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Article

Carbon Dioxide Enrichment Combined with Supplemental Light Improve Growth and Quality of Plug Seedlings of Astragalus membranaceus Bunge and Codonopsis lanceolata Benth. et Hook. f.

by
Ya Liu
1,
Xiuxia Ren
1 and
Byoung Ryong Jeong
1,2,3,*
1
Department of Horticulture, Division of Applied Life Science (BK21 Plus Program), Graduate School of Gyeongsang National University, Jinju 52828, Korea
2
Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Korea
3
Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea
*
Author to whom correspondence should be addressed.
Agronomy 2019, 9(11), 715; https://doi.org/10.3390/agronomy9110715
Submission received: 29 September 2019 / Revised: 2 November 2019 / Accepted: 4 November 2019 / Published: 5 November 2019
(This article belongs to the Section Horticultural and Floricultural Crops)
Browse Figures
Versions Notes

Abstract

:
Astragalus membranaceus Bunge and Codonopsis lanceolata Benth. et Hook. f. are two medicinal species used to remedy inflammation, tumor, and obesity in Eastern medicine. Carbon dioxide (CO2) and supplemental lighting are two methods to enhance the growth, yield, and quality of crops. However, few studies have focused on the synergistic effects of CO2 and the supplemental light source on plug seedlings of medicinal species. In this study, uniform seedlings were grown with no supplemental light (the control) or under one of three supplemental light sources [high pressure sodium (HPS), metal halide (MH), or mixed light-emitting diodes (LEDs)] combined with one of three levels of CO2 (350, 700, or 1050 μmol·mol−1). The supplemental light (100 μmol·m−2·s−1 photosynthetic photon flux density) and CO2 were provided simultaneously from 10:00 pm to 2:00 am every day. The results showed that the supplemental lighting (LEDs, MH, and HPS) greatly improved the seedling quality with greater dry weights (of the shoot, root, and leaf), stem diameter, leaf area, and Dickson’s quality index (DQI) than those of the control in both species. An enriched CO2 at 1050 μmol·mol−1 accelerated the growth and development of plug seedlings, evidenced by the increased root and leaf dry weights, stem diameter, and DQI compared to the those from the other two CO2 enrichment levels. Moreover, LEDs combined with 1050 μmol·mol−1 CO2 not only increased the contents of soluble sugars but also the starch content. However, an enriched CO2 at 700 μmol·mol−1 was more suitable for the accumulation of total phenols and flavonoids. Furthermore, LEDs combined with 700 or 1050 μmol·mol−1 CO2 increased the chlorophyll, quantum yield, and stomatal conductance at daytime and nighttime for A. membranaceus and C. lanceolata, respectively. In conclusion, the data suggest that LEDs combined with CO2 at 1050 μmol·mol−1 is recommended for enhancing the growth and development of plug seedlings of A. membranaceus and C. lanceolata.

1. Introduction

Astragalus membranaceus Bunge and Codonopsis lanceolata Benth. et Hook. f. are two important medicinal herbs, mainly distributed in Asian countries, especially in Korea, China, and Japan [1,2]. In ancient China, those two species were used as the medicinal material for treating physiological disorders and illnesses, including inflammation, tumor, and obesity [3,4]. In addition to its medicinal values, C. lanceolata has been a high-class vegetable consumed in Asian countries, especially in ancient Korea [5,6]. In recent years, more and more phytochemicals with positive effects on remedying diseases have been found in these two species [7,8]. However, most studies have focused on the separation, extraction, and characteristics of the phytochemicals in A. membranaceus and C. lanceolata. Not much research has focused on the cultivation of those two species to obtain high-quality plug seedlings in a glasshouse.
As a substrate of photosynthesis, carbon dioxide (CO2) has a remarkable influence on the plant growth, yield, and quality [9,10,11]. Within a certain concentration, an enrichment in the CO2 concentration leads to an increase in the intercellular CO2 concentration, which leads to higher quality and yield in crops, as observed in rice [12], wheat [13], and maize [14]. However, excess CO2 levels were also shown to have a negative effect on photosynthesis and yield [15,16], partly because of a lower activation of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) or a decreased enzyme content [17,18]. Therefore, providing a suitable concentration of CO2 for specific species is the primary focus in the practice of CO2 fertilization.
It is well known that photosynthesis is a chemical reaction driven by light. In artificial conditions, including those in a glasshouse, light insufficiency is common, resulting in a degradation of quality and a loss in yield [19,20]. Therefore, supplemental lighting has been employed in horticulture to improve crop growth, yield, and quality [21,22,23]. This is usually carried out at night, since the electricity price per unit kilowatt and energy cost can be lower with off-peak time-of-use (TOU). More importantly, a study reported that supplemental lighting during nighttime is more effective to promote plant growth and yield as compared with daytime both in summer and winter [23]. Furthermore, night-interruption not only increase leaf biomass and crop yield but also result in more compact and healthier plants [24,25,26]. In commercial production, supplemental light intensity is usually between 100 and 200 μmol·m−2·s−1 photosynthetic photon flux density (PPFD) [27,28]. Previous studies have reported the synergistic effects of CO2 and supplemental light intensity [10,29,30]. Moreover, the effects of CO2 and temperature on the plant growth and development were also demonstrated [12,29,31]. However, research on the synergistic effects of CO2 and supplemental lighting source was insufficient, especially in medicinal plants.
As direct or indirect photosynthetic products, primary and secondary metabolites are strongly determined by photosynthesis [27]. Previous studies have proven that light intensity [32], photoperiod [33], temperature [34], and CO2 [16,35] affect the biosynthesis and accumulation of primary and secondary metabolites such as sugar, starch, total phenols and flavonoids. However, more studies should be carried out to clarify how CO2 and supplemental lighting source affect primary and secondary metabolites in medicinal plants. As valuable phytochemicals, total phenols and flavonoids have the potential to scavenge reactive oxygen species and protect human against diseases and physiological disorders [8,36]. Thus, contents of total phenols and flavonoids are necessary parameters to evaluate the medicinal quality. Therefore, more research is needed to provide the ideal supplemental CO2 and lighting condition for medicinal plants.
High-quality plug seedlings are the primary objective of commercial production since a high quality means a high income and interest for growers. Previous study has reported a high quality of seedling defined by growth parameters, such as high dry matter, low shoot to root ratio, and high compactness [37,38]. However, no agreement has been achieved, since no single trait can evaluate plug-seedling quality well [39]. Recently, the Dickson’s quality index (DQI) has been regarded as a promising indicator of plug-seedling quality [39,40]. As a parameter integrating the aspects of total biomass, the sturdiness quotient, and the ratio of stem diameter to shoot height, DQI explains overall plant potential for survival and growth in the field. Increasing DQI suggests the growing potential of plug seedlings for field establishment success after transplanting [39,40]. Therefore, in this study, the DQI was used as the most important parameter to evaluate the quality of plug seedlings, followed by biomass and stem diameter.
In this study, it was hypothesized that an enrichment of CO2 and appropriate supplemental lighting could enhance the quality of plug seedlings of A. membranaceus and C. lanceolata. In order to test this hypothesis, uniform plug seedlings were cultured under no supplemental lighting (the control) or under one of three supplemental light sources [high pressure sodium (HPS), metal halide (MH), or mixed light-emitting diodes (LEDs)] combined with one of three levels of CO2 enrichment (350, 700, or 1050 μmol·mol−1). The growth, development and morphological characteristics of plug seedlings affected by the CO2 enrichment and supplemental light source were investigated. Moreover, the contents of the primary and secondary metabolites such as soluble sugars, starch, total phenols, and flavonoids, were also determined. Furthermore, photosynthesis-related parameters were evaluated, such as the chlorophyll content, quantum yield, and stomatal conductance. These data could provide a theoretical and practical basis for improving the plug-seedling quality in A. membranaceus and C. lanceolata, and serve as useful information for growers to manage other medicinal plants.

2. Materials and Methods

2.1. Plant Materials and Treatments

Seeds of A. membranaceus and C. lanceolata were sown in 200-cell plug trays filled with the BioPlug Medium (FarmHannong Co. Ltd., Seoul, South Korea). After germination, uniform plug seedlings (720 seedlings) were selected as the plant material and then cultured under no supplemental light (the control) or under one of three supplemental light sources [high pressure sodium (HPS, BLV Licht- und Vakuumtechnik, Steinhöring, Germany), metal halide (MH, SunLumen Lighting Co. Ltd., Gyeongju, South Korea), or mixed light-emitting diodes (LEDs, red: blue 6:1, FL300, Senmatic A/S, Søndersø, Denmark) combined with one of three levels of CO2 enrichment (350, 700, or 1050 μmol·mol−1). The supplemental light intensity used in this study was set at 100 μmol·m−2·s−1 photosynthetic photon flux density (PPFD) (mean with five places) by using a portable photo/radiometer (HD-2102.2, Delta OHM, Padova, Italy), and the lighting duration (also the CO2 enrichment time) was 4 h from 10:00 pm to 2:00 am every day. The CO2 concentration during daytime is ambient level (350 µmol mol−1) among all treatments. The ambient level was chosen as control, and 2- and 3-fold increase were chosen as enriched concentration, according to previous study [41,42,43]. It was monitored and controlled by a crop-management system (SH-MV510, SOHA TECH Co,.Ltd., Seoul, Korea). This experiment was carried out in Jinju, Gyeongsangnam-do, Korea, from 3 February to 2 March 2019. The mean day length is around 11 h, ranged from 10 h 32 min to 11 h 27 min during experiment. The photosynthetic light period is 15 h with approximate 4 h darkness before lighting treatments. The average daily maximum light intensity coming from the sunlight was about 568.9 µmol⋅m−2⋅s−1 PPFD. The culture environment had set points of 23/17 °C (day/night) temperatures and (70 ± 5)% relative humidity in a controlled environment inside a glasshouse. The plug seedlings were fertilized once two days with a multipurpose greenhouse nutrient solution [in mg·L−1 Ca(NO3)2·4H2O 737.0, KNO3 343.4, KH2PO4 163.2, K2SO4 43.5, MgSO4·H2O 246.0, NH4NO3 80.0, Fe-EDTA 15.0, H3BO3 1.40, NaMoO4·2H2O 0.12, MnSO4·4H2O 2.10, and ZnSO4·7H2O 0.44 (pH 6.5 and electrical conductivity 1.5 mS·cm−1)] throughout the experiment [44]. The experimental design was of completely random design with three replications. Each replication included 20 plug seedlings (total 60 seedlings per treatment). After four weeks’ cultivation, five plug seedlings were harvested at 10 am for measurements of the growth parameters and the others (55 plants per treatment) were frozen immediately in liquid nitrogen for further analysis. For measurements of the growth parameters, the whole plants were harvested and the roots were washed carefully with tap water. Growth parameters, such as fresh weight, length, and stem diameter, were measured directly whereas dry weight of shoot, root, and leaf were measure after 24 h drying at 60 °C in an oven. The Dickson’s quality index was calculated according to a previous formula [45]. The formula is:
Dickson’s quality index = Total DW/((shoot length/stem diameter) + (shoot DW/root DW)),
where the DW means dry weight.

2.2. Contents of Soluble Sugar and Starch

The contents of soluble sugar and starch were measured according to the anthrone colorimetric method [46,47]. In brief, mixed samples (200 mg) of leaves were ground and homogenized in distilled water (14 mL), followed by extraction for 30 min at 100 °C. The solution was centrifuged at 3000 rpm for 15 min, and the supernatant was transferred into a new tube for the measurement of soluble sugars. The residue was collected and extracted in distilled water mixed with perchloric acid (2 mL, 52%) for the measurement of starch. The supernatant (0.5 mL) was mixed with distilled water (1.9 mL), anthrone (0.5 mL, 2%), and concentrated sulfuric acid (5 mL, 98%), and incubated for 15 min at 100 °C. The absorbance was recorded at 630 nm and 485 nm, respectively, by using a ultraviolet (UV)-spectrophotometer (Libra S22, Biochrom Ltd., Cambridge, UK).

2.3. Contents of Total Phenols and Flavonoids

The contents of total phenols and flavonoids in leaves were measured according to the previous methods described by Manivannan et al. [36]. Briefly, total phenols and flavonoids were extracted with 80% methanol. For the assay of total phenols, the extract solution (100 µL) was mixed with distilled water (900 µL), phenol reagent (500 µL, 1:1 water), and sodium carbonate (2.5%, 1 mL). The mixed solution was incubated for 40 min in dark, followed by recording the absorbance at 765 nm. For the assay of the total flavonoids, the extract solution (100 µL) was mixed with methyl alcohol (80%, 900 µL) and aluminum chloride (2%, 1 mL). After incubation for 30 min, the absorbance at 415 nm was recorded by using a UV-spectrophotometer (Libra S22, Biochrom Ltd., Cambridge, UK). The total phenols and flavonoids were calculated from the standard gallic acid and quercetin calibration curve, respectively.

2.4. Assessment of the Chlorophyll Content, Quantum Yield, and Stomatal Conductance

The chlorophyll content was measured by using a Plus Chlorophyll Meter (SPAD 502, Konica Minolta Sensing Inc., Osaka, Japan). The quantum yield was determined with a FluorPen FP 100 (Photon Systems Instruments, PSI, Drásov, Czech Republic). The dark acclimatization (15 min) was conducted before taking measurements, according to the introduction for this instrument. The stomatal conductance was assessed at 10 am during daytime and at 12 am during nighttime, respectively, by using a Decagon Leaf Porometer SC-1 (Decagon Device Inc., Pullman, WA, USA).

2.5. Data Collection and Analysis

Data were collected and reported as the mean ± standard error (n = 5 for growth parameters and n = 3 for other parametes). All data were processed and analyzed using the Statistical Package for the Social Sciences version 21 (SPSS Inc., Chicago, IL, USA). A two-way analysis of variance (two-way ANOVA) was performed to evaluate the significant differences among treatments, followed by a Duncan’s multiple range test (p < 0.05). All figures were made with the OriginPro software version 9.0 (OriginLab Corporation, Northampton, MA, USA).

3. Results and Discussion

3.1. Growth, Development, and Morphology

Plants grown with supplemental lighting had significantly higher shoot and root dry weights, stem diameter, leaf area, and Dickson’s quality index (DQI), in comparison with plants grown without supplemental lighting (Figure 1, Table 1 and Table 2). As an important parameter, DQI is a good indicator of seedling quality since changes in the strength and balance of the distribution of biomass are assessed, and several other important parameters are used to assess the quality [48]. Therefore, a significantly lower value of DQI under no supplemental lighting (Con) suggested any supplemental lighting greatly upgraded the quality of plus seedlings of A. membranaceus and C. lanceolata. Similarly, Lanoue et al. [49] reported that the biomass production of tomato (Solanum lycopersicum L.) and lisianthus (Eustoma grandiflorum (Raf.) Shinners) were increased by all the supplemental light conditions than in the control. The enhancement of seedling quality is not only duo to supplemental lighting but also duo to night interruption.Similarly, Cao et al. [24] reported that night break not only increased stemdiameter, leaf number, and fruit fresh weight, but also resulted in more compact and healthier plant of tomato. Yoneda et al. [25] also found night interruption treatments (4 h) combined with 8 h photoperiod increased leaf biomass of tomato to amounts observed under LD conditions. Among the supplemental light sources, LEDs showed the greatest promotion of stem diameter and DQI in C. lanceolata plug seedlings, especially when combined with CO2 at 1050 μmol·mol−1, followed by MH and HPS, implying more compact and sturdy plug seedlings. Additionally, in A. membranaceus plug seedlings, the root dry weight is higher in LEDs combined with CO2 at 1050 μmol·mol−1 than other treatments, while the stem diameter and DQI was increased by LEDs combined with CO2 at 1050 μmol·mol−1, only following the LEDs combined with 700 μmol·mol−1 CO2. The results were in agreement with those of Park et al. [41] who reported that a mixture of red, blue and white (RBW, 8:1:1) LEDs promoted the vegetative growth of the shoot and root in lettuce (Lactuca sativa L.), compared to treatments with fluorescent lamps. Similar, Randall et al. [50] reported that DQI for Petunia, Salvia, and Violaw was significantly higher under LEDs than under HPS lamps. As a promising light source, LEDs can provide a narrow band of red and blue light, contributing to a high photosynthetic rate, photosynthetic products, yield, and quality [23,51]. Red and blue are two important spectral ranges, which can be absorbed efficiently by two photoreceptors, phytochromes and cryptochromes, respectively. Although plants can also absorb yellow and green light, the efficiency is pretty low, since there is no specific photoreceptor for those two kinds of light. Red and blue light greatly enhance photosynthesis as compared with others. Moreover, blue light can enhance the compactness by inhibiting the elongation of shoots. Compared with the LED used in this study, both MH and HPS possess a high ratio of yellow and green light which is not suited for improving photosynthesis and compactness.
Moreover, a consistent trend between the CO2 level and growth parameters was found under the same light source. An enriched CO2 at 1050 μmol·mol−1 accelerated the growth and development of plug seedlings, evidenced by the increased root and leaf dry weights, stem diameter, and DQI than those of plants grown with the other two levels of CO2 (Table 1 and Table 2). Wohlfahrt et al. [52] also found that the fresh biomass of grapevines were stimulated by elevated CO2. Therefore, among all treatments, LEDs combined with CO2 at 1050 μmol·mol−1 is recommended for the growth and development of A. membranaceus and C. lanceolata plug seedlings, when considering root biomass, stem diameter, and DQI. This result is similar to that of a previous study that found that a combination of RBW LEDs and the highest concentration of CO2 (1000 μmol·mol−1) was the optimal condition for lettuce growth [41].

3.2. Contents of Soluble Sugar and Starch

Supplemental lighting including LEDs, MH, and HPS, increased the accumulation of soluble sugar in both species, compared to the control (Figure 2A,B, Table 3). Moreover, under the same lighting condition, the soluble sugar content presented an increasing trend with elevated CO2 level in C. lanceolata plug seedlings, whereas it was observed to increase first and then decrease in A. membranaceus plug seedlings (except under LEDs). Analysis of variance showed significant interactions between supplemental lighting and CO2 for soluble sugar in A. membranaceus (Table 3). This was in agreement with a report [10] that the interaction of supplementary light and CO2 enrichment improves nutritional quality significantly in tomato. In details, under 350 or 1050 μmol·mol−1 CO2, LED largely increased the content of soluble sugar, followed by MH or HPS. However, HPS enhanced soluble sugar concentration more than two others did under 700 μmol·mol−1 CO2 (Figure 2). The highest values of the soluble sugar content were 17.62 ± 0.43 mg·g−1 fresh weight (FW) for A. membranaceus and 22.10 ± 0.77 mg·g−1 FW for C. lanceolata, under LEDs combined with 1050 μmol·mol−1 CO2. As an important signal, light, especially red and blue, can activate photosynthetic enzymes including Rubisco and improve stomata opening, which is the foundation of photosynthesis. Supplemental CO2 increases CO2 concentration in intercellular space, which lead to more CO2 molecular is fixed by ribulose bisphosphate. This fixation step was catalyzed by Rubisco. Supplemental CO2 and lighting promoted photosynthesis, increased carbohydrate content, and thus improved the quality of plug seedlings. Compared with MH and HPS, LEDs provided a high percentage of red and blue light, showing a greater promotion on soluble sugar content. Similarly, Terfa et al. [53] reported that plants grown under LEDs (red:blue 8:2) showed higher levels of soluble carbohydrates than those grown under HPS in Rosa × hybrida. Dong et al. [54] found that elevated CO2 increased the concentrations of carbohydrates, such as fructose, glucose, and total soluble sugars. During photosynthesis, sufficient CO2 and light are needed as the substrates of photosynthesis, resulting in more photosynthetic products including soluble sugars. For plug seedlings of A. membranaceus and C. lanceolata, LEDs combined with 1050 μmol·mol−1 CO2 were beneficial to the accumulation of soluble sugars.
Regardless of CO2 treatment with A. membranaceus, the supplemental lighting (LED, MH, and HPS) significantly increased starch content compared to that in the control (Figure 2C,D, Table 3). A significant interaction between supplemental lighting and CO2 for starch was also shown in A. membranaceus, which was similar with soluble sugar. In C. lanceolata, the starch contents were increased with enriched CO2 under the same light source (LED, MH, or HPS). The maximum starch contents were 72.57 ± 0.31 mg·g−1 FW in A. membranaceus and 37.40 ± 1.41 mg·g−1 FW in C. lanceolata, when grown under LEDs with 1050 μmol·mol−1 CO2. This is in agreement with the results of Li et al. [31], where elevated CO2 also increased the concentration of carbohydrates including sucrose, sugar, starch and non-structural carbohydrates in dwarf bamboo. Importantly, contents of soluble sugar and starch are related to plug-seedling quality and survival of transplanting. Once transplanted, a plug seedling will suffer a hardest period to adapt its condition and recover growth. High consumption by respiration and low biosynthesis by photosynthesis will deplete the energy and health of plug seedlings. Thus, it is advantageous for plug seedlings with high contents of soluble sugar and starch to survive during a tough time.

3.3. Contents of Total Phenols and Flavonoids

Plug seedlings grown with elevated CO2 and supplemental lighting (except LED combined 350 μmol·mol−1 CO2, Figure 3C) had significantly higher contents of total phenols and flavonoids, in comparison with control (Figure 3, Table 3). Night interruption may contribute to the contents of secondary metabolites. A similar reported showed that night interruption (4 h) coupled with 8 h photoperiod resulted in higher steviol glycoside content than that under 12 h photoperiod [25]. In A. membranaceus, the highest total phenols level and the greatest flavonoids content were observed when grown under LEDs combined with 700 μmol·mol−1 CO2. Phenylalanine ammonia-lyase is the key enzyme controlling the translation from primary metabolites to secondary metabolites. This enzyme has been shown to be upregulated by blue light [55]. LEDs with a high percentage of blue light accounted for the higher contents of total phenols and flavonoids. Moreover, the higher contents of total phenols and flavonoids might be due to the stimulant effects of elevated CO2 on the phenylpropanoid pathway, since more carbon molecules are available to synthesize phenols and flavonoids. [56]. As a non-enzyme antioxidant, total phenols and flavonoids can degrade the reactive oxygen species and reduce harm from abiotic stress, which is helpful for improving the quality of plug seedlings. Similarly, Park et al. [44] found that the total anthocyanin concentration in Perilla frutescens was greater under RBW LEDs than under fluorescent light. Ren et al. [35] reported that the production of anthocyanin, flavonoids and phenols increased with the CO2 enrichment from 450 to 1200 μmol·mol−1, but decreased when the CO2 level was raised from 1200 to 2000 μmol·mol−1. Moreover, in C. lanceolata, elevated CO2 and/or supplemental lighting had improved the contents of total phenols and flavonoids compared to those of the control combined with 350 μmol·mol−1 CO2 (Figure 3B,D). However, no obvious trends were found in the contents of total phenol and flavonoids under different light sources (LEDs, MH, and HPS). Both the maximum total phenol and flavonoid levels were observed under the HPS combined with 700 μmol·mol−1 CO2. Furthermore, analysis of variance for total phenols and flavonoids showed a significant synergy between CO2 and supplemental lighting source in both A. membranaceus and C. lanceolata (Table 3). Therefore, the optimal CO2 for total phenol and flavonoid varied with different light source and species, meanwhile the best light source changed with CO2 concentration and species. More study with various combination in different species should be carried out to demonstrate this issue.

3.4. Chlorophyll Content, Quantum Yield, and Stomatal Conductance

As shown in Figure 4A,B, all supplemental lighting increased the chlorophyll content in A. membranaceus, compared to that of seedlings grown without supplemental lighting, regardless of the CO2 level (Table 4). This result did not agree with a study by Park et al. [26] who reported a decrease in chlorophyll under night interruption treatments (4 h) as compared with control. The discrepancy is from different photosynthetic light period. In their study, the photosynthetic light period of night interruption is 14 h (short day, 10 h), 2 h less than control (long day, 16 h). Analysis of variance showed a significant interactive effect of CO2 and supplemental lighting source on chlorophyll. Under 350 or 700 μmol·mol−1 CO2, the LED increased chlorophyll, whereas MH did under 1,050 μmol·mol−1 CO2. The highest chlorophyll content observed in A. membranaceus was 37.4 ± 0.6 SPAD, grown under LEDs combined with 700 μmol·mol−1 CO2. In C. lanceolata, the chlorophyll content fluctuated with the supplemental CO2 and lighting and was affected not only by supplemental lighting source but also the interaction (Table 4). The highest value was 27.0 ± 0.3 SPAD when grown under LEDs combined with 700 μmol·mol−1 CO2. These results agreed with those of a previous study. Mamatha et al. [9] found that the total chlorophyll content was higher when plants were grown with 380 and 550 ppm CO2 than with 700 ppm CO2. In photosynthesis, chlorophyll is a vital component in the light-harvesting complex (LHC), which absorbs and transports photons to the reaction center of photosystems [57]. Chlorophyll biosynthesis requires light and is affected by the light quality. Blue light has been shown to be favorable to the formation of chlorophyll [58,59]. Compared with MH and HPS, LEDs used in this study provided a narrow band of blue and red lights, resulting in an increase in the chlorophyll content. Elevated CO2 has also increased the chlorophyll content [15]. The data in this study showed an increase in the chlorophyll content by supplemental lighting and/or elevated CO2, implying an enhancement in the light capture capacity, which promotes the synthesis of photosynthetic products in plants [60]. This partly explains the higher contents of soluble sugar, starch, total phenols and flavonoids under LEDs and elevated CO2 (Figure 2 and Figure 3). However, some studies reported a reduction in chlorophyll content when plants are grown under elevated CO2 levels [13,31]. The different results between ours and theirs are due to different species. Diversely, Ksiksi, et al. [61] reported there was no significant increase in total chlorophyll content in alfalfa under enriched CO2 environments. A meta-analysis using 57 articles consisting of 1015 observations showed the concentrations of total chlorophyll, anthocyanins, and carotenoids were not influence by enriched CO2 [54]. Therefore, more studies should be carried out to clarify how enriched CO2 influences the biosynthesis, decomposition, and contents of chlorophyll at molecular level among species.
Plug seedlings of A. membranaceus grown with 1050 µmol mol−1 CO2 showed higher quantum yield (QY) values in comparison with plants grown with 350 µmol mol−1 CO2 in control and under the LEDs (Figure 4C). However, the CO2 at 700 μmol·mol−1 improved the QY values of A. membranaceus grown under MH and HPS and of C. lanceolata grown under all the light sources (Figure 4C,D). Similarly, Moghaddam et al. [62] observed that a high CO2 concentration (800 μmol·mol−1) improved the efficiency of photosystem II (PSII) in Centella asiatica. Ruhil et al. [63] also reported an increase in the quantum efficiency of Photosystem II with elevated CO2 (585 μmol·mol−1) in mustard (Brassica juncea L. cv Pusa Bold). Therefore, supplemental CO2 and lighting improved photosynthesis by increasing quantum yield, consequently contributing to higher contents of primary and secondary metabolites (Figure 2 and Figure 3). However, Tisarum et al. [64] found that the quantum yield in Hevea brasiliensis cv. ‘RRIT413’ sharply declined by 39.0% under a CO2-enriched condition (1500 μmol·mol−1), compared with that in ambient CO2 conditions. The probable reason for the decline in the quantum yield is the difference in the concentration of CO2. In their study, 1500 μmol·mol−1 CO2 was applied, which is much higher than the concentration used in this study. Another reason may be the species, since different species have different responses to the CO2 concentration [42,65].
Importantly, LEDs had increased the QY in both species as compared to the control and MH under the same concentration of CO2, even though the difference was not significant among all treatments. This agrees with a study where it was reported that the light quantum yield of PSII of pepper (Capsicum frutescens L., Sujiao No. 5) was higher (113.70%) under LED lighting than with no supplemental lighting [66]. Similarly, Bergstrand et al. [67] found that the specific photosynthetic capacity and maximum quantum yield of PSII (Fv/Fm) were higher in Rosa × hybrida leaves grown under LEDs than in the control. QY is the ratio of the number of photons emitted to the number of photons absorbed, and thus represents the efficiency of PS II. Therefore, these results imply that LEDs, as well as MH and HPS, have a positive effect on the photosynthetic efficiency as compared with control combined with 350 μmol·mol−1 CO2, resulting in high contents of primary and secondary metabolites. However, the QY presented in this study was lower as compared with a healthy range (0.70–0.85), which implied a stress. This can be explained by light intensity during daytime. In this study, the average daily maximum light intensity coming from the sunlight was about 568.9 µmol⋅m−2⋅s−1 PPFD in winter, lower than that in growth season. Therefore, further study on the supplemental lighting intensity should be carried out in the near future.
Plug seedlings of A. membranaceus grown with control combined with 350 μmol·mol−1 CO2 showed a low value of stomatal conductance as compared with enriched CO2 and supplemental lighting, although the difference was not significant among all treatments (Figure 5A,C). Among all treatments, the daytime stomatal conductance showed the maximum value under LEDs combined with 700 μmol·mol−1 CO2 in A. membranaceus. However, the maximum value in C. lanceolata was under LEDs with 1050 μmol·mol−1 CO2 (Figure 5), although it was not significantly different from LED or MH combined with 700 μmol·mol−1 CO2 and MH or HPS combined with 1050 μmol·mol−1 CO2. A similar feature was found in the nighttime stomatal conductance (Figure 5C,D). Importantly, in C. lanceolata, the stomatal conductance increased with enriched CO2 under all light sources including LEDs, MH, and HPS (Figure 5B,D). This is in agreement with the results from other studies. For example, Wohlfahrt et al. [52] found that the stomatal conductance and transpiration rate were higher under elevated CO2 for Vitis vinifera L. cvs. Riesling and Cabernet Sauvignon in three seasons. Prince et al. [68] reported that elevated CO2 conditions increased the stomatal conductance and transpiration of Phragmites australis. However, in A. membranaceus, the stomatal conductance under LEDs and MH decreased when the CO2 concentration was increased from 700 to 1050 μmol·mol−1. This is because of negative feedback regulation. Under high CO2, a decrease in stomatal opening and reduction in stomatal conductance occurred in order to balance the rate of absorbing CO2 and photosynthesis. Although the stomatal conductance decreased with elevated CO2, the photosynthesis increased with CO2 enrichment [9,13,69]. The different characteristics of stomatal conductance between A. membranaceus and C. lanceolata may be because different species respond differently to the CO2 concentration.
Compared to MH and HPS under the same CO2 concentration, LEDs combined 700 μmol·mol−1 in A. membranaceus or 1050 μmol·mol−1 in C. lanceolata led to larger improvements in the daytime and nighttime stomatal conductance, although difference was not significant among all treatments. The reason is higher percentage of blue light in LEDs than other light source. Blue light increases stomata opening with higher stomatal conductance, and consequently substrate of photosynthesis (water and CO2) exchanges more frequently, leading to more photosynthetic products and secondary metabolites (Figure 2 and Figure 3). Similarly, Song et al. [70] reported that cucumber seedlings cultivated under white LEDs and RB LEDs had higher stomatal conductance values than those grown under conventional light sources (triphosphate fluorescent lamps and high-frequency fluorescent lamps). Borowski et al. [71] also found that leaves of lettuce grown under LEDs (R/B) had a higher stomatal conductance than those in other growth conditions. As conventional light sources, MH and HPS provided a broad-band spectrum for plant growth [72]. However, they have a lower efficiency for seedling growth compared to LEDs, since yellow and green light account for high proportions of the spectrum. LEDs provided a narrow band of red and blue lights, which can be absorbed by phytochromes and cytochromes, and used for photosynthesis efficiently. Moreover, previous studies have shown that blue light controls the opening and closing of stomata [73]. Therefore, a higher proportion of blue light in LEDs contributed to the stomata opening and greater stomatal conductance of seedlings [53].
Taking all treatments into consideration, LEDs combined with CO2 at 1050 μmol·mol−1 is recommended for growing the seedlings of A. membranaceus and C. lanceolata, since it enhanced the stomatal conductance at daytime and nighttime in those two species. Similarly, Park et al. [41] found that the stomatal conductance was the greatest in lettuce grown under RBW LEDs with 1000 μmol·mol−1 CO2. As an estimation of the gas exchange rate (CO2 uptake and water loss), the stomatal conductance indicates the rates of photosynthesis and transpiration. In this study, LEDs combined with CO2 at 1050 μmol·mol−1 increased the stomatal conductance, resulting in a higher photosynthetic rate and higher quality of plus seedlings.

4. Conclusions

In this study, the effects of the CO2 and supplemental light source on the growth and development of plug seedlings of A. membranaceus and C. lanceolata were demonstrated. The data showed that LEDs combined with CO2 at 1050 μmol·mol−1 greatly enhanced the quality of plug seedlings, evident by the higher shoot, root, and leaf dry weights, stem diameter, leaf area, and Dickson’ quality index compared to those in the control in both species. Moreover, a significant interaction of supplemental CO2 and lighting source on metabolites was found in both A. membranaceus and C. lanceolata. The CO2 and supplemental light source had a positive effect on primary metabolites, and the maximum contents of soluble sugar and starch were obtained when seedlings were grown under LEDs combined with 1050 μmol·mol−1 CO2. Furthermore, an enriched CO2 at 700 μmol·mol−1 is more optimal for accumulation of total phenols and flavonoids. In addition, LEDs combined with 700 or 1050 μmol·mol−1 CO2 enhanced the photosynthesis-related parameters such as the chlorophyll content, quantum yield, and stomatal conductance at daytime and nighttime. In conclusion, the data from this study suggest that LEDs combined with CO2 at 1050 μmol·mol−1 is the optimal condition for enhancing the quality of plug seedlings of A. membranaceus and C. lanceolata.

Author Contributions

Conceptualization, Y.L., X.R. and B.R.J.; Funding acquisition, B.R.J.; Investigation, Y.L. and X.R.; Project administration, X.R. and B.R.J.; Supervision, B.R.J.; Writing—original draft, Y.L.; Writing—review and editing, Y.L., X.R. and B.R.J.

Funding

This research was funded by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (Project No. 116057-03). Y.L. and X.R. were supported by a scholarship from the BK21 Plus Program, Ministry of Education, Korea.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Morphological characteristics of plug seedlings of A. membranaceus (A) and C. lanceolata (B) affected by the carbon dioxide (CO2) and supplemental light source after four weeks of cultivation. Con, control without supplemental lighting; LED, mixed light-emitting diodes; MH, metal halite; and HPS, high-pressure sodium. Bar, 10 cm.
Figure 1. Morphological characteristics of plug seedlings of A. membranaceus (A) and C. lanceolata (B) affected by the carbon dioxide (CO2) and supplemental light source after four weeks of cultivation. Con, control without supplemental lighting; LED, mixed light-emitting diodes; MH, metal halite; and HPS, high-pressure sodium. Bar, 10 cm.
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Figure 2. Contents of soluble sugar and starch in plug seedlings of A. membranaceus (A,C) and C. lanceolata (B,D) as affected by the CO2 level and supplemental light source. Data were presented as the mean ± standard error (n = 3). Con, control without supplemental lighting; LED, mixed light-emitting diodes; MH, metal halite; and HPS, high pressure sodium. Different letters (a–g) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
Figure 2. Contents of soluble sugar and starch in plug seedlings of A. membranaceus (A,C) and C. lanceolata (B,D) as affected by the CO2 level and supplemental light source. Data were presented as the mean ± standard error (n = 3). Con, control without supplemental lighting; LED, mixed light-emitting diodes; MH, metal halite; and HPS, high pressure sodium. Different letters (a–g) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
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Figure 3. Contents of total phenols and flavonoids in plug seedlings of A. membranaceus (A,C) and C. lanceolata (B,D) as affected by the CO2 level and supplemental light source. Data were presented as the mean ± standard error (n = 3). Con, control without supplemental lighting; LED, mixed light-emitting diodes; MH, metal halite; and HPS, high pressure sodium. Different letters (a–g) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
Figure 3. Contents of total phenols and flavonoids in plug seedlings of A. membranaceus (A,C) and C. lanceolata (B,D) as affected by the CO2 level and supplemental light source. Data were presented as the mean ± standard error (n = 3). Con, control without supplemental lighting; LED, mixed light-emitting diodes; MH, metal halite; and HPS, high pressure sodium. Different letters (a–g) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
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Figure 4. Effect of CO2 level and supplemental light source on chlorophyll and quantum yield (Fv/Fm) of plug seedlings of A. membranaceus (A and C) and C. lanceolata (B and D). Data were presented as the mean ± standard error (n = 3). Con, control without supplemental lighting; LED, mixed light-emitting diodes; MH, metal halite; and HPS, high pressure sodium. Different letters (a–e) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
Figure 4. Effect of CO2 level and supplemental light source on chlorophyll and quantum yield (Fv/Fm) of plug seedlings of A. membranaceus (A and C) and C. lanceolata (B and D). Data were presented as the mean ± standard error (n = 3). Con, control without supplemental lighting; LED, mixed light-emitting diodes; MH, metal halite; and HPS, high pressure sodium. Different letters (a–e) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
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Figure 5. Effect of CO2 level and supplemental light source on the stomatal conductance of plug seedlings of A. membranaceus (A,C) and C. lanceolata (B,D) at daytime (A,B) and nighttime (C,D). Data were presented as the mean ± standard error (n = 3). Con, control without supplemental lighting; LED, mixed light-emitting diodes; MH, metal halite; and HPS, high pressure sodium. Different letters (a–f) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
Figure 5. Effect of CO2 level and supplemental light source on the stomatal conductance of plug seedlings of A. membranaceus (A,C) and C. lanceolata (B,D) at daytime (A,B) and nighttime (C,D). Data were presented as the mean ± standard error (n = 3). Con, control without supplemental lighting; LED, mixed light-emitting diodes; MH, metal halite; and HPS, high pressure sodium. Different letters (a–f) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
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Table
Table 1. Effect of CO2 and supplemental light source on the growth parameters of A. membranaceus plug seedlings.
Table 1. Effect of CO2 and supplemental light source on the growth parameters of A. membranaceus plug seedlings.
Light (L)Length (cm)Dry Weight (mg)Leaf Area (cm−2)Stem Diameter (mm)Dickson’s Quality Index (×10−4)
ShootRootShootRootLeaf
350Con8.4 ± 0.5 e z5.0 ± 0.4 b39.1 ± 2.5 e6.4 ± 0.8 e8.2 ± 0.6 g4.8 ± 0.3 e z0.99 ± 0.02 d5.1 ± 0.3 d
LED14.9 ± 0.6 a4.9 ± 0.4 b105.5 ± 9.6 bc15.8 ± 1.6 ad17.4 ± 1.6 be7.9 ± 0.5 bd1.37 ± 0.06 bc10.5 ± 1.0 bc
MH14.7 ± 0.5 ab4.7 ± 0.3 b103.8 ± 11.3 bc15.8 ± 2.6 ad16.9 ± 1.6 bf8.0 ± 0.5 bd1.32 ± 0.09 bc10.4 ± 1.8 bc
HPS13.1 ± 0.5 c4.7 ± 0.3 b84.6 ± 8.0 cd11.4 ± 1.4 ce14.1 ± 1.8 df7.6 ± 0.6 bd1.25 ± 0.07 c8.7 ± 1.0 cd
700Con10.7 ± 0.3 d4.5 ± 0.3 b73.2 ± 7.2 d9.5 ± 1.8 de11.8 ± 1.2 fg6.7 ± 0.4 cd1.24 ± 0.04 c8.8 ± 1.1 cd
LED13.3 ± 0.3 bc4.1 ± 0.2 b134.8 ± 9.0 a17.0 ± 3.7 ac25.1 ± 2.0 a10.2 ± 0.6 a1.62 ± 0.06 a16.0 ± 1.7 a
MH13.3 ± 0.5 bc5.9 ± 0.4 a108.2 ± 7.4 bc16.4 ± 1.4 ac19.3 ± 2.2 bd9.3 ± 0.9 ab1.38 ± 0.04 bc12.1 ± 0.8 a-c
HPS13.4 ± 0.2 bc4.1 ± 0.2 b98.3 ± 10.2 bd15.0 ± 2.2 ad16.9 ± 2.5 bf6.5 ± 0.4 d1.28 ± 0.06 c10.4 ± 1.4 bc
1050Con12.2 ± 0.6 c4.9 ± 0.2 b76.8 ± 6.2 d12.8 ± 1.2 be13.5 ± 0.9 ef7.3 ± 0.4 cd1.23 ± 0.06 c8.7 ± 1.2 cd
LED12.7 ± 0.7 c4.5 ± 0.2 b111.2 ± 10.0 ac20.4 ± 3.1 a20.4 ± 1.9 ac8.2 ± 0.5 b-d1.48 ± 0.06 ab15.5 ± 1.5 a
MH13.6 ± 0.3 ac4.0 ± 0.2 b121.4 ± 11.3 ab18.9 ± 2.2 ab21.4 ± 1.4 ab8.4 ± 0.6 bc1.48 ± 0.10 ab14.6 ± 2.3 ab
HPS12.7 ± 0.7 c4.1 ± 0.4 b96.6 ± 5.6 bd14.1 ± 1.1 ad16.1 ± 0.9 cf6.8 ± 0.3 cd1.30 ± 0.03 bc10.8 ± 0.8 bc
F-testCNSNS************
L***NS******************
C×L******NSNSNS****NS
z Different letters indicate significant separation within columns by Duncan’s multiple range test at a 0.05 level. NS, *, **, and ***, represent no significant or significant difference at p < 0.05, 0.01, or 0.001, respectively. Data were presented as the mean ± standard error (n = 6). Con, control without supplemental lighting; LED, mixed light-emitting diodes; MH, metal halite; and HPS, high pressure sodium.
Table
Table 2. Effect of CO2 and supplemental light source on the growth parameters of C. lanceolata plug seedlings.
Table 2. Effect of CO2 and supplemental light source on the growth parameters of C. lanceolata plug seedlings.
CO2 (C)Light (L)Length(cm)Dry Weight (mg)Leaf Area (cm−2)Stem Diameter (mm)Dickson’s Quality Index (×10−4)
ShootRootShootRootLeaf
350Con10.50.4 bdz4.4 ± 0.3 a30.6 ± 4.7 f6.1 ± 1.2 e5.6 ± 0.4 d4.6 ± 0.2 f1.56 ± 0.09 f5.1 ± 0.9 e
LED11.3 ± 0.3 ac3.8 ± 0.4 ab52.9 ± 4.3 bd12.0 ± 2.8 cd8.9 ± 0.8 bc7.2 ± 0.6 ac2.01 ± 0.08 bc10.8 ± 1.9 bc
MH10.0 ± 0.6 d3.0 ± 0.3 b49.1 ± 4.9 ce12.5 ± 2.5 bd8.2 ± 0.7 bc5.6 ± 0.4 df1.69 ± 0.06 ef9.7 ± 1.1 bd
HPS10.5 ± 0.3 bd3.7 ± 0.2 ab46.5 ± 2.2 de12.8 ± 1.4 bd8.3 ± 0.6 bc7.0 ± 0.7 ad1.99 ± 0.07 bc9.9 ± 0.8 bd
700Con10.2 ± 0.3 cd4.4 ± 0.4 a37.2 ± 2.2 ef9.1 ± 0.7 de6.5 ± 0.5 cd5.4 ± 0.5 ef1.73 ± 0.04 df7.3 ± 0.5 ce
LED12.0 ± 0.2 a4.2 ± 0.2 a61.9 ± 6.7 bc16.2 ± 1.9 ac9.7 ± 1.2 b7.3 ± 0.5 ab2.04 ± 0.04 b12.5 ± 1.4 b
MH11.0 ± 0.5 ad4.0 ± 0.2 a63.4 ± 3.8 b15.6 ± 1.8 ac9.8 ± 0.4 b7.1 ± 0.4 ad1.81 ± 0.06 ce12.3 ± 1.2 b
HPS11.6 ± 0.5 ab4.5 ± 0.4 a53.7 ± 4.5 bd13.8 ± 2.3 bd7.5 ± 0.6 bd5.7 ± 0.4 bf1.89 ± 0.05 be10.2 ± 0.7 bd
1050Con11.0 ± 0.5 ad4.5 ± 0.3 a36.5 ± 3.2 ef10.4 ± 1.2 ce6.6 ± 0.6 cd5.7 ± 0.4 bf1.70 ± 0.03 ef6.8 ± 0.6 de
LED12.2 ± 0.2 a4.4 ± 0.3 a80.3 ± 6.8 a20.5 ± 1.4 a12.1 ± 1.4 a7.7 ± 0.9 a2.30 ± 0.10 a17.9 ± 2.1 a
MH11.5 ± 0.2 ab4.1 ± 0.4 a61.7 ± 4.6 bc18.3 ± 2.5 ab9.5 ± 0.7 b6.7 ± 0.3 ae1.96 ± 0.04 bc12.8 ± 1.1 b
HPS10.6 ± 0.5 bd4.5 ± 0.1 a45.1 ± 2.8 de15.0 ± 1.3 ad6.7 ± 0.5 cd5.6 ± 0.4 cf1.90 ± 0.03 bd10.2 ± 0.6 bd
F-testC********NSNS****
L*********************
C×LNSNS*NSNSNSNSNS
z Different letters indicate significant separation within columns by Duncan’s multiple range test at a 0.05 level. NS, *, **, and ***, represent no significant or significant difference at p < 0.05, 0.01, or 0.001, respectively. Data were presented as the mean ± standard error (n = 5). Con, control without supplemental lighting; LED, mixed light-emitting diodes; MH, metal halite; and HPS, high pressure sodium.
Table
Table 3. Analysis of variance for soluble sugar, starch, total phenols, and total flavonoids.
Table 3. Analysis of variance for soluble sugar, starch, total phenols, and total flavonoids.
SpeciesFactorSoluble SugarStarchTotal PhenolsTotal Flavonoids
A. membranaceusCO2 (C) ************
Light(L)******NS***
C × L************
C. lanceolataCO2 (C) ************
Light(L)**********
C × LNSNS******
NS, **, and ***, represent no significant or significant difference at p < 0.01, or 0.001, respectively.
Table
Table 4. Analysis of variance for chlorophyll, quantum yield, and stomatal conductance.
Table 4. Analysis of variance for chlorophyll, quantum yield, and stomatal conductance.
SpeciesFactorChlorophyllQuantum YieldStomatal Conductance
DayNight
A. membranaceusCO2 (C) *****NS*
Light(L)***********
C×L**NS****
C. lanceolataCO2 (C) NSNS****
Light(L)*****NS***
C×L***NSNSNS
NS, *, **, and ***, represent no significant or significant difference at p < 0.05, 0.01, or 0.001, respectively.

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MDPI and ACS Style

Liu, Y.; Ren, X.; Jeong, B.R. Carbon Dioxide Enrichment Combined with Supplemental Light Improve Growth and Quality of Plug Seedlings of Astragalus membranaceus Bunge and Codonopsis lanceolata Benth. et Hook. f. Agronomy 2019, 9, 715. https://doi.org/10.3390/agronomy9110715

AMA Style

Liu Y, Ren X, Jeong BR. Carbon Dioxide Enrichment Combined with Supplemental Light Improve Growth and Quality of Plug Seedlings of Astragalus membranaceus Bunge and Codonopsis lanceolata Benth. et Hook. f. Agronomy. 2019; 9(11):715. https://doi.org/10.3390/agronomy9110715

Chicago/Turabian Style

Liu, Ya, Xiuxia Ren, and Byoung Ryong Jeong. 2019. "Carbon Dioxide Enrichment Combined with Supplemental Light Improve Growth and Quality of Plug Seedlings of Astragalus membranaceus Bunge and Codonopsis lanceolata Benth. et Hook. f." Agronomy 9, no. 11: 715. https://doi.org/10.3390/agronomy9110715

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