Abstract
BackgroundA member of the TaHKT2;1 multigene family was previously identified as a Na+ transporter with a possible role in root Na+ uptake. In the present study, the existing full-length cDNA of this member was used as a basis to query the International Wheat Genome Survey Sequence to identify all members of the TaHKT2;1 family. Individual TaHKT2;1 genes were subsequently studied for gene and predicted protein structures, promoter variability, tissue expression and their role in Na+ and K+ status of wheat.ResultsSix TaHKT2;1 genes were characterized which included four functional genes (TaHKT2;1 7AL-1, TaHKT2;1 7BL-1, TaHKT2;1 7BL-2 and TaHKT2;1 7DL-1) and two pseudogenes (TaHKT2;1 7AL-2 and TaHKT2;1 7AL-3), on chromosomes 7A, 7B and 7D of hexaploid wheat. Variability in protein domains for cation specificity and in cis-regulatory elements for salt response in gene promoters, were identified amongst the functional TaHKT2;1 members. The functional genes were expressed under low and high NaCl conditions in roots and leaf sheaths, but were down regulated in leaf blades. Alternative splicing events were evident in TaHKT2;1 7AL-1. Aneuploid lines null for each functional gene were grown in high NaCl nutrient solution culture to identify potential role of each TaHKT2;1 member. Aneuploid lines null for TaHKT2;1 7AL-1, TaHKT2;1 7BL-1 and TaHKT2;1 7BL-2 showed no difference in Na+ concentration between Chinese Spring except for higher Na+ in sheaths. The same aneuploid lines had lower K+ in roots, sheath and youngest fully expanded leaf but only under high (200 mM) NaCl in the external solution. There was no difference in Na+ or K+ concentration for any treatment between aneuploid line null for the TaHKT2;1 7DL-1 gene and Chinese Spring.ConclusionsTaHKT2;1 is a complex family consisting of pseudogenes and functional members. TaHKT2;1 genes do not have an apparent role in controlling root Na+ uptake in bread wheat seedlings under experimental conditions in this study, contrary to existing hypotheses. However, TaHKT2;1 genes or, indeed other genes in the same chromosome region on 7AL, are candidates that may control Na+ transport from root to sheath and regulate K+ levels in different plant tissues.
Highlights
Among the genes encoding candidate proteins involved in root Na+ influx, TaHKT2;1 is of particular interest in crop breeding because of its high specificity for Na+ and the potential to manipulate TaHKT2;1 genes without largely interfering with the homeostasis of nutritionally important ions (e.g., K+, Ca2+) in plants
Six significant hits having greater than 90% nucleotide identity with the full length cDNA (FL-cDNA) were retrieved that identified three TaHKT2;1 genes on the long arm of chromosome 7A (TaHKT2;1 7AL-1, TaHKT2;1 7AL-2, TaHKT2;1 7AL-3), two genes on the long arm of 7B (TaHKT2;1 7BL-1, TaHKT2;1 7BL-2), and one on the long arm of 7D (TaHKT2;1 7DL-1)
Gene structure was analysed by splice junction prediction software, and subsequently validated in DNA sequence alignments with the FL-cDNA, U16709, revealed three exons interrupted by two introns for each gene (Figure 1) with intron splice junction sites having the conserved motif, 5′ GT- AG 3′
Summary
Channel and transporter proteins are potential targets to reduce Na+ net uptake by roots and have been a major focus to investigate their role in crop tolerance to salinity. Two transporters LCT1 [12,13] and HKT2;1[14] can mediate high and/or low-affinity Na+ transport in wheat While both VICs/NSCCs [15] and LCT1 [13] facilitate Ca2+ dependant unidirectional influx of various cations, TaHKT2;1 mediates Ca2+ independent influx of Na+ and K+ [16,17]. Among the genes encoding candidate proteins involved in root Na+ influx, TaHKT2;1 is of particular interest in crop breeding because of its high specificity for Na+ and the potential to manipulate TaHKT2;1 genes without largely interfering with the homeostasis of nutritionally important ions (e.g., K+, Ca2+) in plants
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