**1. Introduction**

Environmental temperatures are increasing and causing one of the most severe plant abiotic stresses. Global temperatures are projected to increase by 0.2 ◦C per decade, which will result in temperatures ranging from 1.8 to 4.0 ◦C above current levels by 2100 [1]. High-temperature stress is currently the main stress factor affecting plant growth and development. Due to the intensification of the greenhouse effect, the global temperature continues to rise, and agriculture is faced with serious challenges, which puts forward higher requirements for the heat resistance of plants [2]. High temperatures affect crop yield, shorten the life cycle of plants, accelerate senescence and cause a decrease in economic yield [2]. Heat stress leads to multiple changes in plant growth, development, physiological processes and yield, which are usually detrimental [3]. Heat stress affects the stability of various proteins, membranes, RNA species and cytoskeletal structures and alters enzymatic efficiency, impeding major physiological processes and causing metabolic imbalances in cellular responses [4–7].

In recent decades, exogenous applications of protectants, such as osmoprotectants, phytohormones, signaling molecules, and trace elements, have shown beneficial effects

**Citation:** Sun, W.; Hao, J.; Fan, S.; Liu, C.; Han, Y. Transcriptome and Metabolome Analysis Revealed That Exogenous Spermidine-Modulated Flavone Enhances the Heat Tolerance of Lettuce. *Antioxidants* **2022**, *11*, 2332. https://doi.org/10.3390/ antiox11122332

Academic Editor: Nafees A. Khan

Received: 25 October 2022 Accepted: 22 November 2022 Published: 25 November 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

on plants grown at high temperatures because of their growth-promoting and antioxidant abilities [8]. Polyamines, mainly including putrescine (Put), spermidine (Spd), and spermidine (Spm), are low molecular weight natural compounds with aliphatic nitrogenous structures that are present in almost all organisms, from bacteria to plants and animals [9]. For plant growth and development, polyamines are widely involved in cell division and differentiation, root elongation, flower development, fruit ripening, leaf senescence, programmed cell death, DNA synthesis, gene transcription, protein translation, and chromatin organization [10–16]. Spermidine is an endogenous molecule that regulates plant growth and resists unfavorable environments [17]. Spd improves heat tolerance in tomato [18], lettuce [19], wheat [20], tall fescue [21], and rice [22]. Spd pretreatment inhibits membrane lipid peroxidation, increases antioxidant enzyme activities, superoxide dismutase (SOD), peroxidase (POD) catalase (CAT) and ascorbate peroxidase (APX), and reduces the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) [23].

Lettuce (*Lactuca sativa* L.) is one of the most popular leafy vegetables in daily life. It originates from the Mediterranean coast and is highly intolerant to high temperatures, with 15~25 ◦C being the preferred growth temperature and growth being hindered above 30 ◦C. High temperatures are one of the main factors limiting the growth of lettuce. High temperatures always result in thinner leaves and longer internodes of lettuce, which leads to a decrease in its nutritional quality and commercial value [24].

In plants, flavonoids are important compounds that affect the color of leaves, flowers and fruits, and they exhibit antioxidant properties when plants are under stress. Flavonoids are considered to be an effective substance against various abiotic stresses because of their ability to reduce oxidative damage in organisms.

The development of transcriptomic and metabolomic technologies has increased the understanding of the network between genes, and RNA-seq has been widely used for various purposes in plant genetics research, especially transcriptome analysis, which has been used to explore a wide range of changes at the level of gene expression under various biotic and abiotic stresses and used to reveal differential gene expression in various biological processes. Additionally, metabolite analysis has become a new tool that can widely analyze components and is a powerful tool to study the changes in metabolites caused by various environmental changes [25]. Combining the joint analysis of different histologies to establish regulatory networks between genes and metabolites can provide a more comprehensive explanation of changes in species tolerance at multiple levels. The mechanism by which methyl jasmonate regulates monoterpene biosynthesis in grape berry skins was revealed by a combined transcriptome and metabolome analysis [26].

In our previous experiments, we demonstrated that exogenous spermidine could alleviate the damage to lettuce seedlings under high-temperature stress [27]; however, the molecular mechanism by which spermidine mediates the responses of lettuce to stress is not clear, so we attempted to explore the mechanistic model of its action through co-omics analysis. In this study, we further revealed the regulatory network between genes and metabolites by combining changes in the physiological properties of lettuce seedlings treated with spermidine under high-temperature stress with transcriptomic and metabolomic analyses and provided theoretical support for elucidating the mechanism of exogenous spermidine in enhancing the heat tolerance of lettuce.

### **2. Materials and Methods**

#### *2.1. Plant Material and Treatment*

The heat-sensitive lettuce variety 'Beisansheng No. 3 was selected from Beijing University of Agriculture [27]. The full-grained lettuce seeds were placed in filter paperlined Petri dishes for 48 h and then sown in hydroponic seedling trays with built-in Hoagland nutrient solution and placed in an artificial climate chamber with a photoperiod of 14/10 h, diurnal temperature of 23 ◦C/17 ◦C, relative humidity of 70~75%, and light intensity of 350 ± <sup>10</sup> <sup>μ</sup>mol·m−2·s−1, and seedlings with uniform growth were selected. Seedlings were transplanted into 10 L hydroponic tanks with Hoagland nutrient solution, and treatments were started when the seedlings reached four leaves. Lettuce leaves were sprayed with spermidine treatment at 9:00 a.m. each day to the extent that the leaves were moist but not dripping droplets. According to our previous experiments, we set the Spd spray concentration to 1 mmol·L−<sup>1</sup> [27]. The standard for spraying is to spray the leaf surface and leaf back evenly, with the sprayed liquid adhering to the leaf surface so that it is all wet but not dripping.

Experimental material handling can be seen in Table 1. Three treatments were set up in this experiment: normal temperature control (CK): temperature of 22 ◦C/17 ◦C (day/night) and spraying distilled water; high temperature stress (H): temperature of 35 ◦C/30 ◦C (day/night) and spraying distilled water; high temperature stress spraying with spermidine (HS): temperature of 35 ◦C/30 ◦C (day/night) and 1 mmol·L−<sup>1</sup> Spd.

**Table 1.** Experimental treatments.


Each treatment was replicated three times. After 4 days of heat treatment of the lettuce seedlings, healthy plants were randomly selected, and the cotyledon portion of the leaves was collected. Samples were placed in liquid nitrogen at the time of collection and stored in a refrigerator at −80 ◦C for subsequent experiments

#### *2.2. Measurement of the Physiological and Biochemical Parameters*

The malondialdehyde (MDA) content was determined by the thiobarbituric acid method and calculated from the absorption values at 450, 532 and 600 nm. The chlorophyll content was determined by the method of Madhava Rao and Sresty [28]: 0.3 g of the sample was ground well with 2.5 mL of 95% ethanol, filtered and fixed in a 25 mL brown volumetric flask only.

The chloroplast pigment extract was poured into a cuvette, and 95% ethanol was used as a blank control. The optical density (OD) values were measured on a spectrophotometer (UV-5200, Shanghai, China) at 665 and 649 nm. Chlorophylls were calculated according to the following equations:

Chl a content (mg/g) = [13.95OD665 − 6.88OD649] V/1000 W (1)

Chl b content (mg/g) = [24.96OD649 − 7.32OD665] V/1000 W (2)

$$\text{Chl t content (mg/g)} = \text{[18.08OD649 + 6.63OD665] V/1000 V} \tag{3}$$

The flavonoid content was determined by the sodium nitrite-aluminum nitrate chromogenic method. The flavonoid content was determined using a kit (Solarbio, Beijing, China). After the sample was dried to a constant weight, 0.1 g was dissolved in 1 mL of the extraction solution. The sample was extracted by ultrasonication at 60 ◦C for 30 min and then centrifuged at 12,000 rpm for 10 min, and the supernatant was taken for measurement. The absorbance value of the sample extract at 470 nm was measured.

#### *2.3. Determination of Antioxidant Enzyme Activity*

Place 0.5 g of lettuce sample in a cooled mortar and add in 5 mL of phosphate buffer (pH 7.0) for grinding. The homogenate was collected and centrifuged at 4 ◦C for ten minutes at a force of 11,000× *g*. The supernatant was collected, and the above steps were repeated twice.

Superoxide dismutase (SOD) activity was measured by the NBT (nitrogen blue tetrazolium) photochemical reduction method [29], with 50% inhibition of NBT photochemical reduction as one enzyme activity unit (U). Peroxidase (POD) activity was determined

by the guaiacol method, with an increase of 1 per minute of OD 470 nm as one enzyme activity unit (U). Catalase (CAT) activity was determined by the hydrogen peroxide UV spectrophotometric method, with an increase of 0.1 per minute OD240 nm as one enzyme activity unit (U). Ascorbate peroxidase (APX) activity was determined by measuring the rate of ascorbate oxidation at 290 nm (ε = 2.8 mM−<sup>1</sup> cm<sup>−</sup>1) [30].

#### *2.4. Metabolite Measurement and Quantification*

The tissue sample (25 mg) was placed in a 1.5 mL tube with 800 μL of precooled precipitant (methanol: acetonitrile: distilled water = 2:2:1) and two small steel beads, then it was ground in a grinder (60 Hz, 4 min). After the removal of the beads, it was sonicated in an ice bath for 10 min (power 80 HZ), allowed to rest for 120 min at −20 ◦C, and then it was centrifuged for 15 min (25,000× *g*, 4 ◦C) and 600 μL of the supernatant was collected, and it was repeated once. The supernatant was placed in a freeze extractor to drain. Then, 600 μL of 10% methanol solution was added, it was placed in an ice bath and sonicated for 10 min (power 80 HZ) and then centrifuged for 15 min (25,000× *g*, 4 ◦C). The supernatant was collected, and 50 μL of each sample was injected into a QC using an ACQUITYUPLCHSST3 column (100 mm × 2.1 mm, 1.8 μm, Waters, Wilmslow, UK) for chromatographic separation, and the small molecules that eluted from the column were collected in positive and negative ion modes using high-resolution tandem mass spectrometry (Xevo G2-XS QTOF Waters, Wilmslow, UK). Peak extraction was mainly implemented by the commercial software Progenesis QI (version 2.2. Newcastle, UK), including peak alignment, peak extraction, normalization, deconvolution and compound identification steps. Three biological replicates were performed for each sample.

#### *2.5. RNA Extraction and Quality Testing*

The steps of total RNA extraction, RNA purity assessment, library construction, library quality control, and up sequencing were performed at UW (https://www.genomics.cn, accessed on 1 August 2019), following their standard procedures.
