Molecular and functional characterization of SISPL-CNR in tomato fruit ripening and cell death

SlSPL-CNR, an SBP-box transcription factor (TF) gene residing at the epimutant Colourless non-ripening (Cnr) locus, is involved in tomato ripening. This epimutant provides a unique model to investigate the (epi)genetic basis of fruit ripening. Here we report that SlSPL-CNR is a nucleus-localized protein with a distinct monopartite nuclear localization signal (NLS). It consists of four consecutive residues ‘30KRKR33’ at the N-terminal of the protein. Mutation of the NLS abolishes SlSPL-CNR to localize into nucleus. SlSPL-CNR comprises two zinc-finger motifs (ZFMs) within the C-terminal SBP-box domain. Both ZFMs contribute to zinc-binding activity. SlSPL-CNR can induce cell death in tomato and tobacco. Induction of cell death by SlSPL-CNR is dependent on its nuclear localization. However, the two ZFMs have differential impacts on SlSPL-CNR to induce severe necrosis or mild necrotic ringspot. NLS and ZFM mutants cannot complement Cnr fruits to ripen. SlSPL-CNR interacts with SlSnRK1. Virus-induced SlSnRK1 silencing leads to reduction in expression of ripening-related genes and inhibits ripening in tomato. We conclude that SlSPL-CNR is a multifunctional protein that consists of a distinct monopartite NLS, binds to zinc and interacts with SlSnRK1 to affect cell death and tomato fruit ripening.