Normal plant growth requires major and trace elements containing metallic elements, such as K, Ca, Mg, Fe, Na, Cu, Zn, Mn, Ni, and Co, which play various roles in plants. However, these trace elements are highly toxic at excessive amounts. Meanwhile, several non-essential and toxic metal ions, including Cd, Pb, and Hg, have the same chemical structures as some of these trace elements and are thus absorbed by plants (Kim et al., 2004). Salt stress and heavy metal toxicity commonly occur because of the excessive accumulation of various metal ions. Conversely, trace metal element deficiency in poor soils decreases agricultural yield and intake of trace elements in the human body. Therefore, precise metallic element homeostasis is essential in plants (Kim et al., 2004). Several gene families participate in the uptake and transport of metal ions by plants (Lang et al., 2011; Zhang et al., 2013). These metal ion transporters include the cation diffusion facilitator (CDF) family, Zrt/Irt-like protein, P-type ATPase, and ATP-binding cassette transporter, etc., (Zhang et al., 2013).

The CDF family, also called the cation efflux family (Lang et al., 2011), was first described in 1995 (Nies and Silver, 1995). The protein members of the CDF family have been identified in all six kingdoms of living things (Migocka et al., 2015a) and are involved in transport and tolerance to trace elements (Kim et al., 2004). CDF family members have three distinct features, namely, an N-terminal signature sequence, cation efflux domain, and approximately six predicted transmembrane regions (Paulsen and Saier, 1997). Eukaryotic CDFs usually contain a histidine (His)-rich cytoplasmic loop between transmembrane domains 4 and 5 (Paulsen and Saier, 1997). CDF members are classified into three major clusters, namely, Zn-CDFs, Fe/Zn-CDFs, and Mn-CDFs, which differ with respect to selectivity toward the main transported metal ions (Montanini et al., 2007). In plants, CDF members are designated as metal tolerance proteins (MTPs; Migocka et al., 2015b). The MTP family has 12 and 10 members in model plants Arabidopsis and rice, respectively. Unfortunately, their functions remain finitely understood (Migocka et al., 2015b). Recently, MTP proteins from different plant species have been divided into seven groups according to the results of the phylogenetic analysis and annotation performed on Arabidopsis MTPs (Gustin et al., 2011). Among them, groups 1, 5, and 12 belong to the Zn-CDFs, groups 6 and 7 form the Fe/Zn-CDFs, and the Mn-CDF cluster comprises groups 8 and 9 (Gustin et al., 2011). To date, the most studied MTP proteins in plants include the MTP1–MTP4 members of group 1, MTP8 proteins of group 8, and MTP9–MTP11 of group 9 (Migocka et al., 2015b). Most of these MTPs were functionally characterized in diverse plant species. Although group 1 of plant MTPs belongs to Zn-CDFs, its members is capable of transporting different metals, including Zn, Cd, Co, Ni, or Fe, into the vacuole of plant cells (Kim et al., 2004; Xu et al., 2009; Lang et al., 2011; Menguer et al., 2013; Migocka et al., 2015a). Recently, MTP5 has been reported to form a functional complex with MTP12 to transport Zn into the Golgi apparatus in Arabidopsis (Fujiwara et al., 2015). Group 8, comprising only MTP8 proteins, is implicated in Mn homeostasis, which requires a proton gradient when transporting Mn²⁺ across membranes (Migocka et al., 2015b; Eroglu et al., 2017; Li Q.H. et al., 2017). They are necessary for the active sequestration of excess Mn in intracellular organelles or Mn delivery to Mn-dependent enzymes (Migocka et al., 2015b). Meanwhile, group 9 comprises MTP9–MTP11 proteins (Montanini et al., 2007; Gustin et al., 2011), and MTP 9 and MTP 10 are more closely related and thus form an internal separate clade. This relationship indicates some functional differentiation between MTP9/10 and MTP11 proteins (Gustin et al., 2011). Several MTP11 transporters from different species confer Mn²⁺ tolerance (Delhaize et al., 2007; Zhang and Liu, 2017), whereas limited evidence of the involvement of MTP9/10 proteins in Mn transport or tolerance to Mn is currently available (Migocka et al., 2015b), though they are also classified as Mn-CDFs. Current studies generally indicated that MTP proteins contribute to the detoxification of heavy metals and promote their accumulation (Kim et al., 2004; Delhaize et al., 2007; Xu et al., 2009; Lang et al., 2011; Menguer et al., 2013; Migocka et al., 2015a,b; Eroglu et al., 2017; Li Q.H. et al., 2017; Zhang and Liu, 2017). The functions of MTPs or other metal transporters in heavy metal hyperaccumultors may improve phytoremediation efficiency (Kim et al., 2004; Gustin et al., 2009). However, knowledge on the functional assignment of plant MTPs remains insufficient. For example, the functional characteristics of groups 6 and 7 have not been elucidated so far.

The Brassicaceae species has shown apparent advantages in the exploration of metal ion transporter functions. These species include the model plant Arabidopsis and many important vegetables and oilseed crops with easily available genomes (Wang et al., 2011; Liu et al., 2014; Kolaj-Robin et al., 2015; Gan et al., 2016; Yang et al., 2016), and have represented a differential range of tolerance to two important abiotic stresses, namely, salinity and heavy metals (Assunção et al., 2003; Prasad and Freitas, 2003; Inan et al., 2004; Megdiche et al., 2007). For example, over half of the identified Ni hyperaccumulators (more than 300 species) were reportedly from Brassicaceae (Assunção et al., 2003). Turnip (Brassica rapa var. rapa), a Brassicaceae biennial plant, has been widely cultivated in Europe, Asia, and America as a vegetable or fodder. In Asia, one of the cultivation centers of turnip is in the Tibetan Plateau and its surrounding countries and regions. Moreover, turnip has been cultivated at the maximum elevation of 4700 m in Nyima County, China. Thus, this species is faced with diverse soil conditions, indicating that turnips from different populations or cultivated landraces may possess various ion absorption or accumulation characteristics to adapt to the corresponding environments. Turnips from Xinjiang, China, reportedly contain abundant mineral elements (Ma et al., 2016), indicating its eminent absorption ability for metal ions. Additionally, turnip has been classified as a high-Cd accumulation plant (Arthur et al., 2000; Li et al., 2016). We found that turnip landraces from China have strong capacities for Cd accumulation, and several landraces were Cd hyperaccumulators (Li et al., 2016, Li X. et al., 2017). To explore the molecular mechanism of metal ion accumulation in turnip, we focused on several important ion transporters based on the sequencing results of turnip (Cheng et al., 2016). In the present study, we systematically analyzed the sequence and structural characteristics of putative MTPs in turnip and preliminarily investigated the potential roles of each BrrMTP member. This study is expected to improve the understanding of the functions of plant MTPs and provide a basis for the analysis of the functions and mechanisms of BrrMTP proteins.

Although increasing functional studies on plant MTP members have been reported in diverse species, such as Arabidopsis thaliana (Desbrosses-Fonrouge et al., 2005; Delhaize et al., 2007; Eroglu et al., 2016, 2017), Oryza sativa (Menguer et al., 2013; Zhang and Liu, 2017), Brassica juncea (Xu et al., 2009; Lang et al., 2011), Cucumis sativus (Migocka et al., 2015a,b), Thlaspi goesingense (Kim et al., 2004), and Camellia sinensis (Li Q.H. et al., 2017), the understanding of the roles of the MTP family is still significantly limited. On the basis of the increasing genome information, analyzing gene families via comparative genomics is an efficient method for modern functional genomics research (Xu et al., 2016). However, unlike many other gene families, including some transcription factors and kinases (Iftikhar et al., 2017; Ma et al., 2017; Xi et al., 2017), studies on the characteristics and functional analysis of the plant MTP gene family are just reported in few species recently (Fu et al., 2017; Vatansever et al., 2017). As MTP genes have been demonstrated to participate in tolerating and transporting various heavy metals, including plant trace elements and non-essential elements (Kim et al., 2004; Delhaize et al., 2007; Xu et al., 2009; Lang et al., 2011), they might thus play significant roles in plant mineral nutrition maintenance and resistance to stresses caused by metals. Turnip has shown relatively high capacities toward absorbing trace elements and heavy metal Cd (Li et al., 2016; Ma et al., 2016). Thus, exploring the functional characteristics of the metal ion transporters in turnip is of great interest. In the present study, 18 MTP family members in turnip were successfully identified by bioinformatics analysis. These 18 BrrMTPs were located at different positions on 6 chromosomes and were unevenly distributed within the genome. Compared with Arabidopsis, gene family expansion occurred in the BrrMTP gene family. This might be because the polyploidization events occurred in the evolutionary history of Brassica rapa (Yin et al., 2017). These events were followed by chromosomal reduction and rearrangement and numerous gene losses (Yin et al., 2017). In the present results, MTP3 was not observed in turnip; this was likely because of gene loss. However, the incomplete genomes factor was not excluded. The increase in the number of MTP genes during plant evolution was likely related to the functional evolution of metal tolerance and accumulation.

The phylogenetic relationship indicated that BrrMTPs could be divided into three clusters and seven groups as in Arabidopsis (Montanini et al., 2007; Kolaj-Robin et al., 2015). Clusters Zn-CDF, Fe/Zn-CDF, and Mn-CDF contained 7, 3, and 8 members, respectively. This classification was supported by the subsequent analysis of gene structure and protein features. Although the coding regions of most BrrMTPs (except BrrMTP12) encoded polypeptides of 335–464 amino acids in length, the intron numbers of BrrMTPs had significant differences among different groups, ranging from 0 to 12. Variations in intron lengths also existed in the BrrMTPs. However, their functions and evolutionary process require further verification. Like the protein size, the MW, pI, and GRAVY of most BrrMTPs were relatively conserved or similar but closer within groups. Particularly, BrrMTP12 possessed significantly larger protein size (765 amino acids) and MW (86.24 kD) than the other BrrMTP members. This result was consistent with the difference between AtMTP12 and other AtMTPs, indicating the distinctive function and evolutionary process of MTP12. Undoubtedly, all the BrrMTPs contained the conserved cation efflux domain in their sequences. Moreover, a zinc transporter dimerization (ZT dimer) domain was detected in the members of groups 6, 8, and 9, but whether this domain is correlated with the functions of these BrrMTP is unknown. The CDF signature sequence, transmembrane region and His-rich loop are three significant structural features of MTP proteins (Montanini et al., 2007; Kolaj-Robin et al., 2015), which are closely related to their functional characteristics. However, these features showed obvious differences among different groups (or clusters). The complete CDF signature sequences were only observed in the BrrMTPs of group 1. The His-rich loop in the MTP protein sequences has been considered responsible for metal selectivity (Kang and Carey, 1999; Podar et al., 2012; Kolaj-Robin et al., 2015). In the present study, groups 1 and 12 from the Zn-CDF cluster possessed individual His-rich sequences. However, BrrMTP1.1–BrrMTP4 included 17–40 amino acid residues in length, whereas the sequence length of BrrMTP12 was 115 amino acid residues. The results might indicate the differential abilities of different BrrMTPs to transport metal ions. Unlike the above two features, most BrrMTPs contained 4–6 conserved transmembrane regions, except for BrrMTP12, which possessed 14 ones, similar to 15 ones in AtMTP12 (Zhang et al., 2013). Generally, these results were consistent with the structure characteristics of AtMTPs. In Arabidopsis, AtMTP5–AtMTP12 proteins only contained one of the features of the MTP family, such as the transmembrane domains, whereas AtMTP1–AtMTP4 included all the MTP features (Paulsen and Saier, 1997; Zhang et al., 2013). However, three different clusters were divided by the researchers, which included AtMTP6–AtMTP11 (cluster I), AtMTP5 and AtMTP12 (cluster II), and AtMTP1–AtMTP4 (cluster III) (Paulsen and Saier, 1997; Zhang et al., 2013). The results of motif compositions also showed the structural similarity within the BrrMTP groups and the particularity among different groups, indicating the possible functional diversity of the entire family of BrrMTPs.

To date, members of plant MTPs have been reported to be involved in tolerating and transporting different metals, including Zn, Cd, Co, Ni, Fe, and Mn (Kim et al., 2004; Xu et al., 2009; Lang et al., 2011; Menguer et al., 2013; Migocka et al., 2015a,b; Eroglu et al., 2017; Li Q.H. et al., 2017; Zhang and Liu, 2017). Thus, these transporters have shown potential applications in the phytoremediation of heavy metal-polluted soils, especially the members from heavy metal hyperaccumulators (Kim et al., 2004; Gustin et al., 2009). Eroglu et al. (2016) recently has reported that MTP8 in Arabidopsis determines tolerance to iron deficiency-induced chlorosis; this study provides a new insight into roles of MTP proteins in plants. However, whether the MTP family contributes to the plant response toward salt stress is unclear. Moreover, whether the different members have function division or cooperation when transporting various metal ions remains undefined. To expand the information for these issues, we predicted the potential substrate metal ions of each member of the BrrMTPs by performing gene-induced expression. We primarily examined the spatial expression patterns of the BrrMTP genes under normal nutrient conditions. The results showed that all members of groups 1, 5, 6, and 7 and several members of group 9 (BrrMTP9, BrrMTP11.1 and BrrMTP11.2) were mainly expressed in the leaves, whereas most of the others were expressed in the roots, indicating that different BrrMTP members play specific roles or run different mechanisms in turnips. The results are partly consistent with the reports regarding their individual homologous genes (Lang et al., 2011; Chen et al., 2013; Vatansever et al., 2017). This might be related to the special nutrient transport during growth and development of turnips, which form fleshy roots. To investigate the potential roles of BrrMTP members, we detected the expression dynamics of the BrrMTP genes under eight metal ion treatments. These metal ions include a macroelement (Mg), microelement (Zn, Cu, Mn, Fe, Co, or Na) or non-essential element (Cd) for plant growth and also represent heavy metals (e.g., Zn, Cu, Mn, and Cd) or salt ions (e.g., Mg and Na). The results showed that each metal ion induced the expression levels of at least one BrrMTP gene in the roots and leaves, indicating that the BrrMTPs may be involved in the tolerance or transport of corresponding macroelements and microelements. In that case, the BrrMTPs are of significance for turnip plants to maintain mineral element balance under normal condition or to improve tolerance to heavy metals and salts when faced with stresses. Cd is a non-essential element to plants, and plants do not possess specific mechanisms for the uptake of Cd²⁺, but Cd can be absorbed and transported through some of the carriers used for the uptake of essential metals for plant development (Li L.Z. et al., 2017). We found that BrrMTP1.3, BrrMTP2, BrrMTP7.1, and BrrMTP12 were increasingly expressed when treated by Cd, indicating that these genes may be related to the high-Cd accumulation characteristics of turnip (Li et al., 2016). Interestingly, our results are consistent with those of previous studies wherein the MTPs of group 1 were reported to transport multiple metal ions, whereas the members of group 8 or 9 showed a relative specificity to Mn (Kim et al., 2004). However, our results possibly indicated that the MTP genes of turnip have extended functions during the evolution or domestication process. For instance, we found that BrrMTP1.3, an expansion member of group 1 in turnip, showed a positive response (upregulated expression) to seven metal ions in contrast to Arabidopsis. We also found that several metal ions induced the expression levels of groups 6 and 7 in the roots and leaves. Given that the functions of these MTP genes are rarely reported, this study provided a basis for their functional investigation. Additionally, although Cu has not been identified as substrates for MTP transporters even in model plants, we found that Cu induced the expression levels of most BrrMTP genes. The results are supported by a recent study in sweet orange (Fu et al., 2017); thus these findings provide valuable information for future studies on the role of MTPs in Cu detoxification. Nevertheless, we also found that some BrrMTP genes responded to Mg and Na, which have not been used as substrates for MTP transporters in plants either (Kolaj-Robin et al., 2015). Thus, whether our results are related to the special evolution, domestication, or adaptation process of turnips remains unknown. The reasons and mechanisms for these questions need further studies.

Overall, the expression responses to common metal ions of all identified MTP family genes in turnip were explored for the first time in the present study. Our results were partly consistent with those of previous studies (Zhang et al., 2013; Fu et al., 2017). Thus, we provided some original information for the subsequent research of several MTP members that have been seldom studied. However, it is incredible to predict the substrate ions for transporters only from the gene expression response, as ion interaction (e.g., antagonism and synergism) or some physiological or metabolic processes affected by certain ion may indirectly cause changes in other ions and the expression dynamic of their corresponding transporters. Thus, concrete gene function studies by modern molecular biology technology are urgently required for each BrrMTP gene member.

We identified 18 candidate CDF genes from the turnip genome and presented a comparative genomic analysis of CDFs (or MTPs) in turnip, particularly their phylogenetic relationships, gene structures and chromosome distributions, conserved domains, and motifs. The CDF gene family in turnip generated expansions in MTP1, MTP7, MTP8, MTP10, and MTP11 in contrast to those of A. thaliana, although MTP3 might be lost during the evolutionary process. Based on the constructed phylogenetic tree, we divided the BrrMTPs into seven groups (groups 1, 5, 6, 7, 8, 9, and 12), which formed three major clusters (Zn-CDFs, Fe/Zn-CDFs, and Mn-CDFs). The BrrMTP proteins showed similar structural characteristics within groups but significant differences among different groups. Afterward, we performed an expression analysis on all BrrMTP members under eight metal treatments (Mg, Zn, Cu, Mn, Fe, Co, Na, and Cd) to investigate the potential roles of BrrMTPs in turnip. The results showed that the expression levels of all BrrMTP genes can be induced by at least one metal ion, indicating that these genes may be related to the tolerance or and transport of corresponding metal ions. We found first-hand that two metal ions, namely, Mg and Na, significantly induce the expression levels of several BrrMTP genes. According to the different roles of these metal ions for plants, we hypothesized that the BrrMTP family genes are possibly involved in heavy metal accumulation and tolerance to salt stress apart from their role in the maintenance of mineral nutrient homeostasis in turnip. However, these conclusions need further verification by concrete gene function analysis.

YpY and YhY conceived and designed the experiments. XL, YW, and WH analyzed the data. YW, XL, and BL performed the experiments. XL wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.