Hydraulics of high-yield orchard trees: a case study of three Malus domestica cultivars

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1 Tree Physiology 33, doi: /treephys/tpt096 Research paper Hydraulics of high-yield orchard trees: a case study of three Malus domestica cultivars Barbara Beikircher 1,2, Chiara De Cesare 1 and Stefan Mayr 1 1 Institute of Botany, University of Innsbruck, Sternwartestraße 15, 6020 Innsbruck, Austria; 2 Corresponding author (barbara.beikircher@uibk.ac.at) Received June 13, 2013; accepted October 7, 2013; published online December 5, 2013; handling Editor Frederick Meinzer The drought tolerance of three economically important apple cultivars, Golden Delicious, Braeburn and Red Delicious, was analysed. The work offers insights into the hydraulics of these high-yield trees and indicates a possible hydraulic limitation of carbon gain. The hydraulic safety and efficiency of branch xylem and leaves were quantified, drought tolerance of living tissues was measured and stomatal regulation, turgor-loss point and osmotic potential at full turgor were analysed. Physiological measurements were correlated with anatomical parameters, such as conduit diameter, cell-wall reinforcement, stomatal density and stomatal pore length. Hydraulic safety differed considerably between the three cultivars with Golden Delicious being significantly less vulnerable to drought-induced embolism than Braeburn and Red Delicious. In Golden Delicious, leaves were less resistant than branch xylem, while in the other cultivars leaves were more resistant than branch xylem. Hydraulic efficiency and xylem anatomical measurements indicate differences in pit properties, which may also be responsible for variations in hydraulic safety. In all three cultivars, full stomatal closure occurred at water potentials where turgor had already been lost and severe loss of hydraulic conductivity as well as damage to living cells had been induced. The consequential negative safety margins pose a risk for hydraulic failure but facilitate carbon gain, which is further improved by the observed high stomatal conductance. Maximal stomatal conductance was clearly seen to be related to stomatal density and size. Based on our results, these three high-yield Malus domestica Borkh. cultivars span a wide range of drought tolerances, appear optimized for maximal carbon gain and, thus, all perform best under well-managed growing conditions. Keywords: drought-induced embolism, electrolyte leakage, hydraulic safety, leaf conductance, pv-curve, stomatal regulation, turgor. Introduction Trees are long-lived, woody plants that depend on an optimally balanced hydraulic system. A tree must be sufficiently drought tolerant to withstand the level of drought characteristic of its native habitat. Drought tolerance is determined by a number of physiological and anatomical traits operating from the level of the cell to that of the whole plant (e.g., Larcher 2003, Maherali et al. 2006, McDowell et al. 2008, Sperry et al. 2008, Beikircher and Mayr 2009, Nardini et al. 2011). These traits are often interrelated and correlate well with measurements of photosynthesis and growth. Two key traits are hydraulic safety and stomatal regulation. Hydraulic safety is defined as the xylem s ability to avoid embolism formation (Sperry and Tyree 1990, Tyree and Zimmermann 2002). Water transport in the xylem is driven by a pressure gradient pressure becomes increasingly negative as water flows from root to leaf. This gradient is usually associated with a gradient in water potential (Ψ; Tyree and Ewers 1991). Water flow requires that xylem s water columns are hydraulically continuous (intact). Drought and/or freeze thaw events can cause breakage of these water columns (cavitation) The Author Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

2 Drought tolerance of Malus domestica 1297 resulting in embolism where conduits become air-filled, effectively blocking them. The presence of embolism in a volume of wood reduces its hydraulic conductivity (Tyree and Sperry 1989, Sperry and Tyree 1990, Tyree and Ewers 1991). High hydraulic safety is associated with anatomical features that prevent, or at least reduce, hydraulic failure. Owing to the nature of these features, high hydraulic safety is usually associated with low hydraulic efficiency. Small pit pores and narrow conduits increase hydraulic safety but decrease water transport capacity (Tyree and Ewers 1991, Hacke and Sperry 2001, Tyree and Zimmermann 2002, Sperry and Hacke 2004). A certain level of hydraulic efficiency is required if a tissue is to maintain a water content that retains turgor, avoids cell damage and allows optimal photosynthesis and growth (Hubbard et al. 2001, Larcher 2003, Blackman et al. 2010). The balance between hydraulic safety and hydraulic efficiency is not necessarily uniform within a tree. Many species protect critical parts, such as stem and main branches, by features bestowing high cavitation resistance (vulnerability segmentation; Tyree and Sperry 1989, Tyree and Zimmermann 2002, Beikircher and Mayr 2008). In contrast, a leaf is strategically less critical and often lacks these protective features, making it more vulnerable to cavitation (Salleo et al. 2000, Choat et al. 2005, Blackman et al. 2010, Johnson et al. 2011). Hydraulic safety is also strongly dependent on how stomata are regulated. At this stage, two broad regulation strategies have been distinguished isohydric (hydrostable) and anisohydric (hydrolabile). Isohydric plants exhibit sensitive stomatal control and are able to maintain relatively stable values of Ψ under conditions of environmental change. In contrast, anisohydric plants will tolerate quite large decreases in Ψ before their stomata close (Tardieu and Simonneau 1998, Larcher 2003, McDowell et al. 2008). With its early stomatal closure and thus maintenance of high leaf water potential (Ψl), Malus has been defined as an isohydric species (Landsberg et al. 1975, Jones 1992, Lauri et al. 2011). However, recent studies show that this view may be too simplistic as distinctions are not always clear. Also, individual plants can switch their behaviours depending on water supply (Franks et al. 2007, Domec and Johnson 2012, Zhang et al. 2012). The difference in the value of Ψ between the point of stomatal closure and that of cavitation can be defined as the safety margin (Breda et al. 2006, Beikircher and Mayr 2009). Plant species native to habitats experiencing periodic severe drought tend to have larger safety margins than species native to mesic habitats, which can even exhibit negative margins (Pockman and Sperry 2000, Breda et al. 2006, Beikircher and Mayr 2009, Meinzer et al. 2009, Mayr et al. 2010). However, depending on a plant s general strategy for coping with drought, isohydric and anisohydric species, and also species having wide and narrow safety margins, can occur side by side in the same habitat (Vilagrosa et al. 2003, McDowell et al. 2008). At this stage, no apparent associations have been found between phylogeny, leaf phenology, functional group or habitat (Johnson et al. 2009). An advantage of an anisohydric strategy with narrow safety margins is an enhanced carbon gain as the open stomata enable photosynthesis even at low Ψ; the corresponding disadvantage is an increased risk of hydraulic failure under prolonged drought (Jones and Sutherland 1991, Brodribb and Holbrook 2004, Franks et al. 2007, McDowell et al. 2008). Besides physiological regulation, a plant s transpiration is also influenced by its stomatal characteristics. For example, high stomatal density improves the control of transpiration and photosynthetic assimilation rate. Also, leaf conductance is closely related to stomatal pore length (Aasamaa et al. 2001, Sack et al. 2003, 2005, Eensalu et al. 2008). In natural ecosystems, local plant populations (ecotypes) adapt to the prevailing climate and soil conditions over long periods. In agricultural systems, high-yield cultivars are usually grown, which have been selected for maximal fruit quality and yield under the best possible conditions in regard to nutrient and water supply, etc. With Malus, over the millennia innumerable crossings of wild forms have been made with deliberate or inadvertent selection for a wide range of properties, including drought and frost tolerance. This has led to the existence today of tens of thousands of named apple cultivars and varieties, though few are of economic importance (Silbereisen and Kutzelnigg 1995). It is probably safe to assume that drought tolerance varies widely among cultivars. However, there are only a few studies on hydraulic safety in apple; supposed differences in drought tolerance among the major cultivars are based largely on subjective opinion rather than on objective research. The significant increases observed in apple yield and quality in recent decades have been gained as a result of improved agricultural techniques and orchard managements rather than through breeding (Jones 1992). Quality and productivity of apples depend heavily on water supply as water stress strongly reduces photosynthesis (Naor and Girona 2012). Thus, in commercial apple productions, irrigation is widely used to minimize hydraulic limitation of growth and productivity (Naor and Girona 2012). The aim of this study was to investigate hydraulic parameters in three economically important apple cultivars: Golden Delicious, Braeburn and Red Delicious. This work should not only provide detailed information on the variability of drought tolerance in these species but also insight into the hydraulic behaviour of plants growing under optimized conditions in terms of water and nutrient supply. Vulnerability of branch xylem and leaves to drought-induced embolism and stomatal regulation was assessed. Also measured were the specific hydraulic conductivity of branch xylem, stomatal conductance, turgor-loss point, osmotic potential at full turgor and drought tolerance of living tissues. The measured physiological parameters were correlated with observations on xylem and stomatal anatomy. Based on anecdotal information (i.e., Golden Delicious can also be grown Tree Physiology Online at

3 1298 Beikircher et al. on drier sites; Silbereisen and Kutzelnigg 1995, Mühl 2007; also see Plant material and climate ), we hypothesize that Golden Delicious will exhibit more traits associated with drought tolerance than either Braeburn or Red Delicious. Owing to apple s classification as an isohydric species, we anticipate that all three cultivars will exhibit early stomatal closure and high safety margins. Materials and methods Plant material and climate Measurements were made on three Malus domestica Borkh. cultivars: Braeburn, Golden Delicious and Red Delicious. General descriptions classify Braeburn and Red Delicious as being moderately resistant to frost and also as performing best in warm climates and with moist soils. In contrast, Golden Delicious (not closely related to Red Delicious) is frost resistant and can also be grown on drier sites, although for optimal fruit quality Golden Delicious does respond favourably to supplemental water (Silbereisen and Kutzelnigg 1995, Mühl 2007). All three cultivars are grown in commercial orchards in northern Italy (Latsch, South Tyrol, 600 m above sea level; N, E). The orchards are situated close to one another in a dry inner Alpine valley with exceptionally high sunshine duration (315 days per year), high annual mean temperature (9.6 C) and low precipitation ( mm). At the study site, air temperature (daily means) range from 25 C in mid-summer to 10 C in winter (Figure 1). Daily irrigation of the orchards during the vegetation period ensured optimal water supply. From soil thawing in January/February to freezing in November/December, the soil Ψ never fell below 0.20 MPa (Figure 1). Trees were supplied with optimal nutrients and, due to tree shape (spindlebush) and the arrangement of plants within and between rows, grew under favourable light conditions. The study trees were all ~13 years old, 3 m tall and had mean diameters at breast height (DBH) of 3.1 cm (Red Delicious), 3.4 cm (Golden Delicious) and 4.6 cm (Braeburn). At the study site, air temperature at 250 cm height (upper crown) as well as soil temperature and soil Ψ at a depth of 15 cm were recorded at 1-min intervals. Every 15 min, means of these values were stored in a datalogger (ModuLog 3029; sensors and datalogger of Environmental Measuring System, Brno, Czech Republic). Sampling and preparation of branches All measurements were made on ~30 trees per cultivar in the vicinity of the meteorological stations. On each tree, two or three east or west exposed branches (rows aligned northsouth) were chosen at random. Owing to pruning, branches were highly branched and crooked and consisted of several long and short shoots. Branches were cut at the base, then immediately re-cut underwater (above the first annual shoot), wrapped in dark plastic bags and transported to the laboratory. There, branches were promptly re-cut (removing ~5 cm) and saturated for 24 h in clean water. For measurements, only the two most distal shoots per branch were used. With this sampling procedure, artefacts due to air entry during sampling (Melcher et al. 2012, Cochard et al. 2013, Wheeler et al. 2013) could be avoided. In control measurements, no artificial induction of embolism was observed. Figure 1. Daily means of air temperature (grey line) and soil Ψ (black line) from January 2010 to December Dotted lines indicate freezing and thawing soils. Tree Physiology Volume 33, 2013

4 Drought tolerance of Malus domestica 1299 Next, saturated branches were dehydrated to varying extents on the bench. To allow equilibration of Ψ within them so that accurate stem Ψ measurements could be made, branches were again wrapped in dark plastic bags for min prior to measurements. In the following, if not stated otherwise, Ψ refers to measurements on leaves but is assumed to represent the Ψ value of the whole branch. In contrast, measurements of Ψl for stomatal conductance and midday Ψl were made on transpiring leaves and thus are likely to differ from stem Ψ (see Meinzer et al. 2001). Vulnerability analyses of branches For vulnerability analyses, Ψ and respective percentage loss of hydraulic conductivity (PLC) were measured on ~20 branches per cultivar (see Sampling and preparation of branches ). The Ψ values reported are the averages of at least three leaf measurements made with a pressure chamber (Model 1000 Pressure Chamber, PMS Instrument Company, Corvallis, OR, USA). Loss of hydraulic conductivity was quantified by measuring the increase in hydraulic conductivity after removal of xylem embolism by repeated highpressure flushes with a modified Sperry apparatus. The PLC was calculated from the ratio of the initial to the maximum conductivity (Sperry et al. 1988, Beikircher and Mayr 2009). Therefore, ~5-cm-long samples were cut from the branches underwater, decorticated, re-cut with a sharp wood-carving knife and sealed in the silicone tubes of the apparatus. The measurement pressure was set to 4 kpa and the flow rate was determined with a PC-connected balance (Sartorius BP61S, 0.1 mg precision, Sartorius AG, Göttingen, Germany) by weight registration every 10 s and linear regression over 200 s. Flushing (80 kpa, 20 min) was repeated until measurements showed no further increase in conductivity (an additional control measurement was made to exclude problems due to clogging). For measurements, distilled, filtered (0.22 µm) and degassed water containing 0.005% (v/v) Micropur Forte MF 1000F (Katadyn Products, Inc., Wallisellen, Switzerland) was used to prevent microbial growth (Beikircher and Mayr 2009). Specific conductivity (k s ) was calculated as k s Q l = A P c where Q is the volume flow rate (m 3 s 1 ), l is the segment length (m), A c is the xylem cross-sectional area (sapwood minus heartwood; m 2 ) and ΔP is the pressure difference between the segment ends (Pa). On a total of up to 49 measurements per cultivar, vulnerability analyses were then made by plotting PLC versus Ψ and curves were fitted using an exponential sigmoidal equation given in Pammenter and Vander Willigen (1998): (1) 100 PLC = + exp( a( Ψ Ψ )) 1 50 where PLC is the per cent loss of hydraulic conductivity, Ψ is the corresponding xylem pressure (MPa) and a is related to the slope of the curve. Ψ 50 is the Ψ value corresponding to 50% loss of conductivity. The values of Ψ at the onset of cavitation (Ψ at 12%; Ψ 12 ) and at full embolism (Ψ at 88%; Ψ 88 ) were also determined. Vulnerability analyses of leaves and pressure volume analyses Leaf vulnerability analyses were made using the rehydration technique with a total of up to 67 leaf pairs from up to 15 branches per cultivar (see Sampling and preparation of branches ). First, a pressure volume analysis (pv-curve) was carried out to determine the turgor-loss point (TLP). For this, 10 fully saturated leaves per cultivar were taken (from five different branches) and dehydrated while their Ψl and fresh weights were measured at regular intervals. The relative water content (RWC) was calculated from their fresh, saturated and dry weights. For each leaf, the inverse Ψl (1/Ψl) was then plotted versus RWC (pv-curve) and TLP and osmotic Ψ at full turgor (Πo) were determined (Richter et al. 1981, Bartlett et al. 2012) separately for each leaf and the values averaged. Vulnerability analyses of leaves were made following the experimental procedure of Brodribb and Holbrook (2003; see also Charra-Vaskou and Mayr 2011). Initial Ψl was measured on one leaf (Ψl before rehydration; Ψ i ) and a neighbouring leaf was cut under distilled water and allowed to rehydrate via the petiole for s before measurement of final Ψl (Ψl after rehydration; Ψ f ). Leaf hydraulic conductance (K l ; mmol m 2 s 1 MPa 1 ) was then calculated as K l (2) i/ f = C ln Ψ Ψ (3) t where C is leaf capacitance (mmol m 2 MPa 1 ) and t is time (s) of rehydration. The value of C was calculated from the pressure volume relationship according to Brodribb and Holbrook (2003): from the RWC versus Ψ graph, linear equations for data before and after the inflection point (i.e., TLP) were calculated. Leaf capacitance C was calculated as ( RWC/ Ψ) ( DM/LA) ( WW/DM) C = M where ΔRWC/ΔΨ (MPa 1 ) is the slope of the curve before and after TLP, respectively, DM is leaf dry mass (g), LA is leaf area (m 2 ), WW is mass of leaf water at 100% RWC (g) and M is (4) Tree Physiology Online at

5 1300 Beikircher et al. molar mass of water (g mol 1 ). Depending on whether the values of Ψ i and Ψ f were above or below TLP, C pre- or post-turgor loss was used for the calculation of K l. In cases where Ψ i and Ψ f spanned TLP, C pre- and post-turgor loss was averaged for the calculation of K l. Thus we obtained K l values fitted in the curve better than calculation with either pre- or post-turgor loss C, depending on the relative distances of Ψ i and Ψ f from TLP as suggested by Brodribb and Holbrook (2003). For vulnerability analyses, the per cent loss of K l was calculated from the ratio of K l for a given Ψl and the K l maximum value and these were plotted against Ψ i. Curve fitting and calculations of vulnerability thresholds were done similarly to the vulnerability analyses of branches (Eq. (2)), whereby Ψ was substituted by Ψ i and Ψ 50 corresponded to Ψ i at 50% loss of K l. Vulnerability of living tissues to dehydration Drought tolerance of living tissues was analysed using the electrolyte leakage method (Morin et al. 2007) on 12 branches per cultivar. On prepared branches (see Sampling and preparation of branches ), Ψ was measured and ~3 cm long samples were cut off. These were decorticated and periderm was cut into 2-mm-thick strips and the wood into 1- to 2-mm-thick discs. From three leaves per branch, discs (0.6 cm in diameter) were cut with a cork borer, carefully avoiding major veins. Prepared tissues were placed separately into centrifuge tubes filled with 15 ml distilled water and shaken at 5 C for 24 h on a horizontal shaker (ST5 Bidimensional shaker, CAT, Staufen, Germany). After equilibration at room temperature, electrolytic conductivity (C 1 ) was measured with a 4-electrode conductivity sensor (Tetracon 325, WTW, Weilheim, Germany). After this, samples were autoclaved at 120 C for 30 min and then shaken overnight at room temperature before taking the final measurement of electrolytic conductivity (C 2 ). Relative electrolyte leakage (REL) was calculated as C1 REL = 100 C (5) Percentage cellular lysis of each sample (up to 66 samples per cultivar) was calculated as the ratio to REL of the control (REL c ) according to Morin et al. (2007): REL REL Cellular lysis (%) = 100 REL 2 c c 100 (6) Here REL c was calculated from REL values of samples between 0 and 0.2 MPa. Maximum electrolytic conductivity, caused by dehydration, reached only ~80% of that caused by autoclaving. The curve was extrapolated from 80 to 100% using the same exponential sigmoidal equation used for vulnerability curves (Eq. (2)). Curve fitting and calculations of vulnerability thresholds were done by substituting PLC by percentage cellular lysis and Ψ 50 corresponded to the value of Ψ at 50% cellular lysis. The Ψ value at 12% cellular lysis was defined as the starting point for damage to living tissues (Ψ cl12 ). Stomatal conductance and midday Ψl Stomatal conductance (g s ; mol m 2 s 1 ) was measured on sunny days using a steady-state leaf porometer (SC-1, Decagon Devices, Pullman, WA, USA). When stomata were maximally open (between 10:30 and 11:30 h CET), Ψl and maximum g s were measured on six to nine branches per cultivar. Branches were then cut and Ψl and g s were measured at intervals until stomatal closure. As all branches were exposed to similar conditions and measurements were made within a 2-h period, it can be assumed that during dehydration neither light, CO 2 concentration, temperature, nor vapour pressure deficit changed significantly, and so stomatal closure is presumed to have occurred in response to decreasing Ψl (Hubbard et al. 2001, Beikircher and Mayr 2009, Mayr et al. 2010). Percentage stomatal conductance was calculated as the ratio of actual and maximal stomatal conductance and plotted against Ψl. Curve fitting was done using Eq. (2), with PLC being substituted by percentage stomatal conductance and Ψ 50 corresponding to Ψl at 50% g s. Ψl at 88% g s was defined as the value of Ψl at the onset of stomatal closure and Ψl at 12% g s as Ψl at stomatal closure (Ψ sc ). Safety margins were calculated from Ψ sc and Ψ 50 of branch xylem (Eq. (7)) according to Beikircher and Mayr (2009) and Mayr et al. (2010): Safety margin = Ψsc Ψ 50 (7) Initial g s values at Ψ higher than 0.5 MPa for branches analysed per cultivar were averaged to calculate the maximum g s. Midday values of Ψl were measured from April to August in 2010 and again in 2011 on selected sunny days between 10:00 and 11:30 CET. Measurements were made on at least five leaves per cultivar and date. Xylem anatomy Samples used for vulnerability analyses of branches were soaked in an ethanol glycerol water solution (1:1:1, v/v/v) for several weeks. Cross sections were then cut from five samples per cultivar using a microtome (Sledge Microtome G.S.L. 1, Schenkung Dapples, Zurich, Switzerland) and stained with phloroglucinol HCl (stains lignin bright red). Anatomical parameters were analysed with a light microscope (Olympus BX 41, System Microscope, Olympus Austria, Vienna, Austria) interfaced with a digital microscope camera (ProgRes CT3, Jenoptik, Jena, Germany) and image analysis software (ImageJ, 1.37, National Institutes of Health (NIH), Bethesda, MD, USA, public domain). All measurements were made in radial sectors located opposite reaction wood, on a total of 982 to 1256 conduits per cultivar. Individual conduit lumen areas (A) were Tree Physiology Volume 33, 2013

6 Drought tolerance of Malus domestica 1301 measured directly and the respective diameters (d) calculated assuming a circular conduit shape. d = 2 ( A/ π) (8) To avoid bias due to possible over-representation of samples having larger numbers of conduits, d was first averaged per sample and then the mean diameter (d mean ) per cultivar was calculated from the sample averages. The hydraulic conduit diameter (d h ) was calculated from the individual diameters according to Sperry and Hacke (2004) as d dh = Σ 5 (9) 4 Σd Conduit wall reinforcement was analysed by measuring the thickness to span ratio (t/b) h 2 (Hacke et al. 2001). The wall thickness (t) between conduits and the conduit wall spans (b) were measured directly for up to 55 conduit pairs averaging within d h ± 2 µm. Values of d h and (t/b) h 2 were calculated from sample means in the same way as was d mean. Stomatal anatomy For anatomical analyses of stomata, five branches per cultivar were cut, wrapped in black plastic bags with wet filter paper and taken to the laboratory. Next, a total of 10 leaves was excised under water and saturated overnight in distilled water. The next day, trichomes on the adaxial surfaces were removed using adhesive tape and a coat of clear nail varnish was applied. The dry nail varnish layer was then peeled off with adhesive tape, placed on a microscope slide and analysed with a light microscope interfaced with a digital microscope camera and image analysis software (see Xylem anatomy ). As leaves were maintained under well-watered and dark conditions until preparation, it can be assumed that stomata were closed. Stomata were counted on defined areas, to determine the stomatal density (SD; number per mm 2 ) and the pore length (l, µm) was measured directly on a total of up to 170 stomata. Average values per leaf were used for the calculation of mean values per cultivar (see Xylem anatomy ). Statistics Depending on the data set, normal distribution and variance were determined and following tests applied: vulnerability thresholds (branch and leaves) Ψ sc and Ψ cl12 were tested with the Welch test based on mean and standard error values per cultivar calculated from curve fitting at n-2 df obtained with the software FigP (Version 2006, Fig.P Software Corporation, Durham, NC, USA). For all other parameters an analysis of variance (ANOVA) was carried out using SPSS (version 18, SPSS, Chicago, IL, USA). Maximum g s and TLP were tested with the Tamhane test based on measured and calculated values, respectively. Xylem anatomical parameters (d mean, d h, (t/b) h2 ) were tested with the Bonferroni test based on mean and standard error values per branch; leaf anatomical parameters (SD, l) k s and Π o were also tested with the Bonferroni test but based on measured and calculated values, respectively. With the Welch test and the Tamhane test, a pairwise comparison was carried out followed by a Bonferroni correction; the Bonferroni test allowed multiple comparisons. All tests were assessed at a probability level of 5%. Results Hydraulic safety and stomatal regulation Vulnerability to drought-induced embolism differed significantly between the three cultivars, with Golden Delicious being the most tolerant and Red Delicious the most vulnerable. The Ψ at the onset of cavitation in branches (Ψ 12 ) ranged from 1.41 to 2.48 MPa and at 50% loss of hydraulic conductivity (Ψ 50 ) from 2.73 to 3.81 MPa. Except for Golden Delicious, leaves were less vulnerable than branches. In Red Delicious Ψl 50 was as much as 1.1 MPa higher than in branches (Table 1, Figure 2). Table 1. Hydraulic parameters of the three apple cultivars: Golden Delicious, Braeburn and Red Delicious. Hydraulic parameters Golden Delicious Braeburn Red Delicious Branches Ψ 12 (MPa) 2.48 ± 0.12 a 1.53 ± 0.47 b 1.41 ± 0.31 b Ψ 50 (MPa) 3.81 ± 0.12 a 3.46 ± 0.15 a 2.73 ± 0.11 b Ψ 88 (MPa) 5.14 ± 0.12 a 5.40 ± 0.18 a 4.05 ± 0.09 b k s (m 2 s 1 MPa ± 1.19 a ± 1.29 a ± 1.59 b *10 4 ) Ψ cl12 (periderm; 5.68 ± 0.14 a 4.09 ± 0.08 b 3.99 ± 0.10 b MPa) Leaves Ψl 12 (MPa) 2.33 ± 0.08 a 1.98 ± 0.24 a 1.58 ± 0.34 a Ψl 50 (MPa) 3.33 ± 0.08 a 3.81 ± 0.08 b 3.84 ± 0.09 b Ψl 88 (MPa) 4.32 ± 0.08 a 5.63 ± 0.09 b 6.09 ± 0.16 b Ψ sc (MPa) 3.78 ± 0.06 a 3.48 ± 0.06 a 3.99 ± 0.06 a Safety margin (MPa) max g s (mol m 2 s 1 ) 0.77 ± 0.04 a 0.62 ± 0.02 b 0.60 ± 0.02 b TLP (MPa) 2.74 ± 0.04 a 2.48 ± 0.07 a 2.62 ± 0.08 a Πo (MPa) 2.06 ± 0.04 a 2.08 ± 0.04 a 2.05 ± 0.04 a Ψ cl12 (MPa) 3.44 ± 0.16 a 3.04 ± 0.29 a 3.65 ± 0.25 a Water potential at 12, 50 and 88% loss of hydraulic conductivity of branch xylem and leaves (Ψ 12, Ψ 50, Ψ 88 and Ψl 12, Ψl 50, Ψl 88 ), at 12% cellular lysis (Ψ cl12 ) of periderm and leaves and at stomatal closure (Ψ sc ), specific hydraulic conductivity (k s ), maximum stomatal conductance (max g s ), safety margin, TLP and osmotic potential at full turgor (Πo). Different letters indicate significant differences (P < 0.05) between the values among the three cultivars. Means ± SE. Tree Physiology Online at

7 1302 Beikircher et al. Figure 2. (a c) Percentage loss of hydraulic conductivity of branches (closed circles, solid lines) and leaves (open circles, dashed lines) and (d f) percentage stomatal conductance versus Ψ of Golden Delicious, Braeburn and Red Delicious. Solid vertical lines show Ψ at 50% loss of conductivity of branch xylem (Ψ 50 ; upper panels) and at stomatal closure (Ψ sc ; lower panels), dashed lines show Ψ at 50% loss of conductivity of leaves (Ψl 50 ; upper panels). Note that Ψ for stomatal conductance analyses were measured on transpiring leaves and thus may not reflect stem Ψ (see Sampling and preparation of branches ). The onset of cell damage in leaves (Ψ cl12 ) coincided with Ψl 50 in Golden Delicious and Red Delicious, while in Braeburn Ψ cl12 was ~0.8 MPa less negative than Ψl 50 (Table 1). Cell damage of leaves started up to 1 MPa below the TLP, which did not differ significantly between the cultivars (Table 1). Also, for the osmotic potential at full turgor (Πo; about 2 MPa) no significant differences between cultivars were observed. In contrast to leaves, cell damage of the periderm differed significantly between cultivars, ranging from 3.99 MPa in Red Delicious to 5.68 MPa in Golden Delicious (Table 1, Figure 3). Stomata started to close at Ψ near the TLP, while full stomatal closure (Ψ sc ) occurred between 3.48 MPa (Braeburn) and 3.99 MPa (Red Delicious) (Table 1, Figure 4). However, lowest mean values of the midday Ψ were only 1.6 MPa (Figure 4). Maximum stomatal conductance (g s ) was highest in Golden Delicious (0.77 mol m 2 s 1 ) and ~0.61 mol m 2 s 1 in the other two cultivars (Table 1). Hydraulic efficiency and anatomy Despite their larger conduits, Braeburn and Golden Delicious had lower specific conductivity (k s ; ~ m 2 s 1 MPa 1 ) than Red Delicious (Tables 1 and 2). Also cell-wall reinforcement ((t/b) h2 ) was similar in Braeburn and Golden Delicious and slightly higher in Red Delicious (Table 2). Xylem anatomical parameters did not differ significantly between cultivars but significance values for Red Delicious obtained with the conservative Bonferroni test were only slightly above the statistical threshold (Table 2). Stomatal density ranged from 633 to 664 stomata per mm 2 and stomatal pore length (l) was lowest in Braeburn (15.65 µm) and highest in Red Delicious (17.14 µm). For both parameters, no significant differences between cultivars were found (Table 2). Discussion Hydraulic safety of high-yield apple cultivars A tree s drought tolerance is determined by a number of factors, one of which is hydraulic safety. Between the three highyield cultivars under study, hydraulic safety was highest in Golden Delicious. Compared with the most vulnerable cultivar, Red Delicious, both cavitation onset (Ψ 12 ) and Ψ at 50% loss of hydraulic conductivity (Ψ 50 ) were >1 MPa lower (Table 1, Figure 2). This confirms our hypothesis that Golden Delicious is more drought tolerant than either Braeburn or Red Delicious. There are only a few other studies reporting vulnerability thresholds for M. domestica but these point to a relatively high variability in hydraulic safety within this species. Jones et al. Tree Physiology Volume 33, 2013

8 Drought tolerance of Malus domestica 1303 Figure 3. Percentage cellular lysis versus Ψ of leaves (a c) and periderm (d f) of Golden Delicious, Braeburn and Red Delicious. Vertical lines indicate the Ψ at 12% cellular lysis (Ψ cl12 ). Table 2. Anatomical parameters of the three apple cultivars: Golden Delicious, Braeburn and Red Delicious. Anatomical parameters Golden Delicious Braeburn Red Delicious Figure 4. Sequence of characteristic hydraulic parameters during dehydration. Water potentials at 12, 50 and 88% loss of hydraulic conductivity of branches and leaves (light grey bars) and cellular lysis of leaves (dark grey bars). The Ψl at the onset of (dotted lines) and full stomatal closure (Ψ sc ; solid lines), TLP (closed circles), 12% cellular lysis of periderm (Ψ cl12 ; stars), and midday Ψl (open circles) during the vegetation period for the apple cultivars Golden Delicious, Braeburn and Red Delicious. Note that Ψ sc and midday Ψl measurements were made on transpiring leaves and thus may not reflect stem Ψ (see Sampling and preparation of branches ). (1989) found a range in cavitation onset from 0.6 to 1.7 MPa for the apple cultivars Cox s Orange Pippin, Golden Delicious and A120/3. Their values for Golden Delicious ( 0.7 and Branches d mean (µm) ± 0.58 a ± 0.91 a ± 0.19 a d h (µm) ± 0.59 a ± 0.94 a ± 0.34 a (t/b) 2 h ± a ± a ± a Stomata SD (mm 2 ) 664 ± 37 a 633 ± 13 a 657 ± 35 a l (µm) ± 0.85 a ± 1.13 a ± 0.62 a Mean conduit diameter (d mean ), hydraulic conduit diameter (d h ), cellwall reinforcement ((t/b) h2 ), stomatal density (SD) and stomatal pore length (l). Different letters indicate significant differences (P < 0.05) between the values for the three cultivars. Mean ± SE. 1.2 MPa) were at least 1.3 MPa higher than Ψ 12 for Golden Delicious in our study. Nardini and Salleo (2000) observed the cavitation onset in shoots of M. domestica (cultivar not specified) at about 1 MPa. Meanwhile, Lauri et al. (2011) have reported a mean Ψ 50 of 4.3 MPa for an apple progeny of Starkrimson Granny Smith cross, Christensen-Dalsgaard and Tyree (2013) report a range in Ψ 50 from 1.4 to 2.6 MPa for current-year shoots of M. domestica cv. Alberta green, depending on treatment (freeze thaw cycles and bending) and B. Beikircher et al. (unpublished data) found a range in Ψ 50 from 1.96 to 2.42 MPa for three old cultivars of M. domestica. For Malus sylvestris and other different wild forms of Malus, Ψ 50 of Tree Physiology Online at

9 1304 Beikircher et al to 6.1 MPa have been found (Choat et al. 2012, B. Beikircher et al. unpublished data). A similar wide range in Ψ 50 was also reported in the Rosaceae genus Prunus (Cochard et al. 2008). The reason for this high variability could be related to genetic variation within M. domestica, but there may also be effects of root stock and grafting that influence tree vigour, anatomy and thus hydraulics (see Atkinson et al. 2003, Cohen et al. 2007, Bauerle et al. 2011). Accordingly, Jones et al. (1989) found the cavitation onset in M. domestica to vary with the rootstock on which the scions were grafted. To our knowledge this has not been further investigated. Vulnerability to drought-induced embolism is predominately related to pit properties as air seeding occurs at the pits (Hacke and Sperry 2001) but, according to the rare pit hypothesis, it can also be indirectly related to the conduit diameter (Wheeler et al. 2005, Hacke et al. 2006, Christman et al. 2009). As well as the conduit diameter, cell-wall reinforcement can also play an important role as it influences the risk of implosion under low Ψ (Hacke and Sperry 2001). Mean and hydraulic conduit diameter were in the same range as reported by Christensen-Dalsgaard and Tyree (2013), although these authors reported that vulnerability thresholds were much higher than ours (see above) but lower than those reported by Bauerle et al. (2011) for M. domestica cv. Honeycrisp. Among our three cultivars, Red Delicious had the smallest conduit diameters and thickest cell-wall reinforcements but was nevertheless the most vulnerable cultivar (Tables 1 and 2). As Red Delicious also showed the highest specific conductivity (k s, Table 1), it can be assumed that the low hydraulic safety results from increased pit pore size and/or porosity. Although as yet unexplored, the possibility cannot be ruled out that due to crossing and/or grafting effects, developmental mistakes, perhaps extra-large or weak pores (Christman et al. 2009) occur more frequently in high-yield plants. Hydraulic safety is also known to vary within a tree. Here, more dispensable distal parts (current-year shoots and leaves) are more vulnerable than indispensable parts (older branches and stems; Tyree and Zimmermann 2002). In our study, this was true only for Golden Delicious. In Braeburn and Red Delicious, Ψ at 12% and 50% loss of leaf hydraulic conductance (Ψl 12, Ψl 50 ) were below the respective vulnerability thresholds in branches, indicating a higher resistance of leaves (Table 1, Figure 2). As far as we are aware, there are no studies reporting leaf vulnerability thresholds in M. domestica and only one for M. sylvestris (West and Gaff 1976) where on excised leaves, cavitation started at about 1.2 MPa and Ψl 50 (indicated by 50% of cumulative acoustic events) was reported at about 2.7 MPa. Values of Ψl 50 in our apple cultivars were surprisingly low, recognizing that, for many angiosperm species from a range of biomes, Ψl 50 values lie between 1 and 2 MPa (Johnson et al. 2009, Blackman et al. 2010, 2012, Scoffoni et al. 2012). In Golden Delicious, Ψl 50 coincided with the onset of cellular damage in leaf tissues, while initial damage to peridermal cells is at about 5.68 MPa. Strikingly, in Braeburn and Red Delicious, cellular damage to the periderm occurred at between 12 and 50% cellular damage of leaves (Table 1, Figure 3). As with hydraulic safety, the TLP and the osmotic potential at full turgor (Πo) were also relatively low compared with other temperate angiosperm species (Bartlett et al. 2012; Table 1). This suggests a relatively high drought tolerance. Stomatal regulation All three study cultivars closed their stomata at relatively low Ψ. Stem Ψ in this range would already have induced considerable embolism in branches and leaves as well as cell damage due to dehydration (Table 1, Figure 4). However, measurements of stomatal conductance were carried out on transpiring leaves and it is known that Ψl can differ significantly from stem Ψ and xylem pressure (see Meinzer et al. 2001). Despite the low hydraulic safety thresholds, the low Ψ sc resulted in narrow or even negative safety margins (Table 1). In contrast to conifers and ferns, narrow or negative safety margins are not uncommon in angiosperms and represent a strategy for optimizing carbon gain (Brodribb and Holbrook 2004, Breda et al. 2006, Franks et al. 2007, Beikircher and Mayr 2009, Choat et al. 2012, Scoffoni et al. 2012). Indeed, Urli et al. (2013) and Choat et al. (2012) found that the xylem embolism threshold for irreversible hydraulic failure of several angiosperm species was correlated more closely with Ψ 88 than with Ψ 50. However, the onset of stomatal closure had already occurred at higher Ψl around the TLP (Figure 4). This is consistent with the hypothesis that the turgor of the guard cells or neighbouring cells may provide the signal linking stomatal closure to xylem cavitation (hydraulic signalling; Salleo et al. 2000, Brodribb et al. 2003, Brodribb and Holbrook 2003, Sack and Holbrook 2006, Brodribb 2009). Brodribb et al. (2003) found that, depending on the species, the TLP fell between the incipient and final stages of stomatal closure. Guyot et al. (2012) reported that, although leaf hydraulic conductance declined at Ψ values around the TLP, not all species closed their stomata. The authors suggested that maintaining the stomata under an open condition, even when leaf conductance declines, may indicate a degree of hydraulic redundancy in well-hydrated leaves. This would be consistent with findings in this study where leaves were not likely to suffer drought during the vegetation period. Besides late stomatal closure, g s values also indicate that the three cultivars in our study are all programmed to maximize carbon gain. Maximum g s values ranged from 0.60 (Red Delicious) to 0.77 mol m 2 s 1 (Golden Delicious; Table 1) and was thus more than three times higher than the g s values reported for M. domestica (Nardini and Salleo 2000) and also considerably higher than those reported for other temperate angiosperms (e.g., Aasamaa et al. 2001, Larcher 2003, Tree Physiology Volume 33, 2013

10 Drought tolerance of Malus domestica 1305 Eensalu et al. 2008). The relatively high maximum g s values can be explained by stomatal features such as larger stomata and higher SD (Sack et al. 2006, Sellin et al. 2010). According to Aasamaa et al. (2001) and Eensalu et al. (2008), besides SD, there is also a positive relationship between g s and stomatal pore length (l). In our study, both SD and l (Table 2) were relatively high compared with values given for different genotypes of Malus pumila (SD: mm 2, l: 21 µm; Jones 1992) and other deciduous angiosperms (e.g., Aasamaa et al. 2001, Larcher 2003, Sack et al. 2003, 2006, Eensalu et al. 2008). Maximum g s values were highest in Golden Delicious, which also corresponded to the highest SD (Tables 1 and 2). this (B. Beikircher, unpublished data) as an explanation of the severe tree damage (delayed leaf-flush, dieback) observed in some seasons. Increased peridermal conductance of lammas shoots during the leafless season can also increase drought stress (Beikircher and Mayr 2013). This study provides new insights into the hydraulic behaviour of Malus and demonstrates the variability of these properties among some commercial cultivars. Hydraulic properties are likely to be influenced not only by genetics and climatic conditions in a commercial apple orchard but also by a range of other factors such as grafting, pruning, chemical agents and other cultivation practices. Conclusions Measurements reveal an interesting hydraulic behaviour in the three Malus cultivars investigated. For an interpretation, special conditions must be considered. Essentially, plants selected for maximal productivity are grown under optimized conditions in terms of water and nutrient supply. The performance of an apple tree (yield and quality) depends very much on water supply. Water deficits can have direct effects either by limiting cell and fruit expansion or indirect ones on photosynthesis (see Naor and Girona 2012). The high crop load of high-yield cultivars requires an adequate assimilate supply. This becomes even more evident as reducing transpiration by shading can be used as a chemical-free tool for fruit thinning in spring (e.g., Morandi et al. 2011). Thus, it is not surprising that we found traits linking hydraulics to carbon gain such as stomatal regulation, maximum g s and stomatal anatomy, to be adjusted so as to optimize gas exchange and thus productivity. In this context, late closure of stomata may be expected, although this behaviour somewhat contradicts Malus s classification as an isohydric species. On the other hand, midday Ψ during the vegetation period did not decrease to critical values (Figure 4). We are unable to determine whether this behaviour was related to strict stomatal control (isohydric behaviour) or to an optimized water supply (irrigation; Figure 1). Naor and Girona (2012) reported that stomatal conductance in apple, if well-watered, remains relatively stable throughout the day. This may indicate an ability to switch between isohydric and anioshydric modes, as recently reported for Vitis vinifera (Zhang et al. 2012). Late stomatal closure may, at least partially, explain the relatively high hydraulic safety found in our cultivars, as this is the first prerequisite for maintaining carbon gain under decreasing Ψ. Other parameters such as TLP and Π o also point to a relatively high drought tolerance. As expected, we found differences in hydraulic safety among the cultivars, with Golden Delicious being the most drought tolerant. It should be considered that hydraulic safety is not just relevant to summer drought. Owing to the climatic conditions at the study site, plants are prone to frost-induced droughts in spring. We are currently considering Acknowledgments We thank the staff of the South Tyrolean Advisory Service for Fruit and Wine-growing, especially Martin Abler, for helpful support. We also thank Mag. Birgit Dämon for helpful assistance and Dr Johanna Wagner for providing the autoclave. Conflict of interest None declared. Funding This study was financed by the Austrian Science Fund (FWF), project L556-B16. References Aasamaa K, Sober A, Rahi M (2001) Leaf anatomical characteristics associated with shoot hydraulic conductance, stomatal conductance and stomatal sensitivity to changes of leaf water status in temperate deciduous trees. Aust J Plant Physiol 28: Atkinson CJ, Else MA, Taylor L, Dover CJ (2003) Root and stem hydraulic conductivity as determinants of growth potential in grafted trees of apple (Malus pumila Mill.). 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