AtHKT1 is a salt tolerance determinant that controls Na(+) entry into plant roots

Abstract

Two Arabidopsis thaliana extragenic mutations that suppress NaCl hypersensitivity of the sos3-1 mutant were identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1 sos3-1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated that sos3-1 hkt1-1 and sos3-1 hkt1-2 plants have allelic mutations in AtHKT1. AtHKT1 mRNA is more abundant in roots than shoots of wild-type plants but is not detected in plants of either mutant, indicating that this gene is inactivated by the mutations. hkt1-1 and hkt1-2 mutations can suppress to an equivalent extent the Na(+) sensitivity of sos3-1 seedlings and reduce the intracellular accumulation of this cytotoxic ion. Moreover, sos3-1 hkt1-1 and sos3-1 hkt1-2 seedlings are able to maintain [K(+)](int) in medium supplemented with NaCl and exhibit a substantially higher intracellular ratio of K(+)/Na(+) than the sos3-1 mutant. Furthermore, the hkt1 mutations abrogate the growth inhibition of the sos3-1 mutant that is caused by K(+) deficiency on culture medium with low Ca(2+) (0.15 mM) and <200 microM K(+). Interestingly, the capacity of hkt1 mutations to suppress the Na(+) hypersensitivity of the sos3-1 mutant is reduced substantially when seedlings are grown in medium with low Ca(2+) (0.15 mM). These results indicate that AtHKT1 is a salt tolerance determinant that controls Na(+) entry and high affinity K(+) uptake. The hkt1 mutations have revealed the existence of another Na(+) influx system(s) whose activity is reduced by high [Ca(2+)](ext).

Figure 1

The hkt1–1 and hkt1–2 mutations suppress the NaCl hypersensitive phenotypes of sos3–1. Photographs are of representative Col-0 wild-type (SOS3 HKT1), sos3–1 (sos3–1 HKT1), and sos3–1 hkt1–1 and sos3–1 hkt1–2 T₃ seedlings. Four-day-old seedlings were transferred to fresh medium and illustrated is root growth on medium without (0) or supplemented with 75 mM NaCl after 6 days (A) and shoot growth and anthocyanin accumulation on medium with 75 mM NaCl after 15 days (B).

Figure 2

AtHKT1 transcript abundance is greater in the root (R) than the shoot (S) of Col-0 wild-type plants, and expression is functionally disrupted in sos3–1 hkt1–1 and sos3–1 hkt1–2. Two micrograms of total RNA was isolated from shoots or roots of Col-0 wild-type (SOS3 HKT1), sos3–1 (sos3–1 HKT1), or sos3–1 hkt1–1 or sos3–1 hkt1–2 T₃ plants (3 weeks old) to produce first-strand cDNA. One microliter of this product was used as template for the first PCR amplification (20 cycles). One microliter of the reaction product was used for the second PCR (20 cycles).

Figure 3

The hkt1–1 and hkt1–2 mutations suppress sos3–1 Na⁺ hypersensitivity. Root growth of Col-0 wild-type (□), sos3–1 (○), sos3–1 hkt1–1 (⧫), or sos3–1 hkt1–2 (▴) seedlings was determined after 6 days on basal medium with MS salts and supplemented with NaCl (A) or LiCl (B). Values are the mean ± SE, n = 13.

Figure 4

The hkt1–1 and hkt1–2 mutations suppress the K⁺ deficiency phenotype of sos3–1. Root growth of Col-0 wild-type (□), sos3–1 (○), sos3–1 hkt1–1 (⧫), or sos3–1 hkt1–2 (▴) seedlings was evaluated 9 days after their transfer directly onto basal medium with MS micronutrients (KI replaced by NaI), 1/20× macronutrients [KNO₃ eliminated and KH₂PO₄ replaced by (NH₄)₂HPO₄] and supplemented with various amounts of KCl. Illustrated are the mean values ± SE, n = 30.

Figure 5

Suppression of sos3–1 Na⁺ hypersensitivity by hkt1 mutations is Ca²⁺-dependent. Root growth of Col-0 wild-type (□), sos3–1 (○), sos3–1 hkt1–1 (⧫), or sos3–1 hkt1–2 (▴) seedlings was evaluated 9 days after their transfer directly onto basal medium with MS micronutrients and macronutrients with 0 or various amounts of CaCl₂. Data are for seedling root growth on medium supplemented with 50 mM (A) or 120 mM (B) NaCl; mean values ± SE, n = 15.

Figure 6

sos3–1 hkt1–1 accumulates less Na⁺ and maintains intracellular K⁺ relative to sos3–1 in medium supplemented with NaCl. Col-0 wild-type (SOS3 HKT1) or sos3–1 or sos3–1 hkt1–1 seedlings (3 weeks old) were transferred to liquid medium and grown for 2 days. An aliquot of 5 M NaCl stock solution was then added into the flask to supplement the medium with 100 mM NaCl. An equivalent volume of H₂O was added to cultures that were not supplemented with NaCl. Illustrated are the mean values ± SE (n = 3) for Na⁺ (A) or K⁺ (B) content as determined by atomic absorption spectrophotometry or for intracellular K⁺/Na⁺ ratio calculated from the data in A and B (C). Data from one of two experiments with similar results are shown.