MsNRAMP2 Enhances Tolerance to Iron Excess Stress in Nicotiana tabacum and MsMYB Binds to Its Promoter

Iron(Fe) is a trace metal element necessary for plant growth, but excess iron is harmful to plants. Natural resistance-associated macrophage proteins (NRAMPs) are important for divalent metal transport in plants. In this study, we isolated the MsNRAMP2 (MN_547960) gene from alfalfa, the perennial legume forage. The expression of MsNRAMP2 is specifically induced by iron excess. Overexpression of MsNRAMP2 conferred transgenic tobacco tolerance to iron excess, while it conferred yeast sensitivity to excess iron. Together with the MsNRAMP2 gene, MsMYB (MN_547959) expression is induced by excess iron. Y1H indicated that the MsMYB protein could bind to the “CTGTTG” cis element of the MsNRAMP2 promoter. The results indicated that MsNRAMP2 has a function in iron transport and its expression might be regulated by MsMYB. The excess iron tolerance ability enhancement of MsNRAMP2 may be involved in iron transport, sequestration, or redistribution.


Introduction
Alfalfa (Medicago sativa L.), a perennial herb belonging to the legume family, has been used as a worldwide forage crop because it is rich in nutrients, including protein, dietary fiber, vitamins, and minerals [1]. Alfalfa also has potential economic value in the phytoremediation of soil contamination. Soil contamination with toxic metal elements is an increasing environmental problem, posing serious threats to living organisms. Alfalfa has a large biomass productivity, high tolerance, and strong uptake capacity to potentially toxic elements [2]. In the health risk assessment through determining the bioaccumulation of iron (Fe) in forages grown in soil irrigated with city effluent, the maximum value of daily intake of iron was observed in Medicago sativa [3]. Although alfalfa has been widely adopted in phytoremediation technology for the remediation of contaminated soils, there are relatively few studies investigating the heavy metals alleviation possibilities and the mechanisms of heavy metal uptake and excess metal tolerance in alfalfa [4].
Various micronutrients (including iron, copper, zinc, etc.) are essential in plant growth and development and are additionally essential for the basic metabolic processes of forage crops [5]. As an essential trace element for plants, Fe is a cofactor of more than 300 enzymes and plays a role in important metabolic processes such as photosynthesis, respiration, and amino acid biosynthesis [6]. Iron has a crucial role in photosynthetic organisms which is a required cofactor for the operation of photosynthetic functions [7]. However, iron excess significantly inhibits plant growth and development and results in serious metabolic disorders that affect photosynthesis, respiration, and general plant fitness, which have direct consequences on crop production and indirectly affect human health [8]. Excessive iron in plants catalyzes the generation of radicals by the Fenton reaction (Fe 2+ + H 2 O 2 → Fe 3+ + OH − + OH), which attacks and damages cellular macromolecules causing serious oxidative damage [9]. At the same time, the sensitivity of photosystem II to photoinhibition is enhanced, resulting in a decrease in the photosynthetic rate [10].
Iron is not evenly distributed in soil due to geological and human activities; low content is 1-5 mg/kg and high content can be up to 1000 mg/kg [11]. Anthropogenic input, mainly mining activities, has increased the Fe content in the environment which can influence the metal contents in plants and ultimately delivery into the food chain, thereby risking the health of grazing livestock [12]. Further, the acid state of metal-leak impacted area soils may increase the metal contents in plants including forages [13]. Therefore, not only does the mechanism of plants coping with iron deficiency require research, but how plants respond to excess iron also needs attention.
Transporters are involved in the absorption, transportation, redistribution, and detoxification of iron in plants. Dicotyledonous alfalfa belongs to the iron uptake strategy I, in which Fe 3+ is reduced to the form of Fe 2+ by Ferric reduction oxidase (FRO) [14] and absorbed by the iron-regulated transporter (IRT) [15]. Natural resistance-associated macrophage proteins (NRAMPs) are a class of metal transporters widely present in plants. NRAMP family members have been shown to be involved in iron uptake and transport in various plant species ranging from Chlamydomonas reinhardtii to higher plants [16,17]. NRAMPs have been proven to have the function of transporting iron in Arabidopsis thaliana and rice [18][19][20][21][22][23][24]. Additionally, it has been shown that NRAMPs translocate iron in Arachis hypogaea [25], Solanum lycopersicum [26], Nicotiana tabacum [27,28], Medicago truncatula [29], Hordeum vulgare [30], and Prunus persica [31]. Most NRAMP genes are preferentially expressed in vegetative tissues and reproductive organs and are induced by iron deficiency.
NRAMP proteins are subcellularly localized on the polycellular component including the membrane of plasma, tonoplast, trans-Golgi, and vesicles. In NRAMP proteins, the broad selectivity for the transition-metal ions of Mn 2+ , Fe 2+ , Co 2+ and Ni 2+ is emphasized [32]. The crystal structure revealed that NRAMP2 transported only Fe 2+ in a non-electrogenic and proton-independent way [33]. The substrate gradients, protein structure, and physiological voltage determined the directionality of the substrate movement [34,35]. Many apparently quirky properties of NRAMPs may turn out to be more widespread evolutionarily [36].
As upstream genes regulate the expression of metal transporters, chelators, and related enzymes, transcription factors (TFs) also play an important role in plant iron homeostasis.
The v-myb avian myeloblastosis viral oncogene homolog (MYB) TFs contain tandem repeats of a~50 amino acid DNA-binding motif and are involved in the regulation of many aspects of plant development, hormone signaling, and metabolism. Previous studies have also shown that MYB TFs have an indispensable role in the regulation of iron homeostasis, as well as the basic helix-loop-helix (bHLH) TFs [37]. Expression of MYBs upregulated by Fe starvation suggested that MYBs may play a role in iron nutrition. The orchid R2R3-MYB gene DwMYB2 has been shown to affect iron transport and up-regulated AtFRO2, AtIRT1, and AtIRT2 and down-regulated AtIREG1, AtFRD3 and AtNRAMP1 [38,39]. MYB72 and MYB10 are all involved in the expression regulation of an important iron homeostasis-related protein, nicotinamide synthase4 [40]. Iron deficiency in the rhizosphere activates MYB72 in the absence of shoot-derived signals. In response to volatile organic compounds, MYB72 is co-expressed with the iron uptake-related genes FRO2 and IRT1 [41]. β-Glucosidase42 is an MYB72-dependent key regulator of rhizobacteria-induced systemic resistance and modulates iron deficiency responses in Arabidopsis roots. A total of 195 genes was constitutively upregulated in MYB72-overexpressing roots in the absence of Pseudomonas fluorescens WCS417 bacteria [42]. MdMYB58 was shown to transcriptionally repress MdMATE43 in apple plants and its homolog FRD3 in Arabidopsis by directly binding to their promoter [43]. Transcriptomic analysis indicated that the control of Fe homeostasis was crucial to the response to ammonium nutrition and evidenced that MYB28 and MYB29 play a role in this control [44]. Overexpression of MhR2R3-MYB4 enhanced Arabidopsis thaliana tolerance to Fe deficiency and led to multiple biochemical changes [45]. The osmyb36 mutants showed higher calcium levels and lower Fe and other divalent metal-ion levels in shoots [46]. MYB10 and MYB7 were identified as candidates for effecting IRT1-dependent Fe mobilization in roots [47]. In humans, an HBS1L-MYB intergenic region variant associates both with an increased risk of iron overload and a reduced risk of iron deficiency anemia [48]. These results suggest the function as a regulator of iron uptake and storage of MYB TFs in plants.
Interestingly, MYB TFs can also form a regulatory network with bHLH TFs to regulate iron homeostasis. As the upstream genes regulating bHLH, Arabidopsis MYB28/29 interact to negatively regulate bHLH38/39/100/101 and MYB10/72 [49]. MYB10 and MYB72 are also negatively regulated by PYE(bHLH47) [40]. MYB72 is an intrinsic part of the plant's ironacquisition response in a manner that is dependent on the FER-like iron deficiency induced transcription factor (FIT). Additionally, MdMYB58/43-mediated regulation may be related to the PYE-related Fe deficiency regulatory network via MdSAT1, a member of the IVa subfamily of bHLH TFs [43]. This research highlights the fact that MYB is also an important regulator of iron homeostasis.
In this study, we cloned a NRAMP family gene, MsNRAMP2, from alfalfa. To identify the function of MsNRAMP2, the study on its expression, promoter activity, and heterologous expression were performed. In addition, we cloned a 1R-MYB gene, MsMYB, which is co-regulated with MsNRAMP2 under iron excess. The regulatory relationship of MsMYB on MsNRAMP2 was detected by a yeast one-hybrid assay. We hope the study will provide new insights into the tolerance mechanism to iron excess stress in alfalfa and iron homeostasis regulation in plants.

Phylogenetic Analysis of NRAMP Genes
In order to analyze the phylogenetic relationship of MsNRAMP2, a phylogenetic tree was constructed ( Figure 1a). The results show that MsNRAMP2, AtNRAMP2 and MtRNAMP2 have the highest homology ( Figure S1), and belong to the same clade. The promoters of NRAMP family genes contain similar motifs. Except for the four genes contained in clade IV, all of the NRAMP family genes contain a SLC5-6 LIKE_SBD domain (Figure 1b).

Expression of MsNRAMP2 in Alfalfa Treated with Iron Excess
The RT-qPCR was performed on MsNRAMP2 in alfalfa plants treated with iron excess(+Fe) for 0-7 d. The results showed that the expression of MsNRAMP2 was increased by +Fe stress in alfalfa. The expression levels of MsNRAMP2 were significantly higher in alfalfa under +Fe treatment for 1 d, 3 d, and 7 d than that under non-treatment ( Figure 2a). The results showed that MsNRAMP2 expression was a sustained response to +Fe in 7 days. In order to analyze the expression characteristics of MsNRAMP2, RT-qPCR was performed in different tissues of alfalfa under +Fe treatment, with the non-treatment as the control. Compared with the control, the MsNRAMP2 expression was significantly decreased in the roots and significantly increased in the stems and the leaves in alfalfa under +Fe treatment. These results indicate that the expression of MsNRAMP2 was down-regulated in the roots and up-regulated in the stems and leaves by +Fe stress (Figure 2b).

Expression of MsNRAMP2 in Alfalfa Treated with Iron Excess
The RT-qPCR was performed on MsNRAMP2 in alfalfa plants treated with iron excess(+Fe) for 0-7 d. The results showed that the expression of MsNRAMP2 was increased by +Fe stress in alfalfa. The expression levels of MsNRAMP2 were significantly higher in alfalfa under +Fe treatment for 1 d, 3 d, and 7 d than that under non-treatment ( Figure 2a). The results showed that MsNRAMP2 expression was a sustained response to +Fe in 7 days. In order to analyze the expression characteristics of MsNRAMP2, RT-qPCR was performed in different tissues of alfalfa under +Fe treatment, with the non-treatment as the control. Compared with the control, the MsNRAMP2 expression was significantly decreased in the roots and significantly increased in the stems and the leaves in alfalfa under +Fe treatment. These results indicate that the expression of MsNRAMP2 was down-regulated in the roots and up-regulated in the stems and leaves by +Fe stress (Figure 2b).

Cloning and Characterization of MsNRAMP2
The 1 593 bp CDS sequence of MsNRAMP2 (MN_547960) was cloned by PCR a fication. MsNRAMP2 protein was predicted to contain a special functional dom Nramp (natural resistant-associated macrophage protein) and MtnH (Mn 2+ and Fe 2+ porter of NRAMP family), and non-special functional domain of nramp (NRAMP ion transporter), PRK00701 (divalent metal cation transporter MtnH) and Nra (Nramp family divalent metal transporter). MsNRAMP2 contains SLC5-6-LIKE_SB (b) Relative expression of MsNRAMP2 in different tissues of alfalfa under +Fe stress. Alfalfa seedlings at three leaf stage were treated with Hoagland's solution containing 460 mM iron for 7 d, and the relative expression levels of MsNRAMP2 in roots, stems and leaves were determined. Double asterisk indicated a significant difference from the control at p < 0.01 by Tukey's test with three independent biological replicates.
at three leaf stage were treated with Hoagland's solution containing 460 mM iron for 7 d, and the relative expression levels of MsNRAMP2 in roots, stems and leaves were determined. Double asterisk indicated a significant difference from the control at p < 0.01 by Tukey's test with three independent biological replicates.

Subcellular Localization of MsNRAMP2
In order to detect the subcellular localization of the MsNRAMP2 protein, a 35S::MsNRAMP2-GFP expression vector was constructed and transformed into Nicotiana benthamiana epidermal cells. The transient expression and fluorescence detection showed that the green fluorescence signals were throughout the cells in 35S::GFP transformed Nicotiana benthamiana epidermal cells, and the green fluorescence signal appeared in the nucleus and cytoplasm in 35S::MsNRAMP2-GFP transformed Nicotiana benthamiana epidermal cells. The results indicated that the MsNRAMP2 protein was scattered and distributed in the nucleus, cytoplasm, and plasma membrane ( Figure 4).

Physiological Index in MsNRAMP2 Transformed Tobacco
In order to analyze the function of MsNRAMP2 involved in plant resistance to iron stress in tobacco, the plant expression vector pBI121-35S::MsNRAMP2 was constructed and transformed into Nicotiana tabacum by the leaf disc method mediated by Agrobacterium tumefaciens (Figure 5a). After kanamycin was screened and the PCR identified, MsNRAMP2 transgenic tobacco lines were obtained ( Figure S2). The expression level of MsNRAMP2 in L3, L4 and L10 transgenic lines was significantly higher than that of WT (Figure 5b).
35S::MsNRAMP2-GFP expression vector was constructed and transformed into Nicotiana benthamiana epidermal cells. The transient expression and fluorescence detection showed that the green fluorescence signals were throughout the cells in 35S::GFP transformed Nicotiana benthamiana epidermal cells, and the green fluorescence signal appeared in the nucleus and cytoplasm in 35S::MsNRAMP2-GFP transformed Nicotiana benthamiana epidermal cells. The results indicated that the MsNRAMP2 protein was scattered and distributed in the nucleus, cytoplasm, and plasma membrane (Figure 4).

Physiological Index in MsNRAMP2 Transformed Tobacco
In order to analyze the function of MsNRAMP2 involved in plant resistance to iron stress in tobacco, the plant expression vector pBI121-35S::MsNRAMP2 was constructed and transformed into Nicotiana tabacum by the leaf disc method mediated by Agrobacterium tumefaciens (Figure 5a). After kanamycin was screened and the PCR identified, MsNRAMP2 transgenic tobacco lines were obtained ( Figure S2). The expression level of MsNRAMP2 in L3, L4 and L10 transgenic lines was significantly higher than that of WT ( Figure 5b). The MsNRAMP2 overexpression transgenic tobacco lines (L3, L4, and L10) and wildtype tobacco (WT) were treated with +Fe. After +Fe treatments for 7 d, both L3, L4, L10 and WT tobacco showed leaf yellowing. The yellowing degree is lower in L3, L4, and L10 than that in WT (Figure 5c). The chlorophyll content of L3, L4 and L10 was significantly higher than that of WT under +Fe stress (Figure 5d). These results indicated that the dam- The MsNRAMP2 overexpression transgenic tobacco lines (L3, L4, and L10) and wildtype tobacco (WT) were treated with +Fe. After +Fe treatments for 7 d, both L3, L4, L10 and WT tobacco showed leaf yellowing. The yellowing degree is lower in L3, L4, and L10 than that in WT (Figure 5c). The chlorophyll content of L3, L4 and L10 was significantly higher than that of WT under +Fe stress (Figure 5d). These results indicated that the damage degree of chlorophyll synthesis in MsNRAMP2 overexpression tobacco was lower than that in WT under +Fe stress. The MDA, H 2 O 2 and O 2 contents of L3, L4 and L10 were significantly lower than that of WT (Figure 5e-g). The results indicated that MsNRAMP2 overexpression tobacco sustained less membrane damage and oxidative damage under +Fe stress.

Growth of MsNRAMP2 Transformed Yeast
The yeast expression vector p426GAL1-MsNRAMP2 was constructed and transferred into INVSC1 yeast. The p426GAL1 and P426GAL1-MsNRAMP2 transformed yeast were treated with different Fe concentrations. Under no Fe and 2 mM Fe treatments, there was no significant difference in growth between these two kinds of yeast. Under the treatment of 4 mM, 8 mM, and 12 mM Fe, no difference in growth was observed in the two kinds of yeast at 10~10 4 -fold dilutions, whereas the p426GAL1 transformed yeast grew better than P426GAL1-MsNRAMP2 transformed yeast at a 10 5 -fold dilution (Figure 6a). The sum colony area of p426GAL1 transformed yeast was significantly larger than that of p426GAL1-MsNRAMP2 transformed yeast under the treatment of 4 mM, 8 mM, and 12 mM Fe (Figure 6b). Under 2 mM, 4 mM, and no Fe treatment, there was no difference between the two kinds of yeast in OD 600 values. When the Fe treatment concentration increased to 8 mM and 12 mM, the OD 600 values of p426GAL1 transformed yeast liquid culture was significantly higher than that of p426GAL1-MsNRAMP2 transformed yeast liquid culture (Figure 6c). This result implies that MsNRAMP2 increases the sensitivity of yeast to iron.

Cloning and Characterization of MsNRAMP2 Promoter
The 1351 bp fragment of the MsNRAMP2 promoter was cloned by PCR amplification (Figure 7a). Analysis showed that the MsNRAMP2 promoter fragment has core elements and cis-acting elements of hormone response, as well as multiple Myb-binding sites (Figure 7b). To identify the transcriptional activity of the MsNRAMP2 promoter, the pBI121-pMsNRAMP2::GUS expression vector was constructed and stable transformed into Arabidopsis thaliana (Figure 7c). The GUS staining was not obvious in the roots of Arabidopsis thaliana under +Fe treatment and non-stress conditions. There was darker blue staining in stems and leaves of Arabidopsis thaliana under +Fe treatment than those under non-stress conditions (Figure 7d). These results indicate the activity of the MsNRAMP2 promoter and suggest that the MsNRAMP2 responds to excess iron.

Expression Pattern Analysis and Cloning of MsMYB
MsNRAMP2 promoter contains many MYB binding sites and hormone response elements. We selected a MYB family transcription factor MsMYB, which is co-regulated with MsNRAMP2 in alfalfa by iron stress. The qPCR results showed that the expression of MsMYB and MsNRAMP2 was similarly induced by iron excess (Figure 8a). MsMYB and MsNRAMP2 were also similarly induced by salicylic acid, abscisic acid, and methyl jasmonate. The expression of MsMYB and MsNRAMP2 was most significantly induced by ABA (Figure 8b). The CDS of MsMYB (MN_547959) was cloned by PCR amplification ( Figure S3). The obtained MsMYB gene contained 645 nucleotides, encoding 214 amino acids. The bioinformatics analysis showed that MsMYB protein contains a Myb_DNAbinding domain at the amino acids 24 to 75 and has the typical SHAQKYF domain of CCA1-like MYB, indicating that MsMYB belongs to 1R-MYB (Figure 8c). MsMYB protein had a helical, angular, and helical structure. The tertiary structure of MsMYB was consistent with the structure of MYB transcription factors (Figure 8d).  (Figure 6b). Under 2 mM, 4 mM, and no Fe treatment, there was no difference between the two kinds of yeast in OD600 values. When the Fe treatment concentration increased to 8 mM and 12 mM, the OD600 values of p426GAL1 transformed yeast liquid culture was significantly higher than that of p426GAL1-MsNRAMP2 transformed yeast liquid culture (Figure 6c). This result implies that MsNRAMP2 increases the sensitivity of yeast to iron.  The 1 351 bp fragment of the MsNRAMP2 promoter was cloned by PCR amplification (Figure 7a). Analysis showed that the MsNRAMP2 promoter fragment has core elements and cis-acting elements of hormone response, as well as multiple Myb-binding sites (Figure 7b). To identify the transcriptional activity of the MsNRAMP2 promoter, the pBI121-pMsNRAMP2::GUS expression vector was constructed and stable transformed into Arabidopsis thaliana (Figure 7c). The GUS staining was not obvious in the roots of Arabidopsis thaliana under +Fe treatment and non-stress conditions. There was darker blue staining in stems and leaves of Arabidopsis thaliana under +Fe treatment than those under nonstress conditions (Figure 7d). These results indicate the activity of the MsNRAMP2 promoter and suggest that the MsNRAMP2 responds to excess iron.

Expression Pattern Analysis and Cloning of MsMYB
MsNRAMP2 promoter contains many MYB binding sites and hormone response elements. We selected a MYB family transcription factor MsMYB, which is co-regulated with MsNRAMP2 in alfalfa by iron stress. The qPCR results showed that the expression of MsMYB and MsNRAMP2 was similarly induced by iron excess (Figure 8a). MsMYB had a helical, angular, and helical structure. The tertiary structure of MsMYB was sistent with the structure of MYB transcription factors (Figure 8d).

MsMYB Binds to MsNRAMP2 Promoter
Bioinformatics analysis predicted that the MsNRAMP2 promoter fragment tained three kinds of MYB binding sites, with the sequence TGGTTA, TGGTTG CTGTTG ( Figure 9a). In order to identify the interactions between MsMYB MsNRAMP2, three pHIS2-element yeast expression vectors were constructed. Y1H a was performed on MsMYB protein and three elements TGGTTA, TGGTTG, and CTG in pHIS2 (Figure 9b). The results showed that MsMYB could bind to the CTGTTG ele in the MsNRAMP2 promoter, while it could not bind to the other two elements (F 9c). To further verify, a 280 bp fragment of the MsNRAMP2 promoter containing CTG element (pMsNRAMP2f) was constructed into the yeast expression vector (Figure 9b

MsMYB Binds to MsNRAMP2 Promoter
Bioinformatics analysis predicted that the MsNRAMP2 promoter fragment contained three kinds of MYB binding sites, with the sequence TGGTTA, TGGTTG, and CTGTTG ( Figure 9a). In order to identify the interactions between MsMYB and MsNRAMP2, three pHIS2-element yeast expression vectors were constructed. Y1H assay was performed on MsMYB protein and three elements TGGTTA, TGGTTG, and CTGTTG in pHIS2 (Figure 9b). The results showed that MsMYB could bind to the CTGTTG element in the MsNRAMP2 promoter, while it could not bind to the other two elements (Figure 9c). To further verify, a 280 bp fragment of the MsNRAMP2 promoter containing CTGTTG element (pMsNRAMP2f) was constructed into the yeast expression vector (Figure 9b) and hybridized with MsMYB protein. The results showed that MsMYB could also bind to this 280 bp fragment of the MsNRAMP2 promoter (Figure 9d).

Discussion
Plants depend on various metal transporters and transcription factors to tolerate different iron nutrition environments. Iron(Fe) is an important essential heavy metal, while being toxic to plants when it exceeds [5].
Over recent decades, progress has been achieved in understanding the function and regulation of metal transporters in plants' response to iron deficiency, but little is known about how plants deal with the iron excess condition. Although a large amount of research has been conducted on iron toxicity in rice [50], little research has been undertaken on iron toxicity in other crops. The effects of iron excess and the response to iron excess have been determined in soybean [51], common bean [52], radish [53], sweet potato [54,55], and wheat [56]. However, in-depth mechanism studies are still lacking. Alfalfa (Medicago sativa

Discussion
Plants depend on various metal transporters and transcription factors to tolerate different iron nutrition environments. Iron(Fe) is an important essential heavy metal, while being toxic to plants when it exceeds [5].
Over recent decades, progress has been achieved in understanding the function and regulation of metal transporters in plants' response to iron deficiency, but little is known about how plants deal with the iron excess condition. Although a large amount of research has been conducted on iron toxicity in rice [50], little research has been undertaken on iron toxicity in other crops. The effects of iron excess and the response to iron excess have been determined in soybean [51], common bean [52], radish [53], sweet potato [54,55], and wheat [56]. However, in-depth mechanism studies are still lacking. Alfalfa (Medicago sativa L.) is a significant forage legume cultivated worldwide and utilized in animal production. Alfalfa showed beneficial effects in iron-overload conditions and its vegetable sprouts enrich with Fe [57][58][59], which means there is a unique iron absorption and storage mechanism in alfalfa.
The natural resistance-associated macrophage protein (NRAMP) family members in plants are particularly striking for multiple divalent metal cation transporting, accepting ions including iron, aluminum, and cadmium [23,[60][61][62][63]. NRAMP proteins have been reported in a number of plant species. AtNRAMP3, AtNRAMP4, and OsNRAMP1 have been shown to respond to iron deficiency stress [63,64]. The leguminous species remain largely unknown NRAMP proteins. To date, there are few studies on NRAMP gene expression induced by iron excess. In this study, MsNRAMP2 was first isolated and characterized. The MsNRAMP2 has high homology with AtNRAMP2 in Arabidopsis thaliana and MtNRAMP2 in Medicago truncatula. AtNRAMP2 is a trans-Golgi network-localized manganese transporter that is required for Arabidopsis root growth under manganese deficiency and is critical for photosynthesis and cellular redox homeostasis [65,66]. Although manganese and iron have similar properties and often share transporters, AtNRAMP2's involvement in iron deficiency or iron excess tolerance has not been reported.
The RT-qPCR and GUS assay confirmed that the expression of MsNRAMP2 was induced by iron excess and followed the treatment time (Figures 2a and 7d). Most NRAMPs are expressed in the roots and strongly induced by iron deficiency, considered to be transporters involved in iron uptake, such as AhNRAMP1, MtNRAMP1, NtNRAMP1, and NtNRAMP3 [26,[28][29][30]. Interestingly, MsNRAMP2 was upregulated in stems and leaves and downregulated in roots under iron excess (Figure 2b). The expression of TpNRAMP3 is upregulated in leaves, while the expression of TpNRAMP5 is downregulated in roots under iron excess stress [67,68]. The different expression patterns suggest that some potential differences probably exist in MsNRAMP2 and its homologs, which may depend on the plant species. Thus, we hypothesize that MsNRAMP2 may also play a role in the maintenance of iron homeostasis under iron excess in plants.
The NRAMP proteins present a broad spectrum of intracellular localizations and have a variety of potential functions. For example, AtNRAMP3/4 and GmNRAMPs are localized in the vacuolar membrane [22,69]. AtNRAMP6 is localized in the plasma membrane and intima, including the endoplasmic reticulum [70]. OsNRAMP2 and ZmNRAMP2 are tonoplast-localized transporters [71,72]. In this study, The MsNRAMP2 protein is located in the whole cell, including the nucleus and cytoplasm but not in the chloroplast (Figure 4). The results suggested that MsNRAMP2 may have a wider range of functions. Further experiments are needed to exactly determine the subcellular location of MsNRAMP2 in our subsequent studies.
In order to investigate the role of MsNRAMP2 in response to iron excess stress, we constructed MsNRAMP2 overexpressing tobacco lines. Usually, excessive iron in plants will cause an oxidative effect and lead to a decrease in chlorophyll content in plants [73,74]. Similar to NtNRAMP1, the higher chlorophyll content represented the fact that Fe excess did not damage photosynthesis in MsNRAMP2 overexpressing tobacco lines. The lower MDA of O 2 and H 2 O 2 content directly represent the fact that fewer reactive oxygen species accumulated in MsNRAMP2 overexpressing tobacco lines [75]. Different from our results, the contents of MDA and H 2 O 2 increased in tobacco overexpressed with NtNRAMP1 [28]. The results indicated that MsNRAMP2 enhanced tobacco tolerance to excess iron. It is suggested that MsNRAMP2 may participate in iron redistribution and contribute to tolerance to excess iron. In order to further verify the iron tolerance function of MsNRAMP2, we overexpressed MsNRAMP2 in yeast. Yeast that overexpressed MsNRAMP2 showed greater sensitivity to iron supply (Figure 6), suggesting that MsNRAMP2 allows more iron to enter yeast cells. The results are similar to ZmNRAMP4's enhanced plant tolerance to aluminum and yeast sensitivity to aluminum [76]. In brief, MsNRAMP2 could enhance the ability of iron tolerance and may be due to the iron transport function and/or the regulation of iron redistribution in cells.
NRAMP is the major iron transporter in cells and is known to be regulated at the transcriptional level to intracellular iron concentrations. It revealed that the transcription factor INO plays an important role in reducing Fe loading into early-developing seeds via the repression of NRAMP1 expression [77]. The MsNRAMP2 promoter contains many hormone response elements and MYB binding sites (Figure 7b). We cloned a transcription factor MsMYB of the MYB family co-regulated with MsNRAMP2 by iron excess (Figure 8a,b). Y1H results showed that MsMYB could bind to the MsNRAMP2 promoter (Figure 9c,d). This result suggests that MsNRAMP2 expression may be regulated by MsMYB. Current studies have shown that MYB transcription factors could participate in the regulation of iron homeostasis in plants by regulating the expression of transporters [39]. MsNRAMP2 expression was significantly responsive to MeJA and SA (Figure 8b). MeJA and SA play an important role in plant resistance to abiotic stress [2]. Studies have shown that MeJA can affect the absorption of Zn, Mn, and Fe in Arabidopsis thaliana [78]. ABA content in plants can be induced by iron excess and ABA signal transduction pathways can also affect the expression and function of key genes in the regulatory network of iron homeostasis [79][80][81]. It is confusing that none of the known ABA-responsive element occurs in the MsNRAMP2 promoter but MsNRAMP2 and MsMYB both respond to ABA treatment (Figure 8b). This further provided indirect evidence that MsNRAMP2 expression is regulated by MsMYB ( Figure 10). NRAMP is the major iron transporter in cells and is known to be regulated at the transcriptional level to intracellular iron concentrations. It revealed that the transcription factor INO plays an important role in reducing Fe loading into early-developing seeds via the repression of NRAMP1 expression [77]. The MsNRAMP2 promoter contains many hormone response elements and MYB binding sites ( Figure 7B). We cloned a transcription factor MsMYB of the MYB family co-regulated with MsNRAMP2 by iron excess ( Figure  8A,B). Y1H results showed that MsMYB could bind to the MsNRAMP2 promoter ( Figure  9C,D). This result suggests that MsNRAMP2 expression may be regulated by MsMYB. Current studies have shown that MYB transcription factors could participate in the regulation of iron homeostasis in plants by regulating the expression of transporters [39]. MsNRAMP2 expression was significantly responsive to MeJA and SA ( Figure 8B). MeJA and SA play an important role in plant resistance to abiotic stress [2]. Studies have shown that MeJA can affect the absorption of Zn, Mn, and Fe in Arabidopsis thaliana [78]. ABA content in plants can be induced by iron excess and ABA signal transduction pathways can also affect the expression and function of key genes in the regulatory network of iron homeostasis [79][80][81]. It is confusing that none of the known ABA-responsive element occurs in the MsNRAMP2 promoter but MsNRAMP2 and MsMYB both respond to ABA treatment ( Figure 8B). This further provided indirect evidence that MsNRAMP2 expression is regulated by MsMYB ( Figure 10). NRAMP proteins are widely involved in the resistance to iron deficiency stress, but few studies have been conducted on the resistance to iron excess stress. In this study, the function of MsNRAMP2 under iron excess was analyzed and the regulation of its expression by MsMYB was revealed. It provides new insight into the tolerance to excess iron stress in alfalfa. At the same time, MsMYB is involved in the regulation of iron homeostasis, and the regulation function of the MYB transcription factor on iron transporters can also be the focus of future research. These findings will contribute to understanding the function of the NRAMP and MYB genes of iron homeostasis in plants. NRAMP proteins are widely involved in the resistance to iron deficiency stress, but few studies have been conducted on the resistance to iron excess stress. In this study, the function of MsNRAMP2 under iron excess was analyzed and the regulation of its expression by MsMYB was revealed. It provides new insight into the tolerance to excess iron stress in alfalfa. At the same time, MsMYB is involved in the regulation of iron homeostasis, and the regulation function of the MYB transcription factor on iron transporters can also be the focus of future research. These findings will contribute to understanding the function of the NRAMP and MYB genes of iron homeostasis in plants.

Plant Materials and Growth Conditions
Alfalfa (Medicago sativa L. cv. Zhaodong) seeds were donated by the Animal Husbandry Institute of Heilongjiang Academy of Agricultural Sciences. The alfalfa seeds were germinated and then hydroponic cultured in 1/2 Hoagland's solution, which was replaced every 3 d. These alfalfa were used for stress treatment and DNA and RNA extraction. Tobacco (Nicotiana tabacum L. cv. K326) was cultivated in soil and used for genetic transformation and physiological index detection. Nicotiana benthamiana was cultivated in soil and used for subcellular localization. Arabidopsis (Arabidopsis thaliana L. Col-0) was cultured in soil and used for genetic transformation and GUS histochemical staining. All plants were cultured in a 16/8 h (light/ dark, 22/20 • C) greenhouse.

Expression Analysis of MsNRAMP2 and MsMYB
For iron excess (+Fe) treatment, the hydroponic trefoil stage alfalfa seedlings were treated with 1/2 Hoagland's solution containing 460 mM Fe-EDTA for 0 d, 1 d, 3 d, and 7 d. For phytohormone treatment, the hydroponic trefoil stage alfalfa seedlings were treated with 0.25 mM salicylic acid (SA), 0.1 mM abscisic acid (ABA), or 0.2 mM methyl jasmonate (MeJA) for 24 h, separately, with non-phytohormone treatment as the control. The RNA isolated from samples of whole plants, roots, stems, and leaves was used as the RT-qPCR template. The RT-qPCR was performed using SYBR ® Premix Ex Taq ™ (Tli RNaseH Plus) (TaKaRa Co., Ltd., Tokyo, Japan). The primers used are listed in Supplementary Table S1.

Cloning of MsNRAMP2, MsMYB and MsNRAMP2 Promoter
The 0.1 g of plant tissue was ground in liquid nitrogen. The total RNA was extracted using E.Z.N.A. ® Total RNA Kit (OMGAE Co., Ltd., Wood Dale, IL, USA) and reversed into cDNA by ReverTra Ace™ qPCR RT Master MIX (TOYOBO Co., Ltd., Osaka, Japan). The total DNA was extracted using E.Z.N.A. ® SP Plant DNA Kit (OMGAE Co., Ltd., Wood Dale, IL, USA).
The cloning primers are shown in Supplementary Table S1. The CDS sequences of MsNRAMP2 and MsMYB were obtained by PCR amplification using the alfalfa cDNA template under iron excess stress. The MsNRAMP2 promoter fragment was obtained by PCR amplification using alfalfa total DNA as the template. The PCR product was purified and cloned into a pMD18-T vector and sequenced (Sangon Biotech Co., Ltd., Shanghai, China).

Subcellular Localization of MsNRAMP2
The pBI121-MsNRAMP2-GFP recombinant vector was constructed using Xba I and Sma I by an enzyme digestion ligand. The MsNRAMP2 fragment without a stop codon was connected to the GFP and was expressed by the CaMV 35S promoter. The 35S::MsNRAMP2-GFP fusion expression vector and the control 35S::GFP vector were transformed into Agrobacterium tumefaciens GV3101. After PCR identification, the bacterial solution was injected into the 6-8 leaf stage Nicotiana benthamiana leaves. After being cultured for 72 h, the fluorescence of GFP was observed using a Leica TCS SP8 confocal microscope by laser excitation with a wavelength of 488 nm.

Detection of Physiological Indexes in MsNRAMP2 Overexpressing Tobacco
The pBI121-MsNRAMP2 recombinant vector was constructed. The expression of MsNRAMP2 was driven by the CaMV 35S promoter. The pBI121-MsNRAMP2 expression vector was transformed into Agrobacterium tumefaciens GV3101 and the leaf-disc transformation method was used to infect tobacco. The transformed tobacco seedlings were screened with 100 mg/L kanamycin, and identified by PCR and RT-qPCR. The primers were shown in Supplementary Table S1. The MsNRAMP2 transgenic tobacco was sub-generated to T 3 . Two-month-old MsNRAMP2 overexpress tobacco and wild-type tobacco were treated with 460 mM Fe-EDTA for 7 d, renewed every 2 d. The chlorophyll content was determined by the acetone method. The MDA content, H 2 O 2 content and O 2 content were determined using the methods of spectrophotometry.

GUS Staining
The pCAMBIA1301-proMsNRAMP2::GUS recombinant vector was constructed and then transformed into Agrobacterium tumefaciens GV3101 to infect the plant, with the pCAMBIA1301 vector as the control. Arabidopsis was infected by the dipping method. One-week-old proMsNRAMP2::GUS Arabidopsis seedlings were cultured with 460 mM Fe-EDTA for 7d. GUS staining was performed using a GUS stain Kit (Coolaber Co., Ltd., Beijing, China).

Growth Detection of MsNRAMP2 in Yeast
The p426GAL1-MsNRAMP2 recombinant vector was constructed using Sac I and Kpn I by enzyme digestion ligand. The p426GAL1-MsNRAMP2 fusion expression vector and p426GAL1 empty vector were transformed into yeast (Saccharomyces cerevisiae INVSC1). The single colony was inoculated in SC-U liquid medium and incubated at 30 • C for 24 h. After culture centrifugation, 10 mL SC-U (+glucose) induction medium was used to suspend the yeast solution to OD 600 = 0.4 density, and cultured at 30 • C for 36 h. The yeast liquid culture was collected at OD 600 = 1.3. The yeast liquid culture was diluted 10-, 100-, 1000-, 10,000-, and 100,000-fold successively, and the yeast then diluted onto the SC-U+ glucose solid medium (0 mM, 2 mM, 4 mM, 8 mM, and 12 mM Fe). The 3 µL yeast liquid culture was used for each individual inoculation. After incubation at 30 • C for 72 h, the growth state of the colony was observed and the colony area was measured using Image J. Undiluted yeast liquid culture (OD 600 = 1.3) was inoculated respectively into 4 mL SC-U+ glucose liquid medium containing 0 mM, 2 mM, 4 mM, 8 mM, and 12 mM Fe. After being incubated at 30 • C for 36 h, the OD 600 value of the yeast liquid culture was determined.

Yeast One Hybrid
For Y1H, yeast expression vector pGADT7-Rec2-MsMYB, three kinds of pHIS2-elements (TGGTTA, TGGTTG, and CTGTTG) and pHIS2-pMsNRAMP2f were constructed by enzyme digestion and ligand method. The elements (TGGTTA, TGGTTG, and CTGTTG) were synthesized (Sangon Biotech Co., Ltd., Shanghai, China) and connected to an empty pHIS2 vector. pMsNRAMP2f was intercepted from 0 to 280 bp of the MsNRAMP2 promoter 3 to 5 . pGADT7-Rec2-p53 and pHIS2-p53 were co-transferred into yeast Y187 as a positive control. pGADT7-Rec2 was co-transferred separately with three pHIS2-elements and pHIS2-pMsNRAMP2f into yeast Y187 as a negative control. pGADT7-Rec2-MsMYB was cotransferred separately to yeast Y187 with three pHIS2-elements and pHIS2-pMsNRAMP2f, respectively, to verify the binding of elements and the MsNRAMP2 promoter to MsMYB. After SD/-Trp/-Leu screening and PCR identification, the positive clones were diluted to the same concentration. The yeast liquid culture was diluted 10-, 100-, and 1000-fold and inoculated on the surface of the SD/-Trp/-His/-Leu and SD/-Trp/-His/-Leu/3-AT medium. After it had been cultured at 30 • C for 72 h, we observed the growth state. When the 3-AT concentration is sufficient to inhibit the growth of the negative control and the experimental group yeast can still grow, it is considered that the experimental group fragment could interact with the MsMYB protein.

Statistical Analysis and Reproducibility
All treatments were repeated at least three times. Statistical analysis, including a Student's t-test, was performed using the SPSS 20 software (IBM Co., Ltd., Almonk, NY, USA).