Metabolomic Analysis of the Effect of Freezing on Leaves of Malus sieversii (Ledeb.) M.Roem. Histoculture Seedlings

Abstract

Malus sieversii (Ledeb.) M.Roem. is the ancestor of cultivated apples, and is an excellent germplasm resource with high resistance to cold. Artificial refrigerators were used to simulate the low temperature of -3 °C to treat Malus sieversii (Ledeb.) M.Roem. histoculture seedlings. Observations were performed to find the effects of freezing stress on the status of open or closed stomata, photosystems, and detection of metabolomic products in leaves of Malus sieversii (Ledeb.) M.Roem. histoculture seedlings. The percentage of closed stomata in the Malus sieversii (Ledeb.) M.Roem. histoculture seedlings increased, the maximum fluorescence (Fm') excited by a strong light (saturating pulse) was weakened relative to the real-time fluorescence in its vicinity, and the quantum yield of unregulated energy dissipation was increased in PSII under freezing stress. The metabolites in the leaves of the Malus sieversii (Ledeb. M.Roem.) histoculture seedlings were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry using CK, T12h, T36 h, and HF24h. Results demonstrated that cold stress in the Malus sieversii (Ledeb.) M.Roem. histoculture seedlings led to wilting, leaf stomatal closure, and photosystem damage. There were 1020 metabolites identified as lipids (10.2%), nucleotides and their derivatives (5.2%), phenolic acids (19.12%), flavonoids (24.51%), amino acids and their derivatives (7.75%), alkaloids (5.39%), terpenoids (8.24%), lignans (3.04%), organic acids (5.88%), and tannins (0.88%). There were 110 differential metabolites at CKvsT12h, 113 differential metabolites at CKvsT36h, 87 differential metabolites at T12hvsT36h, 128 differential metabolites at CKvsHF24h, 121 differential metabolites at T12hvsHF24h, and 152 differential metabolites at T36hvsHF24h. The differential metabolites in the leaves of the Malus sieversii (Ledeb.) M.Roem. seedlings grown under low-temperature stress mainly involved glycolysis, amino acid metabolism, lipid metabolism, pyrimidine metabolism, purine metabolism, and secondary metabolite metabolism. The Malus sieversii (Ledeb.) M.Roem. seedlings responded to the freezing stress by coordinating with each other through these metabolic pathways. The metabolic network of the leaves of the Malus sieversii (Ledeb.) M.Roem. histoculture seedlings under low temperature stress was also proposed based on the above pathways to deepen understanding of the response of metabolites of Malus sieversii (Ledeb.) M.Roem. to low-temperature stress and to lay a theoretical foundation for the development and utilization of Malus sieversii (Ledeb.) M.Roem. cultivation resources.

Keywords: Malus sieversii (Ledeb.) M.Roem. histoculture seedlings; freezing stress; metabolomics.

Figure 1

Changes in stomata in Malus sieversii (Ledeb.) M.Roem. histoculture seedlings under freezing stress: (a) The stomata of Malus sieversii (Ledeb.) M.Roem. histoculture seedling leaves in each treatment (CK, T12h, T36h, HF24h). The stomata were classified as closed stomata when the ratio ≤ 0.3, and open stomata when the ratio > 0.3. The red crosses labeled in subfigure a are to determine the opening and closing of the stomata; (b) Percentage of the number of open stomata in the leaves of the Malus sieversii (Ledeb.) M.Roem. histoculture seedlings in each treatment (CK, T12h, T36h, HF24h). Means denoted by the same letters did not significantly differ at p < 0.05 (Duncan’s range test) on a given treatment.

Figure 1

Changes in stomata in Malus sieversii (Ledeb.) M.Roem. histoculture seedlings under freezing stress: (a) The stomata of Malus sieversii (Ledeb.) M.Roem. histoculture seedling leaves in each treatment (CK, T12h, T36h, HF24h). The stomata were classified as closed stomata when the ratio ≤ 0.3, and open stomata when the ratio > 0.3. The red crosses labeled in subfigure a are to determine the opening and closing of the stomata; (b) Percentage of the number of open stomata in the leaves of the Malus sieversii (Ledeb.) M.Roem. histoculture seedlings in each treatment (CK, T12h, T36h, HF24h). Means denoted by the same letters did not significantly differ at p < 0.05 (Duncan’s range test) on a given treatment.

Figure 2

Effect of freezing stress on chlorophyll fluorescence of Malus sieversii (Ledeb.) M.Roem. histoculture seedlings: (a) Chlorophyll fluorescence induction curves of Malus sieversii (Ledeb.) M.Roem. histoculture seedling leaves in each treatment (CK, T12h, T36h, HF24h); (b) Y(NO) imaging; (c) Value of Y(NO). Means denoted by the same letters did not significantly differ at p < 0.05 (Duncan’s range test) on a given treatment.

Figure 3

Metabolite class composition ring. Each color represents a metabolite class (where the area of the color block indicates the proportion of that class).

Figure 4

Plot showing PCA scores for each sample data group: PC1 represents the first principal component, PC2 represents the second principal component, PC3 represents the third principal component, and the percentage represents the explanation rate of this principal component to the data set; each point on the graph represents one sample, and samples from the same group are represented by the same color.

Figure 5

Analysis of differential metabolism and trends in its content: (a–f) differential metabolites of CKvsT12h, CKvsT36h, T12hvsT36h, CKvsHF24h, T12hvsHF24h, and T36hvsHF24h. The green dots represent downregulated differential metabolites, and the red dots represent upregulated differential metabolites; VIP + FC dual screening conditions: the horizontal coordinate indicates the logarithmic value of the relative content difference of a metabolite in the two groups of samples, and the larger the absolute value of the horizontal coordinate, the greater the relative content difference of the substance between the two groups of samples. The vertical coordinate indicates the VIP value, and the larger the value of the vertical coordinate, the more significant the difference is, and the more reliable the screened differential metabolite is; (g,h) Venn diagram; (i) K-Means clustering diagram, where the horizontal coordinate indicates the sample grouping, the vertical coordinate indicates the standardized metabolite relative content, and the subclass represents the metabolite class number with the same variation trend. The black line is a fit to the trend of relative content of all metabolites in the sub classes.

Figure 5

Analysis of differential metabolism and trends in its content: (a–f) differential metabolites of CKvsT12h, CKvsT36h, T12hvsT36h, CKvsHF24h, T12hvsHF24h, and T36hvsHF24h. The green dots represent downregulated differential metabolites, and the red dots represent upregulated differential metabolites; VIP + FC dual screening conditions: the horizontal coordinate indicates the logarithmic value of the relative content difference of a metabolite in the two groups of samples, and the larger the absolute value of the horizontal coordinate, the greater the relative content difference of the substance between the two groups of samples. The vertical coordinate indicates the VIP value, and the larger the value of the vertical coordinate, the more significant the difference is, and the more reliable the screened differential metabolite is; (g,h) Venn diagram; (i) K-Means clustering diagram, where the horizontal coordinate indicates the sample grouping, the vertical coordinate indicates the standardized metabolite relative content, and the subclass represents the metabolite class number with the same variation trend. The black line is a fit to the trend of relative content of all metabolites in the sub classes.

Figure 6

Enrichment analysis of KEGG: (a) sugar metabolism; (b) purine metabolism, pyrimidine metabolism, and vitamin 6B metabolic pathway; (c) amino acid metabolism; (d) linolenic and α-linolenic acid metabolism; (e) phenolics metabolism, flavonoid metabolism, and xanthone metabolism. The pentagrams are significant differences.

Figure 6

Enrichment analysis of KEGG: (a) sugar metabolism; (b) purine metabolism, pyrimidine metabolism, and vitamin 6B metabolic pathway; (c) amino acid metabolism; (d) linolenic and α-linolenic acid metabolism; (e) phenolics metabolism, flavonoid metabolism, and xanthone metabolism. The pentagrams are significant differences.

Figure 6

Enrichment analysis of KEGG: (a) sugar metabolism; (b) purine metabolism, pyrimidine metabolism, and vitamin 6B metabolic pathway; (c) amino acid metabolism; (d) linolenic and α-linolenic acid metabolism; (e) phenolics metabolism, flavonoid metabolism, and xanthone metabolism. The pentagrams are significant differences.