Genome-wide analysis of Homeobox (HB) gene in sugarcane and functional analysis of ScHB56 in tillering and leaf development

As a major global industrial crop, sugarcane ( Saccharum spp. hybrids) plays a crucial role in agricultural production, where tillering serves as a crucial agronomic trait determining final yield. Plant growth and morphological development are strongly influenced by the regulatory functions of Homeobox ( HB ) genes. However, the biological functions of ScHB family proteins in sugarcane remain largely uncharacterized. In this study, through genome-wide identification and analysis, we identified 66 ScHB genes. Among them, ScHB56 showed a strong association with tillering based on GWAS results. ScHB56 localizes to the nucleus, shows predominant expression in mature stems, and is up-regulated by auxin (IAA) and 6-BA treatments. Overexpression of ScHB56 in Arabidopsis significantly increased the branch number of transgenic plants and caused irregular leaf margin curling and abnormal leaf area variations. In ScHB56 overexpressing Arabidopsis, differentially expressed genes (DEGs) related to the IAA pathway were identified, with ScGH3 being significantly upregulated and ScARF , ScIAA19 , and ScSAUR27 markedly downregulated. Additionally, Y2H screening revealed interactions between ScHB56 and two proteins: RING-H2 finger protein (ScRUG1) and O-methyltransferase ZRP4 (ScZRP4), which were subsequently confirmed by BiFC and LCI assays. Thus, ScHB56 coordinates tillering and leaf development via IAA signaling and protein interactors, offering key targets for sugarcane improvement. • Genome-wide identification of 66 ScHB genes in sugarcane ( Saccharum spp. hybrids), with ScHB56 strongly associated with tillering via GWAS analysis. • ScHB56 exhibits nuclear localization, stem-predominant expression, and hormonal responsiveness (upregulated by auxin/IAA and 6-BA). • ScHB56 overexpression in Arabidopsis enhances branching and induces leaf morphological abnormalities, linking it to developmental regulation. • Transcriptomic analysis reveals ScHB56 modulates auxin signaling, upregulating GH3 while downregulating ARF/IAA19/SAUR27 Protein interaction networks identify ScHB56 partners: ScRUG1 (RING-H2 finger protein) and ScZRP4 (O-methyltransferase), validated by Y2H/BiFC/LCI. • Proposed model: ScHB56 integrates IAA pathway and protein interactors to coordinate tillering and leaf development, providing molecular tools for sugarcane breeding.