Roles of enzymes in anti-oxidative response system on three species of chenopodiaceous halophytes under NaCl-stress condition

Abstract

Salinity is a major agricultural problem in the world. Reportedly, the scavenging of reactive oxygen species (ROS) plays a pivotal role for tolerance to salinity stress. While sodium chloride (NaCl) is very toxic for most plant species, some chenopodiaceous halophytes show a dependence on sodium (Na) for growth stimulation. This suggests that chenopodiaceous halophytes have an effective anti-oxidative response system (ARS). In this study, we aimed to research the contribution that anti-oxidative enzymes, super oxide dismutase (SOD), catalase (CAT), ascorbic acid peroxidase (APX), and glutathione reductase (GR) have on the growth stimulation of chenopodiaceous halophytes. We cultivated three species of chenopodiaceous halophytes, Suaeda salsa (L.) Pall., Kochia scoparia (L.) Schrad. and Swiss chard (Beta vulgaris (L.) var. cicla) in various NaCl concentration of 5, 50, 200 and 400 mM. Up to 200 mM, the specimens had no significant decrease in dry weight, although the exogenous Na enhanced the content of Na in the leaves and limited the accumulation of other cations. Moreover, at 200 mM NaCl, S. salsa significantly increased dry weight, but K. scoparia and Swiss chard did not show much growth stimulation. ROS-scavenging enzymes might have played pivotal roles, because up to 200 mM NaCl, all of the plant species did not increase their content of the oxidative stress marker, malondialdehyde (MDA). Among all the species, only CAT activity significantly correlated with dry weight. Additionally, APX activity correlated only with dry weight of Swiss chard. However, the growth stimulation of S. salsa at 200 mM NaCl could not be accounted for by water content, MDA and chlorophyll content. This is the first suggestion of CAT-dependent growth stimulation of chenopodiaceous halophytes under NaCl-stress conditions.

Key words:

• anti-oxidative response system (ARS)
• catalase
• chenopodiaceous halophytes
• photosynthesis capacity
• salinity stress

INTRODUCTION

Salinity is one of the most serious problems for agricultural production (Wahid and Ghazanfar 2006; Belkheiri and Mulas 2013), leading to a limit of food production in the world. More than 23% of irrigated lands have been salinized (Tanji 1990) and its adverse effect is increasing (Ghanem et al. 2008). Excessive sodium chloride (NaCl), a major salt in these lands, causes many types of stress symptoms in plants, such as potassium (K) deficiency (Dodd et al. 2010; Fraile-Escanciano et al. 2010) and osmotic stress (Yang et al. 2008). Therefore, preventing crop yield reductions in salinized areas is one of the solutions to avoid a food crisis while the world population is rising. Cultivating highly salt-tolerant plants is considered an effective way to avoid yield reduction in salinized areas.

Chenopodiaceous plants generally tend to possess higher salt tolerance than other families of plants (Maas and Hoffman 1977), and sodium (Na) is an essential element for some of them (Wang et al. 2004). Moreover many chenopodiaceous halophytes are valuable as agricultural products (Mir et al. 1991; Kirkpatrick et al. 1999). Therefore, chenopodiaceous halophytes are promising plants for effective utilization of salinized irrigated lands.

To elucidate mechanisms to maintain or promote dry matter production of halophytes is considered useful to generate new transgenic salt-tolerant plants. Positive physiological responses to Na for dry matter production such as co-transport of pyruvic acid (Furumoto et al. 2011) and osmotic adjustment (Yang et al. 2008) have been reported, but little is known for chenopodiaceous halophytes about mechanisms by which dry matter production is protected against salinized conditions.

This study focused on the anti-oxidative response system (ARS) in chenopodiaceous halophyte under salinity conditions. Excessive Na enhances osmotic pressure in the rhizosphere and the inside of the plant body, and closes stoma causing emergence of reactive oxygen species (ROS)—singlet oxygen, super oxide radical (O₂ ⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (⁻OH)—diminishing plant growth (Hernández 1995; Asada 1999). Oxygen reduction by electrons emerging from photosystem I is the main source of ROS. Therefore, anti-oxidative enzymes scavenging O₂ ⁻ (initial product) and H₂O₂ (product from O₂ ⁻ in the scavenging process) play key roles to protect plant cell from oxidative stress.

Among the enzymes in ARS, super oxide dismutase (SOD), catalase (CAT), ascorbic acid peroxidase (APX) and glutathione reductase (GR) work not only for effective ROS scavenging but also to maintain photosynthesis capacity (Asada 1999). Reportedly, plants with inherently high antioxidative enzyme activities or highly activated enzymes by gene manipulation showed higher salt tolerance (Mittova et al. 2002; Tseng et al. 2007), suggesting that SOD, CAT, APX and GR play an important role to enhance salt tolerance. Therefore, there have been many reports about plant ARS under salinity conditions. However, plants examined for ARS to salinity have been mostly glycophytes, and little is known for chenopodiaceous halophytes under salinity conditions. Moreover, many chenopodiaceous halophytes are stimulated in their growth by NaCl conditions (Lu et al. 2002; Wang et al. 2001, 2004). It appears that chenopodiaceous halophytes have effective ARS supporting the promotion of growth by salt.

In this study, three species of chenopodiaceous halophytes, Suaeda salsa (L.) Pall., Kochia scoparia (L.) Schrad. and Swiss chard (Beta vulgaris var. cicla (L.)), were cultivated with varying concentrations of NaCl in the medium, and antioxidative enzyme activities were measured. This study unveiled that CAT activity had a strong correlation with the dry weight of the three species examined, suggesting that dry matter production was maintained by H₂O₂ scavenging.

MATERIALS AND METHODS

Cultivation and sampling

Seeds of S. salsa, K. scoparia and Swiss chard were sown and germinated in moistened vermiculite. Fourteen days after sowing, seedlings were transplanted into 4-L plastic pots filled with basic nutrient solution. Components of basic nutrient solution were according to the method of Yamanouchi et al. (1987). At 14 d after transplanting, nutrient solution was gradually salinized with daily increments of NaCl concentration in the order of 5, 50, 200, and 400 mM NaCl to accustom plants to NaCl conditions. Specifically, at 14, 15, 16, and 17 d after transplanting, salinization was started for 400, 200, 50, and 5 mM NaCl plots, respectively. Then, at 17 d after transplanting, final NaCl concentrations of 5, 50, 200, and 400 mM were achieved and this time was defined as the initiation of NaCl treatments. S. salsa and K. scoparia were harvested at 11 and 12 d after initiation of NaCl treatments, respectively. Swiss chard was harvested at 4 d after NaCl treatments because leaves then started to wither. All plants were grown in a greenhouse in the Faculty of Agriculture, Tottori University. In this study, 5 mM NaCl was defined as the control imitating cation contents in the natural soil environment and avoiding Na-deficient stress.

Dry weight, leaf water and chlorophyll contents

Plant samples were separated into leaves, stems including petiole, and roots. After fresh weights of the three parts were measured, stems including petiole and roots were dried in an air-forced oven at 70°C and the dry weight was measured. Leaves were frozen in liquid nitrogen immediately after harvest. A part of the frozen leaves was freeze-dried and the dry matter ratio was calculated to determine total dry weight of leaves. Residues of frozen leaves were put into a deep freeze (–80°C) and stocked for further use.

Leaf water content was calculated by following equation:

(1)

To determine the chlorophyll content of the leaf, a leaf sample was homogenized in a mortar with ethanol (99.5%), adding some quartz sand. Absorbance of extracts was measured at 665 nm and 649 nm using a spectrophotometer (U-1800, HITACHI, Tokyo), and chlorophyll (a + b) content was calculated according to the equation by Wintermans and Mots (1965).

Mineral contents

Mineral contents were determined by the wet digestion method. Dried powered samples of 0.05–0.2 g were put into a flask with 15 mL of acid mixture (sulfuric acid (H₂SO₄):nitric acid (HNO₃):perchloric acid (HClO₄) = 1:10:4) and digested (350°C). Phosphorus (P) content was measured by the vanadomolybdate yellow method. K, calcium (Ca), magnesium (Mg), and Na were determined by an atomic absorption spectrophotometer (Z-2310, Hitachi, Tokyo).

Leaf malodialdehyde content

Malondialdehyde (MDA) content in leaves was measured as 2-thiobarbituric acid-reactive substances (TBARs) following the method of Ghanem et al. (2008). We used only leaf samples, because excessive NaCl impairs electron transport in chloroplasts (Asada 1999). High MDA content indicates that plants were exposed to strong oxidative stress. Specific absorbance (532 nm) of TBARs was calculated subtracting non-specific absorbance (600 nm), and then MDA concentration was determined using its molar extinction coefficient (155 mM⁻¹cm⁻¹).

Crude enzyme extraction

A frozen leaf sample (0.3–0.4g) was ground using mortar and pestle with liquid nitrogen and homogenized with 5 mL 50 mM K-P Buffer containing 1 mM ascorbic acid (pH 7.8). After centrifugation of extracts (4°C, 15,000 g, 30 min), the supernatant was used for an enzyme activity assay.

CAT, APX, GR, and SOD activities

CAT, APX and GR activities in leaves were measured according to the modified method of Tanaka et al. (1982). Specific absorbance of H₂O₂ (230 nm) was measured in 1 mL of assay mixture containing 100 mM K-P Buffer (pH 7.8), 20 mM H₂O₂ and 2% [volume/volume (v/v)] crude enzyme extracts, then CAT activity was determined as the amount of scavenged H₂O₂ per minute. Extinction coefficient (0.04 mM⁻¹cm⁻¹) was used in the calculation.

For measurement of APX activity, specific absorbance of L-ascorbic acid (290 nm) was measured in 1 mL of assay mixture containing 100 mM K-P Buffer (pH 7.8), 0.5 mM L-ascorbic acid and 2 % (v/v) crude enzyme extracts, then APX activity was determined as the amount of oxidized L-ascorbic acid per minute. Extinction coefficient (2.8 mM⁻¹cm⁻¹) was used in the calculation.

For GR activity, specific absorbance of oxidized form of NADPH (340 nm) was measured in 1mL of assay mixture containing 50 mM K-P Buffer (pH 7.8), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.02 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH), 0.02 mM oxidized form of glutathione, and 5 % (v/v) crude enzyme extract, then GR activity was determined as the amount of oxidized NADPH per min. Extinction coefficient (6.2 mM⁻¹cm⁻¹) was used in the calculation.

SOD activity in leaf was measured according to the modified method of Tanaka and Sugahara (1980). One milliliter of crude enzyme extracts in a dialysis membrane was dialyzed in 500 mL of 10 mM K-P buffer (pH 7.8) for 12 h. The K-P Buffer (pH 7.8) was renewed every 3 h. Dialyzed enzyme extracts were used for SOD activity. Specific absorbance of oxidized cytochrome c (550 nm) was measured in 1 mL assay mixture containing 50 mM K-P Buffer (pH 7.8), 0.1 mM EDTA, 0.1 mM xanthine, 10 µM cytochrome c, 5 % enzyme liquid (v/v) and 1.6 × 10⁻² U xanthine oxidase, then the ascent rate of absorbance (v) was determined, where V is the ascent rate of absorbance when 5% enzyme liquid (v/v) was replaced by distilled water. One unit was defined as v/V = 0.5. Activity of SOD was calculated by the following expression:

(2)

Protein assay

Protein content in crude enzyme extracts was measured by the Bradford method (Bradford 1976). Enzyme activity per protein was calculated using the protein content.

Statistical analyses

Statistics software, the SPSS statistical program, version 19.0 (IBM SPSS Inc, Tokyo, Japan) was used. Means were compared by Student-Newman-Keuls test at a 5% level of significance. Correlation coefficients were determined by Pearson test at a 5% level of significance.

RESULTS

Fresh and dry weight of shoot

In S. salsa, shoot dry weight significantly increased in 200 mM NaCl (Fig. 1). K. scoparia and Swiss chard significantly decreased at 400 mM NaCl; however, no significant change was observed at 5–200 mM NaCl. The effect of NaCl treatments on shoot fresh weight was almost the same as that on dry weight.

Figure 1 Effect of sodium chloride (NaCl) treatment on shoot fresh and dry weight of (a) and (d) Suaeda salsa (L.) Pall., (b) and (e) Kochia scoparia (L.) Schrad., (c) and (f) Swiss chard (Beta vulgaris (L.) var. cicla). Means of four replicates are shown with standard deviation. Differences are significant at P < 0.05; Student-Newman-Keuls test.

Leaf water content

Leaf water content of S. salsa tended to increase at 50 mM NaCl and decreased significantly at 400 mM NaCl (Table 1). In K. scoparia, it was significantly increased at 200 mM NaCl but was significantly decreased at 400 mM NaCl. Swiss chard decreased leaf water content at 400 mM NaCl.

Table 1  Effect of sodium chloride (NaCl) treatment on leaf water content (%)

NaCl (mM)	5	50	200	400
Suaeda salsa (L.) Pall.	92.9 ± 0.2 a	93.5 ± 0.3 a	92.9 ± 0.3 a	92.1 ± 0.5 b
Kochia scoparia (L.) Schrad.	86.2 ± 1.2 b	87.8 ± 1.6 ab	88.7 ± 0.3 a	84.1 ± 0.3 c
Swiss chard (Beta vulgaris (L.) var. cicla)	90.7 ± 0.7 a	90.8 ± 0.6 a	89.7 ± 0.3 a	86.5 ± 1.8 b
Means of four replicates are shown with standard deviation. Differences are significant at P < 0.05; Student-Newman-Keuls test.

Chlorophyll content

Leaf chlorophyll (a + b) content in S. salsa did not show a significant difference up to 200 mM NaCl but decreased significantly at 400 mM NaCl (Table 2). On the other hand, K. scoparia showed a continuous decrease in leaf chlorophyll (a + b) content with the increase of NaCl concentration. There was no significant difference in chlorophyll (a + b) content in Swiss chard among treatments.

Table 2  Effect of sodium chloride (NaCl) treatment on leaf chlorophyll content

NaCl (mM)	5	50	200	400
chlorophyll (mg g^−1 FW)
Suaeda salsa (L.) Pall.	0.75 ± 0.09 a	0.68 ± 0.07 a	0.77 ± 0.06 a	0.48 ± 0.06 b
Kochia scoparia (L.) Schrad.	1.91 ± 0.16 a	1.66 ± 0.21 a	1.28 ± 0.22 b	1.03 ± 0.14 c
Swiss chard (Beta vulgaris (L.) var. cicla)	0.80 ± 0.18 a	0.79 ± 0.15 a	0.82 ± 0.08 a	0.74 ± 0.20 a
Means of four replicates are shown with standard deviation. Differences are significant at P < 0.05; Student-Newman-Keuls test. FW, fresh weight.

Mineral contents

In S. salsa, Na content was significantly increased with the increase of NaCl concentration in all parts (Table 3). In the leaf, P, K, Ca, and Mg contents decreased significantly in higher NaCl treatments. Changes of the five elements above were intensive in the leaf, but sluggish in lower parts.

Table 3  Effect of sodium chloride (NaCl) treatment on phosphorus (P), sodium (Na), potassium (K), calcium (Ca) and magnesium (Mg) contents

	P	Na	K	Ca	Mg
	mg g ^−1 DW	mg g^−1 DW	mg g^−1 DW	mg g^−1 DW	mg g^−1 DW
Leaf
Suaeda salsa (L.) Pall.
5	9.52 ± 1.50 a	38.02 ± 2.95 d	39.01 ± 6.55 a	11.23 ± 1.40 a	23.69 ± 1.34 a
50	9.80 ± 1.62 a	89.90 ± 3.79 c	32.62 ± 5.91 a	4.18 ± 0.30 b	12.83 ± 1.93 b
200	8.65 ± 0.76 a	128.82 ± 7.63 b	14.65 ± 1.94 b	3.37 ± 1.10 b	9.32 ± 1.96 c
400	5.56 ± 0.34 b	154.23 ± 7.98 a	13.59 ± 1.54 b	4.16 ± 1.31 b	7.23 ± 1.41 d
Kochia scoparia (L.) Schrad.
5	4.23 ± 0.45 a	8.41 ± 0.85 d	46.34 ± 4.92 a	6.36 ± 0.34 a	10.39 ± 0.45 a
50	4.10 ± 0.49 a	30.61 ± 3.17 c	38.06 ± 4.86 b	3.56 ± 0.43 b	6.64 ± 0.38 b
200	4.54 ± 0.33 a	59.65 ± 3.38 b	25.57 ± 2,36 c	3.58 ± 0.79 b	3.75 ± 0.32 d
400	4.30 ± 0.39 a	71.88 ± 3.41 a	48.38 ± 3.42 a	2.29 ± 0.54 c	4.88 ± 0.40 c
Swiss chard (Beta vulgaris (L.) var. cicla)
5	12.46 ± 1.95 a	25.82 ± 3.25 d	43.29 ± 7.91 a	3.65 ± 0.94 a	5.63 ± 1.03 a
50	13.17 ± 1.34 a	48.41 ± 1.95 c	35.75 ± 8.65 a	2.64 ± 0.56 a	4.72 ± 0.73 a
200	15.42 ± 1.48 a	64.86 ± 5.09 b	38.09 ± 5.71 a	3.60 ± 0.36 a	5.84 ± 0.62 a
400	12.56 ± 1.76 a	85.63 ± 9.89 a	43.14 ± 6.74 a	3.55 ± 1.19 a	5.74 ± 1.00 a
Stem including petiole
S. salsa
5	9.69 ± 2.23 a	33.00 ± 7.73 b	29.86 ± 6.40 a	7.95 ± 1.70 a	5.83 ± 1.48 a
50	8.41 ± 2.36 a	44.67 ± 5.72 b	18.44 ± 3.90 b	2.87 ± 0.60 b	3.45 ± 0.32 b
200	7.27 ± 1.13 a	71.78 ± 11.39 a	9.40 ± 2.19 c	2.56 ± 0.48 b	2.67 ± 0.49 b
400	8.00 ± 0.47 a	81.17 ± 6.10 a	8.82 ± 1.76 c	4.41 ± 0.61 b	4.46 ± 1.24 ab
K. scoparia
5	5.02 ± 0.27 b	6.94 ± 1.10 c	50.90 ± 7.65 a	2.36 ± 0.14 ab	2.39 ± 0.28 b
50	4.33 ± 0.34 bc	32.11 ± 3.22 b	27.41 ± 8.17 b	1.77 ± 0.34 b	2.07 ± 0.15 b
200	3.81 ± 1.01 c	57.15 ± 3.65 a	6.33 ± 1.85 c	2.12 ± 0.12 ab	1.30 ± 0.16 b
400	6.42 ± 0.67 a	61.89 ± 3.60 a	25.19 ± 7.37 b	2.90 ± 0.89 a	4.28 ± 1.29 a
Swiss chard
5	10.38 ± 1.43 a	25.82 ± 3.07 d	8.69 ± 1.95 a	1.95 ± 0.08 a	1.98 ± 0.59 a
50	10.67 ± 0.57 a	48.41 ± 2.77 c	6.65 ± 1.18 ab	1.22 ± 0.31 a	2.04 ± 0.42 a
200	12.00 ± 1.35 a	64.86 ± 6.34 b	5.35 ± 0.27 b	1.30 ± 0.37 a	1.65 ± 0.21 a
400	12.56 ± 0.40 a	85.74 ± 4.79 a	6.55 ± 0.75 ab	1.97 ± 0.60 a	2.69 ± 0.76 a
Root
S. salsa
5	5.25 ± 0.56 a	4.72 ± 1.42 c	15.30 ± 2.74 a	2.39 ± 0.21 a	2.43 ± 0.46 a
50	5.68 ± 0.68 a	8.73 ± 3.05 c	13.53 ± 4.27 a	1.75 ± 0.22 b	1.88 ± 0.42 a
200	5.99 ± 0.41 a	19.65 ± 2.03 b	13.80 ± 1.55 a	0.87 ± 0.09 c	2.02 ± 0.24 a
400	5.73 ± 0.56 a	28.52 ± 3.47 a	10.46 ± 1.78 a	0.85 ± 0.23 c	1.78 ± 0.27 a
K. scoparia
5	4.79 ± 0.47 a	2.08 ± 0.17 d	19.25 ± 3.34 a	1.60 ± 0.10 a	2.32 ± 0.79 a
50	4.68 ± 0.50 a	8.46 ± 1.76 c	12.89 ± 0.40 b	1.07 ± 0.23 a	1.94 ± 0.56 a
200	4.43 ± 0.23 a	18.58 ± 0.67 b	6.41 ± 1.59 c	1.12 ± 0.08 a	1.21 ± 0.11 a
400	4.83 ± 0.86 a	30.18 ± 5.38 a	5.23 ± 0.36 c	1.21 ± 0.19 a	1.65 ± 0.28 a
Swiss chard
5	7.21 ± 2.41 a	6.31 ± 1.91 d	5.87 ± 1.43 b	1.24 ± 0.30 a	1.86 ± 0.48 a
50	6.38 ± 1.10 a	13.14 ± 4.05 c	4.79 ± 2.51 b	0.93 ± 0.16 a	1.47 ± 0.13 a
200	8.22 ± 1.65 a	20.88 ± 5.79 b	5.76 ± 0.88 b	0.71 ± 0.17 a	2.19 ± 1.53 a
400	7.68 ± 0.37 a	37.09 ± 3.19 a	10.31 ± 1.88 a	1.01 ± 0.46 a	1.78 ± 0.43 a
5, 50, 200 and 400 indicate NaCl concentration (mM). Means of four replicates are shown with standard deviation. Differences are significant at P < 0.05; Student-Newman-Keuls test. DW, dry weight.

In K. scoparia, Na content was significantly increased with the increase of NaCl concentration inall parts. Changes of P, Ca, and Mg affected by NaCl treatments were similar to those in S. salsa, although K content decreased up to 200 mM and increased again at 400 mM in leaf and stem, and continuously decreased with the increase of NaCl concentration in root.

In Swiss chard, only Na content in all parts was affected and increased significantly with the increase of NaCl concentration, but other elements hardly changed with changing NaCl treatments.

MDA content

In S. salsa, MDA content in leaf tended to increase at 50 mM NaCl, but did not show significant differences among NaCl treatments (Fig. 2). In K. scoparia, MDA content was almost constant up to 200 mM and increased significantly at 400 mM NaCl. In Swiss chard, MDA content significantly increased at 400 mM NaCl.

Figure 2 Effect of sodium chloride (NaCl) treatment on leaf malondialdehyde (MDA) content of (a) Suaeda salsa (L.) Pall., (b) Kochia scoparia (L.) Schrad. and (c) Swiss chard (Beta vulgaris (L.) var. cicla). Means of four replicates are shown with standard deviation. Differences are significant at P < 0.05; Student-Newman-Keuls test. FW, fresh weight.

Antioxidant enzyme activities

In S. salsa, SOD activity was not significantly different among NaCl treatments; however, CAT activity significantly increased at 200 mM NaCl (Fig. 3). Activity of APX peaked at 50 mM and dropped significantly at higher NaCl concentrations, and GR activity also peaked at 50 mM, though activities between 5–50 mM were not significantly different, and then dropped significantly to 400 mM NaCl.

Figure 3 Effect of sodium chloride (NaCl) treatment on antioxidative enzymes (A) super oxide dismutase (SOD), (B) catalase (CAT), (C) ascorbic acid peroxidase (APX), (D) glutathione reductase (GR) activity in leaves. (a), (b) and (c) indicate Suaeda salsa (L.) Pall., Kochia scoparia (L.) Schrad. and Swiss chard (Beta vulgaris (L.) var. cicla), respectively. Means of four replicates are shown with standard deviation. Differences are significant at P < 0.05; Student-Newman-Keuls test.

In K. scoparia, SOD activity increased significantly at 200 mM NaCl. CAT activity decreased gradually and significantly with the increase of NaCl concentration. There was no significant change in APX or GR activity.

In Swiss chard, SOD activities were significantly higher at 50 mM NaCl, and those were decreased gradually and significantly toward higher NaCl concentrations. CAT activities significantly decreased at 200–400 mM NaCl. The activity of APX was almost constant among NaCl treatments and GR activity significantly decreased only at 200 mM NaCl.

Table 4 shows correlation coefficients between leaves or shoot dry weights and activities of anti-oxidative enzymes. Significant correlations were shown in the relationships between both dry weights and CAT activity in all three species, and between those and APX activity in Swiss chard.

Table 4  Correlation coefficients between (a) leaves, (b) shoot dry weights and antioxidative enzyme activities in leaves

(a)
	SOD	CAT	APX	GR
Suaeda salsa (L.) Pall.	–0.121	0.657**	0.051	–0.148
Kochia scoparia (L.) Schrad.	–0.147	0.522*	0.057	–0.259
Swiss chard (Beta vulgaris (L.) var. cicla)	0.181	0.686**	0.620*	0.394
(b)
	SOD	CAT	APX	GR
S. salsa	–0.069	0.609*	–0.019	–0.159
K. scoparia	–0.125	0.515*	–0.055	–0.270
Swiss chard	0.156	0.653**	0.647**	0.376
Crude enzyme was extracted from leaves. Correlation coefficients were calculated from four replicates in four sodium chloride (NaCl) concentrations of 5, 50, 200 and 400 mM respectively (n = 16). * and ** show significant correlation at the P < 0.05 and P < 0.01 level, respectively; Pearson test. SOD, super oxide dismutase; CAT, catalase; APX, ascorbic acid peroxidase; GR glutathione reductase.

DISCUSSION

In this study, we measured activities of antioxidant enzymes, SOD, CAT, APX and GR that play important roles, and observed the relationship between dry matter production and antioxidant enzyme activities. Moreover, mineral contents, chlorophyll content and MDA content were measured to unveil their roles in antioxidant enzymes.

S. salsa significantly increased dry weight at 200 mM NaCl (Fig. 1). Interestingly, water content, however, did not change significantly up to 200 mM NaCl (Table 1). This suggests that growth stimulation at 200 mM NaCl is independent of water content. S. salsa transported more Na to leaf than to stem including petiole (Table 3). Na may have played some role in the leaf. There were, however, competitive inhibitions in nutrient absorption by increasing NaCl concentration in the medium (Table 3). Table 3 shows that leaf P content significantly decreased at 400 mM NaCl, suggesting that a significant decrease in P content was probably a factor to decrease dry weight. Mg is an essential element to synthesize chlorophyll (Cornah et al. 2003), and Mg content decreased continuously up to 200 mM NaCl. Chlorophyll (a + b) content was maintained up to 200 mM (Table 2), suggesting that Mg was not a limiting factor for chlorophyll synthesis.

Guard cells control turgor to open or close stomata loading K into the cells (Hafsi et al. 2011), while H₂O₂ in guard cell closes stomata (Jannat et al. 2012). Lu et al. (2002) showed that S. salsa decreased transpiration rate with the increase of NaCl concentration in the medium. Leaf K content at 200 mM NaCl decreased to 38% of that at 5 mM NaCl (Table 3). It appears that S. salsa closed stomata due to the decrease of leaf K content, possibly resulting in the decrease of transpiration rate. Reportedly, stomatal closure by K deficiency limits carbon dioxide (CO₂)-fixiation and increase of inverse effects by ROS (Hafsi et al. 2011). Therefore, it appears that antioxidant enzymes reduced inverse effects by ROS and protected from decrease of CO₂-fixation capacity.

Since SOD scavenges O₂ ⁻ having a high reactive character, SOD plays an important role to protect cells from oxidation and maintain photosynthesis capacity (Asada 1999). However, S. salsa showed no significant change in SOD activity (Fig. 3), which could be supported by a report that S. salsa increased Fe-SOD activity by NaCl treatment and Cu-SOD compensatively decreased, resulting in no significant change in total SOD activity (Wang et al. 2004). Catalase functions effectively in H₂O₂ scavenging (Jannat et al. 2012). CAT activity of S. salsa correlated significantly with dry weight (Table 4), suggesting that high CAT activity would be very important for dry matter production. Why did CAT activity correlate with dry weight? Interestingly, MDA and chlorophyll content did not show significant change up to 200 mM NaCl, although ROS causes lipid oxidation and chlorophyll degradation. It was reported that H₂O₂ causes not only cell tissue oxidation but also inhibition of CO₂-fixation (Asada 1999). Enzymes relating to the Calvin cycle, nicotinamide adenine dinucleotide phosphate (NADP⁺) glyceraldehyde 3-phosohate dehydrogenase, fructose 1, 6-bisphosphatase, ribulose 5-phosphate kinase and sedoheptulose 1,7-bisphosphatase are sensitive to H₂O₂ (Kaiser 1979; Tanaka et al. 1982).

It was also reported that exogenous CAT scavenged H₂O₂ and recovered these enzyme activities, resulting in recovery of CO₂-fixation capacity (Asada 1992). Moreover, spinach (Spinacia oleracea L.) belonging to the same family as S. salsa decreased CO₂-fixation capacity when APX activity decreased (Tanaka et al. 1982). Therefore, it appears that scavenging H₂O₂ is necessary for CO₂-fixation in growth stimulation of chenopodiaceous halophytes by NaCl. It was reported that S. salsa increased stability of photosystem II and CO₂-fixation capacity by Na absorption (Lu et al. 2003). This suggests that stimulated CAT activity by NaCl treatment played a pivotal role in CO₂-fixation of S. salsa. Activities of APX and GR didn't correlate with dry weight (Table 4). This suggests APX and GR weren't involved in growth stimulation by NaCl treatment. The reduction of APX and GR activities at 50–400 mM NaCl correlated with water content in leaves. This is similar to the behavior of APX and GR activities in seed of Quercus robur L. in drought-stress environments (Pukacka et al. 2011). It appears that APX and GR activity in S. salsa was affected by water content.

K. scoparia maintained dry weight up to 200 mM. However, increase of Na content and decrease of Mg content in leaf were clearer than those in stem including petiole and root (Table 3). Mg content in K. scoparia leaf was much lower than that in S. salsa, suggesting that decrease of Mg content was a factor to decrease chlorophyll content. Significant decrease of K content in root at higher NaCl concentration suggested inhibition of K absorption by Na (Table 3). Our laboratory previously demonstrated that K. scoparia decreased transpiration with the increase of NaCl concentration in the medium as does S. salsa (unpublished data), suggesting that K. scoparia closed stomata due to K deficiency at least up to 200 mM. This condition possibly promoted ROS emergence; therefore, ARS would have been essential for the maintenance of dry weight.

Activity of SOD in K. scoparia significantly increased at 200 mM NaCl (Fig. 3). Since there was no significant change in leaf MDA content (Fig. 2), it appears that SOD contributed to protect cell tissues from oxidative stress. Activity of CAT in K. scoparia also showed significant correlation with dry weight (Table 4), which might be due to protection from oxidative stress and maintenance of CO₂-fixation like S. salsa. Activity of CAT significantly decreased at 400 mM NaCl while MDA content significantly increased (Fig. 2), suggesting that CAT activity was insufficient to scavenge H₂O₂. Moreover, the decrease of chlorophyll content in K. scoparia leaf was correlated with the decrease of CAT activity, suggesting that CAT protected chlorophyll from oxidative stress like S. salsa.

Changes of APX and GR activities did not accompany those of dry weight or decrease of MDA contents. This suggests that APX and GR are less effective than SOD and CAT in salinity.

Swiss chard trended to increase dry weight at 50 mM (Fig. 1), suggesting that Swiss chard would be a halophilic plant. However, a treatment term of 4 d, was not long enough to observe significant changes. Swiss chard showed increase in leaf Na content; however, there were no significant changes in other mineral contents in leaf (Table 3). It might be due to the shorter treatment term than those of S. salsa and K. scoparia. Accordingly, maintenance of Mg contents leaded to stable chlorophyll content (Table 2).

Activity of SOD in Swiss chard correlated with MDA content, suggesting that SOD contributed to alleviate oxidative stress. Activity of CAT had a significant correlation with dry weight as well as S. salsa and K. scoparia (Table 4), which might be due to the anti-oxidative effect and maintenance of CO₂-fixation. At 400 mM NaCl, loss of CAT activity might be a factor to allow MDA content. Activity of APX in Swiss chard leaf also had a significant correlation with dry weight (Table 4). It seemed that APX localizing in chloroplasts scavenged H₂O₂ which maintained CO₂-fixation capacity. Moreover, APX activity correlated with water content; therefore, maintenance of water content might be essential to preserve APX activity.

Activity of CAT significantly correlated with dry weight in the three species examined; however, APX activity only in Swiss chard correlated with dry weight (Table 4). Therefore, it could be obvious that chenopodiaceous halophytes have common features to enhance CAT activity to preserve dry matter production under NaCl conditions. This prompted the next question: why they elevated CAT but not APX. As previously described, CAT is more tolerant to oxidative stress than APX (Asada 1999). It appears that chenopodiaceous halophytes developed a strategy to enhance CAT activity through the course of evolution, because APX easily loses activity in stress condition. Shikanai et al. (1998) reported that Nicotiana tabacum L. introduced with the gene for CAT of Escherichia coli with low Km value showed maintenance of CO₂-fixation capacity under drought stress conditions with the strength to deactivate APX activity, which supports the idea that CAT in this study contributed to maintenance of CO₂-fixation. Moreover, a recent study unveiled that the AM fungus, Glomus mosseae, enhanced CAT activity in S. salsa at 400 mM NaCl and led to higher salt tolerance (Li et al. 2012). These results support us in hypothesizing that CAT plays a pivotal role in dry matter production.

The result of this study showed that dry matter production of chenopodiaceous halophytes significantly correlated with CAT activity under NaCl conditions. We attributed that to efficient H₂O₂ scavenging, mainly by CAT, which enhanced enzyme activities involved with CO₂-fixation in chenopodiaceous halophytes. This study suggests CAT is a new Na-dependent growth stimulation factor. Providing high CAT activity to chenopodiaceous plants using gene modification could possibly lead to much higher salt-tolerance and yield on the future salinized irrigated lands in the world. It must be further investigated whether increased CAT activity can really contribute to the activities of enzymes relating to CO₂-fixation capacity in the Calvin cycle.

ACKNOWLEDGMENTS

We sincerely thank Mr. Bryan Worrell, Department of Computer Science and Engineering, University of Nevada, Reno, for English corrections.