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. 2019 Jan 16;20(2):353.
doi: 10.3390/ijms20020353.

Exogenous Melatonin Counteracts NaCl-Induced Damage by Regulating the Antioxidant System, Proline and Carbohydrates Metabolism in Tomato Seedlings

Manzer H Siddiqui  1 , Saud Alamri  2 , Mutahhar Y Al-Khaishany  3 , M Nasir Khan  4 , Abdullah Al-Amri  5 , Hayssam M Ali  6 , Ibrahim A Alaraidh  7 , Abdulaziz A Alsahli  8
Affiliations

Exogenous Melatonin Counteracts NaCl-Induced Damage by Regulating the Antioxidant System, Proline and Carbohydrates Metabolism in Tomato Seedlings

Manzer H Siddiqui et al. Int J Mol Sci. .

Abstract

Melatonin, a natural agent, has multiple functions in animals as well as in plants. However, its possible roles in plants under abiotic stress are not clear. Nowadays, soil salinity is a major threat to global agriculture because a high soil salt content causes multiple stresses (hyperosmotic, ionic, and oxidative). Therefore, the aim of the present study was to explore: (1) the involvement of melatonin in biosynthesis of photosynthetic pigments and in regulation of photosynthetic enzymes, such as carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco); (2) the role of melatonin in osmoregulation by proline and carbohydrate metabolism; and (3) the function of melatonin in the antioxidant defense system under salinity. Outcomes of the study reveal that under non-saline conditions, application of melatonin (20 and 50 µM) improved plant growth, viz. shoot length, root length, shoot fresh weight (FW), root FW, shoot dry weight (DW), root DW and leaf area and physio-biochemical parameters [chlorophyll (Chl) a and b, proline (Pro) and total soluble carbohydrates (TSC) content, and increased the activity of CA and Rubisco]. However, tomato seedlings treated with NaCl exhibited enhanced Chl degradation, electrolyte leakage (EL), malondialdehyde (MDA) and reactive oxygen species (ROS; superoxide and hydrogen peroxide). ROS were detected in leaf and root. Interestingly, application of melatonin improved plant growth and reduced EL, MDA and ROS levels through upregulation of photosynthesis enzymes (CA, Rubisco), antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase and ascorbate reductase) and levels of non-enzymatic antioxidants [ascorbate (ASC) and reduced glutathione (GSH)], as well as by affecting the ASC-GSH cycle. Additionally, exogenous melatonin also improved osmoregulation by increasing the content of TSC, Pro and Δ¹-pyrroline-5-carboxylate synthetase activity. These results suggest that melatonin has beneficial effects on tomato seedlings growth under both stress and non-stress conditions. Melatonin's role in tolerance to salt stress may be associated with the regulation of enzymes involved in photosynthesis, the antioxidant system, metabolism of proline and carbohydrate, and the ASC-GSH cycle. Also, melatonin could be responsible for maintaining the high ratios of GSH/GSSG and ASC/DHA.

Keywords: ASC-GSH pathway; SOD-CAT pathway; Solanum lycopersicum; antioxidant system; carbohydrate; melatonin; proline; Δ1-pyrroline-5-carboxylate synthetase.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Growth performance of tomato seedlings under melatonin and salinity conditions.
Figure 2
Figure 2
Effect of melatonin on (A) Chl a, (B) Chl b and (C) Chl degradation in leaves of tomato seedlings under salinity. Data represent mean of 4 replicates with bars indicating SE. The bars labelled with different letters are significantly different at p < 0.05%. [DDW (control), 20 µM melatonin (M20), 50 µM melatonin (M50), 100 mM NaCl (NaCl)].
Figure 3
Figure 3
Effect of melatonin on (A) carbonic anhydrase (CA) and (B) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity in leaves of tomato seedlings under salinity. Data represent mean of 4 replicates with bars indicating SE. The bars labelled with different letters are significantly different at p < 0.05%. [DDW (control), 20 µM melatonin (20M), 50 µM melatonin (50M), 100 mM NaCl (NaCl)].
Figure 4
Figure 4
Effect of melatonin on (A) electrolyte leakage (EL), and (B) malondialdehyde (MDA) content in leaves of tomato seedlings under salinity. Data represent mean of 4 replicates with bars indicating SE. The bars labelled with different letters are significantly different at p < 0.05%. [DDW (control), 20 µM melatonin (20M), 50 µM melatonin (50M), 100 mM NaCl (NaCl)].
Figure 5
Figure 5
Effect of melatonin on (A) O2•− content, and (B) H2O2 content in leaves of tomato seedlings under salinity. Data represent mean of 4 replicates with bars indicating SE. The bars labelled with different letters are significantly different at p < 0.05%. [DDW (control), 20 µM melatonin (20M), 50 µM melatonin (50M), 100 mM NaCl (NaCl)].
Figure 6
Figure 6
Under fluorescence microscope imaging of ROS, (A) H2O2 production (H2O2-dependent DCF-DA fluorescence) in root, (B) O2•− production (O2•− dependent DHE fluorescence) in root, (C) Overlay projection image of root stained with FDA (green: viable cells) and PI (red: non-viable cells), (D) H2O2 production in leaf using DAB and (E) O2•− formation in leaf using NBT staining under NaCl and melatonin application. As negative controls, roots and leaves of NaCl and melatonin exposed plants were preincubated with ascorbic acid (1 mM ASC), a H2O2 scavenger and tetramethyl piperidinooxy (1 mM TMP), an O2•— scavenger. [DDW (control), 20 µM melatonin (20M), 50 µM melatonin (50M), 100 mM NaCl (NaCl)].
Figure 7
Figure 7
Effect of melatonin on (A) proline (Pro) content, (B) Pyrroline-5-carboxylate synthase (P5CS) activity and (C) total soluble carbohydrates (TSC) content in leaves of tomato seedlings under salinity. Data represent mean of 4 replicates with bars indicating SE. The bars labelled with different letters are significantly different at p < 0.05%. [DDW (control), 20 µM melatonin (20M), 50 µM melatonin (50M), 100 mM NaCl (NaCl)].
Figure 7
Figure 7
Effect of melatonin on (A) proline (Pro) content, (B) Pyrroline-5-carboxylate synthase (P5CS) activity and (C) total soluble carbohydrates (TSC) content in leaves of tomato seedlings under salinity. Data represent mean of 4 replicates with bars indicating SE. The bars labelled with different letters are significantly different at p < 0.05%. [DDW (control), 20 µM melatonin (20M), 50 µM melatonin (50M), 100 mM NaCl (NaCl)].
Figure 8
Figure 8
Effect of melatonin on the content of (A) reduced glutathione (GSH), (B) oxidized glutathione, and (C) ratio of GSH/GSSG in leaves of tomato seedlings under salinity. Data represent mean of 4 replicates with bars indicating SE. The bars labelled with different letters are significantly different at p < 0.05%. [DDW (control), 20 µM melatonin (20M), 50 µM melatonin (50M), 100 mM NaCl (NaCl)].
Figure 8
Figure 8
Effect of melatonin on the content of (A) reduced glutathione (GSH), (B) oxidized glutathione, and (C) ratio of GSH/GSSG in leaves of tomato seedlings under salinity. Data represent mean of 4 replicates with bars indicating SE. The bars labelled with different letters are significantly different at p < 0.05%. [DDW (control), 20 µM melatonin (20M), 50 µM melatonin (50M), 100 mM NaCl (NaCl)].
Figure 9
Figure 9
Effect of melatonin on the content of (A) ascorbate (ASC), (B) dehydroascorbate (DHA), and (C) ratio of ASC/DHA in leaves of tomato seedlings under salinity. Data represent mean of 4 replicates with bars indicating SE. The bars labelled with different letters are significantly different at p < 0.05%. [DDW (control), 20 µM melatonin (20M), 50 µM melatonin (50M), 100 mM NaCl (NaCl)].
Figure 10
Figure 10
Effect of melatonin on the activities of (A) superoxide dismutase (SOD), (B) catalase (CAT), (C) glutathione reductase (GR) and (D) ascorbate peroxidase (APX) in leaves of tomato seedlings under salinity. Data represent mean of 4 replicates with bars indicating SE. The bars labelled with different letters are significantly different at p < 0.05%. [DDW (control), 20 µM melatonin (20M), 50 µM melatonin (50M), 100 mM NaCl (NaCl)].
Figure 11
Figure 11
Summary of melatonin-induced tolerance against salinity by regulating the antioxidant system, proline and carbohydrate metabolism and photosynthetic pigments synthesis in plants. Ch-chlorophyll, ROS-reactive oxygen species, CA-carbonic anhydrase, ASC-ascorbate, GSH-reduced glutathione, SOD-superoxide dismutase, CAT-catalase, GR-glutathione reductase, APX-ascorbate peroxidase.
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