In cold chain logistics, low temperature plays an important role in transporting perishable foods from production to consumption while ensuring the quality and safety of foods. Low temperature storage could significantly reduce the expression level of numerous genes related to the aroma volatiles synthesis during the storage of tomato fruit (Zou et al. 2018). The postharvest fruit, as independent existence, infinitely depleted nutrient to result in irreversible senescence until death (Wang et al. 2021a). In the present study, the low temperature (11 oC) treatment effectively delayed the senescence of tomato fruit (Fig. 1A-C). The content of total chlorophyll in the low temperature treated fruit was higher than that in the control fruit, and the lycopene content in the low temperature treated fruit was lower than those in control fruit, which cause a noticeable color difference on tomato fruit peel (Fig. 1D and E).
The fruit ripening and senescence connected with degradation of the cell wall, which the enhancement of water-soluble pectin and the reduction cellulose gradually caused fruit to soften (Wang et al. 2021b). Cellulose, as the main component of plant cell walls, can maintain the strength of the cell wall (Wang et al. 2021b). PG was the major hydrolysis enzyme of modifying pectin, which can further decompose pectin acid-generating from that pectin methylesterase (PME) catalyzed pectin demethylation, finally resulting in the structure of cell wall loosening and fruit softening (Rugkong et al. 2010). The better fruit quality was maintained by lower fruit softening enzyme activity (Adhikary et al. 2021). In this study, the PG and cellulase activities in the low temperature treated fruit were also lower than those in the control fruit (Fig. 2C and D), which indicated that low temperature effectively restrained pectin degradation and cell wall injure to delay senescence and soften of tomato fruit.
Similarly, the higher energy consumption can cause the increase of respiratory rate in postharvest fruit to accelerate the development of senescence and disease (Li et al. 2020). ATP was the center of energy conversion within cells, which produce chemical energy through catabolic and anabolic that was used immediately in plant (Vichaiya et al. 2020). The membrane system integrity was connected with cellular energy status. The continuous consumption of cellular energy may lead to membrane damage, while higher-level ATP content can preserve the membrane potential and maintain the membrane system integrity, to delay fruit senescence and to retain qualities (Aghdam et al. 2018). The higher accumulation of energy-matter in kiwifruit could keep an integral cell membrane (Wang et al. 2020). In this study, low temperature treated fruit retained higher ATP content and EC level compared with the control fruit (Fig. 3), which may be contributing to restraining the activities of PG and CE, to delay the fruit senescence. Previous reports showed that MeJA treatment delays the postharvest softening of blueberry and pineapple fruit by altering cell wall modification and energy metabolism (Boonyaritthongchai and Supapvanich 2017). The senescence process of the litchis (Tang et al. 2020), longans (Li et al. 2020) and broccoli (Huang et al. 2021) accompany the reduction of ATP, ADP content and EC level, and the enhancement of AMP.
The process of fruit ripening and senescence is closely related to the biosynthetic and signaling pathway of ethylene. Among the biosynthetic pathways upstream of ethylene, ACS and ACO enzymes are encoded by multigene families. They were differentially expressed in various tissues at different developmental phases (Nakatsuka et al. 1998). The ETR and CTR1 both are a negative regulatory element in the ethylene signaling pathway, which can activate the positive regulatory factor EIN2, pass the downstream EIN3/EILs, promote the expression of the transcription factor ERF and transcript ERT, and ultimately express ethylene-related response genes (Guo et al. 2018; Hu et al. 2012; Trentmann 2000). DNA methylation and demethylation were requested for both the activation of ripening induced genes and the suppression of ripening repressed genes (Lang et al. 2017).
As one of the key enzymes, ACS tightly regulated ethylene biosynthesis (Barry and Grierson, 2000). Among the ACS genes that have been cloned from tomato, LeACS2 and LeACS4 are mainly expressed during the climacteric phase of tomato fruit, resulting in a large amount of ethylene and a characteristic of auto-catalytic ethylene of system II (Anugerah et al. 2015). During the natural ripening of watermelon, the expression of two ACS isoforms, Cla022653 and Cla011522, were significantly up-regulated in 97103 flesh, which play an important role in ethylene biosynthesis and ripening control in watermelon flesh (Zhou et al. 2016). Here, low temperature treatment reduced the expression of the SlACS10 gene (Fig. 5), which might involve inhibiting the ethylene biosynthesis and delaying the senescence of tomato fruit. The DNA methylation level of the CpG island of SlACS10 in low temperature treated fruit was reduced compared with control, the changes of seven DNA methylation sites in the CpG island of SlACS10 might have no critical importance on its expression.
CTR1 is a negative regulator of ethylene signaling, and its interaction with ETR1 is required for the negative regulation of ethylene signaling (Kieber et al. 1993). LeCTR1 silenced tomato plants induce the expression of ethylene-responsive genes, such as ERF5, EIN2, and EIN3 (Chandan et al. 2019). The amino acid sequence of MhCTR1 showed 55% homology to LeCTR1, as well as the changes of MhCTR1 expression in the pulp were closely related to the regulation of the banana senescence process (Hu et al. 2012). Here, the DNA methylation level of CpG island of LeCTR1 in the low temperature treated fruit decreased than those in control fruit at 6 d, and by which raised the expression of the LeCTR1 gene (Fig. 6A-C), and might suppress ethylene signaling. These may be one of the mechanisms of low temperature delayed senescence of tomato fruit.
EIN3 is a key to activate the ethylene transcription factors ERF. EIN3 proteins bind directly to primary ethylene response element (PERE) motifs to regulate gene expression (Solano et al. 1998). In Arabidopsis, EIN3, ORE1 and CCG work together to regulate ethylene-mediated chlorophyll degradation during leaf senescence (Qiu et al. 2015). CpEIN3a was found to increase and participate in carotenoid accumulation during fruit ripening and senescence in papaya (Fu et al. 2017). Compared with the control, low temperature treatment raised the DNA methylation level of LeEIN3 in tomato fruit, reduced the expression of LeEIN3 (Fig. 6D-E), and suppressed ethylene signaling. The increased DNA methylation of LeEIN3 may be involved in low temperature delaying senescence of tomato fruit.
ERFs are DNA-binding proteins belonging to the AP2/ERF family, and coordinate transcription of diverse ethylene-responsive genes (Gu et al. 2002). The expression levels of the four ERF genes in pear increased significantly during fruit senescence. Pbr012024.1 responded to the ethylene signal, and the Pbr022708.1 can be induced by ethylene and is the only ethylene response factor that regulates fruit senescence (Hao et al. 2017; Xu et al. 2018). In tomato, overexpression of LeERF1 accelerated fruit senescence (Li et al. 2007). LeERF2 is induced by ethylene and suppressed in ripening inhibited mutants (Wu et al. 2002). Here, compared with that in control fruit, the DNA methylation levels of SlERF-A1 in low temperature treated fruit reached maximum at 6 d, the expression of SlERF-A1 gene was significantly decreased at 6 and 12 d (Fig. 7A-C), and by which the ethylene signaling might be suppressed. These may be one of the mechanisms of low temperature delayed tomato fruit senescence.
ERT encodes transcripts that regulate ethylene transcription regulation, and participates in the cascade of constitutive cellular factors related to ethylene signal transduction (Trentmann 2000). 49 putative ethylene-responsive transcripts (ERTs) were isolated from etiolated seedlings of Arabidopsis, and ethylene-regulated nuclear protein (ERN) was isolated and cloned from ERT2. Evidence had shown that ERN1 encoded downstream targets of EIN3 protein as did ERF1 (Solano et al. 1998). Hot water treatment delayed the ripening of tomato fruit, which process accompanies the reduction of the expression level of LeERT10 and increase of methylation level (Pu et al. 2020). In the present study, the low temperature treatment enhanced the DNA methylation level of LeERT10 and significantly reduced the expression of the LeERT10 gene in tomato fruit (Fig. 7D-F), and suppressed ethylene signaling. The enhanced DNA methylation level of LeERT10 may be involved in low temperature delaying senescence of tomato fruit.