The tomato SBP-box protein SlCNR negatively regulates genes involved in fruit cutin deposition

Cutin serves as the foundational structure of the plant cuticle and plays a crucial role in determining fruit development and quality. However, the transcriptional regulation of cutin deposition by fruit-specific transcription factors remains largely unknown. This study delves into the regulatory role of the tomato (Solanum lycopersicum) SBP-box protein Colorless Non-ripening (SlCNR), primarily expressed in fruits, in cutin formation. We found that CRISPR/Cas9-induced slcnr mutants exhibited thicker cuticles and elevated contents in total cutin and cutin monomers compared to wild-type fruits, whereas SlCNR overexpression lines displayed the opposite tendency. Transcriptome-wide RNA sequencing identified differentially expressed genes in SlCNR overexpression fruits. Further validation by gene expression, DNA binding, and transcriptional activity assays revealed that SlCNR directly binds to and represses the transcription of thirteen genes associated with cutin synthesis, export, and assembly, including glycerol-3-phosphate acyltransferase 4/6 (SlGPAT4/6), ATP-binding cassette transporter subfamily G protein 36/42 (SlABCG36/42), and cutin synthase (SlCUS1). In addition, SlCNR directly bound to and repressed the transcription activities of the promoter of NON-RIPENING like-1 (SlNOR-like1), which encodes a positive regulator of cutin deposition. Thus, SlCNR emerged as a negative transcription regulator of cutin content and cuticle thickness, ultimately affecting fruit firmness and cuticle permeability. This study sheds light on the molecular mechanisms governing cutin deposition through transcriptional regulatory networks, highlighting the role of SlCNR as an important player in fruit cuticle development.