Journal of Experimental Botany

Journal of Experimental Botany Advance Access published June 13, 2014 Journal of Experimental Botany doi:10.1093/jxb/eru251 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) Research Paper Zn2+-induced changes at the root level account for the increased tolerance of acclimated tobacco plants Nadia Bazihizina1, Cosimo Taiti1, Lucia Marti1, Ana Rodrigo-Moreno1, Francesco Spinelli1, Cristiana Giordano2, Stefania Caparrotta1, Massimo Gori1, Elisa Azzarello1 and Stefano Mancuso1,* 1  LINV - Department of Agrifood Production and Environmental Sciences – University of Florence, Viale delle Idee 30, 50019 Sesto F.no, Florence, Italy 2  Centro di Microscopie Elettroniche ‘Laura Bonzi’ (Ce.M.E.), ICCOM, CNR, Via Madonna del Piano, 50019 Sesto F.no, Florence, Italy Downloaded from http://jxb.oxfordjournals.org/ at Università degli Studi di Firenze on June 17, 2014 *  To whom correspondence should be addressed. E-mail: [email protected] Received 27 March 2014; Revised 7 May 2014; Accepted 12 May 2014 Abstract Evidence suggests that heavy-metal tolerance can be induced in plants following pre-treatment with non-toxic metal concentrations, but the results are still controversial. In the present study, tobacco plants were exposed to increas- ing Zn2+ concentrations (up to 250 and/or 500 μM ZnSO4) with or without a 1-week acclimation period with 30 μM ZnSO4. Elevated Zn2+ was highly toxic for plants, and after 3 weeks of treatments there was a marked (≥50%) decline in plant growth in non-acclimated plants. Plant acclimation, on the other hand, increased plant dry mass and leaf area up to 1.6-fold compared with non-acclimated ones. In non-acclimated plants, the addition of 250 μM ZnSO4 led to transient membrane depolarization and stomatal closure within 24 h from the addition of the stress; by contrast, the acclimation process was associated with an improved stomatal regulation and a superior ability to maintain a nega- tive root membrane potential, with values on average 37% more negative compared with non-acclimated plants. The different response at the plasma-membrane level between acclimated and non-acclimated plants was associated with an enhanced vacuolar Zn2+ sequestration and up to 2-fold higher expression of the tobacco orthologue of the Arabidopsis thaliana MTP1 gene. Thus, the acclimation process elicited specific detoxification mechanisms in roots that enhanced Zn2+ compartmentalization in vacuoles, thereby improving root membrane functionality and stomatal regulation in leaves following elevated Zn2+ stress. Key words:  Acclimation, heavy-metal toxicity, membrane potential, Nicotiana tabacum, stomatal conductance, transporter, vacuole. Introduction Zinc releases into the environment are associated with biotic processes at the micromolar range; however, Zn2+ is toxic to or natural atmospheric processes, but mining and anthropic plants at supra-optimal concentrations, and toxicity occurs activities have resulted in heavy-metal contamination of when leaf concentrations reach 400–500 μg g–1 of dry mass urban and agricultural soils (Friedland, 1990). Furthermore, (Marschner, 1995; Broadley et  al., 2007). Common Zn2+ an increasing acidity of soils liberates the bound pool of met- toxicity symptoms include: reduced plant water content and als, which in turn leads to increased availability of metal ions stunted plant growth (Sagardoy et al., 2009), decreased stoma- for plants (Påhlsson, 1989). Zn2+ is crucial for the metabolism tal conductance and photosynthesis (Sagardoy et al., 2010), of plant cells, being involved in a wide variety of physiological changes in root growth and morphology, severe nutrient Abbreviations: An, net photosynthetic rate; Ci, substomatal CO2 concentrations; gs, stomatal conductance; MTP, metal tolerance protein; PSII, photosystem II; RT-qPCR, quantitative real-time PCR; WC, water content. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Page 2 of 12 | Bazihizina et al. imbalances, and leaf chlorosis (Marschner, 1995; Vaillant (including Zn2+ toxicity) at the shoot level (Perfus-Barbeoch et al., 2005; Broadley et al., 2007; Sagardoy et al., 2009). et al., 2002; Sagardoy et al., 2010). Increasing evidence sug- Acclimation occurs during plant ontogeny and describes gests that exposure to toxic metal concentrations negatively the enhanced stress tolerance of a particular individual plant affects parameters important for plant–water relationships, as a result of the induction of physiological, biochemical, and, in particular, toxic metal has been found to reduce the and molecular adjustments within the plant’s tissues and biomass allocation to the roots (Ryser and Emerson, 2007) cells (Pandolfi et al., 2012). Plant acclimation to a particular reduce cell-wall elasticity (Barceló et al., 1986) increase cell- abiotic stress condition is associated with responses tailored membrane permeability (Llamas et  al., 2008; Michael and to the specific conditions encountered (Mittler, 2006). There Krishnaswamy, 2011); reduce stem and root hydraulic con- is some evidence suggesting that heavy-metal tolerance can ductivity (Przedpelska-Wasowicz and Wierzbicka, 2011; de be induced in plants following a pre-treatment (i.e. acclima- Silva et al., 2012), and reduce xylem-specific and leaf-specific tion) with non-toxic metal concentrations, which improve the hydraulic conductivity (de Silva et  al., 2012). Hence, given plant’s ability to tolerate otherwise toxic metal concentrations that perturbation of leaf stomatal regulation (Sagardoy (Watmough and Dickinson, 1996). For example, heavy-metal et al., 2010) has been considered one of the early causes of resistance traits (e.g. reduced growth inhibition in response heavy-metal toxicity (e.g. within the first 48 h after treatment; to heavy metal) were induced in a cell suspension culture Sagardoy et al., 2010), it is conceivable that the acclimation Downloaded from http://jxb.oxfordjournals.org/ at Università degli Studi di Firenze on June 17, 2014 from shoot explants of mature trees of Acer pseudoplatanus process will result in an improved stomatal regulation upon through repeated exposure to gradually increasing metal con- exposure to heavy-metal stress. centration in the growth medium (Dickinson et  al., 1992). In the present study, we tested whether pre-treatment for Nevertheless, plant acclimation to heavy metals remains a 1 week with a high but non-toxic Zn2+ concentration had controversial topic in the literature, and currently little infor- any effect on the tolerance of tobacco (Nicotiana tabacum) mation is available regarding the eventual mechanism(s) to toxic Zn2+ concentrations. We hypothesized that the accli- underlying the increased tolerance to toxic heavy-metal con- mation process would substantially decrease the symptoms centrations in acclimated plants (e.g. Turner and Dickinson, generally associated with Zn2+ toxicity, and would therefore 1993; Wisniewski and Dickinson, 2003). By contrast, other increase: (i) shoot and root growth, and (ii) the total chlo- examples of acclimation processes in plants, such as cold rophyll and carotenoid concentrations in leaves. In addition, acclimation (i.e. increased freezing tolerance following expo- considering that plant acclimation requires responses tailored sure to low non-freezing temperatures; Thomashow, 1999) or to the specific external environmental conditions (Mittler, the increased ability of plants to tolerate toxic salt concen- 2006), we hypothesized that, following Zn2+ addition, the trations after exposure to non-toxic salinities (Silveira et al., acclimation process to Zn2+ would: (iii) improve stomatal 2001; Djanaguiraman et al., 2006; Pandolfi et al., 2012) are regulation, and (iv) result in a superior ability to maintain well accepted. negative membrane potential in roots, as these are the first Metal ions such as Cd2+ and Zn2+ have been found to organs to encounter the heavy-metal stress. We also expected induce serious and continuous membrane depolarization that this improved root membrane functionality would be in root cells (Kennedy and Gonsalves, 1987; Aidid and associated with (iv) an enhanced sequestration of Zn2+ in the Okamoto, 1992). As the plant plasma membrane and its vacuole. functions have been regarded as the first targets of heavy- metal toxicity, any form of tolerance should involve protec- tion of membrane integrity (Hall, 2002). In support of this hypothesis, the plasma membrane of tolerant plants generally Materials and methods experiences less metal-induced damage than that of sensitive Plant material and growth conditions plants (Kenderesová et al., 2012). Tolerance to high levels of Tobacco plants (N.  tabacum) were germinated and grown in envi- heavy metals is associated with sequestration of ions in meta- ronmentally controlled chambers (25/25 °C day/night, 12 h day/12 h bolically inactive compartments (i.e. vacuoles) (Hall, 2002; night, with an average photosynthetically active radiation at shoot height of 300  µmol m–2 s–1). Seeds of tobacco were sown in plas- Krämer et al., 2007), and the presence of active cytoplasmic tic pots containing standard potting mix, and 2 (Experiments 2 Zn2+ has been found to induce plasma-membrane depolariza- and 3)  or 5 (Experiment 1)  weeks after emergence, seedlings were tion (Kenderesová et al., 2012); it could therefore be hypoth- transferred to an aerated nutrient solution. All plants were supplied esized that, in acclimated plants, prior exposure to non-toxic with half-strength Hoagland’s nutrient solution (pH adjusted to Zn2+ concentrations will induce specific detoxification mech- 5.8 using KOH). The pH of the solution was checked and adjusted (as required) daily and solutions were changed weekly. Two weeks anisms that, following the addition of elevated and toxic Zn2+ after transferring the plants to the aerated nutrient solution, ZnSO4 concentrations, will reduce the build-up of Zn2+ in sensitive was added to the aerated solutions to obtain the required final Zn2+ and metabolically active sites of the cell and ultimately result concentrations. in an improved root membrane functionality. Zinc toxicity inhibits both photosynthesis and stomatal Experimental design conductance (Sagardoy et  al., 2009; Azzarello et  al., 2012). Responses of tobacco to elevated ZnSO4 in three different experi- However, it is still unclear whether photosynthesis inhibi- ments.  Experiment 1 consisted of eight treatments with four tion or a perturbation of the water, and thus stomatal limita- replicates in a completely randomized block design. In six treat- tions, are one of the primary causes of heavy-metal toxicity ments, plants were exposed to increasing Zn2+ concentrations:  Root acclimation improves heavy-metal tolerance  |  Page 3 of 12 1 μM ZnSO4, considered as the control treatment, and three other Tris/MES basal salt medium (BSM: 0.2 mM KCl, 0.1 mM CaCl2, treatments where the appropriate amount of ZnSO4 was added to pH 5.8) for 4 h before the measurements. Cells were impaled with the control solution to reach final concentrations of 30, 250, and conventional KCl-filled Ag/AgCl microelectrodes (Shabala and 500  μM ZnSO4. In the remaining two treatments, 1 week prior to Lew, 2002; Cuin and Shabala, 2005), and membrane potentials the treatment (250 μM ZnSO4), plants were exposed to a high but were recorded for 2 min. Subsequently 1 ml of buffered Tris/MES non-toxic Zn2+ concentration of 30 μM ZnSO4 (cf. Arrivault et al., BSM with 1.25 mM ZnSO4 was added, resulting in a final concen- 2006). Plants were then harvested 3 weeks after imposing the treat- tration of 250 μM ZnSO4 in the bath solution. Measurements were ments. To elucidate the possible mechanism(s) responsible for the continued for another 10 min after addition of the ZnSO4 solu- enhanced tolerance in acclimated plants, two additional experiments tion. Four individual plants for each treatment were measured. (Experiments 2 and 3) were conducted focusing on the responses in Subsequently membrane potentials were measured in treated plants the short-term (within 24 h) to 250 μM ZnSO4. In these two experi- (250 μM ZnSO4) after 24 h of treatment. Four individual plants for ments we tested whether, following the addition of 250 μM ZnSO4, each treatment were measured, with up to five readings from each the acclimation process was associated, at the root level, with an individual root. improved ability to maintain negative membrane potentials and Leaf gas-exchange parameters (Experiment 2) Leaf gas-exchange sequestrate Zn2+ in the vacuoles. The experiments consisted of three parameters were determined simultaneously with chlorophyll fluo- treatments with four replicates in a randomized block design. In two rescence measurements using the open gas-exchange system Li-6400 treatments, plants were exposed to two Zn2+ concentrations (1 and XT (Li-Cor, Lincoln, NE, USA) with an integrated fluorescence 250 μM ZnSO4), and in the remaining treatment, 1 week prior to the chamber head (Li-6400–40; Li-Cor). Leaf gas-exchange measure- addition of 250 μM ZnSO4, plants were exposed to 30 μM ZnSO4. ments were taken on all plants in each treatment, before the treat- Plant sampling (Experiment 1) Plants were sampled 20 d after ment (0 h) and 24 h after adding 250 μM ZnSO4. Measurements of Downloaded from http://jxb.oxfordjournals.org/ at Università degli Studi di Firenze on June 17, 2014 applying the treatments for the determination of shoot and root net photosynthetic rate (An), stomatal conductance (gs), and subs- fresh and dry masses. Shoot and root tissues were harvested and tomatal CO2 concentrations (Ci) were determined on the youngest their fresh weight recorded. Leaves were scanned for surface area fully expanded leaves at ambient relative humidity (40–50%), refer- and leaf area calculated using the Tomato Analyzer software. In ence CO2 of 400  µmol mol–1, flow rate of 400  µmol s–1, chamber addition, root samples were taken for subsequent transmission elec- temperature of 25  °C and photosynthetically active radiation of tron and light microscopy. Shoot and roots were then oven dried at 300 µmol m–2 s–1. 60 °C to determine their dry mass. In addition, plants were sampled Using the integrated fluorescence chamber head (Li-6400–40) of before applying the treatments and then 24 h after the commence- the open gas-exchange system Li-6400 XT, we measured chlorophyll ment of treatments. Shoot and root tissues were harvested and their fluorescence on the same leaves used for gas-exchange measure- fresh mass recorded. Shoot and roots were then oven dried at 60 °C ments at the end of the night period (i.e. when plants had been in to determine their dry mass. Plant fresh and dry masses were used the dark for at least 11 h, before the lights were switched on in the to calculate the plant water content (WC) on a fresh weight basis controlled environment room). The minimal fluorescence level in the using the following equation: WC (%)=[(fresh mass – dry mass)/ dark-adapted state (F0) was measured using a modulated pulse, and fresh mass]×100. maximal fluorescence in this state (Fm) was measured after applying Leaf pigment analyses (Experiment 1)  In Experiment 1, at the end a saturating actinic light pulse of 7000 μmol m–2 s–1. The values of of the experimental period, total chlorophyll and carotenoid concen- the variable fluorescence (Fv=Fm–F0) and maximum quantum effi- trations were determined in all treatments by reading the absorbance ciency of photosystem II (PSII) photochemistry (Fv/Fm) were calcu- at 537, 647, and 664 nm of extracts obtained from two disks of 10 mm lated from F0 and Fm. in diameter taken from randomly selected youngest fully expanded Confocal microscopy (Experiment 3) Confocal imaging was per- leaves from each replicate. Leaf discs were ground in liquid nitrogen formed using an upright Leica laser-scanning confocal microscope and extracted with an acetone and Tris buffer solution for 48 h at SP5 (Leica Microsystems Wetzlar GmbH, Germany) equipped with 4 °C in the dark (Sims and Gammon, 2002). Chlorophyll and carot- a 40× oil-immersion objective. To analyse the intracellular localiza- enoid concentrations were determined according to Wellburn and tion of the Zn2+ ions in root cells, FluoZin-3-AM (acetoxymethyl) Lichtenthaler (1984) using a Tecan Infinite 200 Spectrophotometer cell permeant (Molecular Probes, USA) was used. FluoZin-3-AM (Männedorf, Switzerland). was chosen as it is considered to be a very specific indicator for intra- Transmission electron microscopy (Experiment 1) Samples of the cellular Zn2+ localization and concentration (Gee et al., 2002). Roots control and ZnSO4-treated roots were cut into pieces 3 mm long and were incubated for 60 min in a solution of 15 µM FluoZin-3-AM. immediately fixed in 2.5 % glutaraldehyde in 0.2 M phosphate buffer After incubation, the samples were mounted in a water solution on (pH 7.2) for 2 h at room temperature. Samples were then washed a slide and observed. The excitation wavelength was set at 488 nm, twice in the same buffer and post-fixed in 2% OsO4 in the same buffer and emission was detected at 530 ± 20 nm. for 2 h at room temperature. Following dehydration in a graded etha- Expression of Zn2+ metal tolerance protein 1 (MTP1) protein trans- nol series (30, 40, 50, 60, 70, 80, 95, and 100%), the specimens were porter in root tissues (Experiment 3) After 24 h of treatments, gradually embedded in Spurr resin (Spurr, 1969) and polymerized roots were collected from tobacco seedlings and immediately at 70  °C for 24 h. Ultrathin (70–90 nm) transverse sections of the frozen in liquid nitrogen. Samples were homogenized with a pes- processed tissue were obtained with an LKB IV ultramicrotome, tle and total RNA was extracted with an RNeasy Plant Mini kit collected on Formvar-coated aluminium grids, stained with uranyl (Qiagen). First-strands cDNA was synthesized using a Quantitect acetate and lead citrate, and examined using a Philips CM12 trans- Reverse Transcription kit (Qiagen) according to the manufacturer’s mission electron microscope (Eindhoven, The Netherlands) operat- instructions. The quantity of RNA and cDNA was measured using ing at 80 kV. a Tecan Infinite 200 Spectrophotometer (Männedorf, Switzerland). Light microscopy (Experiment 1)  The root tissue was processed and Transcript levels were determined by quantitative real-time PCR cut as for transmission microscopy. Semi-thin sections of 1–2  μm (RT-qPCR) with a QuantiFast SYBR Green PCR kit (Qiagen) were fixed to glass slides, and observations were carried out in a using a Rotor-Gene 6000 (Corbett Life Science). RT-qPCR was Leica DM LB2 Light Microscope (Leica Microsystems Wetzlar conducted in a 15  μl reaction mixture volume and the protocol GmbH, Germany). was: initial step of 95  °C for 5 min, and 40 cycles of 95  °C for Membrane potential measurements (Experiment 2) Membrane 12 s and 60  °C for 45 s, followed by meltin-curve analysis. Each potentials were measured on cortical cells of excised root segments sample, standard curve and no-template control were run in trip- of plants grown in aerated solution with 1 μM (control conditions) licate. The primer sequences for the target gene were designed in or 30 μM (acclimated plants) ZnSO4. Excised root segments were a common region for the tobacco orthologue of the Arabidopsis immobilized in a Plexiglass chamber filled with 4 ml of buffered thaliana MTP1 gene (NtMTP1a and NtMTP1b genes; for more Page 4 of 12 | Bazihizina et al. details, see Shingu et al., 2005). As housekeeping genes, EF-1α and L25 were used, as these have been found to have the highest stabil- ity under abiotic stresses in tobacco (Schmidt and Delaney, 2010). As results were similar for the two housekeeping genes, only EF-1α data are shown. Primer sets used for RT-qPCR are listed in Table S1 at JXB online. Relative expression data were calculated using the comparative Livak method (2–ΔΔCT; Livak and Schmittgen, 2001). The target fragment was verified by sequencing. Statistical analyses Statistical analyses were conducted using GraphPad for Mac, 6th edn. One-way or two-way analysis of variance, depending on the dataset, was used to identify overall significant differences between treatments. Unless otherwise stated, the significance level was P≤0.05. Results Downloaded from http://jxb.oxfordjournals.org/ at Università degli Studi di Firenze on June 17, 2014 A 1-week acclimation period with 30 μM ZnSO4 increases Zn2+ tolerance in the long term After 3 weeks of Zn2+ treatments, leaf area and plant dry mass gradually declined with increasing Zn2+ concentra- tions in the root zone (Fig.  1a, b). With 30  μM ZnSO4, both the leaf area and the total dry mass remained similar to that in control plants; however, with 250 and 500  μM ZnSO4 both parameters substantial declined (≥50%). For example, with 250 and 500  μM ZnSO4, leaf area respec- tively declined by 52% and 78% compared with values in control plants (Fig. 1a). One week of acclimation reduced the toxic effect of Zn2+, mostly with 250  μM ZnSO4, as with 500  μM ZnSO4, dry mass was similar in acclimated and non-acclimated plants (Fig. 1b). Indeed, with 250 μM ZnSO4, both leaf area and total dry mass in acclimated plants increased by 60% compared with those in the cor- Fig. 1.  Response of tobacco plants to increasing concentration of responding non-acclimated 250  μM ZnSO4 treatment. ZnSO4 in the root zone. (a) Leaf area. (b) Total dry mass. (c) Plant WC. Plant WC declined significantly (P≤0.05) with only 500 μM In six treatments, the plant root systems were exposed increasing Zn2+ ZnSO4 (Fig.  1c), declining from 95% in control plants to concentrations (1, considered as the control treatment, 30, 250, and 500 μM ZnSO4). In the remaining two treatments, 1 week prior to the 88% in non-acclimated plants exposed to 500 μM ZnSO4. treatments (250 and 500 μM ZnSO4), plants were exposed to 30 μM Also for this parameter, the acclimation period had a posi- ZnSO4. Values are mean±standard error (SE) (n=4). Asterisks indicate tive effect, and plant WC in acclimated plants exposed to significant differences between acclimated and non-acclimated treatments. 500 μM ZnSO4 remained similar to that of control plants *P<0.05, **P<0.01. (Fig. 1c). Total chlorophyll and carotenoid concentrations declined with increasing Zn2+ concentration in the root with well-developed cristae and several proplastids with medium, with marked declines at 250 and 500 μM ZnSO4. short lamellae. Cortical and central cylinder cells exhibited Compared with control plants, total chlorophyll concentra- regular shape (Figs 3a and 4a, b). The presence of elevated tion with 250 and 500  μM ZnSO4 was reduced by 65 and Zn2+ in the root zone severely damaged roots. Plants treated 72%, respectively (Fig. 2a); similarly, carotenoid concentra- with 250 μM ZnSO4 had the cells of the cortical layer dam- tions in plants exposed to 250 and 500 μM ZnSO4 declined aged (Fig.  3b), with a tortuous cell wall (Fig.  4c); further- by 54–57% (Fig. 2b). As observed for leaf area and total dry more, some cortical cell had disintegrated cytoplasmic mass, the acclimation period reduced the toxic effect of 250 content with deposits in the cytoplasm (Fig.  4d). Damage and 500 μM ZnSO4, and compared with the values in non- was more evident with 500 μM ZnSO4; at this concentration, acclimated plants, both total chlorophyll and carotenoids roots had the epidermis and most of the cortical cells com- concentrations in acclimated plants increased by 40–90%. pletely destroyed (Fig.  3d), with disintegrated cytoplasmic content. Furthermore, plants exposed to 500 μM ZnSO4 had cells in the central cylinder with collapsed cytoplasmic orga- Acclimation with 30 µM ZnSO4 reduces root damage nelles (Fig. 4g). The acclimation period with 30 μM ZnSO4 In control roots, the cortical cells showed large nuclei and reduced root injuries in response to Zn2+ stress. Acclimated large vacuoles, long endoplasmic reticulum, mitochondria plants exposed to 250 μM ZnSO4 exhibited reduced damage  Root acclimation improves heavy-metal tolerance  |  Page 5 of 12 acclimated plant gs remained similar to gs in control plants (Table 1). Already after 24 h of treatments, the stronger inhi- bition of gs compared with An led to a visible (7%) reduction in the Ci in non-acclimated plants compared with Ci in accli- mated plants (Table 1). It is unlikely that these reductions in gs observed in non-acclimated plants in the first 24 h of treat- ment were associated with specific Zn2+ toxicity at the leaf level; indeed, leaf Zn2+ concentrations in acclimated plants were several fold higher than those in non-acclimated plants, due to the 1-week exposure to Zn2+ prior to the treatment in acclimated plants, while non-acclimated plants were exposed to Zn2+ for only 24 h (Supplementary Fig. S1 and Table S3 at JXB online). Chlorophyll fluorescence results showed that the maximum quantum efficiency of PSII was not affected by 250 μM ZnSO4, independently of the acclimation period (Table 1). Downloaded from http://jxb.oxfordjournals.org/ at Università degli Studi di Firenze on June 17, 2014 Acclimation with 30 μM ZnSO4 is associated with a more negative membrane potential in root cortical cells The effects of the addition of 250 μM ZnSO4 were monitored Fig. 2.  Total chlorophyll and carotenoid concentrations in tobacco to determine whether early changes in the membrane poten- plants in response to increasing ZnSO4 concentrations in the root zone. tial (EM) of root cortical cells could explain the increased tol- In six treatments, the plant root systems were exposed increasing Zn2+ erance in acclimated plants. Immediately after adding ZnSO4 concentrations (1, considered as the control treatment, 30, 250, 500 μM to the medium, the root cortical cells of non-acclimated plants ZnSO4). In the remaining two treatments, 1 week prior to the treatments transiently depolarized by 16.8 ± 3.8 mV (Fig.  5a, c). By (250 and 500 μM ZnSO4) plants were exposed to 30 μM ZnSO4. Values are mean±SE (n=4). Asterisks indicate significant differences between contrast, in acclimated plants, there was an opposite behav- acclimated and non-acclimated treatments. *P<0.05, **P<0.01. iour, and immediately after addition of the stress, there was a transient hyperpolarization of EM (5.6 ± 3.7 mV, Fig.  5b, in cortical cells compared with non-acclimated ones (Fig. 3c), c). We then assessed how EM changed after 24 h of exposure with healthy cells in the central cylinder, although there were to 250  μM ZnSO4, and observed that there were substan- a few cortical cells that had nuclei and cell walls with an tial differences in the EM of cortical cells in acclimated and irregular shape (Fig. 4e, f). With 500 μM ZnSO4, although non-acclimated plants. In the non-acclimated plants, parts in acclimated plants the root epidermis (Fig. 3e) and several of the plant were able to partially repolarize their plasma cortical cells were damaged, in the cortex there were cells membrane, and thus after 24 h of treatment there were no with well-preserved cell organelles (Fig. 4h), and the central significant differences in EM when compared with control cylinder appeared perfectly functional, with cells showing all plants. On the other hand, EM in acclimated plants remained the cellular organelles. hyperpolarized, with values on average 37% more negative compared with those in non-acclimated plants exposed to 250 μM ZnSO4 (–91.2 ± 5.3 mV in non-acclimated plants vs Zinc rapidly affects gs in non-acclimated plants but not –125.1 ± 3.5 mV in acclimated plants, Fig. 5c). in those acclimated with 30 μM ZnSO4 To unravel the mechanism(s) responsible for improved toler- Acclimation with 30 μM ZnSO4 enhances vacuolar ance observed in acclimated plants, two more experiments Zn2+ sequestration in roots were conducted focusing on the 250  μM ZnSO4 treatment. To evaluate the effect of 250 μM ZnSO4 upon CO2 fixation The ability to compartmentalize Zn2+ in the cell vacuole with respect to leaf conductance, these two parameters were provides an effective mechanism to avoid the toxic effects recorded before and after ZnSO4 application (Table 1). After of Zn2+ in the cytoplasm (Hall, 2002; Krämer et al., 2007). 24 h of treatment, although growth was not affected and Given the striking differences in root membrane depolari- there were no visible differences in plant WC (Supplementary zation and hyperpolarization patterns between acclimated Table S2 at JXB online), An declined by 18% in the leaves and non-acclimated plants, we investigated whether these in non-acclimated plants compared with control plants different responses were associated with different abilities (Table 1). By contrast, An in acclimated plants remained sim- to compartmentalize Zn2+ in the roots. In order to evaluate ilar to that of control plants. The negative effects of 250 μM it, confocal laser-scanning microscopy was used to observe ZnSO4 in non-acclimated plants were, however, more evident the intracellular distribution of Zn2+ in root epidermal cells. in the greater inhibition of leaf gs. Indeed, compared with After 24 h of 250  μM ZnSO4, in non-acclimated plants, initial values, in plants without the acclimation period, the most of the accumulated Zn2+ was found to be located prev- addition of 250  μM ZnSO4 decreased gs by 40%, while in alently in the cytosol, with only a few cells showing good Page 6 of 12 | Bazihizina et al. Downloaded from http://jxb.oxfordjournals.org/ at Università degli Studi di Firenze on June 17, 2014 Fig. 3.  Transverse sections of roots of tobacco plants exposed to increasing concentration of ZnSO4 in the root zone. (a) Roots of plants grown in the control nutrient solution for 3 weeks. (b) Roots of plants grown with 250 μM ZnSO4 with no prior acclimation. (c) Roots of plants grown for 1 week with 30 μM ZnSO4 and then exposed to 250 μM ZnSO4. (d) Roots of plants grown with 500 μM ZnSO4 with no prior acclimation. (e) Roots of plants grown for 1 week with 30 μM ZnSO4 and then exposed to 500 μM ZnSO4. Zn2+ compartmentalization in the vacuole (Fig.  6a–c). By (Fig.  6g). Therefore, cytosolic:vacuolar Zn2+ content ratio contrast, in acclimated plants, most of the Zn2+ was located ranged from 0.4 in acclimated plants to 1.9 in non-accli- in the vacuole, indicating an efficient compartmentalization mated plants. of accumulated Zn2+ (Fig.  6d–f). The intracellular spatial distribution of Zn2+ within the epidermal cells was further The acclimation process enhances the expression quantified using ImageJ software (National Institutes of levels of the tobacco orthologue of the A. thaliana Health, Bethesda, MD, USA). In the present work, arbi- MTP1 gene in roots trary but not absolute values for intracellular Zn2+ concen- trations were used, because for comparative purposes this Given that confocal data indicated that acclimated plants had semi-quantitative method has been found previously to be a superior ability to efficiently sequester Zn2+ into root cell perfectly valid (Cuin et al., 2011). As illustrated in Fig. 6g, vacuoles, we measured the transcript levels of the tobacco the cytosolic Zn2+ content in acclimated plants was found orthologue of the A. thaliana MTP1 gene (NtMTP1; for more to be consistently lower (on average by 60%) compared with details, see Shingu et al., 2005). Indeed, in A. thaliana, MTP vacuolar Zn2+, while in non-acclimated plants, the opposite genes have been found to be involved in cellular detoxifica- was observed, and the cytosolic Zn2+ content was found tion and sequestration of Zn2+ in vacuoles, and thus directly to be, on average, double that of the respective vacuole linked to Zn2+ tolerance (Rascio and Navari-Izzo, 2011).  Root acclimation improves heavy-metal tolerance  |  Page 7 of 12 Downloaded from http://jxb.oxfordjournals.org/ at Università degli Studi di Firenze on June 17, 2014 Fig. 4.  Electron micrographs of transverse section of root cells of tobacco plants exposed to increasing concentration of ZnSO4. (a, b) Plants grown in the control nutrient solution for 3 weeks showing a cortical cell (a) and central cylinder cells (b). (c, d) Cortical cells of plants grown with 250 μM ZnSO4 with no prior acclimation period showing a tortuous cell wall (c) and a damaged cell with deposits (d). (e, f) Plants grown for 1 week with 30 μM ZnSO4 and then exposed to 250 μM ZnSO4 showing irregular cell walls (e) and an irregular nucleus (f). (g) Plants grown with 500 μM ZnSO4, with no prior acclimation period, with disintegrated cytoplasmic organules in the cells of the central cylinder. (h) A cortical cell of plants grown for 1 week with 30 μM ZnSO4 and then exposed to 500 μM ZnSO4 with well-preserved cytoplasmic organules. cw, cell wall; de, deposit; nu, nucleus. The transcript levels of NtMTP1 were found to vary in plants whereas it only increased by approximately 1.5-fold in response to increasing Zn2+; thus, in acclimated plants already non-acclimated plants (Fig. 7). at time 0 (i.e. before adding the stress) transcript abundance (arbitrary units) was approximately 2-fold that in control Discussion plants (Fig.  7). The beneficial effects of the acclimation process were still evident following the addition of 250 μM Exposure to non-toxic Zn2+ concentrations resulted in an ZnSO4; in acclimated plants, the transcript abundance fur- improved tolerance of tobacco plants to elevated Zn2+ con- ther increased to approximately 3-fold the values in control centrations. In non-acclimated plants, 250 and 500 μM ZnSO4 Page 8 of 12 | Bazihizina et al. Table 1.  Leaf photosynthetic rate (An ), stomatal conductance (gs ), substomatal CO2 concentration (Ci ), and maximum quantum efficiency of PSII photochemistry (Fv/Fm) in leaves of tobacco plants 24 h after the addition of 250 μM ZnSO4 in non-acclimated plants and in plants acclimated with 30 μM ZnSO4 for 1 week Parameter Treatment ZnSO4 (μM) 1 1–30 1–250 30–250 a,b a,b b –2 An (μmol m s ) –1 4.88 ± 0.22 4.50 ± 0.22 3.98 ± 0.28 4.95 ± 0.18a gs (mmol m–2 s–1) 115 ± 6a 102 ± 0a,b 70 ± 3 b 121 ± 12a –2 Ci (μmol m ) 319 ± 4a 314 ± 3a 297 ± 1b 312 ± 4a Fv/Fm 0.78 ± 0.01a 0.78 ± 0.00a 0.77 ± 0.00a 0.77 ± 0.01a Data are mean±SE (n=4). Different superscript letters within a row indicate significant differences between treatments (P<0.05). Initial values (prior to the treatment) for control and acclimated plants were, respectively: An, 4.9 ± 0.2 and 4.4 ± 0.2 μmol m–2 s–1; gs, 111 ± 2 and 119 ± 8 mmol m–2 s–1; Ci, 312 ± 2 and 301 ± 5 μmol m–3. Downloaded from http://jxb.oxfordjournals.org/ at Università degli Studi di Firenze on June 17, 2014 both treatments roots presented a functioning central cylin- der. By contrast, in non-acclimated plants directly exposed to 250 and 500 μM ZnSO4, roots showed more severe damage, with disruption of the epidermis and cortex, and in the case of 500 μM ZnSO4 also of the central cylinder. In order to elu- cidate the possible mechanism(s) responsible for the enhanced tolerance, short-term experiments were conducted focusing on the responses to 250 μM ZnSO4 in acclimated and non- acclimated plants. These experiments showed that the accli- mation process induced specific detoxification mechanisms at the root level that, following the addition of elevated Zn2+ concentrations in the growth medium, reduced the build-up of Zn2+ in sensitive and metabolically active sites of the cell, ultimately resulting in an improved leaf stomatal regulation and an EM hyperpolarization of the root cortical cells. Root membrane potential data confirmed our hypothesis that the Zn2+ acclimation process led to an improved response at the root level, indicating an improved activity and regu- lation of plasma membrane-located processes. In non-accli- Fig. 5.  (a, b) Zn2+-dependent changes in EM in root cortical cells of mated plants, immediately after the roots were exposed to tobacco plants after the addition of ZnSO4 (with a final concentration of 250 μM ZnSO4, there was a rapid depolarization of the EM, 250 μM ZnSO4 in the medium) in non-acclimated (a) and acclimated (b) with a partial repolarization of the plasma membrane 24 h plants. (c) Mean values±SE (n=15–20) of membrane depolarization and after the treatment. Interestingly, 1 week of acclimation with hyperpolarization in cortical root cells of non-acclimated and acclimated 30 μM ZnSO4 resulted in EM hyperpolarization in root corti- plants. Immediately after adding of ZnSO4 to the medium, transient depolarization occurred in non-acclimated plants (a, c). By contrast there cal cells, immediately after the addition of Zn2+ and for the was a transient hyperpolarization in plants acclimated with 30 μM ZnSO4 1 following 24 h. These results are in agreement with the recent week prior to the addition of ZnSO4, which lasted for the following 24 h (b, finding of a clear link between EM depolarization/hyperpolar- c). Asterisks indicate significant differences between acclimated and non- ization patterns in cortical root cells following Zn2+ addition acclimated treatment. ***P<0.001. (0.1–1 mM) and the overall Zn2+ tolerance in three Arabidopsis species (Kenderesová et  al., 2012). In the above-mentioned was highly toxic over the 3-week treatment period, with a study, the magnitude and duration of the Zn2+-dependent marked decline (>50%) in plant growth. Conversely, a 1-week depolarization was higher in the sensitive A. thaliana than in exposure to non-toxic Zn2+ concentrations prior to the Zn2+ the tolerant Arabidopsis arenosa and Arabidopsis halleri; the treatments substantially reduced the Zn2+ toxic effects both addition of 0.5 mM ZnCl2 depolarized the root plasma mem- at the root and at the shoot level; in acclimated plants, com- brane of A. thaliana by 26 mV, while in the hyperaccumulator pared with non-acclimated ones, there was a 1.6-fold increase A. halleri there was no significant depolarization. in plant dry mass and leaf area and up to a 2-fold increase in The contrasting EM depolarization/hyperpolarization pat- leaf pigment concentrations. Furthermore, at the root level, terns observed in the present study between control and accli- the anatomical analyses clearly showed that the roots of accli- mated plants were probably coupled with the detoxification of mated plants were less damaged by high Zn2+ concentrations. intracellular Zn2+. The intracellular distribution of Zn2+ in root Indeed, although roots of acclimated plants showed ultra- epidermal cells of acclimated and non-acclimated plants sup- structural alterations at the epidermal and cortical level, in ports the hypothesis that the acclimation period elicited specific  Root acclimation improves heavy-metal tolerance  |  Page 9 of 12 Downloaded from http://jxb.oxfordjournals.org/ at Università degli Studi di Firenze on June 17, 2014 Fig. 6.  Zinc compartmentation in root epidermal cells of tobacco plants before (a, d) and after (b,e) the addition of ZnSO4 (with a final concentration of 250 μM ZnSO4 in the medium) in non-acclimated (a, b) and acclimated (d, e) plants. One typical root for non-acclimated (c) and non-acclimated (f) plants is shown. Measurements were made in the mature zone, between 10 and 20 mm from the root apex. In (g), quantification of the cytosolic:vacuolar Zn2+ content ratio in epidermal root cells was evaluated and values are given as means±SE (n=12–16). The Zn2+ content in each cell compartment is proportional to the intensity of FluoZin-3-AM (showed in arbitrary units). Asterisks indicate significant differences between acclimated and non-acclimated treatment. *P<0.05. Page 10 of 12 | Bazihizina et al. analysis using the Conserved Domain Database (http://www. ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). From this analy- sis, it emerged that the functional domains of NtMTP1, AtMTP1, and AtMTP3 are conserved and that these proteins are characterized by a histidine-rich domain, from aa 182 to 232 (Supplementary Fig. S2 at JXB online); this histidine-rich domain has been shown to have a regulatory function on the activity of the protein (Kawachi et  al., 2008). Interestingly, sequence alignments of MTP in A. thaliana and tobacco high- lighted a high similarity between the histidine-rich domain in NtMTP1 and AtMTP3 (Supplementary Fig. S2); it is there- fore plausible that, despite the NtMTP1 sequence being closer to that of AtMTP1, the function of the protein in tobacco may indeed be more similar to that of AtMTP3, thus explain- ing the observed results. However, this statement remains speculative, and further studies are clearly needed to validate Downloaded from http://jxb.oxfordjournals.org/ at Università degli Studi di Firenze on June 17, 2014 Fig. 7.  Relative transcript levels of the tobacco orthologue of the this hypothesis. Furthermore, given that 24 h of exposure to A. thaliana MTP1 gene (NtMTP1) before (prior to the addition of Zn2+) 30 μM ZnSO4 was not sufficient to induce detectable increases and 24 h after the addition of 250 μM ZnSO4 in the root zone. In two in NtMTP1 transcript levels, it would be reasonable to expect treatments, the plant root systems were exposed increasing Zn2+ that a threshold/minimum acclimation period is required for concentrations (1, considered as the control treatment, and 250 μM the pre-exposure to non-toxic levels of Zn2+ to induce accli- ZnSO4). In the remaining treatment, 1 week prior to the treatment (250 μM ZnSO4), plants were exposed to 30 μM ZnSO4. Values are means±SE mation in roots. A similar time- and concentration-dependent (n=4). The mRNA levels of genes for NtMTP1 were determined by real- increase in MTP3 transcript levels has been reported previously time PCR using specific primer pairs and normalized to that of the EF 1-α. in A. thaliana roots exposed to different Zn2+ concentrations, Asterisks indicate significant differences between acclimated and non- with higher transcript levels at higher Zn2+ concentrations and acclimated treatment. **P<0.01. an almost linear increase in the pMTP3::GUS activity during the first 8 d of exposure to 30  μM ZnSO4 (Arrivault et  al., detoxification mechanisms, i.e. enhanced vacuolar Zn2+ seques- 2006). The hypothesis that there is a minimum time required, tration. Conversely, in non-acclimated plants, the addition of depending on the Zn2+ concentration used, to induce acclima- 250 μM ZnSO4 led to larger increases in cytosolic Zn2+ com- tion would explain the contradictory results found in the lit- pared with vacuolar Zn2+. As a result, the cytosolic:vacuolar erature regarding plant acclimation to heavy-metal stress (e.g. Zn2+ content ratio ranged from 0.4 in acclimated plants to Turner and Dickinson 1993; Wisniewski and Dickinson 2003). 1.9 in non-acclimated plants, indicating a correlation between Elevated Zn2+ concentrations in the root zone increased vacuolar Zn2+ sequestration and improved membrane func- the resistance of the CO2 pathway from the atmosphere to tionality following Zn2+ exposure. Indeed compartmentation the sites of carboxylation. Gas-exchange data suggested of metals within the cell and sequestration in the vacuoles is an that Zn2+ treatments, over the 24 h treatment period, mainly effective way to maintain cytoplasmic Zn2+ concentrations as affected stomatal functioning rather than the photosynthetic low as necessary, keeping toxic Zn2+ away from active cellular machinery. Indeed, if the increase in stomatal limitation is the metabolic components (Krämer et al., 2007). dominant cause of the reduction in An (Fig. 2c), then Ci must Maintenance of metal homeostasis in cells and their trans- decrease (Long and Hallgren, 1985; Bednarz et  al., 1998). port across the plasma membrane, tonoplast, and other Therefore, given that in non-acclimated plants the declines in endomembranes is achieved by the activity of specific trans- gs were paralleled by declines in An and Ci, these results would porters and metal pumps. Interestingly, in parallel with the support the view that photosynthesis was limited by the low increases in Zn2+ vacuolar sequestration, we also observed leaf conductance resulting from stomatal closure (Medrano that the acclimation period increased the transcript levels of et al., 2002). Accordingly, when Beta vulgaris was grown with the tobacco orthologue of the A. thaliana MTP1 gene in roots. 100 and 300  μM Zn2+, stomatal limitations accounted for Despite this gene having yet to be characterized in tobacco, in 79–86% of the total photosynthesis reduction, whereas meso- A. thaliana vacuolar MTP has been shown to be involved in phyll conductance accounted only for the remaining 14–21%, the active transport of Zn2+ from the cytosol into the vacu- and non-significant biochemical limitations occurred. Several ole (Kobae et  al., 2004; Arrivault et  al., 2006; Gustin et  al., factors could explain the declines in stomatal opening in 2009; Kawachi et al., 2009). In the present study, the expres- non-acclimated plants. Given that leaf Zn2+ concentrations sion levels of NtMPT1 increased following Zn2+ exposure, in a in acclimated plants were several fold higher than those in dose-dependent manner (Fig. 7), which contrasts with several non-acclimated plants (Supplementary Fig. S1 and Table published studies that clearly show that expression of MTP1 S3), it would appear that the higher gs in acclimated plants, in A. thaliana is not induced by Zn2+ exposure. However, given rather than being associated with an improved detoxification that expression levels of MTP3 have been shown to increase processes, could have been dependent on the improved root following zinc exposure (Kobae et al., 2004; Arrivault et al., plasma-membrane functionality. Disturbed root membrane 2006; Gustin et al., 2009), we performed a protein sequence functionality in the long term may result in increased lipid  Root acclimation improves heavy-metal tolerance  |  Page 11 of 12 peroxidation and increased membrane permeability, which in elongation of Impatiens balsamina. Environmental and Experimental Botany 32, 439–448. turn increase plant water losses or increase membrane resist- Arrivault S, Senger T, Kramer U. 2006. The Arabidopsis metal tolerance ance, thus reducing water uptake (Barceló and Poschenrieder, protein AtMTP3 maintains metal homeostasis by mediating Zn exclusion 1990; Kamaluddin and Zwiazek, 2004; Llamas et al., 2008). from the shoot under Fe deficiency and Zn oversupply. The Plant Journal In a recent study, it was found that heavy metals very quickly 46, 861–879. 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