TpIRT1 is a transition metal transporter in Polish Wheat (Triticum polonicum L.) with a broad substrate specicity

Aims (1) Explore the metal substrate specicity of homologous TpIRT1A and TpIRT1B transporters from dwarf Polish wheat by expressing them in protoplast, yeast, and transgenic Arabidopsis; (2) screen polymorphic residues of IRT1 homologs from tetraploid and diploid ancestral wheat species that change the substrate specicity. Methods Two IRT1 homoeologs were isolated from A (TpIRT1A) and B (TpIRT1B) genomes of a tetraploid crop, polish wheat (Triticum polonicum). Both of them were analysed by expressing them in yeast and Arabidopsis protoplast, respectively. Then we constructed over-expressing transgenic plants of TpIRT1B for metals property analysis in Arabidopsis. We also isolated 22 IRT1 homoeologs from tetraploid and diploid ancestral wheat species and expressed them in yeast for function analysis. Results Our data highlighted the importance of TpIRT1 in the uptake and translocation of Fe, Mn, Co, and Cd with direct implications for wheat yield potential. Both TpIRT1A and TpIRT1B were located at the plasma membrane and internal vesicles in Arabidopsis protoplasts, and responsible for Cd and Co sensitivity in yeast. The over-expression of TpIRT1B in A. thaliana increased Fe, Mn, Co, and Cd concentration in its tissues and improved plant growth under Fe, Mn, and Co deciencies, while causing more sensitivity to Cd than wild-type plant. Functional analysis of IRT1 homoeologs from tetraploid and diploid ancestral wheat species in yeast disclosed four distinct amino acid residues in TdiIRT1B (T. dicoccum) and TtuIRT1B (T. turgidum). Altogether, these results increase the knowledge of IRT1 function in a global crop, wheat.


Introduction
Adequate uptake of micronutrients including iron (Fe), zinc (Zn), copper (Cu) and manganese (Mn) from the soil into plant roots, root-to-shoot delivery and lateral distribution is critical for the growth and development of plants (Kumar et al. 2009;Kobayashi and Nishizawa 2012;Yamaji et al. 2013).Thus, micronutrient de ciency in soils adversely in uences crop growth and yield and results in a poor quality of plant-based food, which, in turn, negatively in uences the health and well-being of the world's population (Grotz and Guerinot 2006).However, the over-accumulation of Fe, Zn, and Mn in plant cells causes toxicity with the detrimental consequences for crop growth and yield as well (Uriu-Adams and Keen 2005; Valko et al. 2005).Additionally, highly toxic and non-essential metals, such as cadmium (Cd) and lead (Pb), compete with micronutrients for uptake and internal transport, posing a threat to plant growth and human health (Benavides et al. 2005;Wierzbicka et al. 2007;Zhai et al. 2014).
Concerning IRT1-like proteins in non-grass species, the A. thaliana genome possesses three IRTs: AtIRT1, AtIRT2, and AtIRT3 (Vert et al. 2001(Vert et al. , 2002;;Lin et al. 2009).AtIRT1 localizes to the plasma membrane and participates in the absorption of essential metals including Fe, Zn, Mn, cobalt (Co), and also toxic metals such as Cd and Ni from the soil to root epidermal cells (Korshunova et al. 1999;Rogers et al. 2000;Nishida et al. 2011;Barberona et al. 2014).AtIRT2 localizes to intracellular vesicles and plays a role mainly in Fe and Zn compartmentalization into internal storage vesicles to alleviate metal toxicity (Vert et al. 2001(Vert et al. , 2009)).AtIRT3, localized to the plasma membrane and facilitates the uptake of Fe and Zn but not of Cd and Mn (Lin et al. 2009).IRT1 homolog from pea (Pisum sativum), PsIRT1 mediates Fe and Zn uptake as evidenced by its ability to complement growth of Saccharomyces cerevisiae Fe and Zn uptake mutants (Cohen et al. 1998(Cohen et al. , 2004)); PsIRT2 localizes to the mitochondria and controls Fe transport in the vasculature (Alagarasan 2016).In tomato, both LeIRT1 and LeIRT2 transport Fe, Zn, Mn, and Cu (Schikora et al. 2006).
Concerning IRT1 homologs in grasses, rice (Oryza sativa), possesses two IRT1-like proteins, OsIRT1 and OsIRT2 that contribute to the uptake of Fe, Zn and Cd (Bughio et al. 2002;Ishimaru et al. 2006) but not Mn, Co, and Cu (Nakanishi et al. 2006;Lee et al. 2009a, b).Maize IRT1 homolog, ZmIRT1, localizes to the plasma membrane and endoplasmic reticulum of silk and embryos, and is involved in Fe and Zn transport (Li et al. 2015).IRT1 from barley (Hordeum vulgare), HvIRT1, plays an essential role in Mn uptake, translocation and grain accumulation (Long et al. 2017).These results indicate that although IRTs have broad transport substrate speci cities and transport both essential and non-essential metals, their transport speci cities and cellular localization differ among different species.Here, we thought to characterize IRT1 homoeologous from wheat.We speci cally focused on Dwarf Polish Wheat (DPW) because it can accumulate high concentrations of Cd, Zn, and Fe in its seedlings without showing toxicity symptoms (Wang et al. 2016).DPW (Triticum polonicum L., 2n = 4x = 28, AABB) was originally collected from Tulufan, Xinjiang province, China, by Prof. Chi Yen of the Sichuan Agricultural University, China.In the present study, two IRT1 homoeologous, TpIRT1A and TpIRT1B have been isolated from DPW and analyzed for the tissue speci city of their expression, the subcellular localization and metal transport capabilities by using functional complementation studies in yeast and over-expressing TpIRT1B in A. thaliana.We have also initiated functional analysis of TpIRT1 homologs from tetraploid (T.dicoccum) and diploid ancestral (T.turgidum) wheat species.

Plant materials and growth conditions
Dwarf Polish wheat (DPW, Triticum polonicum L., 2n = 4x = 28, AABB) were grown either hydroponically in the greenhouse or in the Wenjiang experimental eld of Triticeae Research Institute (30.6822°N, 103.8566°E), Sichuan Agricultural University, Sichuan, China as detailed below.For analyses of the expression pattern of TpIRT1 homoeologs in different tissues, seeds of DPW were sowed on October 29th, 2015 and October 30th, 2016.Field trials were performed in a randomized complete block design with three replications, and each plot included one row with 20 plants.Tissues were collected at the three growth stages including the jointing stage (root, basal stem, leaf sheath, leaf blade, and young leaf), owering stage (root, stem, leaf, leaf I, ag leaf, sheath, node, rachis, rachilla, lemma, palea, awn, ovary, and anther), and lling stage (root, stem, leaf, leaf I, ag leaf, sheath, node, rachis, rachilla, lemma, palea, awn, and immature grain).All tissues were frozen in liquid nitrogen, then stored at -80 °C for RNA isolation.
To impose Fe, Zn, Mn or Co de ciency, or Cd toxicity, the uniform-size seeds were sterilized for 15 min in 5% (m/v) sodium hypochlorite (NaClO

Gene cloning, bioinformatics, and phylogenetic analysis
The full-length cDNAs of TpIRT1A and TpIRT1B were ampli ed from leaves.PCR primers were selected based on the reference sequence of the wheat genome (Wang et al. 2016) were designed using Beacon Designer v7.0 (PREMIER Biosoft International, California, USA) (Table S1).Ten tetraploid wheat and two diploid ancestral species were used to investigate IRT1 homoeologs with the same primers (Table S2).
The subcellular localization of TpIRT1A and TpIRT1B in the leaf protoplast of A. thaliana The ORFs of TpIRT1A and TpIRT1B without stop codon were individually fused at the C terminus with the modi ed green uorescent protein (EGFP) and placed under the control of the cauli ower mosaic virus 35S promoter (CaMV35s) in the pSAT6-N1-EGFP-Gate vector (Jung et al. 2012).TpIRT1A-EGFP and TpIRT1B-EGFP constructs and the vector lacking cDNA inserts were transfected into A. thaliana protoplasts prepared as described by Jung et al. (2012).EGFP-mediated uorescence and chlorophyll auto-uorescence were visualized and collected using FITC (for EGFP) or rhodamine (for chlorophyll) lter sets of the Axio Imager M2 microscope equipped with the motorized Z-drive and the high-resolution 25 Axio Cam MR Camera (Zeiss, Oberkochen, Germany).

The generation and characterization of transgenic A. thaliana ectopically expressing TpIRT1B
The ORF of TpIRT1B with the stop codon was inserted between HindIII and XbaI sites of the pCAMBIA1305.1 vector (Jefferson et al. 1987).The recombined constructs and the vector lacking the cDNA insert were individually transformed into A. thaliana wild-type (cv.Col-0) by oral in ltration (Bent et al. 1994).Transgenic plants were selected on ½ MS medium for the resistance to 25 mg/L hygromycin.
The presence of TpIRT1B was also veri ed by PCR and sequencing using primer pairs TpIRT1B-HindIII-F and TpIRT1A/B-XbaI-R (Table S1).The T4 generation of homozygous lines was used for analyses.The expression level of TpIRT1 in transgenic lines was analyzed by qRT-PCR as described above.
To test the role of TpIRT1B in mineral element de ciency and Cd toxicity, transgenic plants and A. thaliana wild-type were grown hydroponically.To do so, seeds were sterilized and treated with 4 o C for two days, then sowed onto ½ MS media.After one week, the uniform-size seedlings were transplanted into the fresh hydroponic medium containing 1.25 mM KNO 3 , 0.5 mM MgSO 4 , 0.625 mM KH 2 PO 4 , 0.5 mM Ca (NO 3 ) 2 , 2.5 µM NaCl, 10 µM Fe (III)-HBED, 3.5 µM MnCl 2 , 0.25 µM ZnSO 4 , 0.125 µM CuSO 4 , 17.5 µM H 3 BO 3 , 0.05 µM Na 2 MoO 4 , and 0.025 µM CoCl 2 .After two weeks of growth, a subset of plants was transferred either to the same medium or a medium with the reduced concentration of ZnSO 4 (50 nM) or Fe (III)-HBED (2.5 µM), or MnCl 2 (3.5 µM), or CoCl 2 (2.5 nM).A subset of plants was also transferred to a hydroponic medium with 20 µM CdCl 2 .Root lengths were measured after ve days of growth for a subset (eight plants/line) of plants.In addition, roots and leaves were collected after two weeks of growth, dried at 80 o C for two days, and dry weight was analysed.

Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
Different plant lines were grown hydroponically for four weeks as described above.Plants were collected and mineral elements were desorbed from the root surface as described by Zhai et al. (2014).Brie y, plant roots were washed for 10 min in a medium containing 10 µM EDTA (ethylenediaminetetraacetic acid), then transferred for 5 min to a medium containing 0.3 µM BPS (4,7-diphenyl-1,10-phenanthrolinedisulfonic acid) and 5.7 mM Na 2 S 2 O 3 , followed by ve sequential washes in ddH 2 O. Roots were then dried in 80° C, ground, digested in 80% nitric acid at 220-280° C, and plant residue was dissolved in 25 mL of ddH 2 O. Mineral concentrations were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Thermo Fisher Scienti c, Massachusetts, USA).

Perl's staining with DAB/H 2 O 2 Intensi cation
The Perl's staining with DAB/H 2 O 2 (3,3'-diaminobenzidine tetrahydrochloride/Hydrogen peroxide) was performed according to Brumbarova and Ivanov (2014).Brie y, the ve-day-old seedlings grown on ½ MS media were collected and washed with ddH 2 O.Samples were vacuum-in ltrated (500 mbar, 30 min) with the xation solution containing (methanol: chloroform: glacial acetic acid in 6:3:1 ratio) and washed with ddH 2 O for 1 min three times.Fixed samples were incubated with the pre-warmed staining solution (4% K 4 Fe(Cn) 6 : 4% HCl in 1:1 ratio) for 15 min under vacuum (500 mbar) and washed with 0.1 M phosphate buffer (pH 7.4) three times.For the intensi cation reaction, samples were applied with intensi cation solution (0.1 M Phosphate buffer (pH 7.0) containing 0.025% DAB, 0.005% H 2 O 2 , and 0.005% CoCl 2 ) for 10 min, then the reaction was terminated by rinsing with ddH 2 O.Samples were stored in ddH 2 O one week prior to analyses.Images were collected using the Axio Imager M2 microscope equipped with the motorized Z-drive (Zeiss, Oberkochen, Germany).

Cloning and characterization of TpIRT1A and TpIRT1B
TpIRT1A and TpIRT1B cDNAs were cloned from A and B genomes of DPW, respectively.The full-length cDNA of TpIRT1A included a 127 bp 5′-UTR, 1110 bp ORF, and 383 bp 3′-UTR; the full-length cDNA of TpIRT1B included a 124 bp 5′-UTR, 1119 bp ORF, and 104 bp 3′-UTR (Table S3).Alignment of the sequenced TpIRT1A and TpIRT1B to the reference sequence of the hexaploid wheat genome (The International Wheat Genome Sequencing Consortium 2018; Elizabeth 2018) revealed that TpIRT1A and TpIRT1B are localized on the chromosome 4AL (TRIAE_CS42_4AL_TGACv1_290140_AA0982100) and 4BS (TRIAE_CS42_4BS_TGACv1_328611_AA1090980), respectively, and each gene included an intron and two exons.Analysis of the deduced 370 and 373 amino acid sequences of TpIRT1A and TpIRT1B polypeptides, respectively, revealed that TpIRT1A shares 95.44% amino acid identity to TpIRT1B and each polypeptide contained eight predicted transmembrane domains (Table S3).

The expression pattern of TpIRT1A/B in DPW
To analyse the tissue-speci city and dynamics of the expression pattern of TpIRT1A and TpIRT1B we used plants at different developmental stages.Due to the high sequence identity of TpIRT1A and TpIRT1B genes, we were not able to identify speci c regions for distinguishing the expression pattern of TpIRT1A from TpIRT1B.Thus, the results below show the expression pattern for both TpIRT1A and TpIRT1B, and indicated from here on as TpIRT1A/B.We found that TpIRT1A/B was highly expressed in ag leaves, followed by roots and lemma at the owering stage (Fig. 1A).The high expression of TpIRT1A/B in ag leaves and roots was maintained throughout the grain lling stage (Fig. 1B).In this developmental stage, the expression of TpIRT1A/B was also evident in reproductive tissues including lemma, palea, awn, rachilla, and grain (Fig. 1B).At the jointing stage, TpIRT1A/B was highly expressed in old leaves and was also evident in roots with the lowest expression in young leaves (Fig. 1C).The expression of TpIRT1A/B was up-regulated only in response to Mn, but not to Fe, Zn or Co de ciencies (Fig. 1D).In contrast, the expression of TpIRT1A/B was considerably up-regulated by Fe, Mn, Co, and Cd, but not Zn excess (Fig. 1E).

Subcellular localization of TpIRT1A and TpIRT1B
To establish the cellular localization of TpIRT1A and TpIRT1B, TpIRT1A-EGFP, TpIRT1B-EGFP and the empty pSAT6-N1-EGFP-Gate vector were transiently expressed in protoplasts isolated from A. thaliana mesophyll cells.The EGFP signal from the pSAT6-N1-EGFP-Gate vector was found in the entire cell (Fig. 2A-C).The uorescence of TpIRT1A-EGFP and TpIRT1B-EGFP-transfected protoplasts were detected at the cell periphery suggesting that both proteins are associated with the plasma membrane (Fig. 2E-L).The TpIRT1A-EGFP and TpIRT1B-EGFP-mediated uorescence was also found to be associated with internal vesicles.While the nature of these vesicles is unknown, we speculate that they might be related to the endocytic pathway and participate in the recycling of TpIRT1A-EGFP and TpIRT1B-EGFP.
As would be expected for plasma membrane-localized Cd uptake transporters, the expression of both TpIRT1A and TpIRT1B increased the sensitivity of the Δycf1 mutant to 40 µM Cd in a solid medium compared to the Δycf1 mutant expressing the vector without cDNA inserts (EV) (Fig. 3A).The increased sensitivity of Δycf1-TpIRT1A and Δycf1-TpIRT1B strains to Cd compared to the EV-transformed mutant was also observed in a liquid medium (Fig. 3B-C).Under Co stress, the expression of both TpIRT1A and TpIRT1B also enhanced the sensitivity of the COT1 mutant to 250 µM Co on plates and 100 µM Co in liquid media compared to EV (Fig. 3D-F).However, the growth of Δzrc1-TpIRT1A and Δzrc1-TpIRT1B was similar to Δzrc1-EV cell lines on solid or in liquid medium with high Zn (Fig. 3G-I).
Transgenic A. thaliana expressing TpIRT1B accumulates more Fe, Mn, and Co To determine the metal transport properties of TpIRT1 in plants, we over-expressed TpIRT1B in Arabidopsis and generated two independent TpIRT1B-overexpressing lines (TpIRT1B-OE-L1 and TpIRT1B-OE-L2).The expression level of TpIRT1B in TpIRT1B-overexpressing lines was much higher than that in control lines (Fig. S1).Although TpIRT1B-overexpressing lines and control lines were indistinguishable when were grown under control conditions, the over-expression of TpIRT1B signi cantly increased Fe and Mn concentrations in leaves and roots of plants (Fig. 4A-F).The concentration of Co was elevated only in leaves in both TpIRT1B-OE lines compared to wild-type, correspondingly the translocation factor was also improved (Fig. 4G-I).Furthermore, Perls-DAB staining showed that plants ectopically expressing TpIRT1B accumulated more Fe at the root tip, the zone of maturation, the root-to-shoot junction, leaf vasculature, as well as stomatal area including stomatal cavity and guard cells of the leaf (Fig. 5).
A. thaliana transgenic lines expressing TpIRT1B are more tolerant to Fe and Mn de ciencies We then exposed different plant lines to mineral de ciencies and, as expected, root and leaf biomass of control lines was signi cantly lower when they were grown under Fe or Mn de ciency and leaf biomass was lower when plants were grown under Co de ciency compared to plants grown under control conditions.Concerning TpIRT1B expressing plants, while Mn de ciency decreased root biomass, Fe and Co de ciencies decreased shoot biomass, overall, plant growth was less affected compared to wild-type or empty vector expressing plants (Fig. 6).Speci cally, the over-expression of TpIRT1B partly rescued the plant root growth (Fig. 6E) and even increased the root length (Fig. 6D) and the leaf biomass (Fig. 6F) of Mn-de cient plants compared to control lines.Although no signi cant differences were found in root length or biomass between the transgenic and control lines grown under Co de ciency (Fig. 6G-H), leaf biomass was higher in TpIRT1B-overexpressing lines than in the wild-type or vector expressing plants (Fig. 6I).The increased tolerance of TpIRT1B-overexpressing plants to Fe, Mn, and Co de ciencies may result from their increased ability to accumulate these minerals when they are in abundance (Fig. 4) and use them under mineral de ciency.Consistently, as evidenced by the decreased root length of TpIRT1Boverexpressing lines, these plant lines were more sensitive than controls to elevated concentrations of Fe, Mn, and Co (Fig. S2).Interestingly, over-expression of TpIRT1B had no in uence on plant growth under both either low or excess Zn and did not change internal Zn concentration when compared to control lines (Fig. S3).
A. thaliana transgenic lines expressing TpIRT1B are more sensitive to Cd toxicity We then examined the sensitivity of TpIRT1B-overexpressing lines to excess Cd.To do that different plant lines were gown either hydroponically (Fig. 7A-G) or on solid ½ MS medium (Fig. 7H).The over-expression of TpIRT1B caused more serious toxicity symptoms including lesion and chlorosis of leaves, inhibited root lengths and decreased biomass when compared to control lines (Fig. 7A-D, H).Consistently, TpIRT1Boverexpressing plants accumulated more Cd in roots and leaves (Fig. 7E-G).

The identi cation of IRT1 homoeologs from different wheat subgenomes
We then used an in silico analysis to identify IRT1 homoeologs in different wheat genomes.Twenty IRT1 homoeologs that were isolated from ten tetraploid wheat genomes included ten homoeologs from the genome A and ten homoeologs from the genome B (Table S3).In addition, two IRT1 homoeologs, TuIRT1 and AsIRT1, were also identi ed from two diploid wheat species T. urartu and Aegilops speltoides, and classi ed as TuIRT1A and AsIRT1B because T. urartu and A. speltoides are considered as the A and B genome donors, respectively (Haider 2013).The ORF lengths of a total of 22 IRT1 homoeologs ranged from 1094 bp to 1121 bp (Fig. S4), which encoded 363 to 372 amino acids (Fig. S5).According to amino acid alignment, two amino acid substitutions were identi ed in 12 IRT1A members while the other ten proteins shared the same sequence; ve polymorphisms were found in IRT1B members (Fig. S5).Most IRT1 proteins were predicted to possess eight TMs, except for TdiIRT1B that was predicted to have nine TMs (Fig. S6).

Functional characterization of wheat IRT1 homoeologs
To reveal the function of different IRT1 homoeologs, we individually introduced them into Zn, Cd and Co sensitive yeast strains, Δzrc1, Δycf1, and Δcot1 respectively.We found that two homoeologs were distinct from others in their ability to complement yeast mutants.First, the expression of TdiIRT1B increased the sensitivity of the Δzrc1 mutant to Zn toxicity when compared with other wheat IRT1s and empty pYES2 vector (Fig. 9C).Second, while all IRT1s tested increased the sensitivity of Δycf1 to Cd, the expression of TtuIRT1B dramatically increased Cd tolerance of this mutant strain (Fig. 9D-F).Meanwhile, TtuIRT1Bexpression did not affect the growth of Δcot1 mutant under Co stress in comparison to other YK40-IRT1s that increased the Co sensitivity of this mutant (Fig. 9G-I).The comparison of IRT1 sequences disclosed that TdiIRT1B has two SNPs (571 and 773 sites) distinguishing it from other IRT1 homoeologs that converted the Histidine (His/H) to Tyrosine (Tyr/Y) at the positions of 193 (H193Y) and 260 (H260Y) (Fig. 9J).In addition, two SNPs were found in TtuIRT1B at positions 253 and 657 that converted Arginine 89 to His 89 (R89H) and Isoleucine 230 to Tyr 230 (I230T), respectively (Fig. 9J).The nucleotide variations in TdiIRT1B and TtuIRT1B may have altered the function of these genes, causing the functional differentiation of metal transport.

Discussion
TpIRT1B transports Fe, Mn, Co, and Cd transport, but not Zn unlike its homologs from other species In this manuscript, we show that TpIRT1B mediates uptake of Fe, Mn, Co, and Cd, but not Zn.This conclusion was made based on the following ndings: rst, the expression of TpIRT1B increased the sensitivity of Δcot1 and Δycf1 mutant yeast cells to Co and Cd, respectively.This suggests that TpIRT1B promoted the accumulation of Co and Cd in cells and caused toxicity.In contrast, Zn sensitivity of the Δzrc1 did not change (Fig. 3).Second, the over-expression of TpIRT1B enhanced Fe, Mn, and Co concentrations in roots and shoots in A. thaliana and improved the tolerance of plants to these mineral de ciencies (Fig. 4-6).These data imply that Fe, Mn, and Co accumulation in plant tissues during growth under control conditions have helped to sustain normal growth under mineral de ciencies (Fig. 6) and increased the sensitivity to excess Fe, Mn, and Co stresses (Fig. S2).Third, the over-expression of TpIRT1B enhanced Cd concentration in roots and shoots, increasing the sensitivity to Cd stress (Fig. 7).Fourth, the over-expression of TpIRT1B in A. thaliana did not change Zn concentration and the growth of transgenic plants (Fig. S3).
The TpIRT1 is preferentially expressed in roots, leaves, lemma, and palea (Fig. 1A-C).Previous studies demonstrated that OsIRT1 is mainly expressed in roots and leaves; AtIRT1, ZmIRT1, and HvIRT1 are mainly expressed in roots but also found in anthers (Bughio et al. 2002;Vert et al. 2002;Ishimaru et al. 2006;Li et al. 2015;Long et al. 2017).In this study, the TpIRT1 expression in roots was up-regulated by Mn de ciency (Fig. 1D).Surprisingly, TpIRT1 transcript abundance did not change in response to Fe, Zn or Co de ciency; on the contrary, its expression was up-regulated by excess Fe, Mn, and Co (Fig. 1D-E).Our results differ from reports on IRT1s from other species.Speci cally, AtIRT1, OsIRT1, AhIRT1, LeIRT1, and ZmIRT1 are up-regulated by Fe and Zn de ciencies; OsIRT1, AtIRT1, and HvIRT1 are up-regulated by Mn de ciency; AtIRT1 and ZmIRT1 are up-regulated and down-regulated by Fe and Zn de ciency and su ciency, respectively (Vert et al. 2002(Vert et al. , 2003;;Ishimaru et al. 2006;Schikora et al. 2006;Lee and An 2009a;Ding et al. 2010;Shanmugam et al. 2010;Nakanishi et al. 2010;Li et al. 2013;Long et al. 2017;Zheng et al. 2018).Similar to AtIRT1 and OsIRT1, TpIRT1 was up-regulated by Cd toxicity (Fig. 1E).We also found that TpIRT1s were potentially located at plasma membrane and internal vesicles (Fig. 2).This result is in agreement with data showing that AtIRT1 resides at the plasma membrane and AtIRT2 on a periphery of small vesicles (Vert et al. 2001(Vert et al. , 2002)); ZmIRT1, in addition to plasma membrane is also associated with endoplasmic reticulum (ER) (Li et al. 2013).Together, our results suggest that TpIRT1 is involved in Fe, Mn, Co, and Cd uptake and internal distribution in the plant.
It is well-recognized that non-gramineous plants use IRT1 to take up Fe(II) from the soil into root epidermal cells (Eckhardt et al. 2001;Varotto et al. 2002;Vert et al. 2002;Schikora et al. 2006;Hodoshima et al. 2007;Ding et al. 2009;Tan et al. 2015;Shanmugam et al. 2011).The non-gramineous plants absorb Fe-phytosiderophores using transporters from the YSL family, but not IRT1 (Lee et al. 2009c;Inoue et al. 2009).Previous studies have shown that rice and maize use both strategies and rely on IRT1 and YSL transporters to absorb Fe from the soil (Ishimaru et al. 2006;Li et al. 2013Li et al. , 2015Li et al. , 2018)).In this study, we show that TpIRT1B is expressed mainly in roots and transports Fe.These results imply that wheat, similar to rice and maize, uses both strategies for Fe absorption.
Among mineral elements studied here, Co, is bene cial but can be toxic if in excess, while Cd is highly toxic for plant growth and development (Battersby 1993;Palit et al. 1994;Ar n et al. 1995;Komeda et al. 1997;Bakkaus et al. 2005;Järup and Åkesson 2009;Ismael et al. 2018).Thus, uptake and internal transport of these minerals in the plant relies on transporters for essential metals (Thomine et al. 2000;Morel et al. 2009;Cheng et al. 2011;Takahashi et al. 2011Takahashi et al. , 2014;;Wang et al. 2017Wang et al. , 2019)).It is noteworthy that global crop wheat, when grown on Cd-polluted soils, is an entry point of Cd into a daily human diet (Greger and Löfstedt 2004).To date, several essential metal transporters from wheat including TaHMA2, TaVP1, TpNRAMP3, TpNRAMP5 and TpNRAMP6 have been discovered to transport Co and/or Cd (Peng et al. 2008a, b;Khoudi et al. 2012;Tan et al. 2013;Wang et al. 2019).Our results show that TpIRT1B also transports Co and Cd (Fig. 4,6,7).Thus, these transporters may contribute to Cd accumulation in wheat, which therefore are available genes for genetic manipulation to reduce the Cd accumulation in the food chain.
Amino acid residues involved in Zn, Cd, and Co transport in IRT1 homoeologs It has been shown that point substitutions of several pivotal amino acid residues in mineral ion transporters can change their substrate speci city (Zhao and Eide 1996a, b;Rogers et al. 2000;Grossoehme et al. 2006).For example, the replacement of the key lysine (Lys) residue in the intracellular loop of ZRT1 from yeast and AtIRT1 alters their transport speci city and the ability to transport Cd and Co (Gitan et al. 2000;Dubeaux et al. 2018).In this study, two His substitutions (H193Y and H260Y) of TdiIRT1B increased the sensitivity of Δzrc1-TdiIRT1B strain to Zn stress when compared to other wheat IRT1s and the empty pYES2 vector (Fig. 9A-C, J).This result suggested that this polymorphism increased TdiIRT1B speci city to Zn.Meanwhile, the unique substitution H260Y in TdiIRT1B potentially caused an additional TM (TM9) between TM5 and TM6 as predicated by TMHMM v2.0 (Fig. S6).TMs form a potential ion channel and allow metal ions to pass through the cell membrane (Kadir et al. 2018).In the P1B-type ATPase family, the difference in TMs number appears to confer their capability to transport metals (Argüello et al. 2007).Thus, it is tempting to speculate that an additional TM9 in TdiIRT1B might be a central component of Zn transport.
In addition, the expression of TtuIRT1B with two polymorphic residues (R89H and I230T) dramatically increased the tolerance of ∆ycf1 strain to Cd; while, the expression of other IRT1s in ∆ycf1 increased the sensitivity to Cd (Fig. 9D-F, J).Meanwhile, TtuIRT1B-expression did not change the growth of the ∆cot1 strain under Co stress when compared to other YK40-IRT1s that were sensitive to Co (Fig. 9G-I).These results implied that the two polymorphic residues might change the transport properties of TtuIRT1B or its subcellular localization, or the ability to bind Cd or Co.For example, Cd normally binds to various amino acid residues including His, Glu, Cys, Asp and Tyr (Friedman 2014).Thus, His 89 may be a potential Cd-binding site for intercellular mobilization at the IRT1.Consequently, the Ile 230 seemed to be the Cobinding site, and its substitution may result in failing to transport Co in yeast.

Conclusion
Our results highlight the importance of TpIRT1 in the uptake and translocation of Fe, Mn, Co, and Cd.TpIRT1 is preferentially expressed in roots and leaves, and is signi cantly induced by Fe, Mn, Cd and Co excess, and Mn de ciency.TpIRT1A and TpIRT1B localize at the plasma membrane and small internal vesicles in Arabidopsis protoplasts.The expression of TpIRT1A and TpIRT1B increased yeast sensitivity to Cd and Co.The overexpression of TpIRT1B in Arabidopsis has led to Fe, Mn, and Co accumulation in plant tissues and partially rescued the plant defects under these metals de ciencies when compared to control lines.Meanwhile, the over-expression of TpIRT1B enhanced Cd concentration in Arabidopsis, resulting in the increased sensitivity of plants to Cd toxicity.Most of the IRT1 homoeologs that exist in abundant natural variations of wheat species have shown a similar function in Zn, Co, and Cd transport in yeast.However, four SNPs were detected that, we speculate, might change the metal transport speci city.Our future studies will focus on the discovery of amino acid residues that contribute to metal selectivity of IRTs in wheat.

Declarations
Figures

Figure 7 The
Figure 7