Tomatoes not turning red? Shut down SlERF.F12!

Fleshy fruits are attractive and nutritious for human consumption, not that plants care about that. However, from the plant perspective, the development of juicy, eye-catching fruits is critical for seed dispersal by attracting frugivores, a fundamental dispersal strategy found in many seed plants.

Early studies of ripening regulation focused on the role of ethylene, a gaseous plant hormone with a clear and profound effect on fruit maturation. More recently, our understanding of transcription factors and the role of the epigenome in ripening have progressed rapidly and several candidate “master regulators” or essential ripening regulators have been identified (Giovannoni et al., 2017). However, compared with previous reports that relied on spontaneous mutants in tomato (Solanum lycopersicum), recent studies using CRISPR/Cas9-induced mutations of the same loci revealed relatively milder effects on ripening in general. One model to reconcile these observations is that the spontaneous mutations may represent gain-of-function/dominant-negative alleles, rather than loss-of-function alleles (Wang et al., 2020). Further, the underlying regulatory network defined by these genes is not well understood, arguing for their specific roles and mode of action to be revisited. Focusing on the Ethylene Response Factor.F (ERF.F) subfamily, Heng Deng, Yao Chen and colleagues (Deng et al., 2022) identified SlERF.F12 as a putative repressor of tomato fruit ripening (see Figure). ERF.F subfamily members bear one or two ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif(s), a plant-specific transcriptional repressor domain (Kagale and Rozwadowski, 2011). SlERF.F12 boasts two such EAR motifs, but the functional significance and mechanism of EAR motif-containing ERFs in fruit ripening have remained largely unexplored until recently.

Figure .

SlERF.F12 represses the transition to fruit ripening in tomato. A, Wild type (WT), SlERF.F12-OE, and SlERF.F12-RNAi lines ripen at different times. Model for SlERF.F12/TPL2/HAD1/3 tripartite complex (B) before the onset of ripening initiation and (C) during ripening. Adapted from Deng et al. (2022), Figures 2D and 11.

Among the ERF.F family members, SlERF.F12 expression strongly decreases at the transition to ripening in tomato fruit. In immature green fruit and vegetative tissue, SlERF.F12 expression is relatively high, but rapidly declines at the onset of ripening (breaker stage) and remains low thereafter. Using overexpression (OE) and RNA interference (RNAi) lines, the authors nicely demonstrated that SlERF.F12 is a negative regulator of tomato fruit ripening (Figure). The authors confirmed these phenotypes using genome-edited alleles. Interestingly, the delayed onset of fruit ripening in SlERF.F12-OE lines was accompanied by slower fruit softening, a major ripening-associated trait. At the red-ripe (breaker + 7) stage, SlERF.F12-OE lines exhibited enhanced fruit firmness, lower water loss, and delayed wrinkling, underlying a potential application strategy to increase fruit shelf life.

To establish a direct connection between the observed phenotypes and ethylene signaling, the authors performed comprehensive transcriptome analyses, RT-qPCR verification, protoplast transactivation assays, and electrophoretic mobility shift assays. Their results firmly established that SlERF.F12 represses the transcription of ripening-related genes through direct binding to cis-elements in their promoters. Moreover, the EAR2 motif, but not EAR1, was necessary for mediating such repression. To identify more regulatory components, the authors performed a yeast two-hybrid screen and co-immunoprecipitation assays and identified the TOPLESS protein TPL2 and the histone deacetylases HDA1 and HDA3 as forming an in vivo tripartite complex with SlERF.F12. Silencing TPL2 or treating wild-type fruit with the HDA inhibitor trichostatin A accelerated tomato fruit ripening, resembling the phenotype of SlERF.F12-RNAi lines. Finally, using chromatin immunoprecipitation assays, the authors investigated two permissive histone marks (H3K9Ac and H3K27Ac) at the promoter region of several ripening-related genes in SlERF.F12-OE and RNAi lines. Consistently, both permissive histone marks were reduced in SlERF.F12-OE lines and enriched in SlERF.F12-RNAi lines, corresponding to inactivation (Figure) and activation (Figure) of ripening, respectively.

This study brings important insights by connecting epigenetic control and transcriptional dynamics to climacteric fruit ripening. The ERF.F subfamily, particularly SlERF.F12 and its EAR motif, is shown here to be important players. Other questions remain such as: What other transcriptional/epigenetic regulators participate in ripening? Are there any “master regulators”? How is the transcriptional network integrated with the optimal timing of fruit ripening; in particular, how does the promotive ethylene-signaling pathway interplay with the repressive ERF.F-mediated pathway? For instance, SlERF.F12 homologs can be identified in several climacteric fruit species and the expression patterns of the encoding genes also appear similar to that in tomato in these other fruit species (Deng et al., 2022). Apart from understanding and the appreciation of nature, the development of application strategies to improve fruit quality for human benefits is anticipated.