Identification and functional characterization of the ZmCOPT copper transporter family in maize

Copper (Cu) is an essential micronutrient for plant growth and development; Cu homeostasis in plant is maintained by the important functions of Ctr/COPT-type Cu transporters. Although the COPT genes have been identified in Arabidopsis thaliana and rice, little is known about Cu transporters in maize. In this study, three-members of putative maize Cu transporters (ZmCOPT 1, 2 and 3) are identified. ZmCOPT genes have expression in all of the tested tissues, including roots, stems, leaves and flowers (male and female), and their expression levels vary responding to stress due to Cu-deficiency and excess. Functional complementation and overexpression together with Cu uptake measurements in ZmCOPTs-transformed ctr1⊿ctr2⊿mutant strain or the wild type strain of Saccharomyces cerevisiae show that the three ZmCOPT members possess the ability to be Cu transporters. Among these, ZmCOPT1 and ZmCOPT2 have high-affinity while ZmCOPT3 has low-affinity. In addition, ZmCOPT2 tend to specifically transport Cu (I) but no other bivalent metal ions.


Introduction
Copper (Cu) is an essential micronutrient metal required by plants for growth and development. Cu plays essential roles in many biological processes, such as in photosynthesis, respiratory electron-transport chains, oxidative stress protection, hormone signaling and cell wall metabolism. Cu also acts as a structural element in regulatory proteins and cofactors in many enzymes including cytochrome c oxidaxidase, plastocyanin, superoxide dismutase, and amino oxidase [1]. Cu deficiency in plants induces certain symptoms, such as plant chlorosis (especially in young leaves), defects in cell wall formation and lignification which causes insufficient water transport, and defective pollen development and viability [2]. In biochemical reactions, the utility of Cu depends on the cycle between oxidized Cu (II) and reduced Cu (I) states in enzymes and electron carriers [3]. However, when Cu is in excess, cycling between Cu (I) and Cu (II) can produce highly toxic hydroxyl radicals which subsequently damage a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Ctr/COPT-type Cu transporter genes in maize. The genetic assumption of their ability to transport Cu was performed by functionally complementing the growth defects of the yeast mutant ctr1⊿ctr2⊿. Cu uptake assays have also demonstrated these Cu transporters have specificity in Cu absorption. Moreover, the expression profile of maize Cu transporters in different tissues and under different Cu concentrations supports further evidence that these transporters are related to Cu transportation.

Plant growth and treatment
Seeds of the maize inbred line 178 were surface-sterilized in 0.5% (w/v) NaOCl for 15 min and then 75% (w/v) ethanol for 2 min before being germinated for 3 d at room temperature on paper soaked with distilled water. The seedlings were then transferred into containers with Hoagland's nutrient solution [34] and grown in a greenhouse (70% relative humidity, 14 h/ 28˚C and 10 h/22˚Cday-night cycle and 300 μmol m -2 s -1 intense luminosity) for about 7 d. The nutrient solution was renewed every two days. To analyze plants for effects of Cu stress, ten-day-old seedlings were grown in a Hoagland's solution containing 0.5 μM Cu (CuSO4) (Cu excess) or without Cu (Cu deficiency) for 24 h, respectively.

Gene expression analysis
The expression patterns of maize COPT genes in different tissues (roots, stems, leaves, male and female flowers) was detected by semi-quantitative RT-PCR; response to Cu deficiency and Cu excess stress in plant roots and shoots were detected using real-time quantitative RT-PCR. The ZmCOPT gene-specific primers were designed according to the cDNA sequences obtained from the MaizeSequence database, these information are listed in S3 Table. Total RNA isolation, subsequent cDNA synthesis, semi-quantitative RT-PCR and real-time quantitative RT-PCR were performed as the methods of Li [35]. ZmGAPDH was used as the reference gene in our experiment. Each qRT-PCR or RT-PCR assay was carried out in triplicate in each of the three biological repeats. Statistic analysis was assessed by REST [36].

Plasmid constructs
Complete ORFs of the maize ZmCOPT1-3 genes were amplified by using gene-specific primers and cDNA as the template (S4 Table, S1 Text). PCR was then performed using the highfidelity KOD enzyme (TOYOBO, Shanghai, China). The PCR products were separated on a 1.0% agarose gel and purified using a Gel DNA Purification Kit (Tiangen, Beijing, China). The purified PCR products were then cloned into the pMD19-T vector (TaKaRa, Dalian, China) and verified by sequencing. Subsequently, cDNAs of ZmCOPT1, ZmCOPT2 and ZmCOPT3 were individually subcloned into EcoRI/BamHI sites, BamHI/SacI sites and EcoRI/SacI sites of pGADT7 (+Leu) vectors in which gene expression is driven by the constitutive promoter ADH1.
To test Cu transport activities of ZmCOPT members, ZmCOPTs yeast expression constructs and the corresponding control vectors were transformed into the ctr1⊿ctr2⊿mutant and wild cells of S. cerevisiae using the lithium acetate method [38]. The transformed yeast cells were grown in SC-Leu to OD 600 nm = 1.0 and verified by PCR amplication (S5 Table). The cells were harvested by centrifugation (5,000×g, 2 min) and washed five times with sterile water to remove Cu. Several 10-fold diluted clones of the transformed ctr1⊿ctr2⊿yeast cells were then plated as drops on YPEG and YPD media; clones of the transformed wild yeast cells were plated as drops on YPD media with a Cu supplement (0 mM, 0.5 mM and 1 mM CuSO4). The plates were incubated for 3 to 6 days at 30˚C. The growth rate of the transformed ctr1⊿ctr2⊿strains was also monitored using a slightly modified method. In brief, these strains were grown in liquid YPEG to log phase (OD 600nm = 0.6-0.8). The cells were collected by centrifugation (5,000×g, 10 min) and diluted to an OD 600nm of 0.1 to culture in liquid YPEG at 30˚C, 160 rpm. The OD 600nm was measured every 2 h for the first 12 h and then at 24 h.

Copper uptake measurement in yeast cells
S. cerevisiae ctr1⊿ctr2⊿ mutant cells harboring plasmids were incubated in liquid YPD to log phase (OD 600nm = 0.6-0.8), collected by centrifugation (5,000×g, 10 min), and diluted to an OD 600nm = 1.0. CuCl 2 was then added to 100 ml of the culture to a final concentration of 0.5 μM; cells were incubated at 30˚C, 160 rpm for 10 min. To further detect whether the ZmCOPT Cu transporter proteins had absorbed other metal ions, CuCl 2 , AgNO 3 , FeCl 2 and ZnCl 2 at a final concentration of 0.5 μM was independently added to the cell culture containing ctr1⊿ctr2⊿mutant cells harboring the ZmCOPT2 expression vector and the empty vector (OD 600nm = 1.0). The cells were incubated at 30˚C, 160 rpm for 10 min. The cell culture was centrifuged (5,000×g, 10 min) to remove yeast; the supernatant was retained and residual metal ion contents were analyzed by flame spectrophotometer (Pride SpectrumFP650, Shanghai, China). This test was independently performed in triplicate at least twice.

ZmCOPTs of maize has all the conserved features of known Ctr/COPTtype Cu transporters
To identify the gene family members of COPT/Ctr in the maize genome, a systematic TBLASTP search was performed using six known Arabidopsis and seven known rice full-length COPT amino acid sequences. Based on an e-value threshold of 10 −10 and the typical structural characters of COPT, three non-allelic sequences were identified and named as ZmCOPT1 (GRMZM2G003179), ZmCOPT2 (GRMZM2G042412) and ZmCOPT3 (GRMZM2G317696). The predicted proteins were 157-170 amino acid (aa) residues in length which were separately located on the maize chromosome 7, 3 and 4.
The predicted maize ZmCOPTs share an overall hydrophobicity feature of COPT/Ctr transporters: three TMs, an extracellular amino-terminal and a cytoplasmic carboxy-terminal (Fig 1 and S1 Fig). Among these, TM2 is separated from TM3 by only a few residues (Fig 1), and the conserved motif MxxxM in TM2 and GxxxG in TM3(x representing any amino acid) are found. The amino-terminals of ZmCOPTs also have the conserved Met-rich motifs: one in ZmCOPT1 and two in ZmCOPT2 and ZmCOPT3, respectively. Carboxy-terminals, which eventually release Cu into cytoplasm, tend to have Cys-motifs. As expected, the CxC motif was detected in ZmCOPT1, and the CC motif was detected in ZmCOPT2 at the carboxy-terminal. Surprisingly, the Cys-motif has not been found in ZmCOPT3 (Fig 1). In addition, the promoter analysis showed that the ZmCOPT genes probably involved in light responsiveness, stress response, phytohormone response, etc (S2 Table). Interestingly, CuRE box, a promoter element related to copper ion transport, has not been found in the promoter region of ZmCOPTs. Nevertheless, we have found multiple copy of GTAC motif, the core sequence of CuRE box, in all three promoters of ZmCOPTs (S2 Text). SQUAMOSA promoter binding like protein 7 (SPL7) mediates the transcriptional activation of the genes involved in Cu homeostasis, through binding of its SQUAMOSA promoter binding protein (SBP) domain to the GTAC motifs present in the promoters of the target genes. These targets include the highaffinity Cu uptake transporters COPT1 and COPT2 [39].In addition, the promoter region of ZmCOPT2 contains the AT-rich sequence which is the core sequence of MRE (metal regulatory element) (S2 Table). The potent Cu-responsive AMT1 gene, which encodes a copper metalloregulatory transcription factor, its autoactivation was elicited by the single MRE harbored in its promoter [40].
A phylogenetic tree was further established to examine the evolutionary relationship of 24 COPT/Ctr proteins from various species using the software IQ-TREE (Fig 2). As shown in Fig 2, these COPT/Ctr proteins were classified into four groups. Group 1 included all the members of plant species, seven rice COPTs (OsCOPT1 to OsCOPT7), six Arabidopsis COPTs (AtCOPT1 to AtCOPT6) and three maize COPTs (ZmCOPT1 to ZmCOPT3). Group 2 contained the unique member, baker's yeast (ScCtr1). Group 3 contained two members from humans, hCtr1 and hCtr2. Group 4 included Ctr proteins originated from fission yeast (SpCtr4, SpCtr5, and SpCtr6) and baker's yeast (ScCtr2 and ScCtr3). Among the group, Group 1 had two main clades and each clade contains representatives from all three plant species. The phylogenetic analysis using the software MEGA 5.0 obtained the similar result (S2 Fig). This result indicates a common ancestor gene existed in each clade before the divergence of monocotyledonous and dicotyledonous plants. ZmCOPTs are evolutionarily closely related to plants originated from Arabidopsis AtCOPTs and rice OsCOPTs rather than those of other species. Meanwhile, ZmCOPTs are more closely related to OsCOPTs than to AtCOPTs. For example, ZmCOPT1 is clustered together with rice OsCOPT7, ZmCOPT2 with OsCOPT2, and ZmCOPT3 with OsCOPT3 and OsCOPT4 (Fig 2); this is consistent with the close evolutionary relationship between maize and rice.

The expression patterns of ZmCOPT genes were influenced by Cu stress conditions
To learn more about the function of ZmCOPTs, the expression pattern of the three ZmCOPT genes was examined. As shown in Fig 3A, ZmCOPT genes were expressed in all of the tested tissues, including roots, stems, leaves and flowers (male and female). To further characterize the roles of potential Cu transport ZmCOPT in maize plants, the expression of ZmCOPT genes in both roots and shoots under stress conditions (Cu-deficient or Cu-excess) were investigated. The expression of the ZmCOPT1 gene was strongly up-regulated under Cu-deficient conditions whereas it was suppressed by Cu-excess stress in shoots; results from roots indicated no significant variation due to Cu-deficient or excess. The expression level of ZmCOPT2 was stimulated by a deficiency of Cu and suppressed by an excess of Cu in both shoots and roots. Interestingly, the expression of ZmCOPT3 was induced in both shoots and roots regardless of the Cu levels (deficient or excess). These results reveal that the expression levels of ZmCOPT genes respond to some extent by an excess/deficiency of Cu in the environment.

Cloning of ZmCOPT genes and functional complementation in the ctr1⊿ctr2⊿ mutant of S. cerevisiae
To investigate the potential role of the three ZmCOPTs in Cu transport, the complete cDNA of these ZmCOPT genes were amplified by gene-specific primer and confirmed by sequencing (S3 Table,  Among these, the wild type strain BY4742 of S. cerevisiae was used as the positive control while the ctr1⊿ctr2⊿ mutant strain and the ctr1⊿ctr2⊿ mutant strain transformed with the empty pGADT7 vector were used as the negative controls. As shown in Fig 4A, when the three ZmCOPT genes were individually transformed, the growth defect of the ctr1⊿ctr2⊿ mutant was restored on the YPEG media. Results showed that ZmCOPT1 or ZmCOPT2 could efficiently rescue the growth defect of ctr1⊿ctr2⊿ mutant while the complementary effect of ZmCOPT3 was weaker (Fig 4A). To further assess the complementation of each ZmCOPT gene in ctr1⊿ctr2⊿ mutant strains, the growth rate of wild type strains and all transformed mutant strains in liquid YPEG were also monitored. As shown in Fig 4B, the ctr1⊿ctr2⊿ mutant strains transformed with ZmCOPT3 grew more slowly than wild type strains and those strains transformed with ZmCOPT1 or ZmCOPT2; ZmCOPT3 therefore reached the stationary phase before ZmCOPT1 or ZmCOPT2. This result is consistent with the weak rescuing phenotype of ZmCOPT3 observed on agar plates, suggesting that both ZmCOPT1 and ZmCOPT2 can mediate high-affinity Cu transport in maize whilst ZmCOPT3 can mediate low-affinity Cu transport.

Overexpression of ZmCOPT genes conferred growth retardation of wild type S. cerevisiae
Excessive copper uptake will be toxic to the cell. On YPD media with and without Cu, an attempt to clarify this suggestion was undertaken by monitoring the growth of wild type S. cerevisiae and those (wild type background) individually transformed with ZmCOPT genes. As shown in Fig 4C, the growth of wild type strains and those transformed with ZmCOPTs had a similar phenotype without Cu or with the addition of 0.5 mM Cu on YPD media. However the growth of ZmCOPTs overexpressed yeast strains with the addition of 1 mM Cu were severely inhibited compared with the wild type yeast strains. These results firmly support genetic evidence that ZmCOPTs necessarily participate in the process of Cu transport.

Copper uptake measurements in the ctr1⊿ctr2⊿ mutant of S. cerevisiae
To further evaluate the Cu transport function of the maize ZmCOPTs, Cu uptake assays were individually carried out in ctr1⊿ctr2⊿mutant strains transformed with ZmCOPT1, 2 and 3. Cu content obviously decreased in the supernatant of ZmCOPT-transformed yeast strains compared with those transformed with the empty vector (Fig 5). Consistent with our growth complementation results, Cu uptake was much higher in ctr1⊿ctr2⊿mutant strains transformed by ZmCOPT1 or ZmCOPT2 than by ZmCOPT3 (Fig 5). Despite Cu, the content of other metals, such as iron, silver and zinc, were also measured to verify the transport specificity of ZmCOPTs. ZmCOPT2 was selected for the analysis as it was the highest-affinity Cu transporter of the three maize genes. The results demonstrated that in the supernatant of ZmCOPT2 transformed ctr1⊿ctr2⊿ mutant strains, there was only a slight decrease in iron or zinc content against a distinct decline in Ag content (9.65%) and Cu (14.41%) in comparison with the control (Fig 6). Taken together, these results indicated that restoration Cu uptake defects via expression of ZmCOPT1, 2 and 3 are coincidently with growth defects. The results also show that ZmCOPT2 tend to specifically transport Cu (I) (and Ag) but no other bivalent metal ions.

Discussion
Cu is involved in a range of important biological processes which makes it essential for normal plant growth and development. Although COPT genes encoding Cu transporter proteins have been identified in several plant species, their function has only been reported in Arabidopsis and rice [10,15,29]. In this study, we report the identification and characterization of three members of the ZmCOPT gene family in maize.
ZmCOPTs share a conserved structure with Cu transporter homologs from rice and Arabidopsis. For example, each ZmCOPT contains three TMs and the conserved residues were largely restricted to the three TMs, giving the clue of functional conservation of these ZmCOPTs. In addition, as each TM2 of the ZmCOPT genes contains the MxxxM motif, this suggests ZmCOPTs are potential Cu transporters. The MxxxM motif is essential for Cu uptake and may coordinate Cu during intramembraneous transport [17]. Substitution of the methionine of TM2 with alanine or serine in yCtr and hCtr proteins resulted in the proteins losing ZmCOPTs expression vector or the empty vector pGADT7, and copper content in liquid YPD without yeast cells as a control. The error bars represent ± SD. The sign" Ã "and " ÃÃ "presented as statistically significant difference at P < 0.05 and at P < 0.01 level (student t-test). https://doi.org/10.1371/journal.pone.0199081.g005 The ZmCOPT copper transporters of maize the ability to transport Cu [17]. And AtCOPT4 protein which does not possess the MxxxM motif is also incapable of restoring the growth defect of ctr1⊿ctr3⊿ mutant of S. cerevisiae [10]. Furthermore, all three ZmCOPTs have the Met-rich motif in their amino-terminal which can sequester Cu (I) to the Cu transport channel, but the number of this motif varied. ZmCOPT1 and ZmCOPT2 also contain the Cys-rich motif, a motif which controls the release of Cu into the cytoplasm; this motif is not present in ZmCOPT3. In addition, GTAC motif and the ATrich element found in the promoter region of ZmCOPT2 were related to Cu transport [39,40]. These conserved structural features of ZmCOPTs strongly suggest a functional involvement in Cu transport; variation however may occur among the ZmCOPTs to enable them to adapt to fluctuating environmental variation of Cu. The fact that ZmCOPTs clustered together with COPTs originating from plants in the same group of the phylogenetic tree provides further proof that ZmCOPTs are Cu transporters.
Generally, the expression pattern of a gene is closely correlated with its function. In our investigation, the three ZmCOPT genes had displayed a constitutively expression pattern in all of the detected tissues, but the expression level are not the same for all organs (Fig 3), suggesting that maybe each ZmCOPT has its function in a specific part of the plant. This expression pattern has also been reported for the COPT transporters in Arabidopsis (except AtCOPT6), rice and grapevine [10,29,31]. On the other hand, ZmCOPT genes had a distinct response under Cu stress conditions. For example, the expression of ZmCOPT1 in shoots and ZmCOPT2 in shoots and roots was induced by a deficiency of Cu and suppressed with an The ZmCOPT2 showed transport specificity to Cu. Copper, silver, iron and zinc content were measured in supernatant of the ctr1⊿ctr2⊿ yeast mutant transformed with ZmCOPT2 or the empty vector pGADT7. Ion content was compared to control cells (ctr1⊿ctr2⊿yeast mutant transformed with pGADT7) represented as 100% (control bar). The error bars represent ± SD. The sign" Ã "and " ÃÃ "presented as statistically significant difference at P < 0.05 and at P < 0.01 level (student t-test). https://doi.org/10.1371/journal.pone.0199081.g006 The ZmCOPT copper transporters of maize excess of Cu; ZmCOPT3 was induced in both shoots and roots regardless of Cu-deficient or Cu-excess (Fig 3). In the same case, the expression of AtCOPT2 was induced under Cu-deficient conditions and the expression of AtCOPT1 and AtCOPT2 were suppressed with an excess of Cu [10]. And the expression of OsCOPT1, 2, 5 and 7 was induced under Cu-deficient conditions and suppressed with an excess of Cu in rice [29]. An exploring with promoter-fused GUS in plant under Cu deficiency (using Cu chelator) could give more information of the expression pattern of ZmCOPT genes.
Previous investigations have reported that a series of Ctr/COPT-type Cu transporters can complement the phenotype of S. cerevisiae ctr1⊿ctr3⊿ mutant, these include mCtr1 of mice [26], hCtr1 of humans [24], AtCOPT1, 2, 3, and 5 of Arabidopsis [10,41], and OsCOPT7 of rice [29]. In our investigation, all of the three maize ZmCOPT genes also demonstrated their function in Cu transport by genetic complementation of the ctr1⊿ctr2⊿ mutant. Measurement of Cu content in the corresponding transformed mutant strains was in accordance with the function of ZmCOPTs in Cu transport and the complementation test (Figs 4A and 5). On the contrary, the growth of ZmCOPTs-transformed wild type strain was inhibited by excess Cu, the result is similar to the severe growth retardation of yeast strains transformed with the Cu transporter gene TaCT1 in the presence of 0.5 mM Cu [42], thus providing additional supporting evidence for the ability of ZmCOPT for Cu transport.
Interestingly, the growth rate of ZmCOPT3-transformed mutant strains was considerably lower than that of ZmCOPT1-or ZmCOPT2-transformed mutant strains, whether in solid or liquid media, suggesting a different affinity of these ZmCOPTs in the process of Cu transport: ZmCOPT1 and ZmCOPT2 had high-affinity and ZmCOPT3 had low-affinity. This finding seems to be partially attributed to the variation in number of Met-rich motifs in the amino-terminal of the ZmCOPTs. High-affinity Cu transporters with two Met-rich motifs had a higher ability for Cu transport than those with one [10,15]. And deletion of the eight Met-rich motifs of yCtr1 decreased copper uptake rate by one-third compared with the wild type [17]. In our study, the high-affinity Cu transporter ZmCOPT2 which had two Met-rich motifs in the amino-terminal seemed transport more Cu than ZmCOPT1, which only had one Met-rich motif, according to the growth inhibition. However, Met-rich motifs could not be used as a measuring criterion of the affinity of Cu transporters. Otherwise, ZmCOPT3-transformed wild type and mutant strains of S. cerevisiae would have a higher Cu transport ability or Cu content than ZmCOPT1-transformed. Actually, ZmCOPT3 (with two Met-rich motifs) had the weakest ability to transport Cu among the three ZmCOPTs. Similarly, the high-affinity Cu transporter yCtr3 contains no Met-rich motifs and OsCOPT7 has one motif in the amino-terminal; low-affinity Cu transporters OsCOPT3 and OsCOPT4 have three motifs [8,29]. Results for phylogenetic analysis showed that ZmCOPT1 was more related to rice OsCOPT7, this being the only gene which could mediate high-affinity copper transporter alone; ZmCOPT2 was more related to rice OsCOPT2, which could cooperate with OsCOPT6 to mediate high-affinity Cu uptake and ZmCOPT3 is more related to rice OsCOPT3 and OsCOPT4, which could mediate low-affinity Cu transport in rice [29]. As to the low-affinity of ZmCOPT3, at least two factors should be taken into account. Firstly, because the Cys-rich motif in the carboxy-terminal of yeast Ctr1 is dispensable for high affinity uptake, the lack of the Cys-rich motif in carboxy-terminal region of ZmCOPT3 suggests that this does not affect its low-affinity to Cu. Secondly, ZmCOPT3 has the special up-regulated expression pattern whether subjected to Cudeficiency or Cu-excess stress, perhaps caused by a lack of regulation domain (the Cys-rich motif) in the carboxy-terminal which prevents excessive uptake of Cu [43]. These factors, together with the low-affinity of Cu transport of ZmCOPT3, suggested that it may play a role in basal level Cu transport in a fluctuating environment. Otherwise, ZmCOPT3 might be controlled by the feedback regulation of biological requirement in maize. Furthermore, the ZmCOPT2 protein is a high-affinity transporter with specificity for Cu (I), but not for other bivalent ions, such as Mn or Zn, similar to Arabidopsis COPT1 and other Ctr1 family members (Fig 6) [10,11].Moreover, like hCtr1, ZmCOPT2 could also transport Ag due to similar isoelectronic structure of Ag (I) and Cu (I) which can cause competition [11,12]. So far, COPT/Ctr proteins function specifically in Cu transport. Though the endogenous iron, manganese, or zinc may influence Cu homeostasis by influencing the expression of COPTs in rice, none of them could restore the Fe-or Zn-uptake functions of the yeast mutants [29]. Thus, just like other COPTs, whether or not, the ZmCOPT1 and ZmCOPT3 also show specificity in Cu transport, is required further study to determine. Meanwhile, the direct assessment of Cu content in the ZmCOPTs-transformed yeast cells and conduction the subcellular location study in plant cells and promoter analysis in plant system would also be powerful to explain the Cu transport ability of the ZmCOPTs in the future.
In conclusion, our investigation identified three ZmCOPT transporters and suggested that ZmCOPT genes may play essential roles in the uptake and translocation of Cu and that they respond to fluctuating environmental levels of Cu. However, the precise roles of individual ZmCOPT genes require further research.