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Article DOI: 10.1093/treephys/tpp018

Article Number: tpp018

First Author: Barbara Beikircher

Article Title: Intraspecific differences in drought tolerance and Corresponding Author: Barbara Beikircher acclimation in hydraulics of Ligustrum vulgare and Viburnum lantana AUTHOR QUERIES – TO BE ANSWERED BY THE CORRESPONDING AUTHOR Dear Author, During the preparation of your manuscript for typesetting, the queries listed below have arisen. Please answer these queries by marking the required corrections at the appropriate point in the text. Electronic file usage Sometimes we are unable to process the electronic file of your article and/or artwork. If this is the case, we have proceeded by:

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Tree Physiology Page 1 of 12 doi:10.1093/treephys/tpp018

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Intraspecific differences in drought tolerance and acclimation in hydraulics of Ligustrum vulgare and Viburnum lantana BARBARA BEIKIRCHER1,2 and STEFAN MAYR1 1

Institut fu¨r Botanik, Universita¨t Innsbruck, Sternwartestr. 15, A-6020 Innsbruck, Austria

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Corresponding author ([email protected])

Received October 3, 2008; accepted March 5, 2009

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Summary An adequate general drought tolerance and the ability to acclimate to changing hydraulic conditions are important features for long-lived woody plants. In this study, we compared hydraulic safety (water potential at 50% loss of conductivity, W50), hydraulic efficiency (specific conductivity, ks), xylem anatomy (mean tracheid diameter, dmean, mean hydraulic diameter, dh, conduit wall thickness, t, conduit wall reinforcement, (t/b)h2) and stomatal conductance, gs, of forest plants as well as irrigated and drought-treated garden plants of Ligustrum vulgare L. and Viburnum lantana L. Forest plants of L. vulgare and V. lantana were significantly less resistant to drought-induced cavitation (W50 at 2.82 ± 0.13 MPa and 2.79 ± 0.17 MPa) than drought-treated garden plants ( 4.58 ± 0.26 MPa and 3.57 ± 0.15 MPa). When previously irrigated garden plants were subjected to drought, a significant decrease in dmean and dh and an increase in t and (t/b)h2 were observed in L. vulgare. In contrast, in V. lantana conduit diameters increased significantly but no change in t and (t/b)h2 was found. Stomatal closure occurred at similar water potentials (Wsc) in forest plants and drought-treated garden plants, leading to higher safety margins (Wsc  W50) of the latter (L. vulgare 1.63 MPa and V. lantana 0.43 MPa). These plants also showed higher gs at moderate W, more abrupt stomatal closure and lower cuticular conductivity. Data indicate that the development of drought-tolerant xylem as well as stomatal regulation play an important role in drought acclimation, whereby structural and physiological responses to drought are species-specific and depend on the plant’s hydraulic strategy.

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Keywords: embolism, hydraulic efficiency, hydraulic safety, phenotypic plasticity, stomatal conductance, vulnerability, xylem anatomy.

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Introduction

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Environments are highly heterogeneous in both space and time (Valladares et al. 2007). Thus, survival and

distribution of sessile organisms such as plants depend strongly on their ability to adjust to environmental variation. This may be of particular importance for longlived woody plants which cannot survive adverse periods in the form of desiccation-tolerant seed stages (e.g., Larcher 1995, Magnani et al. 2002). Adaptation enables plants to optimize their life processes to prevailing environmental conditions at an evolutionary scale. In contrast, acclimation (i.e., positive response to new conditions) occurs within a plant’s lifetime and without altering the genetic constitution (Batanouny 2000, Gienapp et al. 2008). Acclimation processes (structural or physiological) occur in the range of phenotypic plasticity (Sultan 1995, 2000). They are the long-term responses taking place over the course of weeks to years, and thus differ from the short-term physiological regulations. Drought is an important stress factor and can lead to several physiological and structural responses to maintain balanced water relations (e.g., Jones and Sutherland 1991, Larcher 1995, Maherali and Delucia 2000, Froux et al. 2005, Breda et al. 2006, Poyatos et al. 2007, McDowell et al. 2008). Stomatal closure is by far the most efficient reaction to daily and seasonal water shortage (Froux et al. 2005) as it occurs within a few minutes, and it is dynamic and reversible (modulative response; Jones and Sutherland 1991, Larcher 1995, Breda et al. 2006). By closing their stomata, plants prevent a critical decrease in water potential (W; Tyree and Sperry 1988, Saliendra et al. 1995, Cochard et al. 2002, Sperry et al. 2002), though this goes at the expense of reduced CO2 assimilation (Jones and Sutherland 1991, Tyree and Ewers 1991, Cochard et al. 1996, 2002, Breda et al. 2006). In the long term, increased biomass allocation to roots versus leaves and leaf shedding are additional possibilities to reduce water losses. Furthermore, changes in conductivity related to ion concentrations, water storage and refilling processes might play an important role (see Larcher 1963, Gasco et al. 2007, Nardini et al. 2007) in avoiding critical W. However, due to cuticular transpiration, W can continue to decrease even after complete stomatal closure (e.g., Baig

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ACCLIMATION TO DROUGHT IN TWO WOODY SHRUBS

87 and Tranquillini 1980, Larcher 1995, Mayr et al. 2003, 88 Froux et al. 2005) leading to xylem embolism when air 89 enters the pits and breaks the water columns (cavitation; 90 e.g., Sperry and Tyree 1990, Tyree and Ewers 1991, Sperry 91 and Sullivan 1992, Tyree and Zimmermann 2002). 92 Normally, the surface tension at the porous pit membrane 93 (angiosperms) or the pit apparatus itself (conifers) prevents 94 this ‘air-seeding’ (Sperry and Tyree 1990, Tyree et al. 1994, 95 Tyree and Zimmermann 2002), but when W decreases 96 below specific thresholds, these mechanisms fail. Therefore, 97 a sufficient safety margin (i.e., range of W between the time 98 of stomatal closure and cavitation) is required to protect 99 Q1 the xylem from embolism (see Hacke and Sauter 1996, 100 Pockman and Sperry 2000, Froux et al. 2005, Breda et al. 101 2006). As the hydraulic safety (i.e., the ability of the xylem 102 to prevent embolism) and the hydraulic efficiency (i.e., 103 hydraulic conductivity) correlate with several anatomical 104 parameters (e.g., Sperry and Tyree 1990, Sperry and 105 Sullivan 1992, Sperry and Saliendra 1994, Sperry et al. 106 1994, 2007, Hacke and Sperry 2001, Hacke et al. 2001, 107 Tyree and Zimmermann 2002, Tyree et al. 1994), structural 108 modifications of the xylem enable an increase in its resis109 tance to drought-induced embolism. 110 Many authors demonstrated the impacts of drought on 111 hydraulic parameters of conifers (e.g., Maherali and 112 Delucia 2000, Froux et al. 2005, Breda et al. 2006, Ladjal 113 et al. 2007) and tropical, Mediterranean or desert angio114 sperms (e.g., Kolb and Davis 1994, Fotelli et al. 2000, 115 Hacke et al. 2000, Brodribb et al. 2003, Manes et al. 116 2006, Poggi et al. 2007). Only few studies focused on tem117 perate and boreal angiosperms (e.g., Saliendra et al. 1995, 118 Alder et al. 1996, Vogt 2001, Cochard et al. 2002, Tissier 119 et al. 2004, Breda et al. 2006), although acclimation to 120 drought may be particularly important in these predomi121 nantly humid regions with sporadically occurring drought 122 periods. In these regions, an appropriate phenotypic plas123 ticity in hydraulic traits may become even more important 124 when frequency and intensity of drought periods increase 125 due to climate change (Alcamo et al. 2007). There is also 126 little knowledge on structural changes and intraspecific 127 response in hydraulics upon changing environmental con128 ditions (e.g., Mencuccini 2003, Maseda and Fernandez 129 2006). 130 In this study, we analysed intraspecific differences in 131 hydraulic safety, xylem anatomy and stomatal conductance 132 (gs) of Ligustrum vulgare L. and Viburnum lantana L., sub133 jected to different soil moisture conditions. Both species are 134 woody shrubs and often occur associated in the understorey 135 of temperate forests, but can also be found at drier, even 136 rocky sites. Thus, we hypothesized that they do not develop 137 a high cavitation resistance a priori, but are able to accli138 mate within a broad range when subjected to drought. 139 Our study should also reveal whether these two species, 140 growing under identical environmental conditions, might 141 differ in their strategies regarding transpiration and hydrau142 lic acclimation to avoid drought stress.

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Materials

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Plant material

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Measurements were made on L. vulgare (privet) and V. lantana (snowball bush). Both species are up to 4 m high, deciduous, semi-ring-porous shrubs (Grosser 1977); L. vulgare has small (4–6 cm long and 1–2 cm broad), slightly leathery leaves, whereas V. lantana has broader leaves (10–20 cm long and 4–9 cm broad; see Kollmann and Grubb 2002, Fischer et al. 2005). Both species are characterized as ‘European temperate’ (Preston and Hill 1997, Kollmann and Grubb 2002) and show a similar range of distribution (native to Europe, the temperate zone of East Asia and North Africa; Kollmann and Grubb 2002). In Austria (Central European Alps), L. vulgare and V. lantana often occur associated in colline and submontane dry-warm forests (e.g., Erico-Pinion, Quercion pubescenti-petraeae) on calcareous soils (Fischer et al. 2005).

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Study sites and water conditions

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The study was carried out in a natural pine stand (Erico161 Pinion sylvestris) in Telfs (Tyrol, Austria; 630 m a.s.l.; Q2 162 47° 18 0 N, 11°4 0 O) and in the Innsbruck botanical garden Q3 163 (600 m and a.s.l.; 47°16’2 0 N and 11°23’34 0 O). Precipita164 tion (monthly sum) and temperature (monthly mean) did 165 not differ significantly between both sites (Central Institute 166 for Meteorology and Geodynamics, Regional Office for 167 Tyrol and Vorarlberg). The same can be assumed for light 168 conditions, as on both sites plants grew in the shadow of 169 taller trees. Nevertheless, due to soil and irrigation treat170 ments, plants grew on a gradient from optimal to subopti171 mal moisture conditions (irrigated garden plants > forest 172 plants > drought-treated garden plants) as described in 173 the following: (I) Plants in the botanical garden originated 174 from the cuttings of native individual plants and grew on a 175 loamy sand (< 12% clay; FAO 2006). They were irrigated 176 daily so that despite the low field capacity ( 25%), it can 177 be assumed that the soil water potential (Wsoil) remained 178 constantly high and that the plants grew under optimal 179 water conditions. (II) After vulnerability analysis in August 180 2005, the irrigation was stopped. In the following growing 181 seasons (2006–2008), sufficient soil humidity was reached 182 only for few days after rainfalls, while most of the time 183 the soil dried out and the plants grew under suboptimal 184 conditions. Measurements of Wsoil in the rooting area of 185 the same species on similar sites revealed a decrease below 186 1.4 MPa within 4 days. In 2006, climatic conditions were 187 also overall drier than in 2005; e.g., monthly sum of precip188 itation in July 2006 was 109 mm less compared to that in 189 2005. The longest drought period was observed in June 190 2006 with nine successive days without precipitation. (III) 191 Plants in the natural pine stand grew on a humous, humid 192 forest soil (Rendzina). Several accompanying indicator Q4 193 species for mesic conditions (e.g., Molinia caerulea and 194 Frangula alnus) were found. 195

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196

Experimental design

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Vulnerability analyses and conductivity measurements were made on up to 2-m-tall forest plants and irrigated plants in 2005, and on drought-treated garden plants in 2006. Measurements were done in August, thus plants were subjected to respective conditions for the main growing season. For measurements, whole main shoots were cut, wrapped in dark plastic bags and transported to the laboratory. All individual plants were recut twice (about 5 cm) under water and saturated for 24 h. In 2007, further vulnerability measurements on forest plants and drought-treated garden plants were made to account for possible changes in vulnerability to drought-induced embolism. At the same time, gs of forest plants and drought-treated garden plants was measured. Anatomical analyses were done on samples that were previously used for vulnerability analyses.

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Methods

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Vulnerability analysis and conductivity measurements

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Vulnerability curves were obtained by dehydrating samples to differing extents and plotting the fractional (%) loss of conductivity versus the W. Curves were fitted using the exponential sigmoidal equation given in Pammenter and Vander Willigen (1998):

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carried out on saturated samples with Phloxine B (Sigma 247 Chemical Co., St. Louis, MO; 2% (w/v); see Mayr & 248 Cochard 2003) to prove full conductivity at the beginning 249 of dehydration experiments. The PLC was calculated, 250 assuming that ksat corresponded to 0% loss of conductivity, 251 according to Q5 252   k sd  100: ð2Þ PLC ¼ 1  254 k sat For measurements, samples, free of side branches and up 255 to 6 cm long (mean vessel length was about 5 cm), were 256 immersed in distilled water, decorticated, recut with a sharp 257 wood-carving knife and sealed into the silicone tubes of the 258 apparatus (see Mayr et al. 2002). Measurement pressure was 259 set to 4 kPa, and the flow rate was determined using a PC260 connected balance (Sartorius BP61S, 0.1 mg precision, Sar261 torius AG, Go¨ttingen, Germany) by mass registration every 262 10 s. Flow rates were calculated by linear regression over 263 200 s. For measurements, distilled, filtered (0.22 lm) and 264 degassed water containing 0.005% (v/v) ‘Micropur Forte Q6 265 MF 1000F’ (a mixture containing Ag+ and sodium 266 hypochlorite sold for water sterilization and preservation; 267 Katadyn Products Inc., Wallisellen, Switzerland) was used 268 to prevent microbial growth (Sperry et al. 1988, Mayr 269 et al. 2006). The specific hydraulic conductivity (ks) was cal270 culated as follows: 271 ks ¼

PLC ¼ 100=ð1 þ exp ðaðW  W50 ÞÞÞ;

ð1Þ

where per cent loss of hydraulic conductivity (PLC) is the per cent loss of conductivity, a is related to the slope of the curve, W is the corresponding water potential (MPa) and W50 is the W value corresponding to 50% loss of conductivity. The W was measured using a pressure chamber (Model 1000 Pressure Chamber, PMS Instrument Co., Corvallis, OR). Measurements were made on up to 10 cm long end segments of side-twigs (L. vulgare) or on leaves (V. lantana): PLC was calculated by measuring the loss of specific hydraulic conductivity (ks) after dehydration of the samples and ks was measured using a modified Sperry apparatus (Sperry et al. 1988, Chiu and Ewers 1993, Vogt 2001). In both species, PLC was quantified using the paired-segment-method (Sperry and Tyree 1990, Sperry and Sullivan 1992), as flushing was not possible due to the soft and highly vulnerable pith. Therefore, conductivities of two consecutive segments within a main shoot were compared: these samples were taken when the branch was saturated (ksat) and after dehydration (ksd). Between two segments, at least 3 cm was discarded to avoid embolism induced by cutting. Previous experiments proved that this distance is sufficient for paired segment measurements in the species under study (data not shown). All cuttings were done under water. To avoid an underestimation of drought-induced embolism due to native embolism, dye stainings were

Ql ; Ac P

ð3Þ

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where Q is the volume flow rate (m3 s1), l is the segment length (m), Ac is the xylem cross-sectional area (sapwood less heartwood; m2) and DP is the pressure difference between the segment ends (Pa). Calculations were corrected to 20 °C to account for changes in the viscosity of water with temperature.

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Anatomical measurements

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For anatomical measurements, samples were soaked in an ethanol–glycerol–water solution (1:1:1, v/v/v) for 2 weeks. Cross sections were cut using a microtome (Schlittenmikrotom OME, Reichert, Vienna, Austria) and stained with phloroglucinol–HCl (stains lignin brightly red). Anatomical parameters were analysed with a light microscope (Olympus BX 41, System Microscope, Olympus Austria, Vienna, Austria) interfaced with a digital camera (Sony, Cyber-shot, DSC-W17, Vienna, Austria) and image analysis software (ImageJ, 1.37, National Institutes of Health (NIH), Bethesda, USA, public domain). Anatomical measurements were made in radial sectors of the youngest annual ring. Radial sectors were located opposite to compression wood, which is hydraulically of minor importance (see also Mayr and Cochard 2003). Individual conduit areas (i.e., lumen; A) were measured directly and the respective diameters were calculated from A assuming that the conduits had a circular shape:

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ACCLIMATION TO DROUGHT IN TWO WOODY SHRUBS

300 301 302 303

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d ¼2

rffiffiffi A : p

ð4Þ

The mean hydraulic conduit diameter (dh) was calculated from the individual diameters according to (Sperry and Hacke 2004): dh ¼

Rd 5 : Rd 4

ð5Þ

To characterize the conduit wall reinforcement, we measured the ‘thickness to span ratio’ (t/b)h2 (Hacke et al. 2001). Therefore, the wall thickness between the conduits (t) and the conduit wall span (b) were measured directly on conduit pairs that averaged within dh ± 10 lm. The percentage of conducting area per total xylem area (CA) was calculated from the lumen area and the total xylem area of the annual ring.

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Stomatal conductance

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Stomatal conductance was measured on sunny days using an AP4 Porometer (Delta-T Devices Ltd., Cambridge, UK). In the previous evening and in the morning of measurement days, respectively, all plants were saturated by watering. When stomata were fully open, the main shoots were cut and W and gs were measured at intervals until stomatal closure. Thereby, cut shoots were further exposed to conditions at the study site. As stomatal closure occurred relatively fast (within 1 h), it can be assumed that during dehydration neither light and CO2 concentration nor temperature or vapour pressure deficit changed considerably, and stomatal closure thus occurred in response to decreasing W (see Larcher 1995, Hubbard et al. 2001). The W at 10% gs was defined as W at stomatal closure (Wsc). It was calculated by plotting the per cent stomatal conductance (PSC) versus W. Curves were fitted according to Eq. (1), whereby PLC was substituted by PSC and W50 corresponded to W at 50% gs. The safety margin was defined as the difference in W between Wsc and W50 (also see Brodribb and Hill 1999) and was calculated as follows:

337

Safety margin ¼ Wsc  W50 :

ð6Þ

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Number of samples and statistics

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Vulnerability analyses and conductivity measurements were made on up to 30 individuals per species, site and treatment. For anatomical measurements, 7–10 samples per species, site and treatment were analysed. Mean diameter (dmean) and mean hydraulic diameter (dh) were calculated of a total of 194–1096 conduits per annual ring. The conduit wall thickness (t) and the ‘wall thickness to span ratio’ (t/b)h2 were determined for 12–98 conduit pairs per annual ring. Diurnal courses of gs and desiccation experiments were made on five to six individuals per species and site,

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respectively. For each run, three side-twigs per individual plant were taken for W measurements and 10 leaves per individual plant for measuring gs. The differences between sites and treatments within a species were tested with the Mann–Whitney U test (dmean, t and (t/b)h2), the Welch-test (dh, vulnerability thresholds and Wsc) or Student’s t test (ks, gs and CA). All tests were made at a probability level of 5% (except the Welch test) after testing for normal distribution and for variance of the data. Correlation coefficients were tested with Pearson’s product– moment coefficient.

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Results

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Hydraulic safety and hydraulic efficiency

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In L. vulgare and V. lantana, W at 10%, 50% and 90% loss of 362 conductivity (W10, W50 and W90) was significantly less nega363 tive in irrigated garden plants than in drought-treated garden 364 plants (Table 1; Figure 1). According to soil moisture condi365 tions, vulnerability thresholds of V. lantana forest plants 366 were intermediate. In contrast, L. vulgare forest plants were 367 more vulnerable than irrigated plants (Table 1). In both spe368 cies and on both sites, no significant differences in vulner369 ability thresholds were observed between 2006 and 2007 370 (Figure 1). Parameter ‘a’ (slope of the curve) was similar 371 among all vulnerability curves and indicated a steep increase 372 in loss of conductivity over a small range in W (also see 373 Mayr et al. 2002). Significant differences in specific hydraulic Q7 374 conductivity (ks) were only found for irrigated versus 375 drought-treated garden plants of L. vulgare (Table 1). 376 Anatomy

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In both species, conduit wall thickness (t) increased and, accordingly, percentage of conducting area (CA) decreased after drought treatment. For mean diameter (dmean), mean hydraulic diameter (dh) and conduit ‘wall thickness to span ratio’ ((t/b)h2), different responses to the drought treatment were observed: while dmean and dh decreased and (t/b)h2 increased in L. vulgare, the opposite was found in V. lantana (Table 2; Figure 3). The cross-species analysis revealed an increase in t and (t/b)h2, and a decrease in CA with decreasing W50, though correlations were not statistically significant (Figure 2).

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Stomatal conductance

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Stomatal conductance differed significantly between forest plants and drought-treated garden plants: in drought-treated garden plants, gs at moderate W was higher, stomata closed more abruptly and cuticular conductivity (stomatal conductivity after stomatal closure) was lower compared to forest plants (Figure 4). The peak in gs at 3 MPa of L. vulgare forest plants is statistically not significant: gs at

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Table 1. Water potential at 10%, 50% and 90% loss of conductivity (W10, W50 and W90), parameter a (slope of the vulnerability curve), specific hydraulic conductivity (ks), W at stomatal closure (Wsc) and safety margin (Wsc  W50) of irrigated and drought-treated garden plants as well as forest plants of L. vulgare and V. lantana. Circles and asterisks indicate significant intraspecific differences (P < 0.05) of drought-treated from irrigated garden plants and forest plants, respectively. Mean ± SE. Parameter

Garden plants Irrigated

Forest plants Drought-treated

L. vulgare W10 (MPa) W50 (MPa) W90 (MPa) Parameter a ks (m2 s1 MPa1 104) Wsc (MPa) Safety margin (Wsc  W50)

0.66 3.11 5.55 0.90 13.99 – –

± ± ± ± ±

0.64 0.15° 0.34° 0.17 0.53°

0.90 4.58 8.27 0.60 6.57 2.95 1.63

± ± ± ± ± ±

1.05 0.26 0.54 0.12 0.29 0.10

1.12 2.82 4.53 1.29 8.02 2.48 0.34

± ± ± ± ± ±

0.54 0.13* 0.28* 0.29* 0.53 0.26

V. lantana W10 (MPa) W50 (MPa) W90 (MPa) Parameter a

0.47 1.70 3.87 1.01

± ± ± ±

0.45° 0.14° 0.16° 0.14

1.49 3.57 5.65 1.06

± ± ± ±

0.58 0.15 0.29 0.21

0.95 2.79 4.62 1.20

± ± ± ±

0.71 0.17* 0.36* 0.32

ks (m2 s1 MPa1 104) Wsc (MPa) Safety margin (Wsc  W50)

12.86 ± 0.88 – –

11.07 ± 0.93 3.14 ± 0.27 0.43

14.55 ± 0.84 3.32 ± 0.40 0.53

Figure 1. The per cent loss of conductivity (closed circles and solid curve lines) and per cent gs (open circles and dashed curve lines) versus W of irrigated and drought-treated garden plants as well as forest plants of L. vulgare and V. lantana. Vertical lines show W at 50% loss of xylem conductivity (W50; solid lines) and Wsc (dashed lines), respectively. Triangles show data points measured in 2007. Asterisks indicate statistically significant (P < 0.05) regressions.

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Figure 2. (A) Conduit wall thickness (t), (B) conduit ‘wall thickness to span ratio’ ((t/b)h2) and (C) percentage conducting area per total area (CA; C) versus W at 50% loss of conductivity (W50) for L. vulgare and V. lantana. Values are shown for irrigated (L+ and V+) and drought-treated garden plants (L and V) and forest plants (L and V). For forest plants of V. lantana (V), t and (t/b)h2 could not be calculated as not enough tracheid pairs were available (P < 0.05).

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2, 2.5, 3 and 3.5 MPa do not differ significantly. Maximum gs of drought-treated garden plants was significantly higher in V. lantana, while in L. vulgare the difference were less pronounced. Stomatal closure occurred at similar W in forest plants and drought-treated garden plants (Table 1; Figure 1), although W50 of the latter was significantly lower (Table 1; Figure 4). This led to a higher safety margin in drought-treated garden plants (Table 1).

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Discussion

405

A key component in drought tolerance of woody plants is the ability to avoid the formation of embolism (Maherali et al. 2004). This can be achieved by the avoidance of critical W or by building up a xylem tissue which is resistant to low W (e.g., Jones and Sutherland 1991, Tyree and Ewers 1991, Tyree et al. 1994, Sperry 2004). The xylem’s vulnerability to drought-induced embolism was found to be highly plastic in L. vulgare and V. lantana. In both species, W at 50% loss of conductivity (W50) significantly decreased after drought treatment (Table 1). This demonstrates that both species exhibited a considerable acclimation potential (Table 1; Figure 1). It supports our hypothesis that L. vulgare and V. lantana do not develop a high resistance to drought-induced embolism a priori, but are able to acclimate when extended drought periods occur. We assume that this might be similar in other temperate woody plants, as the development of generally drought-tolerant tissues may be even maladaptive: it is costly (building costs, reduction in net carbon assimilation; Magnani et al. 2000, Pockman and Sperry 2000) and thus can lower a plant’s competitive potential. In any case, the acclimation potential seems to be highly species-specific even in species of the same ecosystem. In this study, V. lantana was less drought tolerant than L. vulgare, but at the same time it was able to acclimate to altered soil moisture conditions within a broader range (Table 1). Intraspecific differences in vulnerability to embolism were reported in several studies (e.g., Cochard et al. 1992, Alder et al. 1996, Pockman and Sperry 2000). Even organs within a plant (e.g., shaded versus sunexposed branches, Cochard et al. 1999) or leaf rachises within a canopy (Cochard et al. 1997) were demonstrated to differ considerably in drought tolerance, and Kolb and Sperry (1999) showed that vulnerability to drought-induced embolism can also change seasonally. These and our findings support the hypothesis of Cochard et al. (1997) that xylem vulnerability is not only species- or organ-specific, but can also depend on the acclimation of the plants to environmental conditions (see also Alder et al. 1996, Brodribb and Hill 1999, Maherali and Delucia 2000, Cornwell et al. 2007). Maherali et al. (2004) reported significant phylogenetically independent correlation between W50 and annual precipitation for conifers and evergreen angiosperms but not for deciduous angiosperms. The authors stated that relatively vulnerable temperate species may have arisen from more resistant ancestors and that high resistance to cavitation may be costly in mesic environments. Cavitation resistance depends mainly on pit properties (e.g., size, permeability and stability of the torus) and conduit wall reinforcement (Sperry and Tyree 1990, Sperry and Sullivan 1992, Sperry and Saliendra 1994, Sperry et al. 1994, Tyree et al. 1994, Hacke and Sperry 2001, Hacke et al. 2001, Tyree and Zimmermann 2002, Pittermann et al. 2006). In L. vulgare, cell wall thickness (t) and ‘cell wall thickness to span ratio’ ((t/b)h2) increased significantly

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Table 2. Mean diameter (dmean), mean hydraulic diameter (dh), conduit wall thickness (t), conduit ‘wall thickness to span ratio’ ((t/b)h2) and percentage of conducting area to total area (CA) of irrigated and drought-treated garden plants of L. vulgare and V. lantana. Asterisks indicate significant intraspecific differences (P < 0.05). Mean ± SE. Parameter

L. vulgare garden plants Irrigated

dmean (lm) dh (lm) t (lm) (t/b)h2 CA (%)

19.22 26.46 2.94 0.016 12.31

± ± ± ± ±

V. lantana Drought-treated

0.21 0.00 0.13 0.001 0.84

17.44 25.55 3.81 0.024 10.52

± ± ± ± ±

*

0.21 0.00* 0.15* 0.002* 1.03

Irrigated 21.29 28.15 1.71 0.004 12.13

± ± ± ± ±

Drought-treated 0.31 0.00 0.08 0.000 1.15

23.55 32.53 1.81 0.002 10.72

± ± ± ± ±

0.35* 0.00* 0.08 0.000 0.68

Figure 3. Transverse section of the xylem of L. vulgare (A and B) and V. lantana (C and D) before (A and C) and after (B and D) drought treatment.

460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477

upon drought treatment, whereas in V. lantana no clear trend was observed (Table 2). In the cross-species analysis, an increase in t and (t/b)h2 with decreasing W50, and a decrease in the percentage conducting area per total area (CA) was observed, but correlations were not significant (Figure 2; see also Hacke et al. 2001, Jacobsen et al. 2007). Recent studies also confirmed an indirect impact of conduit diameter on cavitation resistance as larger conduits have more pits, and a greater total pit area increases by chance the size of the single largest pit membrane pore, which offers the weakest capillary barrier to air-seeding (pit area hypothesis; Wheeler et al. 2005, Hacke et al. 2006, Sperry et al. 2007). Under dry conditions, plants may develop smaller pit pores and thicker walls to increase drought tolerance, but they do not necessarily have to reduce conduit diameter. Accordingly, Sperry and Hacke (2004) found that species with high cavitation resistance and thus low-conductive pits do not automatically have a

low overall conductivity, if conduit width and length 478 increase to overcome the added pit resistance. In L. vulgare, 479 mean diameter (dmean) and mean hydraulic diameter (dh) 480 decreased significantly after drought treatment (Table 2; 481 Figure 3) and, accordingly, a significant decrease in ks 482 483 was observed (Table 1). In contrast, in V. lantana no difference in ks was observed after drought treatment although 484 conduit diameters increased (Tables 1 and 2; Figure 3). 485 We conclude that V. lantana developed wider conduits to 486 compensate for a higher resistance at the pits, which was 487 probably related to smaller pit pores less vulnerable to 488 air-seeding. Anatomical adjustments to increase drought 489 resistance are obviously species-specific and based on con490 Q8 491 certed changes of several structures (Table 3). Despite the potentially higher risk for embolism (accord492 ing to the pit area hypothesis), maintaining or even increas493 ing ks by the formation of larger conduits after water 494 limitation may be advantageous: it can optimize water 495

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Figure 4. Stomatal conductance of L. vulgare (A) and V. lantana (B) forest plants (closed circles) and drought-treated garden plants (open circles). Values are averaged in 0.5 MPa W intervals, error bars show SE. Arrows indicate the W at 50% loss of xylem conductivity (W50) for forest plants (closed arrowhead) and drought-treated garden plants (open arrowhead).

Table 3. Overview of general changes in hydraulic and anatomical parameters upon drought treatment of L. vulgare and V. lantana. Arrows in parentheses indicate nonsignificant intraspecific differences between irrigated and drought-treated plants. Different trends between species are marked by solid arrows.

496 497 498 499 500

Parameter

L. vulgare

V. lantana

W10 (MPa) W50 (MPa) W90 (MPa) ks dmean (lm) dh (lm) t (lm) (t/b)h2 CA (%)

()    ¨ ¨ æ ˙ ()

   () ˙ ˙ (æ) ¨ ()

transport and consequently carbon fixation during periods of sufficient water availability and can facilitate higher transpiration at less negative W (e.g., Breda et al. 2006, Cornwell et al. 2007). This is also consistent with the findings of Maherali et al. (2004), who observed an increase in

9

ks with decreasing rainfall in deciduous angiosperms and concluded that the evolution of increased ks may be an important adaptation to water limitation in this functional group. Recent studies have shown that also short-term physiological regulations of ion concentrations may alter ks (see Gasco et al. 2007, Nardini et al. 2007). Adjustments of ks may therefore play an important role in avoiding critical W and thus embolism (see Tyree et al. 1994), and support transpiration control as the main mechanism to maintain moderate W. Adequate stomatal regulations can stave off significant drought effects in the short term. As stated by Sperry (2004) ‘Walking the tightrope between avoiding hydraulic failure and maximizing gas exchange requires rapid and well-regulated stomatal responses to these factors’. A strong relationship of stomatal regulation and avoidance of xylem cavitation was also observed by other authors (e.g., Cochard et al. 2002, Sperry et al. 2002, Brodribb et al. 2003, Breda et al. 2006). We found forest plants of L. vulgare and V. lantana to potentially exhibit high embolism rates before stomatal closure was evident. In V. lantana, W at stomatal closure (Wsc) was at 3.32 MPa. At this W, vulnerability analysis already indicated about 65% loss of conductivity (Table 1; Figure 1). This might be related to relatively humid conditions at the sample site (see the Materials section), where drought probably is rare and, in consequence, the risk of embolism low. In droughttreated garden plants, Wsc was in the same range as in forest plants (Table 1). Also Martinez-Vilalta et al. (2004) reported that different species of pines, growing in habitats of different water availability, closed their stomata within an only small range of needle W. Little plasticity in Wsc was also found in Fagus, Acer and Rhododendron species (Beikircher, unpublished). Due to the acclimation in cavitation resistance (W50; see above) and constantly high Wsc, safety margins (Wsc  W50) increased upon drought treatment in L. vulgare and V. lantana (Table 1; Figure 1). There are several examples for species with large safety margins, including species periodically experiencing severe drought in their habitat (Pockman and Sperry 2000, Breda et al. 2006), species with low vulnerability thresholds (Froux et al. 2005) or species which have no mechanisms of embolism reversal by refilling (Tyree and Sperry 1988). During severe drought periods, stomatal closure may not be sufficient to avoid critical water losses. In this case, structural and physiological long-term acclimation processes to reduce transpiration are required (see Introduction section). Reducing gs can also be achieved by reducing the number and size of stomata or by altering stomatal sensitivity to environmental conditions (see Jones and Sutherland 1991, Larcher 1995). In L. vulgare and V. lantana, the course of gs differed considerably between forest plants and drought-treated garden plants: in the latter, gs remained high at moderate W, stomata closed more abruptly and cuticular conductivity was lower (Figure 4). This was particularly evident in V. lantana, where maximum gs was also

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significantly higher in drought-treated plants compared to forest plants. This may enable drought-treated garden plants to maintain sufficient CO2 uptake at moderate W and reduce water losses at critical W. The remarkable changes in stomatal regulation of V. lantana indicate that this species changed from an isohydric (forest plants) to an anisohydric strategy (drought-treated plants; see Tardieu and Simonneau 1998, Hubbard et al. 2001, McDowell et al. 2008). Isohydric plants reduce gs to maintain a constant W under dry conditions, whereas anisohydric plants allow W to decline with drought. McDowell et al. (2008) stated that anisohydric species tend to occupy more drought-prone habitats compared to isohydric species, although this is not a general feature. Similar to our study, West et al. (2008) also found both strategies within one vegetation type. Overall, the shift to an anisohydric behaviour and observed changes in xylem anatomy (see above) indicate that V. lantana tried to facilitate transpiration under humid conditions. Despite the resultant higher risk for hydraulic failure, this strategy may be advantageous for woody species in temperate zones where drought periods are not likely to occur for a long time. Our measurements on L. vulgare and V. lantana demonstrate that different hydraulic strategies can be successful in the same ecosystem and that these strategies may even be altered within a species due to the changing conditions. Adaptation, acclimation and short-term physiological regulations enable plants to balance trade-offs between avoidance of hydraulic failure and maintenance of photosynthetic activity.

587

Acknowledgments

588 589 590 591 592 593 594 595 596 597 598

This study was supported by alpS – Centre for Natural Hazard Management (Project 2.8AC ‘Recultivation of Rocky Slopes’), by FWF (Fonds zur Fo¨rderung der Wissenschaftlichen Forschung) and APART (Austrian Program for Advanced Research and Technologies). The authors thank the Central Institute for Meteorology and Geodynamics (ZAMG, Regionalstelle fu¨r Tirol und Vorarlberg) for providing climate data. They also thank Mag. Birgit Da¨mon for helpful assistance and Mag. Dagmar Rubatscher for help in pedology. Last but not the least, the authors thank the three anonymous reviewers for their critical and helpful comments.

Appendix

Abbreviations PLC per cent loss of hydraulic conductivity W water potential Wsoil soil water potential W10, W50 and W90 water potential at 10%, 50% and 90% loss of conductivity Wsc water potential at stomatal closure, corresponding to a water potential at 10% stomatal conductance

gs stomatal conductance ks specific hydraulic conductivity of the xylem ksat specific hydraulic conductivity of the xylem at saturation ksd specific hydraulic conductivity of the xylem after dehydration A lumen area dmean mean conduit diameter dh mean hydraulic diameter t conduit wall thickness b conduit wall span (side of a square with equal area to the conduit) (t/b)h2 conduit ‘wall thickness to span ratio’ CA percentage conducting area per total xylem area

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