Plant growth experiments were conducted using hydroponic tanks with saline solutions at the Salt Laboratory of Agriculture and Agri-Food Canada in Swift Current, Saskatchewan as described in Steppuhn et al. (2012), but with the following variations. We tested transgenic alfalfa genotypes overexpressing microRNA156 (miR156OE) and plants transformed with an empty vector (EV) that were generated by Aung et al. (2015a). In addition to genotypes A8, A11, and A11a described in (Aung et al. (2015a), we also included an additional genotype (A4a) with relatively low miR156 expression in our study (Supplementary Figure 1A). MiR156OE and EV plants were grown starting from foam plugs of rooted cuttings in tanks that featured three cuttings per genotype and five genotypes (EV and 4 miR156OE) randomly distributed in each tank. The overall experimental design included four tanks for each of three electroconductivity (EC) salt levels (i.e., EC 1.4 dSm^-1, 7.0 dSm^-1, and 14 dSm^-1). All 12 tanks were randomized for location in a greenhouse under an average of 16 h daylight and 20°C temperature conditions (Supplementary Figure 1B). EC 1.4 dSm^-1, 7.0 dSm^-1, and 14 dSm^-1 were designated as control, mild and severe salinity stress, respectively.

Physiological data were collected from four experiments (i.e., four re-growth harvests of forage material collected prior to blooming). For the first harvest, data were collected after 70 days of plant transplantation whereas for subsequent second, third, and fourth harvests, data were collected 30–35 days after the preceding harvest. Plant height was measured from the rooted cutting plug to the tip of the longest shoot. For root sample (4th harvest) collections, tanks were flooded to facilitate soil loosening. Alfalfa developmental stages (stage 0; early vegetative, stage 1;mid-vegitative, stage 2; late vegetative, stage 3; early bud, stage 4; late bud, stage 5; early flower) were determined from four harvests by examining the stems as described in Information Bulletin 217 (Fick and Mueller, 1989).

Leaf and root samples for RNA extraction, antioxidant and ion analysis were collected from the fourth harvest. Leaf RNA samples were collected from the tip of two shoots with no flower buds, three plants per genotype from each tank. Leaf samples for antioxidants were collected from two healthy shoots including tiny buds. RNA and antioxidant samples were frozen immediately in liquid nitrogen. Plant samples were first weighed to determine fresh weight or biomass. Dry weight was obtained by incubating samples in a forage drum drier at 40°C for 1–2 weeks. Dried samples from the fourth harvest were then ground for ions and nutrition analysis. Twenty centimeter-long root samples (measuring from the longest tip) were collected for RNA extraction from three plants per genotype per tank. Samples were then frozen immediately in liquid nitrogen.

To assess forage quality, we determined the fiber content in alfalfa leaf material collected from the fourth harvest. Neutral detergent fiber (NDF) levels were measured using an ANKOM200 fiber analyzer (Model 200; ANKOM; Fairport, NY, USA) according to the ANKOM Neutral Detergent Fiber in Feeds Filter Bag Technique (Ferreira and Mertens, 2007). For acid detergent fiber (ADF), we followed a protocol described in Goering and Van Soest (1970). For total Kjeldahl nitrogen (TKN) measurements, we used a H₂SO₄/Se/Na₂SO₄ digestion method as described in Varley (1966), and analysis was performed following a published method (Noel and Hambleton, 1976).

Concentration of ions (Na⁺, Ca²⁺, Mg²⁺, and Zn²⁺) was measured from leaves of 1-month-old plants from the fourth harvest using HClO₄/HNO₃ digestion method (Jones, 1991) in the ICP-OES (Inductively Coupled Plasma – Optical Emission Spectroscopy) on a Fisher Scientific iCAP6300 Duo machine by following manufacturer’s instructions. Briefly, 1 g sample was taken into PTFE high pressure vessels, and 5 mL of concentrated nitric acid and 5 mL water were added for digestion in Milestone Ethos EZ microwave. The digest was diluted with ultra-pure water before running the samples along with blanks for each digestion cycle. For data analysis, the instrument was calibrated, and samples were analyzed. We performed method detection limit study by analyzing three digestion blanks and the average values were taken for detection limits.

Total antioxidant levels were determined from 1-month-old leaf samples (100 mg) using an antioxidant assay kit (Sigma–Aldrich, Oakville ON, Canada; catalog number CS0790) following the manufacturer’s instructions. Antioxidant reactions were performed in a 96-well plate by reading endpoint absorbance at 405 nm using a plate reader (BioTek, Synergy 2, Winooski, VT, USA). Concentration of antioxidants was calculated using a Trolox^TM standard curve (Supplementary Figure 2).

Leaf and root samples were ground using mortar and pestle into fine powder and 100 mg representative sample was used for RNA extraction using the PowerPlant RNA Isolation Kit (Mo Bio Laboratories Inc., San Diego, CA, USA). Extracted RNA was treated with TURBO DNase (Ambion, Austin, TX, USA). One microgram of total RNA was used for cDNA synthesize using an iScript cDNA synthesis kit (Bio-Rad Laboratories, Mississauga, ON, Canada). Gene amplification was performed in a C1000 Touch^TM Thermocycler Real-Time PCR System (Bio-Rad) using SsoFast SYBR Green Master Mix (Bio-Rad). QRT-PCR conditions were 1 cycle at 95°C for 30 s, then 40 cycles at 95°C for 5 s, 60°C for 15 s, followed by melting curve 65–95°C with 5 s/step, +0.5°C/cycle. QRT-PCR data was analyzed using a CFX-Manager (Bio-Rad) with a 2^-ΔCT method (Schmittgen and Livak, 2008). Suitable reference genes (ACC1, Actin) were selected (Supplementary Figure 3) and primers for reference and target genes were designed from M. sativa sequences (Gao et al., 2016) and are listed in Supplementary Table 1.

GraphPad Prism software was used to statistically analyze the data. The t-test or Analysis of Variance (ANOVA) were used to test the significance followed by Tukey’s HSD test for multiple comparisons. Statistical analyses were conducted on a sample size of 3–12 and P-value < 0.05.

Transgenic alfalfa genotypes exhibited variable levels of miR156 expression with A4a and A8 showing low expression, A11a medium and A11 higher expression of miR156 (Supplementary Figure 1A). In our study, alfalfa genotypes overexpressing miR156 accumulated significantly higher shoot dry biomass at least in two miR156OE genotypes (A4a, A8) compared to EV under control (EC 1.4). Three miR156OE genotypes (A4a, A11, and A11a) showed enhanced shoot biomass under severe salt stress (EC 14), while no significant difference was observed under mild salt stress (Figure 1A). Similar to leaves, significantly enhanced root dry biomass was observed in A4a and A8 under control conditions compared to EV (Figure 1B). Medium and high miR156 over-expressers (A11a, A11) exhibited significantly higher accumulation of dry root biomass compared to EV under severe stress conditions (Figure 1B). Furthermore, we observed an increased (statistically or/and numerically) fresh shoot and root biomass accumulation in miR156OE genotypes (except in A8 during severe stress) under control, mild and severe stress conditions (Supplementary Figure 4). We also compared shoot and root biomass gain in EV and miR156OE genotypes during mild and severe stress relative to their corresponding control plants, and results indicated that medium and high miR156 over-expressers (A11, A11a) consistently accumulated biomass that was significantly higher than EV. Low miR156 over-expressers, however, showed insignificant results with EV (Supplementary Figure 5). These results suggest that miR156 improves fresh and dry biomass in alfalfa under control and salinity stress conditions.

Plant physiological responses to salinity stress vary and depend on underlying molecular mechanisms (Ahmed et al., 2013). In our study, we measured plant height, number of stems or branches and plant developmental stages in EV and miR156OE genotypes under control, mild and severe salt stress conditions. Results revealed that under control conditions, low miR156 over-expressing plants were either significantly taller (A4a) or showed a similar height compared with EV (A8), while medium and high miR156 over-expressers (A11 and A11a) were significantly shorter. A similar trend was observed in these genotypes under mild stress conditions (Figure 2A). Under severe stress, we recorded the same plant height for A4a and EV, whereas A8, A11 and A11a genotypes displayed a significantly shorter height than EV (Figure 2A). Interestingly, A11 and A11a genotypes gained significantly more height than EV under mild and severe salt stress conditions when data were presented relative to counterpart plants grown under control conditions (Supplementary Figure 6A), indicating an enhanced ability of medium and high miR156 over-expressers to maintain growth under salinity stress.

Number of stems, which impacts biomass yield, was significantly higher in A8, A11, and A11a than in EV under control and mild stress conditions. Furthermore, medium and high miR156 over-expressers (A11, A11a) showed a significantly higher number of stems compared to EV under severe salt stress, whereas under similar conditions, low miR156 over-expressers (A4a, A8) showed no differences with EV (Figure 2B). When severe stress results were compared relative to the corresponding controls, A11a and A11 exhibited significantly higher number of stem compared to EV (Supplementary Figure 6B), providing additional evidence that genotypes with higher levels of miR156 possess better ability to withstand salinity and maintain stem growth.

To further investigate the physiological responses, we recorded developmental stages in EV and miR156OE genotypes. At the time of data collection, medium and high miR156 over-expresser (A11, A11a) were between developmental stages 2 and 3, which was significantly lower than EV and low miR156 expressers (A4a, A8) that were already between stages 4 and 5 under control and mild stress conditions (Figure 2C). Similar results were observed under severe stress where A11 and A11a were at or below stage 2 (significantly lower than EV) and low miR156 expressers were at or above stage 3 (Figure 2C). Low miR156 expressers (A4a and A8) exhibited developmental stages that were similar to EV under control, mild and severe stress conditions (Figure 2C). These results indicate that higher expression of miR156 reduces plant height, increases number of branches and delays developmental phases under control and salt stress conditions. To our surprise, medium and high (A11, A11a) miR156 expressers completed developmental stages significantly faster than EV under severe salt stress conditions when results were presented relative to the corresponding unstressed control plants (Supplementary Figure 6C). This suggested a positive role of miR156 in maintenance of alfalfa physiology and development under salinity stress conditions. Taken together our results show that the ability of alfalfa plants to maintain stem growth and biomass under salinity stress improves with increasing miR156 expression.

Neutral detergent fiber (NDF) and ADF represent the fiber in plant cell walls and ultimately determine forage quality. Low fiber content indicates higher feed digestibility and nutritional value for animal consumption (Grant et al., 2014). In this study, we observed significantly lower NDF content in three miR156OE genotypes (A8, A11, and A11a) compared to EV under control conditions (Figure 3A). The same three genotypes also showed numerically reduced NDF content during mild and severe salt stress compared to EV but differences were statistically insignificant with EV. NDF content was, however, significantly lower in high miR156 over-expressing genotype (A11) compared to EV during mild stress (Figure 3A). We did not observe significant differences of ADF content between EV and miR156OE genotypes under control or severe salt conditions (Figure 3B). During mild stress, ADF content was significantly reduced in medium and high miR156 over-expressers (A11, A11a) and also in one low miR156 over-expresser (A8) compared to EV controls. In contrast, one of the low miR156 over-expressers (A4a) exhibited significantly higher content of ADF compared to EV (Figure 3B).

Nitrogen is an essential component of chlorophyll and amino acids and hence plays a crucial role in photosynthesis and protein synthesis. Overall, we did not observe significant differences in TKN between control and mild stress plants of EV and miR156OE genotypes. During severe salinity stress, however, TKN content was significantly increased in A4a, A11, and A11a compared to unstressed counterparts (Figure 4A). On comparing EV and miR156OE genotypes, we did not observe differences between EV and three miR156OE genotypes (A8, A11, and A11a) under all conditions. However, a significantly lower level of TKN was observed in A4a compared to EV at control and mild stress (Supplementary Figure 7).

Abiotic stress enhances accumulation of antioxidants in plants to minimize the effect of toxic oxidants produced under stress conditions (Wang et al., 2003). Compared to control plants, mild and severe salt stress slightly induced accumulation of antioxidants in EV and a lower miR156 expresser A8 (Figure 4B). On the other hand, another lower miR156 expresser A4a showed increased antioxidant levels only during mild stress compared to corresponding control plants. Furthermore, we did not observe any significant differences in antioxidant accumulation in medium and high miR156 over-expressers (A11, A11a) in response to either mild or severe stress (Figure 4B).

Our results reveal that with increasing miR156 expression, nitrogen content also increases whereas fiber content decreases in alfalfa under same salinity conditions, contributing toward improved alfalfa quality as a forage crop.

To study alfalfa miR156OE genotypes response to ion uptake under saline conditions, we analyzed sodium (Na⁺), calcium (Ca²⁺), zinc (Zn²⁺), and magnesium (Mg²⁺) ions uptake in EV and miR156OE genotypes under control, mild and severe salt stress conditions. Salt stress increased Na⁺ and Zn²⁺ accumulation in EV and four miR156OE genotypes whereas the same conditions reduced Ca²⁺ and Mg²⁺ levels (Supplementary Figure 8). Furthermore, we observed a reduction in Na⁺ uptake at least in medium and high miR156 expressers (A11a, A11) compared to EV under severe salinity stress. Similarly, we also observed reduced levels of Zn²⁺ in A11a during mild stress, and in A8, A11a under severe stress conditions (Table 1). On the other hand, a miR156OE genotype (A4a) exhibited higher levels of Ca²⁺ than EV under mild stress. Remaining genotypes showed similar levels of Ca²⁺ and Mg²⁺ compared to EV during mild and severe salinity stress (Table 1). These results suggest that alfalfa plants with increased miR156 levels tend to reduce uptake of Na⁺ under severe salt stress conditions.

To assess the effect of miR156 on molecular responses of alfalfa to salinity, we analyzed expression of genes belonging to three main categories; (1) SPL family genes – direct target of miR156, (2) non-SPL transcription factors with known function in salt tolerance and, (3) downstream effector genes with known role in mediating salt responses in plants (see Introduction). We tested the expression of genes in the above-mentioned three categories to determine if miR156 affects these important genes and transcription factors under salt stress for regulation of salinity stress responses in alfalfa.

Alfalfa biomass is an important trait for forage and biofuel industries. In our study, miR156 overexpressing plants exhibited an increase in shoot and root biomass under control conditions as previously reported (Aung et al., 2015a). Normally, salinity reduces plant biomass accumulation (Li et al., 2010) but, interestingly, we recorded a less reduction in biomass accumulation in miR156OE genotypes relative to EV under severe stress condition. In absolute term, an increased branching (shoot number) and slower developmental stage completion by miR156OE genotypes compared to EV may have contributed to the accumulation of higher biomass. Surprisingly, miR156OE genotypes were quick in completing developmental stages, and also exhibited increased plant growth (height, stem, and developmental stages) in relation to their corresponding controls, when compared with EV under stress conditions, indicating a stress avoidance mechanism in transgenic alfalfa plants under salinity stress. Previously, an increased shoot growth was observed in tomato and rice under salt stress (Zhu et al., 1998; Mayak et al., 2004).

Total Kjehldahl nitrogen (TKN), NDF, and ADF contribute to forage quality and digestibility. Protein increases forage quality, and reduction in nitrogen content leads to reduced crude protein content, which ultimately reduces forage quality (Owensby et al., 1996). NDF consists of cellulose, hemicellulose and lignin whereas ADF contains mainly cellulose and lignin but lacks hemicellulose (Van Soest, 1994). Furthermore, NDF and ADF are components of fiber that can reduce digestibility, palatability, and intake of forages leading to degradation of forage quality (Van Soest, 1994). Our study shows a significant increase of TKN in response to severe salt stress in three miR156OE genotypes but not in EV plants. Moreover, we recorded a reduction of NDF and ADF in miR156OE genotypes compared to EV under control and mild salt stress conditions, respectively. An increased TKN and decreased NDF and ADF content in miR156OE genotypes indicates that miR156 also contributes to alfalfa forage quality improvement under normal and saline conditions. A study has shown an improved forage quality of bermudagrass and wheatgrass under salinity stress (Robinson et al., 2004). Similarly, in another study miR156 overexpression led to enhanced forage quality in switchgrass (Fu et al., 2012).

Higher levels of Na⁺ ions can cause ion toxicity resulting in reduced plant growth and performance. Several membrane transporters particularly Na⁺ and K⁺ transporters play a crucial role in salinity tolerance in plants (Schroeder et al., 2013). Most studies have been conducted in Arabidopsis where cytokinin mediates expression of AtHKT1 in a negative manner via type B response regulators ARR1 and ARR12, thereby indicating that cytokinin levels are reduced in response to salinity stress, resulting in induction of AtHKT1 expression (Mason et al., 2010), which supports our HK1 expression results.

Previously, studies have identified a group of HKT transporters that regulate Na⁺/K⁺ co-transport (Rubio et al., 1995). Subsequent studies have suggested that AtHKT1-mediated Na⁺ removal from the xylem stimulates K⁺ loading into xylem, resulting in a high K⁺/Na⁺ ratio in leaves that also alleviates Na⁺ stress (Ren et al., 2005; Sunarpi et al., 2005). There is an additional study showing that HKTs play a crucial role in determining a high K⁺/Na⁺ ratio in plants. For example, a TaHKT1 gene contributed to Na⁺ removal from xylem in the leaf sheath of durum wheat to protect leaf blades from Na⁺ over accumulation and toxicity (Huang et al., 2006). Mutating Arabidopsis HKT gene in (AtHKT1) caused Na⁺ hypersensitivity, as well as enhanced accumulation of Na⁺ upon salinity stress in Arabidopsis leaves (Mäser et al., 2002; Berthomieu et al., 2003; Horie et al., 2006). Subsequent studies further demonstrated the role of AtHKT1, and a rice ortholog OsHKT1, in removing Na⁺ and protecting leaves from Na⁺ toxicity (Ren et al., 2005; Sunarpi et al., 2005; Davenport et al., 2007). Another study conducted on root stellar cells in Athkt1 mutant plants provided additional evidence that AtHKT1 mediates passive Na⁺ channel-like transport (Xue et al., 2011).

In our study, salt stress induced Na⁺ accumulation in EV and all miR156OE genotypes, but this accumulation was reduced in moderate and high miR156 expressers (A11, A11a) compared to EV under severe stress conditions. Reduced accumulation of Na⁺ in A11 and A11a could be due to induced expression of HK1 in these genotypes under severe stress since HKT transporters play key role in Na⁺ efflux from the xylem (Sunarpi et al., 2005). Furthermore, tonoplast and plasma membrane localized NHXs antiporters are essential for maintaining low Na⁺ in the cytoplasm and Na⁺ detoxification via sequestering within the vacuole (Deinlein et al., 2014). Over-expression of NHX led to salinity tolerance in Arabidopsis (Apse et al., 1999). A recent transcriptome study revealed that alfalfa transporter genes HKT1 and anion exchanger maintained high transcript levels in salt tolerant alfalfa genotypes (Gruber et al., 2016). Na⁺ influx can raise sodium concentration inside the cytosol activating K⁺ efflux and disturbing K⁺/Na⁺ ratio (Sun et al., 2009; Britto et al., 2010). The high-affinity K⁺ transporter (HKT) family transports Na⁺ and K⁺ (Benito et al., 2014). Our results showed reduced Na⁺ concentration in medium and high miR156 expressors (A11, A11a) under severe stress, which may have increased K⁺ influx. This is also supported by induced expression of HK1 in leaves of miR156OE genotypes. Overexpression of Arabidopsis H⁺-ATPase and SOS1 enhances salt tolerance in plants (Bao et al., 2009; Yang et al., 2009). In our study, an enhanced expression of NHX1 in root and HK1, H⁺-ATPase and SOS1 in leaves of medium and higher miR156 expressers under severe saline conditions may have contributed to the removal of Na⁺ and a reduction of its toxicity in these genotypes.

Salinity stress induces antioxidant accumulation in plants to counter the damage caused by oxidants under stress conditions (Bose et al., 2014). In our study, we did not observe a slight induction of antioxidants in medium and higher expressers of miR156 (A11, A11a) under mild or severe stress. This may indicate that these genotypes may have low internal stress level compared to EV and low miR156 expressers (A4a, A8) that accumulated significantly higher levels of antioxidants in response to mild and severe stress.

Given that SPL transcription factors (SPL6, SPL12, and SPL13) are targeted by miR156 (Aung et al., 2015a), it is important to analyze their expression patterns to better understand the mechanism of salt tolerance regulated by miR156. Previously, there was one study that revealed that rSPL9 (expressing miR156-insensitive SPL9) and rSPL10 (expressing miR156-insensitive SPL10) plants exhibited hypersensitivity to drought and salt stress (Cui et al., 2014). Their results indicate that SPLs could potentially be negative regulators of stress response. Similarly, genes encoding non-SPL transcription factors such as ZFP1, AP2, ERF, WRKY, and bZIP and other downstream effector genes play vital role in salt tolerance in plants (Singh et al., 2002; Ciftci-Yilmaz and Mittler, 2008; Jiang and Deyholos, 2009; Pandey and Somssich, 2009; Rushton et al., 2010; Mizoi et al., 2012; Shi et al., 2014; Hu et al., 2016), and testing expression of these genes could also be helpful in expanding list of direct or indirect targets of miR156 particularly under salt stress. This information could be helpful in drawing a pathway that miR156 uses to regulate salinity stress responses in alfalfa (Figure 8).

Several studies have established that miR156 modulates abiotic stress responses in various plant species (Wang M. et al., 2013; Cui et al., 2014; Kohli et al., 2014; Stief et al., 2014; Xie et al., 2014; Wang and Wang, 2015). MiR156 controls plant responses through targeting SPL family transcription factors (Aung et al., 2015a), which in turn may alter expression of downstream genes. We observed downregulation of SPL genes in leaves and roots under stress conditions indicating that miR156 expedites the cleavage of SPLs to modulate stress responses in alfalfa. SPL genes respond to abiotic stress and stimulate the synthesis of secondary metabolites (Wang M. et al., 2013; Yu et al., 2015) showing that they may directly or indirectly be involved in stress tolerance.

Under salt stress conditions, several genes and transcription factors including WRKY, ZFP, bZIP, and ERF/AP2 are upregulated to help plants adapt to saline conditions (Golldack et al., 2011; Deinlein et al., 2014). In our study, however, not all the tested genes and transcription factors were upregulated in response to salt stress. An enhanced expression of WRKY and ZFP1 in leaf and root, ERF and AP2 in leaf, and bZIP in root of miR156OE genotypes under salt stress indicates that transcription activities were increased in miR156OE genotypes. We hypothesize that as transcription activators, SPLs may have an inverse relationship with other stress responsive transcription factors (Gou et al., 2011), and a reduced expression of SPLs may have led to enhanced transcriptional activities of WRKY, ZFP, bZIP, and ERF/AP2 transcription factors in leaves and roots of stressed alfalfa plants.

Overexpression of downstream salt responsive genes such as NHX1, VATP, GRP, HK1 RCI2, H⁺-ATPase, and SOS1 improve salt stress tolerance in plants (Coba de la Pena et al., 2008; Silva and Geros, 2009; Leidi et al., 2010; Long et al., 2013, 2015). In our study, upregulation of these genes in leaf and root of miR156OE genotypes under salt stress conditions may have contributed to salt stress tolerance of transgenic alfalfa. This also provides further support to our hypothesis that high transcription activities in miR156OE plants led to altered expression of downstream genes resulting in an adaptive response of alfalfa plants to salt stress.

Overall, three common tendencies can be found in our data; (1) tissue-specific –in case of SPL12, ERF, AP2, GRP, HK1, (show expression differences only in leaf) and WRKY, bZIP, NHX1, RCI2 (show differences only in root), (2) severe stress-specific – show consistent downregulation of SPLs, and upregulation of non-SPLs transcription factor as well as downstream effector genes under severe stress conditions across leaf and root, and (3) miR156 level-specific – show consistently significant results in medium and high miR156 expresssors (A11, A11a). This supports our hypothesis that a relationship exists between miR156 levels, SPLs, non-SPL transcription factors and downstream genes.

Taken together, we report that miR156 positively regulates salinity response in alfalfa partially by altering expression of important transcription factors and downstream genes. Thus, a trend seems to exist where increased expression of miR156 exhibits a salt tolerant phenotype during severe stress. We noted that gene expression in plants is differently regulated and depends on the intensity of stress and the tolerance or sensitivity of the genotypes. Our results are in line with other published studies. For example, a study by (Rodrigues et al., 2009) showed an insignificant expression of several genes at mild water stress in sugarcane cultivars, but their expression was significantly increased under severe stress. In support of genotypic differences, (El Maarouf et al., 1999) observed that a susceptible cowpea genotype showed enhanced expression of Phospholipase D under drought stress conditions whereas a tolerant genotype showed low expression of this gene throughout the experiment. These studies support our results of variable expression in different genotypes at different stress levels.

Several studies in other plant species have revealed that miR156 levels do not only change in response to salt stress but also under various other stresses such as heat, drought, cold, UV-B radiation, and hypoxia (Zhou et al., 2008; Jia et al., 2009; Moldovan et al., 2010; Cui et al., 2014; Stief et al., 2014). However, we cannot rule out the possibility that salt stress response is in part regulated by additional salt-responsive genes and transcription factors not identified in this study, which may directly or indirectly be regulated by miR156. Whereas this study reveals a role for miR156 in salt stress response, further studies involving next generation sequencing, and functional characterization of target SPL genes and downstream SPL-regulated genes would be needed to fully understand how miR156 affects salt tolerance. Our recent study has shown SPL13 as a negative regulator of drought responses in alfalfa (Arshad et al., 2017), and a similar work under salt stress would provide a better understanding of how the miR156-SPL gene regulatory network affects salt tolerance. The detailed analysis of the regulation of miR156, and other salt-responsive genes and transcription factors remains an important area of research, as it will be interesting to see whether miR156 along with identified genes and transcription factors also play a role in improving tolerance to other stresses in various plant species.