Class II knotted‐like homeodomain protein SlKN5 with BEL1‐like homeodomain proteins suppresses fruit greening in tomato fruit

Abstract

SUMMARYKnotted1‐like homeodomain (KNOX) proteins are essential in regulating plant organ differentiation. Land plants, including tomato (Solanum lycopersicum), have two classes of the KNOX protein family, namely, class I (KNOX I) and class II KNOX (KNOX II). While tomato KNOX I proteins are known to stimulate chloroplast development in fruit, affecting fruit coloration, the role of KNOX II proteins in this context remains unclear. In this study, we employ CRISPR/Cas9 to generate knockout mutants of the KNOX II member, SlKN5. These mutants display increased leaf complexity, a phenotype commonly associated with reduced KNOX II activity, as well as enhanced accumulation of chloroplasts and chlorophylls in smaller cells within young, unripe fruit. RNA‐seq data analyses indicate that SlKN5 suppresses the transcriptions of genes involved in chloroplast biogenesis, chlorophyll biosynthesis, and gibberellin catabolism. Furthermore, protein–protein interaction assays reveal that SlKN5 physically interacts with three transcriptional repressors from the BLH1‐clade of BEL1‐like homeodomain (BLH) protein family, SlBLH4, SlBLH5, and SlBLH7, with SlBLH7 showing the strongest interaction. CRISPR/Cas9‐mediated knockout of these SlBLH genes confirmed their overlapping roles in suppressing chloroplast biogenesis, chlorophyll biosynthesis, and lycopene cyclization. Transient assays further demonstrate that the SlKN5–SlBLH7 interaction enhances binding capacity to regulatory regions of key chloroplast‐ and chlorophyll‐related genes, including SlAPRR2‐like1, SlCAB‐1C, and SlGUN4. Collectively, our findings elucidate that the KNOX II SlKN5‐SlBLH regulatory modules serve to inhibit fruit greening and subsequently promote lycopene accumulation, thereby fine‐tuning the color transition from immature green fruit to mature red fruit.