Expression of genes related to auxin biosynthesis, transport and signaling
Tryptophan (Trp) is a precursor for auxin (indole-3-acetic acid, IAA) (Woodward and Bartel, 2005), whose related genes were shown to be differentially expressed under CT and HT in post-harvest peach fruits. Although the expression level of tryptophan synthase alpha chain (TRPA; Prupe.3G144500) was decreased on day 2 in both treatments (Figure 5 ), the expression level of tryptophan synthase beta chain (TRPB; Prupe.4G207900) showed a peak on day 2 and reached the maximum on day 7 for the CT condition but not for the HT condition. The expression level of another auxin biosynthetic enzyme, indole-3-glycerol phosphate synthase (IGPS; Prupe.3G196200), manifested different trends between HT and CT at early stage. Overall, HT reduces tryptophan biosynthetic enzyme expression level compared with CT.
Several Trp-dependent pathways, which are generally named after an intermediate, have been proposed, including indole-3-pyruvic acid (IPA) pathway, indole-3-acetamide (IAM) pathway, tryptamine (TAM) pathway and indole-3-acetaldoxime (IAOx) pathway (Woodward and Bartel, 2005; Zhao, 2012). In IPA pathway [Trp → IPA → IAA], the FPKM value of tryptophan aminotransferase ofArabidopsis(TAA1) was less than 5 in all samples, but the expression of indole-3-pyruvate monooxygenase (YUCCA) significantly increased on day 2 at HT. In IAM pathway [Trp → IAM → IAA], the amidase (AMI; Prupe.6G327700) had increased expression at the early stage under both temperatures. In TAM pathway [Trp → TAM → indole-3-acetaldehyde (IAAld) → IAA], aldehyde dehydrogenase (NAD+) (ALDH2B7; Prupe.1G255200) under HT showed lower expression than CT in the whole storage period. To sum up, IPA pathway is affected most by HT in our work.
The expression patterns of auxin-related genes were evaluated to understand their responses to HT. It was found that the expression of auxin influx carrier 1 (AUX1) decreased most on day 2 (Figure 5 ) under both temperature conditions, but auxin efflux carrier component (Pin-formed, PIN5) had a higher expression level under CT. The auxin response factor (ARF) genes in group II increased at the later stage for CT rather than HT condition (Supplementary Figure S2 ).
These auxin-responsive genes have been classified into three major groups: Aux/IAA, small auxin-up RNA (SAUR), and Gretchen Hagen 3 (GH3) (Guilfoyle, 1999; Liscum and Reed, 2002). Aux/IAAs were sorted into three groups based on cluster analysis (Figure 6 ), which bind transcription factors of ARF family (Supplementary Figure S2 ). Generally, the genes within Group I were down-regulated with time, while the genes in Group III showed a higher level of expression in HT fruits (Figure 6 ). Based on the clustering results, SAUR genes were divided into four groups which had different responses to CT and HT (Supplementary Figure S3 ). In addition, the gene GH3.1 (Prupe.6G226100) showed a higher expression level under CT compared with HT, while the other three members of GH3 gene family (GH3.6, Prupe.8G248400; GH3.1, Prupe.8G137900; GH3.17, Prupe.1G213200) showed higher expression levels under CT in the later stage (Figure 5 ).