Abstract
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-terminus of the protein. Mutation of the NLS abolishes SlSPL-CNR’s ability to localize in the 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, dependent on its nuclear localization. However, the two ZFMs have differential impacts on SlSPL-CNR’s induction of 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.
Highlights
Colourless non-ripening (Cnr) is a naturally occurring epimutant of tomato
potato virus X (PVX) infection of AC leaf tissues was further evidenced by immunodetection of viral coat protein (CP; Fig. 1D).These data demonstrate that the PVX-based transient gene expression system was effective to express SlSPL-CNR:green fluorescent protein (GFP) in tomato cells, and that SlSPL-CNR is a nucleus-localized protein
AC fruits treated with PVX/SlSPL-CNR (Fig. 1G) that are expected to express free SlSPL-CNR protein with full functionality developed necrotic cell death (Fig. 1H–J), whilst control AC fruits treated with PVX/GFP remained normal (Fig. 1K)
Summary
Cnr is a naturally occurring epimutant of tomato. SlSPL-CNR is developmentally regulated, being mainly expressed in ripening fruits (Manning et al, 2006; Salinas et al, 2012), with its expression fine-tuned by SlymiR157 (SlmiR157) to affect fruit ripening (Chen et al, 2015a). SlCMT2, SlCMT3, SlDRM7, and SlMET1, which are key genes in the RNA-directed DNA methylation and methylation maintenance pathways, are required to maintain the Cnr epiallele. Inhibition of these genes by virus-induced gene silencing (VIGS) results in ripening reversion in Cnr fruits (Chen et al, 2015b). Ailsa Craig, AC) to phenocopy the physical, physiological, biochemical, and molecular characteristics of Cnr fruits (Lai et al, 2015)
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