Leaf Hydraulic Vulnerability Is Related to Conduit Dimensions and Drought Resistance Across a Diverse Range of Woody Angiosperms

New Phytologist Research

Leaf hydraulic vulnerability is related to conduit dimensions and drought resistance across a diverse range of woody angiosperms

Christopher J. Blackman, Tim J. Brodribb and Gregory J. Jordan School of Plant Science, University of Tasmania, Private Bag 55, Hobart, Tas. 7001, Australia

Summary

Author for correspondence: • Hydraulic dysfunction in leaves determines key aspects of whole-plant responses Tim Brodribb to water stress; however, our understanding of the physiology of hydraulic dys- Tel: +61 3 62261707 function and its relationships to leaf structure and ecological strategy remains Email: [email protected] incomplete. Received: 23 May 2010 • Here, we studied a morphologically and ecologically diverse sample of angio- Accepted: 16 July 2010 sperms to test whether the water potential inducing a 50% loss in leaf hydraulic

conductance (P50leaf) is predicted by properties of leaf xylem relating to water

New Phytologist (2010) 188: 1113–1123 tension-induced conduit collapse. We also assessed the relationships between P50leaf doi: 10.1111/j.1469-8137.2010.03439.x and other traits considered to reﬂect drought resistance and ecological strategy.

• Across species, P50leaf was strongly correlated with a theoretical predictor of vulnerability to cell collapse in minor veins (the cubed ratio of the conduit wall thick- Key words: cavitation, cell collapse, drought resistance, functional traits, leaf hydraulics, ness to the conduit lumen breadth). P50leaf was also correlated with mesophyll pressure–volume, water stress, xylem traits known to be related to drought resistance, but unrelated to traits associated vulnerability. with carbon economy. • Our data indicate a link between the structural mechanics of leaf xylem and hydraulic function under water stress. Although it is possible that collapse may contribute directly to dysfunction, this relationship may also be a secondary product of vascular economics, suggesting that leaf xylem is dimensioned to avoid wall collapse.

implications for plant function because photosynthesis and Introduction growth are dependent on the efﬁcient supply of water to the The ability of plants to maintain hydraulic conductance sites of evaporation (Hubbard et al., 2001; Brodribb & under conditions of water stress is a central driver of species’ Holbrook, 2007). The vulnerability of the hydraulic path- distribution patterns (Engelbrecht et al., 2007). Because way to dysfunction is typically assessed as P50, or the physical tension increases in the xylem when leaf water tension required to cause a 50% decline in hydraulic con- potentials fall as a result of transpirational water loss, the ductance. In leaves, P50 has been linked to plant survival hydraulic pathway from the roots to the shoots is exposed (Blackman et al., 2009; Brodribb & Cochard, 2009), and to stresses that can compromise the capacity of plants to stem P50 has been shown to be adaptive across broad taxo- transport water. Although this tension-induced loss of nomic groups in relation to gradients in water availability hydraulic conductance is often attributed to cavitation (Brodribb & Hill, 1999; Pockman & Sperry, 2000; resulting from air bubbles entering the water column via pit Maherali et al., 2004). membranes (Zimmermann, 1983; Tyree & Sperry, 1989), Hydraulic vulnerability to dysfunction is a highly inte- it may also be a consequence of xylem wall implosion and grated component of a suite of physiological and anatomical cell collapse (Cochard et al., 2004; Brodribb & Holbrook, traits that reﬂect different patterns of hydraulic response to 2005) or increased extra-xylary resistance (Brodribb & drought. In dry climate environments, P50 in stems is Holbrook, 2004). Hydraulic dysfunction has serious correlated with the minimum seasonal water potential

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experienced by species in the field (Pockman & Sperry, Materials and Methods 2000; Bhaskar et al., 2007; Jacobsen et al., 2007). Coordination between loss of leaf hydraulic conductance Plant species and habitat and the regulation of stomatal conductance also suggests that hydraulic vulnerability to dysfunction in leaves plays an We sampled 20 phylogenetically disparate woody angio- important role in plant responses to short-term water stress sperm species from montane rainforest (15 species) and dry (Brodribb et al., 2003). Others have demonstrated a strong sclerophyll forest (five species) on the island of Tasmania, in positive correlation between wood density (WD) and P50 cool temperate Australia (Table 1). Nineteen of these species in stems (Hacke et al., 2001) and have emphasized the were evergreen, but ranged in their degree of scleromorphy inherent costs of increased resistance to hydraulic dysfunc- (as reflected by leaf mass per unit area, LMA) from ) tion in terms of both xylem cell wall reinforcement and 137 g m 2 in the relatively broad leaves of the rainforest ) narrower xylem conduits that reduce hydraulic efficiency species Atherosperma moschatum to 772 g m 2 in the extre- (Hacke et al., 2006) and affect plant growth (Poorter et al., mely scleromorphic needles of Hakea lissosperma (Table 1). 2010). The sample group also included the winter deciduous spe- ) Most studies of the functional and ecological significance cies Nothofagus gunnii with LMA = 102 g m 2. One of the of hydraulic vulnerability have focused on stems (Hacke evergreen species, Tasmannia lanceolata, was vessel-less. et al., 2001, 2009; Maherali et al., 2004). However, water Climatic limits in terms of minimum water availability, as transport in leaves is functionally distinct from that in stems reflected by the fifth percentile of mean annual rainfall ) and, because of their relatively high hydraulic resistance across each species’ distribution, ranged from 351 mm yr 1 ) (Sack & Holbrook, 2006), leaves impose significant for the dry forest species Bursaria spinosa to 1268 mm yr 1 constraints on maximum stomatal conductance and photo- for the montane rainforest species Orites diversifolia (C. J. synthetic capacity (Brodribb et al., 2005). Compared with Blackman et al., unpublished). These species are known to stems, leaves are often more vulnerable to hydraulic dys- vary widely in leaf xylem vulnerability to hydraulic dysfunc- function (Salleo et al., 2000; Brodribb et al., 2003; Choat tion, which, in turn, is closely correlated with the estimates et al., 2005a; Hao et al., 2008). They also differ in xylem of climatic limits for water availability described above (C. structure. Unlike the xylem in stems (Wagner et al., 1998), J. Blackman et al., unpublished). much of the leaf xylem is not reinforced to withstand mechanical rupture under dynamic or static loads, and Vulnerability to hydraulic dysfunction therefore may be vulnerable to cell collapse under negative pressure. Cell collapse has been linked to leaf hydraulic dys- For each species, leaf hydraulic vulnerability curves were function in some conifers (Cochard et al., 2004; Brodribb constructed by measuring the percentage loss of leaf hydraulic & Holbrook, 2005), although cavitation in the petioles and conductance from maximum values (Kmax) in leaves rehy- midribs of a number of conifer and angiosperm species has drated from a range of leaf water potentials (Wleaf). For the also been reported (Nardini et al., 2001; Johnson et al., purposes of these curves, Kleaf was measured by assessing the 2009a). The large volumes of air inside these leaves create kinetics of Wleaf relaxation upon leaf rehydration (Brodribb maximum pressure differentials across xylem cell walls and & Holbrook, 2003). Briefly, hydrated branches from three create a substantial risk of xylem implosion in the leaf veins. individuals of each species were cut early in the morning and However, this phenomenon has not been observed in angio- immediately bagged to arrest water loss. Having transported sperms, and no studies have examined how leaf xylem them to the laboratory, the branches were allowed to desiccate anatomy may relate to drought resistance across different slowly at light intensities sufficient to ensure light-induced ) ) angiosperm species. hydraulic function (c.50lmol quanta m 2 s 1) over a Here, we examine how interspecific variation in the maximum of 48 h or until the percentage loss of Kleaf vulnerability of leaves to hydraulic dysfunction (P50leaf)is approached 100%. Initial Wleaf was determined by measuring related to a number of structural and functional traits in a leaves neighbouring the sample leaf in a Scholander pressure sample of woody temperate angiosperm species with a chamber (PMS, Albany, OR, USA). The sample leaf was broad range of leaf forms and rainfall preferences. then cut under water and allowed to rehydrate for a period Specifically, we tested whether structural properties of the of between 30 and 300 s depending on the initial Wleaf. leaf xylem were related to hydraulic vulnerability, on the Final Wleaf was measured with the pressure chamber, and basis that the wall thickness and lumen diameter of leaf Kleaf was calculated from the ratio of the initial to final Wleaf xylem conduits determine their capacity to resist implosion and the capacitance of the leaf: by water tension. We also tested for relationships between K ¼ C ln½W =W =T Eqn 1 leaf hydraulic vulnerability and other leaf structural and leaf leaf o f functional traits that are widely recognized to influence [Wo, initial leaf water potential (MPa); Wf, final leaf water drought tolerance and reflect plant ecological strategy. potential (MPa); T, duration of rehydration (s); Cleaf, leaf

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Table 1 Compiled data (mean ± SD) of leaf hydraulic vulnerability (P50leaf), absolute leaf hydraulic conductance (Kleaf), leaf mass per unit area (LMA) and wood density (WD) for all species sampled in the study

P50leaf Kleaf ) ) ) ) Family Species ()MPa) (mmol m 2 s 1 MPa) LMA (g m 2)WD(gcm3)

Montane rainforest Asteraceae Olearia pinifolia (Hook.f.) Benth. 1.71 ± 0.03 2.52 ± 0.40 314.1 ± 13.2 0.643 ± 0.016 Atherospermataceae Atherosperma moschatum Labill. 1.48 ± 0.03 3.14 ± 0.45 137.2 ± 0.6 0.558 ± 0.019 Ericaceae Cyathodes straminea R.Br. 2.00 ± 0.15 2.91 ± 0.88 199.5 ± 11.3 0.702 ± 0.041 Ericaceae Gaultheria hispida R.Br. 1.32 ± 0.04 6.75 ± 1.05 172.3 ± 8.1 0.536 ± 0.02 Ericaceae Richea scoparia Hook.f. 1.41 ± 0.09 3.79 ± 0.65 201.2 ± 20.4 0.550 ± 0.015 Myrtaceae Eucalyptus coccifera Hook.f. 2.65 ± 0.15 8.91 ± 1.66 253.4 ± 17.0 0.639 ± 0.012 Nothofagaceae Nothofagus cunninghamii (Hook.) Oerst. 1.70 ± 0.11 3.88 ± 0.81 186.7 ± 13.7 0.583 ± 0.019 Nothofagaceae Nothofagus gunnii (Hook.) Oerst. 1.53 ± 0.04 13.53 ± 0.62 102.0 ± 4.4 0.643 ± 0.02 Pittosporaceae Pittosporum bicolor Hook. 1.87 ± 0.08 3.25 ± 0.67 251.5 ± 18.5 0.730 ± 0.02 Proteaceae Hakea lissosperma R.Br. 2.85 ± 0.24 13.91 ± 2.81 772.4 ± 71.1 0.656 ± 0.022 Proteaceae Lomatia polymorpha R.Br. 1.57 ± 0.12 4.93 ± 0.78 292.8 ± 17.7 0.700 ± 0.033 Proteaceae Orites diversifolia R.Br. 1.25 ± 0.10 9.94 ± 3.17 380.9 ± 16.4 0.743 ± 0.023 Proteaceae Telopea truncata (Labill) R.Br. 1.58 ± 0.08 9.18 ± 1.75 243.3 ± 9.0 0.689 ± 0.019 Rubiaceae Coprosma nitida Hook.f. 1.95 ± 0.04 12.82 ± 1.70 174.7 ± 17.4 0.720 ± 0.02 Winteraceae Tasmannia lanceolata (Poir) A.C.Sm. 1.56 ± 0.13 3.54 ± 0.64 212.0 ± 19.7 0.698 ± 0.021 Dry sclerophyll forest Asteraceae Olearia hookeri (Sond.) Benth. 2.36 ± 0.11 9.83 ± 1.54 227.9 ± 36.0 0.828 ± 0.005 Myrtaceae Eucalyptus pulchella Desf. 4.31 ± 0.36 8.03 ± 1.69 217.8 ± 13.6 0.681 ± 0.045 Pittosporaceae Bursaria spinosa Cav. 3.20 ± 0.08 3.33 ± 0.54 132.1 ± 11.8 0.757 ± 0.019 Proteaceae Hakea microcarpa R.Br. 3.96 ± 0.22 15.11 ± 3.31 628.1 ± 25.8 0.658 ± 0.019 Proteaceae Lomatia tinctoria (Labill) R.Br. 2.08 ± 0.13 4.22 ± 1.56 177.3 ± 10.9 0.769 ± 0.012

) ) capacitance (mmol m 2 MPa 1) calculated from Eqn 2]. from fully expanded sun-exposed shoots. Leaves were always Leaf vulnerability was analysed by plotting Kleaf against Wo. rehydrated from initial water potentials that fitted within We tested the possibility that Kleaf may have changed the range before incipient loss of conductance. The mean during rehydration (Brodribb & Holbrook, 2006) by com- leaf capacitance for each species was then used in Eqn 1 to paring calculated Kleaf values after rehydration times ranging calculate Kleaf. from 30 to 300 s. Calculated Kleaf remained independent of rehydration time in all species, confirming the Pressure–volume traits suitability of rehydration for the measurement of vulnerability. The vulnerability of leaf hydraulic conduc- For each species, one leaf from each of six neighbouring tance to decreasing water potential (P50leaf) for individual plants was sampled for the determination of leaf turgor species was defined as the Wleaf value at which Kleaf had dynamics from pressure–volume (P–V) analysis (Tyree & declined by 50% from the mean maximum rate (Kmax) for Hammel, 1972). For species with small leaves or reduced each species. For each species, P50leaf was calculated by fit- leaf petioles, small terminal shoots were sampled. Branches ting a three-parameter sigmoidal regression function of the were cut under water in the morning and rehydrated until a( )b) form, Kleaf (%) = 100 ⁄ [1 + e Wleaf ], to the Kleaf vs Wo Wleaf was > 0.05 MPa, after which leaves were detached and data. allowed to slowly desiccate on the laboratory bench. During Leaf capacitance was measured directly by rehydrating this process, the leaf weight (weighed to 0.0001 g) and leaves connected to a flow meter that measured the volume water potential were measured periodically. of water entering the leaf through the petiole. Here, leaf The turgor loss point (TLP) was determined from the capacitance was calculated as the volume of water taken up inflection point of the graph of 1 ⁄ Wleaf vs the relative water by the leaf during a transition from Wo to Wf: content. The calculation of osmotic potential at full turgor (Wp) followed Kirkham (2005). The bulk modulus of elas- Cleaf ¼ RF =ðWo Wf ÞT Eqn 2 ticity (e), meanwhile, was calculated from the change in pressure potential at full turgor: [RF, total water uptake into the leaf during rehydration ) ) adjusted for leaf area (mmol m 2 s 1) and temperature fol- e ¼ DP=DR V Eqn 3 lowing Brodribb & Holbrook (2006); Wo, initial leaf water potential (MPa); Wf, final leaf water potential (MPa); T, [P, pressure potential; R, relative water content; V, volume duration of rehydration (s)]. Leaf samples were collected of symplastic water] (Schulte & Hinckley, 1985).

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for the radial elastic modulus of leaf xylem for angiosperms, Water potentials and estimates for wood xylem vary tremendously, with Predawn and midday water potentials were measured upper values as high as 3000 MPa (Bergander & Salmen, during late summer 2008. Measurements were made on three 2000). However, direct experimental analysis of wood fibres replicate samples collected from montane rainforest species subjected to transverse compression suggests that the elastic co-occurring on Mount Wellington and from the dry forest modulus of lignified woody cells falls in the range 50– species Hakea microcarpa (Table 1). Rainfall for Mount 70 MPa (Shiari & Wild, 2004). Because the xylem conduits Wellington is spread fairly uniformly throughout the year in leaf minor veins are generally helically rather than more (Bureau of Meteorology; http://www.bom.gov.au). However, uniformly thickened, they are likely to have relatively low because samples were collected at the end of several years of elastic moduli. We estimated the elastic modulus for the below-average rainfall, midday water potentials at this time current sample of angiosperms by calculating Eqn 4 using a were measured as an estimate of the minimum water poten- range of E values until qcr corresponded to a 1 : 1 relation- tial (Wmin) experienced by plants in the field. To determine ship with P50leaf. The Poisson ratio for lignin (0.28) was the extent to which these environmental conditions used (Innes, 1995). impacted leaf hydraulics in situ, Wmin was transposed to each species’ vulnerability curve, and the percentage loss (if LMA and WD any) of Kleaf was calculated. The distance (MPa) between Wmin and the hydraulic limit (P50leaf) was defined as the LMA and WD were measured for each species in order to safety margin for each species. test for links between leaf vulnerability and ecologically relevant plant traits. LMA was measured in 5–20 mature, fully expanded, sun-exposed leaves within the most recent Leaf xylem dimensions growth cohort, from five individuals of each species. Leaf Anatomical dimensions of the leaf xylem of the minor veins areas were measured as projected areas with a flatbed scan- were measured from transverse sections using a Nikon DS- ner and image analysis software (Image J; National L1 digital camera connected to a light microscope at ·100 Institutes of Health, Bethesda, MD, USA). Leaves were magnification amplified by a 2.5· magnification tube. Leaf then placed in an oven at 70C for at least 3 d, and LMA sections from three individuals of each species were cut was calculated as the ratio between the dry leaf mass and leaf using a freeze-microtome, stained with 5% toluidine blue, area. and mounted on glass microscope slides in phenol glycerine WD was measured on five individuals of each species. jelly. Lumen breadth (b) and wall thickness (t) were mea- From each species, five 4-cm-long wood samples were taken sured on all or, at most, five adjacent conduits from each of from 3–5-yr-old stems, as inferred from counts of growth three minor veins per leaf section per species, giving a total rings. The pith and bark were removed from each sample, of c. 45 conduits per species (see Fig. 2). Both b and t were and fresh volume was measured by water displacement. The measured by adjusting the microscope focus up and down sample mass was determined after drying for at least 3 d at and clearly defining the thickest part of the helically thick- 70C, and WD was calculated as the ratio between oven- ) ened cell wall; b was calculated as the average of the maxi- dried mass and fresh volume (g cm 3). mum and minimum diameters of each lumen. Leaf minor veins were defined as the highest vein order that retained Trait correlations clearly distinguishable xylem and phloem anatomy. Xylem conduits associated with the leaf minor veins were carefully Linear regression analyses were used to examine key inter- distinguished from cells associated with vein endings, which specific trait correlations (Sigmaplot; SPSS Inc., Chicago, were enlarged and comprised of sclereids in some species. In IL, USA). Regressions or differences were considered to be hydrated leaves, the conduits in the leaf minor veins were significant if P £ 0.05. very close to circular in cross-section, with relatively mini- mal contact between cells, and hence did not conform well Results to the double-wall bending model for stem xylem (Hacke et al., 2001). The theoretical collapse pressure for these con- For all species, the response of leaf hydraulic conductance duits (qcr) was therefore calculated using Timoshenko’s to decreasing water potential was sigmoidal, with an initial equation (Eqn 4) for pipe buckling under pressure: plateau, followed by a decline in Kleaf to a minimum value close to zero as Wleaf declined (Fig. 1 shows two vulnerabil- 2 3 qcr ¼ 2E =ð1 v Þðt=bÞ Eqn 4 ity curves typical of species with low and high leaf hydraulic vulnerability; Supporting Information Fig. S1 shows vul- [E, elastic modulus (MPa); v, Poisson ratio; b, conduit nerability curves for the remaining species). Among the ) lumen diameter; t, wall thickness]. There are no estimates sampled species, mean P50leaf ranged from 1.25 MPa in

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(a) (b)

Fig. 1 Percentage loss of leaf hydraulic conductance from maximum (Kmax) as leaf water potentials decline for two representative species with high (Atherosperma moschatum, a) and low (Eucalyptus coccifera, b) leaf hydraulic vulnerability. Curves ﬁtted are sigmoidal functions. Solid vertical lines indicate the water potential at 50% loss of Kleaf (P50leaf).

Orites diversifolia to )4.3 MPa in Eucalyptus pulchella the leaf minor veins of each species, a strong correspon- (Table 1). dence with P50leaf was found if the cell wall elastic modulus At the cellular level, a strong and highly significant linear was set at 200 MPa (Fig. 3). 2 relationship (r = 0.81) was found between mean P50leaf The in situ minimum water potential (Wmin) varied and the mean ratio (t ⁄ b)3 for cell dimensions in the leaf among the 16 species examined, and was also correlated sig- 2 minor veins for each species. A wide range of values was nificantly with P50leaf (Fig. 4; r = 0.48; P < 0.01). The found for this ratio, with high P50leaf associated with nar- safety margin between Wmin and P50leaf also correlated with 2 rower, thicker walled cells (Fig. 2). Across species, mean P50leaf (r = 0.82; P < 0.001), and was generally lower in wall thickness and lumen breadth were unrelated. Of these species that were more vulnerable to hydraulic dysfunction two cell dimensions, P50leaf was only correlated significantly (Fig. 4). However, according to predicted reductions in 2 with lumen breadth (r = 0.25; P < 0.05). Conduit dimen- Kleaf at Wmin for each species, more vulnerable species were sions in the leaf minor veins varied across species, with values not more exposed than less vulnerable species to hydraulic for lumen breadth ranging from 2.78 ± 0.4 lminRichea dysfunction at Wmin (Fig. S2). The species that was pre- scoparia to 5.38 ± 1.4 lminOrites diversifolia and conduit dicted to suffer the greatest loss in Kleaf (27.8%) under wall thickness ranging from 0.44 ± 0.05 lminRichea drought conditions observed in the field was the moderately scoparia to 0.8 ± 0.2 lminCoprosma nitida (Table S1). vulnerable species Olearia pinifolia (Fig. S2). 3 Substituting the mean ratio (t ⁄ b) for each species in Eqn 4 Significant correlations were found between P50leaf and to calculate the theoretical collapse pressure for conduits in the osmotic and bulk elastic characteristics of the leaves. Strong positive linear relationships between P50leaf and both TLP (Fig. 5a) and osmotic potential at full turgor

Fig. 2 Relationship between leaf hydraulic vulnerability (P50leaf) and the conduit dimensions that dictate cell vulnerability to collapse, assuming that conduit geometry in the minor veins approximates a Fig. 3 Regression lines through the theoretical collapse strength of cylindrical tube. Data points are mean ± SD across all surveyed cells. leaf xylem conduits (qcr) calculated for each species (Eqn 4) using Regression line and signiﬁcance level are shown (***, P < 0.001). estimates of cell wall elastic modulus (E) of 100 and 300 MPa for Inset images show detail of the conduit dimensions in the leaf minor conduits in the leaf minor veins. Data points with error bars show veins for two species typical of high (Atherosperma moschatum;i) the variation of qcr calculated for each species using E = 200 MPa. and low (Hakea microcarpa; ii) leaf hydraulic vulnerability. Bars, Using this value of E, the theoretical collapse strength was found to

10 lm. closely correspond to a 1 : 1 relationship with P50leaf across species.

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(a)

(b)

Fig. 4 Relationship between leaf hydraulic vulnerability (P50leaf) and seasonal minimum water potential (Wmin) for a subset of 16 species. Data points are mean ± SD. Regression line and signiﬁcance level are shown (**, P < 0.01). For each species, the difference

between P50leaf and Wmin was deﬁned as the safety margin (MPa) before signiﬁcant hydraulic dysfunction occurred under drought conditions (dashed line). (c)

(Wp; Fig. 5b) were evident across species means; however, P50leaf was only weakly positively correlated with leaf bulk modulus of elasticity (e; Fig. 5c). A trade-off between leaf hydraulic vulnerability to dysfunction and absolute leaf hydraulic conductance (Kleaf) was not observed across the species sampled (Fig. 6a). In addition, P50leaf was not correlated significantly with either WD (Fig. 6b) or LMA (Fig. 6c). Further to this, neither WD nor LMA was correlated significantly with the climatic limits in terms of minimum water availability across each species’ distribution (Fig. S3). Fig. 5 Relationships between leaf hydraulic vulnerability (P50leaf) and cell turgor traits derived from pressure–volume analysis. P50leaf Discussion correlated significantly with both the turgor loss point (TLP, a) and osmotic potential at full turgor (Wp100, b), but only weakly with the e The strong correlation between P50leaf and the theoretical bulk modulus of elasticity ( , c). Data points are mean ± SD for (a) predictor of xylem collapse across our broad sample of spe- and (b), and species’ means for (c). Regression lines and significance levels are shown (***, P < 0.001; *, P < 0.05). cies indicates an important link between xylem anatomy and P50 that may be either causal, or at least indicate a leaf Leaf vulnerability and cell collapse highly conservative margin between the pressure causing cell collapse and P50leaf. Furthermore, our results suggest The response of leaf hydraulic conductance to declining leaf that P50leaf is coordinated with a suite of other leaf traits water potentials across our sample of species was typical of related to drought resistance. However, P50leaf was unre- other species (Brodribb & Holbrook, 2003; Hao et al., lated to leaf hydraulic conductance and other important 2008) in following a sigmoidal trajectory, whereby the per- traits widely recognized as indicators of ecological strategy centage loss of conductance increased as Wleaf approached (e.g. WD and LMA). Amongst our sample of woody plants, TLP. The most conventional explanation for drought- P50leaf therefore represents an important trait dimension induced dysfunction is xylem cavitation and subsequent describing plant ecological strategy in relation to drought conduit embolism (Nardini et al., 2001; Salleo et al., 2001; resistance, independent of traits correlated with carbon Johnson et al., 2009a). However, the strong and significant 3 economy. This is, to our knowledge, the first study to examine correlation between P50leaf and (t ⁄ b) in our sample of directly how interspecific variation in P50leaf is coordinated plants adds weight to other studies suggesting that conduit with both leaf structural traits and other functional traits collapse may also contribute significantly to decreasing Kleaf relevant to land plant ecology. as leaf water potential declines. In studies of conifers,

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(a) 100–300 MPa (as determined here), conduits in the minor veins would begin to collapse when water potentials in these conduits decline to c. P50leaf. In transpiring leaves, the water potential in these minor veins will be higher than that of the mesophyll, but lower than that in the major veins and petiole. As such, water potential in these minor veins may differ from the bulk leaf water potential reﬂected in P50leaf. This discrepancy is difﬁcult to estimate, but may not be large, given that Sack et al. (2005) suggested that resistances upstream and downstream of the minor veins were compa- (b) rable in magnitude. Further to this, a preliminary survey of leaf xylem dimensions across different plant groups indicates that the ratio of cell wall thickness to cell lumen breadth is constant across different vein orders (C. J. Blackman et al., unpublished), suggesting that xylem throughout much of the leaf is equally vulnerable to collapse- induced dysfunction. Such collapse-induced dysfunction has important implications for the physiology of dehydrated leaves, particularly in terms of hydraulic recovery. For example, hydraulic recovery from wall collapse could occur (c) more readily than recovery from xylem embolism when pressures are largely negative (Tyree & Yang, 1992), which may help explain the rapid recovery of Kleaf observed in plant species exposed to moderate drought stress (Lo Gullo et al., 2003; Blackman et al., 2009). Although we showed a strong relationship between 3 P50leaf and (t ⁄ b) , there was no evidence of a direct link between cell collapse and hydraulic dysfunction in leaves. Alternatively, it may be that the linkage between these traits is the result of an evolved coordination between xylem structural strength and P50leaf. This would suggest that a safety factor is maintained between cavitation-induced hydraulic

Fig. 6 Absolute leaf hydraulic conductance (Kleaf) (a), wood density dysfunction and conduit wall collapse in leaf xylem. (WD) (b) and leaf mass per unit area (LMA) (c) as a function of leaf Maximum economy in terms of vein synthesis will be hydraulic vulnerability (P50leaf). Data points are mean ± SD. achieved if the wall reinforcement of xylem conduits in leaf Regression lines and significance levels are shown (ns, P > 0.05). veins is the minimum required to avoid implosion at negative pressures sufficient to cause conduit cavitation. Hacke evidence of progressive leaf xylem cell collapse was shown to et al. (2001) used this economic argument to explain their mirror leaf hydraulic dysfunction (Cochard et al., 2004; observed relationship between P50 and xylem implosion Brodribb & Holbrook, 2005). These authors also showed resistance in woody stems. Our results would be consistent that the theoretical collapse pressure for these conduits, cal- with such a process if our estimate of the elastic modulus of culated from equations that describe cell wall resistance to the conduit walls was incorrect, leading to biased estimation bending (Hacke et al., 2001) and pipe buckling under tension of collapse pressure, or if the water potential in the minor (Timoshenko, 1930), corresponded to actual cell collapse veins is considerably less negative than Wleaf. observed during leaf dehydration. Other studies have emphasized the role of xylem cavita- The relationship between the theoretical collapse pressure tion and subsequent conduit embolism in driving reduced (qcr) for the conduits of our sampled angiosperms and the leaf hydraulic conductance (Nardini et al., 2001; Bucci measured pressure at which losses in Kleaf occurred, depends et al., 2003; Johnson et al., 2009a). Johnson et al. (2009a), on the elastic modulus used to calculate qcr. The estimates for example, provided good evidence that linked leaf of elastic modulus (E) used in our calculations are in broad hydraulic dysfunction to cavitation events in a small num- agreement with estimates of E for xylem conduits in the ber of conifer and angiosperm species. However, these leaves of a number of conifer species (Cochard et al., 2004; authors detected cavitation solely in the leaf midrib where, Brodribb & Holbrook, 2005). If E for xylem in the leaf relative to the minor veins, xylem is more structurally minor veins across our sample of species was on the order of reinforced by surrounding fibres and may thus be more

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prone to cavitation than collapse. We therefore propose Johnson et al., 2009b), suggesting that plants are able to that future studies aimed at identifying the processes that readily reverse leaf hydraulic dysfunction. Indeed, rapid drive leaf hydraulic dysfunction need to carefully examine recovery of Kleaf is likely if the principal cause of leaf different vein orders and how they behave under drying hydraulic dysfunction is cell collapse in minor veins. If, on conditions. the other hand, slowly reversible cavitation is the process of leaf hydraulic dysfunction, this would suggest that a number of these species on Mount Wellington are at the limit of Leaf vulnerability and traits related to drought their ecological tolerance, and that further climatic change Our result of a strong relationship between P50leaf and the towards increasing drought severity may create conditions minimum water potential experienced by plants in the field for catastrophic hydraulic decline, resulting in local popula- (Wmin) is consistent with previous studies in stems tion extinction. (Pockman & Sperry, 2000; Bhaskar et al., 2007; Jacobsen Our results place P50leaf at the centre of a suite of coordi- et al., 2007). Because Wmin in the current study was largely nated leaf traits related to drought resistance. We observed measured in co-occurring species, interspecific variation in significant relationships between P50leaf and pressure–volume Wmin, and therefore P50leaf, might be related to variation in traits that describe the behaviour of leaves during increasing rooting depth that reflects different water foraging strate- water deficit. Among these was a significant relationship gies. Although this is typical of co-occurring species with a between P50leaf and the water potential associated with broad range of vulnerabilities in Mediterranean climates TLP. This result suggests that, despite the strong link (Pockman & Sperry, 2000), here Wmin was measured in co- between P50leaf and xylem anatomy, leaf hydraulic dysfunc- occurring species from montane rainforest where high tion may be partly the consequence of reduced extra-xylary annual rainfall greatly reduces the probability of strong conductance as TLP is approached (Brodribb & Holbrook, moisture gradients developing through the soil profile. 2004; Knipfer & Steudle, 2008). Further to this, previous Under such mesic conditions, high drought resistance is studies have reported a complex association between unlikely to be advantageous. However, we measured Wmin hydraulic dysfunction, stomatal closure and TLP, suggest- at the end of a multi-year period of below-average rainfall ing that leaf hydraulic vulnerability influences the timing of and thus some species may have been affected by soil dry- stomatal closure during water stress (Brodribb et al., 2003). ing. Furthermore, such episodic drought may have contrib- However, it is clear that species with inherently more nega- uted over time to the ecologically anomalous co-existence of tive TLPs consistently show greater tolerance of lower leaf the observed broad range of tolerances to water stress in this water potentials (Kubiske & Abrams, 1994; Bucci et al., generally high-rainfall site. Equally, the observed variation 2004) and greater ecological amplitude in terms of mini- in Wmin among these species might be related to variation mum water availability (Lenz et al., 2006). in stomatal sensitivity to leaf water potential, whereby the The weak, but significant, relationships between P50leaf stomata of species with xylem that is more resistant to and both osmotic potential at full turgor (Wp100) (Fig. 5b) drought-induced dysfunction remain open at lower water and bulk modulus of elasticity (Fig. 5c) suggest that potentials, thereby allowing Wmin to fall below that of more increased leaf hydraulic vulnerability scales with the leaf tis- sensitive species. sue properties outside the xylem. Although low osmotic Interestingly, we also observed a significant relationship potentials at full turgor are entirely compatible with low leaf between P50leaf and the hydraulic safety margin (Wmin – water potentials, and may enable water uptake from drying P50leaf) across the species examined. This indicates that spe- soil, both increases and decreases in bulk elastic modulus cies with more negative P50leaf in the current sample tend have been attributed to aiding survival during drought to exhibit broader safety margins from hydraulic dysfunc- (Roberts et al., 1980). The negative relationship between tion under drought conditions than more vulnerable leaf hydraulic vulnerability and bulk elastic modulus (e) species. Although Meinzer et al. (2009) reported similar supports data suggesting that high e confers greater drought results between hydraulic safety and daily minimum stem resistance by allowing large adjustments of leaf water water potential, these authors argued that large safety potential with only small changes in leaf water content margins were a consequence of high sapwood capacitance (Niinemets, 2001). and low WD. Despite this, we found no evidence to suggest that species with more vulnerable leaves and narrower safety Leaf vulnerability as an independent trait dimension margins were more susceptible to hydraulic dysfunction during drought (or had lower WD). However, some of the Many previous studies of interspecific variation in stem P50 species sampled are likely to experience reduced Kleaf in the have pointed to the possible costs and trade-offs associated field, and may operate at water potentials sufficiently low to with increased resistance to hydraulic dysfunction. For induce some hydraulic dysfunction. Regular loss of Kleaf has example, a trade-off between xylem safety and efficiency has been reported in several species (Nardini et al., 2003; been demonstrated in the stems of a number of arid-land

New Phytologist (2010) 188: 1113–1123 The Authors (2010) www.newphytologist.com Journal compilation New Phytologist Trust (2010) New Phytologist Research 1121 shrub species (Hacke et al., 2009) on the basis that vulnera- understanding both ecosystem functioning and the combined bility to cavitation is correlated with the diameter of the effect of climate change and increased drought on different largest pores (with the least hydraulic resistance) in intervessel plant communities. pit membranes (Choat et al., 2005b). However, this relationship is inconsistent across species (Maherali et al., Conclusion 2004). According to our data, it is also inconsistent in leaves. Indeed, there is no reason to expect such a trade-off Our results point to leaf hydraulic vulnerability to water in leaves, considering that Kleaf and P50leaf are coordinated stress (P50leaf) as a key functional trait strongly linked to with different aspects of leaf structure and function. Kleaf is leaf structure, anatomy and ecological tolerance. In particu- related to traits such as vein density and hydraulic architec- lar, we provide evidence across a diverse group of species of ture (Sack & Frole, 2006; Nardini et al., 2008; Brodribb & a strong relationship between the conduit dimensions (t ⁄ b)3 Feild, 2010) that influence water flux and gas exchange, in leaf minor veins and P50leaf. Further work is necessary to whereas P50leaf is associated with a different suite of traits determine whether this correlation is causal or a secondary related to the ratio of xylem conduit wall thickness and product of vascular economics. We also show strong rela- lumen breadth (t ⁄ b) and pressure–volume characteristics tionships between P50leaf and traits within the leaf symplast, (Sack & Holbrook, 2006). Although not significant, we suggesting that processes in the mesophyll may also contrib- observed a trend towards increased resistance to hydraulic ute to leaf hydraulic dysfunction. Further examination of dysfunction and increased hydraulic conductance in leaves. these relationships will advance our understanding of plant This pattern may reflect the variety of leaf hydraulic path- physiological responses to water stress. ways and structures that influence conductivity in different species. For example, a high concentration of water-con- Acknowledgements ducting sclereids embedded in the mesophyll of the two Hakea species in the current study may account for the very The Australian Research Council provided support in the high Kleaf values recorded for these species (Brodribb et al., form of a grant (DP0878342) to T.J.B. and G.J.J. and a 2010), which were also characterized by low leaf hydraulic post-graduate scholarship to C.J.B. vulnerability. 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