Effects of harvest time on chilling tolerance and the transcriptome of 'Wonderful' pomegranate fruit

We observed that harvest time markedly affected chilling tolerance of 'Wonderful' pomegranate fruit; early-harvested fruit were extremely chilling sensitive, whereas late-harvested ones were relatively chilling tolerant. Damage to inner membranes is the most obvious phenotypic damage observed in pomegranate fruit exposed to chilling. To elucidate the molecular mechanisms that govern chilling tolerance of pomegranate fruit, we conducted RNA-Seq analysis of inner membrane tissues from early- and late-harvested fruit on harvest day and after a 2-week exposure to a cold quarantine treatment at 1 °C. Pair-wise comparisons revealed that 6853 transcripts were significantly (p ≤ 0.01) induced or repressed by a factor of at least 4 after exposure to chilling in early-harvested fruit, as compared with 8000 transcripts in late-harvested ones. In early-harvested, chilling-sensitive fruit most (63%) differentially expressed transcripts were down-regulated by cold storage, whereas in late-harvested fruit most (62%) differentially expressed transcripts were up-regulated, indicating activation of adaptation processes. The results demonstrate that transcripts related to several regulatory, metabolic, and stress-adaptation pathways were specifically induced in late-harvested fruit while suppressed in early-harvested, chilling-sensitive fruit. These regulatory mechanisms included activation of jasmonic acid and ethylene biosynthesis and signal transduction pathways, induction of various stress-related transcription factors, including AP2/ ERFs, MYBs, WRKYs, bHLH, homeobox, and HSFs. The observed changes in transcripts related to metabolic pathways involved primary and secondary carbohydrate metabolism, including activation of starch degradation and of galactinol and raffinose biosynthesis genes. Finally, we observed up-regulation of transcripts corresponding to stress-tolerance, most notably heat shock proteins.