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.