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 Á NaCl secretion Á ing structures, including salt glands and salt bladders, can Salt gland Á 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 Á Q. Sun Á Y. Deng Á 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 Á 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 Á 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.