Characterization and expression analysis of bHLH transcription factors reveal their putative regulatory effects on nectar spur development in Aquilegia species

Abstract

Nectar spur is a hollow extension of certain flower parts and shows strikingly diverse size and shape in Aquilegia. Nectar spur development is involved in cell division and expansion processes. The basic helix-loop-helix (bHLH) transcription factors (TFs) control a diversity of organ morphogenesis, including cell division and cell expansion processes. However, the role of bHLH genes in nectar spur development in Aquilegia is mainly unknown. We conducted a genome-wide identification of the bHLH gene family in Aquilegia to determine structural characteristics and phylogenetic relationships, and to analyze expression profiles of these genes during the development of nectar spur in spurless and spurred species. A total of 120 AqbHLH genes were identified from the Aquilegia coerulea genome. The phylogenetic tree showed that AqbHLH proteins were divided into 15 subfamilies, among which S7 and S8 subfamilies occurred marked expansion. The AqbHLH genes in the same clade had similar motif composition and gene structure characteristics. Conserved residue analysis indicated nineteen residues with conservation of more than 50% were found in the four conserved regions. In the upstream sequence of AqbHLH genes, the light-responsive element was the most abundant cis-acting element. Eighteen AqbHLH genes showed syntenic relationships, and eight genes from four syntenic pairs underwent tandem duplications. According to the expression profiling analysis by public RNA-Seq data and qRT-PCR results, five AqbHLH genes, including AqbHLH027, AqbHLH046, AqbHLH082, AqbHLH083 and AqbHLH092, were differentially expressed between different tissues in A. coerulea at early developmental stages, as well as between spurless and spurred Aquilegia species. Of them, AqbHLH046 was not only highly expressed in spur compared with blade, but also showed higher expression levels in spurred species than spurless specie, suggesting it plays an essential role in the development of spur by regulating cell division. This study lays a foundation to investigate the function of AqbHLH genes family in nectar spur development, and has potential implications for speciation and genetic breeding in the genus Aquilegia.