Nocturnal stomatal conductance responses to rising [CO2], temperature and drought

Nocturnal stomatal conductance responses to rising [CO2],temperature and drought

Melanie J. B. Zeppel1, James D. Lewis2,3, Brian Chaszar4, Renee A. Smith2, Belinda E. Medlyn1,

Travis E. Huxman4,5 and David T. Tissue2

1Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia; 2Hawkesbury Institute for the Environment, University of Western Sydney, Richmond, NSW 2753,

Australia; 3Louis Calder Center – Biological Field Station and Department of Biological Sciences, Fordham University, Armonk, NY 10504, USA; 4Department of Ecology and Evolutionary

Biology, University of Arizona, Tucson, AZ 85721, USA; 5Biosphere 2, University of Arizona, Tucson, AZ 85721, USA

Author for correspondence:Melanie J. B. Zeppel

Tel: +61 2 9850 9256Email: [email protected]

Received: 5 September 2011

Accepted: 31 October 2011

New Phytologist (2012) 193: 929–938doi: 10.1111/j.1469-8137.2011.03993.x

Key words: drought mortality, elevatedCO2, elevated temperature, Eucalyptussideroxylon, night-time stomatalconductance, nocturnal fluxes, pre-industrialCO2.

Summary

• The response of nocturnal stomatal conductance (gs,n) to rising atmospheric CO2 concen-

tration ([CO2]) is currently unknown, and may differ from responses of daytime stomatal

conductance (gs,d). Because night-time water fluxes can have a significant impact on

landscape water budgets, an understanding of the effects of [CO2] and temperature on gs,n is

crucial for predicting water fluxes under future climates.

• Here, we examined the effects of [CO2] (280, 400 and 640 lmol mol)1), temperature

(ambient and ambient + 4�C) and drought on gs,n, and gs,d in Eucalyptus sideroxylon

saplings.

• gs,n was substantially higher than zero, averaging 34% of gs,d. Before the onset of drought,

gs,n increased by 85% when [CO2] increased from 280 to 640 lmol mol)1, averaged across

both temperature treatments. gs,n declined with drought, but an increase in [CO2] slowed this

decline. Consequently, the soil water potential at which gs,n was zero (W0) was significantly

more negative in elevated [CO2] and temperature treatments. gs,d showed inconsistent

responses to [CO2] and temperature.

• gs,n may be higher in future climates, potentially increasing nocturnal water loss and suscep-

tibility to drought, but cannot be predicted easily from gs,d. Therefore, predictive models using

stomatal conductance must account for both gs,n and gs,d when estimating ecosystem water

fluxes.

Introduction

The combined rise in atmospheric CO2 concentration ([CO2])and global air temperature may significantly alter plant water use.Rising [CO2] often reduces leaf-scale daytime stomatal conduc-tance (gs,d) (Morison, 1987; Medlyn et al., 2001; Lewis et al.,2002a; Ainsworth & Rogers, 2007), and rising air temperatureoften additionally reduces gs,d because it is associated with highervapour pressure deficits (D) (Lewis et al., 2002a; Lloyd &Farquhar, 2008). A number of feedbacks at plant and ecosystemscales can modify these direct effects on leaf-level water use (Fieldet al., 1995; Saxe et al., 1998; Ainsworth & Rogers, 2007). Forexample, theory suggests that rising [CO2] may lead to ‘watersavings’ in the soil by reducing gs,d and transpiration, therebyameliorating drought stress (Wullschleger et al., 2002). However,changes in whole-plant characteristics, such as increased leaf areaor reduced rooting depth, may interact with leaf-level watersavings to determine canopy and ecosystem water use (Bobichet al., 2010; Duursma et al., 2011; Warren et al., 2011). Pred-icting the overall effect of climatic perturbations on vegetation

water use therefore requires careful integration of both direct([CO2], temperature) and indirect (soil water content, plant size)effects.

One component of plant water use that has not yet beenfactored into these predictions is nocturnal water use. There isincreasing awareness that nocturnal water use may be a signifi-cant component of the overall plant water balance, and asizeable fraction (c. 10–20%) of annual water budgets (Cairdet al., 2007b; Dawson et al., 2007; Marks & Lechowicz,2007). However, there is currently little understanding of themechanisms driving nocturnal water use, making it difficult topredict (Caird et al., 2007a). It is not yet known, for example,whether nocturnal water use confers a benefit to plants, is aconstraint associated with additional adaptive functions or ismerely the result of ‘leaky’ stomata (Dawson et al., 2007;Marks & Lechowicz, 2007; Christman et al., 2008, 2009;Howard & Donovan, 2010). Studies of nocturnal water use arecurrently focused on documenting patterns across species or inresponse to environmental variables, because the regulatorymechanisms have not been elucidated (Dawson et al., 2007;

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Marks & Lechowicz, 2007; Christman et al., 2008; Howard &Donovan, 2010).

Predicting nocturnal water use is difficult because responseshave been examined for only a few variables, and nocturnalresponses may differ substantially from daytime responses toimportant functional drivers. For example, it is now establishedthat nocturnal water fluxes decline as soil dries (Bucci et al.,2004; Barbour & Buckley, 2007; Cavender-Bares et al., 2007;Dawson et al., 2007; Howard & Donovan, 2007; Zeppel et al.,2010), which is consistent with the response of daytime waterflux. However, night-time and daytime responses to D have beenfound to differ. Daytime stomatal conductance (gs,d) decreasesconsistently with higher D (Oren et al., 1999; Lewis et al.,2002a; Lloyd & Farquhar, 2008). In comparison, responses ofnocturnal stomatal conductance (gs,n) to D are highly variable,and have been found to rise (Dawson et al., 2007), remainunchanged (Barbour et al., 2005) and decline (Barbour & Buckley,2007) with rising D. This variability among studies mayoccur because short-term and long-term responses of gs,n to Ddiffer. Barbour & Buckley (2007) found that gs,n of plants incontrolled environments declined in response to short-termincreases in D. By contrast, gs,n in field-grown trees increasedwith D when compared across nights with differing D (Dawsonet al., 2007).

Another key factor that may regulate gs,n is [CO2]; yet, to ourknowledge, the effect of rising [CO2] on gs,n has not been quanti-fied. In a recent study, Zeppel et al. (2011) found that the noc-turnal sap flow of well-watered Eucalyptus saligna increased underelevated [CO2], in contrast with the reductions seen in daytimesap flow dynamics with rising [CO2]. These whole-tree sap flowresults suggest that leaf-level gs,n and gs,d may exhibit differentresponses to rising [CO2]. However, the nocturnal sap flowresponse to elevated [CO2] occurred during the night-time stemrecharge period, and therefore may have reflected changes in stemrecharge, rather than transpirational fluxes to the atmosphere.

In this study, the effect of rising [CO2] on gs,n was quantified.Eucalyptus sideroxylon trees were grown at three levels of [CO2](280 lmol mol)1, pre-industrial; 400 lmol mol)1, ambient;and 640 lmol mol)1, elevated). We included a pre-industrial[CO2] treatment to provide a baseline for the assessment of theeffects of rising [CO2] since the beginning of the industrial age,which may also help us to predict future responses to [CO2](Sage & Coleman, 2001; Gill et al., 2002; Gerhart & Ward,2010; Lewis et al., 2010), and allows for an extended responsesurface to more adequately understand correlations among func-tional traits. We tested whether gs,n decreased in response torising [CO2], as might be expected from the effects of rising[CO2] on gs,d, or whether gs,n increased, as suggested by the studyof nocturnal sap flow in Zeppel et al. (2011). In addition to com-paring responses across a [CO2] gradient, this study also investi-gated the potential interactive effects of temperature (ambientand ambient + 4�C) and [CO2] on gs,n over the course of a pro-tracted drought. Across our temperature treatments, relativehumidity was approximately equal, meaning that D was higher athigher temperature; therefore, the elevated temperature treatmentrepresented a combined elevated temperature and elevated D

environment when compared with the ambient conditions. Basedon a study showing that gs,n increased with D (Dawson et al.,2007), we anticipated that gs,n would increase with temperature.We also anticipated that gs,n would decrease with reduced soilwater content, as has been shown in numerous other studies(Bucci et al., 2004; Barbour & Buckley, 2007; Cavender-Bareset al., 2007; Dawson et al., 2007; Howard & Donovan, 2007;Zeppel et al., 2010). It is not known whether these responsesdepend on growth [CO2].

Our main objectives were therefore to determine the following:whether gs,n would decrease in response to rising [CO2], as mightbe expected from the effects of [CO2] on gs,d; whether gs,n wouldincrease with growth temperature, and an associated increase inD; whether gs,n would decrease as soil water content decreased;and whether rising [CO2] would modify these responses to tem-perature, D and soil water content. Overall, we sought to identifyhow nocturnal water use was affected by growth [CO2] toimprove our understanding of the complex effects of climatechange on vegetation water use.

Materials and Methods

Growth conditions

Soil was collected from the A horizon (top 50 cm) of theHawkesbury Forest Experiment site, University of WesternSydney, Richmond, NSW, Australia (Barton et al., 2010). Thesoil is a loamy-sand with low organic matter content, lowwater-holding capacity and low fertility (Ghannoum et al.,2010a). The soil was air dried and c. 95 kg was added to eachof 36 pots (volume, 75 l). Six pots were placed in each of sixadjacent, naturally lit glasshouse compartments. Temperatureand [CO2] conditions in the glasshouse compartments weremaintained as described previously by Ghannoum et al.(2010a). In summary, three glasshouse compartments were setto simulate ambient temperature, defined as the temperature ofa 30-yr average local (Richmond, NSW, Australia) day for themonths of November to May (hereafter ambient temperaturetreatment). Three glasshouse compartments were maintained atambient + 4�C (i.e. elevated temperature treatment), which iswithin the range of temperatures predicted with rising [CO2] inthe next century (IPCC, 2007). Over the course of every 24 h,temperatures were changed five times to simulate natural tem-perature variation. Maximum temperatures during the middleof the day and middle of the night for the ambient and elevatedtemperature treatments were 26 : 18 and 30 : 22�C (day :night), respectively. The vapour pressure deficit (D) varied

between 0.4 and 2.5 kPa in the ambient temperature treatmentand between 1.0 and 3.8 kPa in the elevated temperature treat-ment. Within each temperature treatment, compartments wereautomatically regulated to maintain pre-industrial (280 lmolmol)1, target), ambient (400 lmol mol)1, target) and elevated(640 lmol mol)1, target) [CO2]. Actual average daytime [CO2]values during the study period for the pre-industrial, ambientand elevated treatments were 290, 400 and 650 lmol mol)1,respectively.

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Temperature and relative humidity were measured in 5-s inter-vals using Vaisala thermistors, one per glasshouse room (VaisalaInc., Boston, MA, USA), and logged every 15 min on a Camp-bell CR 1000 data logger (Campbell Scientific Inc., Logan, UT,USA). Relative humidity was not regulated and averaged 57%over the growing season. The vapour pressure deficit (D) was esti-mated from the temperature and relative humidity (Pearcy et al.,1989). Soil moisture was measured using one Time DomainReflectometry (TDR) probe to a depth of 0.30 m within each ofthe 36 pots. Data were measured every 5 min and logged hourlyon a Campbell CR 800 data logger (Campbell Scientific Inc.).The volumetric water content was converted to the soil waterpotential (Ws) using a previously determined calibration curve(Phillips et al., 2010).

Plant material

Six E. sideroxylon (A. Cunn. ex Woolls) trees (one per 75-l pot)were grown in each of the six glasshouse compartments. Seeds ofE. sideroxylon were obtained from Ensis (Australian Tree SeedCentre, ACT, Australia) and sown (mid-September 2009) inseedling tubes filled with seed raising mix (Plugger Custom;Debco Pty Ltd, Berkshire Park, NSW, Australia). Two monthslater, seedlings were transplanted into the 75-l pots and grownfor an additional 7 months in the temperature and [CO2] treat-ments. The drought treatments commenced at this point. Withineach [CO2]–temperature treatment, three pots were randomlyassigned to each of the well-watered and drought treatments. Potsin the well-watered treatment were watered to field capacity(c. 2–3 l) every 3 d, and pots in the drought treatment receivedno water after the start of the drought treatment. Pots containedsmall drainage holes to prevent excessive soil waterlogging.

Tree size measurements

Each tree was destructively harvested at the completion of theexperiment, 60 d after the onset of the drought treatment, whenthe trees were 11 months old. Total leaf area was quantified usinga leaf area meter (LI-3100A; Li-Cor, Lincoln, NE, USA).

Stomatal conductance

A Decagon porometer (Decagon Devices, Pullman, WA, USA),which minimally disturbs the leaf boundary layer and thereforemore accurately reflects ambient atmospheric conditions, wasused to measure nocturnal (gs,n) and daytime (gs,d) stomatal con-ductance under ambient growth conditions. Nocturnal measure-ments were conducted weekly during the drought treatment.Daytime measurements were conducted at the start of thedrought period. Measurements on two to five mature, fullyexpanded leaves per tree were made during the daytime between11:00 and 13:00 h. Nocturnal measurements were made between19:30 and 22:00 h (sunset occurred at approximately 17:30 hduring the study period). For all measurements, the porometerwas equilibrated to room temperature for 2 h before initiatingthe measurements of gs,n and gs,d.

Statistical analyses

All statistical analyses, except partial correlations, were conductedusing R versions 2.11.1 and 2.13.0 (R Development Core Team,2010). In all tests, [CO2] was treated as a continuous variableand temperature was treated as a categorical variable. Results wereconsidered to be significant if P £ 0.05. Normal probability plotsand plots of residuals vs predicted values were used to assesswhether data violated the assumptions of normality and homo-geneity of variances. These assumptions were met for all variables.

Two-way analysis of variance (ANOVA) was conducted on alldata (well-watered and drought plants) on the first day to exam-ine the effects of [CO2] and temperature on gs,n before the onsetof drought. To test whether variation in D could account foreffects of [CO2] and temperature on gs,n, a partial correlationanalysis (SPSS v16.0 for Windows) was conducted on all plants(six plants per treatment for six treatments) whilst holding Dconstant. A partial correlation analysis explains the measure ofvariance in the dependent variable that is explained by anindependent variable (predictor), over and above the effects ofother independent variables in the model (Murray & Hose,2005). The use of this technique allowed an examination of theunique relationships between gs,n, [CO2] and temperature, whilstholding constant potentially confounding effects of D.

To test the interactive effects of [CO2] and temperature onthe response of gs,n to D, we conducted a homogeneity of slopestest using a linear model on gs,n and D data collected from well-watered trees during 10 measurement periods that spanned thedrought period. Further, a homogeneity of slopes test was con-ducted on data from drought plants to test whether the slopes ofthe Ws vs gs,n relationship were different among [CO2] and tem-perature treatments. Finally, for each drought plant, the Ws atwhich gs,n reached zero (W0) was estimated as W0 = gs,nmax ⁄ m,where m is the slope of the relationship between gs,n and Ws,and gs,nmax is the value of gs,n when Ws is zero. Subsequently, weused an ANOVA to test the effects of [CO2] and temperatureon W0.

Results

Environmental conditions – D and soil water potential

Mean D increased with growth temperature, both during the dayand at night. There were some differences in D among [CO2]treatments. At ambient temperature, mean (± SE) daytime(10:00–16:00 h) D values across the study period were 1.4(± 0.01), 1.4 (± 0.01) and 1.3 (± 0.01) kPa in the 280, 400 and640 lmol mol)1 treatments, respectively, whereas the mean noc-turnal (18:00–06:00 h) D value was 0.5 kPa (± 0.01) in all ofthese treatments. At elevated temperature, mean daytime (10:00–16:00 h) D values were 1.9, 2.4 and 2.3 kPa in the 280, 400 and640 lmol mol)1 treatments, respectively, whereas the mean noc-turnal (18:00–06:00 h) D values were 0.9, 1.0 and 1.1 kPa inthese treatments, respectively (± 0.01 kPa in all treatments).Statistical analyses of our data were designed to correct for thesesmall differences in D among treatments.

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The soil water potential (Ws) remained high in well-wateredplants throughout the experiment (Fig. 1). In drought plants, Ws

declined during the dry-down period, as expected. Rising [CO2]increased the rate of depletion of soil water, such that the mostrapid decline occurred in the elevated [CO2] treatment. Elevatedtemperature also increased the rate of depletion of soil water. Theeffect of rising [CO2] on Ws was probably a result of a larger treesize (Fig. 2). Leaf area increased linearly with increasing [CO2](P = 0.07). However, elevated temperature had no effect on leafarea (Fig. 2). Accordingly, the effect of elevated temperature onWs was probably a result of increased evapotranspiration associ-ated with increased D.

Effects of [CO2] and temperature on gs,n and gs,d beforedrought onset

At the beginning of the experiment, when all treatments werewell watered, gs,n increased significantly with rising [CO2] inboth temperature treatments (Fig. 3a, P = 0.02). Averaged acrosstemperature treatments, gs,n increased by 85% as [CO2] increasedfrom 280 to 640 lmol mol)1. The partial correlation analysisshowed that there was a [CO2] effect, even after taking intoaccount differences in D among chambers (P = 0.014). Under

ambient temperature, there was a reduction in gs,d with rising[CO2], as has commonly been found. Under elevated tempera-ture, there was no clear trend (Fig. 3b). Thus, the response of gs,n

Fig. 1 Middle of the day soil water potential (Ws, MPa) ofwell-watered (closed circles) and drought-treated (opencircles) Eucalyptus sideroxylon plants within eachtreatment during the drought period (mean ± SE, n = 3).[CO2] treatments were 280, 400 and 640 lmol mol)1

and temperature treatments were ambient andambient + 4�C.

Fig. 2 Leaf area (m2) at the end of the experiment for well-wateredEucalyptus sideroxylon plants for each [CO2] and temperature treatment(mean + SE, n = 3). Ambient temperature, black bars; ambient + 4�C,grey bars.

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to rising [CO2] did not follow a similar pattern to that of gs,d

within the same environmental context at the onset of this experi-ment. The mean value of gs,n as a percentage of gs,d across alltreatments was 34% (range, 16–50%).

Effects of [CO2] and temperature on the relationshipbetween gs,n and D

Overall, gs,n increased with rising D (P < 0.001; Fig. 4). Therewas a significant effect of growth temperature on the relationshipbetween gs,n and D (P = 0.01; Table 1). At ambient growthtemperatures, the response to rising D was relatively flat, whereasgs,n tended to increase more strongly with rising D at elevated tem-perature (Fig. 4). There was also a significant effect of [CO2] onthe gs,n–D relationship (P = 0.04; Table 1). The slope of the gs,n–D relationship increased with rising [CO2] from 280 to 640 lmolmol)1 (Fig. 4). The effect of increasing [CO2] was marginallyhigher (P = 0.07) in the elevated relative to the ambient tempera-ture treatment. In addition, there were significant effects of [CO2]and temperature (P = 0.01) on gs,n, even after taking into account

(a)

(b)

Fig. 3 Stomatal conductance at night (gs,n) (a) and day (gs,d) (b) for allEucalyptus sideroxylon plants from the well-watered and drought treat-ments combined, before the drought treatment started, allowing addi-tional replication (mean + SE, n = 6). Ambient temperature, black bars;ambient + 4�C, grey bars. P values are provided for the two-way ANOVAon the effects of [CO2] (C) and temperature (T) treatments on gs,n; therewere no significant treatment effects on gs,d.

(a)

(b)

(c)

Fig. 4 The relationship between stomatal conductance at night (gs,n) andvapour pressure deficit (D) at 19:00 h for well-watered Eucalyptus

sideroxylon plants in each [CO2] (280, 400 and 640 lmol mol)1) andtemperature (ambient temperature, black circles; ambient + 4�C, greycircles) treatment. Data points represent the mean (± SE) of eachtreatment on each sampling night across the experiment (n = 8–10).P values are shown in Table 1.






differences in D (P = 0.015; Table 1). This result indicates thatgs,n increased as temperature and [CO2] increased from 280 to640 lmol mol)1 across the entire study period.

Effects of [CO2] and temperature on the relationshipbetween gs,n and Ws

Rising [CO2] modified the response of gs,n to decreasing Ws. Asthe drought progressed, gs,n declined in the drought treatment(Fig. 5). However, there was a significant interaction between[CO2] and Ws on gs,n (P = 0.02; Table 2). As soil dried, gs,n

declined more slowly with lower Ws in rising [CO2] (Fig. 5). Therelative sensitivity of gs,n to elevated [CO2] increased as soilbecame drier, such that in dry soils, gs,n was highest underelevated [CO2] and lowest under pre-industrial [CO2].

There was an interactive effect of [CO2] and temperature treat-ment on W0 (the soil water potential at which gs,n reached zero).W0 increased significantly with rising [CO2] in the elevated tem-perature treatment (P < 0.05), indicating that complete stomatalclosure occurred at progressively more negative Ws with rising[CO2], and in the elevated temperature treatment (Fig. 6).

Discussion

We had four main objectives. We tested whether gs,n decreased inresponse to rising [CO2]; we found that an increase in [CO2]from 280 to 640 lmol mol)1 increased gs,n by 85% in well-watered plants, when averaged across temperature treatments.We asked whether gs,n would increase with growth temperature,and an associated increase in D; we found that gs,n increased withrising D in the ambient + 4�C temperature treatment, and thatthe increase was steeper than in the ambient temperature treat-ment; however, this effect varied with rising [CO2]. We testedwhether gs,n would decrease as soil water potential decreased; wefound that gs,n declined with drought and lower soil water poten-tial. Finally, we asked whether rising [CO2] would modify theseresponses to temperature, D and soil water content; we foundthat rising [CO2] and elevated temperature slowed the decline ings,n with drought, and that the soil water potential at which gs,n

was zero was significantly more negative with rising [CO2]. Insummary, [CO2] modified the response of gs,n to temperature, D

and soil water potential. Therefore, we conclude that there weresynergistic effects of rising [CO2] and temperature on gs,n and onthe responsiveness of gs,n to soil water potential.

Table 1 Results of a test of homogeneity of slopes to examine the effectsof [CO2] and temperature on the relationship between nocturnal stomatalconductance (gs,n) and vapour pressure deficit (D) in well-wateredEucalyptus sideroxylon plants across the entire sampling period

Variable df SS F P

[CO2] 1 1547 6.09 0.015Temperature 1 2909 11.45 < 0.001D 1 4089 16.093 < 0.001[CO2]:Temp 1 31 0.124 0.725[CO2]:D 1 1050 4.134 0.043Temp:D 1 1675 6.592 0.011[CO2]:Temp:D 1 829 3.264 0.073Residuals 140

SS, sum of squares.

(a)

(b)

(c)

Fig. 5 The relationship between nocturnal stomatal conductance (gs,n)and soil water potential (Ws) for drought-treated Eucalyptus sideroxylonplants for all [CO2] and temperature treatments (ambient temperature,black circles; ambient + 4�C, grey circles). Data points represent the mean(± SE) of each treatment on each sampling night across the experiment(n = 8–10). P values are shown in Table 2.

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The effect of temperature treatment on gs,n could partially beaccounted for by changes in D, as gs,n increased with increasing Dover a wide range of D in elevated temperatures. Interestingly,these results suggest that gs,n is not functionally linked to gs,d as asimple fractional formulation. Specifically, the increases in gs,n

with rising [CO2] and D in elevated temperature were opposite tothe commonly observed reductions in other studies of gs,d withrising [CO2] and D. However, our results confirmed those ofDawson et al. (2007), who found increasing gs,n with rising D ina field study. These results generally contradict the hypothesis thatgs,n follows the patterns of gs,d, suggesting that gs,n is not readilypredictable from our current understanding of gs,d responses to[CO2], temperature and drought. Given the growing importanceof night-time water loss to plant and landscape water budgets, ourlack of a mechanistic understanding constrains our ability to fullyconsider the consequences of a changing environment.

Increasing [CO2] increased gs,n

Rising [CO2] significantly increased gs,n at the leaf scale. The85% increase in gs,n from pre-industrial to elevated [CO2],

averaged across temperature treatments, was in contrast with theresponse of gs,d, which did not show a consistent pattern ofresponse to rising [CO2] or temperature, and to previous studieson gs,d, which generally found a reduction in gs,d with rising[CO2] (Medlyn et al., 2001; Lewis et al., 2002a; Ainsworth &Long, 2005; Ghannoum et al., 2010b). However, the increase ings,n with rising [CO2] in wet soils was consistent with a recentwhole-tree study of nocturnal sap flux density, which found that,when soil was wet, nocturnal sap flux in E. saligna was higherunder elevated [CO2] than under ambient [CO2] (Zeppel et al.,2011). What causes these changes in gs,n in E. sideroxylon withrising [CO2] and temperature conditions?

Several factors, including treatment differences in D (Dawsonet al., 2007), higher water fluxes in immature leaves (Phillipset al., 2010) and nutrient transport (Caird et al., 2007a), havebeen proposed to account for changes in nocturnal fluxes. In ourexperiment, when D was held constant, [CO2] still had a signifi-cant influence on gs,n, ruling out D as a potential confoundingfactor. Differences in gs,n among treatments may also arise ifleaves of different ages are sampled among treatments. Rising[CO2] may alter leaf phenology (Lewis et al., 2002b; Warrenet al., 2011), and it is possible that the proportion of newlyflushed leaves may increase with rising [CO2]. However, weselected mature, fully expanded leaves for all of our measure-ments, so that differences in leaf age cannot account for suchfindings. Increased nutrient demand with rising [CO2], as a resultof an increased leaf area of trees, could potentially explain theincrease in gs,n. We did not measure leaf nutrients for this study,and so we cannot discount this mechanism. Results on the effectsof nutrient demand on nocturnal fluxes to date are inconclusive(Howard & Donovan, 2007, 2010; Scholz et al., 2007).

Alternatively, perhaps the increase in [CO2] generated morefavourable leaf and stomatal water status at night. Eucalyptsgrown in higher [CO2] may exhibit higher branch conductance(Atwell et al., 2007), thereby potentially allowing faster refillingof roots, stems and branches. Subsequently, this may lead tomore turgid guard cells and less water-stressed leaves at night,thereby facilitating higher stomatal conductance. However, ameta-analysis has shown that plant hydraulic efficiency (capacityto supply water per unit of leaf area) tends to decrease under ele-vated [CO2] (Mencuccini, 2003). Clearly, more detailed studiesare required to examine the potential roles of nutrient transport(Howard & Donovan, 2007, 2010; Scholz et al., 2007), branchconductance and removal of embolisms in the regulation of noc-turnal water fluxes (Dawson et al., 2007).

Increasing temperature and D increased gs,n

Although it has been observed in field-grown trees that gs,n

increases with rising D (Dawson et al., 2007), these data may beconfounded by concomitant increases in temperature with risingD. For example, when night temperature was held constant (c.18�C) and D was allowed to vary through the night, gs,n inRicinus communis declined in both well-watered and droughtconditions with increasing D (Barbour & Buckley, 2007). Inaddition, in a study on six tree species – Pinus ponderosa, Pinus

Table 2 Results of a test of homogeneity of slopes to examine interactionsamong [CO2], temperature and soil water potential (Ws) on nocturnalstomatal conductance (gs,n) of drought-treated Eucalyptus sideroxylon

plants across the drought period

Variable df SS F P

[CO2] 1 343 1.6631 0.199Temperature 1 1598 7.7552 0.006Ws 1 8389 40.7158 < 0.001[CO2]:Temp 1 61 0.2950 0.588[CO2]:Ws 1 1086 5.2722 0.023Temp:Ws 1 687 3.3332 0.069[CO2]:Temp:Ws 1 279 1.3565 0.245Residuals 158 32 556

Fig. 6 The soil water potential (mean + SE) at which the nocturnal stoma-tal conductance (gs,n) is zero (W0) in drought-treated Eucalyptus sideroxylon

plants, with values of W0 obtained using a linear regression betweensoil water potential (Ws) and gs,n for each plant (see the Materials andMethods section for details; n = 3). ANOVA P values are provided for[CO2] (C) and temperature (T) treatments. Ambient temperature, blackbars; ambient + 4�C, grey bars.






radiata, Dacrydium cupressinum, Weinmannia racemosa, Quintiniaacutifolia and Quercus rubra – in the field under varying night-time temperature conditions, significant gs,n was observed, butthere was no significant relationship between gs,n and D (Barbouret al., 2005). In our study, gs,n generally increased with rising D. Atambient temperatures, night-time D ranged from 0.2 to 0.8 kPaand the relationship with gs,n was relatively flat. At elevatedtemperature, night-time D ranged from 0.6 to 1.6 kPa. gs,n wasincreased relative to the ambient temperature treatment, but alsoincreased significantly with D. Thus, there were increases in gs,n

with rising D when D was confounded with higher temperature,but also when temperature was held constant and differences inD were driven by the variation in absolute humidity from nightto night.

The effects of temperature and D on gs,n in E. sideroxyloncontrast starkly with studies on the response of gs,d to D, whichgenerally show that gs,d decreases at higher D and temperatures(Berryman et al., 1994; Cunningham, 2004; Eamus et al.,2008). Importantly, different responses of gs,n and gs,d to Dfurther demonstrate that gs,n responses to climatic variables cannotbe predicted from gs,d responses. Although there have been manyexperiments on the impact of temperature on stomatal conduc-tance, few have examined the effect of changes in D which typi-cally occur with changing temperature (Lewis et al., 2002a; Way& Oren, 2010). Separating the effects of D from the effects oftemperature is critical because stomatal processes are stronglyinfluenced by D as well as by temperature. Further, becausefuture climate projections predict both rising temperatures andD, in conjunction with increasing frequency and severity ofdrought in many regions (Allen et al., 2010), it is essential toaccount for D when studying the interactive effects of thesevariables on nocturnal water fluxes.

Rising [CO2] influenced the response of gs,n to Ws

Rising temperature and [CO2] both increased gs,n, such that thehighest values of gs,n were observed under elevated [CO2] andelevated temperature conditions. These results suggest that thecombined effect of elevated [CO2] and elevated temperature maylead to increased water loss at the leaf level as a result of increasedgs,n, and may partially account for the more rapid soil dry-downobserved in the elevated [CO2] and elevated temperature treat-ment. The rapid dry-down is also partly explained by the higherleaf area in elevated [CO2] trees, which is commonly observed(Ainsworth & Long, 2005). Higher leaf area under elevated[CO2], combined with higher water loss at night under elevated[CO2] and temperature, suggests that E. sideroxylon may experi-ence greater water stress in future climates. Decreasing gs,d withrising [CO2] has been suggested to lead to ‘water savings’ in thesoil, and reduced drought stress (Wullschleger et al., 2002). How-ever, this effect has not always been observed in trees (Schaferet al., 2002; Wullschleger et al., 2002; Duursma et al., 2011;Warren et al., 2011). The effects of reduced gs,d were offset by alarger tree size and higher water fluxes at night under elevated[CO2]. Therefore, our results clearly indicate that reduced gs,d didnot lead to sufficient ‘water savings’ to reduce drought stress. In

fact, tree size had a greater influence than stomatal closure on soilwater content under elevated [CO2], as plants with the lowest gs,d

were also those that experienced the most rapid soil dry-down.Drought has been observed to reduce both gs,n (Barbour &

Buckley, 2007; Cavender-Bares et al., 2007) and nocturnal sapflow (Dawson et al., 2007; Zeppel et al., 2010). Consistent withthese patterns, soil dry-down was associated with a decrease ings,n in the present study. However, the response of gs,n to dryingsoil differed among [CO2] treatments. As soils dried, gs,n declinedmore slowly under elevated [CO2] relative to pre-industrial andambient [CO2]. Further, the soil water potential at which gs,n waszero (W0) was significantly more negative in elevated [CO2] andelevated temperature. Accordingly, across soil moisture levels, gs,n

was higher under elevated temperature and elevated [CO2] ratherthan pre-industrial [CO2]. One key implication of these results isthat stomata may remain open at night under drier soils in ele-vated [CO2] in conditions similar to the present experiment. Thispotential increase in nocturnal water loss may lead to reducedhydraulic redistribution (Howard et al., 2009), which, in con-junction with the higher leaf area under elevated [CO2], in turnmay lead to greater susceptibility to drought stress and mortality.

It is important to obtain an idea of the magnitude of the[CO2] effect on gs,n on the whole-plant water balance (Meinzeret al., 2010). To achieve this, we carried out a simple estimationof [CO2] effects on plant water fluxes using measured values of gs

and D during the daytime and night-time. We estimated E asgs · D. We assumed that D averaged 2.5 and 1 kPa during theday and night, respectively, that gs at ambient [CO2] averaged125 and 30 mmol m)2 s)1 during the day and night, respec-tively, and that gs,d at elevated [CO2] was 20% lower than inambient [CO2], averaging 100 mmol m)2 s)1. If both day andnight gs decrease by 20% at elevated [CO2], then, all else beingequal, we would expect total 24-h E to decrease by 20% at ele-vated [CO2]. If, however, daytime gs decreases by 20%, butnight-time gs increases by 85%, as observed in this study, total24-h E would still decrease, but the reduction will be 12%. Thus,our results suggest that elevated [CO2] could still lead to watersavings, but these savings are reduced when rising gs,n is included(12% compared with 20%).

These estimates, however, assume that the leaf area remainsunchanged. If the leaf area were to increase with rising [CO2],such that whole-plant daytime E remained unchanged, total 24-hE would increase by c. 9%. These ‘back-of-the-envelope’ calcula-tions provide an idea of the importance of these changes in night-time gs for the overall water budget. Increased leaf gs,n, in con-junction with increased leaf area under elevated [CO2], maycause increases in whole-tree water stress, despite reductions ings,d. These findings have implications for the water availability offorests, which, in conjunction with rising [CO2], may also experi-ence extreme heatwaves and more severe and prolonged droughtsunder future climates (De Boeck et al., 2011).

Conclusions

Nocturnal stomatal conductance (gs,n) was substantial in ourstudy, averaging 34% of daytime stomatal conductance (gs,d).

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Under high soil water content, rising [CO2] (from 280 to640 lmol mol)1) increased gs,n by 85% when averaged acrosstemperature treatments. During drought, gs,n declined moreslowly in elevated [CO2] and elevated temperature. Further, thesoil water potential at which gs,n was zero (W0) was significantlylower (more negative) in elevated [CO2] and elevated tempera-ture. As a result, gs,n was highest in elevated [CO2] and elevatedtemperature across the entire drought period. Taken together,these results indicate that there are synergistic effects of rising[CO2] and temperature on gs,n and on the responsiveness of gs,n

to soil water potential. Critically, gs,n may be higher in futureclimates, potentially increasing nocturnal water loss and increasingthe susceptibility of plants to drought, but this cannot bepredicted from gs,d. Consequently, predictive models utilizingstomatal conductance must account for both gs,n and gs,d whenestimating ecosystem water fluxes.

Acknowledgements

We thank three anonymous reviewers for insightful comments,Kaushal Tewari for technical assistance, and Markus Lowe andHonglang Duan for assistance with data collection. This researchwas funded by an Australian Research Council Discovery grantDP0879531 (D.T.T.), a University of Western Sydney Inter-national Science Research Schemes Initiative (71846; J.D.L.),Fordham University (J.D.L.), the Philecology Foundation of FortWorth, Texas through a gift to the University of Arizona (T.E.H.)and a Macquarie University Research Fellowship (M.J.B.Z.).

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Nocturnal stomatal conductance responses to rising [CO2], temperature and drought