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 .