Please cite this paper as：

Shukla, M., Bajwa, V., Freixas-Coutin, J. and Saxena, P. 2021. Salt stress in Arabidopsis thaliana seedlings: Role of indoleamines in stress alleviation. Melatonin Research. 4, 1 (Jan. 2021), 70-83. DOI:https://doi.org/https://doi.org/10.32794/mr11250082.

Research Article

Salt stress in Arabidopsis thaliana seedlings: Role of indoleamines in stress alleviation

Mukund R. Shukla^¹, Vikramjit S. Bajwa^¹, Jose A. Freixas-Coutin², Praveen K. Saxena^*1

¹Department of Plant Agriculture, Gosling Research Institute for Plant Preservation,

University of Guelph, Guelph, ON, N1G 2W1, Canada.

²Present address: Danone, Analytical Performance Center, 1900 Cherry Street, Louisville CO 80027, USA.

^These authors contributed equally to this work.

*Correspondence: psaxena@uoguelph.ca, Tel: 519-824-4120 ext 52495; Fax 519-767-0755

Running title: Role of indoleamines in salt stress

Received: September 8. 2020; Accepted: October 22, 2020

ABSTRACT

     Salinity is a major environmental stress in agriculture with significantly detrimental effects on crop productivity. The development of strategies to enhance salinity stress tolerance in plants is essential to ensure crop production in saline environments. Melatonin (Mel) and serotonin (Ser) accumulate in response to environmental stresses and are presumed to play protective roles and improve growth of tissues during recovery. In this study, the effects of Mel and Ser were investigated in Arabidopsis under NaCl stress. Exogenous Mel (10 µM) and Ser (10 µM) treatment significantly increased fresh weight, lateral root number, and shoot height in A. thaliana seedlings exposed to NaCl stress (25 mM and 50 mM) compared to the non-treated control seedlings. In order to understand the role of these indoleamines in alleviating salt stress, we investigated the effects of Mel and Ser treatments on the expression of salt stress responsive genes including, transcription factors involved in abscisic acid (ABA) signaling pathway, ABA-INSENSITIVE 3 (ABI3)and ABA-INSENSITIVE 5 (ABI5); ABA responsive gene, RESPONSIVE TO DESSICATION 29B (RD29B), ABA-independent gene, RESPONSIVE TO DESSICATION 29A (RD29A) and Arabidopsis trithorax-like gene (ATX1) which function in stress responses via ABA-dependent and ABA-independent manner. Other genes included, ROS-signaling transcription factor ZAT10 and ZAT12, and the genes encoding ion transporters crucial for maintaining ion homeostasis, HIGH AFFINITY K⁺ TRANSPORTER 5 (HAK5) and SALT OVERLY SENSITIVE 1 (SOS1). Mel (10 µM) pre-treatment for 24 hrs followed by 50 mM salt treatment up-regulated ABI3, RD29B, ZAT12 and HAK5. The Ser (10 µM) pre-treatment significantly up-regulated ZAT12.These results indicate that indoleamine pre-treatment improved plant growth under salt stress with Mel facilitating salt tolerance via upregulation of ABA responsive genes, mediation of antioxidant defense systems to counteract the salt-induced ROS overproduction as well as controlling ion homeostasis. Although Ser displayed no significant effects on ABA signaling, it was found to increase the expression of antioxidant defense gene, ZAT12. This study demonstrates the importance of indoleamine pathway in mediation of salt stress response and provides the first indication of the involvement of Ser in salt stress tolerance.

Key words: Salt stress, melatonin, serotonin, ABA signalling pathway, reactive oxygen species (ROS).

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1. INTRODUCTION

     Soil salinity is among the major environmental stresses that adversely affect productivity in crop plants (1, 2). Worldwide, salt-affected irrigated agricultural land is expected to climb from 33% to up to 50% in the next decade (3, 4). Increasing levels of soil salinity can seriously decrease food production as approximately one third of the world’s food is produced on agricultural land (1, 5, 6). Despite numerous attempts in water management practices to alleviate soil salinity, the excessive salt concentration in arid and semi-arid regions around the globe has been difficult to overcome and new strategies that can enhance salt stress tolerance in crop plants must be developed (7, 8).

     The indoleamines, melatonin (N-acetyl-5-methoxytryptamine; Mel) and serotonin (5-hydroxytryptophan; Ser) are derived from the aromatic amino acid tryptophan; where Ser is synthesized first serving as precursor for Mel formation (9, 10). Mel has been implicated for its role in enhancing abiotic and biotic stress tolerance in plants (11–19). In particular, Mel plays an important role in salt and drought stress tolerance in apple, soybean, cucumber, watermelon and citrus (13, 15, 18, 20, 21). Increased Mel and Ser levels have been reported in sunflower (Helianthus annuus L.) seedlings exposed to salinity stress. Both indoleamines promoted germination significantly and were associated with differential localization in sunflower tissues, a phenomenon which has also been implicated in thermal stress tolerance in St. John’s wort (Hypericum perforatum L.) (22, 23). Diverse mechanisms have been demonstrated for Mel-mediated salt stress tolerance in plants, including prevention of electrolyte leakage, lipid-peroxidation and chlorophyll degradation as well as induction of antioxidant defense systems to prevent tissue damage in plants exposed to salinity stress (18, 20). In watermelon, pre-treatment with Mel on salt-stressed roots enhanced the photosynthetic rate by inhibiting the stomatal closure as well as improving light energy absorption and electron transport in photosystem II. In addition, Mel alleviated the salt-induced oxidative stress damage via enhancing redox homeostasis and activation of antioxidant enzymes (18). Similarly, in salt-stressed rapeseed (Brassica napus L.) seedling roots, the application of Mel together with Nitric Oxide-releasing compounds counteracted seedling growth inhibition and re-established ion homeostasis which was associated with alleviating ROS overproduction, a decrease in Na⁺/K⁺ ratio and modulation of the antioxidant defense genes SODIUM HYDROGEN EXCHANGER (NHX1) and SALT OVERLY SENSITIVE 2 (19).

     Salinity responses involve multiple signaling pathways integrated into a complex regulatory network which also includes ABA metabolic pathway (24–26). ABA biosynthesis is dramatically increased under high salinity and drought conditions and mediates the expression of transcription factor (TF) genes implicated in biotic and abiotic stress responses, including those responding to water dehydration stress in an ABA-dependent and independent manner (27–29). Based on the active role of Mel in alleviating salt stress, we hypothesized that Mel and Ser may effectively mitigate salt stress in Arabidopsis and that the pre-treatment with these compounds will lead to modifications in transcription of several drought, salinity and ABA-related genes. With the aim to understand the metabolic pathways involved in indoleamine-mediated salt stress alleviation, we conducted a targeted gene expression analysis by selecting candidate genes involved in abiotic stress tolerance. The data from this study indicate that exogenous treatment with Mel and Ser significantly improved salt stress tolerance in Arabidopsis seedlings. Mel response appears to be mediated by ABA-responsive and antioxidant defense genes, whereas Ser response seems to be mediated through antioxidant defense genes in an ABA-independent manner. These protective effects with indoleamines suggested that these compounds act as inductive signals in pre-conditioning the plant to tolerate abiotic stress, a phenomenon similar to the role of Mel and Ser in plant morphogenesis and thermal stress (23).

2. MATERIALS AND METHODS

2.1. Plant material and growth conditions.

     The Arabidopsis ecotype Columbia-0 (Col) was obtained from Arabidopsis Biological Resource Center (Columbus, OH, USA). Seeds were surface sterilized with 70% (v/v) ethanol for 1 min followed by 15% Clorox® bleach solution for 20 min and then washed three times with sterile water. Sterile seeds were placed in a single row in Petri dishes (70 mm diameter) containing Murashige and Skoog (MS, Phytotechnology Lab, KS, USA) modified salt mixture (30) with 1% sucrose,  pH adjusted to 5.7 prior to adding 2.2 g/L phytagel (Sigma Aldrich) and autoclaved at 121°C for 20 min. Mel (0, 10, 30 μM) and Ser (0, 10, 30 μM) were filter sterilized and added to the medium cooled to 55 °C under low light conditions (5 μmolm^-2s^-1 ). All Petri dishes were kept inclined at an angle of 65° to allow optimum light penetration for root and shoot growth and kept at 4°C for two days. This cold stratification improves subsequent seed germination and seedling development with minimum impact on plant metabolism (31). Standard growth conditions for all experiments were 25 °C with a 16 h photoperiod and a light intensity of 40 μmolm^-2s^-1, provided by cool white fluorescent tubes (LI-250A, LI-COR® Biosciences, USA).

2.2. Salt stress treatment.

     For salt stress treatment, five-day-old Arabidopsis seedlings, originally grown on Mel (0, 10, 30 μM) and Ser (0, 10, 30 μM) supplemented medium, were transferred to the same MS medium but containing various levels of NaCl (25, and 50 mM), including a control with 0 mM NaCl. Cultures were kept under 16 h photoperiod and a light intensity of 15 μmolm^-2s^-1 at 25 °C. Growth parameters including shoot height, primary root length, lateral root numbers and fresh weight were measured after 14 days of growth.

     For gene expression analyses, sterile Arabidopsis seeds were grown in 125 mL flasks containing 10 mL liquid medium (MS modified salt mixture) to get enough mass for sample analysis and rotated on an orbital shaker at 75 rpm. This liquid culture method is commonly used to grow Arabidopsis seedlings in vitro; the growth medium can be supplemented with abiotic stress treatments for efficient collection of tissues for gene expression studies (32–34). MS medium was supplemented with Mel (0 or 10 μM) and Ser (0 or 10 μM) and 11-day-old seedlings were treated with NaCl by replacing the remaining culture medium with a fresh half strength MS medium containing 50 mM NaCl. Tissues were collected from the flasks after 24 hours of NaCl treatment, immediately flash frozen in liquid nitrogen and stored at -80 °C until gene expression analyses.

2.3. RNA extraction and gene expression analysis.

     Total RNA was extracted from the Arabidopsis seedlings using the cetyltrimethylammonium bromide (CTAB) method (35). The RNA samples were treated with DNase enzyme using the RNase-free DNase Kit (Qiagen, Toronto, ON, Canada) followed by a purification step with the RNAeasy Plant Mini Kit (Qiagen, Toronto, ON, Canada), following to the manufacturer’s instructions. The DNA-free total RNA was quantified at 260 nm using a Synergy H1 Hybrid Reader spectrophotometer (BioTek, VT, USA) and the integrity was measured in agarose gel electrophoresis after the samples were mixed with 3X Ambion® NorthernMax® Formaldehyde Load Dye (Life Technologies Inc., ON, Canada). The cDNA libraries were prepared from 2.5 μg of total RNA using SuperScript^TM VILO^TM cDNA Synthesis Kit (Invitrogen, ON, Canada) as described in the manufacturer’s instructions. Samples of cDNA were mixed with Sso-Fast EvaGreen Supermix (Bio-Rad, Mississauga, ON, Canada) and gene specific primers in triplicate prior to incubation in a CFX Connect Real-Time PCR Detection System (Bio-Rad, Mississauga, ON, Canada) for quantitative RT-PCR. Gene specific primers were designed to target the flanking coding region of each gene. Three technical replicates were assessed from each treatment. Expression of each gene was normalized to that of Arabidopsis β-actin gene and was calculated relative to the NaCl (50mM) - stressed samples without Mel or Ser treatment.

2.4. Statistical analysis.

     For the studies conducted to determine the efficacy of Ser and Mel in improving salt stress tolerance of plants in Petri dishes, the experiment was conducted as a randomised complete block design with three replications, each treatment was applied to six Petri dishes per replication. All growth data were analyzed using JMP 10.0.0 (SAS Institute, Cary, NC) and ANOVA was conducted to determine significance of the model followed by means comparison using Student’s t-test at α = 0.1 significance level.

     For gene expression study, data were compared for significant differences using two different P-values < 0.05 or ≤ 0.1. Data were presented as the mean ± the standard error of two biological replications for all genes except ATX1 for which there was only one biological replication. Each biological replication had three technical replications.

3. RESULTS

3.1. Mel and Ser independently induced salt stress tolerance in Arabidopsis seedlings.

     To compare the effects between exogenous Mel and Ser treatment on plant growth and development under salt stress, Arabidopsis seedlings were grown in different salt concentrations with and without these indoleamines. Overall, better root and shoot growth was observed in the Arabidopsis seedlings treated with Mel or Ser compared to the non-treated seedlings subjected to salt stress, indicating that Mel and Ser treatments reduced the growth inhibition caused by salt stress (Figures 1 and 2).

Fig. 1. Effects of different concentrations of melatonin on the growth of Arabidopsis seedlings.

Arabidopsis seedlings were cultured for 5 days on MS medium supplemented with Melatonin (Mel) (0, 10, and 30 μM) for pre-treatment. The pre-treated seedlings were grown for additional 10 days on fresh MS medium without Mel but supplemented with 25 mM NaCl (A) and 50 mM NaCl (B). 

Fig. 2. Effects of different concentrations of serotonin on the growth of Arabidopsis seedlings.

     Arabidopsis seedlings were cultured for 5 days on MS medium supplemented with Serotonin (Ser) (0, 10, and 30 μM) for pre-treatment. The pre-treated seedlings were grown for additional 10 days on fresh MS medium without Ser but supplemented with 0 mM NaCl (A), 25 mM NaCl (B) and 50 mM NaCl (C).

     However, at high salt concentration (50 mM NaCl) Ser appeared to be a better stress modulating molecule than Mel as demonstrated with higher fresh weight, root number and root length in 10 μM Ser treatment compared to 10 μM Mel (Figure 3). During moderate salt stress treatment (25 mM NaCl), both Mel and Ser regardless of their concentration showed higher indicators of plant growth and development than the control plants without indoleamine treatment (Figure 3). Increased fresh weight (67% and 100) was observed in plants under Ser treatments (10 and 30 µM) relative to the control treatment at high salt concentration of 50 mM. Similarly, Mel treatments at 10 and 30 µM showed increased fresh weight (31 and 25%) compared to the control treatment under salt stress concentration of 50 mM.  In particular, the number of lateral roots was significantly higher in both Mel (55.29%) and Ser (45.88 %) treatments relative to the control with a salt concentration of 25 mM (Figure 3C). For both indoleamines, 10 μM treatment seems to be the ideal concentration to alleviate salt stress in Arabidopsis seedlings as opposed to the treatment with 30 μM. Under no stress condition (0 mM NaCl), Arabidopsis seedlings treated with Ser showed higher fresh weight than the seedlings treated with Mel. Although both indoleamines helped the seedlings escape from the adverse conditions imposed by NaCl treatment, exogenous application of Ser appeared to have a better ameliorating effect than Mel.

Fig. 3. Effect of Melatonin (Mel: 0, 10, and 30 μM) and Serotonin (Ser: 0, 10, and 30 μM) on Arabidopsis seedlings.

     (A) fresh weight, (B) shoot height (C) number of lateral roots and (D) primary root length of Arabidopsis seedlings after 15 days of salt treatment (0, 25 and 50 mM of NaCl). The data are presented as the mean ± standard error of three replications. Means with the same letter are not significantly different at P<0.1

3.2. Mel and Ser modulated expression of salt tolerance genes in Arabidopsis

     To understand the possible molecular mechanisms through which Mel and Ser mediate salt stress tolerance in plants, the expression of salt and drought stress responsive genes, ABI3, ABI5, ATX1, RD29A, RD29B, ZAT10, ZAT12, SOS1 and HAK5 was investigated in Arabidopsis seedlings pre-treated with Mel (10 μM) and Ser (10 μM) and exposed to 50 mM NaCl relative to control seedlings without any indoleamine pre-treatment but exposed to 50 mM NaCl. The relative expression of ABI3 was up-regulated by three-fold in plants pre-treated with Mel after 1-day (1d) of salt treatment compared to the control plants (Figure 4). Conversely, ABI5 expression remained unaltered in response to Mel pre-treatments after salt stress, suggesting that only ABI3, which is a central ABA-induced TF, is capable of responding to Mel treatment in salt-stressed plants. As opposed to Mel pre-treatment, ABI3 and ABI5 appear to be unresponsive to Ser pre-treatment which was reflected in similar expression levels of these genes after NaCl treatment relative to the control plants (Figure 4B).

Fig. 4. Relative normalized expression of ABA-dependant transcription factors (ABI3 and ABI5).

     ABI3 and ABI5implicated in seed maturation and drought stress adaptation after exposure of the seedlings to salt stress (50 mM) for one day. The expression of ABI3 and ABI5 was normalized to that of β-actin and calculated relative to the control treatment (0 μM, Mel or Ser) at the same salt-stress level. Values represent the mean ± standard error of two biological replicates for both ABI3 and ABI5. Student’s t-test was utilized to determine the statistically significant difference between treatments and control at *p≤0.05.

     ATX1 expression remained unchanged in Mel and Ser pre-treated plants (Figure 5A). The downstream genes RD29A and RD29B, which are regulated by ATX1 gene product, showed negligible expression changes in Ser pre-treated plants after salt stress exposure (Figure 5B and 5C). However, RD29B expression was significantly upregulated by Mel pre-treatment after 1d of salt stress (Figure 5 C).

Fig. 5. Relative normalized expression of transcription factors ATX1, RD29A and RD29B.

     ATX1 modulating downstream genes related to water deficit and salt stress RD29A and RD29B, after exposure of the seedlings to salt stress (50 mM) for one day. The expression of ATX1, RD29A and RD29B was normalized to that of β-actin and calculated relative to the control treatment (0 μM, Mel or Ser) at the same salt-stress level. Values represent the mean ± standard error of two biological replicates for RD29A and RD29B and one biological replicate for ATX1. Student’s t-test was utilized to determine the statistical significant difference between treatments and control at *p≤0.1.

    Genes encoding ZAT10 and ZAT12 TF, which mediate salt stress tolerance in plants, were upregulated after 1d of exposure to salt stress treatment in both Ser and Mel pre-treated plants (Figure 6). Although both ZAT10 and ZAT12 transcript levels were upregulated, only ZAT12 shows statistically significant difference in expression with Mel and Ser treatments compared to the control (Figure 6B). Two stress related transcripts, SOS1 and HAK5, required to maintain Na⁺ and K⁺ homeostasis in Arabidopsis were also investigated in this study. The expression of HAK5 was upregulated in Mel pre-treated plants relative to the control after exposure to salt stress (Figure 7B).  In contrast, the expression levels of HAK5 and SOS1 remained unchanged in Ser pre-treated plants exposed to salt (Figure 7).

Fig. 6. Relative normalized expression of transcription factors ZAT10 and ZAT12.

      ZAT10 and ZAT12 implicated in activation of ROS-related antioxidant genes, after exposure of the seedlings to salt stress (50 mM) for one day. The expression of ZAT10 and ZAT12 was normalized to that of β-actin and calculated relative to the control treatment (0 μM, Mel or Ser) at the same salt-stress level. Values represent the mean ± standard error of two biological replicates for both ZAT10 and ZAT12. Student’s t-test was utilized to determine the statistical significant difference between treatments and control at *p≤0.1.

Fig. 7. Relative normalized expression of SOS1 and HAK5 genes.

     SOS1 and HAK5 genes implicated in salt tolerance via ion homeostasis, after exposure of the seedlings to salt stress (50 mM) for one day. The expression of SOS1 and HAK5 was normalized to that of β-actin and calculated relative to the control treatment (0 μM, Mel or Ser) at the same salt-stress level. Values represent the mean ± standard error of two biological replicates for both SOS1 and HAK5. Student’s t-test was utilized to determine the statistical significant difference between treatments and control at *p≤0.1.

4. DISCUSSION

     Water resources, environmental pollution and increased salinization of soils represent three major threats for agricultural sustainability. In particular, soil salinity is one of the most devastating environmental stresses causing serious reductions in cultivated land (29, 36). The accumulation of soluble salts in the soil is known to suppress plant growth and development, ultimately leading to loss in crop productivity (37). Increased soil salinization in arable land is positively correlated with the world population, which is expected to increase by 1.5 billion in the next two decades (1, 38). If soil salinity is not overcome within this period, the increasing demand of major crops such as rice, wheat and maize required to feed the world’s growing population will not be met (1). Different strategies to overcome salinity problems have been developed since the early 90s, including reclamation of salt-affected soils and development of salt-tolerant cultivars (36). However, these strategies have been unable to increase crop production at the pace required to feed the world’s growing population.

     Pre-treatment with Mel has shown to effectively rescue plant growth and development from salt stress in crops such as sunflower, wheat, watermelon and rice and could represent an important mitigation strategy to enhance crop productivity in saline soils (13, 15, 18, 20, 22, 39). However, molecular mechanisms in the role of these indoleamines with respect to inducing salt stress tolerance in plants remains largely unknown.  We investigated the effects of Mel as well as its precursor Ser in promoting salt-stress tolerance. Mel-mediated salt stress mitigation has been described in diverse species including apple, grape, fava bean, cucumber, soybean, rice and bitter orange (13, 15, 20, 40–44). The role of Mel in alleviating salt stress has been attributed to its antioxidant effects such as prevention of electrolyte leakage, lipid peroxidation, pigment degradation as well as regulation of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) (13, 18, 20, 45). In soybean seedlings, Mel alleviated the inhibitory effects of salt stress by up-regulating the expressions of genes that are otherwise inhibited by high salt concentration (44). In contrast, the effects of Ser in salt stress mitigation have not been extensively investigated. Mukherjee et al. (2014) reported that Ser as well as Mel improved survival of sunflower (H. annuus) seedlings under salt stress, which may be associated with tissue specific increases in endogenous indoleamines production (22). Therefore, the investigation of the role of Ser in salt stress mediation together with the expression of associated genes is important to determine if Ser modulated salt stress tolerance is via a Mel independent mechanism.

     ABA is well-known for its role in abiotic stresses and acts through the activation of ABA-responsive genes to induce stress tolerance in planta. Among these genes, ABI3 and ABI5 have been shown to enhance drought stress adaptation in cotton plants (46). In addition, ectopic expression of ABI3 increased the ABA-induced accumulation of cold- and drought- responsive transcripts in Arabidopsis seedlings (47). The gene-expression data from our study indicated that Mel pre-treatment up-regulated ABI3, but not ABI5 in Arabidopsis seedlings exposed to salt-stress (Figure 4). In addition, Mel up-regulated the expression of RD29B after salt stress; however, the expression of its counterpart, RD29A, remained unaffected (Figure 5). The RD 29A is known to be mainly induced via an ABA-independent pathway whereas RD 29B induction depends upon ABA availability (48, 49).  ABI3 is a central TF with a hierarchical role in activating downstream genes via signal transduction cascades (50, 51). In Mel pre-treated Arabidopsis seedlings, one of these ABI3-regulated genes could be encoded by RD29B. In this case, Mel could have activated ABI3 expression that would trigger a signaling cascade involving RD29B. Interestingly, Nakashima et al (2006) showed that ABI3 can strongly up-regulate the expression of RD29B in Arabidopsis, but not that of RD29A, which remained unaltered in our study after salt stress exposure (52). Moreover, RD29B has been shown to have transcriptional stress memory by increasing its rate of transcription upon additional stress treatments in drought-stressed Arabidopsis plants, whereas no signs of transcriptional memory have thus far been observed for RD29A (53). Taken together, these results indicate that Mel pre-treatment induces stress response in ABA-dependent manner and there might be a cross-talk between ABA and Mel. The results also indicate that Mel treatment might induce transcriptional stress memory in salt-stressed plants through the activation of a TF cascade involving ABI3 and RD29B. Mel pre-treatment has previously been shown to stimulate the biosynthesis of ABA in Arabidopsis and grape plants (54, 55). Thus, the possibility remains that Mel pre-treatment could increase ABA biosynthetic and signalling pathways prior to salt stress exposure.

     ATX1, which is involved in dehydration stress responses in Arabidopsis is not up-regulated by Mel or Ser pre-treatment. Although ATX1 can activate the downstream genes RD29A and RD29B in the presence of ABA, this plant hormone can also induce the expression of RD29A and RD29B in atx1 mutants as well as in wild type plants (27), suggesting that RD29A and RD29B activation could also occur independent of ATX1. Taken together, it appears that up-regulation of RB29B in salt exposed Arabidopsis plants pre-treated with Mel could be independent of ATX1.

Mel and Ser pre-treatment also enhanced the expression of ZAT10 and ZAT12 genes which are TFs related to abiotic stress responses in Arabidopsis. Induction of ZAT10 and ZAT12 transcripts by Mel has also been described in Arabidopsis plants subjected to cold stress (17). In other stress-related studies, ZAT10 and ZAT12 stimulated the expression of reactive oxygen‐defense transcripts, and enhanced the tolerance of plants to salinity, heat and osmotic stresses (13, 56). HAK5, a structural gene encoding an ion transporter implicated in stress tolerance was upregulated by Mel after 1d of salt stress. Previously, Mel had been related to ion homeostasis for inducing the expression of ion transporter genes in apple plants under salt stress (57). HAK5 is required to maintain ion homeostasis in plants, which is severely impaired after exposure to high salt concentration (45, 58).

     Overall, the results from this study suggest that Mel and Ser can effectively mitigate salinity stress with both indoleamines acting via modulation of stress-related genes in metabolic pathways of ABA, ROS, and ion homeostasis. It is noteworthy that this study also highlights the role of Ser in salt stress amelioration. This effect may involve shared pathways that are both similar and different than those described for melatonin. Moreover, Ser showed strong antioxidant effect but less influence on ABA related gene upregulation. Both Mel and Ser can act as antioxidants, exhibit wide ranging effects in plant development under normal and stress conditions, and are known to improve survival of plants in response to biotic and abiotic stresses (21, 59–63). In addition to antioxidation, these stress survival mechanisms may include altered biosynthesis, redistribution, interconversions and feedback mechanisms in the indoleamines pathway (23). It is plausible that Mel and Ser act in coordination via interactive pathways to mediate salinity stress in Arabidopsis with Ser balancing Mel-mediated growth and survival response under normal and stress conditions.

ACKNOWLEDGMENTS

     This research was supported by the National Sciences and Engineering Research Council (NSERC) of Canada and the Gosling Research Institute for Plant Preservation (GRIPP).

AUTHORSHIP

     MRS, VSB, and PKS were involved in the development of the hypotheses and experimental design. MRS and VSB conducted the majority of the experiments and the manuscript was developed by MRS, VSB, and JAFC. All authors participated in the final revision of the manuscript.

CONFLICT OF INTEREST

     The authors have no conflict of interest

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