Expression of genes related to ethylene biosynthesis and signaling pathway
Ethylene is biosynthesized by S-adenosylmethionine synthetase (SAMS) from the amino acid methionine to S-adenosyl-L-methionine (SAM), and SAM is then converted by 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) to ACC which is further catalyzed by ACC-oxidase (ACO) to ethylene. Notably, the activity of ACS determines the rate of ethylene production (Yu et al., 1979). Similar to ethylene production (Figure 1b ), the peach fruits at CT were higher than HT in the enzyme activities of ACO and ACS at most time points (Figure 7 ).
Expressions of genes involved in ethylene biosynthesis and signaling were examined to understand the lower ethylene production in HT fruits. Among these, the expression of 5 genes (SAMS5, Prupe.1G107000; SAMS1, Prupe.3G004000; SAMS3, Prupe.7G128500; ACS1, Prupe.2G176900; ACO1, Prupe.3G209900) were significantly higher at CT than HT (Figure 8a ), which agreed with the physiological data of ACO/ACS activities (Figure 7 ).
Ethylene is perceived by a family of five membrane-localized receptors that are homologous to bacterial two-component histidine kinases involved in sensing environmental changes (Stepanova and Alonso, 2009). According to the homology with Arabidopsis , four peach ethylene receptors were identified, including ethylene receptors 1 (ETR1; Prupe.1G556000), ETR2 (Prupe.1G034300), ethylene response sensor 1 (ERS1; Prupe.8G265200) and ethylene insensitive 4 (EIN4; Prupe.6G348000). The expression levels of ETR2 and ERS1 at CT were generally higher than HT, while those of ETR1 and EIN4 were not obvious (Figure 8b ). Constitutive triple response 1 (CTR1; Prupe.7G117700), the downstream element of ethylene receptors, showed significantly decreased expression on day 2 with an average of 2.69-fold change, and then kept in low FPKM value at later time points (Figure 8b ). Especially, EIN2 (Prupe.6G235600), whose function is a positive regulator of the ethylene pathway (Stepanova and Alonso, 2009), displayed reverse trends of expression levels between CT and HT. EIN3 (Prupe.2G058500, Prupe.2G058400) exhibited declining expression level on day 2. Besides, ERFs were sorted into four groups based on hierarchical clustering (Supplementary Figure S4a ). Group I showed a higher level of transcripts in HT condition, indicating that group I genes might play a significant role in heat stress response (Supplementary Figure S4b ). Therefore, temperature caused complicated responses in receptor genes and downstream signaling genes of ethylene in both CT and HT conditions.
Expression of genes related to