*2.7. Quantitative Real-Time PCR*

Total leaf RNA extraction was performed using a kit (Yueyang Hua, Yueyang, China, Cat:0416-50gk). The first cDNA strand was synthesized using a reverse transcription kit (Tiangen, Beijing, China. Cat:KR118-02). The primer sequences of the related genes were downloaded from the GenBank library of NCBI. The primer design is shown in Table S1. qRT-PCR analysis was performed using the reverse transcription product cDNA as the

template and 18S as the internal reference gene. qRT-PCR was performed using the TB Green Premix Ex Taq II (2×) (Tli RNaseH Plus) kit (TaKaRa, Kyoto, Japan) in the CFX96 Real-Time PCR Detection System instrument (Bio-Rad Laboratories, Hercules, CA, USA). The reaction system was 20 μL, consisting of 9 μL TB Green Premix Ex Taq II (TliRNaseH Plus), 7 μL ddH2O, 2 μL cDNA template and 1 μL forward and reverse primers. The cycling progression was as follows: 95 ◦C for 3 min; 95 ◦C for 10 s and 56 ◦C for 30 s, for a total of 39 cycles. Gene expression change ploidy analysis was calculated using 2−ΔΔCt, and relative mRNA expression levels were normalized using 18S. The qRT-PCR validation of the DEGs is shown in Figure S1.

#### *2.8. Statistical Analysis*

One-way ANOVA was performed on all data using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA), and Duncan's test was used to test the significance of the differences between samples (*p* < 0.05).

#### **3. Results**

*3.1. Effect of Exogenous Spermidine on the Growth and MDA and Chlorophyll Contents of Lettuce under High-Temperature Stress*

As shown in Table 2, high-temperature stress significantly reduced the total fresh weight, shoot fresh weight, root length, total dry weight, and root-to-shoot ratio of lettuce, while the application of spermidine alleviated the damage to the total fresh weight, root length, and total dry weight under high-temperature stress, but had no significant effect on shoot fresh weight, root fresh weight, plant height and root-to-shoot ratio.

**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.


CK: 22 ◦C/17 ◦C, distilled water; H: 35 still, distilled water; HS: 35 ◦C/30 ◦C, distilled 1 mM Spd.

From Figure 1A, it can be seen that the leaves of lettuce seedlings under hightemperature stress were elongated, appeared to be twitching, root growth was weak, fewer roots were produced, the main roots were short, biomass accumulation was reduced, which indicated that high temperature stress inhibited the growth condition and organic matter accumulation of lettuce seedlings. At the same time, the MDA content in the leaves increased under high temperature, and the increased MDA content reflected, to some extent, the increase in membrane lipid peroxidation. In the leaves of seedlings sprayed with spermidine, these effects were ameliorated to some extent. In addition, exogenous spermidine also affected the photosynthetic pigment content of the lettuce leaves. Although the total chlorophyll content of the leaves increased under high-temperature stress, the contents of chlorophyll a and chlorophyll b did not change significantly, while the application of spermidine significantly increased the contents of chlorophyll a and total chlorophyll (Figure 1C–E).

In conclusion, exogenous spermidine increased the total fresh weight, total dry weight, and root length, reduced the MDA content, and enhanced the chlorophyllscontent of the lettuce under high-temperature stress, alleviating the damage of high temperature on growth and physiological indexes of lettuce, providing a preliminary basis for us to further explore the possible mechanism of spermidine-mediated enhancement of heat tolerance of lettuce.

**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.

#### *3.2. Effect of Spermidine on the Antioxidant Enzyme Activity of Lettuce under High-Temperature Stress*

Under high temperatures, SOD activity decreased in control plants sprayed with distilled water; however, SOD activity increased in plants treated with spermidine (Figure 2A). Similar to SOD activity, spermidine treatment increased CAT activity in lettuce under high temperature compared with deionized water spray (Figure 2C); compared with the control, high temperature stress had no significant effect on POD activity but spraying with spermidine under high temperature stress still significantly increased its activity compared with deionized water spray (Figure 2B). However, for APX, there was no significant effect of high temperature stress compared to the ambient control, and neither deionized water nor spermine spraying had any significant effect on its activity under high temperature stress (Figure 2D). This suggests that exogenous spermidine can withstand high temperature stress by regulating the activities of antioxidant enzymes such as SOD, CAT and POD, and may therefore attenuate oxidative damage in lettuce leaf cells.

**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.
