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Foliar application of 5-aminolevulinic acid (ALA) alleviates NaCl stress in cucumber (Cucumis sativus L.) seedlings through the enhancement of ascorbate-glutathione cycle
Scientia Horticulturae 257 (2019) 108761
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Foliar application of 5-aminolevulinic acid (ALA) alleviates NaCl stress in cucumber (Cucumis sativus L.) seedlings through the enhancement of ascorbate-glutathione cycle
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Yue Wua, Linli Hua, Weibiao Liaoa, Mohammed Mujitaba Dawudaa,b, Jian Lyua, Jianming Xiea, ⁎ Zhi Fenga, Alejandro Calderón-Urreac,d, Jihua Yua, a
College of Horticulture, Gansu Agricultural University, Lanzhou, 730070, PR China Department of Horticulture, FoA, University for Development Studies, P. O. Box TL 1882, Tamale, Ghana College of Plant Protection, Gansu Agricultural University, Lanzhou, PR China d Department of Biology, College of Science and Mathematics, California State University, Fresno, USA b c
ARTICLE INFO
ABSTRACT
Keywords: Ascorbate-glutathione cycle 5-Aminolevulinic acid (ALA) Ascorbic acid oxidase (AAO) Salt stress Cucumber seedlings
The natural plant growth regulator 5-aminolevulinic acid (ALA) promotes plant growth. The present study assessed the effects of exogenously applied ALA (25 mg L−1) on the growth and H2O2 scavenging system (ascorbate-glutathione cycle, AsA-GSH cycle) under NaCl stress (50 mmol L−1) in cucumber (Cucumis sativus L.) seedlings. Growth of cucumber seedlings was significantly inhibited by NaCl stress. Moreover, NaCl caused accumulation of H2O2 and malonaldehyde (MDA) as well as dehydroascorbic acid (DHA) and oxidized glutathione (GSSG) in cucumber leaves. However, the application of ALA reversed the adverse effect of NaCl on growth of the cucumber seedlings by increasing the shoots and roots biomass. The application of ALA also increased the contents of AsA and GSH of the seedlings under moderate salt stress. The ratios of reduced/ oxidized antioxidant, such as AsA/DHA and GSH/GSSG, in seedlings under NaCl treatment were also improved by ALA, indicating that the regeneration efficiency of antioxidants could be promoted by exogenous ALA. In addition, exogenous ALA also promoted the activities of enzymes involved in the AsA-GSH cycle, including ascorbic acid oxidase (AAO), ascorbate peroxidase (APX), monodehydroascorbic acid reductase (MDHAR), dehydroascorbic acid reductase (DHAR) and glutathione reductase (GR). These results suggested that ALA alleviate the damages caused by NaCl through the enhancement of the AsA-GSH pathway in cucumber seedlings.
1. Introduction Salinity is one of the major environmental factors limiting the yield and productivity of crops across the globe (Tang et al., 2015). Salt stress causes injuries such as leaf scorch and root damage to plants and also retards plant growth or even leads to plant death. Moreover, salt stress causes series of physiological changes in plants, including cell dehydration, ion toxicity and oxidative stress (Mickelbart et al., 2015). Reactive oxygen species (ROS) such as superoxide free radicals (·O2−), hydrogen peroxide (H2O2), hydroxyl radicals (OH−) and singlet oxygen (1O2), are formed as byproducts of various physiological and metabolic pathways in different cellular compartments; and these ROS are indispensable second messengers of stress response mechanism in plants (Deinlein et al., 2014; Batth et al., 2017). However, under stimulated external environmental factors, excess ROS accumulates in cells,
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disturbing cellular homeostasis and causing lipid peroxidation (Mishra et al., 2011). In higher plants, there are multiple pathways to produce H2O2, such as respiratory electron transport chain in mitochondrion, photochemical reaction in chloroplast, and the reactions of superoxide dismutase (SOD) and peroxidase (POD) (Halliwell, 1978; Slesak et al., 2007; Quan et al., 2008). The major pathway of scavenging H2O2 in higher plant cell is the ascorbate-glutathione cycle (AsA-GSH cycle) which exists extensively in many cellular organs (Li et al., 2010). The research on AsA-GSH cycle in higher plants began with the study of mycorrhiza in soybean (Glycine max L.) (Dalton et al., 1991). The AsAGSH cycle has been proven to be closely related to plant resistance to environment stress factors such as the ozone (Wang et al., 2013), salt (Li et al., 2013), drought (Kang et al., 2013), cold (Jiang et al., 2013) and zinc (Tripathi et al., 2017). In recent years, many plant growth regulators (PGRs) have been
Corresponding author at: College of Horticulture, Gansu Agricultural University, No. 1 Yinmen Village, Anning District, Lanzhou, 730070, PR China. E-mail address: [email protected] (J. Yu).
https://doi.org/10.1016/j.scienta.2019.108761 Received 11 February 2019; Received in revised form 21 May 2019; Accepted 8 August 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
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used to improve plants resistance to abiotic stresses. ALA plays an important role in regulating growth and enhancing tolerance to salt stress in plants such as Swiss chard (Beta vulgaris L.), rice (Oryza sativa L.), oilseed rape (Brassica napus L.), tomato (Solanum lycopersicum L.), peach (Prunus persica L.), watermelon (Citrullus lanatus Thunb.) and cucumber (Cucumis sativus L.) (Liu et al., 2014; Nunkaew et al., 2014; Tian et al., 2014; Zhang et al., 2015; Ye et al., 2016; Chen et al., 2017; Wu et al., 2018). Furthermore, foliar application of ALA reduced the levels of H2O2 and malonaldehyde (MDA) in oilseed rape (B. napus L.) under drought stress (Liu et al., 2013). Moreover, exogenous application of ALA significantly enhanced the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) in winter rape (B. napus L.) plants under herbicide stress (Averina et al., 2014). Similarly, in tomato (S. lycopersicum L.) plants under low temperature stress, the application of ALA increased the activities of SOD, APX, monodehydroascorbic acid reductase (MDHAR) and dehydroascorbic acid reductase (DHAR) and it also increased the contents of AsA and GSH (Liu et al., 2018). Finally, the AsA-GSH pathway in Ginkgo biloba L. leaves was enhanced by spraying 100 mg L−1 ALA even under normal growth condition (Xu et al., 2009). In cucumber seedlings under salt stress, exogenous ALA application enhanced photosynthesis through the activation of the chlorophyll biosynthesis pathway (Wu et al., 2018). However, the role of exogenous ALA on ROS scavenging system has not been adequately studied, particularly on the effect of ALA onalleviation of NaCl stress in cucumber with regard to its role on the AsA-GSH cycle. The main objective of this study was to investigate the role of ALA in the AsA-GSH cycle in cucumber seedlings under salt stress.
of the leaves. ALA application was done twice, as soon as the seedlings were exposed to the salt treatment and repeated 24 h later. The plants without ALA treatments were sprayed with distilled water in a similar manner as those that received the ALA treatment. 2.3. Biomass and morphology After 10 days of treatment, 6 seedlings were taken from each treatment to measure fresh weight. Then, the dry weight was measured by drying seedlings in oven, at 105℃ for 15 min and then at 80℃ to constant weight. In addition, the morphology of seedlings was obtained by digital camera. 2.4. H2O2 content The content of H2O2 was measured according to the method of Junglee with some modifications (Junglee et al., 2014). A fresh leaf sample (0.1 g) was homogenized with liquid nitrogen, and then transferred into a 2 mL centrifuge tube which was then kept in an ice bath. Then 1.5 mL 0.1% TCA was added, the homogenate was centrifuged at 12,000 g for 15 min at 4 °C. After that the supernatant (0.5 mL) was fully mixed with 0.5 mL PBS (pH 7.0) and 1 mL of a 1 mol L−1 KI. The mixture was placed under constant temperature (28℃) for 1 h. The absorbance was measured at 390 nm and concentration was calculated using a standard curve of H2O2 reference standards. Specifically, the concentrations of H2O2 standard curve were 0, 1, 2, 3, 4 and 5 mmol L−1.
2. Materials and methods
2.5. MDA content
2.1. Plant material and growth conditions
The content of MDA was determined according to the method of Fazeli and co-authors with some modifications (Fazeli et al., 2007). A fresh leaf sample (0.3 g) was ground in ice an bath with 2 mL 0.05 mol L−1 trichloroacetic acid (TCA). The homogenate was transferred into centrifuge tube and centrifuged at 10,000 g for 5 min at 25 °C. Then, 5 mL of a 0.5% (m/v) thiobarbituric acid (TBA) solution was added into the supernatant. The extract was placed in a boiling water bath for 10 min, and then quickly moved to cold water bath. After cooling, the solution was centrifuged again at 10,000 g for 10 min at 25 °C. The absorbance of the supernatant was measured at 532 nm; and the value for nonspecific absorption at 600 nm was subtracted. Finally, the content of MDA was calculated from the molar extinction coefficient of − MDA-TBA at 155 mmol−1 cm 1.
Cucumber seeds (Cucumis sativus L. cv. Xinchun No. 4) were surface sterilized with potassium permanganate (0.03%) for 10 min and rinsed with distilled water. The seeds were then soaked in distilled water for 6 h and exposed to germination conditions. The moistened seeds were placed on double-layer filter paper and kept at 28 ± 1 °C under dark condition. At 5 days after germination, seedlings with uniform size, fully spread cotyledons, and well-formed roots were transferred to 1-L capacity opaque plastic containers withhalf-strength Yamasaki’s cucumber nutrient solution: the macroelement contained Ca(NO3)2 − 1.75 mmol L−1,KNO33 mmolL 1, NH4H2PO4 0.5 mmol L−1, −1 MgSO4·7H2O 1 mmol L ; and the microelement contained EDTAFeNa·3H2O 35 μmol L−1, MnSO4·4H2O 5 μmol L−1, H3BO3 25 μmol L−1, ZnSO4·7H2O 0.4 μmol L−1, CuSO4·5H2O 0.15 μmol L−1, (NH4)6Mo7O24·4H2O 0.07 μmol L−1. Each container had 4 seedlings. The seedlings were grown in artificial climate chamber throughout the experiment under the following conditions: light intensity at − 350–450 mmol m-2s 1, temperature at 28/18℃ (day/night) and relative humidity at 50–60%. The nutrient solution was changed at 2-day intervals.
2.6. AsA and DHA contents The determination of AsA and DHA were done as described by Arakawa and co-authors with some modifications (Arakawa et al., 1981). A fresh leaf sample (0.5 g) was homogenized with 20 mL 50 g L−1 TCA in an ice bath, then 50 g L−1 TCA was added to make 100 mL. The homogenate was filtered and the filter liquor was taken as ascorbic acid extract. The extract (1 mL) was mixed with 1 mL of a 50 g L−1 TCA solution, 1 mL absolute ethyl alcohol, 0.5 mL 0.4% phosphoric acidalcohol, 1 mL of a 5 g L−1 bathophenanthroline (BP)-alcohol and 0.5 mL 0.3 g L−1 FeCl3-alcohol. The mixture was kept under constant temperature of 30℃ for 60 min. Then, the absorbance was determined at 534 nm. The AsA concentration was calculated using a standard curve of AsA reference standards. The concentrations of AsA standard curve were 0, 10, 20, 30, 40, 50, 60 μg mL−1. For the measurement of DHA content, 1 mL extract was mixed with 0.5 mL 60 mmol L−1 DTTacetic acid. The pH value of homogenate was adjusted to 7–8 by adding some drops of NaHPO-NaOH. The reduction reaction of DHA was done under room temperature for 10 min. Then, a 0.5 mL 0.2 g mL−1 TCA was added to the mixture. Subsequent steps were the same as the reaction system of AsA determination. The total ascorbic acid content was calculated according to AsA standard curve. The content of DHA was
2.2. Treatments and experimental design The 30-day-old uniform seedlings with three fully expanded true leaves were selected for the treatments. Each experimental unit had two 1 L capacity opaque plastic containers and each container was supplied with four seedlings. Each treatment was replicated three times and arranged in a completely randomized design. The treatments were: (1) CK: normal nutrient solution only; (2) NaCl: 50 mmol L−1 NaCl in nutrient solution +0 mg L−1 ALA; (3) NaCl + ALA: 50 mmol L−1 NaCl in nutrient solution +25 mg L−1 ALA, and (4) ALA: normal nutrient solution +25 mg L−1 ALA. The concentrations of NaCl and ALA were selected based on our previous experiment (Wu et al., 2018). The ALA (Sigma Aldrich, US) was applied with a 100 mL capacity hand sprayer. The ALA was thoroughly sprayed on both the upper and lower surfaces 2
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the total ascorbic acid content minus the AsA content.
growth of the cucumber seedlings (Fig. 2D).
2.7. GSH and GSSG contents
3.2. H2O2 and MDA contents
The contents of GSH and GSSG were measured by GSH content detection and GSSG content detection kits (Solarbio, China) following the manufacturer’s instructions, respectively. Briefly, the concrete operation was followed the specification of kit. For the determination of GSH, the 2-nitro-5-mercaptobenzoic acid, a yellow chemical, could be produced by the reaction of GSH with 5,5-dithiobis and 2-nitrobenzoic acid, and has maximum light absorption at wavelength 412 nm. Fresh leaf sample (0.1 g) was quickly grounded in liquid nitrogen, and 1 mL extraction reagent was added. The homogenized mixture was centrifuged at 8000 rpm for 10 min under 4 °C and took the supernatant. The absorbance of reaction system was determined at 412 nm. The content of GSH was calculated according to GSH standard curve. For the determination of GSSG, the 2-vinylpyridine was employed to inhibit the intrinsic GSH in cell, and then GSSG was reduced by glutathione reductase, and measured as the method of GSH.
The results in Fig. 3A shows that the H2O2 content in cucumber leaves under NaCl stress increased significantly by 46.0% compared with the CK. Exogenous application of ALA effectively decreased the H2O2 content to a level similar to that of the CK. In addition, under normal growth condition, H2O2 content in seedlings treated with ALA alone did not differ fromthe CK. Under salt stress, the content of MDA increased greatly by 400.8% in cucumber leaves compared with the control seedlings (Fig. 3B). However, the MDA content of seedlings under NaCl stress was decreased with the application of exogenous ALA. In addition, MDA content in seedlings under normal condition but sprayed with ALA was similar to those of the CK plants. 3.3. AsA-GSH pool Fig. 4A shows that the content of AsA in the leaves of the cucumber seedlings decreased under moderate salt stress. However, the application of ALA significantly increased the content of AsA in the seedlings which were exposed to the NaCl stress compared with seedlings treated with NaCl alone. There was no significant difference in the content of AsA between CK seedlings and the ALA treated seedlings under no NaCl stress condition.However, the DHA accumulated in cucumber leaves under salinity stress (Fig. 4B). Application of ALA to theseedlings under NaCl stress significantly decreased the DHA content compared with seedlings treated with NaCl alone. Moreover, the application of ALA under stress-free condition did not affect the DHA content in the cucumber seedlings. Under salinity condition, GSH content in the leaves reduced significantly in leaves when compared with CK (Fig. 4C and D). However, the GSH content was recovered to a level similar to that of the control seedlings when ALA was applied. Moreover, the content of GSH decreased in control seedlings which sprayed with ALA. In addition, GSSG content in the leaves increased to the maximal value under NaCl stress. Foliar application of ALA to seedlings under NaCl stress condition recovered the content of GSSG to a level similar to that of the CK plants. Besides, without salt stress, there was no significant difference between the contents of GSSG in the CK seedlings and the seedlings treated with ALA only.
2.8. Enzymatic activities The extraction method of enzyme was done as described by Rao and Terry with some modifications (Rao and Terry, 1989). A frozen leaf sample (0.5 g) was ground in liquid nitrogen and then transferred into a centrifuge tube. The homogenate was extracted in pre-chilled extraction buffer (5 mL) which consisted of 100 mmol L−1 Tricine-NaOH (pH 8.0), 10 mmol L−1MgCl2, 1 mmol L−1 EDTA, 10 mmol L−1 DTT, 10 mmol L−1 Na-ascorbate, 0.5% BSA and 1% (w/v) PVP-40. The mixture was centrifuged at 12,000 g for 15 min under 4 °C. The supernatant was used for enzymes activities assay. Activities of the enzymes involved in AsA-GSH cycle, including ascorbic acid oxidase (AAO, EC 1.10.3.3), APX (EC 1.11.1.11), MDHAR (EC 1.6.5.4), DHAR (EC 1.8.5.1) and GR (EC 1.6.4.2) were determined using enzyme activity assay kits (Solarbio, China). 2.9. Statistical analysis The experiment was performed with three replicates and the results were expressed as mean ± SE. Analysis of variance was performed using SPSS 22.0 (SPSS Institute Inc., US) and treatments means were compared using the Tukey’s test at a 0.05 level of probability. All figures were prepared with OriginPro 2017 (OriginLab Institute Inc., US).
3.4. AsA/DHA ratio and GSH/GSSG ratio
3. Results
The effect of NaCl stress on non-enzymatic antioxidant ratios, including AsA/DHA and GSH/GSSG, is shown in Fig. 5. The level of AsA/ DHA decreased significantly in the plants exposed to the salt stress but the application of ALA to the seedlings under the salt stress recovered the level similar to those of the control seedlings (Fig. 5A). The application of ALA to normal seedlings did not affect the value of AsA/DHA compared with CK seedlings. Similarly, exogenous application of ALA recovered the level of GSH/GSSG in the seedlings exposed to the salt stress to levels similar to that of CK (Fig. 5B). Furthermore, the level of GSH/GSSG in the control seedlings decrease when ALA was applied.
3.1. Biomass and plant morphology The shoot and root biomass of the seedlings were measured at 10 d after treatment application (Fig. 1). These indexes decreased significantly under NaCl stress. In comparison with the plants grown in normal condition, the fresh and dry shoot weights of the seedlings under salt stress also decreased by 45.2% and 40.6%, respectively (Fig. 1A, C). The fresh and dry root weights of the seedlings under stress decreased by 54.9% and 43.5% respectively (Fig. 1B, D). Exogenous application of ALA significantly increased the fresh and dry weights of shoots as well as the root fresh weight. However, ALA application did not significantly affect root dry weight of the seedlings under salt stress. Moreover, the application of ALA in the absence of salt stress decreased the dry weight of shoots and roots. NaCl stress caused leaf chlorosis, reduced leaf size and decreased the size of the root systems whiles the seedlings of CK treatment producedgreen and bigger leaves as well as larger root systems (Fig. 2). The application of ALA to the seedlings exposed to NaCl stress alleviated the NaCl-induced stress and the seedlings appear similar to the control plants (Fig. 2C). Moreover, the application of ALA also improved the
3.5. Enzymatic activities of AAO and APX The results of our experiment showed no significant difference in AAO activity of NaCl-treated seedlings and CK seedlings (Fig. 6A). However, the activity of AAO was markedly enhanced by spraying ALA, which reached a level more than 5-fold of the control plants. Moreover, the activity of AAO in the control seedlings was enhanced whenALA was applied. NaCl stress caused a significant inhibition of APX activity but this was enhanced by ALA application (Fig. 6B). Similarly, exogenous ALA enhanced the activity of APX in the control seedlings. 3
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Fig. 1. Effect of exogenous ALA on (A) fresh shoot weight (B) fresh root weight (C) dry shoot weight and (D) dry root weight, of cucumber seedlings under NaCl stress. The data presented are the means of three plants ± SE of means (n = 3, with 6 plants per replication). Treatment means with different lower case letters indicate significant differences according to Tukey’s test (p < 0.05).
3.6. Enzymatic activities of MDHAR, DHAR and GR
8.5-fold more than the CK seedlings. In addition, the activity of DHAR in seedlings under NaCl was similar to those under NaCl stress but treated with ALA. However, the DHAR activities in these two treatments were significantly higher compared to the CK plants (Fig. 7B). There was marked difference of DHAR between the seedlings treated with ALA alone and that of CK. Besides, GR activity in cucumber leaves was significantly suppressed by salinity stress (Fig. 7C). However, exogenous application of ALA significantly enhanced the GR
Fig. 7 shows the changes in the activities of other three key enzymes involved in the AsA-GSH cycle, which include MDHAR, DHAR and GR. The MDHAR activityin the control seedlings, seedlings treated with NaCl and those sprayed with ALA alone were statistically similar (Fig. 7A). However, MDHAR activity of the NaCl treated seedlings was significantly enhanced by the application of ALA, and the increase was
Fig. 2. The morphology of cucumber seedlings under difference treatments. (A) CK: seedlings under normal growth condition. (B) NaCl: seedlings under 50 mmol L−1 NaCl stress. (C) NaCl + ALA: seedlings under 50 mmol L−1NaCl stress and sprayed 25 mg L−1 ALA on the leaves. (D) ALA: seedlings sprayed 25 mg L−1 ALA on the leaves under normal growth condition.
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Fig. 3. Effect of exogenous ALA on (A) hydrogen peroxide (H2O2) and (B) malondialdehyde (MDA) contents in the leaves of cucumber seedlings under NaCl stress. The data presented are the means of three plants ± SE of means (n = 3, with 6 plants per replication). Treatment means with different lower case letters indicate significant differences according to Tukey’s test (p < 0.05).
Fig. 4. Effect of exogenous ALA on contents of (A) ascorbate (AsA), (B) dehydroascorbic acid (DHA), (C) glutathione (GSH) and (D) oxidized glutathione (GSSG) in the leaves of cucumber seedlings under NaCl stress. The data presented are the means of three plants ± SE of means (n = 3, with 6 plants per replication). Treatment means with different lower case letters indicate significant differences according to Tukey’s test (p < 0.05).
activitycompared with the control plants and the plants under NaCl stress.
shoot biomass of seedlings. In addition, root growth of the cucumber seedlings was also inhibited by salt stress but foliar application of ALA significantly increased root fresh weight. It is likely that the effect was a result of regulatory changes of aquaporin gene expression as previously reported: aquaporins CsPIPs1:1 (plasma membrane intrinsic protein) and CsNIP (nodulin-26-like intrinsic membrane protein) in cucumber (C. sativus L.) seedling, under salt stress were down-regulated by exogenous ALA application and this prevented the dehydration of the cells (Yan et al., 2014). Moreover, in the present study, application of ALA to cucumber seedlings under normal condition caused significant decrease in root dry weight compared with CK plants. In a previous study, the application of 50 mg L−1 exogenous ALA on the leaves of salt-sensitive cucumber seedlings resulted in significant reduction of root dry weight, which was similar to that of plants exposed to 75 mmol L−1 NaCl stress (Yan et al., 2014). Besides, pretreatment of ALA significantly enhanced
4. Discussion Salt stress inhibits seed germination, interferes with physiological processes such as water transport, protein synthesis and photosynthesis and also damages both the anatomy and morphology of plants. ALA was reported to have the capacity of improving plant tolerance to abiotic stress (Akram and Ashraf, 2013). In Indian mustard (Brassica juncea L.) plants under salt stress, the fresh weight of seedlings reduced with increases in NaCl concentrations (Pandey and Penna, 2017). In this study, NaCl stress decreased plant growth and the seedlings exhibited leaf chlorosis, reduced leaf sizes and also decreased the size of root systems. However, exogenous application of ALA significantly improved the 5
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Fig. 5. Effect of exogenous ALA on changes of (A) AsA/DHA ratio and (B) GSH/GSSG ratio in the leaves of cucumber seedlings under NaCl stress. The data presented are the means of three plants ± SE of means (n = 3, with 6 plants per replication). Treatment means with different lower case letters indicate significant differences according to Tukey’s test (p < 0.05).
the root respiration of fig plants (An et al., 2016). These results indicate that the application of ALA to plants under normal growth condition may enhance the dry matter consumption of plants, and finally adversely affected dry matter accumulation. In higher plants, AsA-GSH cycle is an indispensable ROS scavenging system in cells and this is mainly located in cytoplasm and many organelles. In this study, H2O2 and MDA accumulated in the leaves of cucumber plants under salt stress, indicating that the cell membrane lipid peroxidation already occurred in the seedlings. H2O2 is known as a crucial signal molecule in plants under normal growth condition, and it plays several important functions in cell, including gene expression, cellular metabolism, programmed cell death, resistance response and cell senescence (Huang et al., 2016). However, under abiotic stress, membrane system and cell organelles can be damaged by excess accumulation of H2O2. In rice (Oryza sativa L.) plants under drought stress, the content of H2O2 increased but the contents of AsA, GSH and the ratios of antioxidants decreased suggesting that drought caused oxidative stress in the plants (Pyngrope et al., 2013). The main pathway of scavenging H2O2 in higher plants is through the AsA-GSH cycle (Jin et al., 2003). Although AsA-GSH cycle is known as the antioxidant defense system in plant cell, its activity can also be regulated by environmental stresses. For example, at 0.5 mmol L−1 cadmium stress, the value of AsA/DHA decreased by 28.5% compared with that of control plants; moreover, at 1.0 mmol L−1 cadmium stress, this index decreased by 57.1% (Hasanuzzaman et al., 2017). These results indicated that oxidative stress can be aggravated with increases in environmental stress. In some studies, the level of reduced antioxidants in plants could be expressed by the ratio of reduced type and oxidized type, like AsA/DHA and GSH/ GSSG (Zhao et al., 2016; Hasanuzzaman et al., 2017; Liu et al., 2018). In the present study, foliar application of ALA to cucumber seedlings under NaCl stress caused an increase in the contents of AsA and GSH.
Moreover, it recovered the decreased contents of AsA/DHA and GSH/ GSSG caused by salinity. These results are in agreement with Liu and co-authors who reported that increased accumulation of ROS and MDA occurred in swede type rape (B. napus L.) plants under drought stress (Liu et al., 2011). In another study, the application of exogenous ALA alleviated the membrane lipid peroxidation caused by oxidative stress and also increased the AsA/DHA and GSH/GSSG contents. The total value of AsA + DHA was increased in poplar (Populus deltoides×Populus maximowiczii) leaves under ozone and Cd stress (Castagna et al., 2015). AsA is the most abundant antioxidant in plants and plays a role in the coping mechanism of plants to oxidative stress (Noctor and Foyer, 1998). The content of AsA is affected by reductase activity (like MDHAR or DHAR), and by its upstream biosynthesis in plants. For example, GDP-d-mannose pyrophosphorylase (GMPase) is a key enzyme in AsA synthesis pathway (Tabata et al., 2002). Overexpression of GMPase coding gene SlGME in tomato (S. lycopersicum L.) showed that AsA content increased significantly in the leaves and seedlings were more resistant to methyl viologen stress and cold stress than the wild type (Zhang et al., 2011). As two antioxidants in higher plant cell, AsA and GSH play very important roles in AsA-GSH cycle in scavenging free ROS, because relatively high level of antioxidant means more electron donor will take part in the reactions of H2O2 reduction and AsA regeneration. In Arabidopsis thaliana, several ascorbate-deficient (vtc) mutants, including VTC1, VTC2, VTC3 and VTC4, AsA content decreasedby 25–30% compared with wild type plants (Colville and Smirnoff, 2008). In addition, the mutants were more sensitive to ozone stress and UV-B stress than the wild type plant (Gao and Zhang, 2008). Similarly, A. thaliana mutant with the γ-glutamyl-cysteine synthetase encoding gene (GSH1) knock-out revealed obstacle in synthesizing GSH as well as embryo development inhibition or embryo death (Cairns et al., 2006; Pasternak et al., 2008). Fig. 6. Effect of exogenous ALA on the activities of (A) ascorbic acid oxidase (AAO) and (B) ascorbic acid peroxidase (APX) in the leaves of cucumber seedlings under NaCl stress. The data presented are the means of three plants ± SE of means (n = 3, with 6 plants per replication). Treatment means with different lower case letters indicate significant differences according to Tukey’s test (p < 0.05).
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Fig. 7. Effect of exogenous ALA on the activities of (A) monodehydroascorbic acid reductase (MDHAR) (B) dehydroascorbic acid reductase (DHAR) and (C) glutathione reductase (GR) in the leaves of cucumber seedlings under NaCl stress. The data presented are the means of three plants ± SE of means (n = 3, with 6 plants per replication). Treatment means with different letters indicate significant differences according to Tukey’s test (p < 0.05).
In this experiment, the activities of AAO and APX in cucumber leaves were both enhanced by exogenous ALA under stressful or nonstressful condition. Although the catalytic reactions of AAO and APX in plant cell are similar, the AAO is located in apoplast whiles APX is located in organelles or cytoplasm. Although AAO does not belong to intracellular AsA-GSH circulatory system, it plays vital role in regulating the redox state of extracellular ascorbic acid pool. When plants suffer from environmental stresses, the value of oxidation/reduction ratio of ascorbic acid pool is regulated by AAO, and this will become the first defense of cells against stress damages (Kao, 2015). The activity of AAO can be suppressed by certain metallic element, such as zinc and cadmium (Morina et al., 2010; Zelinová et al., 2014). In addition, antisense oriented AAO gene in tobacco (Nicotiana tabacum L.) caused AAO activity in the transgenic plants to decreased by about 0.2-fold of that in wild type plants (Yamamoto et al., 2005). However, pretreatment with H2S up-regulated the relative expression of TaAAO gene (encoding AAO) in leaves and roots of wheat (Triticum aestivum L.) under drought stress (Ma et al., 2016). Furthermore, AAO activity could be enhanced by glycinebetaine (GB) (Chen and Heuer, 2013). Similarly, in the present study, exogenous ALA promoted AAO activity in cucumbers seedlings under NaCl stress. Diverse roles of AAO such as cell mitosis, cell elongation and photosynthesis in plants have been reported (Tullio et al., 2004; Batth et al., 2017). The APX is also an antioxidant that is directly involved in the decomposition of H2O2 in the AsA-GSH cycle. Therefore, the enhancement of APX activity indicates the improvement of degradation efficiency of ROS in plant cell. Under low temperature stress, 15 °C/8 °C (day/night), foliar application of 25 mg L−1 ALA enhanced the activities of APX, MDHAR, DHAR and GR in tomato (S. lycopersicum L.) plants and improved their resistance to low temperature (Liu et al., 2018). The activities of several antioxidant enzymes involved in AsA-GSH pathway in the leaves of G. biloba L. were significantly increased with the application of exogenous ALA (Xu et al., 2009). Another key enzyme in AsA-GSH cycle is GR which is a ratelimiting enzyme involved in the last step of AsA-GSH pathway, and catalyzes the reduction of GSSG. Moreover, GR is involved in maintaining high ratio of GSH/GSSG which is essential for reprocessing AsA and stress response (Meloni et al., 2003). In cucumber under salt stress, the relative expression of cAPX (encoding APX) and GR (encoding GR) were both up-regulated with exogenous ALA application. This resulted in increased tolerance to salt and increased biomass accumulation (Zhen et al., 2012). Overexpression of GR gene in tobacco (N. tabacum L.) showed notable increase in GSH level in the leaves as well as enhancement of plant resistance to oxidative stress (Broadbent et al., 1995). In this experiment, the activity of GR was enhanced by the foliar application of ALA to seedlings under NaCl stress. In pepper (Capsicum annuum L.) plants, the application of ABA induced the up-regulation of genes encoding DHAR, including DHAR1 and DHAR2 which enhanced the activity of DHAR. (Guo et al., 2012). Similarly, the activities of
MDHAR, DHAR were enhanced by application of ALA in G. biloba. Thus, the efficiency of AsA-GSH pathway can be improved by ALA to scavenge H2O2 in cucumber cells and protect the plants from the oxidative stress caused by NaCl. 5. Conclusion The results of our experiment showed that foliar application of ALA enhanced the AsA-GSH cycle in cucumber seedlings under NaCl stress. The activities of MDHAR, DHAR and GR were up-regulated, leading to the enhancement of AsA and GSH, which indicates that the ALA improved the antioxidant defense system. Moreover, the activities of APX and AAO were also enhanced by ALA, suggesting that ALA enhanced thedecomposition of H2O2 in the cells. The accumulation of H2O2 and MDA which were caused by NaCl stress were reversed by AsA-GSH cycle and the oxidative stress was mitigated. Therefore, foliar application of ALA to cucumber seedlings enhances the AsA-GSH cycle and improves the plants tolerance to NaCl stress. Author contributions YW, JY and ZF conceived and designed the research. YW and LH conducted the experiments. YW, LH and JL analyzed the data and prepared the figures and illustrations. YW wrote the manuscript. WL, JX,MD and AC-U read the manuscript and made valuable inputs. All authors read and approved the submission of the manuscript. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (31660584), Agriculture Research System of China (CARS-23-C-07), Gansu Province Science and Technology Project (17ZD2NA015-03) and Sheng Tongsheng Technology Innovation Fund (GSAU-STS-1746). References Akram, N.A., Ashraf, M., 2013. Regulation in plant stress tolerance by a potential plant growth regulator, 5-aminolevulinic acid. J. Plant Growth Regul. 32 (3), 663–679. https://doi.org/10.1007/s00344-013-9325-9. An, Y., Qi, L., Wang, L., 2016. ALA pretreatment improves waterlogging tolerance of fig plants. PLoS One 11 (1), e0147202. https://doi.org/10.1371/journal.pone.0147202. Arakawa, N., Tsutsumi, K., Sanceda, N.G., Kurata, T., Inagaki, C., 1981. A rapid and sensitive method for the determination of ascorbic acid using 4, 7-diphenyl-l, 10phenanthroline. Agric. Biol. Chem. 45 (5), 1289–1290. https://doi.org/10.1271/ bbb1961.45.1289. Averina, N.G., Nedved’, E.L., Shcherbakov, R.A., Vershilovskaya, I.V., Yaronskaya, E.B.,
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Foliar application of 5-aminolevulinic acid (ALA) alleviates NaCl stress in cucumber (Cucumis sativus L.) seedlings through the enhancement of ascorbate-glutathione cycle
T
Yue Wua, Linli Hua, Weibiao Liaoa, Mohammed Mujitaba Dawudaa,b, Jian Lyua, Jianming Xiea, ⁎ Zhi Fenga, Alejandro Calderón-Urreac,d, Jihua Yua, a
College of Horticulture, Gansu Agricultural University, Lanzhou, 730070, PR China Department of Horticulture, FoA, University for Development Studies, P. O. Box TL 1882, Tamale, Ghana College of Plant Protection, Gansu Agricultural University, Lanzhou, PR China d Department of Biology, College of Science and Mathematics, California State University, Fresno, USA b c
ARTICLE INFO
ABSTRACT
Keywords: Ascorbate-glutathione cycle 5-Aminolevulinic acid (ALA) Ascorbic acid oxidase (AAO) Salt stress Cucumber seedlings
The natural plant growth regulator 5-aminolevulinic acid (ALA) promotes plant growth. The present study assessed the effects of exogenously applied ALA (25 mg L−1) on the growth and H2O2 scavenging system (ascorbate-glutathione cycle, AsA-GSH cycle) under NaCl stress (50 mmol L−1) in cucumber (Cucumis sativus L.) seedlings. Growth of cucumber seedlings was significantly inhibited by NaCl stress. Moreover, NaCl caused accumulation of H2O2 and malonaldehyde (MDA) as well as dehydroascorbic acid (DHA) and oxidized glutathione (GSSG) in cucumber leaves. However, the application of ALA reversed the adverse effect of NaCl on growth of the cucumber seedlings by increasing the shoots and roots biomass. The application of ALA also increased the contents of AsA and GSH of the seedlings under moderate salt stress. The ratios of reduced/ oxidized antioxidant, such as AsA/DHA and GSH/GSSG, in seedlings under NaCl treatment were also improved by ALA, indicating that the regeneration efficiency of antioxidants could be promoted by exogenous ALA. In addition, exogenous ALA also promoted the activities of enzymes involved in the AsA-GSH cycle, including ascorbic acid oxidase (AAO), ascorbate peroxidase (APX), monodehydroascorbic acid reductase (MDHAR), dehydroascorbic acid reductase (DHAR) and glutathione reductase (GR). These results suggested that ALA alleviate the damages caused by NaCl through the enhancement of the AsA-GSH pathway in cucumber seedlings.
1. Introduction Salinity is one of the major environmental factors limiting the yield and productivity of crops across the globe (Tang et al., 2015). Salt stress causes injuries such as leaf scorch and root damage to plants and also retards plant growth or even leads to plant death. Moreover, salt stress causes series of physiological changes in plants, including cell dehydration, ion toxicity and oxidative stress (Mickelbart et al., 2015). Reactive oxygen species (ROS) such as superoxide free radicals (·O2−), hydrogen peroxide (H2O2), hydroxyl radicals (OH−) and singlet oxygen (1O2), are formed as byproducts of various physiological and metabolic pathways in different cellular compartments; and these ROS are indispensable second messengers of stress response mechanism in plants (Deinlein et al., 2014; Batth et al., 2017). However, under stimulated external environmental factors, excess ROS accumulates in cells,
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disturbing cellular homeostasis and causing lipid peroxidation (Mishra et al., 2011). In higher plants, there are multiple pathways to produce H2O2, such as respiratory electron transport chain in mitochondrion, photochemical reaction in chloroplast, and the reactions of superoxide dismutase (SOD) and peroxidase (POD) (Halliwell, 1978; Slesak et al., 2007; Quan et al., 2008). The major pathway of scavenging H2O2 in higher plant cell is the ascorbate-glutathione cycle (AsA-GSH cycle) which exists extensively in many cellular organs (Li et al., 2010). The research on AsA-GSH cycle in higher plants began with the study of mycorrhiza in soybean (Glycine max L.) (Dalton et al., 1991). The AsAGSH cycle has been proven to be closely related to plant resistance to environment stress factors such as the ozone (Wang et al., 2013), salt (Li et al., 2013), drought (Kang et al., 2013), cold (Jiang et al., 2013) and zinc (Tripathi et al., 2017). In recent years, many plant growth regulators (PGRs) have been
Corresponding author at: College of Horticulture, Gansu Agricultural University, No. 1 Yinmen Village, Anning District, Lanzhou, 730070, PR China. E-mail address: [email protected] (J. Yu).
https://doi.org/10.1016/j.scienta.2019.108761 Received 11 February 2019; Received in revised form 21 May 2019; Accepted 8 August 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Scientia Horticulturae 257 (2019) 108761
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used to improve plants resistance to abiotic stresses. ALA plays an important role in regulating growth and enhancing tolerance to salt stress in plants such as Swiss chard (Beta vulgaris L.), rice (Oryza sativa L.), oilseed rape (Brassica napus L.), tomato (Solanum lycopersicum L.), peach (Prunus persica L.), watermelon (Citrullus lanatus Thunb.) and cucumber (Cucumis sativus L.) (Liu et al., 2014; Nunkaew et al., 2014; Tian et al., 2014; Zhang et al., 2015; Ye et al., 2016; Chen et al., 2017; Wu et al., 2018). Furthermore, foliar application of ALA reduced the levels of H2O2 and malonaldehyde (MDA) in oilseed rape (B. napus L.) under drought stress (Liu et al., 2013). Moreover, exogenous application of ALA significantly enhanced the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) in winter rape (B. napus L.) plants under herbicide stress (Averina et al., 2014). Similarly, in tomato (S. lycopersicum L.) plants under low temperature stress, the application of ALA increased the activities of SOD, APX, monodehydroascorbic acid reductase (MDHAR) and dehydroascorbic acid reductase (DHAR) and it also increased the contents of AsA and GSH (Liu et al., 2018). Finally, the AsA-GSH pathway in Ginkgo biloba L. leaves was enhanced by spraying 100 mg L−1 ALA even under normal growth condition (Xu et al., 2009). In cucumber seedlings under salt stress, exogenous ALA application enhanced photosynthesis through the activation of the chlorophyll biosynthesis pathway (Wu et al., 2018). However, the role of exogenous ALA on ROS scavenging system has not been adequately studied, particularly on the effect of ALA onalleviation of NaCl stress in cucumber with regard to its role on the AsA-GSH cycle. The main objective of this study was to investigate the role of ALA in the AsA-GSH cycle in cucumber seedlings under salt stress.
of the leaves. ALA application was done twice, as soon as the seedlings were exposed to the salt treatment and repeated 24 h later. The plants without ALA treatments were sprayed with distilled water in a similar manner as those that received the ALA treatment. 2.3. Biomass and morphology After 10 days of treatment, 6 seedlings were taken from each treatment to measure fresh weight. Then, the dry weight was measured by drying seedlings in oven, at 105℃ for 15 min and then at 80℃ to constant weight. In addition, the morphology of seedlings was obtained by digital camera. 2.4. H2O2 content The content of H2O2 was measured according to the method of Junglee with some modifications (Junglee et al., 2014). A fresh leaf sample (0.1 g) was homogenized with liquid nitrogen, and then transferred into a 2 mL centrifuge tube which was then kept in an ice bath. Then 1.5 mL 0.1% TCA was added, the homogenate was centrifuged at 12,000 g for 15 min at 4 °C. After that the supernatant (0.5 mL) was fully mixed with 0.5 mL PBS (pH 7.0) and 1 mL of a 1 mol L−1 KI. The mixture was placed under constant temperature (28℃) for 1 h. The absorbance was measured at 390 nm and concentration was calculated using a standard curve of H2O2 reference standards. Specifically, the concentrations of H2O2 standard curve were 0, 1, 2, 3, 4 and 5 mmol L−1.
2. Materials and methods
2.5. MDA content
2.1. Plant material and growth conditions
The content of MDA was determined according to the method of Fazeli and co-authors with some modifications (Fazeli et al., 2007). A fresh leaf sample (0.3 g) was ground in ice an bath with 2 mL 0.05 mol L−1 trichloroacetic acid (TCA). The homogenate was transferred into centrifuge tube and centrifuged at 10,000 g for 5 min at 25 °C. Then, 5 mL of a 0.5% (m/v) thiobarbituric acid (TBA) solution was added into the supernatant. The extract was placed in a boiling water bath for 10 min, and then quickly moved to cold water bath. After cooling, the solution was centrifuged again at 10,000 g for 10 min at 25 °C. The absorbance of the supernatant was measured at 532 nm; and the value for nonspecific absorption at 600 nm was subtracted. Finally, the content of MDA was calculated from the molar extinction coefficient of − MDA-TBA at 155 mmol−1 cm 1.
Cucumber seeds (Cucumis sativus L. cv. Xinchun No. 4) were surface sterilized with potassium permanganate (0.03%) for 10 min and rinsed with distilled water. The seeds were then soaked in distilled water for 6 h and exposed to germination conditions. The moistened seeds were placed on double-layer filter paper and kept at 28 ± 1 °C under dark condition. At 5 days after germination, seedlings with uniform size, fully spread cotyledons, and well-formed roots were transferred to 1-L capacity opaque plastic containers withhalf-strength Yamasaki’s cucumber nutrient solution: the macroelement contained Ca(NO3)2 − 1.75 mmol L−1,KNO33 mmolL 1, NH4H2PO4 0.5 mmol L−1, −1 MgSO4·7H2O 1 mmol L ; and the microelement contained EDTAFeNa·3H2O 35 μmol L−1, MnSO4·4H2O 5 μmol L−1, H3BO3 25 μmol L−1, ZnSO4·7H2O 0.4 μmol L−1, CuSO4·5H2O 0.15 μmol L−1, (NH4)6Mo7O24·4H2O 0.07 μmol L−1. Each container had 4 seedlings. The seedlings were grown in artificial climate chamber throughout the experiment under the following conditions: light intensity at − 350–450 mmol m-2s 1, temperature at 28/18℃ (day/night) and relative humidity at 50–60%. The nutrient solution was changed at 2-day intervals.
2.6. AsA and DHA contents The determination of AsA and DHA were done as described by Arakawa and co-authors with some modifications (Arakawa et al., 1981). A fresh leaf sample (0.5 g) was homogenized with 20 mL 50 g L−1 TCA in an ice bath, then 50 g L−1 TCA was added to make 100 mL. The homogenate was filtered and the filter liquor was taken as ascorbic acid extract. The extract (1 mL) was mixed with 1 mL of a 50 g L−1 TCA solution, 1 mL absolute ethyl alcohol, 0.5 mL 0.4% phosphoric acidalcohol, 1 mL of a 5 g L−1 bathophenanthroline (BP)-alcohol and 0.5 mL 0.3 g L−1 FeCl3-alcohol. The mixture was kept under constant temperature of 30℃ for 60 min. Then, the absorbance was determined at 534 nm. The AsA concentration was calculated using a standard curve of AsA reference standards. The concentrations of AsA standard curve were 0, 10, 20, 30, 40, 50, 60 μg mL−1. For the measurement of DHA content, 1 mL extract was mixed with 0.5 mL 60 mmol L−1 DTTacetic acid. The pH value of homogenate was adjusted to 7–8 by adding some drops of NaHPO-NaOH. The reduction reaction of DHA was done under room temperature for 10 min. Then, a 0.5 mL 0.2 g mL−1 TCA was added to the mixture. Subsequent steps were the same as the reaction system of AsA determination. The total ascorbic acid content was calculated according to AsA standard curve. The content of DHA was
2.2. Treatments and experimental design The 30-day-old uniform seedlings with three fully expanded true leaves were selected for the treatments. Each experimental unit had two 1 L capacity opaque plastic containers and each container was supplied with four seedlings. Each treatment was replicated three times and arranged in a completely randomized design. The treatments were: (1) CK: normal nutrient solution only; (2) NaCl: 50 mmol L−1 NaCl in nutrient solution +0 mg L−1 ALA; (3) NaCl + ALA: 50 mmol L−1 NaCl in nutrient solution +25 mg L−1 ALA, and (4) ALA: normal nutrient solution +25 mg L−1 ALA. The concentrations of NaCl and ALA were selected based on our previous experiment (Wu et al., 2018). The ALA (Sigma Aldrich, US) was applied with a 100 mL capacity hand sprayer. The ALA was thoroughly sprayed on both the upper and lower surfaces 2
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the total ascorbic acid content minus the AsA content.
growth of the cucumber seedlings (Fig. 2D).
2.7. GSH and GSSG contents
3.2. H2O2 and MDA contents
The contents of GSH and GSSG were measured by GSH content detection and GSSG content detection kits (Solarbio, China) following the manufacturer’s instructions, respectively. Briefly, the concrete operation was followed the specification of kit. For the determination of GSH, the 2-nitro-5-mercaptobenzoic acid, a yellow chemical, could be produced by the reaction of GSH with 5,5-dithiobis and 2-nitrobenzoic acid, and has maximum light absorption at wavelength 412 nm. Fresh leaf sample (0.1 g) was quickly grounded in liquid nitrogen, and 1 mL extraction reagent was added. The homogenized mixture was centrifuged at 8000 rpm for 10 min under 4 °C and took the supernatant. The absorbance of reaction system was determined at 412 nm. The content of GSH was calculated according to GSH standard curve. For the determination of GSSG, the 2-vinylpyridine was employed to inhibit the intrinsic GSH in cell, and then GSSG was reduced by glutathione reductase, and measured as the method of GSH.
The results in Fig. 3A shows that the H2O2 content in cucumber leaves under NaCl stress increased significantly by 46.0% compared with the CK. Exogenous application of ALA effectively decreased the H2O2 content to a level similar to that of the CK. In addition, under normal growth condition, H2O2 content in seedlings treated with ALA alone did not differ fromthe CK. Under salt stress, the content of MDA increased greatly by 400.8% in cucumber leaves compared with the control seedlings (Fig. 3B). However, the MDA content of seedlings under NaCl stress was decreased with the application of exogenous ALA. In addition, MDA content in seedlings under normal condition but sprayed with ALA was similar to those of the CK plants. 3.3. AsA-GSH pool Fig. 4A shows that the content of AsA in the leaves of the cucumber seedlings decreased under moderate salt stress. However, the application of ALA significantly increased the content of AsA in the seedlings which were exposed to the NaCl stress compared with seedlings treated with NaCl alone. There was no significant difference in the content of AsA between CK seedlings and the ALA treated seedlings under no NaCl stress condition.However, the DHA accumulated in cucumber leaves under salinity stress (Fig. 4B). Application of ALA to theseedlings under NaCl stress significantly decreased the DHA content compared with seedlings treated with NaCl alone. Moreover, the application of ALA under stress-free condition did not affect the DHA content in the cucumber seedlings. Under salinity condition, GSH content in the leaves reduced significantly in leaves when compared with CK (Fig. 4C and D). However, the GSH content was recovered to a level similar to that of the control seedlings when ALA was applied. Moreover, the content of GSH decreased in control seedlings which sprayed with ALA. In addition, GSSG content in the leaves increased to the maximal value under NaCl stress. Foliar application of ALA to seedlings under NaCl stress condition recovered the content of GSSG to a level similar to that of the CK plants. Besides, without salt stress, there was no significant difference between the contents of GSSG in the CK seedlings and the seedlings treated with ALA only.
2.8. Enzymatic activities The extraction method of enzyme was done as described by Rao and Terry with some modifications (Rao and Terry, 1989). A frozen leaf sample (0.5 g) was ground in liquid nitrogen and then transferred into a centrifuge tube. The homogenate was extracted in pre-chilled extraction buffer (5 mL) which consisted of 100 mmol L−1 Tricine-NaOH (pH 8.0), 10 mmol L−1MgCl2, 1 mmol L−1 EDTA, 10 mmol L−1 DTT, 10 mmol L−1 Na-ascorbate, 0.5% BSA and 1% (w/v) PVP-40. The mixture was centrifuged at 12,000 g for 15 min under 4 °C. The supernatant was used for enzymes activities assay. Activities of the enzymes involved in AsA-GSH cycle, including ascorbic acid oxidase (AAO, EC 1.10.3.3), APX (EC 1.11.1.11), MDHAR (EC 1.6.5.4), DHAR (EC 1.8.5.1) and GR (EC 1.6.4.2) were determined using enzyme activity assay kits (Solarbio, China). 2.9. Statistical analysis The experiment was performed with three replicates and the results were expressed as mean ± SE. Analysis of variance was performed using SPSS 22.0 (SPSS Institute Inc., US) and treatments means were compared using the Tukey’s test at a 0.05 level of probability. All figures were prepared with OriginPro 2017 (OriginLab Institute Inc., US).
3.4. AsA/DHA ratio and GSH/GSSG ratio
3. Results
The effect of NaCl stress on non-enzymatic antioxidant ratios, including AsA/DHA and GSH/GSSG, is shown in Fig. 5. The level of AsA/ DHA decreased significantly in the plants exposed to the salt stress but the application of ALA to the seedlings under the salt stress recovered the level similar to those of the control seedlings (Fig. 5A). The application of ALA to normal seedlings did not affect the value of AsA/DHA compared with CK seedlings. Similarly, exogenous application of ALA recovered the level of GSH/GSSG in the seedlings exposed to the salt stress to levels similar to that of CK (Fig. 5B). Furthermore, the level of GSH/GSSG in the control seedlings decrease when ALA was applied.
3.1. Biomass and plant morphology The shoot and root biomass of the seedlings were measured at 10 d after treatment application (Fig. 1). These indexes decreased significantly under NaCl stress. In comparison with the plants grown in normal condition, the fresh and dry shoot weights of the seedlings under salt stress also decreased by 45.2% and 40.6%, respectively (Fig. 1A, C). The fresh and dry root weights of the seedlings under stress decreased by 54.9% and 43.5% respectively (Fig. 1B, D). Exogenous application of ALA significantly increased the fresh and dry weights of shoots as well as the root fresh weight. However, ALA application did not significantly affect root dry weight of the seedlings under salt stress. Moreover, the application of ALA in the absence of salt stress decreased the dry weight of shoots and roots. NaCl stress caused leaf chlorosis, reduced leaf size and decreased the size of the root systems whiles the seedlings of CK treatment producedgreen and bigger leaves as well as larger root systems (Fig. 2). The application of ALA to the seedlings exposed to NaCl stress alleviated the NaCl-induced stress and the seedlings appear similar to the control plants (Fig. 2C). Moreover, the application of ALA also improved the
3.5. Enzymatic activities of AAO and APX The results of our experiment showed no significant difference in AAO activity of NaCl-treated seedlings and CK seedlings (Fig. 6A). However, the activity of AAO was markedly enhanced by spraying ALA, which reached a level more than 5-fold of the control plants. Moreover, the activity of AAO in the control seedlings was enhanced whenALA was applied. NaCl stress caused a significant inhibition of APX activity but this was enhanced by ALA application (Fig. 6B). Similarly, exogenous ALA enhanced the activity of APX in the control seedlings. 3
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Fig. 1. Effect of exogenous ALA on (A) fresh shoot weight (B) fresh root weight (C) dry shoot weight and (D) dry root weight, of cucumber seedlings under NaCl stress. The data presented are the means of three plants ± SE of means (n = 3, with 6 plants per replication). Treatment means with different lower case letters indicate significant differences according to Tukey’s test (p < 0.05).
3.6. Enzymatic activities of MDHAR, DHAR and GR
8.5-fold more than the CK seedlings. In addition, the activity of DHAR in seedlings under NaCl was similar to those under NaCl stress but treated with ALA. However, the DHAR activities in these two treatments were significantly higher compared to the CK plants (Fig. 7B). There was marked difference of DHAR between the seedlings treated with ALA alone and that of CK. Besides, GR activity in cucumber leaves was significantly suppressed by salinity stress (Fig. 7C). However, exogenous application of ALA significantly enhanced the GR
Fig. 7 shows the changes in the activities of other three key enzymes involved in the AsA-GSH cycle, which include MDHAR, DHAR and GR. The MDHAR activityin the control seedlings, seedlings treated with NaCl and those sprayed with ALA alone were statistically similar (Fig. 7A). However, MDHAR activity of the NaCl treated seedlings was significantly enhanced by the application of ALA, and the increase was
Fig. 2. The morphology of cucumber seedlings under difference treatments. (A) CK: seedlings under normal growth condition. (B) NaCl: seedlings under 50 mmol L−1 NaCl stress. (C) NaCl + ALA: seedlings under 50 mmol L−1NaCl stress and sprayed 25 mg L−1 ALA on the leaves. (D) ALA: seedlings sprayed 25 mg L−1 ALA on the leaves under normal growth condition.
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Fig. 3. Effect of exogenous ALA on (A) hydrogen peroxide (H2O2) and (B) malondialdehyde (MDA) contents in the leaves of cucumber seedlings under NaCl stress. The data presented are the means of three plants ± SE of means (n = 3, with 6 plants per replication). Treatment means with different lower case letters indicate significant differences according to Tukey’s test (p < 0.05).
Fig. 4. Effect of exogenous ALA on contents of (A) ascorbate (AsA), (B) dehydroascorbic acid (DHA), (C) glutathione (GSH) and (D) oxidized glutathione (GSSG) in the leaves of cucumber seedlings under NaCl stress. The data presented are the means of three plants ± SE of means (n = 3, with 6 plants per replication). Treatment means with different lower case letters indicate significant differences according to Tukey’s test (p < 0.05).
activitycompared with the control plants and the plants under NaCl stress.
shoot biomass of seedlings. In addition, root growth of the cucumber seedlings was also inhibited by salt stress but foliar application of ALA significantly increased root fresh weight. It is likely that the effect was a result of regulatory changes of aquaporin gene expression as previously reported: aquaporins CsPIPs1:1 (plasma membrane intrinsic protein) and CsNIP (nodulin-26-like intrinsic membrane protein) in cucumber (C. sativus L.) seedling, under salt stress were down-regulated by exogenous ALA application and this prevented the dehydration of the cells (Yan et al., 2014). Moreover, in the present study, application of ALA to cucumber seedlings under normal condition caused significant decrease in root dry weight compared with CK plants. In a previous study, the application of 50 mg L−1 exogenous ALA on the leaves of salt-sensitive cucumber seedlings resulted in significant reduction of root dry weight, which was similar to that of plants exposed to 75 mmol L−1 NaCl stress (Yan et al., 2014). Besides, pretreatment of ALA significantly enhanced
4. Discussion Salt stress inhibits seed germination, interferes with physiological processes such as water transport, protein synthesis and photosynthesis and also damages both the anatomy and morphology of plants. ALA was reported to have the capacity of improving plant tolerance to abiotic stress (Akram and Ashraf, 2013). In Indian mustard (Brassica juncea L.) plants under salt stress, the fresh weight of seedlings reduced with increases in NaCl concentrations (Pandey and Penna, 2017). In this study, NaCl stress decreased plant growth and the seedlings exhibited leaf chlorosis, reduced leaf sizes and also decreased the size of root systems. However, exogenous application of ALA significantly improved the 5
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Fig. 5. Effect of exogenous ALA on changes of (A) AsA/DHA ratio and (B) GSH/GSSG ratio in the leaves of cucumber seedlings under NaCl stress. The data presented are the means of three plants ± SE of means (n = 3, with 6 plants per replication). Treatment means with different lower case letters indicate significant differences according to Tukey’s test (p < 0.05).
the root respiration of fig plants (An et al., 2016). These results indicate that the application of ALA to plants under normal growth condition may enhance the dry matter consumption of plants, and finally adversely affected dry matter accumulation. In higher plants, AsA-GSH cycle is an indispensable ROS scavenging system in cells and this is mainly located in cytoplasm and many organelles. In this study, H2O2 and MDA accumulated in the leaves of cucumber plants under salt stress, indicating that the cell membrane lipid peroxidation already occurred in the seedlings. H2O2 is known as a crucial signal molecule in plants under normal growth condition, and it plays several important functions in cell, including gene expression, cellular metabolism, programmed cell death, resistance response and cell senescence (Huang et al., 2016). However, under abiotic stress, membrane system and cell organelles can be damaged by excess accumulation of H2O2. In rice (Oryza sativa L.) plants under drought stress, the content of H2O2 increased but the contents of AsA, GSH and the ratios of antioxidants decreased suggesting that drought caused oxidative stress in the plants (Pyngrope et al., 2013). The main pathway of scavenging H2O2 in higher plants is through the AsA-GSH cycle (Jin et al., 2003). Although AsA-GSH cycle is known as the antioxidant defense system in plant cell, its activity can also be regulated by environmental stresses. For example, at 0.5 mmol L−1 cadmium stress, the value of AsA/DHA decreased by 28.5% compared with that of control plants; moreover, at 1.0 mmol L−1 cadmium stress, this index decreased by 57.1% (Hasanuzzaman et al., 2017). These results indicated that oxidative stress can be aggravated with increases in environmental stress. In some studies, the level of reduced antioxidants in plants could be expressed by the ratio of reduced type and oxidized type, like AsA/DHA and GSH/ GSSG (Zhao et al., 2016; Hasanuzzaman et al., 2017; Liu et al., 2018). In the present study, foliar application of ALA to cucumber seedlings under NaCl stress caused an increase in the contents of AsA and GSH.
Moreover, it recovered the decreased contents of AsA/DHA and GSH/ GSSG caused by salinity. These results are in agreement with Liu and co-authors who reported that increased accumulation of ROS and MDA occurred in swede type rape (B. napus L.) plants under drought stress (Liu et al., 2011). In another study, the application of exogenous ALA alleviated the membrane lipid peroxidation caused by oxidative stress and also increased the AsA/DHA and GSH/GSSG contents. The total value of AsA + DHA was increased in poplar (Populus deltoides×Populus maximowiczii) leaves under ozone and Cd stress (Castagna et al., 2015). AsA is the most abundant antioxidant in plants and plays a role in the coping mechanism of plants to oxidative stress (Noctor and Foyer, 1998). The content of AsA is affected by reductase activity (like MDHAR or DHAR), and by its upstream biosynthesis in plants. For example, GDP-d-mannose pyrophosphorylase (GMPase) is a key enzyme in AsA synthesis pathway (Tabata et al., 2002). Overexpression of GMPase coding gene SlGME in tomato (S. lycopersicum L.) showed that AsA content increased significantly in the leaves and seedlings were more resistant to methyl viologen stress and cold stress than the wild type (Zhang et al., 2011). As two antioxidants in higher plant cell, AsA and GSH play very important roles in AsA-GSH cycle in scavenging free ROS, because relatively high level of antioxidant means more electron donor will take part in the reactions of H2O2 reduction and AsA regeneration. In Arabidopsis thaliana, several ascorbate-deficient (vtc) mutants, including VTC1, VTC2, VTC3 and VTC4, AsA content decreasedby 25–30% compared with wild type plants (Colville and Smirnoff, 2008). In addition, the mutants were more sensitive to ozone stress and UV-B stress than the wild type plant (Gao and Zhang, 2008). Similarly, A. thaliana mutant with the γ-glutamyl-cysteine synthetase encoding gene (GSH1) knock-out revealed obstacle in synthesizing GSH as well as embryo development inhibition or embryo death (Cairns et al., 2006; Pasternak et al., 2008). Fig. 6. Effect of exogenous ALA on the activities of (A) ascorbic acid oxidase (AAO) and (B) ascorbic acid peroxidase (APX) in the leaves of cucumber seedlings under NaCl stress. The data presented are the means of three plants ± SE of means (n = 3, with 6 plants per replication). Treatment means with different lower case letters indicate significant differences according to Tukey’s test (p < 0.05).
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Fig. 7. Effect of exogenous ALA on the activities of (A) monodehydroascorbic acid reductase (MDHAR) (B) dehydroascorbic acid reductase (DHAR) and (C) glutathione reductase (GR) in the leaves of cucumber seedlings under NaCl stress. The data presented are the means of three plants ± SE of means (n = 3, with 6 plants per replication). Treatment means with different letters indicate significant differences according to Tukey’s test (p < 0.05).
In this experiment, the activities of AAO and APX in cucumber leaves were both enhanced by exogenous ALA under stressful or nonstressful condition. Although the catalytic reactions of AAO and APX in plant cell are similar, the AAO is located in apoplast whiles APX is located in organelles or cytoplasm. Although AAO does not belong to intracellular AsA-GSH circulatory system, it plays vital role in regulating the redox state of extracellular ascorbic acid pool. When plants suffer from environmental stresses, the value of oxidation/reduction ratio of ascorbic acid pool is regulated by AAO, and this will become the first defense of cells against stress damages (Kao, 2015). The activity of AAO can be suppressed by certain metallic element, such as zinc and cadmium (Morina et al., 2010; Zelinová et al., 2014). In addition, antisense oriented AAO gene in tobacco (Nicotiana tabacum L.) caused AAO activity in the transgenic plants to decreased by about 0.2-fold of that in wild type plants (Yamamoto et al., 2005). However, pretreatment with H2S up-regulated the relative expression of TaAAO gene (encoding AAO) in leaves and roots of wheat (Triticum aestivum L.) under drought stress (Ma et al., 2016). Furthermore, AAO activity could be enhanced by glycinebetaine (GB) (Chen and Heuer, 2013). Similarly, in the present study, exogenous ALA promoted AAO activity in cucumbers seedlings under NaCl stress. Diverse roles of AAO such as cell mitosis, cell elongation and photosynthesis in plants have been reported (Tullio et al., 2004; Batth et al., 2017). The APX is also an antioxidant that is directly involved in the decomposition of H2O2 in the AsA-GSH cycle. Therefore, the enhancement of APX activity indicates the improvement of degradation efficiency of ROS in plant cell. Under low temperature stress, 15 °C/8 °C (day/night), foliar application of 25 mg L−1 ALA enhanced the activities of APX, MDHAR, DHAR and GR in tomato (S. lycopersicum L.) plants and improved their resistance to low temperature (Liu et al., 2018). The activities of several antioxidant enzymes involved in AsA-GSH pathway in the leaves of G. biloba L. were significantly increased with the application of exogenous ALA (Xu et al., 2009). Another key enzyme in AsA-GSH cycle is GR which is a ratelimiting enzyme involved in the last step of AsA-GSH pathway, and catalyzes the reduction of GSSG. Moreover, GR is involved in maintaining high ratio of GSH/GSSG which is essential for reprocessing AsA and stress response (Meloni et al., 2003). In cucumber under salt stress, the relative expression of cAPX (encoding APX) and GR (encoding GR) were both up-regulated with exogenous ALA application. This resulted in increased tolerance to salt and increased biomass accumulation (Zhen et al., 2012). Overexpression of GR gene in tobacco (N. tabacum L.) showed notable increase in GSH level in the leaves as well as enhancement of plant resistance to oxidative stress (Broadbent et al., 1995). In this experiment, the activity of GR was enhanced by the foliar application of ALA to seedlings under NaCl stress. In pepper (Capsicum annuum L.) plants, the application of ABA induced the up-regulation of genes encoding DHAR, including DHAR1 and DHAR2 which enhanced the activity of DHAR. (Guo et al., 2012). Similarly, the activities of
MDHAR, DHAR were enhanced by application of ALA in G. biloba. Thus, the efficiency of AsA-GSH pathway can be improved by ALA to scavenge H2O2 in cucumber cells and protect the plants from the oxidative stress caused by NaCl. 5. Conclusion The results of our experiment showed that foliar application of ALA enhanced the AsA-GSH cycle in cucumber seedlings under NaCl stress. The activities of MDHAR, DHAR and GR were up-regulated, leading to the enhancement of AsA and GSH, which indicates that the ALA improved the antioxidant defense system. Moreover, the activities of APX and AAO were also enhanced by ALA, suggesting that ALA enhanced thedecomposition of H2O2 in the cells. The accumulation of H2O2 and MDA which were caused by NaCl stress were reversed by AsA-GSH cycle and the oxidative stress was mitigated. Therefore, foliar application of ALA to cucumber seedlings enhances the AsA-GSH cycle and improves the plants tolerance to NaCl stress. Author contributions YW, JY and ZF conceived and designed the research. YW and LH conducted the experiments. YW, LH and JL analyzed the data and prepared the figures and illustrations. YW wrote the manuscript. WL, JX,MD and AC-U read the manuscript and made valuable inputs. All authors read and approved the submission of the manuscript. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (31660584), Agriculture Research System of China (CARS-23-C-07), Gansu Province Science and Technology Project (17ZD2NA015-03) and Sheng Tongsheng Technology Innovation Fund (GSAU-STS-1746). References Akram, N.A., Ashraf, M., 2013. Regulation in plant stress tolerance by a potential plant growth regulator, 5-aminolevulinic acid. J. Plant Growth Regul. 32 (3), 663–679. https://doi.org/10.1007/s00344-013-9325-9. An, Y., Qi, L., Wang, L., 2016. ALA pretreatment improves waterlogging tolerance of fig plants. PLoS One 11 (1), e0147202. https://doi.org/10.1371/journal.pone.0147202. Arakawa, N., Tsutsumi, K., Sanceda, N.G., Kurata, T., Inagaki, C., 1981. A rapid and sensitive method for the determination of ascorbic acid using 4, 7-diphenyl-l, 10phenanthroline. Agric. Biol. Chem. 45 (5), 1289–1290. https://doi.org/10.1271/ bbb1961.45.1289. Averina, N.G., Nedved’, E.L., Shcherbakov, R.A., Vershilovskaya, I.V., Yaronskaya, E.B.,
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