Lun Li

Zongzheng Zhu

Yonghui Liao

Changhong Yang

Ni Fan

Jie Zhang

Naoki Yamaji

Léon Dirick

Jian Feng Ma

Catherine Curie

Chao-Feng Huang
email: huangcf@cemps.ac.cn

NRAMP6 and NRAMP1 cooperatively regulate root growth and manganese translocation under manganese deficiency in Arabidopsis

Keywords: manganese transport, NRAMP6, NRAMP1, root growth, manganese translocation, long-distance signaling, Arabidopsis thaliana

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Manganese (Mn) is an indispensable nutrient element for most organisms including plants. Mn is essentially required for photosynthesis and antioxidant defense through acting as a constituent of the oxygen-evolving complex in photosystem II (PSII) and manganese superoxide dismutase (MnSOD), respectively [START_REF] Marschner | Mineral nutrition of Higher plants 3 edn[END_REF]. Mn can also function as a cofactor or activator for over 30 enzymes to regulate diverse biological processes [START_REF] Marschner | Mineral nutrition of Higher plants 3 edn[END_REF]. Mn deficiency in plants normally occurs in calcareous soils or alkaline soil with high pH, which greatly restrict the availability of Mn for plants (Schmidt et al., 2016). The typical symptoms of plant Mn deficiency include diffuse and/or interveinal chlorosis in younger leaves and reduced root and shoot growth [START_REF] Marschner | Mineral nutrition of Higher plants 3 edn[END_REF]. Under serious Mn deficiency, root hair growth can also be inhibited [START_REF] Gao | NRAMP2, a trans-Golgi network-localized manganese transporter, is required for Arabidopsis root growth under manganese deficiency[END_REF].

Plants utilize a number of transporters to mediate Mn uptake, translocation and distribution. Natural Resistance-Associated Macrophage Proteins (NRAMPs) that act as proton/metal symporters have been reported to play crucial roles in Mn nutrition in plants. OsNRAMP5 has been shown to be essentially required for Mn uptake and translocation in rice [START_REF] Sasaki | Nramp5 is a major transporter responsible for manganese and cadmium uptake in rice[END_REF][START_REF] Yang | OsNRAMP5 contributes to manganese translocation and distribution in rice shoots[END_REF], while OsNRAMP3 is involved in the distribution of Mn to young leaves and panicles in low Mn conditions [START_REF] Yamaji | A node-based switch for preferential distribution of manganese in rice[END_REF]. In Arabidopsis, there are six NRAMP family proteins.

NRAMP1 is a plasma membrane-localized high-affinity Mn influx transporter and plays a vital role in root Mn uptake [START_REF] Cailliatte | High-affin ity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions[END_REF]. Mutation of NRAMP1 reduces root and shoot growth in low Mn conditions. NRAMP3 and NRAMP4 are localized to the tonoplast and are redundantly involved in the remobilization of Mn from vacuoles to chloroplasts under Mn deficiency [START_REF] Lanquar | Export of vacuolar manganese by AtNRAMP3 and AtNRAMP4 is required fo r optimal photosynthesis and growth under manganese deficiency[END_REF], although both proteins were initially documented to redundantly regulate iron (Fe) remobilization under Fe-deficient conditions [START_REF] Lanquar | Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron[END_REF]. Recently, NRAMP2 was reported to be localized to the trans-Golgi network (TGN) and plays a critical role in the reutilization of Golgi Mn [START_REF] Alejandro | Intracellular distribution of manganese by the trans-Go lgi network Transporter NRAMP2 is critical for photosynthesis and cellular redo x ho meostasis[END_REF][START_REF] Gao | NRAMP2, a trans-Golgi network-localized manganese transporter, is required for Arabidopsis root growth under manganese deficiency[END_REF].

Mutation of NRAMP2 reduces root growth and leaf chlorophyll accumulation.

NRAMP6 was shown to be able to transport cadmium (Cd) and Fe to modulate Cd tolerance and Fe utilization, respectively [START_REF] Cailliatte | The NRAMP6 metal transporter contributes to cadmium toxicity[END_REF][START_REF] Li | The intracellu lar transporter AtNRAMP6 is involved in Fe homeostasis in Arabidopsis[END_REF].

Nevertheless, whether NRAMP6 plays a role in Mn nutrition is still unknown.

In addition to NRAMP proteins, members of other transporter families are also involved in Mn transport and utilization in low Mn conditions. The Iron Regulated Transporter 1 (IRT1), a member of ZIP family, was initially identified as a high-affinity iron (Fe) transporter that also plays a role in Mn uptake in Arabidopsis in Fe deficient conditions [START_REF] Korshunova | The IRT1 protein fro m Arabidopsis thaliana is a metal t ransporter with a broad substrate range[END_REF][START_REF] Vert | IRT1, an Arabidopsis transporter essential for iron uptake fro m the soil and for p lant growth[END_REF]. In rice, the MTP/CDF family member OsMTP9 is involved in both Mn uptake and translocation [START_REF] Ueno | A polarly localized transporter for efficient manganese uptake in rice[END_REF]. AtZIP1 and AtZIP2, two other ZIP members, were reported to regulate Mn translocation from roots to shoots [START_REF] Milner | Transport properties of members of the ZIP family in plants and their role in Zn and Mn homeostasis[END_REF]. The UPF0016 family members PAM71 (PHOTOSYNTHESIS AFFECTED MUTANT71) and CMT1 (Chloroplast Manganese Transporter 1), which are localized in the thylakoid membrane and the inner chloroplast envelope, respectively, were shown to mediate the uptake of Mn to thylakoid lumen and to the chloroplast stroma [START_REF] Schneider | The evolutionarily conserved protein PHOTOSYNTHESIS AFFECTED MUTANT71 is required fo r efficient manganese uptake at the thylakoid membrane in Arabidopsis[END_REF][START_REF] Eisenhut | The p lastid envelope CHLOROPLA ST MANGANESE TRANSPORTER1 is essential for manganese homeostasis in Arabidopsis[END_REF][START_REF] Zhang | Inner envelope CHLOROPLA ST MANGANESE TRANSPORTER 1 supports manganese homeostasis and phototrophic growth in Arabidopsis[END_REF]. Recently, the Golgi-localized PML3 of the UPF0016 family was demonstrated to mediate Mn uptake into Golgi, which is critical for Golgi glycosylation and cell wall biosynthesis through activating Golgi-localized mannosidases or glycosyltransferases [START_REF] Yang | Go lgi-localised manganese transporter PML3 regulates Arabidopsis growth through modulating Golg i glycosylation and cell wall biosynthesis[END_REF]. The Golgi-localized AtECA3, which belongs to the Ca 2+ -ATPase subfamily of P-type ATPase superfamily, has also been reported to mediate Mn transport into Golgi for Mn utilization and plant growth under Mn deficiency [START_REF] Mills | ECA3, a Golg i-localized P2A-type ATPase, plays a crucial role in manganese nutrition in Arabidopsis[END_REF].

In response to changing nutrient availability in soils, plants develop adaptive systemic responses and exhibit a high degree of plasticity of plant growth and development. Long-distance signaling between roots and shoots has been reported to be crucial for the adaptive responses of plants to the alterations in nutrient status (Liu et al., 2009, Ko andHelariutta, 2017). For instance, nitrate influx and the expression of the high-affinity nitrate transporters-encoding genes in roots can be regulated by shoot-derived signals [START_REF] Forde | Local and long-range signaling pathways regulating plant responses to nitrate[END_REF][START_REF] Liu | The long-distance signaling of mineral macronutrients[END_REF]. Shoot-to-root communication is also important for root growth and development under different nitrate concentrations.

Cytokinins and carboxy-terminally encoded peptides (CEPs) have been demonstrated to be critical players in the shoot-to-root long distance signaling of plants in response to different nitrogen status (Tabata et al., 2014, Ko andHelariutta, 2017).

Shoot-to-root signaling is also critical for plants adapted to the deficiency of other macronutrients such as phosphorus and sulfur [START_REF] Liu | The long-distance signaling of mineral macronutrients[END_REF]. In addition to the macronutrients, root acquisition of the micronutrient iron also involves long-distance signaling, via a shoot-derived signal [START_REF] Grusak | Shoot-to-root signal transmission regulates root Fe(III) reductase activity in the dgl mutant of pea[END_REF]Pezeshgi, 1996, Rogers andGuerinot, 2002). Recently, a peptide family called IMA or FEP was found to be involved in such long-distance Fe signaling in plants [START_REF] Grillet | IRON MAN is a ubiquitous family of peptides that control iron transport in plants[END_REF][START_REF] Hirayama | The putative peptide gene FEP1 regulates iron deficiency response in Arabidopsis[END_REF]. Nevertheless, it is still uncertain whether Mn uptake in plants relies on shoot-to-root signaling.

In the present study, we characterized the role of NRAMP6 in Mn nutrition and found that NRAMP6 plays a cooperative role with NRAMP1 in the regulation of root growth and root-to-shoot Mn translocation under Mn deficient conditions. Interestingly, our grafting experiments showed that mutation of NRAMP6 in shoots is required for the defective root growth and Mn translocation in nramp6nramp1 double mutant, suggesting that NRAMP6 is involved in long-distance Mn signaling. Furthermore, we found that although mRNA expression of NRAMP6 is not affected by Mn deficiency, its protein level and subcellular localization was altered in response to Mn deficiency, implying that NRAMP6 is subjected to post-transcriptional regulation by Mn deficiency.

RESULTS

Knockout of NRAMP6 in nramp1 background re duces root growth in low Mn conditions

To determine whether NRAMP6 is involved in the regulation of Mn homeostasis in Arabidopsis, we used the nramp6-1 knockout mutant previously reported [START_REF] Cailliatte | The NRAMP6 metal transporter contributes to cadmium toxicity[END_REF][START_REF] Li | The intracellu lar transporter AtNRAMP6 is involved in Fe homeostasis in Arabidopsis[END_REF]. We also generated a second mutant allele of NRAMP6, nramp6-2, by the CRISPR/Cas9 technology [START_REF] Ma | CRISPR/Cas9-based multip lex genome edit ing in monocot and dicot plants[END_REF]. The nramp6-2 mutant has a frameshift mutation (1-bp insertion located 15 bp downstream of the start codon) in the NRAMP6 gene (Figure 1a). Both nramp6-1 and nramp6-2 single mutants did not show defective phenotype under Mn deficient conditions (Figure 1b).

Considering the close sequence homolog between NRAMP6 and NRAMP1 in

Arabidopsis, we asked whether both transporters were functionally cooperative by generating nramp6-1nramp1 and nramp6-2nramp1 double mutants. The six genotypes showed no difference of growth in Mn replete conditions (Figure 1b and1c). Under Mn deficient conditions, however, root growth of nramp1 was more inhibited than that of WT as previously shown [START_REF] Cailliatte | High-affin ity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions[END_REF]. Interestingly, this growth defect was strongly exacerbated in the two double mutants (Figure 1b and1c). The short-root phenotype of the double mutants was gradually rescued with the increase of Mn supply and 10 µM Mn was required to fully recover the root growth (Figure 1b and 1c), while 2 µM Mn was able to rescue the defective root growth of nramp1 completely. In addition to increased root growth inhibition, the double mutants also showed shorter root hair than nramp1 under Mn deficiency (Figure 1d and 1e). These results demonstrate that NRAMP6 and NRAMP1 have overlapping functions in modulating root growth and root hair elongation in low Mn conditions.

To confirm that NRAMP6 is required for sustaining the root growth and root hair elongation in nramp1 background under Mn deficiency, we introduced the WT NRAMP6 gene into the nramp6-1nramp1 double mutant. Measurement of root growth under Mn deficiency in two independent transgenic lines showed recovery up to the nramp1 mutant level (Figure S1a andS1b). Defective root hair growth in the double mutant was also rescued in the transgenic lines under Mn deficient conditions (Figure S1c andS1d). These results indicate that the increased inhibition of root growth and root hair elongation in the double mutants compared to nramp1 is induced by the nramp6 mutations.

Mutation of NRAMP2 also causes reduced root growth in low Mn conditions [START_REF] Gao | NRAMP2, a trans-Golgi network-localized manganese transporter, is required for Arabidopsis root growth under manganese deficiency[END_REF]. To investigate whether NRAMP6 plays a cooperative role with NRAMP2 in the regulation of root growth, we generated nramp6-1nramp2 double mutant to analyze the sensitivity to Mn deficiency. The result showed that Mn deficiency-induced inhibition of root growth in nramp6-1nramp2 was similar to that in nramp2 single mutant (Figure S2a and S2b), although both mutants displayed shorter root growth than the WT under Mn deficient conditions. Together, these results demonstrate that NRAMP6 plays a cooperative role with NRAMP1, but not with NRAMP2, to sustain root growth when Mn availability is limiting.

Mutation of NRAMP6 affects Mn translocation from roots to shoots

To analyze the effect of the nramp6 mutation on Mn accumulation, we firstly grew the plants of WT, nramp1, nramp6-1, nramp6-2 and double mutants under Mn sufficient conditions and then exposed the plants to either Mn sufficient or deficient conditions for 9 days. The Mn concentration in roots, young and old leaves was then measured.

Under Mn sufficient conditions, Mn concentration in all the tissues did not differ between the six genotypes analyzed (Figure 2a). Under Mn deficient conditions, however, the double mutants accumulated more Mn in roots and less Mn in young leaves than each of the WT and single mutants (Figure 2b). In the old leaves, there was no difference in Mn concentration among all the lines. We also determined Fe and Zn concentrations in these samples and the results showed that in roots and leaves of the double mutants they were similar to those of WT, nramp1, nramp6-1 and nramp6-2 under both Mn deficient and sufficient conditions (Figure S3). These data suggest that Mn translocation from roots to shoots was only defective in nramp6nramp1 double mutants. We next assessed Mn concentrations in the two complementation lines and observed full rescue under Mn deficient conditions (Figure S1e), indicating that the loss of NRAMP6 in the nramp1 background is responsible for the defect of Mn translocation in the double mutant.

Expression pattern of NRAMP6 and NRAMP1

NRAMP6 was expressed higher in shoots than in roots (Figure 3a and 3b) [START_REF] Cailliatte | The NRAMP6 metal transporter contributes to cadmium toxicity[END_REF][START_REF] Li | The intracellu lar transporter AtNRAMP6 is involved in Fe homeostasis in Arabidopsis[END_REF]. In leaves, NRAMP6 was more expressed in young tissues than in old tissues (Figure 3a). The expression of NRAMP6 was not responsive to Mn deficiency in both WT and nramp1 mutant backgrounds (Figure 3b), nor was it affected by Fe or Zn deficiency (Figure 3c). Compared to NRAMP1, NRAMP6 was expressed at a lower level, particularly in roots (Figure S4a).

To determine the tissue-specific expression pattern of NRAMP6, we made a translational fusion between NRAMP6 and the β-glucuronidase (GUS)-encoding gene uidA under the control of 1.8 kb NRAMP6 promoter and analyzed GUS expression in Arabidopsis transgenic lines. NRAMP6-GUS was expressed at a low level in roots and the GUS activity was only detected in root caps and junction sites between lateral roots and primary roots (Figure 3d and3e). In leaves, NRAMP6-GUS expression was found in whole cotyledons at an early stage (Figure 3f) and was then confined to the major vascular tissues of both cotyledons and true leaves at later stages (Figure 3g) [START_REF] Cailliatte | The NRAMP6 metal transporter contributes to cadmium toxicity[END_REF].

NRAMP1 was previously reported to be mainly expressed in roots [START_REF] Cailliatte | High-affin ity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions[END_REF]. To determine NRAMP1 expression pattern in shoots, we generated pNRAMP1:NRAMP1-GUS transgenic lines. GUS activity was observed in most root tissues with a stronger signal detected in the root tip region (Figure S4b), which is consistent with previous results [START_REF] Cailliatte | High-affin ity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions[END_REF]. In shoots, GUS activity was strong in whole cotyledons (Figure S4c) and faded in true leaves (Figure S4d).

Loss of NRAMP6 is required in both roots and s hoots for the defective phenotype of nramp6nramp1

Although NRAMP6 was mainly expressed in shoots (Figure 3a and 3b), the double mutants showed defective growth and Mn accumulation in roots. To determine whether nramp6-1 mutation in shoots contribute to the observed phenotypes in nramp6-1nramp1, we performed reciprocal grafting between nramp1 and nramp6-1nramp1 mutants. Successfully grafted plants were first cultivated in a nutrient solution with sufficient Mn (10 μM Mn) for 12 days and then subjected to Mn deficiency treatment for 8 days. Mn concentration was then measured in roots, young and old leaves of these grafted plants. Self-grafted nramp6-1nramp1 showed reduced root growth and Mn translocation from roots to young leaves compared to self-grafted nramp1 under Mn deficient conditions (Figure 4a and4b), which is in accordance with the results of nongrafted plants (Figure 1 and 2). Plants with a nramp6-1nramp1 root stock and a nramp1 shoot scion or with a nramp1 root stock and a nramp1nramp6 shoot scion displayed a similar root growth and Mn translocation capacity to the self-grafted nramp1 under Mn deficiency (Figure 4a and4b). These data indicate that mutation of NRAMP6 in both roots and shoots is required for the defective root growth and Mn translocation in nramp6-1nramp1.

We also performed reciprocal grafting between nramp6-1 and nramp6-1nramp1 to determine the contribution of the nramp1 mutation in roots and shoots to the defective phenotypes of nramp6-1nramp1. Results showed that plants with a nramp6-1nramp1 root stock and a nramp6-1 shoot scion were defective in root growth and Mn translocation from the roots to the young leaves, which is similar to self-grafted nramp6-1nramp1 (Figure 4c and4d). This indicates that unlike for NRAMP6, the absence of NRAMP1 expression in the shoot does not contribute to the abnormal phenotype of the double mutant, regardless of Mn supply. Plants with a nramp6-1 root stock and a nramp6-1nramp1 shoot scion did not display the double mutant phenotype, which expectedly requires the absence of NRAMP1 expression in the root.

Intriguingly, these plants accumulated more Mn in young leaves than self-grafted nramp6-1 (Figure 4d).

NRAMP1 can substitute for NRAMP6 to rescue the nramp6 mutation-induced defective root growth

Since NRAMP6 plays a cooperative role with NRAMP1 to promote root growth and Mn translocation under Mn deficiency, we asked whether the two proteins were exchangeable in the mediation of Mn transport. To that aim, we transformed the nramp6-1nramp1 double mutant with either one of the constructs pNRAMP6:NRAMP1-GFP and pNRAMP1:NRAMP6-GFP. Root growth was similar across all the lines under Mn sufficient conditions (Figure 5). Under Mn deficient conditions, nramp6-1nramp1 slow growth was rescued up to the nramp1 level by the introduction of the pNRAMP6:NRAMP1-GFP transgene (Figure 5a

Effect of Mn deficiency on protein accumulation and subcellular localization of NRAMP6

The NRAMP6 protein was recently showed to be addressed to Golgi and TGN in protoplasts [START_REF] Li | The intracellu lar transporter AtNRAMP6 is involved in Fe homeostasis in Arabidopsis[END_REF]. To examine NRAMP6 subcellular localization in Arabidopsis, we first examined GFP fluorescence in Arabidopsis protoplasts transiently expressing the 35S:NRAMP6-GFP construct. We found that NRAMP6-GFP was mainly localized to the ER, which was evidenced by its co-localization with the ER marker RFP-HDEL (Figure S6a). We also fused GFP to the N-terminus of NRAMP6 and investigated the subcellular localization of GFP-NRAMP6. Similarly, GFP-NRAMP6 fluorescence was also predominantly localized to the ER (Figure S6b). To determine the subcellular localization of NRAMP6 in Arabidopsis plants, we used the pNRAMP1:NRAMP6-GFP and 35S:NRAMP6-GFP lines with nramp6-1nramp1 background to observe GFP fluorescence. The expression level of NRAMP6 in roots of these lines was 15-to 25-fold higher than that in WT (Figure S5a andS5b). However, we could not detect the GFP fluorescence in these transgenic lines.

In order to obtain transgenic lines with high expression level of NRAMP6, we changed to use UBQ10 promoter and generated pUBQ10:NRAMP6-YFP transgenic lines in nramp6-1nramp1 background. The expression level of NRAMP6 in roots of the transgenic lines was around 100-fold higher than in those of WT (Figure S7a). In these lines, we were able to detect YFP fluorescence and found that NRAMP6-YFP fluorescence could be detected in the plasma membrane as well as intracellular membranes, co-localizing in part with the plasma membrane marker FM4-64. The intracellular membrane-localized NRAMP6-YFP was largely but not completely co-localized with an ER marker SP-mCherry-HDEL (Figure S6c). Interestingly, Mn deficiency was able to induce the accumulation of NRAMP6-YFP and promote the localization of NRAMP6-YFP to the plasma membrane (Figure 6a and6b).

We also generated pUBQ10:NRAMP6-HA transgenic lines in nramp6-1nramp1

background. The NRAMP6 expression level in roots of the pUBQ10:NRAMP6-HA transgenic lines was nearly 1,000 times higher than in those of WT (Figure S7b).

Immunoblot analysis using anti-HA antibody showed that Mn deficiency increased NRAMP6-HA protein level in the roots, but reduced its accumulation in the shoots (Figure 6c). To confirm this observation, we performed immunoblot analysis of NRAMP6-HA in two pNRAMP6:NRAMP6-HA transgenic lines with nramp1 background. Although NRAMP6-HA protein was not detected in the roots of the two lines under Mn sufficient conditions, it could be detected in the roots under Mn deficient conditions (Figure 6d). In contrast, Mn deficiency reduced the protein accumulation of NRAMP6-HA in the shoots (Figure 6d). These results revealed that Mn plays an opposite role in the regulation of NRAMP6 protein accumulation between roots and shoots.

We then analyzed the subcellular localization of NRAMP6-HA of the pUBQ10:NRAMP6-HA transgenic lines by aqueous two-phase partitioning. Aqueous two-phase partitioning in polyethylene glycol-Dextran mixtures can make around 90% of the plasma membrane and 80-90% of the intracellular membranes partitioned to the upper phase and the lower phase, respectively [START_REF] Larsson | Preparation of h igh-purity plas ma membranes[END_REF][START_REF] Dewitt | Targeting of two Arabidopsis H + -ATPase isoforms to the plasma membrane[END_REF]. Consistently, our aqueous two-phase partitioning experiments showed that the plasma membrane (H + -ATPase) and ER (BiP) markers enriched to the upper phase and the lower phase, respectively (Figure 6e). NRAMP6-HA was found to be present at both the upper and lower phases in roots and shoots under Mn sufficient conditions, albeit preferentially existed at the lower phase especially in the roots (Figure 6e).

Interestingly, Mn deficiency remarkably promoted the accumulation of NRAMP6-HA to the upper phase in the roots (Figure 6e). Together, these results demonstrate that Mn deficiency increases NRAMP6 protein level in the roots while reduces its accumulation in the shoots, and promotes the localization of NRAMP6 to the plasma membrane.

NRAMP6 can rescue the defective growth of yeast Δsmf2 under Mn deficiency

To examine whether NRAMP6 has Mn transport capacity, we expressed NRAMP6 in the yeast strains Δsmf1 and Δsmf2 that are defective in Mn uptake and endomembrane Mn transport, respectively [START_REF] Supek | A yeast manganese transporter related to the macrophage protein involved in conferring resistance to mycobacteria[END_REF][START_REF] Cohen | The family of SMF metal ion transporters in yeast cells[END_REF]. Under Mn sufficient conditions, NRAMP6-expressing yeast cells could grow at a similar speed to control cells harboring the empty vector (Figure 7). Under Mn deficiency, while the expression of NRAMP6 did not rescue growth of Δsmf1 yeast cells, NRAMP6 expression in Δsmf2 cells was able to restore it substantially (Figure 7). Previously, NRAMP6 was demonstrated to be localized to the endomembrane compartment instead of the plasma membrane of the yeast cells [START_REF] Cailliatte | The NRAMP6 metal transporter contributes to cadmium toxicity[END_REF]. These results thus suggest that NRAMP6 can function as an endomembrane Mn transporter in yeast.

Discussion

NRAMP family members in plants are reported to have a broad range of metal substrates, including Mn, Fe and Cd [START_REF] Lanquar | Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron[END_REF][START_REF] Cailliatte | High-affin ity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions[END_REF][START_REF] Lanquar | Export of vacuolar manganese by AtNRAMP3 and AtNRAMP4 is required fo r optimal photosynthesis and growth under manganese deficiency[END_REF][START_REF] Sasaki | Nramp5 is a major transporter responsible for manganese and cadmium uptake in rice[END_REF][START_REF] Castaings | The high-affinity metal transporters NRAMP1 and IRT1 team up to take up iron under sufficient metal provision[END_REF]. NRAMP6 was previously reported to function as a Cd transporter to contribute to Cd toxicity in Arabidopsis [START_REF] Cailliatte | The NRAMP6 metal transporter contributes to cadmium toxicity[END_REF], and recently it was also characterized as a Fe transporter to regulate plant growth under Fe-deficient conditions [START_REF] Li | The intracellu lar transporter AtNRAMP6 is involved in Fe homeostasis in Arabidopsis[END_REF].

However, whether NRAMP6 plays a role in Mn nutrition in Arabidopsis was still unclear. In this study, we demonstrate that NRAMP6 can function as a Mn transporter to modulate root growth and Mn translocation under Mn deficiency. Although nramp6 single mutants do not show any defective phenotypes under Mn deficient conditions, simultaneous inactivation of NRAMP6 and NRAMP1 genes enhances the root growth inhibition phenotype of the nramp1 single mutant in response to Mn deficiency (Figure 1). Since the expression level of NRAMP1 is remarkably higher than that of NRAMP6 (Figure S4a), these results suggest that NRAMP6 and NRAMP1 have overlapping functions in the plant tolerance to Mn deficiency, with NRAMP1 being the major player.

A previous study established that NRAMP1 is required for Mn acquisition by the roots [START_REF] Cailliatte | High-affin ity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions[END_REF]. In this study we demonstrate that NRAMP1 also plays a cooperative role with NRAMP6 in the translocation of Mn from roots to shoots (Figure 2). The exact mechanism as to how NRAMP1 and NRAMP6 cooperatively mediate Mn translocation is still unknown. Grafting experiments reveal that mutation of NRAMP6 in both roots and shoots is required for the defective root growth and Mn translocation in nramp1nramp6 (Figure 4), while mutation of NRAMP1 only in the roots is required for defective phenotypes in the double mutant. These results suggest that the nramp6 mutation in the shoots might elicit and transmit a signal from the shoots to the roots to regulate root growth and Mn transport. Interestingly, plants harboring a root stock of nramp6-1 and a shoot scion of nramp6-1nramp1 accumulated a higher level of Mn in young leaves than self-grafted nramp6-1 (Figrue 4d), which suggests that the shoots of nramp6-1nramp1 might experience Mn deficiency and transmit a signal to the roots and promotes Mn transport to the shoots, although the exact underlying mechanism remains elusive. Our data also imply the existence of long distance shoot-to-root signaling in plants subjected to Mn deficiency.

Although NRAMP6 is mainly expressed in the shoots (Figure 3), the lower expression of NRAMP6 in the roots is required for root growth and Mn translocation under Mn deficient conditions. The mRNA expression of NRAMP6 was not responsive to Mn deficiency in both WT and nramp1 background (Figure 3b and3c).

Nevertheless, we found that the protein level of NRAMP6-HA under the control of both UBQ10 and NRAMP6 native promoters was increased in the roots while decreased in the shoots under Mn deficiency (Figure 6c and6d). Moreover, Mn deficiency promotes the NRAMP6-YFP and NRAMP6-HA accumulation to the plasma membrane (Figure 6e). Together, these results reveal that plants can increase the total level of NRAMP6 protein and its localization to the plasma membrane to compensate the defective Mn uptake and/or translocation of nramp1 when environmental Mn availability is low. On the other hand, the expression of NRAMP6 in vascular tissues of the shoots implies that NRAMP6 in the shoots might play a role in unloading Mn from the roots and/or the remobilization of Mn from old leaves to young tissues. Under Mn deficient conditions, the reduction of NRAMP6 protein level in the shoots may help the retention of more Mn in the roots to preferentially sustain root growth.

NRAMP6-GFP was previously shown to be localized to the Golgi in Arabidopsis protoplasts [START_REF] Li | The intracellu lar transporter AtNRAMP6 is involved in Fe homeostasis in Arabidopsis[END_REF]), but we found that both NRAMP6-GFP and GFP-NRAMP6 are mainly localized to the ER in the Arabidopsis protoplasts (Figure S6). The reason for the different results of NRAMP6-GFP subcellular localization between the previous report and this study is currently unknown. In Arabidopsis plants, NRAMP6 was found to be localized in both the plasma membrane and ER, and its localization to the plasma membrane was enhanced under Mn deficiency (Figure 6a and6b). In accordance with the partial localization of NRAMP6 to the plasma membrane, the plasma membrane-localized NRAMP1 can substitute for NRAMP6 to rescue the defective root growth of nramp6-1nramp1 (Figure 5a and5b)

and NRAMP6 plays a cooperative role with NRAMP1 but not with the Golgi-localized NRAMP2 in the regulation of root growth under Mn deficiency (Figure 1; Figure S2). The replaceable function of NRAMP6 by NRAMP1 also suggests that the plasma membrane-localized portion of NRAMP6 is involved in the regulation of root growth and Mn accumulation under Mn deficient conditions. The dual localization of NRAMP6 might also explain the lack of full recovery of nramp6nramp1 root growth by the pNRAMP1:NRAMP6-GFP transgene under Mn deficiency (Figure 5c and 5d), although we can not exclude the possibility that NRAMP6 has a different Mn transport activity or protein stability from NRAMP1. In the future, it would be interesting to determine how Mn deficiency regulates NRAMP6 at a post-transcriptional level.

EXPERIMENTAL PROCEDURES Plant materials and growth conditions

Arabidopsis thaliana (Columbia ecotype, Col-0) was used as the wild-type for all the control experiments. The T-DNA insertion lines nramp6-1 (GK-550D06) and nramp1 (SALK_053236C) were derived from uNASC (http://szlapncs01.nottingham.ac.uk/).

nramp2 mutant was obtained from our previous work [START_REF] Gao | NRAMP2, a trans-Golgi network-localized manganese transporter, is required for Arabidopsis root growth under manganese deficiency[END_REF]. To generate an additional mutant allele of NRAMP6, the CRISPR/Cas9-based genome editing method was used. Two 20-bp coding sequences (Table S1) of NRAMP6 were constructed into a sgRNA-Cas9 expression vector [START_REF] Ma | CRISPR/Cas9-based multip lex genome edit ing in monocot and dicot plants[END_REF]) and transformed into Col-0 WT background. After sequencing the target region of NRAMP6 in T3 transgenic lines without Cas9-transgene, a mutant allele (nramp6-2) was identified, which has 1-bp insertion at 15 bp from the start codon of NRAMP6.

Seeds were sterilized by 8% sodium hypochlorite and then stratified for 3 d at 4°C

for plant growth in agar plates. The agar medium consists of 2% sucrose, 1% agar (A1296, Sigma-Aldrich), and full-strength Hoagland nutrient solution (5 mM KNO3, 5 mM CaNO3, 2 mM MgSO4, 1 mM NH4H2PO4, 3 μM H3BO3, 1 μM (NH4)6Mo7O24, 0.4 μM ZnSO4, 0.2 μM CuSO4, 20 μM Fe(III)-EDTA) containing various concentrations of MnSO4 at pH 5.7. For hydroponic cultures, seeds were directly grown a plastic mesh floating on one-tenth strength Hoagland nutrient solution and the solution was renewed every two to three days. Plants were grown in a growth chamber with 14 h of light and 10 h of darkness at 22°C.

Evaluation of root growth in response to Mn deficiency

Seedlings were grown on a nutrient agar plate containing 0, 0.5, 2, 5, or 10 μM MnSO4. After growth for 7 d, the seedlings were photographed and compared. The root length was measured using ImageJ software. Root hairs in the mature zones of the roots were photographed by a stereo microscope (SZX12, Olympus) equipped with a camera (DP20, Olympus) and measured for their lengths by ImageJ.

Measurement of cation concentration

Seeds were grown on a 1/10 strength Hoagland nutrient solution containing 10 μM MnSO4 for 18 d and then the plants were exposed to the same Hoagland nutrient solution containing 0 or 10 μM MnSO4 for 9 d. Roots, young leaves (three to four top unexpanded leaves) and old leaves (fully expanded leaves) were sampled after the roots were washed with a solution of 2 mM CaSO4 and 10 mM EDTA for 10 min to remove apoplastic Mn and subsequently rinsed with ultra-pure water for three times.

The samples were dried at 65°C for more than 3 d and digested with concentrated HNO3 and then diluted with 2% HNO3. The concentrations of Mn, Zn or Fe in the diluted solution were determined using inductively coupled plasma mass spectrometry (ICP-MS; PerkinElmer NexION300D).

Grafting experiments

Sterilized and stratified seeds of nramp1, nramp6-1 and nramp6-1nramp1 were grown on an agar medium consisting of 1.2% agar, 1/2 MS medium plus 0.1% vitamins (M519, PhytoTech Labs), 0.04 ppm 6-benzylaminopurine (B3408,

Complementation analysis

To perform complementation test of nramp6-1nramp1 mutant, a DNA fragment harboring 1.8 kb promoter, the gene and 1.0 kb downstream of stop codon of NRAMP6 (AT1G15960) was amplified and cloned into a binary vector pCAMBIA3301. The resultant vector was transformed into nramp6-1nramp1 by Agrobacterium-mediated floral dip method. T3 homozygous plants harboring the transgene were subjected for root growth assay, root hair observation and Mn accumulation analysis.

To examine whether NRAMP6 fused with GFP is functional in vivo, NRAMP6 was amplified and inserted into pCAMBIA1301 vector that harbors 35S promoter and GFP sequence. The resultant vector containing 35S:NRAMP6-GFP was transformed into nramp6-1nramp1 mutant. T3 homozygous plants harboring the transgene were subjected for root growth assay.

To investigate whether NRAMP1 and NRAMP6 proteins can substitute each other to be functional in plants, we constructed pNRAMP1:NRAMP6-GFP and pNRAMP6:NRAMP1-GFP vectors. For the construct of pNRAMP1:NRAMP6-GFP, the 1.65 kb NRAMP1 promoter and NRAMP6 coding sequence without a stop codon were amplified, respectively, and then fused to one fragment by overlapping PCR and cloned into the pCAMBIA1301 vector harboring GFP sequence. For the construct of pNRAMP6:NRAMP1-GFP, the 1.8 kb NRAMP6 promoter and NRAMP1 coding sequence without a stop codon were amplified and fused, and then inserted into the same pCAMBIA1301 vector harboring the GFP sequence. The two constructs were transformed into nramp6-1nramp1 mutant, respectively, and T3 homozygous plants harboring each transgene were subjected for root growth assay. The primers for all the vector constructs can be found in Table S1.

mRNA expression analysis

To examine the expression pattern of NRAMP6, different tissues including roots, Ltd., Nanjing, China) was used. UBQ10 was used as an internal control. The expression levels of NRAMP6 and NRAMP1 was first normalized to UBQ10 control and then normalized to NRAMP6 level in roots under Mn sufficient conditions. The detailed information of the primers used for expression analysis are listed Table S1.

Real-time RT-PCR data were collected in accordance with the CFX96 Touch real-time PCR detection system (BioRad).

GUS analysis

To generate pNRAMP6:NRAMP6-GUS and pNRAMP1:NRAMP1-GUS transgenic lines, 1.8-kb promoter plus the gene of NRAMP6 and 2.0-kb promoter plus the gene of NRAMP1 without their stop codons were amplified and fused in frame to GUS reporter gene in pORE-R2 vector, respectively. T3 transgenic plants containing the homozygous pNRAMP6:NRAMP6-GUS or pNRAMP1:NRAMP1-GUS were selected for GUS analysis. For pNRAMP6:NRAMP6-GUS transgenic lines, roots and shoots were then stained with a commercialized GUS staining solution (161031; o'Biolab Co., Ltd., Beijing, China) for overnight at 37�. For pNRAMP1:NRAMP1-GUS transgenic lines, roots were stained for 45 min to 1 h, while shoots were stained for overnight at 37�. Stained shoot samples were subjected to an ethanol series (20%, 35%, 50% and 70%) to remove chlorophylls. Stained tissues were observed with a stereomicroscope (SZX7, Olympus) and photographed with a DP26 camera (Olympus).

Subcellular localization analysis

To determine the subcellular localization of NRAMP6 in protoplasts, the NRAMP6 EDTA-free (5892791001; Roche). The homogenate was centrifuged at 8,000 g for 15 min at 4 � and the derived supernatant was then centrifuged at 100,000 g to obtain the microsomal pellet. Aqueous two-phase partitioning was performed using a 20% dextran and 40% PEG3350 mixture and centrifuging at 1,500 g for 5 min.

Immunoblot blot analysis was carried out to determine NRAMP6-HA protein level using anti HA-HRP antibody (12013819001; Roche). The Actin, H + -ATPase and BiP proteins were used as controls, which were detected by anti-Actin (CW0264M;

CoWin Biosciences Co., Ltd., China), anti-H + -ATPase [START_REF] Hayashi | Biochemical characterization of in vitro phosphorylation and dephosphorylation of the p lasma membrane H + -ATPase[END_REF] and anti-BiP (PHY2155S; PhytoAB) antibodies, respectively. The sequence information of the primers used for the vector constructs are listed Table S1.

Yeast experiments

The coding sequence of NRAMP6 was amplified and cloned into yeast vector pYC2, which is a low-copy number plasmid and has a galactose-inducible promoter GAL1.

The NRAMP6-pYC2 and pYC2 empty vectors were, respectively, transformed into wild-type yeast strain BY4741 (MATa;his3,leu2,met15,ura3), and two mutant strains Δsmf1 (MATa;his3,leu2,met15,ura3,smf1::kanMX4) andΔsmf2 (MATa;his3,leu2,met15,ura3,smf2::kanMX4). Yeast cells of each strain were grown in 2% glucose-containing SD liquid mediums without uracil at 30� for overnight and then the yeast cells were harvested and washed with water for three times. The derived yeast cells were resuspended with water and adjusted to OD600=0.6. Aliquots of 10-fold serial dilutions were dropped on 2% agar SD-Ura medium supplemented with or without 5 mM EGTA.

Statistical analysis

One-way ANOVA followed by Tukey test was used for the statistical analysis of all the quantitative data except for the data of Figure 6b in which two-tailed student's t test was used for the statistical analysis.

  and 5b). In contrast, introduction of pNRAMP1:NRAMP6-GFP into the double mutant was unable to recover the deficient root growth of the double mutant to the nramp6-1 level (Figure 5c and 5d), although the pNRAMP1:NRAMP6-GFP transgene could improve the root growth of the double mutant to the nramp1 level. Quantitative RT-PCR analysis showed that the expression levels of NRAMP6 in pNRAMP1:NRAMP6-GFP/nramp6-1nramp1 transgenic lines were comparable to the NRAMP1 expression level in WT (Figure S5a). The defective recovery of root growth in the pNRAMP1:NRAMP6-GFP lines was not due to the functional deficiency of NRAMP6 fusion with GFP, because 35S:NRAMP6-GFP could rescue the defective root growth of nramp6-1nramp1 to the nramp1 level under Mn deficiency (Figure S5b-d). These results demonstrate that NRAMP1 can substitute for NRAMP6 to regulate root growth, whereas the function of NRAMP1 is not replaceable by the NRAMP6.

  Sigma-Aldrich), 0.02 ppm IAA (I2886, Sigma-Aldrich), 3 ppm BEN (381586, Sigma-Aldrich), and 2% sucrose. After growth for 4 d, the seedlings were kept in the dark for 2 d to promote hypocotyl elongation. Subsequently, the seedlings with approximately equal hypocotyl diameters and developmental stages were excised with a 15° stab knife (SAR POINT REF 72-1501) and then shoot scions and root stocks were grafted. The grafted seedlings were grown on the agar medium for 6 d for recovery. The successfully grafted plants were then transferred to a 1/10 strength Hoagland nutrient solution containing 10 μM MnSO4 for a hydroponic culture. After growth for 12 d, the plants were treated with the Hoagland nutrient solution containing 0 or 10 μM MnSO4 for 8 d. The Mn concentrations in roots, young and old leaves were determined by ICP-MS.

  young and old leaves were excised for RNA extraction. For the expression analysis of NRAMP6 in response to Mn, seeds of WT and nramp1 mutant were sown on a nutrient agar medium with or without 10 μM MnSO4 for 7 d growth and the roots and shoots were harvested for RNA isolation. For the expression analysis of NRAMP6 under Fe or Zn deficient conditions, 20-day-old plants of WT were exposed to 1/10 strength Hoagland nutrient solution without Zn or Fe for 7 d. Total RNA of each sample was extracted using the TaKaRa M iniBEST plant RNA Extraction Kit (Cat # 9769). Approximately 1 µg of total RNA was first digested with DNase I and then used for the synthesis of first-strand cDNAs by using the HiScript® 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd., Nanjing, China). One-twentieth of the cDNA products were used for RT-PCR and real-time RT-PCR analysis. For real-time RT-PCR analysis, the SYBR ® Green Master Mix kit (Vazyme Biotech Co.,

  coding sequence was amplified and fused in frame to GFP at the N-or C-terminals of NRAMP6 and then the fused sequences were, respectively, inserted into a pBluescript vector harboring the 35S promoter. The resultant vectors containing 35S:NRAMP6-GFP or 35S:GFP-NRAMP6 were co-transformed with the ER marker 35S: RFP-HDEL into Arabidopsis protoplasts for fluorescence observation using a confocal Zeiss microscope (Zeiss LSM 880). Arabidopsis protoplasts were isolated from 14-day-old seedlings according to a previous method[START_REF] Zhai | Isolation of protoplasts from tissues of 14-day-old seedlings of Arabidopsis thaliana[END_REF] and 10 μg of each plasmid DNA was used for protoplast transformation by the polyethylene glycol method. To investigate the subcellular localization of NRAMP6 in plants, we made the pUBQ10:NRAMP6-YFP fusion fragment by PCR using overlapping primers and then inserted the fragment into pCAMBIA1305 vector. The resultant vector was transformed into nramp6-1nramp1 background. T3 transgenic lines harboring the homogzygous transgene were grown on the nutrient agar plate containing 0 or 10μM MnSO4 for 7 d and then subjected to expression analysis and YFP fluorescence observation by the confocal microscope. We also fused pUBQ10:NRAMP6 or pNRAMP6:NRAMP6 fragments with 3×HA and cloned them into the pCAMBIA1305 vector, and the resultant vectors were then transformed into nramp6-1nramp1 or nramp1 mutant backgrounds. T3 seedlings were grown on a 1/10 strength Hoagland nutrient solution containing 10 μM MnSO4 for 18 d and subsequently exposed to the same nutrient solution with or without 10 μM MnSO4 for 9 d. Roots and shoots of the T3 plants were then excised for total protein extraction or microsomal membrane isolation. Microsomal membranes were extracted by using homogenization buffer consisting of 0.29 M sucrose, 25 mM HEPES (pH =8.5), 20 mM EDTA, Polyvinylpyrrolidone K40 (P0507-50g; Sangon), 0.2% BSA, 3 mM DTT, 50 μM MG132, 100 μM PMSF (not AP assay), and 1×complete protease inhibitor mixture

Figure 1 .

 1 Figure 1. Mutation of NRAMP6 in nramp1 background reduces root growth and root hair elongation at low Mn conditions. (a) Gene structure and mutation sites of NRAMP6. Black boxes and horizontal lines between the boxes indicate exons and introns, respectively. nramp6-1 and nramp6-2 have a T-DNA insertion in intron 7 and 1-bp insertion located 15 bp downstream of the start codon of NRAMP6, respectively. (b)-(e) Phenotypes of root growth (b and c) and root hair elongation (d and e) at low Mn conditions. Seeds of WT, nramp1, nramp6-1, nramp6-1nramp1, nramp6-2, and nramp6-2nramp1 were grown at a nutrient agar plate containing 0, 0.5, 2, 5, or 10 µM. After growth for 7 d, the roots were photographed for root length and root hair measurement. Scale bar=500 µm. Data shown are means ± SD of 10 to 12 root lengths (c) and of around 100 root hair lengths (e). Different letters at each treatment indicate values significantly different (P<0.05, ANOVA followed by Tukey test).

Figure 2 .

 2 Figure 2. Mn translocation from roots to shoots is impaired in nramp6nramp1 double mutants.

Figure 3 .

 3 Figure 3. Expression pattern of NRAMP6. (a) Expression analysis of NRAMP6 in roots, young leaves and old leaves. (b) Expression of NRAMP6 in WT and nramp1 backgrounds in response to Mn deficiency. Seedlings of WT and nramp1 were grown on a nutrient agar plate with 0 (-Mn) or 10 μM Mn (+Mn) for 7 d. and then roots and shoots were harvested for RNA isolation and expression analysis. (c) Expression of NRAMP6 in WT background in response to Fe or Zn deficiency. Twenty-day-old plants were exposed to a nutrient solution with or without Fe or Zn for 7 d. (d)-(g) Detection of GUS activity in pNRAMP6:NRAMP6-GUS transgenic lines. GUS expression was detected in root caps (d) and the junction sites of lateral roots and primary roots (e). (f and g) GUS activity was found in whole cotyledons at early stages (f) and the major vascular tissues of both cotyledons and true leaves at later stages (g). Scale bar=200 µm. Values are means ±SD of three (a and b) or four (c) biological replicates. Means with different letters are significantly different (P<0.05, ANOVA followed by Tukey test).

Figure 4 .

 4 Figure 4. Root biomass and Mn accumulation in grafted plants under Mn deficiency. (a), (b) root biomass (a) and Mn concentration (b) in the grafted plants between nramp1 (nr1) and nramp6-1nramp1 (nr6nr1). Successfully grafted seedlings of nr1/nr1, nr6nr1/nr6nr1, nr6nr1/nr1, and nr1/nr6nr1 (shoot/root) were cultured in

Figure 5 .

 5 Figure 5. NRAMP1 can replace NRAMP6 to regulate root growth, but not vice versa. (a), (b) Rescue of the defective root growth of nramp6-1nramp1 by pNRAMP6:NRAMP1-GFP (pNR6:NR1-GFP). Seedlings of WT, nramp1, nramp6-1, nramp6-1nramp1, and two pNR6:NR1-GFP transgenic lines in nramp6-1nramp1 background were grown on a nutrient agar plate with 0 (-Mn) or 10 μM Mn (+Mn) for 7 d. The seedlings were photographed and compared (a), and root lengths were measured and quantified (b). (c), (d) Images (c) and quantitative data (d) of root growth phenotype in WT, nramp1, nramp6-1, nramp6-1nramp1, and two pNRAMP1:NRAMP6-GFP (pNR1:NR6-GFP) transgenic lines in nramp6-1nramp1 background. Data shown are means ± SD of 10 primary root lengths. Different letters at each treatment indicate values significantly different (P<0.05, ANOVA followed by Tukey test).

Figure 6 .

 6 Figure 6. Subcellular localization of NRAMP6 in Arabidopsis plants.(a and b) Representative pictures (a) and quantitative data (b) of subcellular localization of NRAMP6-YFP under the control of UBQ10 promoter in Arabidopsis roots under different Mn conditions. Roots of pUBQ10:NRAMP6-YFP transgenic lines with nramp6-1nramp1 background were grown on a nutrient agar medium

Figure 7 .

 7 Figure 7. NRAMP6 has Mn transport capacity in yeast.NRAMP6 was constructed into a yeast low copy vector pYC2. The NRAMP6-pYC2 and pYC2 empty vectors were, respectively, transformed into wild-type yeast strain BY4741 and two mutant strains Δsmf1 and Δsmf2. Yeast cells were grown on SD medium without uracil containing galactose in the absence or presence of 5 mM Mn-chelator EGTA. Five microliter yeasts (OD600=0.6) of 10-fold serial dilutions were spotted and grown at 30 � for 3 d.

ACKNOWLEDGEMENTS

We thank Prof. Toshinori Kinoshita from Nagoya University for kindly providing the anti-H + -ATPase antibody. This work was supported by the National Natural Science Foundation of China (Grant No. 31900217), the Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, and National Key Laboratory of Plant Molecular Genetics, and by Grant-in-Aid for Specially Promoted Research (JSPS KAKENHI Grant Number 16H06296 to J.F.M.).

DATA AVAILABILITY STATEMENT

Sequence data in this article can be found in the Arabidopsis Information Resource (TAIR) database under the following accession number: At1g15960 for NRAMP6, At1g80830 for NRAMP1 and At1g80830 for NRAMP2.

AUTHOR CONTRIBUTIONS

LL, ZZ, YL, and C-FH designed the experiments; LL, ZZ, YL, CY, NF, JZ, NY, LD, and JFM performed the experiments; JFM, and CC commented on this work; LL, ZZ and C-FH analyzed the data and prepared figures; C-FH wrote the manuscript.

CONFLICT OF INTEREST

The authors declare no competing interests.

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