MeGLYI-13, a Glyoxalase I Gene in Cassava, Enhances the Tolerance of Yeast and Arabidopsis to Zinc and Copper Stresses

Although zinc and copper are the two essential nutrients necessary for plant growth, their excessive accumulation in soil not only causes environmental pollution but also seriously threatens human health and inhibits plant growth. The breeding of plants with novel zinc or copper toxicity tolerance capacities represents one strategy to address this problem. Glyoxalase I (GLYI) family genes have previously been suggested to be involved in the resistance to a wide range of abiotic stresses, including those invoked by heavy metals. Here, a MeGLYI-13 gene cloned from a cassava SC8 cultivar was characterized with regard to its potential ability in resistance to zinc or copper stresses. Sequence alignment indicated that MeGLYI-13 exhibits sequence differences between genotypes. Transient expression analysis revealed the nuclear localization of MeGLYI-13. A nuclear localization signal (NLS) was found in its C-terminal region. There are 12 Zn2+ binding sites and 14 Cu2+ binding sites predicted by the MIB tool, of which six binding sites were shared by Zn2+ and Cu2+. The overexpression of MeGLYI-13 enhanced both the zinc and copper toxicity tolerances of transformed yeast cells and Arabidopsis seedlings. Taken together, our study shows the ability of the MeGLYI-13 gene to resist zinc and copper toxicity, which provides genetic resources for the future breeding of plants resistant to zinc and copper and potentially other heavy metals.


Introduction
Given that heavy metal pollution has had, and will continue to have, a serious impact on the environment and plant, animal and microbial life, leading to the destruction of ecosystems, heavy metal pollution is attracting increasing worldwide attention [1]. There are many reasons for heavy metal pollution in farmland soil during agricultural production, including the use of wastewater for soil irrigation, the application of fertilizers, the spraying of pesticides, herbicides and so on [2]. Especially, the frequent and extensive use of various zinc (Zn)-and copper (Cu)-containing fertilizers and fungicides has resulted in the continuous accumulation of Zn and Cu in farmland soils [3]. Both Zn and Cu are essential micronutrients for plant growth and development. However, when their concentrations are too high, they result in toxicity to organisms [4,5].

MeGLYI-13 Gene Sequence Analysis
The MeGLYI-13 gene was cloned from the genome of the SC8 cassava cultivar using the Manes.12G054100 sequence retrieved from the AM560-2 cultivar as a reference (Manihot esculenta v6.1 dataset). Its cDNA length is 495 bp, and it encodes a protein of 164 amino acids in length. It has 99.3% similarity with the Manes.12G054100 sequence from cassava variety AM560-2 in the cassava genome database. There is one base difference between MeGLYI-13 in SC8 and Manes.12G054100 in AM560-2, which is " 205 C" in SC8 and " 205 T" in AM560-2. At the protein level, this corresponds to " 69 P" in SC8 but " 69 S" in AM560-2 ( Figure S1). The MeGLYI-13 gene from SC8 was registered in NCBI GenBank (MW413417).

Prediction of Zn 2+ and Cu 2+ Binding Sites in MeGLYI-13 Protein Sequence
In order to understand the potential of MeGLYI-13 to bind zinc and copper ions, we predicted the Zn 2+ /Cu 2+ ion binding sites in its protein sequence. The results showed that there were 12 Zn 2+ binding sites and 14 Cu 2+ binding sites in the MeGLYI-13 amino acid sequence. In addition, six sites: 57LYS, 66HIS, 83ASP, 106 ASP, 108GLU and 127GLU bound to both Zn 2+ and Cu 2+ (Figure 1). to broaden our understanding of the function of the MeGLYI-13 gene and its potential application in modern agriculture.

MeGLYI-13 Gene Sequence Analysis
The MeGLYI-13 gene was cloned from the genome of the SC8 cassava cultivar using the Manes.12G054100 sequence retrieved from the AM560-2 cultivar as a reference (Manihot esculenta v6.1 dataset). Its cDNA length is 495 bp, and it encodes a protein of 164 amino acids in length. It has 99.3% similarity with the Manes.12G054100 sequence from cassava variety AM560-2 in the cassava genome database. There is one base difference between MeGLYI-13 in SC8 and Manes.12G054100 in AM560-2, which is " 205 C" in SC8 and " 205 T" in AM560-2. At the protein level, this corresponds to " 69 P" in SC8 but " 69 S" in AM560-2 (Figure S1). The MeGLYI-13 gene from SC8 was registered in NCBI GenBank (MW413417).

Prediction of Zn 2+ and Cu 2+ Binding Sites in MeGLYI-13 Protein Sequence
In order to understand the potential of MeGLYI-13 to bind zinc and copper ions, we predicted the Zn 2+ /Cu 2+ ion binding sites in its protein sequence. The results showed that there were 12 Zn 2+ binding sites and 14 Cu 2+ binding sites in the MeGLYI-13 amino acid sequence. In addition, six sites: 57LYS, 66HIS, 83ASP, 106 ASP, 108GLU and 127GLU bound to both Zn 2+ and Cu 2+ (Figure 1).

MeGLYI-13 Protein Was Localized in the Nucleus
With the intention of assessing the localization of the MeGLYI-13 protein, pCam-bia1300-MeGLYI-13-GFP and pCambia1300-GFP plasmids were transformed into onion epidermal cells by Agrobacterium tumefaciens infection. The green fluorescent signal could be clearly observed in the cytoplasm and nucleus following infection with the control vector, whereas the fluorescent signal could only be detected in the nucleus after infection with the pCambia1300-MeGLYI-13-GFP vector. The nuclear localization was confirmed by DAPI staining (Figure 2).

MeGLYI-13 Protein Was Localized in the Nucleus
With the intention of assessing the localization of the MeGLYI-13 protein, pCambia1300-MeGLYI-13-GFP and pCambia1300-GFP plasmids were transformed into onion epidermal cells by Agrobacterium tumefaciens infection. The green fluorescent signal could be clearly observed in the cytoplasm and nucleus following infection with the control vector, whereas the fluorescent signal could only be detected in the nucleus after infection with the pCambia1300-MeGLYI-13-GFP vector. The nuclear localization was confirmed by DAPI staining (Figure 2).

The Ability of Yeast to Tolerate Cu 2+ and Zn 2+ Toxicity Is Enhanced by MeGLYI-13
The growth characteristics of INVSc1 yeast, containing the pYES2-MeGLYI-13 plasmid or pYES2 control vector, respectively, treated by Zn 2+ or Cu 2+ , were compared. The pYES2-MeGLYI-13-transformed yeast showed better growth ability than the control yeast after the Zn 2+ and Cu 2+ treatments ( Figure 3). No colonies appeared after 10 −5 dilution of the yeast transformed by the control vector after the Zn 2+ treatment. However, under the same conditions, the pYES2-MeGLYI-13-transformed yeast could still grow after 10 −6 dilution. After treatment with Cu 2+ , the growth of the 10 −3 dilution control yeast was completely suppressed, whereas the growth of the 10 −6 dilution pYES2-MeGLYI-13 yeast

The Ability of Yeast to Tolerate Cu 2+ and Zn 2+ Toxicity Is Enhanced by MeGLYI-13
The growth characteristics of INVSc1 yeast, containing the pYES2-MeGLYI-13 plasmid or pYES2 control vector, respectively, treated by Zn 2+ or Cu 2+ , were compared. The pYES2-MeGLYI-13-transformed yeast showed be er growth ability than the control yeas after the Zn 2+ and Cu 2+ treatments ( Figure 3). No colonies appeared after 10 -5 dilution of the yeast transformed by the control vector after the Zn 2+ treatment. However, under the same conditions, the pYES2-MeGLYI-13-transformed yeast could still grow after 10 -6 dilu tion. After treatment with Cu 2+ , the growth of the 10 -3 dilution control yeast was completely suppressed, whereas the growth of the 10 -6 dilution pYES2-MeGLYI-13 yeast was not completely suppressed ( Figure 3). These results indicated that overexpression of the MeGLYI-13 gene raised the resistance of INVSc1 yeast to both Zn 2+ and Cu 2+ toxicity.  2.4. The Ability of Yeast to Tolerate Cu 2+ and Zn 2+ Toxicity Is Enhanced by MeGLYI-13 The growth characteristics of INVSc1 yeast, containing the pYES2-MeGLYI-13 plas mid or pYES2 control vector, respectively, treated by Zn 2+ or Cu 2+ , were compared. The pYES2-MeGLYI-13-transformed yeast showed be er growth ability than the control yeas after the Zn 2+ and Cu 2+ treatments ( Figure 3). No colonies appeared after 10 -5 dilution o the yeast transformed by the control vector after the Zn 2+ treatment. However, under the same conditions, the pYES2-MeGLYI-13-transformed yeast could still grow after 10 -6 dilu tion. After treatment with Cu 2+ , the growth of the 10 -3 dilution control yeast was com pletely suppressed, whereas the growth of the 10 -6 dilution pYES2-MeGLYI-13 yeast was not completely suppressed ( Figure 3). These results indicated that overexpression of the MeGLYI-13 gene raised the resistance of INVSc1 yeast to both Zn 2+ and Cu 2+ toxicity.

Expression of MeGLYI-13 in Arabidopsis
Arabidopsis was transformed with the pCambia1300-MeGLYI-13-GFP vector. A total of six transgenic lines were generated under hygromycin selection, and the presence of MeGLYI-13 was confirmed by PCR ( Figure 4A). The six transgenic lines were named OE1, OE2, OE3, OE4, OE5 and OE6, respectively. To determine the MeGLYI-13 gene expression in the transgenic lines, qRT-PCR was performed. The results revealed that the expression level of the MeGLYI-13 gene varied in different transgenic lines. The transcriptional abundance of the MeGLYI-13 gene was highest in the OE1 and OE5 lines, followed by OE2. The transcriptional abundance of MeGLYI-13 was lowest in the OE4 transgenic line. There was no MeGLYI-13 gene expression in the WT Arabidopsis ( Figure 4B). Because the vector had a GFP tag to further verify the MeGLYI-13 transgenic Arabidopsis, the seedlings of six transgenic Arabidopsis lines were exposed to a LUYOR-3415RG dual-wavelength fluorescent protein excitation light source. The results revealed that the leaves of the WT and OE4 with low MeGLYI-13 expression were red, while the leaves and roots of the other five transgenic lines showed a strong green fluorescence. This indicated that the MeGLYI-13 gene was successfully translated in lines OE1, OE2, OE3, OE5 and OE6 ( Figure 4C).
Arabidopsis was transformed with the pCambia1300-MeGLYI-13-GFP vector. A total of six transgenic lines were generated under hygromycin selection, and the presence of MeGLYI-13 was confirmed by PCR ( Figure 4A). The six transgenic lines were named OE1 OE2, OE3, OE4, OE5 and OE6, respectively. To determine the MeGLYI-13 gene expression in the transgenic lines, qRT-PCR was performed. The results revealed that the expression level of the MeGLYI-13 gene varied in different transgenic lines. The transcriptional abundance of the MeGLYI-13 gene was highest in the OE1 and OE5 lines, followed by OE2. The transcriptional abundance of MeGLYI-13 was lowest in the OE4 transgenic line. There was no MeGLYI-13 gene expression in the WT Arabidopsis ( Figure 4B). Because the vector had a GFP tag to further verify the MeGLYI-13 transgenic Arabidopsis, the seedlings of six transgenic Arabidopsis lines were exposed to a LUYOR-3415RG dual-wavelength fluorescent protein excitation light source. The results revealed that the leaves of the WT and OE4 with low MeGLYI-13 expression were red, while the leaves and roots of the other five transgenic lines showed a strong green fluorescence. This indicated that the MeGLYI-13 gene was successfully translated in lines OE1, OE2, OE3, OE5 and OE6 ( Figure 4C).

MeGLYI-13-Overexpressing Arabidopsis Showed Be er Tolerance to Zinc Shortage and Toxity
To test whether MeGLYI-13 is involved in the tolerance to Zn 2+ or Cu 2+ toxicity, three independent lines-namely, OE1, OE2 and OE5-of MeGLYI-13 transgenic Arabidopsis were selected for study. The wild type and MeGLYI-13-overexpressing in vitro-grown plants were separately treated with different concentrations of Zn 2+ and Cu 2+ . This study revealed that, when grown on normal 1/2 MS medium (15 µmol/L Zn 2+ and 0.05 µmol/L Cu 2+ ), there was no significant difference in the phenotypes of the WT and MeGLYI-13overexpressing lines ( Figure 5A,B). However, following treatment with Zn 2+ , we found that, when there was no Zn 2+ or only low levels of Zn 2+ (4 µmol/L) in the medium, the growth of WT was inhibited compared to the MeGLYI-13-overexpressing lines. Indeed the leaves were smaller, and the taproots and lateral roots were shorter than that of the transgenic lines. After exposure to high levels of zinc (60 µmol/L), the taproots of the WT were obviously shorter than those of the MeGLYI-13-overexpressing lines. The lateral roots of the WT were hard to cultivate, whereas those of the MeGLYI-13-overexpressing lines

MeGLYI-13-Overexpressing Arabidopsis Showed Better Tolerance to Zinc Shortage and Toxity
To test whether MeGLYI-13 is involved in the tolerance to Zn 2+ or Cu 2+ toxicity, three independent lines-namely, OE1, OE2 and OE5-of MeGLYI-13 transgenic Arabidopsis were selected for study. The wild type and MeGLYI-13-overexpressing in vitro-grown plants were separately treated with different concentrations of Zn 2+ and Cu 2+ . This study revealed that, when grown on normal 1/2 MS medium (15 µmol/L Zn 2+ and 0.05 µmol/L Cu 2+ ), there was no significant difference in the phenotypes of the WT and MeGLYI-13-overexpressing lines ( Figure 5A,B). However, following treatment with Zn 2+ , we found that, when there was no Zn 2+ or only low levels of Zn 2+ (4 µmol/L) in the medium, the growth of WT was inhibited compared to the MeGLYI-13-overexpressing lines. Indeed, the leaves were smaller, and the taproots and lateral roots were shorter than that of the transgenic lines. After exposure to high levels of zinc (60 µmol/L), the taproots of the WT were obviously shorter than those of the MeGLYI-13-overexpressing lines. The lateral roots of the WT were hard to cultivate, whereas those of the MeGLYI-13-overexpressing lines grew well. Meanwhile, the leaves of the WT were also smaller than those of the MeGLYI-13-overexpressing lines ( Figure 5C,D). The above consequences indicated that the overexpression of MeGLYI-13 enhanced the survivability towards both the deficiency and toxicity of zinc. grew well. Meanwhile, the leaves of the WT were also smaller than those of the MeGLYI 13-overexpressing lines ( Figure 5C,D). The above consequences indicated that the over expression of MeGLYI-13 enhanced the survivability towards both the deficiency and tox icity of zinc.

MeGLYI-13-Overexpressing Arabidopsis Showed Be er Tolerance to Copper Toxity
When grown on zero Cu 2+ medium, the taproot length of the MeGLYI-13-overexpress ing lines was distinctly shorter than that of the WT. When grown on the medium contain ing 10 µmol/L Cu 2+ , by contrast, the roots of the MeGLYI-13-overexpressing lines becam longer than those of the WT, and the leaves were visibly larger. At high concentrations (6 µmol/L), however, the growth of both the WT and MeGLYI-13-overexpressing lines wer significantly inhibited. However, the MeGLYI-13-overexpressing lines still grew be e than the WT, with larger leaves, longer taproots and more root hair ( Figure 5E,F). There fore, it is speculated that the MeGLYI-13 gene is involved in Cu 2+ tolerance.

Discussion
Zinc and copper are two essential metal elements for plant growth and developmen The lack of these two elements in the environment can inhibit the growth of crops, result ing in, amongst other consequences, reduced yields and quality. However, the presenc of excessive amounts of these two elements can also be toxic to plants [4]. In recent years the extensive use of zinc-containing and copper-containing pesticides and fertilizers, a well as the development of zinc and copper mining, means that the pollution of zinc an

MeGLYI-13-Overexpressing Arabidopsis Showed Better Tolerance to Copper Toxity
When grown on zero Cu 2+ medium, the taproot length of the MeGLYI-13-overexpressing lines was distinctly shorter than that of the WT. When grown on the medium containing 10 µmol/L Cu 2+ , by contrast, the roots of the MeGLYI-13-overexpressing lines became longer than those of the WT, and the leaves were visibly larger. At high concentrations (60 µmol/L), however, the growth of both the WT and MeGLYI-13-overexpressing lines were significantly inhibited. However, the MeGLYI-13-overexpressing lines still grew better than the WT, with larger leaves, longer taproots and more root hair ( Figure 5E,F). Therefore, it is speculated that the MeGLYI-13 gene is involved in Cu 2+ tolerance.

Discussion
Zinc and copper are two essential metal elements for plant growth and development. The lack of these two elements in the environment can inhibit the growth of crops, resulting in, amongst other consequences, reduced yields and quality. However, the presence of excessive amounts of these two elements can also be toxic to plants [4]. In recent years, the extensive use of zinc-containing and copper-containing pesticides and fertilizers, as well as the development of zinc and copper mining, means that the pollution of zinc and copper in soil has become increasingly serious [3,37]. In order to solve the damage caused by soil metal pollution to plants, people have tried many methods. One approach is to use soil amendments to limit the migration of metal ions so as not to affect plant growth [20]. Another approach is to increase a plant's tolerance to metal ion toxicity by spraying it with chemicals [5]. In addition, another method is genetic engineering to improve the metal ion tolerance of plants. The improved plants can efficiently absorb metal ions in the soil and reduce the excessive accumulation of metal ions. Therefore, it is important to cultivate plants resistant to zinc or copper toxicity for the phytoremediation of soil contaminated with copper and zinc. In this respect, a two-phase strategy is required: firstly, the generation of plants that can cope with heavy metals and produce sustainable yields and, secondly, finding a mechanism by which heavy metals are stored in the part of the plants not used for human and/or animal consumption [38,39]. Cassava is a kind of woody shrub, with its tuberous roots full of starch, which is a good resource for producing bioenergy. A previous study indicated that cassava could grow well in Pb-, Cd-, Znand Cu-polluted soils. The soil mobile metal and bioavailable metal content decreased significantly after cassava planting [36]. Thus, cassava can be used as an important plant resource for phytoremediation-on the one hand, to improve the polluted soil environment, and on the other hand, to produce bioenergy raw materials. Furthermore, the key genes for heavy metal resistance in cassava can also be used to improve heavy metal resistance in plants.
To combat the stress of metal toxicity, plants have evolved various protective mechanisms. Since metal toxicity stress can induce cytotoxic MG production in plants, plants have evolved a glyoxalase system to detoxify intracellular MG. Some reports have shown that the physiological mechanism of chemical measures to improve heavy metal resistance in plants involves the mobilization of a glyoxalase system [22,23,29,32,34,35]. Glyoxalase I (GLYI) is in the first step of the glutathione-dependent glyoxalase pathway to scavenge excess MG into S-D-lactoylglutathione. GLYI family genes have been identified and characterized in many species, such as Arabidopsis [40], rice [19], sugarcane [41], Chinese cabbage [42], grapes [43] and date palms [44]. We have also identified 19 GLYI family gene members in cassava [33]. The function of some GLYI genes in abiotic stress has been continuously demonstrated by experiments. For example, a sugar beet M14 glyoxalase I gene could increase E. coli tolerance to MG and increase tobacco tolerance to MG, H 2 O 2 , salt and mannitol [45], whereas an OsGLYI gene from rice was proven to improve rice tolerance to NaCl, ZnCl 2 and mannitol [46]. Similarly, a sugarcane GLYI gene (SoGloI) improved E. coli tolerance to NaCl, CuCl 2 , CdCl 2 and ZnSO 4 stress [47]. Mutants of nuclear-located AtGLYI-2 displayed an increase in the level of MG, a decrease in GLYI activity and were more susceptible to salt stress [19], while nuclear-located OsGLYI-8 could recue the sensitivity of the atglyi-2 mutant to MG and salt stress [19]. In view of previous reports on the role of GLYI genes in plant regulation of abiotic stress resistance, especially resistance to heavy metals, and our previous finding that the cassava MeGLYI-13 gene confers Fe 3+ resistance [33], we speculate that MeGLYI-13 may also confer other heavy metal stress resistance, which is worthy of further study. Here, in our study, we found that the expression of the MeGLYI-13 gene was induced by Zn 2+ stress, and the peak value appeared at 6 h in the leaves and at 24 h in the roots ( Figure S2A). The expression of the MeGLYI-13 gene was also induced by Cu 2+ stress, and the peak value appeared at 12 h in the roots, stem and leaves ( Figure S2B). The function of MeGLYI-13 in high-concentration Zn 2+ and Cu 2+ stress tolerance was further confirmed by gene overexpression in yeast cells and Arabidopsis seedlings ( Figure 5). These results indicate that the MeGLYI-13 gene has a potential role in broader heavy metal stress resistance. However, the specific mechanism by which MeGLYI-13 enhances Zn 2+ and Cu 2+ resistance is still unclear. As evolutionarily ubiquitous metalloenzymes, GLYIs require bivalent metal ions to bind to their active sites to activate the enzyme [30]. The activity of different GLYI enzymes depends on different metal ions. For example, in rice, the OsGLYI-11.2 enzyme was activated by Ni 2+ and Co 2+ but not by Zn 2+ [30], which is similar to two GLYIs (GLXI;1 and GLXI;2) in Arabidopsis [48]. In Arabidopsis, another GLYI (GLXI;3) could be activated by Mn 2+ , Co 2+ and Zn 2+ [48]. Whether Cu 2+ plays a role in promoting GLYI activity is unknown. In our current study, we found that there were more than 10 binding sites for Zn 2+ or Cu 2+ in the MeGLYI-13 amino acid sequence, and there were six common binding sites for both (Figure 1). These consequences forecast that MeGLYI-13 has the binding potential of Zn 2+ and Cu 2+ . Moreover, Zn 2+ and Cu 2+ both might have a role in MeGLYI-13 activity, but the degree to which they activate the MeGLYI-13 enzyme needs to be verified experimentally.
In this study, we tested the heavy metal tolerance function of the MeGLYI-13 gene in model organisms of yeast and Arabidopsis, because these two organisms are commonly used in laboratory studies and are easy to manipulate. Of course, if other organisms have matured genetic transformation systems, they can also be used to produce transgenic lines that overexpress MeGLYI-13 to repair soil contaminated by heavy metals. In addition, virusinduced gene silencing (VIGS) is a powerful tool to trigger transient sequence-specific gene silencing in plants, which is very helpful for studying gene function. VIGS techniques have also been reported in cassava but mostly for cassava leaves and less for the roots [49][50][51][52]. It would be helpful to silence the MeGLYI-13 gene in SC8 cassava roots by VIGS to better understand the function of MeGLYI-13 in the tolerance of plants to Zn 2+ , Cu 2+ and other heavy metals. Whilst the ultimate proof of function needs to be provided in future experiments in which loss-of-function of the MeGLYI-13 gene is assessed directly in cassava, the data obtained in Arabidopsis suggest that it is highly likely that this gene mediates the tolerance of plants to Zn 2+ and Cu 2+ toxicity.
In our study, the MeGLYI-13 gene cloned from the SC8 cassava variety has one nucleotide difference and one amino acid difference compared to the sequence from the AM560-2 cassava variety ( Figure S1). This result suggests that the MeGLYI-13 gene may display genotypic differences. Therefore, it is necessary to conduct sequencing verification in the study of other genotypes of cassava. Interestingly, in rapeseed, the BnGLYI-3 gene from a heat-tolerant rapeseed cultivar was found to improve the heat and cold tolerance of yeast cells, while the BnGLYI-2 gene from a heat-sensitive rapeseed cultivar was not, although there are only two amino acid differences between the BnGLYI-2 and BnGLYI-3 sequences [53]. It can be seen that, sometimes, changes in a few amino acids can lead to large differences in gene function. Therefore, whether the difference in the sequence of cassava MeGLYI-13 will affect the function of the gene remains to be verified.
Protein subcellular localization is very important for the study of protein function. Onion epidermal cells are large, living, transparent monolayer cells, which are suitable for visual fluorescence fusion protein observation. The research presented in our study shows that MeGLYI-13 from SC8 cassava is located in the nucleus of onion epidermal cells (Figure 2). To our knowledge, MeGLYI-13 is the first glyoxalase I protein found to be localized in the nucleus in cassava. The nuclear location of MeGLYI-13 is suggestive of a regulatory role. A few reports also found nuclear localization of GLYIs, such as AtGLYI2-4 in Arabidopsis and OsGLYI-8 in rice [25]. Their nuclear localization signal (NLS) sequences were predicted and experimentally verified [25]. Meanwhile, MG has also been detected in the nucleus, which indicated that the MG detoxification mechanism presents in the nucleus. According to previous studies, we also used the same tool to predict a strong nuclear localization signal in the MeGLYI-13 amino acid sequence ( Figure S3). These results confirmed the nuclear location of MeGLYI-13, suggesting that MeGLYI-13 may play a vital role in nuclear MG detoxification, protecting the nucleus from adverse stress.
It seems strange that MeGLYI-13 is localized in the nucleus and resistant to heavy metal ion toxicity. However, previous studies have confirmed that heavy metal ions can enter the nucleus after crossing the plasma membrane barrier, thus interfering with nucleic acids, chromatin and the internal components [54][55][56]. For example, a higher concentration of copper induced a change in the nucleus structure, higher chromatin density and lower DNA synthesis [56]. DNA damage was found in potatoes after cadmium treatment [55]. In order to reduce the damage of heavy metals to the nucleus, some genes in the nucleus actively respond; in addition to some common transcription factors, there are some other genes. For instance, cysteine synthase 1 (OsCS1) in rice was proven to be localized in the nucleus, and its overexpression transgenic plant obtained a higher tolerance to cadmium stress [57]. A rice heavy metal-associated isoprenylated plant protein gene (OsHIPP17) was localized in the nucleus, and overexpressing it in Arabidopsis enhanced the copper tolerance of transgenic plants [58]. Therefore, we speculate that MeGLYI-13 localization in the nucleus may be related to the clearance of excessive MG caused by heavy metals in the nucleus, and the binding sites for Zn 2+ or Cu 2+ may be used for binding Zn 2+ or Cu 2+ in order to activate MeGLYI-13 enzyme activity.

MeGLYI-13 Gene Analysis
The total RNA from 40-day tissue culture seedlings of SC8 cassava (Manihot esculenta Crantz) was isolated following the instructions of the Plant Total RNA Isolation Kit Plus (FOREGENE). The RTIII Super Mix (with dsDNase) kit (MonScript, Monad, Suzhou, China) was used to clear away possible residual DNA fragments in the total RNA solution, and after, the first strand of cDNA was synthesized. According to the sequence of Manes.12G054100 in the AM560-2 cassava genome data, Primer Premier 6.0 was used to design a pair of specific primers with a predicted length of 556 bp (Table S1). The 50 µL PCR system included PrimeSTAR HS (premix) 25 µL, template cDNA (1 µg/µL) 1 µL, 10 µmol/L upstream and downstream primers 2 µL each of ddH 2 O 20 µL. The PCR procedure included 94 • C pre-degeneration for 5 min, 98 • C degeneration for 10 s, 55 • C annealing for 15 s, 72 • C extension for 1 min for 32 cycles and 72 • C extension for 10 min. PCR production was then detected using 1% agarose gel electrophoresis. The target fragment was purified by the use of a Gel Extraction Kit (Omega) and then sequenced by Sangon Biotech. The sequence alignment was performed using Jellyfish software version 4.1.1.0. The Zn 2+ and Cu 2+ binding sites were predicted by the online tool MIB2 (http://bioinfo.cmu.edu.tw/MIB2/, accessed on 21 November 2022). The nuclear localization signal on the MeGLYI-13 protein sequence was predicted by using the online tool cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi, accessed on 6 January 2023).

Subcellular Localization of MeGLYI-13
SnapGene software version 4.2.4.0 was used to design homologous recombination primers. SpeI restriction sites were introduced upstream and KpnI restriction sites downstream of ORF of the MeGLYI-13 gene. By using the Seamless Assembly Cloning Kit (Clone Smarter, USA), the fusion expression vector pCAMBIA1300-35S-MeGLYI-13: GFP was obtained. The vector was then transferred into DH-5α E. Coli for sequencing. Both the pCAMBIA1300-35S-MeGLYI-13: GFP vector and the control pCAMBIA1300-35S-GFP vector were transformed into living onion epidermal cells by an Agrobacterium Tumefaciens LBA4404-mediated protocol. After incubation at 28 • C on solid MS medium (pH5.8) for 16-24 h, the onion cells were studied using a Laser Scanning Confocal Microscope FV1000 (Olympus, Tokyo, Japan).

Expression Analysis of MeGLYI-13 in Response to Zinc and Copper Stress
To explore the response of the MeGLYI-13 gene in zinc or copper stress, 30-day-old cassava seedlings of the SC8 variety were treated with 60 µmol/L Zn(CH 3 COOH) 2 or 100 µmol/L CuSO4 for 0, 2, 6, 12 and 24 h, respectively. Three plants and three biological replicates were set for each treatment. The leaves, roots and stems were harvested and used to extract RNA and perform a qRT-PCR analysis according to the method in our previous report [33].

Yeast Transformation and Stress Treatments
In order to construct a yeast expression vector, a restriction site (KpnI) was introduced upstream of the MeGLYI-13 ORF, and an additional restriction site (EcoRI) was introduced downstream of the gene. Afterward, a fragment of MeGLYI-13 gene was inserted into the yeast expression vector pYES2 by the double restriction method to generate the pYES2-MeGLYI-13 yeast expression vector. The sequences of the primers pYES2-MeGLYI-13 forward (F) and pYES2-MeGLYI-13 reverse (R) are given in Supplementary Table S1. The empty plasmid pYES2 and recombinant plasmid pYES2-MeGLYI-13 were transformed into E. coli DH5α using the chemical transformation method, and the presence of the transgene was detected by PCR and subsequent sequencing. The pYES2 and pYES2-MeGLYI-13 plasmids in E. coli DH5α with correct sequencing were extracted and transferred into INVSc1 yeast strains using the PEG/LiAc transfer method. In order to investigate the response of MeGLYI-13 transgenic yeast to Zn 2+ and Cu 2+ ion stress, transgenic yeast and control yeast were cultured in SC-Ura liquid medium (2% galactose, 25 mg/L Amp) at 28 • C and 180 r/min for 36 h. The bacteria were then collected by centrifugation and then resuspended to OD600 = 0.2 by SC-Ura liquid medium containing different Zn 2+ (0 mmol/L, 6 mmol/L, 12 mmol/L, 18 mmol/L, 24 mmol/L and 30 mmol/L) and Cu 2+ (0 mmol/L, 0.6 mmol/L, 1.2 mmol/L, 1.8 mmol/L, 2.4 mmol/L and 3.0 mmol/L). Then, 2 mL of each bacterial solution was inserted into a new tube and incubated at 28 • C, 180 r/min for 30 h, and the OD600 value was measured. Three biological replicates were performed per treatment. At the same time, the survival difference between the recombinant yeast and the control yeast after 30 mmol/L Zn 2+ and 3 mmol/L Cu 2+ stress was assessed. Then, 5 mL of bacterial solution cultured for 36 h was centrifuged to collect the bacteria, respectively, and suspended in the treatment solution and cultured for 6 h at 28 • C, 180 r/min. After that, the treated bacterial suspension was attenuated into the six 10 −1 , 10 −2 , 10 −3 , 10 −4 , 10 −5 and 10 −6 gradient dilutions. Then, 5 µL of diluted bacterial suspension was plated on SC-Ura solid medium. The bacteria were cultured at 28 • C, and two days later, the survival difference of the yeast was observed and photographed.

Transformation of Arabidopsis with MeGLYI-13
The Agrobacterium LBA4404 containing the pCAMBIA1300-MeGLYI-13:GFP expression vector was shock cultured until the OD600 value of its suspension reached 0.9~1.0 at 28 • C and with 250 r/min shaking with YEP liquid medium containing 50 mg/L kanamycin and 50 mg/L rifampicin. The suspension was then centrifuged to collect the bacteria at 4000× g and 4 • C. After washing one time in 1/2 MS liquid medium (containing 0.03% Silwet-L77), the bacteria were resuspended to an OD600 value of 0.6~0.8 and then kept away from light for 3~12 h. Inflorescences containing unopened flowers of Arabidopsis were dipped into the above bacteria suspension for 3 min, then cultured in the dark for 16~24 h. After that, the Arabidopsis were cultured under 16 h light/8 h dark at 22 • C conditions for three days. The mature seeds were collected as the T0 generation. The seeds were disinfected with 75% ethanol (containing 0.01% Tween 20) for 5 min and washed by absolute ethanol twice. Next, the seeds were evenly spread on 1/2 MS solid medium (containing 80 mg/L hygromycin) and vernalized at 4 • C under darkness for two to four days and then cultured under 12 h light/12 h dark at 22 • C. Ten days later, the Arabidopsis seedlings that grew well on the resistance medium were transplanted into soil for pot culturing.

Detection of Transgenic Arabidopsis
To detect transgenic Arabidopsis, we performed DNA-level PCR. Then, 2 mm 2 leaves of each Arabidopsis seedling were collected separately and treated with 20 µL lysate (M5 mix Kit, MF848, Mei5bio, Beijing, China) until the liquid turned green. The liquid was kept at 95 • C for 5 min and shortly centrifuged for 1 min at 10,000× g, and then, the supernatant was used as a template for the PCR. The sequences of primer pairs 1300F and 1300R used to detect the transgenic plants are given in Supplementary Table S1. The 20 µL PCR reaction liquid contained 10 µL 2 × M5 HiPer plus Taq HiFi, 0.5 µL 1300F, 0.5 µL 1300R, 2 µL template DNA and 7 µL ddH 2 O. The PCR procedure included 95 • C for 3 min, 94 • C for 25 s, 53 • C for 25 s, 72 • C for 45 s for 34 cycles and a 72 • C extension for 5 min. The PCR product was detected by 1% agarose electrophoresis.

Expression Level Detection of MeGLYI-13 in Transgenic Arabidopsis
To detect the expression level of MeGLYI-13 in PCR-positive lines by qRT-PCR, the total RNA from the PCR-positive Arabidopsis lines was extracted, purified and reversetranscribed by successively using the RNA Isolation Kit (Foregene, Chengdu, China) and RTIII Super Mix with dsDNase kit (Monad, Suzhou, China). To perform the reaction for qRT-PCR, the reaction system was prepared according to the manual of SYBR ® Premix Ex Taq TM II (Takara, Otsu, Japan). For detecting the MeGLYI-13 gene, the primer pairs of QMeGLYI-13F and QMeGLYI-13R were used. For reference, the Actin gene primer pairs ActinF and ActinR were used (see Supplementary Table S1). QRT-PCR was performed and recorded using a 7900 HT Fast Real-Time PCR System (ABI, Foster City, CA, USA). The relative expression levels of MeGLYI-13 in different Arabidopsis lines were calculated using the 2 −∆∆CT method. The wild type Arabidopsis (WT) was used as the control. Three biological replicates were performed in this experiment.

Fluorescence Detection of Transgenic Arabidopsis
The T1 generation seeds were germinated on 1/2 MS medium after vernalization, as described in Section 2.4. Then, a dual-wavelength fluorescent protein excitation light source (Luyor-3415RG, Luyor Corporation, Shanghai, China) was used to irradiate the germinated seedlings. Positive transgenic Arabidopsis seedlings were selected, as they showed green fluorescence under light, while the wild type and transgenic lines with too-low gene expression showed a red color [59].

Znic and Copper Stress Treatment of Transgenic Arabidopsis
After identification by PCR, qRT-PCR and fluorescence, some transgenic lines were selected for further experiments. The WT seeds and T4 generation seeds of three selected transgenic Arabidopsis lines were sterilized and seeded on 1/2 MS medium at 4 • C for 2 days of vernalization and then germinated at 22 • C for 12 h light/12 h dark for 8 days of germination. Then, the Arabidopsis seedlings were transferred to 1/2 MS medium containing diverse concentrations of Zn(CH 3 COOH) 2 (0, 4 and 60 µmol/L) and CuCl 2 (0, 10 and 60 µmol/L) for stress treatment under 22 • C for 12 h light/12 h dark cultural conditions for 12 days. The normal 1/2 MS medium (Zn 2+ 15 µmol/L and Cu 2+ 0.05 µmol/L) was set as the control. After treatment, any phenotypic changes were observed and photographed. The length of the taproot was measured. For each treatment, three plates were set up, and three plants were used per line in each plate.

Statistical Analysis
All data analyses and graphic output were performed using GraphPad Prism 8.0 software. The one-way analysis of variance was performed to analyze the data, and Dunnett's multiple comparisons test was used to assess the significant differences.

Conclusions
In summary, a MeGLYI-13 gene was cloned from SC8 cassava with a cDNA length of 495 bp and protein length of 164 amino acids. The MeGLYI-13 from SC8 cassava germplasm was different to that from AM560 cassava germplasm with one nucleobase difference in the cDNA sequence and one amino acid in the protein sequence. Subcellular localization of the MeGLYI-13 protein was demonstrated in the nucleus. There, six binding sites were predicted to bind to both Zn 2+ and Cu 2+ in the MeGLYI-13 protein sequence. The overexpression of MeGLYI-13 enhanced the Zn 2+ and Cu 2+ toxicity in both yeast cells and Arabidopsis seedlings. The results of this study highlight the function of one MeGLYI-13 gene from cassava in resistance to multiple heavy metals stress, possibly by stabilizing the MG balance in the nucleus. It can thus likely be used as a target for genetic modification to improve crop resistance to heavy metals. Therefore, we will continue to explore the mechanisms by which MeGLYI-13 regulates Zn and Cu toxicity tolerance in the future.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/plants12193375/s1: Figure S1: Sequence alignments of MeGLYI-13 CDS (A) and amino acids (B) between SC8 and AM560-2 cassava genome data. Figure S2: Expression profile of MeGLYI-13 in cassava seedlings after 60 µmol/L Zn 2+ and 100 µmol/L Cu 2+ treatments. Figure S3: Nuclear localization signal (NLS) prediction of MeGLYI-13 in cassava SC8. The red characters indicate the NLS sequence. Table S1: Sequences of the primers. Data Availability Statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.