Ben Amar S et al. (2014), Functional characterization in Xenopus oocytes ...

XB-ART-47822

J Exp Bot 2014 Jan 01;651:213-22. doi: 10.1093/jxb/ert361.

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Functional characterization in Xenopus oocytes of Na+ transport systems from durum wheat reveals diversity among two HKT1;4 transporters.

Ben Amar S , Brini F , Sentenac H , Masmoudi K , Véry AA .

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Plant tolerance to salinity constraint involves complex and integrated functions including control of Na(+) uptake, translocation, and compartmentalization. Several members of the high-affinity K(+) transporter (HKT) family, which comprises plasma-membrane transporters permeable to K(+) and Na(+) or to Na(+) only, have been shown to play major roles in plant Na(+) and K(+) homeostasis. Among them, HKT1;4 has been identified as corresponding to a quantitative trait locus (QTL) of salt tolerance in wheat but was not functionally characterized. Here, we isolated two HKT1;4-type cDNAs from a salt-tolerant durum wheat (Triticum turgidum L. subsp. durum) cultivar, Om Rabia3, and investigated the functional properties of the encoded transporters using a two-electrode voltage-clamp technique, after expression in Xenopus oocytes. Both transporters displayed high selectivity for Na(+), their permeability to other monovalent cations (K(+), Li(+), Cs(+), and Rb(+)) being ten times lower than that to Na(+). Both TdHKT1;4-1 and TdHKT1;4-2 transported Na(+) with low affinity, although the half-saturation of the conductance was observed at a Na(+) concentration four times lower in TdHKT1;4-1 than in TdHKT1;4-2. External K(+) did not inhibit Na(+) transport through these transporters. Quinine slightly inhibited TdHKT1;4-2 but not TdHKT1;4-1. Overall, these data identified TdHKT1;4 transporters as new Na(+)-selective transporters within the HKT family, displaying their own functional features. Furthermore, they showed that important differences in affinity exist among durum wheat HKT1;4 transporters. This suggests that the salt tolerance QTL involving HKT1;4 may be at least in part explained by functional variability among wheat HKT1;4-type transporters.

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Species referenced: Xenopus
Genes referenced: tbx2

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	Fig. 1. Phylogenetic relationships between HKT transporters in rice and wheat. The unrooted phylogenetic tree was constructed using full polypeptide sequences aligned with MUSCLE (http://www.bioinformatics.nl/tools/muscle.html; Dereeper et al., 2008), and the neighbour-joining method with 1000 bootstrap replicates, using PhyML software (http://phylogeny.lirmm.fr). The tree was drawn using Dendroscope (Huson et al., 2007). Bootstrap values (as percentages) are indicated at the corresponding nodes. The protein accession numbers are: OsHKT1;1, Q7XPF8.2; OsHKT1;3, Q6H501.1; OsHKT1;4, Q7XPF7.2; OsHKT1;5, Q0JNB6.1; OsHKT2;1, Q0D9S3.1; OsHKT2;2, BAB61791.1; OsHKT2;3, Q8L481.1; OsHKT2;4, Q8L4K5.1; TaHKT1;5-B1, ABG33943; TaHKT1;5-B2, ABG33944; TaHKT1;5-D, ABG33945; TaHKT2;1, AAA52749; TmHKT1;4-A1, ABK41858; TmHKT1;4-A2, ABK41857; TmHKT1;5, ABG33939; TdHKT1;4-1, KF443078; TdHKT1;4-2, KF443079. Os, Oryza sativa; Ta, Triticum aestivum; Td, Triticum turgidum subsp. durum; Tm, Triticum monococcum.
	Fig. 2. TdHKT1;4-1 and TdHKT1;4-2 function as monovalent cation transporters with a strong preference for Na+ in Xenopus oocytes. Bath solutions contained the standard background supplemented with NaCl, KCl, RbCl, CsCl, or LiCl, at 3mM. The voltage-clamp protocol consisted of 12 pulses of 1 s, with a voltage increment of 15 mV between pulses. (A) Currents from control (H2O-injected) oocytes plotted against applied voltages. (B, C) Currents flowing through TdHKT1;4-1 (B) and TdHKT1;4-2 (C) transporters versus applied voltages. Data are means±standard error (SE) (n=4) and are representative of five experiments performed on different oocyte batches.
	Fig. 3. TdHKT1;4-1 and TdHKT1;4-2 transporters differ in their affinity for Na+. (A, B) Currents flowing through TdHKT1;4-1 (A) or TdHKT1;4-2 (B) transporters versus applied voltages in the presence of varying external Na+ concentrations (0.1, 1, 10, 50, and 100mM). Na+ was provided as glutamate salt. Data are means±SE (n=4 in A and n=5 in B) and are representative of six experiments performed on different oocyte batches. Insets in (A) and (B): I–V relationships in water-injected oocytes belonging to the same batch as HKT-expressing oocytes. Experimental conditions were the same as for HKT-expressing oocytes. Currents are means±SE (n=4 in A and n=3 in B). (C, D) Zero-current potentials through TdHKT1;4-1 (C) and TdHKT1;4-2 (D) versus bath Na+ activity. Current reversal potentials were obtained from the I–V data shown in (A) and (B). (E, F) Variation of TdHKT1;4-1 (E) and TdHKT1;4-2 (F) macroscopic inward conductance with external Na+ concentration. Macroscopic inward conductances were defined as slopes of I–V relationships between the three most negative imposed potentials in each ionic condition. The conductances in (E) and (F) were extracted from the I–V data shown in (A) and (B). Inward conductances plotted against external Na+ concentrations were fitted (solid line) with a Michaelis–Menten equation to determine the apparent half-saturation constant (K M). Fitted parameters were as follows: K M ∼3mM and G max (maximum whole-cell conductance) ~225 µS (E); K M ~12mM and G max ∼490 µS (F).
	Fig. 4. Effect of external K+ and Li+ on Na+ transport by TdHKT1;4-1 (A, C) and TdHKT1;4-2 (B, D). Bath solutions contained 0.1mM (left panels) or 3mM (right panels) Na+ (glutamate salts). They were or were not supplemented with KCl (A, B) or LiCl (C, D), with concentration of the added salt being 30mM (left panels) or 50mM (right panels). Data are means±SE (n=7 in A, n= 6 in B, n= 5 in C, n=8 in D) and are representative of two experiments performed on different oocyte batches.
	Fig. 5. Effect of potential inhibitors on Na+ transport activity in TdHKT1;4-1 (A, C) and TdHKT1;4-2 (B, D). Control (1 Na) external solution contained 1mM Na+ as glutamate salt. Currents were recorded successively in the control solution and in the same solution but supplemented with 1mM spermine or 1mM spermidine (A, B) or with 500 µM amiloride or 500 µM quinine (C, D). Data are means±SE (n=7 in A, n=3 in B, n=5 in C and D).

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