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Plants (Basel)
2019 Dec 21;91:. doi: 10.3390/plants9010016.
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Expression and Ion Transport Activity of Rice OsHKT1;1 Variants.
Imran S
,
Horie T
,
Katsuhara M
.
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OsHKT1;1 in rice, belongs to the high-affinity K+ Transporter family, has been found to be involved in salt tolerance. OsHKT1;1 in japonica rice (Nipponbare) produces mRNA variants, but their functions remain elusive. In salt tolerant rice, Pokkali, eight OsHKT1;1 variants (V1-V8) were identified in addition to the full-length OsHKT1;1 (FL) cDNA. Absolute quantification by qPCR revealed that accumulation of OsHKT1;1-FL mRNA is minor in contrast to that of OsHKT1;1-V1, -V2, -V4, and -V7 mRNAs, all of which are predominant in shoots, while only V1 and V7 mRNAs are predominant in roots. Two electrode voltage clamp (TEVC) experiments using Xenopuslaevis oocytes revealed that oocytes-expressing OsHKT1;1-FL from Pokkali exhibited inward-rectified currents in the presence of 96 mM Na+ as reported previously. Further TEVC analyses indicated that six of eight OsHKT1;1 variants elicited currents in a Na+ or a K+ bath solution. OsHKT1;1-V6 exhibited a similar inward rectification to the FL protein. Contrastingly, however, the rests mediated bidirectional currents in both Na+ and K+ bath solutions. These data suggest possibilities that novel mechanisms regulating the transport activity of OsHKT1;1 might exist, and that OsHKT1;1 variants might also carry out distinct physiological roles either independently or in combination with OsHKT1;1-FL.
Figure 1. OsHKT1;1 transcripts identified in Pokkali plants. (A) A gel image of RT-PCR produces amplified with primers for OsHKT1;1; (B) the transmembrane domain of the full-length OsHKT1;1 protein (OsHKT1;1-FL) predicted by TMHMM Server v.2.0. Nine transmembrane domains (M1-M9) were predicted; (C) schematic diagrams of OsHKT1;1-FL and its variants. Boxes indicate amino acid regions that are the same as FL (black) or different from FL because of the frame shift (grey). The line “─” indicates non-translated regions, and dot line “┉” indicates missing nucleotide regions (gap) compared with the FL sequence.
Figure 2. qPCR analyses on OsHKT1;1 transcripts in Pokkali plants grown under normal growth condition. Expression levels of OsHKT1;1-FL and its variants in shoots (A) and roots (B) of 14 day-old plants were investigated by absolute quantification. Insets; Expanded views of variants showing low expression. Data are means ± SE, n = 3 Three independent experiments were performed, and similar results were obtained. Different letters indicate significant differences (p < 0.05).
Figure 3. qPCR analyses on OsHKT1;1 transcripts in Pokkali seedlings grown under salt stressed conditions. Expression levels of OsHKT1;1-FL and each variant in shoots and roots were investigated by absolute quantification. 14 day-old Pokkali plants were treated with 100 mM NaCl solution for 0, 6, 12, 24, or 48 h prior to the total RNA extraction. (A) OsHKT1;1-FL, (B) OsHKT1;1-V1, (C) OsHKT1;1-V2, (D) OsHKT1;1-V3, (E) OsHKT1;1-V4, (F) OsHKT1;1-V5, (G) OsHKT1;1-V6, (H) OsHKT1;1-V7, (I) OsHKT1;1-V8. Absolute amounts of transcript (copies/μg RNA) were displayed. Data are means ± SE, n = 3. Three independent experiments were performed, and similar results were obtained. Different letters indicate significant differences (p < 0.05).
Figure 4. Ion transport activity of OsHKT1;1-FL and its variants. Two electrode voltage clamp experiments using Xenopus laevis oocytes were conducted. (A,C) Current-voltage relationships from oocytes expressing OsHKT1;1-FL, -V1, -V4, -V6, and water-injection control. (B,D) Current-voltage relationships from oocytes expressing OsHKT1;1-V2, -V3, -V5, -V7, -V8, and water-injection control. External solution contains 96 mM NaCl (A,B) or 96 mM KCl (B,D). All external solutions contain, as background elements, 1.8 mM CaCl2, 1.8 mM MgCl2, 1.8 mM mannitol, and 10 mM HEPES (pH 7.5 with Tris). Water was injected as a negative control. Data represent means ± SE, n = 18–24, from three independent experiments performed on different oocytes batches.
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