MsYSL6, A Metal Transporter Gene of Alfalfa, Increases Iron Accumulation and Benefits Cadmium Resistance

Iron (Fe) is necessary for plant growth and development. The mechanism of uptake and translocation in Cadmium (Cd) is similar to iron, which shares iron transporters. Yellow stripe-like transporter (YSL) plays a pivotal role in transporting iron and other metal ions in plants. In this study, MsYSL6 and its promoter were cloned from leguminous forage alfalfa. The transient expression of MsYSL6-GFP indicated that MsYSL6 was localized to the plasma membrane and cytoplasm. The expression of MsYSL6 was induced in alfalfa by iron deficiency and Cd stress, which was further proved by GUS activity driven by the MsYSL6 promoter. To further identify the function of MsYSL6, it was heterologously overexpressed in tobacco. MsYSL6-overexpressed tobacco showed better growth and less oxidative damage than WT under Cd stress. MsYSL6 overexpression elevated Fe and Cd contents and induced a relatively high Fe translocation rate in tobacco under Cd stress. The results suggest that MsYSL6 might have a dual function in the absorption of Fe and Cd, playing a role in the competitive absorption between Fe and Cd. MsYSL6 might be a regulatory factor in plants to counter Cd stress. This study provides a novel gene for application in heavy metal enrichment or phytoremediation and new insights into plant tolerance to toxic metals.


Introduction
Iron (Fe) is an essential micronutrient playing an active role in plant photosynthesis, antioxidant defense systems, electron respiration, and embryogenesis.Under iron deficiency, plants show typical chlorosis symptoms and hampered crop yield and productivity [1].Plants develop efficient absorption strategies to support the requirement of sufficient iron.Iron abundance in plants is tightly regulated by iron uptake, translocation, and recycling.In addition to playing an important role in the uptake process, iron transporters are responsible for the transport and distribution of iron in plants [2].Cadmium (Cd) is one of the most phytotoxic elements that negatively affects plant metabolism, growth, and development and indirectly contributes to ROS formation by altering the antioxidant system in plants, reducing crop failure [3][4][5][6].Since there is no Cd-specific transporter in plants, Cd is supposed to be transported in plants via cation transport systems.Being closely similar to the essential metal element Fe, Cd is often taken up by Fe transporters.For example, NRAMPs in Arabidopsis thaliana, Oryza sativa [7][8][9], and Sedum alfredii [10], IRTs in Oryza sativa [11], and OPT and ZIP in Arabidopsis thaliana [12,13] transport both Fe and Cd.Cd distribution throughout a plant is an intricate process controlled by root uptake, root-to-shoot translocation through the xylem, and the redistribution of Cd from the leaves to sink tissues [4,14].The contribution of cation transporters to the management of stress is beneficial in plant improvement programs [15].Metal transporter genes can be potentially used for engineering genotypes for phytoremediation or minimizing Cd in crops.The overexpression of AtHMA3 [16], OsNramp1 [8], NtPIC1 [17], and Miscanthus sacchariflorus MsYSL1 [18,19] enhances plant tolerance to Cd.The sodium/calcium exchanger-like gene TaNCL2-A plays a role in alleviating the toxic effects of Cd in conjunction with salinity and osmotic stress in Arabidopsis [20].
Cd competes with essential macro-and microelements at their absorption sites, disrupting the homeostasis of crucial microelements [6,21].It has been postulated that Cd toxicity can be attributed, at least in part, to the perturbation of the metabolism of Fe [22].The chlorosis of plant leaves under Cd stress is similar to the typical symptoms of iron deficiency [23].Cd induces moderate-to-strong iron deficiency in leaves, which strongly affects photosynthesis [24].Previous studies revealed that Cd significantly decreased the Fe content in shoots, but increased the Fe content in roots [22,25].Fe significantly accumulated in root apoplasts and root cell walls under Cd stress [10]; Fe played a significant role in alleviating the damage caused by Cd toxicity through reducing Cd accumulation and increasing Cd detention, photosynthesis protection, antioxidant defense capacity enhancement, electrolyte leakage amelioration, and other ion status regulation [26][27][28][29].Limiting Fe uptake through the downregulation of Fe acquisition mechanisms confers a Si-mediated alleviation of Cd toxicity [30].The Fe regulation transcript factors (FITs) AtbHLH38, AtbHLH39, bHLH104, and BTS are involved in Cd tolerance [22,31,32].Although the Cd-induced increase in root Fe contents has been described, and several possible explanations for such observations including impaired Fe uptake and translocation have been proposed, the underlying mechanism of Cd-induced changes in Fe homeostasis within plants is still not fully understood.
The yellow stripe-like transporter (YSL) family has attracted attention in recent years for its function in iron uptake from soil or iron translocation throughout whole plants [19].During the last two decades, YSLs have been revealed to transport iron complexes with specific Fe chelators and phytosiderophores.YSLs participate in Fe homeostasis in graminaceous and nongraminaceous plants with distinguished functions in each plant type.In graminaceous plants, most of the YSLs take up the Fe 3+ -mugineic acid (MA) complex [33,34].Some YSLs transport Fe 2+ -Niacinamide (NA) or Fe 3+ -2 -deoxymugineic acid (DMA) over long distances and enable the distribution of Fe within the plant leaves, phloem, and reproductive organs [35][36][37].In nongraminaceous plants, YSLs transport the Fe-NA complex over long distances and intracellularly distribute Fe in plants [38][39][40].Beyond the essential functions in Fe transport, evidence supports the notion that YSLs are mediated in the processes of transporting Cu, Mn, Ni, and Cd in plants [19,[41][42][43].YSL1 and YSL3 in Arabidopsis thaliana [40], SnYSL3 in Solanum nigrum [44], BjYSL7 in Brassica junce [45], YSL3 in peanut [46], MsYSL1 in Miscanthus sacchariflorus [18], and ZmYS1 in maize [47] were reported to be involved in the uptake, distribution, and translocation of Cd.
Leguminous plants can grow in poor and degraded soils.Meanwhile, perennial grasses occupy diverse soils worldwide, including many sites contaminated with heavy metals [26], whereas studies on Fe and Cd interaction have minimal information reported in legumes [48].Alfalfa (Medicago sativa L.) is an essential forage of biological nitrogen fixation, biofuel, and animal feed.Investigators have supported the idea that alfalfa reaches a new steady state to acclimate under chronic Cd stress by adequately adjusting its metabolic composition [30,49].An increased abundance of xylogalacturonan of Medicago sativa after long-term exposure to Cd might hinder Cd binding in the cell wall and is an important factor during tolerance acquisition [50].At the same time, reducing Cd bioaccumulation and controlling Cd distribution in alfalfa deserve more attention.The overlap of Fe and Cd transport in previous reports reflects the importance of the transporter's function.The overexpression or elimination of transporter reduces Cd uptake in plants [51].
Moreover, several YSL genes have been found to enhance Cd resistance ability in transgenic plants [18,45].It is beneficial to explore the potential of YSL in metal transport for genetic engineering.Several YSL genes can assist plants in participating in the process of Cd uptake, transport, and accumulation [52].There are also some YSLs that can help the zinc-iron biofortification of wheat [53].It was found that the rice YSL family is closely related to the evolution and function of other plants [54].Whether YSL is involved in Cd uptake and transport has not been investigated in alfalfa yet.Therefore, we isolated iron transport genes MsYSL6 from alfalfa, focusing on the Fe and Cd contents and the Cd-tolerant ability in MsYSL6 transgenic plants.This research will provide new insight into plant metal transporters in heavy metal enrichment or phytoremediation.

MsYSL6 Belongs to the YSL Transporter Family
The open reading frame (ORF) of MsYSL6 (LOC_MG673951.1) was cloned from alfalfa by PCR (Supplementary Figure S1A).The full length of MsYSL6 ORF contains 2 028 bp, encoding 675 amino acids.MsYSL6 protein harbors 12 transmembrane domains (Supplementary Figure S2).The phylogenetic tree was constructed using data of MsYSL6 and other 23 YSL proteins from eight plant species (Figure 1).MsYSL6 belonged to a branch with AtYSL6, OsYSL6, MtYSL6, GmYSL6, GsYSL6, VaYSL6, CcYSL6, and CaYSL6, indicating a close relationship in these YSLs.These findings indicated that MsYSL6 belonged to the YSL transporter family.Moreover, several YSL genes have been found to enhance Cd resistance ability in transgenic plants [18,45].It is beneficial to explore the potential of YSL in metal transport for genetic engineering.Several YSL genes can assist plants in participating in the process of Cd uptake, transport, and accumulation [52].There are also some YSLs that can help the zinc-iron biofortification of wheat [53].It was found that the rice YSL family is closely related to the evolution and function of other plants [54].Whether YSL is involved in Cd uptake and transport has not been investigated in alfalfa yet.Therefore, we isolated iron transport genes MsYSL6 from alfalfa, focusing on the Fe and Cd contents and the Cdtolerant ability in MsYSL6 transgenic plants.This research will provide new insight into plant metal transporters in heavy metal enrichment or phytoremediation.

MsYSL6 Belongs to the YSL Transporter Family
The open reading frame (ORF) of MsYSL6 (LOC_MG673951.1) was cloned from alfalfa by PCR (Supplementary Figure S1A).The full length of MsYSL6 ORF contains 2 028 bp, encoding 675 amino acids.MsYSL6 protein harbors 12 transmembrane domains (Supplementary Figure S2).The phylogenetic tree was constructed using data of MsYSL6 and other 23 YSL proteins from eight plant species (Figure 1).MsYSL6 belonged to a branch with AtYSL6, OsYSL6, MtYSL6, GmYSL6, GsYSL6, VaYSL6, CcYSL6, and CaYSL6, indicating a close relationship in these YSLs.These findings indicated that MsYSL6 belonged to the YSL transporter family.

The Subcellular Localization of MsYSL6
The subcellular localization of MsYSL6 was investigated using a fusion protein with GFP (Figure 2).The pBWA(V)HS-MsYSL6-Glosgfp was transformed into tobacco leaf cells.Apparent green fluorescence signals of MsYSL6-GFP were observed in the cell membrane and protoplast.The tobacco leaf cells expressing GFP alone exhibited weak and dispersive fluorescence inside the cells (Figure 3).Overlay images were created and showed that MsYSL6-GFP-mediated fluorescence does not co-localize with the chlorophyll-mediated fluorescence.

The Subcellular Localization of MsYSL6
The subcellular localization of MsYSL6 was investigated using a fusion protein with GFP (Figure 2).The pBWA(V)HS-MsYSL6-Glosgfp was transformed into tobacco leaf cells.Apparent green fluorescence signals of MsYSL6-GFP were observed in the cell membrane and protoplast.The tobacco leaf cells expressing GFP alone exhibited weak and dispersive fluorescence inside the cells (Figure 3).Overlay images were created and showed that MsYSL6-GFP-mediated fluorescence does not co-localize with the chlorophyll-mediated fluorescence.

The Subcellular Localization of MsYSL6
The subcellular localization of MsYSL6 was investigated using a fusion protein with GFP (Figure 2).The pBWA(V)HS-MsYSL6-Glosgfp was transformed into tobacco leaf cells.Apparent green fluorescence signals of MsYSL6-GFP were observed in the cell membrane and protoplast.The tobacco leaf cells expressing GFP alone exhibited weak and dispersive fluorescence inside the cells (Figure 3).Overlay images were created and showed that MsYSL6-GFP-mediated fluorescence does not co-localize with the chlorophyll-mediated fluorescence.

The Expression of MsYSL6 in Alfalfa
To gather insight into the expression of the MsYSL6 gene in alfalfa, the transcript profiles of MsYSL6 were monitored in alfalfa under iron deficiency or Cd treatment, with nonstress treatment as the control.MsYSL6 showed tissue-specific expression in alfalfa in

The Expression of MsYSL6 in Alfalfa
To gather insight into the expression of the MsYSL6 gene in alfalfa, the transcript profiles of MsYSL6 were monitored in alfalfa under iron deficiency or Cd treatment, with nonstress treatment as the control.MsYSL6 showed tissue-specific expression in alfalfa in the control.The expression level was higher in the leaves than that in the roots and the stems.In alfalfa roots, the expression of MsYSL6 was significantly upregulated by iron deficiency and Cd stress at 0.05 and 0.01 levels, respectively.In particular, MsYSL6 expression increased nearly 60 folds in alfalfa roots under Cd treatment.In alfalfa stems, the expression of MsYSL6 was significantly upregulated by iron deficiency and Cd stress at 0.05 level.Although the expression was higher than that in the roots and the stems, the expression of MsYSL6 showed no significant differences in leaves under these three conditions (Figure 4A).deficiency and Cd stress at 0.05 and 0.01 levels, respectively.In particular, MsY expression increased nearly 60 folds in alfalfa roots under Cd treatment.In alfalfa ste the expression of MsYSL6 was significantly upregulated by iron deficiency and Cd st at 0.05 level.Although the expression was higher than that in the roots and the stems, expression of MsYSL6 showed no significant differences in leaves under these th conditions (Figure 4A).

The GUS Activity
In order to determine the specific expression of MsYSL6, we fused the MsY promoter in-frame to β-glucuronidase (GUS) reporter (Supplementary Figures S3 and The pBI121-MsYSL6pro::GUS construct was transformed in alfalfa and induced ha roots (Supplementary Figure S5).The alfalfa with hairy roots were grown in a medi containing 75 µM CdCl2, with no Cd treatment as the control.The alfalfa hairy ro exhibited blue when the medium contained no Cd, and exhibited strong blue when medium contained Cd.The alfalfa cotyledons exhibited no staining when the medi contained no Cd, and exhibited blue when the medium contained Cd (Figure 4B).GUS activity driven by MsYSL6pro was enhanced by Cd indicating that MsY responded to Cd stress, which is consistent with the upregulated expression of MsY detected by qRT-PCR.

The Growth of MsYSL6 Transgenic Tobacco
To screen the effect of MsYSL6 on Cd tolerance, the T3 seeds of three MsY overexpress lines (MsYSL6OE), namely, L5, L8, L9, and WT, were grown in the medi containing 50 µM CdCl2, 75 µM CdCl2, and 100 µM CdCl2, with no Cd as the contro preliminary observation showed that MsYSL6OE germination was similar to WT pla when grown in no Cd medium.The germination of MsYSL6OE and WT decrea following the increase of Cd concentration, and the germination of MsYSL6OE lines w significantly less affected than that of WT (Supplementary Figure S6, Table S1).The plant grown on 75 µM CdCl2 medium exhibited shorter and smaller roots, w MsYSL6OE exhibited a normal root phenotype with minimal stunted growth.MsYSL6OE plants showed significantly higher fresh weight and longer roots than the (Figure 5).

The GUS Activity
In order to determine the specific expression of MsYSL6, we fused the MsYSL6 promoter in-frame to β-glucuronidase (GUS) reporter (Supplementary Figures S3 and S4).The pBI121-MsYSL6pro::GUS construct was transformed in alfalfa and induced hairy roots (Supplementary Figure S5).The alfalfa with hairy roots were grown in a medium containing 75 µM CdCl 2 , with no Cd treatment as the control.The alfalfa hairy roots exhibited blue when the medium contained no Cd, and exhibited strong blue when the medium contained Cd.The alfalfa cotyledons exhibited no staining when the medium contained no Cd, and exhibited blue when the medium contained Cd (Figure 4B).The GUS activity driven by MsYSL6pro was enhanced by Cd indicating that MsYSL6 responded to Cd stress, which is consistent with the upregulated expression of MsYSL6 detected by qRT-PCR.

The Growth of MsYSL6 Transgenic Tobacco
To screen the effect of MsYSL6 on Cd tolerance, the T 3 seeds of three MsYSL6 overexpress lines (MsYSL6OE), namely, L5, L8, L9, and WT, were grown in the medium containing 50 µM CdCl 2 , 75 µM CdCl 2 , and 100 µM CdCl 2 , with no Cd as the control.A preliminary observation showed that MsYSL6OE germination was similar to WT plants when grown in no Cd medium.The germination of MsYSL6OE and WT decreased following the increase of Cd concentration, and the germination of MsYSL6OE lines was significantly less affected than that of WT (Supplementary Figure S6, Table S1).The WT plant grown on 75 µM CdCl 2 medium exhibited shorter and smaller roots, while MsYSL6OE exhibited a normal root phenotype with minimal stunted growth.The MsYSL6OE plants showed significantly higher fresh weight and longer roots than the WT (Figure 5).

The Chlorophyll Content
The chlorophyll content decreased in MsYSL6OE and WT plants under Cd treatment more than that of the control.The chlorophyll content of MsYSL6OE leaves was significantly higher than that of WT in the control at 0.05 level.The chlorophyll content of L5 and L8 leaves was significantly higher than that of WT under Cd treatment at 0.05 level.The higher chlorophyll content showed that the MsYSL6OE transgenic tobacco was more tolerant to Cd stress (Figure 6A).

The Chlorophyll Content
The chlorophyll content decreased in MsYSL6OE and WT plants under Cd treatment more than that of the control.The chlorophyll content of MsYSL6OE leaves was significantly higher than that of WT in the control at 0.05 level.The chlorophyll content of L5 and L8 leaves was significantly higher than that of WT under Cd treatment at 0.05 level.The higher chlorophyll content showed that the MsYSL6OE transgenic tobacco was more tolerant to Cd stress (Figure 6A).

The Chlorophyll Content
The chlorophyll content decreased in MsYSL6OE and WT plants under Cd treatment more than that of the control.The chlorophyll content of MsYSL6OE leaves was significantly higher than that of WT in the control at 0.05 level.The chlorophyll content of L5 and L8 leaves was significantly higher than that of WT under Cd treatment at 0.05 level.The higher chlorophyll content showed that the MsYSL6OE transgenic tobacco was more tolerant to Cd stress (Figure 6A).

The MDA Content
The malondialdehyde (MDA) content showed no difference in MsYSL6OE tobacco and WT in the control.Cd treatment increased the MDA content in the MsYSL6OE tobacco and WT plants.The MDA content in L5, L8, and L9 was significantly lower than that of WT under Cd treatment at 0.05 level (Figure 6B).The results showed that overexpress of MsYSL6 protects plants from damage and leakage in the membrane caused by Cd stress.

The DAB and NBT Staining
Under Cd treatment, DAB staining showed that the leaf of WT was deeper brown while the leaves of L5, L8, and L9 were shallow brown.The staining was scattered at the leaves of L5, L8, and L9, and the brown color was less than that of WT.NBT staining showed that the leaf of WT was deeper blue, while the leaves of L5, L8, and L9 were shallow blue (Figure 7).The results represent that MsYSL6OE tobacco has less ROS production than WT under Cd stress.

The MDA Content
The malondialdehyde (MDA) content showed no difference in MsYSL6OE tobacco and WT in the control.Cd treatment increased the MDA content in the MsYSL6OE tobacco and WT plants.The MDA content in L5, L8, and L9 was significantly lower than that of WT under Cd treatment at 0.05 level (Figure 6B).The results showed that overexpress of MsYSL6 protects plants from damage and leakage in the membrane caused by Cd stress.

The DAB and NBT Staining
Under Cd treatment, DAB staining showed that the leaf of WT was deeper brown while the leaves of L5, L8, and L9 were shallow brown.The staining was scattered at the leaves of L5, L8, and L9, and the brown color was less than that of WT.NBT staining showed that the leaf of WT was deeper blue, while the leaves of L5, L8, and L9 were shallow blue (Figure 7).The results represent that MsYSL6OE tobacco has less ROS production than WT under Cd stress.

The Cd Content and Translocation Rate
The Cd accumulation in MsYSL6OE plants under 75 µM Cd treatment with a significant increase was observed compared with the WT plants (Figure 8A).The Cd content in L5, L8, and L9 roots was significantly higher than that of the WT at 0.05 level.The Cd content in shoots of L5 and L8 was significantly higher than that of the WT at 0.05 level, which showed no difference in L9 from that of the WT (Figure 8A).The Cd translocation rate was 31%, 34%, 38%, and 33% in L5, L8, L9, and WT, respectively.

The Cd Content and Translocation Rate
The Cd accumulation in MsYSL6OE plants under 75 µM Cd treatment with a significant increase was observed compared with the WT plants (Figure 8A).The Cd content in L5, L8, and L9 roots was significantly higher than that of the WT at 0.05 level.The Cd content in shoots of L5 and L8 was significantly higher than that of the WT at 0.05 level, which showed no difference in L9 from that of the WT (Figure 8A).The Cd translocation rate was 31%, 34%, 38%, and 33% in L5, L8, L9, and WT, respectively.

The Fe Content and Translocation Rate
In the roots, the Fe content of MsYSL6OE lines was significantly higher than that of WT under Cd or non-Cd treatment at 0.05 level.Notably, the Fe content in roots was increased in all lines by Cd treatment, and the increase of MsYSL6OE plants was greater than that of WT (Figure 8B).In the shoots, the Fe content was significantly higher in L8

The Fe Content and Translocation Rate
In the roots, the Fe content of MsYSL6OE lines was significantly higher than that of WT under Cd or non-Cd treatment at 0.05 level.Notably, the Fe content in roots was increased in all lines by Cd treatment, and the increase of MsYSL6OE plants was greater than that of WT (Figure 8B).In the shoots, the Fe content was significantly higher in L8 and significantly lower in L9 than that in WT under non-Cd treatment; the Fe content was significantly higher in L5 and L8 than that in WT under Cd treatment at 0.05 level; the Fe content increased in MsYSL6OE shoots and decreased in WT shoots by Cd treatment (Figure 8C).Under non-Cd treatment, the Fe translocation rate accounted for 15%, 24%, and 13% in L5, L8, and L9, respectively, both lower than 29% in WT.Under Cd treatment, the Fe translocation rate accounted for 24%, 29%, and 16% in L5, L8, and L9, respectively, both lower than 34% in WT.

The Cu, Mn, and Zn Content
The Cu, Mn, and Zn content were also screened in MsYSL6OE plants and the WT plants.In plant roots, Cu contents were significantly higher and Mn contents were significantly lower in MsYSL6OE lines than that of WT plants under no-Cd treatment at 0.05 level; Cu contents and Mn contents in L5 and L8 were significantly higher and Mn content in L9 was significantly lower than that of the WT plants under Cd treatment at 0.05 level; Zn contents of MsYSL6OE lines showed no difference with the WT plants under all treatments (Figure 9A-C).In shoots, the Cu and Zn contents in L8 and L9 were significantly higher and Mn content in L5 shoots was significantly lower than that of the WT plants under no-Cd treatment at 0.05 level; the Mn contents in L5, L8, and L9 shoots were significantly lower than that of the WT plants at 0.05 level (Figure 9D-F).In general, Cu, Mn, and Zn contents in the roots and Cu contents in the shoots decreased, while Mn and Zn contents in the shoots of MsYSL6OE plants and the WT plants increased by Cd treatment.

Discussion
Although not essential for plant growth, Cd is readily taken up by roots and accumulated in plant tissues to high levels [26].Cd uptake and transport in different plant species is linked to Fe due to their similar chemical characteristics.It has been reported that the iron transporter OsNRAMP5 contributes to the uptake of Cd from the soil, and the total Cd content in the OsNRAMP5i plants decreases [9].The upregulation of IRT1 facilitated Cd uptake by roots, thus increasing the Cd concentration in plant tissues [32].Transcript levels of IRT1 are very low and transcript levels of HMA2 are strongly elevated in Arabidopsis halleri from the most highly heavy metal-contaminated soil, which can account for its altered Cd handling [55].ABC, NRAMP, and ZIP genes might play important roles in different levels of Cd accumulation in sunflower cultivars [56].MsYSL1 SnYSL3 and BjYSL7 may be essential transporters for diverse metal-NAs to participate in the Cd detoxification by mediating the reallocation of other metal ions [18,40,45].Studies have shown that most YSLs may function in the intracellular transport, translocation, and distribution of Fe and other metals in Arabidopsis thaliana and Oryza sativa.In this study, we obtained a novel YSL transporter gene, MsYSL6, from alfalfa.MsYSL6 belonged to a branch with AtYSL6, OsYSL6, and several YSL6s in leguminous plants, indicating a close relationship in these YSLs.A fundamental role has been demonstrated for Arabidopsis YSL6 in managing chloroplastic iron [38].OsYSL6 was suggested to be a Mn-NA transporter [42].
Transporters play specific biological roles in mineral transport within the tissues where they are expressed.Data indicated that YSL expression was detected in three main territories: vascular tissues throughout the plant, pollen grains, and seeds [35,39,57,58].MsYSL6 was expressed in all vegetative tissues of alfalfa and was most expressed in the leaf.However, MsYSL6 was induced by iron deficiency and Cd in alfalfa roots, consistent with MsYSL1, SnYSL3, and BjYSL7, suggesting an important role for MsYSL6 in response to Cd stress, possibly functioning in Cd uptake [18,43,44].The altered patterns of MsYSL6 expression might be a response to Cd stress.
In plants, Cd toxicity disrupts photosynthesis, causes the production of reactive oxygen species (ROS), and increases antioxidant enzyme activity [6,21,27,[59][60][61].The present study also found that MsYSL6 overexpression reduced ROS accumulation and mitigated the adverse effects on the growth of tobacco under Cd stress, in accordance with previous studies.The specific mechanism of MsYSL6 conferring tolerance to Cd stress into tobacco might be involved in metal distribution.
The Fe-biofortified lentil genotype exhibited Cd tolerance by inciting an efficient antioxidative response to Cd toxicity [19].Fe significantly reduced Cd transfer towards rice grains, which might be attributed to a sharp decrease in the proportion of bioavailable Cd in leaves [62].The significantly high Fe content in MsYSL6 transgenic tobacco indicated the primary function of MsYSL6 in Fe uptake from the medium.Increasing supply of Fe remarkably reduced Cd accumulation in rice shoots, mainly because of inhibited translocation of Cd from rice roots to shoots [63].Fe deficiency increased Cd uptake and accumulation in peanuts but decreased Cd translocation from roots to shoots [64].In rice, low Fe or excess Fe facilitated the uptake of Cd in rice roots, as low Fe upregulated the expression of Cd-transport-related genes, and excess Fe enhanced Cd enrichment on the root by iron plaque [63].Thus, the accumulation of Cd in MsYSL6 transgenic tobacco might be due to the high expression level of MsYSL6 in this study.
Many studies have found that Cd stress affects Fe concentration in plants.Cd-induced Fe accumulation in roots is mediated by upregulating Fe transporter genes such as IRT1 [65].The Cd and Fe accumulation in plants could be partially due to the crucial ligands for metal chelation [66].Like AhOPT3/6/7 and AhYSL1/3 in peanuts, MsYSL6 might be involved in the transport of Fe and/or Cd and Fe/Cd interactions [67].Cd damages the photosynthetic apparatus, as well as the "Fe-deficiency-like" symptoms, and the supply of Fe nutrients saves the photosynthesis symptoms caused by Cd [68,69].Under Cd stress, high Fe concentrations in MsYSL6 confer the plant tolerance to Cd toxicity.We infer that MsYSL6 might have a dual function in the absorption of Fe and, at the same time, Cd.It plays a role as a regulatory factor in the competition absorption between iron and Cd.MsYSL6 increased the ratio of iron to Cd in the total amount and transferred more Fe to the above-ground part of the plant, thereby reducing the oxidative damage of Cd to the plant and resulting in the improvement of tolerance to Cd toxicity in MsYSL6 transgenic tobacco lines.
The metal translocation rate represents the metal translocate capacity in plants.The Cd translocation ratio showed little difference in all tested plant lines.From the results, we hypothesize that MsYSL6 might possibly contribute to the Cd uptake while not likely involved in Cd transport from roots to shoots.In this study, overexpression of MsYSL6 did not increase the Fe transport rate, suggesting that MsYSL6 might not be involved in the translocation of Fe from roots to shoots, while Cd stress increased the Fe transport rate in MsYSL6OE lines, which is similar to the idea that Cd exposure increases the root-to-shoot translocation ratios of Fe in MsYSL1 and SnYSL3 overexpressing Arabidopsis thaliana.The relationship between Cd stress and Fe uptake ability deserves further investigation [66].Additionally, the absorption and transport of Mn, Zn, and Cu in MsYSL6 transgenic tobacco were affected by Cd stress.Specialized cation transport mechanisms have been developed in plants, which can maintain a balance between the deficiency and toxicity of these ions [15].The state of these metals could also have an effect on plant growth and is worth further study.
The phytosiderophores chelate iron were suggested to prevent graminaceous plants from Cd toxicity.In maize, phytosiderophore provides an advantage under Cd stress relative to Fe acquisition via ferrous Fe [47].The nicotinamide synthase (NAS) synthesizes nicotinamide (NA), which plays an essential role together with YSLs in iron homeostasis in nongraminaceous plants.It has also been reported that Cd induces the expression of the NAS gene in Medicago truncatula [70].MsYSL6 may also achieve the effect of alleviating plant Cd toxicity by co-acting with NAS, despite reports claiming that YSL6 in Arabidopsis thaliana has an essential role in chloroplast development, likely in the transport of Fe required for chloroplast development [36].In another study, AtYSL6 was localized to vacuole membranes and to internal membranes [71].In this study, MsYSL6 was found to have no chloroplast localization properties, so it is unlikely that MsYSL6 is involved in Cd detoxification through this pathway.Our results support the idea that MsYSL6 may function as a Cd/Fe transport gene and may be useful for genetic engineering in cultivating Fe-fortified or Cd-tolerant crops.

Plant Growth Conditions
Alfalfa (Medicago sativa L. cv.Zhaodong) seeds were soaked in 70% ethanol and 10% NaClO for 10 min, then washed with H 2 O three times.Seeds were placed onto sterile Whatman paper and germinated for 3 d in the dark.After germination, seedlings were transferred into 1/2 Hoagland's solution.The solution was renewed every 2 d.The seedlings were grown at 25 • C with 16 h light/8 h darkness.The wild-type tobacco (Nicotiana tabacum) seeds were grown on a sterile MS medium at 25 • C with 16 h light/8 h darkness.For detection of gene expression, 30-day-old hydroponic alfalfa seedlings were treated with iron deficiency (no Fe supply, -Fe), 0.5 mM CdCl 2 (Cd), and the control (no stress, Ck) for 4 days.For detection of the tolerance to Cd stress, T 3 of MsYSL6OE and WT tobacco seedlings were germinated and cultivated in MS medium for four weeks, and then transferred onto MS medium containing 75 µM CdCl 2 (Cd) for 10 d, with no CdCl 2 MS medium as the control (Ck).

RNA and DNA Extraction
The total RNA of plant tissues was isolated using the RNA prep Pure Plant Kit 175 (TianGen Biotech, Beijing, China).RNA derived from the leaf was reverse transcribed into cDNA by using Easy Script One-Step DNA Removal and cDNA Synthesis SuperMix (Trans-Gen 177 Biotech, Beijing, China).The total RNA quality was performed by visualization on a 1 × TBE gel stained with ethidium bromide.Total DNA was extracted using a Plant Kit (OMEGA, New York, NY, USA, D3485-01).

Cloning of MsYSL6 and MsYSL6pro
Alfalfa cDNA was used as the template to amplify the open reading frames (ORFs) of the MsYSL6 gene.The MsYSL6 gene amplified primers were designed according to alfalfa iron deficiency transcriptome data (Table S2).The PCR system was as follows: template 1 µL, each primer 0.4 µL, ExTaq DNA polymerase (5 U/µL) 0.05 µL, dNTP mix (2.5 mM) 0.8 µL, 10 × PCR Buffer 1 µL, add ddH 2 O to 10 µL.PCR was performed using the following program: 94 • C for 5 min, 94 • C for 30 s, 55 • C for 30 s, 72 • C for 2.5 min, 95 • C for 15 s, for 30 cycles; 16 • C for 1 h.The PCR products were purified with a Gel DNA Purification Kit (OMGAE), and then cloned into pMD18T plasmids for sequencing.The amino acid sequence of MsYSL6 was identified using the SMART website (http://smart.embl-heidelberg.de/accessed on 11 June 2022).To elucidate the evolutionary relationship, a BLASTP search was performed in the NCBI nr (nonredundant protein sequences) database.Amino acid sequences were aligned with MEGA7.0 software.An unrooted neighborjoining tree was constructed.The transmembrane domains were predicted using the TMHMM Server v.2.0 website (http://www.cbs.dtu.dk/services/TMHMM/accessed on 11 June 2022).Conserve protein motifs were predicted through the online program MEME (http://memesuite.org/tools/memeaccessed on 11 June 2022).
The amplification primers of the MsYSL6 promoter (MsYSL6pro) fragment were designed according to alfalfa genomic data (Table S2).The PCR system was as follows: template DNA 1 µL, each primer 0.4 µL, ExTaq DNA polymerase (5 U/µL) 0.05 µL, dNTP mix (2.5 mM) 0.8 µL, 10 × PCR Buffer 1 µL, add ddH 2 O to 10 µL.The PCR was performed as follows: 94 • C for 5 min; 30 cycles of 94 • C for 30 s, 58.1 • C for 30 s, and 72 • C for 2 min; and a final extension at 72 • C for 10 min.The PCR products were separated and purified with a Gel DNA Purification Kit (OMEGA).Purified products were then cloned into pMD18T plasmids for sequencing.The cis-elements in MsYSL6pro were predicted online by PlantCARE.

Subcellular Localization of MsYSL6
The MsYSL6 ORF without stop codes was cloned into the pBWA(V)HS-GLosgfp vector to construct a fusion plasmid.Then, the fusion plasmid was introduced into Agrobacterium tumefaciens GV3101 by freeze-thaw method and injected into tobacco leaf through the epidermis with a syringe.After cultivation for 2 d, fluorescence signals were recorded using a confocal laser scanning microscope (Nikon C2-ER Laser Scanning Confocal Microscope (Tokyo, Japan)).The green fluorescent protein was excited at 488 nm, and emissions were collected at 510 nm.The chlorophyll autofluorescence was excited at 640 nm, emissions were collected at 675 nm, and overlay images were created.

qRT-PCR
The qRT-PCR was performed by a Real-Time PCR System (Applied Biosystems 7300 Real-Time PCR System, Foster City, CA, USA), using specific primers (Table S2).MsActin and NtGAPDH were used as internal controls.The qRT-PCR system was as follows: cDNA 2 µL, primer Mix 1 µL, qPCR Mix 10 µL, 50 × ROX 0.4 µL, ddH 2 O 6.6 µL, total 20 µL.The qRT-PCR was performed as follows: 95 • C for 10 min, 95 • C for 15 s, 55 • C for 30 s, 72 • C for 30 s, 95 • C for 15 s, 60 • C for 30 s, for 40 cycles; 95 • C for 15 s.The qRT-PCR results were quantified using the comparative 2 −∆∆Ct method.All of the qRT-PCR experiments were performed with three independent RNA samples.

GUS Assay
The MsYSL6pro was cloned to create a C-terminal translational fusion to the reporter gene GUS.The pBI121-MsYSL6pro::GUS was introduced into the Agrobacterium rhizogenes K599 and then infected on alfalfa roots to induce hairy roots.MsYSL6pro::GUS transformed explants were cultured with MS 1 medium with 50 µM kanamycin for 4 d and then cultured with MS 3 medium.After hairy root emergence, the transformants were transferred to an MS 3 medium supplemented with 75 µM CdCl 2 (Cd) and no stress (Ck) for 15 d.The alfalfa hairy roots were stained for the exam the GUS activity.

Plant Transformation and Selection
MsYSL6 was digested with XbaI and SmaI and cloned into a plant expression vector pBI121.The Agrobacterium tumefaciens GV3101 harboring the pBI121-35S:: MsYSL6 was transformed into tobacco by leaf disc method.Subsequently, the transgenic T 0 plants were consecutively self-crossed and detected by PCR and qRT-PCR analysis with MsYSL6 gene-specific primers.T 3 seeds were used for further analyses.

Phenotypic and Physiological Analysis
The 2-week-old tobacco seedlings of three MsYSL6OE tobacco lines (L5, L8, and L9) and WT were treated with 75 µM CdCl 2 (Cd) and no stress (Ck) for 15 d.The germinated seeds were recorded and used to calculate the germination rate.The fresh weight and root length of seedlings were measured and photographed.The chlorophyll was extracted by acetone according to Lichtenthaler [72] et al., and the absorption value was measured at 649 nm and 665 nm by an ultraviolet-visible spectrophotometer (Metash Instruments CO.UV-5100).The MDA content was measured at 450 nm according to Reshmi et al. [73].

Oxidative Analysis
The leaves from tobacco were infiltrated with NBT and DAB dyeing solution following published procedures.The leaf was infiltrated with 1 mg/mL nitro blue tetrazolium (NBT) (pH 7.8) for 40 to 60 min in the dark to detect O 2 -accumulation.The leaf was infiltrated with 1 mg/mL diaminobenzidine (DAB) (pH 7.0) for 8 h in the dark to detect H 2 O 2 accumulation; the leaf was then decolorized by boiling in ethanol: glycerin (3:1) solution for 10 min, photographed after cooling.

Metal Content Determination
The 30-day-old tobacco seedlings of MsYSL6 transgenic tobacco (MsYSL6OE) lines and wild-type plants (WT) were treated with 75 µM CdCl 2 for 10 d [74].The shoots and roots of MsYSL6OE and WT were collected, and all samples were dried to measure weight.The dried and ground samples were placed into the digestive tube with 4 mL 65% HNO 3 and heated at 120 • C for 90 min.After cooling, 1 mL 30% H 2 O 2 was added and it was heated at 120 • C for 90 min.After cooling, 750 µL 30% H 2 O 2 was added and it was left to stand for 4 h, constant to 50 mL with ddH 2 O.All standard solutions were analyzed with the ICP-OES method using ICP-OES8000 (Perkin Elmer, Waltham, MA, USA).All of the measurements were performed with three independent samples.

Statistical Analysis
All of the experiments were repeated three times independently.Data were analyzed by one-way analysis of variance (ANOVA) with LSD Test (least significant difference).The significance of the data was annotated with asterisks (*) based on the following criteria: *, p < 0.05, **, p < 0.01.

Figure 3 .
Figure 3.The subcellular localization of MsYSL6.The subcellular localization of MsYSL6 was investigated using a pBWA(V)HS-MsYSL6-Glosgfp vector.MsYSL6-GFP fusion protein transiently expressed in tobacco.Left to right: green fluorescence of GFP, red fluorescence of chloroplast spontaneous fluorescence, bright field, and merged microscope images (scale bars: 20 µm).

Figure 3 .
Figure 3.The subcellular localization of MsYSL6.The subcellular localization of MsYSL6 was investigated using a pBWA(V)HS-MsYSL6-Glosgfp vector.MsYSL6-GFP fusion protein transiently expressed in tobacco.Left to right: green fluorescence of GFP, red fluorescence of chloroplast spontaneous fluorescence, bright field, and merged microscope images (scale bars: 20 µm).

Figure 3 .
Figure 3.The subcellular localization of MsYSL6.The subcellular localization of MsYSL6 was investigated using a pBWA(V)HS-MsYSL6-Glosgfp vector.MsYSL6-GFP fusion protein transiently expressed in tobacco.Left to right: green fluorescence of GFP, red fluorescence of chloroplast spontaneous fluorescence, bright field, and merged microscope images (scale bars: 20 µm).

Figure 4 .
Figure 4.The expression of MsYSL6 in response to iron deficiency and Cd.The 30-day hydroponic young alfalfa seedlings were treated with no Fe supply (-Fe), 0.5 mM CdCl2 (Cd), nonstress as the control (Ck) for 4 days.(A) The relative expression of MsYSL6 in the roots, stems, and the leaves of alfalfa.Data are means ± s.d. of three experimental replications.Statis comparison was performed by ANOVA followed by an LSD test.Asterisks indicate signifi differences between the treatment and the control (* p < 0.05; ** p < 0.01).(B) The GUS acti induced by MsYSL6pro in alfalfa hairy roots.The alfalfa hairy roots were treated with 75 µM C (Cd) and no stress as the control (Ck) for 15 days.

Figure 4 .
Figure 4.The expression of MsYSL6 in response to iron deficiency and Cd.The 30-day-old hydroponic young alfalfa seedlings were treated with no Fe supply (-Fe), 0.5 mM CdCl 2 (Cd), and nonstress as the control (Ck) for 4 days.(A) The relative expression of MsYSL6 in the roots, the stems, and the leaves of alfalfa.Data are means ± s.d. of three experimental replications.Statistical comparison was performed by ANOVA followed by an LSD test.Asterisks indicate significant differences between the treatment and the control (* p < 0.05; ** p < 0.01).(B) The GUS activity induced by MsYSL6pro in alfalfa hairy roots.The alfalfa hairy roots were treated with 75 µM CdCl 2 (Cd) and no stress as the control (Ck) for 15 days.

Figure 5 .
Figure 5.The growth of MsYSL6OE tobacco under Cd stress.The 4-week-old tobacco seedlings of three MsYSL6OE tobacco lines (L5, L8, and L9) and WT plants were treated with 75 µM CdCl2 (Cd) and no stress as the control (Ck) for 10 d.The fresh weight and root length were detected.(A) The fresh weight of in MsYSL6OE tobacco lines and WT.(B,C) The root length of in MsYSL6OE tobacco lines and WT.The data presented are the means ± s.d. of three experimental replications.Statistical comparison was performed by ANOVA followed by an LSD test.Asterisks indicate significant differences (* p < 0.05; ** p < 0.01).

Figure 6 .
Figure 6.The chlorophyll and MDA content of MsYSL6OE tobacco under Cd stress.The 4-week-old seedlings of three MsYSL6OE tobacco lines (L5, L8, and L9) and WT plants were treated with 75 µM CdCl2 (Cd) and no stress as the control (Ck) for 10 d. (A) The chlorophyll content in MsYSL6OE tobacco lines and WT.(B) The MDA content in MsYSL6OE tobacco lines and WT.The data presented are the means ± s.d. of three experimental replications.Statistical comparison was performed by ANOVA followed by an LSD test.Asterisks indicate significant differences (* p < 0.05).

Figure 5 .
Figure 5.The growth of MsYSL6OE tobacco under Cd stress.The 4-week-old tobacco seedlings of three MsYSL6OE tobacco lines (L5, L8, and L9) and WT plants were treated with 75 µM CdCl 2 (Cd) and no stress as the control (Ck) for 10 d.The fresh weight and root length were detected.(A) The fresh weight of in MsYSL6OE tobacco lines and WT.(B,C) The root length of in MsYSL6OE tobacco lines and WT.The data presented are the means ± s.d. of three experimental replications.Statistical comparison was performed by ANOVA followed by an LSD test.Asterisks indicate significant differences (* p < 0.05; ** p < 0.01).

Figure 5 .
Figure 5.The growth of MsYSL6OE tobacco under Cd stress.The 4-week-old tobacco seedlings of three MsYSL6OE tobacco lines (L5, L8, and L9) and WT plants were treated with 75 µM CdCl2 (Cd) and no stress as the control (Ck) for 10 d.The fresh weight and root length were detected.(A) The fresh weight of in MsYSL6OE tobacco lines and WT.(B,C) The root length of in MsYSL6OE tobacco lines and WT.The data presented are the means ± s.d. of three experimental replications.Statistical comparison was performed by ANOVA followed by an LSD test.Asterisks indicate significant differences (* p < 0.05; ** p < 0.01).

Figure 6 .
Figure 6.The chlorophyll and MDA content of MsYSL6OE tobacco under Cd stress.The 4-week-old seedlings of three MsYSL6OE tobacco lines (L5, L8, and L9) and WT plants were treated with 75 µM CdCl2 (Cd) and no stress as the control (Ck) for 10 d. (A) The chlorophyll content in MsYSL6OE tobacco lines and WT.(B) The MDA content in MsYSL6OE tobacco lines and WT.The data presented are the means ± s.d. of three experimental replications.Statistical comparison was performed by ANOVA followed by an LSD test.Asterisks indicate significant differences (* p < 0.05).

Figure 6 .
Figure 6.The chlorophyll and MDA content of MsYSL6OE tobacco under Cd stress.The 4-week-old seedlings of three MsYSL6OE tobacco lines (L5, L8, and L9) and WT plants were treated with 75 µM CdCl 2 (Cd) and no stress as the control (Ck) for 10 d. (A) The chlorophyll content in MsYSL6OE tobacco lines and WT.(B) The MDA content in MsYSL6OE tobacco lines and WT.The data presented are the means ± s.d. of three experimental replications.Statistical comparison was performed by ANOVA followed by an LSD test.Asterisks indicate significant differences (* p < 0.05).

Figure 7 .
Figure 7.The DAB and NBT staining of MsYSL6OE tobacco under Cd stress.The 4-week-old tobacco seedlings of MsYSL6OE tobacco lines (L5, L8, and L9) and WT plants were treated with 75 µM CdCl2 (Cd) and no stress as the control (Ck) for 10 d.The leaf was stained with nitro blue tetrazolium chloride (NBT) and diaminobenzidine (DAB).The leaf of WT showed deeper staining.The results represent that MsYSL6OE tobacco has less ROS than WT under Cd stress.

Figure 7 .
Figure 7.The DAB and NBT staining of MsYSL6OE tobacco under Cd stress.The 4-week-old tobacco seedlings of MsYSL6OE tobacco lines (L5, L8, and L9) and WT plants were treated with 75 µM CdCl 2 (Cd) and no stress as the control (Ck) for 10 d.The leaf was stained with nitro blue tetrazolium chloride (NBT) and diaminobenzidine (DAB).The leaf of WT showed deeper staining.The results represent that MsYSL6OE tobacco has less ROS than WT under Cd stress.

Plants 2023 , 17 Figure 8 .
Figure 8.The Cd and Fe contents in MsYSL6OE tobacco and WT.The 4-week-old tobacco seedlings of three MsYSL6OE tobacco lines (L5, L8, and L9) and WT plants were treated with 75 µM CdCl2 (Cd) and no Cd treatment as the control (Ck) for 10 d. (A) The Cd content in the roots and shoots of MsYSL6OE tobacco lines and WT.(B) The Fe content in the roots of MsYSL6OE tobacco lines and WT.(C) The Fe content in the shoots of MsYSL6OE tobacco lines and WT.Data are means ± s.d. of three experimental replications.Statistical comparison was performed by ANOVA followed by an LSD test.Asterisks indicate significant differences (* p < 0.05).

Figure 8 .
Figure 8.The Cd and Fe contents in MsYSL6OE tobacco and WT.The 4-week-old tobacco seedlings of three MsYSL6OE tobacco lines (L5, L8, and L9) and WT plants were treated with 75 µM CdCl 2 (Cd) and no Cd treatment as the control (Ck) for 10 d. (A) The Cd content in the roots and shoots of MsYSL6OE tobacco lines and WT.(B) The Fe content in the roots of MsYSL6OE tobacco lines and WT.(C) The Fe content in the shoots of MsYSL6OE tobacco lines and WT.Data are means ± s.d. of three experimental replications.Statistical comparison was performed by ANOVA followed by an LSD test.Asterisks indicate significant differences (* p < 0.05).

Plants 2023 , 17 Figure 9 .Figure 9 .
Figure 9.The Cu, Mn, and Zn contents in MsYSL6OE tobacco and WT.The 4-week-old tobacco seedlings of three MsYSL6OE tobacco lines (L5, L8, and L9) and WT plants were treated with 75 µM CdCl2 (Cd) and no stress as the control (Ck) for 10 d. (A) The Cu content in the roots of MsYSL6OE tobacco lines and WT.(B) The Mn content in the roots of MsYSL6OE tobacco lines and WT.(C) The Zn content in the roots of MsYSL6OE tobacco lines and WT.(D) The Cu content in the shoots of MsYSL6OE tobacco lines and WT.(E) The Mn content in the shoots of MsYSL6OE tobacco lines andFigure 9.The Cu, Mn, and Zn contents in MsYSL6OE tobacco and WT.The 4-week-old tobacco seedlings of three MsYSL6OE tobacco lines (L5, L8, and L9) and WT plants were treated with 75 µM