ZmNRAMP4 Enhances the Tolerance to Aluminum Stress in Arabidopsis thaliana

Aluminum (Al) toxicity causes severe reduction in crop yields in acidic soil. The natural resistance-associated macrophage proteins (NRAMPs) play an important role in the transport of mineral elements in plants. Recently, OsNrat1 and SbNrat1 were reported specifically to transport trivalent Al ions. In this study, we functionally characterized ZmNRAMP4, a gene previously identified from RNA-Seq data from Al-treated maize roots, in response to Al exposure in maize. ZmNRAMP4 was predominantly expressed in root tips and was specifically induced by Al stress. Yeast cells expressing ZmNRAMP4 were hypersensitive to Al, which was associated with Al accumulation in yeast. Furthermore, overexpression of ZmNRAMP4 in Arabidopsis conferred transgenic plants with a significant increase in Al tolerance. However, expression of ZmNRAMP4, either in yeast or in Arabidopsis, had no effect on the response to cadmium stress. Taken together, these results underlined an internal tolerance mechanism involving ZmNRAMP4 to enhance Al tolerance via cytoplasmic sequestration of Al in maize.


Introduction
Aluminum (Al) is the most abundant metal element in the Earth's crust [1]. About 50% of the Earth's arable land is acidic. In acidic soil (pH < 5.0), Al exists in the form of trivalent Al (Al 3+ ), which is toxic to plants, especially at high concentrations [2]. Therefore, Al toxicity is an important limiting factor for crop yield in acidic soil [3].
To cope with the high availability of soil Al, plants have evolved two mechanisms involving external exclusion and internal tolerance [4,5]. External exclusion of Al via exudation of organic acid anions from roots is an important Al resistance mechanism in many plants. External exclusion of Al via exudation of organic acid anions from the roots is an important Al tolerance mechanism in many plant species. Malate and citrate transporters have been functionally characterized in many species, including AtMATE and AtMLT1 in Arabidopsis, OsFRDL2 and OsFRDL4 in rice, SbMATE in sorghum, and TaALMT1 in wheat [6]. With regard to an internal detoxification mechanism, some transporters associated with Al tolerance can be so classified. For example, STAR1 and STAR2 form a transporter protein complex that is responsible specifically for efflux of UDP-glucose, which is likely to be used to modify the cell wall [7]. In Arabidopsis, AtSTAR1 and ALS3 are involved in Al tolerance [8,9]. Overexpression of OsPIN2, which encodes an auxin transporter, can decrease the binding of Al to the cell wall [10]. Certain transcription factors Int. J. Mol. Sci. 2022, 23, 8162 2 of 12 (AtSTOP1 [11], OsART1 [12], VuNAR1 [13], WRKY47 [14], HvHOX9 [15], GsMAS1 [16] and abscisic acid (ABA)-stress and ripening (ASR5) [17]) and ROS-related genes (ZmAT6) [18] are also involved in plant Al stress response.
NRAMP proteins comprise a group of highly conserved integral membrane proteins widespread among bacteria, animals, and plants [19]. The genomes of sorghum, rice, maize, and Arabidopsis contain 7, 7, 8, and 6 NRAMP members, respectively [20]. NRAMP proteins play a major role in the transport of mineral elements in plants, including the metal ions Fe 2+ , Mn 2+ , Cd 2+ , Zn 2+ , Co 2+ , and Cu 2+ [21]. For example, OsNRAMP1 transports Cd, As, and Fe, but not Mn [22]; OsNRAMP5 is the main transporter for Cd 2+ influx in rice [23,24]; AtNRAMP3 and AtNRAMP4 transport Fe 2+ and Mn 2+ in Arabidopsis [25]; BcNRAMP1 is a plasma membrane-localized protein and transports Mn and Cd [26]; and FeNRAMP5 is a transporter for the uptake of Mn and Cd [27]. Interestingly, two NRAMP members, OsNrat1 and SbNrat1, transport Al 3+ specifically but not divalent metal ions, such as Fe 2+ , Mn 2+ , and Cd 2+ , and are required for a prior step of final Al detoxification through vacuolar sequestration of Al [3,28,29]. The aim of the present study was to functionally characterize the role of ZmNRAMP4, which was previously identified from RNA-Seq data for Alstressed maize roots, in Al transport in maize. The results showed that ZmNRAMP4 was upregulated by Al treatment. Combined heterologous expression in yeast and Arabidopsis indicated that ZmNRAMP4 is an Al transporter and its expression is stimulated by Al. Overexpression of ZmNRAMP4 enhanced the Al tolerance of transgenic Arabidopsis plants via Al influx in the roots.

Cloning and Phylogenetic Analysis of ZmNRAMP4
ZmNRAMP4 was cloned from the Al-tolerant maize inbred line 178 ( Figure S1). The gene encoded a putative protein of 556 amino acids, which belongs to the NRAMP family of maize. The multiple sequence alignment showed that ZmNRAMP4 included all conserved elements of NRAMP members, such as Motif A and Motif B, and had the highest identity with SbNrat1 of 96% ( Figure S2). A phylogenetic tree for NRAMP members was composed of five main clades. Notably, ZmNRAMP4 was placed in the same clade as OsNrat1 and SbNrat1, two previously reported transporters of trivalent Al ions, but was separated from divalent ion transporters ( Figure 1).

Expression Patterns of ZmNRAMP4
To explore if the eight ZmNRAMP members were stimulated by Al stress, the expression patterns of all eight ZmNRAMP genes were investigated. The results revealed that ZmNRAMP4 was the only ZmNRAMP gene that was induced by Al stress (Figure 2A). Therefore, the expression pattern of ZmNRAMP4 in response to Al stress was studied further. The Al-induced expression of ZmNRAMP4 was predominantly detected in the root and was highest in the root tip ( Figure 2B). A time-course experiment showed that ZmN-RAMP4 expression increased after Al exposure and peaked at 24 h, and thereafter decreased with prolonged treatment ( Figure 2C). The expression of ZmNRAMP4 was significantly affected by the AlCl 3 concentration; a broad range of AlCl 3 concentrations (40-100 µM) stimulated ZmNRAMP4 expression effectively and 80 µM AlCl 3 induced the optimum stimulation ( Figure 2D). In addition, ZmNRAMP4 expression was specifically affected by Al rather than other metal ions, such as Zn, Cu, La, or Cd, as well as low pH ( Figure 2E).  The information of accession number and species for NRAMPs is as follows, including AtNRAMP1 to AtNRAMP6 (AT1G80830, AT1G47240, AT2G23150, AT5G67330, AT4G18790, AT1G15960), OsNRAMP1 to OsNRAMP6 (Os07g15460, Os03g11010, Os06g46310, Os07g15370, Os12g39180, Os01g0503400), OsNrat1 (Os02g0131800), SbNRAMP1 to SbNRAMP6 (XP_002459640, XP_002465667, XP_002438846, XP_021317241, XP_002461772, XP_002464246), SbNrat1 (XP_002451480), ZmN-RAMP1 to ZmNRAMP8 (Zm00001d005479, Zm00001d007397, Zm00001d014391, Zm00001d015133, Zm00001d019327, Zm00001d030846, Zm00001d033367, Zm00001d048129). nificantly affected by the AlCl3 concentration; a broad range of AlCl3 concentratio 100 µM) stimulated ZmNRAMP4 expression effectively and 80 µM AlCl3 induced timum stimulation ( Figure 2D). In addition, ZmNRAMP4 expression was specific fected by Al rather than other metal ions, such as Zn, Cu, La, or Cd, as well as l ( Figure 2E).  (E), Expression of ZmNRAMP4 in response to other metals. Seedlings were exposed to a solution containing 60 µM AlCl 3 , 10 µM LaCl 3 , 100 µM ZnSO 4 , 2 µM CuCl 2 , and 30 µM CdCl 2 or pH 4.2 for 12 h. Asterisk indicated significant difference from control at p < 0.05 by Tukey's test with three independently biological replicates.

Subcellular Localization of ZmNRAMP4
To visualize the subcellular localization of ZmNRAMP4, ZmNRAMP4-GFP was transiently expressed in Nicotiana benthamiana leaves. The green fluorescent signal of GFP of the control vector was ubiquitous throughout the cell ( Figure 3A). In contrast, the ZmNRAMP4-GFP signal was only detected at the plasma membrane ( Figure 3B), consistent with the prediction of ZmNRAMP4 as a putative metal ion transporter.

Subcellular Localization of ZmNRAMP4
To visualize the subcellular localization of ZmNRAMP4, ZmNRAMP4-GFP was transiently expressed in Nicotiana benthamiana leaves. The green fluorescent signal of GFP of the control vector was ubiquitous throughout the cell ( Figure 3A). In contrast, the ZmNRAMP4-GFP signal was only detected at the plasma membrane ( Figure 3B), consistent with the prediction of ZmNRAMP4 as a putative metal ion transporter.

Functional Expression of ZmNRAMP4 in Yeast
To investigate the Al transport ability of ZmNRAMP4, ZmNRAMP4 was expressed in the yeast strain BY4741. OsNrat1, an Al transporter in rice, its expression vector, and the empty vector were used as a positive control and negative control, respectively. In the absence of Al, all yeast cells, either carrying ZmNRAMP4, OsNrat1, or the empty vector, showed similar growth rates ( Figure 4A). However, compared with the negative control, the growth of yeast cells expressing ZmNRAMP4 or OsNrat1 were slightly inhibited in the presence of Al ( Figure 4A), suggesting that Al influx into the yeast cells occurred. Moreover, the yeast cells expressing ZmNRAMP4 or OsNrat1 accumulated a higher concentration of Al ( Figure 4C), accompanied by stronger inhibition of growth ( Figure 4B) when compared with the negative control. We also investigated the Cd transport activity of ZmNRAMP4 in yeast because some NRAMP proteins are involved in the transport of other metal ions. The pYES2-ZmNRAMP4 construct or the empty vector pYES2 were expressed both in a Cd-sensitive mutant strain Δycf1 and the wild-type strain BY4741. No growth difference was observed among the transformed yeasts in the absence of Cd stress ( Figure S3). In response to treatment with 50 µM CdCl2, BY4741 yeast cells carrying ZmNRAMP4 grew similarly to those carrying the empty vector. In contrast, Cd severely inhibited the growth of Δycf1 yeast cells transformed with the empty vector but not cells transformed with pYES2-ZmNRAMP4 ( Figure S3). These results revealed that ZmNRAMP4 has transport activity for Al but not Cd in yeast.

Functional Expression of ZmNRAMP4 in Yeast
To investigate the Al transport ability of ZmNRAMP4, ZmNRAMP4 was expressed in the yeast strain BY4741. OsNrat1, an Al transporter in rice, its expression vector, and the empty vector were used as a positive control and negative control, respectively. In the absence of Al, all yeast cells, either carrying ZmNRAMP4, OsNrat1, or the empty vector, showed similar growth rates ( Figure 4A). However, compared with the negative control, the growth of yeast cells expressing ZmNRAMP4 or OsNrat1 were slightly inhibited in the presence of Al ( Figure 4A), suggesting that Al influx into the yeast cells occurred. Moreover, the yeast cells expressing ZmNRAMP4 or OsNrat1 accumulated a higher concentration of Al ( Figure 4C), accompanied by stronger inhibition of growth ( Figure 4B) when compared with the negative control. We also investigated the Cd transport activity of ZmNRAMP4 in yeast because some NRAMP proteins are involved in the transport of other metal ions. The pYES2-ZmNRAMP4 construct or the empty vector pYES2 were expressed both in a Cd-sensitive mutant strain ∆ycf1 and the wild-type strain BY4741. No growth difference was observed among the transformed yeasts in the absence of Cd stress ( Figure S3). In response to treatment with 50 µM CdCl 2 , BY4741 yeast cells carrying ZmNRAMP4 grew similarly to those carrying the empty vector. In contrast, Cd severely inhibited the growth of ∆ycf1 yeast cells transformed with the empty vector but not cells transformed with pYES2-ZmNRAMP4 ( Figure S3). These results revealed that ZmNRAMP4 has transport activity for Al but not Cd in yeast.

Overexpression of ZmNRAMP4 Enhanced Al Tolerance of Arabidopsis Plants
To determine whether ZmNRAMP4 affected Al tolerance in other plants, ZmNRAMP4 was overexpressed in Arabidopsis under the control of the 35S promoter. The Al-tolerant phenotype conferred by ZmNRAMP4 was investigated in two transgenic lines that highly expressed ZmNRAMP4 ( Figure S4). Under normal condition, root growth of transgenic plants was comparable with that of the wild type, whereas the roots were longer than those of the wild type after Al exposure ( Figure 5A). Similarly, the relative root growth of the transgenic lines was significantly higher than that of the wild type under Al stress ( Figure 5B). In addition, the transgenic lines accumulated greater concentrations of Al in the root than the wild type ( Figure 5C). However, overexpression of ZmNRAMP4 did not affect the root growth of the transgenic Arabidopsis plants under Cd stress ( Figure 6). These results demonstrated that overexpression of ZmNRAMP4 in Arabidopsis conferred a notable increase in Al tolerance, but had no effect on tolerance of Cd stress.

Overexpression of ZmNRAMP4 Enhanced Al Tolerance of Arabidopsis Plants
To determine whether ZmNRAMP4 affected Al tolerance in other plants, ZmNRAMP4 was overexpressed in Arabidopsis under the control of the 35S promoter. The Al-tolerant phenotype conferred by ZmNRAMP4 was investigated in two transgenic lines that highly expressed ZmNRAMP4 ( Figure S4). Under normal condition, root growth of transgenic plants was comparable with that of the wild type, whereas the roots were longer than those of the wild type after Al exposure ( Figure 5A). Similarly, the relative root growth of the transgenic lines was significantly higher than that of the wild type under Al stress ( Figure 5B). In addition, the transgenic lines accumulated greater concentrations of Al in the root than the wild type ( Figure 5C). However, overexpression of ZmNRAMP4 did not affect the root growth of the transgenic Arabidopsis plants under Cd stress ( Figure 6). These results demonstrated that overexpression of ZmNRAMP4 in Arabidopsis conferred a notable increase in Al tolerance, but had no effect on tolerance of Cd stress.

Discussion
Most NRAMP proteins are capable of transporting Cd and essential metals, such as Mn and Fe [30]. Recent studies have shown that OsNrat1 and SbNrat1 have transport activity for Al [3]. ZmNRAMP4 is a candidate gene for Al tolerance QTL co-localization and upregulated after 6 h of Al exposure in the root tip [31]. However, the functional characterization of ZmNRAMP genes in response to Al stress remains unclear. To verify the function of ZmNRAMP4, we investigated the effects of ZmNRAMP4 expression on metal tolerance and accumulation in yeast and Arabidopsis.
In rice, OsNrat1 expression is stimulated rapidly by Al, particularly in the root, and expression is maintained at a low level in the shoot. Furthermore, the stimulation by Al is specific because other metals do not induce OsNrat1 expression. The expression pattern of ZmNRAMP4 was consistent with that of OsNrat1 but differed notably from that of SbNrat1, which was not responsive to Al regardless of the Al concentration and exposure duration [3]. Given that ZmNRAMP4 was placed in the same subclade as OsNrat1 and SbNrat1 in the present phylogenic analysis (Figure 1), the difference in expression is probably attributable to differences among cereal species in the fine-tuning of environmental adaptation during evolution. This suggestion is consistent with the evolution of diverse

Discussion
Most NRAMP proteins are capable of transporting Cd and essential metals, such as Mn and Fe [30]. Recent studies have shown that OsNrat1 and SbNrat1 have transport activity for Al [3]. ZmNRAMP4 is a candidate gene for Al tolerance QTL co-localization and upregulated after 6 h of Al exposure in the root tip [31]. However, the functional characterization of ZmNRAMP genes in response to Al stress remains unclear. To verify the function of ZmNRAMP4, we investigated the effects of ZmNRAMP4 expression on metal tolerance and accumulation in yeast and Arabidopsis.
In rice, OsNrat1 expression is stimulated rapidly by Al, particularly in the root, and expression is maintained at a low level in the shoot. Furthermore, the stimulation by Al is specific because other metals do not induce OsNrat1 expression. The expression pattern of ZmNRAMP4 was consistent with that of OsNrat1 but differed notably from that of SbNrat1, which was not responsive to Al regardless of the Al concentration and exposure duration [3]. Given that ZmNRAMP4 was placed in the same subclade as OsNrat1 and SbNrat1 in the present phylogenic analysis (Figure 1), the difference in expression is probably attributable to differences among cereal species in the fine-tuning of environmental adaptation during evolution. This suggestion is consistent with the evolution of diverse functions among NRAMP members in the transport of metal ions, such as Fe 2+ , Mn 2+ , Cd 2+ , Zn 2+ , Co 2+ , and Cu 2+ .
Different from other Nramp members that could transport divalent metals, OsNRAT1 specifically transports trivalent aluminum [32]. In the present study, NRAMP transporters were resolved into five clades in a phylogenetic analysis, of which ZmNRAMP4 was placed in the same clade as the previously reported Al 3+ transporters OsNrat1 and SbNrat1 (Figure 1). The metal binding site of NRAMP proteins is characterized by two conserved sequence motifs, i.e., the Asp-Pro-Ser-Asn motif (motif A) and the Ala-Ile-Ile-Thr motif (motif B) [33]. The Nrat1-type motif B is both effective and essential for Al transport by OsNrat1, as one crucial determinant of Al selectivity [34]. Missense mutations of amino acids in motif B of Al-tolerant rice lines reduce Al uptake in yeast cells, and the full function of OsNrat1 in Al influx is dependent on the most closely matched sequence of motif B [32]. Therefore, the exactly matched sequence of motif B in ZmNRAMP4 may be the predominant determinant of Al selectivity ( Figure S2). In the present study, ZmNRAMP4expressing yeast cells exhibited transport activity for Al but not Cd, which was consistent with accumulation of a higher concentration of Al. This was also the case for the Al influx transporters OsNrat1 and SbNrat1 when expressed in yeast [3]. In addition, the plasma membrane localization of ZmNRAMP4 was compatible with its function as a metal ion transporter.
NRAMP genes have been reported to be widely involved in the transport of metal ions. Recent studies have shown that overexpression of SbNrat1 recovers the Al tolerance of the rice mutant osnrat1 [3]. Similarly, overexpression of OsNRAT1 improves the Al tolerance of transgenic Arabidopsis and results in a significant increase in Al content [32]. In the present study, overexpression of ZmNRAMP4 in Arabidopsis conferred significantly elevated Al tolerance compared with the control, but had no effect on Cd tolerance. Consistent with the Al influx transport function of ZmNRAMP4, the roots of transgenic Arabidopsis plants accumulated higher concentrations of Al. The expression of endogenous AtALS1 is elevated ( Figure S5), consistent with significantly enhanced expression of NRAT1 and Al accumulation in transgenic Arabidopsis roots [32]. Given that AtALS1 has the capacity to sequester the cytosolic Al in the vacuole, it is speculated that orthologs of AtALS1 coordinate with ZmNRAMP4 to enhance the Al tolerance of maize via Al sequestration.

Plant Materials and Growth Conditions
The Al-tolerant maize inbred line 178 was used in this experiment [18]. Seeds were sterilized in 10% NaClO for 15 min, washed three times with deionized water, then cultured in an incubator at 28 • C. After germination, the seedlings were transplanted to pots filled with Hoagland's nutrient solution and grown in a greenhouse maintained at 28/20 • C under a 16 h/8 h (day/night) photoperiod. The nutrient solution was replaced every 3 days.

Phylogenetic Analysis
The sequences of NRAMP family members were retrieved from the ARAMEMNON plant membrane protein database (http://aramemnon.botanik.uni-koeln.de/index.ep; accessed on 12 December 2018). A phylogenetic tree was constructed using the neighborjoining algorithm with MEGA 5.0.

Subcellular Localization of ZmNRAMP4
The cDNA of ZmNRAMP4 was subcloned into the expression vector pC2300-GFP under the control of the 35S promoter using the primers listed in Supplementary Table  S2. The pC2300-GFP construct was introduced into Agrobacterium tumefaciens strain EHA105. Agrobacterium-mediated transient expression assays were conducted in Nicotiana benthamiana leaves [35].

Mental Ion Transport Activity of ZmNRAMP4 in Yeast
The coding sequence of ZmNRAMP4 was cloned into the pYES2 vector (negative control) to generate the construct pYES2-ZmNRAMP4. The construct pYES2-OsNrat1 was generated to serve as a positive control. The yeast strains used in this study were BY4741 (MATa his2∆0 met15∆0 ura3∆0) and the Cd-sensitive strain ∆ycf1 (MATa his2∆0 met15∆0 ura3∆0 YOL122c::KanMX4). Aluminum and Cd sensitivity was tested on agar, whereas Al uptake was measured in liquid culture. For Al-sensitivity evaluation, the constructs pYES2-ZmNRAMP4 and pYES2-OsNrat1 and the empty vector pYES2 were transformed into yeast strain BY4741 and cultured on solid medium (LPM with galactose as the carbon source) supplemented with 0 or 100 µM Al buffered with 5 mM succinic acid. For Cd-sensitivity evaluation, pYES2-ZmNRAMP4 and pYES2 were transformed into yeast strains BY4741 and ∆ycf1, and cultured in liquid SD-U medium supplemented with 0 or 50 µM Cd. For estimation of Al uptake in liquid culture, transformants were selected on uracil-deficient medium and grown in synthetic complete yeast solution using glucose as the carbon source. Cells at the mid-exponential phase were harvested, transferred to LPM medium using galactose as the carbon source, and were cultured for 2 h. Next, AlCl 3 was added to the medium at the final concentration of 60 µM Al. After incubation for 4 h with shaking, the cells were harvested by centrifugation, washed three times with deionized water, and then digested with 2 M HCl. The concentration of Al in the digest solution was determined by inductively coupled plasma-mass spectrometry.

Overexpression of ZmNRAMP4 in Arabidopsis thaliana
ZmNRAMP4 was inserted into the pC2300 vector under the control of the 35S promoter. Transgenic plants were screened with kanamycin (40 mg/L) and confirmed by PCR analysis. Two dependent T 3 transgenic lines with elevated expression levels of ZmNRAMP4 were used for assessment of Al tolerance ( Figure S4).

Evaluation of Al Tolerance
To quantify the effect of Al toxicity, the wild type and two transgenic lines were surface sterilized and germinated for 4 days on solid Murashige and Skoog (MS) medium. Seedlings of uniform growth were transferred to half-strength MS solid medium supplemented with AlCl 3 (0 or 100 µM) or CdCl 2 (0 or 25 µM) and cultured for 7 days. The root length of the seedlings was measured individually with a ruler before and after treatment.

Statistical Analysis and Reproducibility
All treatments were repeated at least three times. Statistical analysis including student's t test was performed using the SPSS software. The figures were drawn using the Origin 8.0 software (OriginLab Corporation, Northampton, MA, USA).

Conclusions
We investigated a ZmNRAMP4 gene encoding a plasma membrane-localized Al influx transporter from the Al-tolerant maize inbred line 178. Its expression is significantly upregulated in response to Al exposure, but not by other metals, in maize roots. ZmN-RAMP4 confers Al influx activity in yeast cells. Ectopic overexpression of ZmNRAMP4 in Arabidopsis enhances the Al tolerance of transgenic plants. The results suggested that ZmNRAMP4 may increase resistance to Al toxicity via cytoplasmic sequestration of Al in transgenic Arabidopsis. Data Availability Statement: All data generated or analyzed during this study are available within the article or upon request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.