Diversity and redundancy of the ripening regulatory networks revealed by the fruitENCODE and the new CRISPR/Cas9 CNR and NOR mutants

Abstract

Tomato is considered as the genetic model for climacteric fruits, in which three major players control the fruit ripening process: ethylene, ripening transcription factors, and DNA methylation. The fruitENCODE project has now shown that there are multiple transcriptional circuits regulating fruit ripening in different species, and H3K27me3, instead of DNA methylation, plays a conserved role in restricting these ripening pathways. In addition, the function of the core tomato ripening transcription factors is now being questioned. We have employed CRISPR/Cas9 genome editing to mutate the SBP-CNR and NAC-NOR transcription factors, both of which are considered as master regulators in the current tomato ripening model. These plants only displayed delayed or partial non-ripening phenotypes, distinct from the original mutant plants, which categorically failed to ripen, suggesting that they might be gain-of-function mutants. Besides increased DNA methylation genome-wide, the original mutants also have hyper-H3K27me3 in ripening gene loci such as ACS2, RIN, and TDR4. It is most likely that multiple genetic and epigenetic factors have contributed to their strong non-ripening phenotypes. Hence, we propose that the field should move beyond these linear and two-dimensional models and embrace the fact that important biological processes such as ripening are often regulated by highly redundant network with inputs from multiple levels.

Knockout of VvCCD8 gene in grapevine affects shoot branching

“…Intriguingly, targeted mutagenesis of SBP-CNR and NAC-NOR transcription factors, which are thought to be master regulators of tomato ripening, resulted in tomato delayed ripening or partial non-ripening. This phenotype was surprisingly distinct from the previously used original tomato mutants [31], suggesting a great potential of CRISPR/Cas9 system in gene functional research. SLs were found to retard bud outgrowth [5,6], and SL biosynthetic genes CCD7 (MAX3) and CCD8 (MAX4) had been demonstrated to be involved in branching control in multiple herbaceous plants, including Arabidopsis, rice and tomato [8,[32][33][34].…”

Genetic dissection of climacteric fruit ripening in a melon population segregating for ripening behavior

“…MELO3C024516 has 10 non-synonymous variants between Ved and PS. ROS1 encodes a demethylase, and demethylation has been reported as one of the main mechanism regulating fruit ripening 15 , 38 . In tomato, the ROS1 orthologue DML2 is a demethylase governing fruit ripening 14 .…”

RNA directed DNA methylation and seed plant genome evolution

“…Furthermore, during fruit ripening in Solanum lycopersicum among other species, DNA demethylases (DML & DME) are upregulated leading to gradual demethylation at loci of ripening genes such as CNR (Zhang et al 2018) which encodes a transcription factor CNR (colorless non-ripening). Mutants of cnr gene display hypermethylation at the promoter preventing binding of RIN (ripening inhibitor) (Gao et al 2019) transcription factor and lowered gene expression ( Fig. 6b).…”