*3.6. GO Enrichment Analysis*

To further understand the DEG functions of H and HS, GO functional annotation of DEGs was performed (Figure 5). A Q value (padj) < 0.05 was set as the threshold for significant enrichment. According to the GO database, genes can be classified according to biological process (BP), cytological component (CC) and molecular function (MF) categories. GO functional enrichment reveals the enrichment of distinct functional entries in differentially expressed genes, annotating differentially expressed genes to individual biological functions, so that we can understand the linkage between analyzing different differentially expressed genes and biological functions.

In this analysis, we significantly enriched the subcategories of differentially expressed genes in the GO database. In the DEGs of CK compared with H, 28.48%, 38.77% and 32.75% were categorized as biological processes, cellular components and molecular functions, respectively (Figure 5A). However, 30.32%, 37.29% and 32.39% of the DEGs in the HS versus H comparison were categorized as biological processes, cellular components and

molecular functions, respectively (Figure 5B), these values were observed in 28.87%, 39.04% and 32.09% of the DEGs in the CK versus HS comparison (Figure 5C).

**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.

#### *3.7. KEGG Enrichment Analysis*

To understand the metabolic or signaling pathways of spermidine involved in hightemperature stress, all differentially expressed genes were compared with the Kyoto encyclopedia of genes and genomes (KEGG) database to get the matched KEGG Orthology (KO). The KEGG enrichment analysis of different genes in each treatment group is shown in Figure 6. The vertical coordinates in the figure represent the KEGG pathway, and the horizontal coordinates are the enrichment factors. The larger the enrichment factor, the greater the degree of enrichment; the larger the point, the greater the number of differentially expressed genes enriched in the pathway; the bluer the point, the more significant the enrichment. The KEGG enrichment analysis of DEGs in the H and CK treatments is shown in Figure 6A. The DEGs between the H and CK treatments mainly focused on the biosynthesis of sesquiterpenes and triterpenes; biosynthesis of keratin, sulfites and waxes; fatty acid metabolism; biosynthesis of unsaturated fatty acids; starch and sucrose

metabolism; degradation of other polysaccharides; phytohormone signaling; fatty acid degradation and biosynthesis; and biotin metabolism.

**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.

The KEGG analysis of DEGs between the HS and CK enrichment analyses is shown in Figure 6C. The DEGs between HS and CK were mainly involved in plant-pathogen interactions, sesquiterpene and triterpene biosynthesis, the MAPK signaling pathway, keratin, sulfite and wax biosynthesis, other polysaccharide degradation, phytohormone signaling, and unsaturated fatty acid biosynthesis.

The KEGG enrichment analysis of DEGs between HS and H is shown in Figure 6B. As shown in the figure, the DEGs between HS and H were mainly focused on unsaturated fatty acid biosynthesis, plant-pathogen interactions, sesquiterpene and triterpene biosynthesis, fatty acid metabolism, photosynthesis-antennal proteins, the MAPK signaling pathway, linolenic acid metabolism, and flavonoid biosynthesis. These results suggest that exogenous spermidine may protect against high temperature stress by improving photosynthesis, affecting and participating in signal transduction, and regulating flavonoid biosynthesis in lettuce.

## *3.8. Transcription Factors*

The different transcription factors of differentially expressed genes (DEGs) were different in the different treatment comparisons (Figure 7). Seventeen transcription factor families, including 160 TF genes, were identified in the high-temperature treatment compared with control-treated lettuce seedlings, and the family with the highest number of transcription factors among the differentially expressed genes (DEGs) was AP2-EREBP, followed by the MYB, NAC, MADS, WRKY, SBP, and bHLH families (Figure 7A). In addition, a total of 172 TF genes from 28 transcription factor families, mainly including 37 AP2- EREBPs, 19 MYBs, 16 WRKYs, 14 NACs, and 12 bHLH, were found in the comparison of high-temperature spermidine treatment with the control treatment, while 75 TFs from 13 TF families were found in the comparison of the high-temperature spermidine treatment with the high-temperature control treatment. The family with the highest number of transcription factors among the differentially expressed genes (DEGs) was AP2-EREBP, followed by

the WRKY, MYB, and NAM families (Figure 7B,C). Our experimental results revealed that most of the transcription factors were upregulated after spraying with spermidine under high-temperature stress (Figure 7D), suggesting that it may alleviate the damage caused by high-temperature stress mainly by upregulating the expression of AP2, MYB, WRKY and other transcription factors and thus further regulate the expression of the corresponding genes in lettuce.

**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.

#### *3.9. Spermidine Regulates the Metabolism of Flavonoids under High-Temperature Stress*

As shown in Figure 8A, spraying spermidine increased the total flavonoid content in leaves under high-temperature stress. A total of 19 DEGs rich in flavonoid-related metabolic pathways were identified in HS vs. H, encoding six enzymes related to flavonoid synthesis, including hydroxycinnamoyltransferase (HCT), flavonol synthase (FLS), caffeoyl coenzyme A methyltransferase (CCoAOMT) dihydroflavonol reductase (DFR), chalcone synthase (CHS), and colorless anthocyanin dioxygenase (LDOX), which are involved in the regulation of the synthesis of flavonoid substances such as dihydroflavonol, flavonol, colorless anthocyanin, and chalcone (Figure 8B). Among them, 12 genes significantly increased their expression under high-temperature stress after the application of spermidine, and half of these 12 genes enhanced their expression by more than 2-fold, which may be responsible for the increase in total flavonoid content. These results, in agreement with our previous results, suggest that exogenous spermidine regulates the synthesis of flavonoid substances in lettuce leaves, thereby affecting their tolerance to high-temperature stress.

**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.

#### **4. Discussion**

High temperature (HT) is a widespread environmental stress that affects most plants at all periods of growth and can limit plant growth and development and reduce productivity. Plant growth and development involve many temperature-sensitive biochemical responses [31]. One approach to dealing with the adverse effects of heat stress may involve exploring some molecules that have the potential to protect plants from the deleterious effects of HT. Polyamines (PAs) are low molecular weight aliphatic amines and organic polycations found in a variety of organisms from bacteria to plants and animals [32]. They also play an important role in plant responses to abiotic stresses. Our previous studies have shown that high-temperature stress limits normal growth and leads to the accumulation of unwanted ROS, altering the enzymatic and nonenzymatic antioxidant activity of lettuce seedlings [27], while exogenous spermidine restores growth and photosynthesis by improving carbon metabolism, thereby increasing stress tolerance [33].

In our experiments, heat stress resulted in wilting of lettuce, curling of leaves, a reduction in biomass, and an increase in malondialdehyde content. The application of spermidine improved the growth of lettuce leaves, reduced the malondialdehyde content under heat stress, and increased the chlorophyll content. Previous studies showed that Spd treatment significantly promoted FW and DW in white clover seedlings under PEG-simulated water stress conditions [34]. Zeng found a significant decrease in aboveground FW and DW of hybrid rice under low-temperature stress, while Spd-treated plants showed a significant increase in these indices by 30.48% and 18.19% under low temperature conditions [35]. Similar to these reports, our results confirm the role of spermidine in the protection of lettuce biomass.

Chlorophyll is an important indicator of the ability of leaves to maintain their green color, and the decrease in chlorophyll concentration is generally considered to be a response mechanism to reduced light uptake by chloroplasts under stress; furthermore, the decrease in chlorophyll content during stress may be related to impaired chlorophyll synthesis or pigment protein degradation and could be a result of ROS production [36]. The results in Figures 2 and 3 indicate that exogenous spermidine promoted the enhancement of antioxidant enzyme activity with increased chlorophyllin lettuce seedlings, which may be one of the reasons for their increased tolerance of heat stress. Our previous study also confirmed that spraying exogenous spermine at high temperatures increased chlorophyll a and b content in lettuce leaves and was able to avoid oxidative damage to chloroplasts [37]. These results suggest that Spd spraying can maintain chlorophyll and content to support light energy capture and transport in lettuce under high temperature stress. Higher photosynthesis rates promote flavonoid accumulation because flavonoids are synthesized in chloroplasts and flavonoids are hypothesized to be positively correlated with photosynthesis [38–40]. Therefore, we hypothesized that enhanced photosynthetic activity in lettuce leaves increased the production of primary and secondary metabolites, including flavonoids.

Crop tolerance to HT stress is associated with an increase in antioxidant capacity [41]. Previous studies have shown that Spm pretreatment increased antioxidant enzyme activities under HT and drought stress conditions, while the activity levels of CAT, POD and SOD were consistently higher in Spm pretreated seedlings than in the control [42]. In our experiments, high-temperature stress affected POD and SOD activities in lettuce leaves, while the application of spermidine increased CAT, POD and SOD activities without a significant effect on APX. This may be due to the fact that antioxidant enzymes differ in their perception of the degree and duration of temperature, and thus different antioxidant enzymes vary over different temperature ranges.

Upregulation of many genes has been reported to help plants resist stress conditions that lead to plant adaptation [43]. Under various biotic and abiotic stresses, plants are able to receive external signals and respond to the stress by associating various internal pathways to transmit information to downstream molecules through their respective methods. The main molecular mechanisms underlying the response to salt stress were previously revealed by transcriptome sequencing analysis [44]; physiological and transcriptomic approaches were used in grapes to explore the effect of exogenous monocrotaline lactones on drought stress at the transcriptional level, among others [45]. A combined transcriptomic and metabolomic analysis also provided an enhanced understanding of the regulatory network between certain specific genes and compounds in broccoli species under selenate treatment [46]. Therefore, in our study, we attempted to investigate the response of spermidine to differential gene expression patterns, expression of different transcription factors, and plant secondary metabolites and related genes in lettuce seedlings under high temperature stress by studying RNA-sequence analysis and metabolite components.

Signaling molecules are involved in the activation of many stress-responsive genes, and various signal transduction molecules associated with the activation of stress-responsive genes exist depending on the plant type and type of stress. Among them, widely used are Ca-dependent protein kinases (CDPKs), mitogen-activated protein kinases (MPKs), NO, sugar (as signaling molecules), and phytohormones [47]. These molecules, along with transcription factors, activate stress response genes. Once activated, stress response genes reactivate essential enzymes and structural proteins, which contribute to the detoxification of ROS (by activating detoxifying enzymes and free radical scavengers) to maintain cellular homeostasis [48]. The available data suggest that some signaling molecules may lead to an increase in cellular antioxidant capacity [49,50].

TFs stimulate and regulate multiple stress response pathways in plants subjected to high-temperature stress [48,49]. Flavonoids are plant polyphenol secondary metabolites with a wide range of physiological functions [51]. The flavonoid pathway is derived from the general phenylpropane pathway, and flavonoid biosynthetic genes are regulated by interactions between different TF families [44,46]. For example, genes involved in the anthocyanin and condensed tannin pathways are regulated by R2R3MYB, bHLH, and WD40 proteins (MYB-bHLH-WD40, MBW complex) [52]. Flavonoid synthesis genes, such as F3H, F3 H and FLS, are regulated by MYB genes [53]. In the present study, AP2- EREBP, WRKY, MYB, and NAM were the most abundantly expressed transcription factor families after spermidine treatment (Figure 6), and in agreement with previous studies, spermidine may have regulated the MYB family and thus the expression of genes related to the flavonoid metabolic pathway and, in this way, coordinated the total flavonoid content. However, the regulatory functions of these TFs in flavonoid biosynthesis need to be further investigated.

Flavonoids may protect plants from oxidative stress by blocking ROS production through their ability to chelate metal ions and scavenge ROS, thus achieving their antioxidant function [54–56]. Moreover, since the synthesis of flavonoids is performed in chloroplasts, the increased chlorophyll content and antioxidant enzyme activity in our experimental results suggest that spermidine may be able to maintain high light energy capture and transport by increasing chlorophyll content and avoiding oxidative loss of chloroplasts to ensure smooth synthesis of flavonoids, which in turn may protect organelles from oxidative damage [39,40]. It has been shown that excessive accumulation of flavonoids with higher free radical scavenging activity in transgenic Arabidopsis enhances tolerance to drought stress [57]. In our study, KEGG enrichment analysis of DEGs showed significant enrichment in the "flavonoid biosynthesis" pathway (Figure 6B) and six enzymes related to the flavonoid synthesis pathway (oxalate hydroxycinnamyltransferase (HCT), flavonol synthase (FLS), CCoAMOT (caffeoyl-coenzyme A methyltransferase), chalcocyanine, and chalcocyanine), chalcone synthase (CHS), and colorless anthocyanin dioxygenase (LDOX)) were differentially expressed. Similarly, most of the representative genes involved in these pathways were downregulated under high-temperature stress and upregulated under high-temperature spermidine treatment (Figure 8 and Supplementary Table S3), which may be responsible for the elevated total flavonoid concentration (Figure 8A). In other words, the elevated flavonoid concentration may have mitigated the effect of high-temperature stress on lettuce leaves to some extent.

#### **5. Conclusions**

Exogenous spermidine increased the content of chlorophylls and antioxidant enzymes and decreased the content of malondialdehyde under high-temperature stress. By integrating transcriptome and metabolite analysis, we found that spermidine may be involved in or induce the expression of TF families such as AP2-EREBP, WRKY, MYB, and NAM to transmit information, and combined with the metabolic pathways identified in KEGG enrichment, spermidine influenced the biosynthesis of flavonoid synthesis and ultimately alleviated the damage to lettuce from high-temperature stress. A hypothetical model of exogenous spermidine-mediated heat stress tolerance in lettuce is shown in Figure 9, but its specific regulatory mechanism needs further exploration and experimental corroboration. Exogenous spermidine may be a promising approach and may enhance the heat tolerance

of lettuce by regulating various factors such as growth, physiology, molecular activities and metabolite accumulation under high temperature conditions.

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

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11122332/s1, Figure S1: Validation of the RNA-Seq results by qRT-PCR. Data are presented as the means of three replicates; Table S1: Sequence information of primers of genes of interest used in qRT-PCR assays; Table S2: Sequence reads of the transcriptome and their alignment with the reference genome; total of 307 differential metabolites in HSvsH.

**Author Contributions:** W.S.: investigation, writing—original draft; J.H.: writing—review and editing; S.F.: resources; C.L.: conceptualization, writing—review and editing, funding acquisition; Y.H.: conceptualization, funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Natural Science Foundation of China (Grant number: 32172607) and Beijing Joint Research Program for Germplasm Innovation and New Variety Breeding (Grant number: G20220628003).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All of the data is contained within the article.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

