Dynamic control of osmolality and ionic composition of the xylem sap ...

American Journal of Botany 101(6): 1013–1022. 2014.

DYNAMIC CONTROL OF OSMOLALITY AND IONIC COMPOSITION OF THE XYLEM SAP IN TWO MANGROVE SPECIES1

JORGE LÓPEZ-PORTILLO2,6, FRANK W. EWERS3, RODRIGO MÉNDEZ-ALONZO2, CLAUDIA L. PAREDES LÓPEZ4, GUILLERMO ANGELES2, ANA LUISA ALARCÓN JIMÉNEZ5, ANA LAURA LARA-DOMÍNGUEZ2, AND MARÍA DEL CARMEN TORRES BARRERA5 2Red

de Ecología Funcional, Instituto de Ecología, A. C., Carretera antigua a Coatepec 351 El Haya Xalapa 91070 Veracruz, México; 3Biological Sciences Department, California State Polytechnic University, Pomona, 3801 West Temple Avenue, Pomona, California 91768 USA; 4Posgrado en Ecología y Manejo de Recursos, Instituto de Ecología, A. C. Carretera antigua a Coatepec 351 El Haya Xalapa 91070 Veracruz, México; and 5Centro de Ciencias de la Atmósfera, UNAM, Circuito Exterior, Ciudad Universitaria, México, D.F. 14510 México • Premise of the study: Xylem sap osmolality and salinity is a critical unresolved issue in plant function with impacts on transport efficiency, pressure gradients, and living cell turgor pressure, especially for halophytes such as mangrove trees. • Methods: We collected successive xylem vessel sap samples from stems and shoots of Avicennia germinans and Laguncularia racemosa using vacuum and pressure extraction and measured their osmolality. Following a series of extractions with the pressure chamber, we depressurized the shoot and pressurized again after various equilibration periods (minutes to hours) to test for dynamic control of osmolality. Transpiration and final sap osmolality were measured in shoots perfused with deionized water or different seawater dilutions. • Key results: For both species, the sap osmolality values of consecutive samples collected by vacuum extraction were stable and matched those of the initial samples extracted with the pressure chamber. Further extraction of samples with the pressure chamber decreased sap osmolality, suggesting reverse osmosis occurred. However, sap osmolalities increased when longer equilibration periods after sap extraction were allowed. Analysis of expressed sap with HPLC indicated a 1 : 1 relation between measured osmolality and the osmolality of the inorganic ions in the sap (mainly Na+, K+, and Cl−), suggesting no contamination by organic compounds. In stems perfused with deionized water, the sap osmolality increased to mimic the native sap osmolality. • Conclusions: Xylem sap osmolality and ionic contents are dynamically adjusted by mangroves and may help modulate turgor pressure, hydraulic conductivity, and water potential, thus being important for mangrove physiology, survival, and distribution. Key words: Avicennia germinans; ion-mediated hydraulic conductivity; Laguncularia racemosa; mangroves; sap ionic composition; sap salinity; turgor pressure; water potential; xylem sap osmotic potential; xylem sap solutes.

For more than 50 years, mangroves and other “salt-loving” plants (halophytes) have been near the center of debate regarding the osmotic potential of the apoplast, the nonliving component of plants (Scholander et al., 1962, 1964; James et al., 2006). How high is the osmotic potential and salinity of the apoplast and how does the apoplast interact with the symplast, the living cell system, to generate water potential gradients? If the osmotic potential in the apoplast, and more specifically in the xylem vessel sap, is due mainly to inorganic ions, water flow efficiency would be impacted through an ionic effect (Nardini et al., 2011). It would 1 Manuscript received 9 December 2013; revision accepted 5 May 2014. This work was supported by the CONACyT research grant 25935-N to J.L.P. The participation of C.L.P. was in partial fulfillment of a PhD degree at INECOL. The authors thank H. Bravo for HPLC facilities at the CC A-UNAM, V. Néquiz Castillo, R. Bautista Benítez and V. M .Vásquez Reyes for assisting in the chemical analyses and field work, I. I. Ortiz and A. LópezPortillo for artwork in Fig. 1, and the anonymous reviewers for constructive criticisms and suggestions. J.L.P. thanks N. Koedam and F. Dahdouh-Guebas for stimulating discussions and hospitality at ULB-VLB supported by the Coastal Research Network on Environmental Changes (CREC), classified as a Marie Curie Action (FP7-PEOPLE-2009-IRSES #247514). 6 Author for correspondence (e-mail: [email protected])

doi:10.3732/ajb.1300435

also reduce the turgor pressure in living cells (in contrast to pure water) and the freezing point of the xylem vessel sap, which would be advantageous for plants growing in colder environments. Xylem vessel sap salinity, extracted by means of the pressure chamber, varies inter- and intraspecifically according to the soil substrate, the time of day, and the rate of transpiration and growth (Drennan and Pammenter, 1982; Sobrado, 2004). High amounts of solutes, reaching as much as 20% of the soil pore water solute concentrations (mainly due to Na+, K+, and Cl−), have been reported in the xylem sap of halophytes (Flowers, 1977; Sobrado, 2001, 2004; Sobrado and Ewe, 2006). However, other reports argue that in halophytes the concentration of ions in the apoplast and in xylem vessels must be as low as in the saltintolerant glycophytes; otherwise, cell dysfunction would occur due to the accumulation of salts carried by the transpiration stream (Scholander et al., 1965; Ball, 1986, 1988; Flowers et al., 1991; Shabala, 2007). In a previous study (López Portillo et al., 2005), we reported xylem vessel sap osmolarities between −0.21 and −1.36 MPa (49–316 mmol·L−1 NaCl) in Avicennia germinans and −0.14 to −0.56 MPa (32–130 mmol·L−1 NaCl) in Conocarpus erectus. In contrast, using cryo-SEM energy dispersive x-ray microanalysis, Stuart et al. (2007) reported that the osmolarities of xylem vessel sap in the mangrove species Avicennia marina and Aegiceras

American Journal of Botany 101(6): 1013–1022, 2014; http://www.amjbot.org/ © 2014 Botanical Society of America

1013

1014

AMERICAN JOURNAL OF BOTANY

corniculatum were at least one order of magnitude less, a value similar to glycophytes and to the values obtained by Rokitta et al. (2004) for A. marina using 23Na nuclear magnetic resonance. Such low osmolarities and concentrations of solutes matched those estimated by Ball (1988) through a mass balance method for the same two species. Stuart et al. (2007) considered that our values were due to contamination via cell disruption along the cut surface of the shoot. Even more, there is a widespread assertion that the severed living cells of a cut stem will release the contents of vacuoles and organic osmolytes from the pith, parenchyma, and phloem, thus contaminating the xylem vessel sap (Flowers, 1985; Popp et al., 1985; Yeo, 2007). Caution is recommended against the use of the pressure chamber and vacuum techniques to obtain accurate samples of xylem vessel sap (Schurr, 1998), but we were unable to find protocols that test directly whether such contamination occurs. An opposite effect is also possible if the xylem vessel sap is extracted with a pressure chamber, since water from living cells from the undamaged section of the shoot would move into the apoplast as pressure is applied (a reverse osmosis effect) and thus decrease the ionic concentration of xylem vessel sap (Scholander et al., 1962, 1966). For the present study, we developed original protocols to test whether mangroves have high apoplastic ion concentrations, using direct measurements of xylem vessel sap osmolality in two salt-secreting mangrove species: Avicennia germinans (L.) L. (Scholander et al., 1966) and Laguncularia racemosa (L.) C.F. Gaertn. (Sobrado, 2004). Specifically, we explored whether the vacuum extraction technique (Bollard, 1953; Scholander et al., 1966; Améglio et al., 2004; Charrier et al., 2013) and the pressure chamber technique (Scholander et al., 1966; Sobrado, 2002, 2004) can give similar results. Further, based on the observation that sap flux densities measured in mangroves with heat dissipation probes peak at midday and decrease to zero at night (Krauss et al., 2007; Muller at al., 2009), we manipulated the pressure chamber technique to determine whether plants can re-establish their xylem sap salinity when equilibration periods of increasing time are allowed (effectively mimicking zero sap flux). To this end, we proposed the following hypotheses to test whether high xylem vessel sap osmolalities are artifacts of sampling: (1) the osmolality of the initial sap sample extracted with the pressure chamber and the vacuum method will be similar, but the differences between both methods will increase as more sap is extracted due to the reverse osmosis effect exerted by the pressure chamber. (2) If there is significant contamination by the contents of the severed living cells, recutting of stems and shoots will increase xylem vessel sap osmolality. (3) If contamination by organic osmolytes occurs after recutting, the correlation between the directly measured osmolality and the osmolality calculated as the sum of analytically inorganic ions determined by high pressure liquid chromatography (HPLC), will depart from a 1 : 1 proportion. Finally, (4) in shoots where osmolality has been reduced due to excessive sap extraction or to perfusion with deionized water, high sap solute concentrations will be re-established by the living cells of the shoot and leaves. This work aims to provide a wider understanding of the mechanisms of sap regulation and suggest new ways in which plants control for high salinity in their water pathways.

[Vol. 101

annual temperature 25°C). Based on previous field observations, we chose this hypersaline site to maximize the opportunity to find high xylem sap osmolalities. The Laguncularia plants were located at the west end of the 60 m × 50 m study plot, where there was some fresh water seepage, whereas the Avicennia plants were sampled from the northwest to the northeast ends of the site. Soil pore water samples were collected on 30 August 2008 near the base of the 11 sampled plants per species at a depth of 10 cm, which is the main rooting zone for feeder roots in these species (Angeles et al., 2002), using an arrow with three 1–2-mm slots carved 1 cm above the arrowhead and connected to a 60-mL syringe (McKee et al., 1988). Soil pore water and seawater samples were also collected on 10 March 2011 to determine their ionic composition. The study site was naturally flooded with brackish water during the collection periods. A total of 11 stems and 11 shoots were cut with a clean sharp clipper at atmospheric pressure. Three were collected per day from 25 to 28 August 2008. Stems were 1 m long, and all leaves cut away in situ. One shoot per stem with at least six expanded leaves (leaf areas: 224 ± 11 cm2 and 143 ± 11 cm2 for Laguncularia and Avicennia, respectively) was chosen. The stems and shoots were placed in plastic bags, carried to the laboratory within 15 min, and placed in a dark, insulated box. Experiment 1: Vacuum extraction—In this experiment (Fig. 1, upper left sampling sequence), the stem segments were completely debarked to remove phloem and vascular cambium tissue (this is not completely possible in Avicennia germinans, which has successive cambia; Robert et al., 2011), and then washed with deionized water and dried with a paper towel. The distal end of the stem was recut with a clipper to about 10-mm xylem diameter, and the surface was shaved with a fresh razor blade, washed, and dried. A Wescor (Logan, Utah, USA) filter paper sample disc was placed inside of a new disposable 2-mL Costar snap-cap microcentrifuge tube (Corning, Corning, New York, USA) to which the distal end of the stem segment was then introduced. The distal 10 cm of the stem, with the attached tube and inserted disc, was exposed to a vacuum of between −80 to −93 kPa in a Kitasato flask (Bollard, 1953; Scholander et al., 1966; Améglio et al., 2004) (upper section, Fig. 1). Preliminary experiments indicated visible xylem sap extraction started when the stems of Laguncularia were cut to 50 cm, whereas such extraction was not visible in Avicennia until stems were cut to about 35 cm. Therefore, for Laguncularia, the first cut was made at 50 cm, followed by a series of cuts, each 3 cm more distal. For Avicennia, the first cut was made at 35 cm, followed by cuts each 2 cm more distal. Vacuum extraction was allowed to proceed for up to 45 s between cuts. After each cut, the filter paper was observed for any copious fluid, in which case, the vacuum was released. The microcentrifuge tube with the sample was then removed from the stem, and the integrated cap was closed to prevent evaporation (collection technique A, Fig. 1). The next microcentrifuge tube and filter paper sample disc was then attached to the stem for the next extraction using this collection technique A. In this way, five successive samples containing more than 0.01 mL of xylem sap (enough to saturate the sample disc) were collected per stem.

MATERIALS AND METHODS

Experiment 2: Pressure chamber extraction and recuts—In this experiment (Fig. 1, lower left sampling sequence), a shoot was sampled from each of the 11 stems used for the vacuum extraction. The bark was removed from the proximal 3 cm of the shoot axis, and the cut end was shaved, washed, and dried. The shoot was placed in a pressure chamber (Model 1000 PMS, Corvallis, Oregon, USA), leaving 3–5 cm of the shoot stump outside. Pressure (using compressed nitrogen gas as the source) was gradually applied until the balancing pressure was reached, and 0.01 mL of the expressed sap was collected in a filter paper disc, and placed in a microcentrifuge tube that was then closed to prevent evaporation (collection technique B, Fig. 1). Five consecutive samples (samples 1 through 5) were collected in this manner as the balance pressure was gradually increased by 0.05 to 0.1 MPa for the continued extrusion of sap. Next, tubing was attached to the exposed stem base (Scholander et al., 1965; Paliyavuth et al., 2004; Sobrado, 2004; collection technique C, Fig. 1) to collect 0.15 mL of sap in a microcentrifuge tube (sample 6). The pressure was gradually increased by more than 0.5 MPa to ensure the continued flow of the sap. After the collection of 0.15 mL, the exposed stem was recut, washed, and dried. Three consecutive 0.01-mL samples of sap (samples 7–9) were then collected with technique B, followed by two more 0.15-mL samples (samples 10, 11) collected with technique C.

Study site, pore water, and plant collection—This study was performed at the north fringe of the La Mancha mangrove forest in Veracruz, in the Gulf of Mexico (19°35′35″N; 96°23′13″W; mean annual precipitation 1200 mm; mean

Experiment 3: Pressure chamber extraction and equilibration periods— To test the hypothesis of a dynamic equilibration between the symplast and the apoplast, we modified Experiment 2 by introducing cyclic extractions separated

June 2014]

LÓPEZ-PORTILLO ET AL.—CONTROL OF SAP OSMOLALITY AND IONS IN MANGROVES

1015

Fig. 1. Collection techniques and protocols used in three sap extraction experiments. (A) Vacuum extraction: 0.01 mL of xylem sap is collected in a filter paper disc inside a microcentrifuge tube at the proximal part of the stem after cutting two or three stem segments from the distal part. Pressure chamber extractions: (B) 0.01 mL of xylem sap collected directly from the shoot with a paper disc, (C) 0.15 mL collected into a microcentrifuge tube, with the shoot connected to a dialysis tube. The protocol for each experiment specifies the collection technique and number of xylem sap samples of either 0.01 or 0.15 mL.

by equilibration periods, in which shoots were kept for increasing amounts of time inside the chamber (no light) at atmospheric pressure (Fig. 1, sampling sequence at right). Four shoots per species were collected on 7–9 March 2011 from the site already described, and two pressure chambers were used for simultaneous extractions in Avicennia and Laguncularia. The next pair of shoots was collected after processing the previous pair. Shoots, prepared as in Experiment 2, were introduced in the chamber and three 0.1-mL samples were collected using technique B (Fig. 1), then the chamber was slowly depressurized to atmospheric pressure and after an equilibration period, pressure was applied and 0.15 mL were collected using technique C, after which other three 0.01-mL samples were collected using technique B. Three sap collecting cycles were

performed at increasing time periods (0.2–0.6 h, 2.8–4 h, and 5.6–7.1 h). All sap samples were kept in their microcentrifuge tubes and stored in a freezer inside a sealed plastic bag. Experiment 4: Potometer experiment—To explore the effect of osmolality on shoot transpiration and final xylem sap osmolality, 24 shoots of Laguncularia racemosa and 24 of Avicennia germinans, similar in size and leaf number to the previous experiments, were collected at the same study site on 23 March 2010 within 1 h, starting at 0600 hours. The shoots were cut under deionized water and transported to the laboratory in sealed plastic bags with the cut surface under water. Bark was removed from the proximal 5 cm of the shoot axis, and

1016

[Vol. 101

AMERICAN JOURNAL OF BOTANY

the exposed transversal xylem surface was recut with a new safety blade and cleaned with a paper towel under water to avoid embolisms. Each shoot was connected under water to a 20-cm-long tubing attached to a 2-mL graduated pipette and perfused with different solutions taking advantage of the transpiration pull. Volume loss and time of day were annotated on a masking tape alongside the pipette, and a new tape was applied before the meniscus reached the end of the graduated scale. Each pipette was refilled with the assigned solution as needed with tubing attached to a syringe. There were four perfusion treatments: deionized (D), isosmotic (I, the osmolality of the native sap for each species), twice isosmotic (2I), and 100% seawater (SW, collected at the beach, 100 m from the laboratory). Seawater was passed through a 2-µm filter. The dilutions were prepared by mixing deionized water with seawater and degassing under vacuum. The average of the first 0.01 mL sample extracted with the pressure chamber from 11 shoots per species was used as the target value of both isosmotic solutions. There were six replicate shoots per treatment, and the position and treatment of each shoot in the 48 vertical supports that occupied a 36-m2 shaded area was assigned at random. The experiment was run simultaneously in both species for 24 h, starting at 0200 hours on 23 March 2010 to have a similar evaporative gradient among species and treatments. Solutions were replenished as needed after measuring the amount of transpired solution in the graduated pipettes. After the experiment was finished, all shoots were carried to the laboratory in an insulated cooler box, 10 m from the experimental site. Each shoot was separated from the tubing, and the cut end was washed in deionized water and dried with a paper towel before inserting it in the pressure chamber. Pressure was applied at a very low rate, and the first two 0.01-mL samples were collected on paper discs. Each sample was placed in a microcentrifuge tube and stored in a freezer inside sealed plastic bags. Osmolality measurements—Xylem sap osmolality was measured in a Vapor Pressure Osmometer (VAPRO 5520, Wescor) using fully saturated discs containing ca. 0.01-mL of fluid. The osmometer was checked for contamination approximately every 30 samples and calibrated with standard reference solutions or cleaned if needed. Results are presented as osmolality in mmol·kg−1. For comparative purposes with other publications and based on our calibration curves using sodium chloride solutions, we calculated that 1 mmol·L−1 ≈ 1.6 mmol·kg−1 and that 1 MPa ≈ 375 mmol·kg−1 (N = 9; r2 > 0.99; P < 0.0001). We used sodium chloride for the calibration curves because it is the major component of xylem sap and of the soil pore water associated with mangroves (Sobrado and Greaves, 2000; Naidoo, 2006).

shoots to determine the most probable match in each plant species. A linear regression was used to compare between the measured (by osmometer) and calculated (by adding ionic species) osmotic potential. The proportion of each ion to the total calculated osmolality was also used to compare between species. Finally, one way-repeated measures ANOVAs were used to analyze the effect on shoot transpiration of the different artificial xylem sap solutions in each species. Analyses and multiple comparisons were performed using the program Sigma Plot ver. 10 (Systat Software, San Jose, California, USA) or JMP 6.0.0 (SAS Institute, Cary, North Carolina, USA).

RESULTS Soil pore water and xylem sap osmolality— On average, Avicennia occurred in more saline soils (1399 ± 108 mmol·kg−1) than Laguncularia (644 ± 100 mmol·kg−1) and had more negative xylem sap water potentials. The xylem sap water potentials, calculated for comparison purposes from calibration curves with osmolality values, ranged from −5 to −1.5 MPa for Avicennia and from −3 to −0.2 MPa for Laguncularia (Fig. 2). The ANCOVA indicated that the slopes of the two regression lines were not significantly different (F1, 18 = 2.7; P = 0.12), but that the intercepts were (F1, 19 = 25.9; P < 0.0001), indicating consistently higher stem osmolality values in Avicennia for the range of soil pore water osmolalities. The linear model with no interactions accounted for 85% of the total variability in the data (F2, 19 = 60, P < 0.001; Fig. 2). The least square mean values (LSM) in the ANCOVA (i.e., the means accounting for the variation in soil osmolality) were 359 ± 28 and 120 ± 28 mmol·kg−1 for Avicennia and Laguncularia (about −0.9 and −0.3 MPa), respectively, and the mean soil osmolality used by the ANCOVA to calculate the LSMs for both species was 1021 ± 109 mmol·kg−1, equivalent to −2.5 MPa, the osmotic potential of seawater (Fig. 2). The average extent of solute “exclusion” at that soil osmolality was lower in Avicennia than in Laguncularia (64% vs. 88%).

Ionic composition of sap—After measuring the osmolality of the samples from the xylem sap solute equilibration experiment, we used the 0.01-mL samples with highest osmolality and all the 0.15-mL samples to determine the concentration of the inorganic anions and cations. Due to the sensitivity of the HPLC, all samples were diluted with 25 mL of deionized water and mechanically shaken for 3 h before the analysis. The anions SO4−2, NO3− and Cl− were analyzed using a PerkinElmer HPLC (Waltham, Massachusetts, USA) equipped with a Hamilton (Reno, Nevada, USA) PRP-X100 cationic separation column (100 mm × 4.1 mm), an isocratic 250 pump, and a contactless conductivity detector. The injection was 0.1 mL, and the flow rate was 2 mL·min−1. The eluent was 2 mmol·L−1 phthalic acid at 10% acetone adjusted to pH 5 with NaOH using a flux of 2 mL·s−1. Detection limits (in mg·L−1) were 0.11 for SO4−2 and NO3−, and 0.08 for Cl−. Bicarbonate (HCO−3) was determined by titration as total acidity using Gran’s method (Gran, 1988). The cations Na+, NH4+, K+, Mg+2, and Ca+2 were analyzed with a Waters HPLC (Milford, Massachusetts, USA) equipped with an M/D cationic separation column (150 mm × 4.1 mm), an isocratic 550 pump, and a conductivity detector. The injection was 0.1 mL, and the flow rate was 1 mL·min−1. The eluent was 3 mmol·L−1 EDTA with 2 mmol·L−1 HNO3. High purity certified standards (0.1 mg·mL−1 in ultrapure H2O) were used for ion identification and quantification. Statistical analyses—We used an analysis of covariance (ANCOVA) to relate the stem xylem sap osmolality extracted with the vacuum technique with soil pore water osmolality and the mangrove species. For the data related to vacuum extraction, we used one-way repeated measures ANOVAs to compare the sequence of sap samples in each species and carried out all pairwise multiple comparison procedures using the Holm-Sidak method when required. We carried out two-way repeated measures ANOVA to compare xylem sap osmolalities between species and with the sequence of stem xylem sap samples. We also used one-way repeated measures ANOVA to compare the first sample from the stem as the control against all subsequent samples obtained from the

Fig. 2. Xylem sap osmolality as a function of soil water osmolality. Results shown for the first extracted 0.01-mL sample from stems of Laguncularia racemosa (open symbols) and Avicennia germinans (solid symbols) using the vacuum technique vs. soil water osmolality. For comparison purposes, the corresponding estimated osmotic potential of both axes is shown on the right and upper axes. Each symbol represents one individual.

June 2014]

LÓPEZ-PORTILLO ET AL.—CONTROL OF SAP OSMOLALITY AND IONS IN MANGROVES

1017

Experiment 1: Vacuum extraction— The xylem vessel sap osmolality was significantly lower in Laguncularia than in Avicennia (F1, 11 = 45, P < 0.001), and there were no significant intraspecific differences among the five sequential samples (F4, 44 = 0.24, P = 0.9). Thus, there was no significant change in sap osmolality in either species as sampling progressed from the first to the fifth sample (Fig. 3), indicating no contamination after recuts.

5 extracted with the pressure chamber method. Thus, the mean osmolality (±SE) of the vessel xylem sap of Laguncularia where both extraction techniques match is 75 ± 12 mmol·kg−1. In Avicennia, there was a significant match between the first sample extracted with the vacuum method and the first sample extracted with the pressure chamber method, so the mean osmolality of xylem vessel sap in which both techniques match is 404 ± 32 mmol·kg−1.

Experiment 2: Pressure chamber extraction and recuts— Sap osmolality was consistently higher in Avicennia than in Laguncularia (note different y-axis scales in Figs. 4A, B), and decreased significantly with successive extractions in both species (F10, 100 = 52 and 26 for Avicennia and Laguncularia, respectively, P < 0.001). Multiple comparisons indicated that in Laguncularia and Avicennia, the osmolality of the first xylem sap sample was significantly higher than the rest, samples 2–5 had intermediate values, and samples 6–11 (including recuts and 0.15 mL samples) were lower in osmolality and similar among them. As sap is being extracted and shoots are thus dehydrating, more pressure is required for further sap extraction. In Laguncularia, the applied pressures for continued xylem sap extractions were gradually increased from about 2.3 to 3.5 MPa (F10, 100 = 138, P < 0.001). Multiple comparisons indicated that the balancing pressure was distributed among three tiers of samples: 1–5, 6–9, and 10 and 11 (Fig. 4C). In Avicennia, the applied pressures were gradually increased from 3.8 to 5.9 MPa to allow for continued extractions (F10, 100 = 321; P < 0.001). Three tiers were also found (Fig. 4D). The difference in the required pressure between the initial and final extractions was 1.2 MPa in Laguncularia and 2.1 MPa in Avicennia.

Experiment 3: Pressure chamber extraction and equilibration periods— As with Experiment 2, the osmolality of the xylem sap decreased in both species with subsequent 0.01-mL extractions (samples 1–3, Fig. 5A–H), and it was lowest after the extraction of 0.15-mL samples (sample 4, empty triangle) and the three consecutive 0.01-mL samples 5–7 (empty circles in Fig. 5A–H). However, in each of the cycles, after the chambers were depressurized and the shoots allowed to equilibrate, sap osmolality consistently increased to the initial values or exceeded them as more equilibration time was allowed (Fig. 5A–H).

Vacuum vs. pressure chamber sap extractions— In Laguncularia, the first xylem vessel sap sample extracted with the vacuum method was statistically similar to samples 2 through

Fig. 3. Xylem vessel sap osmolality in Laguncularia racemosa (open circles) and Avicennia germinans (solid circles) of the first five 0.01-mL samples extracted from 10-mm-wide stems by the vacuum method. Error bars indicate ±1 SE. The osmolality is significantly higher in Avicennia when compared to Laguncularia. There are no significant differences among sap collections within either species.

Ionic content of xylem sap— There was a significant linear regression between the osmolality measured with the osmometer and the calculated osmolality using HPLC, equal to the sum of the osmolalities of all determined ions (Fig. 6). The slope and 95% confidence intervals indicated a 1 : 1 correspondence between values and the model accounted for 96% of the total variability in the data, with the ordinate not significantly different from zero. The ion Cl− accounted for ca. 46% of the 50% osmolality due to anions in both plant species (Table 1). However, Na+ was significantly higher and K+ significantly lower in Avicennia (41% and 9% of cations, respectively) when compared with Laguncularia (30% and 18%, respectively, Table 1), and the two cations added up to 50% and 48% of the 50% of the osmolality due to cations. NaCl accounted for 77% of the total amount of salts in Laguncularia and 85.4% in Avicennia. The concentration of all other ions was statistically similar between mangrove species. The Na/K and Na/Cl proportions were significantly higher, and the K/Cl proportion was significantly lower in Avicennia than in Laguncularia (Table 1). Nevertheless, the Na/K proportions were much lower than those obtained in chemical analyses of seawater and soil pore water samples collected at the study site (Na/K = 30.1 ± 1.1; with no significant differences between soil pore water and seawater). Experiment 4: Water perfusion experiment—The transpiration rate at midday, one order of magnitude lower than the data published by Sobrado (2000) for the same species, tended to be higher in the shoots perfused with deionized or isosmotic solutions than in the shoots perfused with the twice isosmotic and sea water solutions (Fig. 7). The maximum transpiration was observed between 1000 hours and 1400 hours, and decreased in both species toward the evening. When comparing the osmolality of the artificial sap solutions with the sap extracted from the shoots with the pressure chamber after the experiment was finished, significant differences were only found in the deionized water treatment, where sap osmolality increased from 26 ± 4 mmol·kg−1 (this nonzero value of deionized solution probably due to the resolution of the osmometer) to 196 ± 69 mmol·kg−1 in Laguncularia (inset, Fig. 7A) and to 209 ± 95 mmol·kg−1 in Avicennia (inset, Fig. 7B). The osmolality of sap samples from the other treatments were statistically similar to their perfused solution.

1018

AMERICAN JOURNAL OF BOTANY

[Vol. 101

Fig. 4. (A, B) Xylem vessel sap osmolality of 11 sequential samples and (C, D) chamber pressure used to extract them in (A, C) Laguncularia racemosa and (B, D) Avicennia germinans. Solid circles, sequential 0.01-mL samples; open triangle, 0.15-mL sample; open circles, 0.01-mL samples after the shoot was cut to induce contamination; following open triangles, 0.15-mL samples. The F values correspond to the effect of the sample on osmolality or chamber pressure after the analysis of each data set by means of one-way repeated measures ANOVA. Horizontal lines within each graph mark statistically similar values.

DISCUSSION Within a hypersaline site, we determined the sap osmolality values of two mangrove species using two techniques of direct sap extraction and different configurations to test for possible contamination. In addition to validating these two experimental techniques, our results gave us original insights on the ionic equilibrium between symplast and apoplast in halophytes. Our first hypothesis, that both methods are reliable for quantifying sap osmolality, was supported for both species since the first samples obtained with the pressure chamber and the vacuum techniques were statistically similar. As predicted, the osmolality of successive samples obtained by the pressure chamber tended to decrease due to reverse osmosis (Scholander et al., 1964), which was more evident when relatively large volumes of sap (0.15 mL) were extracted. The pressures applied for the extraction of all samples were within the range required for measurements of midday water potentials at our study site, which at the peak of the dry season are around −5.9 MPa in A. germinans (López-Portillo et al., 2005). Our second hypothesis, that contamination due to repeated cutting would be evident as an increase in overall osmolality, was not supported since repeated cuts while using the vacuum and pressure chamber techniques did not increase the sap osmolality, which had been a serious concern regarding both methods of sap extraction (Schurr, 1998). With the vacuum technique, the osmolalities were similar in the five consecutive samples, even when cutting two or three segments per stem to obtain the 0.01 mL sap sample. This was also true for Avicennia germinans,

a species with successive cambia (Robert et al., 2011) impossible to remove for the experiment. Our results suggest that the first or second 0.01 mL sample obtained by either method should not be discarded since they truly reflect the solute contents in the xylem vessel sap. The range of values found for Laguncularia are within those observed in some high altitude trees (Charrier et al., 2013) and in Populus nigra (Secchi and Zwieniecki, 2012), both extracted with the vacuum technique, although the compounding osmolytes probably differ. The vacuum technique has recently being used by Secchi and Zwieniecki (2012) to determine the concentration of ions and sugars in embolized and nonembolized vessels in several species, with no reported contamination issues. Our third hypothesis was also not supported, since the correlation between osmolality measurements and the osmolality calculated as the sum of the inorganic ions was not statistically different from a 1 : 1 proportion, which would not occur if there was a significant contribution of organic osmolytes. The ions that accounted for the bulk osmotic potential were Na+, K+, and Cl−, with no evidence of contamination from organic solutes. Our fourth hypothesis, that osmolality will increase if equilibration periods are allowed after being reduced to very low values by high volumes of sap extraction, was supported. In our equilibration experiments, the xylem sap osmolality (and salinity, as indicated by the analysis of the extracted sap) of the cut shoots increased even though there were no new fresh cuts through the xylem. Our results suggest that Na+, K+, Cl−, and other ions are not strictly sequestered in the vacuoles of living cells (Flowers, 1985; Blumwald, 2000; Tester and Davenport,

June 2014]

LÓPEZ-PORTILLO ET AL.—CONTROL OF SAP OSMOLALITY AND IONS IN MANGROVES

1019

Fig. 5. Xylem vessel sap osmolality of samples extracted with a pressure chamber from the shoots of four individuals of (A–D) Laguncularia racemosa and (E–H) Avicennia germinans. Except for the three initial samples, solid and open symbols indicate the values of samples obtained before and after the extraction of 0.15 mL, respectively. Sample volumes: circles, 0.01 mL; triangles, 0.15 mL. Numbers above solid circles indicate the time (hours) after the shoots were depressurized.

2003). We suggest that living cells may download solutes into the xylem vessels for osmotic adjustment between symplast and apoplast. This is supported by the fact that in our water perfusion experiment diurnal transpiration was greater when shoots were perfused with solutions that were osmotically similar to the native sap. Furthermore, the xylem vessel sap osmolality extracted after the experiment was statistically similar to the perfused solutions in all treatments except the deionized water treatment, where there was a match between the extracted sap and the original native sap. Thus, the relatively high salinity of the apoplast that we report appears to be a normal part of the physiology of the mangroves. Downloading of ions from living

cells to the xylem sap has also been reported for stems of Solanum lycopersicum (Trifilò et al., 2013). The discrepancies between the osmolality results found here and those reported with other methods may be due to the nature of the experiments involved and the condition of the plants. Stuart et al. (2007) compared our previously reported xylem vessel sap osmolarity values (López-Portillo et al., 2005) with their own values obtained from xylem vessels by cryo-SEM energy dispersive x-ray microanalysis and concluded that our values were overestimated. Their results for xylem ion concentrations seem not be to statistically different from zero due to their relatively high standard errors (Stuart et al., 2007, table 1).

1020

AMERICAN JOURNAL OF BOTANY

[Vol. 101

Fig. 6. Measured vs. calculated osmolality of vessel xylem sap extracted from shoots of Laguncularia racemosa (open symbols) and Avicennia germinans (solid symbols) subjected to the equilibration experiment. Measured osmolality did not depart from the 1 : 1 correspondence with calculated osmolality (discontinuous line). The linear regression and 95% confidence intervals are represented by the solid and dotted lines, respectively.

Although useful to determine the ionic distribution in the tissues, the cryo-SEM EDX technique seems imprecise to determine overall osmolality of sap in this case. Since tissues immersed in liquid nitrogen are not simultaneously freezing, the contents of xylem vessels are prone to dilution with fresh water pouring from living cells before they were placed in the cryo-SEM (Tyree and Cochard, 2003). This could also help explain the wide variability in the osmolarity in the vacuoles of the pith and cambial cells reported by Stuart (2003). Another study using 23Na nuclear magnetic resonance reported low concentrations of sodium in the xylem vessel sap and high concentrations in the central part of the pith and the cortex of Avicennia marina saplings, but these plants were maintained in tap water (i.e., very low salinity) for 6 mo (Rokitta Percentage ± SE contribution of anion and cation species to the total osmolality in xylem sap samples extracted with the pressure chamber from shoots of Laguncularia racemosa (N =11) and Avicennia germinans (N = 11). Cl− is the dominant anion in both species whereas Na+ and K+ account for most of the cations. The Na/K, Na/Cl, and K/ Cl ratios are shown in the last three rows. Asterisks indicate significant differences between species at P < 0.05.

Fig. 7. Leaf transpiration rates during 24 h for shoots of (A) Laguncularia racemosa and (B) Avicennia germinans connected to potometers. Shoots were fed with deionized water (triangles up, D) or solutions of deionized water and seawater representing isosmotic osmolality (triangles down, I), twice isosmotic (squares, 2I), and full seawater (circles, SW). Each symbol is the mean of six shoots; vertical lines represent ±1 SE. The insets show the osmolality (in mmol·kg−1) of the initial solutions (solid symbols) compared to the sap extracted from the cut shoots after the experiment was finished (open symbols). NXS = native xylem sap osmolality.

TABLE 1.

Ionic species or ratio Cl− HCO3− SO4− NO3− OH− Na+ K+ Mg++ Ca++ NH4+ H+ Na/K Na/Cl K/Cl

Laguncularia racemosa

Avicennia germinans

47.2 ± 1.1 0.99 ± 0.23 0.38 ± 0.07 0.21 ± 0.06 7.8 ± 2.5 (10−6) 29.9 ± 1.7* 18.0 ± 1.1* 1.9 ± 0.2 1.09 ± 0.15 0.21 ± 0.10 1.5 ± 0.3 (10−3) 1.77 ± 0.19* 0.64 ± 0.04* 0.38 ± 0.03*

44.3 ± 1.6 2.09 ± 0.62 0.37 ± 0.06 0.35 ± 0.14 1.4 ± 0.4 (10−5) 41.1 ± 0.7* 9.0 ± 0.5* 1.3 ± 0.2 0.97 ± 0.15 0.50 ± 0.17 1.2 ± 0.2 (10−3) 4.77 ± 0.32* 0.95 ± 0.05* 0.21 ± 0.02*

et al., 2004). It would be informative to compare these methods with direct measurements of sap osmolality in hypersaline conditions, such as in the plants we are reporting. By using a combination of direct and indirect methods and models such as those used by Ball (1988) and Vandegehuchte et al. (2014), it would be possible to determine the capacitance and flow of ions and to perform new determinations of salt mass balance in halophytes, improving our measurements of the ionic concentration of xylem contents and providing valuable insights into the mechanisms that allow the interactive regulation of transpiration at the interphase between symplast and apoplast. A mechanism to explain high osmolalities in the transpiration stream of mangroves— An adaptive interpretation for the observed downloading of inorganic solutes is that each species has a maximum ideal sap concentration, which is a function of soil salinity (Downton, 1982; Drennan and Pammenter, 1982; Li et al., 2008; present study), water availability (e.g., Evans et al., 1992) and of the amount that can be secreted or diluted by

June 2014]

LÓPEZ-PORTILLO ET AL.—CONTROL OF SAP OSMOLALITY AND IONS IN MANGROVES

the leaves after some of the ions are used for development of new tissues (Boon and Allaway, 1986; Fitzgerald et al., 1992; Sobrado, 2002, 2004). However, if the xylem sap goes below a minimum level during rehydration, the parenchyma cells of the xylem may respond by downloading some of their stored salt into the apoplast, and the water potential would decrease due to the amount of solutes in the xylem vessel sap (Scholander et al., 1962, 1966; Boyer, 1967; James et al., 2006). The concentration of solutes in the xylem sap may have four other key impacts: (1) If the transpiration stream were virtually pure water, a more negative sap pressure would be required for transport due to the high salinity of the soil pore water. This would increase the risk of cavitation and require investment in a stronger and less efficient vascular system (i.e., smaller conduits, thicker walls) to withstand the higher tensions that must be sustained to reach high transpiration rates (Becker et al., 1997; Sobrado, 2001). Therefore, there should be a trade-off between salt exclusion and transport, and in this context, it is significant that Avicennia germinans, generally growing in more saline substrates, allows more salt into its sap than Laguncularia racemosa does. (2) A higher osmolality in the vessel sap will reduce the symplastic turgor pressure of living cells adjacent to the transport pathway in roots, stem, and leaves. This reduction would be potentially advantageous to balance osmotic potential when the vacuoles compartmentalize high concentrations of salt. Hypothetically, pure apoplastic water, totally lacking salts, would increase the turgor pressure significantly, challenging the tensile strength of the cell walls. Relative to a situation with pure xylem sap, and based upon the osmotic potential of the xylem vessel sap and the pressure needed to extract it, the solutes in the xylem vessels would reduce tensions by 28% in Avicennia and 13% in Laguncularia. In the cytoplasm, the balance of osmotic potential to mediate between the vessel xylem sap and the vacuoles could be reached by the synthesis and accumulation of organic osmolytes as well as ions such as K+ that do not interfere with the biochemical reactions (Popp et al., 1985; Glenn et al., 1999; Tester and Davenport, 2003; Gil et al., 2013). (3) The variation in vessel sap salinity could impact the hydraulic conductivity of the plant through the ionic effect (Zwieniecki et al., 2001; Nardini et al., 2011; Zwieniecki and Secchi, 2012). For example, Trifilò et al. (2008) found that residual xylem hydraulic conductivity (Kh) could be upregulated in plants suffering from cavitation and embolism when the concentration of K+ in the xylem vessel sap is higher, increasing water availability to the transpiration stream. In four species of Acer, upregulation was greater in species that lived in exposed and dry habitats compared to species of shady and humid habitats (Nardini et al., 2012). Perhaps the use of K+ or Na+ by the plant for ion-mediated regulation in hydraulic conductivity may depend on its availability in the environment where the species has evolved. In Avicennia germinans, the xylem specific conductivity (Ks) of Avicennia germinans was maximal at artificial (NaCl-based) xylem sap osmotic potentials of −0.8 MPa and decreased to around 70% of Ks-max at extremely low or high sap osmotic potentials (−2.4 and 0 MPa; López-Portillo et al., 2005). The range of the native xylem sap osmotic potentials was from −1.5 to −0.6 MPa for Avicennia, which should result in hydraulic conductivity values ranging from 82 to 100% of Ks-max.

1021

(4) Xylem sap salinity may affect the latitudinal distribution of species. Based on our measurements, the freezing point depression due to the xylem sap salinity would be about −0.75°C in A. germinans and −0.14°C in L. racemosa. It should be noted that actual freezing events can occur even below the freezing point due to the effect of supercooling (Ashworth et al., 1985). The effect of xylem sap salinity may thus be significant at the margins of each species’ latitudinal distribution, where drought is also an issue (Quisthoudt et al., 2012). Both Laguncularia and Rhizophora have lower xylem sap salinities than Avicennia, and, when coupled with anatomical changes related to high salinity, drought and low temperatures (Stuart et al., 2007; Robert et al., 2009) would help explain the greater latitudinal extent of Avicennia. Thus, understanding the mechanisms that regulate the osmolality of xylem sap has critical physiological, ecological, and biogeographical implications in mangroves. LITERATURE CITED AMÉGLIO, T., M. DECOURTEIX, G. ALVES, V. VALENTIN, S. SAKR, J. L. JULIEN, G. PETEL, A. GUILLIOT, AND A. LACOINTE. 2004. Temperature effects on xylem sap osmolarity in walnut trees: Evidence for a vitalistic model of winter embolism repair. Tree Physiology 24: 785–793. ANGELES, G., J. LÓPEZ-PORTILLO, AND F. ORTEGA-ESCALONA. 2002. Functional anatomy of the secondary xylem of roots of the mangrove Laguncularia racemosa (L.) Gaertn. (Combretaceae). Trees 16: 338–345. ASHWORTH, E. N., G. A. DAVID, AND J. A. ANDERSON. 1985. Factors affecting ice nucleation in plant tissues. Plant Physiology 79: 1033–1037. BALL, M. C. 1986. Photosynthesis in mangroves. Wetlands (Australia) 6: 12–22. BALL, M. C. 1988. Salinity tolerance in the mangroves Aegiceras corniculatum and Avicennia marina. I. Water use in relation to growth, carbon partitioning, and salt balance. Functional Plant Biology 15: 447–464. BECKER, P., A. ASMAT, J. MOHAMAD, M. MOKSIN, AND M. T. TYREE. 1997. Sap flow rates of mangrove trees are not unusually low. Trees (Berlin) 11: 432–435. BLUMWALD, E. 2000. Sodium transport and salt tolerance in plants. Current Opinion in Cell Biology 12: 431–434. BOLLARD, E. G. 1953. The use of tracheal sap in the study of apple-tree nutrition. Journal of Experimental Botany 4: 363–368. BOON, P. I., AND W. G. ALLAWAY. 1986. Rates and ionic specificity of salt secretion from excised leaves of the mangrove Avicennia marina Vierh. Aquatic Botany 26: 143–153. BOYER, J. S. 1967. Leaf water potential measured with a pressure chamber. Plant Physiology 42: 133–137. CHARRIER, G., H. COCHARD, AND T. AMÉGLIO. 2013. Evaluation of the impact of frost resistances on potential altitudinal limit of trees. Tree Physiology 33: 891–902. DOWNTON, W. J. S. 1982. Growth and osmotic relations of the mangrove Avicennia marina, as influenced by salinity. Functional Plant Biology 9: 519–528. DRENNAN, P., AND N. W. PAMMENTER. 1982. Physiology of salt excretion in the mangrove Avicennia marina (Forsk.) Vierh. New Phytologist 91: 597–606. EVANS, R. D., R. A. BLACK, W. H. LOESCHER, AND R. J. FELLOWS. 1992. Osmotic relations of the drought-tolerant shrub Artemisia tridentata in response to water stress. Plant, Cell & Environment 15: 49–59. FITZGERALD, M. A., D. A. ORLOVICH, AND W. G. ALLAWAY. 1992. Evidence that abaxial leaf glands are the sites of salt secretion in leaves of the mangrove Avicennia marina (Forsk.) Vierh. New Phytologist 120: 1–7. FLOWERS, T. J. 1985. Physiology of halophytes. Plant and Soil 89: 41–56. FLOWERS, T. J. 1977. The mechanism of salt tolerance in halophytes. Annual Review of Plant Physiology 28: 89–121.

1022

AMERICAN JOURNAL OF BOTANY

FLOWERS, T. J., M. A. HAJIBAGHERI, AND A. R. YEO. 1991. Ion accumulation in the cell walls of rice plants growing under saline conditions: Evidence for the Oertli hypothesis. Plant, Cell & Environment 14: 319–325. GIL, R., M. BOSCAIU, C. LULL, I. BAUTISTA, A. LIDÓN, AND O. VICENTE. 2013. Are soluble carbohydrates ecologically relevant for salt tolerance in halophytes? Functional Plant Biology 40: 805–818. GLENN, E. P., J. J. BROWN, AND E. BLUMWALD. 1999. Salt tolerance and crop potential of halophytes. Critical Reviews in Plant Sciences 18: 227–255. GRAN, G. 1988. Equivalence volumes in potentiometric titrations Analytica Chimica Acta 206: 111–123. JAMES, J. J., N. N. ALDER, K. H. MÜHLING, A. E. LÄUCHLI, K. A. SHACKEL, L. A. DONOVAN, AND J. H. RICHARDS. 2006. High apoplastic solute concentrations in leaves alter water relations of the halophytic shrub, Sarcobatus vermiculatus. Journal of Experimental Botany 57: 139–147. KRAUSS, K. W., P. J. YOUNG, J. L. CHAMBERS, T. W. DOYLE, AND R. R. TWILLEY. 2007. Sap flow characteristics of neotropical mangroves in flooded and drained soils. Tree Physiology 27: 775–783. LI, N., S. CHEN, X. ZHOU, C. LI, J. SHAO, R. WANG, E. FRITZ, A. HÜTTERMANN, AND A. POLLE. 2008. Effect of NaCl on photosynthesis, salt accumulation and ion compartmentation in two mangrove species, Kandelia candel and Bruguiera gymnorhiza. Aquatic Botany 88: 303–310. LÓPEZ-PORTILLO, J., F. W. EWERS, AND G. ANGELES. 2005. Sap salinity effects on xylem conductivity in two mangrove species. Plant, Cell & Environment 28: 1285–1292. MCKEE, K. L., I. A. MENDELSSOHN, AND M. W. HESTER. 1988. Reexamination of pore water sulfide concentrations and redox potentials near the aerial roots of Rhizophora mangle and Avicennia germinans. American Journal of Botany 75: 1352–1359. MULLER, E., L. LAMBS, AND F. FROMARD. 2009. Variations in water use by a mature mangrove of Avicennia germinans, French Guiana. Annals of Forest Science 66: 803. NAIDOO, G. 2006. Factors contributing to dwarfing in the mangrove Avicennia marina. Annals of Botany 97: 1095–1101. NARDINI, A., F. DIMASI, M. KLEPSCH, AND S. JANSEN. 2012. Ion-mediated enhancement of xylem hydraulic conductivity in four Acer species: Relationships with ecological and anatomical features. Tree Physiology 32: 1434–1441. NARDINI, A., S. SALLEO, AND S. JANSEN. 2011. More than just a vulnerable pipeline: Xylem physiology in the light of ion-mediated regulation of plant water transport. Journal of Experimental Botany 62: 4701–4718. PALIYAVUTH, C., B. CLOUGH, AND P. PATANAPONPAIBOON. 2004. Salt uptake and shoot water relations in mangroves. Aquatic Botany 78: 349–360. POPP, M., F. LARHER, AND P. WEIGEL. 1985. Osmotic adaption in Australian mangroves. Vegetatio 61: 247–253. QUISTHOUDT, K., N. SCHMITZ, C. F. RANDIN, F. DAHDOUH-GUEBAS, E. M. ROBERT, AND N. KOEDAM. 2012. Temperature variation among mangrove latitudinal range limits worldwide. Trees 26: 1919–1931. ROBERT, E. M. R., N. KOEDAM, H. BEECKMAN, AND N. SCHMITZ. 2009. A safe hydraulic architecture as wood anatomical explanation for the difference in distribution of the mangroves Avicennia and Rhizophora. Functional Ecology 23: 649–657. ROBERT, E. M., N. SCHMITZ, I. BOEREN, T. DRIESSENS, K. HERREMANS, J. DE MEY, E. VAN DE CASTEELE, ET AL. 2011. Successive cambia: A developmental oddity or an adaptive structure? PLoS ONE 6: e16558. ROKITTA, M., D. MEDEK, J. M. POPE, AND C. CRITCHLEY. 2004. 23Na NMR microimaging: A tool for non-invasive monitoring of sodium distribution in living plants. Functional Plant Biology 31: 879–887. SCHOLANDER, P. F., E. D. BRADSTREET, H. T. HAMMEL, AND E. A. HEMMINGSEN. 1966. Sap concentrations in halophytes and some other plants. Plant Physiology 41: 529–532. SCHOLANDER, P. F., E. D. BRADSTREET, E. A. HEMMINGSEN, AND H. T. HAMMEL. 1965. Sap pressure in vascular plants. Science 148: 339–346.

SCHOLANDER, P. F., H. T. HAMMEL, E. HEMMINGSEN, AND E. D. BRADSTREET. 1964. Hydrostatic pressure and osmotic potential in leaves of mangroves and some other plants. Proceedings of the National Academy of Sciences, USA 52: 119–125. SCHOLANDER, P. F., H. T. HAMMEL, E. HEMMINGSEN, AND W. GAREY. 1962. Salt balance in mangroves. Plant Physiology 37: 722–729. SECCHI, F., AND M. A. ZWIENIECKI. 2012. Analysis of xylem sap from functional (nonembolized) and nonfunctional (embolized) vessels of Populus nigra: Chemistry of refilling. Plant Physiology 160: 955–964. SHABALA, S. 2007. Transport from root to shoot. In A. R. Yeo and T. J. Flowers [eds.], Plant solute transport, 214–234. Blackwell Publishing, Singapore. SCHURR, U. 1998. Xylem sap sampling—New approaches to an old topic. Trends in Plant Science 3: 293–298. SOBRADO, M. A. 2000. Relation of water transport to leaf gas exchange properties in three mangrove species. Trees 14: 258–262. SOBRADO, M. A. 2001. Hydraulic properties of a mangrove Avicennia germinans as affected by NaCl. Biologia Plantarum 44: 435–438. SOBRADO, M. A. 2002. Effect of drought on leaf gland secretion of the mangrove Avicennia germinans L. Trees 16: 1–4. SOBRADO, M. A. 2004. Influence of external salinity on the osmolality of xylem sap, leaf tissue and leaf gland secretion of the mangrove Laguncularia racemosa (L.) Gaertn. Trees 18: 422–427. SOBRADO, M. A., AND S. M. EWE. 2006. Ecophysiological characteristics of Avicennia germinans and Laguncularia racemosa coexisting in a scrub mangrove forest at the Indian River Lagoon, Florida. Trees 20: 679–687. SOBRADO, M. A., AND E. D. GREAVES. 2000. Leaf secretion composition of the mangrove species Avicennia germinans (L.) in relation to salinity: A case study by using total-reflection X-ray fluorescence analysis. Plant Science 159: 1–5. STUART, S. A. 2003. What sets the latitudinal limit of the mangrove habit? B.A. thesis, Harvard University, Cambridge, Massachusetts, USA. STUART, S. A., B. CHOAT, K. C. MARTIN, N. M. HOLBROOK, AND M. C. BALL. 2007. The role of freezing in setting the latitudinal limits of mangrove forests. New Phytologist 173: 576–583. TESTER, M., AND R. DAVENPORT. 2003. Na+ tolerance and Na+ transport in higher plants. Annals of Botany 91: 503–527. TRIFILÒ, P., M. A. LO GULLO, S. SALLEO, K. CAELLA, AND A. NARDINI. 2008. Xylem embolism alleviated by ion-mediated increase in hydraulic conductivity of functional xylem: Insights from field measurements. Tree Physiology 28: 1505–1512. TRIFILÒ, P., M. A. LO GULLO, F. RAIMONDO, S. SALLEO, AND A. NARDINI. 2013. Effects of NaCl addition to the growing medium on plant hydraulics and water relations of tomato. Functional Plant Biology 40: 459–465. TYREE, M. T., AND H. COCHARD. 2003. Vessel contents of leaves after excision: A test of the Scholander assumption. Journal of Experimental Botany 54: 2133–2139. VANDEGEHUCHTE, M. W., A. GUYOT, M. HUBEAU, T. DE SWAEF, D. A. LOCKINGTON, AND K. STEPPE. 2014. Modelling reveals endogenous osmotic adaptation of storage tissue water potential as an important driver determining different stem diameter variation patterns in the mangrove species Avicennia marina and Rhizophora stylosa. Annals of Botany 10.1093/aob/mct311. YEO, A. R. 2007. Water limited conditions. In A. R. Yeo and T. J. Flowers [eds.], Plant solute transport, 314–339. Blackwell Publishing, Singapore. ZWIENIECKI, M. A., AND F. SECCHI. 2012. Getting variable xylem hydraulic resistance under control: interplay of structure and function. Tree Physiology 32: 1431–1433. ZWIENIECKI, M. A., P. J. MELCHER, AND N. M. HOLBROOK. 2001. Hydrogel control of xylem hydraulic resistance in plants. Science 291: 1059–1062.