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

Authors: GAO, YING - China Agricultural University; ZHU, NING - The Chinese University Of Hong Kong (CUHK); ZHU, XIAOFANG - China Agricultural University; WU, MENG - China Agricultural University; Jiang, Cai-Zhong; GRIERSON, DONALD - University Of Nottingham; LUO, YUNBO - China Agricultural University; SHEN, WEI - The Chinese University Of Hong Kong (CUHK); ZHONG, SILIN - The Chinese University Of Hong Kong (CUHK); FU, DA-QI - China Agricultural University; QU, GUIQIN - China Agricultural University

Submitted to: Horticulture Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/23/2019
Publication Date: 2/11/2019
Citation: Gao, Y., Zhu, N., Zhu, X., Wu, M., Jiang, C., Grierson, D., Luo, Y., Shen, W., Zhong, S., Fu, D., Qu, G. 2019. Diversity and redundancy of the ripening regulatory networks revealed by the fruitENCODE and the new CRISPR/Cas9 CNR and NOR mutants. Horticulture Research. 6:39. https://doi.org/10.1038/s41438-019-0122-x.
DOI: https://doi.org/10.1038/s41438-019-0122-x

Interpretive Summary: For flowering plants, fruits serve as seed dispersal vehicles that have evolved usually from carpels or adjacent floral tissues. Fruit ripening is often described as a developmental process that alters the physiological and biochemical properties of a seed-bearing organ to aid seed dispersal. This enables plants to interact with the coevolving animals that consume the fruits and disperse the defecated seeds to distant locations, and hence increase plant reproductive success. Charles Darwin has also acknowledged its evolutionary advantage as “beauty serves merely as a guide to birds and beasts in order that the fruit may be devoured and the matured seed disseminated”. However, at the molecular level, transforming an unappealing carpel to a tasty fruit is not a simple task. It requires a complete reprograming of the carpel gene expression network, during which hundreds if not thousands of genes have to be switched on and off in a highly coordinated manner. It is the combined action of these so called “ripening genes” at a precise developmental timing that has transformed nearly every aspect of the carpel tissue, such as color, aroma, flavor, texture and nutritional content, in order to attract frugivorous as seed dispersers. This process must also be kept under strict regulatory control, as any premature transformation ahead of seed maturation is highly detrimental. This ripening regulatory network has fascinated scientists for decades, and also has important implications in agriculture worldwide as it affects yield, nutritional value and shelf life of our horticultural produces. Significant progress has been made using tomato (Solanum lycopersicum) as a model to elucidate its genetic and epigenetic basis. The tomato ripening model has three key components: the hormone ethylene, ripening transcription factors such as MADS-box RIPENING INHIBITOR (RIN), SBP-box COLORLESS NON-RIPENING (CNR) and NAC transcription factor NON-RIPENING (NOR) and DNA methylation. However, recent findings from the fruitENCODE project has shown that this model is not universal, as there are at least three different types of transcriptional positive feedback circuits controlling ripening in seven climacteric species. Only tomato has a genome-wide DNA CG and CHG demethylation, and CHH hypermethylation, while most species used H3K27me3 to regulate the ripening circuits. In addition, gene silencing and genome editing technology have now enabled us to re-examined the tomato fruit ripening transcription factors, resulting in controversial and sometimes conflicting views of its biological functions. In this study, we examined the diversity of the ripening regulatory systems from a genomic and evolutionary perspective. We also provided new evidence showing that CRISPR/Cas9-induced mutations in the core tomato ripening transcription factor SBP-CNR and NAC-NOR failed to abolish ripening, suggesting that the ripening transcriptional regulatory network is highly robust and has few single points of failure. In a robust system, the lack of mutant phenotypes does not necessarily mean that the gene is not involved in the biological process, while the presence of phenotypes might suggest that the process is not important enough for plants to evolve a backup plan. It is about time for plant biologists to re-evaluate those linear and two-dimensional models generated from traditional genetic studies and often developed solely based on single species studies. After all, complex and important biological processes such as ripening are often regulated by highly redundant transcriptional network with inputs from multiple epigenome levels.

Technical 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.