Plant Physiology and Biochemistry 77 (2014) 15e22

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Research article

Cellular proton dynamics in Elodea canadensis leaves induced by cadmium M. Tariq Javed*, Sylvia Lindberg, Maria Greger Department of Ecology, Environment and Plant Sciences, Stockholm University, SE-10691 Stockholm, Sweden

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 October 2013 Accepted 18 January 2014 Available online 28 January 2014

Our earlier investigations showed that Elodea canadensis shoots, grown in the presence of cadmium (Cd), caused basification of the surrounding medium. The present study was aimed to examine the proton dynamics of the apoplastic, cytosolic and vacuolar regions of E. canadensis leaves upon Cd exposure and to establish possible linkage between cellular pH changes and the medium basification. The changes in cytosolic calcium [Ca2þ]cyt was also investigated as the [Ca2þ]cyt and [pH]cyt homeostasis are closely linked. The cellular Hþ and Ca2þ concentrations were monitored by fluorescence microscopy and ionspecific fluorescent dyes. Cadmium concentration of leaf-cell walls was measured after plant cultivation at different fixed levels of starting pH. The protoplasts from E. canadensis leaves were isolated by use of a newly developed enzymatic method. Upon Cd addition, both cytosolic and vacuolar pH of leaf protoplasts increased with a concomitant rise in the cytosolic Ca2þ concentration. Time course studies revealed that changes in [Ca2þ]cyt and [pH]cyt followed similar dynamics. Cadmium (0.5 mM) exposure decreased the apoplastic pH by 0.85 units. The maximum cell wall bound Cd-contents were obtained in plants grown at low starting pH. It is concluded that Cd treatment causes apoplastic acidosis in E. canadensis leaves associated with enhanced Cd binding to the cell walls and, consequently, reduced Cd influx into the cytosol. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Apoplastic pH Cadmium Cytosolic Ca2þ Cytosolic pH Proton dynamics Protoplast Vacuolar pH

1. Introduction Cadmium (Cd) is a divalent, highly toxic heavy metal that is ubiquitous in sewage sludge, industrial wastes and mining sites. Presence of Cd in biotic systems is a potential hazard for plants, animals and humans. Although non-essential, it is readily taken up by vascular plants both through their roots and shoots (Jarvis et al., 1976). For leaves of most plants, Cd concentrations higher than 5e 10 mg/g dry matter are considered to be toxic (White and Brown, 2010). Research on the toxicity of Cd to plant growth, metabolism and enzyme activity has been extensively reported for terrestrial plants, which take up Cd only through roots (Lux et al., 2011). In contrast, the response of aquatic macrophytes to Cd stress is

Abbreviations: 6-CFDA, 6-Carboxyfluorescein diacetate; BCECF AM, Acetoxymethyl ester of Bis (carboxyethyl) carboxyfluorescein; CPW, Cell protoplast washing medium; DMSO, Dimethyl sulfoxide; Fura-2 AM, Acetoxymethyl ester of calcium binding benzofuran; PC, Phytochelatin; PEG, Polyethylene glycol; PVP, Polyvinyl pyrolidone. * Corresponding author. Present address: Department of Botany, Faculty of Science and Technology, Government College University, Faisalabad 38000, Pakistan. Tel.: þ92 41 9201488. E-mail address: [email protected] (M. Tariq Javed). 0981-9428/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2014.01.009

scarcely documented particularly at the cellular level, although the aquatic ecosystems are major reservoirs for metal contamination (Lavid et al., 2001). Moreover, aquatic macrophytes are remarkably effective in sequestering metals from polluted waters (Dushenkov et al., 1995) and it has been reported that shoots of Elodea canadensis can accumulate high amounts of metals (Cd, Cu, Zn and Pb) from the water as well as sediment (Fritioff and Greger, 2007). Leaves of E. canadensis consist of only two cell layers, which exhibit light-induced polar pH reactions, where the upper leaf layer alkalizes while the lower leaf layer acidifies the surrounding medium (Elzenga and Prins, 1989). Shoots of E. canadensis take up Cd directly from the water phase, and Cd deposition in apoplastic regions is its special feature as demonstrated both by short- (Nyquist and Greger, 2009) and long-term studies (Dalla Vecchia et al., 2005). Cadmium accumulation in the vicinity of the cell membrane may initiate interactions between Cd and cellular components leading to alterations of plasma membrane properties. At the plasma membrane, the Cd passage is known to occur via ion channels or specialized membrane proteins that mediate ion transport (Perfus-Barbeoch et al., 2002). Intracellular Cddetoxification processes lead to an imbalance in the production and consumption of protons leading to cytosolic pH modulations. Cadmium treatment causes cytosolic acidosis in plants as reported

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for wheat (Triticum aestivum L.) leaf protoplasts (Lindberg et al., 2007) and maize (Zea mays L.) root cells (Nocito et al., 2008). However, plants also possess mechanisms to buffer cytoplasmic pH achieved either by buffering agents e.g. phosphoric acids, amino acids, organic acids (Javed et al., 2013), by bio-chemical pH stat (Davies, 1986) or bio-physical pH stat (Kurkdjian and Guern, 1989). Such buffering systems enable the plant to cope with abiotic stresses as reported for Oryza sativa (Kader et al., 2007). The Hþ-ATPase creates a proton motive force across the plasma membrane and is the main active system involved in the transport of ions. The cytosolic acidosis or alkalinization may depend on the activity of Hþ-ATPase. The inhibitory effects of Cd on Hþ-ATPase pumping activities were reported in Beta vulgaris (Lindberg and Wingstrand, 1985) and Avena sativa (Astolfi et al., 2003). However, Cd was also shown to increase Hþ-ATPase activity as reported in cucumber (Cucumis sativus L.) root cells (Janicka-Russak et al., 2012). Calcium acts as a 2nd messenger in several signal-transduction pathways and cytosolic [Ca2þ] level is usually increased in response to various external stimuli, including Cd (Gao et al., 2004). Cellular pH is also involved in cell signaling and transduces the incoming message, either directly, or in cross talk with Ca2þ or plant hormones (Gao et al., 2004; Kader and Lindberg, 2010). Thus the cytosolic calcium and pH homeostasis in plant cells are closely linked. The changes in pH and/or Ca2þ act as indicators of stress and transient shifts in intracellular, as well as apoplastic pH, have been observed in several signal-transduction processes. Plant cells may avoid entry of toxic metals into their metabolic pathways by binding them to their cell wall (Haynes, 1980), where the cations are trapped either by polysaccharides (Crist et al., 1990) or certain un-protonated groups, such as a carboxyl oxygen or sulfate (Campbell and Stokes, 1985). Redjala et al. (2009) showed that increasing Cd concentration in the exposure solution led to retaining a higher concentration of Cd in the cell walls of Zea mays and Noccaea caerulescens roots. For both species, high plant-Cd content was reflecting an increase in the apoplastic Cd uptake and a decrease in the symplastic absorption (Sterckeman et al., 2011). Negative charges carried by pectins could be determined by methyl-esterification of carboxylic groups of galacturonic acid units which account for the quantity of metal it can bind (Sattelmacher, 2001). In Flax (Linum usitatissimum L.), Cd induces enzymatic reorganization of pectin epitopes by releasing low methyl esterified pectin epitopes, which in turn enhance Cd binding to the cell walls (Douchiche et al., 2007). Our earlier investigations showed that E. canadensis shoots increased the surrounding medium pH in the presence of Cd, when the medium pH was initially low (pH 3.5e5.0) (Nyquist and Greger, 2009; Javed, 2011; Javed and Greger, 2011). Current studies were designed to clarify why Cd induces a pH increase in the media of E. canadensis. It was hypothesized that Cd-induced external medium basification was due to the enhanced cytosolic proton influx depending on a negative impact of Cd on proton-extruding activity of plasma membrane Hþ-ATPase, which should lead to a cytosolic pH decrease. Little information is available about Cd effects on cellular proton dynamics in aquatic plants. Thus, the apoplastic, cytosolic and vacuolar pH changes in E. canadensis upon Cd exposure were investigated. 2. Methods 2.1. Plant material 2.1.1. Plant cultivation for protoplast extraction Elodea canadensis Michx. shoots were grown in a greenhouse pond (width 1.5 m, length 3 m, depth 1 m) carrying aerated water.

The greenhouse was equipped with supplementary lamps (Osram Daylight, HQ1-BT 400 W) to provide 18 h/6 h light/dark periods while the temperature and relative humidity were maintained at 20  2  C and 65%. After about 8 weeks, 10 cm long shoot tips were excised and planted in 45 L black plastic boxes, containing a 3 cm thick soil layer, covered with 2 cm thick sand layer and aerated tap water (ca. 40 L). The water surface was covered with the water fern Azolla spp to provide shade for the plants. The plastic containers were placed in the same greenhouse as described earlier. 2.1.2. Plant cultivation for apoplastic pH measurements Ten centimeters long E. canadensis shoot tips were transferred to twenty black pots (2 shoots per pot), each containing 500 ml Hoagland solution (1%), and were acclimatized for 72 h in a growth chamber (Conviron CMP 3000). Subsequently, half of the pots were treated with 0.5 mM CdCl2 for 72 h and the other half was left untreated. The used Cd concentration was chosen from a toxicity test, based on chlorophyll contents and was in the least toxic range. Solutions were aerated with micro-tubes during acclimatization as well as during the course of the experiment. To provide the natural summer conditions for growth, temperature in the chamber was set to 20  C, relative humidity to 70% with 16 h/8 h light/dark conditions. Photosynthetic photon flux density at the water surface was 100 mmol m2 s1 and was measured by ILT 1400 radiometer/ photometer (International Light Technologies). 2.2. Protoplast extraction Fresh shoot tips (10 cm) of E. canadensis were collected from the greenhouse and washed thrice with distilled water. The young leaves were pre-plasmolysed in 0.7 M sorbitol for 30 min before extracting protoplasts. Leaves were then sliced transversely in pieces smaller than 1 mm, and treated for 2 h at 30  C in darkness with 1.5% cellulase from Trichoderma reesei (Sigma e St Louis, MO, USA EC 3.2.1.4), 1.5% hemicellulase from Aspergillus niger (Sigma, EC 3.2.1.8), 0.75% macerozyme R-10 from Rhizopus (SERVA, EC 3.2.1.15) and 2.5% (v/v) b-glucuronidase from Helix pomatia (Sigma, EC 3.2.1.31) (modified after Staal et al., 1988) in a medium (CPW- salt medium) containing 0.7 M sorbitol, 1 mM calcium chloride, 0.05% (w/v) PVP (Sigma), 0.05% (w/v) bovine serum albumin (BSA; Sigma) and 5 mM MES (Sigma) at pH 5.5. At the end of incubation, the suspension was gently shaken to release the protoplasts and then filtered through a tea strainer. The filtrate was washed twice with 1 ml of the buffer solution (CPW-salt medium) without enzymes, to release more protoplasts and then filtered through nylon net with 60 mm pores. The protoplast suspension was centrifuged for 6 min at 60  g. The supernatant was removed with a suction pump. The protoplasts were extracted from E. canadensis plants obtained from seven independent cultivations. They were used for dye loading, Cd treatment and fluorescence measurements. 2.3. Dye loading For the measurement of cytosolic pH, protoplasts were loaded with the pH-specific dye, BCECF-AM (Sigma, EC 200-664-3). The BCECF-AM dye solution was prepared by mixing 5 ml of a stock solution (1.6 mM) with 5 ml of ethanol (99%) and 1.25 ml pluronic F127 (Sigma). From the above mixed dye solution of BCECF-AM, 5 ml was added to 1 ml of protoplast suspension containing 0.7 M sorbitol, 1 mM CaCl2, 0.2% (w/v) PVP and 5 mM Tris-MES buffer (pH 5.5; medium A). Loading was performed for 2 h at 4  C in darkness. After loading, protoplasts were washed in a solution similar to medium A, but with 5 mM Tris/HEPES buffer at pH 7 (medium B). Afterwards, the protoplast samples were kept in darkness at room temperature for about half an hour before starting the fluorescence

M. Tariq Javed et al. / Plant Physiology and Biochemistry 77 (2014) 15e22

measurements, which were carried out within 1e2 h after the loading procedure was finished. For cytosolic Ca2þ measurement, the protoplasts were loaded with Fura 2-AM in the acetoxymethyl ester form (Fura 2-AM, Molecular Probes, Leiden, Netherlands). The Fura 2-AM dye solution was prepared by mixing 2 ml of Fura 2-AM stock solution (5 mg/ml) in dry (