Re-evaluation of the rin mutation and the role of RIN in the induction of tomato ripening

Abstract

Tomato (Solanum lycopersicum) rin mutants completely fail to ripen: they do not produce red pigmentation, soften or induce an ethylene burst. Therefore, RIN has long been believed to function as a major regulator that is essential for the induction of ripening. Here, we provide evidence contradicting this concept of RIN function, showing induction of fruit ripening in the absence of RIN. A CRISPR/Cas9-mediated RIN-knockout mutation did not repress initiation of ripening and the mutant fruits showed moderate red colouring. Moreover, inactivation of the rin mutant allele partially restored the induction of ripening. Therefore, RIN is not required for the initiation of ripening and rin is not a null mutation, but rather is a gain-of-function mutation that produces a protein that actively represses ripening. Since the discovery of the rin mutant a half-century ago, many models have depicted RIN as indispensable for the induction of ripening; these models should be reconsidered in light of these results.

A critical evaluation of the role of ethylene and MADS transcription factors in the network controlling fleshy fruit ripening

“…b; reproduced from Figs 2a and 5a in Ito et al ., ), and the lycopene and beta‐carotene content, ethylene production and fruit firmness were similar to WT fruits. Expression of related genes such as ACS2 , ACS4 , and PL showed no distinct difference (Ito et al ., ), although PSY1 and PG2a were expressed at slightly lower levels compared to WT. These results clearly indicate that full ripening progression is blocked if MADS‐RIN function is lost and recovered if MADS‐RIN expression is restored.…”

“…The reason for its effectiveness is simple: although the changes in color, flavor, texture and taste and aroma function to make the fruit attractive to eat, the inevitable consequence of ripening is a continuing decrease in firmness (Tucker et al ., ), which is undesirable because it renders the fruit susceptible to microbial attack, deterioration and rotting. The presence of RIN‐MC in hybrids reduces this adverse softening but unfortunately also reduces other quality attributes as well (Ito et al ., ). All of these observations were thought to be consistent with the conclusion that normal RIN is required for ripening and that RIN‐MC is a loss‐of‐function mutation.…”

“…Ito et al . () used homozygous CRISPR/Cas9 mutants and Li et al . () used homozygous RNAi plants to investigate the effect on tomato ripening of reducing or eliminating MADS‐RIN levels in WT fruit.…”

“…Ito et al . () concluded that fruit ripening could initiate without MADS‐RIN, whereas Li et al . () proposed that downregulation of RIN expression inhibited initiation and development of ripening.…”

Application and future perspective of CRISPR/Cas9 genome editing in fruit crops

“…However, tomato has been a model for studying fleshy fruit development and fruit ripening for decades (Vrebalov et al ; Giovannoni ). CRISPR/Cas9 genome editing was used to investigate gene function as shown in ARGONAUTE7 ( SlAGO7 ) (Brooks et al ), SHORT ROOT (SHR) (Ron et al ), RIPENING INHIBITOR (RIN) (Ito et al ; ), RNA Editing Factor SlORRM4 (Yang et al ), SlMAPK3 in drought tolerance (Wang et al ), auxin signaling SlIAA9 in parthenocarpic fruit (Ueta et al ), and PROCERA , a DELLA protein, in fruit development (Tomlinson et al ). High mutation frequency (83.56%) was observed at SlPDS (phytoene desaturase) and SlPIF4 (Phytochrome interacting factor) in the T 0 lines, and mutations were stably transmitted to the T 1 and T 2 generations (Pan et al ).…”