Plant materials, growth conditions, and experimental design.

Experiments were conducted in an artificial climatic chamber at Jiangsu University in China (32.2°N, 119.5°E). Romaine lettuce (L. sativa L.) was selected as the experimental plant. Lettuce seeds were sown in a plug tray filled with peat-based compost (peat : vermiculite at 3:1; v/v) at a light intensity of 200 μmol·m⁻²·s⁻¹ and at a temperature of 22 ± 1 °C/18 ± 1 °C (mean ±sd, day/night). When the seedlings had three true leaves, they were transplanted into plastic basins (15.5 cm [d] × 13 cm [h]) at a density of one plant per basin. The plastic basins were filled with vinegar residue, peat, and vermiculite at a ratio of 2:1:1 (by volume) with the following composition (percentage of dry matter): total N, 0.96%; total P, 0.19%; and total K, 0.43%. A photoperiod of 12 h, relative humidity (of air) of 60% to 70%, and CO₂ concentration of 400 ± 50 ppm were maintained throughout the experiments.

Red–blue light-emitting diode (LED) arrays (DR/W120; Philips Lighting Inc., Maarheeze, the Netherlands) and fluorescent lamps (T5-28 watt; Nonghui Biotechnology Co., Ltd., Shanghai, China) were used as light sources. The climate chamber had two cultivation shelves, each comprising three layers. The LED and fluorescent lamps were placed 35 cm above each layer. The spectral distribution of lamps was determined to identify light wavebands from 400 to 800 nm by using a field spectroradiometer (FieldSpec®3; Analytical Spectral Devices, Inc., Boulder, CO), as depicted in Fig. 1. Both types of light sources exhibited energy aggregation areas at ≈440 and 650 nm. These two spectral regions are crucial for plant growth. The light intensity value of the LED was twice that of the fluorescent lamp.

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Experiments were arranged in three batches as follows: Expt. 1: 30 °C/25 °C (Sept. 26, 2015 to Oct. 25, 2015); Expt. 2: 23 °C/18 °C (10 Apr. 2016 to 9 May 2016); and Expt. 3: 15 °C/10 °C (5 Dec. 2016 to 5 Jan. 2017). Each experimental set comprised five light intensity levels of 100, 200, 350, 500, and 600 μmol·m⁻²·s⁻¹, which were achieved by combining varying numbers of LED and fluorescent lamps on each layer of the cultivation shelves, as illustrated in Table 1. Photosynthetic photon flux density measured at the upper extremity of plants between 6 and 18 h was obtained by using a quantum sensor (ZDR-24; Zeda Instrument Co., Ltd., Hangzhou, China). Each level of light level treatment housed 28 samples.

Table 1.Light intensity levels achieved by combining various numbers of fluorescent and LED lamps.

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Chlorophyll fluorescence measurements.

Chlorophyll fluorescence parameters were measured 2, 3, 4, and 5 weeks after transplantation using an imaging pulse amplitude-modulated fluorometer (Imaging PAM; Heinz Walz, Effeltrich, Germany); three plants were used per treatment for gas exchange measurements. Plants were dark adapted for 30 min before the measurement, and the upper six fully expanded leaves of lettuce plants were selected for measurements. The minimum fluorescence (F_o) and maximum fluorescence (F_m) were obtained by applying measuring light pulses at a low frequency (1 Hz) from an LED and a 600-ms saturating blue pulse (10 Hz), respectively. The maximum PSII quantum yield (F_v/F_m) was calculated according to the following formula: F_v/F_m = (F_m − F_o)/F_m. Actinic illumination (500 μmol photons/m²/s) was then switched on. Saturating pulses were applied at 20 s intervals to measure the maximum fluorescence yield during actinic illumination (F^′_m) as well as the chlorophyll fluorescence yield during actinic illumination (F^′). NPQ was calculated according to the following formula: NPQ = (F_m –F^′_m)/F^′_m. Φ_PSII was calculated as the quotient (F^′_m − F^′)/F^′_m (Janka et al., 2015; Yang et al., 2018).

Gas exchange measurements.

Photosynthetic characteristics were measured on the same upper six fully expanded leaves of lettuce plants in each treatment at 2, 3, 4, and 5 weeks after transplantation. Three plants were used per treatment for gas exchange measurements. The P_n, g_S, and T_r were measured using a portable photosynthesis instrument (LI-6400 XT; LI-COR Biosciences, Lincoln, NE) as reported previously (O’Carrigan et al., 2014). The irradiance and temperature of the leaf chamber were set according to each treatment, and results were recorded when P_n reached a steady state. The gas exchange system allowed control of the CO₂ concentration at 400 μmol·m⁻²·s⁻¹ using an integrated CO₂ mixer (LI-COR Biosciences), and the flow rate was adjusted to 500 μmol·s⁻¹. All measurements were performed between 0900 and 1100 hr.

Lettuce nutrition measurements.

Three plants per treatment were randomly selected and used to analyze the total N, P, and K content 5 weeks after transplantation. After drying, all leaves were treated with H₂SO₄–H₂O₂ for heat digestion. The N content in lettuce leaves was determined using a Continuous Flow Auto Analyzer III (AA3-HR; SEAL Analytical, Southampton, UK) at 660 nm. The P content was measured using a spectrophotometer (ultraviolet-2100; Beifen-Ruili Analytical Instrument Co., Ltd, Beijing, China), and absorbance was read at 880 nm. The K content was quantified using a flame photometer (BWB-XP; BWB Technologies, Newbury, UK) in accordance with the method of a previous study (Mudau et al., 2007). The N, P, and K uptake of lettuce plants were calculated by multiplying the DW of lettuce plants by their N, P, and K content, respectively.

Growth and biomass measurements.

Three lettuce samples were randomly selected to count the LN and calculate LA by scanning the leaves of each plant using an LA2400 scanner. Images were then acquired using the WinFOLIA software program (Regent Instruments Inc., Quebec). At the end of each experiment, all the shoots of the lettuce plants were harvested; and the leaves were counted and weighed collectively to determine their FW, by using an electronic analytical balance with an accuracy of 0.1 mg. The DW of leaves was obtained by drying them in an oven first at 105 °C for 1 h and then at 80 °C for another 72 h.

Statistical analysis.

Results are expressed as the mean ±sd of three replicates in each of three individuals. Data were analyzed using two-way ANOVA. Multiple comparisons between treatment means were conducted using the least significant difference (lsd) test at P < 0.05. Pearson’s analysis (two-tailed) was used to evaluate correlations between light intensity and the variables pertaining to the lettuces’ properties. An independent (unpaired) t test (two-tailed) was used to test the significance of differences between the two means (P < 0.05).

Chlorophyll fluorescence characteristics.

F_v/F_m was used as a sensitive indicator of the original light-energy capture efficiency of PSII reaction centers and plant health status (Wong et al., 2012; Zhang et al., 2017). High values of F_v/F_m indicated high PSII maximum light conversion efficiencies. In healthy organisms, the F_v/F_m value is ≈0.8–0.84 in most C3 plant species, but the value decreases significantly when plants are exposed to stress (da Silva Branco et al., 2017; Kalaji et al., 2012; Wang et al., 2004). In this study, we observed that F_v/F_m was closely related to light intensity (P < 0.05, ANOVA), temperature (P < 0.05, ANOVA), and the interaction between light intensity and temperature (P < 0.05, ANOVA) (Fig. 2). Under the low-temperature condition, a negative correlation was observed between the F_v/F_m value and light intensities (T15) (Pearson’s r = −0.646, P < 0.05). The F_v/F_m value was significantly lower at a high light intensity (600 μmol·m⁻²·s⁻¹) than at a low light intensity (100 μmol·m⁻²·s⁻¹). The F_v/F_m value at 600 μmol·m⁻²·s⁻¹ was the smallest, and values at 600 and 500 μmol·m⁻²·s⁻¹ were below 0.80 for all four determination times. Under the low temperature condition, results indicated that the light intensities of 500 and 600 μmol·m⁻²·s⁻¹ may exceed the optimal light intensity range required by lettuce plants, resulting in photoinhibition. The curves of F_v/F_m at the five light intensity treatments were synchronous at T15; the highest reading was observed in week 4 (28 d after transplantation). Nevertheless, the correlations between F_v/F_m and light intensity were positive at the medium temperature (T23) (Pearson’s r = 0.610, P < 0.05) and at the high temperature (T30) (Pearson’s r = 0.908, P < 0.001). At T23, the dynamics of F_v/F_m at light intensities of 350, 500, and 600 μmol·m⁻²·s⁻¹ were completely consistent. All these treatments reached their respective peaks at 3 weeks, and the F_v/F_m values were >0.80. The curve of F_v/F_m at 500 μmol·m⁻²·s⁻¹ was the highest, followed by the curves at 200, 350, and 600 μmol·m⁻²·s⁻¹ for all treatment times. The F_v/F_m value at 500 μmol·m⁻²·s⁻¹ did not differ significantly from the F_v/F_m values at 200, 350, and 600 μmol·m⁻²·s⁻¹, according to the results of the t test (P > 0.05). However, the F_v/F_m values at 100 and 200 μmol·m⁻²·s⁻¹ reached their peak at 4 weeks. The F_v/F_m values were the lowest at a light intensity of 100 μmol·m⁻²·s⁻¹ and were less than 0.8. These results indicated that photoinhibition may have occurred when light intensity was below 100 μmol·m⁻²·s⁻¹ at T23. Similar changes were observed at T30. The F_v/F_m values at 100 μmol·m⁻²·s⁻¹ were the smallest, together with those for 200 and 350 μmol·m⁻²·s⁻¹ treatments. F_v/F_m values at 100, 200, and 350 μmol·m⁻²·s⁻¹ were all below 0.8. Severe stress occurred at 100 μmol·m⁻²·s⁻¹, whereas mild stress occurred at 200 and 350 μmol·m⁻²·s⁻¹ (P < 0.05, t test). However, the effects of the light intensities of 500 and 600 μmol·m⁻²·s⁻¹ on F_v/F_m were significantly different from those of low light intensities. F_v/F_m at intensities of 500 and 600 μmol·m⁻²·s⁻¹ were higher and were all greater than 0.8. The F_v/F_m value was the highest at 500 μmol·m⁻²·s⁻¹ at all four determined times, indicating this light intensity is optimal for lettuce growing at T30. In addition, the curves of F_v/F_m at the five light intensities were synchronous, and the highest reading was obtained at week 3.

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Light energy is absorbed by PSII through three pathways: photochemistry, thermal dissipation, and nonphotochemical quenching. Because those processes are competitive, the responses of the photosynthetic apparatus to different environmental conditions can be indicated by complementary changes in the yield of chlorophyll fluorescence. These responses are widely described using NPQ and Φ_PSII (Demmig-Adams et al., 1996; Kramer et al., 2004). NPQ measures the proportion of light energy lost through regulatory thermal dissipation, and Φ_PSII measures the proportion of light energy absorbed by antenna pigments in PSII used in photochemistry. In the present study, we observed that NPQ values were negatively correlated with light intensities at T15, T23, and T30 (Pearson’s r = −0.703, −0.632, and −0.395, respectively; all P < 0.05), and Φ_PSII was positively correlated with light intensity at T15, T23, and T30 (Pearson’s r = 0.704, 0.750, and 0.808, respectively; all P < 0.05) (Fig. 3). At T15, NPQ values at 100 and 200 μmol·m⁻²·s⁻¹ were the highest, and these values were significantly higher than those at other light intensity treatments (P < 0.05, t test). However, Φ_PSII was the lowest at 100 μmol·m⁻²·s⁻¹, followed by 200 μmol·m⁻²·s⁻¹ during the treatment times (all P < 0.05, t test). This coincided with earlier findings that poor light with low temperature caused a decrease of photochemical efficiency by increasing the thermal deexcitation of PSII (Hovenden and Warren, 1998; Verhoeven, 2014). The Φ_PSII value was highest at 350 μmol·m⁻²·s⁻¹, followed by 500 μmol·m⁻²·s⁻¹; and no significant difference was noted between the two light intensities (P > 0.05, t test). This result demonstrated that these plants at 350 and 500 μmol·m⁻²·s⁻¹ allocated more of the absorbed light energy to photochemistry than to dissipation processes. This allocation helped the plants to optimize photosynthesis and growth. However, NPQ and Φ_PSII values both decreased at 600 μmol·m⁻²·s⁻¹, indicating that the consumption of excess light energy decreased simultaneously through photochemical reactions and heat dissipation. Therefore, more excess light energy could be used to generate large numbers of reactive oxygen species, which aggravated photooxidative risks (Niyogi, 1999). The curves of Φ_PSII at 350, 500, and 600 μmol·m⁻²·s⁻¹ were synchronous, with the peak at week 4. At T23 and T30, NPQ was the highest at a light intensity of 100 μmol·m⁻²·s⁻¹, and it was not significantly different from those observed at the other four light intensities (P > 0.05, t test). At T23, Φ_PSII value was the highest at 500 μmol·m⁻²·s⁻¹, followed by 350, 600, and 200 μmol·m⁻²·s⁻¹; and these light intensities had similar effects on the light-use efficiencies of lettuce plants. The Φ_PSII value at 100 μmol·m⁻²·s⁻¹ was significantly lower than those at other light intensities (all P < 0.05, t test). These findings are consistent with those of a previous study, which reported that a light intensity of 200 to 600 μmol·m⁻²·s⁻¹ was suitable for the growth of lettuce and did not result in light stress (Fu et al., 2012a). The curves of Φ_PSII at 350, 500, and 600 μmol·m⁻²·s⁻¹ were synchronous, with the peak at week 3, whereas those at 100 and 200 μmol·m⁻²·s⁻¹ were synchronous, with the peak at week 4. At T30, Φ_PSII values were relatively high at 350, 500, and 600 μmol·m⁻²·s⁻¹, and the optimum light intensity was 500 μmol·m⁻²·s⁻¹. No significant differences in Φ_PSII were observed among the three light intensities. The Φ_PSII value was the lowest at 100 μmol·m⁻²·s⁻¹, followed by 200 μmol·m⁻²·s⁻¹. These results indicated that at T30, lettuce grown under light intensities of 350 to 600 μmol·m⁻²·s⁻¹ more effectively used absorbed light in the photosynthetic processes than lettuce grown under low light (below 200 μmol·m⁻²·s⁻¹). In addition, the curves of Φ_PSII at the five light intensities were synchronous, and the highest reading was obtained at week 3.

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Photosynthetic characteristics.

As detailed in Fig. 4, light intensities exerted significant effects (P < 0.05, ANOVA) on P_n and exhibited strong interactions with temperature (P < 0.05, ANOVA) during treatment. The variable P_n was positively correlated with light intensity at all three temperatures (Pearson’s r = 0.606, 0.859, and 0.913, respectively; all P < 0.05). Values of P_n at 100 μmol·m⁻²·s⁻¹ and 200 μmol·m⁻²·s⁻¹ were all lower than those at other light intensities during the treatment times. At T15, the P_n values at 350 μmol·m⁻²·s⁻¹ and 500 μmol·m⁻²·s⁻¹ were the highest, and values were significantly higher than that at 600 μmol·m⁻²·s⁻¹ (all P < 0.05, t test). The decrease in P_n at 600 μmol·m⁻²·s⁻¹ was probably due to photoinhibition, as indicated by the decrease in F_v/F_m. The major reason for this photoinhibition was that the light intensity was higher than the light saturation point (500–520 μmol·m⁻²·s⁻¹), thus limiting carbon assimilation in lettuce (Li and Gong, 2002). This was consistent with a previous study that revealed photoinhibition in lettuce after exposure to high light (Zhou et al., 2009). However, at T23 and T30, P_n reached an optimum at 500 μmol·m⁻²·s⁻¹ at all four determined times. At T23, no significant difference was noted among P_n values for treatments of 350, 500, and 600 μmol·m⁻²·s⁻¹ (all P > 0.05, t test). The results indicated that the lettuce plants under light intensities of 350 to 600 μmol·m⁻²·s⁻¹ were more efficient in using light energy, which played an important role in maximizing the number of photoassimilates stored in the growing plant. At T30, no difference was observed between the P_n values at 500 and 600 μmol·m⁻²·s⁻¹, and they were significantly higher than P_n at 350 μmol·m⁻²·s⁻¹ (P < 0.05, t test). This result demonstrated that at high temperatures, high light intensity was beneficial to maintaining high photosynthetic rates. At such rates, the quantity of Rubisco and PSII reaction center increased with the expansion of the light-harvesting complex II (Hikosaka and Terashima, 1995).

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The g_S and the T_r showed parallel changes with P_n under different combinations of light intensity and temperature for all treatments. Similar to P_n, g_S and T_r exhibited a significantly positive correlation with light intensity at T15, T23, and T30 (all P < 0.05, Pearson’s). This confirmed the prior finding that the effect of light intensity on photosynthesis was positively related to stomatal limitations (Fu et al., 2017).

Total uptake of N, P, and K.

N, P, and K are crucial mineral elements for growing lettuce. As detailed in Table 2, light intensity exhibited a significant effect (P < 0.05, ANOVA) on N, P, and K content, as well as the N, P, and K uptake of lettuce. It also had a strong interaction with temperature (P < 0.05, ANOVA). N, P, and K content exhibited parallel changes under different combinations of light intensity and temperature. As light intensities decreased, N, P, and K content increased and were the highest at 100 μmol·m⁻²·s⁻¹ at each temperature. This result is consistent with the finding of a previous study that low light intensity markedly weakened the photosynthetic capacity of plants, resulting in a significant decrease in dry matter accumulation, thus increasing nutrient content (Kazuo and Nobutoshi, 1998). However, low light intensity reduced nutrient uptake. N, P, and K uptake at 100 μmol·m⁻²·s⁻¹ were significantly lower than those at the other four light intensities. Nitrogen is an integral constituent of proteins, nucleic acids, and various coenzymes; and a large proportion of N in leaves is present in chloroplasts, with most of it being in the photosynthetic machinery (Evans, 1987). At T15, N uptake was highest at 350 μmol·m⁻²·s⁻¹, relatively high at 200 and 500 μmol·m⁻²·s⁻¹, and low at 600 μmol·m⁻²·s⁻¹. At T23 and T30, N uptake was highest at 350 μmol·m⁻²·s⁻¹, followed by 500 μmol·m⁻²·s⁻¹; both were significantly higher than those at 200 and 600 μmol·m⁻²·s⁻¹. Light intensities of 350 to 500 μmol·m⁻²·s⁻¹ at T23 and T30 markedly increased N uptake, a result probably associated with an increase in chlorophyll content and electron transport capacity (Evans, 1989). Phosphorus is an indispensable component in nucleic acids and plays a structural role in cellular membranes. Potassium participates in the activation of numerous enzymes required in the metabolism and transport of carbohydrates (Maathuis, 2009). At T15, P and K uptake at 350 μmol·m⁻²·s⁻¹ were the highest, and they were slightly higher than those at 200, 500, and 600 μmol·m⁻²·s⁻¹. The effect of light intensity on P and K uptake was not significant at low temperatures. However, at T23 and T30, compared with other light intensities, those of 350 and 500 μmol·m⁻²·s⁻¹ significantly enhanced P and K uptake. P uptake values were both the second highest at 600 μmol·m⁻²·s⁻¹, and K uptake values were the second highest for P200-T23 and P600-T30 treatments, respectively. Regarding photosynthetic parameters, light intensity of 350 to 600 μmol·m⁻²·s⁻¹ at T23 and T30 caused the opening of stomata and more transpiration. Thus, the plants grew at higher rates, and the demand for P and K increased.

Table 2.Effects of light intensity and temperature on nitrogen (N), phosphorus (P), and potassium (K) content and N, P, and K uptake in lettuce plants 5 weeks after transplantation. Mean values with standard error of mean (n = 3). Letters indicate significant differences at P < 0.05 according to the least significant difference test. * indicates significant difference at P < 0.05.

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Plant growth and yield.

As detailed in Table 3, obvious morphological differences were observed among the treatment groups. The results of the lsd multiple comparison test revealed a significant interaction effect between light intensity and temperature on the LN and LA of lettuce plants (P < 0.05). Low light intensity generally increases LA (Hou et al., 2010; Pires et al., 2011). However, our results differed from other studies. LA values at 100 and 200 μmol·m⁻²·s⁻¹ were low among all the light intensities at three temperature levels because of the inherent photosynthetic physiological characteristics of lettuce. At T15, LA and LN at 350 and 500 μmol·m⁻²·s⁻¹ were the highest, and they were significantly higher than those at other light intensities. Although LN had no significant difference among all light intensities at T23, the LA reached highest at 500 μmol·m⁻²·s⁻¹, followed by 350 μmol·m⁻²·s⁻¹. This result indicates that at T23, light intensity of 350 to 500 μmol·m⁻²·s⁻¹ can enhance leaf expansion, allowing plants to make better use of photosynthetic active radiation by increasing their surface area. LA and LN decreased significantly at 600 μmol·m⁻²·s⁻¹, which may benefit lettuce by decreasing the exposure of plant tissues to high light intensity and reducing water loss (Matos et al., 2009). At T30, the LN and LA both increased with increasing light intensity, and they reached their highest values at 600 μmol·m⁻²·s⁻¹, followed by 500 μmol·m⁻²·s⁻¹. This finding showed that at T30, light intensity of 500 to 600 μmol·m⁻²·s⁻¹ had a greater effect on leaves formed and expansion.

Table 3.Effects of light intensity and temperature on the leaf number (LN), leaf area (LA), dry weight (DW), and fresh weight (FW) of lettuce 5 weeks after transplantation. Mean values with standard error of mean (n = 3). Letters indicate significant differences at P < 0.05 according to the least significant difference test. * indicates significant difference at P < 0.05.

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Table 3 shows that the biomass of lettuce plants was significantly associated with both light intensity and the interaction between light intensity and temperature (P < 0.05). The DW and FW were lower under 100 and 200 μmol·m⁻²·s⁻¹ at all the temperatures, indicating a lack of low light adaptive response by the lettuce plants. At T15, the values of DW at 350, 500, and 600 μmol·m⁻²·s⁻¹ were maintained at high levels, whereas FWs at 350 and 500 μmol·m⁻²·s⁻¹ were the highest; and they were significantly higher than those at other light intensities. Similar to LN and LA, this result indicates that light intensity of 300 to 500 μmol·m⁻²·s⁻¹ led to the highest photosynthetic efficiency of lettuce plants, which is optimal for lettuce growth and biomass accumulation. At T23, the DW was the highest at 600 μmol·m⁻²·s⁻¹, and it was significantly higher than those at other light intensities; whereas FW at light intensities of 500 and 600 μmol·m⁻²·s⁻¹maintained high levels, and it was significantly higher than those at other light intensities. At T30, DW and FW increased considerably with increases in light intensity, and the highest DW and FW were obtained at a light intensity of 600 μmol·m⁻²·s⁻¹, followed by 500 μmol·m⁻²·s⁻¹. Therefore, at medium and high temperatures, higher light intensity favored higher biomass accumulation and was most likely to cause the increase in the carbohydrates used for physiological metabolism and growth.