A SOS3 homologue maps to HvNax4, a barley locus controlling an environmentally sensitive Na+ exclusion trait

Abstract

Genes that enable crops to limit Na(+) accumulation in shoot tissues represent potential sources of salinity tolerance for breeding. In barley, the HvNax4 locus lowered shoot Na(+) content by between 12% and 59% (g(-1) DW), or not at all, depending on the growth conditions in hydroponics and a range of soil types, indicating a strong influence of environment on expression. HvNax4 was fine-mapped on the long arm of barley chromosome 1H. Corresponding intervals of ∼200 kb, containing a total of 34 predicted genes, were defined in the sequenced rice and Brachypodium genomes. HvCBL4, a close barley homologue of the SOS3 salinity tolerance gene of Arabidopsis, co-segregated with HvNax4. No difference in HvCBL4 mRNA expression was detected between the mapping parents. However, genomic and cDNA sequences of the HvCBL4 alleles were obtained, revealing a single Ala111Thr amino acid substitution difference in the encoded proteins. The known crystal structure of SOS3 was used as a template to obtain molecular models of the barley proteins, resulting in structures very similar to that of SOS3. The position in SOS3 corresponding to the barley substitution does not participate directly in Ca(2+) binding, post-translational modifications or interaction with the SOS2 signalling partner. However, Thr111 but not Ala111 forms a predicted hydrogen bond with a neighbouring α-helix, which has potential implications for the overall structure and function of the barley protein. HvCBL4 therefore represents a candidate for HvNax4 that warrants further investigation.

Fig. 1.

Frequency distributions for shoot [Na⁺], in Clipper×Sahara 3771 DH lines carrying the Clipper (black) or Sahara 3771 (grey) alleles of HvNax4, in experiment 1 (A), 2 (B), 3 (C), and 4 (D).

Fig. 2.

Rice–barley comparative map. Lines connect putatively orthologous genes in rice and barley, with colour/shading indicating chromosomal segments separated by ancestral rice–barley inversion breakpoints. Sequences of markers on the right showed strong similarity to rice chromosomes other than chromosome 5, except for the wheat genomic RFLP probe WG184, for which there was no significant match in rice. BCD808=BCD265 and ABC261=AWBMA4 represent homologous RFLP probes. Positions on rice chromosome 5 refer to the sequence NC_008398.1. The bracket on the left indicates the deduced rice HvNax4 interval.

Fig. 3.

Mapping with DH lines. Recombinants for the HvNax4 interval were assigned HvNax4 genotypes, based on the [Na⁺] ranges for line carrying Clipper (A) or Sahara 3771 (B) alleles defined by the frequency distributions in Fig. 1. Shading indicates [Na⁺] values falling within either of the designated ranges (bottom right). Line 46 likely carries the Clipper allele because it was designated Clipper in two out of the three experiments and in the experiments that gave the clearest definition (1 and 2).

Fig. 4.

Mapping with recombinant DNA83×DNA89 F₂ plants and their F₃ progeny. Markers on the left were scored in the F₂ recombinants, which defined the illustrated chromosome types (black=Clipper derived; white=Sahara 3771 derived). F₃ plants from each family were grown in soil, scored individually for marker ABC257 (families 6, 19, 457, 578, 796, and 885), BCD304 (families 6, 19, 26, 135, 601, and 1472) or cMWG733 (family 990), and shoot [Na⁺]. For each F₃ family, mean [Na⁺] for the two homozygous classes (outer numbers) and heterozygotes (middle number) are presented, together with the family standard error of the means (SEM). Within each family, means that were not significantly different at P=0.05 are denoted by the same letters. Inferred HvNax4 position relative to each recombination point is indicated by an arrow. In control families 6 and 19, individuals found to be recombinant between ABC257 and BCD304 were excluded from the analysis. Analysis of each F₃ family was based on 5–16 plants of each homozygote class and 7–24 plants of each heterozygous class.

Fig. 5.

Alignment of barley HvCBL4 proteins from Clipper (c) and Sahara 3771 (s), maize ZmCBL4 (Wang et al., 2007), rice OsCBL4, OsCBL7, and OsCBL8 (Hwang et al., 2005) and Arabidopsis AtCBL4 (SOS3). Dashes represent gaps introduced into the alignment. Shading indicates >50% amino acid sequence identity (black) or similarity (grey). EF hand motifs are boxed and calcium binding residues marked with asterisks (X, Y, Z, –Y, –X, –Z residues, respectively). Residues with predicted N-myristoylation or S-acylation are indicated by green and red arrows, respectively. The single amino acid residue difference between HvCBL4c and HvCBL4s is marked by the green triangle.

Fig. 6.

3D structure of SOS3 and molecular models of HvCBL4. (A), Stereoview of ribbon representations of the superposed Arabidopsis SOS3 structure (green) and the Clipper HvCBL4 model (steel blue), showing the disposition of secondary structure elements. The four bound Ca²⁺ ions are shown as yellow spheres. Side chains of Val111 (SOS3) and Ala111 (Clipper HvCBL4) are shown in sticks and are arrowed. (B), Stereoview of partial ribbon representations of the superimposed Clipper (steel blue) and Sahara 3771 (pink) HvCBL4 models, detailing the region containing the Ala111Thr substitution. It is an enlargement of the boxed section in (A) and is rotated 45° North with respect to (A). Amino acid residue Thr111 (cpk green colour) in Sahara 3771 forms a hydrogen bond with Trp163 (dashed line). Side chains of residues surrounding the polymorphic site are included to indicate local environment. The last 12 (C-terminal) residues are omitted. (C), Surface representations of SOS3 (left) and the Clipper HvCBL4 model (right), rotated by about 90° North and 90° clockwise with respect to (A). Arrows (and grey) highlight residues 110–112 of HvCBL4 containing the polymorphic site, and the corresponding residues in SOS3. Yellow indicates EF hand domains, cyan and green-cyan indicate the two sections of the molecule that would move apart to expose a hydrophobic SOS2-binding crevice, magenta indicates the 12 C-terminal residues, and orange indicates the remainder of the surface. The dimerization surface in each protein is dotted.

Fig. 7.

HvCBL4 transcript analysis. Quantitative RT-PCR was used to monitor HvCBL4 transcript abundance in roots of BC₁F₂ derived lines, homozygous for HvNax4 alleles from Clipper (five lines) or Sahara 3771 (four lines), grown in supported hydroponics containing 150 mM NaCl. Means ±SEM are shown, with individual BC₁F₂ derived lines being treated as replicates for each allele and time point.