Unraveling tomato ripening: Roles and contributions of transcription factors in ripening regulation

Ripening is important for fleshy fruit-bearing plants as it is an important trait in both physiological and agricultural contexts. Over the past two decades, numerous transcription factors (TFs) have been identified as regulators of ripening, with several acting as upstream regulators of ripening, and thus regarded as ripening master regulators.. However, more recent evidence has challenged this model, showing that knockout mutations in some of these key TFs do not completely abolish ripening. This has led to the hypothesis that ripening is regulated by a complex and potentially redundant network of multiple TFs. This thesis investigates the roles of selected transcription factors from the NAC, MADS, and GRAS families in the regulation of tomato fruit ripening. Chapter 1 provides an overview of climacteric ripening, emphasizing ethylene’s central role in both the initiation and progression of the ripening process. The molecular basis of ethylene biosynthesis and perception is discussed, providing the necessary context for subsequent chapters. Chapter 2 explores the additive function of two NAC TFs—NAC-NOR (NOR) and NOR-like1 (NL1). Double knockout mutants exhibit a strong non-ripening phenotype, with suppressed ethylene production, inhibited color development, and limited softening. Transcriptome analyses show a broad downregulation of ripening-related genes, supporting the view that NOR and NL1 act together to regulate early ripening events. Chapter 3 focuses on GRAS38, previously implicated in ripening and shelf life regulation. Contrary to earlier reports, the GRAS38 knockout mutant in the Moneyberg cultivar shows minimal phenotypic deviation from the wild type. Further investigation reveals that GRAS38 may act indirectly through the regulation of GA20ox3, a key gibberellin biosynthesis gene. Knockout of GA20ox3 results in accelerated ripening and elevated ethylene production, explained by decreased expression of ethylene receptor genes and increased ethylene sensitivity. Chapter 4 examines the interaction between NAC4 and NAC1. Interestingly, these genes appear to act in opposition—nac4 mutant show delayed ripening, while nac1 mutant ripen earlier than wild type. However, the double mutant restores normal ripening initiation. Expression analysis of key ethylene biosynthesis and receptor genes suggests that NAC4 and NAC1 modulate ripening by coordinating ethylene production and perception. Finally, Chapter 5 integrates these findings into a broader model of climacteric ripening regulation. It proposes that ripening is initiated by early-acting TFs such as NL1 and NAC4, which activate system 2 ethylene biosynthesis. This triggers a positive feedback loop involving ethylene signaling and downstream TFs like NOR, RIN, and FUL1. The conservation of these regulatory modules in other fruit species, including non-climacteric and dry fruits, suggests an evolutionarily conserved mechanism. Collectively, this work supports a model in which tomato fruit ripening is regulated not by a single master switch but by a dynamic and interconnected transcriptional network.