A Root-Expressed Magnesium Transporter of the MRS2/MGT Gene Family in Arabidopsis thaliana Allows for Growth in Low-Mg2+ Environments

Michael Gebert; Karoline Meschenmoser; Soňa Svidová; Julian Weghuber; Rudolf Schweyen; Karolin Eifler; Henning Lenz; Katrin Weyand; Volker Knoop

doi:10.1105/tpc.109.070557

. 2009 Dec;21(12):4018–4030. doi: 10.1105/tpc.109.070557

A Root-Expressed Magnesium Transporter of the MRS2/MGT Gene Family in Arabidopsis thaliana Allows for Growth in Low-Mg²⁺ Environments^[W]

Michael Gebert ^a, Karoline Meschenmoser ^a, Soňa Svidová ^b, Julian Weghuber ^b, Rudolf Schweyen ^b,¹, Karolin Eifler ^a, Henning Lenz ^a, Katrin Weyand ^a, Volker Knoop ^a,²

PMCID: PMC2814501 PMID: 19966073

Abstract

The MRS2/MGT gene family in Arabidopsis thaliana belongs to the superfamily of CorA-MRS2-ALR-type membrane proteins. Proteins of this type are characterized by a GMN tripeptide motif (Gly-Met-Asn) at the end of the first of two C-terminal transmembrane domains and have been characterized as magnesium transporters. Using the recently established mag-fura-2 system allowing direct measurement of Mg²⁺ uptake into mitochondria of Saccharomyces cerevisiae, we find that all members of the Arabidopsis family complement the corresponding yeast mrs2 mutant. Highly different patterns of tissue-specific expression were observed for the MRS2/MGT family members in planta. Six of them are expressed in root tissues, indicating a possible involvement in plant magnesium supply and distribution after uptake from the soil substrate. Homozygous T-DNA insertion knockout lines were obtained for four members of the MRS2/MGT gene family. A strong, magnesium-dependent phenotype of growth retardation was found for mrs2-7 when Mg²⁺ concentrations were lowered to 50 μM in hydroponic cultures. Ectopic overexpression of MRS2-7 from the cauliflower mosaic virus 35S promoter results in complementation and increased biomass accumulation. Green fluorescent protein reporter gene fusions indicate a location of MRS2-7 in the endomembrane system. Hence, contrary to what is frequently found in analyses of plant gene families, a single gene family member knockout results in a strong, environmentally dependent phenotype.

INTRODUCTION

Magnesium is essential for a vast number of fundamental biochemical processes in all living cells. For example, Mg²⁺ ions are involved in the interaction of the ribosome subunits, are the counter-ions of ATP and the central ions in chlorophylls, and act as cofactors in numerous enzymes, most notably those involved in nucleotide metabolism (Maguire and Cowan, 2002) and photosynthetic carbon fixation (Lilley et al., 1974; Marschner, 2002). The Mg²⁺ ion has a unique physico-chemistry: of all biologically relevant cations, it has the smallest ionic radius, yet the largest hydration shell. This results in a 400-fold difference in volume between the hydrated and nonhydrated states. Accordingly, the proteins for the transport of magnesium across biological membranes likewise appear to be unique in nature (Shaul, 2002; Gardner, 2003; Moomaw and Maguire, 2008).

Best studied among the membrane transport systems for Mg²⁺ are the bacterial CorA proteins, which were named after the cobalt resistance phenotype observed in the respective bacterial mutants (Kehres et al., 1998; Moncrief and Maguire, 1999; Niegowski and Eshaghi, 2007). CorA proteins are characterized by a unique topology of two closely spaced, C-terminal transmembrane (TM) domains, the first of which invariably ends with a GMN (Gly-Met-Asn) tripeptide motif. The crystal structure of the Thermotoga maritima CorA protein has recently been determined (Eshaghi et al., 2006; Lunin et al., 2006; Payandeh and Pai, 2006). These studies have shown that CorA assembles as a pentamer protein complex in which the respective first TM domains of each protein subunit line the channel's pore within the membrane, while the five N termini form a large, cone-shaped funnel inside the cell.

Homologous proteins of the CorA type can be identified in all domains of life: in archaea, in eubacteria, and in all kingdoms of eukaryotes (Knoop et al., 2005). The best studied of the eukaryotic CorA homologs is the yeast Mrs2p protein, which is located in the inner mitochondrial membrane and was named for the impaired mitochondrial RNA splicing phenotype of mutants that was initially observed (Wiesenberger et al., 1992; Bui et al., 1999; Gregan et al., 2001a; Kolisek et al., 2003; Weghuber et al., 2006; Schindl et al., 2007). Structural and functional CorA/MRS2 homologs in the yeast plasma membrane are the ALR proteins, named for the aluminium resistance phenotype of mutants that initially led to their identification (MacDiarmid and Gardner, 1998; Graschopf et al., 2001; Liu et al., 2002; Lee and Gardner, 2006; Wachek et al., 2006). Only single, mitochondrial CorA-type homologs are identified in metazoan genomes, and the human homolog has been shown to complement the yeast mrs2 mutant (Zsurka et al., 2001).

In plants, CorA homologs have been identified as members of extended gene families. In Arabidopsis thaliana, this gene family has 10 members, and the family was initially named At MRS2 (Schock et al., 2000), and alternatively At MGT (Li et al., 2001) for magnesium transport. In the studies cited above, several members of the CorA-MRS2-ALR superfamily (or 2-TM-GMN-type proteins; Knoop et al., 2005) were shown to complement respective mutants across wide phylogenetic distances to varying degrees, indicating structural and functional homology in spite of low overall sequence similarities (except for the conserved GMN motif).

The extension of the MRS2/MGT gene families in plants may on the one hand be explained by adapting an evolutionary very old invention to the many different membrane systems of the plant cell. Indeed, the most distant and phylogenetic basal gene family member MRS2-11/MGT10, for example, could be localized to the chloroplast (Drummond et al., 2006). On the other hand, because land plants are sessile organisms that cannot actively choose between alternative environments, they may require a larger range of magnesium transport functionality (e.g., in adapting to Mg²⁺ availability in the soil).

Given the diversity of the MRS2/MGT gene family, it is fundamental to clearly evaluate the individual capacities for magnesium transport and the tissue-specific expression patterns. The recently established method of directly measuring the uptake of magnesium using the fluorescent magnesium binding dye, mag-fura-2, has motivated us to study all members of the plant family. Here, we used the mag-fura-2 system (Kolisek et al., 2003) to characterize the transport properties of the whole gene family via heterologous expression in the yeast mrs2 mutant.

Fusions of the β-glucuronidase (GUS) reporter gene to the promoter region of each MRS2/MGT gene were tested to investigate whether tissue- or organ-specific expression patterns are present in Arabidopsis. The physiological functions of MRS2/MGT proteins were addressed by characterizing gene knockout mutants. Viable homozygous knockout lines were raised for four genes of the family. No significant phenotypes were observed for single-gene knockouts (KOs) of three genes (MRS2-1, MRS2-5, and MRS2-10). Likewise, no impairment of plant growth and development was observed for two double KO lines that were created (mrs2-1 mrs2-5 and mrs2-5 mrs2-10), even in spite of strong and overlapping gene expression early in seedling development. However, when substrate magnesium supply was lowered to 50 μM Mg²⁺, we found a strong magnesium-dependent phenotype in planta for three independent single-gene KOs of the root-expressed MRS2-7 gene. This KO mutant phenotype of mrs2-7 is complemented and overcompensated with a cauliflower mosaic virus (CaMV) 35S promoter–driven MRS2-7 construct, which additionally leads to an increase in biomass accumulation under magnesium-limiting conditions when compared with wild-type Arabidopsis.

RESULTS

Ancestry of the Plant MRS2/MGT Gene Family

The Arabidopsis MRS2 gene family was originally described as encompassing 10 genomic loci, including one presumptive pseudogene, MRS2-9 (Schock et al., 2000). A nearly simultaneous report of the gene family (Li et al., 2001) introduced the alternative MGT nomenclature and left the MRS2-9 pseudogene unaccounted for but added a member with more distant similarity, MGT10, now also referred to as MRS2-11 (Drummond et al., 2006). The complete rice (Oryza sativa) genome sequence allows comparative inferences concerning differential diversification of the gene family in this monocot compared with the dicot Arabidopsis. The members of the At MRS2/MGT gene family have their counterparts in nine proteins encoded in the genome of rice (Figure 1).

Five clades of Arabidopsis MRS2/MGT genes were observed (Figure 1), and for these we here suggest labels A through E, with characteristic intron insertion sites for each type of gene. We found that all 12 introns in MRS2-11 (and its rice ortholog Os03g48000) are unique to clade A as can be inferred from different intron phases of closely spaced introns for which we suggest a nomenclature (see Supplemental Figure 1 online). Only clades B through E share three ancient introns in phase 0, which are occasionally lost later in evolution of the gene family (Figure 1).

Complementation of the Yeast mrs2 Mutant

Given that MRS2-1, the founding member of the Arabidopsis gene family, was initially characterized by its ability to complement the yeast mrs2 mutant when targeted to mitochondria (Schock et al., 2000), we reasoned that other members of the gene family would behave similarly, if possibly to differing degrees. Accordingly, we have now cloned a series of constructs containing the core coding regions of all MRS2 genes (except for the pseudogenes MRS2-8 and MRS2-9). These constructs were fused to the yeast Mrs2p mitochondrial targeting sequence and are driven from the native yeast MRS2 promoter in the vector YEp351.

Complementation of the mrs2 respiratory deficiency is easily monitored by restoration of the growth defect on nonfermentable medium with glycerol as the main carbon source (YPdG). Growth of yeast transformants containing Arabidopsis constructs was indistinguishable from growth of the yeast mrs2 mutant transformed with the native Mrs2p coding sequence or the empty vector on nonselective YPD medium, indicating that these constructs do not interfere with yeast metabolism (Figure 2, left panels). We found that members of the MRS2 gene family were able to complement the mrs2 phenotype to similar degrees (Figure 2, right panels). MRS2-6 reconstituted growth to the highest level among the Arabidopsis proteins but was clearly less efficient compared with wild-type Mrs2p.

Figure 2. — Complementation of the Yeast *mrs2* Mutant Strain Transformed with the *Arabidopsis MRS2* Genes.

Genes were translationally fused downstream of the native yeast MRS2 mitochondrial target sequence and were transcribed from the native yeast *MRS2* promoter. Five microliters of yeast cultures grown to an OD₆₀₀ of 1.0 were each trickled in three dilutions (from left to right: 1.0, 0.1, 0.01, and 0.001, respectively) in parallel on nonselective YPD medium (two left panels) and on nonfermentable YPdG medium (two right panels).

Mg²⁺ Transport Measurements

The molecular analysis of mrs2 complementation using MRS2-1 showed that splicing of mitochondrial group II introns was restored (Schock et al., 2000). This phenomenon is now known to be an indirect effect through providing appropriate Mg²⁺ levels for group II intron ribozyme function in the yeast mitochondria (Gregan et al., 2001b). An indication for a direct involvement of MRS2-1 in mitochondrial Mg²⁺ uptake, however, had been obtained through measurements of mitochondrial magnesium content, which reached nearly the wild-type level upon complementing the yeast mutant (Schock et al., 2000). In the meantime, a system for direct Mg²⁺ uptake measurements in yeast mitochondria has been developed using the fluorescent dye mag-fura-2, allowing a more direct confirmation of Mg²⁺ transport over biological membranes in real time, has been set up (Kolisek et al., 2003). Mag-fura-2 is a UV-excitable, Mg²⁺-dependent fluorescent indicator that undergoes a blue shift from 380 to 340 nm upon Mg²⁺ binding. Accordingly, mitochondria were isolated from all yeast strains transformed with the Arabidopsis MRS2 constructs to measure the magnesium uptake levels after external application of increasing Mg²⁺ concentrations in mag-fura-2–loaded mitochondria.

Mg²⁺ uptake was indeed measurable for all constructs to different extents, confirming their role as proteins mediating magnesium transport. Representative Mg²⁺ uptake recordings are shown in Figure 3. High magnesium uptake efficiencies were observed for MRS2-1, MRS2-7, and MRS2-10, whereas the other proteins proved somewhat less efficient uptake. A discrepancy was observed for MRS2-3, which appeared to complement well in the growth assay (Figure 2) but showed magnesium uptake that was not considerably higher than the background mutant level. Possibly, MRS2-3 acts as a comparatively slow transporter for Mg²⁺ (at least in the foreign yeast mitochondrial membrane environment), allowing for ion homeostasis over periods of hours as in the growth assays but not in measurable amounts over shorter time intervals, such as minutes as in the uptake experiments.

Figure 3. — Measurements of Mg²⁺ Import into Isolated Mitochondria Using the Fluorescent Mag-fura-2 Dye.

Mitochondria isolated from the complemented yeast strains were exposed to stepwise increased external concentrations of magnesium (1, 3, and 9 mM MgCl₂) at time points of 100, 200, and 300 s of incubation, respectively. The 340/380-nm ratio of fluorescence was continuously recorded by the FL-WinLab program (v4.0; Perkin-Elmer), and the formula established by Grynkiewicz et al. (1985) was used to calculate the Mg²⁺ concentrations inside the mag-fura-2–loaded mitochondria ([Mg²⁺]_m). Curves were averaged over at least two independent recordings.

Tissue-Specific MRS2 Gene Family Expression

To address their potential differential functions in planta, the upstream regions of all MRS2 genes were fused to the GUS reporter gene to investigate the tissue specificities of their promoters. We chose to clone >1000 bp of upstream noncoding regions, including the first coding exon of the MRS2 genes, as translational fusions in front of GUS to maximize the inclusions of targets for regulatory influence on gene expression. Highly different tissue-specific patterns of gene expression during development were observed for the members of the MRS2 gene family (Figure 4, Table 1). Several genes showed expression already at very early developmental stages of the seedlings. Nearly ubiquitous expression was observed in 3-d-old seedlings of MRS2-1 and MRS2-5 (Figures 4A and 4B), while expression was restricted to the radicle excluding the tip in the case of MRS2-10 (Figure 4C).

Table 1.

Summary of MRS2/MGT Gene Activities as Determined from GUS Activities in Transgenic Promoter-GUS Fusion Lines (Figure 4)

Tissue	MRS2-1	MRS2-2	MRS2-3	MRS2-4	MRS2-5	MRS2-6	MRS2-7	MRS2-10	MRS2-11
Early seedling
Cotyledons	+	+^a	+^a	+	+	−	−	−	++
Hypocotyl	+	+^a	++	−	+	−	−	−	−
Radicle	+	+^a	+^a	−	+	−	+^h	++	−
Inflorescence
Flower
Anthers	−	+	−	−	+	++^c	−	−	−
Filament	+	±^a	+^a	±^a	−	−	−	+	−
Pistil	−	+^b	−	+	−	−	±	−	−
Sepals	+	−	−	+	−	−	−	±	±^a
Petals	−	−	−	−	−	−	−	−	−
Silique	+	+^b	+^f	++	−	−	±^f	+^f	+^f
Cauline leaf	+	+^a	+^a	++	+	−	+^a	±^d	+^a
Stem	−	+^a	+^a	+^a	++^a	−	+^a,i	+^g	+ⁱ
Rosette
Juvenile leaf	+	+^a	+^a	++	+^a,e	−	+ⁱ	+^d,e	++
Adult leaf	+	±^a	±^a	++	+	−	+^a	+^a,g	++
Roots
Lateral root	+^a	−	+^a	−	+^a	−	++^a	−	−
Root hair zone	+^a	+^a	+^a	−	+^a	−	++^a	++	−
Elongation zone	+^a	+^a	+^a	−	+^a	−	−	++	−
Meristematic zone	++	−	+	−	++	−	++	−	−
Root tip	−	−	−	−	++	−	−	−	−

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Letters indicate distinct staining in vascular tissues (a), ovules (b), pollen grains (c), hydathodes (d), leaf primordia (e), abscission zones (f), trichomes (g), quiescent centers (h), or stomata cells (i).

Expression of other genes began somewhat later in development (e.g., in the case of MRS2-3 in the central cylinder of the root; Figure 4D), interestingly with additional strong expression in the meristematic zone and in the hypocotyl (Figure 4E). Likewise, expression of MRS2-2 was strictly restricted to the central cylinder (Figure 4F) and the veins (Figure 4G) at the early seedling stage. The absence of MRS2-2 expression in the root tip (Figure 4F) similar to MRS2-10 (Figure 4C) was merely complementary to the more widespread expression of MRS2-1 in the different tissues of the root, most dominantly in the root tip (Figure 4B).

In the photosynthetic organs, the early ubiquitous expression of MRS2-1 and MRS2-5 (Figure 4A) became more localized to the vascular tissues of the expanded cotyledons during development, notably with a strong focus of MRS2-5 gene expression in the early development of the first postcotyledon leaf pair (Figure 4H). This contrasted the highly localized expression of MRS2-10 in the hydathodes of the cotyledons (Figure 4I) and the epicotyl (Figure 4J). Whereas expression of MRS2-7 was entirely restricted to the root at the seedling stage (Figure 4K), a completely contrasting picture was observed for MRS2-4 and MRS2-11 with strong expression in photosynthetic tissues from the earliest stages of cotyledon development onwards (Figures 4L and 4M).

A unique particularity of MRS2-11 was its pronounced expression in stomata guard cells (Figures 4M and 4N). Expression of MRS2-4 in aboveground organs continued through plant development (e.g., in older leaves; Figure 4O) and the sepals of the developing flower (Figure 4P). In strong contrast, expression of the phylogenetically related MRS2-6 gene (Figure 1) was particularly restricted and could only be observed in pollen grains (Figures 4Q and 4R). Complementary gene activities in the male flower parts were observed in the filaments for MRS2-3 and MRS2-10 (Figures 4S and 4T). Expression in the maturing seeds was observed for MRS2-2 (Figures 4U and 4V). At later stages of development, pronounced gene activity was seen for MRS2-1 in the style and coat of the silique (Figure 4W). Particularly strong overall gene expression was again observed for MRS2-4 at an early stage of silique development with the exception of the stigma (Figure 4X).

Later, in the fully developed silique, MRS2-4 gene expression was restricted to the abscission zone, as also observed for MRS2-11 (Figure 4Y). The focus of MRS2-2 expression in vascular tissue in early stages of development continued later through development as, for example, in the veins during early bud development (Figure 4Z). Observation of shoot cross sections after xylem counterstaining with safranin demonstrated expression in the phloem (Figure 4Za). MRS2-10, phylogenetically very close to MRS2-1, interestingly showed a very cell type–specific expression not only in early stages of development (Figures 4C, 4I, and 4J) but also a pronounced expression in trichomes (Figure 4Zb).

Publicly available microarray data (www.genevestigator.com) so far give no indication for environmental regulation of the MRS2 genes, and, similarly, we could not observe any obvious variability in gene expression when the MRS2-GUS lines were grown on different magnesium concentrations in hydroponic culture systems (see below). To address the issue of potential magnesium dependent gene regulation yet more explicitly, we performed RT-PCR analyses using Arabidopsis plantlets raised on 50, 500, or 1500 μM Mg²⁺ to detect potential changes in transcript amounts. No evidence for magnesium-dependent regulation for any of the MRS2 genes was observed (see Supplemental Figure 2 online).

Gene to Function: KO Phenotypes

To further address the function of MRS2 proteins in planta, we searched for gene-specific inactivation lines to investigate mutant phenotypes. Candidates for 17 transgenic Arabidopsis lines with T-DNA insertions in (or close to) members of the MRS2 gene family and potentially affecting the respective gene functions were identified in the SALK (and GABI-KAT) collections (http://signal.salk.edu/cgi-bin/tdnaexpress; see Supplemental Table 1 online) for all genes except MRS2-4. Seeds for all insertion lines except those of the pseudogenes MRS2-8 and MRS2-9 were ordered, and the progeny were investigated to first verify the genomic insertion through a PCR-based strategy.

Subsequently, we tried to raise homozygous insertion lines and confirm the knockout of transcription. Among the insertion lines, one could not be verified as harboring the insertion, and among the remaining lines, five could only be obtained in the heterozygous state. A transcriptional knockout was not observed for any of the other lines having insertions outside of the coding regions but could be confirmed for a total of six lines with insertions within the coding regions of four genes in the family (see Supplemental Figure 3 online): one each for MRS2-1, MRS2-5, and MRS2-10 and three independent KO lines for MRS2-7. Furthermore, double KO mutant lines of MRS2-1 + MRS2-5 and MRS2-5 + MRS2-10 could be obtained via crossing and screening for progeny homozygous in both insertion alleles.

All six single-gene KO lines [mrs2-1, mrs2-5, mrs2-7(1), mrs2-7(2), mrs2-7(3), and mrs2-10] and the two double KO lines mrs2-1 mrs2-5 and mrs2-5 mrs2-10 appeared fully vital without obvious phenotypes under standard growth conditions and went through a normal reproductive cycle, initially suggesting strong genetic redundancy in the gene family or its subclades, respectively.

To explore the possibility that a certain mrs2-related phenotype may be seen only in conditions of Mg²⁺ stress, we set up two different culture systems: a hydroponic and a liquid culture system. For the hydroponic culture system, we allowed plants to complete their life cycle under defined growth conditions. We found that wild-type Arabidopsis (ecotype Columbia [Col-0]) tolerates the full range of Mg²⁺ concentrations between 50 μM and 5 mM under the otherwise unaltered ion concentrations of the Siegenthaler culture medium (Siegenthaler and Depéry, 1976). Hence, MgSO₄ concentrations ranging between micro- and millimolar levels were used to scan for Mg²⁺-dependent phenotypes among the T-DNA insertion lines. While the knockout lines mrs2-1, mrs2-5, and mrs2-10 and the double knockouts mrs2-1 mrs2-5 and mrs2-5 mrs2-10 remained as unaffected by the varying Mg²⁺ concentrations as the wild type, a striking phenotype was observed for the knockout line mrs2-7(2). Plantlets were severely retarded in development under 50 μM Mg²⁺ (Figure 5A). Raising the Mg²⁺ concentrations to slightly higher levels (150 μM) immediately restored this phenotype practically to wild-type growth, and the mutant plants remained unaffected by yet higher magnesium concentrations.

Figure 5. — Hydroponic Cultivation of KO and Wild-Type Plants in Two Alternative Systems.

**(A)** Effect of Mg²⁺ concentration on growth of *Arabidopsis* wild type (Col-0) and the homozygous KO *mrs2-7(2)* T-DNA insertion line. Plants were grown on Siegenthaler medium with either 50 or 150 μM MgSO₄ and are shown 28 d after germination in the Araponics culturing system.

**(B)** Twenty to thirty seedlings each of *Arabidopsis* wild-type and three independent (*1-3*) homozygous T-DNA insertion *mrs2-7* KO lines 35 d after germination in a pipette tip–based high-density hydroponic culturing system established in the laboratory.

To exclude that any additional genomic rearrangements were responsible for the low Mg²⁺ phenotype observed, we also investigated the two alternative KO lines in MRS2-7, which could also be obtained in the homozygous state. These lines, mrs2-7(1) and mrs2-7(3), also carry, like the initially characterized mutant mrs2-7(2), T-DNA insertions in introns of the MRS2-7 gene. Exactly the same phenotype as for the mrs2-7(2) KO line was observed (Figure 5B), confirming that inactivity of MRS2-7 indeed is the fundamental cause for the low Mg²⁺ phenotype. To determine whether KO of MRS2-7 affects ion homeostasis more globally, we used the Purdue Ionomics service (Baxter et al., 2007; Salt et al., 2008; www.ionomicshub.org), offering comprehensive ion content analyses. For all three independent mrs2-7 KO lines, no significant imbalances in homeostasis of Mg²⁺ or any one of 17 other ions were observed under normal growth conditions in the ionomics measurements other than a possible 10 to 20% reduction of K⁺ in comparison to the wild type (see Supplemental Figure 4 online).

As no similar phenotypes could be observed for the KO lines mrs2-1, mrs2-5, and mrs2-10 and even for the double KO mutants, we tried to investigate potentially more subtle differences in these mutants using a liquid culture system containing Murashige and Skoog (MS) medium with 50 and 100 μM of MgSO₄. The liquid media were supplemented with sucrose to allow enhanced seedling growth, while any other complex compounds that might contribute spurious amounts of Mg²⁺ were excluded. Additionally, a stable transgenic complementation line harboring the MRS2-7 coding sequence under control of the constitutive CaMV 35S promoter within the mrs2-7(2) background was investigated. As observed in the hydroponic culture, KO lines mrs2-1, mrs2-5, and mrs2-10 never showed significant differences compared with the wild type under any condition tested (data not shown). By contrast, the mrs2-7 KO line failed to germinate under these liquid culture conditions, whereas the MRS2-7–overexpressing line revealed a strong increase in biomass production under 50 μM and a slight increase under 100 μM MgSO₄ compared with wild-type seedlings (Figure 6). This germination phenotype of mrs2-7 prompted us to reinvestigate the earliest stages of MRS2-7 gene expression. Indeed, the MRS2-7:GUS reporter line showed very specific expression of MRS2-7 very early in the quiescent zone of the emerging radicle (Figure 7). This finding is in full accord with the high-resolution transcriptional profiling of the Arabidopsis root quiescent center (Nawy et al., 2005).

Figure 6. — Biomass Accumulation of *Arabidopsis* Seedlings.

Photos were taken 14 d after incubation of ∼20 seeds of each individual line in liquid shaking cultures (24°C, 16 h light long-day regime) containing 100 mL of MS medium supplemented with 1% sucrose with MgSO₄. Plants shown are *Arabidopsis* wild-type ecotype Col-0 (**[A]** and **[D]**), *mrs2-7(2)* KO line (**[B]** and **[E]**), and this KO line transformed with *MRS2-7* cDNA driven by the CaMV 35S promoter (**[C]** and **[F]**).

**(A)** to **(C)** Plants grown in 50 μM MgSO₄.

**(D)** to **(F)** Plants grown in 100 μM MgSO₄.

Figure 7. — GUS Assay for Promoter Activity of the *MRS2-7:GUS* Transgenic Line in the Earliest Stage of Seedling Development Showing Transcriptional Activity in the Tip of the Radicle at Day 1.

Subcellular Localization of MRS2-7

Given that a first magnesium-related phenotype is now identified in planta for a member of the MRS2/MGT gene family, we wished to determine the subcellular localization of MRS2-7. The full-length coding sequence of MRS2-7 was cloned as a translational fusion upstream of the green fluorescent protein (GFP) under control of the CaMV 35S promoter. The construct was used for transient transformation of Nicotiana benthamiana leaves via Agrobacterium tumefaciens followed by laser scanning confocal fluorescence microscopy (Figure 8). GFP fluorescence was observed in the endomembrane system, suggesting targeting to the endoplasmatic reticulum (ER). As a control experiment, we used the ER-targeted HDEL:DsRED construct (Höfer et al., 2008) in a cotransformation assay. Nearly perfectly overlapping expression with the MRS2-7:GFP construct was observed.

Figure 8. — Subcellular Localization of MRS2-7.

**(A)** and **(B)** Confocal laser scanning microscopy images taken 2 d after transient *N. benthamiana* leaf transformation with the AtMRS2-7:GFP fusion construct **(A)** or the HDEL:Ds-Red construct **(B)** to visualize the ER.

**(C)** Overlay of **(A)** and **(B)**.

DISCUSSION

We were able to raise homozygous T-DNA insertion knockout lines for four genes of the MRS2/MGT family: MRS2-1, MRS2-5, MRS2-7, and MRS2-10. Based on the phylogeny shown in Figure 1, this can be nicely explained, given that all four genes belong to clades B and E containing closely related homologs, which may contribute genetic redundancy, as commonly observed for members of Arabidopsis gene families (Briggs et al., 2006). By contrast, we were unable to identify, raise in a homozygous state, or ultimately verify on transcriptional level any KO lines for the more isolated members of the gene family, most notably MRS2-11/MGT10 or MRS2-3/MGT4, which are the respective single Arabidopsis members of clades A or C (Figure 1). Likewise, no KOs could be obtained for MRS2-4/MGT6, for MRS2-6/MGT5 with its unique, exclusive pollen-specific expression (Figure 4), or for MRS2-2/MGT9. This is in full accord with recent reports finding that only heterozygous T-DNA insertion lines of MRS2-6/MGT5 and MRS2-2/MGT9 are viable (Li et al., 2008; Chen et al., 2009).

Here, we did not consider the pseudogenes MRS2-8 (dysfunctional in ecotype Col-0) and MRS2-9 (dysfunctional in ecotypes Col-0 and Landsberg) of clade E for functional studies. It should be noted, however, that the cluster of neighboring genes MRS2-7/MRS2-8/MRS2-9 on Arabidopsis chromosome 5 may be an interesting object of study for ecotype variation (and possible functional evolutionary adaptation) in the future. It came as a surprise to us that just the knockout of MRS2-7 belonging to this group of probably recently duplicated young and rapidly evolving genes resulted in the observable magnesium-dependent phenotype in planta for a member of the MRS2/MGT gene family. The dramatic phenotype observed under very low magnesium conditions of 50 μM is easily complemented both by a moderate increase in Mg²⁺ to 150 μM in the substrate and by ectopic overexpression of MRS2-7 under control of the CaMV 35S promoter in the mutant background. Surprisingly, the complemented transgenic Arabidopsis line additionally shows a striking gain in vitality and biomass accumulation compared with wild-type plants. As a side note, we could not observe a similar effect when a genomic instead of a cDNA construct for MRS2-7 was used. This turned out to be due to aberrant mis-splicing of MRS2-7 mRNAs in that transgenic line, also including a natural splice alternative that lacks an exon and was reported previously as nonfunctional (Mao et al., 2008).

Very obviously, the overexpression of MRS2/MGT genes in planta may have significant influences on ion homeostasis. Three genes of the MRS2/MGT gene family have indeed been identified as candidate loci in a search for quantitative trait loci affecting seed mineral concentrations in general and Mg²⁺ in particular: MRS2-2, MRS2-3, and MRS2-11 (Waters and Grusak, 2008). Overexpression of MRS2-11, localizing to the chloroplast envelope membrane, however, showed no difference in magnesium content, neither in whole Arabidopsis plants nor isolated chloroplasts (Drummond et al., 2006).

Aside from the single KO lines for the three MRS2/MGT genes of clade B (Figure 1), we were also able to obtain double KO lines for mrs2-5 mrs2-1 and mrs2-5 mrs2-10. Like the corresponding single-gene KO lines, these lines had no detectable phenotype. This observation is somewhat surprising in the light of the strong and overlapping expression, notably of MRS2-1 and MRS2-5 early in seed development (Figure 4). Moreover, in the context of proteome analyses of cellular compartments, it was found that MRS2-1/MGT2 was localized to the tonoplast (Carter et al., 2004), whereas MRS2-5/MGT3 was found either in the plasma membrane (Alexandersson et al., 2004) or the tonoplast (Whiteman et al., 2008). This allows no reasonable assignment as to whether these two proteins are redundant in function or not. The magnesium-proton exchanger MHX, which is unrelated to the MRS2/MGT gene family, was also localized in the tonoplast membrane (Shaul et al., 1999). Assuming that the MRS2-1/MGT2 channel would allow Mg²⁺ flow from the positively charged, acidic vacuole into the cytosol, it is unlikely that a knockout could be functionally complemented by MHX, which transports Mg²⁺ into the vacuole in exchange for protons. On the other hand, MRS2-10/MGT1, like MRS2-5/MGT3, was also found to be localized to the plasma membrane (Li et al., 2001). Hence, the double KO line mrs2-5 mrs2-10 described here shows that two membrane proteins of identical localization may be missing simultaneously without strongly interfering with Mg²⁺ homeostasis in the plant.

Certainly, observations and reports regarding protein localizations in the cell should not be overstated and instead should be considered carefully. At least some MRS2/MGT proteins, including MRS2-7, here found to be ER localized (Figure 8), may actually be localized to more than one membrane type (possibly in variable stoichiometries), and this is certainly very obvious for the endomembrane system extending from the ER up to the plasma membrane. Clearly set apart with respect to subcellular localizations are the MRS2/MGT family members localized to the endosymbiotic organelles: MRS2-6/MGT5 in mitochondria (Li et al., 2008) or MRS2-4/MGT6 and MRS2-11/MGT10 in chloroplasts (Froehlich et al., 2003; Drummond et al., 2006; this study).

The drastic phenotype of growth retardation in mrs2-7 KO lines in environments of very low magnesium concentrations may, in the light of an ER localization of the protein, suggest that the endomembrane system is highly sensitive to Mg²⁺ deficiency. Notably, among the many functions of magnesium ions in the cell is the stabilization of biological membranes, which may at the same time be a possible explanation for the enhanced viability of Arabidopsis lines upon overexpression of MRS2-7 here observed. Alternatively, Mg²⁺ has also been shown to participate in Ca²⁺-based signal transduction processes (Baumann et al., 1991; Wiesenberger et al., 2007), and low magnesium concentrations may become a limiting factor for functional intracellular communication.

The GUS indicator gene expression data of the MRS2 gene family revealed partially overlapping expression for some genes but also highly specific patterns of expression for others (Figures 4 and 7). Publicly available whole-genome expression data from chip hybridization experiments are largely consistent as far as comparable; unfortunately, a probe set for MRS2-5 is missing on the widely used Arabidopsis whole-genome Affymetrix probe array ATH1 (Redman et al., 2004). A gene expression map of the Arabidopsis root distinguishing 15 tissue stages based on data from the microarray data (Birnbaum et al., 2003) is likewise in accord (e.g., reflecting comparatively strong overall expression of MRS2-1 and MRS2-3 [again, MRS2-5 is lacking] in the root). The strong stage 1 expression observed for MRS2-7 in those experiments is fully congruent with the focused expression in the meristematic zone observed here. Available microarray expression data (www.genevestigator.com) so far give no conclusive hints on significantly divergent expression patterns of MRS2/MGT genes in different environmental setups, and the same can be said for GUS assays after plant cultivation on different media (data not shown).

Direct proof for channel-like Mg²⁺ transport through the yeast mitochondrial homolog Mrs2p by use of the fluorescence indicator mag-fura-2 had previously been shown (Kolisek et al., 2003). The measurements reported here of Mg²⁺ uptake into isolated mrs2 yeast mutant mitochondria after expression of Arabidopsis MRS2/MGT proteins show their principal capability of magnesium transport in this nonhomologous membrane system. The complementation of mutants lacking some of their endogenous Mg²⁺ uptake systems has previously been demonstrated for Arabidopsis MRS2-1/MGT2 and MRS2-11/MGT10 in yeast (Schock et al., 2000; Li et al., 2001) and for Arabidopsis MRS2-2/MGT9, MRS2-6/MGT5, MRS2-7/MGT7, and MRS2-10/MGT1 in Salmonella enterica (Li et al., 2001, 2008; Mao et al., 2008; Chen et al., 2009). Somewhat in contrast with this apparent functional complementation across wide phylogenetic distances, only MRS2-1, MRS2-10, and MRS2-11 but not the other members of the gene family were found to complement the alr1 alr2 yeast mutant with a defect of plasma membrane magnesium transport (Drummond et al., 2006). Those observations may possibly be explained by mistargeting of the Arabidopsis MRS2/MGT proteins in the yeast cells.

Direct demonstration of Mg²⁺ transport through the yeast plasma membrane homolog ALR1 had previously also been achieved electrophysiologically (Liu et al., 2002). In an alternative approach, patch clamp experiments using giant lipid vesicles have recently demonstrated that the yeast MRS2 protein is a Mg²⁺-regulated Mg²⁺ channel of high conductance with lower conductance for Ni²⁺ but none for Co²⁺, Mn²⁺, or Ca²⁺ (Schindl et al., 2007). This may be reasonably well explained by an increasing ion radius, although there is only a minor difference between the one for Ni²⁺ (0.72 Å) versus Co²⁺ (0.74 Å), compared with (0.65 Å) for Mg²⁺. However, these findings stand in contrast with the bacterial CorA proteins obviously also transporting Co²⁺ (Niegowski and Eshaghi, 2007). Certainly, this may reflect actual differences in eukaryotic protein structure affecting ion selectivity, and it will be highly interesting to perform comparative studies.

The membrane topology of the 2-TM-GMN type of proteins has been clarified through crystallization and x-ray structural analysis of the T. maritima CorA homolog (Eshaghi et al., 2006; Lunin et al., 2006; Payandeh and Pai, 2006), showing the protein to form a pentamer in the membrane. It is likely that the eukaryotic homologs of the MRS2 or ALR type will assemble very similar structures in the membranes. Indeed, there is already biochemical evidence (through cross-linking) and genetic evidence using the yeast mating-based split-ubiquitin system (mbSUS; Obrdlik et al., 2004) for oligomerization of ALR1 and ALR2, the yeast cytoplasmic type of 2-TM-GMN–type membrane proteins. As a consequence, mutations in one of the two proteins can exert dominant-negative effects on magnesium transport (Wachek et al.. 2006).

METHODS

Plant Material and Growth Conditions

Arabidopsis thaliana ecotype Col-0 and T-DNA insertion lines in MRS2/MGT genes were obtained from the European Arabidopsis Stock Centre. T-DNA insertions were confirmed, homozygous lines raised, and gene knockout on the transcript level was finally confirmed for six lines in four of the MRS2/MGT genes (see Supplemental Table 1 and Supplemental Figure 2 online). Additionally, two double KO lines were successfully obtained via crossing: mrs2-1 mrs2-5 and mrs2-5 mrs2-10. Arabidopsis seeds were surface sterilized (Swinburne et al., 1992) and sown on MS medium (Duchefa Biochemie) supplemented with 1% sucrose, 0.05% MES, pH 5.7 (KOH), and 0.8% bacto agar. Transgenic plants were selected in the presence of 50 μg/mL kanamycin. All plants were stratified for 2 d at 4°C and grown under 16 h of light photoperiods at 24°C and ∼100 μmol/m²/s light intensity.

Hydroponic cultivation of Arabidopsis plants (Norén et al., 2004) was modified, using either opaque filter tip containers or the Araponics growing system (Araponics) and nutrient solutions with varying concentrations of magnesium. Nutrient solutions were replaced every 2 weeks or immediately upon indication of emerging algal growth. Liquid cultivation of germinating Arabidopsis seeds was done in conical flasks incubated on a rotary shaker at 100 rpm. Liquid media were based on MS medium mentioned above, which was adjusted to different magnesium concentrations. Comprehensive ionomics measurements with inductively coupled plasma mass spectrometry were performed using the Purdue Ionomics Information Management System (Baxter et al., 2007).

Plasmid Constructs and Plant Transformation

All plasmid constructs were cloned by recombination of PCR products into destination vectors using Gateway technology (Invitrogen). GUS (uidA) reporter gene constructs comprised the noncoding upstream region and the first exon of each MRS2 gene cloned in frame upstream of GUS into vector pKGWFS7 (Karimi et al., 2002). The full-length MRS2-7 (At5g09690.1) coding sequence was cloned behind the CaMV 35S promoter in vector pK7WG2D (Karimi et al., 2002) for overexpression and upstream of GFP in translational fusion into vector pMDC83 (Curtis and Grossniklaus, 2003), respectively. Agrobacterium tumefaciens strain GV3101 pMP90 (Koncz and Schell, 1986) was transformed with the binary vector constructs (Höfgen and Willmitzer, 1988) and used to transform developing floral tissues of 4-week-old Arabidopsis plants using the floral dip method (Clough and Bent, 1998).

Transcript Analyses

Total RNAs were prepared from 2-week-old Arabidopsis plantlets grown in liquid cultures or rosette leafs from adult plants grown on soil using the NucleoSpin RNA plant kit (Macherey-Nagel), and cDNA was synthesized with the Transcriptor High Fidelity cDNA synthesis kit (Roche Diagnostics) applying anchored-oligo (dT)₁₈ primer with equalized amounts of template RNA. Expression levels of the target genes were determined after 30, 35, and 40 cycles of gene-specific PCR amplification via gel electrophoresis and ethidium bromide staining.

GUS Assays, Histochemical Staining, and Microscopy

To detect GUS activity, Arabidopsis plant samples or entire seedlings were infiltrated with substrate solution (1 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid, 100 mM sodium phosphate buffer, at pH 7.0, 10 mM EDTA, 3 mM ferrocyanide, 0.5 mM ferricyanide, and 0.1% Triton X-100) at 37°C for 16 h (Jefferson et al., 1987). After staining, chlorophyll was removed with 70% ethanol at 37°C. For sectioning before microscopy, plant samples were placed in fixation buffer (50 mM PIPES, 1.5% glutaraldehyde, and 0.5% glucose) and dehydrated through a dilution series of ethanol incubations up to absolute ethanol. Tissues were embedded in Spurr Low-Viscosity Embedding Media (Polysciences Europe) and sliced with a microtome (Ultramikrotom Om U43; Reichert Optische Werke).

Transient Expression of GFP Fusions in Nicotiana benthamiana

Agrobacterium strains containing the MRS2-7:GFP or a HDEL:DsRED construct (Jach et al., 2001) for ER colocalization studies (kindly provided by the cell biology department at the Institut für Zelluläre und Molekulare Botanik, Bonn) were grown overnight in 4 mL Luria-Bertani medium containing the appropriate antibiotics at 28°C to stationary phase. Bacteria were harvested by centrifugation and pellets were resuspended in 2 mL infiltration medium (10 mM MgCl₂, 10 mM MES, and 150 μM acetosyringone, pH 5.7) and incubated at 28°C for 3 h. The bacterial suspensions were adjusted to an optical density (OD₆₀₀) of 1.0 and coinfiltrated into the abaxial spaces of young but fully expanded leaves of 6- to 8-week-old N. benthamiana plants with a needleless syringe. Infiltrated leaves were examined after 48 h with an Olympus FV1000 confocal microscope, and serial confocal optical sections were taken. Images were analyzed using the Olympus FV1000 Viewer software.

Functional Complementation and Mg²⁺ Uptake Measurements in the Yeast mrs2 Mutant

To express the Arabidopsis MRS2/MGT proteins in yeast, an upstream fragment of the yeast MRS2 gene encoding the first 94 amino acids for mitochondrial targeting and the native promoter region was amplified by PCR from the plasmid YEp351-MRS2 (Wiesenberger et al., 1992). The amplified fragment was fused to the respective N-terminally shortened MRS2 coding sequences at the first conserved protein motif (RDLR) of the Arabidopsis MRS2 genes with a single hemagglutinin tag at the C termini by overlap extension PCR and cloned into vector YEp351 (Hill et al., 1986). Yeast strain DBY mrs2-1 (Wiesenberger et al., 1992) was transformed using the LiOAc/SS Carrier DNA/PEG method (Gietz and Woods, 2002) and trickled in parallel in a 10-fold serial dilution on normal (YPD) and nonfermentable media (YPdG) to test complementation of the mrs2 growth defect (Bui et al., 1999). Magnesium uptake was determined via the fluorescent dye mag-fura-2 (Invitrogen) in isolated mitochondria as previously described (Kolisek et al., 2003).

Phylogenetic Tree Construction

Accession numbers for protein sequences used for alignment are listed in the following section. Arabidopsis and rice (Oryza sativa) MRS2/MGTprotein sequences were aligned using the CLUSTAL algorithm (gap creation and extension penalties of 10 and 1, respectively) with minor manual adjustments in MEGA (Kumar et al., 2008). Alignments are provided in Supplemental Data Set 1 online. The protein phylogeny was inferred using the neighbor-joining method (Saitou and Nei, 1987) and Poisson-corrected amino acid distances with MEGA with the pairwise deletion option eliminating indels only in pairwise comparisons. Node reliability was determined with 10,000 bootstrap replicates.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL or Arabidopsis Genome Initiative data libraries under the accession numbers that follow. The following nucleotide sequence accessions were used for cloning of the yeast and the Arabidopsis constructs: NM_101469 (MRS2-1, At1g16010), NM_125852 (MRS2-2, At5g64560), NM_112854 (MRS2-3, At3g19640), NM_115759 (MRS2-4, At3g58970), NM_126412 (MRS2-5, At2g03620), NM_119000 (MRS2-6, At4g28580), NM_121006 (MRS2-7, At5g09690), NM_106738 (MRS2-10, At1g80900), and NM_122188 (MRS2-11, At5g22830). The following protein sequence accessions were used for construction of the phylogenetic tree: CAC13981 (At MRS2-1), AAN73212 (At MRS2-2), AAN73213 (At MRS2-3), AAN73214 (At MRS2-4), AAN73215 (At MRS2-5), AAN73216 (At MRS2-6), NP_196531 (At MRS2-7), AAN73218 (At MRS2-8), NP_196533 (At MRS2-9), AAN73219 (At MRS2-10), AAG45213 (At MRS2-11), BAB89805 (Os01g64890), BAB92573 (Os01g68040), Os03g04480, as given in the Aramemnon database (Schwacke et al., 2003), AAR87307 (Os03g39790), AAK14424 (Os03g48000), CAE03029 (Os04g35160), CAE01634 (Os04g42280), BAD38112 (Os06g44150), and AAK20062 (N-terminally modified; Os10g39790). Accession numbers for T-DNA insertion lines are as follows: SALK_006797 (mrs2-1), SALK_105475 (mrs2-5), SALK_064741 [mrs2-7(1)], SALK_090559 [mrs2-7(2)], SALK_063452 [mrs2-7(3)], and SALK_100361 (mrs2-10).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Alignment of Arabidopsis MRS2 Proteins -11, -3, -5, -6, and -2 as Representatives of Clades A through E.
Supplemental Figure 2. Expression Levels of All Functional MRS2/MGT Genes Expressed in Vegetative Tissue in 2-Week-Old Plantlets Grown in Liquid Culture on Different MgSO₄ Concentrations.
Supplemental Figure 3. Investigation of Transcriptional Status for Homozygous T-DNA Insertion Lines.
Supplemental Figure 4. Ion Profiles of the Three mrs2-7 KO Lines in Reference to the Col-0 Wild-Type Background.
Supplemental Table 1. List of Arabidopsis T-DNA Insertion Lines Investigated in the Course of This Study.
Supplemental Data Set 1. Text File of the Alignment Used for the Phylogenetic Analysis Shown in Figure 1.

Supplementary Material

[Supplemental Data]

tpc.109.070557_index.html^{(910B, html)}

Acknowledgments

We thank Diedrik Menzel and coworkers at the cell biology department of the Institut für Zelluläre und Molekulare Botanik, especially Martina Beck for access to and help with confocal laser scanning microscopy, and Ursula Mettbach for help with thin sections for light microscopy. For skillfull technical assistance, we thank Monika Polsakiewicz, Johanna Schmitz, and Daniel Serwas. We are grateful for helpful comments and constructive criticisms by referees and editors on the initial version of this article. Finally, we gratefully acknowledge support of this work in an early phase through the Deutsche Forschungsgemeinschaft DFG (Kn411/4) in the framework of Schwerpunktprogramm SPP1108 on plant membrane transport.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instruction for Authors (www.plantcell.org) is: Volker Knoop (volker.knoop@uni-bonn.de).

^[W]

Online version contains Web-only data.

www.plantcell.org/cgi/doi/10.1105/tpc.109.070557

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Supplemental Data]

tpc.109.070557_index.html^{(910B, html)}

tpc.109.070557_1.pdf^{(335KB, pdf)}

tpc.109.070557_Supplemental_Dataset_1.txt^{(22.6KB, txt)}

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PERMALINK

A Root-Expressed Magnesium Transporter of the MRS2/MGT Gene Family in Arabidopsis thaliana Allows for Growth in Low-Mg2+ Environments[W]

Michael Gebert

Karoline Meschenmoser

Soňa Svidová

Julian Weghuber

Rudolf Schweyen

Karolin Eifler

Henning Lenz

Katrin Weyand

Volker Knoop

Abstract

INTRODUCTION

RESULTS

Ancestry of the Plant MRS2/MGT Gene Family

Figure 1.

Complementation of the Yeast mrs2 Mutant

Figure 2.

Mg2+ Transport Measurements

Figure 3.

Tissue-Specific MRS2 Gene Family Expression

Figure 4.

Table 1.

Gene to Function: KO Phenotypes

Figure 5.

Figure 6.

Figure 7.