Mutation confirmation, subcellular localization, and expression
patterns of OsMTP1
In order to confirm the mutation in OsMTP1 , genomic DNA was
isolated and PCR-based molecular cloning was conducted. This was
followed by Sanger DNA sequencing for analyzing the regions to see where
the mutations predicted in gDNA of OsMTP1 were present.
Consistent with the WGS analysis, we identified the insertion of T
between nucleotide 4601 and 4062 in exon 5 of the mOsMTP1 gene
(mutated Os01g47460 ) in the sitl1 (Figure 8a). Next, we
explored whether this T insertion can lead to the incorrect splicing of
the OsMTP1 mRNA in the sitl1 . Sanger sequencing results
showed that the T insertion was found between nucleotide 1044 and 1045
in mRNA of mOsMTP1 as expected but we did not find any other
splicing forms of OsMTP1 (Figure 8a). The predicted protein
sequence of mOsMTP1 in the sitl1 showed that this T insertion led
to a frameshift in CorA-like ZntB cation transporter domain region of
the mOsMTP1 protein sequence (LVSIILNQEIRRLATQVIRV to
SSQHHIESRNQKVSNTGNQS, 349 – 368 amino acids) and the appearance of a
premature stop codon (TAA) (Figure S11).
Gene expression levels of OsMTP1 were analyzed using two primer
sets, which were designed in the N- or C- terminal region of theOsMTP1 . Real-time qRT-PCR results with qRT1 primer set showed
that the expression level of OsMTP1 was not changed between thesitl1 and WT (Figure 8b). However, gene expression levels of
OsMTP1 in the sitl1 significantly lower than that of WT in both
roots and leaves when we used qRT2 primer set (Figure 8c). One possible
explanation for this is the premature termination codon containing
aberrant mRNAs of the mOsMTP1 caused degradation due to eukaryotic
quality control system (Mekawy et al., 2015; Nyiko et al., 2017). These
results suggest that a single nucleotide insertion in codon 349 of themOsMTP1 gene might disrupt existing gene functions and gene
expression of OsMTP1 in the sitl1 .
OsMTP1 protein contains putative metal ion transporter CorA-like ZntB
cation transporter domain with 2 transmembrane (TM) domains in the
C-terminal regions. The alignment between OsMTP1 and its one paralog in
rice (Os01g41720) and other orthologs from sorghum, maize, and
Arabidopsis evidenced highly conserved the CorA-like ZntB magnesium
transporter domain containing conserved GIN-motif (Figure S12 and S13).
A phylogeny from the previous studies of MRS2/MGT gene family in rice
and Arabidopsis (Lalonde et al., 2017) with the OsMTP1 and its
orthologs showed a clear distinction between the MRS2/MGT and OsMTP1
with its orthologs, suggesting divergence in function (Figure S14). We
also confirmed that subcellular localization of the35S::OsMTP1-sGFP was clearly co-localized with the plasma
membrane marker (pm-rk) in rice protoplasts (Figure 8d)
Lastly, we evaluated OsMTP1 expression patterns using various
tissues from different developmental stages in WT rice (Figure 8e). TheOsMTP1 was rarely expressed in leaf sheath compared to other
tissues, whereas it was highly induced in root and leaf blade tissues at
young seedling stage. The expression levels of OsMTP1 were
gradually reduced in all tissues at late vegetative stage and the
reproductive stage. Further examination of the expression levels ofOsMTP1 in response to salinity stress in 1-week-old root and leaf
tissues was evaluated (Figure 8f-g). In roots, the OsMTP1 gene
was upregulated at 30 m after NaCl treatment and highly induced with
peak expression occurring at 1 h. The induced mRNA abundances of theOsMTP1 were dramatically reduced at 6 to 24 h NaCl treatment in
roots (Figure 8f). Similar expression pattern of the OsMTP1 was
observed in leaf tissues. The OsMTP1 exhibited increased
transcript abundance at 30 m until 6 h by NaCl treatment and then
reduced dramatically (Figure 8g). Collectively, these results showed
that OsMTP1 mainly functions in roots and leaves in the early response
to salinity stress and T insertion mutation in the OsMTP1 led to
decrease in Na+ and Mg2+ uptake at
plasma membrane resulting in salinity tolerance in the sitl1 .