Figure 1. Effects of exogenous spermidine on the morphology, MDA content and chlorophyll content of lettuce under high temperature. (A) Phenotypic map. (B) MDA content. (C) Chlorophyll a content. (D) Chlorophyll b content. (E) Total chlorophyll content. Values above each vertical bar followed by different letters show significant differences (p < 0.05). The highest value was labeled as a, and those with significant differences were labeled as b in that order. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 1. Effects of exogenous spermidine on the morphology, MDA content and chlorophyll content of lettuce under high temperature. (A) Phenotypic map. (B) MDA content. (C) Chlorophyll a content. (D) Chlorophyll b content. (E) Total chlorophyll content. Values above each vertical bar followed by different letters show significant differences (p < 0.05). The highest value was labeled as a, and those with significant differences were labeled as b in that order. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 2. Effects of exogenous spermidine on the activities of antioxidant enzymes in lettuce under high-temperature stress. (A) SOD activity, (B) POD activity, (C) CAT activity, and (D) APX activity. Values above each vertical bar followed by different letters show significant differences (p < 0.05). The highest value was labeled as a, and those with significant differences were labeled as b in that order. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 2. Effects of exogenous spermidine on the activities of antioxidant enzymes in lettuce under high-temperature stress. (A) SOD activity, (B) POD activity, (C) CAT activity, and (D) APX activity. Values above each vertical bar followed by different letters show significant differences (p < 0.05). The highest value was labeled as a, and those with significant differences were labeled as b in that order. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 3. Differential metabolome analysis in HSvsH. (A) Differential metabolite composition. (B) PCA model for metabolic data. (C) Volcano plot of differential metabolites. (D) Heat map for cluster analysis of differential metabolites. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 3. Differential metabolome analysis in HSvsH. (A) Differential metabolite composition. (B) PCA model for metabolic data. (C) Volcano plot of differential metabolites. (D) Heat map for cluster analysis of differential metabolites. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 4. Analysis of DEGs in lettuce under high-temperature stress with exogenous spermidine. (A) Transcriptome sample correlation map. (B) Venn diagrams of DEGs. (C) Numbers of DEGs. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 4. Analysis of DEGs in lettuce under high-temperature stress with exogenous spermidine. (A) Transcriptome sample correlation map. (B) Venn diagrams of DEGs. (C) Numbers of DEGs. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 5. Gene Ontology (GO) classification and distribution of GO annotated genes. (A) H vs. CK; (B) HS vs. H; (C) HS vs. CK. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 5. Gene Ontology (GO) classification and distribution of GO annotated genes. (A) H vs. CK; (B) HS vs. H; (C) HS vs. CK. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 6. KEGG pathway enrichment analysis of DEGs in response to different stress treatments. (A) H vs. CK; (B) HS vs. H; (C) HS vs. CK. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 6. KEGG pathway enrichment analysis of DEGs in response to different stress treatments. (A) H vs. CK; (B) HS vs. H; (C) HS vs. CK. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 7. Regulation of DEGs of transcription factors. (A) H vs. CK; (B) HS vs. H; (C) HS vs. CK; (D) Expression trends of transcription factors in HS vs. H. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 7. Regulation of DEGs of transcription factors. (A) H vs. CK; (B) HS vs. H; (C) HS vs. CK; (D) Expression trends of transcription factors in HS vs. H. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 8. Flavonoid metabolic pathway association with differentially expressed genes (DEGs). (A) Total flavonoid content; (B) Expression of related DEGs in flavonoid metabolic pathways. Values above each vertical bar followed by different letters show significant differences (p < 0.05). The highest value was labeled as a, and those with significant differences were labeled as b, c in that order. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 8. Flavonoid metabolic pathway association with differentially expressed genes (DEGs). (A) Total flavonoid content; (B) Expression of related DEGs in flavonoid metabolic pathways. Values above each vertical bar followed by different letters show significant differences (p < 0.05). The highest value was labeled as a, and those with significant differences were labeled as b, c in that order. CK: 22 °C/17 °C, distilled water; H: 35 °C/30 °C, distilled water; HS: 35 °C/30 °C, distilled 1 mM Spd.

Figure 9. Hypothetical model of exogenous Spd to improve the heat tolerance of lettuce.

Figure 9. Hypothetical model of exogenous Spd to improve the heat tolerance of lettuce.

Table 1. Experimental treatments.

Table 1. Experimental treatments.

TreatmentTemperature (Day/Night)Type of SprayingCK22 °C/17 °Cdistilled waterH35 °C/30 °Cdistilled waterHS35 °C/30 °C1 mmol·L−1 Spd

Table 2. Effects of exogenous spermidine on lettuce growth under high-temperature stress. Values above each vertical bar followed by different letters show significant differences (p < 0.05). The highest value was labeled as a, and those with significant differences were labeled as b, c in that order.

Table 2. Effects of exogenous spermidine on lettuce growth under high-temperature stress. Values above each vertical bar followed by different letters show significant differences (p < 0.05). The highest value was labeled as a, and those with significant differences were labeled as b, c in that order.

TreatmentsFresh Weight (g)Shoot Fresh Weight (g)Root Fresh Weight (g)Root Length (cm)Dry Weight (g)Plant Height (cm)Root-Shoot RatioDry Weight (g)CK38.60 ± 1.46 a22.78 ± 2.90 a12.28 ± 0.68 a25.83 ± 2.25 a1.01 ± 0.23 a15.33 ± 2.52 a1.71 ± 0.25 a1.01 ± 0.23 aH19.01 ± 0.93 c7.66 ± 0.91 b11.04 ± 1.26 a9.61 ± 1.57 c0.32 ± 0.02 c16.57 ± 0.40 a0.58 ± 0.10 b0.32 ± 0.02 cHS24.32 ± 1.54 b10.98 ± 0.85 b11.22 ± 0.24 a14.14 ± 1.36 b0.61 ± 0.04 b17.50 ± 1.00 a0.81 ± 0.03 b0.61 ± 0.04 b

Table 3. PLS-DA model parameters.

Table 3. PLS-DA model parameters.

GroupR2Q2H vs. CK0.9920.735HS vs. H0.9520.105HS vs. CK0.9970.811