Proteomic and metabolic profile analysis of low-temperature storage responses in Ipomoea batata Lam. tuberous roots

doi:10.21203/rs.2.22545/v1

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Proteomic and metabolic profile analysis of low-temperature storage responses in Ipomoea batata Lam. tuberous roots

https://doi.org/10.21203/rs.2.22545/v1

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published 21 Sep, 2020

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Background: Sweetpotato ( Ipomoea batatas L.) is one of the seven major food crops grown worldwide, with 70-75% of production in China.Cold stress often can cause protein expression pattern and substance contents variations for tuberous roots of sweetpotato during low-temperature storage. Recently, we developed proteometabolic profiles of the fresh sweetpotatoes (cv. Xinxiang) in an attempt to discern the cold stress-responsive mechanism of tuberous root crops during post-harvest storage.

Results: For roots stored under 4℃ condition, the activities of SOD, CAT, APX, O 2 .- producing rate, proline and especially soluble sugar contents were significantly increased as compared to sweetpotatoes exposed to room temperature (13℃). Most of the differentially expressed proteins (DEPs) were implicated in pathways related to metabolic pathway (~22%), especially phenylpropanoids (~10%) and followed by starch and sucrose metabolism (~3%). α-amylase, sucrose synthase and fructokinase were significantly up-regulated in starch and sucrose metabolism, while β-glucosidase, glucose-1-phosphate adenylyl-transferase and starch synthase were opposite. Furthermore, metabolome profiling revealed that glucosinolate biosynthesis, tropane, piperidine and pyridine alkaloid biosynthesis as well as protein digestion and absorption played a leading role in metabolic pathways of sweetpotato roots. More importantly, leucine, tryptophan, tyrosine, isoleucine and valine were all significantly up-regulated in glucosinolate biosynthesis.

Conclusions: Our proteomic and metabolic profile analysis of sweetpotato roots stored at low temperature reveal that the antioxidant enzymes activities, proline and especially soluble sugar content were significantly increased. Most of the DEPs were implicated in phenylpropanoids and followed by starch and sucrose metabolism. Glucosinolate biosynthesis played a leading role in metabolic pathways of sweetpotao roots. More importantly, leucine, tryptophan, tyrosine, isoleucine and valine were all significantly up-regulated in glucosinolate biosynthesis.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

Sweetpotato

Low-temperature storage

Proteometabolomic

Starch metabolism

Chilling tolerance

Sweetpotato (Ipomoea batatas L.), a dicotyledonous plant which belongs to the Convolvulaceae family, ranks as the seventh-most important food crop in the world. As a major nutrition and harvesting organ, storage root (SR) of sweetpotatoes possess a mass of starch and photoassimilate. Starch accounts for 50–80% proportion of the dry matter which is the major component of the SR [1, 2]. Since soluble sugar content is very low in freshly harvested sweetpotatoes during general production process, a certain time of post-harvest storage at 13–15 ̊C is imperative to facilitate starch-sugar interconversion and boost the sweetness to increase the tuberous food quality before sale. It is noticeable, however, that exposure to low temperature (5 ̊C) for 20 d has been observed to increase sweetness of ‘Kokei 14’ roots, this treatment also caused freezing injury such as rottenness and high rate of carbohydrate loss [3]. Therefore, a better understanding of the biochemical and molecular response mechanisms to chilling stress is essential for extending tuberous crops storage time under low temperature condition.

Compared to the model plants, it is more difficult for sweetpotatoes to find out genes implicated in various stress tolerance because of its complicated genetic background. Although some genomic [4, 5] and proteomic [6, 7, 8] resources of sweetpotatoes have been available now, these pieces of information are still limited to explicate the mechanism of chilling resistant molecular. With the development of sequencing technique, metabolomics has been considered as a powerful complementary tool to acquire the biological information associated with the metabolites. Metabolites are not only the end-products of some genes expressions, but also the consequence of interaction between the genome and its milieu. Therefore, it is probable to envisage the functional genomics assembly by connecting gene expressions to the metabolomic knowledge [9].

It is now generally accepted that the normal storage temperature for sweetpotatoes is 13–15 ̊C. As a chilling-sensitively tropical crop, sweetpotato can be irreparably damaged when the temperature drops below 10 ̊C. A main reason for this damage is oxidative injuries caused by an increased accumulation of reactive oxygen species (ROS) [10, 11, 12, 13, 14, 15, 16]. In plants, stress-induced ROS scavenging is usually implemented by both enzymatic and non-enzymatic low molecular metabolic antioxidants [17, 18]. As we all know, sweetpotato is a tuberous crop which is rich in starch. The starch content in fresh roots of sweetpotatoes is about 15–30% [8]. Soluble sugar not only serve as substrates for starch production, but also may also function as a signal involved in chilling defense for tuberous roots during low-temperature storage.

To better explore the proteins and metabolic pathways under chilling condition, we carried out the proteometabolomic profile of fresh sweetpotatoes to clarify the cold stress-responsive mechanism. Integration of proteomic and metabolomic profiles information resources would give new insights into the molecular functions of tuberous root crops during post-harvest storage. This would provide a basis for future comparative proteomic efforts for this important crop including gene discovery and improvement of chilling stress tolerance.

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 13 °C (CK) and 4 °C incubator for 14 days. As shown in Fig. 1, sweetpotatoes exposed to 4 °C (Fig. 1B) were significantly smaller, spotted and thinner than those stored at 13 °C (Fig. 1A). In addition, the color under cold storage was also markedly darker compared to those in normal condition.

Antioxidant enzymes and soluble sugar contents

The activities of SOD, CAT, APX, O₂⁻ producing rate, proline and soluble sugar contents in the roots of I. batata L. cv. Xinxiang exposed to cold storage condition have been shown in Fig. 2. The low temperature (4℃) significantly increased the activities of antioxidant enzymes (Fig. 2A, B, C) and the producing rate of O₂.⁻ (Fig. 2D) as compared to the control roots (13℃). Not only that, chilling stress also enhanced the proline (Fig. 2E), glucose, fructose and sucrose (Fig. 2F) 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, as compared to the specific control under freezing temperature storage condition.

Segregation and identification of proteins

Compared to the control roots, 266 and 158 proteins were found significantly up- and down-regulated by > 1.5 fold, respectively in sweetpotato roots under 4℃ storage (Supplementary Table S2, Additional file 1 and Additional file 2). 30 µg protein were loaded into SDS-PAGE and separated by 1-DE. The protein bands showed that clear, uniform and not degraded in each lane (Supplementary Figure S1). The molecular masses of identified proteins were distributed between 5 and 275 kDa, with majority of proteins (96%) distributed in the range of < 50 to 100 kDa (Supplementary Figure S2). These results showed that extracted proteins were suitable for LC-MS/MS analysis when lacerated from the gel and subjected to trypsin lysis.

Annotation of differentially expressed proteins in GO classification, subcellular localization and pathway enrichment

Annotation of differentially expressed protein (DEP) function and their cellular location is necessary to understand their roles at molecular level and therefore, the identified tuberous roots proteins under 4 °C and 13 °C were subjected to Blast2Go annotation to understand their molecular roles (Additional file 3). The analysis 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. 3A, B, C). Most of them were associated with catalytic activities (~ 47%), followed by binding (~ 43%), metabolic process (~ 40%), cell (~ 34%) and organelle (~ 23%). Proteins whose function could not be ascertained in chilling storage condition, were designated as unknown or uncharacterized proteins (~ 4%) as compared to room temperature.

In addition, wolfpsort software was used to predict subcellular localization of the proteins. The proteins 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. 3D).

The identified proteins were further analyzed via KEGG database for interpretation of their involvement in different metabolic pathways (Additional file 5). Most of the proteins were implicated in pathways related to metabolic pathway (~ 22%), followed by biosynthesis of secondary metabolites (~ 16%), and phenylpropanoid biosynthesis. Starch and sucrose metabolism ranked the sixth (Fig. 3E).

Differentially expressed proteins involved in phenylpropanoid biosynthesis

As previously mentioned, most of proteins were involved in metabolic pathway and biosynthesis of secondary metabolites. Phenolic compounds regulated by differentially expressed proteins (DEPs) such as phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), Hydroxycinnamoyl transferase (HCT) were listed in Table 1. The p value of these proteins was negatively corelated with their significances in phenylpropanoid biosynthesis pathway. Hence, the significance order of DEP was shikimate and peroxidase4 > 4-coumarate-CoA ligase > Cytochrome P450 (cytochrome P450 monooxygenases) > PAL > CAD.

Table 1

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 tuberous roots stored at 13℃, there were 11 differentially expressed proteins (DEPs) participated in starch and sucrose metabolism of roots under low-temperature condition (4℃) (Fig. 4). 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 DEP. 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. These results appeared to indicate that these two proteins play a more important role in starch and sucrose metabolism (Fig. 4).

Functional network of the DEPs in starch and sucrose metabolism

Two-tailed Fisher’s exact test was conducted to reveal proteins association network. The network for tuberous roots under chilling stress are illustrated in Fig. 5 as compared to the controls in room temperature. There are three up- and three down-regulated differentially expressed proteins (DEPs). EC: 3.2.1.1 (red) protein, α-amylase which 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) protein played a significantly up-regulated role in amino and nucleotide sugar metabolism in the chilling tolerance of sweetpotato roots. On the other hand, EC: 3.2.1.21, EC: 2.7.7.27 and EC: 2.4.1.21 proteins (green) were significantly down-regulated in starch and sucrose metabolism of roots stored at 4℃, which was named as β-glucosidase, glucose-1-phosphate adenylyl-transferase and starch synthase, respectively. They were mainly involved in phenylpropanoid biosynthesis, biosynthesis of starch and secondary metabolites as well as polysaccharide accumulation. These findings thus showed that the degradation of starch into soluble sugar can not only boost the sweetness, but also significantly improve the resistance of sweetpotato roots 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). PLS-DA statistic method was applied to investigate the fold change among the different metabolic components. The absolute value level of fold change (FC) was closely related to significance of the metabolic component. The results (Fig. 6) showed that in the up-regulated metabolites, the absolute Log₂FC value of 4 components were more than 10, including glutaric acid (16.69), 3-hydroxy-3-methylpentane-1,5-dioic acid (14.97), apigenin O-malonylhexoside (14.1) and apigenin 7-O-glucoside (cosmosiin) (13.56). Nevertheless, 9 components were more than 10 in down-regulated DEM, namely sinapoylcholine (14.38), followed by D-glucoronic acid (14.08), N-acetyl-5-hydroxytryptamine (14.5), 5-Methylcytosine (13.32) etc. We can speculate that the metabolic activities of a large proportion of identified components may drop off for tuberous roots under low temperature storage (Fig. 6).

Screening and distribution of DEMs in tuberous roots under chilling stress

Metabolites among different varieties or organizations can be preliminarily screened based on the OPLS-DA (Partial Least Squares-Discriminant Analysis) statistic results. However, it is more feasible to screen out metabolites accurately with the combined application of fold change and VIP (Variable Importance in Project) value obtained from OPLS-DA model. Compared to the absolute value level of fold change, 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 the members of nucleotide and its derivates and amino acid derivatives group. The screened metabolic compounds on the basis of VIP and Log₂FC value were demonstrated in Table 2. The results illustrated that most of components were down-regulated except glutaric acid and 3-hydroxy-3-methylpentane-1,5-dioic acid. The VIP and Log₂FC value of glutaric acid, which belonged to organic acids, were the highest (4.01 and 16.69, respectively), then 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 2 and Fig. 7A). Carbohydrates were represented by D-glucoronic acid, which was an important member of sugar metabolism. Organic acids and nucleotide/amino acid derivates played a vital role in sweetpotato roots response to chilling stress.

Furthermore, KEGG pathway enrichment was conducted to in terms of their P-value and rich factor. P-value and rich factor had negative and positive correlation with enrichment significance of metabolic compounds, respectively. As Table 3 and Fig. 7B shown, 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). Therefore, these three metabolic pathways may play a key role for sweetpotao roots in the response improvement to chilling stress.

Network of the differential metabolic compounds in glucosinolate biosynthesis

As previously mentioned, glucosinolate biosynthesis, comprised of amino acid such as Leucine, Tryptophan, Tyrosine, Isoleucine and Valine, 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. 8). Leucine, Isoleucine and Valine were involved in branched-chain amino acids. Tryptophan and Tyrosine were imperative for aromatic amino acids pathway. All these amino acids were significantly up-regulated in glucosinolate biosynthesis (Fig. 8).

Table 2

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 3

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)

ROS scavenging and osmotic adjustment substances

Higher plants growth, productivity and distribution are severely limited by environmental stresses including freezing, drought and salinity. Plants have evolved in the presence of ROS and have acquired dedicated pathways to protect themselves against oxidative damage and fine modulation of low levels of ROS for signal transduction [19, 20, 21, 22, 23, 24]. The enzymatic systems of ROS scavenging mechanisms mainly include SOD, POD, CAT, APX and GPX [25]. Among the enzymatic systems, SOD is able to rapidly convert ·OH to H₂O₂, and the generated H₂O₂ is then converted to water and dioxygen by POD and CAT [26, 27, 28]. Kim et al. (2013) [29] reported that 11 genes involved in ROS scavenging enzymatic system were isolated and identified from sweetpotato roots. The expression of intracellular genes CuZnSOD, swAPX1 and GST was significantly correlated with low temperature stress (4℃). Furthermore, the tolerance of transgenic sweetpotatoes to salt stress was significantly improved after CuZnSOD and APX transferred into salt-sensitive sweetpotatoes. Induction of osmoprotectants biosynthesis is another type of the plant response to low temperature. Several studies have attributed an antioxidant feature to proline, suggesting ROS scavenging activity and proline acting as a singlet oxygen quencher [30]. Increasing amounts of data suggested that increased abiotic stress tolerance was obtained by introducing simple metabolic traits from other organisms such as the production of trehalose and proline [31] to plants. Furthermore, proline can protect and stabilize ROS scavenging enzymes and activate alternative detoxification pathways. In salt-stressed tobacco cells, proline increased the activities of methylglyoxal detoxification enzymes, enhanced POD, SOD and CAT activities [32]. Recent data suggested that overexpression of DlICE1 (Dimocarpus longan L.) in transgenic Arabidopsis conferred enhanced cold tolerance via increased proline content and antioxidant enzyme such as SOD, CAT, APX [33]. In our research, the low temperature (4℃) significantly increased the activities of antioxidant enzymes (Fig. 2A, B, C), the producing rate of O₂.⁻ (Fig. 2D) and proline content (Fig. 2E) as compared to the control roots (13℃). Thus, less damage from membrane lipid peroxidation enabled the sweetpotato roots to continue normal metabolism under low-temperature condition, contributing to their higher cold tolerance.

The role of endogenous phenolics compounds and glucosinolate biosynthesis under abiotic stress

Phenylpropanoids are a group of secondary metabolites synthesized from the amino acid phenylalanine [34, 35, 36]. In plants, the phenylpropanoid pathway underlying abiotic stress tolerance is tightly connected with physiological and molecular mechanisms. Phenolic accumulation is usually activated when plants face multiple abiotic stresses [37]. Increased phenolic levels play crucial role in plants protection against chilling stress [38]. Gao et al (2016) [39] confirmed that stimulated phenolic biosynthesis was owing to the enhanced expression of PAL, CAD and HCT by carrying out the experiments with peach under low-temperature stress. In our research, the phenylpropanoid biosynthesis, a main metabolic pathway, were regulated by lots of proteins, especially HCT, PAL and CAD (Fig. 3E and Table 1). Thus, our results were coincidence with former research. More importantly, the thickness of plant cell walls is enhanced due to phenolic accumulation, which is beneficial for the prevention of chilling injury [40, 41].

Glucosinolates, also known as mustard oil glucosides, are nitrogen- and sulfur-containing compounds found in the Arabidopsis and oilseed rape (Brassica napus L.). They mainly function as defense molecules [42]. Over 100 glucosinolates are found in plants [43]. Depending on their precursor amino acids, glucosinolates can be categorized into indole glucosinolates derived from Trp, aliphatic glucosinolates from Ala, Leu, Ile, Val, or Met, and aromatic glucosinolates from Phe and Tyr [42]. These results were consistent with our research (Table 3 and Fig. 8). Studies with two Arabidopsis mutants, reduced epidermal fluorescence2 (ref2) and ref5, demonstrated that the accumulation of glucosinolate biosynthetic intermediates can limit the production of phenylpropanoids [44, 45]. However, it seems like that there was no obviously crosstalk between phenylpropanoids and glucosinolates biosynthesis, which may be needed to confirm with further experiments.

Sugar as antioxidants in sweetpotato roots

Sweetpotato has been known as one of the highest starch producing crops due to their higher sink strength [46]. Starch was synthesized from photoassimilate sucrose, which is the most common form of storage of carbohydrates in cells [47]. The metabolism of sucrose and other soluble sugars thus not only acted as a key indicator to evaluate the quality of sweetpotatoes, but also affected various metabolic processes [48]. This protection by soluble sugars may occur if sugar fluxes can be maintained or targeted towards cellular zones of ROS stress [49, 50]. Soluble sugars were linked with the production rates of ROS by regulating its producing metabolic pathways, such as mitochondrial respiration or photosynthesis [51]. It was known that starch content has significantly negative relationship with sucrose in tuber crops, because the catabolism of starch been impaired with respiratory rate decrease in low storage temperature. In our study, three types of soluble sugar contents were obviously increased (Fig. 2F). Moreover, α-amylase, β-amylase, sucrose synthase and fructokinase were significantly up-regulated, which boosted the sweetness of the tuber roots under cold temperature as compared to the control roots (Fig. 4 and Fig. 5). These results confirmed that sweetness could enhance the chilling stress tolerance of sweetpotato roots.

Nevertheless, the concept ‘sugar as antioxidant’ has been put forward in recent years [52], and it is increasingly clear that water-soluble carbohydrates (glucose, fructose and sucrose) are regarded as key regulators in plant responses under abiotic or biotic stress. Sperdouli and Moustakas (2012) [53] reported an accumulation of increased soluble sugars maintaining a high antioxidant protection in Arabidopsis thaliana leaves under drought stress. Furthermore, some genes encoding different sugar compounds has been confirmed to enhance freezing tolerance in petunia, tobacco and rice [54, 55, 56, 57, 58]. Hence, by definition, soluble carbohydrates are synthesized in response to osmotic stress, acting as osmoprotectants that stabilize cellular membranes and maintain turgor [59]. Both simple sugars and polysaccharides are able to protect cellular membranes, which is a prerequisite for survival under stress conditions [60, 61].

In summary, our proteomic and metabolic profile analysis of sweetpotato roots stored at low temperature reveal that the increased activities of SOD, CAT, APX, O₂.⁻ producing rate, proline and especially soluble sugar contents as compared to controls (13℃). Most of the proteins were implicated in pathways related to metabolic pathway, especially phenylpropanoids and followed by starch and sucrose metabolism. α-amylase, sucrose synthase and fructokinase were significantly up-regulated in starch and sucrose metabolism, while β-glucosidase, glucose-1-phosphate adenylyl-transferase and starch synthase were opposite. Furthermore, glucosinolate biosynthesis played a leading role in metabolic pathways of sweetpotao roots. More importantly, leucine, tryptophan, tyrosine, isoleucine and valine were all significantly up-regulated in glucosinolate biosynthesis. These results would expand our understanding of the proteome as well as metabolome and give new insights into how to resist the freezing stress for sweetpotato tubers.

Plant materials and storage condition

Sweetpotatoes (Ipomoea batatas L. cv. Xinxiang), obtained from Zhejiang Academy of Agricultural Sciences of China, see supplementary Table S1, were grown in the greenhouse at 25–30℃ under a long-day photoperiod (16/8 h, light/dark) according to standard agricultural practices in 2018. Mature tuberous roots were selected with 5–7 cm in central diameter. Then, they were divided into two groups and stored in 4℃ and 13℃ (CK) incubator for 14 days, respectively. Each treatment has four individual tubers with three replications. The tubers were sliced to 1 mm thickness, then put into liquid nitrogen and stored at -80℃ for further analysis.

Determination of lipid peroxidation and antioxidant enzyme

For enzyme activities, the fresh roots (0.1–0.5 g) were homogenized in 10 ml of 50 mM precooled potassium phosphate buffer (PBS; pH 7.8) under ice cold conditions. Homogenate was centrifuged at 8,000 rpm for 20 min at 4℃ and the supernatant was remained for the determination of following enzyme activities. Superoxide anions (O₂^·−) producing rate was determined according to Jiang & Zhang (2001) [62] method with some modifications. Superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC1.11.1.11) activity was determined according to Dhindsa & Matowe (1981) [63], Aebi (1984) [64], and Nakano & Asada (1981) [65], respectively. Moreover, proline content was measured according to Bates et al. (1973) [66]. Total soluble sugar content was determined by using the method of anthrone colorimetry, and glucose solution was used as a standard. The absorbance in the supernatant was read at 630 nm.

Protein extraction and 1-DE SDS-PAGE

The flesh of sweetpotato was grinded by liquid nitrogen into cell powder and four volumes of lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail) were added to sonication extract three times on ice using a high intensity ultrasonic processor. The remaining debris was removed by centrifugation at 12,000 g at 4 °C for 10 min. Then, the supernatant was collected and the protein concentration was determined with Enhanced BCA kit (Beyotime, P0009) according to the manufacturer’s instructions. The aliquots of 30 µg protein samples were denatured for 5 min with 5 µl 4 × loading buffer and 2% SDS to 20 µl final volume. The electrophoresis was performed with 12% SDS PAGE gels and stained by Coomassie Blue R-250.

LC-MS/MS of digested peptides

The tryptic peptides were loaded onto a home-made reversed-phase analytical column (15 cm length, 75 µm i.d.). The mass ratio of trypsin-to-protein was 1:50 and 1:100 for the first digestion overnight and a second 4 h-digestion, respectively. The digested peptide was subjected to NSI source followed by tandem mass spectrometry (LC-MS/MS) in Q Exactive™ Plus (Thermo Scientific) coupled online to the UPLC. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion.

Database searching of proteins

The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8) in uniport Toxoplasma gondii database. Tandem mass spectra were searched against Vert_tom_20141002 database (117,248 entries). Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in First search and 5 ppm in Main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cysteine was specified as fixed modification and oxidation on Met was specified as variable modifications.

Bioinformatics analysis

The Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). Identified proteins domain functional description were annotated by InterProScan (http://www.ebi.ac.uk/interpro/) based on protein sequence alignment method used InterPro domain database. The KEGG (Kyoto Encyclopedia of Genes and Genomes) database was used to annotate protein pathway. Localization of proteins was predicted with wolfpsort software (PSORT/PSORT II). For functional enrichment, a two-tailed Fisher’s exact test was conducted to test the enrichment of the differentially expressed protein (DEP) against all identified proteins.

Metabolome profiling of tubers

100 mg flesh powder was extracted overnight at 4 °C with 1.0 ml 70% aqueous methanol. The extracted proteins were analyzed using an LC-ESI-MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30A, MS, Applied Biosystems 6500 Q TRAP). LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (Q TRAP), equipped with an ESI Turbo Ion-Spray interface, operating in a positive ion mode and controlled by Analyst 1.6.1 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 500℃; ion spray voltage (IS) 5500 V; ion source gas I (GSI), gas II (GSII), curtain gas (CUR) were set at 50, 60, and 25 psi, respectively. Instrument tuning and mass calibration were performed with 10 and 100 µmol/l polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. DP and CE for individual MRM transitions was done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period.

Protein and metabolite data analysis

Proteins absent in two storage treatments of six (3 × 2) samples were filtered out prior to analysis. Each metabolite was searched on metware database (MWDB) (https://www.metware.cn) and PubChem (https://pubchem.ncbi.nlm.nih.gov/) was used for their classifications. Significance analysis of possible comparisons (p value) were tested at p < 0.05. The Filtered p value matrix (p < 0.05) transformed by the function x=-lg (p value) was used to evaluate the ratio of celesius4 to celesius13, which was positively corelated with the expression multiple of the DEP (x > 1.5). A set of significant proteins in each combination were further analyzed for Gene Ontology Enrichment of Biological Process (BP). The analysis of variance and mean separation of all metabolites were performed with Analyst 1.6.1, Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) model. Figures were drawn using origin2018.

APX: ascorbate peroxidase; Arg: Arginine; CAD: cinnamyl alcohol dehydrogenase; CAT: catalase; DEMs: differentially expressed metabolites; DEPs: differentially expressed proteins; FC: fold change; GO: Gene Ontology; HCT: Hydroxycinnamoyl transferase; Ile: Isoleucine; KEGG: Kyoto Encyclopedia of Genes and Genomes; Leu: Leucine; Lys: Lysine; MWDB: metware database; OPLS-DA: Orthogonal Partial Least Squares-Discriminant Analysis; PAL: phenylalanine ammonia lyase; PLS-DA: Partial Least Squares-Discriminant Analysis; ROS: reactive oxygen species; O₂^·−: Superoxide anions; SOD: Superoxide dismutase; Try: Tryptophan; Tyr: Tyrosine; Val: Valine; VIP: Variable Importance in Project.

Ethics approval and consent to participate

The voucher specimen of sweetpotato was deposited in Zhejiang Academy of Agricultural Sciences. Their taxon, variety, voucher, geographic origin and identifier were listed in supplementary Table S1.

Consent to publish

Not applicable.

Availability of data and materials

The data generated during this study are all included in the supplementary information files.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the National Natural Science Foundation of China (No. 31871857), Zhejiang Provincial Natural Science Foundation of China (No. LY19C200015), and the Earmarked Fund for China Agriculture Research System (No. CARS-10-B19) and the Scientific Research Fund of Zhejiang A&F University (2017FR026).

Authors’ contributions

PC and HY conceived the research plan, analysed the data and wrote the manuscript. CC and YH did the sugar content analysis. YL and GL performed antioxidant enzymes measurements. All authors read and approved the final manuscript.

Acknowledgements

We thank Prof. Liehong Wu who kindly provided the sweetpotato tubers.

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Additional file 1: The peptides information of proteins identified in I. batata roots stored at 4℃ compared to tubers under 13℃ (CK) were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC.

Additional file 2: Protein description of DEPs in I. batata roots by >1.5 fold.

Additional file 3: Functional classification of Gene ontology (GO) annotation of I. batata proteins. Proteins were assigned into three categories: biological process, cellular components and molecular functions.

Additional file 4: Subcellular location of differentially expressed proteins (DEPs) identified by >1.5 fold in I. batata roots under cold stress (4℃) compared to CK.

Additional file 5: KEGG annotations of DEPs in I. batata roots under cold stress (4℃) compared to CK.

Additional file 6: Differentially expressed metabolites (DEMs) information in I. batata roots under cold stress (4℃) compared to CK.

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Proteomic and metabolic profile analysis of low-temperature storage responses in Ipomoea batata Lam. tuberous roots

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Results

Morphological variations under cold storage

Antioxidant enzymes and soluble sugar contents

Segregation and identification of proteins

Differentially expressed proteins involved in phenylpropanoid biosynthesis

Differential multiple of the DEPs participated in starch and sucrose metabolism

Functional network of the DEPs in starch and sucrose metabolism

Metabolome profiling and its fold change analysis

Screening and distribution of DEMs in tuberous roots under chilling stress

Network of the differential metabolic compounds in glucosinolate biosynthesis

Discussion

ROS scavenging and osmotic adjustment substances

The role of endogenous phenolics compounds and glucosinolate biosynthesis under abiotic stress

Sugar as antioxidants in sweetpotato roots

Conclusions

Methods

Plant materials and storage condition

Protein extraction and 1-DE SDS-PAGE

LC-MS/MS of digested peptides

Database searching of proteins

Bioinformatics analysis

Metabolome profiling of tubers

Protein and metabolite data analysis

Abbreviation

Declarations

References

Supplementary Files Legend

Supplementary Files

Status:

Journal Publication

Version 1