# Molecular and Hormonal Mechanisms Regulating Fleshy Fruit Ripening
## 3 Ripening Is Influenced by Multiple Hormones and Light
### Auxin Delays Ripening and Antagonises the Effects of Ethylene  
Physiological studies suggested that auxin plays an inhibitory role in fruit ripening [137; 138], and reverse genetics experiments support this idea [139; 140; 141; 142; 143]. Low internal auxin concentration or reduced auxin signalling activity are believed to increase the sensitivity of fruit tissue to ethylene, promoting the transition from system-1 to system-2 autocatalytic ethylene production. Figure 4: Ethylene response of WT and RIN-deficient tomato fruits (reproduced with the permission from [PERSON] et al. [15]). Effect of exogenous ethylene and ethylene perception inhibitor 1-MCP treatment on ripening progression of RIN-CRISPR tomato fruit. WT and RIN-CRISPR tomato fruits were picked at MG stages and treated and replenished daily with ethylene (100 ppm) and 1-MCP (10 ppm) or air continually for up to 15 days. Fruits in horizontal rows are biological replicates. Enlarged photos of representative samples are shown compared to WT fruits on the right. The red scale bar represents 2 cm. Auxin can be synthesised in the chloroplasts from tryptophan and then via either indole-3-acetamide or indole-3-pyruvate acid, to generate IAA [144]. There is also an important tryptophan-independent auxin biosynthesis pathway in the cytosol, involving TAA and a multigene family of _YUCCA_ genes, which encode flavin-containing monooxygen-nases [145,146]. There are three types of auxin transcriptional regulators, ARFs, Aux/IAA and TOPLESS (TPS) proteins. ARFs play a key role in regulating the expression of auxin response genes and act in concert with Aux/IAAs to control auxin-dependent transcriptional activity of target genes. There are 47 _ARF_ genes in banana [147], 22 in tomato [148] and 19 in sweet orange [149]. Most have an N-terminal DNA-binding domain, a variable central transcriptional regulatory region, which can function as an activator or repressor domain, and a carboxy-terminal dimerization domain, that allows formation of either ARF/ARF homodimers or ARF/Aux/IAA hetero-dimers. Experiments with several ARFs have indicated that they play a role in tomato fruit development and ripening. The over-expression of tomato _ARF2A_ resulted in a blotchy ripening pattern, with some parts of the fruit ripening faster than others [140]. The downregulation of tomato _ARF4_ also altered ripening-associated phenotypes, such as firmness, sugar and chlorophyll content, leading to dark green fruit and blotchy ripening [143,150], and [PERSON] et al. [151] found a similar phenotype with tomato _ARF10_, which is involved in regulating chlorophyll and sugar accumulation during tomato fruit development [151]. _ARF2A_ expression is reduced in the _nor_, _rin_ and _Nr_ ripening mutants and responds to exogenous application of ethylene, auxin and ABA [140]. ARF2A homodimerizes and also interacts with the ABA STRESS RIPENING (ASR1) protein, suggesting that ASR1 could link ABA and ethylene-dependent ripening [140]. Both auxin and ethylene _cis_-regulatory elements are present in the promoter regions of a number of _ARFs_, suggesting that they may be regulated by both hormones [148,152]. Furthermore, AtARF7 and AtARF19 are believed to be involved in the ethylene response in Arabidopsis [153] and several _ERF_ genes are also induced by auxin [154,155]. The two _ARF2_ paralogs in the tomato genome, _SIARF2A_ and _SIARF2B_, are nuclear localised where they repress auxin-responsive genes. In fruit tissues, _SIARF2A_ is ethylene-regulated, while _SIARF2B_ is auxin-induced [141]. Taken together, these results indicate substantial interactions between ethylene and auxin in the regulation of ARFs and ERFs. SAURs (small auxin-upregulated RNAs) are believed to be involved in various auxin-related actions [156], and _SI-SAUR69_, which shows reduced expression in the _rin_ mutant, is involved in changing auxin signalling or transport. Overexpression of _SI-SAUR69_ in tomato causes premature initiation of ripening, whereas its downregulation delays ripening initiation [139,142].