Acta Physiol Plant (2013) 35:741747 DOI 10.

1007/s11738-012-1114-8

ORIGINAL PAPER

Effects of salinity and nitric oxide donor sodium nitroprusside (SNP) on development and salt secretion of salt glands of Limonium bicolor
Feng Ding

Received: 23 June 2012 / Revised: 22 August 2012 / Accepted: 19 September 2012 / Published online: 10 October 2012 rski Institute of Plant Physiology, Polish Academy of Sciences, Krako w 2012 Franciszek Go

Abstract NO, as a signaling molecule, is involved in abiotic stresses. Limonium bicolor seedlings were treated with 200 mM NaCl combined with 0.05 mM SNP for 20 days to study the effects of NO on development and salt-secretion rates of salt glands. It was shown that the total number of salt glands on adaxial surfaces under condition of 200 mM NaCl containing 0.05 mM SNP treatment increased signicantly compared with that under 200 mM NaCl treatment. Na? secretion rate per leaf under 200 mM NaCl containing 0.05 mM SNP was signicantly higher than that under 200 mM NaCl without SNP. However, there was no signicant difference in salt-secretion rate of individual salt glands between 200 mM NaCl containing 0.05 mM SNP treatment and 200 mM NaCl treatment. Although there was no signicant difference in salt-secretion rate of individual glands, Na? concentration in the leaves treated with 200 mM NaCl solution containing SNP was signicantly lower than that treated with 200 mM NaCl solution. Treatment with 200 mM NaCl solution containing SNP caused a remarkable increase in Na? concentration in salt glands. Obviously, the efciency of the secretion process per gland was enhanced by adding SNP to NaCl. The results showed NO may enhance the salt secretion by inducing more dermatogen cells to develop into salt glands and by enhancing the efciency of the secretion process per gland.

Keywords Limonium bicolor Nitric oxide Number of salt glands Sodium secretion rate

Introduction Salt injury to a plant may arise from a decrease in the water potential of the soil, from specic ions accumulated to injurious concentrations, and from nutritional imbalances caused by the presence of high concentrations of a few ions that compete for sites of uptake of essential elements (Ramadan and Flowers 2004). In some halophytes, salt excretion represents an avoidance strategy that permits control and regulation of salt content in plant organs, and especially in photosynthetic ones (Atkinson et al. 1967). Salt excretion has been shown to be mediated by specic glands scattered on the leaf surfaces. During the secretion of salt by glands, both symplastic and apoplastic movement of ions takes place toward the gland: secretion itself, although not well understood, appears to be an active process. It is clear that secretion increases with external concentration of, and time of exposure to, salt (Liphschitz and Waisel 1974; Marcum and Murdoch 1992; Pollak and Waisel 1970). However, it is difcult to nd in the literature an answer to the question whether or not glandular function is inducible (Thomson et al. 1988). Liphschitz and Waisel (1974) found no increase in the number of glands in Chloris gayana over a 5-day exposure to salt, although, interestingly, spraying with benzyl adenine (BA) did increase the number of glands. Rozema and Riphagen (1977) found that the number of salt glands of Glaux maritime in high saline environments was significantly higher than that in low-salinity. Ramadan and Flowers (2004) reported BA to increase salt loss from

Communicated by S. Weidner. F. Ding (&) College of Food and Bioengineering, Shandong Polytechnic University, Jinan 250353, Shandong, Peoples Republic of China e-mail: bio_ding@126.com

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maize plants due to its inuence on the number of microhairs and on leaf area, but this loss was unrelated to salt excretion per se. Increasing evidence proved that NO is involved in many physiological and metabolic processes in higher plants (Bethke et al. 2004). NO is a small lipophilic and chemically unstable molecule that is well suited to early out the multiple biological functions. NO readily permeates through biological membranes to elicit effects in target cells adjacent to the cells where it is formed. NO has also been suggested to act as a signal molecular mediating responses to biotic and abiotic stresses in the plant kingdom. It could induce germination instead of red right (Giba et al. 1998; Beligni and Lamattina 2000), affect growth and development of plant tissue (Leshem and Haramaty 1996; a et al. 1997; Durner and Klessing 1999), and Gouve enhance plant cell senescence (Pedroso and Durzan 2000; Pedroso et al. 2000a, b). Also, NO was suggested to be involved in the responses to drought stress, heat stress, disease resistance, and apoptosis (Delledonne et al. 1998; Leshem et al. 1998; Durner and Klessing 1999; Ribeiro et al. 1999; Beligni and Lamattina 2000; Pedroso et al. 2000b; Mata and Lamattina 2001). In salt-tolerant reed calluses, expression and activity of the plasma membrane H?-ATPase protein were activated by SNP (Zhao et al. 2004). Chen et al. (2010) found that nitric oxide enhanced salt secretion in the leaves of Avicennia marina through increasing the expression of H?-ATPase and Na?/H? antiporter under high salinity. However, it is still unknown how NO regulates the salt-secretion process of salt glands. In the present study, nitric oxide donor SNP was used to study the effects of exogenous NO on the salt-secretion rate and the number of salt glands.

full-strength Hoagland solution containing 200 mM NaCl. When the fth leaves appeared, one pot treated with NaCl was selected and irrigated with full-strength Hoagland solution containing 200 mM NaCl and 0.05 mM SNP. Plants were then irrigated with the same solutions once a day for 20 days. Measurement of Na? secreted by leaves To determine the quantity of Na? secreted from the leaves, ions were collected according to Zhou and Zhao (2003). The adaxial and abaxial surfaces of the 4th, 5th, 6th leaves were thoroughly washed with distilled water after 20 days. The chosen leaves were washed again with a xed volume of distilled water (5 mL) after 1 day, and the Na? in the washing solution was measured with ame photometer (Flame Photometer 410, Sherwood). Determination of Na? in leaves Leaves were quickly washed with distilled water, surfacedried with lter paper, and dried in an oven for 2 days at 80 C. Dry samples were placed in a mufe furnace to be ashed at 550 C. The ash was dissolved with concentrated nitric acid, and then distilled water was added to obtain a nal volume of 20 ml. Na? and K? was determined by a ame photometer (Flame Photometer 410, Sherwood). Measurement of leaf area (S) The whole leaf was excised from the plant, traced onto scale paper, cut out (the area was represented with S), and weighed (the weight was represented with G). The whole scale paper was weighed (the weight was represented with G) and its area was measured (the area was represented with S). The area (S) was calculated from the weight (G) and the known weight of a unit area (G/S) as follows: (S) = G 9 (S/G). Calculation method of salt-secretion rate and density of salt glands Leaf-prints were prepared according to the method described by Hilu and Randall (1984). Clear nail varnish was applied to the leafs surface for about 0.5 h and then the dry lm peeled off using ultra clear Sellotape and placed on a clean slide. Salt glands were counted with a light microscope (Olympus BX51) using a magnication of 100-fold (an area of 0.59 mm2). Salt glands were counted in 10 elds selected randomly. The density of salt glands was calculated according to the average of these 10 elds. The total number of salt glands (the number was represented with N) on adaxial and abaxial surfaces of

Materials and methods Plant growth and treatment conditions Limonium bicolor seeds were collected from native soil salinealkaline land in Yellow River Delta. After being sterilized with 0.1 % HgCl2 for 10 min and washed with tap water, plump seeds were selected and sowed in plastic pots lled with ne sand. They were irrigated with tap water. After germination they were irrigated with half-strength Hoagland solution (pH 5.7) in controlled growth chambers (photoperiod: 10 h; light intensity: 600 lmol m-2 s-1; relative humidity of 5060 %; day temperature: 20 2 C; night temperature: 15 2 C). When the third leaves came out, uniform seedlings were selected and transferred to plastic pots (5 plants in a pot). They were divided into two groups (2 pots in a group) and irrigated with full-strength Hoagland solution and

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L. sinense leaves was calculated according to the leaf area and the density of salt glands. The total Na? secreted by salt glands (the total Na? was represented with M) on adaxial and abaxial surfaces of L. sinense leaves for 3 days was calculated according to Na? concentrations measured with ame photometer. Na? secretion rate per salt gland (the rate was represented with V) was calculated according to following formula, V (nmol gland-1 day-1) = M/3N. CoroNa Green AM loading Staining to reveal Na? content was performed as described (Meier et al. 2006; Kanai et al. 2007). Abaxial peels were soaked in 500 lM vanadate, 1 mM bumetanide, and 500 lM amiloride for 45 min and then they were thoroughly washed with distilled water to wash out the extracellular inhibitors and Na?. In order to visualize intracellular free Na? localization, the intracellular Na? -specic uorescent indicator CoroNa Green AM (Invitrogen Corp, Carlsbad, CA, USA) was used. The peels were incubated in 20 mM MES-KOH (pH 6.5) containing 0.5 M mannitol and 2 lM CoroNa Green AM at 37 C for 30 min. In order to easily load the CoroNa Green AM into the salt glands and to efciently contact of the inhibitors with salt gland cells, peels in the chambers were subjected to vacuum to remove air bubbles adhering to the surface of the peels for 5 min. The incubated peels were observed with a confocal laser scanning microscope (TCS SPE; Leica, Germany). Statistical analysis Statistical analyses were carried out with one-way ANOVA followed by Duncans test at P \ 0.05 (SPSS 10.0). gland on adaxial surface of the 4th leaf was signicantly higher than that of the 3rd or 5th leaf but there was no signicant difference in Na? secretion rate per gland on abaxial surface among the 3rd, 4th, and 5th leaves. SNP signicantly expanded the leaf area SNP signicantly enhanced Na? secretion per leaf When plants were treated with NaCl, Na secretion rate per leaf was signicantly higher than that of control (Fig. 1a). Adding 0.05 mM SNP to NaCl solution resulted in a signicant increase in Na? secretion rate per leaf. At 200 mM NaCl, Na? secretion rate of adaxial surface was signicantly higher than that of abaxial surface. Na? secretion rate of the 4th or 5th leaf was signicantly higher than that of the 3rd leaf. There was no signicant difference in Na? secretion rate per gland when 0.05 mM SNP was added to NaCl solution (Fig. 1b). In 200 mM NaCl, Na? secretion rate per gland of adaxial surface was signicantly higher than that of abaxial surface, whether or not plants were treated with SNP; Na? secretion rate per
?

Fig. 1 a Effect of SNP on Na? secretion rate per leaf on adaxial and abaxial surface of L. bicolor leaves under 200 mM NaCl treatment. b Effect of SNP on salt-secretion rate per salt gland on adaxial and abaxial surfaces of L. bicolor leaves under 200 mM NaCl treatment. Means in the same treatment with different letters are signicantly different at P \ 0.05. Vertical bars represent standard deviations (n = 5)

Results

For the 3rd and 4th leaves, leaf areas of L. bicolor treated with 200 mM NaCl were obviously larger than those treated with Hoagland solution only (Fig. 2). In 200 mM NaCl, leaf areas of L. bicolor treated with SNP were signicantly greater than those treated without SNP. There was no signicant difference in the 5th leaf areas of L. bicolor among plants treated with Hoagland solution, 200 mM NaCl solution, and 200 mM NaCl solution containing 0.05 mM SNP. SNP increased the number of the glands per leaf There was no signicant difference in the densities of salt glands between plants treated with NaCl solution and NaCl

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Number of salt glands on adaxial and abaxial surfaces of the leaves treated with 200 mM NaCl was signicantly higher than that in Hoagland solution (P \ 0.05) (Fig. 3b). Number of salt glands both on adaxial and abaxial surfaces of the leaves treated with 200 mM NaCl solution containing SNP was signicantly higher than that treated with 200 mM NaCl solution. In 200 mM NaCl, number of salt glands on adaxial and abaxial surfaces of leaf 4 and leaf 5 was signicantly higher than that of leaf 3. SNP decreased Na? concentration in the leaves
Fig. 2 Effect of SNP on the 3rd, 4th and 5th leaf areas of L. bicolor under 200 mM NaCl treatment. Means in the same leaf identied by different letters are signicantly different at P \ 0.05. Vertical bars represent standard deviations (n = 5)

Na? concentration in the leaves treated with 200 mM NaCl was signicantly higher than that in Hoagland solution (P \ 0.05); Na? concentration in the leaves treated with 200 mM NaCl solution containing SNP was signicantly lower than that treated with 200 mM NaCl solution. In 200 mM NaCl, Na? concentration in leaf 3 was signicantly higher than that in leaf 4 and leaf 5 (Fig. 4). SNP signicantly increased the accumulation of Na? in the salt glands Arrows in transmitted light images (Fig. 5b, d, f) indicate the salt glands. In the leaf peel of L. bicolor of the controls, uorescence was hardly detectable (Fig. 5a). However, in that of L. bicolor exposed to NaCl for 20 days, uorescence was visualized indicating that free Na? was localized in salt glands (Fig. 5c). Treated with 200 mM NaCl solution containing SNP, stronger uorescence in salt glands was observed (Fig. 5e) indicating that more free Na? accumulated in salt glands.

Fig. 3 a Effect of SNP on densities of salt glands on adaxial and abaxial surfaces of L. bicolor leaves under 200 mM NaCl treatment. b Effect of SNP on total number of salt glands on adaxial and abaxial surfaces of L. bicolor leaves under 200 mM NaCl treatment. Means in the same treatment identied by different letters are signicantly different at P \ 0.05. Vertical bars represent standard deviations, n = 50 for a and n = 5 for b

solution containing 0.05 mM SNP (Fig. 3a). However, the densities of salt glands of plants in those conditions were signicantly higher than those of plants treated with Hoagland solution. There was no signicant difference in the densities of salt glands among the 3rd, 4th, and 5th leaves in the same treatment.

Fig. 4 Effect of SNP on concentration of Na? in L. bicolor leaves under 200 mM NaCl treatment. Means in the same leaf with different letters are signicantly different at P \ 0.05. Vertical bars represent standard deviations (n = 5)

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Acta Physiol Plant (2013) 35:741747 Fig. 5 Visualization of intracellular Na? present in the salt glands in the leaves of L. bicolor stained with CoroNa Green AM. a, c, e Fluorescent light images; b, d, f transmitted light images. a, b Exposure to Hoagland solution; c, d exposure to 200 mM NaCl; e, f exposure to 200 mM NaCl containing 0.05 mM SNP. Bars 75 lm for all panels. Arrows indicate the salt glands

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Discussion Plants exposed to 200 mM NaCl, Na? concentration in leaf 3 was signicantly higher than that in leaf 4 and leaf 5. However, Na? secretion rate of the 4th or 5th leaf was signicantly higher than that of the 3rd leaf. It suggested that the ability of young leaves to secret Na? was greater than that of older leaves. In all NaCl treatments, the ability

of salt glands on adaxial surfaces of leaves to secret Na? was greater than that of abaxial surfaces of leaves. However, there was no signicant difference in gland numbers between adaxial surfaces and abaxial surfaces. It suggested that the salt-secretion rate of individual glands on adaxial surfaces is greater than that on abaxial surfaces. NO, as a signaling molecule, is involved in various processes, such as disease resistance, stomatal movement,

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response to abiotic stresses, as well as various developa-Mata and mental process (Delledonne et al. 1998; Garc Lamattina 2001; Lamotte et al. 2005; Ling et al. 2005). However, information about whether or how NO is involved in salt stress response is very limited. This paper rst reported that SNP, an NO donor enhanced the secretion and number of salt glands in recretohalophyte L. bicolor leaves signicantly. Since SNP treatment increased leaf areas, NO could lead to bigger cell sizes and thus lower frequencies of specialized cell types. However, the total number of salt glands on adaxial surfaces under condition of 200 mM NaCl containing 0.05 mM SNP treatment increased signicantly compared with that under 200 mM NaCl treatment. Obviously, SNP caused more dermatogen cells to develop into salt glands. Ramadan and Flowers (2004) found BA increased salt secretion from maize plants due to its inuence on the number of microhairs, but not due to its effect on the efciency of the secretion process per microhair. Na? secretion rate per leaf under 200 mM NaCl containing 0.05 mM SNP was signicantly higher than that under 200 mM NaCl without SNP. However, there was no signicant difference in salt-secretion rate of individual salt glands between 200 mM NaCl containing 0.05 mM SNP treatment and 200 mM NaCl treatment. These results showed that NO increased secretion per leaf due to its inuence on the number of salt glands, rather than its effect on the secretion rate per gland. Although there was no signicant difference in salt-secretion rate of individual glands, Na? concentration in the leaves treated with 200 mM NaCl solution containing SNP was signicantly lower than that treated with 200 mM NaCl solution. Confocal uorescence microscopy revealed that in 200 mM NaCl treatment, Na? concentration in salt glands was signicantly higher than that in Hoagland solution (Fig. 5a, c). Treatment with 200 mM NaCl solution containing SNP caused a remarkable increase in Na? concentration in salt glands (Fig. 5e). These results suggest the efciency (it means efciency of secretion from cells to gland, and does not equal with efciency of secretion per gland to environment) of the secretion process per gland was enhanced by adding SNP to NaCl. The enhancement of the efciency of secretion process per gland was caused by accumulation of Na? in the salt glands, but there was no enhancement in the process of salt secretion from salt glands to outside environment. Zhang et al. (2006) found that NO treatment stimulated vacuolar H?-ATPase and H?-PPase activities, resulting in increased H?-translocation and Na?/H? exchange. Generally, salt glands in recretohalophytes lack a large central vacuole but contain many microvacuoles, these microvacuoles may have a relationship with active ttge 1967; metabolism of salt glands (Ziegler and Lu Shimony and Fahn 1968; Vassilyev and Stepanova 1990).

Li (2008) found there were Na?/H? exchangers locating on these small vacuoles in glands of Limonium sinense. So NO may enhance the salt secretion of individual glands by increasing the activities of H?-ATPase and H?-PPase in these microvacuoles. In conclusion, NO acts as a signal molecule in the saltsecretion process by inducing more dermatogen cells to develop into salt glands and by enhancing the efciency of the secretion process per gland.

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