Introduction

Salinity is a major constraint of crop productivity and can cause both hyperionic and hyperosmotic stress effects resulting in the plant demise (Flowers and Yeo 1995; Hasegawa et al. 2000; Yamaguchi and Blumwald 2005). Most commonly, the stress is caused by high Na+ concentration in the soil solution (Shabala and Cuin 2008; Shabala 2009). Under salinity condition, intracellular Na+ to K+ homeostasis is critical for cell metabolism, and overaccumulation of Na+ to toxic levels may cause the attenuated acquisition of nutrients and metabolic toxicity (Hasegawa et al. 2000; Shabala and Cuin 2008). In plants, as in animals, cell death is a crucial process during development and the stress responses (Pennell and Lamb 1997). Plant programmed cell death (PCD) shares some typical characteristics with mammal apoptosis, such as organelle dysfunction, caspase-like activation, chromatin condensation, and finally, the nuclear and DNA fragmentation (Affenzeller et al. 2009; Wang et al. 2010). Only recently, it has been shown that high salinity leads to PCD in higher plants (Huh et al. 2002; Lin et al. 2005; Shabala 2009).

Production of reactive oxygen species (ROS), which are versatile molecules mediating a diversity of cellular responses and signaling pathways in plant cells, is one of the earliest hallmarks of plant responses to various stresses (Apel and Hirt 2004; Tsanko and Jacques 2005). Higher plants possess nonenzymatic (including ascorbate, glutathione, carotenoids, and tocopherols) and enzymatic antioxidant defense systems [including superoxide dismutase (SOD), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR)] to minimize these deleterious reactions and involve in the detoxification of ROS and the avoidance of resulting damage (Gao et al. 2003; Sharma et al. 2012).

In plants, ROS can be generated by various processes occurring in different cellular compartments (Romero-Puertas et al. 2004; Ashtamker et al. 2007). The spatial, temporal, and quantitative components of ROS signaling may couple to the mechanism of oxidative damage to cellular function and dictate the specificity of stress response (Garnier et al. 2006; Ashtamker et al. 2007). As a plant-specific organelle that is responsible for energy capture, chloroplast could generate superoxide anion (O2 −) and singlet oxygen from photosynthesis system (PS) I and PS II, respectively (Vitaly et al. 2003; Baier and Dietz 2005; Tanaka and Tanaka 2006). Yamane et al. (2004) suggested that the accumulation of excess hydrogen peroxide (H2O2) and hydroxyl free radical (OH·) was responsible for the ultrastructural distortion in rice chloroplasts, and Mitsuya et al. (2003) suggested that NaCl-induced damages in chloroplasts were due to light-dependent oxidative stress. Mitochondria, as the common organelle in animals and plants, have received considerable attention in the investigation of ROS-dependent PCD (Yao et al. 2002, 2004). Intracellular ROS generated from the mitochondrial electron transport chain can directly interact with mitochondrial proteins and lipids, causing their dysfunction (Yao et al. 2002; Chen et al. 2003; Hiroko et al. 2007).

Peppermint (Mentha × piperita L.) is a specialty crop of considerable economic value because its oil is widely used as an additive in cosmetics, pharmaceuticals, and confectionery food (Behn et al. 2010; Santoro et al. 2013). In the soils that contain more than 0.3 % NaCl (about 75 mM), peppermint cannot maintain normal growth, and the biomass and essential oil yield are severely decreased (Clark and Menary 1980). Through years of cultivation and domestication in the saline land, a salt-tolerant peppermint (Keyuan-1) was selected by our laboratory. Our preliminary studies demonstrate that this salt-tolerant peppermint is able to maintain normal growth on saline soil with NaCl concentration of 0.8 % (about 150 mM) (Li et al. 2014a, b) and can be cultivated on the large scale in the Yellow River Delta region of China (Fig. S1). However, the cellular responses and molecular mechanisms of peppermint to NaCl stress have not yet been well clarified, which is extremely important to the development and utilization of peppermint in saline areas.

The aim of our present work is to elucidate some of the cellular events which can be used for the tolerance screening in peppermint under different concentrations of NaCl stress. Based on the results obtained from fluorescence techniques and biochemical approaches, this work provides a new insight into the cellular responses and molecular mechanisms of peppermint to NaCl stress.

Materials and methods

Plant material and chemical reagents

The peppermint (Keyuan-1) used in our experiment is a salt-tolerant species that was cultivated and selected in the saline soil by our lab (Fig. S1). Peppermint (M. × piperita L.) seedlings used for all experiments were grown in pots with 1/2 Murashige & Skoog (MS) solutions in a growth chamber (model E7/2; Conviron, Winnipeg, MB, Canada) with 16-h light photoperiod (120 μmol quanta m−2 s−1) and a relative humidity of 75/80 % at 23/21 °C (light/dark) for 4 weeks.

2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) and MitoTracker Red CMXRos were obtained from Molecular Probes (Eugene, OR, USA). Fluorescein diacetate (FDA) and ascorbic acid (AsA) were purchased from Sigma-Aldrich (Shanghai, China). Caspase-3 inhibitor Ac-DEVD-CHO was purchased from Beyotime Institute of Biotechnology (Zhejiang, China).

Isolation of peppermint mesophyll protoplasts

Peppermint leaves with or without NaCl treatment were collected. Small leaf strips (0.5 to 1 mm) in the enzyme solution (0.4 M mannitol, 20 mM KCl, 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), 10 mM CaCl2, pH 5.7) including cellulase R10 (1–1.5 %) and macerozyme R10 (0.2–0.4 %) (Yakult Honsha, Tokyo, Japan) were vacuum infiltrated for about 30 min and then incubated in the dark for 3 h. After filtration through a 75-μm nylon mesh, the crude protoplast filtrates were sedimented by centrifugation for 3 min at 100g. The purified protoplasts were suspended in W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose, 1.5 mM MES-KOH, pH 5.6) and counted in a hemocytometer.

NaCl treatment

To treat peppermint seedlings, the seedlings of uniform size were transferred to the plastic pots filled with 500 mL 1/2 MS solutions. NaCl treatments were initiated by adding NaCl to 1/2 MS solution to achieve final concentrations of 100, 150, 200, or 250 mM. The nutrient solution was changed every other day. The leaves and roots were harvested at indicated times of NaCl treatment for the protoplast isolation and subsequent experiments. Unstressed plants grown in parallel served as the control and were harvested at the same time.

To treat peppermint mesophyll protoplasts isolated from untreated leaves, NaCl solutions at the indicated concentrations were added to 100 μL of protoplast suspension in 96-well plates and incubated for the required time at room temperature. The control protoplasts were incubated in W5 solution without NaCl treatment and maintained in growth chamber conditions at room temperature during the experimental period.

Confocal microscopy and in vivo imaging of organelles

All microscopic observations were performed using a Zeiss LSM 510 confocal laser scanning microscope (LSM 510/ConfoCor2; Carl-Zeiss, Jena, Germany). H2DCFDA, FDA, and Rh123 signals were visualized with excitation at 488 nm and emission at 500–550 nm using a band-pass filter, and chloroplast autofluorescence (488 nm exCitation) was visualized at 650 nm with a long-pass filter. MitoTracker Red CMXRos signals were visualized in another detection channel using a 543-nm excitation light from a He-Ne laser and a 565–615-nm band-pass filter. All of the quantitative analyses of the fluorescence images were performed using Zeiss Rel4.2 image-processing software.

Viability assay

Isolated mesophyll protoplasts or root tips of NaCl-treated peppermint were incubated with 50 μM FDA for 5 min at room temperature in the dark to determine cell viability. The fluorescence of FDA was observed under the Zeiss LSM 510. For protoplasts, approximately 100 cells were measured for each treatment. For root tips, the intensity of FDA fluorescence was quantified based on an auto microplate reader (BioTek, USA) using an excitation wavelength of 488 nm and an emission wavelength of 521 nm. Twenty-milligram root strips (5~10 mm) were used for each measurement.

Hoechst 33258 staining

Isolated mesophyll protoplasts or root tips of NaCl-treated peppermint were stained with Hoechst 33258 staining solution according to the manufacturer’s instructions (Beyotime Institute of Biotechnology, China). They were observed under a Nikon fluorescence microscope (Nikon USA, Melville, NY, USA) attached to a hydrargyrum lamp with an excitation filter of 330–380 nm and an emission filter of 450–490 nm.

Protein extraction and caspase-3-like activity assay

Caspase-3-like activity was measured using the fluorogenic substrates Ac-DEVD-pNA. At the indicated time points, 0.4-g peppermint seedlings were ground to powder in liquid nitrogen. Then, the samples were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 15 mM NaCl, 1 % Triton X-100, and 100 mg mL−1 phenylmethylsulfonyl fluoride) and incubated on ice with gentle shaking on a level shaker for 30 min. Samples were centrifuged for 5 min at 12,000g and 4 °C, and the supernatants were transferred to new 1.5-mL tubes. Protein concentrations were determined using the Bradford method. Caspase-3-like activity was measured by determining the cleavage of the fluorogenic caspase-3-like substrate Ac-DEVD-pNA using prepared supernatant. The extent of Ac-DEVD-pNA cleavage was measured as the change in A405 because of the release of free fluorescent pNA.

Assay of O2 −, H2O2, and malondialdehyde contents

The rate of O2 − generation was measured by estimating the blue-colored formazan formed by nitroblue tetrazolium (NBT) dye with O2 − (Kholova et al. 2010). Absorbance was recorded at 540 nm with a microplate reader (BioTek, USA), and O2 − content was calculated according to its extinction coefficient (12.8 mM−1 cm−1). H2O2 content was measured via the formation of colored titanium-H2O2 complex (Omoto et al. 2013). Absorbance was measured at 410 nm using a microplate reader (BioTek, USA). The concentration of H2O2 was determined by comparing the absorbance to a standard curve. Malondialdehyde (MDA) content was determined by the thiobarbituric acid (TBA) reaction (Zhang et al. 2006). Absorbance at 532 and 600 nm was determined, and MDA concentration was estimated by subtracting the nonspecific absorption at 600 nm from the absorption at 532 nm, using an absorbance coefficient of extinction (155 mM−1 cm−1).

Antioxidant enzyme activities

APX (EC 1.11.1.11) activity was determined by the absorbance of the reaction mixture at 290 nm. Enzyme activity was calculated by using the extinction coefficient of 2.8 mM−1 cm−1. One unit of APX was defined as the amount of enzyme required to oxidize 1 μM ascorbate min−1. SOD (EC 1.15.1.1) activity was determined by the absorbance of the reaction mixture at 560 nm. One unit of SOD was defined as the amount of enzyme required to inhibit 50 % of the reduction of NBT. GR (EC 1.6.4.2) activity was determined by the absorbance of the reaction mixture at 340 nm. Enzyme activity was calculated using the extinction coefficient of 6.2 mM−1 cm−1. One unit of GR was defined as the amount of enzyme required to oxidize 1 μM NADPH min−1. DHAR (EC 1.8.5.1) activity was determined by the absorbance of the reaction mixture and monitored at 265 nm with a spectrophotometer. Enzyme activity was calculated by using the extinction coefficient of 14 mM−1 cm−1. One unit of DHAR was defined as the amount of enzyme required to form 1 μM ascorbate min−1 (Omoto et al. 2013).

Determination of ascorbate and glutathione contents in isolated chloroplasts

Total ascorbate was determined by the absorption of the supernatant at 525 nm. Total ascorbate content was estimated from the standard curve of l-ascorbate determined using the above methods. Total glutathione content was measured spectrophotometrically by following the change in absorbance at 412 nm (Anderson et al. 1992).

Isolation of mitochondria and measurement of mitochondrial membrane permeability

Mitochondria were isolated from NaCl-treated peppermint leaves using the method described previously (Moller and Rasmusson 2005). Isolated mitochondria were suspended with 0.2 % (w/v) bovine serum albumin (BSA), and the concentration of mitochondrial proteins was adjusted to approximately 0.5 mg mL−1. For mitochondrial membrane permeability detection, the absorbance at 540 nm was determined with a UV spectrophotometer (Li and Xing 2011).

Preparation of isolated chloroplasts

Chloroplasts were isolated from NaCl-treated peppermint leaves using the method described previously (Li et al. 2012). The isolated protoplasts were suspended in 2 mL of 0.05 M Tricine-NaOH buffer (pH 7.5) containing 0.5 M sucrose and 0.1 % BSA and were then ruptured through a syringe (0.5 × 4 cm, Termo). The ruptured protoplast preparations (0.5 mL) were directly layered on top of 15 mL of the linear sucrose gradient (35–60 %, w/w) dissolved in 0.02 M Tricine-NaOH buffer (pH 7.5) and run at 24,000 rpm for 3 h at 4 °C using a Beckman-Spinco SW 25-3 rotor. At the end of the run, 0.5-mL fractions were collected.

Measurement of ROS production in isolated chloroplasts and isolated mitochondria

Isolated chloroplasts or mitochondria (0.5 mg mL−1) were incubation with 5 μM H2DCFDA for 30 min, and the DCF fluorescence intensity was measured with a microplate reader (BioTek, USA) at room temperature. The values at 525 nm were used to determine the fluorescence intensity of DCF.

Chlorophyll content determination

The DMSO extraction technique was used for chlorophyll extraction. Absorbance of the DMSO-chlorophyll extractions and blank (pure DMSO) was measured at 645 and 663 nm, using a microplate reader (BioTek, USA). Total chlorophyll content was calculated by Arnon’s equations.

Net photosynthetic rate and electron transport rate measurement

The net photosynthetic rate (Pn) and electron transport rate (ETR) of the peppermint leaves was measured using a commercially available system (LI-6400; LI-COR, Inc., Lincoln, NE, USA) equipped with the 6400-15 Chamber (1.0 cm in diameter) and artificial illumination (irradiated by a modulated tungsten lamp) in the morning (08:30–10:30). Pn and ETR of the leaves were determined at a leaf chamber with CO2 concentration of 400 ppm after the leaves in the leaf cuvette were irradiated for about 15 min by saturated irradiation of 1000 μmol photon m−2 s−1. The relative humidity (RH) and temperature of the leaf cuvette were about 85 and 22 °C, respectively.

Imaging and measurement of chlorophyll fluorescence parameters

A portable version of an Imaging-PAM Chlorophyll Fluorometer (PAM-MINI, Walz, Effeltrich, Germany) connected to a computer with data acquisition software (ImagingWin v2.0 m, Walz) was used. After NaCl treatment, leaves were dark adapted for 15 min for precise determination of the minimal and maximal fluorescence levels in the dark (Fo and Fm, respectively). The maximum PS II quantum yield (Fv/Fm) was automatically calculated by the ImagingWin software (Walz). Images of the fluorescence parameters were displayed with the help of a false color code, ranging from 0.000 (black) to 1.000 (purple).

Results

Concentration- and time-dependent effects of NaCl treatment on the cell viability of peppermint

The effects of NaCl treatment on cell viability were first investigated in peppermint leaves and roots by using FDA staining (Fig. 1). In mesophyll protoplasts, cell viability declined in a concentration- and time-dependent manner. Under the NaCl concentration of 100 and 150 mM, cell viability was essentially unaffected (Fig. 1a, b). However, compared with the control protoplasts, only about 56 ± 7 and 37 ± 4 % of the mesophyll protoplasts could be stained by FDA at 6 and 12 days after 200 mM NaCl treatment, respectively. Moreover, up to nearly 78 ± 9 and 91 ± 3 % of the protoplasts were scored dead when treated with 250 mM treatment for 6 and 12 days (Fig. 1a, b). In the root cells, the changes of cell viability exhibited the similar results. As shown in the Fig. 1c confocal images, the viable root cells could be stained by FDA, and quantitative analysis of FDA fluorescence was shown in Fig. 1d; 100 and 150 mM NaCl treatment exerted no obvious effect on the cell viability, whereas the FDA intensity of 200 mM NaCl-treated root cells decreased to 44 ± 3 and 25 ± 6 % at 6 and 12 days, respectively, compared to the control (Fig. 1c, d). Furthermore, the FDA intensity decreased by up to 89 ± 5 and 95 ± 2 % at 6 and 12 days under 250 mM NaCl treatment (Fig. 1c, d).

Fig. 1
figure 1

Cell viability of peppermint under different concentrations of NaCl treatment. a, b Mesophyll protoplast viability of peppermint. After various treatments, the mesophyll protoplasts were isolated from peppermint leaves and incubated with 50 μM FDA and observed with a LCSM (a). Scale bars = 100 μm. The viable protoplasts were counted at the indicated times after NaCl treatments (b). c, d Root cell viability of peppermint. After various treatments, the roots were subjected to 50 μM FDA and fluorescence images were obtained by LCSM (c). Scale bars = 50 μm. The FDA fluorescence intensity of viable root cells under the indicated treatment was analyzed (d). Each data point is the mean ± SD of five replicates. Differences were evaluated by two-way ANOVA (Bonferroni posttest, P < 0.05) and are shown by different letters

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Chromatin condensation and caspase-3-like activation under different concentrations of NaCl treatment in peppermint

To further evaluate the status of cell death, morphological examination was performed with Hoechst 33258. Hoechst 33258 stains the condensed chromatin in apoptotic cells more brightly than normal chromatin because of enhanced membrane permeability in apoptotic cells (Affenzeller et al. 2009). As shown in Fig. 2, points with bright blue fluorescence were clearly observed in mesophyll protoplasts and root cells after the exposure to 200 and 250 mM NaCl treatment for 6 and 12 days. Compared with the control group, samples treated with 100 and 150 mM NaCl exhibited the normal appearance containing uniformly stained chromatin with light blue fluorescence (Fig. 2a, b). Subsequently, the caspase-3-like activity in peppermint seedlings was detected by fluorogenic substrate Ac-DEVD-pNA. The results demonstrated that 100 and 150 mM NaCl treatment did not effectively activate the caspase-3-like protease. However, the induction of caspase-3-like activity was detected under 200 and 250 mM NaCl treatment. Under 200 and 250 mM NaCl treatment, the caspase-3-like activity both peaked at 9 days and subsequently decreased with the increase of the treatment time (Fig. 2c). Moreover, the addition of the caspase-3 inhibitor Ac-DEVD-CHO notably alleviated the caspase-3-like activation and the cell viability induced by 200 and 250 mM NaCl treatment (Fig. 2d, e).

Fig. 2
figure 2

NaCl-induced PCD in peppermint. a, b NaCl-induced nuclear condensation in peppermint. After various treatments, the isolated protoplasts (a) and roots (b) were subjected to Hoechst 33258 staining. Fluorescent images were collected via a Nikon fluorescence microscope with an attached hydrargyrum lamp. The points with bright blue fluorescence indicate condensed chromatin. c NaCl-induced caspase-3-like activity tested in an in vitro assay using the caspase-3 substrate. The extracts from peppermint seedlings treated with different concentrations of NaCl at the indicated times were used. Error bars indicate SD values for five replicates. (d, e) Effect of caspase-3 inhibitor Ac-DEVD-CHO on the caspase-3-like activity (d) and cell viability (e) of peppermint under different concentrations of NaCl treatment. After incubation with 100 μM Ac-DEVD-CHO for 3 days, peppermint seedlings were treated with different concentrations of NaCl for 12 days. Then, the protein extracts and isolated protoplast were used for analysis. Each data point is the mean ± SD of five replicates. Differences were evaluated by two-way ANOVA (Bonferroni posttest, P < 0.05) and are shown by different letters

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NaCl-induced ROS production in peppermint

Generation of ROS, such as H2O2 and O2 −, occurs in different cellular compartments and plays crucial roles in the stress response (Apel and Hirt 2004). In NaCl-treated peppermint seedlings, changes of O2 − and H2O2 contents were in a concentration- and time-dependent manner. Under the concentration of 100 and 150 mM NaCl, O2 − and H2O2 contents were essentially unaffected compared with the control, whereas under 200 and 250 mM NaCl treatment, O2 − and H2O2 contents were significantly increased (Fig. 3a, b). At 12 days of 200 mM NaCl treatment, O2 − and H2O2 contents increased by nearly fourfold and tenfold, respectively, compared with control values (Fig. 3a, b). Furthermore, O2 − and H2O2 contents increased by more than 5-fold and 16-fold at 12 days of 250 mM NaCl treatment, respectively (Fig. 3a, b).

Fig. 3
figure 3

Effects of different concentrations of NaCl on the H2O2 (a), O2 − (b), and MDA (c) contents in peppermint. Each data point is the mean ± SD of five replicates. Differences were evaluated by two-way ANOVA (Bonferroni posttest, P < 0.05) and are shown by different letters

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The ROS-induced peroxidation of lipid membranes is a reflection of stress-induced damage at the cellular level (Zhang et al. 2006). An enhanced level of lipid peroxidation, as indicated by MDA content, was observed in NaCl-treated peppermint, clearly indicating an oxidative stress under the effect of NaCl stress. Under the concentration of 100 and 150 mM NaCl, MDA content was essentially unaffected, whereas under 200 and 250 mM NaCl treatment, MDA content was significantly increased compared with the control (Fig. 3c). At 12 days of 200 and 250 mM NaCl treatment, MDA content increased by nearly 3.4-fold and 5.3-fold, respectively, compared with control values (Fig. 3c).

Changes of antioxidation capacities in peppermint under NaCl treatment

NaCl treatment induced the significant increase in the activities of antioxidant enzymes (including SOD, APX, GR, and DHAR) in concentration- and time-dependent manners. Under the concentration of 100 and 150 mM NaCl, activities of SOD, APX, GR, and DHAR all significantly increased. At 9 days of 100 mM NaCl treatment, 1.63-fold, 1.18-fold, 1.91-fold, and 1.53-fold increase was obtained for SOD, APX, GR, and DHAR activities, respectively. At 9 days of 150 M NaCl treatment, 1.72-fold, 1.12-fold, 2.06-fold, and 1.67-fold increase was detected for SOD, APX, GR, and DHAR activities compared with the control, respectively (Fig. 4a–d), whereas under 200 and 250 mM NaCl treatment, the activities initially exhibited slight increase and then notably decreased. At 12 days of 200 M NaCl treatment, SOD, APX, GR, and DHAR activities decreased to 77 ± 4, 103 ± 6, 78 ± 3, and 75 ± 7 % of the control levels, respectively. At 12 days of 250 M NaCl treatment, APX, SOD, GR, and DHAR activities decreased to 28 ± 6, 37 ± 5, 26 ± 3, and 42 ± 7 % of the control levels, respectively (Fig. 4a–d).

Fig. 4
figure 4

Effect of different concentrations of NaCl on the activities of SOD (a), APX (b), GR (c), DHAR (d), and the contents of total ascorbate (e) and glutathione (f) in peppermint. Values represent mean ± SD of five replicates. Different letters indicate statistical difference at P < 0.05 (two-way ANOVA with Bonferroni posttest)

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The increase of total ascorbate and glutathione contents in NaCl-treated peppermint was also detected (Fig. 4e, f). Within the 12-day treatment of 100 and 150 mM NaCl, total ascorbate content was significantly increased along with the extension of time. At 12 days of 100 and 150 mM NaCl treatment, 1.5-fold and 2.5-fold increases were detected compared with the control, respectively; 200 and 250 mM NaCl treatment induced an initial increase and a subsequent decline of total ascorbate content. Under 200 and 250 mM NaCl treatment, total ascorbate content peaked at 6 and 3 days after treatment, respectively, at which 108 ± 3 and 34 ± 5 % increases were obtained, whereas total ascorbate content decreased to 98 ± 3 and 41 ± 4 % at 12 days of 200 and 250 mM NaCl treatment compared with the control, respectively (Fig. 4e). Total glutathione content also exhibited a significant increase within 12 days of 100 and 150 mM NaCl treatment. The total glutathione content peaked at 9 days after 100 and 150 mM NaCl treatment, increasing by 18.5 and 24.4 % of the control. During the 6-day treatment of 200 and 250 mM NaCl, an initial increase and a subsequent decline of total glutathione content were detected. Under 200 and 250 mM NaCl treatment, total ascorbate content peaked at 6 and 3 days after treatment, respectively, increasing by 16 ± 4 and 7 ± 2 %, whereas the total glutathione content decreased to 95 ± 3 and 71 ± 4 % at 12 days of 200 and 250 M NaCl treatment compared with the control, respectively (Fig. 4f).

Effects of exogenous antioxidant on ROS level and protoplast viability of NaCl-treated peppermint

To further establish the function of ROS in the response of peppermint to NaCl stress, the effects of AsA, a natural antioxidant, on ROS amount and cell survival were examined. As shown in Fig. 5, mesophyll protoplasts and roots preincubated with AsA exhibited an apparent decrease in DCF fluorescence than samples without antioxidant treatment at 12 days of 200 mM NaCl treatment (Fig. 5a). Quantitative analysis also showed that, in the presence of AsA, DCF fluorescence intensity in mesophyll protoplasts at 12 days of 200 mM NaCl treatment was much lower than that in mesophyll protoplasts without AsA pretreatment (Fig. 5b, P > 0.05). Furthermore, preincubation of protoplasts with AsA effectively alleviated the NaCl-induced cell death. At 12 days of 200 mM NaCl treatment, nearly 70 ± 6 % of protoplast survival could be observed for protoplasts preincubated with AsA (P > 0.05), whereas protoplast survival was only 34 ± 7 % (P < 0.01) in the absence of AsA compared with control protoplasts (Fig. 5c). This result demonstrated that ROS burst participated and played a vital role in the onset and execution of NaCl-induced cell death.

Fig. 5
figure 5

ROS production and its effect on the cell viability in peppermint under 200 mM NaCl treatment. a Imaging of the effects of AsA on the ROS production in mesophyll protoplasts and roots of peppermint using H2DCFDA staining. Scale bars = 50 μm. b DCF fluorescence intensity of mesophyll protoplasts with or without pretreatment of AsA under 200 mM NaCl treatment. c Effect of AsA on the protoplasts viability under 200 mM NaCl treatment. Before NaCl treatment, peppermint seedlings were pretreated with AsA at the final concentration of 1 mM for 3 days. Values represent mean ± SD of five replicates. Asterisks indicate a significant difference from the control at *P < 0.05 or **P < 0.01 by t test

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Subcellular locations of ROS in NaCl-treated peppermint

To monitor the intracellular ROS localization and dynamics by laser confocal scanning microscopy (LCSM) at the single-cell level in vivo, the mesophyll protoplasts, mitochondria, and chloroplasts of peppermint were first isolated and then subjected to NaCl treatment for 12 h. Under LCSM, an increase in DCF fluorescence was exhibited in mesophyll protoplasts under 200 mM NaCl treatment (Fig. 6a). The double-staining experiments with the mitochondrion-specific marker MitoTracker Red CMXRos and the ROS probe H2DCFDA showed that DCF-stained regions were almost in the mitochondria at 6 h after 200 mM NaCl treatment and then, to some extent, in the cytosol; subsequently, the large DCF-stained regions were found to be mainly localized in the chloroplasts (Fig. 6a). To further clarify the results obtained with these co-localization assays, kinetics of ROS accumulation as reported by DCF were monitored by collecting data separately from the mitochondria and chloroplast regions in individual immobilized protoplast under the time-series scan mode of LCSM. Measurements were collected every 20 min from indicated regions. Data showed that ROS accumulation during 200 mM NaCl treatment occurred in the mitochondria and chloroplast in tandem (Fig. 6b). Taken together, the results above showed that after 200 mM NaCl treatment, DCF reported the real-time accumulation of ROS in the areas of mitochondria and chloroplasts, and the DCF fluorescence first accumulated in the area of mitochondria and, finally, to the region of chloroplasts. Moreover, measurement of the DCF fluorescence intensity in isolated mitochondria and chloroplasts under 200 mM NaCl treatment indicated that mitochondria and chloroplasts were sources of NaCl-induced ROS production in peppermint (Fig. 6c, d).

Fig. 6
figure 6

Spatiotemporal changes in NaCl-induced subcellular accumulation of ROS. a Images of ROS subcellular accumulation. Samples of isolated protoplasts treated with 200 mM NaCl for the indicated time period were double-stained with H2DCFDA and MitoTracker Red CMXRos or chloroplast autofluorescence, respectively, and observed with a LCSM. b Kinetics graphs of DCF signal intensity after 200 mM NaCl application. DCF signals were recorded by Zeiss Rel3.2 image-processing software from the designated subcellular compartments of images with a LCSM in the mode of time series. Scale bars = 10 μm. c, d Measurement of ROS production in isolated mitochondria (c) and chloroplasts (d) under 200 mM NaCl treatment. Data are means ± SD of five independent experiments. Asterisks indicate a significant difference from the control at *P < 0.05 or **P < 0.01 by t test

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Effect of NaCl stress on mitochondrial function in peppermint

As described above, ROS production induced by NaCl occurred in mitochondria. Also, the mitochondria may be important targets for ROS, causing mitochondrial dysfunction (Yao et al. 2004). Thus, alterations in mitochondrial position or shape, one of the indicators of mitochondrial activity, were monitored in Al-treated peppermint (Fig. 7a, b). To visualize the mitochondrial dynamics, protoplasts were loaded with MitoTracker Red CMXRos; a cell-permeant dye contained a mildly thiol-reactive chloromethyl moiety for labeling mitochondria. In control protoplasts, the majority of mitochondria appeared as typical elongated rods or filamentous structures and was evenly distributed in the cytoplasm. However, after 200 mM NaCl treatment, most of the mitochondria had undergone morphological transition with prolonged exposure to NaCl. The mitochondria first became swollen and spherical in shape, then irregularly clumped or clustered, and finally aggregated to be fused within the cytoplasm, which could be alleviated by the addition of AsA to eliminate ROS (Fig. 7a). Mitochondrial swelling can also be detected as a rapid absorbance loss at 540 nm (Li and Xing 2011). Our result showed that treating isolated mitochondria of peppermint protoplasts with 200 mM NaCl led to a progressive decline in their absorbance at 540 nm (Fig. 7b). At 12 h after 200 mM NaCl treatment, the absorbance at 540 nm decreased to 59 ± 4 % of the control sample, and pretreatment with AsA effectively delayed and inhibited the absorbance decrease.

Fig. 7
figure 7

Effect of NaCl on mitochondrial function in peppermint. a, b Transition of mitochondrial morphology and swelling of isolated mitochondria during 200 mM NaCl treatment. Three-dimensional reconstructed images of MitoTracker-stained mesophyll protoplasts treated with 200 mM NaCl with or without pretreatment of 1 mM AsA (a). Scale bars = 10 μm. Mitochondrial swelling is indicated by the changes in absorbance at 540 nm (b). Error bars are ±SD values for five replicates. c, d Disruption of the MTP after NaCl treatment. Images of Rh123 fluorescence in 200 mM NaCl-treated mesophyll protoplasts in the presence or absence of 1 mM AsA (c). Curve of fluorescence intensity of Rh123 in protoplasts treated with 200 mM NaCl with or without pretreatment of 1 mM AsA (d). Scale bars = 10 μm. Error bars are ±SD values for five replicates. Asterisks indicate a significant difference from the control at *P < 0.05 or **P < 0.01 by t test

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As another indicator of mitochondrial activity, the changes in mitochondrial transmembrane potential (MTP) induced by NaCl were examined using Rh123, a specific fluorescent probe to monitor active mitochondria. The mitochondria-specific marker MitoTracker Red CMXRos was also used to confirm that Rh123 was mainly localized to the mitochondria. Under LCSM, control protoplasts were stained extensively with Rh123, the fluorescence of which co-localized with MitoTracker, thus establishing the specificity of Rh123 for mitochondria (Fig. 7c). Under 200 mM NaCl treatment, the protoplasts showed a time-dependent MTP decrease compared with control protoplasts (Fig. 7c). After treatment with 200 mM NaCl for 6 and 12 h, the fluorescence intensity of Rh123 decrease by 31 ± 3 and 59 ± 6 % of control samples, respectively, which can be obviously alleviated by pretreatment with AsA (Fig. 7d).

Effect of NaCl stress on photosynthetic capacity in peppermint

Previous studies demonstrate that altered Pn values can accurately reflect photosynthetic damage under adverse stresses (Zhang and Xing 2008). Pn, which is quantified by measuring rates of CO2 consumption, can reflect the efficiency of CO2 fixation. The results showed that both Pn values exhibited a time-dependent decrease under 200 mM NaCl treatment (Fig. 8a). At 6 and 12 days after 200 mM NaCl treatment, the Pn value decreased to 61 ± 5 and 26 ± 3 % of the control sample, and pretreatment with AsA effectively delayed and inhibited its decrease (Fig. 8a). As parallel experiments, the change of ETR was investigated under 200 mM NaCl stress. In Fig. 8b, ETR value decreased by 39 ± 6 and 62 ± 7 % of the control sample at 6 and 12 days after 200 mM NaCl treatment, which can also be obviously alleviated by pretreatment with AsA (Fig. 8b).

Fig. 8
figure 8

Effect of NaCl on chloroplast function and photosynthetic capacity in peppermint. a, b Pn and ETR values of peppermint leaves with or without pretreatment of 1 mM AsA under 200 mM NaCl treatment. c Three-dimensional reconstructed images of chloroplasts treated with 200 mM NaCl with or without pretreatment of 1 mM AsA. Scale bars = 10 μm. d Total chlorophyll content of peppermint leaves with or without pretreatment of 1 mM AsA under 200 mM NaCl treatment. e, f Imaging and quantitative analysis of chlorophyll fluorescence parameter Fv/Fm of peppermint leaves with or without pretreatment of 1 mM AsA under 200 mM NaCl treatment. The false color code depicted at the right of bottom ranged from 0.000 (black) to 1.000 (purple). Photographs were taken at the same magnification. Data are means ± SD of five independent experiments. Asterisks indicate a significant difference from the control at *P < 0.05 or **P < 0.01 by t test

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Then, changes in chloroplasts morphology and chlorophyll content were monitored. The 3D reconstructed images produced from optical sections of cells showed that at 6 days after 200 mM NaCl treatment, chloroplasts started to become rounded and cells showed unusual morphology (Fig. 8c). Moreover, the transition of chloroplast morphology could be alleviated by pretreatment with the ROS scavenger AsA (Fig. 8c). Under 200 mM NaCl treatment, chlorophyll content exhibited a progressive decline. At 6 and 12 days after 200 mM NaCl treatment, chlorophyll contents decreased by 46 ± 3 and 77 ± 6 % of the control sample, which can also be obviously alleviated by pretreatment with AsA (Fig. 8d).

Subsequently, the photochemical activity of PS II was investigated using chlorophyll fluorescence. The fluorescence parameters Fv/Fm which represent the capacity for the photon energy absorbed by PS II to be utilized in photochemistry under dark-adapted conditions (Chaerle and Van Der Straeten 2000; Baker and Rosenqvist 2004) were analyzed with 200 mM NaCl-treated peppermint leaves. Under 200 mM NaCl treatment, Fv/Fm value exhibited an obvious decline. At 6 and 12 days after 200 mM NaCl treatment, Fv/Fm value decreased to 77 ± 5 and 65 ± 4 % of the control sample, which can also be obviously alleviated by pretreatment with AsA (Fig. 8e, f).

Discussion

This study is an attempt to understand some of the cellular events of peppermint under different concentrations of NaCl stress through the investigation of a cascade of phenomena in NaCl-exposed peppermint cells using fluorescence techniques and biochemical approaches. The results shown in this work provide new insight into the cellular responses and molecular mechanisms of peppermint to NaCl stress.

High concentrations of salts in the soil have a strong inhibitory effect on the growth and harvestable yield of all major crop species (Flowers and Yeo 1995; Yamaguchi and Blumwald 2005). Estimations indicate that more than 50 % of all the arable land will be affected by serious salinization by the year 2050 (Hasegawa et al. 2000). Both ecologists and plant physiologists have long been interested in the effect of salinity on plants, and researches on the mechanisms of salt stress response could be important for future advances (Yamaguchi and Blumwald 2005). The specialty crop, peppermint (M. × piperita L.), is of considerable economic value (Santoro et al. 2013), and a salt-tolerant peppermint species (Keyuan-1) has been screened out by our lab, which can be potentially promoted and cultivated in the saline land on the large scale (Fig. S1). For the further development and utilization of peppermint in saline areas, the cellular signaling and molecular mechanisms of peppermint to cope with NaCl stress are required to be clarified.

General symptoms of damage by salt stress are growth inhibition, accelerated senescence, and the final death (Hasegawa et al. 2000). Previous studies have investigated salt-induced PCD in plants, including apoptotic-like morphological changes (Huh et al. 2002; Lin et al. 2005; Shabala 2009). PCD plays important roles to maintain the proper development and appropriate stress responses, and extensive research has provided increasing evidence that proteases with caspase-like activity exist in plants and mediate processes of cell death in the development and stress responses (Affenzeller et al. 2009; Wang et al. 2010). The present study observed the distinct chromatin condensation and obvious caspase-3-like activation in the cell death of peppermint induced by 200 mM NaCl treatment (Figs. 1 and 2).

Excess salinity leads to oxidative stress in plants through an increase in ROS, such as O2 − and H2O2, which are highly destructive to lipids, nucleic acids, and proteins (Alscher et al. 2002). Our results showed that 100 and 150 mM NaCl essentially unaffected the contents of O2 −, H2O2, and MDA, which were significantly increased in peppermint treated with 200 mM NaCl (Fig. 3). Plants have a number of complex and refined antioxidant regulatory mechanisms to minimize the formation of ROS, thus establishing redox homeostasis under unfavorable environmental conditions (Apel and Hirt 2004). The antioxidation capacities were significantly increased by 100 and 150 mM NaCl in peppermint, while after 200 mM NaCl treatment, antioxidant regulatory mechanisms did not keep in high levels resulting in the severe oxidative burst in peppermint (Fig. 4).

In plants, ROS can be generated by various processes occurring in different cellular compartments (Romero-Puertas et al. 2004; Ashtamker et al. 2007). The spatial, temporal, and quantitative components of ROS signaling may couple to the mechanism of oxidative damage to cellular function and dictate the specificity of stress response (Garnier et al. 2006; Ashtamker et al. 2007). In the present study, mitochondria and chloroplasts were identified as subcellular sites of ROS accumulation in 200 mM NaCl-treated peppermint mesophyll protoplasts, and the accumulation process emerged in the area of mitochondria and regions of chloroplasts with the chronological order (Fig. 6). In the study of adverse stresses, ROS have been recognized as playing crucial roles both in animal and plant cells (Apel and Hirt 2004; Tsanko and Jacques 2005). Our results showed that the elimination of ROS effectively alleviated NaCl-induced cell death (Fig. 5).

Mitochondria, as the common organelles in animals and plants, have received considerable attention in the investigation of ROS-dependent PCD. Intracellular ROS generated from the mitochondrial electron transport chain can directly interact with mitochondrial proteins and lipids, causing their dysfunction (Yao et al. 2002; Chen et al. 2003; Hiroko et al. 2007). In animal and plant cells, the significance of mitochondrial morphology and MPT, the two indicators of mitochondrial activity, has been well documented during PCD (Kroemer 1999; Jeffrey 2006). Here, our results showed that the mitochondrial swelling and decrease in MTP occurred in peppermint mesophyll protoplasts treated with 200 mM NaCl, which could be effectively alleviated by the antioxidant molecule AsA (Fig. 7).

As the specific organelle that is responsible for energy capture in plants, chloroplasts, on the one hand, are the prime site of ROS production; on the other hand, they are the sensitive targets of ROS attack and may generate intermediate signals involved in plant stress responses and programmed cell death (Vitaly et al. 2003; Baier and Dietz 2005; Tanaka and Tanaka 2006). In our case, peppermint mesophyll protoplasts treated with 200 mM NaCl exhibited a decline in photochemical efficiency and alteration in the structure of chloroplasts and cell morphology, which could be inhibited by preincubating the protoplasts with AsA (Fig. 8).

In conclusion, our data elucidated that the salt-tolerant peppermint can tolerate 150 mM NaCl treatment and showed the execution of PCD, the ROS dynamics, and the behaviors of organelles especially mitochondria and chloroplasts which can be used for the tolerance screening in the cellular responses of peppermint to 200 mM NaCl stress. Our study contributed to the understanding of the cellular signaling events and molecular mechanisms of NaCl-induced biological responses in peppermint, providing the theoretic basis for the further development and utilization of peppermint in saline areas.