Dynamic Surface Tension of Xylem Sap Lipids

Tree Physiology 40, 433–444 doi:10.1093/treephys/tpaa006

Research paper

Dynamic surface tension of xylem sap lipids Downloaded from https://academic.oup.com/treephys/article-abstract/40/4/433/5728682 by guest on 24 April 2020 Jinlong Yang1, Joseph M. Michaud2, Steven Jansen3, H. Jochen Schenk2,5 and Yi Y. Zuo1,4,5

1Department of Mechanical Engineering, University of Hawaii at Manoa, 2540 Dole Street, Holmes Hall 302, Honolulu, HI 96822, USA; 2Department of Biological Science, California State University, 800 N. State College Blvd., Fullerton, CA 92831, USA; 3Institute of Systematic Botany and Ecology, Ulm University, Albert-Einstein-Allee 11, Ulm D-89081, Germany; 4Department of Pediatrics, John A. Burns School of Medicine, University of Hawaii, 1319 Punahou Street, Honolulu, HI 96826, USA; 5Corresponding authors H.J. Schenk ([email protected]); Y.Y.Zuo ([email protected])

Received August 10, 2019; accepted January 13, 2020; handling Editor Kathy Steppe

The surface tension of xylem sap has been traditionally assumed to be close to that of the pure water because decreasing surface tension is thought to increase vulnerability to air seeding and embolism. However, xylem sap contains insoluble lipid-based surfactants, which also coat vessel and pit membrane surfaces, where gas bubbles can enter xylem under negative pressure in the process known as air seeding. Because of the insolubility of amphiphilic lipids, the surface tension influencing air seeding in pit pores is not the equilibrium surface tension of extracted bulk sap but thelocal surface tension at gas–liquid interfaces, which depends dynamically on the local concentration of lipids per surface area. To estimate the dynamic surface tension in lipid layers that line surfaces in the xylem apoplast, we studied the time-dependent and surface area-regulated surface tensions of apoplastic lipids extracted from xylem sap of four woody angiosperm plants using constrained drop surfactometry. Xylem lipids were found to demonstrate potent surface activity, with surface tensions reaching an equilibrium at ∼25 mN m-1 and varying between a minimum of 19 mN m-1 and a maximum of 68 mN m-1 when changing the surface area between 50 and 160% around the equilibrium surface area. It is concluded that xylem lipid films in natural conditions most likely range from nonequilibrium metastable conditions of a supersaturated compression state to an undersaturated expansion state, depending on the local surface areas of gas–liquid interfaces. Together with findings that maximum pore constrictions in angiosperm pit membranes are much smaller than previously assumed, low dynamic surface tension in xylem turns out to be entirely compatible with the cohesion–tension and air-seeding theories, as well as with the existence of lipid-coated nanobubbles in xylem sap, and with the range of vulnerabilities to embolism observed in plants.

Keywords: apoplastic xylem lipids, constrained drop surfactometry, DGDG, embolism, galactolipids, MGDG, phospholipids, xylem surfactant.

Introduction Outside vascular plants, negative pressure in aqueous solutions Vascular plants are the only organisms that transport water also exists in other systems that possess nanocapillaries from by taking advantage of cohesion between water molecules to which water evaporates, such as soils and rocks (Graham et al. pull it through a hydraulic system rather than pushing it under 1997, Or and Tuller 2002), and such conditions have also positive pressure. Pulling stretches the distance between water been created in engineered devices that are often referred to as molecules slightly and thereby creates negative pressure in the synthetic trees (Thut 1928, Wheeler and Stroock 2008, Duan water. The pull is caused by evaporation of water from nanocap- et al. 2012, Vincent et al. 2012). All such systems encompass illaries in the cell walls of leaves (Askenasy 1895, Dixon and Joly multiple phases and surfaces between them, including rigid 1895, Dixon 1914b), and the negative pressure is generated solid walls that do not collapse under the negative pressure, by the weight of the water column and the hydraulic resistance a continuous aqueous phase, solid–liquid surfaces, gas bub- in the hydraulic system (Hacke et al. 2006, Lucas et al. 2013). bles and gas–liquid menisci in nanocapillaries. There has been

Table 1. Composition of xylem sap lipids (in % of molar composition) for the four studied species determined by direct-infusion electrospray ionization triple-quadrupole mass spectrometry, arranged by headgroups and expressed as mean ± standard error percentage of total lipids (Schenk et al. 2019). Acyl chains (not shown here) most commonly had 34 or 36 carbon atoms, and >98% of acyl chains were unsaturated, mostly featuring 1–3 double bonds for 34 carbon atom acyl chains and 3–5 double bonds for 36 carbon atom acyl chains (Schenk et al. 2019).

Percent of all lipids L. tulipifera G. parviflora D. buccinatoria E. farinosa Galactolipids DGDG 7.8 ± 3.1 3.3 ± 0.7 9.1 ± 1.3 2.9 ± 0.9 MGDG 26.6 ± 12.6 7.1 ± 1.5 11.0 ± 2.1 4.0 ± 1.1 Phospholipids PA 21.7 ± 9.3 48.9 ± 15.8 33.9 ± 11.5 49.6 ± 15.8 PC 21.2 ± 5.3 15.3 ± 3.9 19.3 ± 4.1 13.5 ± 5.8 ± ± ± ± PE 8.4 4.2 11.1 2.1 10.2 2.3 8.7 6.0 Downloaded from https://academic.oup.com/treephys/article-abstract/40/4/433/5728682 by guest on 24 April 2020 PI 4.8 ± 2.9 7.8 ± 3.5 11.3 ± 3.8 13.4 ± 8.8 PS 2.3 ± 1.1 3.4 ± 0.8 2.9 ± 1.1 2.8 ± 1.6 PG 1.2 ± 0.5 0.7 ± 0.4 0.6 ± 0.2 0.8 ± 0.5 LysoPG 4.4 ± 0.8 1.8 ± 0.8 1.2 ± 0.3 1.9 ± 0.3 LysoPC 1.5 ± 1.0 0.3 ± 0.1 0.2 ± 0.1 1.4 ± 0.4 LysoPE 0.3 ± 0.2 0.2 ± 0.1 0.2 ± 0.1 0.9 ± 0.5

Galactolipids: DGDG = digalactosyldiacylglycerol, MGDG = monogalactosyldiacylglycerol. Phospholipids: PA = phosphatidic acid, PC = phosphatidylcholine, PE = phosphatidylethanolamine, PI = phosphatidylinositol, PS = phosphatidylserine, PG = phosphatidylglycerol, LysoPG = lysophos- phatidylglycerol, LysoPC = lysophosphatidylcholine, LysoPE = lysophosphatidylethanolamine. surprisingly little research into surface phenomena in negative associated with pit membranes would decrease surface tension pressure systems, except for studies on heterogeneous bubble and thereby increase the likelihood of gas entry through this nucleation at solid surfaces that are in touch with the liquid porous medium (Cochard et al. 2009, Domec 2011, Schenk under negative pressure (Wheeler and Stroock 2009, Chen et al. 2015, 2017, 2018, 2019, Losso et al. 2017). To date, et al. 2016, Menzl et al. 2016). Understanding the behavior only limited data are available to indicate what the effects of of surfaces under negative pressure is complicated further natural xylem surfactants on surface tension in xylem may be by the presence of surface-active molecules (surfactants) in (Christensen-Dalsgaard et al. 2011, Losso et al. 2017, Schenk the system (Atchley 1989, Yount et al. 1977, Schenk et al. et al. 2017). 2015). The behavior of lipid coatings in xylem under fluctuating The hydraulic system of a vascular plant encompasses the negative pressure remains subject to speculation until more entire plant, but the negative pressure is restricted to the parts is known about their surface tension properties. Amphiphilic outside living cells, including cell walls, intercellular spaces and lipids aggregate on surfaces and are essentially insoluble in the dead cells of the xylem, including vessels and tracheids. water, with very low critical micelle concentrations (CMCs; Together, these nonliving parts are referred to as the apoplast. Marsh and King 1986). Thus, surface tension imparted by Surfactants in xylem sap were first documented by Christensen– these lipids depends strongly on their local concentration at Dalsgaard et al. (2011) and were found to include amphiphilic a surface, i.e., the surface density of lipid molecules, which lipids, including phospholipids, and proteins (Gonorazky et al. may change over time, a phenomenon known as dynamic 2012, Schenk et al. 2017, 2018). A recent lipidomic anal- surface tension (Figure 2). Even at very low concentrations ysis of lipids extracted from xylem sap found phospholipids in xylem, amphiphilic lipids would minimize their free energy (25–93%) and galactolipids (7–75%) (see Table 1), with total by quickly accumulating on gas–liquid surfaces (Gibbs 1906, lipid concentrations ranging from 0.18 to 0.63 μmol−1 of xylem Rice 1926) and imparting a surface tension that depends on sap (Schenk et al. 2019). concentration of lipids per interfacial area and not on the overall Xylem lipids are concentrated on the surfaces of xylem lipid concentration per water volume (Figure 2). This dynamic conduits and on the cellulosic and nanoporous pit membranes surface tension concept is very different from the surface serving as connections between conduits (Figure 1). Since tension imparted by soluble surfactants in water, such as Tween, the 3D structure of pit membrane pores is characterized by Triton-X or alcohols, which below their CMCs depends on the various pore constrictions, which are typically below 20 nm concentration per volume of water and is essentially constant in diameter (Choat et al. 2003, Zhang et al. 2017, 2019), above CMC. Soluble surfactants have been used experimentally these pit membranes prevent the entry of large gas bubbles to test for effects of lowering surface tension in xylemSperry ( into water-filled conduits that are under negative pressure and Tyree 1988, Cochard et al. 2009, Hölttä et al. 2012, Losso (Zimmermann 1983, Schenk et al. 2015). However, surfactants et al. 2017).

Tree Physiology Volume 40, 2020 Dynamic surface tension of xylem sap lipids 435

Figure 1. Confocal microscopy images of transverse and tangential cell walls in secondary xylem after vacuum infiltration with FM 1-43 fluorophores Downloaded from https://academic.oup.com/treephys/article-abstract/40/4/433/5728682 by guest on 24 April 2020 (yellow color) to detect amphiphilic lipids, in vessel walls and bordered intervessel pits. Image (A) is a longitudinal view, while all other images (B-D) are transverse views. Because the secondary xylem area scanned was some distance below the injection point, the fluorophores do not occur in all conduits. Blue-colored walls show lignin autofluorescence. Lt, L. tulipifera;Gp,G. parviflora;Db,D. buccinatoria;Ef,E. farinosa; V, vessel; short arrows, intervessel walls with intervessel pits. All scale bars represent 20 μm. Reproduced and slightly modified from Schenk et al. (2018) with permission.

Figure 2. Conceptual illustration of dynamic surface tension shown for the example of amphiphilic lipid films on gas bubbles in water, depending on both surface area and time. Sizes of lipid molecules and bubbles are roughly to scale. A = collapsed lipid film, showing both outward and inward folding of the lipid layer; B = lipid monolayer at equilibrium surface tension; C = bubble expansion leading to stretched monolayer with dense and stretched lipid domains and some gaps, which can potentially be filled in by the adjacent lipid micelles, if they are present and given enough time; D = bubble compression leading back to equilibrium surface tension. I = compression, II = expansion, III = compression. Note that bubbles A–D are each coated by the same number of lipid molecules.

It is impractical to collect lipids directly from coatings on salts. In addition, using a newly developed in situ Langmuir– xylem surfaces, but fortunately they are also found in low Blodgett (LB) transfer technique (Yang et al. 2018), we revealed concentrations in xylem sap. Here, we report research on the the ultrastructure of natural xylem lipid films using atomic force dynamic surface tension properties of lipids extracted from microscopy (AFM). xylem sap of four woody angiosperm species measured using We hypothesized that purified xylem sap lipid films would constrained drop surfactometry (CDS). The CDS is a novel show similar dynamic surface tension to what is found in droplet-based surface tensiometry technology developed in other natural lipid mixtures, such as pulmonary surfactants the Zuo laboratory (Figure 3) for studying the surface activity (Zuo et al. 2008, Parra and Pérez-Gil 2015), with nonconstant of pulmonary surfactants (Valle et al. 2015). Using CDS, but surface area-regulated dynamic surface tension upon film we directly measured the time-dependent and surface area- compression and expansion (Schürch et al. 2010). Galac- regulated dynamic surface tensions of lipids extracted from tolipids in xylem lipid mixtures may affect the behavior of xylem sap, which cannot be measured using standard methods films especially at low surface tension, because their galac- such as the pendant drop technique. The technique was used tose headgroups strongly interact with each other, promoting previously for studying dynamic surface tension of freeze- lipid stacking and layering (Lee 2000, Bottier et al. 2007, dried xylem sap extract (Schenk et al. 2017), i.e., non-purified Kanducˇ et al. 2017). Together with the xylem sap lipidomic mixtures of proteins, amino acids, carbohydrates, lipids and analysis in a companion study (Schenk et al. 2019), our

Tree Physiology Online at http://www.treephys.oxfordjournals.org 436 Yang et al.

between species, but such comparisons were not the goal of this study. Most collections were done at dawn to ensure full hydration of the stems. The extraction protocol was described in detail in Schenk et al. (2019). Branches were cut from the plants at a length exceeding the longest vessel for each species, transported immediately to the lab and cut under water, with the final cuts made with a fresh razor blade. The bark was removed from the proximal end for ∼4 cm length to expose the xylem cylinder and to avoid phloem contamination. The cut surface

was thoroughly cleaned with deionized water using a high- Downloaded from https://academic.oup.com/treephys/article-abstract/40/4/433/5728682 by guest on 24 April 2020 pressure dental flosser (WP-100 Ultra Water Flosser, Water Pik Inc., Fort Collins, CO, USA) for 2 min to remove cell debris and cytoplasmic content from the surface. Xylem sap was extracted from the proximal end of the stem under vacuum into a glass test tube embedded in ice inside a 1-l Buchner flask. The flask was subjected to lab vacuum for ∼30 s, after which the distal end of the branch was cut back by ∼4cm,followedbysuccessive Figure 3. Schematic of the CDS for studying the surface activity of lipid films. A liquid droplet is ‘constrained’ on a 3- to 5-mm carefully machined 2-cm cuts made to all the side branches until sap was observed pedestal with a knife-sharp edge. The lipid film can be periodically dripping into the test tube. Further 1-cm cuts were made every compressed and expanded by precisely regulating liquid flow into and minute to allow for slow, continuous extraction of xylem sap. out of the droplet with a motorized syringe. The surface tension and Depending on stem size, 1–2 ml of sap were extracted from surface area of the lipid film are simultaneously determined from the shape of the droplet using axisymmetric drop shape analysis. ADSA, each stem, moved in a glass pipette into pre-weighed 1.5-ml axisymmetric drop shape analysis. LoBind Eppendorf microcentrifuge tubes, flash-frozen in liquid nitrogen and stored in the freezer until further processing. Samples were also taken to test for contamination of sap data provide a novel insight into the understanding of surface samples by lipids from living cell remnants (Schenk et al. 2017). phenomena in xylem of vascular plants and their potential This was done prior to xylem sap extraction by inserting the impacts on hydraulic function at the organ to whole-plant cleaned, cut ends of xylem into a glass vial containing 1 ml level. of nanopure water. Lipid concentrations in these controls for the four study species ranged from 1.1 to 6.7% of the lipids Materials and methods foundinxylemsap(Schenk et al. 2019), which was too low to be included in surface tension measurements. Other tests have Extraction of xylem lipids shown that xylem sap lipids are not contaminants that originate Natural xylem lipids were extracted from xylem sap of four from repeated cuts of stems made during the vacuum extraction woody plant species, Distictis buccinatoria (DC.) A.H. Gentry (Schenk et al. 2019), and the ordered nature of lipid layers (syn. Amphilophium buccinatorium (DC.) L.G. Lohmann, ever- observed on xylem surfaces (Figure 1)(Schenk et al. 2017, green vine, Bignoniaceae), Encelia farinosa A. Gray ex Torr. 2018) also rules out contamination as the source of xylem sap (semi-deciduous desert shrub, Asteraceae), Geijera parviflora lipids. Lindl. (evergreen tree, Rutaceae) and Liriodendron tulipifera L. Xylem sap samples in LoBind Eppendorf microcentrifuge (deciduous tree, Magnoliaceae). These species represent differ- tubes were partially lyophilized in a freeze dryer (FreeZone ent plant growth forms from four major clades distributed across 1 Liter Benchtop Freeze Dry System, Labconco, Kansas City, the angiosperm phylogeny. Stems for xylem sap extraction MO, USA) until only ∼100 μl of sample remained in each were collected in August 2017. Because lipid concentrations tube. The remaining aqueous samples were combined into one in sap are very low, as lipids are mostly attached to xylem sample for each species using a glass pipette and weighed. surfaces, samples (from two stems for Encelia and four stems Methanol:chloroform 1:1 (both HPLC grades, Fisher Scientific, for Distictis, Geijera and Liriodendron) were combined into one Waltham, MA, USA) was added to the combined aqueous sam- composite sample per species to ensure a sufficiently high ple to create an approximate 5:5:1 methanol:chloroform:water concentration of lipids per sample for surface tension analysis one-phase mixture. The mixture was then vortexed and cen- and to create the same lipid composition as in other subsamples trifuged, and the supernatant was collected using a 1-ml glass analyzed via mass spectrometry (Schenk et al. 2019). Analysis syringe with PrecisionGlide needle. A new one-phase 5:5:1 of composite samples does not allow statistical comparisons mixture of methanol:chloroform:water was then added to the

Tree Physiology Volume 40, 2020 Dynamic surface tension of xylem sap lipids 437 residue, the mixture was vortexed and centrifuged, and the supernatant again was collected and combined with the previously collected supernatant. Samples were dried in a desiccator outfitted with an in-line carbon filter capsule (model 6704- 7500, Whatman, GE Healthcare Life Sciences, Marlborough, MA, USA) and sent to the Zuo lab at the University of Hawaii at Manoa for analysis. Other samples from these same collections extracted the same way were sent out for mass spectrometry analyses to determine the lipid compositions, which are reported in a separate paper (Schenk et al. 2019).

Xylem lipids extracted via this protocol also include very Downloaded from https://academic.oup.com/treephys/article-abstract/40/4/433/5728682 by guest on 24 April 2020 low concentrations of hydrophobic proteins determined by Tris- Tricine SDS-PAGE to be between 5 and 18 kDa in size (data not shown). The very low concentrations have prevented the identification of these proteins to date.

Constrained drop surfactometry Figure 4. Time-dependent dynamic surface tension of natural xylem Dynamic surface tension measurements and surfactant film lipids from four woody angiosperm species, measured via CDS and imaging were carried out with CDS (Valle et al. 2015). Con- ADSA (Figure 3) starting with a pure water droplet at time zero (72 -1 strained drop surfactometry is a new generation of drop- mN m ) and after adding xylem lipids to the droplet at ∼3 s, leading to an immediate drop in surface tension to around 37 mN m-1 and then based surfactometer. As shown in Figure 3, it uses a sessile -1 reaching equilibrium surface tension, γ e,around25mNm within a few drop (∼3 mm in diameter) to generate the air–water surface, seconds. where the natural surfactant sample is spread and studied. The sessile drop is confined on a carefully machined pedestal that LB-transferred from the air–water surface to a small piece of uses a knife-sharp edge to prevent the droplet from spreading freshly peeled mica sheet by lifting the mica through the drop at low surface tensions. The spread surfactant film can be surface at a rate of 1 mm min as described in Yang et al. (2018). compressed and expanded by withdrawing and injecting liquid Topographic images of the xylem lipid films were obtained into the droplet using a motorized syringe. Surface tension using an Innova AFM (Bruker, Santa Barbara, CA, USA). Samples is determined photographically from the shape of the droplet were scanned in air in a contact mode using a silicon nitride using axisymmetric drop shape analysis (ADSA) (Yang et al. cantilever with a spring constant of 0.12 N m-1 andatipradius 2017). of 2 nm. Lateral structures were analyzed using Nanoscope For CDS measurement, each freeze-dried xylem lipid sample Analysis (version 1.5). was dissolved in 50 μl of chloroform. The samples were subjected to 5 min of ultrasonic treatment, followed by 1 min Results of vortex treatment to create a homogeneous solution, after which 1 μl of xylem lipid sample was spread at the surface Time-dependent dynamic surface tension of xylem lipids of the pure water droplet (Milli-Q ultrapure water; Millipore, Figure 4 shows the time-dependent dynamic surface tensions Billerica, MA, USA) of the CDS instrument, where it was left of xylem lipids extracted from D. buccinatoria, E. farinosa, G. undisturbed for 1 min to allow film spreading and evaporation parviflora and L. tulipifera. Reproducibility of these dynamic of the chloroform. Time-dependent dynamic surface tension surface tension measurements within each composite sample was recorded with ADSA until an equilibrium surface tension canbefoundinFigure S1 available as Supplementary Data at (γ e) was reached. Subsequently, the xylem surfactant film was Tree Physiology Online. After spreading at the air–water surface, -2 compressed and expanded at a rate of 0.1 cm min. The the xylem lipids immediately reduced surface tension below surface area-regulated surface tension was recorded during 40 mN m-1. They further reduced the surface tension to an three compression–expansion cycles for each composite sam- -1 equilibrium value (γ e)around25mNm within 2–3 s. The γ e ple. All measurements were conducted at room temperature of for xylem lipids of all four species is very close and is consistent ± ◦ 20 1 C. with that of the adsorbed film from fully hydrated phospholipid bilayers (Zuo et al. 2008). Film imaging with AFM Surface area-regulated surface tension of xylem lipids To determine if the lipid films were smooth monolayers at γ e, the molecular organization and lateral structure of the spread Figure 5 shows the surface area-regulated dynamic surface ten- xylem lipid films at γ e were visualized via AFM. The films were sion of the xylem lipids for the four plant species. Reproducibility

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Figure 5. Surface area-regulated surface tension of xylem lipids from four woody angiosperm species. Film compression is indicated by solid symbols and film expansion is indicated by open symbols. Lipids from all four species show similar compression and expansion cycles. Cycles start at100% relative surface area, followed by compression to 50–70% and expansion to 140–170% of surface area. Explanation of symbols: γ max = maximum surface tension on expanded surface area, γ e = equilibrium surface tension at 100% relative surface area, γ min = minimum surface tension of collapsed lipid layers. of these compression–expansion cycles within composite sam- be found in Figure S3 available as Supplementary Data at Tree ples can be found in Figure S2 available as Supplementary Data Physiology Online. At γ e, the lipid film at the air–water surface at Tree Physiology Online. In these experiments, the equilibrium reaches a dynamic equilibrium with lipid micelles and vesicles xylem surfactant films at γ e were first compressed to ∼60% of in the subphase. As shown in Figure 6, all xylem lipid films the original area and subsequently expanded to 160% of the maintain a lateral structure of coexisting interfacial monolayer original area. When an insoluble surfactant film at equilibrium with randomly distributed protrusions. Although the thickness of is compressed, it enters a metastable compression regime. All the background layer is unknown, its smoothness shows it to be four xylem lipid films were remarkably similar between species, a monolayer, as lipids prone to multilayer formation would form despite the differences in lipid composition (Table 1). Lipid a much bumpier surface. Height analysis indicates that the sizes films quickly reach a minimum surface tension (γ min) around of most protrusions are multiples of 4 nm, which is the thickness 19 mN m-1 when the films are compressed by <5% of the of a fully hydrated lipid bilayer (Nagle and Tristram-Nagle original area. This γ min is in line with the collapse surface 2000). Hence, these protrusions most likely represent multilay- tension reported for the two galactolipids in xylem lipid, mono- ers of lipids attached to the interfacial monolayer of the xylem galactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol surfactant under the equilibrium condition. The number and (DGDG) (Gzyl-Malcher et al. 2008, Hoyo et al. 2016). The γ min height of the protrusions are different for the xylem lipid from the does not change significantly with further area reduction by40% four plant species. Lipids from Distictis and Liriodendron,which of the lipid film. Upon expansion, all four xylem lipid films show have the largest amounts of galactolipids (Table 1), have the -1 an abrupt surface tension increase to γ e, i.e., around 25 mN m . fewest peaks, and most are around 8 nm in height. Although the The surface tension is stabilized at γ e until the film is expanded number of 8-nm peaks is highest in Distictis and Liriodendron, beyond its original surface area. With further expansion, the a 12-nm peak was found for Encelia. These images support lipid film enters a metastable expansion regime, in which the the conclusion that xylem lipids form monolayers at equilibrium surface tension increases continuously with area expansion to surfacetensionbutbegintocreatefoldsandstacksthatare 160% of its original area. The film is then compressed again likely to increase upon further compression of the layer. with decreasing surface tension to complete a cycle.

Ultrastructure of xylem lipid films Discussion Figure 6 shows the AFM topographic images of the four xylem lipid films at the equilibrium surface tensionγ ( e). Reproducibility An aqueous liquid under negative pressure is in a metastable of these AFM topographic images within composite samples can state and is prevented from turning into vapor by the nucleation

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Figure 6. Atomic force microscopy topographic images of four natural xylem lipid films close to equilibrium surface tension (see Figure 5). The films were created by moving a clean mica sheet through a lipid film at equilibrium. (a) D. buccinatoria,(b)E. farinosa;(c)G. parviflora and (d) L. tulipifera. The AFM images in the first row have the same scanning area of10 × 10 μm and the z-range of 20 nm. Images in the second row show the surface render of the zoom-in xylem surfactant film as indicated by the white boxes. White arrows indicate the heights of the multilayered structures. energy required to break hydrogen bonds between water further compression could further reduce the surface tension molecules and form a void (Wheeler and Stroock 2009, Chen toward zero, as the lipid layers stack and fold. When expanded et al. 2016, Menzl et al. 2016). Pure water has a very high above γ e, the xylem surfactant films maintain an undersaturated tensile strength, up to a theoretical maximum of −190 MPa or metastable state and increase the surface tension to a maximum -1 -1 at least to an experimentally achievable maximum of −30 MPa (γ max)of52mNm for E. farinosa and 68 mN m for D. (Caupin et al. 2012), which prevents homogeneous nucleation buccinatoria, i.e., closer to the surface tension of pure water under negative pressure in plants (Oertli 1971, Stroock et al. (Figure 5). The shapes of surface tension curves as function 2014). Xylem sap, however, is not pure water, and the presence of drop surface area are remarkably similar between species of salts, organic solutes and especially surface-active molecules (Figure 5), despite considerable differences in lipid composition would affect nucleation thresholds (Dixon 1914b, Hemmingsen (Table 1). 1978). Because of the perception that low surface tension Our hypothesis that xylem lipids would show similar dynamic would increase vulnerability to embolism, the surface tension of surface tension to other biological lipid mixtures, such as pul- xylem sap has been traditionally assumed to be equal to that monary surfactants, was supported by these findings. Dynamic of the pure water (Oertli 1971, Tyree and Zimmermann 2002, changes in surface tension are crucial for the function of Meyra et al. 2007). That assumption, however, has proven to alveoli in the lungs (Parra and Pérez-Gil 2015), but it remains be incorrect (Christensen-Dalsgaard et al. 2011, Domec 2011, to be investigated what their functional importance is in the Losso et al. 2017, Schenk et al. 2017). Moreover, the functional angiosperm xylem, where changes in pressure that affect gas– significance of surface-active insoluble lipids on xylem surfaces liquid interfaces typically show diurnal and seasonal fluctuations, demands the consideration of not just the equilibrium surface not many fluctuations per minute, as in the lungs. Effects of tension of bulk xylem sap but also the concentration-dependent, compression and expansion rates on dynamic surface tension local surface tension at phase interfaces in xylem. (see Figure S2 available as Supplementary Data at Tree Phys- Here, we determined both time-dependent and surface area- iology Online) merit further investigation, as not all pressure regulated dynamic surface tensions of natural lipids extracted changes in plants are slow. Embolism formation in a xylem from xylem sap. It was found that when spread at the air– conduit creates a sudden, local pressure increase (Hölttä et al. water surface, all xylem lipids quickly reach an equilibrium 2009), and it remains to be investigated how lipid layers on (γ e) by reducing the surface tension of the air–water surface air–water interfaces would respond to such an extremely rapid from 72 to ∼25 mN m-1 within a few seconds (Figure 3), change in surface conditions. indicating a potent surface activity. When compressed below The presence of substantial amounts of galactolipids in xylem γ e, the xylem lipid films maintain a supersaturated metastable sap (6.9% in Encelia to 34.4% in Liriodendron; Table 1)was state without collapse and reduce the surface tension to a very unexpected, as both are plastid lipids, and MGDG has -1 minimum surface tension (γ min)around19mNm .Presumably never been found outside of plastids before (Dörmann and

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Benning 2002, Kalisch et al. 2016). Apoplastic galactolipids surface tension of xylem sap can be reduced to the range of in xylem vessels most likely originate from the original cell 52–58 mN m-1, most likely caused by a reduction of surface content of the living vessels before programmed cell death, area due to evaporation from the drop, which was not in a although this requires further research (Esau 1965, Esau et al. vapor-saturated environment. In a later study, the surface tension 1966, Schenk et al. 2019). The role of galactolipids in the of xylem sap extracted from two conifers was measured using dynamic surface tension patterns shown in Figures 4 and 5 the sessile drop method (Losso et al. 2017), finding that the requires further investigation. Galactolipids are typically highly equilibrium surface tension showed seasonal variations and unsaturated (Dörmann and Benning 2002, Kalisch et al. 2016), fluctuated between 50 and 68-1 mNm over the year. Minimum and unsaturated MGDG, which made up between 4.0 and surface tensions reported by these researchers are closer to 26.6% of all xylem lipids in this study, do not form bilayers by the maximum surface tension (γ max) determined in the present

themselves, because their small headgroup and spreading acyl study. Downloaded from https://academic.oup.com/treephys/article-abstract/40/4/433/5728682 by guest on 24 April 2020 chains give them a cone shape that favors curved shapes of The differences between the present study and previous lipid layers (Lee 2000). However, strong headgroup interactions measurements can be explained by the following two facts. between galactolipids favor stacking, such as seen in the First, both previous measurements (Christensen-Dalsgaard et grana of thylakoids in chloroplasts (Lee 2000, Simidjiev et al. al. 2011, Losso et al. 2017) did not study the surface area- 2000, Bottier et al. 2007, Kanducˇ et al. 2017). Galactolipids regulated, dynamic surface tension. Second, both previous may be especially prone to stacking and folding and could measurements (Christensen-Dalsgaard et al. 2011, Losso et al. be responsible for the distinct inward folding of lipid coats 2017) used extracted xylem sap without further purification seen in freeze-fracture electron micrographs of lipid-coated and concentration. In contrast, the xylem lipids studied here nanobubbles from xylem sap in Figure 8C in Schenk et al. are purified (by organic solvent extraction) and concentrated (2017) and the multilayered structures seen under AFM, even (by pooling multiple xylem sap samples). Consequently, the at equilibrium surface tension, γ e (Figure 6). xylem lipids studied here show significantly more potent surface Most xylem sap lipids are phospholipids, especially phospha- activity than the xylem sap studied before, as indicated by tidic acid (PA) and phosphatidylcholine (PC) (Table 1), and the a much faster adsorption (reaching equilibrium within 5 s vs -1 composition of phospholipid headgroups is likely to affect some >30 min) and a much lower γ e (25 vs > 50 mN m ). A look of the differences in dynamic surface tension between species. at the xylem conduits shown in Figure 1 (also see Schenk et al. For example, the headgroup of PA is small and negatively 2018) demonstrates why it makes sense to measure surface charged, with a tendency toward forming nanodomains and tension of lipid films rather than that of extracted xylem sap. concave (inward) curvature of lipid layers (Kooijman et al. Lipid layers on inner vessel wall surfaces and in bordered pits 2003). However, surface tension is more strongly affected by create high local lipid concentrations at locations where bubble acyl chain characteristics, and these were very similar between nucleation is most likely to occur, while most of the surface- species, with ∼84% of acyl chain lengths of 17 and 18 carbon attached lipids are unlikely to appear in extracted and non- atoms, > 98% unsaturated, and with mostly 1–3 double bonds purified xylem sap. Therefore, the equilibrium surface tension for chain lengths of 17 and 3–5 double bonds for chain lengths measurements of extracted bulk xylem sap do not provide any of 18. The similarity in chain lengths and degree of saturation information about the functional significance of surfactants in could explain why minimum and equilibrium surface tension water-conducting cells of xylem tissue. On the other hand, while was remarkably similar between species. Further research will lipid extraction from sap is necessary to concentrate the lipids be required to determine the effects of lipid composition on for realistic surface tension measurements, extraction removes the differences in surface tension between species, including other components found in sap, such as ions, proteins and their effects on folding, stacking and formation of nanodomains carbohydrates, that may interact with lipids in xylem. Future by phase separation, best studied via AFM at minimum and research will have to compare the behavior of lipids in the maximum surface tension. presence and absence of these other xylem components. Our data therefore indicate that the xylem surfactant films Comparison with previous surface tension measurements of studied previously were most likely at an undersaturated xylem sap expansion state, as they were conducted with xylem sap with Both γ e and γ min determined here are significantly lower than very low surfactant concentrations (Schenk et al. 2017, 2019), the traditionally estimated and measured surface tension of bulk and using static drop-based methods that did not include drop xylem sap (Tyree and Zimmermann 2002, Christensen-Dals- compression (Christensen-Dalsgaard et al. 2011, Losso et al. gaard et al. 2011, Losso et al. 2017). A previous study of the 2017), except for potential evaporation from a pendant drop, surface tension of xylem sap of three angiosperm tree species potentially causing it to shrink and decreasing surface tension using the pendant drop method (Christensen-Dalsgaard et al. (Christensen-Dalsgaard et al. 2011). The droplet for surface 2011) found that given a long-enough time (i.e., 30 min), the tension measurements usually has a diameter of 3–4 mm,

Tree Physiology Volume 40, 2020 Dynamic surface tension of xylem sap lipids 441 corresponding to a surface area of 0.15–0.4 cm2 (Figure S2 tension of an air–water meniscus is reduced to 25 mN m-1,the available as Supplementary Data at Tree Physiology Online). equilibrium surface tension found in this study, a meniscus could This drop size is required by the measurement principle of pass through a 20-nm pore with a shape correction factor of drop shape analysis. When the drop/bubble is smaller than 0.5 under a pressure difference of 2.5 MPa. This is a far more the capillary length (e.g., 2.7 mm for pure water), it cannot realistic value for air seeding to cause embolism in angiosperms be effectively deformed by gravity and thus maintains a nearly than 7.2 MPa (Choat et al. 2012). spherical shape that fails the surface tension measurements The assumption that a rare pit membrane with a much larger from shape analysis (Yang et al. 2017). The difference pore will trigger embolism formation at a similar air-seeding between our study and previous ones is not in the size of pressure is frequently suggested in the literature (Wheeler et the droplet but in the changing concentration of surface- al. 2005, Plavcová et al. 2013). This hypothesis, however, does

active molecules on the droplet’s surface, which is needed not consider the presence of xylem sap surfactants and the 3D Downloaded from https://academic.oup.com/treephys/article-abstract/40/4/433/5728682 by guest on 24 April 2020 to understand the concentration-dependent local surface structure of the pit membrane pore space, which consists of tension. various pore constrictions with highly variable pore volumes, depending on the overall pit membrane thickness and the Implications of dynamic surface tension of lipids for xylem number of cellulose microfibril layersJansen ( et al. 2018, Kaack function et al. 2019, Zhang et al. 2019). Low surface tension in xylem is Dynamic surface tension commonly shows hysteresis between therefore entirely compatible with the cohesion–tension and air- compression and expansion (Figure 5)(Ingenito et al. 1999, seeding theories and with the range of observed vulnerabilities Zuo et al. 2008), due to processes such as the formation, to embolism in plants, while xylem sap with the high surface dissolution and tearing of lipid domains (Kwan and Borden tension of pure water is incompatible with both the theories 2010), lipid layer folding during compression and unfolding and observations. during expansion and replenishment of lipids from micelles into Xylem lipids not only coat solid surfaces but have also been stretched lipid filmsFigure ( 2). Hysteresis may be important found to coat nanobubbles in extracted xylem sap (Schenk et in the face of pressure fluctuations in xylem conduits. Xylem al. 2017). It is impossible to know for certain if lipid-coated commonly experiences diurnal fluctuations in negative pressure nanobubbles exist in sap that is under negative pressure in that can exceed 2 MPa (Meinzer et al. 2016), with the most intact xylem, but their existence in aqueous solution under negative pressures occurring in the middle of the day and fluctuating negative pressure is theoretically possible if their less negative ones often just before dawn. Large fluctuations in surface tensions were sufficiently lowAtchley ( 1989, Schenk pressure would affect phase interfaces in xylem and cause area et al. 2015, 2017). In fact, closely packed insoluble surfactant variations of the lipid-covered surface, thus actively regulating monolayers and the resultant regulation of surface tension due the surface tension. Hysteresis effects are most likely short-lived, to surface area variations may be the only way to allow very as lipids can migrate quickly between micelles in bulk and phase small gas bubbles to exist in a liquid (Atchley 1989, Yount interfaces, but hysteresis phenomena in lipid films could play an et al. 1977, Yount 1979, 1997). Otherwise, a small gas important role in response to sudden pressure fluctuations, such bubble in water would either dissolve instantaneously because as a sudden local tension relief caused by the formation of an of the excess Laplace pressure inside the bubble or expand embolism (Hölttä et al. 2009). into an embolism if the differences between the sap pressure The most likely sources for emboli in xylem are pores in pit and the internal gas pressure in the bubble exceeded the membranes between sap-filled and gas-filled conduits (Zimmer- pressure originating from the surface tension (Atchley 1989, mann 1983, Hacke and Sperry 2001, Meyra et al. 2007, Jansen Schenk et al. 2017). Lipid-coated nanobubbles could originate and Schenk 2015). Estimated pore constrictions in fresh (fully from liquid–lipid–gas interfaces moving through highly variable hydrated, non-dried) pit membranes of angiosperms are much pore volumes with multiple pore constrictions. The resulting smaller (<20 nm in diameter) than previously assumed, while nanobubbles need to be smaller than the critical size that causes previous estimates of maximum pore sizes were based on a bubble expansion depending on surface tension (Schenk et al. simplistic, 2D view of pores and affected by drying artifacts 2017). Bubbles larger than that critical size would expand and (Zhang et al. 2017, 2019). According to the Young–Laplace either continue to be stabilized by increasing area-dependent equation, the pressure difference required to force a bubble surface tension (Figures 2 and 5) or continue expanding to form through a 20-nm pore, assuming a contact angle of zero a xylem embolism. (Meyra et al. 2007 Caupin et al. 2008), and a pore shape Obviously, xylem in a living plant experiences constant correction factor for non-cylindrical pores of 0.5 (Schenk et al. changes in temperature, pressure and dissolved gas con- 2015), would be 7.2 MPa in pure water. Most angiosperms centrations (Schenk et al. 2016), and simple equilibrium are vulnerable to air seeding far below that value (Choat et models cannot do justice to ever-changing conditions. The al. 2012). With lipids in pit membrane pores, if the surface dynamic surface tension data presented in this study will

Tree Physiology Online at http://www.treephys.oxfordjournals.org 442 Yang et al. allow the development of more realistic dynamic models to Acknowledgments predict the formation and behavior of lipid-coated nanobubbles The authors thank Tatiana Melendres for assistance with xylem and of other phase interfaces in xylem, especially at pit sap extractions, Greg Pongetti at the Fullerton Arboretum for membranes. allowing sampling of plants, and Aissa Do and Alison Miyamoto Remarkably, the first one to suggest a role of colloids for for conducting protein analyses of xylem lipid mixtures. enabling water transport under negative pressure in plants was no other than Henry Dixon, one of the originators of the cohesion–tension theory (Dixon 1914a, 1914b). The evidence Conflict of interest presented in this study sheds a novel light on the long-standing Wedeclarethatwehavenoconflictofinterest. question of how plants transport water under negative pressure, which remains incompletely understood, especially with respect Funding Downloaded from https://academic.oup.com/treephys/article-abstract/40/4/433/5728682 by guest on 24 April 2020 to the mechanisms that prevent embolism formation and the temporal frequency of hydraulic failure. The research was funded by the National Science Foundation IOS-1558108 and IOS-1754850 (H.J.S., S.J. and Y.Y.Z.) and Conclusions CBET-1604119 (Y.Y.Z.). S.J. acknowledges funding from the German Research Foundation (Individual Research Grant No. Contrary to long-standing assumptions, the apoplast of 383393940). angiosperm xylem contains amphiphilic lipids, which are insoluble and accumulate on surfaces, including at air–water References interfaces. Using constrained drop surfactometry, we were able to study the time-dependent and surface area-regulated Askenasy E (1895) Ueber das Saftsteigen. Verhandlungen des Naturhistorisch-medizinischen Vereins zu Heidelberg. NF 5: dynamic surface tensions of apoplastic lipids. These were 325–345. extracted from xylem sap, because it is impractical to collect Atchley AA (1989) The Blake threshold of a cavitation nucleus having lipids directly from surfaces in the xylem. It is found that xylem a radius—dependent surface tension. J Acoust Soc Am 85:152–157. lipids extracted from all four tree species demonstrate potent Bottier C,GéanJ, Artzner F,DesbatB, Pézolet M, Renault A, D V surface activity. Surface tensions of the xylem surfactants reach Marion ,Vié (2007) Galactosyl headgroup interactions con- trol the molecular packing of wheat lipids in Langmuir films and -1 an equilibrium at 25 mN m and vary between a minimum in hydrated liquid-crystalline mesophases. Biochim Biophys Acta value of 19 mN m-1 and a maximum value of 68 mN m-1 Biomembr 1768:1526–1540. when changing the surface area between 60 and 160% around Caupin F, Cole MW,BalibarS, Treiner J (2008) Absolute limit for the the surface area at equilibrium. We therefore concluded that capillary rise of a fluid. Europhys Lett 82:1–6. Caupin F, Arvengas A,DavittK,AzouziMEM, Shmulovich KI,RambozC, xylem lipid films in natural conditions vary most likely between Sessoms DA, Stroock AD (2012) Exploring water and other liquids nonequilibrium conditions of a supersaturated metastable at negative pressure. J Phys Condens Matter 24:284110. compression state and an undersaturated metastable expansion Chen IT, Sessoms DA,ShermanZM,ChoiE, Vincent O,Stroock state, depending on the local surface area available at phase AD (2016) Stability limit of water by metastable vapor-liquid equilibrium with nanoporous silicon membranes. 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Cochard H, Hölttä T, Herbette S,DelzonS, Mencuccini M (2009) New xylem lipids both affect air seeding through the tiny pore insights into the mechanisms of water-stress-induced cavitation in constrictions of angiosperm pit membranes and can also conifers. Plant Physiol 151:949–954. stabilize resulting nanobubbles, thus collectively reducing the Dixon HH (1914a) On the tensile strength of sap. Sci Proc R Dublin vulnerability to embolism at the organ and whole-plant level. Soc NS 14:229–234. Dixon HH (1914b) Transpiration and the ascent of sap in plants. Macmillan and Co., London, UK, p 216. Supplementary Data Dixon HH, Joly J (1895) On the ascent of sap. Philos Trans R Soc Lond B 186:563–576. Supplementary Data for this article are available at Tree Domec JC (2011) Let’s not forget the critical role of surface tension in Physiology online. xylem water relations. Tree Physiol 31:359–360.

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