Discussion
Peach fruits are usually harvested in hot summer, and in transport, softening, decay and browning are accelerated. Responses to high temperature during fruit ripening and senescence involve a series of physiological and molecular changes. Little information is available on the transcriptomic responses to high temperature in postharvest peach fruits. Here, HT conditioning after cold treatment was performed and it showed beneficial effects to the ‘Tianxianhong’ peach fruits (flesh firmness without cold injury). The factors affecting fruit quality during storage were identified as ethylene metabolism, cell wall enzymes and cellular oxidase which showed different responses to high temperature. We summarized our transcriptomic findings into a rough model for the high temperature response process in peach fruits (Figure 10 ). First, MEKK1-MKK2-MPK4/6 are highly expressed, which was regarded as the stress signal transduction process following HT. Then, the low expression levels of ACS1 and ACO reduced biosynthesis of ethylene and the decreased expression of TRPB, IGPS and ALDH2B7 reduced auxin production. A series of genes involved in flesh softening and membrane stability were influenced by these response factors such as ARFs, GH3 and ERFs. Finally, fruit quality in storage was improved under HT.
MAPK cascades play a key role in various cellular processes with accurate signal transduction via phosphorylation of their substrate proteins (Ichimura et al., 2002). In plants, MAPKs have been identified in abiotic stress and hormonal responses, innate immunity, disease resistance and developmental programs (Cristina et al., 2010). Among them, abiotic stress induction of MAPK genes and increased MAPK kinase activity have been detected when plants are subjected to salt, drought, cold, ozone and oxidative stresses (Mizoguchi et al., 1996), but there have been few studies of the MAPKs under high temperature stress in plants. In our study, the MEKK1-MKK2-MPK4/6 displayed high transcript abundances under HT. This is similar to the findings in the research of plant MAPK signaling during abiotic stress. In A. thaliana , MKK2 plays a pivotal role in cold and salt stress responses following the stress-induced MEKK1 and followed by the downstream MAPK4 and MAPK6 (Teige et al., 2004). MAPK4 and MAPK6 are also rapidly activated by wounding and touch associated with tyrosine phosphorylation (Ichimura et al., 2002). In addition, high temperature up-regulates the expression of MPK6 and AtMPK6-phosphorylated HSFA2 might participate in the response inA. thaliana (Li et al., 2012b). While in tomato, SIMPK1, a close homolog to AtMPK6, showed a negative effect on thermotolerance by regulating antioxidant defense via its substrate SISPRH1 involved in this pathway (Ding et al., 2018). To sum up, these data identified that the MEKK1-MKK2-MPK4/6 cascades mediate high temperature response in peach. Analyzing loss and over-expression of these genes in peach fruit would help to determine their potential effects in HT stress.
Ethylene is a major plant hormone involved in the regulation of many fruit developmental processes from maturation to ripening and senescence (Bapat et al., 2010; Pech et al., 2012; Kumar et al., 2014) . It was identified as a trigger and promoter in typical climacteric fruits ripening and can crosstalk to other phytohormones including auxin (Aux), ABA, jasmonic acid (JA) through controlling their biosynthesis pathway and signaling pathway (Kumar et al., 2014). However, there are few studies on the effect of temperature on ethylene metabolism and the corresponding molecular mechanism. In our study, the expression levels of ACO and ACS genes and their enzyme activities were both decreased in HT samples, resulting in lower ethylene production than CT fruits, which indicated that the ethylene biosynthesis that is affected by heat stress plays an important role in peach fruit ripening process. Similarly, in grapevine berry, ACO and ACS genes were also less expressed under the high temperature regime of 41.7 °C (the maximum air temperature) (Pastore et al., 2017). In Kiwifruit, fruit ripening was inhibited by decreased ACS and ACO activities at high temperature above 30 °C (Antunes and Sfakiotakis, 2000). These showed that temperature has direct effect on the process of ethylene biosynthesis especially on its ACO and ACS enzymes. But what exact reason the maturity inhibition effect is attributed to has not been determined; for example, it can be a lack of ethylene production, the inability to react to ethylene, or other reasons (Burg, 1962). EIN3 and ERFs, the downstream elements of ethylene signaling pathway, play important roles in development, defense, and environment related responses in fruits (Gutterson and Reuber, 2004; Mizoi et al., 2012; Licausi et al., 2013). The ERF proteins, specifically binding to an AGCCGCC element (GCC box), were found in the promoter region of ethylene-regulated genes and could respond to ethylene, pathogens, and wounding (Ohme-takagi and Shinshi, 1995). In this study, the expression levels of ERFs under HT condition were clustered into several groups (Supplementary Figure S4 ) and co-expressed with several genes related to membrane oxidation and cell wall metabolism (Supplementary Figure S10 ). In Arabidopsis , ERF genes were involved in cell expansion which requires the proteins EXP and the actin modeling factor ADF5 (Marsch-Martinez et al., 2006), and also link to ethylene signaling and auxin biosynthesis (Mao et al., 2016). Ethylene response element binding protein (EREBP, Prupe.1G432000) in group Ⅱ (Supplementary Figure S4 ) was co-expressed with PE, POD and AUX genes in peach fruits, and its ortholog APD1(AT4G13040.1) in Arabidopsis has been reported as an important regulator for basal plant defense and abiotic stress response (Giri et al., 2014). This raises a possibility that several downstream genes associated with flesh softening and membrane oxidation could be involved in the ERFs response to heat stress. Clarifying the relationship between them should be a goal of future work on heat response.
It has been reported that MAPK cascades regulate ethylene biosynthesis and signal transduction by protein phosphorylation and the expression of ACS genes (Meng and Zhang, 2013; Li et al., 2017). ACS proteins are substrates of MPK3 and MPK6, and the phosphorylation of ACS results in an increase in ethylene production in tobacco and Arabidopsis(Liu and Zhang, 2004; Han et al., 2010; Li et al., 2012a). In our results, the expression of MAPK4 and MAPK6 were induced by high temperature at early stage (Figure 4b ) and meanwhile low ACS1 expression and ethylene production were observed. This implies the potential association between MAPK cascades and ACS genes in peach fruits, which needs further experimental verification.
In summary, we proposed a model of the molecular response mechanism of peach fruit at high temperature: under HT condition, MAPK cascade acts as a signal sensor and triggers the down-regulation of ethylene production and auxin synthesis; ERF/ARF proteins regulate cell wall enzymes and membrane enzymes; these processes ultimately help to maintain fruit quality. A series of direct or indirect possible regulations are proposed in the model, such as ACS1/ACO for ethylene production and EXP for fruit hardness. Future validation of these regulations will enhance our understanding on the high temperature response of fruits. The findings of this study provide new insights into the molecular mechanism of temperature adaptation and have implications for high temperature domestication of fruits.