# Unravelling the Molecular Regulation Mechanisms of Slow Ripening Trait in _Prunus persica_
## 3 Discussion
### NAC072 and Direct Possible Target Genes  
_NAC072_ presented a peak of expression in T2 (Figure 1), probably affecting the S1/S2 transition or altering the seed/embryo development. We know that NR and SR individuals are similar in phenotype in T1 and T2 (Figure 1), and considering the transcriptomic information, no significant differences were observed in T1 and T2 (Figure 2). These results reinforce the hypothesis that _NAC072_ is responsible for the slow ripening phenotype. Similar to _NAC072_, another 51 genes with descriptions displayed expression only in the normal ripening phenotype. Among them, five were described as SAUR family proteins and one as a 1-aminocyclopropane-1-carboxylate synthase 1 (_ACSI_), associating _NAC072_ with auxin and the ethylene signaling pathways. None of them presented an expression pattern like _NAC072_ and for the most part they were genes that, in normal ripening fruits, are expressed only in late stages of development (Table 1), suggesting the indirect _NAC072_ regulation on these genes. On the other hand, unlike _NAC072_, 64 genes with descriptions were only expressed in slow ripening fruit. Of them, two \(\alpha\)-farnesene synthases 1 (_AFS1_) and two genes related to cell number regulation were distinguished, where the two AFS (terpene biosynthesis-related proteins) showed expression patterns like that of _NAC072_ (Table 1).  
The plant hormone auxin, or indole-3-acetic acid (IAA) is responsible for various aspects of plant development [25], including organ initiation from the shoot apical meristem (SAM) and flower/fruit development [26]. The auxin response is mediated by auxin response factors (ARF) that may activate or repress the expression of early auxin-responsive genes like the small auxin up RNA (SAUR) genes mentioned before [25]. In general, these SAUR family genes have an unknown function, but some of them have been associated with cell expansion on the hypocotyl and leaves (SAUR19) [27], auxin synthesis regulation (SAUR39) [28], and leaf senescence (SAUR36) [29]. In fruit, a crucial role of endogenous IAA was reported as controlling the onset of ripening in fleshy fruit and regulate fruit growth and development together with GAs [7]. On the other hand, an association between _NAC072_ and ethylene was identified through the differential expression of the gene _ACS1_ between the normal and slow ripening individuals. _ACS1_ was identified as a critical enzyme in ethylene biosynthesis [30], using S-adenosylmethionine (AdoMet) to produce 1-aminocyclopropane-1-carboxylate (ACC). Therefore, its regulation at the transcriptional level is an important factor regulating ethylene production in response to different stimuli. ACS protein stability also plays a significant role in controlling ethylene biosynthesis [31].  
However, as _ACS1_ presents its expression only in late stages of development (Figure 3, blue section), we believe that it could be a consequence of the loss of the _NAC072_ gene in earlier stages of fruit development, and not a direct regulation between _NAC072_ and _ACS1_.  
Moreover, \(\alpha\)-farresene synthase 1 (_AFS1_) is an enzyme that uses farnesyl diphosphate (FPP) to produce \(\alpha\)-farresene [32]. This \(\alpha\)-farresene is a sesquiterpene described as a lepidopteran attractant and an oviposition inducer [33]. It was reported to be produced during the storage of apple fruit and its oxidation is hypothesized to be the causal agent of superficial scald [34]. No correlations have been reported between _AFS1_ and the slow ripening phenotype, with respect to either maturity date _NAC072_. However, the precursor FPP, used to produce \(\alpha\)-farresene, is also used to produce geranylgeranyl diphosphate (GGPP), a key substrate for ABA and GA biosynthesis, two plant hormone biosynthesis [33]. Thus, our results suggest a possible correlation between the expression of _AFS1_ in SR individuals and ABA or GA production, but the regulation mechanisms involved remain unknown.