Red leaf lettuce seeds (Lactuca sativa L. cv. Red Wave; Sakata Seed Co., Japan) were pre-germinated for 1 day at 20°C under 200 μmol∙m⁻²∙s⁻¹ photosynthetic photon flux (PPF) for 12 h by using fluorescent lamps (FL40SBR-A; NEC Co., Japan). The germinated seeds were sown in sponge cubes of 2 × 2 × 2 cm where they were grown under the same prior conditions. The nutrient solution was based on a half-strength culture solution of the Otsuka House A-recipe (Otsuka Chemical Co., Japan). Plants were grown under a 12-h day/night cycle throughout the experiments. After 14 days from the date of sowing, the night temperature treatment was initiated by transferring plants to different temperature conditions at nighttime. The temperature of the day period was set at 20˚C in all plants. MJ (5 mM) was sprayed onto the whole leaves for the last 1 h of night temperature treatment and all the plants were kept in the dark for 1 h at 20˚C. The control plants were sprayed with distilled water. These treatments were repeated for 3 days. After 17 days from the date of sowing, and just after exposure to light for 12 h, the plants were harvested, and the growth and metabolite analyses were conducted. All experiments were repeated at least four times―all obtained similar results.

The anthocyanin content was spectrophotometrically measured as described elsewhere [25] . The second true leaves (50 mg) were homogenized with 1 mL methanol containing 1% hydrochloric acid. After storage at 4˚C overnight, each sample was centrifuged at 10,000 g for 5 min at room temperature. The absorbance of the supernatant was measured at 533 nm, and a standard curve was prepared by using cyanidin-3-glucoside.

The total phenol content was measured using the modified Folin-Ciocalteu method, as described previously [25] . The second true leaves (50 mg) were homogenized with 500 µL of 90% methanol. After storage at 4˚C overnight, each sample was centrifuged at 10,000 g for 5 min at room temperature. The supernatant (50 µL) was diluted with distilled water to 650 µL, and a 50 µL of phenol reagent was mixed with it. After adding 300 µL of 5% sodium carbonate the mixture was incubated at 25˚C for 30 min. The absorbance of the supernatant was measured at 765 nm, and a standard curve was prepared by using gallic acid.

The hydrogen peroxide (H₂O₂) content was spectrophotometrically measured as described previously [26] but with slight modifications. The second true leaves (100 mg) were homogenized in 600 µL of 1% trichloroacetic acid (TCA). Then each sample was centrifuged at 10,000 g for 10 min. The supernatant (250 µL) was added to 250 µL of a 10 mM K-phosphate buffer (pH 7.0) and 500 µL of 1 M KI. The absorbance of the mixture was measured at 390 nm, and a standard curve was prepared by using H₂O₂ in the range from 25 - 500 μmol mL⁻¹.

The level of malondialdehyde―MDA, a lipid peroxidation product―was determined via the thiobarbituric acid reaction (TBAR) method [25] [27] . The second true leaves (100 mg) were homogenized in 0.5 mL of a 0.1% TCA solution. After centrifuging at 10000 g for 5 min, 0.2 mL of the supernatant was added to 0.8 mL of 0.5% thiobarbituric acid in 20% TCA. The mixture was incubated at 95˚C for 30 min; the reaction was stopped by cooling on ice. After centrifuging at 10,000 g for 5 min at room temperature, the absorbance of the supernatant was measured at 532 nm and the nonspecific absorbance at 600 nm was subtracted. The concentration of MDA was quantified by using a molar extinction coefficient of 155 mM⁻¹∙cm⁻¹.

Superoxide dismutase (SOD) activity was spectrophotometrically measured as described previously [28] , but with slight modifications. The second true leaves (100 mg) were homogenized in 0.5 mL of a 50 mM phosphate buffer (pH 7.0, 0.1 mM EDTA). The sample was centrifuged at 10,000 g for 10 min. The supernatant (2.5 µL) was added to 1 mL of a SOD reaction mixture (50 mM phosphate buffer [pH 7.7], 0.1 mM EDTA, 13 mM methionine, 75 µM nitroblue tetrazolium [NBT], 2 µM riboflavin). The reaction mixture was illuminated for 15 min at a light intensity of 300 µmol∙m^?2∙s^?1, and the absorbance was then monitored at 550 nm. One unit of SOD activity was defined as the amount of enzyme required to inhibit the NBT reduction by 50%. The total soluble proteins were determined spectrophotometrically by using the reagent Protein Assay Rapid Kit (Wako Ltd., Japan) and by applying the Bradford method in which bovine serum albumin was used as the standard.

Three-day treatments of five different night temperatures (5˚C, 10˚C, 20˚C, 30˚C, and 37˚C) did not significantly influence the leaf biomass of red leaf lettuce (Figure 1, Figure 2(A) and Figure 2(B)). By contrast, the water content of the leaves was significantly decreased by subjecting the lettuce plants to 5˚C and 10˚C low night temperatures as compared with the non-shifted control temperature (20˚C), while the high night temperature shifts did not significantly influence the leaf water content (Figure 2(C)). Water contents of plants grown at

night temperature of 5˚C, 20˚C, and 37˚C were 90.1%, 92.2%, and 92.6%, respectively.

Red pigmentation of the leaves was enhanced in those plants treated with low (5˚C and 10˚C) or high (37˚C) night temperatures, especially in second true leaves (Figure 1). In agreement with this result, the production of anthocyanin in second true leaves was significantly increased by the treatment of low night temperatures as compared with the control plants grown at 20˚C (Figure 3(A)). In plants grown at 5˚C and 10˚C, their total leaf anthocyanin content increased about 4.0- and 2.8-fold, respectively, when compared with the control plants. The plants grown at a 37˚C night temperature also displayed a 1.6-fold increase

in their amount of anthocyanin, though this difference was not significant (Figure 3(A)). Consistent with these results, the total phenol content of leaves exposed to either low or high temperatures at night was comparably increased as well (Figure 3(B)). The exposure of plants to temperatures of 5˚C, 10˚C, and 37˚C at night caused about 2.1-, 1.7-, and 1.6-fold increase in their total phenol content, respectively, as compared with the control plants. However, no apparent differences in the anthocyanin and total phenol contents were observed between 20˚C (control) and the 30˚C treated plants (Figure 3).

Next, we tested the combined effects of either a low (5˚C) or high (37˚C) night temperature with MJ on the leaf biomass and metabolites in red leaf lettuce. After the 3-day treatment, MJ tended to reduce the leaf biomass regardless of the night temperature (Figure 4(A) and Figure 4(B)). The MJ-induced biomass reduction was greater in plants exposed to the low night temperatures as compared with plants exposed to the high night temperature. Similarly, those plants exposed to MJ showed a tendency of decreased leaf water content irrespective of the night temperature treatment (Figure 4(C)).

The application of MJ increased the anthocyanin content in leaves regardless of the night temperature (Figure 5(A)). The effect of enhanced anthocyanin content by MJ was higher in those plants that were exposed to the high night temperature as compared with those that were co-treated with the low night temperature. The amounts of anthocyanin in leaves that were exposed to 5˚C, 20˚C, and 37˚C night temperatures and co-treated with MJ respectively were approximately 1.8-, 2.9-, and 2.7-fold higher than that in corresponding leaves un- treated with MJ. Consistent with these results, the production of total phenol was up regulated by the MJ treatment at all tested temperatures (Figure 5(B)).

The total phenol contents of leaves that were exposed to 5˚C, 20˚C, and 37˚C night temperatures and co-treated with MJ respectively were about 4.0-, 3.3-, and 3.5-fold higher than that in corresponding leaves untreated with MJ.

Anthocyanin accumulation is triggered by several oxidative stresses in plants [29] [30] . Therefore, we investigated the leaf H₂O₂ content in the experimental plants treated with night temperatures in combination with MJ. Low or high night temperatures triggered the production of H₂O₂ in the absence of MJ (Figure 6(A)). The MJ treatment up regulated the production of H₂O₂ at any of the night temperatures tested. The level of H₂O₂ was significantly higher in those plants exposed to a high night temperature in combination with MJ as compared with those exposed to a low night temperature in combination with MJ. The amounts of H₂O₂ in leaves that were exposed to 5˚C, 20˚C, and 37˚C night temperature and co-treated with MJ respectively were about 1.4-, 1.5-, and 2.0-fold higher than that in corresponding leaves untreated with MJ.

To monitor the marker for oxidative stress status we measured a lipid peroxidation products, MDA using the TBAR method. Consistent with the above changes found in the amount of leaf H₂O₂, MDA was induced by either low or high night temperatures, and it was accelerated by the MJ treatment (Figure 6(B)). The amount of MDA in leaves that were exposed to 5˚C, 20˚C, and 37˚C night temperatures and co-treated with MJ respectively were approximately1.3-, 1.2-, and 2.0-fold greater than that in corresponding leaves without MJ.

Plants are able to mitigate oxidative stresses by activating antioxidative enzymes, such as SOD [31] . Hence, we also monitored the SOD activity of leaves in plants that were exposed to different night temperatures in combination with MJ. The SOD activity tended to increase at either a low or high night temperature treatment in the absence of MJ (Figure 7). Co-treatment with MJ up regulated the SOD activity, irrespective of any night temperature.

Low temperature induces the production of plant secondary metabolites, including anthocyanin, in various plants [17] . Similar to this view, our present re-

sults show that the exposure of red leaf lettuce plants to a low night temperature triggers anthocyanin accumulation in their leaves. When stressed by low temperatures, plants can activate the expression of a variety of genes, some of which are involved in the biosynthesis of anthocyanin, such as PAL, CHS, and CHI in many plant species [32] [33] [34] [35] [36] . In lettuce, these anthocyanin biosynthesis genes can be transcriptionally regulated by the Arabidopsis MYB transcription factor AtMYB60 [37] . Given that MYB transcriptional factors are common regulators of the anthocyanin synthesis pathway in many plant species [38] , the modulation of the temperature-responsive transcriptional factors that orchestrate the expression of the involved anthocyanin biosynthesis genes may offer a strategy for increasing the content of this valuable metabolite in lettuce crops. In this study, the plants underwent the temperature stresses during the night period only, which did not significantly affect their leaf biomass. This suggests that the method of a periodical temperature stress treatment may allow for the efficient production of valuable metabolites.

High temperature also modulates the production of anthocyanin, a process that is well studied, especially in fruit vegetables [9] [39] [40] . In apples and grapes, the colors of their fruits decrease under high temperatures due to the down- regulation of anthocyanin accumulation [39] [40] . In accord with this, increasing the growing temperature decreases the expression of several genes encoding the transcription factors that regulate the anthocyanin synthetic genes [39] [41] . In prior studies, anthocyanin production in lettuce decreased when the growing temperatures were increased [18] [20] [42] . In contrast to that finding, here we found that a high night temperature enhances the anthocyanin content in red leaf lettuce leaves. Because the high temperature treatment in those previous studies was set 25˚C - 30˚C lower than that used in our present study (37˚C), those temperatures might be insufficient to activate the heat stress signaling associated with the anthocyanin synthesis. It is also possible that the modest high temperature stimuli used in previous studies may have caused alternations of the growth and metabolites as a result of a plant acclimation response to the modest temperature shift. Indeed, when 1-month-old sugarcane sprouts were exposed to more severe condition of heat stress (40˚C), within 24 h this treatment had triggered anthocyanin accumulation in them [43] . Given that exposure to 35˚C increased the phenolic compounds in tomato though this same treatment did not do so in watermelon plants [44] , the particular temperature required for the induction of heat stress responses is likely species- and cultivar-dependent.

Previously, we demonstrated that MJ induced the anthocyanin production in red leaf lettuce in a light-dependent manner [21] . Here, we show that MJ accelerated the accumulation of anthocyanin in leaves triggered by the treatment with low or high night temperatures. Jasmonic acid (JA) is produced in response to biotic and abiotic stresses, and it acts as a signal molecule in the synthesis of flavonoids, such as anthocyanin [10] [45] . Alone, both JA and MJ can induce anthocyanin production in Arabidopsis by activating the expression of the anthocyanin biosynthesis genes PAL, CHS, DFR, LDOX, and UF3GT [15] . In guava fruits, an MJ treatment in combination with a 5°C-low temperature treatment increased PAL activity, which in turn led to the accumulation of total phenols [46] . Given that a low temperature stress is known to activate the anthocyanin biosynthetic genes in several plants [32] [33] [34] [35] [36] , and that the low temperature- induced expressions of these genes were dependent on light [47] , the augmentation of a low night temperature-induced anthocyanin accumulation by MJ in the red leaf lettuce plants may be caused by the cumulative upregulation of gene expression related to anthocyanin synthesis.

Chilling and heat stresses can trigger oxidative stress responses, including the production of reactive oxygen species which could play a key role in mediating important signal transduction responses involved in plant tolerance to changing temperatures [48] . Similar to this, in the present study, either a high or low night temperature treatment was able to induce H₂O₂ production and lipid peroxidation in the red leaf lettuce plants. These oxidative stress parameters were also upregulated by the MJ treatment regardless of the night temperature condition, and they were closely related to the production of anthocyanin. Anthocyanins are involved in several stress responses, namely as antioxidants that alleviate the oxidative damage in plants [49] [50] [51] [52] [53] . Considering that JA is also a mediator of oxidative stress responses and is involved in plant adaptation to mitigate several stresses [54] , the increased production of anthocyanin in our experiments is among the antioxidative responses that resulted from the heat, chilling, and JA-mediated stress responses. The augmentation effects of MJ on anthocyanin synthesis, H₂O₂ accumulation, and lipid peroxidation, were more evident in those lettuce plants exposed to high night temperature as compared with those exposed to the low or control (20˚C) night temperatures. Interestingly, in rice plants, the endogenous JA content is markedly increased under cold stress but it is reduced by heat stress [55] . In soybean plants, ethylene synthesis is activated under high temperature stress, which triggers premature leaf senescence by increasing the superoxide radical and H₂O₂ content, as well as membrane damage [56] [57] . Given that the color pigmentation of apple skin is enhanced by both MJ and ethylene, and enhanced by their combined treatment [58] , the augmentation effect of the oxidative responses triggered by the high night temperature in combination with the MJ treatment may be driven by the dual induction of ethylene-dependent and JA-dependent signaling for anthocyanin synthesis.

In the present study, the low night temperature and the MJ treatment significantly decreased the leaf water content in red leaf lettuce, thus indicating a modest drought stress to leaves. Anthocyanin is produced in response to drought stress and it is involved in tolerance to this stress [17] [59] [60] . In Arabidopsis, the oxidative and drought tolerances are enhanced by an overaccumulation of antioxidant flavonoids, including anthocyanins [59] . Given that anthocyanin production is negatively related with the leaf water content in the present study, the low night temperature and MJ treatments may induce anthocyanin production in red leaf lettuce in part via the activation of drought stress signaling. Thus, the regulation of plant water status by manipulating the hydroponic cultivation system might be a useful way to further modulate the productionof anthocyanin in crops of red leaf lettuce.

Our experimental results indicate that the combined treatment of multiple stimuli, such as the night temperature shift and the MJ treatment, could be a useful strategy to modulate plant secondary metabolite production in red leaf lettuce. Even if a single stress treatment is effective at increasing the content of valuable plant metabolites, this effect seems to be less than that arising from multiple stimuli treatments. Given that a single stimulus is required for a relatively long- term period to accumulate anthocyanin in plants, this may eventually affect the plant biomass of lettuce. Therefore, providing multiple short-term stimuli just before harvesting potentially offers a simple and effective way for many plants of a crop to gain more valuable metabolites while minimizing the stimuli’s influence on plant biomass.