Genome-Wide Investigation and Functional Verification of the ZIP Family Transporters in Wild Emmer Wheat

The zinc/iron-regulated transporter-like protein (ZIP) family has a crucial role in Zn homeostasis of plants. Although the ZIP genes have been systematically studied in many plant species, the significance of this family in wild emmer wheat (Triticum turgidum ssp. dicoccoides) is not yet well understood. In this study, a genome-wide investigation of ZIPs genes based on the wild emmer reference genome was conducted, and 33 TdZIP genes were identified. Protein structure analysis revealed that TdZIP proteins had 1 to 13 transmembrane (TM) domains and most of them were predicted to be located on the plasma membrane. These TdZIPs can be classified into three clades in a phylogenetic tree. They were annotated as being involved in inorganic ion transport and metabolism. Cis-acting analysis showed that several elements were involved in hormone, stresses, grain-filling, and plant development. Expression pattern analysis indicated that TdZIP genes were highly expressed in different tissues. TdZIP genes showed different expression patterns in response to Zn deficiency and that 11 genes were significantly induced in either roots or both roots and shoots of Zn-deficient plants. Yeast complementation analysis showed that TdZIP1A-3, TdZIP6B-1, TdZIP6B-2, TdZIP7A-3, and TdZIP7B-2 have the capacity to transport Zn. Overexpression of TdZIP6B-1 in rice showed increased Zn concentration in roots compared with wild-type plants. The expression levels of TdZIP6B-1 in transgenic rice were upregulated in normal Zn concentration compared to that of no Zn. This work provides a comprehensive understanding of the ZIP gene family in wild emmer wheat and paves the way for future functional analysis and genetic improvement of Zn deficiency tolerance in wheat.


Introduction
Zinc (Zn) is one of the most essential micronutrients for plants and humans and plays a critical role in diverse biochemical processes [1,2]. Zn is irreplaceable for plant normal growth and development [3]. Both deficient and excess Zn has negative effects on the physiological and biochemical processes of the plant [4]. The Zn availability in agriculture soils of many parts of the world is low, which leads to yield reduction and poor nutritional quality in harvested grains [5]. It was estimated that one-third of the world's population suffers from inadequate intake of Zn, resulting in various health problems [5][6][7]. A foodbased strategy (biofortification) is considered the most cost effective and sustainable option for improvement of human health [8]. Thus, improving the Zn nutrition in crop varieties

Phylogenetic Relationships and Comparative Analysis of the ZIP Gene Family in Wild Emmer Wheat
A total of 33 putative ZIP genes were identified and confirmed from the wild emmer reference genome. These ZIP genes were tentatively designated as TdZIP1A-1 to TdZIP7B-3 (Table 1) according to their locations on chromosomes. We found that these ZIPs were unevenly distributed on the chromosomes, with 16 and 17 genes positioned on the A and B chromosomes, respectively (Table 1, Figure S1). The first chromosome group had the largest number of TdZIPs (10 genes), followed by the second (7 genes), the sixth (5 genes), and the seventh chromosome groups (5 genes). The remaining chromosomes had one TdZIP gene each ( Figure S1). Gene structure analysis revealed that the number of introns varied from 0 to 11 ( Figure S2A), and exons ranged from 1 to 12 among the 33 TdZIP genes. The amino acid lengths of the TdZIPs varied from 68 (TdZIP2A-2) to 577 (TdZIP1A-3 and TdZIP1B-3), the PIs ranged from 5.05 (TdZIP2A-2) to 9.8 (TdZIP2A-3), and these proteins had 1 to 13 TM domains. Using the MEME tool, we identified 15 conserved motifs with length varied from 6 to 50 amino acids ( Figure S2B). Most of the TdZIP proteins were predicted to be localized on the plasma membrane, except three TdZIPs (TdZIP6A-2, TdZIP6B-2, and TdZIP7B-1) were predicted to be localized on the endoplasmic reticulum (ER) ( Table 1), suggesting that these three genes may be responsible for transporting Zn from the ER to the cytoplasm.
We constructed a phylogenetic tree using 33 TdZIPs and 29 ZIPs from rice, maize, and Arabidopsis to further study the phylogenetic relationships between TdZIPs and other ZIPs in plants. The result is presented in Figure 1. In the phylogenetic tree, these ZIPs were divided into three clades: clade I (1 OsZIP, 2 ZmZIPs, 2 AtZIPs, and 6 TdZIPs), clade II (5 OsZIPs, 2 ZmZIPs, 4 AtZIPs, and 13 TdZIPs), and clade III (5 OsZIPs, 3 ZmZIPs, 5 AtZIPs, and 14 TdZIPs). We further investigated the collinearity relationship of ZIP genes between wild emmer wheat and common wheat. The 33 TdZIPs genes were blasted against the genome of Chinese Spring (CS). Among them, 32 ZIPs had homologous genes/sequences in the CS genome, and 15 genes were able to be detected with 82-100% identities. One gene TdZIP5B-1 was not matched with CS genes ( Figure S3, Table S1).

Functional Annotation and Promoter Analysis of TdZIP Genes
To investigate the potential functions of ZIPs, we performed GO, KOG, and Swiss_Prot_ annotation analyses for the 33 TdZIPs. GO terms for those ZIP genes can be classified into three categories: biological process (BP), cellular component (CC), and molecular function (MF). In the BP annotation, TdZIP proteins predicted their functions in cellular process (GO:0009987), single-organism process (GO:44699), location (GO:0051179), and response to stimulus (GO:0050896). In the CC annotation, TdZIP proteins percentage annotated with the membrane (GO:0016020), membrane part (GO:0044425) and cell (GO:0005623), cell part (GO:0044464), organelle (GO:0043226), and organelle part (GO:0044422). Transporter activity (GO:0005215), catalytic activity (GO:0003824), and binding (GO:0005488) were annotated in the MF annotation ( Figure S4A). KOG annotation revealed that all TdZIPs were involved in inorganic ion transport and metabolism ( Figure S4B). In addition, Swiss_Prot_annotation showed that most of TdZIPs (27 genes) were orthologous to OsZIP genes in rice (Table S2). The remaining TdZIPs were orthologous to AtZIP genes in Arabidopsis thaliana. These results were consistent with the phylogenetic analysis data.

Functional Annotation and Promoter Analysis of TdZIP Genes
To investigate the potential functions of ZIPs, we performed GO, KOG, and Swiss_Prot_annotation analyses for the 33 TdZIPs. GO terms for those ZIP genes can be classified into three categories: biological process (BP), cellular component (CC), and molecular function (MF). In the BP annotation, TdZIP proteins predicted their functions in cellular process (GO:0009987), single-organism process (GO:44699), location (GO:0051179), and response to stimulus (GO:0050896). In the CC annotation, TdZIP proteins percentage annotated with the membrane (GO:0016020), membrane part (GO:0044425) and cell (GO:0005623), cell part (GO:0044464), organelle (GO:0043226), and organelle part (GO:0044422). Transporter activity (GO:0005215), catalytic activity (GO:0003824), and binding (GO:0005488) were annotated in the MF annotation ( Figure  S4A). KOG annotation revealed that all TdZIPs were involved in inorganic ion transport and metabolism ( Figure S4B). In addition, Swiss_Prot_annotation showed that most of To further understand the role of TdZIPs, we used the 2000 bp upstream sequence from the translation initiation site (ATG) of TdZIP genes and analyzed in the PlanCARE for cis-element prediction. The results showed that hormone, stresses, and grain-filling, and developmental responsive cis-elements were widely predicted in TdZIPs promoters. In hormone category, abscisic acid (ABRE), MeJA (CGTCA_motif), auxin (TGA_element), salicylic acid (TCA), and gibberellin (GARE_motif) responsive cis-elements were dominant (Table S3). Abscisic acid-responsive element was the most frequent in TdZIPs promoters. Category for stresses had a different type of cis-regulatory elements. For instance, LTR involved in low-temperature responses, MBS in drought-inducibility, ARE in anaerobic induction, and TC_rich_repeats in defense and stress responses ( Figure S5). In grain-filling and developmental category, zein metabolism regulation (O2_site) was the most frequent elements in TdZIP promoters, followed by meristem expression (CAT_box), light responsive element (TCCC_motif), seed-specific regulation (RY_element), and endosperm expression (GCN4_motif). These results showed that the TdZIPs may undergo different types of transcriptional regulation and the potential diversity functions of TdZIPs in wild emmer.

Yeast Complementation Analysis
To determine the biological function of TdZIPs, we chose six TdZIP genes (TdZIP1A-2, TdZIP1A-3, TdZIP6B-1, TdZIP6B-2, TdZIP7A-3, and TdZIP7B-2) for yeast complementation analysis. The coding sequences of these genes were cloned and expressed in the wildtype yeast strain DY1457 and extracellular Zn transporter (zrt1/zrt2) double mutant. The zrt1/zrt2 mutant cells transformed with TdZIP genes were grown on SD media containing different Zn concentrations. Among them, cells transformed with TdZIP6B-1 had the strongest growth on SD media with ZnSO 4 . TdZIP genes TdZIP1A-2, TdZIP1A-3, TdZIP6B-1, and TdZIP7B-2 were able to fully complement and TdZIP6B-2 and TdZIP7A-3 were able to partially complement the growth phenotypes of the double zrt1/zrt2∆ yeast mutant ( Figure 3). These results suggest that the six TdZIPs genes have the ability to transport Zn. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 15

Figure 2. Expression levels of 15
TdZIP genes in the shoots and roots in response to Zn-deficient stress. Error bars indicate the mean values between three replicates ± standard deviation (SD). * denotes the statistically differences at p < 0.05 (Student's t-test). Error bars indicate the mean values between three replicates ± standard deviation (SD). * denotes the statistically differences at p < 0.05 (Student's t-test).
type yeast strain DY1457 and extracellular Zn transporter (zrt1/zrt2) double mutant. The zrt1/zrt2 mutant cells transformed with TdZIP genes were grown on SD media containing different Zn concentrations. Among them, cells transformed with TdZIP6B-1 had the strongest growth on SD media with ZnSO4. TdZIP genes TdZIP1A-2, TdZIP1A-3, TdZIP6B-1, and TdZIP7B-2 were able to fully complement and TdZIP6B-2 and TdZIP7A-3 were able to partially complement the growth phenotypes of the double zrt1/zrt2Δ yeast mutant ( Figure 3). These results suggest that the six TdZIPs genes have the ability to transport Zn.

Analysis of Phenotypes and Metal Concentrations in TdZIP6B-1 Overexpression Rice Plants
The yeast complementation experiment showed that TdZIP6B-1 has the highest ability to complement the growth phenotypes of the double zrt1/zrt2Δ yeast mutant. The cDNA sequence of TdZIP6B-1 shares 95.77% identity with that of TaZIP6B-1 in CS. TdZIP6B-1 was phylogenetically close to Arabidopsis gene AtZIP11, maize gene ZmZIP2, and rice gene OsZIP2 (Figure 1). We constructed transgenic plants overexpression TdZIP6B-1 to further verify the function of TdZIP6B-1. The TdZIP6B-1 overexpression line (TdZIP6B) together with wild-type (WT) cultivar (Oryza. Sativa L. spp. Japonica) were exposed to no Zn (0 mg/L ZnSO4) and normal Zn conditions (8.6 mg/L ZnSO4) ( Figure 4A). The TdZIP6B-1 overexpression lines had higher fresh weight of roots and shoots than those of WT plants after 14 days growth under normal Zn conditions ( Figure 5B, C). The total roots length of overexpression lines was significantly longer than that of WT plants ( Figure 5A,D). The total roots area and volume were higher than those of WT plants, but the differences were not significant ( Figure 5E,F). In roots, the expression levels of TdZIP6B-1 were significantly higher in normal Zn condition than those of in no Zn condition at 5 days post-treatment ( Figure 4B). No significant differences were observed for the expression levels of TdZIP6B-1 at 7 and 9 days. In shoots, the expression levels of TdZIP6B-1 were significantly higher in normal Zn condition than those of in no Zn condition at 7 days post-treatment ( Figure 4C). We further examined the Zn, Mn, and Fe concentrations in roots and shoots after 14 days of growth under normal Zn condition. The results

Analysis of Phenotypes and Metal Concentrations in TdZIP6B-1 Overexpression Rice Plants
The yeast complementation experiment showed that TdZIP6B-1 has the highest ability to complement the growth phenotypes of the double zrt1/zrt2∆ yeast mutant. The cDNA sequence of TdZIP6B-1 shares 95.77% identity with that of TaZIP6B-1 in CS. TdZIP6B-1 was phylogenetically close to Arabidopsis gene AtZIP11, maize gene ZmZIP2, and rice gene OsZIP2 (Figure 1). We constructed transgenic plants overexpression TdZIP6B-1 to further verify the function of TdZIP6B-1. The TdZIP6B-1 overexpression line (TdZIP6B) together with wild-type (WT) cultivar (Oryza. Sativa L. spp. Japonica) were exposed to no Zn (0 mg/L ZnSO 4 ) and normal Zn conditions (8.6 mg/L ZnSO 4 ) ( Figure 4A). The TdZIP6B-1 overexpression lines had higher fresh weight of roots and shoots than those of WT plants after 14 days growth under normal Zn conditions ( Figure 5B, C). The total roots length of overexpression lines was significantly longer than that of WT plants ( Figure 5A,D). The total roots area and volume were higher than those of WT plants, but the differences were not significant ( Figure 5E,F). In roots, the expression levels of TdZIP6B-1 were significantly higher in normal Zn condition than those of in no Zn condition at 5 days post-treatment ( Figure 4B). No significant differences were observed for the expression levels of TdZIP6B-1 at 7 and 9 days. In shoots, the expression levels of TdZIP6B-1 were significantly higher in normal Zn condition than those of in no Zn condition at 7 days post-treatment ( Figure 4C). We further examined the Zn, Mn, and Fe concentrations in roots and shoots after 14 days of growth under normal Zn condition. The results showed that the transgenic plants had higher Zn, Fe, and Mn concentrations compared with that of WT plants in roots ( Figure 4D), while those of in shoots between overexpression lines and WT plants were not significantly different ( Figure 4E,F). showed that the transgenic plants had higher Zn, Fe, and Mn concentrations compared with that of WT plants in roots ( Figure 4D), while those of in shoots between overexpression lines and WT plants were not significantly different ( Figure 4E,F).

Discussion
Zn is a micronutrient element that is necessary for all living organisms [8,27]. Zn deficiency in major food crops could lead to yield reduction and poor nutritional quality [2]. Zn homeostasis is tightly regulated by kinds of proteins, with Zn transporter proteins being particularly important [27,28]. The ZIP transporters contribution to Zn homeostasis showed that the transgenic plants had higher Zn, Fe, and Mn concentrations compared with that of WT plants in roots ( Figure 4D), while those of in shoots between overexpression lines and WT plants were not significantly different ( Figure 4E,F).

Discussion
Zn is a micronutrient element that is necessary for all living organisms [8,27]. Zn deficiency in major food crops could lead to yield reduction and poor nutritional quality [2]. Zn homeostasis is tightly regulated by kinds of proteins, with Zn transporter proteins being particularly important [27,28]. The ZIP transporters contribution to Zn homeostasis

Discussion
Zn is a micronutrient element that is necessary for all living organisms [8,27]. Zn deficiency in major food crops could lead to yield reduction and poor nutritional quality [2]. Zn homeostasis is tightly regulated by kinds of proteins, with Zn transporter proteins being particularly important [27,28]. The ZIP transporters contribution to Zn homeostasis has been widely reported in many plant species, including common wheat [21]. To our knowledge, however, the ZIP family has not been well studied in wild emmer wheat. In the present study, we performed a genome-wide investigation of ZIP family in wild emmer and identified 33 TdZIP genes. Although most (97%) TdZIPs had homologous genes/sequences in common wheat cv. CS genome, large sequences differences in ZIP genes were detected between wild emmer and CS. These TdZIP genes in wild emmer can provide new candidate genes for improving the nutritional quality of wheat.
The phylogenetic tree of the TdZIPs genes was clustered into three clades. In clade I, six TdZIPs were closely related to OsZIP10, AtZIP4, AtZIP9, ZmZIP5, and ZmZIP7. The OsZIP10 was found to be associated with grain Zn content in rice [20]. A previous study had shown that AtZIP9 complemented a yeast Mn uptake-deficient mutant, while it did not complement the Zn uptake-deficient mutant [29]. ZmZIP5 played an important role in Zn and Fe uptake and root-to-shoot translocation in maize [30]. In clade II, 13 TdZIPs were clustered with OsZIP1, AtZIP2, and AtZIP11, which were previously shown to have ability to transport Zn and response to Zn deficiency [21,29,31]. A total of 14 TdZIPs in clade III were clustered together with OsZIP3, OsZIP4, OsZIP5, OsZIP8, OsZIP9, ZmZIP3, ZmZIP4, ZmZIP8, AtZIP1, AtZIP3, AtZIP5, AtZIP7, and AtZIP12. The OsZIP3, OsZIP4, OsZIP5, OsZIP8, AtZIP1, AtZIP3, AtZIP7, and AtZIP12 had been verified to have ability to transport Zn in plants [11,12,20,29,32,33], while the ZmZIPs could complement the transport of Zn and Fe in yeast mutants [34]. Taken together, these data indicate that the TdZIPs closely related to OsZIP, ZmZIP, and AtZIP genes are likely to have the ability to absorb and transport Zn and Mn and respond to Zn deficiency.
The cis-regulatory elements present in the promoter region have an important role in plant regulation control and in different stimulus responsive genes [35]. On the basis of ciselement analysis, many TdZIPs were predicted to have hormone, stress, grain-filling, and developmentally responsive cis-elements. We have identified different types of cis-elements such as the hormones ABRE, which participates in ABA responses [36]; CGTCA_motif, which has a role in MeJA responses [37]; and TGA_element, which participates in auxin responses [38]. The cis-element for stress responses is LTR, which is involved in lowtemperature responses [39]; meanwhile, MBS functions in drought-inducibility [40], ARE is involved in anaerobic induction, and TC_rich_repeats participate in defense and stress responses [41]. For grain-filling and developmental, O2_site has a role in zein metabolism regulation [42], CAT_box functions in meristem expression [38], TCCC_motif participates in light responsiveness [43], RY_element functions in seed-specific regulation [44], and GCN4_motif is involved in endosperm expression [45]. The presence of different types of cis-elements in TdZIPs implies the different transcriptional regulatory mechanisms in which the TdZIPs genes may be involved.
The expression pattern of genes is often tightly correlated with their functions [30]. The expression data of TdZIPs in different tissues at different time points are important for understanding genes' functions in which they participate. Previous studies have reported that ZIP genes were mainly expressed in roots and shoots of rice, Arabidopsis, and common wheat [29,34,46,47]. In this study, six TdZIPs (TdZIP1A-1, TdZIP1A-2, TdZIP1A-3, TdZIP1B-3, TdZIP6B-2, and TdZIP7B-2) were mainly expressed in root and/or leaf tissues, suggesting their functions in these tissues, which is consistent with previous studies [8,46]. We found some TdZIP genes were highly expressed in developing spike and glume (e.g., TdZIP1B-5, TdZIP2B-3, and TdZIP2A-3), flower (TdZIP6B-3), and grain (TdZIP1A-1 and TdZIP6B-2), suggesting their functions in spike developmental and grain filling processes. Interestingly, TdZIP6B-2 had high expression in most tissues sampled at different time points, implying the potential diverse functions of this gene.
Several ZIP genes related to Zn transportation were upregulated in different tissues under Zn deficiency. For example, OsZIP1 was upregulated in roots and the transcripts were not detected in shoots under Zn deficiency [11,32,46,48]. Zn deficiency induced the expression of HvZIP6 in roots, while it did not in shoots [8]. TaZIP3, TaZIP5, TaZIP6, TaZIP7, and TaZIP13 were upregulated in both roots and shoots under Zn deficiency [21]. In the present study, different expression patterns of TdZIPs in roots and shoots responding to Zn deficiency were determined. We observed that 11 TdZIPs were significantly induced in roots under Zn deficiency, of which three genes (TdZIP1A-4, TdZIP6B-1, and TdZIP7B-1) were highly induced in both roots and shoots of Zn-deficient plants, suggesting that these genes are involved in Zn deficiency responses and enhanced absorbance and root-to-shoot translocation of Zn. Three genes (TdZIP1B-3, TdZIP6A-2, and TdZIP6B-2) showed little or no sensitivity to Zn deficiency or the normal Zn concentration, suggesting that they contribute little to the uptake and root-to-shoot translocation of Zn.
High-affinity Zn transporter Zrt1 is required for yeast growth in Zn-limiting media. Low-affinity transporter Zrt2 can transport Zn under mild or abundant Zn concentrations, whereas it cannot transport Zn under more severe Zn-limiting media [49,50]. Mutations on each of them will lead to yeast growth abnormal under Zn deficiency. Therefore, the yeast zrt1/zrt2∆ mutant has been widely used to demonstrate the complement ability of Zn transporter genes in rice, maize, barely, Arabidopsis, and common wheat under Zn-deficient conditions [18,19,21,51]. In wheat, TdZIP1 and TaZIP3, -5, -6, -7, and -13 have been identified to have the ability to transport Zn by functional complementation study of the zrt1/zrt2 yeast mutant [21,25]. In this study, six genes that belong to three clades of TdZIPs ( Figure 3) were randomly selected to rescue the growth defects in the zrt1/zrt2∆ mutant under Zn-deficient conditions. The results revealed that four genes TdZIP1A-2, TdZIP1A-3, TdZIP6B-1, and TdZIP7B-2 rescued the growth defects of the zrt1/zrt2∆ mutant, suggesting that these TdZIPs could transport Zn effectively.
In the present study, we further verified the Zn transport capability of TdZIP6B-1 by rice transformation. We found that the expression levels of TdZIP6B-1 in transgenic rice lines were higher under normal Zn condition than that of no Zn condition in both the roots and shoots. The Zn concentrations in roots of TdZIP6B-1 overexpression lines were higher than the WT plants. Previous reports showed that the overexpression of OsZIP4 and OsZIP5 in rice [12,52] and TaZIP13-B [53] in Arabidopsis could enhance the concentration of Zn in roots and shoots. Our preliminary results verified that the TdZIP6B-1 has the ability to uptake and probably induce root-to-shoot translocation of Zn. In addition, the accumulated Fe and Mn concentrations in roots and shoots of overexpression lines indicate that TdZIP6B-1 may have pleiotropic effects on metal ion absorption in plants.

Plant Materials and Zn-Deficient Treatments
The advanced wheat line BAd7-209 (T. aestivum CN16/T. dicoccoides D97 F 12 ) [61] and transgenic rice (Oryza. Sativa L. spp. Japonica) were used in the present study. The seeds were germinated on sterile culture dish with filter paper for 7 days, and seedlings were transplanted into Hogland nutrient solution with two concentrations of Zn, supplemented in the form of ZnSO 4 , including no Zn (Zn deficiency) and normal Zn supply (8.6 mg/L ZnSO 4 .7H 2 O). Leaves and roots of the individuals were harvested at 1, 3, 5, 7, and 9 days after Zn-deficient treatments. All samples were stored at −80 • C for further analysis.

RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
Total RNA from leaf and root samples was isolated using plant extraction kit v1.5 (Biofit Biotechnologies, Chengdu, China). First-strand cDNA synthesis was performed using the TaKaRa PrimeScriptTMRT Reagent Kit (Takara, Dalian, China) according to the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed using the Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) in 10 µL reaction volume containing 2 ng of cDNA, 5 µL of 1 × SYBR Premix Ex Taq (TaKaRa), and 0.5 µL (300 nM) of each primer. The GAPDH gene was used as the internal reference. The gene expression was quantified using the 2 −∆∆CT method [62]. Three biological replicates were used for each data point.

Rice Transformation
The cDNA of TdZIP6B-1 was cloned into the overexpression vector pCAMBIA2300-GFP (pCAMBIA2300-GFP-TdZIP6B-1). The construct had KpnI and SpeI on the 3 side of the CaMV 35S promoter. An Agrobacterium tumefaciens strain (AGL1) carrying the above construct was used to transform rice (Oryza. Sativa L. spp. japonica) following the method of [11]. The T 1 seeds obtained from the transformants were germinated on MS medium containing 50 mg L −1 hygromycin to select resistant plants. In addition, the hygromycinresistant lines were further confirmed by PCR using gene-specific primer and qRT-PCR to detect the expression of TdZIP6B-1 in transgenic lines. The OsActin gene was used as the internal reference. Homozygous T 3 transgenic lines were selected for subsequent experimental analysis.

Measurement of Metal Concentration
The roots and shoots were sampled and dried at 70 • C for 3 days. The samples were wet-ashed by HNO 3 (60%) as described previously [63]. After dilution, the Zn, Fe, and Mn concentrations were determined by inductively coupled plasma atomic emission spectrometry (SPS1200VR; Seiko, Tokyo, Japan) [52]

Conclusions
Zn homeostasis is important for plant development and adaptation to diverse stresses. The plant ZIP family proteins play critical role in uptake and transport of Zn ion. In this study, a genome-wide analysis of ZIP family gene in wild emmer was performed. A total of 33 TdZIPs genes were identified and compared with CS TaZIP genes. We found large sequence differences for most of them, and that TdZIP5B-1 could not match TaZIP genes. We performed the phylogenetic, gene structure, chromosomal localization, expression, conserved motif, gene annotation (GO, KOG, and Swiss_Prot_annotation), cis-element, qRT-PCR, and functional analyses of TdZIP genes. TdZIP1A-2, TdZIP1A-3, TdZIP6B-1, and TdZIP7B-2 have the ability to rescue the growth defects of the zrt1/zrt2∆ mutant. The overexpression of TdZIP6B-1 in rice improved the Zn, Fe, and Mn concentrations in roots under normal Zn condition. Our results demonstrate that wild emmer can sever as a valuable reservoir of genetic variation in the study of Zn transporters and lay a significant foundation for future functional analysis and genetic improvement of Zn deficiency tolerance in wheat and TdZIP6B-1 is a candidate Zn transporter that responsible for absorption and probably root-to-shoot translocation of Zn.

Conflicts of Interest:
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.