Genome-wide identification and expression pattern analysis of HD-Zip Ⅰ transcription factor family in loquat

【Objective】Homologous structural domain-leucine zip (HD-Zip) transcription factors are involved in a variety of plant abiotic stress response processes. However, the HD-Zip I gene family has not been identified in loquat.【Methods】A genome-wide identification and analysis of the loquat HDZip Ⅰ transcription factor were carried out using bioinformatic methods for identification. The expression patterns of HD-Zip I family members in different tissues and by various drought treatments were examined by qPCR.【Results】A total of 20 putative loquat HD-Zip I family members were identified by searching the Big Seven Stars loquat genome database. The HD-Zip I members were further named EjHB1- EjHB20 according to their positions on 10 different chromosomes. We performed covariance analysis within the loquat genome and found 25 duplicate gene pairs in the HD-Zip I family, including 3 tandem duplicate gene pairs and 22 fragment duplicate gene pairs. The nucleotide sequence identity of the HD-Zip I duplicate pairs ranged from 42.04% to 93.71%, and the Ka/Ks ratios ranged from 0.08to 0.43. To further investigate the phylogenetic relationships among HD-Zip I family members in different plant species, phylogenetic trees were constructed for HD-Zip I protein sequences in loquat, apple, Arabidopsis thaliana and rice. The HD-Zip I proteins were classified into nine clades, namely α, β1, β2, γ, δ, ε, φ1, φ2 and ζ. Among them, the φ1 and ζ clades contained only the family members of Arabidopsis thaliana and rice, respectively, and were not clustered with the HD-Zip I genes of apple and loquat. The members of the loquat HD-Zip I clades clustered closer to the apple homologues and further away from the rice homologues. In addition, loquat, apple, Arabidopsis and rice had the most members in the α clade, followed by the γ clade. Multiple sequence comparison of 20 loquat HD-Zip I proteins using DNAMAN software revealed that all HD-Zip I proteins had HD and Zip conserved structural domains. To further investigate the relationship between loquat HD-Zip I proteins, we constructed a phylogenetic tree of all loquat HD-Zip I protein sequences and analysed their gene structures and motifs. Similar to the results of the phylogenetic analysis described above, the loquat HD-Zip I gene family was divided into seven clades: α, β1, β2, γ, δ, ε and φ2. Since the intron-exon structure of genes played a crucial role in the evolution of multigene families, we examined the intron-exon structures of 20 loquat HD-Zip I genes to better understand their structural evolution. The γ clade members had one intron, the β1 clade members had three introns, and the other branch members contained two introns. Combined with phylogenetic analysis, we found that genes in the same branch had similar intron-exon structures, whereas the intron-exon structures of different branches differed. To gain insight into the differences and functions of the loquat HD-Zip I protein, we used the MEME programme to identify its motifs. We identified 10 motifs ranging from 20 to 50 residues in length. All predicted motifs were identified only once in each HD-Zip I protein. Except for motif 1, which was present in all HD-Zip I proteins, the remaining nine motifs were only present in certain branches. Tissue expression analysis showed that HD- Zip Ⅰ was found in loquat roots, stems, leaves, flowers and fruits. The results showed that EjHB3, EjHB6, EjHB8, EjHB15 and EjHB20 were mainly expressed in leaves, and EjHB9, EjHB16 and EjHB18 were mainly expressed in roots. Most members had high expression levels in stems and low expression levels in fruits. In addition, EjHB11, EjHB12 and EjHB13 were expressed at higher levels in flowers than in other tissues, while other members were also generally expressed at lower levels in flowers. Cis-acting element analysis revealed that most HD-Zip I promoters contained ABRE elements, which were normally involved in ABA- related responses. And HD- Zip I promoters contained drought- inducible elements (MBS), as well as defence and stress- responsive elements (TC- rich repeats). In addition, there were a number of cis-elements associated with stress response and stress- related hormone signalling, such as MYB, MYC, SA and MeJA. The HD- Zip I family contained cis- acting elements associated with drought stress. To identify the role of HD-Zip I in the regulation of drought tolerance in loquat, we analysed the expression of 20 HD-Zip I genes under drought stress. It was shown that the expression levels of EjHB9, EjHB10, EjHB17 and EjHB18 in the γ-clade and EjHB20 in the ε- branch significantly increased after drought treatment, whereas EjHB2 and EjHB19 in the β2-branch were significantly downregulated by drought.【Conclusion】In this study, 20 members of the HD-Zip I transcription factor family were identified from the complete loquat protein sequence, and promoter prediction analysis indicated that they responded to drought stress. Expression analysis after drought treatment also confirmed that loquat HD-Zip I transcription factors may play an important role in drought stress response. The present study may provide a reference for the future analysis of the mechanism of loquat HD- Zip I genes and the development of drought-resistant breeding in loquat.