Abstract

Aims:

Effects of salt stress on the physiology of Salvinia auriculata were investigated.

Method

Plants were supplemented with 0, 50, 100 and 150 mmol L^-1 NaCl and incubated for 5 days. NO content was evaluated after 2 hours and 5 days. Photosynthetic pigments, proline and nutrients were analyzed after 5 days.

Major Results

Higher chlorophyll a content was observed in plants treated with 50 mmol L^-1, decreasing in higher NaCl concentrations, while chorophyll b content decreased with increasing NaCl concentrations. Exposure to 50 mmol L^-1 NaCl increased biomass, while higher concentrations caused loss of biomass. Ca, K and Mg decreased with increasing NaCl concentrations, and the Na/K ratio was significantly increased at 150 mmol L^-1 NaCl. Proline increased significantly at 150 mmol L^-1. Extracellular NO content increased after 2 hours, with significantly higher NO concentrations in roots observed at 50 mmol L^-1. Decreases in NO content were observed after 5 days.

Conclusions

The results indicate that moderate salinity induces NO production earlier during incubation, probably associated to signaling for the production of compounds that assist in stress tolerance. At higher concentrations, this tolerance is reduced. This allows for further understanding of the physiological and biochemical mechanisms associated with the adaptation of this macrophyte to saline conditions, which, in turn, affect this species ecology and distribution in coastal areas.

Keywords:
Salvinia auriculata; salinity stress; nitric oxide; clorophyll; NaCl; macrophyte

Resumo

Objetivos:

Efeitos do estresse salino sobre a fisiologia de Salvinia auriculata foram investigados.

Metodologia

As plantas foram expostas a 0, 50, 100 e 150 mmol de NaCl L^-1 e incubadas durante 5 dias. O conteúdo de NO foi avaliado após 2 horas e 5 dias. Pigmentos fotossintéticos, prolina e nutrientes foram analisados após 5 dias.

Resultados Principais

Observou-se maior teor de clorofila a em plantas tratadas com 50 mmol L^-1, diminuindo em concentrações mais altas, enquanto o conteúdo de clorofila b diminuiu com o aumento das concentrações de NaCl. A exposição a 50 mmol L^-1 de NaCl aumento a biomassa, enquanto concentrações mais elevadas causaram perda de biomassa. Ca, K e Mg diminuíram com o aumento das concentrações de NaCl, e a razão Na/K foi significativamente aumentada em 150 mmol L^-1 NaCl. A prolina aumentou significativamente a 150 mmol L^-1. O conteúdo extracelular de NO aumentou após 2 horas, e diminuiu após 5 dias. Após 2 horas, concentrações significativamente maiores nas raízes foram observadas a 50 mmol L^-1, enquanto após 5 dias diminuições foram observadas.

Conclusões

Os resultados indicam que a salinidade moderada induz a produção de NO durante a incubação, possivelmente associada à sinalização para a produção de compostos que auxiliem na tolerância à salinidade. Em concentrações superiores esta tolerância é reduzida. Com isso, é possível compreender melhor os mecanismos fisiológicos e bioquímicos associados a essa adaptação em macrófitas sob condições salinas, que afetam sua ecologia e distribuição em áreas costeiras

Palavras-chave:
Salvinia auriculata; estresse salino; NO; clorofila; NaCl; macrófita

1. Introduction

Salinity is the major environmental factor that limits plant growth and primary productivity in aquatic ecosystems (Moradi et al., 2013MORADI, S., YOSEFI, R. and GHADERI, O. Bioconcentration factor and relative growth rate of Azolla (Azolla caroliniana) in arsenic and salinity stress conditions. International Journal of Agronomy and Plant Production, 2013, 4(10), 2617-2623.). In coastal water bodies, salinity can vary seasonally and can be influenced by changes in water levels, precipitation, evaporation (Schallenberg et al., 2003SCHALLENBERG, M., HALL, C.J. and BURNS, C.W. Consequences of climate-induced salinity increases on zooplankton abundance and diversity in coastal lakes. Marine Ecology Progress Series, 2003, 251, 181-189. http://dx.doi.org/10.3354/meps251181.
http://dx.doi.org/10.3354/meps251181... ), hydrological alterations (Howard & Mendelssohn, 1999HOWARD, R.J. and MENDELSSOHN, I.A. Salinity as a constraint on growth of oligoaline marsh macrophytes.I. Species variation in stress tolerance. American Journal of Botany, 1999, 86(6), 785-794. PMid:10371721. http://dx.doi.org/10.2307/2656700.
http://dx.doi.org/10.2307/2656700... ) and anthropogenic activities (Roache et al., 2006ROACHE, M.C., BAILEY, P.C. and BOON, P.I. Effects of salinity on the decay of the freshwater macrophyte, Triglochin procerum. Aquatic Botany, 2006, 84(1), 45-52. http://dx.doi.org/10.1016/j.aquabot.2005.07.014.
http://dx.doi.org/10.1016/j.aquabot.2005... ).

Exposure to salinity may cause several morphological, physiological and biochemical changes in plants, due to excess ions and water deficit (Greenway & Munns, 1980GREENWAY, H. and MUNNS, R. Mechanisms of salt tolerance in nonhalophytes. Annual Review of Plant Physiology, 1980, 31(1), 149-190. http://dx.doi.org/10.1146/annurev.pp.31.060180.001053.
http://dx.doi.org/10.1146/annurev.pp.31.... ; Maskri et al., 2010MASKRI, A., AL-KHARUSI, L., AL-MIQBALI, H. and KHAN, M.M. Effects of salinity stress on growth of lettuce (Lactuca sativa) under closed-recycle nutrient film technique. International Journal of AAgriculture and Biology, 2010, 12, 377-380.). The most common effects in plants are toxicity, diminished CO₂ assimilation and enhanced generation of reactive oxygen species (Chawla et al., 2013CHAWLA, S., JAIN, S. and JAIN, V. Salinity induced oxidative stress and antioxidant system in salt-tolerant and salt-sensitive cultivars of rice (Oryza sativa L.). Journal of Plant Biochemistry and Biotechnology, 2013, 22(1), 27-34. http://dx.doi.org/10.1007/s13562-012-0107-4.
http://dx.doi.org/10.1007/s13562-012-010... ). Changes in fundamental processes have also been observed, such as growth, photosynthesis, protein synthesis and lipid metabolism (Parida & Das, 2004PARIDA, A.K. and DAS, A.B. Effects of NaCl stress on nitrogen and phosphorus metabolism in a true mangrove Bruguiera paeviflora grown under hydroponic culture. Journal of Plant Physiology, 2004, 161(8), 921-928. PMid:15384403. http://dx.doi.org/10.1016/j.jplph.2003.11.006.
http://dx.doi.org/10.1016/j.jplph.2003.1... ). High salinity concentrations in plants also generate changes in plant productivity (Doganlar et al., 2010DOGANLAR, Z.B., DEMIR, K., BASAK, H. and GUL, I.H. Effects of salt stress on pigment and total soluble protein contents of three different tomato cultivars. African Journal of Agricultural Research, 2010, 5(15), 2056-2065.; Hasegawa et al., 2000HASEGAWA, P.M., BRESSAN, R.A., ZHU, J.K. and BOHNERT, H.J. Plant cellular and molecular responses to hight salinity. Annual Review of Plant Physiology, 2000, 51(1), 463-499. PMid:15012199. http://dx.doi.org/10.1146/annurev.arplant.51.1.463.
http://dx.doi.org/10.1146/annurev.arplan... ), nutrient imbalances (Ashraf, 2009ASHRAF, M. Biotechnological approach of improving plant salt tolerance using antioxidants as markers. Biotechnology Advances, 2009, 27(1), 84-93. PMid:18950697. http://dx.doi.org/10.1016/j.biotechadv.2008.09.003.
http://dx.doi.org/10.1016/j.biotechadv.2... ), accumulation of osmoprotective compounds, such as proline (Bohnert et al., 1995BOHNERT, H.J., NELSON, D.E. and JENSEN, R.G. Adaptation to environmental stress. The Plant Cell, 1995, 7(7), 1099-1111. PMid:12242400. http://dx.doi.org/10.1105/tpc.7.7.1099.
http://dx.doi.org/10.1105/tpc.7.7.1099... ), and changes in nitric oxide (NO) content (Zhang & Blumwald, 2001ZHANG, H.X. and BLUMWALD, E. Transgenic salt tolerant tomato plant accumulate salt in the foliage not in the fruit. Nature Biotechnology, 2001, 19(8), 765-768. PMid:11479571. http://dx.doi.org/10.1038/90824.
http://dx.doi.org/10.1038/90824... ).

Na⁺ acts on the activation of a wide range of enzymes in plants, is involved in membrane osmosis, and can also replace K⁺ in some osmotic and metabolic functions. Cl^- plays an important role in photosynthesis, enzyme activation, osmotic regulation and cell division (Ashari-Esna & Gholami, 2010ASHARI-ESNA, M. and GHOLAMI, M. The effect of increased chloride (Cl-) content in nutrient solution on yield and quality of strawberry (Fragaria ananassa Duch.) fruits. Journal of Fruit and Ornamental Plant Research., 2010, 18(1), 37-44.). Excessive Na⁺ and Cl^- concentrations affect the absorption of many essential nutrients such as K, Ca Mg and N (Abdallah et al., 2016ABDALLAH, S.B., AUNG, B., AMYOT, L., LALIN, I., LACHÂAL, M. and KARRAY-BOURAOUI, N. Salt stress (NaCl) affects plant growth and branch pathways of carotenoid and flavonoid biosyntheses in Solanum nigrum. Acta Physiologiae Plantarum, 2016, 38(3), 1-13.; Iqbal et al., 2015IQBAL, N., UMAR, S. and KHAN, N.A. Nitrogen availability regulates proline and ethylene production and alleviates salinity stress in mustard (Brassica juncea). Journal of Plant Physiology, 2015, 178, 84-91. PMid:25800225. http://dx.doi.org/10.1016/j.jplph.2015.02.006.
http://dx.doi.org/10.1016/j.jplph.2015.0... ). This occurs through competitive interactions affecting the ionic selectivity of cell membranes (Stoeva & Kaymakanova, 2008STOEVA, N. and KAYMAKANOVA, M. Effect of salt stress on the growth and photosynthesis rate of bean plants (Phaseolus vulgaris L.). Journal of Central European Agriculture, 2008, 9(3), 385-392.) and photosynthetic activity (Parida et al., 2002PARIDA, A., DAS, A.B. and DAS, P. NaCl stress causes changes in photosynthetic pigments, proteins and other metabolic components in the leaves of a true Mangrove, Bruguiera parviflora, in hydroponics cultures. Journal of Plant Biology, 2002, 45(1), 28-36. http://dx.doi.org/10.1007/BF03030429.
http://dx.doi.org/10.1007/BF03030429... ), reducing stomata opening and leading to decreases in intracellular CO₂ (Munns & Tester, 2008MUNNS, R. and TESTER, M. Mechanisms of salinity tolerance. Annual Review of Plant Physiology, 2008, 59, 651-681. PMid:18444910.).

Biomarkers are used to indicate an exposure to or the effect of xenobiotics present in the environment and in organisms. Biomarkers of exposure provide functional measures of exposure that are characterized at a sub-organism level (Brain & Cedergreen, 2009BRAIN, R.A. and CEDERGREEN, N. Biomarkers in aquatic plants: selection and utility. Reviews of Environmental Contamination and Toxicology, 2009, 198, 49-109. PMid:19253039. http://dx.doi.org/10.1007/978-0-387-09647-6_2.
http://dx.doi.org/10.1007/978-0-387-0964... ). The use of species-specific biomarkers have been utilized in a diverse array of studies aimed at assessing organismal, population, or ecosystem health (Ashraf, 2009ASHRAF, M. Biotechnological approach of improving plant salt tolerance using antioxidants as markers. Biotechnology Advances, 2009, 27(1), 84-93. PMid:18950697. http://dx.doi.org/10.1016/j.biotechadv.2008.09.003.
http://dx.doi.org/10.1016/j.biotechadv.2... ).

Two main plant metabolites used as biomarkers to salinity stress are proline and nitric oxide (NO). Proline is an osmotic regulator, enzyme denaturation protector and a macromolecule or molecular assembly stabilizer, as well as a nitrogen and carbon source reservoir and/or a hydroxyl radical scavenger in aquatic macrophytes (Bagdi & Shaw, 2013BAGDI, D.L. and SHAW, B.P. Analysis of proline metabolic enzymes in Oryzasativa under NaCI stress. Journal of Environmental Biology, 2013, 34(4), 667-681. PMid:24640240.). NO, on the other hand, is a signaling molecule produced as a physiological response in plants under stress conditions (Kausar & Shahbaz, 2013KAUSAR, F. and SHAHBAZ, M. Interactive effect of foliar application of nitric oxide (NO) and salinity on wheat (Triticum aestivum L.). Pakistan Journal of Botany, 2013, 45, 67-73.; Lamattina et al., 2003LAMATTINA, L., GARCÍA-MATA, C., GRAZIANO, M. and PAGNUSSAT, G. Nitric oxide: the versatility of an extensive signal molecule. Annual Review of Plant Physiology, 2003, 54, 109-136. PMid:14502987.). It is involved in several physiological processes that include germination, root growth, stomatal closing, and adaptive response to biotic and abiotic stresses (Delledonne, 2005DELLEDONNE, M. NO news is good news for plants. Current Opinion in Plant Biology, 2005, 8(4), 390-396. PMid:15922651. http://dx.doi.org/10.1016/j.pbi.2005.05.002.
http://dx.doi.org/10.1016/j.pbi.2005.05.... ; Delledonne et al., 1998DELLEDONNE, M., XIA, Y., DIXON, R.A. and LAMB, C. Nitric oxide functíons as a signal in plant disease resistance. Nature, 1998, 394(6693), 585-588. PMid:9707120. http://dx.doi.org/10.1038/29087.
http://dx.doi.org/10.1038/29087... ; Neill et al., 2008NEILL, S., BARROS, R., BRIGHT, J., DESIKAN, R., HANCOCK, J., HARRISON, J., MORRIS, P., RIBEIRO, D. and WILSON, I. Nitric oxide, stomatal closure, and abiotic stress. Journal of Experimental Botany, 2008, 59(2), 165-176. PMid:18332225. http://dx.doi.org/10.1093/jxb/erm293.
http://dx.doi.org/10.1093/jxb/erm293... ). NO acts as an antioxidant during different stress situations, increasing under NaCl stress. This indicates that nitrosative stress could participate in damage mechanisms produced by abiotic toxic conditions (López-Carrión et al., 2008LÓPEZ-CARRIÓN, A.I., CASTELLANO, R., ROSALES, M.A., RUIZ, J.M. and ROMERO, L. Role of nitric oxide under saline stress: implicatíons on proline metabolism. Biologia Plantarum, 2008, 52(3), 587-591. http://dx.doi.org/10.1007/s10535-008-0117-1.
http://dx.doi.org/10.1007/s10535-008-011... ), although not many studies are available in this regard (Siddiqui et al., 2011SIDDIQUI, M.H., AL-WHAIBI, M.H. and BASALAH, M.O. Role of nitric oxide in tolerance of plants to abiotic stress. Protoplasma, 2011, 248(3), 447-455. PMid:20827494. http://dx.doi.org/10.1007/s00709-010-0206-9.
http://dx.doi.org/10.1007/s00709-010-020... ).

Salvinia auriculata is a freshwater free-floating aquatic macrophyte that, under favorable conditions (e.g. high P and N concentrations), colonizes large water areas in short periods of time (Peixoto et al., 2005PEIXOTO, P.H.P., PIMENTA, D.S. and ANTUNES, F. Efeitos do flúor em folhas de plantas aquáticas de Salvinia auriculata. Pesca e Agropecuária Brasileira, 2005, 40(8), 727-734. http://dx.doi.org/10.1590/S0100-204X2005000800001.
http://dx.doi.org/10.1590/S0100-204X2005... ). This species is a pollution bioindicator in aquatic ecosystems, since it shows great ability to remove and accumulate various organic and inorganic elements and compounds present in water and sediment (Henry-Silva & Camargo, 2002HENRY-SILVA, G.G. and CAMARGO, A.F.M. Valor nutritivo de macrófitas aquáticas flutuantes (Eichhornia crassipes, Pistia stratiotes e Salvinia molesta) utilizadas no tratamento de efluentes de aqüicultura. Acta Scientiarum, 2002, 24(2), 519-526.; Soares et al., 2008SOARES, D.C.F., OLIVEIRA, E.F., SILVA, G.D., DUARTE, L.P., POTT, V.J. and VIEIRA FILHO, S.A. Salvinia auriculata: Aquatic bioindicator studied by instrumental neutron activation analysis (INAA). Applied Radiation and Isotopes, 2008, 66(5), 561-564. PMid:18191404. http://dx.doi.org/10.1016/j.apradiso.2007.11.012.
http://dx.doi.org/10.1016/j.apradiso.200... ).

In order to improve fishing activities in the northern region of the State of Rio de Janeiro, Brazil, the implementation of artificial sandbars separating coastal lagoons from the sea has become a common practice (Suzuki et al., 2002SUZUKI, M.S., FIGUEIREDO, R.O., CASTRO, S.C., SILVA, C.F., PEREIRA, E.A., SILVA, J.A. and ARAGON, G.T. Sand bar opening in a coastal lagoon (Iquipari) in the northern region of Rio de Janeiro state: hydrological and hydrochemical changes. Brazilian Journal of Biology = Revista Brasileira de Biologia, 2002, 62(1), 51-62. PMid:12185923. http://dx.doi.org/10.1590/S1519-69842002000100007.
http://dx.doi.org/10.1590/S1519-69842002... ). However, these sandbar openings can cause radical changes in the physicochemical properties and physical and chemical conditions of these areas, including drastic reductions in water volume and profound changes in the biota, including aquatic macrophytes, such as S. auriculata, due to excess brackish water and increased salt water influx (Suzuki et al., 2002SUZUKI, M.S., FIGUEIREDO, R.O., CASTRO, S.C., SILVA, C.F., PEREIRA, E.A., SILVA, J.A. and ARAGON, G.T. Sand bar opening in a coastal lagoon (Iquipari) in the northern region of Rio de Janeiro state: hydrological and hydrochemical changes. Brazilian Journal of Biology = Revista Brasileira de Biologia, 2002, 62(1), 51-62. PMid:12185923. http://dx.doi.org/10.1590/S1519-69842002000100007.
http://dx.doi.org/10.1590/S1519-69842002... ). In addition, intrusion processes of saline or brackish water in these coastal ecosystems may cause an indirect salinization process of other water bodies not directly connected to the sea, by groundwater (Gomes et al., 2011GOMES, M.A.D.C., SUZUKI, M.S., CUNHA, M. and TULLII, C.F. Effect of salt stress on nutrient concentration, photosynthetic pigments, proline and foliar morphology of Salvinia auriculata Aubl. Acta Limnologica Brasiliensia, 2011, 23(2), 164-176. http://dx.doi.org/10.1590/S2179-975X2011000200007.
http://dx.doi.org/10.1590/S2179-975X2011... ).

Thus, the aim of this study is to verify salinity effects on S. auriculata by evaluating chlorophyll a and b, carotenoids, proline and NO content after exposure to different salinity concentrations.

2. Material and Methods

2.1. Plant material

Approximately 20 kg fresh weight (FW) of S. auriculata were sampled from the Jacú Lagoon, a freshwater lagoon in the municipality of Campos dos Goytacazes, Rio de Janeiro, Brazil. At the laboratory, the specimens were washed several times to eliminate solid residues and maintained for five days at 25 °C and 12 hour-photoperiods (100 µmol.m^-2.s^-1) in plastic boxes. Plants were maintained in a nutritive solution containing several trace-nutrients such as K, Mn and Mg, according to Hoagland & Arnon (1950)HOAGLAND, D.R. and ARNON, D.I. The water-culture method for growing plants without soil. California Agricultural Experiment Station Circular, 1950, 347, 1-32. and Smart & Barko (1985)SMART, R.M. and BARKO, J.W. Laboratory culture of submersed freshwater macrophytes on natural sediments. Aquatic Botany, 1985, 21(3), 251-263. http://dx.doi.org/10.1016/0304-3770(85)90053-1.
http://dx.doi.org/10.1016/0304-3770(85)9... .

2.2. NaCl assays

Approximately 50 g FW of S. auriculata were transferred to a box containing 3 L of the nutritive solution and supplemented with different NaCl concentrations (0, 50, 100 and 150 mmol L^-1), in triplicate. These concentrations were chosen after pilot studies in our lab demonstrated that 50 mmol L^-1 caused no stress on the samples, while data on concentrations above of 150 mmol L^-1 have already been published (Gomes et al., 2011GOMES, M.A.D.C., SUZUKI, M.S., CUNHA, M. and TULLII, C.F. Effect of salt stress on nutrient concentration, photosynthetic pigments, proline and foliar morphology of Salvinia auriculata Aubl. Acta Limnologica Brasiliensia, 2011, 23(2), 164-176. http://dx.doi.org/10.1590/S2179-975X2011000200007.
http://dx.doi.org/10.1590/S2179-975X2011... ). Samples were incubated for five days in controlled conditions, in 12- hour photoperiods (100 µmol.m^-2.s^-1) at 20 °C (dark period) and at 25 °C (light period). NO content was evaluated after 2 hours (since NO is rapidly formed during signaling) and 5 days. The remaining parameters (photosynthetic pigments, proline and nutrients) were analyzed after 5 days. This limit was set due to the death of most plants when exposed to the highest concentration.

2.3. Determination of photosynthetic pigments

Photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) were analyzed, in triplicate, according to Wellburn (1994)WELLBURN, A.R. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. Journal of Plant Physiology, 1994, 144(3), 307-313. http://dx.doi.org/10.1016/S0176-1617(11)81192-2.
http://dx.doi.org/10.1016/S0176-1617(11)... . S. auriculata leaves (0.2 g FW each) were cut into strips and transferred to polypropylene tubes containing dimethylsulfoxide (DMSO). After digestion, 1 mL of each sample was separated and chlorophyll a, chlorophyll b and carotenoids were measured at 480, 649 e 665 nm, respectively, on a UV-Vis spectrophotometer (model UV-160A, Shimadzu, São Paulo, Brazil). All procedures were carried out in a low-light environment. Values were expressed in nmol.cm⁻² of dry weight (DW).

2.4. Proline quantification

Proline content was determined according to Bates et al. (1973)BATES, L.S., WALDREN, R.P. and TEARE, I.D. Rapid determination of free proline for water-stress studies. Plant and Soil, 1973, 39(1), 205-207. http://dx.doi.org/10.1007/BF00018060.
http://dx.doi.org/10.1007/BF00018060... . Leaf and root samples (300 mg FW each, in triplicate), were homogenized in 6 mL of sulfosalicilic acid 3%, at 4 °C. Samples were then transferred to polypropylene tubes, incubated and centrifuged at 5,000 rpm for 20 minutes. Subsequently, 1 mL of the supernatant of each sample was incubated with 1 mL of an acid ninhydrin solution containing 2.5% ninhydrin, 60% phosphoric acid (v/v) and 1 mL of glacial acetic acid (100%) in a boiling water bath (CT 246, Cientec, Belo Horizonte, Brazil) during 1 hour. After incubation, the samples were rapidly cooled on ice and absorbances were determined at 518 nm on a UV-Vis spectrophotometer (UV-160A, Shimadzu, Kyoto, Japan).

2.5. NO determinations

The NO determinations were carried out in the samples incubated for two hours and 5 days using fluorescence microscopy and fluorometry, according to Tun et al. (2006)TUN, N.N., SANTA-CATARINA, C., BEGUM, T., SILVEIRA, V., HANDRO, W., FLOH, E.I. and SCHERER, G.F. Polyamines induce rapid biosynthesis of nitric oxide (NO) in Arabidopsis thaliana seedlings. Plant & Cell Physiology, 2006, 47(3), 346-354. PMid:16415068. http://dx.doi.org/10.1093/pcp/pci252.
http://dx.doi.org/10.1093/pcp/pci252... , with minor modifications. For quantification of endogenous NO by microscopy fluorescence, root segments (10 segments/sample, in triplicate) were incubated with 2 mL of a nutritive solution containing 0, 50, 100 and 150 mmol L^-1 NaCl, during two hours in the light (100 µmol.m^-2.s^-1) at 25 °C and during 5 days in 12-hour photoperiods at 20 °C in the dark and at 25°C in the light. After the NaCl treatments, the samples were incubated during 1 hour with 15 µmol L^-1 4,5-diaminofluorescein diacetate (DAF-FM-DA, Calbiochem, Darmstadt, Germany, a cell-permeable fluorescent dye. Samples were then washed twice with the respective NaCl concentrations and plates were prepared. Samples were visualized using an Axioplan-Zeiss fluorescence microscope (Carls Zeiss, Jena, Germany) with the filter set for DAF-FM-DA excitation at 515 nm and emission at 525 nm. Images were acquired using a digital AxioCam MRC5 camera (Carl Zeiss, Jena, Germany) and the fluorescence intensity of the root tips was determined using the AxioVisionLE software version 4.8 package (Carls Zeiss) as number of pixels per area. All experiments were conducted at least twice.

For quantification of NO release by fluorometry, plants (100 mg FW each sample, in triplicate) were incubated in 3 mL of nutritive solution containing 0, 50, 100 and 150 mmol L^-1 NaCl, during two hours in the light (100 µmol.m^-2.s^-1) at 25 °C and during 5 days in 12-hour photoperiods at 20 °C in the dark and at 25 °C in the light. Subsequently, the samples were incubated for 1 hour with 10 µmol L^-1 4,5-diaminofluorescein (DAF-FM, Calbiochem), a cell-impermeable fluorescent dye to analyze released NO. After incubation, 2 mL of the supernatant were separated and analyzed on a spectrofluorometer (Shimadzu RF-5301, Kyoto, Japan) using a DAF-FM filter set at excitation at 515 nm and emission at 525 nm. The data was presented as relative fluorescence.

2.6. Nutrient determinations

Nutrient (Ca⁺², K⁺, Na⁺, Mg⁺², Cl^-, P (phosphate) and N (nitrate)) determinations were conducted according to Malavolta et al. (1997)MALAVOLTA, E., VITTI, G.C. and OLIVEIRA, S.A. Avaliação do estado nutricional das plantas: princípios e aplicações. 2nd ed. Piracicaba: Associação Brasileira para Pesquisa da Potassa e do Fosfato, 1997.. Samples were collected after the five days of treatment with NaCl and dried at 60 °C during four days. The samples were then grounded to a fine powder and samples of 500 mg DW, in triplicate, were used for the nutrient determinations. Ca⁺², K⁺, Na⁺ and Mg⁺² were analyzed by atomic absorption spectrophotometry (AAS4, Carl Zeiss). Total P content was obtained by complete digestion of the samples (500 mg FW, in triplicate) with nitric acid (100%) and perchloric acid (100%) (1:1, v/v) and subsequent colorimetric quantification using the molybdate method (Malavolta et al., 1997MALAVOLTA, E., VITTI, G.C. and OLIVEIRA, S.A. Avaliação do estado nutricional das plantas: princípios e aplicações. 2nd ed. Piracicaba: Associação Brasileira para Pesquisa da Potassa e do Fosfato, 1997.). Cl^- was determined in aqueous extracts by titration with silver nitrate, also according to Malavolta et al. (1997)MALAVOLTA, E., VITTI, G.C. and OLIVEIRA, S.A. Avaliação do estado nutricional das plantas: princípios e aplicações. 2nd ed. Piracicaba: Associação Brasileira para Pesquisa da Potassa e do Fosfato, 1997.. N determinations (100 mg DW, in triplicate) were conducted using the methodology proposed by Nessler (Jackson, 1965JACKSON, M.L. Soil chemical analysis. New Jersey: Prentice Hall, 1965. 498 p.), by sulfuric digestion and subsequent absorbance readings at 480 nm on a UV-Vis spectrophotometer UV-160A, Shimadzu).

2.7. Statistical analyses

With the exception of the extracellular and intracellular NO results, the data were evaluated by a two-way ANOVA (p < 0.05) with a posteriori Bonferroni post-test. All other data were analyzed using the non-parametric Kruskal-Wallis test (p<0.05) and a retrospective Dunn's test to compare the results between treatments, since data followed a non-normal distribution. All data were analyzed using the GraphPad Prism 4 statistical software package.

3. Results

Figure 1 shows results after 5 days of incubation with different NaCl concentrations. Color changes in the plants were observed during the assays, especially at the higher NaCl concentration of 150 mmol L^-1.

Corroborating the color changes, the different NaCl treatments significantly affected photosynthetic pigment content in S. auriculata (Table 1).

Higher chlorophyll a content was observed in plants treated with 50 mmol L^-1, decreasing in plants incubated with higher concentrations (100 and 150 mmol L^-1). On the other hand, chorophyll b content decreased with increasing NaCl concentrations, and statistically significant differences between controls and plants incubated with 150 mmol L^-1 NaCl were observed (Table 1). The chlorophyll a to chlorophyll b ratio was higher in plants treated with 50 mmol L^-1 NaCl, with no statistical differences between plants incubated with 100 and 150 mmol L^-1 NaCl. Total chlorophyll content (chlorophyll a + chlorophyll b) was not statistically different (p<0.05) between treatments. Carotenoid content increased with increasing NaCl concentrations (Table 1), albeit non-significantly. Plant biomass also varied significantly between the NaCl treatments. Plants exposed to 50 mmol L^-1 showed significantly higher FW and DW when compared to the other NaCl concentrations. Loss of biomass occurred in plants submitted to 100 and 150 mmol L^-1 NaCl (Figure 2).

Nutrient content in S. auriculata was also affected by the NaCl treatments after 5 days (Table 2). Statistically significant differences between controls and 150 mmol L^-1 NaCl were observed for Ca⁺², K⁺, Na⁺, Mg⁺² and Cl^-, as well as for the Na/K ratio. The nutrients Ca⁺², K⁺ and Mg⁺² decreased with increasing NaCl concentrations, while N and P showed no significant differences among saline treatments. A significant increase in the Na/K ratio was observed between controls and plants incubated with 150 mmol L^-1 NaCl, expected due to the increase of Na⁺ concentrations in the incubation solutions. K⁺ content was the most affected when compared to the other analyzed nutrients, decreasing significantly with increases in NaCl concentrations.

The proline content in S. auriculata increased significantly in the 150 mmol L^-1 NaCl treatment compared to controls (Figure 3).

Extracellular NO content increased after 2-hour incubations with NaCl, with significant differences (p < 0.05) observed between controls and exposure to 150 mmol L^-1 NaCl (Figure 4A). On the other hand, plants showed decreases in NO releases with increasing NaCl concentrations after 5 days of incubation (Figure 4B), with significant differences observed between controls and plants exposed to 100 mmol L^-1 NaCl.

Intracellular NO results are displayed in Figure 5. After two hours of incubation, significantly higher NO concentrations in S. auriculata roots were observed in plants incubated with 50 mmol L^-1 NaCl (Figure 5c and Figure 6A) when compared to the other investigated concentrations (Figure 5). After 5 days, decreases in intracellular NO were observed in roots, with significant differences between the treatments (Figure 5 and Figure 6B). Plants incubated during two hours showed higher induction of NO synthesis (Figure 5 a-h and Figure 6A) when compared to incubation for 5 days (Figure 5i-p and Figure 6B), where lower NO content was observed. Control roots presented significantly lower (p<0.05) intracellular NO when compared to the 50 mmol L^-1 NaCl treatment (Figures 5a and 5b, 6A and 6B).

4. Discussion

Na⁺ and Cl^- are known to cause injuries to plant leaves (Kozlowski, 1997KOZLOWSKI, T.T. Responses of woody plants to flooding and salinity. Tree Physiology Monographs, 1997, 1, 1-17.). In the present study, total chlorophyll pigment content decreased with increasing NaCl concentrations, resulting in decreased photosynthetic rate (Table 1). These results corroborate other studies, that indicate that plants subjected to increased salinity show decreased photosynthetic pigments (Aghaleh et al., 2009AGHALEH, M., NIKNAM, V., EBRAHIMZADEH, H. and RAZAVI, K. Salt stress effects on growth, pigments, proteins and lipid peroxidation in Salicornia persica and S. europaea. Biologia Plantarum, 2009, 53(2), 243-248. http://dx.doi.org/10.1007/s10535-009-0046-7.
http://dx.doi.org/10.1007/s10535-009-004... ; Centritto et al., 2003CENTRITTO, M., LORETO, F. and CHARTZOULAKIS, K. The use of low [CO2] to estimate dissusional and non-diffusional limitatíons of photosynthetic capacity of salt- stressed olive saplings. Plant, Cell & Environment, 2003, 26(4), 585-594. http://dx.doi.org/10.1046/j.1365-3040.2003.00993.x.
http://dx.doi.org/10.1046/j.1365-3040.20... ; Chaves et al., 2009CHAVES, M.M., FLEXAS, J. and PINHEIRO, C. Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Annals of Botany, 2009, 103(4), 551-560. PMid:18662937. http://dx.doi.org/10.1093/aob/mcn125.
http://dx.doi.org/10.1093/aob/mcn125... ; Jampeetong & Brix, 2009JAMPEETONG, A. and BRIX, H. Effects of NaCl salinity on growth, morphology, photosynthesis and proline accumulation of Salvinia natans. Aquatic Botany, 2009, 91(3), 181-186. http://dx.doi.org/10.1016/j.aquabot.2009.05.003.
http://dx.doi.org/10.1016/j.aquabot.2009... ; Netondo et al., 2004NETONDO, G.W., ONYANGO, J.C. and BECK, E. Sorghum and salinity.II. Gas exchange and chlorophyll fluorescence of sorghum under salt stress. Crop Science, 2004, 44(3), 806-811. http://dx.doi.org/10.2135/cropsci2004.8060.
http://dx.doi.org/10.2135/cropsci2004.80... ). In addition, increases in chlorophyll a, carotenoids and the chlorophyll a/chlorophyll b ratio with increasing salinity were also observed, whereas chlorophyll b content decreased (Table 1). These results also corroborate previous studies indicating that NaCl stress has more effects on chlorophyll b than chlorophyll a (Houimli et al., 2010HOUIMLI, S.I.M., DENDEN, M. and MOUHANDES, B.D. Effects of 24-epibrassinolide on growth, chlorophyll, electrolyte leakage and proline by pepper plants under NaCl-stress. Eurasian Journal of Biosciences, 2010, 4, 96-104. http://dx.doi.org/10.5053/ejobios.2010.4.0.12.
http://dx.doi.org/10.5053/ejobios.2010.4... ). This implies in an increase in the chlorophyll a/chlorophyll b ratio, since the first step of chlorophyll b degradation results in its conversion into chlorophyll a (Fang et al., 1998FANG, Z., BOUWKAMP, J.C. and SOLOMOS, T. Chlorophyllase activies and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L. Journal of Experimental Botany, 1998, 49, 503-510.) and therefore, a decrease in total chlorophyll content (Pinheiro et al., 2008PINHEIRO, H.A., SILVA, J.V., ENDRES, L., FERREIRA, V.M., CÂMARA, C.A., CABRAL, F.F., OLIVEIRA, J.F., CARVALHO, L.W.T., SANTOS, J.M. and SANTOS FILHO, B.G. Leaf gas exchange, chloroplastic pigments and dry matter accumulation in castor bean (Ricinus communis L) seedlings subjected to salt stress conditions. Industrial Crops and Products, 2008, 27(3), 385-392. http://dx.doi.org/10.1016/j.indcrop.2007.10.003.
http://dx.doi.org/10.1016/j.indcrop.2007... ). This is also in accordance to other studies, such as those by Upadhyay & Panda (2005)UPADHYAY, R.K. and PANDA, S.K. Salt tolerance of two aquatic macrophytes, Pistia stratiotes and Salvinia molesta. Biologia Plantarum, 2005, 49(1), 157-159. http://dx.doi.org/10.1007/s10535-005-7159-4.
http://dx.doi.org/10.1007/s10535-005-715... , that observed the same regarding Salvinia molesta (Mitchell) and Pistia stratiotes (Linn) subjected to NaCl salinity. Total chlorophyll content was also higher in plants incubated with 50 mmol L^-1 NaCl compared with the other NaCl treatments, suggesting that this concentration can stimulate S. auriculata development and growth. This would suggest that this species would be able to broadly colonized coastal oligohaline wetlands, due to its adaptability to higher salinity conditions via physiological and biochemical changes.

Carotenoids are important pigments that can also act as antioxidants (Edge et al., 1997EDGE, R., MCGARVEY, D.J. and TRUSCOTT, T.G. The carotenoids a antioxidantes: a review. Journal of Photochemistry and Photobiology. B, Biology, 1997, 41(3), 189-200. PMid:9447718. http://dx.doi.org/10.1016/S1011-1344(97)00092-4.
http://dx.doi.org/10.1016/S1011-1344(97)... ), protecting plasma membrane lipids from oxidative stress generated in plants exposed to salinity (Falk & Munné-Bosch, 2010FALK, J. and MUNNÉ-BOSCH, S. Tocochromanol functíons in plants: antioxidation and beyond. Journal of Experimental Botany, 2010, 61(6), 1549-1566. PMid:20385544. http://dx.doi.org/10.1093/jxb/erq030.
http://dx.doi.org/10.1093/jxb/erq030... ). The data from the present study also corroborate with other data reported for other species, such as rice specimens (Oriza sativa L.) subjected to salinity also showing increases in carotenoid content (Misra et al., 1997MISRA, A., SAHU, A.N., MISRA, M., SINGH, P., MEERA, I., DAS, N., KAR, M. and SAHU, P. Sodium chloride induced changes in leaf growth, and pigment and protein contents in two rice cultivars. Biologia Plantarum, 1997, 39(2), 257-262. http://dx.doi.org/10.1023/A:1000357323205.
http://dx.doi.org/10.1023/A:100035732320... ).

A biomass increase was observed in a study with Phragmites karka (Retz.) exposed to 100 mmol L^-1 NaCl, with leaf osmotic adjustment conducted primarily by balance of inorganic solutes (K⁺ and Na⁺), while soluble sugar and proline content remained unchanged (Abideen et al., 2014ABIDEEN, Z., KOYRO, H.W., HUCHZERMEYER, B., AHMED, M.Z., GUL, B. and KHAN, M.A. Moderate salinity stimulates growth and photosynthesis of Phragmites karka by water relations and tissue specific ion regulation. Environmental and Experimental Botany, 2014, 105, 70-76. http://dx.doi.org/10.1016/j.envexpbot.2014.04.009.
http://dx.doi.org/10.1016/j.envexpbot.20... ). In another study, Salicornia europaea growth and net photosynthetic rate were stimulated, rather than inhibited, by 100-400 mmol L^-1 NaCl (Lv et al., 2012LV, S., JIANG, P., CHEN, X., FAN, P., WANG, X. and LI, Y. ultiple compartmentalization of sodium conferred salt tolerance in Salicornia europaea. Plant Physiology and Biochemistry, 2012, 51, 47-52. PMid:22153239. http://dx.doi.org/10.1016/j.plaphy.2011.10.015.
http://dx.doi.org/10.1016/j.plaphy.2011.... ). In S. auriculata, 50 mmmol L^-1 exposure induced higher FW and DW (Figure 1), suggesting that this concentration affects biomass accumulation in this species, while lower biomasses were observed at 100 and 150 mmol L^-1.

Regarding nutrient content, Na⁺ and Cl^- showed increases, while Ca²⁺, K⁺ and Mg²⁺ reduced with increasing NaCl concentrations (Table 2). Osmotic stress induced by high salinity (NaCl) has been shown to affect the absorption and translocation of Ca²⁺ in plants (Lee & Liu, 1999LEE, T.M. and LIU, C.H. Correlation of decreases calcium contents with proline accumulation in the marine green macroalga Ulva fasciata exposed to elevated NaCl contents in seawater. Journal of Experimental Botany, 1999, 50(341), 1855-1862. http://dx.doi.org/10.1093/jxb/50.341.1855.
http://dx.doi.org/10.1093/jxb/50.341.185... ), as observed herein. Similarly, Eichhornia crassipes (Mart.) and Pistia stratiotes (Linn.) maintained under salt stress conditions also showed decreased Ca²⁺ content (Niaz & Rasul, 1998NIAZ, M. and RASUL, E. Aquatic macrophytes as biological indicators for pollution management studies. Pakistan Journal of Science, 1998, 1(4), 332-334.) in high salinity conditions. Salinity can also affect K⁺ absorption and transport in plants (Shabala & Cuin, 2008SHABALA, S. and CUIN, T.A. Potassium transport and plant salt tolerance. Physiologia Plantarum, 2008, 133(4), 651-669. PMid:18724408. http://dx.doi.org/10.1111/j.1399-3054.2007.01008.x.
http://dx.doi.org/10.1111/j.1399-3054.20... ; Xu et al., 2010XU, J., WANG, W., YIN, H., LIU, X., SUN, H. and MI, Q. Exogenous nitric oxide improves antioxidative capacity and reduces auxin degradation in roots of Medicago truncatula seedlings under cadmium stress. Plant and Soil, 2010, 326(1-2), 321-330. http://dx.doi.org/10.1007/s11104-009-0011-4.
http://dx.doi.org/10.1007/s11104-009-001... ). In the present study, the decreases in K⁺ content compared to the controls probably occurred as a consequence of the antagonistic relationship between same-charged ions, as well as the competition between Na⁺ and K⁺ during nutrient absorption (Niaz & Rasul, 1998NIAZ, M. and RASUL, E. Aquatic macrophytes as biological indicators for pollution management studies. Pakistan Journal of Science, 1998, 1(4), 332-334.; Niu et al., 1995NIU, X., BRESSAN, R.A., HASEGAWA, P.M. and PARDO, J.M. lon homeostasis in NaCl stress environments. Plant Physiology, 1995, 109(3), 735-742. PMid:12228628. http://dx.doi.org/10.1104/pp.109.3.735.
http://dx.doi.org/10.1104/pp.109.3.735... ). Consequently, the Na⁺/K⁺ ratio also increased with increasing salinity, corroborating other reports (Jampeetong & Brix, 2009JAMPEETONG, A. and BRIX, H. Effects of NaCl salinity on growth, morphology, photosynthesis and proline accumulation of Salvinia natans. Aquatic Botany, 2009, 91(3), 181-186. http://dx.doi.org/10.1016/j.aquabot.2009.05.003.
http://dx.doi.org/10.1016/j.aquabot.2009... ). This decrease in K⁺ content and increase of the Na⁺/K⁺ ratio in the cytosol is characteristic of Na-induced toxicity (Maathuis & Amtmann, 1999MAATHUIS, F.J.M. and AMTMANN, A. K+ Nutrition and Na+ toxicity: the basis of cellular k+/Na+ ratios. Annals of Botany, 1999, 84(2), 123-133. http://dx.doi.org/10.1006/anbo.1999.0912.
http://dx.doi.org/10.1006/anbo.1999.0912... ). According to Esteves & Suzuki (2008)ESTEVES, B.S. and SUZUKI, M.S. Efeito da salinidade nas plantas. Oecologia Brasiliensis, 2008, 12(4), 662-679., excess salinity also reduces Mg²⁺ absorption by plants, corroborated herein. Decreases in Mg²⁺ content have also been described in other species, such as E. crassipes and P. stratiotes (Niaz & Rasul, 1998NIAZ, M. and RASUL, E. Aquatic macrophytes as biological indicators for pollution management studies. Pakistan Journal of Science, 1998, 1(4), 332-334.), Hydrilla verticillata (Rout & Shaw, 2001ROUT, N.P. and SHAW, B.P. Salt tolerance in aquatic macrophytes: Ionic relation and interaction. Biologia Plantarum, 2001, 44(1), 95-99. http://dx.doi.org/10.1023/A:1017978506585.
http://dx.doi.org/10.1023/A:101797850658... ), Typha domingensis (Esteves & Suzuki, 2008ESTEVES, B.S. and SUZUKI, M.S. Efeito da salinidade nas plantas. Oecologia Brasiliensis, 2008, 12(4), 662-679.) and S. natans (Jampeetong & Brix, 2009JAMPEETONG, A. and BRIX, H. Effects of NaCl salinity on growth, morphology, photosynthesis and proline accumulation of Salvinia natans. Aquatic Botany, 2009, 91(3), 181-186. http://dx.doi.org/10.1016/j.aquabot.2009.05.003.
http://dx.doi.org/10.1016/j.aquabot.2009... ) subjected to different salinity concentrations.

A slight decrease in P content was also observed, although with no significant difference between salinity treatments (Table 2). This suggests that salinity stress in this species may be associated with decreased P content, although the mechanism by which NaCl influences P absorption is not fully elucidated (Silva et al., 2008SILVA, C., MARTÍNEZ, V. and CARVAJAL, M. Osmotic versus toxic effects of NaCl on pepper plants. Biologia Plantarum, 2008, 52(1), 72-79. http://dx.doi.org/10.1007/s10535-008-0010-y.
http://dx.doi.org/10.1007/s10535-008-001... ) and further NaCl exposure assays are warranted.

Ammonia showed a small progressive increase with increasing of NaCl concentrations, although no significant differences were observed. Decrease in nitrogen uptake in roots have been attributed to the ionic antagonism between NH₄⁺ and Na⁺ (Naidoo, 1987NAIDOO, G. Effects of salinity and nitrogen on growth and plant water relatíons in the mangrove Avicennia marina (Forssk.). The New Phytologist, 1987, 107(2), 317-326. http://dx.doi.org/10.1111/j.1469-8137.1987.tb00183.x.
http://dx.doi.org/10.1111/j.1469-8137.19... ) or Cl^- and NO₃^- (Parida & Das, 2004PARIDA, A.K. and DAS, A.B. Effects of NaCl stress on nitrogen and phosphorus metabolism in a true mangrove Bruguiera paeviflora grown under hydroponic culture. Journal of Plant Physiology, 2004, 161(8), 921-928. PMid:15384403. http://dx.doi.org/10.1016/j.jplph.2003.11.006.
http://dx.doi.org/10.1016/j.jplph.2003.1... ). On the other hand, Jampeetong & Brix (2009)JAMPEETONG, A. and BRIX, H. Effects of NaCl salinity on growth, morphology, photosynthesis and proline accumulation of Salvinia natans. Aquatic Botany, 2009, 91(3), 181-186. http://dx.doi.org/10.1016/j.aquabot.2009.05.003.
http://dx.doi.org/10.1016/j.aquabot.2009... observed an increase in N content in the leaves and a decrease in the roots of S. natans at concentrations of 50, 100 and 150 mmol L^-1 NaCl.

Several studies have shown proline synthesis and accumulation in response to salt stress (Ahmad et al., 2012AHMAD, P., JOHN, R., SARWAT, M. and UMAR, S. Responses of proline, lipid peroxidation and antioxidative enzymes in two varieties of Pisum sativum L. under salt stress. International Journal of Plant Production, 2012, 2(4), 353-366.; Cheng et al., 2013CHENG, T.S., HUNG, M.J., CHENG, Y.I. and CHENG, L.J. Calcium-induced proline accumulation contributes to amelioration of NaCl injury and expression of glutamine synthetase in greater duckweed (Spirodela polyrhiza L.). Aquatic Toxicology, 2013, 144-145, 265-274. PMid:24200992. http://dx.doi.org/10.1016/j.aquatox.2013.10.015.
http://dx.doi.org/10.1016/j.aquatox.2013... ; Singh et al., 2015SINGH, D., RAM, P.C., SINGH, A. and SRIVASTAVA, S. Alleviating adverse effect of soil salinity on biomass production and physiological changes in wheat (Triticum aestivum L.) through application of zinc fertilizer. Research in Environment and Life Sciences, 2015, 8(2), 251-254.). This compound acts in the protection of cellular components by dehydration, maintaining the membrane structure and acting as a free radical scavenger (Hasegawa et al., 2000HASEGAWA, P.M., BRESSAN, R.A., ZHU, J.K. and BOHNERT, H.J. Plant cellular and molecular responses to hight salinity. Annual Review of Plant Physiology, 2000, 51(1), 463-499. PMid:15012199. http://dx.doi.org/10.1146/annurev.arplant.51.1.463.
http://dx.doi.org/10.1146/annurev.arplan... ). Proline synthesis and accumulation was corroborated herein, since proline content in S. auriculata increased significantly with salinity. This has also been observed for S. natans (Jampeetong & Brix, 2009JAMPEETONG, A. and BRIX, H. Effects of NaCl salinity on growth, morphology, photosynthesis and proline accumulation of Salvinia natans. Aquatic Botany, 2009, 91(3), 181-186. http://dx.doi.org/10.1016/j.aquabot.2009.05.003.
http://dx.doi.org/10.1016/j.aquabot.2009... ).

NO is a free, gaseous, lipophilic compound with high power diffusion through membranes (Crawford, 2006CRAWFORD, N.M. Mechanisms for nitric oxide synthesis in plants. Journal of Experimental Botany, 2006, 57(3), 471-478. PMid:16356941. http://dx.doi.org/10.1093/jxb/erj050.
http://dx.doi.org/10.1093/jxb/erj050... ). It is a versatile cell flag that plays important roles in physiological processes in animals and plants, acting against oxidative stress (Lamotte et al., 2005LAMOTTE, O., COURTOIS, C., BARNAVON, L., PUGIN, A. and WENDEHENNE, D. Nitric oxide in plants: the biosynthesis and cell signalling properties of a fascinating molecule. Planta, 2005, 221(1), 1-4. PMid:15754190. http://dx.doi.org/10.1007/s00425-005-1494-8.
http://dx.doi.org/10.1007/s00425-005-149... ). Studies show that NO can prevent the oxidative damage caused by salt stress on leaves (Haihua et al., 2002HAIHUA, R., WENBIAO, S., MAOBING, Y. and LANGLAI, X. Protective effects of nitric oxide on salt stress-induced oxidative damage to wheat (Triticum aestivum L.) leaves. Chinese Science Bulletin, 2002, 47(8), 677-681. http://dx.doi.org/10.1360/02tb9154.
http://dx.doi.org/10.1360/02tb9154... ) and root rot (Shi et al., 2007SHI, Q., DING, F., WANG, X. and WEI, M. Exogenous nitric oxide protect cucumber roots against oxidative stress induced by salt stress. Plant Physiology and Biochemistry, 2007, 45(8), 542-550. PMid:17606379. http://dx.doi.org/10.1016/j.plaphy.2007.05.005.
http://dx.doi.org/10.1016/j.plaphy.2007.... ). In our study, the NaCl treatments affected the levels of intra and extracellular NO, suggesting that salinity stimulates the release of NO from S. auriculata roots, decreasing endogenous NO levels (Figure 6) at the higher NaCl concentrations.

Salt stress has been shown to induce a rapid increase in NO levels in plants soon after salt exposure, since this compound is involved in salt tolerance (Zhang et al., 2006ZHANG, Y., WANG, L.L., LIU, Y., ZHANG, Q., WEI, Q. and ZHANG, W. Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast. Planta, 2006, 224(3), 545-555. PMid:16501990. http://dx.doi.org/10.1007/s00425-006-0242-z.
http://dx.doi.org/10.1007/s00425-006-024... ; Zhao et al., 2004ZHAO, L., ZHANG, F., GUO, J., YANG, Y., LI, B. and ZHANG, L. Nitric oxide functíons as a signal in salt resistance in the calluses from two ecotypes of reed. Plant Physiology, 2004, 134(2), 849-857. PMid:14739346. http://dx.doi.org/10.1104/pp.103.030023.
http://dx.doi.org/10.1104/pp.103.030023... , 2007ZHAO, M.G., TIAN, Q.T. and ZHANG, W.H. Nitric oxide synthase-dependent nitric oxide production is associated with salt tolerance in Arabidopsis. Plant Physiology, 2007, 144(1), 206-217. PMid:17351048. http://dx.doi.org/10.1104/pp.107.096842.
http://dx.doi.org/10.1104/pp.107.096842... ). In the present study, 50 mmol L^-1 NaCl in S. auriculata seems to be a moderate concentration, allowing for increases in NO concentrations after 2 hours. This, in turn, can be related to activation of physiological adjustments, such as biomass (Figure 2) and chlorophyll a and carotenoid content (Table 1), enabling the plants to grow in saline conditions and tolerate the salt stress.

According to Beligni & Lamattina (2001)BELIGNI, M.V. and LAMATTINA, L. Nitric oxide in plants: the history is just beginning. Plant, Cell & Environment, 2001, 24(3), 267-278. http://dx.doi.org/10.1046/j.1365-3040.2001.00672.x.
http://dx.doi.org/10.1046/j.1365-3040.20... , protection of chlorophyll mediated by NO may be due to NO ability to eliminate reactive oxygen species produced in plants under stress. Thus, it can be suggested that the higher endogenous NO after two hours of incubation with 50 mmol L^-1 NaCl observed in the present study may have acted as a signal, stimulating the preservation/biosynthesis of photosynthetic pigments through reduction of reactive oxygen species after five days. In comparison, higher NaCl concentrations, lead to plant chlorosis after 5 days. This suggests that after five days NO content is not due to an induced signal caused by salt stress, but a morphological and physiological response of the plant to stress conditions, including yellowing and bronze staining with increasing NaCl exposure (Figure 1). These results emphasize the importance of analyzing NO content in roots just after salinity exposure (i.e. 2 hours), to identify salinity effects on NO signaling during early stress induction compared to the end of salinity stress.

Studies have shown that NO is also associated with leaf expansion in several species. The increases observed in S. auriculata FW and DW associated to higher NO content have been demonstrated in other studies, resulting in leaf expansion, for example (An et al., 2005AN, L., LIU, Y., ZHANG, M., CHEN, T. and WANG, X. Effects of nitric oxide on growth of maize seedling leaves in the presence or absence of ultraviolet-B radiation. Journal of Plant Physiology, 2005, 162(3), 317-326. PMid:15832684. http://dx.doi.org/10.1016/j.jplph.2004.07.004.
http://dx.doi.org/10.1016/j.jplph.2004.0... ; Beligni & Lamattina, 2001BELIGNI, M.V. and LAMATTINA, L. Nitric oxide in plants: the history is just beginning. Plant, Cell & Environment, 2001, 24(3), 267-278. http://dx.doi.org/10.1046/j.1365-3040.2001.00672.x.
http://dx.doi.org/10.1046/j.1365-3040.20... ; Neill et al., 2008NEILL, S., BARROS, R., BRIGHT, J., DESIKAN, R., HANCOCK, J., HARRISON, J., MORRIS, P., RIBEIRO, D. and WILSON, I. Nitric oxide, stomatal closure, and abiotic stress. Journal of Experimental Botany, 2008, 59(2), 165-176. PMid:18332225. http://dx.doi.org/10.1093/jxb/erm293.
http://dx.doi.org/10.1093/jxb/erm293... ).

Regarding NO diffusion, NO can also diffuse into the cell from production sites, such as the mitochondria, to other regions of the cell, which can then produce an effect by interaction with target proteins (Freschi, 2013FRESCHI, L. Nitric oxide and phytohormone interactions: current status and perspectives. Frontiers in Plant Science, 2013, 4, 398. PMid:24130567. http://dx.doi.org/10.3389/fpls.2013.00398.
http://dx.doi.org/10.3389/fpls.2013.0039... ). It is also possible that NO can diffuse out of the cell through the plasma membrane to adjacent cells to stimulate its effect (Neill et al., 2008NEILL, S., BARROS, R., BRIGHT, J., DESIKAN, R., HANCOCK, J., HARRISON, J., MORRIS, P., RIBEIRO, D. and WILSON, I. Nitric oxide, stomatal closure, and abiotic stress. Journal of Experimental Botany, 2008, 59(2), 165-176. PMid:18332225. http://dx.doi.org/10.1093/jxb/erm293.
http://dx.doi.org/10.1093/jxb/erm293... ). In the present study, higher concentrations of NO in the extracellular medium were observed (Figure 5a) when compared to the intracellular environment (Figure 6A) after two hours of incubation. These results suggest that there may have been transport from the intracellular to extracellular medium in the higher NaCl treatments (100 and 150 mmol L^-1) after two hours of incubation. Equilibrium between the production and maintenance of NO within the cell is an important factor for cellular signaling in S. auriculata, and herein, the best adaptation was observed in plants exposed to 50 mmol L^-1 NaCl.

High salt concentrations can also induce damage in the structure of the plasma membrane of cells through changes in fundamental constituents, such as Ca⁺, as discussed previously (Schapire et al., 2008SCHAPIRE, A.L., VOIGT, B., JASIK, J., ROSADO, A., LOPEZ-COBOLLO, R., MENZEL, D., SALINAS, J., MANCUSO, S., VALPUESTA, V., BALUSKA, F. and BOTELLA, M.A. Arabidopsis synaptotagmin 1 is required for the maintenance of plasma membrane integrity and cell viability. The Plant Cell, 2008, 20(12), 3374-3388. PMid:19088329. http://dx.doi.org/10.1105/tpc.108.063859.
http://dx.doi.org/10.1105/tpc.108.063859... ). This element plays an essential role in the processes that preserve the structural and functional integrity of the membranes and regulate transport and ion selectivity in plants (Shoresh et al., 2011SHORESH, M., SPIVAK, M. and BERNSTEIN, N. Involvement of calcium-mediated effects on ROS metabolism in the regulation of growth improvement under salinity. Free Radical Biology & Medicine, 2011, 51(6), 1221-1234. PMid:21466848. http://dx.doi.org/10.1016/j.freeradbiomed.2011.03.036.
http://dx.doi.org/10.1016/j.freeradbiome... ). In this sense, changes in the structure of the plasma membranes induced in higher concentrations of NaCl may also have contributed to the increased extracellular release of NO in S. auriculata after two hours of incubation. Studies carried out with the addition of an exogenous NO donor, demonstrated that NO may increase plant tolerance to salinity by increasing DW, reducing oxidative damage, and maintaining a high Na⁺/K⁺ ratio in the cytoplasm (Shi et al., 2007SHI, Q., DING, F., WANG, X. and WEI, M. Exogenous nitric oxide protect cucumber roots against oxidative stress induced by salt stress. Plant Physiology and Biochemistry, 2007, 45(8), 542-550. PMid:17606379. http://dx.doi.org/10.1016/j.plaphy.2007.05.005.
http://dx.doi.org/10.1016/j.plaphy.2007.... ; Zhang et al., 2006ZHANG, Y., WANG, L.L., LIU, Y., ZHANG, Q., WEI, Q. and ZHANG, W. Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast. Planta, 2006, 224(3), 545-555. PMid:16501990. http://dx.doi.org/10.1007/s00425-006-0242-z.
http://dx.doi.org/10.1007/s00425-006-024... ). Thus, it has been suggested that NO acts as a signal for salt tolerance, causing increased Na⁺ secretion (Chen et al., 2010CHEN, J., XIAO, Q., WU, F., DONG, X., HE, J., PEI, Z. and ZHENG, H. Nitric oxide enhances salt secretion and Na+ sequestration in a mangrove plant, Avicennia marina, through increasing the expression of H+-ATPase and Na+/H+ antiporter under high salinity. Tree Physiology, 2010, 30(12), 1570-1585. PMid:21030403. http://dx.doi.org/10.1093/treephys/tpq086.
http://dx.doi.org/10.1093/treephys/tpq08... ).

5. Conclusions

At 50 mmol L^-1 NaCl exposure, S. auriculata induced NO production early in the stress process, which is probably associated to signaling to produce compounds that assist in stress tolerance, whereas at 150 mmol L^-1 this tolerance is reduced. Thus, it seems that S. auriculata can develop very well in environments with moderate salinity (50 mmol L^-1) such as oligohaline environments, as is the case of the study area, that can show excess brackish water and increased salt water influx due to the implementation of artificial sandbars in the region. This, in turn, may directly influence this species distribution in the coastal areas of Northern Rio de Janeiro. Thus, these results are fundamental in understanding the physiological and biochemical mechanisms associated with the adaptation of this macrophyte to saline conditions, which, in turn, affect this species ecology.

References

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