CATION EXCHANGER1 Cosegregates with Cadmium Tolerance in the Metal Hyperaccumulator Arabidopsis halleri and Plays a Role in Limiting Oxidative Stress in Arabidopsis Spp

Abstract

Arabidopsis halleri is a model species for the study of plant adaptation to extreme metallic conditions. In this species, cadmium (Cd) tolerance seems to be constitutive, and the mechanisms underlying the trait are still poorly understood. A previous quantitative trait loci (QTL) analysis performed on A. halleri × Arabidopsis lyrata backcross population1 identified the metal-pump gene Heavy Metal ATPase4 as the major genetic determinant for Cd tolerance. However, although necessary, Heavy Metal ATPase4 alone is not sufficient for determining this trait. After fine mapping, a gene encoding a calcium(2+)/hydrogen(+) antiporter, cation/hydrogen(+) exchanger1 (CAX1), was identified as a candidate gene for the second QTL of Cd tolerance in A. halleri. Backcross population1 individuals displaying the A. halleri allele for the CAX1 locus exhibited significantly higher CAX1 expression levels compared with the ones with the A. lyrata allele, and a positive correlation between CAX1 expression and Cd tolerance was observed. Here, we show that this QTL is conditional and that it is only detectable at low external Ca concentration. CAX1 expression in both roots and shoots was higher in A. halleri than in the close Cd-sensitive relative species A. lyrata and Arabidopsis thaliana. Moreover, CAX1 loss of function in A. thaliana led to higher Cd sensitivity at low concentration of Ca, higher sensitivity to methylviologen, and stronger accumulation of reactive oxygen species after Cd treatment. Overall, this study identifies a unique genetic determinant of Cd tolerance in the metal hyperaccumulator A. halleri and offers a new twist for the function of CAX1 in plants.

Figure 1.

Identification of CAX1 as a candidate gene for the QTL CdTol2. A, Densification of the QTL CdTol2. Five additional markers (red) were used to confirm the localization and reduce the CdTol2 locus to a 3-centiMorgan region [for marker descriptions, see Supplemental Table S3; all other markers are defined in Courbot et al. (2007)]. The vertical dashed line represents the LOD score threshold (1.5) for QTL detection at an error level of α = 0.05. Bars indicate the one-LOD (10-fold) support interval, and whiskers (lines extending beyond bars) indicate the two-LOD (100-fold) support intervals. BC1 individuals were phenotyped at 0.5 mm CaNO₃ in the study by Courbot et al. (2007). B, CAX1 transcript levels in BC1 individuals displaying the Ah/Alp (h) or Alp/Alp (a) allelic combinations at the CdTol2 locus. At least three cuttings for each BC1 genotype were transferred in hydroponic solution according to the work by Courbot et al. (2007) for 4 weeks. Clones were pooled together, and CAX1 expression levels were assessed through qPCR analysis. Data are means ± sd (n = 9). *, P < 0.05 (Student’s t test).

Figure 2.

Expression analysis of CAX1. CAX1 transcript levels at low (A) and moderate (B) Ca concentration in control condition (gray bars) and upon 72 h of 10 µm CdSO₄ treatment (white bars) of A. halleri, A. lyrata ssp. petraea, and A. thaliana. Data are means ± sd (n = 8–12). Lowercase letters are for comparison between conditions in the same species, and capital letters are for comparison between species in the same condition (Kruskall Wallis test). C, Western blot showing relative levels of CAX1 protein in root from A. halleri, A. lyrata, and A. thaliana. Samples have been collected from plants grown at low-Ca concentration in control condition (lanes 1, 3, and 5) or upon 72 h of 10 µm CdSO₄ treatment (lanes 2, 4 and 6). As negative control, we loaded total protein fraction extracted from A. thaliana cax1-1 mutant (lane 7). Equal amounts of total protein (35 ng) were loaded onto the gel.

Figure 3.

QTL mapping of Cd tolerance in the CdTol2 region at two different Ca concentrations. Red line, LOD score obtained with 125 BC1 individuals phenotyped for Cd tolerance at 2 mm CaNO₃ and genotyped for six markers in the CdTol2 region. Green line, LOD score obtained with the phenotyping at 0.5 mm CaNO₃ (Courbot et al., 2007) and the six markers. The vertical dashed line represents the LOD score threshold for QTL detection at an error level of α = 0.05. LG4, LINKAGE GROUP4.

Figure 4.

Analysis of Cd tolerance in the A. thaliana wild type and cax1-1 mutant. Comparison of Cd tolerance between the A. thaliana wild type (Col-0; gray bars) and cax1-1 mutant (white bars) was based on primary root length (A) and total shoot surface (C). Plants were sown in vitro on one-half-strength MS containing 1% Suc solidified with agar. After 1 week, plantlets were transferred to one-half-strength MS agar plates containing low (0.5 mm) or moderate (2 mm) Ca concentrations and 0 or 100 µm CdCl₂ for 1 week. Data are means ± sd (n = 58–92 for primary root length measurements and n = 25–47 for shoot surface analysis). *, P < 0.05 (Student’s t test). The representative phenotypes for root (B) and shoot (D) at low Ca are reported. Bars = 2 cm (B; roots) and 1 cm (D; shoots).

Figure 5.

Cd tolerance assessed in A. thaliana k.o. mutant cax3-1 at low Ca concentration. Comparison of Cd tolerance was performed between the A. thaliana wild type (WT; gray bars), cax1-1 (white bars), and cax3-1 (striped bars). Tolerance was assessed in vitro by measuring primary root length as a parameter for growth. One week after germination, plantlets were transferred at low (0.5 mm) Ca concentration in control condition or with 100 µm CdSO₄ for another 1 week. Data are means ± sd (n = 15–27). *, P < 0.05 (Student’s t test).

Figure 6.

H₂O₂ detection and quantification in DAB-stained samples. A, A. thaliana (the wild type and cax1-1), A. halleri, and A. lyrata roots were stained with DAB reagent in control condition (upper) or after 72 h at 10 µm CdSO₄ treatment (lower) at low Ca. Polymerization of DAB is visible as a brown precipitate in the presence of H₂O₂. Bars = 50 µm. B, The amount of H₂O₂ was measured in roots of the A. thaliana wild type (WT; gray bars), cax1-1 (white bars), A. halleri (black bars), and A. lyrata (checked bars). H₂O₂-DAB content in the samples was determined using a standard curve prepared with known amounts of DAB. Plants were grown in hydroponic solution with low or moderate Ca contaminated for 72 h with 0 or 10 µm CdSO₄. To obtain sufficient material for H₂O₂ quantification, roots (n = 3) were pooled before measurements.

Figure 7.

In vitro tolerance to ROS-inducing reagent MV. Comparison between the A. thaliana wild type (Col-0; gray bars) and cax1-1 mutant (white bars) was based on primary root length (A) and total shoot surface (B). Plants were grown at low (0.5 mm) and moderate (2 mm) Ca concentrations in control condition or with 0.1 µm MV. Data are means ± sd (n = 20–30). *, P < 0.05 (Student’s t test). Pictures of A. thaliana wild-type and cax1-1 roots and shoots are reported in C and D, respectively. Bars = 2 cm.