Response of mitochondrial thioredoxin PsTrxo1, antioxidant enzymes, and respiration to salinity in pea (Pisum sativum L.) leaves.

Mitochondria play an essential role in reactive oxygen species (ROS) signal transduction in plants. Redox regulation is an essential feature of mitochondrial function, with thioredoxin (Trx), involved in disulphide/dithiol interchange, playing a prominent role. To explore the participation of mitochondrial PsTrxo1, Mn-superoxide dismutase (Mn-SOD), peroxiredoxin (PsPrxII F), and alternative oxidase (AOX) under salt stress, their transcriptional and protein levels were analysed in pea plants growing under 150 mM NaCl for a short and a long period. The activities of mitochondrial Mn-SOD and Trx together with the in vivo activities of the alternative pathway (AP) and the cytochrome pathway (CP) were also determined, combined with the characterization of the plant physiological status as well as the mitochondrial oxidative indicators. The analysis of protein and mRNA levels and activities revealed the importance of the post-transcriptional and post-translational regulation of these proteins in the response to salt stress. Increases in AOX protein amount correlated with increases in AP capacity, whereas in vivo AP activity was maintained under salt stress. Similarly, Mn-SOD activity was also maintained. Under all the stress treatments, photosynthesis, stomatal conductance, and CP activity were decreased although the oxidative stress in leaves was only moderate. However, an increase in lipid peroxidation and protein oxidation was found in mitochondria isolated from leaves under the short-term salinity conditions. In addition, an increase in mitochondrial Trx activity was produced in response to the long-term NaCl treatment. The results support a role for PsTrxo1 as a component of the defence system induced by NaCl in pea mitochondria, providing the cell with a mechanism by which it can respond to changing environment protecting mitochondria from oxidative stress together with Mn-SOD, AOX, and PrxII F.

Introduction

Salinity in soil and irrigation water is a serious problem for agriculture in arid and semi-arid regions, and appears to be one of the most important factors of abiotic stress that severely limits plant productivity (Flowers, 2004). Physiological and molecular aspects of plants under salt stress have been widely studied in terms of its effects on plant growth, stomatal conductance, water relations, ion metabolism, photosynthesis, and respiration (Munns, 2005; Ashraf and Foolad, 2007). In particular, the response of photosynthesis to salinity has been extensively studied and shown to depend on the severity and duration of the imposed stress, and its impairment under salinity has been directly related to limitations in CO2 diffusion (stomatal and mesophyll limitations) and also to photosynthetic biochemical limitations (Flexas ; Chaves ). On the other hand, the effect of salt stress on plant mitochondrial respiration has received less attention and there is a lack of consistency in the described effects of salinity as several studies have shown that specific rates of respiration are either enhanced (Schwarz and Gale, 1981; Revenui et al., 1997; Khavari-Nejad and Chaparzadeh, 1998), diminished, or unaffected (Keiper ; Epron ; Koyro ). Moreover, changes in the properties of the mitochondrial electron transport chain in mitochondria isolated from plants under salt stress were studied with the use of specific inhibitors of the respiratory chain (Jolivet ; Fernandes de Melo ). However, the use of these inhibitors has been questioned for measuring the activities of both the cytochrome pathway (CP) and the alternative pathway (AP) (Day ), oxygen isotope fractionation being the most reliable technique for studying the regulation of the electron partitioning between the two respiratory pathways (Lambers ). AP respiration bypasses electron transport through complexes III and IV of the CP, oxidizing ubiquinol directly by alternative oxidase (AOX). It has been hypothesized that AOX may prevent over-reduction of the ubiquinone pool, avoiding the formation of reactive oxygen species (ROS) (Maxwell ; Møller, 2001). Therefore, the AP could have a role in salt stress tolerance or avoidance, but there is no information about changes on the in vivo electron partitioning between the AP and the CP under salt stress conditions.

Under salt stress as well as in other stress situations, the formation of ROS may occur (Hasegawa and Bressam, 2000; Hernández ). This should have negative consequences depending on the intensity and duration of the stress. ROS affect cellular structures and metabolism (Bartels and Sunkar, 2005), but they can also have positive implications due to the possible signalling nature of some of the species produced (Dat ; Gechev ; Noctor ). Plants with high levels of either constitutive or induced antioxidants have been reported to provide resistance against oxidative damage, and several studies have described a correlation between up-regulation of specific antioxidant enzymes and tolerance to abiotic stress, including salinity (Hernández , 2001; Mittova ; Demiral and Turkan, 2005).

Mitochondria have been described as organelles where ROS generation has an important role under different physiological and stress situations including salinity (Hernández ; Jiménez ; Vanacker ). Thus, the existence of different antioxidant systems such as Mn-SOD enzymes, that control O2·– content in the organelle, and the complete ascorbate–glutatione pathway, previously described (Jiménez ), allow this organelle to regulate the internal H2O2 concentration, including that produced in other cell compartments (Chew ). Reports on SOD activity in different plant species under salinity as well as under other stress conditions suggest that different mechanisms operate in oxidative stress injury. It is also noticeable that different isoforms of the same antioxidant enzyme present different activities in lines/cultivars differing in salt tolerance (Olmos ; Ashraf, 2009).

In addition, a thioredoxin/peroxiredoxin (Trx/Prx) system which includes an NADPH-dependent thioredoxin reductase (NTR) and glutaredoxin has been described in plant mitochondria, similar to that in chloroplasts (Bernier-Villamor ; Rouhier ; Finkemeier ). This system is involved in redox homeostasis and may also act as an antioxidant by eliminating hydroperoxides, including H2O2 (Laloi ; Barranco-Medina ). Recently the presence of a new PsTrxo1 in pea leaves localized in mitochondria and the nucleus was reported. Moreover, AOX has been shown to be regulated by the mitochondrial PsTrxo1 (Martí ), and a strong interaction occurs between PsTrxo1 and PsPrxII F (Barranco-Medina ). Further analysis showed that additional mitochondrial proteins are targets of Trxo1, suggesting that PsTrxo1 may also control their redox status. The possible role of Trx and Prx in the response of plants to abiotic stress including salinity and their involvement in plant tolerance to stress has been less studied (Barranco-Medina ; Pulido ; Tripathi ), although a role in redox sensing and signal transduction has been proposed (Rouhier and Jaquot, 2005). It has been shown that the drought-induced chloroplastic Trx CDSP32 was accumulated in chloroplast stroma in response to photooxidative stress (Broin ), reducing the plastidic BAS1 Prx, and limiting the accumulation of alkyl hydroperoxides in the chloroplast (Broin and Rey, 2003). Consequently, Trx may participate in the repair of oxidized proteins during environmental constraints (Dos Santos and Rey, 2006). It has also been described that the light-, ascorbate-, and oxidative stress-dependent regulation of different genes encoding Prxs exhibit distinct patterns, being related to their proposed subcellular location (Horling ). On the other hand, mitochondrial PrxII F transcript and protein levels were up-regulated in leaves of Arabidopsis thaliana and poplar, but not in roots, under cold, salinity, and cadmium stress (Finkemeier ; Barranco-Medina ). These results suggest a role for PrxII F in antioxidant defence. However, no information about changes in mitochondrial Trxo1 under salt stress is available. Moreover, it should be noted that the real function of this Trx in plant mitochondria still remains to be established.

In order to gain more insight into the physiological function of PsTrxo1, transcript and protein levels of the PsTrxo1/PsPrxII F system as well as of AOX and Mn-SOD antioxidant enzymes were determined in pea plants under short- (5 d) and long-term (14 d) salt stress conditions. Simultaneously, leaf stomatal conductance, net photosynthesis, and growth parameters, as well as inorganic ion accumulation and oxidative stress indicators were determined in order to evaluate the physiological status of the plants after the stress imposition. Finally, the in vivo activities of the mitochondrial respiratory pathways were determined as well as those of PsTrxo1 and Mn-SOD.

Materials and methods

Plant material and growing conditions

Pea seeds (Pisum sativum L. cv. Lincoln) were surface-sterilized and germinated in vermiculite as described in Hernández . Plants were grown in growth chambers at 350 μmol quanta m−2 s−1 under a 16/8 h photoperiod and day/night temperatures of 25 °C/20 °C. Relative humidity was ∼40–50%. Five days after sowing, plants were watered with Hoagland nutrient solution. NaCl treatment was applied progressively by supplementing 50, 100, and 150 mM NaCl to Hoagland solution 8, 9, and 10 d after sowing, respectively. From day 10 after sowing, plants were watered with 150 mM NaCl for 5 d or 14 d.

Leaf gas exchange measurements

Maximum net CO2 assimilation (AN) and stomatal conductance (gs) were measured with an open infrared gas-exchange analyser system (Li-6400; Li-Cor Inc., Lincoln, NE, USA) equipped with a leaf chamber fluorometer (Li-6400-40, Li-Cor Inc.). Eight measurements on a fully expanded leaf from eight different plants were carried out on days 5 and 14 after starting the salt treatment at 6–8 h of the light period under a light-saturating photon flux density (PFD) of 1000 mol m−2 s−1 (provided by the light source of the Li-6400 with 10% blue light).

Analysis of mineral content

Oven-dried leaf and root tissues were digested in a microwave Ethos 1 (Milestone, Italy) with HNO3:H2O2 (4:1), and the mineral concentration was determined by inductively coupled plasma spectrometry (ICP) (Iris Intrepid II; Thermo Electron Corporation, Franklin, MA, USA).

Determination of lipid peroxidation, protein oxidation, and H2O2

The extent of lipid peroxidation in fully expanded leaf, root, and isolated mitochondria from fully expanded leaves was estimated by determining the concentration of thiobarbituric acid-reactive substances (TBARS) (Cakmak and Horst, 1991). Protein oxidation (carbonyl protein content) was measured by reaction with 2,4-dinitrophenylhydrazine, as described by Levine . The concentration of H2O2 in a fully expanded leaf and in the root was determined in fresh extract by a peroxidase-coupled assay with 4-aminoantipyrine and phenol as donor substrates as previously described by Frew . Protein was measured by the protein dye-binding method of Bradford (1976) using bovine serum albumin (BSA) as standard.

Quantitative real-time PCR (qPCR)

After 5 d and 14 d of 150 mM NaCl treatment, tissue samples (fully expanded leaves and roots) were taken from control and treated plants. Three biological replicates were analysed in three different experiments. Tissue samples were collected, placed immediately in liquid N2, and stored at –80 °C. Total RNA was extracted from each tissue sample using an RNeasy Plant Mini Kit (Qiagen, Germany) following the manufacturer's protocol. cDNA was obtained from RNA subjected to reverse transcriptase reactions using an oligo(dT) primer and M-MLV Reverse Transcriptase (Promega) followed by RNase H (Invitrogen, USA) treatment according to the manufacturer's instructions. RNA and cDNA were quantified in an ND-1000 spectrophotometer (NanoDrop, USA).

qPCR was performed on a 7500 Real Time PCR System (Applied Biosystems, USA) with SYBR Green Supermix (Bio-Rad, Spain). Each reaction was performed in triplicate using the following conditions: 2 min at 50 °C, 10 min activation at 95 °C, and 40–45 cycles of amplification, depending on the primer pair (15 s at 95 °C; 1 min at 57 °C), followed by the melting curve. The actin gene (GeneBank: X90378) was used as an internal control for the normalization of results. Relative quantity (ΔCT) was calculated using the comparative CT method:

Primers were designed for qPCR using Primer Express v 2.0 (Applied Biosystems, USA) employing the following sequences from the data bank: Trxo1 (accession number AM235208), PrxII F (AJ717306), AOX (X68702), and Mn-SOD (X60170). Primers used are listed in Supplementary Table S1 available at JXB online.

Purification of mitochondria

Mitochondria were purified from pea fully expanded leaves by differential and density gradient centrifugation in discontinuous gradients of Percoll as previously described (Jiménez ). All operations were performed at 0–4 °C.

Western blot analysis

For western blot analysis, equal amounts of proteins from mitochondria isolated from fully expanded leaves of control and salt-treated plants were resolved using SDS–PAGE as described by Laemmli (1970) and transferred onto a nitrocellulose membrane using a semi-dry blotting apparatus (BioRad, Spain). Immunoreaction was carried out by using rabbit polyclonal antibodies against Trxo1 (1:2000) (Martí ), PrxII F (1:3000) (Barranco-Medina ), and Mn-SOD (1:5000), and a mouse monoclonal antibody against AOX (1:50) (Martí ), all of them diluted in TRIS-buffered saline (TBS) containing 1% (w/v) BSA and 0.1% (v/v) Tween-20. The polyclonal antibody against the C-terminal sequence INWKHASEVYEKES of pea Mn-SOD (accession number P27084) was raised in a New Zealand white rabbit by Sigma-Genosys (UK). Goat anti-rabbit and goat anti-mouse antibodies conjugated to alkaline phosphatase (Boehringer Mannheim, Germany) were used as secondary antibodies. The antigen was detected by a colorimetric assay using NBT/BCIP (Roche, Germany) following the manufacturer's protocol. Densitometry of the different bands was performed using an image analyser (Gen Tools, Syngene).

Respiration and oxygen isotope fractionation measurements

Prior to the start of measurement, one or two fully expanded leaves were dark incubated for 30 min to avoid light-enhanced dark respiration. Then, leaves were placed in a 3 ml stainless-steel closed cuvette maintained at a constant temperature of 25 °C using a copper plate and a serpentine around the cuvette with a temperature-controlled water bath.

Changes in the 18O/16O ratios and oxygen consumption were determined with a dual-inlet mass spectrometer system (Delta XPlus, Thermo LCC, Bremen, Germany) as described in Florez-Sarasa . Calculations of the oxygen isotope fractionation were performed as described in Ribas-Carbó , and the electron partitioning between the two pathways in the absence of inhibitors was calculated as described in Guy . The r2 values of all unconstrained linear regressions between –ln f and ln (R/Ro), with a minimum of five data points, were at least 0.995, considered minimally acceptable (Ribas-Carbó ). The electron partitioning to the AP (τa) was calculated as follows:

where Δn, Δc, and Δa are the oxygen isotope fractionation in the absence of inhibitors, in the presence of salicylhydroxamic acid (SHAM), and in the presence of KCN, respectively. In order to obtain Δa, leaves were incubated for 30 min by sandwiching between medical wipes soaked with a water solution of 10 mM KCN. In addition, a piece of medical wipe wetted with 10 mM KCN was placed in the cuvette. In order to obtain Δc, leaves were cut into slices and incubated for 45 min in a 25 mM SHAM solution prepared from a stock of 1 M SHAM in dimethylsulphoxide. All stock solutions were freshly prepared before use. A minimum of three replicates were performed and the values obtained were 30.7±0.2‰ for Δa and 20.1±0.2‰ for Δc.

The individual activities of the CP (vcyt) and AP (valt) were obtained by multiplying the total respiration rate (Vt) by the partitioning to each pathway as follows:

Trx activity assay

The insulin reduction method was used to measure the Trx activity in purified mitochondria. The processing of 300 g of leaves was necessary to obtain enough isolated mitochondria (from six gradients as described above) to assay the activity. The method is based on the protein disulphide bond reductase activity of the Trx system. Insulin disulphide bonds can be reduced by Trx, leading to the precipitation of reduced insulin which was followed by measuring the increase in turbidity at 650 nm for 1.5 h at 30 °C as described by Martí . In brief, the reaction mixture (224 μl) contained 0.1 M potassium phosphate buffer (pH 6.5), 2 mM EDTA, 0.5 mM NADPH, 50 nM of the mitochondrial Trr2 from Saccharomyces cerevisiae, and 0.8 μg of insulin in a 1 cm semi-micro quarz cuvette. The reaction was started by the addition of the mitochondrial sample (100–200 μg of protein).

SOD isoenzymes assay

Separation of SOD isoenzymes from pea mitochondria was performed by non-denaturing PAGE, as previously described (Gómez ). The activities of the different isoenzymes were quantified on an image analyser (Gen Tools, Syngene).

Statistical analysis

The experiment was conducted in a completely randomized design. All experiments were repeated a minimum of three times. The results are the mean of at least three different samples from each experiment. Statistical analysis of the results was carried out according to the Student's t-test.

Results

Effect of salinity on growth parameters, net photosynthesis, and stomatal conductance

The effect of 150 mM NaCl on plant growth was assessed from changes in the length, fresh weight, and dry weight of both the aerial part and roots (Table 1). After 5 d of salt treatment, growth was slightly inhibited, although only significant in terms of length (∼20% for the aerial part and 17% for the root). Changes were more noticeable after continuing the salt treatment for 14 d. At that point, growth decreased by 49% and 21% in the aerial part and roots, respectively. Similarly, fresh weight decreased ∼46% in the aerial part and 30% in roots, whereas their dry weight decreased ∼24% in the aerial part of these plants. Net photosynthesis (AN) and light-saturated stomatal conductance (gs) were measured in leaves from control and 150 mM NaCl-treated plants on days 5 and 14 after the start of treatment. After 5 d of salt treatment, AN and gs were reduced by 51% and 78%, respectively, and further reduced after 14 d to 87% and 93%, respectively (Table 1).

Table 1.

Growth parameters of pea plants and net photosynthesis (AN) and stomatal conductance (gS) of fully expanded leaves of plants irrigated with or without 150 mM NaCl during 5 d and 14 d

		5 d Control	150 mM NaCl	14 d Control	150 mM NaCl
Length (cm)	Aerial part	8.65±0.23	6.96±0.17(*)	14.44±0.35	7.42±0.22(*)
	Root	15.73±0.82	13.04±0.55(*)	17.41±0.87	13.70±0.60(*)
Fresh weight (g plant−1)	Aerial part	0.68±0.03	0.61±0.04	1.55±0.05	0.83±0.04(*)
	Root	0.56±0.04	0.53±0.04	0.66±0.04	0.46±0.02(*)
Dry weight (g plant−1)	Aerial part	0.083±0.006	0.076±0.011	0.17±0.009	0.13±0.008(*)
	Root	0.067±0.014	0.068±0.007	0.054±0.003	0.055±0.004
Water content (%)	Aerial part	88.0	89.2	89.2	87.4
	Root	82.7	78.1	88.4	86.4
AN (μmol CO2 m−2 s−1)	Fully expanded leaves	23.8±1.3	11.6±1.2(*)	23.9±0.5	3.3±1.1(*)
gS (mol H2O m−2 s−1)		0.418±0.030	0.092±0.005(*)	0.459±0.018	0.032±0.007(*)

Values are means ±SE, n≥15. Differences are significant at P <0.05 (*).

Mineral content in fully expanded leaves and roots under salt stress

The analysis of macro- and micronutrients in fully expanded leaves and in roots did not reveal any significant change after 5 d of 150 mM NaCl treatment (Table 2). Longer exposure to salt treatment produced a significant increase in Na concentration both in fully expanded leaves and in roots (∼18-fold and 3.5-fold, respectively), combined with a significant decrease in the Cu, P, and Ca concentration in leaves (∼1.5-fold) and in S, K, and Ca concentration in roots (∼1.9-, 1.5-, and 1.9-fold, respectively) (Table 2).

Table 2.

Nutrient content in fully expanded leaves and in roots of pea plants irrigated with or without 150 mM NaCl during 5 d and 14 d

	Fe (ppm)	Cu (ppm)	Mn (ppm)	Zn (ppm)	P (%)	S (%)	Na (%)	K (%)	Ca (%)	Mg (%)
Leaf 5 d
Control	276.1±56.1	18.0±1.5	100.1±30.0	38.2±10.7	0.74±0.05	0.47±0.05	0.15±0.03	2.82±0.20	0.45±0.09	0.47±0.06
150 mM NaCl	225.1±37.5	14.5±1.5	107.9±16.9	30.6±6.2	0.61±0.09	0.38±0.03	1.78±0.75	2.55±0.22	0.41±0.04	0.60±0.09
Root 5 d
Control	1300.0±196.9	41.6±3.2	265.3±82.6	32.6±10.2	0.88±0.10	0.97±0.13	0.50±0.03	6.10±0.17	0.29±0.06	0.67±0.11
150 mM NaCl	914.3±91.0	40.1±3.3	101.1±18.2	39.3±14.2	0.99±0.08	1.08±0.12	2.25±0.42	5.67±0.60	0.20±0.02	0.62±0.10
Leaf 14 d
Control	1072.3±733.0	15.3±2.1	144.8±17.0	45.7±5.4	0.67±0.02	0.84±0.18	0.28±0.06	3.11±0.15	0.96±0.16	1.23±0.37
150 mM NaCl	629.0±179.5	9.9±1.1(*)	121.5±16.4	37.3±3.6	0.45±0.03(*)	0.35±0.01	5.08±0.87(*)	2.39±0.06	0.49±0.01(*)	1.07±0.04
Root 14 d
Control	1237.9±237.4	49.4±8.0	298.7±87.0	35.5±4.1	1.12±0.05	1.44±0.11	0.60±0.12	6.53±0.30	0.48±0.06	0.87±0.10
150 mM NaCl	2153.0±504.5	44.0±6.1	70.2±7.2	50.6±9.7	1.06±0.10	0.75±0.07(*)	2.10±0.19(*)	4.36±0.29(*)	0.25±0.02(*)	0.84±0.10

Values are means ±SE, n≥3. Differences are significant at P <0.05(*)

Changes in H2O2 content, lipid peroxidation, and protein oxidation by salinity

H2O2 content did not show any significant change with the short-term salt treatment either in leaves or in roots. However, with the longer salt treatment, H2O2 showed a tendency to increase in leaves whereas a significant increase was observed in roots (∼2.2-fold). The extent of lipid peroxidation, measured as the formation of malondialdehyde, revealed a significant decrease in roots after 5 d of treatment, whereas this parameter did not change in plants treated for 14 d when compared with controls. The level of protein oxidation, measured as the content of carbonyl proteins, only revealed a significant increase in leaves from plants growing for 14 d in the presence of 150 mM NaCl (Table 3). However, in isolated leaf mitochondria, the short NaCl treatment but not the long one provoked an increase in both lipid peroxidation and protein oxidation (Table 4).

Table 3.

H2O2 content, lipid peroxidation, and protein oxidation in fully expanded leaves and in roots of pea plants irrigated with or without 150 mM NaCl during 5 d and 14 d

		5 d Leaf	Root	14 d Leaf	Root
H2O2 (nmol g−1 FW)	Control	0.39±0.10	0.07±0.01	0.27±0.04	1.20±0.17
	150 mM NaCl	0.36±0.10	0.17±0.04	0.40±0.03	2.61±0.26(*)
Lipid peroxidation (nmol g−1 FW)	Control	1.4±0.1	1.9±0.2	1.7±0.2	1.1±0.1
	150 mM NaCl	1.2±0.1	1.1±0.1(*)	1.6±0.2	0.9±0.1
CO-proteins (nmol mg−1 protein)	Control	5.8±0.54	16.8±2.6	2.8±0.2	8.8±0.8
	150 mM NaCl	4.8±0.15	15.9±1.8	4.9±0.8(*)	6.4±0.8

Values are means ± SE, n≥3. Differences are significant at P <0.05 (*).

Table 4.

Lipid peroxidation and protein oxidation in mitochondria isolated from leaves of pea plants irrigated with or without 150 mM NaCl during 5 d and 14 d

		5 d	14 d
Lipid peroxidation (nmol ml−1)	Control	4.40±0.24	5.56±0.53
	150 mM NaCl	5.30±0.33(*)	5.11±0.32
CO-proteins (nmol mg−1 protein)	Control	1.04±0.05	0.82±0.30
	150 mM NaCl	1.52±0.17(*)	1.22±0.46

Values are means ±SE, n≥3. Differences are significant at P <0.05 (*).

Response of antioxidant enzyme transcript levels to salt stress

The study of Mn-SOD, PrxII F, Trxo1, and AOX gene expression in fully expanded leaves by quantitative PCR after 5 d of salt treatment revealed that PrxII F and Trxo1 were significantly up-regulated (∼1.5-fold and 2.5-fold in their expression, respectively) in fully expanded leaves (Fig. 1). At 14 d of salt treatment, Trxo1, AOX, and PrxII F were down-regulated, especially the last two genes (Fig. 1).

Fig. 1.

Expression pattern of several mitochondrial genes in fully expanded pea leaves from plants irrigated with 150 mM NaCl during 5 d and 14 d. Determination of mRNA levels was performed by real-time RT-PCR. Values are expressed as relative expression against the actin gene and normalized to control treatment plants. Bars show means ±SE (n=4) and asterisks denote significant differences with a P-value <0.05.

Effect of salinity on mitochondrial protein levels

Immunoblotting was used to assess the relative abundance of Trx, Prx, Mn-SOD, and AOX in mitochondria isolated from fully expanded leaves of plants irrigated with 150 mM NaCl during 5 d and 14 d. The reduced AOX form (∼35 kDa) was detected as a double band in the isolated mitochondria (Fig. 2), possibly corresponding to different isoforms of the AOX protein. The quantification of these bands revealed that both of them increased after 5 d and were more pronounced at 14 d of salt treatment, although the band with higher mobility was the one with a higher increase (∼1.5-fold at 5 d and 2.5-fold at 14 d, relative to control). The analysis of the Trxo1 protein level after immunoblotting revealed an increase only at 14 d of salt treatment (∼1.5-fold), while Mn-SOD and PrxII F protein levels did not change either with short- or with long-term salt treatment.

Fig. 2.

Immunoblotting of Trxo1, Mn-SOD, AOX, and PrxII F proteins from mitochondria isolated from fully expanded pea leaves of plants irrigated with 150 mM NaCl during 5 d and 14 d. Mitochondrial proteins were separated by SDS–PAGE, immunoblotted with the relevant antibodies, and visualized by colorimetry.

Effect of salinity on the mitochondrial thioredoxin activity

The capacity for reduction of insulin in the presence of NADPH/TR was used to assay the Trx activity in isolated mitochondria from leaves of plants grown in the presence and absence of NaCl. The short salt treatment (5 d) provoked no change in the Trx activity (Fig. 3), whereas a significant increase in this activity (20% increase related to the value in the control plants) was observed in mitochondria isolated from plants grown in the presence of 150 mM NaCl for 14 d.

Fig. 3.

Trx activity in mitochondria isolated from pea plants irrigated with or without (control) 150 mM NaCl during 5 d and 14 d. Values are a percentage of control activity and asterisks denote significant difference with a P-value <0.05.

Changes in the mitochondrial Mn-SOD activity after NaCl treatments

The activity of the Mn-SOD isoenzyme was visualized in polyacrylamide gels (Fig. 4) after electrophoresis of the mitochondrial proteins isolated from leaves of plants grown in the presence and absence of 150 mM NaCl for 5 d and 14 d. The quantification of the band revealed that neither short- nor long-term salt treatment provoked changes in the activity of this isoenzyme.

Fig. 4.

Mn-SOD activity in mitochondria isolated from pea plants irrigated with 150 mM NaCl during 5 d and 14 d.

Leaf respiration and electron partitioning

Oxygen isotope fractionation during respiration was measured in leaves of control and 150 mM NaCl-treated plants on days 5 and 14 after the start of treatment. After 5 d of 150 mM NaCl treatment, leaf total respiration (Vt) and electron partitioning through the AP (τa) were not significantly affected (Fig. 5A). However, Vt decreased by 35% after long-term (14 d) exposure to salt treatment (Fig. 5B). At that point, τa was significantly increased, mainly due to a 50% reduction of the CP activity (vcyt) while the activity of the AP (valt) remained constant (Fig. 5B). In parallel, the capacity of the AP was measured in leaf discs as cyanide-resistant respiration and was found to increase from 0.3 μmol O2 m−2 s−1 in control plants to 0.5 μmol O2 m−2 s−1 in plants grown for 14 d in the presence of salt.

Fig. 5.

Total respiration (Vt), electron partitioning to the alternative pathway (τa), cytochrome pathway activity (vcyt), and alternative pathway activity (valt) of fully expanded leaves from pea plants irrigated with (black bars) or without (white bars) 150 mM NaCl during 5 d (A) and 14 d (B). Bars represent means ±SE of five replicates and asterisks denote significant differences between control and 150 mM NaCl-treated plants with a P-value <0.05.

Discussion

Plant growth limitation under salinity can be due to either the effect of ions on metabolism or adverse water relations. The response of plants to an excess of salt is quite complex and implies several changes in morphology, physiology, and metabolism, depending on many factors including the intensity of the stress, the natural capacity of plants to cope with stress situations, and the response or acclimation by induction of defence systems and metabolites which diminish dangerous or even deleterious effects (Munns and Tester, 2008). Salinity induced a significant decrease in growth, especially after 14 d, which can be due to changes in the ion balance, water status, mineral nutrition, lower efficiency of photosynthesis, and the amount and use of carbon (Munns, 1993; Flexas ; Taylor ). In this situation, a decrease in absorption of calcium, potassium, and sulphur ions and an increase in sodium was observed. A similar decrease in Ca2+ and K+ and increase in Na+ have been described in leguminous plants including pea and cowpea under salinity, and this accumulation could be involved in the turgor adjustment as previously reported, although such accumulation of Na+ may produce important toxic effects and cell damage (Hernández , 1999). Ca2+ has been described to be involved in the signalling response to abiotic stress including drought and salinity and affecting important processes such as those involved in the regulation of inward K+ channels in stomatal guard cells (Zou et al., 2010). It was also described that Na+ more than Cl– produced, among others, the inhibition of specific SOD isoforms in protoplasts (Hernández ). Although changes in nutrients could affect the stomatal closure, a direct action under the present conditions cannot be attributed to the stomatal regulation. Previous studies have shown that the salt-induced reduction of gs can be used as an indicator of the level of the stress (Hernández , 2000; Flexas ). After 5 d of NaCl treatment, gs was reduced to levels indicating a mild stress, whereas after 14 d, gs was further decreased to <0.05 mol H2O m−2 s−1, indicating a severe situation (Flexas ). It is also well documented that photosynthesis is one of the main processes affected by salinity (Lawlor, 1995; Munns, 2002). According to this, AN was reduced by 51% and 87% after 5 d and 14 d of NaCl treatment, respectively. Similar reductions in gs were previously observed by Hernández et al. (1999) in the Puget variety of pea plants subjected to 150 mM of NaCl for 14 d, with similar growth reductions, suggesting that cv. Lincoln, used in the present study, has similar salt tolerance to the moderately tolerant cv. Puget.

Previous studies showed that NaCl provoked oxidative stress in different pea leaf cell compartments, with significant differences in oxidative parameters based on the compartment studied (mitochondria, chloroplasts, and apoplast), the intensity of the salt stress applied, and the pea variety (Hernández , 1994, 1995, 2001; Gómez , 2004). An excess of ions introduced into cells together with a decrease in stomatal conductance can induce the formation of ROS, disrupting cellular homeostasis and causing oxidative stress. The latter can be determined by increases in lipid peroxidation, protein oxidation, and H2O2 content (del Río ; Corpas ; Olmos ; Hernández , 2001; Mittler, 2002; Gómez ). Although after 14 d of salt treatment photosynthesis and gs indicated that a severe stress was applied, leaf oxidative stress was only moderate as no significant increases in leaf H2O2 content and lipid peroxidation were observed. Similarly, no important oxidative stress was observed in roots in spite of the H2O2 increase in the long-term treatment, this H2O2 being involved in other processes related to growth relay or signalling, although this aspect requires further studies. This is in agreement with growth parameters, indicating that the cultivar studied is moderately tolerant to the applied salt stress. When the oxidative stress indicators were analysed in mitochondria, the increased protein oxidation and lipid peroxidation found under the short-term treatment was not further augmented by a longer duration of the stress, supporting that the cultivar was adapted to the NaCl conditions, as previously reported (Hernández ).

Redox regulation of enzyme activity is essential for the mitochondrial function together with the antioxidant capacity because it provides a versatile mechanism to adapt mitochondria metabolism and signalling to environmental changes. Mitochondrial Trxs are key players in this regulation (Pedrajas ) and may also act as an antioxidative molecule scavenging free radicals, reducing H2O2, and reactivating proteins inactivated by oxidation (Spector ). PsTrxo1 has recently been described as a protein with a dual targeting to pea mitochondria and the nucleus, and several proteins have been described as its targets, including PrxII F and AOX (Martí ). However, the role of PsTrxo1 and PrxII F in abiotic stress including salinity is still unknown. Salt stress induced PrxII F mRNA levels after 5 d of treatment. The alterations that occur in gene expression following oxidative stress are frequently associated with a regulation of the cellular redox and antioxidant systems. However, the levels of mRNA of all proteins analysed, including PrxII F, decreased after 14 d of treatment. The fact that PrxII F protein levels remained constant throughout the salt treatment despite the observed changes at the level of mRNA highlights the importance of post-transcriptional mechanisms in the regulation of these redox proteins, including mRNA stability, the rate of protein translation, and its stability. In this sense, a strong regulation of PrxII F by unknown post-transcriptional mechanisms was suggested to explain the 3-fold increase in PrxII F levels in response to treatment of A. thaliana with high H2O2 concentration (Finkemeier ). Horling observed no significant changes in Prx II F mRNA levels of A. thaliana with oxidative or light treatments. Furthermore, it has been reported that this PrxII F protein appears to be much more constitutively expressed than other Prxs (Dietz ). However, the results obtained by Barranco-Medina in pea plants only confer a constitutive role for PrxII F in roots, while the expression of the enzyme is more dynamic, at both the protein and transcript levels, in leaves under long-term and moderate NaCl (25–50 mM) and CdCl2 (10 μM) treatments. It is important to note that although transgenic plants of A. thaliana lacking PrxII F presented a decreased in growth in the presence of Cd, in wild-type A. thaliana the levels of PrxII F did not increase in the presence of Cd (Horling ). These controversial results demonstrate the lack of information about the mechanism controlling the behaviour of PrxII F in plants under oxidative stress-inducing conditions, as pointed out by Barranco-Medina . The expression of PrxII F as well as of PsTrxo1 was induced in a transitory way at the beginning of the salt treatment, which could represent an adaptive behaviour against the moderate oxidative stress induced by NaCl at this time and may help in the acclimation of plants to longer NaCl treatment. In fact, mitochondria from plants grown in the presence of 150 mM NaCl for 5 d and not 14 d showed significant increases in the indexes of lipid peroxidation and protein oxidation, which may be a signal for the induced expression of both proteins. In this sense, PsTrxo1 and PrxII F may be involved in preventing the establishment of a more severe oxidative stress in mitochondria during long-term growth under these saline conditions. PrxII F is an important component of plant defence against oxidative stress because of its ability to detoxify H2O2, alkyl hydroperoxides, and peroxynitrite (Horling ; Barranco-Medina ), but also possibly by acting, together with Trxo1, as a ROS receptor in the context of NaCl acclimation. Thus, under the present salt conditions, PsPrxII F could participate in impeding the accumulation of lipid peroxides as observed since Trxo1 would not limit its activity. In this sense, a stronger inhibition of root growth in KO-AtPrxII F seedlings under stress conditions induced by CdCl2 has been observed (Finkemaier et al., 2005), and a principal role for PrxII F in antioxidant defence and possibly redox signalling in plants cells was assigned.

As mentioned above, AOX is another target protein of PsTrxo1, which has been described to regulate the reduction of the disulphide bonds of this protein responsible for the alternative mitochondrial respiration, as well as its capacity (Martí ). Two isoforms of the AOX protein were presumably detected in immunoblots. Although Lennon showed only one band in AOX inmunoblots of mitochondria isolated from pea leaves, different bands have been shown to correlate with the expression of different isoforms of the AOX protein (Finnegan ). Noticeably, Lennon used pea leaves from seedlings at a very early stage of development, which contrasts with the fully expanded pea leaves used in the present study, suggesting that other isoforms could be expressed in mature leaves as previously observed in leaves of bean and tobacco plants (Gonzalez-Meler et al., 2001; Galle ). Nevertheless, the parallel induction of AOX and PsTrxo1 proteins found under salinity (14 d) suggests that the redox modulation of AOX would be active under salt stress and thus its activity would not be limited under these conditions. The effects of salinity on respiration and partitioning between the two main respiratory pathways were only visible after 14 d of salt treatment. It has been described that the respiratory costs of ion compartmentation (i.e. Na+ and Cl–), excretion by salt glands, and synthesis and accumulation of organic solutes increase in leaves under salinity conditions (Munns, 2002), thus increasing the maintenance component of respiration (Schwarz and Gale, 1981; Epron ). The AP has been shown to make an important contribution to maintenance respiration (Florez-Sarasa ), and this is in agreement with the accumulation of Na+ and an increased contribution by the AP to total respiration observed in leaves under longer term salinity conditions, as reflected by an increase in τa. This increase in τa was also observed by Ribas-Carbó under severe water stress conditions where gs decreased below 0.05 mol H2O m−2 s−1, similar to the value found in the plants used in the present study. However, in the present study, Vt decreased after 14 d of salt treatment mainly by a decrease in CP activity (vcyt), whereas AP activity remained constant. This decrease in vcyt is likely to be due to the control exerted by an increase in the ATP/ADP ratio as a result of reduced energy demand for growth (Munns, 2002) concomitant with the observed significant reduction in leaf growth under saline conditions. This observation is in full agreement with the observation by Florez-Sarasa that growth respiration is mainly dependent on vcyt. Nevertheless, vcyt can also be inhibited by natural inhibitors such as carbon monoxide, cyanide, sulphide, allelochemicals, or nitric oxide (·NO) (Peñuelas ; Lambers ). Nitrositative stress and ·NO production occur under salt stress conditions (Valderrama ) and ·NO and ·NO-derived products could regulate mitochondrial respiration (Cassina and Radi, 1996; Millar and Day, 1996) mainly affecting vcyt. On the other hand, the activity of the AP was not increased but maintained, despite an increase in its capacity that was parallel to an increase in Trx activity in the mitochondria, due to the presence of PsTrxo1, without excluding the possibility of the existence of other mitochondrial Trxs as has been described in Arabidopsis (AtTrxo1) and poplar (PtTrxh2) (Laloi ; Gelhaye ). Increased AOX capacity in overexpressing plants has been correlated to greater salt tolerance in Arabidopsis (Smith ), but discrepancies in AOX expression and in vivo activity have also been reported (Lennon ; Guy and Vanlerberghe, 2005; Ribas-Carbó ; Vidal ) and recently discussed (Rasmusson ). In the present study, the reason for this discrepancy could be the existence of a post-translational regulation of AOX activity such as interaction with α-ketoacids (Millar ). Nevertheless, the fact that the AP activity is maintained under salinity conditions may diminish the further accumulation of ROS in mitochondria and reflects the presence of the sustainable active form of AOX, in which, PsTrxo1 could have a role through the reduction of its disulphide bonds. In this sense, the increase in Trx activity observed at 14 d of treatment implied that this protein is active in this condition, and may regulate its target proteins such as PrxII F and AOX under salt stress, or at least is not limiting their functions possibly through redox regulation. In this sense, PsTrxo1, PrxII F, AOX, and the maintained Mn-SOD activity, together with specific enzyme components of the ascorbate–glutathione cycle, which are enhanced under similar NaCl concentrations in mitochondria of pea leaves (Gómez ), can contribute to avoidance of ROS formation and oxidative damage and thus allow the moderate tolerance to salt stress observed in this pea cultivar. Together with this function, mitochondrial Trx may also participate in the regulation of other target proteins involved in the process of photorespiration, such as components P and T of the glycine decarboxylase complex and serine hydroxymethyltransferase, previously described as Trx targets (Martí ).

Taken together, the results pointed to mitochondrial Trx as providing the cell with a mechanism by which it can respond to a changing environment through the modulation of the activity of its target enzymes and probably also protecting mitochondria from oxidative stress together with Mn-SOD, AOX, and PrxII F. The activity level of the Trx was parallel to a decrease in oxidative protein and lipid modification in mitochondria under NaCl stress, whereas the impairment of the Trx function by a reduction of its activity may enhance the susceptibility of plants to stress-induced oxidation under these stress conditions, where an increase in ROS production in mitochondria was previously demonstrated (Jolivet ; Hernández ).

Supplementary data

Supplementary data are available at JXB online.

Table S1.† Primers used in the qPCR experiments.