer, and inferred values were acquired as described in the text. Inferred and measured ψl values were linearly related with a slope close to unity [(inferred ψl) = 0.97 × (measured ψl) + 0.03 MPa; n = 8, r2 = 0.91]. The regression line is solid; a dotted 1:1 line is shown for reference.

Estimation of χM

As described in Materials and methods and shown by Eqn 7, one constraint on the parameters in Eqn 3 (which relates gs to Pe and Pg) may be estimated from the initial slope of the trend between gs and water potential after leaf excision. Figure 6 presents a sample phase plot of gs versus ψl, showing the linear trend that obtains for a period after excision. The initial linear phase is also shown for all other excision experiments in which ψl could be estimated. The minimum, average, maximum and standard deviation of the slopes of these lines were −0.0151, −0.0186, −0.0225 and 0.0027 mol m−2 s−1 MPa−1, respectively. Using our estimate of 0.5 for M, this gives an average value for χ of 0.037 mol m−2 s−1 MPa−1.

Figure 6.

Phase plots of stomatal conductance (gs) and bulk leaf water potential (ψl) after leaf excision. The temporal sequence of points is from right to left. Data and linear regressions are shown for the initial linear phase of the gs versus ψl relationship for all excision experiments in which ψl could be estimated; the entire data time course is also shown for one experiment for reference (large open diamonds; for comparison with Figs 1 and 2, this is the 25 June experiment, which used an outdoor plant). The slope of the initial linear phase provides an estimate of the quantity (–χM), a product of two parameters. The average slope among these lines was − 0.0186 ± 0.0027 mol m−2 s−1 MPa−1.

Inferred dynamics of stomatal water relations after leaf excision

To generate hypotheses to explain why the WWR to leaf excision differed between outdoor and glasshouse plants, we inferred the dynamics of ψl, Pe, Pg and πg in the experiments described above. Figure 2 shows these inferred time courses for two sample excision experiments: one outdoor plant (25 June) and one glasshouse plant (01 July). In all cases, inferred Pe declined to zero in less than 8 min. Inferred ψl declined steadily, slowing gradually as stomatal closure reduced water loss. Inferred Pg also declined steadily after excision, generally following a roughly sigmoidal time course. The most distinct difference between outdoor and glasshouse plants, however, was the dynamic behaviour of πg after excision. The time that it took for πg to complete 25% and 75% of its eventual total decline (t25 and t75, respectively) was much longer in outdoor plants than in glasshouse plants, but the time required for the middle 50% of the πg decline (t(25−75) = t75 − t25) was similar in both groups of plants (Table 2). All of the inferred πg time courses are compiled and presented on a relative basis in Fig. 3 to permit comparison between outdoor and glasshouse plants.

Sensitivity analysis for the parameter M

The results described above were based on an arbitrarily assumed value of 0.5 for the parameter M, the net mechanical advantage of the epidermis. To assess the effect of uncertainty in M, we repeated all simulations for M, which ranged from 0.3 to 1.0 (mod 0.1). The qualitative results (Table 3) were unaffected: outdoor plants had a longer lag time for, but not a slower rate of πg adjustment. However, the maximum inferred values of Pg increased strongly at higher M, from a mean of 5.7 ± 0.9 MPa to 10.4 ± 1.7 MPa for glasshouse plants. The fact that such large Pg values have never been observed, to our knowledge, might suggest that M in P. × fraseri is closer to the low end of this range.

Table 3.  Sensitivity of inferred results to the value of the unknown parameter M (net mechanical advantage, unitless), which was assumed equal to 0.5 for the simulations described in Table 2 and shown in Figs 2 and 3. ‘od’ and ‘gh’ refer to outdoor plants and glasshouse plants, respectively. Sensitivities are shown for the maximum inferred value of guard cell turgor pressure (Pg) during the experiment, and for three parameters describing the kinetics of adjustment in guard cell osmotic pressure (πg) following excision: t25, t(25−75) and t75, the time required for πg to complete the first 25%, the middle 50%, and the first 75% of its eventual total decline
 M0.30.40.50.60.70.80.91.0
max Pg/MPaod 2.1 ± 0.2 2.6 ± 0.33.1 ± 0.4 3.6 ± 0.4 4.1 ± 0.5 4.5 ± 0.6 5.0 ± 0.6 5.5 ± 0.7
gh 3.9 ± 0.5 4.8 ± 0.75.7 ± 0.9 6.6 ± 1.0 7.6 ± 1.2 8.5 ± 1.4 9.4 ± 1.510.4 ± 1.7
t25 min−1od20.1 ± 3.917.6 ± 4.215.7 ± 3.214.9 ± 2.914.4 ± 2.714.1 ± 2.613.9 ± 2.513.7 ± 2.5
gh 8.3 ± 2.8 8.2 ± 2.68.2 ± 2.3 8.2 ± 2.2 8.1 ± 2.1 8.1 ± 2.1 8.0 ± 2.0 8.0 ± 2.0
t(25−75) min−1od 6.2 ± 2.5 7.5 ± 2.58.1 ± 2.4 8.3 ± 2.4 8.5 ± 2.4 8.6 ± 2.4 8.5 ± 2.3 8.7 ± 2.3
gh 8.4 ± 2.7 8.6 ± 2.68.7 ± 2.4 8.6 ± 2.4 8.6 ± 2.2 8.6 ± 2.2 8.7 ± 2.1 8.7 ± 2.1
t75 min−1od26.3 ± 3.325.2 ± 3.323.8 ± 2.823.2 ± 2.722.9 ± 2.622.7 ± 2.722.4 ± 2.722.4 ± 2.6
gh16.7 ± 4.616.7 ± 4.216.7 ± 3.916.7 ± 3.816.7 ± 3.616.7 ± 3.616.7 ± 3.516.7 ± 3.5

DISCUSSION

This study examined pressure–volume relations and stomatal responses to leaf excision in an evergreen shrub (P. × fraseri) and compared the behaviour of plants that had been kept in a glasshouse for at least 9 d before measurement (‘glasshouse’ plants) or had been kept outdoors and transferred to a glasshouse three or fewer days before measurement (‘outdoor’ plants). A transient opening response (WWR) was always observed for gs following leaf excision. That is, gs initially increased after excision before subsequently declining towards zero (Fig. 1). The initial trend between measured gs and inferred ψl after leaf excision was linear and had a conserved, negative slope (Fig. 6). Furthermore, in all experiments, inferred values of πg eventually began to decline exponentially after a variable lag period, whereas Pg and Pe declined immediately after excision. Together, these results are consistent with the hypothesis that stomatal responses to leaf excision involve two distinct phases: an initial ‘hydropassive’ phase during which πg is constant or changes only passively (as a result of water loss from guard cells), followed by a ‘hydroactive’ phase that involves metabolic reduction of πg (Darwin 1898; Darwin & Pertz 1911; Stålfelt 1929; Ehret & Boyer 1979; Meidner 1986; Buckley et al. 2003).

The duration of the WWR was substantially longer in outdoor plants than in glasshouse plants (Fig. 1). Our analysis of the dynamics of stomatal water relations during these excision responses suggests that the lag period between the time of leaf excision and the time when the hydroactive response of πg begins in earnest was much longer in outdoor plants, but that the halftime for the subsequent exponential decline in πg did not differ between outdoor and glasshouse plants. This suggests that the induction, rather than the execution, of guard cell osmoregulation is slower in the outdoor plants. In contrast, most dynamic models of gs have predicted WWRs and stomatal oscillations by assuming a longer time constant for πg adjustment than for passive hydraulic adjustments (Rand et al. 1981; Haefner, Buckley & Mott 1997; Jarvis et al. 1999), as opposed to a long lag time for πg. Cowan & Farquhar (1977) introduced a lag time but did not discuss whether or how it affected the predicted dynamics of gs. It is unclear what effect the distinction between a slow time constant and a long lag may have on the stability of stomatal control, but the matter would appear to warrant further study, and future efforts to model gs dynamically should consider this distinction.

An alternative view on stomatal hydraulics holds that a large water potential gradient exists between guard and epidermal cells. According to that view, M is overcome – and right-way hydraulic responses are achieved – by changes in the magnitude of this gradient, not by active adjustment of πg in relation to local water status (any change in πg is strictly passive). There are numerous arguments against this hypothesis (Buckley 2005). One is that it is difficult to explain the archetypal two-phase response to hydraulic perturbations with this hypothesis. The correct pattern would result if Pg responded very slowly to changes in ψl, but our analysis indicated that both Pe and Pg began to decline immediately after excision (Fig. 2). Furthermore, for this hypothesis to explain the observed decline in gs to near zero without active down-regulation of πg, guard cell water potential would have to decline during the excision response by an amount approaching the initial magnitude of πg. That magnitude, however, was generally much greater than the magnitude of ψl late in the excision response (≈ 3–6 MPa for πg versus 2–3 MPa for ψl; see Fig. 2). This generates a contradiction: guard cell water potential first lags behind the decline in ψl because guard cells are downstream from the bulk of leaf tissue in the transpiration stream, but guard cell water potential later overtakes ψl despite this fact.

A related study (Buckley & Mott 2002a) combined a model with stomatal aperture time courses measured with a microscope to infer the dynamics of πg for single pairs of guard cells during humidity responses. That study also inferred values for the effective resistance between guard and epidermal cells needed to explain the observed stomatal responses if πg were assumed constant. The inferred resistance changed dramatically during the humidity responses – first decreasing, then increasing and finally stabilizing at a value that was larger than the initial value in some cases and smaller in others. In contrast, πg varied monotonically in time during the humidity response, and with humidity in the steady-state. The authors concluded that πg regulation was a more parsimonious explanation than a varying water potential gradient between guard and epidermal cells for the observed responses. Assmann & Gershenson (1991) likewise concluded that an exponential decay model suggesting metabolic adjustment of πg best described the kinetics of stomatal adjustment to changes in VPD, and Grantz & Zeiger (1986) found that the humidity response was kinetically similar to the light response, which is known to involve πg adjustment.

Ecological implications of variable WWR kinetics

Other experiments have also found a large degree of variation in the kinetics of stomatal responses to light, which are known to involve guard cell osmotic adjustment (Woods & Turner 1971; Saxe 1979; Kirschbaum, Gross & Pearcy 1988; Meidner 1990; Tinoco-Ojanguren & Pearcy 1992; Mott, Shope & Buckley 1999; Buckley & Mott 2000). It is well established that the rate of stomatal opening can, in many conditions, be the dominant limitation on photosynthetic induction in light flecks. Allen & Pearcy (2000a,b) found that photosynthetic induction was slower at lower initial conductances (pre-light fleck). When initial conductance was high, gs began to increase almost immediately after illumination, but when initial gs was low, the stomatal response was preceded by a lag time on the order of 5 min (e.g. figure 1b & e in Allen & Pearcy 2000a). However, most of this trend occurred across a narrow range of quite low initial gs values, so the lag may have resulted from parts of the leaf still having been in the ‘Spannungsphase’– the period during which πg and Pg have begun to increase after illumination, but before Pg has increased enough to overcome epidermal backpressure (Stålfelt 1929). The variable lag times reported in the present study are unrelated to the Spannungsphase, because they preceded stomatal closure, not opening. Nonetheless, they may influence carbon–water balance: to the extent that photosynthesis remains induced during dark periods, rapid stomatal closure after light flecks can be undesirable (for review, see Pearcy et al. 1994).

Our analysis suggested that Pe declined to zero in less than 8 min after leaf excision in all cases. It is unclear whether loss of epidermal turgor would have occurred in response to a more moderate and repeatable hydraulic insult, such as xylem cavitation or a change in ambient humidity. The available data do suggest steady-state that Pe can decline dramatically – by nearly two-thirds in some cases – across a physiological range of evaporative gradient (Shackel & Brinkmann 1985; Nonami, Schulze & Ziegler 1990; Mott & Franks 2001), and is it likely that Pe declines further still while E is elevated during the WWR following a reduction in humidity. Klein et al. (1996) concluded that Pe was close to zero throughout the day in V. faba. If, as suggested by those results and by our data, the water lost during WWRs is of the same order as the leaf's initial water content, then variations in WWR duration may determine whether loss of epidermal turgor occurs in the course of normal leaf functioning. This possibility is supported by data of Brodribb & Holbrook (2003), who found that stomata remained open at bulk leaf water potentials low enough to cause leaf turgor loss, and that substantially lower ψl was required to induce total stomatal closure.

Cowan (1972) has discussed the possibility that the dual-feedback control mechanism believed to underlie both WWRs and stomatal oscillations – positive feedback from passive water loss and negative feedback from guard cell osmoregulation – might serve an adaptive function by exploring the space of possible steady-states in order to find the optimal state (i.e. that which balances water loss and carbon gain with diurnally varying conditions as needed to maximize daily carbon gain for the available transpirable water supply; Cowan & Farquhar 1977). Furthermore, because WWRs and oscillations allow ψl transiently to decline farther than it would in the steady-state, they may also permit ψl to cross the cavitation threshold transiently. The resulting reduction in xylem hydraulic conductance can provide a kind of feedforward control (Oren et al. 1999; Buckley & Mott 2002b) by informing stomata of the proximity of the cavitation threshold. Variation in WWR length could therefore help to explain ‘apparent feedforward’ and isohydric behaviour (Buckley 2005). It may also play a role in defining cavitation safety margins, because the magnitude of the transient deviation of ψl below steady-state should depend on the duration of WWRs. Finally, because the relative time constants for hydraulic and osmotic adjustments are major determinants of the tendency for gs to oscillate (Cowan 1972; Farquhar & Cowan 1974; Cowan & Farquhar 1977; Rand et al. 1981), osmoregulatory lag time should affect the stability of the stomatal control system. The variation reported here in lag time could therefore also help to explain why oscillations and patchy gs are so difficult to replicate in different leaves despite similar experimental conditions (Mott & Buckley 2000).

Assumptions of the analysis

Our procedure for inferring πg assumed epidermal and guard cells were hydraulically quasi-static with respect to ψl and to one another. That is, changes in bulk leaf, epidermal and guard cell water potentials occurred simultaneously. It is therefore possible that the inferred delay in πg adjustment was not caused by a delayed metabolic response to a change in water status, but instead by a delayed response of epidermal water status to the change in bulk leaf water status. The fact that gs began to increase immediately after excision implies that Pe declined immediately, consistent with quasi-stasis between Pe and ψl, at least on the time scale of our measurements (≈ 15 s). Our data do not rule out the possibility that guard cell water potential and turgor (Pg) respond slowly. However, there are two reasons to doubt this. First, Pg was inferred from gs and Pe using Eqn 3, which contains no assumptions about guard cell hydraulic kinetics, and these inferred Pg values also began to decline immediately after leaf excision. Second, recent experiments found that the halftime for adjustment of guard cell volume following a change in local water potential in epidermal peels of V. faba was typically much less than 1 min, unless the peels were pre-treated with membrane trafficking inhibitors (J.C. Shope and K.A. Mott, unpublished results).

Two other untested assumptions of our analysis are that πe is similar to πs, and that πe is constant during the excision response. The latter assumption is supported by earlier experiments in which πe was found to vary only slightly with E (Meidner & Edwards 1975; Frensch & Schulze 1988; Nonami et al. 1990). The former assumption, however, is neither supported nor refuted by any evidence of which we are aware. If πe and πs differ, it seems more likely that πe < πs, because mesophyll cells comprise a large fraction of the total cellular volume in most broad leaves and they often contain substantial stores of osmotically active photosynthate.

It bears mentioning that one curious feature of our results could be explained by a failure of the assumption that πs is a reliable proxy for πe. Whereas inferred values of πg remained roughly constant during the lag period after excision in glasshouse plants, inferred πg increased substantially during this period in outdoor plants (Fig. 3). Mathematically, this increase can be explained by the fact that inferred Pe declined to zero long before the WWR had ended, requiring elevated πg to explain the still-elevated gs. If, however, we delayed the loss of epidermal turgor in the analysis by assuming πe = 1.5 MPa for outdoor plants – instead of 1.0 MPa, the average value of πs measured in outdoor plants – then the inferred increase in πg was greatly reduced or eliminated in all cases (not shown). It is also possible that the inferred πg increase was real and resulted from the concentration of solutes in the guard cell because of volume loss before the induction of active osmotic efflux. We are unable to distinguish these alternatives on the basis of our data.

ACKNOWLEDGMENTS

We thank S. Chin Wong for technical assistance, Keith Mott for comments, and Susanne von Caemmerer, Missy Holbrook and two anonymous referees for thoughtful suggestions on earlier versions of this work. This research was supported by the Cooperative Research Centre for Greenhouse Accounting at the Research School of Biological Sciences of the Australian National University, Canberra.

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