**4. Discussion**

Sweet potato is now globally recognized as a functional food due to its preventive and therapeutic effects against chronic diseases [1]. However, the sweetness of freshly harvested sweet potato tuberous roots is not enough, which limits the acceptance of consumers. Cold storage can improve the sweetness of sweet potato tuberous roots, but long-term storage will cause CI. The development and likelihood of CI can also depend on the sweet potato origin, variety, cultivar, harvest season, temperature, and chilling stress duration [20]. Therefore, precise control of storage temperature and time may improve tuberous root sweetness without resulting in CI. Our study demonstrated that the sweetness of mature "Xinxiang" sweet potato tuberous roots increased 2-fold after 14 d of storage at 5 ◦C, which significantly improved their taste according to our sensory tests. Moreover, no CI was detected at this time point. During storage at 13 ◦C, the soluble sugar and sweetness index of the sweet potato tuberous roots did not change significantly, which contrasted with the findings of Ru et al. [21], who demonstrated that the levels of sugars in tuberous roots stored at both 13 and 4 ◦C increased after 15 d. We speculate that these differences were related to the maturity of the sweet potatoes. In this study, sweet potatoes were harvested 135 d after planting, which was different from our previous study (Ru et al.) [21]. More mature tuberous roots appeared to be less sensitive to low-temperature stress [21]. Therefore, there were no observable differences in the soluble sugar contents of tuberous roots stored at 13 ◦C even after 21 d.

Phenolic compounds are naturally occurring chemicals that defend sweet potatoes and other plants against biotic and abiotic stressors [22–24]. Chlorogenic acid (5-Ocaffeoyliquinic acid, CGA) is the predominant component of phenolic acids in sweet potato tuberous roots and is the primary contributor to its antioxidative, antimutagenic, and radical-scavenging properties [25–27]. The increase in phenolic compound contents due to low-temperature exposure may enhance the nutraceutical value of sweet potato tuberous roots. The chlorogenic acid content in "Xinxiang" sweet potato tuberous roots increased markedly after 14 d of storage at 5 ◦C. This is consistent with the findings of Ishiguro et al. [28], who detected a significant increase in total phenolic content in sweet potato tuberous roots after two weeks of low-temperature exposure.

Low temperature also increases the concentration of some free amino acids in sweet potato tuberous roots, thus enhancing the nutritional value of cold-stored tuberous roots. However, tyrosine is one of the main substrates of enzymatic browning. Specifically, the enzyme tyrosinase oxidizes tyrosine to produce quinones. This, in turn, leads to melanin production, resulting in browning [29]. Therefore, a large amount of tyrosine accumulated during long-term low-temperature storage can lead to browning/blackening in fruits and vegetables [30]. This is consistent with the internal browning of sweet potato tuberous roots with surface cuts after 21 d of storage at 5 ◦C and 3 d at room temperature.

Several studies have assessed the effects of low temperature on transcript profiles in stored sweet potato tuberous roots. For instance, Ji et al. [8] analyzed the transcriptome changes of sweet potato tuberous roots stored at an optimal (13 ◦C) and low temperature (4 ◦C) for 6 weeks and found that the tuberous roots could resist CI by inducing genes related to the biosynthesis of unsaturated fatty acids, pathogen defense, and phenylalanine metabolism. These results indicate that phenylalanine metabolism can affect the production of phenolic compounds at low temperatures, which is consistent with the results of the present study. Additionally, their study demonstrated that membrane damage may be the main cause of chilling injury, thus highlighting the critical importance of lipid metabolism to improve the stress resistance of tuberous roots under low-temperature storage conditions. Xie et al. [31] also conducted transcriptomic analyses on "xushu18" sweet potato tuberous roots and found that the expression of genes associated with carbohydrate metabolism was regulated during cold storage, leading to the accumulation of sucrose in sweet potato tuberous roots, which was consistent with the results of this study. There have been reports on long-term cold storage of sweet potato tuberous roots to study the molecular mechanism of CI in tuberous roots, but there are no transcriptome reports on short-term cold storage of sweet potato tuberous roots. In this study, RNA-Seq was used to explore the molecular mechanisms through which short-term low-temperature storage enhances sweet potato tuberous roots' quality. The concentrations of soluble sugars, phenolic compounds, and amino acids increased significantly after short-term low-temperature storage.

Sucrose synthase (SUS; EC 2.4.1.13), sucrose phosphate synthase (SPS; EC 2.4.1.14), and acid convertase (AI; EC 3.2.1.26) are key enzymes in sucrose metabolism (Figure 7). Among them, sucrose synthase can catalyze the reversible reaction of sucrose synthesis and decomposition [32]. Previous studies have reported that low temperatures increase the activity of sucrose synthase in wheat [33]. In this study, low-temperature storage upregulated the expression of two sucrose synthase genes. SPS is a rate-limiting enzyme in the synthesis of sucrose [34]. In this study, SPS expression was downregulated in the CS treatment, suggesting that the regulation of SPS activity is highly complex and may not be affected at the transcript level [21].

In plants, CGA biosynthesis occurs downstream of the phenylpropanoid pathway (red and blue color represents upregulation and downregulation, respectively) (Figure 7). Phenylalanine generates P-coumaroyl-CoA, which in turn is catalyzed by key enzymes in the biosynthesis of chlorogenic acid, including phenylalanine ammonia-lyase (PAL; EC 4.3.1.24), cinnamic acid 4-hydroxylase (C4H; EC 1.14.14.91), 4-coumarate-CoA ligase (4CL; EC 6.2.1.12), hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT; EC 2.3.1.133), β-coumaroylester 3-hydroxylases (C3H; EC 1.14.14.96), and hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferase (HQT; EC 2.3.1.99) [35,36]. In this study, several key genes belonging to the chlorogenic acid synthesis pathway, such as 3 PAL, 1 4CL, and 1 HQT, were regulated by low temperature. The expression levels of PAL in the control and CS groups increased by 5.69- and 22.71-fold, 4CL increased by 1.17 and 2.72-fold, respectively, and HQT increased by 2.20- and 4.47-fold, respectively. Further, some key genes in the shikimic acid pathway were also upregulated by low temperature, which might have contributed to the accumulation of tyrosine in CS tuberous roots.

**Figure 7.** Color, sucrose content, and chlorogenic acid content changes in sweet potato tuberous roots during 14 d of storage. The figure contains the metabolite map of the sucrose and chlorogenic acid synthesis pathway. The relative expression levels of C0, C14, and CS14 are shown as heat maps.
