Genetic diversity at the Dhn3 locus in Turkish Hordeum spontaneum populations with comparative structural analyses

Abstract

We analysed Hordeum spontaneum accessions from 21 different locations to understand the genetic diversity of HsDhn3 alleles and effects of single base mutations on the intrinsically disordered structure of the resulting polypeptide (HsDHN3). HsDHN3 was found to be YSK2-type with a low-frequency 6-aa deletion in the beginning of Exon 1. There is relatively high diversity in the intron region of HsDhn3 compared to the two exon regions. We have found subtle differences in K segments led to changes in amino acids chemical properties. Predictions for protein interaction profiles suggest the presence of a protein-binding site in HsDHN3 that coincides with the K1 segment. Comparison of DHN3 to closely related cereals showed that all of them contain a nuclear localization signal sequence flanking to the K1 segment and a novel conserved region located between the S and K1 segments [E(D/T)DGMGGR]. We found that H. vulgare, H. spontaneum, and Triticum urartu DHN3s have a greater number of phosphorylation sites for protein kinase C than other cereal species, which may be related to stress adaptation. Our results show that the nature and extent of mutations in the conserved segments of K1 and K2 are likely to be key factors in protection of cells.

Figure 1. General structure of the Dhn3 locus in H. vulgare (Choi et al. 1999) that is conserved in H. spontaneum.

Arrows show the location of primers used to amplify Dhn3 alleles in this study.

Figure 2. Multiple sequence alignment of the deduced amino acid sequences of the DHN3 proteins from H. spontaneum (9 haplotypes) and two H. vulgare genotypes (cvs TK157/37 and Dicktoo).

The conserved segments (Y-, S-, and K-segments) are shown in yellow shade. The NLS segments are denoted in red and framed by a black line. The PKC phosphorylation sites are in boldface letters and the CK2 sites are underlined. The SNPs are shown in purple. The intron position is indicated by an arrow. Asterisks (*) indicates fully conserved residues, while colons (:) and periods (.) indicate less conserved residues.

Figure 3. Structural characteristics of DHN3 in H. spontaneum genotyes and H. vulgare cvs. TK157/37 and Dicktoo.

(A) Hydrophobicity values according to Kyle-Doolittle (1982). (B) Disorder probability predicted by DISOclust via the IntFOLD server. (C) Probability of protein binding amino acids predicted using DISOPRED3.

Figure 4. Multiple alignments of the predicted amino acid sequences of the DHN3 protein from H. spontaneum (consensus sequence), along with the ortholog proteins in other cereal species.

The conserved segments are shown in yellow shade. The NLS segments are denoted in red and framed by a black line. A novel conserved sequence is shown in blue shade. Amino acid substitutions in conserved regions (Y-, S- , and K-segments) are denoted in blue. Asterisks (*) indicates fully conserved residues, while colons (:) and periods (.) indicate less conserved residues.