Duplication of the class I cytosolic small heat shock protein gene and potential functional divergence revealed by sequence variations flanking the alpha-crystallin domain in the genus Rhododendron (Ericaceae)

Positive selection in the alpha-crystallin domain (ACD) of the chloroplast small heat shock protein (CPsHSP) gene was found in a previous study and was suggested to be related to the ecological adaptation of Rhododendron species in the subgenus Hymenanthes. Consequently, it was of interest to examine whether gene duplication and subsequent divergence have occurred in other sHSP genes, for example class I cytosolic sHSP genes (CT1sHSPs) in Rhododendron in Taiwan, where many endemic species have evolved as a result of habitat differentiation. A phylogeny of CT1sHSP amino acid sequences was built from Rhododendron, Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Vitis vinifera and other species for elucidation of the phylogenetic relationships among CT1sHSPs. Phylogenies of Rhododendron CT1sHSP nucleotide and amino acid sequences were generated for positive selection and functional divergence analysis, respectively. Positively selected sites and amino acid differences between types of Rhododendron CT1sHSPs were mapped onto the wheat sHSP16 center dot 9 protein structure. Average genetic distance (D-xy) and d(N)/d(S) ratios between types of Rhododendron CT1sHSP genes were analysed using sliding window analysis. Gene conversion was also assessed between types of Rhododendron CT1sHSPs. Two types of Rhododendron CT1sHSP were identified. A high level of genetic similarity and diversity within and flanking the ACD, respectively, between types of Rhododendron CT1sHSP were found. Main differences between the two types of Rhododendron CT1sHSPs were: (1) increased hydrophobicity by two positively selected amino acid sites and a seven-amino-acid insertion in the N-terminal arm; and (2) increased structural flexibility and solubility by a seven-amino-acid insertion in the N-terminal arm and one positively selected amino acid site in the C-terminal extension. Functional conservation of the ACD of Rhododendron CT1sHSP genes was inferred because of strong purifying selection. However, sequence variations flanking the ACD in Rhododendron CT1sHSP gene duplicates may have resulted in functional divergence and played important roles in chaperon function enhancement.