Prohydrojasmon Promotes the Accumulation of Phenolic Compounds in Red Leaf Lettuce

Prohydrojasmon (PDJ) is a synthetic jasmonate derivative that is primarily used as a growth regulator, but its mechanism of action is unclear. In this study, we elucidated the effects of PDJ on phytochemical production in red leaf lettuce. The PDJ treatments promoted the accumulation of phenolic compounds in aerial plant parts. An LC-MS analysis revealed that these accumulated compounds were identified as cyanidin-3-O-glucoside, cyanidin-3-O-(6″-O-malonyl)-glucoside and cyanidin-3-O-(6″-O-malonyl)-glucoside methyl ester. The abundance of these compounds in lettuce extracts increased significantly in response to the PDJ treatment. Additionally, the LC-MS analysis also identified the accumulated phenolic compounds in the extracts of PDJ-treated lettuce, including caffeoyltartaric acid, chlorogenic acid, caffeoylmalic acid, chicoric acid, and dicaffeoylquinic acid. Gene expression analyses indicated the PDJ treatments upregulated the expression of PAL, F3H, and ANS genes in lettuce. These results suggest that PDJ treatments enhance the expression of genes involved in the synthesis of anthocyanins and phenolic compounds, resulting in an increase in the quantities of these compounds, which reportedly have various functions affecting human physiology.


Introduction
The demand for foods rich in phytochemicals has recently increased [1]. There is considerable epidemiological evidence that polyphenols promote health and decrease the risk of various diseases, including cancers and cardiovascular diseases [2,3]. Diverse vegetables and fruits are sources of polyphenols, and diets rich in polyphenols can help protect against several diseases [4,5].
Lettuce is a popular vegetable included in various foods to enhance human health. Its health-promoting properties are associated with its substantial abundance of antioxidants, primarily vitamin C and polyphenols, and fiber [6]. A previous study involving humans and rats revealed the protective effects of lettuce against cardiovascular diseases [7]. In red leaf lettuce (Lactuca sativa L.), quercetin, luteolin, cyanidin, chicoric acid, and caffeoylmalic acid are the major phytochemicals [8]. Phytochemical production in lettuce increases in response to environmental stresses, such as ultraviolet radiation, as well as injuries and postharvest processing [9].
Enhancing flavonoid biosynthesis pathways by modulating environmental conditions may be useful for promoting phytochemical production in crops. For example, cultivating lettuce plants in a greenhouse under low-temperature conditions reportedly induces the production of some types of phytochemicals [10]. Recent studies examined the utility of high-intensity artificial light, such as light-emitting diodes or ultraviolet light sources, for increasing lettuce anthocyanin contents [11,12]. Some genes encoding key enzymes in the phenylpropanoid pathway, including phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), are light-inducible genes [13][14][15]. Additionally, some genes affecting anthocyanin synthesis pathways, including those encoding dihydroflavonol 4reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT), are jasmonic acid (JA)-inducible genes regulated by the phytochrome-mediated pathway in Arabidopsis [16].
Although altering light conditions is a convenient way to promote crop growth and phytochemical production during commercial crop production, installing new equipment and the associated power consumption may increase crop production costs.
Jasmonic acid and its derivatives are plant hormones and/or plant growth regulators in higher plants that control fruit ripening and coloration (including pigment accumulation). They can also induce the biosynthesis of secondary compounds, including phenylpropanoid derivatives, caffeic acids, flavonols, and anthocyanins [17,18]. Moreover, JA can improve crop quality by increasing secondary metabolite contents.
In this study, we evaluated the effects of PDJ treatments on production of phenolic compounds in hydroponically grown red leaf lettuce. Red leaf lettuce (Lettuce) plants were cultivated in PDJ-supplemented culture media. We also quantitatively analyzed the components of methanolic extracts from PDJ-treated lettuce plants via colorimetric assays and high-performance liquid chromatography (HPLC). Furthermore, we investigated the compounds and molecular mechanisms in lettuce affected by PDJ using liquid chromatography-mass spectrometry (LC-MS)-based methods to identify the unknown compounds. We conducted gene expression analyses to clarify the regulation of enzymes involved in the phenylpropanoid derivative synthesis pathway in PDJ-treated lettuce.

PDJ Treatments Increase the Phenolics Contents in Aerial Parts of Lettuce Plant
Lettuce plants were grown hydroponically for 14 days after sowing and PDJ was supplied by irrigation for another 2 days. The first and second leaves were seen on the 16th day after sowing. The PDJ-treated lettuce showed red color compared to the lettuce without PDJ treatment (Supplementary Figure S1). We determined the contents of metabolites in the extracts from the aerial parts of lettuce plants treated with 0, 100 or 200 µM PDJ by measuring the absorbance at 540 and 305 nm. The metabolites absorbed at 540 nm containing the anthocyanin, were, respectively, 2.7-and 4.2-times higher in the lettuce plants treated with 100 and 200 µM than in the control plants ( Figure 1A). The metabolites absorbed at 305 nm containing UV-light absorbing pigments (i.e., caffeic acid and flavonoid derivatives) were, respectively, 1.6-and 2.3-times higher in the lettuce plants treated with 100 and 200 µM than in the control plants ( Figure 1B). We also determined the total phenolic content of the extracts from the aerial parts of lettuce plants treated with 0, 100 or 200 µM PDJ. Total phenolic content was 1.3-and 2.2-times greater in the lettuce plants treated with 100 and 200 µM PDJ than in the control plants, respectively ( Figure 1C). with 100 and 200 µM than in the control plants ( Figure 1B). We also determined the total phenolic content of the extracts from the aerial parts of lettuce plants treated with 0, 100 or 200 µM PDJ. Total phenolic content was 1.3-and 2.2-times greater in the lettuce plants treated with 100 and 200 µM PDJ than in the control plants, respectively ( Figure 1C). Amounts of compounds in extracts from aerial parts of PDJ-treated lettuce plant. Extracts from aerial parts of plants treated with (black) or without (white) 100 µM or 200 µM PDJ were measured the absorbance at 540 nm (A) and 305 nm (B). The extracts were reacted with phenolic reagents (C), for measurement of total phenolic content, measured the absorbance at 750 nm, respectively. Each value was calibrated with the fresh weight of the aerial parts. Each value indicated as mean ± standard error. Statistically significant differences are indicated by an asterisk (**p < 0.01).

Figure 1.
Amounts of compounds in extracts from aerial parts of PDJ-treated lettuce plant. Extracts from aerial parts of plants treated with (black) or without (white) 100 µM or 200 µM PDJ were measured the absorbance at 540 nm (A) and 305 nm (B). The extracts were reacted with phenolic reagents (C), for measurement of total phenolic content, measured the absorbance at 750 nm, respectively. Each value was calibrated with the fresh weight of the aerial parts. Each value indicated as mean ± standard error. Statistically significant differences are indicated by an asterisk (** p < 0.01).

Extracts of PDJ-Treated Lettuce Plants Increased in Antioxidant Activities
We conducted the DPPH radical scavenging assay to measure the antioxidant activities of the lettuce extracts. DPPH radical scavenging activities of the extracts from lettuce plants treated with 200 µM PDJ were higher than that of the control plants ( Figure 2).

Extracts of PDJ-Treated Lettuce Plants Increased in Antioxidant Activities
We conducted the DPPH radical scavenging assay to measure the antioxidant activities of the lettuce extracts. DPPH radical scavenging activities of the extracts from lettuce plants treated with 200 µM PDJ were higher than that of the control plants ( Figure 2).

Figure 2.
Radical scavenging activity in extracts from aerial parts of PDJ-treated lettuce plant. Extracts from aerial parts of plants treated with (black) or without (white) 100, 150, or 200 µM PDJ were reacted with DPPH reagents, for radical scavenging activity, and then measured the absorbance at 520 nm, respectively. Each value indicated as mean ± standard error. Statistically significant differences are indicated by an asterisk (*p < 0.05).

Quantification and Identification of the Pheolics in the Leaf Extracts from Lettuce Plants Treated with PDJ
To clarify the changes to phenolics in PDJ-treated lettuce plants, we analyzed the extracts from the aerial parts of lettuce plants at 2 days after the PDJ treatment using an HPLC system. The chromatograms (at 520 nm) for the anthocyanins in the extracts from lettuce plants treated with 100 and 200 µM PDJ included three peaks ( Figure 3).
The anthocyanins with increased contents induced by the PDJ treatments were analyzed by LC-MS. We determined all three peaks in Figure  549 m/z), respectively, on the basis of the mass spectra, UVvis spectra, and previous reports [28][29][30][31]. One of the three peaks was detected at the same retention time as the standard compound for cyanidin-3-O-glucoside using the HPLC system ( Figure S7).
The Cy3G content of the PDJ-treated lettuce increased 2.5-and 6.39-times in responses to the 100 and 200 µM PDJ treatment, respectively. The Cy3MG content of the PDJ-treated lettuce increased 2.31-and 10.5-times in responses to the 100 and 200 µM PDJ treatment, respectively. The Cy3MG-Me content of the PDJ-treated lettuce increased 1.95-and 8.05times in responses to the 100 and 200 µM PDJ treatment, respectively (Table 1 and Figure  3).

Figure 2.
Radical scavenging activity in extracts from aerial parts of PDJ-treated lettuce plant. Extracts from aerial parts of plants treated with (black) or without (white) 100, 150, or 200 µM PDJ were reacted with DPPH reagents, for radical scavenging activity, and then measured the absorbance at 520 nm, respectively. Each value indicated as mean ± standard error. Statistically significant differences are indicated by an asterisk (* p < 0.05).

Quantification and Identification of the Pheolics in the Leaf Extracts from Lettuce Plants Treated with PDJ
To clarify the changes to phenolics in PDJ-treated lettuce plants, we analyzed the extracts from the aerial parts of lettuce plants at 2 days after the PDJ treatment using an HPLC system. The chromatograms (at 520 nm) for the anthocyanins in the extracts from lettuce plants treated with 100 and 200 µM PDJ included three peaks ( Figure 3).
The anthocyanins with increased contents induced by the PDJ treatments were analyzed by LC-MS. We determined all three peaks in Figure  549 m/z), respectively, on the basis of the mass spectra, UV-vis spectra, and previous reports [28][29][30][31]. One of the three peaks was detected at the same retention time as the standard compound for cyanidin-3-O-glucoside using the HPLC system ( Figure S7).
The Cy3G content of the PDJ-treated lettuce increased 2.5-and 6.39-times in responses to the 100 and 200 µM PDJ treatment, respectively. The Cy3MG content of the PDJtreated lettuce increased 2.31-and 10.5-times in responses to the 100 and 200 µM PDJ treatment, respectively. The Cy3MG-Me content of the PDJ-treated lettuce increased 1.95and 8.05-times in responses to the 100 and 200 µM PDJ treatment, respectively (Table 1 and Figure 3).    The chromatograms (at 325 nm) for the phenolics in the extracts from lettuce plants treated with 100 and 200 µM PDJ ( Figure 4 and Table 2) comprised six peaks.
We determined all six peaks in Figure     The chromatograms (at 325 nm) for the phenolics in the extracts from lettuce plants treated with 100 and 200 µM PDJ ( Figure 4 and Table 2) comprised six peaks. on the basis of the mass spectra, UV spectra and previous reports [34,38,39]. It was suggested that peak 6 in Figure 4 may be the quercetin-3-O-(6″-O-malonyl)-glucoside (Q3MG; [M − H] − , 549 m/z) from the mass spectra and previous reports [34,38,39]. However, since no further evaluation data were available, we could not be identified as the Q3MG. Two of the six peaks were detected at the same retention times as each of the standard compounds for chlorogenic acid and chicoric acid using the HPLC system (Supplementary Figure S8).   We determined all six peaks in Figure 4  515 m/z), respectively, on the basis of the mass spectra, UV spectra and previous reports [34,38,39]. It was suggested that peak 6 in Figure 4 may be the quercetin-3-O-(6"-O-malonyl)-glucoside (Q3MG; [M − H] − , 549 m/z) from the mass spectra and previous reports [34,38,39]. However, since no further evaluation data were available, we could not be identified as the Q3MG. Two of the six peaks were detected at the same retention times as each of the standard compounds for chlorogenic acid and chicoric acid using the HPLC system (Supplementary Figure S8).
The chicoric acid content increased 1.09-and 3.86-times in response to the 100 and 200 µM PDJ treatment, respectively. The content of CTA was 1.2-and 3.75 times higher in the extracts from lettuce plants treated with 100 and 200 µM PDJ, respectively, than in the control extract. The chlorogenic acid contents increased 1.17-and 4.56-times in response to the 100 and 200 µM PDJ treatment, respectively. The CMA contents increased 0.87-and 3.18-times in response to the 100 and 200 µM PDJ treatment, respectively. The diCQA contents increased 0.77-and 2.56-times in response to the 100 and 200 µM PDJ treatment, respectively (Table 2 and Figure 4).

Expression Analysis of Lettuce Genes Involved in the Phenylpropanoid Derivative Synthesis Pathway in PDJ-Treated Lettuce
We detected an increase in the contents of several anthocyanins and phenolic acids in PDJ-treated lettuce plants. In previous studies, some lettuce genes in the phenylpropanoid synthesis pathway were identified and investigated [11,40]. To clarify the mechanisms in lettuce affected by PDJ treatments, we analyzed the PDJ-induced gene expression changes in lettuce plants. We observed a slight difference in the degree of red color between the first and second leaf in the lettuce plantlets ( Figure S1). Therefore, we determined the PDJ-induced gene expression changes in the first and second leaf of the lettuce aerial parts.
At 48 h after the PDJ treatment, the expression of most of the analyzed genes was upregulated in the first and second leaves of PDJ-treated lettuce plants. In the first leaf, the PAL (2.5-times), F3H (8.75-times), DFR (2.62-times), and ANS (2.89-times) expression levels were higher in the PDJ-treated lettuce plants than in the control plants, although the difference was significant only for PAL and F3H. Additionally, the UFGT expression level (3.29-times) also tended to be higher in PDJ-treated lettuce, relative to the control level ( Figure 5). In the second leaf, the PAL (1.93-times), F3H (1.95-times), DFR (7.3-times), and ANS (5.04-times) genes tended to be more highly expressed in the PDJ-treated lettuce plants than in the control plants, but the difference was significant only for ANS ( Figure 6). In contrast, there were no differences in the CHS expression levels between the PDJ-treated and control plants. These results suggested that the genes induced by the PDJ treatment influenced lettuce plant metabolism and promoted the production of caffeic acid derivatives and anthocyanin derivatives within 48 h.

Discussion
Prohydrojasmon, which is a synthetic JA derivative, has been used as a bio-stimulant to increase anthocyanin accumulation in crops [27]. Although PDJ is commercially available as a plant growth regulator useful for enhancing the coloration of various fruits, such as apple and grape, its mechanism of action has not been comprehensively characterized. Thus, in this study, we clarified its effects using leaves from hydroponically grown lettuce plants. In our preliminary experiments, we detected significant increases in the metabolites absorbing at 305 and 540 nm and the total phenolic content in the first and second leaf of lettuce plants at 1, and 2 days after a 200 µM PDJ treatment, especially in the first and second leaf at 2 days after the treatment (data not shown). Significant increases in the metabolites absorbing at 305 and 540 nm and the total phenolic content were detected in

Discussion
Prohydrojasmon, which is a synthetic JA derivative, has been used as a bio-stimulant to increase anthocyanin accumulation in crops [27]. Although PDJ is commercially available as a plant growth regulator useful for enhancing the coloration of various fruits, such as apple and grape, its mechanism of action has not been comprehensively characterized. Thus, in this study, we clarified its effects using leaves from hydroponically grown lettuce plants. In our preliminary experiments, we detected significant increases in the metabolites absorbing at 305 and 540 nm and the total phenolic content in the first and second leaf of lettuce plants at 1, and 2 days after a 200 µM PDJ treatment, especially in the first and second leaf at 2 days after the treatment (data not shown). Significant increases in the metabolites absorbing at 305 and 540 nm and the total phenolic content were detected in the lettuce plants treated with 100 µM PDJ, but they were lower than that of 200 µM PDJ treatment and no significant difference in the antioxidant activity at the 100 µM PDJ treatment. These differences between 100 and 200 µM PDJ may depend on the amounts of these compounds.
In this study, Cy3MG was the most abundant anthocyanin following the PDJ treatment. This anthocyanin is synthesized via the malonylation of Cy3G by anthocyanin 3-malonyl transferase [41].
We identified some malonylated compounds in the current study (e.g., Cy3MG). Malonylation enables the storage of phenolic compounds in vacuoles because it leads to increased water solubility and helps to stabilize the labile structure, which is conducive to the transport of compounds into the vacuole [42]. After entering cells, naphthol is mainly accumulated as a malonylglucoside, and some of it is released from cells in a methoxylated form and as a glycoside [42]. A treatment with PDJ may promote the malonylation of compounds, which are then transported and stored in vacuoles. Earlier investigations proved that Cy3MG can function as a superoxide scavenger [43] and that red leaf lettuce plants cultivated under cool conditions accumulate Cy3MG [44]. Because JA is a stressinducible plant growth regulator, stress-induced responses may accelerate the accumulation of these malonylated compounds.
On the other hand, it was reported that the Cy3MG-Me was observed as a form of the methylated cyanidin malonylglucoside during elution of the anthocyanin with acidified MeOH [28,29]. In this process, Cy3MG reacted with the acidified MeOH and then esterification formed the methyl ester of Cy3MG. Additionally, finally, it converted to cyanidin-3-O-glucoside [29]. Since we used the acidified MeOH to extract the anthocyain, a part of Cy3MG might convert to the Cy3MG-Me and/or Cy3G.
CMA has the same m/z that of coutaric acid (CoTA). The retention time of the CoTA was 5.72 min. This was earlier than the retention time of 5-CQA (9.11 min) [38]. The retention time of CMA was later (13.04 min) than 5-CQA and CTA (3.75 min) [38]. In the present results, peak 3 in Figure 4 showed a later retention time than chlorogenic acid. Additionally, in the UV spectrum of this compound showed, the λmax was around 328 nm (Table 2 and Supplementary Figure S6), which was different from the λmax of CoTA (313 nm) [38]. Therefore, we determined that peak 3 in Figure 4 was the CMA.
The pathways involved in the synthesis of caffeic acid derivatives, such as chicoric acid, have not been thoroughly elucidated. However, a previous study on Arabidopsis confirmed that the pathway involved in the synthesis of caffeic acid derivatives diverges from p-coumaryl-CoA of the phenylpropanoid derivative synthesis pathway [45]. Phenylalanine ammonia-lyase is an early and key enzyme in the phenylpropanoid derivative synthesis pathway, and functions upstream of the synthesis of p-coumaryl-CoA. Methyl jasmonate treatments reportedly enhance PAL activity and increase the abundance of some phenolic compounds in radish [46]. We determined that PAL gene expression in lettuce is upregulated by PDJ treatments. A methyl jasmonate treatment can promote PAL activity and the expression of the PAL gene [47]. Exogenously applied PDJ can also increase the PAL gene expression level in red pear [26]. Thus, in lettuce plants, PDJ may enhance PAL expression, resulting in an increase in caffeic acid derivative contents. However, future studies regarding the regulated expression of other caffeic acid synthesis-related genes will need to clarify the PDJ-induced mechanisms.
We observed that the PAL, F3H, and ANS gene expression levels in PDJ-treated lettuce tend to increase after 48 h. In Arabidopsis, JA upregulates the expression of the late anthocyanin biosynthesis genes [48]. In the current study, we did not detect anthocyanin and flavonol intermediates, possibly because PDJ also induced the expression of the late anthocyanin biosynthesis genes (e.g., DFR, ANS, and UFGT genes), leading to the production of anthocyanin derivatives. Regarding the ANS and DFR genes, JA promotes anthocyanin ac-  [48]. Furthermore, these transcription factors have a synergistic effect under far-red light conditions, which is mediated by the far-red light receptor phytochrome A (PHYA) [16]. A recent study indicated that endogenous JA is inactivated by a sulfotransferase (ST2a) in a process that is dependent on the PHYB-PIF pathway under low red:far-red light conditions [49]. In the present study, the artificial light sources used for our experiments emitted far-red light (700-780 nm). Thus, the transcriptional regulation of MYB through PHYA and COI1-JAZ as well as ST2a via the PHYB-PIF-mediated pathway may contribute to the anthocyanin accumulation in lettuce plants treated with PDJ.
In our preliminary experiments, the total phenolic content and chicoric acid contents in lettuce plants grown under low-light conditions (photosynthetic photon flux density < 90 µE) did not increase following the PDJ treatment (data not shown). These results suggest that high-intensity light is required for the production of phenolic metabolites in lettuce plants. Moreover, an exposure to PDJ may enhance the production of these compounds via the upregulated expression of the corresponding genes (Figure 7). Becker et al. reported that the contents of some flavonoids, including Cy3G and Q3MG, increase in red leaf lettuce depending on the photosynthetic photon flux density, whereas phenolic acid contents, including chicoric acid, chlorogenic acid, and caffeoylmalic acid, are influenced by the leaf developmental stage rather than the photosynthetic photon flux density [44]. In future studies, we will investigate the relationship between light intensity and PDJ effects to determine the most appropriate way to improve phytochemical production in crops.
Plants 2021, 10, x FOR PEER REVIEW 11 of 17 ANTHOCYANIN PIGMENT 1) transcription factors [48]. Furthermore, these transcription factors have a synergistic effect under far-red light conditions, which is mediated by the far-red light receptor phytochrome A (PHYA) [16]. A recent study indicated that endogenous JA is inactivated by a sulfotransferase (ST2a) in a process that is dependent on the PHYB-PIF pathway under low red:far-red light conditions [49]. In the present study, the artificial light sources used for our experiments emitted far-red light (700-780 nm). Thus, the transcriptional regulation of MYB through PHYA and COI1-JAZ as well as ST2a via the PHYB-PIF-mediated pathway may contribute to the anthocyanin accumulation in lettuce plants treated with PDJ. In our preliminary experiments, the total phenolic content and chicoric acid contents in lettuce plants grown under low-light conditions (photosynthetic photon flux density <90 µE) did not increase following the PDJ treatment (data not shown). These results suggest that high-intensity light is required for the production of phenolic metabolites in lettuce plants. Moreover, an exposure to PDJ may enhance the production of these compounds via the upregulated expression of the corresponding genes (Figure 7). Becker et al. reported that the contents of some flavonoids, including Cy3G and Q3MG, increase in red leaf lettuce depending on the photosynthetic photon flux density, whereas phenolic acid contents, including chicoric acid, chlorogenic acid, and caffeoylmalic acid, are influenced by the leaf developmental stage rather than the photosynthetic photon flux density [44]. In future studies, we will investigate the relationship between light intensity and PDJ effects to determine the most appropriate way to improve phytochemical production in crops.

Plant Materials and Growth Conditions
Lettuce plants (Lactuca sativa L. cv. Red-fire) were grown under hydroponic conditions. Lettuce seeds were purchased from Takii and Co. Ltd. (Kyoto, Japan). The lettuce seeds were sown and germinated and the resulting seedlings were grown on Rockwool (Grodan, Roermond, The Netherlands) moistened with tap water in a growth chamber (HD-type Koitotron, Koito-Kogyo Co., Tokyo, Japan) at the National Institute for Environmental Studies, Japan. The temperature and relative humidity were maintained at 23 • C and 60% to 70%, respectively. Plants were exposed to a 14 h light/10-h dark cycle under fluorescent lights (National FPR96EX-N/A, Panasonic Co., Osaka, Japan) with a photosynthetic photon flux density of approximately 94 µmol m −2 s −1 at the top of each plant for 16 days.

PDJ Treatments
The lettuce seedlings were treated with a commercially available plant growth regulator, Jasmoneito Ekizai (Meiji Seika Pharma Co., Ltd., Tokyo, Japan), which contains 5% active PDJ (equivalent to 200 mM). Working solutions at specific concentrations (100, 150 and 200 µM) were prepared by dilution with tap water. Regarding the control treatment, tap water containing 30% (v/v) 1-propanol (Fujifilm-Wako Chemical Co., Osaka, Japan) and 0.03% (v/v) Tween 20 was used. At 14 days after sowing, PDJ and control solutions were supplied by irrigation only once under hydroponic conditions, and grown for another two days. For extraction of compounds absorbing at 305 and 540 nm and the total phenolic content, and for determination of antioxidant activity, aerial parts of lettuce plants (containing first and second leaves) were harvested 16 days after sowing. The aerial parts (fresh weight of each sample was about 0.13 g) of harvested lettuce were immediately frozen in liquid nitrogen and stored at −80 • C, after which they were lyophilized. In the aerial parts of the lettuce plant, water content was predicted about 92.7% calculated from fresh weight (just after harvested sample) and dry weight (lyophilized samples) (n = 18-20).

Extraction of Phenolics from Lettuce Leaves
Lyophilized lettuce samples (at 16 days after sowing) were ground to a powder and transferred to 15 mL tubes. The ground material was resuspended in a 10-fold volume of methanolic hydrochloric acid (80% methanol and 1% hydrochloric acid). The tubes were gently shaken to ensure the ground material was completely soaked. The tubes were incubated in darkness at 4 • C for 48 h. The obtained extracts were used for measuring amounts of the compounds absorbing at 305 and 540 nm and the total phenolic content.

Measurement of the Total Phenolic Contents of Lettuce Extracts
The total phenolic contents were measured using the Folin-Ciocalteu (FC) reagent as previously described [27]. The FC reagent was then added and the sample was incubated in darkness for 3 min. Next, 10% sodium carbonate (Tokyo Kasei Industry Co. Ltd., Tokyo, Japan) was added and the sample was incubated in darkness for 30 min. The absorbance (at 750 nm) of each extract per gram fresh weight of plant tissue was measured using a microreader (Varioskan LUX, Thermo Fisher Scientific Inc., Waltham, MA, USA). The total phenolic content, which was expressed as gallic acid equivalents (GAE), was calculated based on a standard curve generated using gallic acid solutions. The data are presented herein as milligrams of gallic acid equivalents per gram of plant tissue fresh weight (mg GAE/g FW). The analysis was conducted three times using samples obtained from three independent experiments.

Measurement of the Compounds Aabsorbing at 305 and 540 nm Contents of Lettuce Extracts
The compounds absorbing at 305 and 540 nm were measured as previously described, with some modifications [27]. Briefly, the absorbance at 305 nm and 540 nm of the methanolic hydrochloric acid extracts of plant tissues (whole aerial lettuce plant parts) were measured using the Varioskan LUX microreader. Each extract was transferred to a 96-well Plants 2021, 10,1920 13 of 17 microplate. The compounds absorbing at 305 and 540 nm contents are herein expressed as the absorbance of the extract per gram of plant tissue fresh weight (Absorbance at 305 nm/g FW and Absorbance at 540 nm /g FW). The analysis was conducted three times using samples obtained from three independent experiments.

Measurement of the Antioxidant Activity of Lettuce Extracts
The antioxidant activity was measured using the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay as previously described [27,34]. Briefly, a DPPH working solution was prepared and Trolox was used to generate a standard curve. A 10 µL aliquot of the extract or Trolox was mixed with a 190 µL DPPH working solution. The sample was added to a microplate, which was incubated in darkness for 10 min at ambient temperature to allow the reaction to proceed. The absorbance at 520 nm was measured in triplicate for each DPPH (+) and DPPH (−) sample using the Varioskan LUX microreader. The DPPH radical scavenging activity was expressed as the percentage absorbance inhibition (%) calculated according to the following:

HPLC and LC-MS Analyses of the Extracts from PDJ-Treated Lettuce
The methanolic hydrochloric acid extracts were passed through filter paper (ADVAN-TEC, Tokyo, Japan), after which the filtrates were heated in a water bath at 40 • C and then dried using a rotary evaporator (EYELA Tokyo Rikakikai Co. Ltd., Tokyo, Japan). The volume was adjusted with methanolic hydrochloric acid for a sample concentration of 5 mg/mL. Extracts were stored at −30 • C until analyzed. The extracts were filtered using a 0.45 µm Syringe Driven Filter (Milex-LH, Merck Millipore Ltd., Tullagreen, Ireland) for the HPLC analysis, which was completed using the Ultimate 3000 system (Thermo Fisher Scientific, Waltham, MA, USA). More specifically, the extracts were separated and analyzed using the Acclaim C30 stationary phase column (pore size 3 µm, 3.0 mm × 150 mm; Thermo Fisher Scientific, Waltham, MA, USA). The mobile phases were 0.1% (v/v) trifluoroacetic acid (Fujifilm-Wako Chemical, Richmond, VA, USA) as solvent A and 100% acetonitrile (Fujifilm-Wako Chemical) as solvent B. The initial gradient conditions were 90% A and 10% B. In 10 min, the gradient reached 75% A and 25% B. In the following 10 min, the gradient was adjusted to 90% A and 10% B. The sample injection volume was 1 µL. The column flow rate was 0.5 mL/min at 30 • C. The absorbance was measured at 520 and 325 nm for anthocyanin and phenolic acids (hydroxycinnamic acids), respectively, using a diode array detector system (DAD-3000, Thermo Fisher Scientific, Waltham, MA, USA). As standard compounds, cyanidin-3-O-glucoside chloride (Nagara Science Co. Ltd., Gifu, Japan; purity on HPLC ≥ 99%), chlorogenic acid (Fujifilm-Wako Chemical Co., Osaka, Japan), and chicoric acid (Tokyo Chemical Industry Co. Ltd., Tokyo, Japan; purity on ≥ 98%). The cyanidin-3-O-glucoside chloride was diluted in distilled water. The chlorogenic acid and the chicoric acid were diluted in methanol. To quantify the amounts of anthocyanins and phenolic compounds, we made the standard curve of cyanidin-3-Oglucoside and chicoric acid. To construct the calibration curves, appropriate 10 mg/mL of cyanidin-3-O-glucoside chloride and chicoric acid were diluted with water and methanol, respectively. For cyanidin-3-O-glucoside chloride, four concentration levels (0.1, 0.15, 0.2 and 0.25 mg/mL) were analyzed. For chicoric acid, five concentration levels (0.05, 0.1, 0.15, 0.2 and 0.25 mg/mL) were analyzed. The calibration curve showed linearly, with correlation coefficients 0.9979 and 0.9863 for cyanidin-3-O-glucoside chloride and chicoric acid, respectively. For quantification, peak areas were correlated with concentrations in accordance with the calibration curves. For Cy3G, Cy3MG, and Cy3MG-Me, cyanidin-3-Oglucoside chloride was used for calibration. For CTA, chlorogenic acid, CMA, chicoric acid, diCQA, chicoric acid equivalent was used.
The amounts of compounds were expressed µg Cy3G equivalent (for anthocyain) or mg chicoric acid equivalent (for phenolic acids or flavonol derivative) per gram of plant material fresh weight.
The HPLC analysis was conducted three times using samples obtained from three independent experiments.

Gene Expression Analysis of Phenylpropanoid Derivative Synthesis Genes in PDJ-Treated Lettuce
We analyzed the expression-level changes to the phenylpropanoid synthesis genes in PDJ-treated lettuce by conducting a qPCR assay. The first and second leaves of five lettuce plants treated with or without 200 µM PDJ were harvested at 0, 24 and 48 h after the treatments. The collected leaves were stored in RNAlater solution (Thermo Fisher Scientific, Waltham, MA, USA) at −20 • C until used. Total RNA was extracted from the harvested leaves using the RNAprep Pure Plant Plus Kit with RNase-Free DNase I (TIANGEN Biotech Co. Ltd., Beijing, China). Specifically, leaves in RNAlater solution were transferred to 1.5 mL microtubes, which were weighed before the leaves were ground using a pestle homogenizer. The RNA was extracted according to the manufacturer's instructions. The total RNA quantity was determined using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
The extracted RNA served as the template for a reverse transcription using the Superscript IV VILO Master Mix kit (Thermo Fisher Scientific, Waltham, MA, USA). The total RNA (1 µg) was mixed with the DNase solution comprising 1 µL 10× ezDNase Buffer, 1 µL ezDNase enzyme, and nuclease-free water (up to 10 µL) to digest genomic DNA at 37 • C for 2 min. The mixture was then placed on ice. After adding 4 µL SuperScript IV VILO Master mix and 6 µL nuclease-free water, the solution was incubated at 25 • C for 10 min and then at 50 • C for 10 min. To inactivate the enzyme, the mixture was incubated at 85 • C for 5 min. The synthesized cDNA quantity was measured, after which the cDNA was stored at −20 • C until used.
The qPCR was completed using the KOD SYBR qPCR kit (TOYOBO Co. Ltd., Osaka, Japan). The synthesized cDNA (500 ng in 5.6 µL) was mixed with 10 µL KOD SYBR qPCR Mix, 4 µL primer solution (forward and reverse primers, 0.2 µM each), and 0.4 µL 50× ROX reference dye. The PCR conditions were as follows: 50 • C for 2 min and 95 • C for 10 min; 45 cycles of 95 • C for 15 s and 60 • C for 60 s. Expression data were obtained from three independent experiments. The expression levels of all analyzed genes were normalized against the ACT expression level. The expression data were analyzed using the comparative Ct (∆∆Ct) method of the 7500 Fast Real-Time System software (version 2.0.5) (Thermo Fisher Scientific, Waltham, MA, USA). The qPCR assay was conducted three times using RNA samples obtained from three independent experiments.

Statistical Analysis
All data were analyzed using Microsoft Excel. Significant differences were estimated based on an ANOVA test (p < 0.05 or 0.01).

Conclusions
We identified the lettuce compounds affected by PDJ treatments. Additionally, we revealed that the expression of some lettuce genes related to the synthesis of anthocyanins and phenolics is upregulated by exogenous PDJ. We determined that PDJ treatments enhance the expression of genes encoding enzymes involved in phenylpropanoid synthesis pathways, thereby promoting the production of caffeic acid derivatives and anthocyanin derivatives in lettuce.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/plants10091920/s1, Figure S1: Red leaf lettuce seedling treated without or with prohydrojasmon (PDJ), Figure S2: Mass spectrometry chromatograms (Selected Ion Monitoring: SIM) of the extracts from lettuce leaves not treated with PDJ, Figure S3. Mass spectrometry chromatograms (SIM) of the extracts from lettuce leaves treated with 100 µM PDJ, Figure S4: Mass spectrometry chromatograms (SIM) of the extracts from lettuce leaves after 200 µM PDJ treatments, Figure S5: Mass spectrometry spectra (Scan) of the extracts from lettuce leaves with 200 µM PDJ treatments, Figure S6: UV-Vis spectra of the peaks found in the extracts from lettuce leaves with 200 µM PDJ treatments, Figure S7: Chromatograms of the metabolites absorbing at 520 nm from lettuce plants treated with or without PDJ, Figure S8: Chromatograms of the metabolites absorbing at 325 nm from lettuce leaves with or without PDJ treatments, Table S1: Sequences of gene-specific qPCR primers. Funding: This research was supported by funds from the Zeon Corporation, Tokyo, Japan (Industry-University Collaboration between University of Tsukuba and Zeon Corporation, No. CDA29177). The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All our data, tables and figures in this manuscript are original. We have added our data availability statement. The data presented in this study are available in the article and its supplementary material.