Overexpression of Mtr-miR319a Contributes to Leaf Curl and Salt Stress Adaptation in Arabidopsis thaliana and Medicago truncatula

Salt stress is a worldwide agronomic issue that limits crop yield and quality. Improving salt stress tolerance via genetic modification is the most efficient method to conquer soil salinization problems in crops. Crop miRNAs have been declared to be tightly associated with responding and adapting to salt stress and are advantageous for salt tolerance modification. However, very few studies have validated vital salt tolerance miRNAs and coupled potent target genes in Medicago species, the most economically important forage legume species. In this study, Mtr-miR319a, a miRNA that was identified from the previous next-generation sequencing assay of salt-treated Medicago truncatula, was overexpressed in M. truncatula and Arabidopsis thaliana, inducing the curly leaves and salt stress tolerance phenotypes. Combining the elevated expression level of Mtr-miR319a in the M. truncatula overexpression lines under normal and salt-treatment conditions, the regulatory roles of Mtr-miR319a in leaf development and salt stress adaptation were demonstrated. Several predicted target genes of Mtr-miR319a were also regulated by Mtr-miR319a and were associated with the aforementioned phenotypes in M. truncatula plants, most notably MtTCP4. Our study clarified the functional role of Mtr-miR319a and its target genes in regulating leaf development and defending salt stress, which can help to inform crop breeding efforts for improving salt tolerance via genetic engineering.


Introduction
Soil salinization is a worldwide issue affecting crop yield, quality, and distribution [1,2]. When plants are exposed to a salt circumstance, various biological processes are negatively impacted by osmotic stress, ion toxicity, and oxidative damage due to the salinity [3,4]. It is calculated that more than half of the arable land in the world will suffer from salinity in 30 years [5]. Hence, studying salt stress adaptation mechanisms and enhancing the salt tolerance of plants are important and imperative for crop productivity and agricultural sustainable development around the world.
MicroRNAs (miRNAs), which are widely distributed in diverse plant species, are endogenous non-coding RNA transcripts with a small length of 19-24 nt [6,7]. miRNAs have a vital role in the regulation of gene transcription, cell proliferation, and protein translation [6,8]. Under salt stress, miRNAs function by activating rapid and synchronized changes at post-transcriptional levels for responding to the detrimental circumstance [9,10]. Currently, there are significant efforts to identify stress-inducible miRNAs and miRNA target genes via high-throughput sequencing approaches [6,9,11,12]. For instance, several salt stress-related miRNAs, which could have a broad range of target mRNAs and perform important roles in salt stress regulation, were documented in alfalfa (M. sativa) and M. truncatula [6,9], whereas the overexpression of miR393 can enhance salt stress adaptation

Sequence Analysis of Mtr-miR319a
To explore the sequence conservation and variation of miR319 in A. thaliana and M. truncatula, multiple sequence alignment of the mature sequences and the secondary structure prediction of the precursors were analyzed. The results of the multiple sequence alignment showed that the mature sequences of miR319 in A. thaliana and M. truncatula were conserved ( Figure 1A), indicating that the overexpression of Mtr-miR319a in A. thaliana might work normally. However, the results also showed that there were differences among the sequences as well as the secondary structures of Mtr-miR319 precursors in M. truncatula ( Figure 1B).
To explore the sequence conservation and variation of miR319 in A. thaliana and M truncatula, multiple sequence alignment of the mature sequences and the secondary struc ture prediction of the precursors were analyzed. The results of the multiple sequence alignment showed that the mature sequences of miR319 in A. thaliana and M. truncatula were conserved ( Figure 1A), indicating that the overexpression of Mtr-miR319a in A. tha liana might work normally. However, the results also showed that there were differences among the sequences as well as the secondary structures of Mtr-miR319 precursors in M truncatula ( Figure 1B).

Leaf Curl Phenotypes in Mtr-miR319a-Overexpressed A. thaliana and M. truncatula Plants
The Mtr-miR319a-overexpressed A. thaliana lines of At-OE-7 and At-OE-17 and M truncatula lines of Mt-OE-22 and Mt-OE-32 were used for further functional clarification ( Figure 2). In the Mtr-miR319a-overexpressed A. thaliana seedlings, both young and ma ture leaves were curled with an impaired cotyledon boundary and serration formation which could also be found in the Mtr-miR319a-overexpressed M. truncatula seedlings

Leaf Curl Phenotypes in Mtr-miR319a-Overexpressed A. thaliana and M. truncatula Plants
The Mtr-miR319a-overexpressed A. thaliana lines of At-OE-7 and At-OE-17 and M. truncatula lines of Mt-OE-22 and Mt-OE-32 were used for further functional clarification ( Figure 2). In the Mtr-miR319a-overexpressed A. thaliana seedlings, both young and mature leaves were curled with an impaired cotyledon boundary and serration formation, which could also be found in the Mtr-miR319a-overexpressed M. truncatula seedlings ( Figure 2). Except for this leaf curl phenotype, no other growth or developmental differences were found in comparison to the WT plants in both A. thaliana and M. truncatula. The leaf development performance in the overexpression plants might be caused by the overexpression of Mtr-miR319a.
( Figure 2). Except for this leaf curl phenotype, no other growth or dev ences were found in comparison to the WT plants in both A. thaliana The leaf development performance in the overexpression plants migh overexpression of Mtr-miR319a.

Overexpression of Mtr-miR319a and Its Impacts on Salt Tolerance in A. truncatula
To test the salt tolerance ability of the Mtr-miR319a-overexpress and physiological assays were conducted in A. thaliana and M. truncatu stress (Figures 3 and 4). After exposure to high salt conditions for 24 an and M. truncatula, respectively, the growth status of all of the overexp better than their corresponding WT plants ( Figures 3A and 4A). The o before salt treatment at 0 d is shown in Figure 2. Specifically, in A. thali tent of At-OE-7 and At-OE-17 was significantly higher than that of th salt stress, which showed no difference under the control conditions (F ative proline content was higher in the At-OE-7 and At-OE-17 plants co plants under salinity ( Figure 3C). In M. truncatula, the MDA content in lines did not change after salt stress, whereas it was significantly high ( Figure 4B), and the relative proline content was significantly increas while the two overexpression plants had higher levels than the WT p The growth and physiological performances of the WT and Mtr-miR31 plants after salt stress demonstrated that overexpressing Mtr-miR319a i

Overexpression of Mtr-miR319a and Its Impacts on Salt Tolerance in A. thaliana and M. truncatula
To test the salt tolerance ability of the Mtr-miR319a-overexpressing plants, growth and physiological assays were conducted in A. thaliana and M. truncatula plants under salt stress (Figures 3 and 4). After exposure to high salt conditions for 24 and 35 d in A. thaliana and M. truncatula, respectively, the growth status of all of the overexpressed plants was better than their corresponding WT plants ( Figures 3A and 4A). The original plant status before salt treatment at 0 d is shown in Figure 2. Specifically, in A. thaliana, the water content of At-OE-7 and At-OE-17 was significantly higher than that of the WT plants under salt stress, which showed no difference under the control conditions ( Figure 3B). The relative proline content was higher in the At-OE-7 and At-OE-17 plants compared to the WT plants under salinity ( Figure 3C). In M. truncatula, the MDA content in the overexpression lines did not change after salt stress, whereas it was significantly higher in the WT plant ( Figure 4B), and the relative proline content was significantly increased after salt stress while the two overexpression plants had higher levels than the WT plants ( Figure 4C). The growth and physiological performances of the WT and Mtr-miR319a overexpression plants after salt stress demonstrated that overexpressing Mtr-miR319a is beneficial for improving salt tolerance in A. thaliana and M. truncatula.

High Expression of Mtr-miR319a Was Maintained before and after Salt Stress in Mtr-miR319a-Overexpressed M. truncatula Plants
To explore the expression pattern of Mtr-miR319a after salt circumstance, the expression level of Mtr-miR319a in M. truncatula WT and Mtr-miR319a-overexpressing plants was determined after exposure to a time course of salt treatment ( Figure 5). The results showed that the expressions of Mtr-miR319a in Mt-OE-22 and Mt-OE-32 were significantly higher than that of the WT seedlings at 0 h, showing five-fold differences. The expression of Mtr-miR319a in the WT plants increased five-fold after 2 h of salt treatment, which showed no difference compared to the overexpression plants. However, the expression of

Mtr-miR319 Acts on the Target Genes Involved in Leaf Development and Salt-Resp Roles in M. truncatula
To examine the transcriptional expression of the possible targets for verif functional roles of Mtr-miR319, eleven genes, including MYBs, CUC, TCPs, an which are involved in leaf development and salt stress adaptation, were determ fore and after salt stress in M. truncatula plants ( Figure 6). Specifically, the expr MYB2 and TCP3 did not show a significant difference between the WT and the pression lines under both control and salinity circumstances. Under normal co the expression of LOX13 in Mt-OE-22 and MYB1, TCP4, and LOX9 in the overex lines were significantly suppressed by Mtr-miR319, while TCP10 in the overex lines and LOX13 in Mt-OE-32 did not change. In addition, the CUC2 levels in M significantly increased. However, after 2 h of salt treatment, the expression of T TCP10 in the overexpression lines sharply declined, MYB1 and CUC2 in the two pression lines did not show any difference with the WT, and LOX9 in Mt-OE LOX13 in the two overexpression lines were significantly higher than in the W Mtr-OE-32 were exposed to 150 mM NaCl for 0, 2, 12, and 48 h. The values are shown as the mean ± standard error (SE); n = 3 for all groups. The bars represent the SE. Bars with different lowercase letters indicate statistically significant differences at p < 0.05 based on ANOVA.

Mtr-miR319 Acts on the Target Genes Involved in Leaf Development and Salt-Responsive Roles in M. truncatula
To examine the transcriptional expression of the possible targets for verifying the functional roles of Mtr-miR319, eleven genes, including MYBs, CUC, TCPs, and LOXs, which are involved in leaf development and salt stress adaptation, were determined before and after salt stress in M. truncatula plants ( Figure 6). Specifically, the expression of MYB2 and TCP3 did not show a significant difference between the WT and the overexpression lines under both control and salinity circumstances. Under normal conditions, the expression of LOX13 in Mt-OE-22 and MYB1, TCP4, and LOX9 in the overexpression lines were significantly suppressed by Mtr-miR319, while TCP10 in the overexpression lines and LOX13 in Mt-OE-32 did not change. In addition, the CUC2 levels in Mt-OE-32 significantly increased. However, after 2 h of salt treatment, the expression of TCP4 and TCP10 in the overexpression lines sharply declined, MYB1 and CUC2 in the two overexpression lines did not show any difference with the WT, and LOX9 in Mt-OE-22 and LOX13 in the two overexpression lines were significantly higher than in the WT plants. According to the expression changes, it can be found that MYB1, TCP4, TCP10, and LOX9 were suppressed under normal or salt stress conditions, indicating the inhibitory role that Mtr-miR319a plays on these target genes.

Prediction and Verification of the Target Genes of Mtr-miR319a
MtTCP was found to be a primary target gene of miR319 by using the psRNATarget miRNA target gene analysis server (http://plantgrn.noble.org/psRNATarget/home, accessed on 22 January 2021) [29]. Specifically, MtTCP4 (MTR_8g463380) was strongly conserved with the predicted binding region, whereas the combination of Mtr-miR319a and MtTCP4 (MTR_8g463380) completely matched. The results of 5′ RLM-RACE showed that the cleavage sites occurred at the 9th to 10th base sites of Mtr-miR319a in MtTCP4 ( Figure  7A). Furthermore, the Western blot assay showed that the protein translation of MtTCP4 was suppressed by Mtr-miR319a ( Figure 7B). Thus, this work predicted and verified the target gene MtTCP4 of Mtr-miR319a.

Prediction and Verification of the Target Genes of Mtr-miR319a
MtTCP was found to be a primary target gene of miR319 by using the psRNATarget miRNA target gene analysis server (http://plantgrn.noble.org/psRNATarget/home, accessed on 22 January 2021) [29]. Specifically, MtTCP4 (MTR_8g463380) was strongly conserved with the predicted binding region, whereas the combination of Mtr-miR319a and MtTCP4 (MTR_8g463380) completely matched. The results of 5 RLM-RACE showed that the cleavage sites occurred at the 9th to 10th base sites of Mtr-miR319a in MtTCP4 ( Figure 7A). Furthermore, the Western blot assay showed that the protein translation of MtTCP4 was suppressed by Mtr-miR319a ( Figure 7B). Thus, this work predicted and verified the target gene MtTCP4 of Mtr-miR319a.

Discussion
Salinity is a widespread problem that threatens the yield and production of crop across the world [30]. Exploring salt response and tolerance mechanisms can lay the the oretical foundation for salt tolerance genetic improvement and modification of economi cally important crops. MicroRNAs have been shown to play an essential role in the regu lation of gene transcription, protein translation, and cell proliferation [6,8]. Herein, ou study firstly verified that the overexpression of Mtr-miR319a can regulate leaf develop ment and salt tolerance via targeting several critical genes (Figure 8), such as TCP, MYB CUC, and LOX, in M. truncatula plants, which is discussed below.

Discussion
Salinity is a widespread problem that threatens the yield and production of crops across the world [30]. Exploring salt response and tolerance mechanisms can lay the theoretical foundation for salt tolerance genetic improvement and modification of economically important crops. MicroRNAs have been shown to play an essential role in the regulation of gene transcription, protein translation, and cell proliferation [6,8]. Herein, our study firstly verified that the overexpression of Mtr-miR319a can regulate leaf development and salt tolerance via targeting several critical genes (Figure 8

Discussion
Salinity is a widespread problem that threatens the yield and production of crop across the world [30]. Exploring salt response and tolerance mechanisms can lay the the oretical foundation for salt tolerance genetic improvement and modification of econom cally important crops. MicroRNAs have been shown to play an essential role in the regu lation of gene transcription, protein translation, and cell proliferation [6,8]. Herein, ou study firstly verified that the overexpression of Mtr-miR319a can regulate leaf develop ment and salt tolerance via targeting several critical genes (Figure 8), such as TCP, MYB CUC, and LOX, in M. truncatula plants, which is discussed below.  In plants, leaves are the most essential plant organs for energy acquisition and carbohydrate generation. Crinkled and curly phenotypes were found in the leaves of the Mtr-miR319a-overexpressing A. thaliana and M. truncatula plants in our results. This conspicuous leaf morphology was also reported in miR319-overexpressing petunia (Petunia × hybrida) [19] and tomato (Solanum lycopersicum) [31] and in tcp A. thaliana mutants [32]. In our study, the verified Mtr-miR319a target gene, TCP4, was detected to be suppressed under normal conditions in the Mtr-miR319a-overexpressed M. truncatula plants. This is in accordance with the observation that overexpressing miR319 can reduce the expression levels of TCPs and trigger leaf serration in A. thaliana, whereas the mir319a/b mutant enhanced the expression of TCPs, resulting in the formation of smooth leaves [15,18]. Therefore, our results suggest that the robust and intricate roles of Mtr-miR319a and its targets, namely, TCP transcription factors, play a crucial role in the development process of the central and marginal regions of the leaf of A. thaliana and M. truncatula [19,32]. In addition, TCP4 negatively regulates the expression of CUC genes and directly interacts with CUC2, which is also involved in leaf serration formation [18,33]. The expression of CUC2 was significantly higher in one of the overexpression lines. This further indicates that the regulatory networks of Mtr-miR319a and its target genes act on leaf formation and development [33].
Notably, the leaf phenotype caused by Mtr-miR319a can contribute to improving salt tolerance. Wider, thicker leaves with increased weight-to-area ratios have been reported in miR319-overexpressing creeping bentgrass plants [26] and switchgrass [24], and they were tightly associated with enhanced salt tolerance by means of maintaining higher water contents, photosynthetic activity, and stomatal conductance, reducing ROS levels (represented by H 2 O 2 content) and accumulating less Na + when coping with salt stress [24,26].
Though the miR319 network in plants is evolutionarily conserved, miR319-targeted genes might have conflicting functions between dicotyledonous and monocotyledonous plant species [34]. Therefore, this could explain why the leaf phenotypes in the A. thaliana and M. truncatula plants in this study were not identical to those in creeping bentgrass and switchgrass, which are monocotyledons in the Poaceae family and are inherently anatomically different from the plant materials used in the present study [34]. Despite these morphological differences, the physiological and biochemical parameters also exhibited improved salt tolerance in the Mtr-miR319a-overexpressing A. thaliana and M. truncatula plants in our study, indicating that the regulatory role of Mtr-miR319a could also be associated with salt tolerance but is not limited to impacting leaf development and morphology [34].
We noticed a delayed flowering time and the suppression of leaf senescence in the Mtr-miR319a-overexpressed A. thaliana and M. truncatula plants when exposed to salt stress. This phenotype was also reported in miR319-overexpressed creeping bentgrass [26] and tomato plants [31]. In this study, the delayed flowering and leaf senescence phenotypes under salt stress could indicate a role of Mtr-miR319a in flowering time and leaf senescence regulation [35], which also contribute to the salt tolerance performance caused by Mtr-miR319a, as manifested in A. thaliana [36,37].
Furthermore, the expression of LOX9 was found to be repressed in Mtr-miR319aoverexpressing M. truncatula plants under normal conditions, whereas the expression of LOX9 in Mt-OE-22 and LOX13 in the two overexpression lines were identified to be significantly higher under salt stress compared to the WT plants. LOX, which encodes the key enzymes of JA biosynthesis and catalyzes the reaction of α-linoleic acid to hydroperoxyoctadecadienoic acid, was induced in MIR319b overexpression rice plants upon blast disease exposure [38]. For the M. truncatula plants coping with salt stress in our study, the expression levels of LOX9 in Mt-OE-22 and LOX13 in the overexpression plants were also higher than that of the WT plant. This induction could be explained by the role of Mtr-miR319a targeting genes used in salt stress defense in M. truncatula plants [38]. In addition, a previous study [39] demonstrated that TCP4 adjusts leaf senescence via binding to the LOX2 promoter and commanding the transcriptional level of LOX2. In this study, the roles of improving salt tolerance and regulating leaf senescence by interacting with TCP4 in LOX9 and LOX13 warrant further clarification.
Moreover, GaMYBs, gibberellin and abscisic acid-regulated MYBs, are reported to be targeted by miR319 and can improve chilling [40] and heat [41] stress tolerance in tomato. The expression of the predicted Mtr-miR319a target gene, MYB1, was found to be suppressed in the Mtr-miR319a-overexpressed M. truncatula plant (Mt-OE-32) under normal conditions compared to the WT plants. The expression of OsGAmyb was significantly lower in the miR319a overexpression plants [42], which is in accordance with our determined expression changes of MYB1, indicating that MYB1 could also be a target gene of Mtr-miR319a in the defense against salt stress in M. truncatula plants.

Salt Treatments and Sampling
For seedling growth under salt stress tests, four-week-old WT and Mtr-miR319aoverexpressing A. thaliana seedlings were irrigated with 20 mL of 200 mM NaCl solution every 3 d for 24 d, and the leaves were sampled at 24 d for the physiological measurements; the four-week-old WT and Mtr-miR319a-overexpressed M. truncatula plants were exposed to 20 mL of 200 mM NaCl solution every 3 d for 35 d, and the leaves were sampled at 35 d for the physiological measurements. For the transcriptional analysis, the hydroponic solution was supplemented with 150 mM NaCl. Twenty-eight-day-old M. truncatula plants were treated with 150 mM NaCl, and leaf samples were collected at 0, 2, 12, and 24 h, respectively; rinsed with deionized water; frozen in liquid nitrogen immediately; and kept at −80 • C until nucleic acid isolation. Each time point was duplicated three times, and each replicate pooled three individual plants.

RNA/DNA Isolation, cDNA Synthesis, and 5 RLM-RACE
The total RNA was isolated from the M. truncatula leaves using Trizol solution (Invitrogen, Carlsbad, CA, USA). The total RNA used for the reverse transcription was first treated with DNase I enzyme (MBI Fermentas, Hanover, MD, USA). The PrimeScript Reverse Transcriptase Kit (Takara, Japan) was used for the cDNA synthesis reaction, referring to the manufacturer's instructions. The genomic DNA extraction was carried out using a DNA isolation kit (Kangweishiji, Beijing, China) according to the manufacturer's instructions. The harvested complementary cDNA and DNA samples were well stored at −20 • C in a freezer. The cleavage sites of the miRNA targets in M. truncatula were conducted with 5 RNA ligase-mediated rapid amplification of the cDNA ends (5 RLM-RACE) [43] using the SMARTer ® RACE 5 /3 Kit (Takara, Kyoto, Japan). The synthesized cDNA was taken as a template for the PCR with 5 primer and gene-specific primers. The gene-specific primers (GSPs) were validated at the 5 end of the RNA adaptor, and the amplified universal primers complementary to the adaptor were designed, as listed in Table S1. The procedures for the RNA adaptation, reverse transcription, and amplification followed the recommended instructions (Kangweishiji, Beijing, China). The product was purified and transformed into the EASY-T5 vector (TransGen, Beijing, China) for sequencing (Zhongchuanhongda, Beijing, China) and was preserved for further usage after the sequence alignment and verification.

Overexpression Vector Construction and Genetic Transformation
The precursor sequences of Mtr-miR319a were searched using the miRbase database (http://www.mirbase.org/, accessed on 22 January 2021). The cloning primers, Mtr-miR319a-F/R, were designed using Pimer6.0 software according to the sequences of the plasmid and precursor, as displayed in Table S1. The amplification products were detected on 1% agar gel stained with gold view (Transgen, Beijing, China) and then purified with a DNA gel extraction kit (Transgen, Beijing, China). The purified fragment was concatenated into the pCAMBIA3301 plasmid that was previously digested with the NcoI restriction enzyme. The pCAMBIA3301-Mtr-miR319a recombinant plasmid was preserved after sequencing (Qingkexinye, Beijing, China). MtTCP4 was cloned by GXL DNA Polymerase (TaKaRa, Kusatsu, Shiga, Japan), ligated into the PCR-TOPO vector (Invitrogen, Waltham, MA, USA), and converted into competent E. coli. After sequencing and vector extraction, TOPO-MtTCP4 was obtained. The recombination of TOPO-MtTCP4 and the pMDC83 vector was performed by Gateway ® LR Clonase ® II (Invitrogen, Waltham, MA, USA) following the operational steps. The pMDC83-TCP4 recombinant plasmid was finally obtained and well preserved after sequencing (Qingkexinye, Beijing, China).
The pCAMBIA3301-Mtr-miR319a plasmid was transformed into A. thaliana following the Agrobacterium (GV3101)-mediated floral-dipping method [44]. The harvest-transformed A. thaliana seeds were further spread on 1/2 MS medium with glufosinate (PPT) (4 mg/L) application and verified by PCR and qRT-PCR assays for the positive lines. The pCAMBIA3301-Mtr-miR319a vector was transformed into M. truncatula using a unified Agrobacteriummediated genetic transformation protocol [45]. The callus was induced and cultured on SH3a medium with PPT (3 mg/L) and cephalosporin (CEP) (450 mg/L) for de-the differentiation, MSBK with PPT (3 mg/L) and CEP (450 mg/L) for the embryo induction, and SH9a with PPT (1 mg/L) and CEP (200 mg/L) for the shoot and root induction. The generated M. truncatula seedlings were also positively selected using PCR and qRT-PCR assays. The pMDC83-TCP4 vector was transiently transformed into the tobacco leaves mediated by Agrobacterium GV3101 [46], and the proteins of the GFP fusion and control were examined by applying a confocal scanning microscope system (Leica TCS SP8, Germany).

Physiological Measurements for Salt Tolerance Evaluation
The physiological parameters, such as relative water content [47], malondialdehyde (MDA) content [48], and relative proline content [49], of A. thaliana and M. truncatula plants after the treatments were determined. The samples for the relative water content and relative proline content measurements were taken from 3 independent biological duplicates for each sampling, while four were used for the MDA content.

Western Blot Analysis for the Mtr-miR319a Target Protein
The total protein was separated from the transformed tobacco leaves through the adoption of the isolation buffer, and the protein concentration was measured using the blood alcohol concentration method. The protein samples were boiled for 12 min after mixing with the buffer. The lysates were split by SDS-PAGE and checked by immunoblotting against the rabbit anti-GFP antibody (Abcam, ab290, Cambridge, MA, USA) for GFP-MtTCP4. As a loading control, actin was detected with the rabbit anti-Actin antibody (Abcam, ab197345, Cambridge, MA, USA). The HRP-conjugated goat anti-rabbit secondary antibody (Beyotime, A0216, Beijing, China) was adopted for anti-GFP or anti-actin immunoblotting.

RT-qPCR Assay of Mtr-miR319a and Its Targets
Relative quantification analyses of Mtr-miR319a and its target genes of MYB1 (MTR_3g011610), MYB2 (MTR_8g042410), CUC2 (MTR_2g078700), TCP3 (MTR_2g078200), TCP4 (MTR_8g463380), TCP10 (MTR_2g090960), LOX9 (MTR_8g018690), and LOX13 (MTR_3g479460) were conducted on a real-time fluorescent quantitative ABI 7300 PCR system (Applied Biosystems, Foster City, CA, USA) and estimated referring to the comparative Ct method. The MtU6 snRNA and MtActin2 genes were taken as the house-keeping reference genes of Mtr-miR319a and its target genes, respectively. The qRT-PCR primers were prepared at the NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 15 June 2021) (Table S1). Reactions were conducted using the miCute enhanced miRNA fluorescence quantitative assay kit (Tiangen, Beijing, China) and SYBR Premix Ex TaqTM II (TaKaRa, Kusatsu, Shiga, Japan), and the quantitative tests were replicated three times. The PCR cycling conditions were set on the basis of the manufacturer's guidance, with a melting curve to affirm the product specificity and avert the primer dimers in the end. The relative expressions of Mtr-miR319a and each gene were analyzed referring to the 2 −∆∆Ct formula [50] and presented as the fold change.

Statistics
The data analysis was conducted using EXCEL 2010 (Microsoft Corporation, Redmond, WA, USA), GraphPad Prism 9.0 (GraphPad, San Diego, CA, USA), and IBM SPSS 20.0 (IBM, Armonk, NY, USA). The significant differences (p < 0.05) shown in this study were calculated using statistical method of analysis of variance (ANOVA) tests. The data are presented as the mean ± stand error.

Conclusions
In this study, Mtr-miR319a, a miRNA considered to participate in salt response and tolerance in Medicago species, as per our prior report, was overexpressed in A. thaliana and M. truncatula plants. Our results showed that overexpressing Mtr-miR319a induced leaf curling phenotypes and enhanced salt tolerance in both the A. thaliana and M. truncatula overexpression plants. The expression of Mtr-miR319a was determined to be maintained at a high level before and after salt stress, indicating the definite roles that Mtr-miR319a played in the process of leaf development and salt stress adaptation. Furthermore, the expressions of potential target genes, such as MYBs, CUC, TCPs, and LOXs, were detected, and among them TCP4, TCP10, MYB1, CUC2, LOX9, and LOX13 were found to be regulated by the overexpression of Mtr-miR319a and were suggested to be associated with the phenotypes observed in the M. truncatula plants; TCP4, especially, might be involved in controlling leaf margin formation and salt tolerance. These findings contribute to the molecular theoretical basis of miR319 in legume plants and further provide important knowledge on developmental and stress tolerance traits for crop genetic engineering.