Functional analysis of AtHKT1 in Arabidopsis shows that Na(+) recirculation by the phloem is crucial for salt tolerance

Abstract

Two allelic recessive mutations of Arabidopsis, sas2-1 and sas2-2, were identified as inducing sodium overaccumulation in shoots. The sas2 locus was found (by positional cloning) to correspond to the AtHKT1 gene. Expression in Xenopus oocytes revealed that the sas2-1 mutation did not affect the ionic selectivity of the transporter but strongly reduced the macro scopic (whole oocyte current) transport activity. In Arabidopsis, expression of AtHKT1 was shown to be restricted to the phloem tissues in all organs. The sas2-1 mutation strongly decreased Na(+) concentration in the phloem sap. It led to Na(+) overaccumulation in every aerial organ (except the stem), but to Na(+) underaccumulation in roots. The sas2 plants displayed increased sensitivity to NaCl, with reduced growth and even death under moderate salinity. The whole set of data indicates that AtHKT1 is involved in Na(+) recirculation from shoots to roots, probably by mediating Na(+) loading into the phloem sap in shoots and unloading in roots, this recirculation removing large amounts of Na(+) from the shoot and playing a crucial role in plant tolerance to salt.

Fig. 1. sas2 plants overaccumulate Na⁺ in shoots. Twelve-day-old plants grown on compost in the greenhouse were irrigated twice within an additional 6-day period, with tap water supplemented or not with 50 mM NaCl. The shoot Na⁺ content of sas2-1 (left) and sas2-2 (right) plants (initially named sas2 and 444B, respectively; see main text) were compared with those of the corresponding wild types in two independent experiments. Means ± SD, n ≥ 30.

Fig. 2. Positional cloning of the sas2 locus. (A) Genetic and physical mapping of the sas2 locus. Top: genetic map of the region of chromosome IV encompassing the sas2 locus. Below: physical map (to scale) including the five BACs, F17A8, T5L19, F28M11, T9A4 and F24G24 (data from the AGI consortium sequencing effort, www.arabidopsis.org). On both maps, numbers in bold italics are the numbers of recombination events (identified by the analysis of 107 recombinant plants) between the corresponding adjacent genetic markers, or between the sas2 locus and the neighboring genetic markers. The physical distance separating the two genetic markers most closely flanking sas2 (F17A8M and F24G24SE) and the position of the AtHKT1 gene are indicated on the physical map. (B and C) Protein sequence alignments of plant members of the HKT1 family in the regions containing the sas2-1 (B) and sas2-2 (C) mutations. Accession numbers of the A.thaliana, eucalyptus-1 and -2, rice-1 and -2, wheat and Mesembryanthemum crystallinum sequences are AAF68393, AAF97728, AAD53890, BAB61790, BAB61791, AAA52749 and AAK52962, respectively. The sas2-1 and sas2-2 mutations (S282 to L282 and G325 to E325, respectively) are marked with circles. A light gray background highlights the amino acid positions common to at least all the members of the family but one. Numbers above the sequence alignments relate to the position of the corresponding amino acids in the Arabidopsis AtHKT1 sequence. (D) The sas2-1 and sas2-2 mutations are marked by stars on the structural model (Kato et al., 2001) of the AtHKT1 protein.

Fig. 3. Comparison of Na⁺, K⁺, Li⁺, Ca²⁺ and Mg²⁺ accumulation in shoots between sas2-1 and wild-type plants. Twelve-day-old in vitro grown plants were cultivated for 6 additional days on standard medium supplemented with NaCl (Na⁺), KCl (K⁺), CaCl₂ (Ca²⁺) or MgCl₂ (Mg²⁺) at a final concentration of 35 mmol/l, or with LiCl (Li⁺) at a final concentration of 4 mmol/l. For each treatment, the shoot content of the cation that had been added to the standard culture medium was determined from a pool of 20 plants. Ion content in sas2-1 shoots was expressed as a percentage of the wild-type value, which was standardized to 100. Wild-type ion contents were 1.6 ± 0.1 mmol/g DW for Na⁺, 3.5 ± 0.3 mmol/g DW for K⁺, 19.8 ± 1.2 µmol/g DW for Li⁺, 0.7 ± 0.1 mmol/g DW for Ca²⁺ and 0.8 ± 0.08 mmol/g DW for Mg²⁺. Means ± SD; n = 6.

Fig. 4. Functional characterization of SAS2-1 in Xenopus oocytes. Experi ments were performed in parallel on SAS2-1 and on wild-type AtHKT1. Recordings were carried out 2 days after cRNA injection. Oocytes (all from the same batch) were successively voltage clamped in bath solutions containing either 0.3 mM Na⁺ plus varying K⁺ concentrations (0.3, 3 and 10 mM), or 0.3 mM K⁺ plus varying Na⁺ concentrations (1 or 10 mM). Applied membrane potentials ranged from –20 to –140 mV. (A–C) Mean steady-state currents (± SE) recorded in oocytes injected with 50 ng of either wild-type AtHKT1 (A, n = 6) or sas2-1 (B, n = 11) cRNA, or with H₂O (C, n = 5). Note the difference in scale of current between (A) and (B and C). (D) Comparison of currents recorded at –140 mV in oocytes expressing either AtHKT1 (wt) or SAS2-1, or in control oocytes injected with H₂O (cont) in the presence of 0.3 mM K⁺ plus 10 mM Na⁺. Means ± SE [data from (A), (B) and (C)].

Fig. 5. AtHKT1 is expressed in the phloem tissues. (A–G) β-glucuronidase staining in excized organs from transgenic Arabidopsis plants expressing the GUS coding sequence under the control of the AtHKT1 promoter. Three-week-old plant (A); leaf (B), flower (D), root (F) and flower peduncle (G) cross-sections; flower (C); roots (E). The cross-sections were counterstained with Schiff reagent. (H–J) In situ hybridization performed with sense (H) or antisense (I and J) AtHKT riboprobes on root longitudinal (H and I) or cross (J) sections. Plants were grown in vitro (A, B, E, F, H, I and J) or in hydroponics (C, D and G). c, cortex; e, endodermis; ep, epidermis; f, filament; ms, mesophyll; p, petal; ph, phloem; s, sepal; st, stele; sty, style; x, xylem. Scale bars = 25 µm (B, F, H, I and J), 50 µm (G) and 200 µm (D).

Fig. 6. The sas2-1 mutation results in decreased Na⁺ concentration in the phloem sap. Eight-week-old hydroponically grown plants were cultivated for 4 additional days on standard medium supplemented with 5 mM NaCl (sas2-1 plants) or 50 mM NaCl (wild-type plants) before phloem sap was collected in EDTA solutions. The Na⁺ to glutamine molar ratios in the EDTA extracts were plotted versus the leaf Na⁺ content. Each symbol corresponds to a single plant.

Fig. 7. sas2-1 and wild-type plants display similar Na⁺ content in xylem sap. Eight-week-old hydroponically grown plants were cultivated for 4 additional days on standard medium supplemented with 10 mM NaCl. Na⁺ and K⁺ concentrations were determined in xylem sap collected as leaf exudate using a pressure chamber. Each symbol corresponds to a single plant.

Fig. 8. The sas2-1 mutation results in NaCl hypersensitivity. Four-week-old plants grown in hydroponics were either subjected to NaCl treatment (standard solution supplemented with 25 or 50 mM NaCl) or further cultivated on standard solution. (A–C) Shoot DW (A), shoot Na⁺ content (B) and shoot K⁺ content (C) during the 18 days following the application of NaCl treatment. Open and closed symbols correspond to wild-type and sas2-1 plants, respectively. Squares, circles and triangles correspond to plants grown on media supplemented with 0, 25 and 50 mM NaCl, respectively. Means ± SE, n = 10. (D) sas2-1 (top) and wild-type (bottom) rosettes after 17 days of growth on the standard culture medium supplemented with 0 (left) or 50 (right) mM NaCl. Scale bars = 1 cm.

Fig. 8. The sas2-1 mutation results in NaCl hypersensitivity. Four-week-old plants grown in hydroponics were either subjected to NaCl treatment (standard solution supplemented with 25 or 50 mM NaCl) or further cultivated on standard solution. (A–C) Shoot DW (A), shoot Na⁺ content (B) and shoot K⁺ content (C) during the 18 days following the application of NaCl treatment. Open and closed symbols correspond to wild-type and sas2-1 plants, respectively. Squares, circles and triangles correspond to plants grown on media supplemented with 0, 25 and 50 mM NaCl, respectively. Means ± SE, n = 10. (D) sas2-1 (top) and wild-type (bottom) rosettes after 17 days of growth on the standard culture medium supplemented with 0 (left) or 50 (right) mM NaCl. Scale bars = 1 cm.