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Methyl jasmonate improves metabolism and growth of NaCl-stressed Glycyrrhiza uralensis seedlings
Scientia Horticulturae 266 (2020) 109287
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Methyl jasmonate improves metabolism and growth of NaCl-stressed Glycyrrhiza uralensis seedlings
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Duoyong Langc,1, Xiaxia Yua,1, Xiaoxia Jiad, Zhixian Lie, Xinhui Zhanga,b,* a
College of Pharmacy, Ningxia Medical University, Yinchuan, 750004, China Ningxia Engineering and Technology Research Center of Hui Medicine Modernization, Ningxia Collaborative Innovation Center of Hui Medicine, Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education, Ningxia Medical University, Yinchuan, 750004, China c Laboratory Animal Center, Ningxia Medical University, Yinchuan, 750004, China d Potato Research Institute, Gansu Academy of Agricultural Sciences, Lanzhou, 730000, China e Hunan Provincial Key Laboratory of Coal Resource Clean Utilization and Mine Environmental Protection, Hunan University of Science and Technology, Xiangtan, Hunan, 411201, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: G. uralensis Methyl jasmonate Antioxidant system Carbon and nitrogen metabolisms Salt stress
Background and Aims: Salinity is one of the major factors that affect growth and metabolism in plants. However, limited information is available on the influence of methyl jasmonate (MeJA) on antioxidant defense, carbon (C) and nitrogen (N) metabolisms in Glycyrrhiza uralensis Fisch. (G. uralensis) under salt stress. Here, this experiment was conducted to investigated the responses of antioxidant defense, C and N metabolisms of salt-stressed G. uralensis to MeJA at different concentrations. Methods: G.uralensis plant seeds were grown under six conditions: control, 200 mM NaCl, and 200 mM NaCl combined with 15, 30, 45 and 60 μM MeJA. Key Results: Results showed that salt stress inhibited G. uralensis growth and this adverse effect was reversed by MeJA at different concentrations. MeJA also affected antioxidant system and C and N metabolisms. In general, MeJA from 45 to 60 μM significantly enhanced superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione peroxidase (GPX) activities in salt-stressed G. uralensis seedlings, MeJA from 15 to 30 μM increased peroxidases (POD) and catalase (CAT) activities, and ascorbate peroxidase (AsA) and glutathione (GSH) contents. Thus, these changes caused a decrease in hydrogen peroxide (H2O2), superoxide radical (O2−) and malondialdehyde (MDA) contents correspondingly. Moreover, 60 μM MeJA enhanced sucrose synthase (SS), sucrose phosphate synthase (SPS) and nitrate reductase (NR) activities in salt-stressed G. uralensis seedlings. Conclusions: MeJA promoted growth of salt-stressed G. uralensis seedlings by alleviating oxidative stress and strengthening C and N metabolism, these effect depended on the applied concentrations of MeJA.
1. Introduction Salinity is one of the most severe problems in agriculture worldwide and NaCl is the predominant salt in most saline environments, especially in arid and semi-arid regions (Ahmadi and Souri, 2018). High concentration of salts can impair the growth, physiological and metabolic activities of plant by causing oxidative and osmotic stresses, or a combination of these factors (Zhang et al., 2008; Slama et al., 2015). Specifically, salt stress accelerates production of reactive oxygen species (ROS) and causes lipid peroxidation in plants (Debouba et al., 2006; Ahmadi and Souri, 2019). In order to ameliorate the damaging effects of ROS, plants have developed several defense mechanisms. One
of the mechanisms is antioxidant system, high activities of antioxidant enzymes and high contents of non-enzymatic constituents are two important factors that contributed to tolerance against salt stress (Sharma et al., 2013). In the antioxidant enzymes and non-enzymatic constituents, superoxide dismutase (SOD) mainly scavenges superoxide radical (O2－), catalase (CAT) and peroxidases (POD) eliminate hydrogen peroxide (H2O2). Moreover, glutathione reductase (GR), ascorbate peroxidase (APX), glutathione peroxidase (GPX) and glutathione (GSH) play important roles in ascorbate-glutathione cycle, which clears hydrogen peroxide (H2O2) in different ways. Bor et al. (2003) reported that salt-stressed Beta vulgaris L. plant had better protection against oxidative damage by enhancing SOD, CAT and GR activities.Azevedo
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Corresponding author at: College of Pharmacy, Ningxia Medical University, Yinchuan, 750004, China. E-mail address: [email protected] (X. Zhang). 1 These authors contributed equally in this work. https://doi.org/10.1016/j.scienta.2020.109287 Received 9 December 2019; Received in revised form 17 February 2020; Accepted 17 February 2020 0304-4238/ © 2020 Elsevier B.V. All rights reserved.
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effects of MeJA at different concentrations on plant growth, antioxidant defense, and C and N metabolisms. The purpose is to provide a better understanding about the significant effect and mechanism of MeJA on alleviating the adverse effect caused by salt stress on G. uralensis
Neto et al. (2006)found that the activities of SOD, APX, GPX and GR increased with time in leaves of salt-stressed maize. Therefore, the increase in the content of antioxidants and the activity and/or expression of antioxidant enzymes is a common adaptive response of plants to oxidative stress (Fidalgo et al., 2015; Souri and Hatamian, 2019). In addition, carbon (C) and nitrogen (N) metabolisms are fundamental processes in plants and closely coordinated in nearly every biochemical pathway (Coruzzi and Bush, 2001). C metabolism processes involve photosynthetic carbon assimilation, sucrose and starch metabolisms and carbohydrate transport. N metabolic processes include N uptake, transport, reduction and assimilation, and amino acid metabolism (Kusano et al., 2011; Jie et al. 2013). Like other abiotic stresses, salt stress also induces the alterations of C and N metabolisms. When plants are grown under abiotic stresses, a series of physiological processes associated with C metabolism are altered, such as inhibition of photosynthesis, changes in carbohydrate content and carbohydrate metabolic enzyme activity (Theocharis et al., 2012). Silva et al. (2014) observed that salt stress enhanced soluble proteins content and reduced nitrate reductase (NR) activity in sunflower plant. Salt stress decreased glutamine synthetase (GS) and glutamate synthase (GOGAT) activities in Populus simonii roots and leaves (Meng et al., 2016; Souri and Tohidloo, 2019), but increased NO3－ content in rice old leaves (Wang et al., 2012). Interestingly, many studies observed correlations between antioxidant activity and salinity tolerance are thought to be conveyed by plant hormones (Salimi et al., 2016). Jasmonates are a class of plant hormones that mediate defense mechanisms and stress responses. Among these, the role of Methyl jasmonate (MeJA) in protecting plants from different abiotic stresses has been considerable (Guo et al., 2012). Walia et al. (2006) showed that MeJA diminished the inhibitory effect caused by salt stress on the photosynthesis rate in rice plant. Jiang et al. (2016) found that MeJA improved growth of salinity-stressed Robinia pseudoacacia plant through increasing the activities of SOD, POD and APX that eliminate the excessive O2－and H2O2 induced by salt stress. Furthermore, MeJA enhanced the activities of antioxidant enzymes (CAT, POD and SOD) to improve the ROS scavenging ability in Artemisia annua and strawberry, both in non-stressed and stressed plants (Tariq et al., 2011; Faghih et al., 2017). These results confirmed that MeJA can alleviate oxidative stress induced by salinity by improving the activity and/or pools of antioxidant enzymes (Poonam et al., 2013). However, there is little research on the role of MeJA on carbon and nitrogen metabolism processes in plants subjected to salt stress, only Hanik et al. (2010) reported that MeJA promoted the transcriptional modification of numerous genes that can impact whole plant resource allocation of both nitrogen- and carbon-containing substrates and increased the export of newly acquired carbon and nitrogen in tomato leaves, further increased the proportion of nitrogen allocated in tomato roots (Gómez et al., 2010). These results undoubtedly suggested that exogenous MeJA may be involved in the defense against salinity stress and their role in manifesting salinity tolerance has recently been acknowledged (Iqbal et al., 2014). However, the precise mechanisms involved are up for debate and depend on the plant species involved. Glycyrrhiza uralensis Fisch. (G. uralensis) is one of the ancient herbal plants of the legume family native to Asia, which comprises 18 wild and cultivated species and widely used herbal medicine (Pan et al. 2014). Its also frequently used as additives in tobacco and food worldwide and as a beneficial desert plant resource for hampering wind, fixing sand, and improving soil conditions. However, production and availability of wild G. uralensis are insufficient for human consumption because of irresponsible over exploitation and adverse agronomic conditions (Zhang et al., 2017). Despite the growing body of evidence supporting the role of MeJA on growth of several plant species under salt stress, however, not yet been well investigated in G. uralensis plants. Moreover, our previous experiments have shown that MeJA has an positive effect on salt-stressed G. uralensis plants during seed germination stage (Yu et al., 2019). Thus, this experiment was conducted to further investigated the
2. Materials and methods 2.1. Plant material G. uralensis seeds were obtained from the wild plants in Yan-chi county, the Ningxia Hui Autonomous Region, China, in August 2017. 2.2. Experimental design and treatments Petri dish germination experiment was conducted at the laboratory. Specifically, the seeds were steeped with 85 % H2SO4 for 2.5 h, then surface sterilized with 0.1 % H2O2 for 10 min, rinsed three-times in distilled water, and imbibed in distilled water for 12 h at room temperation. A total of 50 seeds were sown in 90 mm diameter petri dishes with two layers of Whatman No.2 filter paper to receive different treatments. According to the results of preliminary experiment, the treatments include (i) control (CK), with distilled water only, (ii) 200 mM NaCl, (iii) 200 mM NaCl with 15 μM MeJA, (iv) 200 mM NaCl with 30 μM MeJA; (ⅴ) 200 mM NaCl with 45 μM MeJA and (ⅵ) 200 mM NaCl with 60 μM MeJA. MeJA chemical was purchased as a 95 % aqueous solution and dissolved in Tween 20. Each treatment was replicated six times. 2.3. Seed germination conditions All petri dishes were placed in a plant growth chamber where maintained at 28℃/20℃ (day/night) with a 12 h photoperiod at a light intensity of 37.5 μmol m−2 s−1 PAR. Germination was considered to occur when the radicle had emerged by at least 1.0 mm. The number of germinated seeds was recorded every day. After germinating for 10 days, uniform sized and healthy seedlings of G. uralensis were collected from each of the 24 petri dishes, and analyzed immediately to measure their physiological and biochemical characteristics; For remaining plants in each of the 24 petri dishes, length of hypocotyl and radicle, diameter of hypocotyl and radicle were recorded. 2.4. Determination of antioxidant and lipid peroxidation Each 0.5 g of whole seedling tissue was homogenized in 10 mL of an extraction buffer containing 50 mM phosphate buffer, pH 7.8, 1.0 mM EDTA, 1.0 g polyvinylpyrrolidone (PVP), and 0.5 % (v/v) Triton X-100, then the extract was centrifuged for 20 min at 12,000 rpm and the supernatant was used for analysis of activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), and in concentration of malondialdehyde (MDA). All operations were carried out at 0-4℃. SOD activity was measured by its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT), recorded at 560 nm. One unit of SOD was defined as the amount of enzyme that inhibits NBT reduction by 50 % (Meng et al., 2016). CAT activity was assayed according to the method of Sohn et al. (2005) with minor modifications. The CAT reaction solution comprised 50 mM phosphate buffer (pH 7.8), 0.1 M H2O2 and 0.1 mL enzyme extract. Changes in the absorbance of the reaction solution at 240 nm were recorded for 5 min. POD activity was assayed according to the method of Pan et al. (2006) with minor modifications. 0.2 mL of the enzyme extract was mixed with 2.7 mL 50 mM phosphate buffer (pH 7.8) and 0.1 mL 50 mM of guaiacol and 1 mL 2% H2O2 and the change in absorbance was measured at 470 nm for 5 min. Membrane lipid peroxidation was determined by measuring the MDA content using the method of Zhang et al. (2017) with minor modifications. The enzyme extract (1 mL) was mixed with 2.5 mL of 0.5 2
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2.8. Determination of the activities of enzymes related carbon and nitrogen metabolism
% thiobarbituric acid (TBA) and was then heated at 100℃ for 20 min. The absorbance was determined at 450 nm, 532 nm, and 600 nm, respectively.
Each 0.5 g of fresh shoot and root was homogenized at 4℃ in 3 mL of extraction buffer [100 mM Tris−HCl buffer (pH 7.2) containing 10 mM MgCl2, 1 mM EDTA-Na2, 10 mM β-hydrophobic base ethanol, 2% ethylene glycol and 1% polyvinyl pyrrolidone] with a mortar and pestle. The homogenate was then centrifuged at 12,000 rpm for 10 min and the supernatant was used as the crude extract for the sucrose synthase SS and sucrose phosphate synthase SPS assay. For SS activity, reaction mixtures contained 100 mM Tris−HCl buffer (pH 7.2), 10 mM MgCl2, 5 mM uridine diphosphate glucose (UDPG), 5 mM fructose, and the 0.3 mL of sample. For SPS activity, 5 mM fructose was substituted for 5 mM fructose-6-phosphate. Data are expressed as micromoles of sucrose or sucrose-P produced per gram of fresh weight per hour (Golldack et al., 2014). For the nitrate reductase (NR) activity assay, approximately 0.5 g samples of fresh tissue were ground in a pre-chilled mortar and pestle on ice in 4 mL of extraction buffer. The extraction 0.025 M buffer (pH 8.7) consisted of 10 mM cysteine and 1 mM EDTA. The mixture was centrifuged at 4000 rpm at 4℃ for 15 min and then 0.5 mL supernatant was added to the reaction mixture. The reaction mixture comprised 1.2 mL of 0.1 M KNO3-phosphate buffer (pH 7.5) and incubated at 25℃ for 30 min. The reaction was terminated by adding 1 mL 1% (w/v) sulfanilamide in 3 M HCl. For nitrite determination, 1 mL 0.02 % w/v Nnaphthyl-1-dihydrochloride was added, and the color was allowed to develop for 15 min at 25℃. The mixture was centrifuged at 12,000 rpm at 4℃ for 5 min, and then the absorbance of the supernatant at 540 nm was measured. NR activity is expressed as l mol NO2− g-1 fresh weight (FW) h-1 (Golldack et al., 2014).
2.5. Determination of reactive oxygen species contents Superoxide radicals (O2－) were measured using the method described in Bu et al. (2016) with minor modification. Where, 0.2 g fresh seedlings were allowed to react with 1 mL of hydroxylamine hydrochloride for 1 h; then, 1 mL of p-aminobenzene sulfonic acid and 1 mL of α-naphthylamine were added, and the solution was maintained at 25℃ for 20 min. The absorbance of the mixture was measured at 530 nm, and NaNO2 was used to prepare a standard curve. The hydrogen peroxide (H2O2) content was determined as described by Bu et al. (2016) with minor modification. Briefly, 0.2 g fresh seedling was centrifuged at 10,000 rpm for 10 min after being homogenized in 5 mL of ice-cold acetone. The collected supernatant (1 mL) was subsequently added to a concentrated HCl solution containing 0.1 mL of 20 % Ti(SO4)2 and 0.2 mL of concentrated ammonia, and the mixture was recentrifuged at 8000 rpm for 15 min. The obtained pellets were then added to 3 mL of 1 M H2SO4. The absorbance was observed at 410 nm, and H2O2 was used to prepare a standard curve. 2.6. Determination of ascorbate peroxidase (APX), glutathione reductase (GR) and glutathione peroxidse (GPX) activities Each 0.5 g of fresh shoot and root were homogenized at 4℃ in 8 mL of extraction buffer [50 mM phosphate buffer (pH 7.0) containing 1 mM ascorbic acid (AsA), 1 mM dithiothreitol (DTT), 1 mM glutathione (GSH) and 1 mM MgCl2] with mortar and pestle. The homogenate was then centrifuged at 12,000 rpm for 20 min and the supernatant was used as the crude extract for the activities of GR, APX and GPX. APX activity was determined according to the method of Kang and Saltveit (2001) with minor modification. The 3 mL reaction mixture was composed of 50 mM phosphate buffer (pH 7.0) containing 0.1 mM EDTA, 0.3 mM AsA, 0.06 mM H2O2 and 0.2 mL of enzyme extract. The oxidation of ascorbate was followed by the decrease in the absorbance at 240 nm. GR activity was assayed by Zhang et al. (2017) and measured the decrease in absorbance at 340 nm due to the oxidation of NADPH. The 3 mL reaction mixture comprised 50 mM phosphate buffer (pH 8.2) containing 1 mM EDTA, 0.2 mM NADPH, 1 mM oxidized glutathione (GSSG) and 0.2 mL of enzyme extract. GPX activity was determined as described by Azevedo Neto et al. (2006) in a reaction mixture (2.0 mL) containing 100 mM phosphate buffer (pH 7.0), 0.4 mM EDTA, 0.4 mM glutathione (GSH), 0.4 mM NADPH, 0.2 mM H2O2 and 50 μL enzyme extract. The addition of enzyme extract started the reaction and the increase in absorbance was recorded at 340 nm for 1 min.
3. Statistical analysis All data were present as mean + SE. The difference was analyzed by ANOVA using SPSS 17.0 software (SPSS Inc, Chicago, IL, USA). Significant differences were tested using the Least Significant Difference (LSD) test at P ≤ 0.05.
4. Results 4.1. Effect of MeJA on growth of salt-stressed G. Uralensis seedlings NaCl stress significantly reduced radicle length and radicle diameter of G. uralensis seedlings compared with the control. MeJA addition markedly changed growth of G. uralensis seedling at different concentrations. Specifically, 45 μM MeJA significantly increased radicle length, hypocotyl length and hypocotyl diameter; 30 μM MeJA significantly increased radicle length and hypocotyl diameter; MeJA from 15 to 30 μM also significantly increased radicle diameter (Fig. 1). These results indicate that MeJA can alleviate the harmful effect caused by salt stress in G. uralensis seedling.
2.7. Determination of ascorbate peroxidase (AsA) and glutathione (GSH) contents
4.2. Effect of MeJA on reactive oxygen species and membrane lipid peroxidation in salt-stressed G. Uralensis seedlings
AsA content was determined according to the method of Liu et al. (2014) using 0.2 g fresh seedling. Standards for AsA was prepared in the range of 0−15 mg L−1 in 1 mL 5% (w/v) trichloroacetic acid (TCA), 1 mL alcohol, 0.5 mL 0.4 % H3PO4-alcohol, 1 mL 0.5 % bathophenanthroline (BP)-alcohol and 0.5 mL 0.03 % FeCl3-alcohol at 30℃ for 90 min and measured at 534 nm. GSH was determined by the method of Zhang et al. (2017) and extracted from 0.2 g fresh seedling with 4 mL of 5 mM EDTA-TCA and estimated by 5,5′-dithio-bis (2-nitrobenzoic acid) (TDNB). Changes in absorbance of the reaction mixture were measured at 420 nm and total GSH concentration was calculated from a standard curve with GSH.
NaCl stress significantly increased O2－production rate, and H2O2 and MDA contents in G. uralensis seedlings compared with the control. Interestingly, MeJA addition significantly reduced O2－production rate at low concentrations of 30 μM or below, and H2O2 content at all concentrations; MeJA also significantly decreased MDA content at concentrations of ≥ 30 μM (Fig. 2), indicating that MeJA can mitigate oxidative stress caused by salt stress in G. uralensis seedling.
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Fig. 1. Effect of methyl jasmonate (MeJA) at different concentrations (15, 30, 45, and 60 μM) on root length, stem length, root diameter and stem diameter of G. uralensis seedlings under salt stress (200 mM). The different letters within the different treatments indicate the significant difference at P ≤ 0.05. Values are means ± SE (n = 6).
4.3. Effect of MeJA on antioxidant enzymes activities MeJA on and nonenzymatic antioxidants contents in salt-stressed G. Uralensis seedlings
4.4. Effect of MeJA on carbon and nitrogen metabolizing enzymes activities in salt-stressed G. Uralensis seedlings
NaCl stress significantly increased SOD and GR activities, but decreased GPX activity in G. uralensis seedlings compared with the control. Compared with NaCl treatment, MeJA addition significantly enhanced SOD activity at 45 μM, POD activity at 30 μM, CAT activity at 15 μM, and GPX activity at high concentrations of 30 μM or above, while MeJA addition significantly reduced APX activity at 30 μM (Fig. 3). As to non-enzymatic antioxidants, NaCl stress remarkably decreased AsA content in G. uralensis seedlings compared with the control, while MeJA significantly increased AsA content at 15 and 30 μM and GSH content at 30 μM compared with the NaCl treatment (Fig. 4). These results indicate that MeJA alleviated oxidative stress are associated with the improved the activities of antioxidant enzymes and the contents of non-enzymatic antioxidants in G. uralensis seedlings subjected to NaCl stress.
NaCl stress significantly increased SS and SPS activities but decreased NR activity in G. uralensis seedlings compared with the control. Compared with NaCl treatment, MeJA from 15 to 45 μM significantly reduced SS and SPS activities, but significantly enhanced NR activity at 60 μM (Fig. 5). 5. Discussion 5.1. MeJA improved growth of G. Uralensis seedling under salt stress Salinity stress can change most physiological and biochemical processes and lead to a inhibition in growth and reduction in biomass (Ji et al., 2009; Souri and Tohidloo, 2019). Salt stress inhibited growth of strawberry seedlings (Faghih et al., 2017) and Ammodendron argenteum seedlings (Zhuang et al., 2010) and remarkably decreased the root length of rice even at low NaCl concentration (Kang et al., 2005). Our
Fig. 2. Effect of methyl jasmonate (MeJA) at different concentrations (15, 3, 45, and 60 μM) on O2− production rate, H2O2 and MDA contents of G. uralensis seedlings under salt stress (200 mM). The different letters within the different treatments indicate the significant difference at P ≤ 0.05. Values are means ± SE (n = 6). 4
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Fig. 3. Effect of methyl jasmonate (MeJA) at different concentrations (15, 30, 45, and 60 μM) on SOD, POD, CAT, APX, GR and GPX activities of G. uralensis seedlings under salt stress (200 mM). The different letters within the different treatments indicate the significant difference at P ≤ 0.05. Values are means ± SE (n = 6).
5.2. MeJA mitigated oxidative stress caused by salt-stressed in G. Uralensis seedling
results showed 200 mM NaCl significantly decreased root length and root diameter of G. uralensis seedlings compared with the control. Interestingly, studies have found that application of exogenous MeJA markedly increased growth parameters of Robinia pseudoacacia (Jiang et al., 2016), Artemisia annua (Tariq et al., 2011) and strawberry under slat stress conditions (Faghih et al., 2017). In this study, MeJA significantly increased root length and stem diameter of G. uralensis seedlings at the concentrations of 45 and 60 μM, and root diameter at the concentrations of 15 and 30 μM. MeJA also remarkably increased stem length at 45 μM. These results indicates that 200 mM NaCl significantly caused damage on growth of G. uralensis seedlings and the addition of MeJA at different concentrations promoted these growth parameters of salt-stressed G. uralensis. Therefore, MeJA has beneficial effect on seedling growth of salt-stressed G. uralensis, and this beneficial effect varied depending on the applied concentrations of MeJA.
Salt stress causes the accumulation of reactive oxygen species (ROS) like H2O2 and O2－ that leads to cellular damage through oxidation of lipids, proteins and nucleic acids (Ji et al., 2009). MDA is often used as a major index to judge membrane lipid peroxidation and its concentration represents damaged degree of the membrane lipid (Zhuang et al., 2010). Previous studies found that slat stress increased O2− production rate and H2O2 content in in Tagetes erecta L. (Garg and Bhandari, 2016) and alfalfa (Wang et al., 2009). Accordingly, salt stress increased MDA concentrations in Ammodendron bifolium and Gleditsia sinensis seedling (Liu et al., 2014), Glycyrrhiza uralensis Fisch. (Pan et al., 2006) and Nicotiana tabaccum L.(Babajani et al., 2010). In the present study, 200 mM NaCl significantly increased O2－production rate, H2O2 and MDA contents of G. uralensis seedlings, indicating that 200 mM NaCl induced oxidative stress in G. uralensis seedlings and then G. uralensis cell membrane has been damaged under this level of salt
Fig. 4. Effect of methyl jasmonate (MeJA) at different concentrations (15, 30, 45, and 60 μM) on AsA and GSH contents of G. uralensis seedlings under salt stress (200 mM). The different letters within the different treatments indicate the significant difference at P ≤ 0.05. Values are means ± SE (n = 6). 5
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Fig. 5. Effect of methyl jasmonate (MeJA) at different concentrations (15, 30, 45, and 60 μM) on SPS, SS and NR activities of G. uralensis seedlings under salt stress (200 mM). The different letters within the different treatments indicate the significant difference at P ≤ 0.05. Values are means ± SE (n = 6).
stress. Fortunately, our result also found that the increase of O2－ production rate, H2O2 and MDA contents caused by NaCl stress can be reversed by MeJA addition which reduced O2−, H2O2 and MDA accumulations (Fig. 2), indicating that MeJA can mitigate oxidative stress caused by salt stress in G. uralensis seedling. Which is supported by previous studies for banana (Sun et al., 2013) and diploid and tetraploid Robinia pseudoacacia (Jiang et al., 2016). To mitigate the oxidative damage induced by excessive ROS, plants have developed a complex defense antioxidative system, including lowmolecular mass antioxidants as well as antioxidative enzymes, such as SOD, CAT, POD, APX, GPX and GR (Azevedo Neto et al., 2006). Among these, SOD firstly converts superoxide O2－ into O2 and H2O2 i,n waterwater cycle. Then, CAT and/or POD, primarily converts H2O2 that produced by photorespiration and fatty acid β oxidation into water and O2 in CAT cycle, while APX, GPX and GR catalyze H2O2 to H2O in ascorbate-glutathione cycle (Fig. 6) (Yoon et al., 2008). Previous studies showed that salt stress increased SOD and POD activities in Brassica napus L. seedlings (Qilin et al. 2009), and SOD, CAT and APX activities in wheat seedlings (Rasool et al., 2013), while salt stres reduced the activities of APX, GPX and GR in roots of salt-sensitive maize genotype (Azevedo Neto et al., 2006). Which suggest that salt stress can altered the activities of enzymes involving in scavenging ROS in various species and these ROS-scavenging enzymes were co-regulated in plants. In the
present study, 200 mM NaCl significantly increased SOD and GR activities, but significantly decreased GPX activity in G. uralensis seedlings (Fig. 3), indicating that 200 mM NaCl altered ROS-scavenging system by focusing on water-water cycle and ascorbate-glutathione cycle rather than on CAT cycle, which may be related to CAT mainly scavenges H2O2 in the photosynthesis process. Thus we speculated that POD, APX and GPX coordinated with SOD play a central protective role in the excessive O2－and H2O2 scavenging process and the active involvement of these enzymes is related, at least in part, to salt-induced oxidative stress tolerance in G. uralensis seedlings, which is supported by the previous report on Nicotiana tabacum L. (Badawi et al., 2004). Interestingly, our results also found that MeJA significantly further enhanced SOD activity at 45 μM, POD activity at 30 μM, CAT activity at 15 μM, and GPX activity at high concentrations of 30 μM or above (Fig. 3), indicating that MeJA enhanced ROS-scavenging ability of G. uralensis seedlings subjected to salt stress is involved in the water-water cycle, CAT cycle and ascorbate-glutathione cycle. Which is similar to the results obtained in Artemisia annua (Tariq et al., 2011), strawberry plants (Faghih et al., 2017) and Robinia pseudoacacia plant (Jiang et al., 2016). Moreover, AsA and GSH serve as two important non-enzyme antioxidants which involve ROS-scavenging processes. Previous studies showed that salt stress significantly decreased AsA and GSH contents in
Fig. 6. Effect of methyl jasmonate (MeJA) on production of ROS and some major antioxidant systems in G. uralensis seedlings under salt stress. 6
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most antioxidant enzymes and non-enzymatic antioxidants, while MeJA with 60 μM enhanced C and N metabolizing enzymes. Future research should further investigate deep mechanisms by which MeJA at different concentrations affect G. uralensis plant under salt stress conditions.
grape leaves (Zhao et al., 2015), and Oryza sativa L. (Wang et al., 2014), and AsA content in Limonium aureum L. (Yang et al., 2016). In the present study, 200 mM NaCl significantly reduced AsA content but had no significant effect on GSH content, which may be related to the decrease of GPX activity that due to GSH is oxidized to GSSG by GPX (Fig. 6). Our results also showed that MeJA significantly increased AsA and GSH contents at the concentrations of 15 and 30 μM, but had no significant effect at the concentrations of 45 and 60 μM (Fig. 4), indicating that MeJA enhanced ROS-scavenging ability of G. uralensis seedlings subjected to salt stress at lower concentrations mainly by coregulating both of antioxidant enzymes activities and non-enzymatic antioxidants contents, while this effect of MeJA at higher concentrations mainly by regulating antioxidant enzymes activities. Thus, MeJA can coordinately regulate antioxidant enzymes activities and non-enzymatic antioxidants contents that could decrease O2－ production rate and H2O2 content and finally mitigate oxidative stress induced by salt stress in G. uralensis seedlings.
Author contribution Author contribution statement: Xinhui Zhang conceived and designed the experiments, revised the manuscript. Duoyong Lang and Xiaxia Yu conducted the experiments and drafted the manuscript. Xiaoxia Jia and Zhixian Li helped conduct parts of the experiments and analyses. Compliance with ethical standards In this manuscript, the authors declare that they have no conflict of interest. This work doesn’t involve Human Participants and/or Animals. The authors have informed consent for this manuscript.
5.3. MeJA enhanced carbon and nitrogen metabolisms in G. Uralensis seedling under salt stress
CRediT authorship contribution statement Xinhui Zhang conceived and designed the experiments, revised the manuscript and funding acquisition. Duoyong Lang and Xiaxia Yu conducted the experiments and drafted the manuscript. Xiaoxia Jia and Zhixian Li helped conduct parts of the experiments and analyses.
One of the important adaptive response of plants to salt stress is accumulation of soluble sugars that is closely associated with two key enzymes: SPS and SS. In these, SPS plays an important role in sucrose synthesis, while SS is involved in sucrose-cleaving (Li et al., 2008; Verma et al., 2011). Previous studies showed that salt stress significantly increased SS and SPS activities in cucumber leaves (Yan et al., 2014) and SPS activity in maize plants (Abdel-Latif, 2007). In the present study, 200 mM NaCl markedly increased SPS and SS activities in G. uralensis seedlings, indicating that SS and SPS exerted an important function in carbon metabolism process in salt-stressed G. uralensis plants, and which may be inducing to the accumulation of soluble sugars for better responding to salt stress. Salinity also influence the activities of enzymes related to N metabolism. NR is a limiting factor in plant growth and biological functions in the first step of nitrogen assimilation, which reduced nitrate (NO3－) to nitrite (NO2－) in the cytosol, and NO3－ taken up by plant root from soil using a variety of transporters (Pourranjbari Saghaiesh et al., 2019; Souri and Hatamian, 2019). Previous research found that salt stress significantly decreased NR activity in various crops such as bean, maize and sugar beet (Reda et al., 2011), and tomato (Hayat et al., 2012). Our result showed that 200 mM NaCl significantly decreased NR activity in G. uralensis seedlings, indicating that 200 mM NaCl stress may be weakened N metabolism by reducing NR activity and strengthened C metabolism by increasing SS and SPS activities in G. uralensis seedlings. Up to now, there is few report about the role of MeJA on C and N metabolisms in plants (Yu et al., 2019). In the present study, MeJA at 60 μM significantly increased NR activity in G. uralensis seedlings, while MeJA significantly decreased SPS and SS activities in G. uralensis seedlings at the concentrations of 45 μM or below (Fig. 5) compared with the NaCl treatment, which is differed to our previous results in G. uralensis seedlings grown on 100 mM NaCl stress with 25 and 50 μM MeJA (Yu et al., 2019). These results indicated that MeJA had significant effect on C and N metabolizing processes in salt-stressed G. uralensis seedlings, and this effect was differed depending on the applied concentrations of MeJA and the levels of salt stress. In conclusion, the accumulation of ROS in G. uralensis seedlings under salt stress could increase the membrane lipid peroxidation and further affect C and N metabolisms, which may be one of the main reasons for inhibiting plant growth. Interestingly, MeJA could alleviate adverse effects induced by salt stress on G. uralensis by enhancing antioxidant enzyme activities and non-enzymatic antioxidants, and further regulating C and N metabolizing enzyme activities in G. uralensis subjected to salt stress. Moreover, this effect of MeJA on salt-stressed G. uralensis seedling was differed depending on the applied concentrations of MeJA, that is MeJA from 15 to 30 μM has more positive effects on
Declaration of Competing Interest In this manuscript, the authors declare that they have no conflict of interest. This work doesn’t involve Human Participants and/or Animals. The authors have informed consent for this manuscript. Acknowledgements The authors are grateful for the financial support provided by the National Natural Science Foundation of China (31860343and 31460330), the Key National Research and Development Programs of China (2017YFC1700706) and the project of College students’ innovation of Ningxia Medical University (YJSCXCY2018021). References Abdel-Latif, A., 2007. Response of maize leaf sucrose phosphate synthase to salinity. Res. J. Agri. Biol. Sci. 3, 930–933. Ahmadi, M., Souri, M.K., 2018. Growth and mineral elements of coriander (Corianderum sativum L.) plants under mild salinity with different salts. Acta Physiol. Plant. 40, 94–999. https://doi.org/10.1007/s11738-018-2773-x. Ahmadi, M., Souri, M.K., 2019. Nutrient uptake, proline content and antioxidant enzymes activity of pepper (Capsicum annuum L.) under higher electrical conductivity of nutrient solution created by nitrate and chloride salts of potassium and calcium. Acta Sci. Pol-Hortoru. 18, 113–122. https://doi.org/10.24326/asphc.2019.5.11. Azevedo Neto, A.D., Prisco, J.T., Eneas-Filho, J., Abreu, C.D., Gomes-Filho, E., 2006. Effect of salt stress on antioxidative enzymes and lipid peroxidation in leaves and roots of salt-tolerant and salt-sensitive maize genotypes. Environ. Exp. Bot. 56, 87–94. https://doi.org/10.1016/j.envexpbot.2005.01.008. Babajani, S., Sarmad, J., Norastehnia, A., Sajedi, R.H., Alavi, M.N., 2010. Study of the effect of chloride on malondialdehyde content and antioxidant enzymes activity in three varieties of tobacco plant leaf (Nicotiana tabaccum L.). 2th National Conference of Plant Physiology. Badawi, G.H., Yamauchi, Y., Shimada, E., Sasaki, R., Kawano, N., Tanaka, K., Tanaka, K., 2004. Enhanced tolerance to salt stress and water deficit by overexpressing superoxide dismutase in tobacco (Nicotiana tabacum L.) chloroplasts. Plant Sci. 166, 919–928. https://doi.org/10.1016/j.plantsci.2003.12.007. Bor, M., Özdemir, F., Türkan, I., 2003. The effect of salt stress on lipid peroxidation and antioxidants in leaves of sugar beet Beta vulgaris L. And wild beet Beta maritima L. Plant Sci. 164, 77–84. https://doi.org/10.1016/S0168-9452(02)00338-2. Bu, R., Xie, J., Yu, J., Liao, W., Xiao, X., Lv, J., 2016. Autotoxicity in cucumber (Cucumis sativus L.) seedlings is alleviated by silicon through an increase in the activity of antioxidant enzymes and by mitigating lipid peroxidation. J. Plant Biol. 59, 247–259. https://doi.org/10.1007/s12374-016-0526-1. Coruzzi, G., Bush, R., 2001. Nitrogen and carbon nutrient and metabolite signaling in plants. Plant Physiol. 125, 61–64. https://doi.org/10.1104/pp.125.1.61. Debouba, M., Gouia, H., Suzuki, A., Ghorbel, M.H., 2006. NaCl stress effects on enzymes
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Methyl jasmonate improves metabolism and growth of NaCl-stressed Glycyrrhiza uralensis seedlings
T
Duoyong Langc,1, Xiaxia Yua,1, Xiaoxia Jiad, Zhixian Lie, Xinhui Zhanga,b,* a
College of Pharmacy, Ningxia Medical University, Yinchuan, 750004, China Ningxia Engineering and Technology Research Center of Hui Medicine Modernization, Ningxia Collaborative Innovation Center of Hui Medicine, Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education, Ningxia Medical University, Yinchuan, 750004, China c Laboratory Animal Center, Ningxia Medical University, Yinchuan, 750004, China d Potato Research Institute, Gansu Academy of Agricultural Sciences, Lanzhou, 730000, China e Hunan Provincial Key Laboratory of Coal Resource Clean Utilization and Mine Environmental Protection, Hunan University of Science and Technology, Xiangtan, Hunan, 411201, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: G. uralensis Methyl jasmonate Antioxidant system Carbon and nitrogen metabolisms Salt stress
Background and Aims: Salinity is one of the major factors that affect growth and metabolism in plants. However, limited information is available on the influence of methyl jasmonate (MeJA) on antioxidant defense, carbon (C) and nitrogen (N) metabolisms in Glycyrrhiza uralensis Fisch. (G. uralensis) under salt stress. Here, this experiment was conducted to investigated the responses of antioxidant defense, C and N metabolisms of salt-stressed G. uralensis to MeJA at different concentrations. Methods: G.uralensis plant seeds were grown under six conditions: control, 200 mM NaCl, and 200 mM NaCl combined with 15, 30, 45 and 60 μM MeJA. Key Results: Results showed that salt stress inhibited G. uralensis growth and this adverse effect was reversed by MeJA at different concentrations. MeJA also affected antioxidant system and C and N metabolisms. In general, MeJA from 45 to 60 μM significantly enhanced superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione peroxidase (GPX) activities in salt-stressed G. uralensis seedlings, MeJA from 15 to 30 μM increased peroxidases (POD) and catalase (CAT) activities, and ascorbate peroxidase (AsA) and glutathione (GSH) contents. Thus, these changes caused a decrease in hydrogen peroxide (H2O2), superoxide radical (O2−) and malondialdehyde (MDA) contents correspondingly. Moreover, 60 μM MeJA enhanced sucrose synthase (SS), sucrose phosphate synthase (SPS) and nitrate reductase (NR) activities in salt-stressed G. uralensis seedlings. Conclusions: MeJA promoted growth of salt-stressed G. uralensis seedlings by alleviating oxidative stress and strengthening C and N metabolism, these effect depended on the applied concentrations of MeJA.
1. Introduction Salinity is one of the most severe problems in agriculture worldwide and NaCl is the predominant salt in most saline environments, especially in arid and semi-arid regions (Ahmadi and Souri, 2018). High concentration of salts can impair the growth, physiological and metabolic activities of plant by causing oxidative and osmotic stresses, or a combination of these factors (Zhang et al., 2008; Slama et al., 2015). Specifically, salt stress accelerates production of reactive oxygen species (ROS) and causes lipid peroxidation in plants (Debouba et al., 2006; Ahmadi and Souri, 2019). In order to ameliorate the damaging effects of ROS, plants have developed several defense mechanisms. One
of the mechanisms is antioxidant system, high activities of antioxidant enzymes and high contents of non-enzymatic constituents are two important factors that contributed to tolerance against salt stress (Sharma et al., 2013). In the antioxidant enzymes and non-enzymatic constituents, superoxide dismutase (SOD) mainly scavenges superoxide radical (O2－), catalase (CAT) and peroxidases (POD) eliminate hydrogen peroxide (H2O2). Moreover, glutathione reductase (GR), ascorbate peroxidase (APX), glutathione peroxidase (GPX) and glutathione (GSH) play important roles in ascorbate-glutathione cycle, which clears hydrogen peroxide (H2O2) in different ways. Bor et al. (2003) reported that salt-stressed Beta vulgaris L. plant had better protection against oxidative damage by enhancing SOD, CAT and GR activities.Azevedo
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Corresponding author at: College of Pharmacy, Ningxia Medical University, Yinchuan, 750004, China. E-mail address: [email protected] (X. Zhang). 1 These authors contributed equally in this work. https://doi.org/10.1016/j.scienta.2020.109287 Received 9 December 2019; Received in revised form 17 February 2020; Accepted 17 February 2020 0304-4238/ © 2020 Elsevier B.V. All rights reserved.
Scientia Horticulturae 266 (2020) 109287
D. Lang, et al.
effects of MeJA at different concentrations on plant growth, antioxidant defense, and C and N metabolisms. The purpose is to provide a better understanding about the significant effect and mechanism of MeJA on alleviating the adverse effect caused by salt stress on G. uralensis
Neto et al. (2006)found that the activities of SOD, APX, GPX and GR increased with time in leaves of salt-stressed maize. Therefore, the increase in the content of antioxidants and the activity and/or expression of antioxidant enzymes is a common adaptive response of plants to oxidative stress (Fidalgo et al., 2015; Souri and Hatamian, 2019). In addition, carbon (C) and nitrogen (N) metabolisms are fundamental processes in plants and closely coordinated in nearly every biochemical pathway (Coruzzi and Bush, 2001). C metabolism processes involve photosynthetic carbon assimilation, sucrose and starch metabolisms and carbohydrate transport. N metabolic processes include N uptake, transport, reduction and assimilation, and amino acid metabolism (Kusano et al., 2011; Jie et al. 2013). Like other abiotic stresses, salt stress also induces the alterations of C and N metabolisms. When plants are grown under abiotic stresses, a series of physiological processes associated with C metabolism are altered, such as inhibition of photosynthesis, changes in carbohydrate content and carbohydrate metabolic enzyme activity (Theocharis et al., 2012). Silva et al. (2014) observed that salt stress enhanced soluble proteins content and reduced nitrate reductase (NR) activity in sunflower plant. Salt stress decreased glutamine synthetase (GS) and glutamate synthase (GOGAT) activities in Populus simonii roots and leaves (Meng et al., 2016; Souri and Tohidloo, 2019), but increased NO3－ content in rice old leaves (Wang et al., 2012). Interestingly, many studies observed correlations between antioxidant activity and salinity tolerance are thought to be conveyed by plant hormones (Salimi et al., 2016). Jasmonates are a class of plant hormones that mediate defense mechanisms and stress responses. Among these, the role of Methyl jasmonate (MeJA) in protecting plants from different abiotic stresses has been considerable (Guo et al., 2012). Walia et al. (2006) showed that MeJA diminished the inhibitory effect caused by salt stress on the photosynthesis rate in rice plant. Jiang et al. (2016) found that MeJA improved growth of salinity-stressed Robinia pseudoacacia plant through increasing the activities of SOD, POD and APX that eliminate the excessive O2－and H2O2 induced by salt stress. Furthermore, MeJA enhanced the activities of antioxidant enzymes (CAT, POD and SOD) to improve the ROS scavenging ability in Artemisia annua and strawberry, both in non-stressed and stressed plants (Tariq et al., 2011; Faghih et al., 2017). These results confirmed that MeJA can alleviate oxidative stress induced by salinity by improving the activity and/or pools of antioxidant enzymes (Poonam et al., 2013). However, there is little research on the role of MeJA on carbon and nitrogen metabolism processes in plants subjected to salt stress, only Hanik et al. (2010) reported that MeJA promoted the transcriptional modification of numerous genes that can impact whole plant resource allocation of both nitrogen- and carbon-containing substrates and increased the export of newly acquired carbon and nitrogen in tomato leaves, further increased the proportion of nitrogen allocated in tomato roots (Gómez et al., 2010). These results undoubtedly suggested that exogenous MeJA may be involved in the defense against salinity stress and their role in manifesting salinity tolerance has recently been acknowledged (Iqbal et al., 2014). However, the precise mechanisms involved are up for debate and depend on the plant species involved. Glycyrrhiza uralensis Fisch. (G. uralensis) is one of the ancient herbal plants of the legume family native to Asia, which comprises 18 wild and cultivated species and widely used herbal medicine (Pan et al. 2014). Its also frequently used as additives in tobacco and food worldwide and as a beneficial desert plant resource for hampering wind, fixing sand, and improving soil conditions. However, production and availability of wild G. uralensis are insufficient for human consumption because of irresponsible over exploitation and adverse agronomic conditions (Zhang et al., 2017). Despite the growing body of evidence supporting the role of MeJA on growth of several plant species under salt stress, however, not yet been well investigated in G. uralensis plants. Moreover, our previous experiments have shown that MeJA has an positive effect on salt-stressed G. uralensis plants during seed germination stage (Yu et al., 2019). Thus, this experiment was conducted to further investigated the
2. Materials and methods 2.1. Plant material G. uralensis seeds were obtained from the wild plants in Yan-chi county, the Ningxia Hui Autonomous Region, China, in August 2017. 2.2. Experimental design and treatments Petri dish germination experiment was conducted at the laboratory. Specifically, the seeds were steeped with 85 % H2SO4 for 2.5 h, then surface sterilized with 0.1 % H2O2 for 10 min, rinsed three-times in distilled water, and imbibed in distilled water for 12 h at room temperation. A total of 50 seeds were sown in 90 mm diameter petri dishes with two layers of Whatman No.2 filter paper to receive different treatments. According to the results of preliminary experiment, the treatments include (i) control (CK), with distilled water only, (ii) 200 mM NaCl, (iii) 200 mM NaCl with 15 μM MeJA, (iv) 200 mM NaCl with 30 μM MeJA; (ⅴ) 200 mM NaCl with 45 μM MeJA and (ⅵ) 200 mM NaCl with 60 μM MeJA. MeJA chemical was purchased as a 95 % aqueous solution and dissolved in Tween 20. Each treatment was replicated six times. 2.3. Seed germination conditions All petri dishes were placed in a plant growth chamber where maintained at 28℃/20℃ (day/night) with a 12 h photoperiod at a light intensity of 37.5 μmol m−2 s−1 PAR. Germination was considered to occur when the radicle had emerged by at least 1.0 mm. The number of germinated seeds was recorded every day. After germinating for 10 days, uniform sized and healthy seedlings of G. uralensis were collected from each of the 24 petri dishes, and analyzed immediately to measure their physiological and biochemical characteristics; For remaining plants in each of the 24 petri dishes, length of hypocotyl and radicle, diameter of hypocotyl and radicle were recorded. 2.4. Determination of antioxidant and lipid peroxidation Each 0.5 g of whole seedling tissue was homogenized in 10 mL of an extraction buffer containing 50 mM phosphate buffer, pH 7.8, 1.0 mM EDTA, 1.0 g polyvinylpyrrolidone (PVP), and 0.5 % (v/v) Triton X-100, then the extract was centrifuged for 20 min at 12,000 rpm and the supernatant was used for analysis of activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), and in concentration of malondialdehyde (MDA). All operations were carried out at 0-4℃. SOD activity was measured by its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT), recorded at 560 nm. One unit of SOD was defined as the amount of enzyme that inhibits NBT reduction by 50 % (Meng et al., 2016). CAT activity was assayed according to the method of Sohn et al. (2005) with minor modifications. The CAT reaction solution comprised 50 mM phosphate buffer (pH 7.8), 0.1 M H2O2 and 0.1 mL enzyme extract. Changes in the absorbance of the reaction solution at 240 nm were recorded for 5 min. POD activity was assayed according to the method of Pan et al. (2006) with minor modifications. 0.2 mL of the enzyme extract was mixed with 2.7 mL 50 mM phosphate buffer (pH 7.8) and 0.1 mL 50 mM of guaiacol and 1 mL 2% H2O2 and the change in absorbance was measured at 470 nm for 5 min. Membrane lipid peroxidation was determined by measuring the MDA content using the method of Zhang et al. (2017) with minor modifications. The enzyme extract (1 mL) was mixed with 2.5 mL of 0.5 2
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2.8. Determination of the activities of enzymes related carbon and nitrogen metabolism
% thiobarbituric acid (TBA) and was then heated at 100℃ for 20 min. The absorbance was determined at 450 nm, 532 nm, and 600 nm, respectively.
Each 0.5 g of fresh shoot and root was homogenized at 4℃ in 3 mL of extraction buffer [100 mM Tris−HCl buffer (pH 7.2) containing 10 mM MgCl2, 1 mM EDTA-Na2, 10 mM β-hydrophobic base ethanol, 2% ethylene glycol and 1% polyvinyl pyrrolidone] with a mortar and pestle. The homogenate was then centrifuged at 12,000 rpm for 10 min and the supernatant was used as the crude extract for the sucrose synthase SS and sucrose phosphate synthase SPS assay. For SS activity, reaction mixtures contained 100 mM Tris−HCl buffer (pH 7.2), 10 mM MgCl2, 5 mM uridine diphosphate glucose (UDPG), 5 mM fructose, and the 0.3 mL of sample. For SPS activity, 5 mM fructose was substituted for 5 mM fructose-6-phosphate. Data are expressed as micromoles of sucrose or sucrose-P produced per gram of fresh weight per hour (Golldack et al., 2014). For the nitrate reductase (NR) activity assay, approximately 0.5 g samples of fresh tissue were ground in a pre-chilled mortar and pestle on ice in 4 mL of extraction buffer. The extraction 0.025 M buffer (pH 8.7) consisted of 10 mM cysteine and 1 mM EDTA. The mixture was centrifuged at 4000 rpm at 4℃ for 15 min and then 0.5 mL supernatant was added to the reaction mixture. The reaction mixture comprised 1.2 mL of 0.1 M KNO3-phosphate buffer (pH 7.5) and incubated at 25℃ for 30 min. The reaction was terminated by adding 1 mL 1% (w/v) sulfanilamide in 3 M HCl. For nitrite determination, 1 mL 0.02 % w/v Nnaphthyl-1-dihydrochloride was added, and the color was allowed to develop for 15 min at 25℃. The mixture was centrifuged at 12,000 rpm at 4℃ for 5 min, and then the absorbance of the supernatant at 540 nm was measured. NR activity is expressed as l mol NO2− g-1 fresh weight (FW) h-1 (Golldack et al., 2014).
2.5. Determination of reactive oxygen species contents Superoxide radicals (O2－) were measured using the method described in Bu et al. (2016) with minor modification. Where, 0.2 g fresh seedlings were allowed to react with 1 mL of hydroxylamine hydrochloride for 1 h; then, 1 mL of p-aminobenzene sulfonic acid and 1 mL of α-naphthylamine were added, and the solution was maintained at 25℃ for 20 min. The absorbance of the mixture was measured at 530 nm, and NaNO2 was used to prepare a standard curve. The hydrogen peroxide (H2O2) content was determined as described by Bu et al. (2016) with minor modification. Briefly, 0.2 g fresh seedling was centrifuged at 10,000 rpm for 10 min after being homogenized in 5 mL of ice-cold acetone. The collected supernatant (1 mL) was subsequently added to a concentrated HCl solution containing 0.1 mL of 20 % Ti(SO4)2 and 0.2 mL of concentrated ammonia, and the mixture was recentrifuged at 8000 rpm for 15 min. The obtained pellets were then added to 3 mL of 1 M H2SO4. The absorbance was observed at 410 nm, and H2O2 was used to prepare a standard curve. 2.6. Determination of ascorbate peroxidase (APX), glutathione reductase (GR) and glutathione peroxidse (GPX) activities Each 0.5 g of fresh shoot and root were homogenized at 4℃ in 8 mL of extraction buffer [50 mM phosphate buffer (pH 7.0) containing 1 mM ascorbic acid (AsA), 1 mM dithiothreitol (DTT), 1 mM glutathione (GSH) and 1 mM MgCl2] with mortar and pestle. The homogenate was then centrifuged at 12,000 rpm for 20 min and the supernatant was used as the crude extract for the activities of GR, APX and GPX. APX activity was determined according to the method of Kang and Saltveit (2001) with minor modification. The 3 mL reaction mixture was composed of 50 mM phosphate buffer (pH 7.0) containing 0.1 mM EDTA, 0.3 mM AsA, 0.06 mM H2O2 and 0.2 mL of enzyme extract. The oxidation of ascorbate was followed by the decrease in the absorbance at 240 nm. GR activity was assayed by Zhang et al. (2017) and measured the decrease in absorbance at 340 nm due to the oxidation of NADPH. The 3 mL reaction mixture comprised 50 mM phosphate buffer (pH 8.2) containing 1 mM EDTA, 0.2 mM NADPH, 1 mM oxidized glutathione (GSSG) and 0.2 mL of enzyme extract. GPX activity was determined as described by Azevedo Neto et al. (2006) in a reaction mixture (2.0 mL) containing 100 mM phosphate buffer (pH 7.0), 0.4 mM EDTA, 0.4 mM glutathione (GSH), 0.4 mM NADPH, 0.2 mM H2O2 and 50 μL enzyme extract. The addition of enzyme extract started the reaction and the increase in absorbance was recorded at 340 nm for 1 min.
3. Statistical analysis All data were present as mean + SE. The difference was analyzed by ANOVA using SPSS 17.0 software (SPSS Inc, Chicago, IL, USA). Significant differences were tested using the Least Significant Difference (LSD) test at P ≤ 0.05.
4. Results 4.1. Effect of MeJA on growth of salt-stressed G. Uralensis seedlings NaCl stress significantly reduced radicle length and radicle diameter of G. uralensis seedlings compared with the control. MeJA addition markedly changed growth of G. uralensis seedling at different concentrations. Specifically, 45 μM MeJA significantly increased radicle length, hypocotyl length and hypocotyl diameter; 30 μM MeJA significantly increased radicle length and hypocotyl diameter; MeJA from 15 to 30 μM also significantly increased radicle diameter (Fig. 1). These results indicate that MeJA can alleviate the harmful effect caused by salt stress in G. uralensis seedling.
2.7. Determination of ascorbate peroxidase (AsA) and glutathione (GSH) contents
4.2. Effect of MeJA on reactive oxygen species and membrane lipid peroxidation in salt-stressed G. Uralensis seedlings
AsA content was determined according to the method of Liu et al. (2014) using 0.2 g fresh seedling. Standards for AsA was prepared in the range of 0−15 mg L−1 in 1 mL 5% (w/v) trichloroacetic acid (TCA), 1 mL alcohol, 0.5 mL 0.4 % H3PO4-alcohol, 1 mL 0.5 % bathophenanthroline (BP)-alcohol and 0.5 mL 0.03 % FeCl3-alcohol at 30℃ for 90 min and measured at 534 nm. GSH was determined by the method of Zhang et al. (2017) and extracted from 0.2 g fresh seedling with 4 mL of 5 mM EDTA-TCA and estimated by 5,5′-dithio-bis (2-nitrobenzoic acid) (TDNB). Changes in absorbance of the reaction mixture were measured at 420 nm and total GSH concentration was calculated from a standard curve with GSH.
NaCl stress significantly increased O2－production rate, and H2O2 and MDA contents in G. uralensis seedlings compared with the control. Interestingly, MeJA addition significantly reduced O2－production rate at low concentrations of 30 μM or below, and H2O2 content at all concentrations; MeJA also significantly decreased MDA content at concentrations of ≥ 30 μM (Fig. 2), indicating that MeJA can mitigate oxidative stress caused by salt stress in G. uralensis seedling.
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Fig. 1. Effect of methyl jasmonate (MeJA) at different concentrations (15, 30, 45, and 60 μM) on root length, stem length, root diameter and stem diameter of G. uralensis seedlings under salt stress (200 mM). The different letters within the different treatments indicate the significant difference at P ≤ 0.05. Values are means ± SE (n = 6).
4.3. Effect of MeJA on antioxidant enzymes activities MeJA on and nonenzymatic antioxidants contents in salt-stressed G. Uralensis seedlings
4.4. Effect of MeJA on carbon and nitrogen metabolizing enzymes activities in salt-stressed G. Uralensis seedlings
NaCl stress significantly increased SOD and GR activities, but decreased GPX activity in G. uralensis seedlings compared with the control. Compared with NaCl treatment, MeJA addition significantly enhanced SOD activity at 45 μM, POD activity at 30 μM, CAT activity at 15 μM, and GPX activity at high concentrations of 30 μM or above, while MeJA addition significantly reduced APX activity at 30 μM (Fig. 3). As to non-enzymatic antioxidants, NaCl stress remarkably decreased AsA content in G. uralensis seedlings compared with the control, while MeJA significantly increased AsA content at 15 and 30 μM and GSH content at 30 μM compared with the NaCl treatment (Fig. 4). These results indicate that MeJA alleviated oxidative stress are associated with the improved the activities of antioxidant enzymes and the contents of non-enzymatic antioxidants in G. uralensis seedlings subjected to NaCl stress.
NaCl stress significantly increased SS and SPS activities but decreased NR activity in G. uralensis seedlings compared with the control. Compared with NaCl treatment, MeJA from 15 to 45 μM significantly reduced SS and SPS activities, but significantly enhanced NR activity at 60 μM (Fig. 5). 5. Discussion 5.1. MeJA improved growth of G. Uralensis seedling under salt stress Salinity stress can change most physiological and biochemical processes and lead to a inhibition in growth and reduction in biomass (Ji et al., 2009; Souri and Tohidloo, 2019). Salt stress inhibited growth of strawberry seedlings (Faghih et al., 2017) and Ammodendron argenteum seedlings (Zhuang et al., 2010) and remarkably decreased the root length of rice even at low NaCl concentration (Kang et al., 2005). Our
Fig. 2. Effect of methyl jasmonate (MeJA) at different concentrations (15, 3, 45, and 60 μM) on O2− production rate, H2O2 and MDA contents of G. uralensis seedlings under salt stress (200 mM). The different letters within the different treatments indicate the significant difference at P ≤ 0.05. Values are means ± SE (n = 6). 4
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Fig. 3. Effect of methyl jasmonate (MeJA) at different concentrations (15, 30, 45, and 60 μM) on SOD, POD, CAT, APX, GR and GPX activities of G. uralensis seedlings under salt stress (200 mM). The different letters within the different treatments indicate the significant difference at P ≤ 0.05. Values are means ± SE (n = 6).
5.2. MeJA mitigated oxidative stress caused by salt-stressed in G. Uralensis seedling
results showed 200 mM NaCl significantly decreased root length and root diameter of G. uralensis seedlings compared with the control. Interestingly, studies have found that application of exogenous MeJA markedly increased growth parameters of Robinia pseudoacacia (Jiang et al., 2016), Artemisia annua (Tariq et al., 2011) and strawberry under slat stress conditions (Faghih et al., 2017). In this study, MeJA significantly increased root length and stem diameter of G. uralensis seedlings at the concentrations of 45 and 60 μM, and root diameter at the concentrations of 15 and 30 μM. MeJA also remarkably increased stem length at 45 μM. These results indicates that 200 mM NaCl significantly caused damage on growth of G. uralensis seedlings and the addition of MeJA at different concentrations promoted these growth parameters of salt-stressed G. uralensis. Therefore, MeJA has beneficial effect on seedling growth of salt-stressed G. uralensis, and this beneficial effect varied depending on the applied concentrations of MeJA.
Salt stress causes the accumulation of reactive oxygen species (ROS) like H2O2 and O2－ that leads to cellular damage through oxidation of lipids, proteins and nucleic acids (Ji et al., 2009). MDA is often used as a major index to judge membrane lipid peroxidation and its concentration represents damaged degree of the membrane lipid (Zhuang et al., 2010). Previous studies found that slat stress increased O2− production rate and H2O2 content in in Tagetes erecta L. (Garg and Bhandari, 2016) and alfalfa (Wang et al., 2009). Accordingly, salt stress increased MDA concentrations in Ammodendron bifolium and Gleditsia sinensis seedling (Liu et al., 2014), Glycyrrhiza uralensis Fisch. (Pan et al., 2006) and Nicotiana tabaccum L.(Babajani et al., 2010). In the present study, 200 mM NaCl significantly increased O2－production rate, H2O2 and MDA contents of G. uralensis seedlings, indicating that 200 mM NaCl induced oxidative stress in G. uralensis seedlings and then G. uralensis cell membrane has been damaged under this level of salt
Fig. 4. Effect of methyl jasmonate (MeJA) at different concentrations (15, 30, 45, and 60 μM) on AsA and GSH contents of G. uralensis seedlings under salt stress (200 mM). The different letters within the different treatments indicate the significant difference at P ≤ 0.05. Values are means ± SE (n = 6). 5
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Fig. 5. Effect of methyl jasmonate (MeJA) at different concentrations (15, 30, 45, and 60 μM) on SPS, SS and NR activities of G. uralensis seedlings under salt stress (200 mM). The different letters within the different treatments indicate the significant difference at P ≤ 0.05. Values are means ± SE (n = 6).
stress. Fortunately, our result also found that the increase of O2－ production rate, H2O2 and MDA contents caused by NaCl stress can be reversed by MeJA addition which reduced O2−, H2O2 and MDA accumulations (Fig. 2), indicating that MeJA can mitigate oxidative stress caused by salt stress in G. uralensis seedling. Which is supported by previous studies for banana (Sun et al., 2013) and diploid and tetraploid Robinia pseudoacacia (Jiang et al., 2016). To mitigate the oxidative damage induced by excessive ROS, plants have developed a complex defense antioxidative system, including lowmolecular mass antioxidants as well as antioxidative enzymes, such as SOD, CAT, POD, APX, GPX and GR (Azevedo Neto et al., 2006). Among these, SOD firstly converts superoxide O2－ into O2 and H2O2 i,n waterwater cycle. Then, CAT and/or POD, primarily converts H2O2 that produced by photorespiration and fatty acid β oxidation into water and O2 in CAT cycle, while APX, GPX and GR catalyze H2O2 to H2O in ascorbate-glutathione cycle (Fig. 6) (Yoon et al., 2008). Previous studies showed that salt stress increased SOD and POD activities in Brassica napus L. seedlings (Qilin et al. 2009), and SOD, CAT and APX activities in wheat seedlings (Rasool et al., 2013), while salt stres reduced the activities of APX, GPX and GR in roots of salt-sensitive maize genotype (Azevedo Neto et al., 2006). Which suggest that salt stress can altered the activities of enzymes involving in scavenging ROS in various species and these ROS-scavenging enzymes were co-regulated in plants. In the
present study, 200 mM NaCl significantly increased SOD and GR activities, but significantly decreased GPX activity in G. uralensis seedlings (Fig. 3), indicating that 200 mM NaCl altered ROS-scavenging system by focusing on water-water cycle and ascorbate-glutathione cycle rather than on CAT cycle, which may be related to CAT mainly scavenges H2O2 in the photosynthesis process. Thus we speculated that POD, APX and GPX coordinated with SOD play a central protective role in the excessive O2－and H2O2 scavenging process and the active involvement of these enzymes is related, at least in part, to salt-induced oxidative stress tolerance in G. uralensis seedlings, which is supported by the previous report on Nicotiana tabacum L. (Badawi et al., 2004). Interestingly, our results also found that MeJA significantly further enhanced SOD activity at 45 μM, POD activity at 30 μM, CAT activity at 15 μM, and GPX activity at high concentrations of 30 μM or above (Fig. 3), indicating that MeJA enhanced ROS-scavenging ability of G. uralensis seedlings subjected to salt stress is involved in the water-water cycle, CAT cycle and ascorbate-glutathione cycle. Which is similar to the results obtained in Artemisia annua (Tariq et al., 2011), strawberry plants (Faghih et al., 2017) and Robinia pseudoacacia plant (Jiang et al., 2016). Moreover, AsA and GSH serve as two important non-enzyme antioxidants which involve ROS-scavenging processes. Previous studies showed that salt stress significantly decreased AsA and GSH contents in
Fig. 6. Effect of methyl jasmonate (MeJA) on production of ROS and some major antioxidant systems in G. uralensis seedlings under salt stress. 6
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most antioxidant enzymes and non-enzymatic antioxidants, while MeJA with 60 μM enhanced C and N metabolizing enzymes. Future research should further investigate deep mechanisms by which MeJA at different concentrations affect G. uralensis plant under salt stress conditions.
grape leaves (Zhao et al., 2015), and Oryza sativa L. (Wang et al., 2014), and AsA content in Limonium aureum L. (Yang et al., 2016). In the present study, 200 mM NaCl significantly reduced AsA content but had no significant effect on GSH content, which may be related to the decrease of GPX activity that due to GSH is oxidized to GSSG by GPX (Fig. 6). Our results also showed that MeJA significantly increased AsA and GSH contents at the concentrations of 15 and 30 μM, but had no significant effect at the concentrations of 45 and 60 μM (Fig. 4), indicating that MeJA enhanced ROS-scavenging ability of G. uralensis seedlings subjected to salt stress at lower concentrations mainly by coregulating both of antioxidant enzymes activities and non-enzymatic antioxidants contents, while this effect of MeJA at higher concentrations mainly by regulating antioxidant enzymes activities. Thus, MeJA can coordinately regulate antioxidant enzymes activities and non-enzymatic antioxidants contents that could decrease O2－ production rate and H2O2 content and finally mitigate oxidative stress induced by salt stress in G. uralensis seedlings.
Author contribution Author contribution statement: Xinhui Zhang conceived and designed the experiments, revised the manuscript. Duoyong Lang and Xiaxia Yu conducted the experiments and drafted the manuscript. Xiaoxia Jia and Zhixian Li helped conduct parts of the experiments and analyses. Compliance with ethical standards In this manuscript, the authors declare that they have no conflict of interest. This work doesn’t involve Human Participants and/or Animals. The authors have informed consent for this manuscript.
5.3. MeJA enhanced carbon and nitrogen metabolisms in G. Uralensis seedling under salt stress
CRediT authorship contribution statement Xinhui Zhang conceived and designed the experiments, revised the manuscript and funding acquisition. Duoyong Lang and Xiaxia Yu conducted the experiments and drafted the manuscript. Xiaoxia Jia and Zhixian Li helped conduct parts of the experiments and analyses.
One of the important adaptive response of plants to salt stress is accumulation of soluble sugars that is closely associated with two key enzymes: SPS and SS. In these, SPS plays an important role in sucrose synthesis, while SS is involved in sucrose-cleaving (Li et al., 2008; Verma et al., 2011). Previous studies showed that salt stress significantly increased SS and SPS activities in cucumber leaves (Yan et al., 2014) and SPS activity in maize plants (Abdel-Latif, 2007). In the present study, 200 mM NaCl markedly increased SPS and SS activities in G. uralensis seedlings, indicating that SS and SPS exerted an important function in carbon metabolism process in salt-stressed G. uralensis plants, and which may be inducing to the accumulation of soluble sugars for better responding to salt stress. Salinity also influence the activities of enzymes related to N metabolism. NR is a limiting factor in plant growth and biological functions in the first step of nitrogen assimilation, which reduced nitrate (NO3－) to nitrite (NO2－) in the cytosol, and NO3－ taken up by plant root from soil using a variety of transporters (Pourranjbari Saghaiesh et al., 2019; Souri and Hatamian, 2019). Previous research found that salt stress significantly decreased NR activity in various crops such as bean, maize and sugar beet (Reda et al., 2011), and tomato (Hayat et al., 2012). Our result showed that 200 mM NaCl significantly decreased NR activity in G. uralensis seedlings, indicating that 200 mM NaCl stress may be weakened N metabolism by reducing NR activity and strengthened C metabolism by increasing SS and SPS activities in G. uralensis seedlings. Up to now, there is few report about the role of MeJA on C and N metabolisms in plants (Yu et al., 2019). In the present study, MeJA at 60 μM significantly increased NR activity in G. uralensis seedlings, while MeJA significantly decreased SPS and SS activities in G. uralensis seedlings at the concentrations of 45 μM or below (Fig. 5) compared with the NaCl treatment, which is differed to our previous results in G. uralensis seedlings grown on 100 mM NaCl stress with 25 and 50 μM MeJA (Yu et al., 2019). These results indicated that MeJA had significant effect on C and N metabolizing processes in salt-stressed G. uralensis seedlings, and this effect was differed depending on the applied concentrations of MeJA and the levels of salt stress. In conclusion, the accumulation of ROS in G. uralensis seedlings under salt stress could increase the membrane lipid peroxidation and further affect C and N metabolisms, which may be one of the main reasons for inhibiting plant growth. Interestingly, MeJA could alleviate adverse effects induced by salt stress on G. uralensis by enhancing antioxidant enzyme activities and non-enzymatic antioxidants, and further regulating C and N metabolizing enzyme activities in G. uralensis subjected to salt stress. Moreover, this effect of MeJA on salt-stressed G. uralensis seedling was differed depending on the applied concentrations of MeJA, that is MeJA from 15 to 30 μM has more positive effects on
Declaration of Competing Interest In this manuscript, the authors declare that they have no conflict of interest. This work doesn’t involve Human Participants and/or Animals. The authors have informed consent for this manuscript. Acknowledgements The authors are grateful for the financial support provided by the National Natural Science Foundation of China (31860343and 31460330), the Key National Research and Development Programs of China (2017YFC1700706) and the project of College students’ innovation of Ningxia Medical University (YJSCXCY2018021). References Abdel-Latif, A., 2007. Response of maize leaf sucrose phosphate synthase to salinity. Res. J. Agri. Biol. Sci. 3, 930–933. Ahmadi, M., Souri, M.K., 2018. Growth and mineral elements of coriander (Corianderum sativum L.) plants under mild salinity with different salts. Acta Physiol. Plant. 40, 94–999. https://doi.org/10.1007/s11738-018-2773-x. Ahmadi, M., Souri, M.K., 2019. Nutrient uptake, proline content and antioxidant enzymes activity of pepper (Capsicum annuum L.) under higher electrical conductivity of nutrient solution created by nitrate and chloride salts of potassium and calcium. Acta Sci. Pol-Hortoru. 18, 113–122. https://doi.org/10.24326/asphc.2019.5.11. Azevedo Neto, A.D., Prisco, J.T., Eneas-Filho, J., Abreu, C.D., Gomes-Filho, E., 2006. Effect of salt stress on antioxidative enzymes and lipid peroxidation in leaves and roots of salt-tolerant and salt-sensitive maize genotypes. Environ. Exp. Bot. 56, 87–94. https://doi.org/10.1016/j.envexpbot.2005.01.008. Babajani, S., Sarmad, J., Norastehnia, A., Sajedi, R.H., Alavi, M.N., 2010. Study of the effect of chloride on malondialdehyde content and antioxidant enzymes activity in three varieties of tobacco plant leaf (Nicotiana tabaccum L.). 2th National Conference of Plant Physiology. Badawi, G.H., Yamauchi, Y., Shimada, E., Sasaki, R., Kawano, N., Tanaka, K., Tanaka, K., 2004. Enhanced tolerance to salt stress and water deficit by overexpressing superoxide dismutase in tobacco (Nicotiana tabacum L.) chloroplasts. Plant Sci. 166, 919–928. https://doi.org/10.1016/j.plantsci.2003.12.007. Bor, M., Özdemir, F., Türkan, I., 2003. The effect of salt stress on lipid peroxidation and antioxidants in leaves of sugar beet Beta vulgaris L. And wild beet Beta maritima L. Plant Sci. 164, 77–84. https://doi.org/10.1016/S0168-9452(02)00338-2. Bu, R., Xie, J., Yu, J., Liao, W., Xiao, X., Lv, J., 2016. Autotoxicity in cucumber (Cucumis sativus L.) seedlings is alleviated by silicon through an increase in the activity of antioxidant enzymes and by mitigating lipid peroxidation. J. Plant Biol. 59, 247–259. https://doi.org/10.1007/s12374-016-0526-1. Coruzzi, G., Bush, R., 2001. Nitrogen and carbon nutrient and metabolite signaling in plants. Plant Physiol. 125, 61–64. https://doi.org/10.1104/pp.125.1.61. Debouba, M., Gouia, H., Suzuki, A., Ghorbel, M.H., 2006. NaCl stress effects on enzymes
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