Water supply and demand remain balanced during leaf acclimation of Nothofagus cunninghamii trees

Water supply and demand remain balanced during leafacclimation of Nothofagus cunninghamii trees

Timothy J. Brodribb and Gregory J. Jordan

School of Plant Science, University of Tasmania, Private Bag 55, Hobart, TAS 7001, Australia

Author for correspondence:Timothy J. Brodribb

Tel: +61 362261707

Email: [email protected]

Received: 4 April 2011

Accepted: 16 May 2011

New Phytologist (2011) 192: 437–448doi: 10.1111/j.1469-8137.2011.03795.x

Key words: economics, leaf hydraulics,optimization, stomata, vein density, xylem.

Summary

• Higher leaf vein density (Dvein) enables higher rates of photosynthesis because

enhanced water transport allows higher leaf conductances to CO2 and water. If

the total cost of leaf venation rises in proportion to the density of minor veins, the

most efficient investment in leaf xylem relative to photosynthetic gain should

occur when the water transport capacity of the leaf (determined by Dvein) matches

potential transpirational demand (determined by stomatal size and density).

• We tested whether environmental plasticity in stomatal density (Dstomata) and

Dvein were linked in the evergreen tree Nothofagus cunninghamii to achieve a bal-

ance between liquid and gas phase water conductances. Two sources of variation

were examined; within-tree light acclimation, and differences in sun leaves among

plants from ecologically diverse populations.

• Strong, linear correlations between Dvein and Dstomata were found at all levels of

comparison. The correlations between liquid- and vapour-phase conductances

implied by these patterns of leaf anatomy were confirmed by direct measurement

of leaf conductance in sun and shade foliage of an individual tree.

• Our results provide strong evidence that the development of veins and stomata

are coordinated so that photosynthetic yield is optimized relative to carbon invest-

ment in leaf venation.

Introduction

Higher rates of photosynthesis are inevitably linked tohigher rates of transpirational water loss from leaves as aresult of the exchange of CO2 and water through stomatalpores. However, transpiration carries substantial energeticcosts involved in constructing and maintaining the special-ized xylem tissue that supplies water to the leaves, therebycreating a trade-off between transpirational cost and photo-synthetic benefit (Cowan & Farquhar, 1977). Evidence ofthis trade-off is apparent in many aspects of plant form andfunction, from the behaviour of stomata (Farquhar et al.,1980) to the architecture of the water transport system.However, the complicated process of calculating costs andbenefits associated with transpiration has prevented a pre-dictive analysis of how whole plants should adapt toenvironmental conditions. Leaves provide a simplified set ofcost ⁄ benefit parameters that could provide a quantitativeinsight into how plants optimize the balance between tran-spirational costs and photosynthesis.

Within leaves, the major cost of transpiration is in theprovision of water supply to evaporating tissues, and herevein density (measured as the total length of leaf vasculartissue per unit leaf area; Dvein) is crucial because of its rolein determining the efficiency of water transport (Brodribbet al., 2007; Boyce et al., 2009; McKown et al., 2010).Additional costs are associated with regulating the demandfor water, as a consequence of the construction, mainte-nance and leakiness (Jordan et al., 2008) of stomata. Thisraises the expectation that plants should coordinate watersupply and demand (Kuppers, 1984; Meinzer & Grantz,1991; Brodribb & Feild, 2000) by maintaining a balancebetween Dvein and stomatal density (Dstomata) to maintainhomeostasis in leaf water content. Furthermore, it shouldbe possible to extend stomatal optimization theory (Cowan,1986) to predict the magnitude of leaf anatomical responsesto changes in key parameters such as light and humidity.

Within leaves, the responses of Dvein and Dstomata to arange of environmental cues raise the possibility thatsupply ⁄ demand coordination exists. For example, leaves

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expanded under low light produce fewer stomata and lowergas exchange rates than leaves grown in the sun (Ashton &Berlyn, 1994; Poole et al., 1996; Kurschner, 1997). Thereis also evidence that plants exhibit plasticity in vein densityin response to different light environments and other envi-ronmental factors (Wylie, 1951; Uhl & Mosbrugger, 1999and references therein; Zwieniecki et al., 2004), suggestingthat leaves are able to control the expression of minor veindevelopment to suit the photosynthetic and hydraulicdemands (Nardini et al., 2005; Sack et al., 2005). The pro-duction of relatively high Dvein in sun leaves should resultin a higher hydraulic efficiency than shade leaves, but thisshould come at a cost proportional to Dvein (Brodribb et al.,2007; McKown et al., 2010).

Despite evidence that the density of veins and stomataare correlated among genotypes (Oguro et al., 1985;Tanaka & Shiraiwa, 2009) and respond similarly to changesin light, it remains to be seen whether plasticity in Dvein andDstomata leads to coordinated modification of the demandand supply of water in the leaf. Furthermore, it is unknownwhether the similar responses of veins and stomata occur asa result of co-ordinated development, or merely a correlatedresponse through pleiotropy (the control of two or moretraits by the same genes), or secondary association via a dif-ferent functional trait, such as leaf expansion. One means ofdiscriminating between these alternative explanations is totest whether the responses to different inductive processesare quantitatively equivalent, and whether there is evidencefor independent control of stomatal and vein density. Giventhe prominence of Dstomata as a proxy for leaf function, andthe emerging potential of Dvein for functional reconstruc-tion of living and fossil plants, it is important to understandthe linkage between these distinct leaf characters during ac-climatory and adaptive modification of the leaf.

Here, we examine the magnitude and direction of plastic-ity of Dvein and Dstomata to test the hypothesis that changesin the architecture of leaf xylem and stomatal density shouldbe coordinated such that hydraulic and stomatal conductivi-ties remain balanced. Using a common temperate treespecies (Nothofagus cunninghamii), we compared withinplant responses to sun and shade with responses to the dis-parate environmental cues embodied in differences in fullsun leaves within populations, among populations andwithin plants. The comparison between sun and shadeleaves on the same plants reflects the effects on developmentof a simple inductive cue, whereas the other comparisonsare more complex, integrating adaptive and developmentalresponses to the macro- and micro-environment. In particu-lar; within plant responses are purely developmental,responses among plants within populations reflect a combi-nation of genetic differences and plastic responses tomicrosite variation, and variation among populationsreflects adaptation combined with plastic responses to themacro-environment. Finally, we examined the possibility

that plasticity in Dvein enables maximum photosyntheticyield relative to leaf xylem investment. Examining sun ⁄ -shade plasticity in a single tree, we extended the gasexchange optimality criteria of Cowan & Farquhar (1977)to the relationship between Dvein and assimilation and com-pared observed plasticity in Dvein with the predictedoptimal condition where photosynthetic yield per unitinvestment is at a maximum.

Materials and Methods

Sampling

Leaves were collected in 2008 from eight naturally occur-ring populations of Nothofagus cunninghamii (Hook.)Oerst. in Tasmania. Nothofagus cunninghamii is a commonevergreen tree that dominates cool-temperate rainforest inTasmania. The sampled populations were ecologicallydiverse, ranging from tall lowland cool-temperate rainforestto subalpine shrubland (Supporting Information Table S1).From each population, we collected one upper branchgrowing in full sun from each of nine trees. From five of thepopulations we also collected one branch in full shade andtwo branches in full sun – one from low on the tree and onefrom high on the tree – from an additional tree (making atotal of 10 trees sampled from these populations). Thebranches were chosen so that we could be confident that theleaves on the shade branches developed in the shade under aclosed canopy and those on the sun branches developed inthe sun. We measured vein density, stomatal density, guardcell length and leaf area from leaves randomly sampled fromthe branches previously described.

Sample preparation and measurement

We measured vein density (Dvein) from leaf paradermal sec-tions prepared as follows: a sharp, double-sided razor wasused to remove the upper epidermis and palisade mesophyllfrom whole leaves. The leaf was then placed in commercialhousehold bleach (50 g l)1 sodium hypochlorite and13 g l)1 sodium hydroxide) until clear, rinsed in water, andreplaced in water for c. 20 min to allow residual bleach todissipate. The resulting section was stained with 1% tolui-dine blue for c. 2 min, rinsed gently but thoroughly in water,and mounted on microscope slides in phenol glycerin jelly.Dvein and the longest distance between veins were measuredusing IMAGEJ (http://rsbweb.nih.gov/ij/) from digitalphotomicrographs of the paradermal sections at ·25 magni-fication. For assessment of Dvein, three fields of view weremeasured from each leaf, while the maximum distancebetween veins was measured in 10 randomly selected areolesper leaf. In each areole the vein edge was traced in IMAGEJand then pen size was increased until no white spaceremained within the areole. The maximum pen size was

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converted to the maximum inter-vein distance (div). For eachtree sampled for sun and shade comparisons, we preparedsections from five shade leaves, five lower sun leaves and fiveupper sun leaves. In addition, from all eight populations onesun leaf was prepared from each of five other plants, andthree leaves from another tree.

Leaf areas were measured from the prepared whole-leafparadermal sections by scanning the leaves at 300 pixels perinch with a flatbed scanner, then measuring the area withimage analysis (IMAGEJ). Three replicate measurements ofeach leaf were made by rescanning the leaves at differentorientations and positions on the flatbed scanner.

Stomatal density (Dstomata) was measured on abaxial cuti-cles (stomata were absent from adaxial surfaces of leaves)prepared from the same leaves on which Dvein and leaf areawere measured. The cuticles were prepared by dismountingthe paradermal section by gently heating it on a hot plate, cut-ting it lengthways, rinsing one half in warm water to removeresidual jelly, and then soaking in warm 10% aqueous Cr2O3

until clear, rinsing thoroughly, staining with dilute (< 0.1%)crystal violet, rinsing, if necessary cleaning with a single-hairpaintbrush, and then mounting on microscope slides, inphenol glycerin jelly. The remaining half leaf was remountedfor future reference. Dstomata was also measured on oldercuticle preparations from the same populations. These prepa-rations included three leaves from each tree from eachpopulation. The trees included all those from which measure-ments of Dvein were made. In all cases, stomatal densities weremeasured from digital photomicrographs of the cuticlepreparation at ·50 magnification (giving 20–50 stomata perfield of view) using the counting tool in IMAGEJ. At least threefields of view were measured from each section.

Because maximum stomatal conductance is determinednot only by Dstomata, but also by stomatal aperture size(Parlange & Waggoner, 1970), we also measured porelength to determine if the relationships of Dstomata to Dvein

may be biased by variation in stomatal size.

Hydraulic and stomatal function of sun and shadeleaves

A single adult tree of N. cunninghamii was used to examineacclimation between vein anatomy, leaf hydraulic conduc-tance (Kleaf) and gas exchange. The tree was located oncampus at the University of Tasmania in Hobart(147�18¢30¢¢E, 42�54¢10¢¢S) growing with tree ferns thatshaded the lower canopy. During spring (October 2010) fivefully sun-exposed and five deeply shaded branches weresampled at c. 11:00 h. Branches were immediately baggedand transferred to the laboratory where they were recutunderwater, leaving a small segment bearing c. 20 well-hydrated, healthy, even-aged leaves. These small branchletswere connected to a flowmeter to measure the transpirationalflux (Brodribb & Holbrook, 2006). Laboratory conditions

were controlled at 22�C and 50% relative humidity and afibre-optic light source was used to provide 800 lmol quantam)2 s)1 at the leaf surface. Leaf temperature was monitoredby two K-type thermocouples pressed against the abaxial sur-face of the leaf. During the establishment of a transpirationalsteady state, a stirred layer was maintained around the leavesby an electric fan. After 3 min at a maximum transpirationalsteady state, branchlets were quickly removed, immediatelywrapped in plastic and foil and transferred to a pressure cham-ber (PMS, Albany, OR, USA) where leaf water potential wasmeasured. The leaf hydraulic conductance was calculated asthe ratio of transpirational flux to leaf water potential, andstandardized to the viscosity of water at 20�C. Logged readingsof humidity, leaf temperature and transpirational steady statewere used to calculate mean stomatal conductance for eachbranchlet. Following Kleaf measurements, a subsample of twoleaves from each measured branchlet was obtained and countsof Dvein and div carried out as detailed in the section ‘Samplepreparation and measurement’.

Optimal vein density

To test whether the observed changes in vein densitybetween sun and shade were optimal in terms of carboninvestment, we established the relationship between assimi-lation rate (A) and stomatal conductance (gs) under sun(1500 lmol quanta m)2 s)1) and shade (150 lmol quan-ta m)2 s)1) conditions for canopy leaves of the sameindividual used above for hydraulic measurements. We thenused a leaf hydraulic model to express gs and A in terms ofDvein according to the constraint of maintaining homeosta-sis in leaf water potential. Finally, we used the sameeconomic model employed to determine optimum gs byCowan & Farquhar (1977) to calculate optimum Dvein forN. cunninghamii leaves in the sun and shade. Rather thanthe optimization criterion being constant ¶E ⁄ ¶A we definedconstant ¶Dvein ⁄ ¶A as the optimal vein investment. Clearly,this dynamic definition of optimality does not take intoaccount important contributing factors to long-term opti-mality (such as leaf lifespan), but we considered this to be agood first approximation for optimal Dvein assuming veincosts increase linearly with Dvein (McKown et al., 2010).

Gas exchange: The relationship between A and gs was deter-mined by fitting curves to instantaneous gas exchange datacollected from the same N. cunninghamii tree used in theprevious section. Measurements of the dynamic response ofstomata to sun ⁄ shade transitions were made on fullyexpanded canopy leaves inside the cuvette of a Li-6400(Licor, Lincoln, NE, USA) gas exchange apparatus.Temperature, vapour pressure and CO2 concentrations weremaintained at 22�C, 1.5 kPa and 380 lmol mol)1 CO2,respectively, while leaves were exposed to light transitionsfrom 1500 lmol quanta m)2 s)1 (sun equivalent) to

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150 lmol quanta m)2 s)1 (shade ⁄ sunfleck equivalent).Leaves were initially allowed to reach equilibrium stomatalconductance in the dark followed by illumination at1500 lmol quanta m)2 s)1 and then 150 lmol quantam)2 s)1 while the dynamic response of stomata was recorded.Final measurements of dark respiration and photosynthesisunder 50 lmol mol)1 CO2 were made in order to determinethe CO2 compensation point and maximum carboxylationcapacity. Four light transitions were performed on each of fiveindividual shoots and A, gs and internal CO2 concentrationlogged every 5 s. Nonlinear and linear portions of the gs vs Afunction could be differentiated empirically, with a carboxyl-ation limited portion of the curve fitted using the Caemmerer& Farquhar (1981) model of photosynthesis and a linear por-tion at low light where electron transport is likely to limit A.The transition from linear to nonlinear portions of the A vs gs

function was determined manually by maximizing r2 valuesfor the two partial regressions.

Hydraulic model: A previous study of extant species fromacross the evolutionary spectrum of vascular plants(Brodribb et al., 2007) demonstrated that the hydraulicconductance of mesophyll tissue in leaves is conservative,and hence that Kleaf could be defined by Eqn 1.

Kleaf ¼ 12 670dm�1:27 Eqn 1

where dm = p ⁄ 2 (div2 + dy

2)1 ⁄ 2; assuming hydraulic flowfrom the vein endings to the sites of evaporation in the leafis apoplastic and that mesophyll cells are capsular in shape,div is the longest horizontal distance from the vein terminalsto the stomata and dy is the distance from the vein terminalsto the epidermis. dm, div and dy are expressed in lm.

Vein density has a major influence on dm because higherDvein yields shorter hydraulic distances between the veinand stomata. As a result of the fundamentally similar geom-etry in reticulate veined leaves, the relationship betweenDvein and div is highly conservative across species, enablingdiv and hence Kleaf to be expressed in terms of Dvein (Eqn 2)(Brodribb & Feild, 2010),

div ¼ 650=Dvein Eqn 2

A mean vein-epidermal thicknesses for N. cunninghamiiof 82 lm was used for the parameter dy.

Under steady-state gas exchange, the water flow intoleaves (F) and the vapour flow out of leaves (E) are equaland can be summarized by Eqns 3 and 4.

F ¼ Kleaf DWleaf Eqn 3

E ¼ tgs Eqn 4

where F and E are hydraulic and evapo-transpirationalfluxes (mmol m)2 s)1), DWleaf is the water potential gradi-ent within the leaf (MPa), t is the leaf to air vapourpressure deficit (kPa) and boundary layer effects areassumed to be relatively small.

Rearranging Eqns 1–4 enables gs to be expressed in termsof Dvein. We parameterized equations with values that aretypical for temperate-tropical environments; 2 kPa for tand 0.25 MPa for DWleaf (Brodribb & Holbrook, 2003).Finally, we used the relationship between gs and A describedin the gas exchange methodology to express A in terms ofDvein under the conditions of water potential homeostasis.

Data analysis

For all analyses, data were tested for heteroscedascity andnormality of residuals. No transformations were deemednecessary. Probabilities were adjusted for multiple compari-sons with the Dunn–Sidak method (Sokal & Rohlf, 1995).

Overall relationship between traits among sunleaves: The variation in sun leaves was analysed usingbivariate, hierarchical analyses of variance, optimized usingrestricted maximum likelihood (REML), and implementedin ASREML (Gilmour et al., 1995). This tool identifies vari-ance components (including covariances between traits),which means that it is possible to identify variances and co-variances uniquely attributable to different levels of asampling hierarchy. We used bivariate REML to identifycorrelations between traits at the among populations,among plants within populations, and among leaves withinplants levels. The method is extremely robust with regard toimbalanced sampling, and the estimated correlations areindependent of other levels of the sampling hierarchy,unlike conventional correlations which are biased by theeffects of variation at lower levels of the hierarchy.

The bivariate analyses were performed on pairwise com-binations of Dstomata, Dvein and leaf area. The analysesfollowed a model with populations, plants within popula-tions and leaves within plants as random effects for eachtrait. To minimize the effects of rounding errors, each vari-able was standardized to a variance of 1. The significance ofcorrelations was tested using the likelihood ratio test, inwhich the whole model is compared to one in which the rel-evant correlation is constrained to be zero (Stram & Lee,1994).

For each trait, least squares means were calculated forplants and populations. These were calculated using univar-iate models, the former with leaf within plant as a randomeffect and plant as a fixed effect, the latter with leaf withinplant and plant within population as random effects, andpopulation as a fixed effect. These analyses were imple-mented in JMP7 (SAS Institute Inc., Cary, NC, USA).Conventional means were calculated for individual leaves.

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Analyses were also performed on the inverse of leaf areaand the inverse of the square of leaf area (which are para-meters that, under an assumption of passive responses toleaf expansion, would be expected to be correlated withDstomata and Dvein, respectively). The partitioning ofvariance components and correlations of these parameterswith Dstomata and Dvein were very similar to those of leafarea, except that (as expected) correlations showed reversedsigns.

Effect of sun ⁄ shade on traits: The effects of sun and shadeon Dstomata, Dvein, guard cell length and leaf area wereassessed using two-way factorial fixed effect analysis of vari-ance with site and light conditions (sun or shade) as the twofactors, and based on leaf means of the parameters. The sunleaves included leaves from both upper and lower sunbranches, because preliminary analysis showed that therewere no differences in these traits between leaves from thesetwo areas, either overall or within any site. These analyseswere implemented in JMP7.

Whether the shade-induced changes in Dstomata and Dvein

were consistent with the variation among sun plants wastested as follows. The relationship between Dstomata andDvein induced by shade for each of the five plants for whichshade leaves were available was estimated as the change inDvein divided by the change in Dstomata (i.e. the slope of therelationship). The slope of the relationship between Dvein

and Dstomata for sun leaves was estimated using standardizedmajor axis regression (a form of model 2 regression), imple-mented in SMATR (Daniel S. Falster, David I. Warton andIan J. Wright; Macquarie University, Sydney, Australia).This method was used because, unlike conventional model1 regression, it provides slopes that are relatively unaffectedby the strength of the correlation. Such slopes were calcu-lated at the among population, among plants withinpopulations and among leaves within population levelsbased, respectively, on population least squares means,residuals from a one-way analysis of variance among sitesusing least squares means for plants, and residuals fromone-way analysis of variance among plants using leaf means.

Results

Variation among sun leaves

In sun leaves, 38–52% of the observed variation in stomataldensity, vein density and leaf area occurred among popula-tions, but there was also large variation among plants withpopulations (30–35% of the total variation), and less varia-tion (9–16%) among leaves within plants (Fig. 1;Table S2). The total variation in guard cell size was rela-tively small compared with the other traits (coefficient ofvariation of only 0.06), and it showed much higher contri-butions at the leaves within plant level than the other traits(Table S2).

Stomatal density and vein density were very strongly cor-related at the among population level (r = 0.99), and lessstrongly (r > 0.5) but still significantly at the among plantswithin populations and among leaves within plant levels

Fig. 1 Percentage of variation (± SE) attributable to different levelsof the sampling hierarchy, for leaf traits within Nothofagus

cunninghamii. Values for the inverse and square root of the inverseof leaf area are very similar to those given for leaf area.

Table 1 Relationships in Nothofagus cunninghamii between pairs of traits in sun leaves at population, plant within population and leaf withinplant levels and contrasting sun and shade leaves of the same plants

Stomatal density and veindensity Vein density and leaf area Stomatal density and leaf area

r ± SE Slope r ± SE Slope r ± SE Slope

Among populations 0.99 ± 0.05 0.016*** )0.85 ± 0.21 )0.025** )0.82 ± 0.24 )1.59**Plants within population 0.61 ± 0.13 0.019** )0.53 ± 0.15 )0.029** )0.17 ± 0.19 )1.51 NSSun leaves within plants 0.50 ± 0.14 0.015*** )0.26 ± 0.16 )0.032 NS 0.12 ± 0.13 2.18 NSSun ⁄ shade responses 0.014*** 0.065 NS 2.90 NS

Within-stratum correlations, slopes of the relationships and tests of significance (***, P < 0.001; **, P < 0.01) are given where applicable.The slope for sun vs shade leaves was calculated as the mean of the ratio of the differences in each trait.






(Table 1; Fig. 2). Stomatal density and vein density wereboth strongly correlated with leaf area traits at the amongpopulation level (r2 > 0.5), but not at the leaf within plantlevel. In each case, the relationships among populationswere approximately linear with intercepts significantlygreater than zero (P < 0.01). At the plant within populationlevel, Dstomata and Dvein showed strongly contrasting corre-lations with leaf area (r = 0.17 vs r = 0.53, respectively).Furthermore, the regressions of vein density on 1 ⁄ �leafarea, stomatal density on 1 ⁄ leaf area and vein density on

stomatal density all had significant positive intercepts(P < 0.01; Fig. 2).

Responses to light environment

Dstomata and Dvein, minor vein thickness and guard cell sizewere all very significantly greater (P < 0.001) in sun leavesthan in shade leaves (Fig. 3). These differences were similarat all sites, as indicated by the lack of a significant interactionbetween the site and sun ⁄ shade effects. The effects of shade

(a)

(b)

(c)

Fig. 2 Associations between traits in sun leaves within Nothofagus cunninghamii. For each pair of traits, scatter plots of population meanswith a linear regression (closed circles and line) and leaves (grey crosses) are given on the left, and within-stratum correlations (± SE) are givenon the right. (a) Vein density vs stomatal density (regression: vein density = 3.4 + 0.0160 · stomatal density; r2 = 0.99), (b) vein density vs1 ⁄ leaf area (regression: stomatal density = 233 + 5919 · 1 ⁄ leaf area; r2 = 0.53), and (c) stomatal density vs 1 ⁄ �leaf area (regression: veindensity = 5.4 + 26.9 · 1 ⁄ �leaf area; r2 = 0.61). Results of significance tests for correlations are given (***, P < 0.001; **, P < 0.01; *,P < 0.05; NS, P > 0.05).

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on Dstomata, Dvein and minor vein thickness were large(Figs 3, 4), whereas the changes in stomatal size (as measuredby guard cell size) were small. Leaf area was much greater inshade leaves at the lowest altitude site (Black River) but dif-fered little (and not significantly) at the other sites. This

altitude-dependent response to shade is consistent with thatobserved by Hovenden & Vander Schoor (2006). For all fiveplants, the change in Dvein in response to shade was in pro-portion to the change in Dstomata, as predicted from theoverall relationships among sun plants. This is indicated bythe close similarity of slopes for the sun ⁄ shade response tothe slope of the overall relationship both among populations,among plants within populations and among leaves withinplants (Fig. 5; Table 1). Z-tests comparing these slopesshowed no significant differences among them. By contrast,the relationships between stomatal or vein density and leafarea were not consistent among these different levels ofresponse, with different slopes apparent.

A strong relationship between the spacing of veins (adeterminant of hydraulic conductance) and Dstomata (anindex of transpiration rate) was demonstrated by the highlysignificant linear correlations between 1 ⁄ div and Dstomata

(Fig. 6). Correlations were observed in both sun(y = 21 422x + 53.783; r2 = 0.45) and shade (y = 26163x ) 23.085; r2 = 0.66) sampled leaves, and in bothcases regression intercepts were not significantly differentfrom zero (Fig. 6a). Despite the different ranges of 1 ⁄ div

and Dstomata in sun and shade leaves, the slopes of the tworegressions were similar.

Acclimation of anatomical and hydraulic properties

Measurements of liquid- and gas-phase leaf conductancesfrom sun and shade leaves of an N. cunninghamii treeshowed that stomatal conductances in shade leaves wereon average 70% lower (30.0 ± 11 compared with 101 ±16 mmol m)2 s)1, respectively; n = 5) and Kleaf was 62%lower (3.24 ± 0.6 compared with 8.55 ± 1.1 mmolm)2 s)1 MPa)1, respectively; n = 5) than in sun leaves.Among all leaves from sun and shade, gs and Kleaf werestrongly correlated (Fig. 7a) with a nonsignificant intercept.The corresponding anatomical variation between sun andshade leaves in this individual was similar to that observedacross populations, with a 35% reduction of Dvein in shadeleaves relative to sun leaves and a 10% decline in the dis-tance from veins to epidermis (113 ± 15 to 127 ± 16 lm,respectively). Although vein spacing was strongly correlatedwith stomatal conductance, both Kleaf and gs were lowerthan predicted according to the expected proportionalitywith 1 ⁄ div (Fig. 7b).

Optimization

From the dynamics of stomatal responses to sun and shadelight intensities (1500–150 lmol quanta m)2 s)1), wedefined the optimum sun and shade gs (58 and96 mmol m)2 s)1, respectively) from the inflection pointof the relationship between A and gs. Using a hydraulicmodel to determine the influence of Dvein on A, we found a

(a)

(b)

(c)

(d)

(e)

Fig. 3 The effect of shade on leaf traits of a single Nothofagus

cunninghamii plant from each of five populations (arranged in orderof increasing altitude). Least squares means (± SE) for shade (shadedcolumns) and sun (open columns) leaves are shown for each plant.For stomatal density (a), vein density (b), vein thickness (c) andguard cell length (d), there were significant differences between sunand shade, and among populations (P < 0.001), and no significantinteraction between the two factors (P > 0.05). For leaf area (e),there was a highly significant interaction (P < 0.001) between thetwo factors, but when the Black River plant was excluded, the shadeand interaction effects were insignificant (P > 0.05).






similar inflection point was evident, reflecting the transitionfrom light to CO2 limitation of photosynthesis (Fig. 8).The vein densities at the two inflection points are theoreti-cally the optimum values for the two light intensities(approximating sun and shade). The observed shift in Dvein

from sun to shade (8.9 to 5.7 mm mm)2) was similar tothe shift in optimum Dvein, although in both sun and shadeleaves we found Dvein was slightly higher (20–35%) thanthe optimum value.

Discussion

It is well known that the densities of stomata and leafveins (Dstomata and Dvein) are plastic, particularly in

response to light. Our data illustrate for the first time thatthe responses of Dstomata and Dvein to environmentalconditions achieve a homeostatic balance between hydrau-lic and stomatal conductivities. Proportionality betweenDstomata and Dvein was strong at different levels of thegenetic hierarchy ranging from within plants to withinand among populations (Table 1). Furthermore, we con-firmed the functionality of the observed anatomicalconnection between veins and stomata by demonstratingthat conductances to liquid and water vapour in sun andshade leaves of N. cunninghamii remain tightly correlated(Fig. 7) and proportional to assimilation rate, therebymaintaining optimal vein allocation relative to photo-synthetic gain.

(a) (b)

(c) (d)

Fig. 4 Representative paradermal sections(a, b) and cuticle preparations (c, d) of sun(a, c) and shade (b, d) leaves of Nothofagus

cunninghamii. Note the higher density ofveins and stomata in the sun leaves. Bar,400 lm.

(a) (b) (c)

Fig. 5 The effect of shade on vein density, stomatal density and leaf area, showing pairwise comparisons within a single Nothofagus

cunninghamii plant from each of five populations for sun (open symbols) and shade (closed symbols) leaves, with standard errors. Linesconnect means from the same plant. Leaf means from all sun leaves are shown as grey circles, with the linear regressions through these leafscores indicated by a dashed line. (a) Vein density vs stomatal density; (b) stomatal density vs leaf area; (c) vein density vs leaf area.

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Optimization and coordination of water supply anddemand in response to irradiation level

Studies of leaf gas exchange demonstrate that angiospermsdynamically regulate the apertures of their stomata tominimize transpirational costs while maximizing CO2

assimilation (Cowan, 1986). Our results comparing sun andshade leaves show that these optimization principles extendto developmental control of veins and stomata in the leaf(Fig. 8). Reduced energy for electron transport means thatshade leaves achieve much lower photosynthetic yields thansun leaves, and coordination between photosynthetic rateand stomatal conductance (Wong et al., 1979) results inmuch lower stomatal conductances in shade leaves.Accordingly we found N. cunninghamii shade leaves pro-duced on average 30% fewer stomata than sun leaves of thesame plants. Assuming both stomatal and vein synthesisincur significant costs to the plant, then the most efficient useof resources would occur if Dvein was reduced to match thelower Dstomata in the shade, and this is what we observed inthe plasticity of these parameters within a single tree (Fig. 8).Although the sequence here is described as a stomata-firstresponse to light intensity, the veins may equally be theprimary sensor, or both cell types simultaneously, withoutaltering the significance of the resultant coordination.

Other papers have demonstrated that vein density isrelated to maximum photosynthesis, stomatal anatomy andtranspiration in sun leaves, and that shade-adapted speciestend to produce lower vein density (Schuster, 1908; Wylie,1951; Nardini et al., 2005; Sack et al., 2005). However, thedata here provide strong evidence that, at least in N.cunninghamii, reduced demand for water in the shade isquantitatively matched by a reduction in hydraulic supply

associated with a substantial reduction in Dvein. Measuredconductances in sun and shade leaves confirmed that paral-lel acclimation of vein and stomatal density to light resultedin a very close coordination between water supply anddemand (Fig. 7a). Optimal allocation of carbon to leafveins occurs if Dvein and maximum assimilation rate vary inproportion, thereby maintaining a constant ratio of yield toxylem investment (Cowan & Farquhar, 1977). We foundthat the observed changes in stomatal and vein densityresulted in a photosynthetic rate that remained close to theoptimal value in terms of instantaneous cost vs benefit(Fig. 8). The observed correlations between vein density,stomatal density and leaf gas exchange therefore invoke adevelopmental coordination of tissues responsible for tran-spiration and water delivery to achieve optimal carbonallocation (Figs 6, 8).

Fig. 6 Regressions of leaf means of stomatal density on1 ⁄ interveinal distance for sun (open symbols, dashed line) and shade(closed symbols, unbroken line) leaves of Nothofagus cunninghamii.Both regressions had highly significant slopes (P < 0.01 in bothcases) but neither had an intercept significantly different from 0,indicating a strong proportionality between vein spacing andstomatal density.

(a)

(b)

Fig. 7 (a) Measured hydraulic and stomatal conductances in sun(open) and shade (closed) leaves from an individual tree ofNothofagus cunninghamii. Data for individual leaves as well asmeans for sun and shade (± SD) are shown. (b) Relationshipsbetween vein spacing and stomatal conductance and Kleaf (insert).Dotted lines show expected proportional relationships. Alldifferences between sun and shade are highly significant (P < 0.01).






It is noteworthy that, although the reduction in Kleaf fromsun to shade was proportional to the reduction in stomatalconductance (Fig. 7), Kleaf in the shade was lower thanexpected given the reduction in Dvein (Brodribb et al.,2007). This lower than expected Kleaf may be a consequenceof a decline in the conductivity of the leaf xylem itself, or inthe conductivity of the mesophyll connection between veinsand the sites of evaporation. Changes in leaf thickness cannotexplain the lower than expected Kleaf in shade leaves becausethe vein-to-epidermal distance in shade leaves was c. 10%shorter than in sun leaves, meaning shade leaves should havedisplayed slightly higher, not lower than expected Kleaf

(Noblin et al., 2008). Alternatively if the distribution ofresistances within the venation had changed such that majorveins had substantially lower conductances in the shade, thenthis also could explain low Kleaf in the shade (McKown et al.,2010). However, this would have required a very largechange in the conductances of the midrib and first veinorders, and we noted only minimal changes in the diameterof these lower vein orders between sun and shade (data notshown). A 15% reduction in minor vein diameter (Fig. 3c)was observed between sun and shade leaves, and this mayhave contributed to reducing Kleaf relative to vein density,but most studies show that the mesophyll hydraulic pathwaydominates leaf resistance (Sack & Holbrook, 2006). Underthese circumstances it seems most likely that reduced meso-phyll conductance in shade plants may augment the effect oflower Dvein. Decreased mesophyll hydraulic conductance in

the shade may have been a result of differences in aquaporindensity or mesophyll anatomy, but considering that all leaveswere measured at light intensities > 500 lmol quan-ta m)2 s)1 it is assumed that aquaporin production was atmaximum levels in both sun and shade leaves (Cochardet al., 2007).

Within and among population responses in sun leaves

Strong patterns of correlation between stomatal density andvein density were not only evident between sun and shadeleaves of N. cunninghamii but also among the sample offully sunlit leaves. Highly significant correlations betweenDvein and Dstomata of sun leaves at population, plant withinpopulation and leaf within plant levels reinforce the argu-ment for vein–stomatal coordination. In particular, thevirtually identical slopes in the stomatal density ⁄ vein den-sity relationship regardless of whether the variation wasinduced by shade or by other factors that vary among andwithin populations provide evidence that multiple extrinsicfactors induce parallel responses in stomatal and vein densi-ties. It is difficult to explain such parallel responses except interms of coordinated responses, particularly given that thelack of within plant association between leaf size and veindensity suggests that variation in vein density is not a pas-sive result of differential whole-leaf development (Fig. 2). Itis not clear what the specific drivers of the among andwithin population variation were, but there were majorenvironmental differences among the sample sites, as aresult of both altitude, which spanned c. 1100 m, and otherfactors including geology and regional climatic patterns.Several factors that vary with altitude, such as decreasingatmospheric pCO2, increasing irradiation (Korner, 1999)and possibly increasing vapour deficits (e.g. Leuschner,2000), may have been implicated. A combination of lowambient pCO2 and high vapour deficit at high altitudesshould favour increased stomatal and vein densities byincreasing demand for water for a given rate of CO2 uptake(Gale, 1972; McElwain, 2004). Indeed, systematic increasesin stomatal density with altitude have been observed,including in one Nothofagus species (Kouwenberg et al.,2007). As little of the observed variation in stomatal densityand leaf size among populations along an altitudinal gradi-ent appears to be genetically based (Hovenden & VanderSchoor, 2006), the strong associations between leaf area,vein density and stomatal density at the population levelmay be largely the consequence of correlated morphologicalacclimation.

Developmental coordination

The coordinated plasticity in veins and stomata seen herewas only partially related to leaf size (Figs 3, 5), meaningthat either a common growth hormone stimulates both vein

Fig. 8 Modelled relationships between vein density and assimilationfor sun (bold curve) and shade conditions (thin curve). The inflectionpoint in both curves is attributable to the transitions from light-limited to diffusion-limited photosynthesis, and this transition pointdefines the optimum gas exchange and vein density for sun andshade conditions. Symbols indicate mean (± SD) measured valuesfrom sun (closed circle) and shade leaves (open circle) from a singletree. Although 20–35% higher than optimum, the observed shift inDvein was similar in magnitude to the predicted values.

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and stomatal development, or one developmental processaffects the other. There is a possibility that auxin might actas a coordinating hormone in this respect. Auxin is funda-mentally important for the differentiation and patterning ofleaf veins (Sachs, 1993) and there is evidence that mutationsaffecting auxin carriers also result in abnormal stomatal dif-ferentiation (Mayer et al., 1993; Spitzer et al., 2009). Acommon role for auxin in vascular and stomatal develop-ment would allow integration of these two developmentalprocesses, but as yet no direct evidence associates auxin instomatal developmental processes. However, high lightintensity appears to directly up-regulate stomatal differenti-ation (Boccalandro et al., 2009), and it is possible thatincreased epidermal porosity in expanding leaves mightincrease auxin fluxes in the leaf lamina and simulate branch-ing of minor veins. Clearly this subject is ripe forinvestigation.

Conclusions

We found very strong coordination of vein density and sto-matal density, in spite of independent control of theseparameters, implying that some linking developmental pro-cess may guide the optimal development of veins to matchthe stomatal demand for water. Similar patterns of coordi-nation occur within and between N. cunninghamii plants,emphasizing the fundamental nature of this developmentalorganizing principle. Other plant species are known to showdiffering degrees of plasticity in vein and stomatal charac-ters, including patterns opposite to those shown here(Schuster, 1908), and future research will show whethercoordination is a universal characteristic or an ecologicallyvariable trait. Given the strong emphasis on stomatal andvein densities as proxies of plant function and atmosphericconditions, it is critical to understand the degree of inde-pendence of the two leaf attributes. In this context, our dataprovide a basis for understanding and predicting the coordi-nated plasticity of stomatal and vein density traits inresponse to environment.

Acknowledgements

This work was supported by ARC Discovery GrantDP0559226 to T.B. and G.J. We thank Scott McAdamand Madeline Murphy for collecting material, and prepar-ing and measuring cuticles and paradermal sections.

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Supporting Information

Additional supporting information may be found in theonline version of this article.

Table S1 Description of collection sites

Table S2 Variance components for stomatal density, veindensity and square root of leaf area based on the nine 2008sites

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Water supply and demand remain balanced during leaf acclimation of Nothofagus cunninghamii trees