Ripening of the model fruit tomato (Solanum lycopersicum) is controlled by a transcription factor network including NAC (NAM, ATAF1/2, and CUC2) domain proteins such as No-ripening (NOR), SlNAC1, and SlNAC4, but very little is known about the NAC targets or how they regulate ripening. Here, we conducted a systematic search of fruit-expressed NAC genes and showed that silencing NOR-like1 (Solyc07g063420) using virus-induced gene silencing (VIGS) inhibited specific aspects of ripening. Ripening initiation was delayed by 14 days when NOR-like1 function was inactivated by CRISPR/Cas9 and fruits showed obviously reduced ethylene production, retarded softening and chlorophyll loss, and reduced lycopene accumulation. RNA-sequencing profiling and gene promoter analysis suggested that genes involved in ethylene biosynthesis (SlACS2, SlACS4), color formation (SlGgpps2, SlSGR1), and cell wall metabolism (SlPG2a, SlPL, SlCEL2, and SlEXP1) are direct targets of NOR-like1. Electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), and dual-luciferase reporter assay (DLR) confirmed that NOR-like1 bound to the promoters of these genes both in vitro and in vivo, and activated their expression. Our findings demonstrate that NOR-like1 is a new positive regulator of tomato fruit ripening, with an important role in the transcriptional regulatory network.

Journal of Experimental Botany, Advance Access publication: 27 April 2020, doi: 10.1093/jxb/eraa131

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.

The NAC transcription factor (TF) family is one of the largest plant-specific TF families and its members are involved in the regulation of many vital biological processes during plant growth and development. Recent studies have found that NAC TFs play important roles during the ripening of fleshy fruits and the development of quality attributes. This review focuses on the advances in our understanding of the function of NAC TFs in different fruits and their involvement in the biosynthesis and signal transduction of plant hormones, fruit textural changes, color transformation, accumulation of flavor compounds, seed development and fruit senescence. We discuss the theoretical basis and potential regulatory models for NAC TFs action and provide a comprehensive view of their multiple roles in modulating different aspects of fruit ripening and quality.

Fruit development and ripening is regulated by genetic and environmental factors and is of critical importance for seed dispersal, reproduction and fruit quality. Tomato rin mutant fruit have a classic ripening-inhibited phenotype, which is attributed to a genomic DNA deletion resulting in the fusion of two truncated transcription factors, RIN and MC. In wild-type fruit, RIN, a MADS-box transcription factor, is a key regulator of the ripening gene expression network with hundreds of gene targets controlling changes in color, flavor, texture and taste during tomato fruit ripening; MC, on the other hand, has low expression in fruit, and the potential functions of the RIN-MC fusion gene in ripening remain unclear. Here, over-expression of RIN-MC in transgenic wild-type AC tomato fruits impaired several ripening processes, and down-regulating RIN-MC expression in the rin mutant was found to stimulate the normal yellow mutant fruit to produce a weak red color, suggesting a distinct negative role for RIN-MC in tomato fruit ripening. By comparative transcriptome analysis of rin and rin 35S::RIN-MC RNAi fruits, a total of 1168 and 1234 genes were identified as potential targets of RIN-MC activation and inhibition. Further, the RIN-MC fusion gene was shown to be translated into a chimeric transcription factor that was localized to the nucleus and was capable of protein interactions with other MADS-box factors. These results indicated that RIN-MC is functional and controls expression of many ripening genes, thereby contributing to the rin mutant phenotype.