Growth and Bioactive Compound Content of Glehnia littoralis Fr. Schmidt ex Miquel Grown under Different CO2 Concentrations and Light Intensities

This study aims to determine the effect of different CO2 concentrations and light intensities on the growth, photosynthetic rate, and bioactive compound content of Glehnia littoralis Fr. Schmidt ex Miquel in a closed-type plant production system (CPPS). The plants were transplanted into a deep floating technique system with recycling nutrient solution (EC 1.0 dS·m-1 and pH 6.5) and cultured for 96 days under a temperature of 20 ± 1 °C, a photoperiod of 12/12 h (light/dark), and RGB LEDs (red:green:blue = 7:1:2) in a CPPS. The experimental treatments were set to 500 or 1500 µmol∙mol−1 CO2 concentrations in combination with one of the three light intensities: 100, 200, or 300 µmol∙m−2∙s−1 photosynthetic photon flux density (PPFD). The petiole length of G. littoralis was the longest in the 500 µmol∙mol−1 CO2 concentration with the 100 µmol∙m−2∙s−1 PPFD. The fresh weight (FW) and dry weight (DW) of shoots and roots were the heaviest in the 300 µmol∙m−2∙s−1 PPFD regardless of the CO2 concentration. Higher CO2 concentrations and light intensities produced the greatest photosynthetic rates. However, the SPAD value was not significantly different between the treatments. Higher light intensities produced greater content per biomass of chlorogenic acid and total saponin, although the concentration per DW or FW was not significantly different between treatments. The first and second harvest yields were the greatest in the 300 µmol∙m−2∙s−1 PPFD, regardless of the CO2 concentration. These results show that the 300 µmol∙m−2∙s−1 PPFD enhanced the growth, photosynthetic rate, and bioactive compound accumulation of G. littoralis, regardless of the CO2 concentration in a CPPS.


Introduction
Medicinal plants are those plants where the whole leaves, stems, flowers, fruits, roots, seeds, or whole plants are used as raw materials for medicine. Currently, medicinal plants are being used in various ways as herbal medicines and as the raw materials of cosmetics, functional health foods, and natural medicines; the interest in these products from consumers has increased, thus increasing the demand and related market size   The root growth characteristics, such as the root length, root diameter, FW, and DW of the roots are shown in Table 2. The root growth was closely associated with the light condition for the shoot growth [26]. The root length was the shortest in the 100 µmol•m −2 •s −1 PPFD, regardless of the CO2 concentration, at 10.7 and 11.3 cm for the 500 and 1500 µmol•mol −1 concentration, respectively. The root diameter was the thinnest at 6.50 mm in the 1500 µmol•mol −1 with 100 µmol•m −2 •s −1 PPFD. Furthermore, the FW and DW of roots displayed a positive correlation in which the weight increased as the light intensity increased. The root growth was not affected by the CO2 concentration but, a change in the light intensity produced a significant difference. Nager et al. [27] reported that a high light intensity (300 µmol•m −2 •s −1 PPFD) enhanced the FW of the roots of Nicotiana tabacum. Similarly, Olschowski et al. [28] obtained a heavier root DW of Calibrachoa in a high light intensity when cutting. Kitaya et al. [29] suggested that an optimal PPFD can rapidly produce high-quality lettuce plug seedlings.  The root growth characteristics, such as the root length, root diameter, FW, and DW of the roots are shown in Table 2. The root growth was closely associated with the light condition for the shoot growth [26]. The root length was the shortest in the 100 µmol·m −2 ·s −1 PPFD, regardless of the CO 2 concentration, at 10.7 and 11.3 cm for the 500 and 1500 µmol·mol −1 concentration, respectively. The root diameter was the thinnest at 6.50 mm in the 1500 µmol·mol −1 with 100 µmol·m −2 ·s −1 PPFD. Furthermore, the FW and DW of roots displayed a positive correlation in which the weight increased as the light intensity increased. The root growth was not affected by the CO 2 concentration but, a change in the light intensity produced a significant difference. Nager et al. [27] reported that a high light intensity (300 µmol·m −2 ·s −1 PPFD) enhanced the FW of the roots of Nicotiana tabacum. Similarly, Olschowski et al. [28] obtained a heavier root DW of Calibrachoa in a high light intensity when cutting. Kitaya et al. [29] suggested that an optimal PPFD can rapidly produce high-quality lettuce plug seedlings.

Photosynthetic Rate and the SPAD Value
The CO 2 concentration and light intensity significantly affected the photosynthetic rate in G. littoralis (Figure 2A). The photosynthetic rate was the highest in the 1500 µmol·mol −1 CO 2 concentration with the 300 µmol·m −2 ·s −1 PPFD in 6.8 µmol CO 2 m −2 ·s −1 , while the lowest was found in the 500 µmol·mol -1 CO 2 concentration with 100 µmol·m −2 ·s −1 PPFD in 0.5 µmol CO 2 m −2 ·s −1 . The photosynthetic rate showed a positive correlation with CO 2 concentration and the light intensity. The SPAD did not show a significant difference ( Figure 2B). Zheng et al. [30] reported that a high light intensity (350 µmol·m −2 ·s −1 PPFD) increased the photosynthetic capacity of the mother plant and the primary runner plant of strawberry. Furthermore, CO 2 enrichment increased the photosynthetic rate of Gerbera jamesonii [31]. G. littoralis is a crop native to coastal dunes and grows in high-light natural environments. Similar results showed that the growth of Peucedanum japonicum Thunberg, which is native to the seashore, was also efficient at increasing growth and production in a high light intensity (200 µmol·m −2 ·s −1 PPFD) than in a low light intensity (60 µmol·m −2 ·s −1 PPFD) [32].

Photosynthetic Rate and the SPAD Value
The CO2 concentration and light intensity significantly affected the photosynthetic rate in G. littoralis (Figure 2A). The photosynthetic rate was the highest in the 1500 µmol•mol −1 CO2 concentration with the 300 µmol•m −2 •s −1 PPFD in 6.8 µ mol CO2 m −2 •s −1 , while the lowest was found in the 500 µmol•mol -1 CO2 concentration with 100 µmol•m −2 •s −1 PPFD in 0.5 µ mol CO2 m −2 •s −1 . The photosynthetic rate showed a positive correlation with CO2 concentration and the light intensity. The SPAD did not show a significant difference ( Figure 2B). Zheng et al. [30] reported that a high light intensity (350 µmol•m −2 •s −1 PPFD) increased the photosynthetic capacity of the mother plant and the primary runner plant of strawberry. Furthermore, CO2 enrichment increased the photosynthetic rate of Gerbera jamesonii [31]. G. littoralis is a crop native to coastal dunes and grows in high-light natural environments. Similar results showed that the growth of Peucedanum japonicum Thunberg, which is native to the seashore, was also efficient at increasing growth and production in a high light intensity (200 µmol•m −2 •s −1 PPFD) than in a low light intensity (60 µmol•m −2 •s −1 PPFD) [32].

Total Sugar and Starch
The high light intensity increased the total sugar and starch contents in G. littoralis. However, the CO2 concentration did not influence the total sugar and starch contents. The total sugar and starch

Total Sugar and Starch
The high light intensity increased the total sugar and starch contents in G. littoralis. However, the CO 2 concentration did not influence the total sugar and starch contents. The total sugar and starch contents were the highest in the 300 µmol·m −2 ·s −1 PPFD regardless of the CO 2 concentration (Figure 3). In a CO 2 concentration of 500 µmol·mol −1 , the total sugar and starch contents were higher in the 300 µmol·m −2 ·s −1 PPFD by 5.4 and 2.2 times compared to the 100 µmol·m −2 ·s −1 PPFD, respectively. Many studies have reported that CO 2 enrichment increases the sugar and starch Plants 2020, 9, 1581 5 of 12 contents [33][34][35], which is inconsistent with this study. G. littoralis, a halophyte with developed water storage tissue that stores a lot of water in the cell, is considered to be less sensitive to CO 2 concentrations because of its thick leaves. The leaf is a photosynthetic organ, and the area and thickness are the major factors affecting the growth of the plant. To absorb sufficient light energy, the leaves are as wide as possible, and at the same time to facilitate gas exchange (CO 2 , O 2 , and H 2 O), the leaves are as flat and thin as possible [36]. The higher light intensity can positively affect the accumulation of assimilates, such as proteins, and the starch, and sugar of spinach increased in the 300 µmol·m −2 ·s −1 PPFD than those in the 100 µmol·m −2 ·s −1 PPFD [37]. In Glycine max (Linn.) Merr., a high light intensity induced photosynthetic activity, increasing the soluble sugar, sucrose, and starch contents in the shoots and roots [38].
Plants 2020, 9, x FOR PEER REVIEW 5 of 12 contents were the highest in the 300 µmol·m −2 ·s −1 PPFD regardless of the CO2 concentration ( Figure  3). In a CO2 concentration of 500 µmol·mol −1 , the total sugar and starch contents were higher in the 300 µmol·m −2 ·s −1 PPFD by 5.4 and 2.2 times compared to the 100 µmol·m −2 ·s −1 PPFD, respectively. Many studies have reported that CO2 enrichment increases the sugar and starch contents [33][34][35], which is inconsistent with this study. G. littoralis, a halophyte with developed water storage tissue that stores a lot of water in the cell, is considered to be less sensitive to CO2 concentrations because of its thick leaves. The leaf is a photosynthetic organ, and the area and thickness are the major factors affecting the growth of the plant. To absorb sufficient light energy, the leaves are as wide as possible, and at the same time to facilitate gas exchange (CO2, O2, and H2O), the leaves are as flat and thin as possible [36]. The higher light intensity can positively affect the accumulation of assimilates, such as proteins, and the starch, and sugar of spinach increased in the 300 µmol·m −2 ·s −1 PPFD than those in the 100 µmol·m −2 ·s −1 PPFD [37]. In Glycine max (Linn.) Merr., a high light intensity induced photosynthetic activity, increasing the soluble sugar, sucrose, and starch contents in the shoots and roots [38].

Harvest Yield
G. littoralis can be harvested cyclically by taking a leaf. The CO2 enrichment did not have a significant effect on the harvest yield or the change in the number of leaves from the harvest to the re-harvest. However, the low light intensity condition affected the harvest yield. The first and second harvest yields, at 61 days and 96 days of treatment, are shown in Figure 4A,B, respectively. It was observed that the yield of G. littoralis was high in the light intensity of more than the 200 µmol·m −2 ·s −1 PPFD, where the harvest yield increased by more than twice as much during the second harvest compared to the first harvest in the 1500 µmol·mol −1 CO2 concentration with 300 µmol·m −2 ·s −1 PPFD. However, regardless of the CO2 concentration, there was no significant difference between the first and second harvest yields in the 100 µmol·m −2 ·s −1 PPFD. This was because the high light intensity significantly affected the root development of G. littoralis and sufficient roots were produced for new leaf production. The change in the number of leaves from the harvest to the re-harvest is shown in Figure 5. The number of leaves was only affected by the light intensity, and in particular, leaves were developed the most in the 300 µmol·m −2 ·s −1 PPFD. Lee et al. [39] reported that new leaf emergence and biomass accumulation were promoted at a higher apparent daily light integral level. Furthermore, the higher light intensity produced more primary and total plant runners of strawberry.

Harvest Yield
G. littoralis can be harvested cyclically by taking a leaf. The CO 2 enrichment did not have a significant effect on the harvest yield or the change in the number of leaves from the harvest to the re-harvest. However, the low light intensity condition affected the harvest yield. The first and second harvest yields, at 61 days and 96 days of treatment, are shown in Figure 4A,B, respectively. It was observed that the yield of G. littoralis was high in the light intensity of more than the 200 µmol·m −2 ·s −1 PPFD, where the harvest yield increased by more than twice as much during the second harvest compared to the first harvest in the 1500 µmol·mol −1 CO 2 concentration with 300 µmol·m −2 ·s −1 PPFD. However, regardless of the CO 2 concentration, there was no significant difference between the first and second harvest yields in the 100 µmol·m −2 ·s −1 PPFD. This was because the high light intensity significantly affected the root development of G. littoralis and sufficient roots were produced for new leaf production. The change in the number of leaves from the harvest to the re-harvest is shown in Figure 5. The number of leaves was only affected by the light intensity, and in particular, leaves were developed the most in the 300 µmol·m −2 ·s −1 PPFD. Lee et al. [39] reported that new leaf emergence and biomass accumulation were promoted at a higher apparent daily light integral level. Furthermore, the higher light intensity produced more primary and total plant runners of strawberry.

Bioactive Compound
The chlorogenic acid and total saponin concentrations per DW or FW of G. littoralis were not significantly different between the treatments (Figures 6A and 7A). On the other hand, the chlorogenic acid and total saponin contents per biomass were the greatest in the 300 µmol•m −2 •s −1 PPFD regardless of the CO2 concentration ( Figures 6B and 7B). In a CO2 concentration of 500 µmol•mol −1 , the chlorogenic acid and total saponin contents per biomass were higher in the 300 µmol•m −2 •s −1 PPFD by 1.6 and 6.3 times compared to in the 100 µmol•m −2 •s −1 PPFD, respectively. In other studies, CO2 enrichment improved the nutritional qualities but the total free phenolic acids and chicoric acid contents of lettuce significantly decreased; the reaction to the elevated CO2 concentration was found to be dependent on the plant species [40,41]. The accumulation of bioactive compounds is induced by the increased soluble sugar acting as a precursor that promotes the synthesis and accumulation of antioxidants [42][43][44]. The polyphenol content of lettuce grown in a 350 µmol•m −2 •s −1 PPFD was significantly higher than that of plants grown in a 180 µmol•m −2 •s −1 PPFD [45]. In this study, there was no difference in the concentration of total chlorogenic acid per DW or total saponin per

Bioactive Compound
The chlorogenic acid and total saponin concentrations per DW or FW of G. littoralis were not significantly different between the treatments (Figures 6A and 7A). On the other hand, the chlorogenic acid and total saponin contents per biomass were the greatest in the 300 µmol•m −2 •s −1 PPFD regardless of the CO2 concentration ( Figures 6B and 7B). In a CO2 concentration of 500 µmol•mol −1 , the chlorogenic acid and total saponin contents per biomass were higher in the 300 µmol•m −2 •s −1 PPFD by 1.6 and 6.3 times compared to in the 100 µmol•m −2 •s −1 PPFD, respectively. In other studies, CO2 enrichment improved the nutritional qualities but the total free phenolic acids and chicoric acid contents of lettuce significantly decreased; the reaction to the elevated CO2 concentration was found to be dependent on the plant species [40,41]. The accumulation of bioactive compounds is induced by the increased soluble sugar acting as a precursor that promotes the synthesis and accumulation of antioxidants [42][43][44]. The polyphenol content of lettuce grown in a 350 µmol•m −2 •s −1 PPFD was significantly higher than that of plants grown in a 180 µmol•m −2 •s −1 PPFD [45]. In this study, there was no difference in the concentration of total chlorogenic acid per DW or total saponin per

Bioactive Compound
The chlorogenic acid and total saponin concentrations per DW or FW of G. littoralis were not significantly different between the treatments (Figures 6A and 7A). On the other hand, the chlorogenic acid and total saponin contents per biomass were the greatest in the 300 µmol·m −2 ·s −1 PPFD regardless of the CO 2 concentration (Figures 6B and 7B). In a CO 2 concentration of 500 µmol·mol −1 , the chlorogenic acid and total saponin contents per biomass were higher in the 300 µmol·m −2 ·s −1 PPFD by 1.6 and 6.3 times compared to in the 100 µmol·m −2 ·s −1 PPFD, respectively. In other studies, CO 2 enrichment improved the nutritional qualities but the total free phenolic acids and chicoric acid contents of lettuce significantly decreased; the reaction to the elevated CO 2 concentration was found to be dependent on the plant species [40,41]. The accumulation of bioactive compounds is induced by the increased soluble sugar acting as a precursor that promotes the synthesis and accumulation of antioxidants [42][43][44]. The polyphenol content of lettuce grown in a 350 µmol·m −2 ·s −1 PPFD was significantly higher than that of plants grown in a 180 µmol·m −2 ·s −1 PPFD [45]. In this study, there was no difference in the concentration of total chlorogenic acid per DW or total saponin per FW, but the production of bioactive compounds increased in the 300 µmol·m −2 ·s −1 PPFD, which displayed high photosynthesis and superior growth. FW, but the production of bioactive compounds increased in the 300 µmol•m −2 •s −1 PPFD, which displayed high photosynthesis and superior growth.
Plants 2020, 9, 1581 8 of 12 was controlled by connecting a liquefied carbon dioxide tank and CO2 regulator. The CO2 concentration, temperature, and RH were monitored during the cultivation period using a data logger (TR-76Ui, T&D Co. Ltd., Nagano, Japan). The light intensity was set using a photometer (HD2101.2, Delta Ohm SrL, Caselle, Italy).

Growth Characteristics
After 56 days of treatment, the petiole length, crown and root diameters, root length, FW and DW of the shoots and roots, number of leaves, and leaf area were measured. The FW was investigated using an electronic balance (EW220-3NM, Kern & Sohn GmbH., Balingen, Germany) and the DW was investigated after drying in an oven (Venticell-220, MMM Medcenter Einrichtunger GmbH., Planegg, Germany) at 70 °C for 72 h. The crown and root diameter were measured using Vernier calipers (CD-20CPX, Mitutoyo Co. Ltd., Kawasaki, Japan). The leaf area was measured using a leaf area meter (LI-3000, LI-COR Inc., Lincoln, NE, USA). Photosynthetic rate was measured using a portable photosynthesis system (CIRAS-3, PP Systems International Inc., Amesbury, MA, USA) on the fully unfolded fifth leaf from the top. The measurement conditions were controlled as follows: leaf area 4.5 mm 2 ; leaf temperature 20 °C ; air flow rate 150 mL·min −1 ; 500 or 1500 µmol•mol −1 CO2 concentration; 100, 200, or 300 µmol•m −2 •s −1 PPFD. The chlorophyll content was expressed as the SPAD, and measured using a portable chlorophyll meter (SPAD-502, Konica Minolta Inc., Tokyo, Japan). The first and second harvests were performed after treatment for 61 and 96 days, respectively, and the harvest yield was measured by weight of marketable leaves (over 15 cm 2 of leaf area).

Total Sugar and Starch
For the total sugar content determination, the leaves of G. littoralis were ground with liquid nitrogen and stored in a deep freezer (NF-140SF, Nihon Freezer Co. Ltd., Yushima, Japan) at -70 °C. A 0.3 g sample was taken for each treatment. The samples were mixed with 10 mL of 80% ethanol and then ground for 1 min. After being heated in a 60 °C water bath, the supernatant was separated via centrifugation (908× g, 20 °C, 30 min). Then, 10 mL of 80% ethanol was added to the remaining precipitate, heated in a waterbath at 60 °C for 30 min, and centrifuged under the same conditions. After the two centrifugations, the supernatant was diluted with a total of 40 mL of 80% ethanol. Then, 0.5 mL of 5% phenol reagent was added to the sample solution, vortexed, and 2.5 mL of 98% sulfuric acid were added and vortexed. After cooling at room temperature, the absorbance was measured at

Growth Characteristics
After 56 days of treatment, the petiole length, crown and root diameters, root length, FW and DW of the shoots and roots, number of leaves, and leaf area were measured. The FW was investigated using an electronic balance (EW220-3NM, Kern & Sohn GmbH., Balingen, Germany) and the DW was investigated after drying in an oven (Venticell-220, MMM Medcenter Einrichtunger GmbH., Planegg, Germany) at 70 • C for 72 h. The crown and root diameter were measured using Vernier calipers (CD-20CPX, Mitutoyo Co. Ltd., Kawasaki, Japan). The leaf area was measured using a leaf area meter (LI-3000, LI-COR Inc., Lincoln, NE, USA). Photosynthetic rate was measured using a portable photosynthesis system (CIRAS-3, PP Systems International Inc., Amesbury, MA, USA) on the fully unfolded fifth leaf from the top. The measurement conditions were controlled as follows: leaf area 4.5 mm 2 ; leaf temperature 20 • C; air flow rate 150 mL·min −1 ; 500 or 1500 µmol·mol −1 CO 2 concentration; 100, 200, or 300 µmol·m −2 ·s −1 PPFD. The chlorophyll content was expressed as the SPAD, and measured using a portable chlorophyll meter (SPAD-502, Konica Minolta Inc., Tokyo, Japan). The first and second harvests were performed after treatment for 61 and 96 days, respectively, and the harvest yield was measured by weight of marketable leaves (over 15 cm 2 of leaf area).

Total Sugar and Starch
For the total sugar content determination, the leaves of G. littoralis were ground with liquid nitrogen and stored in a deep freezer (NF-140SF, Nihon Freezer Co. Ltd., Yushima, Japan) at −70 • C. A 0.3 g sample was taken for each treatment. The samples were mixed with 10 mL of 80% ethanol and then ground for 1 min. After being heated in a 60 • C water bath, the supernatant was separated via centrifugation (908× g, 20 • C, 30 min). Then, 10 mL of 80% ethanol was added to the remaining precipitate, heated in a waterbath at 60 • C for 30 min, and centrifuged under the same conditions. After the two centrifugations, the supernatant was diluted with a total of 40 mL of 80% ethanol. Then, 0.5 mL of 5% phenol reagent was added to the sample solution, vortexed, and 2.5 mL of 98% sulfuric acid were added and vortexed. After cooling at room temperature, the absorbance was measured at 490 nm using a spectrophotometer and the total sugar content was calculated using glucose as the standard.
For the starch determination, the leaves were ground with liquid nitrogen and stored in a deep freezer at −70 • C. Total of 0.3 g of sample was used for each treatment. To solubilize the sample, 10 mL of 80% ethanol was added, shaken for 30 min, and then centrifuged at 300× g for 30 min at 20 • C. Then, 40% ethanol was added to the residue, and centrifuged again under the same conditions. Then, 2 mL of 30% HClO 4 and 1 mL of dimethyl sulfoxide were added to the residue and kept at Plants 2020, 9, 1581 9 of 12 room temperature for 30 min. Then, 2 mL of distilled water and 5 mL of H 2 SO 4 were added, and the waterbath was used to maintain the temperature at 100 • C for 1 h, then centrigugation was performed under 300× g for 30 min at 20 • C. Around 0.5 mL of this sample solution was mixed with 0.5 mL of 5% phenol reagent, then vortexed, followed by the addition of 2.5 mL of 98% sulfuric acid and the vortexed. After cooling at room temperature, the absorbance was measured at 470 nm using a spectrophotometer, and the starch content was calculated using glucose as the standard.

Chlorogenic Acid Concentration
For the quantitative analysis of chlorogenic acid, 500 mg of each powdered plant material were first mixed with 20 mL of 80% methanol and then shaken at 100 rpm on an orbital shaker for 24 h at room temperature. Afterward, all of the supernatant was centrifuged at 4250× g for 5 min. The supernatant was filtered through a 0.2 µm syringe filter (25HP020AN, Advantech Co. Ltd., Asan, Korea) before being injected into a high-performance liquid chromatography device (Nexera, Shimadzu Corp., Kyoto, Japan) system equipped with an 4.6 × 150 mm, 5 µm column (Agilent Eclipse plus-C18, Agilent Technology Co Ltd., Santa Clara, CA, USA) and a guard column maintained at 30 • C. Solvents A (methanol) and B (trifluoroacetic acid) were used as the mobile phases. The gradient was as follows: 0 min, 100% A; 3 min, 10% B; 8 min, 30% B; 30 min, 50% B; 40 min, 60% B; 50 min, 100% B; held constant for 10 min. The flow rate was 0.8 mL·min−1 and the injection volume was 10 µL. The chromatogram was monitored at 270 nm using photodiode array detection. To present the chlorogenic acid, concentrations of the derivatized samples, standard curves were prepared using 3-(3,4-dihydroxycinnamoyl) quinic acid (chlorogenic acid, Sigma-Aldrich Co. Ltd., St. Louis, MO, USA). Then, the calculated values were converted to the concentrations in terms of milligrams of chlorogenic acid, per grams of DW of the samples.

Total Saponin Concentration
To measure the total saponin concentration of the root, the total saponin content was extracted using a method modified from [47]. About 0.5 g root powder was defatted with 10 mL of petroleum ether by shaking it for 4 h, and then the residues were extracted twice, each with 5 mL of 80% aqueous methanol, by shaking for 4 h each time on an orbit shaker. The extracts were stored at 4 • C in the dark for later use. Approximately 100 µL of the extract was mixed with 400 µL of 80% methanol, 500 µL of 8% vanillin solution, and 5 mL of 72% sulfuric acid. After the mixture was heated in a water bath at 60 • C for 10 min, it was cooled in ice-cold water. The absorbance of the supernatant was measured using a spectrophotometer at 544 nm to determine the total saponin concentration, which was expressed as milligrams of saponin equivalent (SE) per grams of fresh weight.

Statistical Analysis
The experiment involved three replicates and ten plants per replicate, and was laid out in a completely randomized block design. After selecting uniform plants, three plants per replicate were used to determine the plant growth parameters and three plants per replicate were used to determine the photosynthetic rate; the total sugar, starch, and bioactive compounds; and the harvest yield. The statistical analyses were carried out using the SAS program (SAS 9.4, SAS Institute Inc., Cary, NC, USA). The experimental results were subjected to an analysis of variance (ANOVA) and Tukey's multiple range tests. Graphing was performed with the SigmaPlot program (SigmaPlot 12.0, Systat Software Inc., Palo Alto, CA, USA).

Conclusions
This study focused on the effects of CO 2 concentration (500 or 1500 µmol·mol −1 ) and light intensity (100, 200, or 300 µmol·m −2 ·s −1 PPFD) on the growth, photosynthetic rate, and bioactive compound content of G. littoralis to find an appropriate CO 2 concentration and light intensity for the high-quality, mass production of medicinal plants grown in a CPPS. The G. littoralis was not affected by the CO 2 concentration, while a high light intensity increased the growth, bioactive compound content, and harvest yield. The data showed that a 300 µmol·m −2 ·s −1 PPFD greatly enhanced the plant production in a CPPS.