The Salt-secretion Mechanism Of Salt Glands In The Leaves Of Limonium

Halophytes are divided into euhalophytes, recretohalophytes and pseuhalophytes according to their physiological mechanisms of salinity tolerance, morphologic structures and ecological characteristics. Recretohalophytes include exo-recretohalpphytes having salt glands and endo-recretohalophytes having salt bladders. The most remarkable morphological characteristics of recretohalophytes are the salt-secreting structures (salt glands and salt bladders) that can excrete or sequestrate excessive salts from metabolically active tissues. They play an important role in enhancing the salinity tolerance of plants. Limonium bicolor, a typical recretohalophyte, with multicellular salt glands, is reputed as the"pioneer plant for the bioremediation of saline-alkaline land". Therefore, it is very important to investigate the salt-secretion mechanism of salt glands in the leaves of Limonium bicolor.The net Na⁺, K⁺, Cl^- and H⁺ fluxes of individual salt glands in the leaves of Limonium bicolor were investigated using the Scanning Ion-selective Electrode Technique (SIET). Treatment with 200 mM NaCl or KCl resulted in significant increases in net Na⁺ or K⁺ efflux and significantly enhanced the activity of the plasma membrane H⁺-pump. Both events were effectively prevented by pretreatment with vanadate, a specific inhibitor of the plasmalemma H⁺-ATPase. The Na⁺ secretion rate sharply decreased by pretreatment with bumetanide, a specific inhibitor of vertebrate Na⁺: K⁺: Cl^- cotransporters, and amiloride, a specific inhibitor of the Na⁺/H⁺ exchanger. At 200 mM for both NaCl and KCl, K⁺ concentration was significantly higher than Na⁺ concentration in the leaves, while the Na⁺ secretion rate of individual salt glands was significantly higher than the K⁺ secretion rate. The results suggested that Na⁺, K⁺ and Cl^- are excreted via Na⁺: K⁺: Cl^- cotransporter; In addition, plasmalemma H⁺-ATPase in secretory cells establishes an electrochemical H⁺-gradient across the membrane that drives the efflux of Na⁺ via Na⁺/H⁺ exchanger, which led to the efficiency of gland secretion in Limonium bicolor was Na⁺>K⁺. Surprisingly, Na⁺ effux was significantly decreased while K⁺ efflux was remarkably increased by pre-treatment with ouabain, a specific inhibitor of Na⁺/K⁺-ATPase in animal. Confocal fluorescence microscopy further revealed that pretreatment with ouabain caused a remarkable increase in Na⁺ concentration in salt glands. These results suggested a Na⁺/K⁺-ATPase may exist in salt glands of Limonium bicolor and be involved in salt secretion process. Moreover, it is the coordinate work of Na⁺/H⁺ exchanger (SOS1), Na⁺: K⁺: Cl^- cotransporter and Na⁺/K⁺-ATPase of plasma membrane of salt glands that led to the efficiency of gland secretion in Limonium bicolor being Na⁺>K⁺.The effects of 200 mM NaCl on the morphology and salt secretion rates of a special type of epidermal cells surrounding salt glands of Limonium bicolor (Bunge) Kuntze were studied. Compared with controls, these epidermal cell have the following characteristics: (1) epidermal cells surrounding salt glands were more salient than salt glands at 200 mM NaCl and both of them were covered with a lot of salt crystal; (2) 200 mM NaCl treatment markedly enhanced the enlargement of the epidermal cells surrounding salt glands; (3) Na⁺ secretion rate of the epidermal cell surrounding salt glands gradually declined and H⁺ efflux turn to influx with the increasing distance from the juncture of salt glands and epidermal cells to the site being determined. This is also the first report that epidermal cells surrounding salt glands of Limonium bicolor were a special type of epidermal cells that could secrete salts.Limonium bicolor seedlings were treated with modified Hoagland solution containing 200 mM NaCl combined with three levels of Ca²⁺ (0 mM, 5 mM and 20 mM) for 15 days to study the effects of Ca²⁺ on development and salt-secretion rates of salt glands. It was shown that the 4th leaf areas of Limonium bicolor seedlings under 20 mM Ca²⁺ treatment were significantly higher than those under 0 mM and 5 mM Ca²⁺ treatments. The relative areas of 4th leaves treated with 20 mM Ca²⁺ increased to 234% compared with plants treated with 0 mM Ca²⁺. No significant difference in the relative areas of 4th leaves was observed between 0 mM and 5 mM Ca²⁺ treatments. Both the total number per leaf and diameters of salt glands increased markedly with increased Ca²⁺ concentrations. With 20 mM Ca²⁺ treatment, the total number per leaf increased to 610% compared with plants treated with 0 mM Ca²⁺, the difference was significant. The diameters of salt glands increased by 59% and 63% as Ca²⁺ concentration increased from zero to 5 mM and 20 mM, respectively. Under 20 mM Ca²⁺ treatment, the salt-secretion rate per leaf was obviously higher than that treated with 5 mM Ca²⁺, but there was no significant difference in the salt-secretion rates per salt gland between the two groups. Under 0 mM Ca²⁺ treatment, leaf-cell membrane permeability increased significantly, which led to serious leakage of ions and a significant increase in Na⁺ loss rate. The results suggests that the increase of Ca²⁺ concentration markedly enhanced development and salt-secretion rates of salt glands in the leaves of Limonium bicolor, the increase of salt secretion per leaf is due to the efficiency of the secretion process per salt gland and the number of salt glands, the salt-secretion rates per salt gland have a relationship with the diameters of salt glands. The main innovation points of this study were generalized as follows:1 SIET is first used to study the salt secretion of individual salt glands.2 Plasma membrane H⁺-ATPases, Na⁺/H⁺ exchangers and Na⁺: K⁺: Cl^- cotransporter are involved in salt secretion of salt glands; Na⁺/K⁺-ATPase may exist in salt glands of Limonium bicolor and be involved in salt secretion process. It is the coordinate work of Na⁺/H⁺ exchanger (SOS1), Na⁺: K⁺: Cl^- cotransporter and Na⁺/K⁺-ATPase of plasma membrane of salt glands that led to the efficiency of gland secretion in Limonium bicolor being Na⁺>K⁺.3 Epidermal cells surrounding salt glands of Limonium bicolor were a special type of epidermal cells that could secrete salts.4 Ca²⁺ plays an important role in salt secretion process of salt glands in leaves of Limonium bicolor.