# Molecular and Hormonal Mechanisms Regulating Fleshy Fruit Ripening
## 3 Ripening Is Influenced by Multiple Hormones and Light
### Ethylene Initiates and Promotes Ripening  
Ethylene production occurs at a low level throughout plant growth and development but increases during biotic and abiotic stresses, abscission, ripening and senescence. During ripening onset and progression, there is a massive burst of ethylene production, which occurs in two stages, called system-1 and system-2 ethylene synthesis [107; 108; 109], and when system-2 is activated, ethylene synthesis becomes autocatalytic. In tomato,  
Figure 3: Biochemical origin of volatile compounds in fruits (redrawn from Aragüez and Valpuesta [86]; [PERSON] and [PERSON] [88]). Key aspects of primary metabolism, which provides the substrates for secondary metabolism, are shown on the left. Solid lines indicate a validated step in a pathway, with the responsible enzyme indicated in red. Volatiles are indicated in blue. Broadly, volatiles are derived from the fatty acid (for example, Z-3-hexenol), carotenoid cleavage (for example, geranylacetone) or phenylpropanol (the C6–C3 compounds) pathways. In addition, volatile alcohols can be reversibly converted to esters by the action of an alcohol acetyltransferase(AAT1) and a carboxymethylesterase (carboxylesterase 1 (CXE1)). LoxC, lipoxygenase C; HPL, fatty acid hydroperoxide lyase; ADH2, alcohol dehydrogenase 2; BCAT, branched-chain amino acid aminotransferase; AADC, aromatic amino acid decarboxylase; CCD1, carotenoid cleavage deoxygenase 1. 14 _ACC synthase_ (_ACS_) genes (_ACSIA/B-13_) and 7 _ACC oxidase_ (_ACO_) genes (_ACO1-7) have been identified [110; 111]. _ACS1A_ and _ACS6_ are mainly involved in system-1 ethylene production, and _ACS2_ and _ACS4_ in system-2 [110; 111]. _ACO1_ participates in system-1 ethylene synthesis and _ACO1_ and _ACO4_ are involved in system-2 ethylene in tomato, but the _ACO_ gene family has not been as well-studied as the _ACS_ family [12; 110]. _ACO1_ expression increases strongly and correlates well with the autocatalytic rise in ethylene production. _ACO2_ expression drops to a basal level during further fruit development and ripening. At the onset of ripening, _ACO3_ expression strongly increases. There is a temporal increase in _ACO4_ expression during the breaker stage, mainly in the columella and placenta tissue. _ACO5_ expression increases slightly after antisens and remains at a similar level during further fruit development and ripening. _ACO6_ has a low expression during fruit development but a temporary high expression during the breaker stage, followed by a gradual decline during further ripening. _ACO7_ is only basally expressed during fruit development and ripening [110]. Antisense gene silencing was used to test the function of _ACS_ and _ACO_ genes expressed during tomato ripening, and this resulted in the inhibition of ethylene synthesis and slowed or prevented both fruit ripening and leaf senescence [107; 108; 109], which validated the conclusions drawn about the role of ethylene from experiments with Ag+ (plant ethylene responses inhibitor [112]) and confirmed using 1-MCP (a specific inhibitor of ethylene perception and action [113]). Theologis [114] also showed that the respiratory climacter was abolished in tomatoes in which _ACS_ genes were inhibited. They did not produce ethylene, but ripening was restored by supplying ethylene externally. The roles of ethylene biosynthesis genes have been functionally verified in other fruits as well, such as melon [115], pear [116], kiwifruit [117] and apple [118]. Several TFs have been shown to influence ethylene synthesis, including MADS-RIN [7; 119], HB-1 [120], NAC1/4 [121; 122] and NAC4/9 [123; 124] in tomato, and also apple MdERF2 [125], kiwifruit AdNAC6/7 [20; 126] and banana MaERF9/11 [127]. The relationship between MADS-RIN and ethylene in controlling ripening has recently been clarified Figures 4 and 5). Treatment of RIN-deficient fruit (engineered using CRISPR/Cas9) with propylene (an ethylene analogue used to study system-1 and system-2 ethylene [128]) showed that they were unable to initiate system-2 ethylene production [15].