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The Arabidopsis Mg Transporter, MRS2-4, is Essential for Mg
Homeostasis Under Both Low and High Mg Conditions
Koshiro Oda1,3, Takehiro Kamiya1,3, Yusuke Shikanai1, Shuji Shigenobu2, Katsushi Yamaguchi2 and
Toru Fujiwara1,*
1
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657
Japan
2
National Institute for Basic Biology, Okazaki, Aichi, 444-8585 Japan
3
These authors contributed equally to this work.
*Corresponding author: E-mail, [email protected]; Fax, +81-3-5841-8032.
(Received September 3, 2015; Accepted November 25, 2015)
Magnesium (Mg) is an essential macronutrient, functioning
as both a cofactor of many enzymes and as a component of
Chl. Mg is abundant in plants; however, further investigation
of the Mg transporters involved in Mg uptake and distribution is needed. Here, we isolated an Arabidopsis thaliana
mutant sensitive to high calcium (Ca) conditions without
Mg supplementation. The causal gene of the mutant encodes MRS2-4, an Mg transporter. MRS2-4 single mutants
exhibited growth defects under low Mg conditions, whereas
an MRS2-4 and MRS2-7 double mutant exhibited growth
defects even under normal Mg concentrations. Under
normal Mg conditions, the Mg concentration of the MRS24 mutant was lower than that of the wild type. The
transcriptome profiles of mrs2-4-1 mutants under normal
conditions were similar to those of wild-type plants grown
under low Mg conditions. In addition, both mrs2-4 and
mrs2-7 mutants were sensitive to high levels of Mg. These
results indicate that both MRS2-4 and MRS2-7 are essential
for Mg homeostasis, even under normal and high Mg conditions. MRS2-4–green fluorescent protein (GFP) was mainly
detected in the endoplasmic reticulum. These results indicate that these two MRS2 transporter genes are essential for
the ability to adapt to a wide range of environmental Mg
concentrations.
Keywords: Calcium Double mutant Endoplasmic reticulum Mg transporter.
Abbreviations: CaMV, Cauliflower mosaic virus; EMS, ethylmethane sulfonate ER, endoplasmic reticulum; GFP, green
fluorescence protein; ICP-MS, inductively coupled plasma
mass spectrometry; RNAi, RNA interference; RNA-Seq, RNA
sequencing.
Introduction
Magnesium (Mg) is the most abundant divalent cation in the
cell (Wolf and Cittadini 2003). Cytosolic free Mg ions are present at approximately 0.4 mM, and are a cofactor of >300 types
of enzymes, such as polymerase and hexokinase, in numerous
biological reactions (Karley and White 2009, Bose et al. 2011).
In plants, Mg functions as a reaction center in photosynthetic
reactions. In leaf cells, 15–20% of Mg ions are present in Chl
(Karley and White 2009).
Both low and high levels of soil Mg affect plant growth. High
levels of Mg are found in serpentine soil, which is characterized
by a low calcium (Ca) to Mg ratio (Brady et al. 2005, Chiarucci
and Baker 2007). Adaptation to serpentine soils can be considered a model case of plant adaptation to different soil environments. Although the molecular mechanism underlying
this adaptation is not clear, the genomic sequencing of
Arabidopsis lyrata plants grown in either serpentine or nonserpentine soils shows that polymorphisms are found in the Ca
and Mg transport loci (Turner et al. 2010). In addition to serpentine soils, low soil Mg is a global agricultural problem
(Adams and Henderson 1962, Bernier and Brazeau 1988). Mg
deficiency occurs universally when there is an excess of cations
such as Ca and potassium (K) in the soil. The presence of high
levels of these cations competitively inhibits the uptake of Mg,
leading to Mg deficiency in the plants. Typical Mg deficiency
symptoms in many crops include late-season yellowing between the leaf veins. It has also been reported that some
plant species become more susceptible to pathogen infestation
when they are Mg deficient (Rude and Singer 1981).
Plants are capable of maintaining their cellular Mg concentration within a certain range. The Mitochondrial RNA splicing2
(MRS2; also called MGT in Arabidopsis thaliana) transporter
family has been studied extensively in plants. MRS2, originally
isolated from Saccharomyces cerevisiae (Gregan et al. 2001),
exhibits structural similarities to the CorA family, a group of
Mg transporters found in bacteria (Smith and Maguire 1998).
The plant MRS2 family was determined to be a homolog of
yeast Mrs2 (Schock et al. 2000). In A. thaliana, nine MRS2
genes are present in the genome (Gebert et al. 2009), and the
Mg transport activity of these MRS2 genes has been described
using yeast and Salmonella typhimurium mutants with disrupted Mg transporters (Schock et al. 2000, Li et al. 2001,
Drummond et al. 2006, Mao et al. 2008).
It has been shown that the MRS2 family is required for plant
growth under low Mg conditions. An RNA interference (RNAi)
knock down of MRS2-4 (AtMGT6) confers low Mg sensitivity,
causing the Mg concentration of the shoot to be lower than
Plant Cell Physiol. 57(4): 754–763 (2016) doi:10.1093/pcp/pcv196, Advance Access publication on 8 January 2016,
available online at www.pcp.oxfordjournals.org
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Plant Cell Physiol. 57(4): 754–763 (2016) doi:10.1093/pcp/pcv196
that of the wild type under low Mg conditions (Mao et al. 2014).
This suggests that MRS2-4 has great physiological importance
under low Mg conditions. MRS2-4 fused with green fluorescent
protein (GFP) was localized to the plasma membrane of
A. thaliana protoplasts, and its expression in roots was induced
under low Mg conditions. These findings indicate that MRS2-4
is important for the maintenance of Mg concentrations under
low Mg conditions (Mao et al. 2014). A T-DNA knock-out line
of MRS2-7 also exhibited sensitivity to low Mg in hydroponic
culture (Gebert et al. 2009). MRS2-7 was localized to the endoplasmic reticulum (ER), suggesting that the ER plays a role in Mg
maintenance (Gebert et al. 2009). The subcellular localization of
other transporter members was also reported. MRS2-1, MRS2-5
(Conn et al. 2011), MRS2-10 (Li et al. 2001) and MRS2-11
(Drummond et al. 2006) are localized to the plasma membrane,
tonoplast, plasma membrane and chloroplast membrane of
A. thaliana, respectively. Triple or double knock-out mutants
of MRS2-1/2-5/2-10 and MRS2-1/2-10 exhibited sensitivity to
low Mg conditions, indicating that these transporters are
involved in Mg transport (Lenz et al. 2013). MRS2-2 and
MRS2-3 were shown to be involved in pollen development
using an RNAi line, as homozygous T-DNA insertion lines are
lethal (Chen et al. 2009, Li et al. 2015).
A great deal of work has focused on Mg transport mechanisms under low Mg conditions, and it has been demonstrated
that the MRS2 family is essential for plant survival under low
Mg conditions. However, the identities of the Mg transporters
that mediate Mg uptake under Mg-sufficient conditions and
those required for adaptation to excess Mg remain unknown. In
this study, we isolated low Mg/excess Ca- (–Mg/+Ca) sensitive
mutants and characterized one whose causal gene is MRS2-4.
We established a knock-out mutant of MRS2-4 and demonstrated that MRS2-4 is important for successful adaptation to
both low and high Mg conditions, as well as for Mg uptake
under normal Mg conditions.
Results
The causal gene of kudo7 and kudo8 is MRS2-4
To isolate A. thaliana gene(s) involved in Mg homeostasis, we
carried out a screening of an ethylmethane sulfonate (EMS)mutagenized M2 population of A. thaliana Col-0 seeds under
low Mg conditions. We found that the agar was contaminated
with Mg, which made it difficult to produce low Mg agar substrate (Kamiya et al. 2011, T. Kamiya unpublished data).
To overcome this, we added excess Ca to the MGRL medium
without Mg, as it has been reported that Ca competes
with Mg uptake and high Ca causes Mg deficiency
(Marschner 1995). Hereafter, medium with excess Ca
(15 mM Ca) and without Mg supplementation is referred to
as –Mg/+Ca. As expected, medium with excess Ca enhanced
the low Mg sensitivity of plants of the mrs2-7 T-DNA line
(Gebert et al. 2009, T. Kamiya unpublished data). Using this
medium, we isolated mutants named kudo (‘kudo’ means Mg
in Japanese); we focused on two kudo mutants, kudo7 and
kudo8 (Fig. 1A).
To identify the causal gene of kudo7 and kudo8, we crossed
these mutants with Landsberg erecta (Ler), and a selfed F2
population was obtained. The segregation ratio of the F2
plants was 138 : 50 wild-type to mutant phenotype in kudo7,
and 148 : 52 wild-type to mutant in kudo8. According to a 2
test, this did not differ significantly from the expected segregation ratio of 3 : 1, indicating that the phenotypes of kudo7 and
kudo8 are caused by a single recessive mutation. The mutation
was mapped using simple sequence length polymorphism
(SSLP) markers. The results of the mapping indicate that the
causal genes of these two mutants are located on chromosome
3 between F27K19 and T20O10 (20.7–23.3 Mbp) (Fig. 1B). We
then carried out a genome re-sequencing of kudo7 using nextgeneration sequencing and found mutations in the mapped
region. AtMRS2-4 (At3g58970), an Mg transporter gene, is the
only gene in the mapped region carrying a non-synonymous
mutation. The mutation causes an amino acid substitution
from Glu120 to lysine in MRS2-4 (Fig 1B). kudo8 carries the
same mutation as kudo7. Since kudo7 and kudo8 were derived
from the same batches of EMS seeds used for the screening,
these mutants could be identical lines. We used kudo7 in the
following experiment.
To confirm that MRS2-4 is the causal gene of kudo7, we
obtained an MRS2-4 T-DNA insertion line, SALK_145997.
In this line, T-DNAs are inserted into the third exon and the
30 -untranslated region (UTR) (Supplementary Fig. S1).
Transcripts of MRS2-4 were not detected in this line
(Fig. 1C), indicating that the T-DNA line is a knockout allele.
Hereafter, kudo7 and SALK_145997 are referred to as mrs2-4-1
and mrs2-4-2, respectively. Using this line, we performed an
allelism test. F1 crosses between mrs2-4-1 and mrs2-4-2
developed the –Mg/+Ca-sensitive phenotype (Fig. 1D).
Furthermore, we performed a complementation test using an
MRS2-4 genomic fragment with a promoter region (–2.5 kbp
from ATG) fused with GFP (Fig. 1E). The MRS2-4 genomic
fragment rescued the –Mg/+Ca-sensitive phenotype of mrs24-1 (Fig. 1F). These results demonstrate that the causal gene of
this phenotype is MRS2-4.
To confirm further whether the mrs2-4-1 phenotypes we
observed are caused by the disruption of MRS2-4, we performed
a complementation analysis. A genomic fragment fused with
GFP (Fig. 1E) was introduced into mrs2-4-2 (Supplementary
Fig. S2), and two independent homozygous lines were established. Shoot fresh weight and the Mg concentration of the
introduced lines under low Mg conditions were similar to
those of the wild type. Further, a genomic fragment without
GFP was introduced into mrs2-4-1 and mrs2-4-2, and the
mutant phenotype was completely complemented
(Supplementary Fig. S3). These data show that mrs2-4-2
phenotypes are caused by a defect in MRS2-4 function.
Through the analysis, we obtained two mrs2-4 mutant alleles
with different sensitivity to low Mg.
MRS2-4 is essential for growth under both low
and normal Mg conditions
Using an RNAi line, which is a knock-down line, Mao et al.
(2014) showed that MRS2-4 is essential for growth under low
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K. Oda et al. | MRS2-4 essential for both low and high Mg conditions
Fig. 1 MRS2-4 is the causal gene of kudo7 and kudo8. (A) kudo7 and kudo8 mutant phenotype. The plants were grown for 3 weeks under normal
(1.5 mM Mg/2 mM Ca) and –Mg/+Ca (0 mM Mg/15 mM Ca) conditions. (B) Genetic mapping and mutation site of kudo7. The number of
recombinant F2 plants per total plants is shown under each marker. Black and white boxes represent exons and untranslated regions, respectively. T-DNA insertion sites of mrs2-4-2 are also indicated. (C) MRS2-4 mRNA was not detected by PCR. cDNA prepared from each plant
was used for the PCR. ACTIN8 was used as a positive control. (D) F1 crosses of mrs2-4-1 and 2-4-2 showed –Mg/+Ca sensitivity. Plants were grown
in –Mg/+Ca medium for 3 weeks and shoot fresh weights were measured. Different letters indicate a significant difference at P < 0.05 according
to Tukey–Kramer test. Data are represented as means ± SD (n = 15–20). (E) A schematic diagram of the construct used for the complementation
analysis. Genomic MRS2-4 with the promoter region (–2.5 kbp) was fused to GFP. tNOS. nopaline synthase terminator. (F) Complementation of
mrs2-4-1 with the construct in (E). Two homozygous lines were grown under –Mg/ + Ca conditions for 3 weeks. Scale bars = 1 cm.
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To confirm the role of MRS2-4 in Mg uptake, we measured
the Mg concentration of the wild type and mutants using inductively coupled plasma mass spectrometry (ICP-MS). Plants
were grown on agar plates providing several different Mg conditions for 3 weeks. Under normal Mg conditions, the MRS2-4
mutant lines exhibited lower Mg concentration in their roots
(Fig. 3). Shoots of mrs2-4-2 also exhibited lower Mg concentrations. Taken together with the shoot fresh weight, these results
demonstrate that MRS2-4 is essential for Mg transport under
normal Mg conditions, as well as under low Mg conditions.
The transcriptome profile of mrs2-4-1 under
normal Mg conditions is similar to that of the
wild type under low Mg conditions
Fig. 2 The knock-out allele, mrs2-4-2, exhibited severe sensitivity to
low Mg. (A) Shoot phenotypes of wild-type and mutant plants under
various Mg conditions. Plants were grown under Ctrl (1.5 mM Mg and
2 mM Ca), –Mg (0 mM Mg and 2 mM Ca) and –Mg/+Ca (0 mM Mg
and 15 mM Ca) conditions for 3 weeks. Scale bar = 1 cm. (B) Shoot
fresh weights of the wild-type and mutant plants in (A). Different
letters indicate significant differences at P < 0.05 according to
Tukey–Kramer test. Data are means ± SD (n = 15–20). (C) Shoot
fresh weights of the wild type, mrs mutants and the double mutant.
Plants were grown under normal (Ctrl) and 75 mM (1/20 Mg) Mg
conditions. Different letters indicate significant differences at
P < 0.05 according to Tukey–Kramer test. Data are represented as
the means ± SD (n = 15–20).
Mg conditions. To determine whether MRS2-4 is required for
growth under normal Mg conditions, we grew mrs2-4 mutants
under different Mg conditions. We found that while mrs2-4-1
showed sensitivity to –Mg/+Ca only, mrs2-4-2 exhibited
growth defects in both the 150 mM Mg and –Mg/+Ca treatments. Under normal conditions, the shoot fresh weight was
similar between wild-type and mutant lines. It has been reported that MRS2-7 is required for growth under low Mg conditions (Gebert et al. 2009). To test the redundancy of MRS2-4
and MRS2-7, we established a double mutant (Fig. 2C). Under
both low and normal Mg conditions, the shoot fresh weight of
the double mutant was lower than those of the single mrs
mutant lines. These data suggest that MRS2-4 and MRS2-7
are essential for growth under both normal and low Mg
conditions.
Next, we performed RNA-sequencing (RNA-Seq) analysis of the
wild type and mrs2-4-1 to observe changes in the transcriptome
profile. Total RNA was prepared from the shoots of two-weekold wild-type and mrs2-4-1 plants grown under normal (Ctrl)
and low Mg conditions (–Mg). The total RNA was subjected to
RNA-Seq analysis. We obtained four sets of transcriptomes:
wild-type (Ctrl), wild-type (–Mg), mrs2-4-1 (Ctrl) and mrs2-41 (–Mg). We compared the expression profiles in four combinations: wild-type (Ctrl) vs. wild-type (–Mg), mrs2-4-1 (Ctrl) vs.
mrs2-4-1 (–Mg), wild-type (Ctrl) vs. mrs2-4-1 (Ctrl) and wildtype (–Mg) vs. mrs2-4-1 (–Mg) (Fig. 4A; Supplementary Table
S1). The number of genes significantly (q-value <0.05) altered
in each comparison is shown in a Venn diagram (Fig. 4A). The
accumulation of mRNAs corresponding to 399 genes varied
between the wild-type (Ctrl) and wild-type (–Mg): 389 and
10 genes were induced or repressed by the –Mg treatment,
respectively. We considered these genes to be a low Mg
marker gene set. We then compared the transcription patterns
of this set in four different conditions. A heatmap based on the
log2 value of the normalized counts showed that the transcription patterns of mrs2-4-1 (Ctrl) are similar to those of the wildtype (–Mg) (Fig. 4B). Based on this result, we generated a scatter plot of the normalized count between wild-type (–Mg) and
mrs2-4-1 (Ctrl) (Fig. 4C). The plot showed a strong positive
correlation (R2 = 0.86) between the two variables. These results
suggest that mrs2-4-1 exhibited symptoms of Mg deficiency
even in the presence of sufficient Mg, supporting the conclusion that MRS2-4 is essential for maintaining Mg homeostasis
under both normal and low Mg conditions.
mrs2-7(1) and mrs2-4 mutants exhibited
sensitivity to high Mg conditions
It has been shown that the MRS2 family is important for adaptation to low Mg conditions (Drummond et al. 2006, Gebert
et al. 2009, Conn et al. 2011, Lenz et al. 2013, Mao et al. 2014),
and we showed that MRS2-4 and MRS2-7 are also important for
Mg uptake under normal conditions. However, there have been
no reports concerning the function of MRS2 under high Mg
conditions. To test whether MRS2-4 is required for adaptation
to high Mg conditions, mrs2-4 and mrs2-7(1) mutants were
grown under various high Mg conditions (5, 10 and 15 mM).
Surprisingly, two mrs2-4 mutants showed growth inhibition in
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K. Oda et al. | MRS2-4 essential for both low and high Mg conditions
Fig. 3 The mrs2-4-2 mutant exhibited decreased Mg concentrations under normal Mg conditions. Plants were grown with MGRL hydroponic
solution (see the Materials and Methods) supplemented with 1,500, 300, 150, 75 or 0 mM Mg. The Mg concentrations of shoots (black bars) and
roots (gray bars) were determined using ICP-MS. Different letters indicate significant differences at P < 0.05 according to Tukey–Kramer test.
Data are represented as the means ± SD of three biological replicates.
the 5–15 mM Mg treatment, and mrs2-7(1) exhibited reduced
growth under the 15 mM Mg condition (Fig. 5A). Next, we
tested the mutants for sensitivity to low Ca conditions. It has
been reported that low concentrations of Ca in the medium
increase the Mg concentration of the leaves of Brassica plants
(Rios et al. 2012). Thus, low Ca is expected to increase the Mg
concentration of plants, mimicking high Mg conditions. mrs2-4
and 2–7 mutants experienced growth inhibition under onetenth of the normal Ca level (0.2 mM) (Fig. 5B). These results
show that MRS2-4 and MRS2-7 are required for adaptation to
both high Mg and low Mg conditions.
MRS2-4 is localized to the ER in Arabidopsis
roots and BY-2 cells.
To determine the subcellular localization of MRS2-4, GFP fluorescence was observed in the complemented line carrying the
genomic MRS2-4 with GFP fused to the promoter region
(Fig. 1E). The complemented line of mrs2-4-1 was grown
under normal conditions for 10 d and the root was observed
with a confocal microscope. The fluorescence was observed
broadly within the cell, with the exception of the nuclear
space (Fig. 6A). This distribution pattern is similar to that of
the ER-localized membrane proteins (Wu et al. 2011, Ding et al.
2012). The GFP fluorescence was co-localized with ER-Tracker, a
marker of ER membrane localization (Fig. 6B). We also
observed this localization pattern in tobacco BY-2 cells transiently expressing GFP fusion protein. For transient expression,
MRS2-4 cDNA was fused with the N- or C-terminus of GFP,
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driven by the Cauliflower mosaic virus (CaMV) 35S promoter.
The green fluorescence of GFP alone was detected in both the
nucleus and cytosol (Fig. 6C). In contrast, MRS2-4 fused with
GFP was co-localized with the ER-Tracker. These data suggest
that MRS2-4 is mainly localized to the ER.
Discussion
Phenotypic differences between mrs2-4-1
and mrs2-4-2
We have identified a new MRS2-4 allele, mrs2-4-1, by screening
for a mutant sensitive to –Mg/+Ca conditions. We also identified another knock-out allele, mrs2-4-2, which is a T-DNA
mutant line. mrs2-4-1 and mrs2-4-2 exhibited different phenotypes in response to Mg (Fig. 2A, B). mrs2-4-1 exhibited growth
defects under –Mg/+Ca but not under low Mg (without Mg
supplementation) conditions, whereas mrs2-4-2 exhibited
severe growth defects under both conditions. One possibility
is that the E120K substitution in mrs2-4-1 changes the substrate
specificity of MRS2-4 against Ca, which may allow MRS2-4 to
transport Ca. Based on the CorA structure, Glu120 is predicted
to be localized to the cytoplasm distant from the GMN (GlyMet-Asn) motif in the C-terminal transmembrane domain
required for Mg2+ transport and substrate specificity
(Payandeh and Pai 2006, Eshagi et al. 2006). Thus, it is unlikely
to alter substrate specificity via E120K mutation. Another possibility is that the E120K mutation affects the substrate
Plant Cell Physiol. 57(4): 754–763 (2016) doi:10.1093/pcp/pcv196
Fig. 4 Transcriptome profiles of the mrs2-4-1 mutant under normal Mg conditions are similar to those of wild-type plants exposed to –Mg
conditions. (A) Venn diagram of genes with altered expression analyzed via RNA-Seq analysis. Genes showing significant differences (q-value
<0.05) were selected. (B) Heatmap of log2-normalized counts for genes differentially expressed in response to low Mg (Ctrl vs. –Mg; 399 genes).
The expression levels of 399 genes are compared in four samples. Red and green colors represent high and low expression levels in each
row, respectively. (C) Scatter plot of normalized counts, compared between mrs2-4-1 plants under Ctrl [mrs2-4-1 (Ctrl)] and wild-type plants
under –Mg [wild-type (–Mg)] conditions. The differentially expressed genes (399 genes) are plotted in (B). Five representative genes induced by
low Mg are shown as red points (Kamiya et al. 2011): AtDTX3 (At2g04050), UGT72E (At1g05680), AtDTX1 (At2g04070), CAX3 (At3g51860) and
ACA13 (At3g22910). The line indicates R2 = 1.
transport activity of MRS2-4, making mrs2-4-1 a weak allele. In
fact, mrs2-4-1 exhibited lower Mg concentrations than the wild
type (Fig. 3). In addition, the mrs2-4-1 transcriptome profile
under normal Mg conditions is similar to that of the wild type
under low Mg conditions (Fig. 4). These results suggest that
mrs2-4-1 could experience lower Mg transport activity, causing
the growth phenotype to differ from that of mrs2-4-2.
Possible function of MRS2-4
It has been reported that the mrs2-4 RNAi line and mrs2-7
single knock-out mutant exhibit growth defects only under
low Mg conditions (Gebert et al. 2009, Mao et al. 2014). In
our experiments using the mrs2-4 knock-out allele and the
mrs2-4 and mrs2-7 double mutant (Fig. 2), we revealed that
both MRS2-4 and MRS2-7 are important for normal growth
under normal Mg conditions. Triple or double mutants of the
MRS2 gene family that belongs to clade B (mrs2-1/mrs2-10
double and mrs2-1/mrs2-5/mrs2-10 triple mutant) exhibits a
growth defect under both normal and low Mg conditions, although the single gene knock-out mutant does not exhibit the
defect under either of the conditions (Gebert et al. 2009, Lenz
et al. 2013). MRS2-4 and MRS2-7 belong to clade D and E,
respectively (Lenz et al. 2013). These data suggest that the
physiological function of the MRS2 family in not limited to
low Mg adaptation, and maintains Mg homeostasis under sufficient Mg conditions.
In addition to normal Mg conditions, we found that MRS2-4
and MRS2-7 are required for growth under both high Mg conditions and low Ca conditions, which are both expected to
increase Mg concentrations in the cytosol (Rios et al. 2012).
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K. Oda et al. | MRS2-4 essential for both low and high Mg conditions
Fig. 5 High Mg and low Ca sensitivity of mrs2-4 and mrs2-7 mutants.
Plants were grown on MGRL agar plates for 3 weeks in different concentrations of Mg (A) and Ca (B). The pictures of plants grown under
15 mM Mg and 0.2 mM Ca are shown at the bottom. Different letters
indicate a significant difference at P < 0.05 according to Tukey–
Kramer test. Data are represented as the means ± SD (n = 15–20).
Our current and previous results show that plant genes belonging to the MRS2 family code for an Mg uptake transporter,
which is especially important for growth under low Mg conditions. For example, plasma membrane-localized MRS2s can mediate Mg transport from outside the cell to the cytosol, while
tonoplast-localized MRS2s can mediate Mg transport from the
vacuolar lumen to the cytosol. If this were true, under low Mg,
mrs2-4 and mrs2-7 mutants could be expected to have sensitivity to or tolerance of the high Mg conditions similar to that of
the wild type. Unexpectedly, mrs2-4 and mrs2-7 showed severe
growth defects under high Mg conditions (Fig. 5A). These
phenotypes could be explained by the assumption that
MRS2s is a bi-directional transporter and functions as an Mg
efflux transporter under high Mg conditions, as has been shown
in the bacterial MRS homolog, CorA (Gibson et al. 1991).
Disruption of CorA of S. typhimurium decreases the Mg efflux
from the cell. In our experiments, MRS2-4 is mainly localized to
the ER (see discussion below), where MRS2-7 is localized
(Gebert et al. 2009). Although there are no available data for
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Fig. 6 Localization of MRS2-4 in roots and BY-2 cells. (A) GFP fluorescence of MRS2-4:GFP/mrs2-4-1 (Fig. 1F). Magnified image of the
dotted box is shown on the right. Scale bars = 50 mm. (B) Roots of
complemented lines (A) were stained with ER-Tracker. (C) GFP alone
(GFP), C- (C-GFP) or N-terminal (N-GFP) GFP fusions of MRS2-4
under the control of the 35S CaMV promoter were introduced into
tobacco BY-2 cells. GFP, ER-Tracker fluorescence and merged images
are shown. Scale bars = 30 mm.
the Mg concentration of the ER, the ER can serve as a storage
organelle of Mg and serve to buffer Mg in the cell. This has been
suggested to occur in animal cells (Mooren et al. 2011). Under
low/high Mg concentrations, Mg would be released/sequestered in the cytosol/ER. We propose that both MRS2-4 and
MRS2-7 maintain cytosol Mg concentrations using ER as a storage location.
mrs2-4 mutants showed lower Mg concentration in the root
and shoot, although MRS2-4 is localized to the ER and is unlikely to be involved in Mg uptake in the plasma membrane.
This might suggest that the Mg concentration in the ER is
sensed and its information is transmitted to the plasma
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membrane to promote Mg uptake through unknown mechanisms. In the mutant ER, the Mg concentration would be high, as
MRS2-4 could export Mg to the cytosol, which prevents Mg
uptake in the plasma membrane, leading to a low Mg concentration in the root and shoot. In mammals, such a mechanism is
present, which is referred to as store-operated Ca2+ entry
(SOCE): the Ca2+ channel in the plasma membrane is activated
in response to the depletion of Ca2+ in the ER (Parekh and
Putney 2005, Cahalan 2009). The presence of such a mechanism
in plants will be tested in the future.
Subcellular localization of MRS2-4
In our analysis, MRS2-4 was found to be localized to the ER,
which is not consistent with previous results (Mao et al. 2014).
Previous studies have shown MRS2-4 to be localized to the
plasma membrane, with transient expression in a protoplast
prepared from rosette leaves and suspension cells. These experiments used expression of a C-terminal GFP fusion with
MRS2-4 cDNA in the protoplast, driven by the CaMV 35 S promoter. In our experiments, the genomic fusion of GFP with
MRS2-4 was introduced into mrs2-4-2, which rescued the
mutant phenotype (Fig. 1F). This suggests that MRS2-4 fused
with GFP is functional and likely to reflect endogenous MRS2-4
protein localization. In addition, we expressed N- and Cterminal GFP fusion proteins in BY-2 cells. Both experiments
revealed ER localization (Fig. 6). Based on the localization of
GFP observed in Arabidopsis and BY-2 cells in our studies, we
believe that MRS2-4 is mainly localized to the ER.
In conclusion, we showed that MRS2-4 and MRS2-7 are important for plant survival under both low and high Mg conditions. We propose that MRS2 transporters carry out the
bi-directional transport of Mg depending on the Mg concentration inside or outside the ER. This function may allow plants
to adapt to different soil Mg concentrations. It has been shown
in A. lyrata that MRS2-2 and MRS2-7 are among the genes
associated with serpentine adaptation (low Ca to Mg ratio), a
model system of plant adaptation to an environment (Turner
et al. 2010). The MRS family would provide a good explanation
for this adaptation.
Materials and Methods
Plant materials and growth conditions
EMS-mutagenized A. thaliana Col-0 seeds were purchased from Lehle seeds
(http://www.arabidopsis.com/) and used in the screening (T. Kamiya et al. unpublished). Col-0 and Ler were taken from our laboratory stock. T-DNA mutants were purchased from the Arabidopsis Biological Resource Center (ABRC;
https://abrc.osu.edu/) and the Nottingham Arabidopsis Stock Centre (NASC;
http://arabidopsis.info/). The accession numbers of the T-DNA mutants are as
follows: SALK_145997 (mrs2-4-2), SALK_064741 [mrs2-7(1)]. The WKO plant
was generated by crossing mrs2-4-2 and mrs2-7(1). The primers used for the
genotyping of T-DNA mutants are as follows: for mrs2-4-2, 50 -AGAGGCGAGAA
AGCAGAAGAG-30 (left) and 50 -TACAGCATCAAACAGCATTGC-30 (right); for
mrs2-7(1), 50 -GGCAAATATCAAGGAACCCTC-30 (left) and 50 -ACAAATAGCT
TCCAAGGCCAC-30 (right). The right primer and LB1.3 primer (50 -ATTTTGCC
GATTTCGGAAC-30 ; the sequence was provided by the ABRC) were used in
combination with the right primer to check for the insertion of T-DNA. MGRL
medium (Fujiwara et al. 1992) supplemented with 1% sucrose (Sigma) was used
to grow the plants. MgSO4 (Wako) and CaCl27H2O (Wako) were used as Mg
and Ca sources in the medium, respectively. For the gelling agent, purified agar
(Nacalai tesque) was used at 1.5%. Seeds were sterilized in 10% commercial
bleach and washed briefly. After vernalization for 2–4 d at 4 C, the plants were
grown vertically under long-day conditions (16/8 h light/dark cycle) at 23 C for
3 weeks.
Genetic mapping
For the genetic mapping, kudo7 and kudo8 were crossed with Ler. The
self-fertilized F2 population was used for the mapping. F2 plants were grown
in –Mg/+Ca medium, after which genomic DNA was prepared from those
plants showing the mutant phenotype. SSLP markers were used for the mapbased cloning. After rough mapping, the genome was re-sequenced by SOLiD
(Life Technologies) to determine the mutation in the mapped region, as
described previously (Tabata et al. 2013). The mutation in the mapped
region was re-sequenced using the Sanger method for confirmation.
RNA extraction and analysis of expression level
Total RNA was prepared from plant material using an RNeasy Plant Mini kit
(Qiagen) followed by RNase-free DNase (Qiagen) treatment. Total RNA was
reverse-transcribed to cDNA using a PrimeScript RT reagent kit (TAKARA).
After 10-fold dilution, cDNA was used for quantitative PCR analysis with the
Dice system (TAKARA) and SYBR Premix Ex Taq II (TAKARA). The primers
used for the expression analysis were as follows: MRS2-4, 50 -GAGACATTGTTGG
CCAGCTT-30 and 50 -TGAGCCTAGCAGCTTCTTCC-30 ; ACTIN8, 50 -GCCAGAT
CTTCATCGTCGTG-30 and 50 -TCTCCAGCGAATCCAACCTT-30 . ACTIN8 was
used as an internal control.
RNA sequencing and analysis
Plants were grown on MGRL agar plates for 2 weeks under normal or –Mg
conditions. Total RNA was prepared from plant shoots using Nucleospin RNA
plant (TAKARA) following the manufacturer’s protocol, and was used for RNASeq analysis. RNA-Seq was performed by BGI (http://www.bgitechsolutions.
com/ja.html) using HiSeq 2000 (Illumina).
Sequence files were generated in the FASTAQ format, and aligned to an
Arabidopsis reference genome (TAIR10) using TopHat2 (Trapnell et al. 2009)
via the DDBJ pipeline (Nagasaki et al. 2013). Processed SAM files were converted
to count files using HTseq (Anders et al. 2014). The calculation of q-values in
each comparison and normalization of Trimmed mean of M values (TMM) was
perfumed using R software (http://www.r-project.org/) implemented with
EdgeR
(http://master.bioconductor.org/packages/release/bioc/html/edgeR.
html). These normalized count files were used to generate both the heatmap
and the scatter plot. The Venn diagram was generated using Venny 2.0 (http://
bioinfogp.cnb.csic.es/tools/venny/).
Element measurement in plant
Three-week-old-plants were briefly washed with H2O and dried up for 2 d at
60 C. The dried samples were digested with 1 ml of concentrated HNO3 (Wako)
at 180 C for 2 h. Digested samples were dissolved in 0.08 N HNO3 containing
2 p.p.b. indium (Wako) as an internal standard. The elemental concentrations
were determined by ICP-MS (SPQ9700, SII).
Plasmid constructions and transformations
For the construction of the GFP-fused MRS2-4 genome, a genomic fragment of
MRS2-4 containing the –2.5 kbp region upstream of the first ATG was amplified
via PCR with primers 50 -CGACTCTAGACAGAGTTTTGGCTTTGCAGG-30 and
50 -TCGAGGCGCGCCATGAGCCTAGCAGCTTCTTC-30 (the XbaI and AscI sites
are underlined). The fragment was digested with XbaI and AscI, and then cloned
into the XbaI–AscI site of the pMDC107 vector (Curtis and Grossniklaus 2003).
For the construction of the MRS2-4 genome without a tag, a genomic fragment
of MRS2-4 was amplified with primers, 50 -TCGAGCATGCGAGCTGGCTCAATC
TGTGGA-30 and 50 -CTAGTTAATTAATACTACGGGAGAGCCGAGAG-30 (the
SphI and PacI sites are underlined). The fragment was digested with SphI and
PacI, and then cloned into the SphI–PacI site of the pMDC32 vector (Curtis and
Grossniklaus 2003). These plasmids were introduced into A. thaliana via the
761
K. Oda et al. | MRS2-4 essential for both low and high Mg conditions
floral dip method (Clough and Bent 1998) via Agrobacterium tumefaciens
GV3101::pMP90. Transformants were selected with hygromycin, and the homozygous lines were established. For the transient assay of the tobacco BY-2 cell,
MRS2-4 cDNA was amplified using the following primers: 50 -CTAGTTAATTAA
ATGGGGAAGGGCCCCTTAT-30 and 50 -TCGAGGCGCGCCATGAGCCTAGCA
GCTTCTTCC-30 (PacI and AscI sites are underlined) for pMDC83, and
50 -CAAAGGCGCGCCAAGCTATCACAAGTTTGTACAAAAAATGGGGAAGG
GCCCCTTAT-30 and 50 -TCGATTAATTAAATTATGAGCCTAGCAGCTTCT-30
(AscI and PacI sites are underlined) for pMDC45. The amplified fragments
were digested by PacI and AscI, and cloned into the PacI–AscI site of the
pMDC83 and pMDC45 vector (Curtis and Grossniklaus 2003) to obtain a
CaMV 35 S promoter-driven C- and N-terminal GFP fusion of MRS2-4. The
35 S:GFP plasmid was obtained from a previous study (Uraguchi et al. 2011).
The transformation and culture of BY-2 cells was carried out as reported previously (Mayo et al. 2006), with some modifications. Agrobacterium tumefaciens
EHA101 carrying the plasmids was cultured with BY-2 cells for 2 d at 23 C, to
facilitate infection. After washing the BY-2 cells three times with fresh medium,
the cells were cultured for 2 weeks with hygromycin to select transformants.
Then BY-2 cells were cultured in antibiotic-free medium for a week, and used
for the confocal observation.
Fluorescence observation under a confocal
microscope
To observe GFP and ER-Tracker fluorescence in plant roots and BY-2 cells, a
confocal laser scanning microscope (Fluoview 1000, Olympus) was used.
Detached Arabidopsis root and BY-2 cells were soaked in 1 mM ER-Tracker
Red (Life Technologies) for 30 min at 37 C. The excitation and emission
wavelengths are as follows: 488 nm and 505–540 nm band pass filter for
GFP, and 552 and 600 nm long pass filter for ER-Tracker Red. To observe
GFP and ER fluorescence, the plants were grown under normal conditions
for a week.
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the Japan Society for the
Promotion of Science [grants to K.O. and T.K.]; the Japan
Society for the Promotion of Science [grant to T.F.
(No. 25221202)].
Acknowledgements
We thank the ABRC and NASC for providing the materials used
in this study. We are also grateful to E. Yokota and Y. Kawara for
technical support, S. Arimura and Y. Hayashi for providing BY-2
cells, and M.D. Curtis for providing pMDC vectors.
Disclosures
The authors have no conflicts of interest to declare.
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