Acta Physiol Plant (2014) 36:2729–2741 DOI 10.1007/s11738-014-1644-3

ORIGINAL PAPER

Study on pathway and characteristics of ion secretion of salt glands of Limonium bicolor

Zhongtao Feng • Qiuju Sun • Yunquan Deng • Shufeng Sun • Jianguo Zhang • Baoshan Wang

Received: 16 February 2014 / Revised: 23 June 2014 / Accepted: 21 July 2014 / Published online: 30 July 2014 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2014

Abstract Recretohalophytes with specialized salt-secret- Keywords Limonium bicolor Kuntze NaCl secretion ing structures, including salt glands and salt bladders, can Salt gland Secretory pore secrete excess salts from plant tissues and enhance salinity tolerance of plants. However, the pathway and property of Abbreviations salt secretion by the salt gland has not been elucidated. In DM Dry mass the article, Limonium bicolor Kuntze was used to investi- EDS Energy dispersive spectroscopy gate the pathway and characteristics of salt secretion of salt ESEM Environmental scanning electron gland. Scanning electron microscope micrographs showed microscope that each of the secretory cells had a pore in the center of FS Freeze substitution the cuticle, and the rice grain-like secretions were observed HPF High-pressure freezing above the pore. The chemical composition of secretions NMT Non-invasive micro-test from secretory pores was mainly NaCl using environmental technology scanning electron microscope technique. Non-invasive SD Standard deviation micro-test technology was used to directly measure ion SEM Scanning electron microscope secretion rate of salt gland, and secretion rates of Na? and TEM Transmission electron microscope Cl- were greatly enhanced by a 200-mmol/L NaCl treat- ment. However, epidermal cells and stoma showed little secretion of ions. In conclusion, our results provide evi- dence that the salt glands of L. bicolor have four secretory pores and that NaCl is secreted through these pores of salt Introduction gland. Soil salinity is an increasing problem worldwide. More than 950 million hectares of land are salt affected (Munns Communicated by J. Kovacik. 2002; Tan et al. 2013) and the ability to grow crops on saline land is increasingly important (Rozema and Flowers Z. Feng Q. Sun Y. Deng B. Wang (&) Key Laboratory of Plant Stress Research, College of Life 2008). A high concentration of salt in soil causes oxidative Science, Shandong Normal University, Jinan 250014, China damage, water deficit and nutrient deficiency, which dis- e-mail: [email protected] rupts the intracellular ionic homeostasis and leads to retarded plant growth and development and sometimes S. Sun J. Zhang National Laboratory of Biomacromolecules, death, causing great losses in agricultural production Institute of Biophysics, Chinese Academy of Sciences, (Munns and Tester 2008; Chen et al. 2010; Tarchoune et al. Chaoyang, China 2012). Salt stress damages the semi-permeability of the plasma membrane allowing intracellular ions such as K? to S. Sun J. Zhang ? Center for Bio-imaging, Institute of Biophysics, Chinese move out of cells and extracellular ions such as Na to Academy of Sciences, Chaoyang 100101, China move into cells, which leads to adverse effects on 123 2730 Acta Physiol Plant (2014) 36:2729–2741 potassium nutrition, cytosolic enzyme activities, photo- collecting cells and the outer cup cells. This non-cuticu- synthesis, and metabolism (Kingsbury and Epstein 1986; larized wall region is termed the transfusion area, where Tester and Davenport 2003; Carter and Nippert 2011). ions move from mesophyll cells to salt gland cells According to salinity tolerance, plants are divided into (Campbell and Thomson 1975). Furthermore, early indi- halophytes and nonhalophytes. Halophytes can survive to cations of the presence of the secretory pores can be found reproduce in an environment where the salt concentration in the works of a few researchers who studied the salt is around 200-mmol/L NaCl or more, and they can be glands, and these pores were considered to be a pathway of further divided into recretohalophytes, euhalophytes, and salt release out of the salt gland. Using light microscopy, pseudo-halophytes (Breckle 1995; Flowers and Colmer Ruhland (1915) observed pores in the outer cuticle cover- 2008). Characteristic features of recretohalophytes are ing the salt glands of Statice gmelini. Based on the specialized salt-secreting structures, including salt glands observations of the structure of the salt glands of Spartina and salt bladders, which can secrete or sequester excess townsendii, Skelding and Winterbotham (1939) postulated salts from the cells of metabolically active tissues (Lu¨ttge the presence of pores. Pores in the cuticular layer were 1971; Ding et al. 2010). This ability allows the plants to observed in electron microscopic examinations of Tamarix adapt to salinity-affected habitats. (Campbell and Strong 1964; Wilkinson 1966; Thomson Salt glands in plants play an important role in regulating and Liu 1967; Thomson et al. 1969; Campbell and ion balance, maintaining the stability of osmotic pressure, Thomson 1975; Fahn 2000), Limonium (Ziegler and Lu¨ttge and enhancing salinity tolerance (Ding et al. 2009; Tan et al. 1966, 1967; Hill and Hill 1973; Faraday et al. 1986), 2013). They are excretory organs scattered on leaf surfaces Spartina (Levering and Thomson 1971; Bradley and and stems that are adapted for dealing with salt accumula- Morris 1991), Avicennia (Shimony et al. 1973; Fahn 2000), tion in plants. These excretory organs are highly evolved Distichlis (Oross and Thomson 1982), and Ceratostigma structures, which by their ability to secrete salt, enable (Faraday and Thomson 1986a). In electron microscopic plants to grow on saline–alkali soil without adversely suf- study of the localization of chloride, Ziegler and Lu¨ttge fering from salt damage (Flowers and Colmer 2008; Munns (1967) observed heavy precipitates of AgCl in the pore and Tester 2008). Salt glands represent a typical strategy for regions both under and on the outer surface of the cuticle. ion extrusion in several orders of plants, such as the Poales, William (1974) concluded that secretion of chalk Solanales, Lamiales, Caryophyllales, Ericales, and Myrtales occurred through a pore in the surface of each secretory (Flowers et al. 2010; Tan et al. 2013). cell. After examining chalk secreted by leaf salt glands of Previous studies on the function of salt glands have Plumbago capensis Thunb using polarized reflected light utilized whole plants, excised branches, excised leaves, and microscopy, scanning electron microscopy, X-ray diffrac- leaf discs. Most of these studies have involved applying a tion and energy dispersive X-ray analysis, he reported the challenging salt solution to the roots, to the petioles of presence of magnesium, silicon, phosphorus, sulfur, chlo- excised leaves, or to leaf discs, and collecting and ana- rine, potassium, and calcium in the epidermal cells. How- lyzing the ions secreted by the salt glands. Such studies ever, only calcium and magnesium and traces of silicon have shown that salt glands of some plants can secrete a were detected in the secreted material. While these obser- ? ? 2? 2? - - variety of ions such as Na ,K ,Ca ,Mg ,Cl ,NO3 , vations seem contradictory to the high selectivity of salt 2- 3- - - SO4 ,PO4 , HCO3 , and CO3 (Waisel 1961; Berry glands for sodium over other ions, they do not clarify the 1970; Faraday and Thomson 1986b; Balsamo et al. 1995; involvement of the pores in salt secretion. Sua´rez and Medina 2008). In many instances, the ions were Some other classical electrode techniques such as the apparently secreted in different ratios from those present in patch-clamp technique were also used to measure ion the challenging solutions. Studies have also shown that salt fluxes of plant salt glands. However, these methods could glands in recretohalophytes selectively secrete Na? and not truthfully reflect the ions’ flux characters due to their Cl- as the predominant ions from multi-cation and multi- invasive disadvantage (Dschida et al. 1992; Balsamo et al. anion challenges (Berry 1970; Faraday and Thomson 1995). Non-invasive micro-test technology (NMT) is par- 1986b; Sua´rez and Medina 2008; Ma et al. 2011). How- ticularly useful for in vivo quantification of net ions’ flux ever, all these results of salt gland secretion were based on across plant cell membranes (Yang et al. 2010; Kong et al. the washing fluid from the leaf surface instead of direct 2012) as it has the advantage of being non-invasive, which analyzing ion secretion of salt gland. is desirable because of the presence of cell walls (Sun et al. Based on the ultrastructure of salt glands, the mecha- 2012). Another advantage of NMT is that it provides high nism of movement of salt and other ions out of the salt temporal and spatial resolution (5 s and 10 lm, respec- glands to the surface of the leaves has been proposed. The tively). NMT has been used in plant research and was first external surfaces of salt gland cells are encapsulated by a reported to measure Na? secretion from the salt glands of thick cuticle, except for the region of the wall between the Avicennia marina (Chen et al. 2010). 123 Acta Physiol Plant (2014) 36:2729–2741 2731

In the present study, we investigated the fine ultrastruc- nutrient solution containing the respective concentrations ture of salt glands of the plant Limonium bicolor, a typical of NaCl twice daily and allowed to drain. The experiment recretohalophyte with a typical salt excretory structure in was terminated 28 days after the final salinity concentra- the leaf epidermis. We used NMT to measure Na?,K?, and tions had been reached, and the fully expanded sixth leaf Cl- secretion rates from the salt glands of L. bicolor, from each plant was harvested for experiments. Five rep- combined with environmental scanning electron micro- licate pots were used for each treatment. scope (ESEM) to analyze the surface element compositions of the secretions. Our aims were to use new technologies to TEM (transmission electron microscope) observation determine whether secretion of NaCl occurs through the secretory pores of salt glands located on the surface of Healthy leaves of L. bicolor were selected and tissue seg- L. bicolor leaves and ions secretion characteristics. ments (1.5-mm diameter and 200-lm thick) were dissected from each leaf. Each segment was placed in a 3-mm- diameter copper carrier, which was filled with hexadecane as the cryoprotectant. The carrier with the sample was Materials and methods immediately frozen using a Leica EM PACT2 high-pres- sure freezer, which subjected the sample to a pressure of Plant materials and growth conditions 210 MPa at -196 °C for 30 ms. Freeze substitution (FS) with acetone was carried out in Limonium bicolor Kuntze (Plumbaginaceae) seeds were a Leica EM AFS2 system. The carriers with specimens collected from native soil saline–alkaline land (N37°200, were placed in 1.5-mL vials containing the acetone sub- E118°360) in the Yellow River Delta, China. Dry seeds were stitution medium, 2 % (w/v) osmium tetroxide in acetone, stored in a refrigerator at \4 °C before being used. The which had been frozen in liquid nitrogen in advance. The seeds were surface disinfected in 0.1 % (w/v) HgCl for 2 vials with specimens were then transferred into the FS unit 10 min and then thoroughly washed with deionized water. pre-cooled to -140 °C. Fully filled seeds were selected and planted in plastic pots The FS system was programmed as follows. The tem- (22 cm high 9 20 cm diameter) filled with river sand that perature in the chamber was raised to -90 °C within had been thoroughly rinsed with deionized water. After 30 min then maintained at -90 °C for 84 h, after which it germination, the seedlings were grown in a controlled was raised at a rate of 5 °C per hour to -60 °C then greenhouse (15-h photoperiod; 600 lmol/m2/s light inten- maintained at -60 °C for 24 h. The temperature was then sity; 30 ± 3 °C in the day and 20 ± 2 °C at night; raised at a rate of 5 °C per hour to -30 °C and maintained 70 ± 10 % relative humidity) and were irrigated every day, at -30 °C for 24 h. This was followed by further raising until they reached the sixth-leaf stage, with full-strength the temperature to 0 °C, at a rate of 5 °C per hour, and Hoagland nutrient solution (pH adjusted to 5.7 ± 0.1 with maintaining it at 0 °C for 4 h. At this stage, the acetone NaOH and HCl) with the following composition: 2.5 mmol/ substitution medium was replaced with anhydrous acetone. L Ca(NO ) , 2.5 mmol/L KNO , 1 mmol/L MgSO , 3 2 3 4 On completion, samples were removed from the FS 0.5 mmol/L KH PO ,45lmol/L Fe-EDTA, 23 lmol/L 2 4 machine and placed at room temperature. All subsequent H BO , 4.55 lmol/L MnCl , 0.16 lmol/L CuSO , 3 3 2 4 steps were carried out at room temperature (20 °C). Sam- 0.38 lmol/L ZnSO , and 0.28 lmol/L Na MoO . 4 2 4 ples were rinsed with anhydrous acetone three times, each time for 20 min. Samples were then carefully removed Treatments from the carriers and embedded gradually in Spurr resin without accelerator. A graded resin/acetone (v/v) series Following emergence of the sixth leaf (from the shoot was used: 10, 25, 50, 75, and 100 % resin, with each step base), uniform and vigorous seedlings were selected and lasting 2 h. Samples were then placed in 100 % resin transferred into plastic pots containing well-washed river without accelerator for 8 h and in 100 % resin with sand (three plants per pot). Control plants were irrigated accelerator for 8 h; this step was repeated five times. with full-strength Hoagland nutrient solution (0 mmol/L Samples were then placed in molds containing fresh resin NaCl), while others were treated with 50-, 100-, 200-, and and polymerized for 9 h at 70 °C (Smart et al. 2010). 300-mmol/L NaCl. The NaCl was dissolved in the nutrient Ultrathin sections (70 nm thick) were cut and mounted solution described above. To avoid osmotic shock, the onto copper grids coated with formvar for TEM analysis. NaCl concentration was stepped up by 50 mmol/L per day Samples were stained in 1 % (w/v) uranyl acetate in eth- until the final concentration (50, 100, 200, or 300 mmol/L) anol for 15 min and in lead citrate for a further 5 min. was achieved. To avoid salt accumulation in the sand due Micrographs were obtained with a FEI TECNAI20 TEM at to evaporation, each pot was flushed with 2,000-mL 120 kV using a Gatan UltraScan 1000 CCD camera. 123 2732 Acta Physiol Plant (2014) 36:2729–2741

Determination of ions secretion rate in leaf discs USA) after freezing with liquid nitrogen at -190 °C (Zhou et al. 2013) at the National Laboratory of Biomacromole- Fully expanded sixth leaves (0 mmol/L NaCl treated) were cules, Institute of Biophysics, Chinese Academy of thoroughly rinsed with distilled water to remove all salt Sciences. from the surface that had been secreted previously, and 10-mm-diameter discs were punched from the leaves. The ESEM analysis discs were dried quickly on both sides using absorbent paper then placed in 70-mm-diameter Petri dishes con- The abaxial surface morphologies of L. bicolor leaves were taining 30-mL 0-, 50-, 100-, 200-, and 300-mmol/L NaCl observed by ESEM (Quanta 200F, FEI, Hillsboro, Oregon, solution (pH 6.0), respectively. The abaxial leaf disc sur- USA) equipped with the EDAX energy dispersive spec- face was covered with mineral oil to allow volumetric troscopy (EDS) attachment at Peking University. The EDS analyses of the secretion fluids (Faraday et al. 1986; attachment was employed to detect the elemental compo- Dschida et al. 1992; Balsamo et al. 1995). With time, sitions of the sample surfaces (Li et al. 2013). Only fresh secretory droplets appeared under the oil above the salt fully expanded sixth leaves of plants subjected to 0 and glands. The experiment was conducted over a 24-h period 200 mmol/L NaCl were observed (Barhoumi et al. 2007). at room temperature (20 °C) with a 12-h photoperiod. Secretory droplets were collected with a micropipette. The Preparation for abaxial epidermal peels volume of secretory droplets (V) per leaf disc was deter- mined and their ionic concentration (C) measured by the The fully expanded sixth leaves from treated plants were Dionex ICS-1100 ion chromatography system (Dionex washed five times with distilled water to remove ions from Corp., Sunnyvale, CA, USA). The ion secretion rate (pmol/ the surface and were dried quickly on both sides using gland/h) was calculated as [V 9 C/(number of salt gland absorbent paper. Next, leaves were cut into 2-cm squares per leaf disc 9 time)], where number of salt gland per leaf with the abaxial surface facing up and transferred to a small disc was calculated as density of salt gland (number/ Petri dish (70-mm diameter) filled with specific buffer cm2) 9 area of leaf disc (cm2) and time was duration of the solution (0.1-mmol/L NaCl, 0.1-mmol/L KCl, 0.15-mmol/L secretion (from covering with mineral oil to collecting the CaCl2, and 0.3-mmol/L MES pH 6.0 for NMT). To separate droplets: hours). Five leaf discs were examined for each the abaxial epidermis, a pair of sharp forceps was used to fix treatment. each peel in the buffer and another pair of sharp forceps was used to split the abaxial epidermis from the leaf edge. The Determination of leaf inorganic ions content ion fluxes were measured from salt glands, stoma, and epidermal cells. The fully expanded sixth leaves of L. bicolor under NaCl concentration treatments (0, 50, 100, 200, or 300 mmol/L) Selective ion flux measurements were harvested separately. After the leaves were thor- oughly rinsed with distilled water to remove all salt from Net fluxes of Na?,K?, and Cl- were measured non- the surface that had been secreted previously, they were invasively in YoungerUSA (Xuyue, Beijing, China) NMT heated at 105 °C for 15 min to deactivate enzyme activity, Service Center using a non-invasive micro-test technique and then dried at 80 °C to a constant dry mass (DM). A (NMT-YG-100, YoungerUSA LLC, Amherst, MA01002, 50-mg dry sample was processed in a muffle stove at USA) with ASET 2.0 (Sciencewares, Falmouth, MA02540, 550 °C for 2 days, and the ash was dissolved in 1 mL of USA) and iFluxes 1.0 (YoungerUSA, LLC, Amherst, concentrated nitric acid. After the volume was increased to MA01002, USA) Software (Yang et al. 2010; Kong et al. 25 mL with deionized water, the solution was filtered and 2012; Sun et al. 2012). The NMT measures ion concen- the cations (Na?,K?,Mg2?, and Ca2?) were analyzed by trations using non-invasive ion-selective microelectrodes in the Dionex ICS-1100 ion chromatography system (Dionex both static and dynamic ways. The absolute concentration Corp., Sunnyvale, CA, USA). The Cl- anion was deter- of a specific ion or ion concentration gradient can be mined according to the spectrophotometric method (gela- measured by moving the electrode repeatedly between two tin–alcohol water solution as protective colloid) (Mello positions in a predefined excursion (5–30 lm) at a pro- et al. 2013). grammable frequency in the range 0.01–10.00 Hz with a range of 0.3–0.5 Hz being typical for many types of elec- SEM (scanning electron microscope) observation trodes. The ion-selective electrode was constructed as follows: glass micropipettes (2-lm aperture) were pulled The abaxial surface morphologies of L. bicolor leaves were from 1.5-mm-diameter glass capillaries (TW150-4, World observed by dual beam SEM (Helios Nanolab 600i, FEI, Precision Instruments, Inc., Florida, USA) with an 123 Acta Physiol Plant (2014) 36:2729–2741 2733 electrode puller (P-97, Sutter Instrument). Pulled micropi- pettes were silanized with dimethyl-dichlorosilane (D3879, Sigma, St Louis, MO, USA) at 250 °C for 50 min. For all electrodes, an Ag/AgCl wire electrode holder (EHB-1, World Precision Instruments) was inserted in the back of each electrode to create an electrical contact with the electrolyte; the ground reference electrode was an Ag/ AgCl half-cell (World Precision Instruments); and only electrodes with Nernstian slopes between 53 and 65 mV were used. Isolated abaxial peels were soaked in a test bath solution

(0.1 mmol/L NaCl, 0.1 mmol/L KCl, 0.15 mmol/L CaCl2, 0.3 mmol/L MES, pH 6.0) for 5 min. The ion-selective electrodes were mounted on a manipulator providing 3D positioning, and positioned 20 lm above the salt gland, stoma, and epidermal cell. A peel in the chamber con- taining experimental solution was placed on the micro- Fig. 1 Transmission electron microscope (TEM) image of salt scope stage, and ion fluxes were measured between 10 and glands of Limonium bicolor leaves prepared by HPF (high-pressure 30 lm above the salt gland, stoma, and epidermal cell at freezing) followed by FS (freeze substitution) and then embedded, intervals of 5.5 s. Net ion fluxes were measured for 15 min sectioned, and stained. General view of salt gland after challenge with for each sample. At least eight samples were measured for 200-mmol/L NaCl. Outer cup cell (oc), inner cup cell (ic), accessory ? cell (ac), secretory cell (sc) and collecting cell (co) are noted. each treatment (0 and 200 mmol/L NaCl). Fluxes of Na , c cuticle, n nucleus, nu nucleolus. Bars 2.5 lm K?, and Cl- were calculated using MageFlux software developed by Yue Xu (http://www.youngerusa.com/ mageflux). Secretion property of ions out of the leaves via the secretory pore using detached leaf or leaf discs Statistical analysis By exposing the detached leaves to differing salt concen- The data were analyzed using SPSS 16.0 (SPSS Inc., trations, we observed that salt glands secreted salts out of Chicago, IL, USA) for Windows and all values reported are the leaves, and secreted precipitates could be seen on the the mean ± standard deviation (SD). Treatment signifi- leaf surfaces (Fig. 2). After leaf stalks were incubated in cance was determined with one-way analyses of variance 10-mmol/L NaCl solution for 24 h, salt crystal deposits on (ANOVA), and means denoted by different lower-case leaf surfaces were observed (Fig. 2b). However, no obvi- letters are significantly different at p \ 0.05 based on ous salt crystals were observed on leaf surfaces when the Duncan’s multiple range test. leaves were incubated concurrently in 0-mmol/L NaCl (Fig. 2a). Based on the ultrastructure and the organization of the Results salt glands, it appears that active salt glands are highly turgid and force salt solutions out through pores under Ultrastructure features of L. bicolor salt glands pressure. If this hypothesis is correct, if leaf discs are covered with mineral oil, secretory droplets will form The use of high-pressure freezing (HPF) and FS to fix the under the layer of oil above the salt glands. As shown in leaves was successful, and outstanding preservation of salt Fig. 2, initially no droplets were detected on the surface of glands ultrastructure was obtained, as demonstrated by the the leaf discs (Fig. 2c), but many spherical secreted drop- clearly identifiable intact salt glands in the image shown lets were observed after the discs were incubated in in Fig. 1. There are 16 cells in the salt gland of L. 200-mmol/L NaCl for 24 h (Fig. 2e). Isolated leaf discs bicolor, including four outer cup cells (oc), four inner cup also showed small droplets on the surface after incubation cells (ic), four accessory cells (ac) and four secretory cells in distilled water (0-mmol/L NaCl) for 24 h (Fig. 2d). (sc). The nuclei in these cells are typically large with very To investigate the synergistic secretion of other ions ? 2? 2? 2- - prominent nucleoli (Fig. 1). The salt gland is encapsulated (namely, K ,Mg ,Ca ,SO4 ,NO3 ) with NaCl by a thick cuticular layer (c) except along region of the secretion, secretory droplets were carefully collected with a walls between the collecting cells (co) and the outer cup micropipette and the ion secretion rate of a single salt gland cells. was determined using the ion chromatography system 123 2734 Acta Physiol Plant (2014) 36:2729–2741

Fig. 2 Salt secretion on the abaxial leaf surface of Limonium bicolor the salt glands. Salt crystals were observed on the surface of leaves in treated with 0 (a) and 10 (b) mmol/L NaCl for 24 h. Leaf stalks were 10-mmol/L NaCl solution for 24 h (b, arrows), but no crystals in cut under distilled water to prevent air blockages in the xylem and 0-mmol/L NaCl solution (a). Isolated leaf discs showing droplets on then placed in vials containing 0- or 10-mmol/L NaCl solution for the abaxial surface after incubation in 0 (d) and 200 (e) mmol/L NaCl 24 h under conditions that maintained the transpiration stream up to for 24 h, but no droplets at 0 h (c). Bars 1mm(a, b), 2.5 mm (c–e) under an NaCl concentration gradient (0, 50, 100, 200, and under a range of NaCl concentration gradient (0, 50, 100, 300 mmol/L). Secretion rate of different ions showed dif- 200, and 300 mmol/L). As shown in Table 2, the ions with ferent patterns with increasing salinity. As shown in highest accumulation in leaves were Na? and Cl- for salt Table 1, the secretion rates of Na? and Cl- ions were treatments, consistent with the high Na? and Cl- secretion much higher than those of other ions under the same rates shown in Table 1. The leaf Na? and Cl- contents concentration of NaCl and gradually increased with increased with increasing salinity from 50- to 200-mmol/L increasing concentrations of NaCl. The secretion rate of NaCl with no further increase at 300-mmol/L NaCl. In K? was maximum at 0-mmol/L NaCl and decreased with control plants, K? was the predominant cation at 0-mmol/L increasing concentration of NaCl to 200 mmol/L, and then NaCl, but its content decreased with increasing NaCl remained relatively constant in treatments with salt added. concentration to 100-mmol/L NaCl with no further The Mg2? secretion rate was unchanged from 0 to decrease above this level. Also leaf Mg2? and Ca2? content 50 mmol/L NaCl, then declined with increasing NaCl decreased with increasing salinity (Table 2). concentrations above 50 mmol/L. The secretion rate of Ca2? was very low in the 0 mmol/L NaCl treatment, but Visible secretions and pores on the surface of each increased with added NaCl, particularly at higher treatment secretory cell concentrations. Compared with control, 100-mmol/L NaCl 2- significantly decreased SO4 secretion rate but 200-mmol/ Scanning electron microscope micrographs revealed that - L NaCl increased it. The secretion rate of NO3 increased each salt gland consisted of four secretory cells surrounded with increasing NaCl concentration to a maximum at by collecting cells, and each of the secretory cells had a 200 mmol/L. pore on its surface (Fig. 3a). The initial secretions had a To examine the relationship between the ion secretion rice grain-like appearance (Fig. 3b). Subsequent secretions rate and ion content in leaves, we determined the content of remained primarily over the pores of the secretory cells. ions using ion chromatography and spectrophotometry The build-up of secreted material was obvious as few salt

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Table 1 Ion secretion rates Ion Secretion rate (pmol/gland/h) from salt glands of Limonium bicolor leaf discs treated with NaCl (mmol/L) different concentrations of NaCl (0, 50, 100, 200, and 0 50 100 200 300 300 mmol/L) Na? 18.75 ± 3.90a 39.46 ± 7.25b 100.51 ± 6.56c 140.17 ± 10.62d 222.54 ± 9.75e K? 19.08 ± 1.43a 10.12 ± 1.43b 6.50 ± 0.78c 4.37 ± 0.89d 2.99 ± 1.13d Mg2? 11.20 ± 0.92a 11.55 ± 0.84a 7.63 ± 0.82b 5.03 ± 0.62c 4.11 ± 0.39c Values are mean ± SD (n = 5). Ca2? 0.03 ± 0.02a 1.12 ± 0.26b 0.80 ± 0.17b 3.35 ± 0.15c 10.23 ± 0.65d Values in a line followed by - different lower-case letters are Cl 23.42 ± 1.94a 47.19 ± 3.20b 103.49 ± 8.68c 159.14 ± 7.49d 225.91 ± 7.61e 2- significantly different at SO4 2.68 ± 0.18a 2.74 ± 0.17a 2.61 ± 0.21a 3.02 ± 0.14b 2.80 ± 0.16ab p \ 0.05 according to Duncan’s NO - 0.50 ± 0.12a 0.65 ± 0.13a 1.33 ± 0.12b 1.68 ± 0.11c 1.53 ± 0.09c multiple range test 3

Table 2 Leaf ions content of Limonium bicolor seedlings treated with 0-, 50-, 100-, 200-, and 300-mmol/L NaCl for 28 days Ion Content (mmol/g DM) NaCl (mmol/L) 0 50 100 200 300

Na? 0.15 ± 0.017a 0.43 ± 0.045b 0.61 ± 0.030c 0.85 ± 0.036d 0.88 ± 0.038d K? 1.22 ± 0.038a 0.62 ± 0.029b 0.42 ± 0.031c 0.40 ± 0.031c 0.43 ± 0.042c Mg2? 0.30 ± 0.019a 0.29 ± 0.018a 0.19 ± 0.018b 0.15 ± 0.012c 0.15 ± 0.017c Ca2? 0.49 ± 0.032a 0.31 ± 0.021b 0.24 ± 0.019c 0.22 ± 0.020c 0.15 ± 0.027d Cl- 0.14 ± 0.008a 0.36 ± 0.049b 0.51 ± 0.030c 0.75 ± 0.036d 0.78 ± 0.039d Values, which are means (n = 5) ± SD, indicate the change in the indicated variables. Values in a line followed by different lower-case letters are significantly different at p \ 0.05 according to Duncan’s multiple range test glands were observed with little or moderate deposits. The matching of the elemental compositions of sodium and bulk of the secreted salts accumulated over the pores. chlorine with the scanning electron micrographs showed Secretion of material continued until the entire salt gland that the rice grain-like secretions were mainly composed of and adjacent collecting cells were covered by the deposits sodium and chlorine. (Fig. 3c, d). Selective secretion of Na? and Cl- by salt glands, The secretions being mainly composed of sodium but little secretion of ions by stoma and epidermal cells and chlorine Non-invasive micro-test technology provides a method for Samples were analyzed by ESEM to determine the ele- non-invasively obtaining dynamic information on specific mental composition of the secreted material from salt ionic flux of salt glands. This technique incorporates dif- glands (Fig. 4). Figure 4a, c showed the position of salt ferent temporal and spatial resolution domains from other glands and secreted salt crystals on the leaf surface of traditional methods, and its three-dimensional measure- control and 200-mmol/L NaCl-treated plants, respectively. ment capability enables observation of ion flux character- The surface chemical compositions of deposits were istics of biological phenomena that would be difficult or detected by energy dispersive spectroscopy as shown in even impossible with other techniques (Fig. 5a). The pre- Fig. 4b, d, respectively. The element compositions showed pared peels consisted of the epidermal cells, salt glands, that the sample surface was mainly composed of elements and stoma. The salt glands in the peels were intact and containing C and O, with Na, Mg, S, Cl, K, and Ca also secreted at a steady rate when the abaxial peels were placed presented (Fig. 4b, d). Analysis of the single deposit in buffer solution (Fig. 5b). The abaxial peels and NMT revealed that the concentrations (wt%) of Na, Mg, S, Cl, K were, therefore, considered as a viable experimental system and Ca in the control were similar in a range of for exploring functions of salt glands. 0.31–0.55 %, but the 200-mmol/L NaCl treatment mark- Secreted material was observed over the secretory pores. edly increased Na and Cl element concentrations (14.2 and In order to compare the secretion rates from different leaf 8.7 times, respectively, greater than the control). Careful cells, the NMT system was used to measure ions’ flux

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Fig. 3 SEM (scanning electron microscopy) micrographs of salt glands on the abaxial surface of leaves of Limonium bicolor seedlings grown in 0 (a) and 200 (b–d) mmol/L NaCl for 28 d. a SEM of the leaf salt gland with no secretion. Note the pore present in each of the four secretory cells. b SEM of leaf salt gland with secreted deposit apparently extruding from the pores. c, d SEM of the leaf salt gland(s) showing secretions. d Showed a high magnification view of the rice grain-like appearance of the secretion. Bars 5 lm(a, b, d), 50 lm(c). Arrows indicate secretory pore (a) and secretions (b–d)

directly from the secretory cells, stoma and epidermal cells. Tester 2008; Tan et al. 2013). Recretohalophytes with As shown in Fig. 6a, c, the Na? and Cl- secretion rates of specialized salt-secreting salt glands can secrete excess individual salt glands, stoma and epidermal cells were salts out of plants. This specialized salt exclusion system lower in the control than in the 200-mmol/L NaCl treat- effectively reduces the salt content in plant cells, aids the ment. Under the same conditions, the Na?,K? and Cl- maintenance of relatively constant ion concentrations, and secretion rates of individual salt glands were higher than enhances the salinity tolerance of the plant for tolerating a those of stoma and epidermal cells. The Na? secretion rate saline environment (Munns and Tester 2008; Ding et al. was greatly enhanced by 200-mmol/L NaCl treatment, and 2010). Therefore, salt glands play an important role in the Na? secretion rate of individual salt glands was 6,220 maintaining the normal physiological function of plant times higher than the K? secretion rate, which indicated cells in a saline environment (Bosabalidis 2012; Tan et al. that salt glands have much more secretion for Na? than K? 2013). Limonium bicolor, with multicellular salt glands, is (Fig. 6a, b). The Cl- secretion rate of individual salt glands reputed to be the ‘‘pioneer plant for the bioremediation of was also markedly enhanced by treatment with 200-mmol/ saline-alkaline land’’ because it has well-developed salt L NaCl, being 35 times higher with the NaCl treatment glands (Ding et al. 2009). Therefore, it is important to than for the control treatment (Fig. 6c). Due to the position investigate the salt secretion mechanism and structure of of the electrode close to the secretory pore, we concluded salt glands in the leaves of L. bicolor. that salt glands mainly secrete NaCl via the pore when The external surface of the salt glands and epidermal treated with NaCl. This confirms that the secretion is an cells were covered by a thick cuticle, which extended over actively selected process allowing protection of plant tis- the secretory cells creating a cavity between the cuticular sues against toxic ions without losing essential nutrients. layer and the cell wall of the secretory cell (Fig. 1). Each of the secretory cells has a pore, the secretory pore, on its surface (Fig. 3a). The secretion of salts to the outer surface Discussion of the leaf may occur through the secretory pores of the salt gland within the cuticle. These likely correspond to the Salt secretion is an important mechanism for recretohalo- pores described in the salt glands of Limonium latifolium phytes to adjust to a salinized environment (Munns and (Arisz et al. 1955) and are the site of salt secretion.

123 Acta Physiol Plant (2014) 36:2729–2741 2737

Fig. 4 The abaxial surface morphologies of Limonium bicolor leaves 28 days; c leaf surface after exposure to Hoagland nutrient solution using ESEM (environmental scanning electron microscopy) (a), containing 200-mmol/L NaCl for 28 days. Bars 25 lm. Arrows (c) and EDS (energy dispersive spectroscopy) spectrum (b), d of salt indicate the position of salt gland and secreted salt crystals (a, c) glands. a Leaf surface after exposure to Hoagland nutrient solution for

Fig. 5 Schematic diagram of NMT (non-invasive micro-test tech- b A light micrograph illustrating selective test points designed to nology) system for measuring ions’ flux of Limonium bicolor salt determine ion fluxes from individual salt glands, stoma and epidermal glands. a A diagram showing the system used to measure selective ion cells. e electrode, ec epidermal cell, s stoma, sg salt gland. Bars fluxes from a salt gland using a non-invasive ion-selective electrode. 25 lm

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Fig. 6 Effects of different NaCl treatments on the Na?,K?, and Cl- stoma or epidermal cells at intervals of 5.5 s. The mean fluxes ± SD secretion rate of individual salt glands (SG), stoma (S) and epidermal of the 15-min measuring period are shown on the right. Values cells (EC) in leaf epidermis of Limonium bicolor. A continuous flux followed by the different lower-case letters are significantly different for 15 min was performed to measure net ion flux rates of Na? (a), at p \ 0.05 as determined by Duncan’s multiple range test. Control K? (b), and Cl- (c) for each salt gland, stoma and epidermal cell in plants were treated with Hoagland nutrient solution (0-mmol/L NaCl) corresponding measuring solutions (pH 6.0) on the left, where ion for 28 days; other plants were treated with Hoagland nutrient solution fluxes were measured between 10 and 30 lm above the salt gland, containing 200-mmol/L NaCl for 28 days

Pore-like structures in the overlying cuticle and sub- mesophyll cells of leaves (Campbell and Strong 1964). cuticular cavities occur in most salt glands that have been Thus, all secretions from salt glands pass through these studied. According to Arisz et al. (1955), the salt glands are four small pores (Thomson and Liu 1967). The cuticular- primarily desalting organs capable of reducing salt levels in ized band covering the inner secretory cells probably

123 Acta Physiol Plant (2014) 36:2729–2741 2739 furnishes a resistance to backflow into the underlying salt, the main ions accumulated in the leaves were also Na? mesophyll. Such resistance, at this point, would be particu- and Cl- (Table 2). ESEM is a technique that allows larly important if the collecting compartment or cavity below imaging of organic samples in the fresh or living state (e.g., the outer cuticle is a site of ion accumulation in which con- uncoated, wet, and hydrated tissue) without the need for siderable hydrostatic pressure develops before the salt conventional preparation techniques (Danilatos 1981; solution is released through the cuticular pores. The possi- Manero et al. 2003; Jin et al. 2013). ESEM can be used to bility that considerable hydrostatic pressure develops within measure the microstructure accurately without invasively this compartment, and that the salt solution is emitted altering it (Danilatos 1990; Lu et al. 2013), thus allowing through a valve-like cuticle pore and pressure release, lend specimens to be studied at high resolution and close to their credence to Ruhland’s (1915) observations that the emission natural states (Dang and Copeland 2004). ESEM analysis was due to the build-up of pressure within the salt glands. It confirmed that secretions were also mainly composed of appears that active salt glands are highly turgid and force salt sodium and chlorine (Fig. 4). solutions out through pores under pressure. If ions are Although separating the epidermal layers from the leaf transported into the collecting chamber, resulting in the had the potential to damage the salt gland cells, the NMT osmotic flow of water into the chamber, osmotic equilibrium results (Fig. 6) indicate that the salt glands in the abaxial within the tissue could be reached before a sufficient peels were active and therefore suitable for analysis of salt hydrostatic pressure develops to force the salty fluid to be gland function. Dschida et al. (1992) reported that the salt secreted through the cuticular pores (Campbell and Thomson glands actively secreted when peels were floated on dis- 1975). Then pressure will be created and the highly con- tilled water. We also found that the salt glands secreted centrated salt solution will passively leak through the small steadily when the abaxial peels were placed in various pores on the external surface of the secretory cells (Skelding buffer solutions, thereby demonstrating that the abaxial and Winterbotham 1939; Levering and Thomson 1971; So- peels were suitable experimental material for this research. maru et al. 2002; Semenova et al. 2010; Tyerman 2013). This In earlier studies, secretion of ions was measured mainly proposal is consistent with the conclusion of Arisz et al. by washing leaves. With this approach, in addition to ions (1955), that secretion was dependent on an increase of turgor secreted from salt glands, the detected ions may have pressure within the salt gland. The studies of Thomson and included those excreted by stoma or epidermal cells or ions Liu (1967) and Thomson et al. (1969)onTamarix aphylla deposited on leaves from external sources (Liphschitz and indicated that salt accumulates in small microvacuoles in the Waisel 1974; Ramadan and Flowers 2004). Thus, the cytoplasm. Subsequently these microvacuoles migrate and composition of ions secreted by individual salt glands is fuse with the plasmalemma, discharging the salt solution into not determined. Faraday et al. (1986) and Dschida et al. the walls of the secretory cells. The salt solution then (1992) used leaf discs with an oil cover floating on chal- apparently moves along the walls to the apex of the salt gland lenge buffers and X-ray or electric poles to detect the ions and through pores in the cuticle to the leaf surface. If a secreted from salt glands. However, this method only coupled solute–water–transport system is assumed, water excluded the interference from stoma transpiration, and did moving passively into the extracellular channels in the basal not accurately reflect the dynamic secretion profile of the cell would exert sufficient pressure to cause movement of individual salt glands. ions into the cap–cell wall and out through the pores of the In the present study, secretion rates of Na? and Cl- were ? 2? 2? 2- cuticle (Ding et al. 2010). Our observations in this study not much higher than those of K ,Mg ,Ca ,SO4 and - only provide evidence that the salt glands of L. bicolor have NO3 under the same concentration of external NaCl using four secretory pores, but also indicate that NaCl is secreted leaf disc (Table 1). For example, the secretion rates of K? through these pores of the salt gland. and Na? were similar for the control treatment, but Na? was By exposing detached leaves and leaf discs to incubation secreted at *75-fold rate greater than K? for 300-mmol/L solutions containing graded NaCl concentrations, we NaCl treatment. Moreover, the Na?,K?, and Cl- secretion demonstrated that salt glands were able to fulfill their rates of individual salt glands, epidermal cells, and stoma fundamental role of salt secretion (Fig. 2). But, Na? were further measured using NMT. For the individual salt secretion rate under 200-mmol/L NaCl treatment was 7.5 glands, the Na? secretion rate was significantly higher than times faster than the control (0 mmol/L NaCl) (Table 1). the K? secretion rate for the same concentration of NaCl. These results indicate that NaCl treatment markedly When plants were treated with 200-mmol/L NaCl, the enhanced the salt secretion of salt gland, and secretion as secretion rate per gland was significantly higher for Na? observed is a result of salt treatment rather than secretion than for K? (Fig. 6a, b), and the Na? secretion rate of appearing with time (Fig. 2c–e). Salt glands present in the individual salt glands was 6,220 times higher than the K? leaves, secreted a variety of ions, but the secreted deposits secretion rate. Similar results have been obtained using mainly consisted of NaCl (Table 1). With the addition of whole leaves or leaf discs of other recretohalophytes 123 2740 Acta Physiol Plant (2014) 36:2729–2741

(Rozema et al. 1981; Wieneke et al. 1987). Compared with Barhoumi Z, Djebali W, Smaoui A, Chaıbi W, Abdelly C (2007) individual salt glands, stoma and epidermal cells showed Contribution of NaCl excretion to salt resistance of Aeluropus ? ? - littoralis (Willd) Parl. J Plant Physiol 164:842–850 very little secretion of Na ,K and Cl , and treatment with Berry WL (1970) Characteristics of salts secreted by Tamarix NaCl did not significantly affect their secretion rates. In this aphylla. Am J Bot 57:1226–1230 study, the Cl- secretion rates of individual salt glands were Bosabalidis AM (2012) Programmed cell death in salt glands of markedly enhanced by treatment with 200-mmol/L NaCl, Tamarix aphylla L.: an electron microscope analysis. Cent Eur J - Biol 7:927–930 and the Cl secretion rate was 35 times higher in NaCl Bradley PM, Morris JT (1991) Relative importance of ion exclusion, treatment than for the control (Fig. 6c). These results sug- secretion and accumulation in Spartina alterniflora Loisel. J Exp gest that salt glands secreted ions through secretory pores Bot 42:1525–1532 and show that the predominantly secreted ions were Na? Breckle SW (1995) How do halophytes overcome salinity. In: Khan - MA, Ungar IA (eds) Biology of salt tolerant plants. Book and Cl . Secretion is, thus, a selective phenomenon Crafters, Chelsea, pp 199–213 allowing protection of plant tissues against toxic ions Campbell CJ, Strong JE (1964) Salt gland anatomy in Tamarix without losing essential nutrients under NaCl treatment. pentandra (Tamaricaceae). Southwest Nat 9:232–238 The increase in secretion rates contributed to counteracting Campbell N, Thomson WW (1975) Chloride localization in the leaf of Tamarix. Protoplasma 83:1–14 the increase in salt concentration in the leaf (Table 2). With Carter JM, Nippert JB (2011) Physiological responses of Tamarix increasing salinity, higher secretion rates kept the internal ramosissima to extreme NaCl concentrations. Am J Plant Sci ion concentrations in the leaf relatively low. The selective 2:808–815 Chen J, Xiao Q, Wu F, Dong XJ, He JX, Pei ZM, Zheng HL (2010) character of the secretion contributes to maintaining ? ? ? - Nitric oxide enhances salt secretion and Na sequestration in a favorable Na /K and Cl /anion ratios in the leaf cells. The mangrove plant, Avicennia marina, through increasing the precise molecular mechanism that regulates efflux through expression of H?-ATPase and Na?/H? antiporter under high the pore during salt secretion is currently the subject of salinity. Tree Physiol 30:1570–1585 further study. Dang JMC, Copeland L (2004) Studies of the fracture surface of rice grains using environmental scanning electron microscopy. J Sci Author contribution B.S. Wang conceived the study Food Agr 84:707–713 and revised the manuscript. Z.T. Feng designed experi- Danilatos GD (1981) The examination of fresh or living plant material in an environmental scanning electron microscope. ments, performed the experiments and wrote the manu- J Microsc 121:235–238 script. Q.J. Sun and Y.Q. Deng did ion measurements and Danilatos GD (1990) Mechanisms of detection and imaging in the plant cultivation. S.F. Sun and J.G. Zhang were responsible ESEM. J Microsc 160:9–19 for TEM and SEM work. All authors read and approved the Ding F, Song J, Ruan Y, Wang BS (2009) Comparison of the effects of NaCl and KCl at the roots on seedling growth, cell death and final manuscript. the size, frequency and secretion rate of salt glands in leaves of Limonium sinense. Acta Physiol Plant 31:343–350 Acknowledgments We thank Dr. Li Chen (Electron Microscopy Ding F, Yang JC, Yuan F, Wang BS (2010) Progress in mechanism of Laboratory, School of Physics, Peking University, Haidian, Beijing, salt excretion in recretohalophytes. Front Biol 5:164–170 100871, P.R. China) for technical assistance in ESEM analysis. Our Dschida WJ, Platt-Aloia KA, Thomson WW (1992) Epidermal peels TEM and SEM work was performed at the Center for Bio-imaging, of Avicennia germinans (L.) Stearn: a useful system to study the Institute of Biophysics, Chinese Academy of Sciences. We also thank function of salt glands. Ann Bot 70:501–509 Dr. Yue Xu and his team from Xuyue (Beijing) Sci. and Tech. Co., Fahn A (2000) Structure and function of secretory cells. Adv Bot Res Ltd. (http://xuyue.net) for their professional advice and technical 31:37–75 support for Na?,K?, and Cl- flux measurements using NMT. This Faraday CD, Thomson WW (1986a) Structural aspects of the salt research was supported by the NSFC (National Natural Science glands of the Plumbaginaceae. J Exp Bot 37:461–470 Research Foundation of China, project No. 30870138 and No. Faraday CD, Thomson WW (1986b) Functional aspects of the salt 31070158), key projects in the National Science and Technology glands of the Plumbaginaceae. J Exp Bot 37:1129–1135 Pillar program during the eleventh five-year plan period Faraday CD, Quinton PM, Thomson WW (1986) Ion fluxes across the (2009BADA7B05), Programs Foundation of Ministry of Education of transfusion zone of secreting Limonium salt glands determined China (20123704130001) band program for scientific research inno- from secretion rates, transfusion zone areas and plasmodesmatal vation team in colleges and universities of Shandong province. frequencies. J Exp Bot 37:482–494 Flowers TJ, Colmer TD (2008) Salinity tolerance in halophytes. 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