Introduction

 

Purslane (Portulaca oleracea L.) is a kind of common weeds on the earth (Jin et al. 2016; Library 2018). Purslane is rich in proteins, polysaccharides, organic acids, mineral elements, and has unique nutritional value. Purslane is an annual herb, rich in omega-3 fatty acids and other components, and is an important medicinal plant (Jin et al. 2015). Purslane, as halophyte in the Haloph database, grows readily in soils that may be arid and saline (Yazici et al. 2007).

Salinity is the most serious abiotic factor of abiotic stresses (Mansour 2000). Soil salinization is one of the major limitations of growth and development and produce toxicity to plants, which is a worldwide problem. Purslane has strong adaptability to climate, soil and other environmental conditions, and has a certain salt tolerance. After purslane seedlings were treated by 140 mmol∙L-1 NaCl for 18 days, the activity of glutathione reductase was 3.5 times higher than that of the control (Yazici et al. 2007), indicating that purslane has strong salt tolerance. Kafi and Rahimi (2011) found that K+ content in leaves and stems of purslane decreased by the salt stress. Purslane was able to grow under salt stress. Parvaneh et al. (2012) showed that under 150 mmol∙L-1 and 200 mmol∙L-1 NaCl stress, the chlorophyll contents of purslane was increased, and the corresponding increase of proline and sugar was beneficial to plant photosynthesis (Yazici et al. 2007).

The mechanisms of salt stress such as metabolism, photosynthesis and growth of plant under salt stress have been extensively studied. Salt stress affects chloroplast components of the main organs of plant photosynthesis, such as pigments, enzymes, proteins and membrane lipids (Parida et al. 2003; Zaman et al. 2018), which reduces plant photosynthesis and consequently reduces plant productivity. Thus, the antioxidant physiological characteristics of purslane under salt stress were systematically studied in this paper, which could provide technical reference for further improving saline-alkali soil and developing salt-resistant plant germplasm resources.

 

Materials and Methods

 

The seeds of purslane (P. oleracea L.) were collected from the beach of Yancheng, Jiangsu province, China. The plants of purslane (P. oleracea L.) were identified by the professor Jiao Demao from Institute of germplasm resources and biotechnology, Jiangsu Academy of Agricultural Sciences, China. Seeds were sown into the pots, grown in an illuminating and incubated with 30/23ºC of day/night, 70% relative humidity.

The seeds of purslane were germinated in vermiculite and sprayed with tap water every day. Seedlings of purslane grew in pots in moistened sand. Different levels of NaCl (0, 50, 150, 200 mmol∙L-1) were applied to the pots. Thereafter, the seedlings were watered with tap water every two days and with corresponding salt solution every three days. Measurements were taken 30 days after salinity treatments. Fresh weights of roots and shoots were measured. Purslane seedlings were dried in an oven at 75ºC for 72 h, and the dry weight was determined.

 

Measurement of gas exchange parameters and chlorophyll contents

 

The gas exchange parameters of 4–5 branches of seedlings were measured using a CIRAS-2 PPS (PP Systems, Ltd.) at the 1000 μmol·m-2·s-1 light intensity, the CO2 concentration of 380 μmol·mol-1 and 60% relative humidity. The leaf chamber was a PLC6 (U) standard chamber (2.5 cm2). In this study, the leaf area was measured by LI-3000C Portable Area Meter (Li-Cor Biosciences, Lincoln, NE, USA). The fresh weight of the leaves was determined with a balance with a sensitivity of 0.1 mg.

Chlorophyll pigment contents were extracted from purslane leaves with methanol (30 mg fresh weight·mL-1). The extraction process was carried out in darkness at room temperature for 24 h. The chlorophyll content is expressed as the fresh weight mg·g-1 by Wellburn (1994).

 

Indexes of active oxygen metabolism

 

The activity of superoxide dismutase (SOD), which can be expressed as the ability to inhibit the photochemical reduction of nitroblue tetrazolium, was determined according to Giannopolitis and Ries (1977). Peroxidase (POD) activity was measured according to the method of Kochba et al. (1992). A unit of peroxidase activity was expressed as the change in absorbance per minute. The activity of catalase (CAT), lipid peroxidation and the free proline contents were measured according to Cakmak and Marschner (1992), Heath and Packer (1968) and Bates et al. (1973), respectively.

 

Statistical analysis

 

The data in this test are mean ± standard deviation (SD). LSD test was used for data analysis (P < 0.05). The diffirent letters of each point in the charts and Figures indicated that there are significant differences between groups at the level of 0.05.

 

Results

 

Effect of salt stress on the growth parameters of purslane

 

From Table 1 and 2, it can be seen that the germination rate of purslane decreased gradually under a certain low concentration of salt water (50 mmol·L-1). From the data of root and stem growth, it can be seen that, the growth of stem and root of purslane was significantly inhibited at high salt concentration . The dry weight of purslane reached the maximum at the 50 mmol·L-1 NaCl concentration. The plant height and dry weight did not decrease significantly, which had little effect on the normal growth of purslane. The higher the NaCl concentration, the shorter the plant height (Fig. 1) and root length, the more serious the water loss of leaves. Purslane can not adapt to the salt stress environment at this time. It can be inferred that if NaCl concentration is further increased, it may cause plant death.

With the increase of NaCl concentration, the more parts of purslane root tips stained blue by Trypan Blue (Fig. 2). This phenomenon indicates that under salt stress, the content of dead cells in root tip cells increased due to the damage of salt to root tip cells. When the NaCl concentration increased, the resistance of purslane to salt stress gradually decreased.

 

Effect of salt stress on the gas exchange parameters of purslane

 

According to Fig. 3, chlorophyll a/b increased slightly (P > 0.05) at NaCl concentration of 0–100 mmol·L-1, while chlorophyll a/b ratio increased at NaCl concentration of 100–200 mmol·L-1. The contents of chlorophyll a+b decreased due to the increase NaCl concentration. When NaCl concentration was 50 mmol·L-1, the chlorophyll contents did not decrease significantly (P > 0.05), but had little effect on the photosynthetic capacity of purslane. When salt concentration reached 100 mmol·L-1, the chlorophyll contents decreased significantly (P < 0.05), which had a significant effect on the normal development and growth of purslane and was not suitable for the growth of purslane.

When the NaCl concentration increased, the net photosynthetic rate (Pn) (Fig. 4A) and transpiration rate (Tr) (Fig. 4B) of purslane decreased gradually. When the NaCl concentration reached 200 mmol·L-1, the net photosynthetic rate of purslane was negative, indicating that high salt stress significantly inhibited the photosynthesis and transpiration of purslane. With the increase of seawater concentration, intercellular CO2 concentration (Ci) (Fig. 4C) showed a trend opposite to net photosynthetic rate. As can be seen from Fig. 4D, stomatal conductance (Gs) was inhibited by the salt ions, which was not conducive to the absorption of carbon dioxide in photosynthesis. With the increase of NaCl concentration, the transpiration rate, the stomatal conductance and net photosynthetic rate all decreased, due mainly to the utilization of CO2 reduction.

 

Antioxidant characteristics of purslane under the salt stress

 

Table 1: The seedling growth and seed germination of purslane under salt stress

 

seed germination index

NaCl concentrations (mmol.L-1)

0

50

100

150

200

Prcentage of germination (%)

95.25±4.17 a

90.25±5.13 a

55.33±3.55 b

25.33±2.62 c

5.25±0.65 d

Germination energy (%)

65.33±3.21 a

50.50±4.68 b

25.50±3.45bc

5.33±0.91d

2.12±0.55e

Shoot length(mm)

7.92±0.74 a

6.44±0.92 b

3.83±0.64 c

2.55±0.71d

1.02±0.36 e

Root length(mm)

8.43±1.08 a

4.46±0.68 b

2.34±0.42 c

1.16±0.18 d

0.57±0.12 e

 

Table 2: The growth of purslane seedlings under salt stress

 

NaCl concentration (mmol.L-1)

Fresh (FW) weight (mg)

Dry (DW) weight (mg)

Plant length (cm)

Leaf relative water content (RWC)%

0

3225±127 a

267±31 a

11.2±1.4 a

73.2±6.3 a

50

2932±108 a

281±27 a

10.5±0.8 a

68.4±6.9 a

100

1012±79 b

196±16 b

8.1±0.8 b

59.3±4.1 b

150

863±82 c

157±15 b

7.4±0.6 bc

53.2±5.2 b

200

562±50 d

92±7 c

6.7±0.5 c

36.3±2.8 c

Values are mean±S.E. based on three replicates (n = 3)

 

 

Fig. 1: Effect of salt stress on purslane phenotype. The age of seedling is 30 d

 

 

Fig. 2: Trypan blue staining of cells in root tips of purslane under different NaCl concentrations

From Fig. 5A, it can be seen that the SOD activity of purslane was affected by different concentrations of salt stress. At the 50 mmol·L-1 NaCl concentration, the SOD activity reached its maximum, and then showed a downward trend. POD activity of Portulaca oleracea increased significantly as the results of NaCl stress (Fig. 5C). The MDA content of purslane increased (Fig. 5D), which indicated that the lipid peroxidation of cell membrane of purslane increased and the damage of cell membrane was more serious. The change of CAT activity after salt treatment was consistent with the trend of SOD (Fig. 5B). It showed that low concentration of salt ion induced the activities of protective enzymes to increase in order to remove the active oxygen and MDA (a lipid peroxidation product of membrane) accumulated under stress. Soluble protein and proline are both related to osmotic potential regulation of plant cells. Owing to the NaCl sress, soluble protein and proline increase significantly, which caused the damage of membrane and the enhancement of reactive oxygen. So the growth of purslane was inhibited.

 

Discussion

 

Plant growth and yield are directly related to photosynthetic performance. Chlorophyll is the main photosynthetic pigment and plays an important role in plant photosynthesis (Zhang et al. 2009). Salt stress decreased chlorophyll synthesis and inhibited photosynthesis (Barhoumi et al. 2007). With the increase of salt stress, the degradation of chlorophyll in purslane leaves aggravated in the present study. Chlorophyll a was more susceptible to salt stress than chlorophyll b. The increase of a/b showed that the decrease of chlorophyll b content was one of the main reasons for the decrease of photosynthesis in purslane.

 

Fig. 3: Effects of NaCl stress on chlorophyll contents and chlorophyll a/b in purslane

 

 

Fig. 4: Effect of NaCl stress on net photosynthetic rate (Pn) (A), transpiration rate (Tr) (B), intercellular CO2 concentration (Ci) (C) and stomatal conductance (Gs) (D) in purslane

Purslane is a typical C4 plant with vascular bundle sheath cells and kranz type structure. The roots of purslane also have developed ventilation tissue (Ding et al. 2012), which ensures the metabolic needs of purslane. The result of purslane root tips stained by Trypan Blue showed that purslane had a certain adaptability and tolerance to salt environment. When the concentration of salt in nutrient solution was 50 mmol·L-1, Pn and Gs of purslane decreased slightly, the water use efficiency ,chlorophyll contents and photosynthesis changed little. At the 100 mmol·L-1 NaCl concentration, the photosynthesis of purslane was greatly affected, the physiological functions of various aspects were limited, and the normal development of purslane was slowed down. Purslane had high photosynthetic capacity under low salt stress, alleviated the damage of salt stress to the light system, thus maintaining a higher net photosynthetic rate, which may be a protective mechanism of purslane adapting to salt environment.

Under salinity and other stress conditions, the distribution of membrane lipids and proteins in plant cell membranes would be disturbed, and the activity of plant peroxidase will increase. Soluble substances, such as MDA, protein and proline, were accumulated to reduce the intracellular osmotic potential and ensure the supply of water (Valentina et al. 2002; Zhang et al. 2005). The protective enzymes SOD, CAT and POD in purslane increased to eliminate the damage caused by NaCl stress, and a large amount of protein and proline were accumulated (Fig. 5), which helped maintain the low osmotic potential of plant cells and resist the stress caused by stress. It can be seen that purslane still has strong photoinhibition and photooxidation resistance under salt stress, and maintains a higher photosynthetic capacity, thus reducing the damage of salt stress on plant growth. By studying the response characteristics of key enzymes in C4 efficient carbon assimilation pathway of purslane under above salt stress, the mechanism of high photosynthetic efficiency of purslane was clarified, which was of great significance for the improvement of saline-alkali land and the sustainable development of recycling.

 

Fig. 5: The activities of superoxide dismutase (SOD) (A), peroxidase (POD) (B), and calatase (CAT) (C) and malonyldialdehyde (MDA) (D), soluble protein (E) and proline (Pro) (F) contents in leaves of purslane under NaCl stress

Purslane is known as one of the most promising medicinal and edible plants in the 21st century (Kafi and Rahimi 2011; Jin et al. 2015; Zaman et al. 2018). Because of its strong salt tolerance, purslane could be a promising candidate to be used in ecosystem restoration in arid and semi-arid regions (Jin et al. 2016). It was of significant strategy that purslane was planted on coastal beach and developing saline soil agriculture for further accelerating the sustainable development of agricultural economy and expanding the space for agricultural development in China.

 

Conclusion

 

These findings suggest that Purslane plants responded to NaCl stress by enhancing their antioxidative capacity and proline accumulation. Under salt stress, purslane had high photosynthetic capacity, alleviated the damage of salt stress to the light system, thus maintaining a higher net photosynthetic rate. This study provides theoretic basis and technical approach for studying and exploring salt-tolerant plant resources.

Acknowledgement

 

We acknowledge the financial supports of the Natural Science Foundation of Jiangsu Province in China under Grant No. SBK2015040083.

 

Author Contributions

 

Tang Ning and Yang Ping conceived and designed the experiments; Tang Ning performed the experiments; Wang Like and Yang Ping analyzed the data; Chen Quanzhan contributed materials; Zhang Bianjiang wrote the paper.

 

References

 

Bates LS, RP Waldern, ID Teare (1973). Rapid determination of free proline for water-stress studies, Plant Soil 39:205‒207

Barhoumi Z, W Chaibi, A Smaoui (2007). Salt impact on photosynthesis and leaf ultrastructure of Aeluropus littoralis. J Plant Res 120:529‒537

Cakmak I, H Marschner (1992). Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase in bean leaves. Plant Physiol 98:1222‒1227

Ding H, W Wang, X Zhu, C Liu, J Xing, B Zhao, C Chen, L Hong (2012). Effects of NaCl Stress on the Ion Distribution and Chlorophyll Fluorescence in Portulaca oleracea L. Acta Agric Bor-Sin 27:213‒217

Giannopolitis CN, SK Ries (1977). Superoxide dismutase I occurrence in higher plants. Plant Physiol 59:309‒314

Heath RL, L Packer (1968). Photoperoxidation in isolated chloroplasts, I. kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189‒198

Jin R, H Shi, C Han, B Zhong, Q Wang, Z Chan (2015). Physiological changes of purslane (Portulaca oleracea L.) after progressive drought stress and rehydration. Sci Hortic 194:215‒221

Jin R, Y Wang, R Liu, J Gou, Z Chan (2016). Physiological and metabolic changes of purslane (Portulaca oleracea L.) in response to drought, heat, and combined stresses. Front Plant Sci 6; Article 1123

Kafi M, Z Rahimi (2011). Effect of salinity and silicon on root characteristics, growth, water status, proline content and ion accumulation of purslane (Portulaca oleracea L.). Soil Sci Plant Nutr 57:341–347

Kochba J, ES Lave, P Spiegel-Roy (1992). Difference in peroxidase activity and isozymes in embryogenic and non-embryogenic ‘Shamouti’ organ ovular callus lines, Plant Cell Physiol 18:463‒467

Library WP (2018). Germplasm Resources Information Network (GRIN). Germplasm Resources Information Network Online Database, USDA, Washington DC, USA

Mansour MMF (2000). Nitrogen containing compounds and adaptation of plants to salinity stress. Biol Plantarum 43:491–500

Parida AK, AB Das, B Mittra (2003). Effects of NaCl stress on the structure, pigment complex composition and photosynthetic activity of mangrove Bruguiera parviflora chloroplasts. Photosynthetica 41:191‒200

Parvaneh R, T Shahrokh, MH Seyed (2012). Studying of salinity stress effect on germination, proline, sugar, protein, lipid and chlorophyll content in purslane (Portulaca oleracea L.) leaves. J Stress Physiol Biochem 8:182‒193

Valentina M, T Moshe, V Micha, G Micha (2002). Salt stress induces up-regulation of an efficient chloroplast antioxidant system in the salt-tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species. Physiol Plantarum 115:393‒400

Wellburn AR (1994). The Spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144:307‒313

Yazici I, I Türkan, AH Sekmen, T Demiral (2007). Salinity tolerance of purslane (Portulaca oleracea L.) is achieved by enhanced antioxidative system, lower level of lipid peroxidation and proline accumulation. Environ Exp Bot 61:49–57

Zaman S, SB Shah, YT Jiang, SQ Che (2018). Saline conditions alter morpho-physiological intensification in purslane (Portulaca oleracea L.). J Biol Reg Homeos Agric 32:635‒639

Zhang RH, J Li, SR Guo, T Tezuka (2009). Effects of exogenous putrescine on gas-exchange characteristics and chlorophyll fluorescence of NaCl-stressed cucumber seedlings. Photosynth Res 100:155‒162

Zhang Q, Y Li, C Pang, C Lu, B Wang (2005). NaCl enhances thylakoid-bound SOD activity in the leaves of C3 halophyte Suaeda salsa L. Plant Sci 168:423‒430