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RESEARCH ARTICLE

INVITED SPECIAL ARTICLE

For the Special Section: Wood: Biology of a Living Tissue

The effect of polyploidization on tree hydraulic functioning Niels J. F. De Baerdemaeker1,*, Niek Hias2,*, Jan Van den Bulcke3, Wannes Keulemans2, and Kathy Steppe1,4

Manuscript received 8 September 2017; revision accepted 11 December 2017. 1 Laboratory of Plant Ecology, Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000, Ghent, Belgium 2 Laboratory for Fruit Breeding and Biotechnology, Division of Crop Biotechnics, Katholieke Universiteit (KU) Leuven, Willem de Croylaan 42, B-3001, Heverlee, Belgium 3 Laboratory of Wood Technology, Department of Environment, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000, Ghent, Belgium 4

Author for correspondence (e-mail: [email protected])

* These authors contributed equally to this work. Citation: De Baerdemaeker, N. J. F., N. Hias, J. Van den Bulcke, W. Keulemans, and K. Steppe. 2018. The effect of polyploidization on tree hydraulic functioning. American Journal of Botany 105(2): 161–171. doi:10.1002/ajb2.1032

PREMISE OF THE STUDY: Recent research has highlighted the importance of living tissue in wood. Polyploidization can impact amounts and arrangements of living cells in wood, potentially leading to increased drought tolerance. Tetraploid variants were created from the apple cultivar Malus ×domestica ‘Gala’ (Gala-4x), and their vulnerability to drought-induced cavitation and their hydraulic capacitance were compared to those of their diploid predecessors (Gala-2x). Assuming a positive correlation between polyploidy and drought tolerance, we hypothesized lower vulnerability and higher capacitance for the tetraploid. METHODS: Vulnerability to drought-induced cavitation and the hydraulic capacitance were quantified through acoustic emission and continuous weighing of shoots during a bench-top dehydration experiment. To underpin the hydraulic trait results, anatomical variables such as vessel area, conduit diameter, cell wall reinforcement, and ray and vessel-associated parenchyma were measured. KEY RESULTS: Vulnerability to drought-induced cavitation was intrinsically equal for both ploidy variants, but Gala-4x proved to be more vulnerable than Gala-2x during the early phase of desiccation as was indicated by its significantly lower air entry value. Higher change in water content of the leafy shoot, higher amount of parenchyma, and larger vessel area and size resulted in a significantly higher hydraulic capacitance and efficiency for Gala-4x compared to Gala-2x. CONCLUSIONS: Both ploidy variants were typified as highly sensitive to drought-induced cavitation, with no significant difference in their overall drought vulnerability. But, when water deficit is short and moderate, Gala-4x may delay a drought-induced decrease in performance by trading hydraulic safety for increased release of capacitively stored water from living tissue. KEY WORDS acoustic vulnerability curves; desorption curves; drought stress; embolism; Malus; Polyploidy; water transport; wood; xylem vulnerability.

Horticulture in temperate regions of the world covers a wide variety of cultivation practices. Despite the smaller percentage attributed to fruit production, cultivation of domesticated apple (Malus ×domestica) is one of the main exploits (Velasco et al., 2010). Because of climate change, horticultural practices become more challenging as apple trees have to cope with elevated CO2, increasing air temperatures, aberrant precipitation, and increasing drought stress (Ramírez and Kallarackal, 2015). Combined, these factors challenge the tree’s hydraulic system, which may result in a negative impact on fruit quality and productivity (Naor and Girona, 2012). Proper irrigation management is crucial to maintain healthy apple

plantations, but must be adequately applied to support plant physiological behavior and result in an optimal income–expenditure balance (Black et al., 2008). To this end, it becomes increasingly important to quantify tolerance to drought in order for fruit growers to sustain a viable yield. To combat negative effects associated with drought stress, plants can use two strategies: either they avoid drought, aiming at preserving hydraulic integrity and tissue hydration to delay desiccation (Maherali et al., 2004), or they tolerate drought, allowing a certain degree of desiccation by utilizing, for instance, water storage buffers to maintain plant functioning (Goldstein et al., 1998; Phillips et al.,

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2003). Both responses are affected by a network of morphological, physiological, and molecular alterations throughout the plant, and promoting traits of interest is vital to create desirable cultivars. The living tissue embedded in wood contributes to both strategies as it stores and radially distributes water, carbon, and nutrients and plays an important role in controlling and repairing hydraulic connectivity. Because genome doubling often results in an enhancement of functionality of the wood’s living tissue (e.g., drought tolerance, pathogen resilience) and desired horticultural properties (e.g., increased fruit bearing, more flavor), artificial polyploidization is regularly used in plant breeding programs. Nuclear enlargement, genetic changes through an altered gene dosage and potential epigenetic effects caused by artificial genome doubling result in similar anatomical and physiological changes as in naturally occurring polyploids (Comai, 2005; Maherali et al., 2009; van Laere et al., 2011; Allario et al., 2011; Hao et al., 2013). These alterations are often associated with an increased ecological tolerance as polyploids have been reported to thrive better in more stressful environments such as subarctic, elevated, and xeric regions (Madlung, 2013). To combat harsh conditions, polyploids have larger cell sizes and stomata (Melaragno et al., 1993; Hodgson et al., 2010), suggesting an effect of genome duplication on plant–water relations (Madlung, 2013). Polyploidization also expresses itself in altered morphology such as thicker leaves, increased leaf hairs, and fewer but larger stomata per unit area, and results in physiological adjustments of the polyploid’s hydraulic function such as a more negative osmotic water potential at full turgor, further modifying its water relations to reduce water loss and maintain sufficient gas exchange rates (Li et al., 1996; Hao et al., 2013). Previous studies comparing artificially created polyploids with their diploid predecessor frequently reported a higher tolerance to drought: Betula papyrifera (Li et al., 1996), Chamerion angustifolium (Maherali et al., 2009), Lonicera japonica (Li et al., 2009), Populus sp. (Hennig et al., 2015), and Spathiphyllum wallisii (van Laere et al., 2011). In Malus sp., Zhang et al. (2015) recently showed an increased tolerance of autotetraploid apple seedlings exposed to drought stress. Although a high prevalence exists of triploid apple cultivars, proving their benefits (Einset, 1944), the formation of a higher ploidy in apple as in many other plant species is limited (Ramsey and Schemske, 1998). The use of tetraploid genotypes is very rare and predominantly aimed at the creation of new triploid varieties (Sedysheva and Gorbacheva, 2013; Sedov et al., 2014). Because the effective use of irrigation is crucial for reliable production of high quality fruit, especially in semi-arid regions (Naor et al., 2008), use of polyploidization to increase tolerance to water deficit in apple could lower irrigation demands and is thus a promising and useful research topic. To quantify the different responses of polyploid apple cultivars to drought stress, we need to examine the impact on hydraulic functioning through a framework of physiological and anatomical variables (Beikircher and Mayr, 2009; De Micco and Aronne, 2012; Steppe et al., 2015; Epila et al., 2017). The most frequent impact of drought stress on overall plant functioning is the formation of embolisms, where water-filled conduits become gas-filled due to a resulting increase in xylem tension (Tyree and Sperry, 1989a; Tyree and Zimmermann, 2002). Embolisms will further spread as a function of maximum pit pore size (Zimmermann, 1983; Loepfe et al., 2007; Mayr et al., 2014), with the size of the largest pore determining the air-seeding threshold (Tyree et al., 1994), a theory known

as the air-seeding hypothesis (Shen et al., 2002). The air-seeding threshold will directly influence the safety of a plant’s hydraulic system, which represents the ability of xylem to avoid embolism formation (Sperry and Tyree, 1990). According to a new theory, above that threshold, the presence of lipid surfactants in xylem may be crucial for avoiding embolism formation, as surfactants could coat nanobubbles that form in xylem and keep them below the critical size at which they would expand into an embolism (Schenk et al., 2015, 2017; De Baerdemaeker et al., 2017). High hydraulic safety will help protect the plant against the risks associated with drought stress, but could simultaneously decrease its hydraulic efficiency (Lauri et al., 2011). Physiological assessment of hydraulic safety can be done with two techniques: (1) via a vulnerability curve (VC), which relates loss of xylem hydraulic conductivity to increasing drought stress (Sperry et al., 1988), and (2) via quantification of hydraulic capacitance to determine the capacity to buffer water shortage via internally stored water (Steppe and Lemeur, 2007; Vergeynst et al., 2015a; Epila et al., 2017). A trade-off between hydraulic safety and efficiency will be desirable to ensure optimal plant growth (Sperry et al., 2008; Blackman et al., 2010), but is not as straightforward because some species do not have this trade-off and combine low hydraulic efficiency with high embolism vulnerability (Gleason et al., 2016). The objective of this study was to gain insights into the effects of polyploidization on the wood and, hence, hydraulic system of Malus ×domestica ‘Gala’ Borkh. by comparing tetraploid (Gala-4x) with diploid (Gala-2x) seedlings. As both variants share the same genotype, we could investigate the use of genome doubling in the development of more drought-tolerant cultivars. We measured for both Gala seedlings xylem vulnerability to drought-induced cavitation and hydraulic capacitance of shoots during a bench-top dehydration experiment and complemented these hydraulic measurements with anatomical measurements, including vessel area, total number of vessels, conduit diameter, cell wall reinforcement, and quantification of ray and vessel-associated parenchyma. We hypothesized that Gala-4x would be more drought tolerant because polyploidization has been shown to decrease vulnerability to drought-induced cavitation (Hao et al., 2013). Because of the observed increased cell size (adaxial epidermis, palisade and spongy parenchyma) in the leaves of Gala-4x (Hias et al., 2017), and the higher water content per living cell due to its higher ploidy level (Kehr, 1996), we also hypothesized that Gala-4x would have a higher hydraulic capacitance. MATERIALS AND METHODS Plant material

In vitro Malus ×domestica Gala plants were artificially doubled from nodal segments using colchicine. Ploidy levels were assessed on a flow cytometer (Cyflow Space, Partec, Münster, Germany) as described by Dhooghe et al. (2009). After ex vitro rooting and acclimatization own-rooted di-and tetraploid Gala plantlets were transferred to a 2 L container of commercial soil (DCM Pepi 3, Grobbendonk, Belgium) and grown for 10 months in the KU Leuven faculty greenhouse complex (50°51′34.7″N 4°40′49.1″E) under controlled climate conditions: day/night temperature 18°/14°C, day/night relative humidity 70%/60% and additional lighting (120 μmol PAR·m−2·s−1) during the day when light intensity dropped

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below 450 μmol PAR·m−2·s−1. Shortly after winter chilling outside the greenhouse, the plants were cut back to three buds above the ground and then put back in the greenhouse under the aforementioned conditions. After budburst, one shoot per plant was left to grow for 7 months until the start of the experiment. Sampling procedure and hydraulic trait measurements

Twelve well-watered 1-yr-old seedlings with a shoot ~120 cm long and an average diameter of 7.7 ± 1.0 mm were measured, six each of Gala-2x and Gala-4x. Per variant, three cut shoots were used to determine xylem vulnerability to drought-induced cavitation via acoustic emission detection, and the remaining three were placed on continuous weight balances after cutting to calculate hydraulic capacitance. For fast and equal dehydration, all shoots had leaves attached to their stem to speed dehydration by incorporating water loss via leaf transpiration. Shoots were transported from the greenhouse at KU Leuven to the greenhouse of the Laboratory of Plant Ecology, Ghent University (51°03′10.3″N 3°42′32.3″E) on 23 September 2015, where they were kept well watered. Just before sunset, the shoots were relocated to the experimental room. Sites for sensor installation were marked on the intact plants to ensure that shoots had sensors at equal distance from the cut end. For Gala-2x, the distance was ~40 cm for the dendrometer and ~50 cm for the acoustic emission (AE) sensor. For Gala-4x, the distance for each, respectively, was ~30 and ~40 cm. Shoots of Gala-2x were analyzed on 24 September, and those of Gala-4x the next day. During the following preparation steps, the experimental room was darkened, and an artificial green light created a workable lit environment. This type of light source was chosen because it limits photosynthesis and transpiration during the setup of the experiment (De Baerdemaeker et al., 2017; Epila et al., 2017). Six shoots were cut at the base under water and recut under water twice to avoid artifacts due to air entry (Cochard et al., 2013). To ensure that dehydration did not start during sensor installation, the cut end of each of the shoots was wrapped in wet cloth to keep the end moist and avoid air entry. Three shoots were equipped with a broadband point-contact AE sensor (KRNBB-PC; KRN Services, Richland, WA, USA) and a dendrometer (DD-S; Ecomatik, Dachau, Germany). The shoots were fixed in a custom-built holder to allow easy installation of the sensors and create an unbiased link between them (De Baerdemaeker et al., 2017; Epila et al., 2017). The AE sensor was pressed to the surface of the shoot via a compression spring (D22050; Tevema, Amsterdam, Netherlands) in a small polyvinylchloride tube. A droplet of High-Vacuum Grease (Dow Corning, Seneffe, Belgium) was added between the sensor tip and shoot to ensure good acoustic contact, which was validated via the pencil lead break test (Tyree and Sperry, 1989b; Vergeynst et al., 2015b). To measure xylem shrinkage, a section of bark (1.5 × 0.5 cm2) was removed with a scalpel after which the dendrometer was installed. To prevent evaporation from the wound, petroleum jelly was applied between dendrometer tip and exposed xylem (Vergeynst et al., 2016). The three remaining shoots were placed on continuous weight balances (2x DK 6200 with 0.01 g accuracy and 1x PS 4500/C/1 with 0.1 g accuracy; Henk Maas, Veen, Netherlands), after which wet cloths were removed and normal light turned on in order to start the bench-top dehydration experiment. Readings from dendrometers and balances were registered every minute via custom-built acquisition boards. The AE signals were

amplified by 35.6 decibels (dB) (AMP-1BB-J, KRN Services) and waveforms of 7168 samples length were acquired at 10 MHz sample rate. The signals were collected using two 2-channel PCI boards and redirected to the software program AEwin (PCI-2, Aewin E4.70; Mistras Group BV, Schiedam, Netherlands). A 20-1000 kHz electronic band pass filter was applied, and only waveforms above the noise level of 28 dB were registered (Vergeynst et al., 2016). Xylem water potential (ψ, MPa) in excised leaves wrapped in aluminium foil was measured using a pressure chamber (PMS Instrument Co., Corvalis, OR, USA; Scholander et al., 1965) to achieve equilibrium between leaf and stem water potential. Wrapping with aluminium foil was deemed sufficient because porometer measurements on enclosed leaves showed that stomata were completely closed. The frequency of the readings was guided by the appearance speed of AE signals, which were visualized as dots in the range 28–95 dB and monitored in real-time with the AEwin program. During the first 30 min of dehydration, no AE signals were captured, corresponding with a 0 MPa pressure chamber reading. Frequency of the AE readings then rapidly increased, and the frequency of pressure chamber readings increased accordingly to 5-min intervals. As soon as the frequency of AE signals decreased, water potential readings were reduced to one every 30 min, and at the end of dehydration, measurement frequency further decreased to one every 2 h. To ensure fast dehydration (Rosner, 2012, 2015), not more than eight leaves per shoot were wrapped at any given time (De Baerdemaeker et al., 2017). When new leaves were covered, AE signal detection was put on hold to avoid noise disturbance from the wrapping process. Acoustic vulnerability curves

The obtained AE signals were used to construct an acoustic vulnerability curve (VCAE) (Vergeynst et al., 2016; De Baerdemaeker et al., 2017). The AE signals were cumulated over the measurement period from which the first derivative was calculated to construct an AE activity curve. According to the formulated definition of Vergeynst et al. (2016), the endpoint of the vulnerability curve (AE100) was determined as the point that the decrease in AE activity, following the AE activity peak, decreased most strongly. Mathematically, this point corresponds to the local maximum of the third derivative (Vergeynst et al., 2016). Due to fast dehydration, shoots were completely dehydrated after 8 h so that AE activity was numerically calculated using a 1-min interval, whereas the third derivative was determined using a 2-h interval, based on the timing of the AE peak (Vergeynst et al., 2016). Absolute cumulative acoustic emissions were then rescaled between zero and the defined endpoint to obtain a relative percentage of cumulative AE. In contrast to Vergeynst et al. (2016), the x-axis of the vulnerability curve (ψ, MPa) was defined by linear interpolation of the measured xylem water potentials. This interpolation was justified because the dehydration period was 8 h, and 20 water potential values on average per shoot were available for analysis. Values corresponding with the air entry value (ψ at 12% AE [AE12]), 50% cumulative AE (ψ at 50% AE [AE50]) and full embolism (ψ at 88% AE [AE88]) were also calculated. Desorption curves and hydraulic capacitance

The combined loss in mass of shoot and leaves measured by the balances were used as a surrogate for the overall change in water


content (ΔWC, g). Similar to the VCAE, the desorption curve was obtained by plotting this mass loss against the linearly interpolated water potential data. Within this curve, two regions of interest were determined based on two defined breakpoints, calculated via the Segmented package in R (Muggeo, 2008). The zone between first and second breakpoint is known as the elastic shrinkage phase or phase I. The zone after the second breakpoint until the end of the curve is known as the cavitation or inelastic shrinkage phase or phase II (Vergeynst et al., 2015a). Hydraulic capacitance of both distinct phases was then calculated as the slope of the linear regression between mass loss and interpolated water potential (Vergeynst et al., 2015a). A wood sample of ~5 cm was taken before dehydration from the exposed end of the shoots measured using the continuous weight balances. Diameter and length of the samples were measured with an electronic caliper, and mass was determined with a precision balance (Sartorius precision balance with 0.001 g accuracy; Sartorius Weighing Technology GmBH, Goettingen, Germany). Each sample was then oven-dried (80°C) to a constant mass. Length, diameter, and mass were then remeasured and considering a cylindrical form, basic wood density (ρb, kg·m−3) (oven-dry mass/green volume) was obtained (Vergeynst et al., 2015a). The continuous data obtained by the dendrometers was used to calculate the relative radial diameter shrinkage (Δd/di, μm·mm−1), where Δd/di is the ratio of radial diameter shrinkage (Δd) to the initial diameter before dehydration (di). Plotting the point measurements of water potential versus Δd/di produces the stress–strain curve from which the apparent modulus of elasticity (E′r, MPa; Irvine and Grace, 1997) is calculated as the slope of the linear regression (Eq. 1): d𝜓 (Eq. 1) . Er� = d(Δd∕di ) Wood anatomy

Samples ~3 cm long were taken from the shoots used for vulnerability analysis and stored in an ethanol–glycerol solution (1:1 v/v) for about a month. After briefly heating the samples in an oven at 60°C, cross sections were cut from the samples via a microtome (HM 440 E; MICROM Laborgeräte GmbH, Walldorf, Germany) and stained with 1% aqueous safranin and subsequently with 1% aqueous astra- blue solution to distinguish cellulose (blue) from lignified (red) cells (Von Aufsess, 1973). Cross sections were 30 μm thick, and the best section from each of the six shoots was retained to assess the anatomical variables with a 4× objective lens. Images were captured using a Nikon Ni-U microscope equipped with a Nikon DS-Fi1c camera. One-fourth of the entire cross section was further analyzed with the open source image analysis software Fiji (Schindelin et al., 2012). To distinguish vessels from other anatomical elements such as fibers, an area-based filtering method in Fiji was applied so that only elements with an area greater than 270 μm2 were retained. As a result, 2391 to 3245 vessels per Gala variant were measured. The area of individual vessels (A, μm2) was calculated directly from the processed images, and presuming a circular vessel shape, the diameter of individual vessels could be calculated as follows (Eq. 2):

√ d = 2 (A∕𝜋).

(Eq. 2)

From the individual vessel diameters, a mean diameter (dmean, μm) per variant was calculated. Using the technique described by

Steppe and Lemeur (2007), we calculated hydraulic diameter (dh, μm) as: √ 1 ∑n 4 (Eq. 3) dh = 4 d . n i=1 i Conduit wall reinforcement was calculated by determining the thickness to span ratio (t∕b)2h as described by Hacke et al. (2001). Wall thickness (t) and conduit wall span (b) were measured on 10 μm thick transverse sections with a 20x objective lens. For each variant, 60 images were analyzed to obtain (t∕b)2h so that the overall value per variant was averaged over approximately 150 adjacent vessels. To quantify the amount of ray and vessel-associated parenchyma, a xylem section between pith and cambium from a single cross section of both Gala variants was analyzed with a 20x object lens. Automated quantification was based on color threshold in Fiji and manually checked and complemented to reduce errors from the algorithm. Ray parenchyma was distinguished from vessel- associated parenchyma by fitting an ellipse through the demarcated cells. Percentage parenchyma was derived from the specified xylem section, which had a total area of 1.2 × 106 μm2 for Gala-2x and 1.6 x 106 μm2 for Gala-4x. Statistical analyses

To determine significant differences between the two Malus ×domestica Gala variants, average VCAE and desorption curves were constructed. For smoothing the VCAE, the increase in percentage cumulative AE was fitted via the smooth.spline function in the stats library in R software (RStudio version 0.99.447, RStudio, Boston, MA, USA). Analysis of variance (ANOVA) was carried out on the calculated parameters with the R software, followed by a pairwise comparison. Tests were assessed at a probability level of 5%. All calculated parameters were validated for normality and homogeneity via Wilcoxon signed rank test and Levene test. RESULTS Hydraulic functioning

Overall, Gala-4x experienced slightly greater xylem vulnerability to drought-induced cavitation than Gala-2x (Fig. 1). The air entry value (AE12, MPa) differed significantly between both Gala seedlings (−0.41 and −0.72 MPa for Gala-4x and Gala-2x, respectively; Table 1). Water potential values corresponding to 50% cumulative acoustic emissions (AE50, MPa) and at full embolism (AE88, MPa) were not significantly different (Table 1), but tilted slightly toward less vulnerable in favor of Gala-2x (Fig. 1). Overall mass of Gala- 4x was higher during the entire dehydration experiment (Fig. 2), but initial mass was not significantly different between the two variants (Table 1). Elastic capacitance (Cel, g·MPa−1) and inelastic capacitance (Cinel, g·MPa−1) were both significantly higher in Gala- 4x compared to Gala-2x: 3.01 vs. 1.76 g MPa−1 for Cel and, 8.74 vs. 4.07 g MPa−1 for Cinel (Table 1). The calculated apparent modulus of elasticity (E′r, MPa) (Gala-2x: R2 = 0.983, and Gala-4x: R2 = 0.978) was significantly higher for the tetraploid (Fig. 3, Table 1). Basic wood density (ρb, kg·m−3) did not significantly differ between both variants, but tended to be lowest for Gala-4x (Table 1).


FIGURE 1. Average acoustic vulnerability curve (VCAE) of Gala-2x (blue) and Gala-4x (red) with standard error bands (1 SE). Vulnerability thresholds AE12 (▽), AE50 (♢), AE88 (○), and AE100 (dashed line) are indicated as well.

TABLE 1. Hydraulic variables of the two variants Malus ×domestica Gala-2x and Gala-4x. Variable AE12 (MPa) AE50 (MPa) AE88 (MPa) AE100 (MPa) Cel (g·MPa−1) Cinel (g·MPa−1) Winit (g) E′r (MPa) ρb (kg·m−3)

Gala-2x

Gala-4x

−0.72 ± 0.17 −0.99 ± 0.23a −1.40 ± 0.14a −1.77 ± 0.29a 1.76 ± 0.48a 4.07 ± 0.87a 67.65 ± 9.92a 79.09 ± 26.24a 485.48 ± 15.25a a

FIGURE 2. Average desorption curve of Gala-2x (blue) and Gala-4x (red) with standard error bands (1 SE). The left vertical dashed line indicates the start of the elastic shrinkage phase or phase I, the mid vertical dashed line represents the start of the inelastic shrinkage phase or phase II, and the right vertical dashed line indicates the end of the inelastic shrinkage phase. Hydraulic capacitance (C, g·MPa−1) is calculated as the slope of the linear regression fitted to the curve for both distinct phases and is shown for Gala-2x and Gala-4x.

ANOVA

−0.41 ± 0.08 −0.85 ± 0.14a −1.33 ± 0.26a −1.67 ± 0.28a 3.01 ± 0.28b 8.74 ± 1.90b 76.35 ± 14.85a 134.49 ± 21.76b 442.90 ± 36.60a b

F1, 4 = 8.18, P < 0.05 F1, 4 = 0.73, P > 0.1 F1, 4 = 0.16, P > 0.1 F1, 4 = 0.16, P > 0.1 F1, 4 = 15.38, P < 0.05 F1, 4 = 14.98, P < 0.05 F1, 4 = 0.71, P > 0.1 F1, 4 = 7.93, P < 0.05 F1, 4 = 3.46, P > 0.1

Notes: Water potential at 12, 50, 88, and 100% cumulative acoustic emissions of shoot xylem (AE12, AE50, AE88, and AE100), elastic and inelastic hydraulic capacitance (Cel and Cinel), initial mass (Winit), apparent modulus of elasticity (E′r) and basic wood density (ρb). Different letters indicate significant differences (P < 0.05) between the values of the two Gala variants. Means ± SD.

Anatomy

Average vessel area (A, μm²), derived mean vessel diameter, as well as the frequency of larger vessels were significantly higher in Gala- 4x (Table 2, Fig. 4). These results are in line with the observed significantly higher hydraulic vessel diameter of Gala-4x compared to Gala-2x (Table 2). Conduit wall reinforcement [(t∕b)2h, unitless] was not significantly different between the variants, but tended to be higher in Gala-2x (Table 2). Ray and vessel-associated parenchyma constituted 19.8% of the xylem area for Gala-2x, and 23.6% for Gala- 4x. The difference was mainly due to the amount of ray parenchyma (11.4% for Gala-2x vs. 14.1% for Gala-4x), and less to the amount of vessel-associated parenchyma (8.4% for Gala-2x vs. 9.5% for Gala- 4x). Microscopic wood images also illustrate wider and greater amount of conduits and higher percentage of parenchyma for Gala-4x (Fig. 5).

DISCUSSION Polyploidization and xylem vulnerability

VCAE of seedlings of both variants did not support the original hypothesis that tetraploids have a higher tolerance for drought than diploids (Fig. 1). Their xylem vulnerability to drought-induced cavitation was intrinsically similar (Table 1). Overall, Gala-2x showed a slightly lower vulnerability, and was significantly different from Gala-4x at the onset of embolism formation (AE12, Table 1). The stronger increase in percentage cumulative AE at the beginning of the VCAE of Gala-4x (Fig. 1), might be partially attributed to the stronger elastic shrinkage of bark and wood living tissue (Kikuta, 2003; Vergeynst et al., 2016), which is in accordance with its higher elastic hydraulic capacitance (Table 1, Fig. 2). Jones et al. (1989), who found leaf water potential thresholds similar to our AE12 values (Table 3), associated an early embolism onset to a less vigorous rootstock. The dwarfing rootstock M.9 started cavitating at less negative water potential than the vigorous rootstock M.25. Concurring our reported AE12, we also observed decreased growth vigor in the neopolyploid variants compared to their diploid predecessor. Earlier studies comparing cytotypes with differing ploidy levels also found altered hydraulic characteristics. Hao et al. (2013) observed a positive correlation between a decreased vulnerability to drought-induced cavitation and an increasing ploidy level in Atriplex canescens, but Maherali et al. (2009) only found a marginal increase in hydraulic conductivity in the neotetraploid Chamerion angustifolium, with no significant differences in drought vulnerability. The effect of polyploidization on xylem vulnerability to drought-induced cavitation is clearly not straightforward and will


FIGURE 3. Average stress–strain curve of Gala-2x (blue, R2 = 0.9827) and Gala-4x (red, R2 = 0.9777). The apparent modulus of elasticity (E′r, MPa) is calculated as the slope of the linear regression fitted through the curve. TABLE 2. Anatomical variables of the two variants Malus × domestica Gala-2x and Gala-4x. Variable A (μm²) (t∕b)2h (unitless) dmean (μm) dh (μm)

Gala-2x

Gala-4x

553.04 ± 42.63 0.012 ± 0.005a 26.15 ± 1.01a 27.23 ± 1.06a a

ANOVA

790.27 ± 53.74 0.005 ± 0.002a 31.01 ± 0.99b 32.95 ± 1.22b

b

F1,4 = 35.88, P < 0.01 F1,4 = 6.37, P > 0.05. F1,4 = 35.28, P < 0.01 F1,4 = 37.62, P < 0.01

Notes: Vessel area (A), vessel cell-wall reinforcement ((t∕b)2h), mean vessel diameter (dmean) and hydraulic vessel diameter (dh). Different letters indicate significant differences (P < 0.05) between the values of the two Gala variants. Means ± SD.

not necessarily translate into more resistant xylem, but can contrastingly result in more efficient xylem. Plavcová et al. (2013) found that hybrid poplars exposed to better nitrogen fertilizer conditions showed greater hydraulic efficiency, and hence lower hydraulic safety. These authors also found that the resulting changes in xylem structure were associated with differences in gene expression (Plavcová et al., 2013). Quantification of xylem vulnerability to drought-induced cavitation in Malus ×domestica is limited to just a handful of studies, illustrating a high variability in hydraulic safety between different cultivars (Beikircher et al., 2013; Table 3). According to Beikircher et al. (2013), this divergence in values might be related to genetic variation within the species or to the chosen rootstock or grafting method (Bauerle et al., 2011). AE50-values of our Gala variants (Table 1) were less negative compared to the values listed by Beikircher et al. (2013) (Table 3), and also less negative compared to the range of −3.54 to −6.27 MPa reported for 10 species of the Rosaceae genus Prunus (Cochard et al., 2008). However, vulnerability to drought-induced cavitation of our Gala variants was determined on seedling shoots instead of on branches from mature trees, which was the case for the species of Beikircher et al. (2013), Cochard et al. (2008) and Lauri et al. (2011). Johnson et al. (2016) found that ψ50 of angiosperm branches were between 0.8 and 1.7 MPa more negative than ψ50 values of on average 18 cm-diameter

FIGURE 4. Vessel size distribution for Gala-2x (blue) and Gala-4x (red). Only anatomical elements with an area above 270 μm² were considered to be vessels.

trunks. Domec and Gartner (2003) obtained a similar result in gymnosperms species, and additionally found that the difference appeared to be smallest with age. Both the difference in age and investigated portion of the tree can thus help clarifying the lower hydraulic safety of our two study Gala variants. Polyploidization and xylem anatomy

To underpin the VCAE-results, anatomical variables were also calculated. The tetraploid was characterized by a significantly higher mean vessel diameter, hydraulic diameter, and vessel area compared to Gala-2x (Table 2). This also coincided with vessel size distribution, with overall larger vessels for Gala-4x compared to Gala-2x (Figs. 4 and 5). Polyploidization thus had a clear effect on xylem structure, and because both variants were grown under the same environmental conditions, it supports the idea that polyploidization can influence the trade-off between xylem structure and function (Guet et al., 2015). To explain this trade-off, quantifying pit properties is essential, as air-seeding occurs at interconduit pits (Hacke and Sperry, 2001). In case pit properties are lacking, evidence for the trade-off between hydraulic efficiency and safety can be obtained by applying the “rare pit hypothesis” (Wheeler et al., 2005; Guet et al., 2015). Christman et al. (2009) explained that vessels with a greater total area of intervessel pits have a tendency to be more vulnerable as the chance of having more leaky pits with a lower air-seeding threshold increases. Tendency toward a greater total area of intervessel pits increases with vessel diameter (Christman et al., 2012; Scholz et al., 2013), so that genotypes with larger vessels tend to be more vulnerable to embolism formation, but more efficient at transporting water (Guet et al., 2015). Because orchard trees are typically bred under well-watered and well-fertilized conditions to maximize yield, gene expression in Gala-4x primordially translated to increased hydraulic efficiency (Plavcová et al., 2013). The rare pit hypothesis can also explain why the Malus ×domestica cultivars of Beikircher et al. (2013) and Lauri et al. (2011) were more drought tolerant than our


FIGURE 5. Transverse cross section 30 μm thick under a 4× object magnification of 1-yr-old Malus ×domestica (A) Gala-2x and (B) Gala-4x showing pith, xylem, cambium, and bark. A detailed section of the xylem is shown to better visualize the amount of parenchyma. The green and orange rectangle in the cross section of Gala-2x and Gala-4x, respectively, shows the position where the detail was taken under a 20× object magnification.

variants, as their hydraulic diameter was on average 11 and 13 μm narrower. Likewise, the average hydraulic diameter of 20 μm supports the more negative ψ50-values of the 10 Prunus species reported by Cochard et al. (2008). Vessel cell-wall reinforcement [(t∕b)2h] is positively correlated with pit membrane thickness (Jansen et al., 2009), and negatively correlated with vessel diameter, and hence, hydraulic efficiency (Li et al., 2016), and diminishes the risk of potential implosion under low water potentials (Hacke and Sperry, 2001). Awad et al. (2010) found that the most drought-tolerant poplars had the highest (t∕b)2h and that there is a positive correlation between vessel diameter and ψ50. Cell-wall reinforcement was highest for Gala-2x, which coincided with its narrower conduit diameters (Table 2), confirming Gala-2x’s slightly lower vulnerability. Values of (t∕b)2h reported for the cultivars of Beikircher et al. (2013) were higher (up to 63%) than

those of Gala-4x, confirming that gene expression in our examined tetraploid led to an increased hydraulic efficiency. Wood living tissue was also influenced by polyploidization, resulting in a 4% increase in ray and vessel-associated parenchyma for Gala-4x compared to Gala-2x (Fig. 5). Parenchyma is an important living cell type in secondary xylem, where it fulfills a diversity of roles and provides dynamic responses to mechanical damage and pathogen infection (Morris et al., 2016). The importance of living tissue in tree hydraulic functioning is clear from its role in refilling embolized conduits (Zwieniecki and Holbrook, 2009; Brodersen et al., 2010, 2018), preventing the spread of cavitation by isolating air-filled conduits (Schenk et al., 2008), and providing an important water storage location to buffer drought effects (Phillips et al., 2003). The amount of parenchyma also directly contributes to the overall capacitance to maintain hydraulic transport (Meinzer et al.,


TABLE 3. Comparison of vulnerability thresholds for different Malus domestica cultivars. Threshold Ψ12

Ψ50

VC technique

Study

AE

Jones et al. (1989)

AE Sperry apparatus

Nardini and Salleo (2000) Beikircher et al. (2013)

HPFM Sperry apparatus

Lauri et al. (2011) Beikircher et al. (2013)

Cultivar Cox Orange Pippin M.9 Golden Delicious M.9 A120/3 M.9 Cox Orange Pippin M.25 Golden Delicious M.25 A120/3 M.25 unspecified Golden Delicious Braeburn Red Delicious Starkrimson × Granny Smith cross Golden Delicious Braeburn Red Delicious

Value (MPa) −0.6 −0.7 −0.6 −1.7 −1.2 −1.0 −1.07 −2.48 −1.53 −1.41 −4.3 −3.81 −3.46 −2.73

Notes: Water potential at 12 and 50% loss of hydraulic conductivity (ψ12 and ψ50) for studies examining a variety of Malus ×domestica cultivars by applying different vulnerability curve techniques.

2009), and confirms the increased hydraulic capacitance of the Gala-4x leafy shoot (Fig. 2; Table 1). The effect of polyploidization could also be derived from the apparent modulus of elasticity (E′r), which was higher for Gala-4x compared to Gala-2x (Fig. 3, Table 1). A high E′r relates to stiffer cell walls (Niklas, 1992), for which a small decrease in water content is accompanied by a greater decrease in water potential (Kozlowski et al., 1991; Steppe and Lemeur, 2007). This translates into a lower tolerance towards drought for species with inelastic cell walls (Sanchez-Diaz and Kramer, 1971). Compared to known elasticity modulus range from 0 to −30 MPa for higher plant tissues (Steppe et al., 2006), both our variants have very inelastic cell walls, which coincides with their high vulnerability to drought-induced cavitation (Fig. 2). Polyploidization and hydraulic capacitance

Using xylem vulnerability as criterion, both Gala variants can be classified as drought-sensitive species due to their low AE50 (Table 1). However, such a conclusion could be premature as the species may rely on hydraulic capacitance of living tissue and stomatal control to prolong the negative effect of drought stress (McCulloh et al., 2014). Hydraulic capacitance is defined as a species’ ability to store water as a means to buffer water potential fluctuations and its significance toward drought tolerance has been widely accepted (Scholz et al., 2007; Meinzer et al., 2008; Steppe et al., 2012; Vergeynst et al., 2015a). To ensure an equal fast dehydration in shoots used for VCAE analysis and for hydraulic capacitance, leaves were kept attached to the shoots. Overall measured decrease in water content (ΔWC, g) was hence the combined mass loss of shoot plus leaves. Desorption curves (Fig. 2) and calculated hydraulic capacitances (Table 1) supported our second hypothesis that tetraploidy enhances hydraulic capacitance. This corresponds with the reported larger living cells in leaves (Hias et al., 2017) as well as the higher amount of ray and vessel-associated parenchyma cells in Gala-4x (Fig. 5B). This was further confirmed by the lower ρb value, which points to a higher water storage capacity and thus higher hydraulic capacitance (Scholz et al., 2007, Table 1). This might be a vital strategy for Gala- 4x to tolerate drought stress as its wider conduits allow for a greater hydraulic efficiency (Plavcová et al., 2013; Gleason et al., 2016).

The higher apparent modulus of elasticity (E′r) of the shoot tissue of Gala-4x (Table 1) seems to be in contradiction with its higher capacitance, as Lambers et al. (1998) demonstrated that a higher E′r is indicative for a smaller water storage capacity. Shoot E′r was high for both variants compared to previously published values, indicating that Gala’s shoot hydraulic capacitance values were low as well. The higher capacitance values for Gala-4x compared to Gala-2x derived from the desorption curves were therefore mainly attributed to the leaves, illustrating that polyploidization influenced not only xylem transport efficiency but also water storage capacity of leaves (Hias et al., 2017). Vulnerability thresholds can also be deduced from desorption curves because branch physiology shows that the end of phase I (elastic shrinkage) in the desorption curve correlates with AE12 of the acoustic VC, and the end of phase II (inelastic shrinkage or water released by embolisms) with AE100 (Vergeynst et al., 2016). However, transposing the VC- derived AE12 and AE100 values (Table 1) onto the desorption curves did not make sense in our study, as the desorption curves report the combined dehydration of shoot plus leaves. The higher capacitance of leafy shoots of Gala-4x thus provided an estimate of what should be a higher xylem water storage capacity and buffer against drought stress compared to Gala-2x. By equalizing the dehydration procedure between xylem vulnerability and hydraulic capacitance, weight loss data cannot be normalized to shoot level, but indirect information about the effect of polyploidization on leaves could be derived. To avoid such a combined effect of shoot plus leaves in the desorption curve, xylem vulnerability and hydraulic capacitance should be determined on identical shoots without leaves. Tetraploids clearly had an increased hydraulic capacitance of leafy shoots. Based on observations that Gala-4x had higher relative leaf water content and higher chlorophyll fluorescence parameters compared to Gala-2x under osmotic stress (Zhang et al., 2015), we suggest that the capacitive water released through embolisms sustains leaf water status. It is indeed suggested that hydraulic capacitance can play an important role in xylem water relations of a plant and that embolism formation in some cases can be beneficial in maintaining transpiration (Holbrook and Zwieniecki, 1999; Hölttä et al., 2009; Meinzer et al., 2009). Phillips et al. (2003) also found that a higher water storage capacity resulted in an increased photosynthetic rate. Moreover, polyploidization has been


shown to result in larger stomata at a lower density (Hao et al., 2013), and in some cases, polyploids can maintain photosynthesis under negative water potentials at which photosynthesis of diploids has already ceased (Li et al., 1996). It is hence likely that the faster increase in percentage cumulative AE of Gala-4x at the onset of dehydration (Fig. 1; Table 1) is due to the greater use of stored water possibly to maintain photosynthesis, whereas Gala-2x likely reduces leaf water loss by closing its stomata. Furthermore, Gleason et al. (2016) highlighted that hydraulic capacitance might be crucial to reduce the need for a high hydraulic safety, even in dry habitats. In horticultural crops, where we aim at increasing yield, maintaining gas exchange during drought is beneficial as long as drought-induced damages are limited or reversible. It is suggested that in some specific climatic scenarios it might be better to trade an increased sensitivity under severe water deficits for a higher potential of biomass accumulation through maintenance of transpiration under drought (Tardieu, 2005). We speculate that during a moderate and short drought period the lower air-seeding threshold (AE12) together with the increased hydraulic capacitance in Gala-4x may provide the buffer to drought-induced decreases in photosynthesis. Moreover, higher hydraulic efficiency of the tetraploid may enable the plant to maintain higher productivity rates than its diploid predecessor. A severe and longer drought stress would however result in irreversible damages to the hydraulic system of seedlings of both ploidy levels. CONCLUSIONS Polyploidization has a pivotal impact on tree hydraulic functioning. Previous studies reported an increased tolerance to water deficit after artificial genome doubling in different plant species. We demonstrate that conclusions may not always be straightforward when including only one hydraulic trait in this assessment. Acoustic emissions illustrated that Malus ×domestica Gala-4x was significantly more vulnerable during the early phase of desiccation compared to Gala-2x, but overall xylem vulnerability of both variants to drought-induced cavitation was similar. By also including in the assessment wood and living tissue properties of the leafy shoots, changes in water content and hydraulic capacitance, Gala- 4x showed greater hydraulic efficiency and water storage capacity. Considering all these factors, and the added value of living cells in wood, in particular, is thus crucial to better understand tree hydraulic functioning. Maintaining a desirable yield of trees bred for maximum productivity, such as apple trees, boils down to maintaining hydraulic function during drought. Greater hydraulic capacitance and higher efficiency may facilitate Gala-4x to perform longer under mild drought stress. Polyploidization may not have the expected increased drought tolerance effect, but it may assist in maintaining a viable yield within a changing climate as long as drought conditions do not result in irreversible damage to the species’ hydraulic system. Further research on the impact of polyploidization on plant hydraulic traits and functioning is therefore strongly recommended. ACKNOWLEDGEMENTS We express sincere gratitude to Geert Favyts of the Laboratory of Plant Ecology, Ghent University for constructing the sample

holders. We also thank two members of the Laboratory of Wood Technology, Ghent University, Piet Dekeyser for his aid in preparing and cutting the transverse cross sections from the wood samples and lic. Sam Colpaert for partially analyzing the wood images. We are also grateful to the anonymous reviewers and the special section editor who helped improve the quality of the manuscript. Sincere gratitude is extended to Dr. Olivier Leroux of the Department of Biology, Ghent University for improving the quality of the wood images to fulfill the revision requirements. We are also grateful to ILVO for developing and providing the polyploid plant material. This work was supported by the Research Foundation Flanders (FWO) (research program G.0319.13N and G.0941.15N granted to K.S. and supporting the Ph.D. of N.D.B.) and IWT Flanders (Ph.D. funding granted to N.H.). LITERATURE CITED Allario, T., J. Brumos, J. M. Colmenero-Flores, F. Tadeo, Y. Froelicher, M. Talon, L. Navarro, et al. 2011. Large changes in anatomy and physiology between diploid Rangpur lime (Citrus limonia) and its autotetraploid are not associated with large changes in leaf gene expression. Journal of Experimental Botany 62: 2507–2519. Awad, H., T. Barigah, E. Badel, H. Cochard, and S. Herbette. 2010. Poplar vulnerability to xylem cavitation acclimates to drier soil conditions. Physiologia Plantarum 139: 280–288. Bauerle, T. L., M. Centinari, and W. L. Bauerle. 2011. Shifts in xylem vessel diameter and embolisms in grafted apple trees of differing rootstock growth potential in response to drought. Planta 234: 1045–1054. Beikircher, B., C. De Cesare, and S. Mayr. 2013. Hydraulics of high-yield orchard trees: a case study of three Malus domestica cultivars. Tree Physiology 33: 1296–1307. Beikircher, B., and S. Mayr. 2009. Intraspecific differences in drought tolerance and acclimation in hydraulics of Ligustrum vulgare and Viburnum lantana. Tree Physiology 29: 765–775. Black, B., R. Hill, and G. Cardon. 2008. Orchard irrigation: apple. Utah State University Cooperative Extension, Logan, UT, USA. Available at Horticulture/ Fruit/2008-01pr https://digitalcommons.usu.edu/cgi/viewcontent.cgi?referer=https://www.google.be/&httpsredir=1&article=1645&context=extension_ curall. Blackman, C. J., T. J. Brodribb, and G. J. Jordan. 2010. Leaf hydraulic vulnerability is related to conduit dimensions and drought resistance across a diverse range of woody angiosperms. New Phytologist 188: 1113–1123. Brodersen, C. R., T. Knipfer, and A. J. McElrone. 2018. In vivo visualization of the final stages of xylem vessel refilling in grapevine (Vitis vinifera) stems. New Phytologist 217: 117–126. Brodersen, C. R., A. J. McElrone, B. Choat, M. Matthews, and K. Shackel. 2010. The dynamics of embolism repair in xylem: in vivo visualizations using high- resolution computed tomography. Plant Physiology 154: 1088–1095. Christman, M. A., J. S. Sperry, and F. R. Adler. 2009. Testing the ‘rare pit’ hypothesis for xylem cavitation resistance in three species of Acer. New Phytologist 182: 664–674. Christman, M. A., J. S. Sperry, and D. D. Smith. 2012. Rare pits, large vessels and extreme vulnerability to cavitation in a ring-porous tree species. New Phytologist 193: 713–720. Cochard, H., E. Badel, S. Herbette, S. Delzon, B. Choat, and S. Jansen. 2013. Methods for measuring plant vulnerability to cavitation: a critical review. Journal of Experimental Botany 64: 4779–4791. Cochard, H., S. T. Barigaha, M. Kleinhentz, and A. Eshelc. 2008. Is xylem cavitation resistance a relevant criterion for screening drought resistance among Prunus species? Journal of Plant Physiology 165: 976–982. Comai, L. 2005. The advantages and disadvantages of being polyploid. Nature Reviews. Genetics 6: 836–846.


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