Flavor-associated volatile chemicals make major contributions to consumers' perception of fruits. Although great progress has been made in establishing the metabolic pathways associated with volatile synthesis, much less is known about the regulation of those pathways. Knowledge of how those pathways are regulated would greatly facilitate efforts to improve flavor. Volatile esters are major contributors to fruity flavor notes in many species, providing a good model to investigate the regulation of volatile synthesis pathways.Here we initiated a study of peach (Prunus persica L. Batsch) fruits, and identified that the alcohol acyltransferase PpAAT1 contributes to ester formation. We next identified the transcription factor (TF) PpNAC1 as an activator of PpAAT1 expression and ester production. These conclusions were based on in vivo and in vitro experiments and validated by correlation in a panel of 30 different peach cultivars. Based on homology between PpNAC1 and the tomato (Solanum lycopersicum) TF NONRIPENING (NOR), we identified a parallel regulatory pathway in tomato. Overexpression of PpNAC1 enhances ripening in a nor mutant and restores synthesis of volatile esters in tomato fruits. Furthermore, in the NOR-deficient mutant tomatoes generated by CRISPR/Cas9, lower transcript levels of SlAAT1 were detected. The apple (Malus domestica) homolog MdNAC5 also stimulates MdAAT1 expression via binding to this gene's promoter. In addition to transcriptional control, epigenetic analysis showed that increased expression of NACs and AATs is associated with removal of the repressive mark H3K27me3 during fruit ripening. Our results support a conserved molecular mechanism in which NAC TFs activate ripening-related AAT expression, which in turn catalyzes volatile ester formation in multiple fruit species.

Peach fruit volatile acetate esters impact consumer sensory preference and contribute to defense against biotic stresses. Previous studies showed that alcohol acyltransferase (AAT) family PpAAT1 is correlated with volatile ester formation in peach fruits. However, fruits also contain carboxylesterase (CXE) enzymes that hydrolyze esters. The functions of this family with regard to volatile ester content has not been explored. Here, we observed that content of acetate ester was negatively correlated with expression of PpCXE1. Recombinant PpCXE1 protein exhibited hydrolytic activity toward acetate esters present in peach fruit. Kinetic analysis showed that PpCXE1 showed the highest catalytic activity toward E-2-hexenyl acetate. Subcellular localization demonstrated that PpCXE1 is present in the cytoplasm. Transient expression in peach fruit and stable overexpression in tomato fruit resulted in significant reduction of volatile esters in vivo. Taken together, the results indicate that PpCXE1 expression is associated with catabolism of volatile acetate esters in peach fruit.

The essential role of ethylene in fruit ripening has been thoroughly studied. However, the involvement of brassinosteroids (BRs) in the regulation of fruit ripening and their relationship with the ethylene pathway are poorly understood. In the current study, we found that BRs were actively synthesized during tomato fruit ripening. We then generated transgenic lines overexpressing or silencing SlCYP90B3, which encodes a cytochrome P450 monooxygenase that catalyzes the rate-limiting step of BR synthesis. The expression level of SlCYP90B3 was positively related to the contents of bioactive BRs as well as the ripening process in tomato fruit, including enhanced softening and increased soluble sugar and flavor volatile contents. Both carotenoid accumulation and ethylene production were strongly correlated with the expression level of SlCYP90B3, corroborated by the altered expression of carotenoid biosynthetic genes as well as ethylene pathway genes in transgenic tomato fruits. However, the application of the ethylene perception inhibitor 1-methycyclopropene (1-MCP) abolished the promotion effect of SlCYP90B3 overexpression on carotenoid accumulation. Taken together, these results increase our understanding of the involvement of SlCYP90B3 in bioactive BR biosynthesis as well as fruit ripening in tomato, thus making SlCYP90B3 a target gene for improvement of visual, nutritional and flavor qualities of tomato fruits with no yield penalty.

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Carboxylesterases (CXE) and methylesterases (MES) are hydrolytic enzymes that act on carboxylic esters and are involved in plant metabolic processes and defense responses. A few functions of plant CXE and MES genes have been identified but very little information is available about the role of most members. We made a comprehensive study of this gene family in a commercially important species, peach (Prunus persica L. Batsch). A total of 33 peach CXE genes and 18 MES genes were identified and shown to be distributed unevenly between the chromosomes. Based on phylogenetic analysis, CXEs and MESs clustered into two different branches. Comparison of the positions of intron and differences in motifs revealed the evolutionary relationships between CXE and MES genes. RNA-seq revealed differential expression patterns of CXE/MESs in peach flower, leaf, and ripening fruit and in response to methyl jasmonate (MeJA) and ultraviolet B treatment. Transcript levels of candidate genes were verified by real-time quantitative PCR. Heterologous expression in Escherichia coli identified three CXEs that were involved in the hydrolysis of volatile esters in vitro. Furthermore, two recombinant MES proteins were identified that could hydrolyze MeJA and methyl salicylate. Our results provide an important resource for the identification of functional CXE and MES genes involved in the catabolism of volatile esters, responses to biotic and abiotic stresses and activation of signaling molecules such as MeJA and methyl salicylate.