Genes for sodium exclusion in wheat.

Abstract

Salinity stress limits the growth and productivity of agricultural crops in many regions of the world. Whole plant tolerance to soil salinity involves numerous processes in many different tissues and cell types. For many cereals, sensitivity to salinity is due to the accumulation of sodium (Na⁺) to toxic concentrations in the leaves. This thesis investigates a mechanism of control of Na⁺ accumulation in leaves of wheat. Bread wheat excludes sodium from the leaves better than durum wheat. Bread wheat is hexaploid (AABBDD) whereas durum wheat is tetraploid (AABB). The D-genome in bread wheat carries a major locus for sodium exclusion, Kna1, which may contribute to the differences in sodium exclusion between bread wheat and durum wheat. An unusual durum wheat, Line 149, excludes sodium to a similar degree as bread wheat. Line 149 was derived from a cross between a Triticum monococcum (accession C68-101; AA) and a durum wheat (T. turgidum ssp. durum cv. Marrocos; AABB). Line 149 had been found to contain two major genes for sodium exclusion, named Nax1 and Nax2, which appeared to retrieve sodium from the xylem sap in the roots and so prevent it reaching the leaves. Line 149 had been crossed with the durum wheat cv. Tamaroi, which accumulates high concentrations of Na⁺ in the leaves, and near-isogenic single-gene mapping populations had been developed for Nax1 and Nax2. Nax1 had been located on chromosome 2A. The objective of this thesis was to map Nax2 and identify a candidate gene. Nax2 mapped to chromosome 5AL based on linkage to microsatellite markers. A high-affinity potassium (K⁺) transporter (HKT)-like gene, HKT1;5 was considered as a candidate gene for Nax2, based on similarity of the phenotype to a rice orthologue. Sequence information from a wheat HKT1;5-like expressed sequence tag in the public database was used to develop a probe for use in Southern hybridsation. A HKT1;5-like fragment was identified in Line 149 and T. monococcum C68-101, but was absent in Tamaroi. The HKT1;5-like gene, named TmHKT1;5-A, co-segregated with Nax2 in the Nax2 single-gene mapping population. The HKT1;5 probe identified three putative HKT1;5-like genes on the long arm of chromosome 4B, and one HKT1;5-like gene on the long arm of chromosome 4D, in Langdon (T. turgidum ssp. durum) substitution lines, and in Chinese Spring (T. aestivum) ditelomeric lines. No A-genome HKT1;5 like gene was identified in Langdon or Chinese Spring. The D-genome HKT1;5 gene, named TaHKT1;5-D, was found to co-locate with Kna1, the gene for sodium exclusion in bread wheat, in Chinese Spring chromosome 4D deletion lines. Nax2 (TmHKT1;5-A) was found to be homoeologous with the gene for sodium exclusion in bread wheat, Kna1 (TaHKT1;5-D). TmHKT1;5-A and TaHKT1;5-D, and their promoters, were 94% identical, and both were expressed in the roots of wheat plants. This is consistent with the genes being located in the stele of the roots and retrieving Na⁺ from the xylem sap as it flows towards the shoot, and so excluding Na⁺ from the leaves. A marker for TmHKT1;5-A was developed to track this gene in durum wheat breeding programs. A study of the HKT1;5 gene in diploid ancestors of wheat indicated that this gene is present in most Triticum monococcum accessions, some T. boeoticum accessions, but not present in any T. urartu accessions. T. urartu is the likely A genome ancestor of modern wheat. This may explain the absence of HKT1;5 in the A genome of modern wheat. The protein encoded by TaHKT1;5-D transported sodium when expressed in Xenopus laevis oocytes. The inward currents were specific to Na⁺, but at particular mole fractions of Na⁺ and K⁺ outward currents were observed that were consistent with outward K⁺ transport. These data were consistent with the putative physiological function, of retrieving Na⁺ from the xylem sap as it flows to the leaves, and resulting in a net exchange with K⁺. A construct designed to silence the expression of TaHKT1;5-D was introduced to bread wheat cv. Bob White. Nineteen putative transgenic plants were developed. The leaf Na⁺ concentrations and genotype of the T1 individuals were assayed. The data from two of the transgenic plants indicated that TaHKT1;5-D may have been silenced and that this may have lead to the increase in Na⁺ accumulation in the leaves. However, this data is not conclusive at this time. The information gained from this study will assist the introduction of the Na⁺ exclusion trait into current durum cultivars, which are poor at excluding Na⁺ and are salt sensitive. This information will also contribute to the body of knowledge of ion transport in plants and salinity tolerance in wheat.