Regulation of lignin biosynthesis by an atypical bHLH protein CmHLB in Chrysanthemum

Stem mechanical strength is one of the most important agronomic traits that affects the resistance of plants against insects and lodging, and plays an essential role in the quality and yield of plants. Several transcription factors regulate mechanical strength in crops. However, mechanisms of stem strength formation and regulation remain largely unexplored, especially in ornamental plants. In this study, we identified an atypical bHLH transcription factor CmHLB (HLH PROTEIN INVOLVED IN LIGNIN BIOSYNTHESIS) in chrysanthemum, belonging to a small bHLH sub-family - the PACLOBUTRAZOL RESISTANCE (PRE) family. Overexpression of CmHLB in chrysanthemum significantly increased mechanical strength of the stem, cell wall thickness, and lignin content, compared with the wild type. In contrast, CmHLB RNA interference lines exhibited the opposite phenotypes. RNA-seq analysis indicated that CmHLB promoted the expression of genes involved in lignin biosynthesis. Furthermore, we demonstrated that CmHLB interacted with Chrysanthemum KNOTTED ARABIDOPSIS THALIANA7 (CmKNAT7) through the KNOX2 domain, which has a conserved function, i.e. it negatively regulates secondary cell wall formation of fibres and lignin biosynthesis. Collectively, our results reveal a novel role for CmHLB in regulating lignin biosynthesis by interacting with CmKNAT7 and affecting stem mechanical strength in Chrysanthemum. CmHLB, an atypical bHLH protein, interacts with a class II KNOX transcription factor CmKNAT7 to regulate lignin biosynthesis, which affects stem mechanical strength in Chrysanthemum.