Morphological variations under cold storage
To investigate the effect of chilling stress on the storage of sweetpotatoes, freshy harvested ones (cv. Xinxiang) were stored in the storage chamber of 13°C (CK) and 4°C for 14 days (d). As shown in Fig. 1 and Table 1, roots at 13°C showed no chilling injury (CI) symptoms, while the epidermis of roots exposed to 4°C (Fig. 1B) were significantly spotted and shriveled than those stored at 13°C (Fig. 1A). The CI index was also significantly higher than that of control roots. In addition, the water content exhibited significantly decreases under 13°C, and no differences were found under low temperature (4°C).
Table 1 CI index and water content of sweetpotatoes after storage at different temperatures
Storage time (d)
|
CI index
|
Water content (%FW)
|
13°C
|
4°C
|
13°C
|
4°C
|
0
|
0.0±0.0a
|
0.0±0.0b
|
64.5±2.5a
|
64.5±3.1a
|
14
|
0.0±0.0a
|
0.7±0.1a
|
60.7±1.6b
|
64±2.7a
|
Effects of low-temperature storage on oxidative stress
The relative electrical conductivity (REC) level and malondialdehyde (MDA) content were significantly higher in the roots exposed to 4℃ condition than that at 13℃ (Fig. 2). The activities of SOD, CAT, APX, O2.- producing rate, proline and soluble sugar contents have been shown in Fig. 3. Similarly, the low temperature (4℃) significantly increased the activities of antioxidant enzymes (Fig. 3A, B, C) and the production rate of O2.- (Fig. 3D). Moreover, chilling stress also enhanced the proline (Fig. 3E), glucose, fructose and sucrose (Fig. 3F) contents. It's worth mentioning that three types of soluble sugar contents were increased most among above of physiological indexes, by 112.4%, 145.6% and 139.4%, respectively.
Segregation and identification of proteins
Compared to the control, 266 and 158 proteins were found significantly up- and down-regulated by >1.5 fold, respectively in roots under 4℃ storage (Supplementary Table S2, Additional file 1 and Additional file 2). The protein bands were clear, uniform and not degraded in each lane (Supplementary Figure S1). The molecular masses of identified proteins were distributed 5-275 kDa, with majority of proteins (96%) distributed in the range of <100 kDa (Supplementary Figure S2). The extracted proteins were suitable for further LC-MS/MS analysis.
Annotation of DEPs in GO classification, subcellular localization and pathway enrichment
Annotation of differentially expressed protein (DEPs) function and their cellular location is necessary to understand their roles at molecular level (Additional file 3). The results demonstrated that they were grouped into 15 distinct categories. These proteins were mainly implicated in metabolic processes, cellular components, catalytic activities and binding (Fig. 4A, B, C). Most of them were associated with catalytic activities (~47%), followed by binding (~43%), metabolic process (~40%), cell (~34%) and organelle (~23%).
In addition, the DEPs were delegated based on their presence in a particular compartment (Additional file 4). Most of them were localized in the chloroplast/cytoplasm (~30%), followed by nucleus (~15%) and plasma membrane (~5%) (Fig. 4D).
The identified proteins were further analyzed via KEGG database for interpretation of their involvement in different metabolic pathways (Additional file 5). Most of the DEPs were implicated in pathways related to metabolic pathway (~22%), followed by biosynthesis of secondary metabolites (~16%), and phenylpropanoid biosynthesis (Fig. 4E).
DEPs involved in phenylpropanoid biosynthesis
As previously mentioned, most of proteins were involved in metabolic pathway and biosynthesis of secondary metabolites. Phenolic compounds regulated by phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), Hydroxycinnamoyl transferase (HCT) were listed in Table 2. The p value of these proteins was negatively corelated with their significances in phenylpropanoid biosynthesis pathway. Hence, the significance order of DEPs was shikimate>peroxidase4>4-coumarate-CoA ligase>Cytochrome P450 (cytochrome P450 monooxygenases)>PAL>CAD.
Table 2. Part of DEPs participated in phenylpropanoid biosynthesis
Differentially expressed proteins
|
p value
|
Phenylalanine ammonia lyase
|
5.6×10-9
|
Cinnamyl alcohol dehydrogenase
|
4.3×10-8
|
Peroxidase 4
|
1×10-32
|
Cytochrome P450
|
3.7×10-13
|
4-coumarate-CoA ligase
|
1.1×10-16
|
shikimate O-hydroxycinnamoyl transferase
|
1×10-32
|
|
|
|
Differential multiple of the DEPs participated in starch and sucrose metabolism
As compared to the roots stored at 13℃, there were 11 DEPs participated in starch and sucrose metabolism under 4℃ (Fig. 5). The filtered p value matrix (p<0.05) transformed by the function x=-lg (p value) was conduct to evaluate the celesius4/celesius13 ratio, which was positively corelated with the differential multiple of DEPs. Three proteins (x>1.5) were up regulated, while others (x<1.5) presented an opposite trend in this metabolic pathway. The ratio of sucrose synthase (P11) and β-glucosidase (P3) was 7.19 and 0.56, significantly higher and lower than other proteins, respectively (Fig. 5).
Functional network of the DEPs in starch and sucrose metabolism
The functional network under chilling stress for roots was illustrated in Fig. 6. There were three up- and three down-regulated DEPs. α-amylase (EC: 3.2.1.1, red), associated with starch metabolism and carbohydrate digestion or absorption, was significantly up-regulated when maltodextrin or starch was hydrolyzed to maltose. Furthermore, it was homologous with K01177 (β-amylase: EC: 3.2.1.2), K05992 (maltogenic α-amylase: EC:3.2.1.133) in terms of the orthology analysis. Similarly, both of EC: 2.4.1.13 (sucrose synthase) and EC: 2.7.1.4 (fructokinase) were significantly up-regulated in amino and nucleotide sugar metabolism. On the other hand, EC: 3.2.1.21, EC: 2.7.7.27 and EC: 2.4.1.21 proteins, named as β-glucosidase, glucose-1-phosphate adenylyl-transferase and starch synthase, respectively, were significantly down-regulated in starch and sucrose metabolism pathway. They were mainly involved in phenylpropanoid biosynthesis, biosynthesis of starch and secondary metabolites as well as polysaccharide accumulation. The degradation of starch into soluble sugar can not only boost the sweetness, but also significantly improve the resistance to chilling stress.
Metabolome profiling and its fold change analysis
The metabolome profiling of sweetpotato tubers led to the identification of 76 differentially expressed metabolites (DEMs) in the roots stored at 4℃ as compared to them at 13℃. There were 31 up- and 45 down-regulated metabolites (Supplementary Table S3 and Additional file 6). The absolute value level of fold change (FC) was closely related to significance of the metabolic component. The results (Fig. 7) showed that the absolute Log2FC values of 4 components in up-regulated metabolites were more than 10.00, including glutaric acid (16.69), followed by 3-hydroxy-3-methylpentane-1,5-dioic acid (14.97), apigenin O-malonylhexoside (14.1) and apigenin 7-O-glucoside (cosmosiin) (13.56). Nevertheless, the absolute values of 9 components were more than 10 in down-regulated DEMs, namely sinapoylcholine (14.38), D-glucoronic acid (14.08), N-acetyl-5-hydroxytryptamine (14.5), 5-Methylcytosine (13.32) etc. The metabolic activities of a large proportion of identified components dropped off in roots under 4℃.
Screening and distribution of DEMs in roots under chilling stress
Compared to the absolute value level of fold change, Variable Importance in Project (VIP) value (>1) was extremely associated with the significance of metabolic compound in the corresponding class. All the identified DEMs were categorized into 20 classes. Most of them (~33%) were belonging to nucleotide, its derivates and amino acid derivatives group. On the basis of VIP and Log2FC value, the results (Table 3) illustrated that most of components were down-regulated except 3-hydroxy-3-methylpentane-1,5-dioic acid and glutaric acid. The VIP and Log2FC value of glutaric acid, belonged to organic acids, were the highest (4.01 and 16.69, respectively), followed by D-glucoronic acid (3.69 and 14.08), N-acetyl-5-hydroxytryptamine (3.66 and 14.05) and 5-Methylcytosine (3.58 and 13.32) (Table 3 and Fig. 8A). Carbohydrates were represented by D-glucoronic acid, which was an important member of sugar metabolism..
Furthermore, KEGG pathway enrichment was conducted in terms of their P-values and rich factors. P-value and rich factor had negative and positive correlation with enrichment significance of metabolic compounds, respectively. The P-value of glucosinolate biosynthesis, tropane, piperidine and pyridine alkaloid biosynthesis (9.94×10-3) was obviously lower than protein digestion and absorption (3.56×10-2) (Table 4 and Fig. 8B).
Network of the differential metabolic compounds in glucosinolate biosynthesis
As previously mentioned, glucosinolate biosynthesis, comprised of amino acid such as leucine (Leu), tryptophan (Try), tyrosine (Tyr), isoleucine (Ile) and valine (Val), was significant in metabolic pathways for increasing the chilling tolerance of sweetpotato roots. The glucosinolate can be synthesized from methionine, branched-chain amino acids or aromatic amino acids process (Fig. 9). Leu, Ile and Val were involved in branched-chain amino acids. Try and Tyr were imperative for aromatic amino acids pathway. All these amino acids were significantly up-regulated in glucosinolate biosynthesis (Fig. 9).
Table 3. Screening of differential expressed metabolic components
Compounds
|
Class
|
VIP
|
Log2FC
|
Type
|
Glutaric acid
|
Organic acids
|
4.01
|
16.69
|
up
|
D-glucuronic acid
|
Carbohydrates
|
3.69
|
14.08
|
down
|
N-acetyl-5-hydroxytryptamine
|
Tryptamine derivatives
|
3.66
|
14.05
|
down
|
5-Methylcytosine
|
Nucleotide and its derivates
|
3.58
|
13.32
|
down
|
Esculin
|
Coumarins
|
3.26
|
11.67
|
down
|
3-Hydroxy-3-methylpentane-1,5-dioic acid
|
Amino acid derivatives
|
2.66
|
14.97
|
up
|
O-sinapoyl quinic acid
|
Quinate and its derivatives
|
2.61
|
2.75
|
down
|
Acetyl tryptophan
|
Amino acid derivatives
|
2.45
|
13.08
|
down
|
Sinapic acid
|
Hydroxycinnamoyl derivatives
|
2.39
|
1.72
|
down
|
L-Epicatechin
|
Catechin derivatives
|
2.35
|
11.84
|
down
|
Protocatechuic aldehyde
|
Catechin derivatives
|
2.26
|
10.67
|
down
|
Pantetheine
|
Vitamins
|
2.21
|
5.40
|
down
|
D-arabitol
|
Alcohols and polyols
|
2.15
|
9.97
|
down
|
Table 4. KEGG pathway enrichment of significantly DEMs
KEGG pathway enrichment
|
P-value
|
Compounds
|
glucosinolate biosynthesis
|
9.94×10-3
|
Leu; Try; Tyr; Ile; Val
|
tropane, piperidine and pyridine alkaloid biosynthesis
|
9.94×10-3
|
Putrescine; piperidine; pipecolic acid; Ile; Lys
|
protein digestion and absorption
|
3.56×10-2
|
Putrescine; piperidine; Indole; Val; Ile; Tyr; Try; Arg; Lys; Leu
|
Abbreviation: Leu (Leucine), Try (Tryptophan), Tyr (Tyrosine), Ile (Isoleucine), Val (Valine), Arg (Arginine) and Lys (Lysine)