Growth and Bioactive Compound Content of Glehnia Littoralis Fr

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

Hye Ri Lee 1, Hyeon Min Kim 1, Hyeon Woo Jeong 1, Myung Min Oh 2 and Seung Jae Hwang 1,3,4,5,*

1 Division of Applied Life Science, Graduate School of Gyeongsang National University, Jinju 52828, Korea; dgpﬂ[email protected] (H.R.L.); [email protected] (H.M.K.); [email protected] (H.W.J.) 2 Division of Animal, Horticulture and Food Sciences, Chungbuk National University, Cheongju 28644, Korea; [email protected] 3 Department of Agricultural Plant Science, College of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Korea 4 Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Korea 5 Research of Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea * Correspondence: [email protected]; Tel.: +82-055-772-1916

Received: 8 October 2020; Accepted: 10 November 2020; Published: 15 November 2020

Abstract: 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 · − CO concentrations in combination with one of the three light intensities: 100, 200, or 300 µmol m 2 s 1 2 · − · − photosynthetic photon flux density (PPFD). The petiole length of G. littoralis was the longest in the 500 µmol mol 1 CO concentration with the 100 µmol m 2 s 1 PPFD. The fresh weight (FW) and dry · − 2 · − · − weight (DW) of shoots and roots were the heaviest in the 300 µmol m 2 s 1 PPFD regardless of the CO · − · − 2 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 CO concentration. · − · − 2 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.

Keywords: chlorogenic acid; medicinal plant; photosynthetic rate; total saponin

1. 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 [1]. In the Republic of Korea, various medicinal plants are native in the wild because of Korea’s geographical characteristics and the four distinct seasons. However, only about 50 kinds of medicinal plants are grown in domestic farms, and there are fewer cultivation

Plants 2020, 9, 1581; doi:10.3390/plants9111581 www.mdpi.com/journal/plants Plants 2020, 9, 1581 2 of 12 areas and productions than can supply the increasing demand; therefore, the dependence on imports is also increasing [2]. Because medicinal plants are mainly grown outdoors, the cultivation period is long, the price fluctuation is severe, and the supply of medicinal plants is inelastic. In addition, the harvest time is limited; they are difficult to store or transport; the quality varies between varieties, cultivation environment, and harvest time. Also, the study of the cultivation process for the mass production of medicinal plants (seed germination, growing seedlings, cultivation after transplanting, etc.,) is insufficient, and it is necessary to find suitable conditions for the cultivation environment and develop the optimal cultivation technology for the stable production of high-quality medicinal plants. Glehania littoralis Fr. Schmidt ex Miquiel is a perennial plant belonging to the Umbelliferae family and is commonly found in coastal dunes. The petiole is long and scarlet, and the leaves are divided into three branches, each having three small leaves, where the small leaves have sawtooth edges, and the young leaves are used as vegetables. It has several functional components, such as saponin, coumarin, bergapten, β-sitosterol, and imperatorin [3,4], which are known to be effective against sweating, fever, and labor pains, and have diuretic, antiviral, anti-cancer, and immunosuppression properties [5–7]. CO2 is a very important factor in crop cultivation as a raw material for photosynthesis. The maintenance of CO2 concentrations during growth enhances crop growth [8,9]. CO2 enrichment can enhance CO2 fixation, yield, and quality [10,11]. Previous results showed that the biomass increased by about 50% in C3 plants [12], 35% in CAM (crassulacean acid metabolism) plants [13], and 12% in C4 plants [14] under CO2 enrichment conditions. Moreover, CO2 enrichment is effective in enhancing vegetable quality by promoting the accumulation of antioxidants in vegetables. Dong et al. [15] showed that CO2 enrichment increased the ascorbic acid, chlorophyll b, total antioxidant activity, total phenols, and total flavonoids by 9.5, 42.5, 59.0, 8.9, and 45.5%, respectively. However, the studies on CO2 enrichment in medicinal plants are relatively limited compared to those on the other plants. Light is an energy source for plant photosynthesis and an essential for plant growth, development, and bioactive compound accumulation. To achieve significant levels of plant cultivation in artificial facilities, it is important to set appropriate environmental conditions, especially with regard to light intensity. Plants grown under low light intensity have frequently been found to be more susceptible to photoinhibition than in high light intensity [16]. Previous studies have shown a decrease in the photosynthesis of eggplant [17] and reduced dry weight of wheat [18] under the weak light conditions. The long-term weak lighting conditions resulted in smaller leaf areas and thinner leaves [19]. On the other hand, too high light intensity also has negative effect on plant growth. This induces leaf wilting and reduces leaf area, chlorophyll content, and photosynthesis efficiency [20]. It can also destroy photosynthetic systems and cause serious oxidative damage to leaf tissue [21]. Therefore, the objective of this study is to investigate the growth, photosynthesis, and bioactive compound content of medicinal plants, and consequently, find a suitable combination of CO2 concentration and light intensity for high-quality mass production in a closed-type plant production system (CPPS).

2. Results and Discussion

2.1. Growth Characteristics

The different shoot growth characteristics of G. littoralis, as affected by various CO2 concentrations and light intensities after 56 days of treatment, are shown in Figure1 and Table1. The petiole length was the longest at 8.8 cm in the 500 µmol mol 1 CO concentration with 100 µmol m 2 s 1 PPFD, while · − 2 · − · − the leaf area was the widest at 142.78 cm2 in the 500 µmol mol 1 with 300 PPFD (Figure1B and Table1). · − The crown diameter, number of leaves, fresh weight (FW), and dry weight (DW) of the shoots were the lowest in the 500 µmol mol 1 with 100 µmol m 2 s 1 PPFD. The growth of shoots was more affected · − · − · − by the light intensity than by the CO2 concentration, and the growth of G. littoralis in higher light intensity (300 µmol m 2 s 1 PPFD) was higher compared lower light intensity (100 µmol m 2 s 1 PPFD). · − · − · − · − An appropriate light intensity is a major factor for growth, morphogenesis, and other physiological PlantsPlants 20202020,, 99,, x 1581 FOR PEER REVIEW 33 of of 12 12 other physiological responses [22,23]. Plants grown in a low light intensity have frequently been responses [22,23]. Plants grown in a low light intensity have frequently been shown to display more shown to display more photoinhibition than those grown under a high light intensity [24]. A low photoinhibition than those grown under a high light intensity [24]. A low light intensity condition light intensity condition may lead to stretching of leaf length, leaf width, and plant height [25]. The may lead to stretching of leaf length, leaf width, and plant height [25]. The G. littoralis showed an G. littoralis showed an insignificant difference in the specific leaf area (SLA) (data are not shown) but insignificant difference in the specific leaf area (SLA) (data are not shown) but the petiole length the petiole length was stretched. was stretched.

Figure 1. Images of the growth of the whole plant (A) and leaf (B) of Glehnia littoralis Fr. Schmidt ex Figure 1. Images of the growth of the whole plant (A) and leaf (B) of Glehnia littoralis Fr. Schmidt ex Miquel as affected by different CO2 concentrations and light intensities. Miquel as affected by different CO2 concentrations and light intensities. Table 1. The different shoot growth characteristics of Glehnia littoralis Fr. Schmidt ex Miquel, as affected

Tableby various 1. The CO different2 concentrations shoot growth and light characteristics intensities after of Glehnia 56 days littoralis of treatment Fr. Schmidt (n = 3). ex Miquel, as affected by various CO2 concentrations and light intensities after 56 days of treatment (n = 3). CO2 Concentration Light Intensity Petiole Leaf Area Crown No. of Fresh Weight Dry Weight (µmol mol 1) (A) (µmol m 2 s 1) (B) Length (cm) (cm2/plant) Diameter (mm) Leaves (g/plant) (g/plant) · − Light· − · − CO2 Concentration 100 8.8 0.9 a z 82.65 13.1 bc 8.0 0.6 bc 8.0 0.6 bc 5.93 1.1 bc 1.28 0.2 bc Intensity Petiole± Leaf Area± Crown± ± Fresh ±Weight Dry ±Weight 500 −1 200 7.0 0.7 ab 112.23 19.7 ab 13.5 2.1 ab 12.2 2.1 ab 8.19 1.7 ab 1.61 0.4 ab (µmol∙mol ) ± ± ± No. of Leaves± ± ± (µmol∙m−3002∙s−1) Length 5.3(cm)0.4 b(cm2/plant) 142.78 13.4Diameter a 13.8 (mm)1.7a 14.2 3.0 ab 10.85(g/plant)0.7 a 2.17(g/plant)0.2 a (A) ± ± ± ± ± ± (B) 100 6.6 0.7 ab 46.60 9.5 c 7.6 0.5 c 6.8 1.4 c 3.12 0.6 c 0.58 0.1 c ± ± ± ± ± ± 1500 200 5.0 z0.3 b 104.33 15.1 ab 14.7 1.4 a 16.5 1.2 a 7.55 1.2 ab 1.53 0.2 ab 100 8.8 ± 0.9 a± 82.65 ± 13.1± bc 8.0 ± 0.6 bc± 8.0 ± 0.6± bc 5.93 ±± 1.1 bc 1.28 ±± 0.2 bc 300 5.2 0.2 b 107.04 17.7 ab 13.8 0.6 a 12.7 1.4 ab 9.15 1.1 ab 2.01 0.2 ab 500 200 7.0 ± 0.7 ab± 112.23 ± 19.7± ab 13.5 ± 2.1 ±ab 12.2 ± 2.1± ab 8.19 ±± 1.7 ab 1.61 ±± 0.4 ab A ** * NS NS NS NS Significance y 300 B5.3 ± 0.4 b *** 142.78 ± 13.4 ** a 13.8 ± 1.7 *** a 14.2 ± 3.0 *** ab 10.85 ***± 0.7 a 2.17 ***± 0.2 a A B NS NS NS NS NS NS 100 × 6.6 ± 0.7 ab 46.60 ± 9.5 c 7.6 ± 0.5 c 6.8 ± 1.4 c 3.12 ± 0.6 c 0.58 ± 0.1 c z1500Mean separation200 within columns5.0 ± 0.3 using b Tukey’s104.33 ± multiple15.1 ab range14.7 ± test 1.4 ata p 0.05.16.5 ±y 1.2NS, a *, **,7.55 *** Nonsignificant± 1.2 ab 1.53 ± or 0.2 ab ≤ significant at p 0.05,300 0.01, or5.2 0.001, ± 0.2 respectively. b 107.04 ± 17.7 ab 13.8 ± 0.6 a 12.7 ± 1.4 ab 9.15 ± 1.1 ab 2.01 ± 0.2 ab ≤ The root growthA characteristics,** such as* the rootNS length, rootNS diameter, FW,NS and DWNS of the Significance y B *** ** *** *** *** *** roots are shown inA Table × B 2. TheNS root growthNS was closelyNS associated withNS the lightNS condition forNS the shoot growth [26]. The root length was the shortest in the 100 µmol m 2 s 1 PPFD, regardless of the z Mean separation within columns using Tukey’s multiple range test· −at ·p− ≤ 0.05. y NS, *, **, *** CO concentration, at 10.7 and 11.3 cm for the 500 and 1500 µmol mol 1 concentration, respectively. 2Nonsignificant or significant at p ≤ 0.05, 0.01, or 0.001, 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 root growth the FW characteristics, and DW of roots such displayed as the root a positive length, correlation root diameter, in which FW, and the weightDW of increasedthe roots are shown in Table 2. The root growth was closely associated with the light condition for the shoot as the light intensity increased. The root growth was not affected by the CO2 concentration but, −2 −1 growtha change [26]. in theThe lightroot intensitylength was produced the shortest a significant in the 100 di ffµmol∙merence.∙s Nager PPFD, et al.regardless [27] reported of the that CO a2 concentration, at 10.7 and 11.3 cm 2for the1 500 and 1500 µmol∙mol−1 concentration, respectively. The high light intensity (300 µmol m− s− PPFD) enhanced the FW of the roots of Nicotiana tabacum. · · −1 −2 −1 rootSimilarly, diameter Olschowski was the et thin al. [28nest] obtained at 6.50 a mm heavier in the root 1 DW500 ofµmol∙molCalibrachoa within a high 100 µmol∙m light intensity∙s PPFD. when Furthermore,cutting. Kitaya the et FW al. [and29] suggestedDW of roots that displayed an optimal a positive PPFD correlation can rapidly in produce which the high-quality weight increase lettuced asplug the seedlings. 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.

Plants 2020, 9, x FOR PEER REVIEW 4 of 12

Table 2. The different root growth characteristics of Glehnia littoralis Fr. Schmidt ex Miquel, as affected Plants 2020, 9, 1581 4 of 12 by various CO2 concentrations and light intensities after 56 days of treatment (n = 3).

CO2 Light Intensity Root Length Root Diameter Fresh Weight Dry Weight TableConcentration 2. The di fferent(µmol∙m root− growth2∙s−1) characteristics of Glehnia littoralis Fr. Schmidt ex Miquel, as affected (cm) (mm) (g/plant) (g/plant) −1 by(µmol∙mol various) CO(A) 2 concentrations(B) and light intensities after 56 days of treatment (n = 3). 100 10.7 ± 0.4 b z 9.6 ± 0.5 ab 4.53 ± 0.2 cd 1.20 ± 0.1 c CO Concentration Light Intensity 2 500 200 Root Length29.9 ± (cm)1.5 a Root Diameter11.0 ± 0.3 (mm) a Fresh11.6 Weight0 ± 2.4 (g/ plant)ab Dry1.68 Weight ± 0.4 (gbc/plant) (µmol mol 1) (A) (µmol m 2 s 1) (B) · − · − ·300− 26.3 ± 2.2 a 12.0 ± 0.7 a 16.60 ± 0.7 a 2.71 ± 0.1 a 100 10.7 0.4 b z 9.6 0.5 ab 4.53 0.2 cd 1.20 0.1 c 100 11.3± ± 0.5 b 6.5± ± 0.8 b 3.81± ± 0.7 d 0.80 ± ±0.2 c 500 200 29.9 1.5 a 11.0 0.3 a 11.60 2.4 ab 1.68 0.4 bc ± ± ± ± 1500 300200 26.325.52.2 ± a1.6 a 12.010.40.7 ± 1.3 a a 10.12 16.60 ± 0.71.3 abc 1.51 2.71 ± 0.40.1 bc a 300 31.7± ± 1.5 a 12.3± ± 0.4 a 14.83 ± 1.8 ab 2.20 ± 0.2± ab 100 11.3 0.5 b 6.5 0.8 b 3.81 0.7 d 0.80 0.2 c ± ± ± ± 1500 200A 25.5 1.6NS a 10.4 1.3NS a 10.12 NS1.3 bc 1.51NS 0.4 bc ± ± ± ± Significance y 300B 31.7 1.5*** a 12.3 0.4*** a 14.83 ***1.8 ab 2.20*** 0.2 ab ± ± ± ± AA × B NS** NSNS NSNS NS NS y Significancez Mean separation B within columns ***using Tukey’s multiple *** range test at *** p ≤ 0.05. y NS, **, *** *** A B ** NS NS NS Nonsignificant or significant× at p ≤ 0.01 or 0.001, respectively. z Mean separation within columns using Tukey’s multiple range test at p 0.05. y NS, **, *** Nonsignificant or significant at p 0.01 or 0.001, respectively. ≤ 2.2. Photosynthetic≤ Rate and the SPAD Value 2.2. Photosynthetic Rate and the SPAD Value The CO2 concentration and light intensity significantly affected the photosynthetic rate in G. littoralisThe CO (Fig2 concentrationure 2A). The andphotosynthetic light intensity rate significantly was the highest affected in the the 1500 photosynthetic µmol∙mol−1 rateCO2 in 1 G.concentration littoralis (Figure with2 A).the 300 The µmol∙m photosynthetic−2∙s−1 PPFD ratein 6.8 was µmol the CO highest2 m−2∙s−1, inwhile the the 1500 lowestµmol wasmol found− COin 2 2 1 2 1 · concentrationthe 500 µmol∙mol with- the1 CO 3002 concentrationµmol m− s −withPPFD 100 inµmol∙m 6.8 µ−2mol∙s−1 PPFD CO2 min− 0.5s− µmol, while CO the2 m lowest−2∙s−1. The was -1 · · 2 1 · 2 1 foundphotosynthetic in the 500 µ ratemol showedmol CO a positiveconcentration correlation with with 100 COµmol2 concentrationm s PPFD and inthe 0.5 lightµmol intensity CO m. Thes . · 2 · − · − 2 − · − TheSPAD photosynthetic did not show rate a showedsignificant a positivedifference correlation (Figure 2B). with Zheng CO2 etconcentration al. [30] reported and that the lighta high intensity. light Theintensity SPAD did (350 not µmol∙m show−2 a∙s significant−1 PPFD) increased difference the (Figure photosynthetic2B). Zheng capacity et al. [of30 the] reported mother thatplant a and high the light intensityprimary (350 runnerµmol plantm 2ofs strawberry.1 PPFD) increased Furthermore, the photosynthetic CO2 enrichment capacity increased of thethe motherphotosynthetic plant and rate the · − · − primaryof Gerbera runner jamesonii plant [3 of1]. strawberry. G. littoralis Furthermore,is a crop native CO to 2coastalenrichment dunes increasedand grows the in high photosynthetic-light natural rate of environments.Gerbera jamesonii Similar[31]. resultsG. littoralis showedis a that crop the native growth to coastalof Peucedanum dunes andjaponicum grows Thunberg, in high-light which natural is environments.native to the seashore, Similar results was also showed efficient that at increasing the growth growth of Peucedanum and productio japonicumn in a highThunberg, light intensity which is −2 −1 −2 −1 native(200 toµmol∙m the seashore,∙s PPFD) was than also in e ffia lowcient light at increasing intensity (60 growth µmol∙m and∙s production PPFD) [32 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]. · − · − · − · −

A B 500 mol.m-1 80 8 1,500 mol.m-1 a

) 60

-1

s 6

-2

m b

2 40

4 SPAD c

mol CO mol



( d

Photosynthetic rate 2 d 20

e 0 0 100 200 300 100 200 300 . -2. -1 Light intensity (mol.m-2.s-1) Light intensity (mol m s )

FigureFigure 2. 2.Photosynthetic Photosynthetic rate rate ( A(A)) and and SPADSPAD ((BB)) ofof Glehnia littoralis littoralis Fr.Fr. Schmidt Schmidt ex ex Miquel Miquel,, as asaffected aﬀected

byby various various CO CO2 concentrations2 concentrationsand and lightlight intensitiesintensities 56 days days of of treatment. treatment. The The vertical vertical bars bars represent represent thethe standard standard deviation deviation of of the the mean mean ( n(n= = 3).3). Different Different letters letters above above bars bars indicate indicate significant significant differences differences at pat p 0.05,≤ 0.05, using using Tukey’s Tukey’s multiple multiple range range test.test. ≤ 2.3. Total Sugar and Starch 2.3. Total Sugar and Starch TheThe high high light intensity intensity increase increasedd the total the totalsugar sugarand starch and contents starch in contents G. littoralis in. However,G. littoralis . However, the CO concentration did not influence the total sugar and starch contents. The total the CO2 concentratio2 n did not influence the total sugar and starch contents. The total sugar and starch 2 1 sugar and starch contents were the highest in the 300 µmol m− s− PPFD regardless of the CO2 · · concentration (Figure3). In a CO concentration of 500 µmol mol 1, the total sugar and starch contents 2 · − 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 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 Plants300 µmol·m2020, 9, 1581−2·s−1 PPFD by 5.4 and 2.2 times compared to the 100 µmol·m−2·s−1 PPFD, respectively.5 of 12 Many studies have reported that CO2 enrichment increases the sugar and starch contents [33–35], which is inconsistent with this study. G. littoralis, a halophyte with developed water storage tissue contents [33–35], which is inconsistent with this study. G. littoralis, a halophyte with developed water that stores a lot of water in the cell, is considered to be less sensitive to CO2 concentrations because storageof its thick tissue leaves. that storesThe leaf a lot is ofa photosynthetic water in the cell, organ, is considered and the toarea be and less sensitivethickness to are CO the2 concentrations major factors becauseaffecting of the its growth thick leaves. of the Theplant. leaf To is absorb a photosynthetic sufficient light organ, energy, and the the area leaves and are thickness as wide are as thepossible, major factors affecting the growth of the plant. To absorb sufficient light energy, the leaves are as wide as and at the same time to facilitate gas exchange (CO2, O2, and H2O), the leaves are as flat and thin as possible,possible [36]. and atThe the higher same timelight tointens facilitateity can gas positively exchange affect (CO2 the,O2 accumu, and H2lationO), the of leaves assimilates, are as flatsuch and as thinproteins, as possible and the [36 starch,]. The and higher sugar light of intensityspinach increased can positively in the a ff300ect µmol·m the accumulation−2·s−1 PPFD ofthan assimilates, those in such as proteins, and the starch, and sugar of spinach increased in the 300 µmol m 2 s 1 PPFD than the 100 µmol·m−2·s−1 PPFD [37]. In Glycine max (Linn.) Merr., a high light· intensity− · − induced 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 photosyntheticroots [38]. activity, increasing the soluble sugar, sucrose, and starch contents in the shoots and roots [38].

Figure 3. The different total sugar (A) and starch (B) contents of Glehnia littoralis Fr. Schmidt ex Miquel, Figure 3. The different total sugar (A) and starch (B) contents of Glehnia littoralis Fr. Schmidt ex as affected by various CO2 concentrations and light intensities after 56 days of treatment. The vertical Miquel, as affected by various CO2 concentrations and light intensities after 56 days of treatment. The bars represent the standard deviation of the mean (n = 3). Different letters above bars indicate significant vertical bars represent the standard deviation of the mean (n = 3). Different letters above bars indicate differences at p 0.05, using Tukey’s multiple range test. significant differences≤ at p ≤ 0.05, using Tukey’s multiple range test. 2.4. Harvest Yield 2.4. Harvest Yield G. littoralis can be harvested cyclically by taking a leaf. The CO2 enrichment did not have a significantG. littoralis effect can on thebe harvestharvested yield cyclically or the changeby taking in thea leaf. number The ofCO leaves2 enrichment from the did harvest not have to the a re-harvest.significant effect However, on the the harvest low light yield intensity or the conditionchange in a fftheected number the harvest of leaves yield. from The the first harvest and second to the harvestre-harvest. yields, However, at 61 daysthe low and light 96 days intensity of treatment, condition are affected shown the in harvest Figure 4yield.A,B, respectively.The first and second It was harvest yields, at 61 days and 96 days of treatment, are shown in Figure 4A,B, respectively. It 2was1 observed that the yield of G. littoralis was high in the light intensity of more than the 200 µmol m− s− · −2· −1 PPFD,observed where that the yield harvest of G. yield littoralis increased was high by morein the thanlight twiceintensity as muchof more during than the the 200 second µmol·m harvest·s PPFD, where the harvest yield increased by more1 than twice as much during the second2 harvest1 compared to the first harvest in the 1500 µmol mol− CO2 concentration with 300 µmol m− s− PPFD. 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 However, regardless of the CO2 concentration,2 there1 was no significant difference between the first and second harvest yields in the 100 µmol m− s− PPFD. This was because the high light intensity · −2· −1 significantlyand second harvest affected yields the root in developmentthe 100 µmol·m of G.·s littoralisPPFD.and This su wasfficient because roots the were high produced light intensity for new leafsignificantly production. affected The the change root indevelopment the number of of G. leaves littoralis from and the sufficient harvest roots to the were re-harvest produced is shown for new in Figureleaf production.5. The number The change of leaves in wasthe onlynumber a ff ectedof leaves by the from light the intensity, harvest andto the in re-harvest particular, is leaves shown were in Figure 5. The number of leaves was only2 affected1 by the light intensity, and in particular, leaves were developed the most in the 300 µmol m− s− PPFD. Lee et al. [39] reported that new leaf emergence · −2· −1 anddeveloped biomass the accumulation most in the were300 µmol·m promoted·s atPPFD. a higher Lee apparent et al. [39] daily reported light integral that new level. leaf Furthermore, emergence theand higherbiomass light accumulation intensity produced were morepromoted primary at anda higher total plant apparent runners daily of strawberry. light integral level. Furthermore, the higher light intensity produced more primary and total plant runners of strawberry.

Plants 2020, 9, x FOR PEER REVIEW 6 of 12

Plants 2020, 9, x FOR PEER REVIEW 6 of 12 Plants 2020, 9, 1581 6 of 12

. -1 14 A B 500 mol mol 14 1,500 mol.mol-1 12 a 12 . -1 14 A B 500 mol mol 14 10 1,500 mol.mol-1 10 a 12 ab 12 8 ab 8 10 10 6 6

8 Yield (g/plant) a ab 8 Yield (g/plant) a a ab ab 4 ab 4 6 b b b 6 Yield (g/plant) 2 b a 2 Yield (g/plant) a a ab 4 ab 4 0 0 b100 200 300 b b100 200 300 2 b 2 Light intensity (mol.m-2.s-1) 0 0 100 200 300 100 200 300 Figure 4. The different first (A) and secondLight intensity (B) harvest (mol .yieldm-2.s-1s) of Glehnia littoralis Fr. Schmidt ex Miquel , as affected by various CO2 concentrations and light intensities after 56 days of treatment. The vertical Figurebars 4. representTheThe different different the f first standardirst ( A) and and deviation second second ( (B of)) harvest harvest the mean yield yields (ns of of= GlehniaGlehnia 3). Different littoralis littoralis letters Fr. Schmidt Schmidt above ex exbars Miquel Miquel, indicate, as affected by various CO concentrations and light intensities after 56 days of treatment. The vertical as affectedsignificant by variousdifferen COces2 2at concentration p ≤ 0.05, usings and Tukey’s light multipleintensities range after test.56 days of treatment. The vertical bars represent represent the the standard standard deviation deviation of the of mean the mean (n = 3). (n Di =ff erent 3). Different letters above letters bars above indicate bars significant indicate differences at p 0.05, using Tukey’s multiple range test. significant differen≤ ces at p ≤ 0.05, using Tukey’s multiple range test.

500 mol.mol-1, 100 mol.m-2.s-1 14 500 mol.mol-1, 200 mol.m-2.s-1 *** 500 mol.mol-1, 300 mol.m-2.s-1 . -1 . -2. -1 12 500 1,500mol.mol mol-1, 100mol mol, 100.m -2.mols-1 m s . -1 . -2. -1 14 500 1,500mol.mol mol-1, 200mol mol, 200.m -2.mols-1 m s *** . -1 . -2. -1 500 1,500mol.mol mol-1, 300mol mol, 300.m -2.mols-1 m s 10 . -1 . -2. -1 ** 12 1,500 mol mol , 100 mol m s 1,500 mol.mol-1, 200 mol.m-2.s-1 8 1,500 mol.mol-1, 300 mol.m-2.s-1 10 * ** 6 * 8

Number of leaves Number * 4 * 6 *

Number of leaves Number 2 4 * 0 2 1 2 3 4 5

0 Weeks Figure 5. Changes in the number1 of leaves2 on 3Glehnia littoralis4 Fr.5 Schmidt ex Miquel, as affected by Figure 5. Changes in the number of leaves on Glehnia littoralis Fr. Schmidt ex Miquel, as affected by various CO2 concentrations and light intensitiesWeeks. The error bars represent the standard deviation of various CO2 concentrations and light intensities. The error bars represent the standard deviation of the Figurethe mean5. Changes (n = 3). in *, the **, ***number Significant of leaves at p on≤ 0.05, Glehnia 0.01, littoralis or 0.001, Fr. respectively. Schmidt ex Miquel, as affected by mean (n = 3). *, **, *** Significant at p 0.05, 0.01, or 0.001, respectively. various CO2 concentrations and light≤ intensities. The error bars represent the standard deviation of 2.5.2.5. Bioactivethe Bioactive mean Compound(n Compound= 3). *, **, *** Significant at p ≤ 0.05, 0.01, or 0.001, respectively. TheThe chlorogenic chlorogenic acid aci andd and total total saponin saponin concentrations concentration pers per DW DW or FWor FW of G.of littoralisG. littoralis were were not not 2.5. Bioactive Compound significantlysignificant dilyff erentdifferent between between the treatments the treatment (Figuress 6(FigA andure7sA). 6A On and the 7 otherA). On hand, the the other chlorogenic hand, the 2 1 −2 −1 acidchlorogenic andThe totalchlorogenic saponin acid and a contentsci dtotal and saponin pertotal biomass saponin content were concentrations theper greatestbiomasss inper were the DW 300 the orµ greatestmol FW mof− G ins.− littoralisthePPFD 300 regardless µmol∙mwere not ∙s · · 1 ofsignificant thePPFD CO 2 regardlesslyconcentration different of between (Figuresthe CO2 6 B tconcentrationhe and treatment7B). In a CO s (Fig(Fig2 ureconcentrationures s6 B6 A and and 7 ofB 5007).A). Inµ mol Ona CO mol the2 concentration − other, the chlorogenic hand, ofthe 500 −1 ·2 1 −2 −1 acidchlorogenicµmol∙mol and total acid, saponin the and chlorogenic total contents saponin peracid contentbiomass and totals wereper saponin biomass higher content inwere the sthe 300 per greatestµ mol biomassm −in swerethe− PPFD300 higher µmol∙m by 1.6in the and∙s 300 −2 −1 2 1 · −2 −1· 6.3PPFDµmol∙m times regardless compared∙s PPFD of to theby in the1.6 CO 1002and concentrationµ 6.3mol timesm− scompared− (FigPPFD,ures respectively.to6B in and the 7100B). In µmol∙mIn other a CO studies,2∙s concentrationPPFD CO, 2respectivelyenrichment of 500. In −1 · · improvedµmol∙molother studies, the, the nutritional chlorogenicCO2 enrichment qualities acid improved but and the total total the saponin free nutritional phenolic content qualities acidss per and biomassbut chicoric the total were acid free contentshigher phenolic in of the lettuceacids 300 and −2 −1 −2 −1 significantlyµmol∙mchicoric∙s acidPPFD decreased; content by 1.6s the of and lettuce reaction 6.3 significantlytimes to the compared elevated decreased COto in2 concentration the; th e100 reaction µmol∙m wasto the∙s found elevatedPPFD to, be respectivelyCO dependent2 concentration. onIn theotherwas plant studies, found species COto [be402 enrichment dependent,41]. The accumulation improvedon the plant the of species nutritional bioactive [40, compounds4 1qualities]. The accumulation but is inducedthe total byoffree bioactive the phenolic increased compounds acids soluble and is sugarchicoricinduced acting acid bycontent as thea precursor increaseds of lettuce that solublesignificantly promotes sugar thedecreased acting synthesis as; th ae and precursorreaction accumulation to thatthe elevated promotes of antioxidants CO the2 concentration synthesis [42–44 ]. and Thewasaccumulation polyphenolfound to be dependent contentof antioxidants of lettuceon the [4 plant grown2–44]. species The in a polyphenol 350 [40µ,mol41]. Them content2 accumulations 1 PPFD of lettuce was of significantlygrown bioactive in a compounds 350 higher µmol∙m than is−2 ∙s−1 · − · − thatinducedPPFD of plants was by the significantly grown increased in a higher 180 solubleµmol than sugarm that2 s actingof1 plantsPPFD as grown [ a45 precursor]. Inin a this 180 study, that µmol∙m promotes there−2∙s−1 wasPPFD the no [4 synthesis di5].ff Inerence this and study, in · − · − theaccumulationthere concentration was noof antioxidantsdifference of total chlorogenic in [4the2– 44concentration]. The acid polyphenol per DWof total or content total chlorogenic saponin of lettuce acid per grown FW,per butDW in theaor 350 total production µmol∙m saponin−2∙s of−1 per 2 1 −2 −1 bioactivePPFD was compounds significantly increased higher than in the that 300 of µplantsmol m grown− s− inPPFD, a 180 which µmol∙m displayed∙s PPFD high [45 photosynthesis]. In this study, there was no difference in the concentration of· total· chlorogenic acid per DW or total saponin per and superior growth.

PlantsPlants 2020 20, 920, x, 9FOR, x FOR PEER PEER REVIEW REVIEW 7 of 712 of 12

−2 −1 FW,FW, but but the the production production of of bioactive bioactive compounds compounds increased inin thethe 300 300 µmol∙m µmol∙m∙s−2∙sPPFD,−1 PPFD, which which displayed high photosynthesis and superior growth. displayedPlants 2020, 9 high, 1581 photosynthesis and superior growth. 7 of 12

A B 500 mol.m-1 60 . -1 A B 1,500500 mol molm .m-1 60 . -1 a 1,500a mol m 3 50 a a 3 ab 50 ab 40

DW) 40 -1 2 ab

. 30

DW)

-1 2 ab (mg/plant)

(mg . 30 20

(mg/plant)

(mg 1

20Chlorogenic acid content b b 10

Chlorogenic acid concentration 1

Chlorogenic acid content b b 10

Chlorogenic acid concentration 0 0 100 200 300 100 200 300 0 Light intensity (mol.m-2.s-1) Light intensity (mol.m-2.s-1) 0 100 200 300 100 200 300 Figure 6. The differentLight intensity chlorogenic (mol.m-2 .acids-1) concentrations (A) andLight content intensitys ( permol .mbiomass-2.s-1) (B) of Glehnia littoralis Fr. Schmidt ex Miquel shoots, as affected by various CO2 concentrations and light intensities Figure 6. 6. TheThe different different chlorogenic chlorogenic acid acidconcentration concentrationss (A) and (A) content and contentss per biomass per biomass (B) of Glehnia (B) of after 56 days of treatment. The vertical bars represent the standard deviation of the mean (n = 3). littoralisGlehnia Fr. littoralis SchmidtFr. ex Schmidt Miquel shoot ex Miquels, as affected shoots, by as various affected CO by2 concentration various COs andconcentrations light intensit andies Different letters above bars indicate significant differences at p ≤ 0.05, using Tukey’s2 multiple range alightfter 5 intensities6 days of aftertreatment. 56 days The of treatment.vertical bars The represent vertical the bars standard represent deviation the standard of the deviation mean (n of = the3). test. Differentmean (n =letters3). Di abovefferent bars letters indicate above significant bars indicate differences significant at p di≤ ff0.05,erences using at Tukey’sp 0.05, multiple using Tukey’s range ≤ test.multiple range test.

500 mol.m-1 A B . -1 6 1,500 mol m 100 a

a 500 mol.m-1 A B . 80-1 6 1,500 mol m 100 a FW) 4

-1

g 60 . a b 80

FW) 4 b 40 SE/plant)(mg

(mg SE (mg -1 2 g 60

Total saponin content b b Total saponin concentration b 20

b 40 SE/plant)(mg (mg SE (mg 2 0 0

Total saponin content 100 200 300 100 b 200 300 Total saponin concentration b 20 Light intensity (mol.m-2.s-1) Light intensity (mol.m-2.s-1) 0 0 FigureFigure 7. The 7. The100 di ffdifferenterent total total200 saponin saponin concentration concentration300 ( A(A)) and and100 content content perper biomassbiomass200 ( B)) of of GlehniaGlehnia300 littoralis littoralis Fr. . -2. -1 . -2. -1 SchmidtFr. Schmidt ex Miquel exLight Miquel shoots, intensity shoots, as ( amolff ectedasm affecteds by) various by various CO2 COconcentrations2 concentrationsLight intensity and and ( lightmol lightm intensities intensitiess ) after after 56 56 days of treatment.days of treatment. The vertical The v barsertical represent bars represent the standard the standard deviation deviation of the of mean the mean (n = (3).n = Di3).ff Differeerent lettersnt Figureaboveletters bars7. Theabove indicate different bars significantindicate total saponinsignificant differences concentration differences at p 0.05, at ( Ap )≤using and0.05, content using Tukey’s Tukey’s per multiple biomass multiple range (B )range of test. Glehnia test. littoralis ≤ Fr. Schmidt ex Miquel shoots, as affected by various CO2 concentrations and light intensities after 56 3. Materials3.days Materials of treatment. and and Methods Methods The vertical bars represent the standard deviation of the mean (n = 3). Different letters above bars indicate significant differences at p ≤ 0.05, using Tukey’s multiple range test. 3.1.3.1. Plant Plant Materials Materials and and Growth Growth Condition Condition 3. MaterialsTheThe G. G.littoralisand littoralis Methodsseeds seeds were were sown sown inin aa PetriPetri dish and and the the seedling seedlingss were were placed placed in in128 128-hole-hole plug plug traystrays filled filled with with urethane urethane sponge sponge (Hydroponic (Hydroponic Sponge,Sponge, Easyhydro Easyhydro Co. Co. Ltd., Ltd., Chuncheon, Chuncheon, Korea) Korea) in a in a 3.1.CPPS CPPSPlant (C1200H3, (MaterialsC1200H3, FC and FC Poibe GrowthPoibe Co. Co. Condition Ltd.,Ltd., Seoul,Seoul, Korea) at at a a temperature temperature of of 20 20 ± 1 °C1 ◦, C,relative relative humidity humidity (RH) of 60 ± 10%, a photoperiod of 12/12 h (light/dark), and a 150 µmol∙m±−2∙s2−1 PPFD1 using RGB (RH)The of 60G. littoralis10%, a seeds photoperiod were sown of 12 in/12 a Petri h (light dish/dark), and the and seedling a 150 µsmol werem −placeds− PPFD in 128 using-hole plug RGB (red:green:blue± = 7:1:2) LEDs (ES LEDs Co. Ltd., Seoul, Korea). The 60-·day-old· seedlings were trays(red:green:blue filled with =urethane7:1:2) LEDs sponge (ES (Hydroponic LEDs Co. Ltd.,Sponge, Seoul, Easyhydro Korea). Co. The Ltd., 60-day-old Chuncheon, seedlings Korea) were in a transplantedtransplanted into into a deepa deep floating floating technique technique systemsystem with with recycling recycling Hoagland Hoagland nutrient nutrient solution solution [46] [ 46in ] in CPPSa CPPS. (C1200H3, The plants FC Poibe were culturedCo. Ltd., for Seoul, 96 days Korea) at a temperatureat a temperature of 20 ±of 1 20°C ,± RH 1 °C of, 60relative ± 10%, humidity and a a CPPS. The plants were cultured for 96 days at a temperature of 20 1 ◦C,−2 RH−1 of 60 10%, and a (RH)photoperiod of 60 ± 10%, of 12/12 a photoperiod h (light/dark). of The12/12 experimental h (light/dark), treatments and a were150 µmol∙mset± to 500 ∙sor 1500 PPFD µmol∙mol± using RGB−1 photoperiod of 12/12 h (light/dark). The experimental treatments were set to 500 or 1500 µmol mol 1 (red:green:blue = 7:1:2) LEDs (ES LEDs Co. Ltd., Seoul, Korea). The 60-day-old seedlings−2 were−1 − CO2 concentrations in combination with one of three light intensities: 100, 200, or 300 µmol∙m ·∙s 2 1 CO2 concentrations in combination with one of three light intensities: 100, 200, or 300 µmol m− s− transplantedPPFD, which into were a deep provided floating by technique RGB (red:green:blue system with = 7:1:2)recycling LEDs Hoagland (Figure 8 ).nutrient In CPPS solution where· CO [462 ·] in aPPFD, CPPS. which The plants were providedwere cultured by RGB for (red:green:blue96 days at a temperature= 7:1:2) LEDs of 20 (Figure ± 1 °C8, ).RH In of CPPS 60 ± where10%, and CO a2 photoperiodconcentration, of photoperiod, 12/12 h (light/dark). and temperature The experimental are automatically treatments controlled, were set theto 500 CO 2orconcentration 1500 µmol∙mol was−1 controlled by connecting a liquefied carbon dioxide tank and CO regulator. The CO concentration, CO2 concentrations in combination with one of three light intensities:2 100, 200, or 3002 µmol∙m−2∙s−1 temperature, and RH were monitored during the cultivation period using a data logger (TR-76Ui, PPFD, which were provided by RGB (red:green:blue = 7:1:2) LEDs (Figure 8). In CPPS where CO2 T&D Co. Ltd., Nagano, Japan). The light intensity was set using a photometer (HD2101.2, Delta Ohm SrL, Caselle, Italy). Plants 2020, 9, x FOR PEER REVIEW 8 of 12

concentration, photoperiod, and temperature are automatically controlled, the CO2 concentration 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). Plants 2020, 9, 1581 8 of 12

100 100 mol.m-2.s-1 200 mol.m-2.s-1 . -2. -1 80 300 mol m s

Relative light intensity (%) 20

0 300 400 500 600 700 Wavelength (nm) Figure 8. Relative spectral distribution of the RGB LEDs (red:green:blue = 7:1:2) used in a closed-type Figure 8. Relative spectral distribution of the RGB LEDs (red:green:blue = 7:1:2) used in a closed-type plant production system. plant production system. 3.2. Growth Characteristics 3.2. Growth Characteristics After 56 days of treatment, the petiole length, crown and root diameters, root length, FW and DW of theA shootsfter 56 and days roots, of treatment, number ofthe leaves, petiole and length, leaf areacrown were and measured. root diameter Thes FW, root was length, investigated FW and usingDW of an the electronic shoots and balance roots, (EW220-3NM,number of leaves, Kern and & leaf Sohn area GmbH., were measured. Balingen, The Germany) FW was and investigated the DW wasusing investigated an electronic after balance drying (EW220 in an oven-3NM, (Venticell-220, Kern & Sohn MMMGmbH., Medcenter Balingen, EinrichtungerGermany) and GmbH.,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 calipersPlanegg, (CD-20CPX, Germany) at Mitutoyo 70 °C for Co. 72 Ltd.,h. The Kawasaki, crown and Japan). root diameter The leaf were area measured was measured using using Vernier a leafcaliper areas meter(CD-20CPX, (LI-3000, Mitutoyo LI-COR Co. Inc., Ltd., Lincoln, Kawasaki, NE, USA). Japan). Photosynthetic The leaf area rate was was measured measured using using a leaf a portablearea meter photosynthesis (LI-3000, LI- systemCOR Inc., (CIRAS-3, Lincoln, PP NE, Systems USA). International Photosynthetic Inc., rate Amesbury, was measured MA, USA) using on a theportable fully unfolded photosynthesis fifth leaf system from (CIRAS the top.-3, The PP measurementSystems International conditions Inc., were Amesbury, controlled MA, as follows:USA) on the fully unfolded2 fifth leaf from the top. The measurement conditions1 were controlled as follows:1 leaf area 4.5 mm ; leaf temperature 20 ◦C; air flow rate 150 mL min− ; 500 or 1500 µmol mol− CO2 leaf area 4.5 mm2; leaf temperature 20 °C2 ; 1air flow rate 150 mL·min· −1; 500 or 1500 µmol∙mol· −1 CO2 concentration; 100, 200, or 300 µmol m− s− PPFD. The chlorophyll content was expressed as the · −·2 −1 SPAD,concentration; and measured 100, using200, or a portable 300 µmol∙m chlorophyll∙s PPFD. meter The (SPAD-502, chlorophyll Konica content Minolta was Inc., expressed Tokyo, Japan). as the TheSPAD, first and secondmeasured harvests using were a portable performed chlorophyll after treatment meter (SPAD for 61 and-502, 96 Konica days, respectively,Minolta Inc., and Tokyo, the harvestJapan).yield The first was and measured second byharvests weight were of marketable performed leaves after treatment (over 15 cm for2 61of leafand area).96 days, respectively, and the harvest yield was measured by weight of marketable leaves (over 15 cm2 of leaf area). 3.3. Total Sugar and Starch 3.3. Total Sugar and Starch For the total sugar content determination, the leaves of G. littoralis were ground with liquid nitrogenFor and the storedtotal sugar in a deep content freezer determination, (NF-140SF, Nihonthe leaves Freezer of G Co.. littoralis Ltd., Yushima, were ground Japan) with at 70liquidC. − ◦ Anitrogen 0.3 g sample and stored was taken in a deep for each freezer treatment. (NF-140SF, The Nihon samples Freezer were Co. mixed Ltd., with Yushima, 10 mL Japan) of 80% at ethanol ‒70 °C. andA 0.3 then g sample ground w foras 1taken min. for After each being treatment. heated inThe a 60samples◦C water were bath, mixed the supernatantwith 10 mL wasof 80% separated ethanol viaand centrifugation then ground (908for 1 min.g, 20 AfterC, 30 being min). heated Then, in 10 a mL60 °C of water 80% ethanol bath, the was supernatant added to the was remaining separated × ◦ precipitate,via centrifugation heated in(908 a× waterbath g, 20 °C, 30 at 60min).◦C The for 30n, 10 min, mL and of 80% centrifuged ethanol underwas added the same to the conditions. remaining Afterprecipitate, the two heated centrifugations, in a waterbath the supernatantat 60 °C for 30 was min, diluted and centrifuged with a total under of 40 mLthe same of 80% conditions. ethanol. Then,After0.5 the mL two of centrifugations5% phenol reagent, the supernatant was added wa to thes diluted sample with solution, a total of vortexed, 40 mL of and 80% 2.5 ethanol. mL of The 98%n, sulfuric0.5 mL of acid 5% were phenol added reagent and was vortexed. added to After the sample cooling solution, at room vortexed, temperature, and 2.5 the mL absorbance of 98% sulfuric was measuredacid were atadded 490 nm and using vortexed. a spectrophotometer After cooling at room and thetemperature, total sugar the content absorbance was calculated was measured using at 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% HClO4 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 H2SO4 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.

3.4. Bioactive Compounds

3.4.1. 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.

3.4.2. 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.

3.5. 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).

4. Conclusions This study focused on the eﬀects of CO concentration (500 or 1500 µmol mol 1) and light 2 · − intensity (100, 200, or 300 µmol m 2 s 1 PPFD) on the growth, photosynthetic rate, and bioactive · − · − Plants 2020, 9, 1581 10 of 12

compound content of G. littoralis to ﬁnd an appropriate CO2 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 CO2 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.

Author Contributions: Conceptualization, S.J.H.; methodology, H.R.L. and S.J.H.; software, H.R.L.; validation, H.R.L., H.M.K., and H.R.L.; formal analysis, H.R.L.; investigation, H.R.L., H.M.K., and H.W.J.; resources, S.J.H.; data curation, H.R.L.; writing—original draft preparation, H.R.L.; writing—review and editing, S.J.H.; visualization, H.R.L.; supervision, S.J.H.; project administration, H.R.L., M.M.O.; funding acquisition, M.M.O., S.J.H. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01385201)” Rural Development Administration, Korea. Acknowledgments: This work was carried out with the support of the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01385201)” Rural Development Administration, Korea. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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