The permeability of leaf tissue to water has been reported to increase under illumination, a response reputed to involve aquaporins. We studied this ‘light response’ in red oak (Quercus rubra L.), the species in which the phenomenon was first detected during measurements of leaf hydraulic conductance with the high-pressure flow meter (HPFM). In our HPFM measurements, we found that pre-conditioning leaves in darkness was not sufficient to bring them to their minimum conductance, which was attained only after an hour of submersion and pressurization. However, pre-conditioning leaves under anoxic conditions resulted in an immediate reduction in conductance. Leaves light- and dark-acclimated while on the tree showed no differences in the time course of HPFM measurement under illumination. We also studied the effect of light level and anoxia on rehydration kinetics, finding that anoxia slowed rehydration, but light had no effect either in the lab (rehydration under low light, high humidity) or on the tree (acclimation under high light, 10 min of dark prior to rehydration). We conclude that the declines in conductance observed in the HPFM must involve a resistance downstream of the extracellular air space, and that in red oak the hydraulic conductivity of leaf tissue is insensitive to light.
A number of studies have reported light induced changes in the hydraulic conductance of leaves (Sack et al. 2002; Tyree et al. 2005; Cochard et al. 2007; Kim & Steudle 2007), a response widely hypothesized to arise from light induced changes in plant membrane water channel (aquaporin) activity and density. Still, the phenomenon and its physical basis remains poorly understood. For example, light effects reported from the high-pressure flow meter (HPFM) (Sack et al. 2002; Tyree et al. 2005; Cochard et al. 2007) are typically much greater than those observed with other methods (Kim & Steudle 2007; Scoffoni et al. 2008), while other studies have failed to find a link between aquaporin expression and apparent changes in leaf conductance to liquid water (Voicu, Cooke & Zwiazek 2009), or found declines in permeability at light levels approaching full sunlight (Kim & Steudle 2009). In addition, we note that the phenomenon is better described as a ‘dark response’, as, in general, the difference between ‘non light-responding’ species and ‘responding species’ depends on differences in the values of conductances under the dark rather than light treatments (Sack et al. 2002; Scoffoni et al. 2008). An exception to this pattern is seen in the comparison of Acer saccharum (small to no response) and Quercus rubra (large response) in Tyree et al. (2005), in which the dark conductances of the two species appear identical. However, in that study, the conductance of A. saccharum was half the value reported in Sack et al. (2002) for this species, and the pattern of large light responses being driven by low dark conductances relative to non- or low-responders held for the remaining three temperate and six tropical species reported. Thus, what needs to be explained is why, for some species, hydraulic conductance appears to decline in the dark.
We undertook an intensive study of the dark response of Q. rubra, the species for which light level effects were first reported based on HPFM measurements of leaf conductance (Sack et al. 2002; Tyree et al. 2005). Our primary strategy was to use pre-treatments of different light levels for periods long enough to saturate reported light responses, and then test the effects of these treatments using both HPFM and transient rehydration measurements of leaf permeability to water. In addition, we carried out experiments to determine whether anoxic conditions depress leaf permeability, motivated by the observation that flooding, and the anoxic conditions that follow, results in declines in root cell permeability (Tournaire-Roux et al. 2003). In roots, the declines in conductance appear to result from a pH shock arising from a lack of ATP to drive proton pumps (Tournaire-Roux et al. 2003). While the response of leaves to anoxia has not, to our knowledge, been previously investigated, in tissues bearing chloroplasts light has the potential to mitigate the metabolic effects of anoxia through the production of O2 by PSII, as well as by directly driving ATP synthesis in PSI (Nobel 2005). We also imaged the stomata of leaves frozen under different experimental conditions with cryo-SEM to assess an alternative hypothesis, originally put forth by Sack et al. (2002), that the observed light sensitivity derives from the action of well-described light actuated valves known to exist in leaves: the stomata.
At the core of this study is the comparison between the light sensitivity of water potential relaxation times defined by rehydration kinetics and the whole leaf conductance as defined by the HPFM. As the flow path measured by the HPFM includes the stomata, but that measured by rehydration kinetics does not, we reasoned that a comparison of the two methods would either implicate or exonerate stomata in the light sensitivity observed in HPFM measurements. That is, if the light sensitivity observed in the HPFM derives from changes in membrane permeability, we would expect to see similar degrees of light sensitivity in whole-leaf water potential relaxation times. The latter expectation, that relaxation times for leaves are sensitive to membrane properties, arises from the observation that (at least for angiosperms) the resistivity of the tissue dominates that of the xylem (Cochard, Nardini & Coll 2004), and that change in leaf water content (capacitance) is dominated by the gain and loss of volume by living cells (Kramer & Boyer 1995).
The utility of rehydration kinetics is that as long as the pre- and post-rehydration potential measurements are made at equilibrium, and the experiments are conducted over a range of water contents where the capacitance of the tissue and conductivity of the xylem may be treated as constant, the ratio of ‘final’ post-hydration whole-leaf water potential to ‘initial’ pre-hydration water potential provides a consistent, well-defined measure of treatment effects on tissue transport properties. We leave the question of how one may extract effective tissue material properties from rehydration measurements on leaves to forthcoming work, but note that the general problem of modelling the rehydration kinetics of homogeneous tissues based on cell-level material properties has been previously discussed (Philip 1958a,b; Molz 1976).
Finally, we investigated the phenomenon of light and dark effects seen with the HPFM, and not other methods, as it is specifically with the HPFM that these effects have been shown to occur for red oak (Sack et al. 2002; Tyree et al. 2005). However, we believe the results are of general interest in that they sound a note of caution as to whether reported effects of light level on various measures of liquid phase transport constitute a single coherent phenomenon.
MATERIALS AND METHODS
Experiments were conducted from July through September from 2006 through 2009, on five red oak (Q. rubra L.) trees on the campus of the Harvard Divinity School, Cambridge MA, USA, and three at Harvard Forest, Petersham, MA, USA.
High pressure flow meter (HPFM)
The HPFM was constructed according to the description in Sack (Sack et al. 2002), with the modifications described in further discussion. Degassed water (stirred for 3 h under vacuum) was supplied from a 1 L acrylic reservoir pressurized to 0.2 MPa. A balloon at the interface helped to slow the diffusion of gases into the degassed water. Bev-a-line 1/4″ ID tubing supplied the pressurized, degassed water to two lines, each consisting of two parallel paths connected by polyether ether ketone (PEEK) valves (Upchurch Scientific, Oak Harbor, WA, USA), a PEEK tubing high resistance pathway (1 m black PEEK, 0.004 ID) and a low resistance bypass (tan PEEK, 0.055 ID). Rigid PEEK tubing was used to minimize the compliance of the measurement system. Prior to each experiment, we checked the time constant for the response to pressurization of the HPFM through the PEEK resistor, absent a leaf, and verified that it was less than 5 s. The capacity of the delivery system was designed to be sufficiently high that open flow in one line did not result in a drop in delivery pressure in the parallel line, and therefore allowed two leaves to be run in parallel without affecting each other.
The principle underlying the measurement is simple: since in steady-state, the flux through the system must be equivalent everywhere, an unknown conductance may be calculated from knowledge of the pressure drop across a known conductor somewhere in the system and the pressure drop across the unknown conductor. For each measurement line, the pressure drop across the PEEK resistor was read by a pressure transducer (PX26-030GV, Omega Engineering, Inc.), allowing the conductance per unit leaf area, H, to be calculated as
where the ΔPs refer to the pressure drop across the PEEK resistor and the leaf, respectively, and Hpeek is the conductance of the PEEK resistor. The former quantity is the difference between the driving pressure source (compressed air applying 0.2 MPa to the degassed water column through the balloon) and the pressure recorded by a transducer downstream of the known conductor. The latter represents the difference of the same transducer and the pressure outside the submerged leaf, taken to be atmospheric pressure (Sack et al. 2002), which neglects the small contribution from the weight of the fluid in the bath. The symbol H is used, rather than Kleaf, in order to refer explicitly to the conductance defined by the HPFM, and avoid confusion with other methods and definitions of effective whole-leaf conductances. Data from the pressure transducers and a thermocouple in the experimental bath were collected by a USB DAQ board (USB 6009, National Instruments, Austin, TX, USA), logged at 1 Hz, and monitored in real time using the software Labview 7.0 (National Instruments). Immersed leaf temperatures were maintained by circulating water from a water bath (VWR model 1157) through stainless steel coils submerged in the experimental bath, and kept to within ±1 degree of 22 °C. Ambient air temperature ranged from 21 to 24 °C, and Hpeek was corrected for temperature dependent changes in viscosity. The experimental bath was aerated with a flow of one LPM, which also provided enough stirring to keep temperatures in the bath uniform. Light was provided by a Sylvania mgc1000 ballast with an S52 high-pressure sodium bulb (GTE Products, Fall River MA, USA). Leaf areas were either measured with a LI-3100c (Li-Cor, Lincoln NE, USA) or, where leaves were sampled for cryo-SEM, calculated from digital photographs using ImageJ (NIH) software.
If the light response reported for HPFM experiments also affects the hydraulic conductivity of leaf tissue in nature, we would expect that after controlling the light level experienced by leaves either in the lab or still on the tree during a pre-treatment period sufficiently long to saturate the expected effect, subsequent measurement with the HPFM should find the leaves operating at their minimum or maximum H. To test whether H responds to anoxia, we also employed pre-treatments with air versus N2.
Pre-conditioning on the tree: light levels
Dark conditions were imposed on leaves still on the tree using tubes made of black plastic bags, surrounded by tin foil. The tubes were folded closed around the petiole at the top, but open at the bottom to allow free gas exchange; the tubes extended ≈ 15 cm below the leaf, far enough that light levels in the tube, checked with a LI-190 Quantum sensor (Li-Cor), were <1 µmol m−2 s−1 PPFD. Leaves were covered for 2 h in the morning from approximately 0900 to 1100 h, before harvesting and transport to the HPFM. All leaves were harvested, still covered, by cutting the petioles under water with a razor blade. Leaves under ambient light (>600 µmol m−2 s−1 PPFD) were harvested from nearby shoots. Light and dark adapted leaves were connected to the HPFM under treatment light conditions, a process that took about 10 min, and then measured under ≈1000 µmol m−2 s−1 PPFD.
Pre-conditioning experiments in the lab: light and oxygen
In the pre-treatment experiments, paired leaves were harvested between 1000 and 1100 h, cut under water simultaneously from a single shoot, and connected to the HPFM. The leaves were not pressurized, but were able to draw degassed 1 mM KCL, filtered to 0.2 µm, freely from a reservoir vented to ambient atmospheric pressure. One litre conical flasks were fitted around each of the leaves, and gases delivered to the flasks through a second tube through a stopper; a third tube provided a vent to atmospheric pressure, which remained under water to slow back diffusion into the flask from ambient air. Gases (air as a control, versus N2 or a CO2-free 79% N2, 21% O2 mix as the experimental treatment) were then delivered at a rate of 1 L per minute (LPM), bubbled through 10 mL of water in the bottom of the flask. Relative humidity (RH) in the flasks in the dark ranged from 75 to 79%, and 85 to 89% in the light, as measured by diverting a 200–300 cm3 min−1 air stream from a subset of flasks into the cuvette of a LI-1600 porometer (Li-Cor). Oxygen levels in the flasks were checked through the use of a fluorescent probe (Foxy, Ocean Optics, Dunedin, FL, USA), and were non-detectable in under two minutes of gas flow in the N2 treatments. The flasks were then submerged in the experimental bath under either ≈1000 (hereafter light) or <1 (dark) µmol m−2 s−1 PPFD, for a pre-treatment period of three hours. Leaf temperatures in the light were monitored with a thermocouple attached with surgical tape to the underside of the leaf, and did not exceed 26 °C. After 3 h, the flasks were pulled off, the leaves immersed and the delivery lines to the leaves pressurized to 0.2 MPa to begin measurement by the HPFM. Initial light conditions during measurement were the same as pre-treatment conditions in all experiments; once a stable minimum or maximum was achieved, the light was switched on or off, and the response to the opposite light regime recorded.
Three different experiments were performed on paired leaves: (1) pre-conditioning with N2 versus air in the dark; (2) pre-conditioning with N2 versus air in the light; and (3) pre-conditioning with CO2-free air versus air in the light. Note that the key aspect of these pre-treatment experiments was that the leaves had 3 h to acclimate to the experimental light levels prior to their actual submersion and pressurization, the requisite conditions for measurement with the HPFM. This design was adopted to study the effect on H of the HPFM measurement conditions themselves, holding light level constant.
Response to light quantity
Leaves were harvested and fitted into the HPFM in the dark, and allowed to reach their dark minimum conductance, after about 50 min. The light was switched on, and stepped up through 10, 50, 300, 500 and 1000 µmol m−2 s−1 PPFD at the leaf surface by removing a sequence of filters constructed of plastic mesh shade cloth and translucent film. At each level, the response was allowed to saturate before the next increase.
In addition to the HPFM experiments, rehydration experiments were conducted to test whether a flow path through the tissue that does not include the stomata would show any sensitivity to light. Two types of light experiments were conducted, one in the lab and one on the tree. In the lab, experiments were conducted to test light effects where acclimation and rehydration occurred under uninterrupted treatment light levels. As this approach was limited to 50 µmol m−2 s−1 PPFD, a second experiment was developed on intact trees, which tested the residual effect of ambient light conditions on fully exposed leaves of the exterior canopy after a period of 10 min in darkness, required to infer the pre-rehydration potential of the leaf. In addition, the effect of anoxia and darkness on relaxation time was assayed in the lab.
As noted in the introduction, the question of an appropriate model for calculating tissue conductivity from leaf rehydration data is addressed elsewhere (Rockwell 2010). In general, we may expect whole-leaf potential relaxation, Ψ = ψf/ψo, to be a function of the characteristic time of the tissue, τ = L2c/k, where L is a length that characterizes the tissue, k is a conductivity with units of mol m−1 Pa−1 s−1, c the volumetric capacitance of the tissue in mol m−3 Pa−1, and the precise functional form depends on the boundary conditions and geometry of the tissue (Philip 1958b). Note that when capacitance is normalized to leaf area a, and L is taken as the half-thickness of a leaf, τ is given by Lca/(2k), where 2k/L defines a quantity dimensionally consistent with the leaf conductance K typically reported when single exponential kinetics are assumed. Here, we present the relaxation data as simply ψf/ψo, or final potential normalized by initial potential, rather than as derived parameters such as k or K, as these add no further information for the purposes of judging treatment effects. Variation in individual leaf capacitances (the change in water content for a change in potential) will create some scatter in the data even if conductivity is uniform within the experimental population. However, since all experiments were done on the same branches, we expect that all treatment groups were drawn from the same population of leaves, such that differences in capacitance were randomly distributed. In the case of the anoxia experiments, individual pressure-volume curves of four to five points over the range of rehydration were constructed for each leaf, to check for any persistent effects of anoxia on leaf capacitance following rehydration.
The relaxation of negative leaf water potentials toward equilibrium with a reservoir of water at ψr = 0 (henceforth, two-point rehydrations) was studied in the following manner. Branches were cut from a tree in the early morning, prior to direct illumination, bagged and brought into the lab. A leaf was cut from the branch to furnish a rough estimate of leaf water potential, and the branch was allowed to slowly dry over about an hour to the desired initial potential. Very hydrated branches were placed for short periods (≈10 min) in higher light near a window to bring them to the target potential, ≈−1.2 to −1.4 MPa; this potential was chosen such that rehydration would then occur over a highly linear range of the pressure volume curve, well above the turgor loss point at ≈−2.4 MPa (Fig. 1). Each point in the pressure-volume diagram was determined as follows. After weighing, a leaf was enclosed in a plastic bag, pre-humidified with a breath and closed around the petiole with a rubber band. The walls of the pressure chamber were lined with moistened paper towel to further limit evaporative losses during measurement. For 21 such measurements, weights were checked following removal of the leaf from the bomb and indicated an average loss of 7.8 × 10−4g, equivalent to a change of 0.013 MPa. There was no trend between the amount of water lost and pressure measured, indicating that the losses most likely occurred as evaporation from the cut surface following pressure determination, and were not a function of time in the chamber. Therefore, we regarded the balancing pressure as occurring at the water content given by the initial weight alone.
All changes in leaf weight were assumed to represent evaporative losses of water. Leaf weight was expressed as equivalent moles of water (FW = 18.02 gram/mole) Na, normalized to leaf area as determined by a LI-3100c leaf area meter (Li-Cor), such that the slope of the pressure–volume (PV) relationship represented the change in moles of water per change in Pa of balancing pressure, per meter squared of leaf area. Between measurements, leaves were placed in unsealed ziplock bags and kept in less than one µmol m−2 s−1 PPFD, which resulted in rates of potential change sufficiently slow (≈0.2 MPa per hour) to regard the measurements as representative of the equilibrium PV relationship.
In preliminary tests, no depression of rehydration was observed in leaves until initial potentials fell below −2.1 MPa, which corresponds to the threshold for reductions in conductance previously observed for red oak, according to a vulnerability curve constructed with the method of Brodribb & Holbrook (2003) (unpublished data, N.M. Holbrook, M.A. Zwieniecki). For 132 leaves from eight trees hydrated for between 25 and 30 s in 2007 and 2008, the degree of rehydration (Ψ = ψf/ψo) was found to be independent of initial potential over the range tested, −0.8 to −1.6 MPa (Fig. 2). Therefore, we do not expect initial water stress to be a significant factor influencing potential relaxation in these experiments.
Once at the target potential, each ‘whorl’ of leaves (here defined for convenience as the terminal cluster of leaves on a stem all produced by a single meristem, although the phyllotaxy is not whorled) was enclosed within a heavy weight black plastic bag lined with a damp paper towel to create a local environment of high humidity and low light for at least 2 h to allow equilibration among leaves. Branches were then fitted into 27″ × 27″ glove bags (Glas-Col, Terre Haute, IN, USA), with an input saturated atmosphere bubbled through a flask of water at a rate of 2 L per minute; an output tube for the controlled atmosphere bubbled through another flask. Resistance to the efflux of gas was sufficient that the glove bags remained inflated. A humidity probe (Mannix SAM990DW, General tools, New York, NY, USA) inside each glove bag was checked prior to each experiment to verify that relative humidity was in excess of 99%. Water potentials were measured by the pressure chamber method, using a 7″ chamber (Soilmoisture Equipment, Santa Barbara, CA, USA), retrofitted with a digital gauge with a range of 0 to 500 psi, 1 psi resolution (DPG500, Omega Engineering, Inc., Stamford, CT, USA), calibrated against a 0 to 1000 psi digital test gauge with an accuracy of ±0.05% full scale (DPG4000-1k, Omega Engineering, Inc.).
Two types of experiments were conducted, the first testing the effect of light level on potential relaxation, the second the effect of anoxia. For the light experiments, half the whorls on each branch were shielded from light by black plastic sleeves that maintained light levels <1 µmol m−2 s−1 PPFD, but allowed the free exchange of atmosphere (henceforth ‘dark’ acclimated leaves); uncovered whorls were subjected to 50 µmol m−2 s−1 PPFD from an overhead bank of full-spectrum fluorescent lights (‘light’ acclimated leaves). While humidification of the glove bag sufficed to prevent measurable transpiration-induced water potential gradients at 50 µmol m−2 s−1 PPFD, initial tests at 100 µmol m−2 s−1 PPFD showed within whorl leaf-to-leaf variation in potential in excess of 0.1 MPa, and so the higher light level experiments were abandoned.
After 2 h pre-treatment, dark and light acclimated leaves were rehydrated under their respective light levels. Light leaves were rehydrated first, after which the lights were switched off. The light blocking sleeves were then removed to allow rehydration of dark adapted leaves at < 1 µmol m−2 s−1 PPFD.
All rehydration measurements were conducted in the following manner. The petioles of one or two leaves were slipped under a bracket glued to the bottom of a Petri dish filled with DI water, such that a portion of the petiole was immersed, then cut underwater. Simultaneous with the cutting of the immersed petiole(s), two leaves were cut in air from the same whorl to provide both estimates of the initial potential of the whorl and a test for water potential equilibrium among the leaves of a whorl prior to rehydration. Experiments where the potential of initial leaves differed by more than 0.1 MPa were discarded. In general, agreement between the un-hydrated initial leaves was within 0.05 MPa. The total error arising from estimating initial potential based on two adjacent leaves was found previously to be ±0.056 MPa, which includes an expected repeatability of ±0.034 MPa for potential measurements with the pressure chamber (Rockwell 2010). The difference between initial and post-rehydration water potentials was typically 0.6 to 0.8 MPa, in relation to which our total error was less than 10%.
The rehydration time of 25 s was pre-set on a digital dark room timer activated by foot switch. As time expired, the petioles were dabbed with a paper towel to remove any droplets and wrapped with one or two turns of parafilm. The leaves were then placed in freezer-weight ziplock bags with the ends of the petioles wrapped in parafilm, removed through a port in the glove bag, the ziplocks humidified with a breath, and allowed to equilibrate for one to two hours, a time chosen to be an order of magnitude longer than that expected to be necessary. We previously found this equilibration procedure conserves the water content and leaf water potential of red oak leaves within the measurement error of the pressure chamber (Rockwell 2010).
Anoxia experiments were conducted on branches bearing two equal-sized daughter branches, each placed in their own glove bag; N2 was bubbled through a flask as above and delivered to one bag, while the second bag received air in the same manner. The flow rate of two litres per minute was sufficient to reduce the oxygen level to less than 1% within half an hour, followed by a 1 h treatment period. Light levels at all times were <1 µmol m−2 s−1 PPFD, to maintain stomatal closure. Rehydrations were performed as described earlier. Note that while rehydration took place in the treatment atmosphere, post-hydration equilibration took place in the presence of oxygen. Rehydration time for both treatments was 30 s.
‘On the tree’ method
As noted previously, attempts to test re-hydration in the glove bags at higher than 50 µmol m−2 s−1 PPFD light levels failed due to an inability to achieve water potential equilibrium between leaves in a whorl, presumably due to excessive stomatal opening and transpiration. A variant of the lab rehydration experiment was, therefore, developed for use on intact trees. In these experiments, the canopies of two trees on the Harvard campus were accessed by an 80′ boom lift. In the morning, leaves destined for harvest as initials or hydration as dark-conditioned leaves (‘three hour leaves’) were enclosed in plastic bags with most excess air squeezed out, and sealed with parafilm around the petiole; aluminium foil was then folded around the bagged leaf to block light and prevent excessive heating of the leaf. In the afternoon, after a minimum of 3 h from the end of morning preparations, new leaves were bagged in the same manner and allowed to come to equilibrium with the stem for 10 min (‘10 min leaves’). The time of 10 min was chosen since a 10 min period of darkness has been reported to be too short to reduce the conductivity of light-acclimated leaves (Scoffoni et al. 2008). In an initial test, we sought to determine whether ten minutes was sufficient to bring a transpiring leaf to equilibrium with the stem potential. For five sets of paired (i.e. from the same whorl) leaves, we found the water potential of ten minutes leaves to be 0.033 MPa more negative than the three hour leaves (1.46 versus 1.43 MPa), but the difference was not significant (paired t-test, P = 0.34), and was within the expected accuracy of our pressure chamber measurements. Therefore, we judged 10 min to be sufficient time for transpiring leaves to recover to the water potential of the parent branch following enclosure in a saturated atmosphere (i.e. inside the bag). Prior to the rehydration experiments, transpiration, assimilation and leaf temperature were measured on adjacent leaves using a LI-6400 (Li-Cor). Following the gas exchange measurements, each leaf was collected for water potential measurement in the lab by placing a pre-humidified ziplock over the blade and simultaneously cutting the petiole. While the resulting water potential measurement represents a volume average (Tyree & Hammel 1972) for the transpiring leaf, with an uncertain relationship to the driving force for liquid flow in the transpiring leaf, it provides some constraint on the degree of water stress experienced by the light acclimated and transpiring leaves prior to rehydration.
Rehydration on the tree was then accomplished for an individual leaf by cutting the petiole, bent underwater in a Petri dish, with a single-edge razor blade; the rehydration time of 25 s was monitored with a digital wristwatch. Three hour leaves were alternately harvested as initials or rehydrated, with each leaf taken from a separate whorl in the same vicinity of the crown; for each such pair a third leaf from a new whorl in the same vicinity was chosen for the ten minute treatment, rehydrated and harvested. In this design, an initial leaf does not give an estimate of the initial potential of any particular leaf. Rather, if all the initial leaves are within less than 0.1 MPa of each other, the mean of all the initial leaves provides a robust estimate of each rehydrated leaf's initial potential. Once harvested, all leaves were sealed inside an additional ziplock bag and stored on the lift in a styrofoam box covered in aluminium foil tape. A large thermal mass (two gallons of water) was inside the cooler to help maintain constant conditions. Pressure chamber measurements of potential were then made in the lab, after the usual 1 h equilibration period.
To assess whether pressurization and submersion in the HPFM had effects on stomatal aperture beyond those of light, leaves were taken from the bath while still under pressure from the HPFM (except those leaves sampled to explicitly check the disposition of stomata pre-pressurization), clamped with copper cryo-pliers cooled to liquid nitrogen temperatures, and immediately plunged into liquid nitrogen (LN2). Samples were cut on a copper block cooled to <−140 °C by immersion in LN2 (similar in principal to the N2-vapour-filled stage of a cryomicrotome) and stored in LN2 until they could be viewed. Immediately prior to viewing, the samples were trimmed as above, mounted on stubs with a drop of Tissue-Tek and immediately re-immersed in LN2 to avoid thawing due to contact with the higher thermal mass of the stub. Samples were sputter coated with palladium for 3 min, and viewed at either 5 or 10 KV, with a spot size of 3. Following Cochard et al. (2007), one leaf from each treatment was sampled and imaged. Aperture length and width were measured on 11–24 stomates per sample, at 10–12,000×, with a resolution of 0.029 and 0.025 µm per pixel, respectively.
Replication of the phenomena
Our experiments successfully replicated the light response on H as reported for red oak measured in the HPFM by Sack et al. (2002) and Tyree et al. (2005). For five leaves acclimated and measured in the light (Fig. 3a), an average maximum value for H of 13.39 ± 2.44 SD mmol m−2 MPa−1 s−1 was reached after 5000 s of measurement in the light. After switching off the light, minimum conductance in the dark was <1 mmol m−2 MPa−1 s−1, too small a value to accurately resolve; the change from dark to light saw changes in H as measured by the HPFM of over an order of magnitude. The light response proved repeatable on a single leaf, showing no decay over repeated cycles for over 24 h (data not shown), indicating there were no declines in conductance due to wounding effects. For five leaves acclimated and measured in the dark (Fig. 3b) H declined from an initial average of 7.52 ± 0.63 SD mmol m−2 MPa−1 s−1 in two phases; the first phase saw a drop to an average of 5.08 ± 1.3 SD mmol m−2 MPa−1 s−1 over 200 s, followed by a slower decline to the minimum dark value by 6000 s. Fig. 3c shows that the early values of H for dark (7.52 ± 0.63 SD mmol m−2 MPa−1 s−1) and light (9.8 ± 1.34 SD mmol m−2 MPa−1 s−1) acclimated leaves began to diverge within 100 s. In neither case, despite 3 h of acclimation under treatment light levels, had the apparent light response saturated. Indeed, most of the difference between the treatments arose only after more than an hour under the conditions imposed by measurement with the HPFM, pressurization and submersion.
Preconditioning on the tree: light levels
Leaves dark or light adapted on the tree and then measured under 1000 µmol m−2 s−1 PPFD showed no significant differences in initial response (Fig. 4). Both treatments resulted in early declines in H, behaviour not seen in the light pre-treatment in the lab experiments discussed above. Indeed, increase in H occurred only after 2000 or more seconds in the HPFM, with leaves dark adapted on the tree beginning to increase earlier and more steeply than the light treated leaves. Most notably, even after 3 h of dark adaptation the initial conductance of leaves measured directly off the tree did not approach the minimum value measured by the HPFM, as would be expected if darkness depressed H in nature to the degree predicted by the steady-state dark HPFM value.
Preconditioning experiments in the lab: light and oxygen
Whereas pre-adaptation to darkness in the HPFM or on the tree failed to dramatically depress initial values of H, pre-treatment with the combination of an anoxic N2 atmosphere and darkness resulted in early values for H of only 2 to 3 mmol m−2 MPa−1 s−1 (Fig. 5a & b), less than half the value in dark and air. Furthermore, these values remained stable from within twenty seconds of submersion to over 2000 s, before declining to the typical minimum values observed for all leaves of <1 mmol m−2 MPa−1 s−1. That stable values for the N2 leaves were reached within 20 s demonstrates that the system composed of the HPFM and attachments to the leaf did not constitute an inherently large capacitance upstream of the leaf, which would have had the effect of making H appear artificially high until the upstream capacitance was satisfied. This in turn implies that the initial values of H (7.52 ± 0.63 SD mmol m−2 MPa−1 s−1) measured for the leaf in the dark and air, though only stable for about a minute, did in fact represent real leaf conductances, rather than capacitive artefacts.
As in the case of light and air pre-treatment, pre-treatment with light and N2 did not drive initial H to the maximum H achieved in the HPFM (Fig. 5). Unique to light and N2, the rise to maximum was in all cases interrupted by a period of decline that later reversed, a reaction never seen in air treated leaves. Finally, pre-treatment of leaves with CO 2-free air in light had no effect versus air and light leaves (data not shown).
The results of the three rehydration experiments, which offer a test of light responses on a transport path that does not include the stomata, did not support any effect of light on leaf permeability, but did indicate an effect of anoxia (Fig. 6). For leaves from branches cut from three trees pre- treated for two hours and then hydrated in either an N2 atmosphere or air, the rehydrated potential of leaves in the anoxic treatment were 0.17 MPa more negative than in air (−0.72 N2 versus −0.55 MPa air, n = 16 each group, P < 0.001). There was no difference in the average initial potential of each group (−1.15 N2 versus −1.16 MPa air, P = 0.97). Mean leaf capacitance normalized to leaf area (Ca) was the same for each treatment (361 versus 362 mmol m−2 MPa−1 for N2 and air, respectively, P = 0.91), and did not explain any of the difference in relaxation. In terms of the conductance values calculated by Brodribb & Holbrook (2003) and Scoffoni et al. (2008) (e.g. Kleaf), our data correspond to a decrease from 9.2 to 5.7 mmol m−2 MPa−1 s−1, or 38%. These data suggest that, as in the HPFM experiments, leaf tissue conductivity under dark conditions is indeed sensitive to anoxia, as would be expected based on the behaviour of roots (Tournaire-Roux et al. 2003). Such anoxia sensitivity is furthermore consistent with the expectation that oak leaf rehydration kinetics should be sensitive to membrane permeability.
Rehydration experiments on branches from five different trees show that a light level of 50 µmol m−2 s−1 PPFD during, and for two hours prior to, rehydration had no effect on relaxation, compared to <µmol m−2 s−1 PPFD (Fig. 6b, P = 0.725, n = 19 each treatment). Furthermore, the power (β = 0.8) was sufficient to expect to detect an effect of 0.053 or greater (≈15% change in permeability) in 95 out of 100 such experiments. There was no significant difference in the mean initial potential of either group (−1.20 MPa light, versus −1.22 MPa dark (P = 0.78), offering assurance both that the glove bags were sufficiently humid to prevent the light treatment from inducing water potential disequilibrium that might arise from even a small degree of stomatal opening, and that the order of hydration experiments (light first, then dark) did not bias the results.
Figure 7 shows that a leaf in the HPFM under a light level of 50 µmol m−2 s−1 PPFD is at 40 to 80% of the maximum conductance measured by the HPFM, or more than five times greater than its dark adapted value. Therefore, if the dark minimum conductance of <1 mmol m−2 MPa−1 s−1 was indeed an effect of light on tissue properties, we would have expected a five fold difference in the time constant of rehydration between the 50 µmol m−2 s−1 PPFD and the dark treatments. From these data we therefore conclude that whatever is limiting flow in the HPFM to the dark minimum conductance does not significantly affect rehydration.
Leaves on the tree were exposed to far higher light levels, but showed the same degree of rehydration whether rehydrated after ten minutes of darkness or three hours. The first day of the experiment was cloudless and light levels, recorded by the external sensor of the LI-6400 during the gas exchange measurements taken just prior to the rehydration experiments, averaged 1708 ± 98 µmol m−2 s−1 PPFD (n = 20). Mean stem potential was −1.3 ± 0.03 MPa for tree one (n = 6). On the second day, the weather was overcast, and light levels were 514 ± 103 µmol m−2 s−1 PPFD (n = 22); stem potential for tree two reached −1.01 ± 0.06 MPa (n = 3). Despite the differences in ambient light level, there were no significant differences in the ratio of final to initial water potential (ψf/ψo) on either day (and therefore either tree) for either the light or dark treatments, allowing us to combine the data from the 2 d (Fig. 6c, P = 0.96, n = 9 in each group). These data tell us that if high light levels do affect the permeability of oak tissue, the effect must fully decay within 10 min of imposing darkness, 5 to 100 times faster than the declines in apparent conductance observed in the HPFM here, or in previous HPFM experiments (Cochard et al. 2004; Tyree et al. 2005).
Gas exchange results for leaves on the first day, conducted just prior to the rehydration experiments, showed that adjacent leaves were transpiring at what, in our experience, are healthy rates for red oak. Assimilation was 19.69 ± 1.5 µmol m−2 s−1, transpiration 5.59 ± 0.36 mmol m−2 s−1, and leaf temperature 28.7 °C (means ± SD, n = 20, four measurements x five leaves). Water potentials for the five transpiring leaves were −1.73 ± 0.14 MPa; these are the potentials obtained after all gradients in the leaves that existed at the time of harvest had collapsed. We found the difference between stem and transpiring leaf potentials to be 0.44 MPa (transpiring leaves more negative); stem potentials were given by the initial leaves from the ‘on the tree’ rehydration experiments immediately following. Thus, there is no indication that fluid transport in the leaves exposed to light was impaired by water stress.
Cryomicroscopy: stomatal aperture
Estimates of stomatal aperture at the surface of the leaf were taken from cryo-SEM images of leaves frozen under different light levels and either pressurized by the HPFM, or simply connected to a water source at atmospheric pressure, in order to check for interactions between light and pressurization on the degree of stomatal opening. However, surface measurements of stomatal aperture cannot tell us about the area available for flow as a function of depth in the pore. Apparent aperture at the surface is heavily influenced by the sculpting of surface waxes, and depth is very difficult to judge based on SEM images; for these reasons, we refrained from estimating stomatal conductance to liquid water based on an assumption of Hagen-Poiseuille, or pipe flow, based on pore dimensions as they appear at the surface. Rather, we checked the behaviour of the minor axis dimension visible at the surface in response to light and source pressure.
For leaves frozen at different light levels while pressurized in the HPFM (Fig. 8a–c), we report the conductance measured by the HPFM and the mean minor axis aperture, and find that stomatal aperture does indeed decline with light level as expected. However, the mean aperture of stomata in leaves in full darkness and air (0.26 µm, n = 24, solid line D) was not significantly different (P = 0.17) from the mean aperture of leaves in the HPFM at 100 µmol m−2 s−1 PPFD (0.33 µm, n = 11, triangle C). In addition, mean aperture of leaves in full darkness and pressurized by the HPFM at their minimum conductance (0.05 µm, n = 14, circle A) was one-fifth of the aperture observed for leaves in the air and dark, a difference that was significant (P < 0.001). While these measurements cannot reliably tell us whether the apparent apertures explain the observed conductances in the HPFM, they do provide evidence that the combination of dark, pressurization, and submersion creates an interaction that results in greater stomatal constriction than dark alone; and that higher light levels allow stomata to overcome this constriction.
The light response of red oak leaves is independent of the permeability of the leaf tissue. The question of why it takes so long for HPFM measurements to stabilize in experiments with long pre-treatments at the experimental light level is a critical one. Previous studies have noted that H declines upon introduction of a leaf to the HPFM. Melcher et al. (2001) noted similar declines in Rhizophora mangle, which they suggested could represent infiltration of high resistance compartments within the leaf, raising questions about the interpretation of steady-state HPFM values. More often, observed declines have been ascribed to the capacitance of the leaf as well as the experimental system (Sack et al. 2002; Tyree et al. 2005). In this study, we note that the capacitance of the measurement system was negligible. For a typical oak leaf with an area 100 cm2, and an area normalized capacitance of 300 mmol m−2 MPa−1 above the turgor loss point, the absolute change in water content for a change in potential on the order of 0.1 MPa is just 0.3 mmol. One may then calculate the flow rate into the leaf directly from the pressure drop across the known resistance of the HPFM, and ask how much flow would have to absorbed into storage, if all initial flow rates in the dark in the HPFM higher than that observed at the dark, minimum value of H were due to capacitive effects. In the case of the N2 leaves in the dark, integration of the flow over the first 6000 s, when H reaches its minimum dark value, minus the flow observed at that minimum integrated over the same time interval, indicates that for Hmin to have been in effect from the beginning of the experiment over 10 mmol of water would have to have been absorbed into storage; more than an order of magnitude more than can be accounted for by the leaf tissue. Even to explain only the initial drop in H of air and dark treated leaves from ∼8 to ∼5 mmol m−2 MPa−1 s−1 would require an absorption on the order of 3 mmol.
As the N2 treatments in the HPFM resulted in a measurement of H that was steady within 20 s of measurement and remained so for half an hour, and as the rehydration experiments found no effect of N2 treatment on capacitance, we conclude that the early changes in the apparent leaf conductance H do not arise from the capacitance of the cells or the HPFM apparatus. The order of magnitude differences in the dark minimum and light maximum H observed in the HPFM then only arise following extended submersion and pressurization of the leaf; light level alone is not sufficient to induce them. If we consider the initial differences in H observed in the HPFM between light and dark acclimated leaves (Fig. 3) as the result of a light response, we find only a 20% decrease in the dark. However, even this difference does not appear to arise strictly from tissue properties, as shown by the rehydration experiments.
The rehydration experiments in the lab found no significant effects of light at 50 µmol m−2 s−1 PPFD, and the results on the tree at higher light levels preclude any effects that do not decay within 10 min; this appears to exclude for red oak light responses of the type reported for rehydrating leaves previously, which explicitly claim effects that persist through 10 min of imposed semi-darkness (<5 µmol m−2 s−1 PPFD, Scoffoni et al. 2008). Thus, these experiments have narrowed the range of possible effects of light on liquid transport in red oak to those that only occur at >50 µmol m−2 s−1 PPFD and fully decay in less than 10 min, which excludes all effects reported to date in any species.
Taken together, the lack of light response in rehydration kinetics, and the failure of the apparent conductance in the HPFM to saturate at its high and low values under light level pre-treatments both in the lab and on the tree, suggest that the limiting conductance observed in the HPFM must be downstream of the tissue. Furthermore, this variable conductance only becomes limiting through the interaction of dark, submersion, and pressurization. Finally, it must become non-limiting in the light.
All of these stipulations point to a dominant role for the stomata in producing the changes in H observed in the HPFM; they constitute a known light-responsive variable resistance located at the end of the flow path measured by the HPFM (after the intercellular space). Based on the cryo-SEM images, the stomata both retain their light responsiveness in the HPFM, and show evidence of an interaction between darkness and the measurement conditions imposed by the HPFM that results in depressed aperture. The declines in flow that occur initially in the HPFM could arise from changing stomatal aperture in response to pressurization and submersion in conjunction with a capacitive effect of the intercellular air spaces, acting as a sink downstream of the tissue, such that the effects of stomatal limitation of flow only appear gradually.
Indeed, the declines observed for the leaves dark adapted in the HPFM are consistent with infiltration of the air spaces; for an oak leaf of 100 cm2 area and 200 µm thickness (typical for these leaves), an air fraction of 0.3 (normal for heterobaric leaves, Pieruschka, Schurr & Jahnke 2005) creates a sink large enough to hold 0.6 cm3 or 33.3 mmol of water. Thus the intercellular air space represents a potential source of capacitance large enough to explain the declines in H as a result of the filling of a reservoir in front of a constant resistance equal to H at its dark minimum. The only possible sources of such a resistance downstream of the extracellular air space are the stomata. If we look again at the declines in N2 and dark treated leaves, we see that they are initially stable longer than dark and air leaves, but ultimately take longer to reach the dark minimum H. This is consistent with both treatments filling the same extracellular sink over time, with only a minimal flow out of the stomata. In the case of the N2 treated leaves, the permeability of the tissue upstream of the sink is truly depressed as a result of the treatment, at least initially, as we would expect it to be from the rehydration experiments.
Given this view, the declines in light in the HPFM observed for leaves of both light treatments taken directly from the tree are consistent with the effects of trapped air-water interfaces inside the leaf, which together with the hydrophobicity of the walls of the sub-stomatal chambers (Esau 1977; Nobel 2005), may constrict liquid phase flow until the air is pushed out or dissolved into solution under pressure. That we do not see these declines in air and light treated leaves acclimated to ψ = 0 in the HPFM under ∼ 80% RH may be because the smaller intercellular spaces in the leaves are already flooded.
Relationship of these findings to previous work
The potential for stomatal limitation of flow
Sack et al. (2002) hypothesized that, in the dark, red oak leaves experienced stomatal limitation of flow, which they suggested could occur at reductions of stomatal aperture to less than 1% of the maximum. As noted by the authors of that study, variation in the degree to which stomata close in the dark is well documented (Franks, Cowan & Farquhar 1998). Indeed, for some taxa, to overcome the mechanical advantage of the epidermal cells over the guard cells and open stomata via direct injection of turgor through a microcapillary, a detached epidermis must be floated in a buffer with high solute potential sufficient to reduce epidermal turgor (Franks et al. 1998). However, a subsequent study by Tyree et al. (2005) argued against stomatal limitation of flow, based on a comparison of the stomatal kinetics of intact plants with that of the light response in the HPFM, as well as the observation that treatment of leaves with ABA, an inhibitor of stomatal opening, failed to prevent the light response. Yet, as noted by Sack et al. (2002), in a study measuring the responses of submerged stomata in detached epidermal strips of Commelina communis using viscous flow porometry, Fricker, Grantz & Willmer (1991) found that floating epidermal strips had pore widths four times greater than submerged, un-aerated strips, suggesting that the conditions imposed by the HPFM may influence stomatal behaviour, thereby calling into question the expectation that stomatal kinetics in light and air should match the kinetics observed in the HPFM, if stomata play a role in the latter. Furthermore, Fricker et al. (1991) reported that incubation with 100 µm ABA only reduced apertures in C. communis 50 to 75%, with some recovery over time. At apertures greater than 0.2 µm, stomata may no longer act as a limiting conductance (Tyree et al. 2005), but this is far smaller than the degree of stomatal closure achieved by ABA in Fricker et al.'s study; thus ABA should not necessarily be expected to inhibit the small increase in aperture necessary to produce a light effect in the HPFM.
Cochard et al. (2007) calculated the Hagen-Poiseuille conductance of walnut (Juglans regia) stomata based on cryo-SEM images of leaves frozen under different light levels to argue they could not explain the observed light response in the HPFM. Yet, apparent aperture at the surface may not provide an accurate representation of the geometry of the flow path through the depth of the pore; nor were the leaves frozen while pressurized, which we have shown can affect observed aperture. The preferred hypothesis of Cochard et al. (2007) was that the light response arose from increased aquaporin density, and indeed they reported increased transcript abundance for PIP1,2 and PIP2,2 in walnut at high irradiance (Fig. 5). Yet, the kinetics of the increase transcript levels in fact lagged behind increases in conductance. Taking into account the fact that transcript abundance was plotted on a log percent scale versus conductance on a linear scale, 50% of the maximum conductance corresponded to only 10% of peak transcript abundance (maximums were scaled to the same height). More recent work on another species that shows a light response in the HPFM, Q. macrocarpa, found no relation between the activity of four aquaporin genes and hydraulic conductance defined by the HPFM (Voicu et al. 2009).
Observed light responses vary between methods
Stronger evidence of a light response within the tissue could come from methods in which stomata are excluded from the measured flow path. Kim & Steudle (2007, 2009) report light effects on the permeability of maize midrib epidermal cells using the cell pressure probe, though the effects of light in the 2007 study were highly variable. Halftimes for the decay of a pressure pulse applied to a cell through a microcapillary decreased upon illumination of the leaf; however, once steady state transpiration at the whole leaf level was reached, cellular halftimes appeared to increase four-fold. Under dark conditions, only cells with halftimes longer than 2 s showed a shortening of halftime after 10 min illumination. How common such cells were relative to cells that showed fast kinetics under both conditions was not reported, and cells that dropped in turgor more than 0.2 MPa upon illumination were excluded from the analysis, based on a hypothesized confounding turgor effect. Indeed, the lengthening of half times in the dark became much more consistent after pressurization of the roots; however root pressurization under dark, non-transpiring conditions could have led to flooding of the leaf. Kim & Steudle (2009) added higher light levels, and reported a decline in permeability, a phenomenon not reported for light responses seen with other methods (Sack et al. 2002; Tyree et al. 2005; Cochard et al. 2007; Scoffoni et al. 2008). Thus, it is not clear how these results might relate to the light effects seen in the HPFM.
Scoffoni et al. (2008) reported a light response of 60% to 100% increase in three of six species tested using evaporative flux and a rehydration method, more modest responses than reported for the HPFM. While we found no evidence of an effect of light level on rehydration in red oak, this does not exclude the possibility of a response of rehydration to light in other taxa. However, we note that the experiments of Scoffoni et al. (2008) appear to have been conducted within a range of water potentials where the behaviour of leaves may become dominated by the divergence of the water content and potential relationship (Boyer 1968; Parker & Pallardy 1987; Kubiske & Abrams 1991; Boyer et al. 2008), which is manifested as a sharp increase in capacitance as a leaf approaches equilibrium with a source of water at 0 MPa potential. As a result of this diverging behaviour, a leaf submerged in water (zero transpiration) will continue to uptake water over a time scale of hours to tens of hours at a very slow rate (Weatherley 1963; Zwieniecki, Brodribb & Holbrook 2007), retaining a water potential as negative as −0.1 MPa (Boyer 1968). In the context of rehydration experiments, this behaviour becomes manifest in many leaves as a non-constant capacitance, and a transition to order of magnitude slower kinetics (Weatherley 1963; Zwieniecki et al. 2007). For evaporative flux methods, the effect is that apparent conductance is low at low flux rates, but increases and be- comes linear in the flux at higher water loss rates, independent of the forcing variable chosen, be it leaf temperature, light level, or humidity [see discussion in Boyer (1985) in terms of leaf growth, but this may also be observed for leaves that flood extracellularly as potentials in any part of the leaf tissue approach zero]. Ideally, treatment effects on conductances defined by evaporative flux methods should be assayed at the same levels of flux, which might have been achieved in this case by manipulating ambient humidity, although problems with the definition of the potential gradient in this method would, of course, still remain (Sack et al. 2002).
Returning to the discussion of the data presented here, we have not attempted to explain every aspect of the HPFM time courses. Rather we have simply endeavoured to point out that the observations are most parsimoniously explained by stomatal action. In particular, the exact interactions between stomata and the HPFM remain enigmatic; submersion may lead to low H through the trapping of air-water interfaces, and/or anoxic effects. Pressurization of the blade could also restrict stomatal aperture, due to mechanical interactions between epidermal and guard cells. Nor have we addressed what happens in species that do not show apparent light responses, other than to suggest that such species may not close their stomata as tightly. Rather, our particular interest has been to establish whether light is sufficient to produce non-negligible effects on the conductivity of red oak leaf tissues. Clearly it is not. The alternative hypothesis of stomatal action, originally offered by Sack et al. (2002) is preferred here. While the particular focus of this work has been on red oak, that the light response appears to derive from something other than tissue conductivity in one species – notably the very species in which the phenomenon was first reported – suggests that the concept of aquaporin-mediated, light-induced changes in bulk leaf tissue conductivity requires further careful examination.