The regulatory module consisting of KNOTTED1-like homeobox 6, Homeobox-leucine zipper 14, and BLADE-ON-PETIOLE 2 is involved in shoot development and lateral branching in citrus

Abstract

KNOTTED1-like homeobox (KNOX) family transcription factors (TFs) are important regulatory factors in plant growth and development, participating in various aspects of developmental regulation. In this study, we demonstrate that the citrus KNOX gene 6 (CiKN6) regulates shoot branch development. Overexpression of CiKN6 in citrus plants resulted in a significant phenotype characterized by increased branching and shortened shoots. Expression analysis revealed that CiKN6 was predominantly expressed in the stems. Protein interaction studies showed that CiKN6 interacted with Homeobox-leucine zipper 14 (CiHOX14). Furthermore, we discovered that CiHOX14 specifically binds to the BLADE-ON-PETIOLE 2 (CiBOP2) promoter and enhances its expression. Overexpression of CiBOP2 led to increased branching and shortened shoots in Arabidopsis and citrus, while suppression of CiBOP2 expression resulted in increased plant height in citrus. Additionally, we identified a TEOSINTE BRANCHED1/CYCLOIDEA/PCF TF, CiTCP15, that promotes CiBOP2 expression by binding to its promoter. Lastly, qRT-PCR assays demonstrate that the expression of CiKN6 and CiHOX14 was upregulated by CiBOP2 through feedback regulation. These findings suggest that the CiKN6-CiHOX14 complex, along with CiBOP2, interact to regulate shoot development and branching in citrus, providing new insights into the roles of homeobox proteins in citrus.