Genome-wide identification of five fern bHLH families and functional analysis of bHLHs in lignin biosynthesis in Alsophila spinulosa.

Background: The basic helix-loop-helix (bHLH) transcription factors are involved in the biosynthesis of various secondary metabolites. However, genome-wide studies on the bHLH gene family in ferns and their role in lignin biosynthesis remain limited. As the second largest group of vascular plants, ferns are of significant interest for understanding plant evolution and secondary metabolism. Among ferns, Alsophila spinulosa stands out as one of the few tree ferns with a distinctive trunk structure. Investigating the genes potentially regulating lignin biosynthesis in A. spinulosa offers valuable insights into the growth and development mechanisms of its trunk, which is pivotal for the overall architecture and function of the plant.

Results: In this study, we conducted a systematic study of bHLH gene families in five ferns, including 186 in A. spinulosa, 130 in A. capillus, 107 in A. filiculoides, 71 in S. cucullata, and 67 in C. richardii. Based on phylogenetic analysis, all bHLH genes were classified into 28 subgroups. The number of bHLH members in different ferns was closely related to their growth patterns and life habits, with the number in tree ferns being much larger than in other ferns. In addition, we identified tandem duplication in C. richardii and A. capillus as a key driver of their bHLH gene diversity, whereas in A. spinulosa, segmental duplication contributed more to gene expansion and evolution. Most of the bHLH genes in ferns are in a state of purifying selection. Additionally, tissue-specific expression patterns of AspbHLH genes suggest diverse functional roles in plant growth, development, and metabolite synthesis. We further focused on three genes, AspbHLH80, AspbHLH120, and AspbHLH185, which are specifically highly expressed in xylem. Results from weighted gene co-expression network analysis (WGCNA) and downstream target gene prediction indicate their potential regulatory roles in lignin biosynthesis.

Conclusion: This study presents a comprehensive genomic analysis of the bHLH gene family in five fern species. We found a strong correlation between bHLH gene number and fern growth morphology, with tree ferns exhibiting a significantly higher number of bHLH genes. Tandem duplications were key to bHLH gene diversity in C. richardii, A. capillus, and A. spinulosa, while segmental duplications contributed more to bHLH gene expansion in A. spinulosa. Evolutionary analysis indicated most fern bHLH genes are under purifying selection. Tissue-specific expression patterns of AspbHLH genes suggest roles in growth, development, and secondary metabolism. Furthermore, WGCNA and target gene predictions highlight three genes (AspbHLH80, AspbHLH120, and AspbHLH185) potentially involved in lignin biosynthesis. Overall, this work provides key insights into the mechanisms of wood formation in ferns and advances our understanding of plant secondary metabolism.