Light Quality A ﬀ ects Growth and Physiology of Carpesium triste Maxim. Cultured In Vitro

: The objective of this study was to investigate the e ﬀ ect of light quality on the growth and contents of photosynthetic pigments and total ﬂavonoids, the parameters of chlorophyll a ﬂuorescence, antioxidant capacities, and enzyme activities. It was intended to preliminarily explore the internal mechanisms involved for the e ﬀ ect of light quality on the growth and physiology of Carpesium triste Maxim. Stem apex explants were cultured on the Murashige and Skoog (MS) medium under white (W), red (R), blue (B), or a 1:1 mixture of red and blue (RB) light-emitting diodes (LEDs) for four weeks. The RB induced sturdy plantlets and increased the contents of photosynthetic pigments and total ﬂavonoids, photosynthetic electron transport and e ﬃ ciency, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging capacity, and activities of antioxidant enzymes in the plantlets. Taken together, combinations of monochromatic red and blue LEDs could be used for the best production of high-quality C . triste plantlets in vitro.


Introduction
Carpesium triste Maxim. is a rare perennial herbal species of the Carpesium genus that belongs to the Asteraceae family, which is widely distributed in South Korea, China, and Japan. It has a slender stem and is usually 40-100 cm in height. Its root, stem, leaf, and fruit have long been used as a traditional medicine in the treatment of fevers, mastitis, toothache, diarrhea, and even cancer [1] due to the anti-inflammatory, antifebrile, cytotoxic, and styptic properties of the biologically active compounds [2]. The usage and plant parts extracted for use as herbs in the Carpesium genus vary, according to the oral and literature records in different countries. For example, the shoots of Carpesium abrotanoides L. were used to treat fever and to inhibit inflammation in Korea [3]. The dried seeds of Carpesium abrotanoides L., also called Carpesii Fructus, were prescribed to treat diseases caused by parasites, such as ascariasis, cestodiasis, and enterobiasis in China [4,5]. Previous studies on the Carpesium genus have shown that the effective pharmacological ingredients mainly include flavonoids, sesquiterpene lactones (germacranolide and guaianolide type) [6][7][8], acyclic diterpenoids [9,10], thymol derivatives [11], and terpenoids [12,13]. In the Carpesium genus, previous studies were mainly focused on chemical compounds, officinal substances, and clinical applications. There has been no study that investigates plant regeneration and growth regulation of the Carpesium genus in in vitro conditions until now. antioxidant enzymes were investigated and analyzed. The ultimate aim of this research was to provide an effective approach and the basis of production of high-quality plantlets for a large scale and fast reproduction as a mean to conserve this wild germplasm resource.

Plant Materials and Sterilization Methods
Dried seeds of C. triste were obtained from the Korea National Arboretum (KNA) in Pocheon, Gyeonggi-do, South Korea. Healthy and uniform seeds were selected for surface sterilization. Dust and impurities were cleaned up with running tap water for 5 min, and then, the seeds were dipped in 70% ethanol for 5 min for preliminary disinfection. After being rinsed 5-6 times in distilled water, the seeds were immersed in a 3% (v/v) NaClO solution for 15 min. Afterward, the seeds were washed adequately with distilled water 5 times to remove the residual disinfectants and then dried on sterile tissue papers. After surface sterilization, seeds were cultured on the plant growth regulator (PGR)-free Murashige and Skoog (MS) medium supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar.

Light Treatments and Growth Parameters
Stem apex explants from the 2-month-old plantlets derived from seed germination were cultured on the PGR-free MS medium and incubated in the plant growth chamber for four weeks with day/night temperatures of 24/18 • C and 16 hours of daily photoperiod under one of the following four light environments: white (W, 40 W tubes, Philips, The Netherlands), red (R, 621-710 nm), blue (B, 450-475 nm), or a 1:1 mixture of red and blue (RB) LEDs (LED, 400-700 nm, PSLED-1203-50A, Force Lighting Co.; Ltd., Hwaseong, Korea) at an intensity of 50 µmol·m −2 ·s −1 photosynthetic photon flux density (PPFD) measured with a photo/radiometer (HD 2102.2, Delta OHM, Padova, Italy). The spectral distributions were measured with a portable spectroradiometer (Spectra Light ILT 950, International Light Technologies, Inc., Peabody, MA, USA). The spectral quality of each light treatment is shown in Figure 1. Each treatment was repeated in three sets of 9 plantlets. Shoot and root lengths, number of leaves and roots, shoot diameter, and fresh and dry weights of shoots and roots were recorded after four weeks of exposure to the different lights. The dry weights of the shoots and roots were determined after drying for 48 hours at 60 • C.
Agriculture 2020, 10, x FOR PEER REVIEW 3 of 20 provide an effective approach and the basis of production of high-quality plantlets for a large scale and fast reproduction as a mean to conserve this wild germplasm resource.

Plant Materials and Sterilization Methods
Dried seeds of C. triste were obtained from the Korea National Arboretum (KNA) in Pocheon, Gyeonggi-do, South Korea. Healthy and uniform seeds were selected for surface sterilization. Dust and impurities were cleaned up with running tap water for 5 minutes, and then, the seeds were dipped in 70% ethanol for 5 minutes for preliminary disinfection. After being rinsed 5-6 times in distilled water, the seeds were immersed in a 3% (v/v) NaClO solution for 15 minutes. Afterward, the seeds were washed adequately with distilled water 5 times to remove the residual disinfectants and then dried on sterile tissue papers. After surface sterilization, seeds were cultured on the plant growth regulator (PGR)-free Murashige and Skoog (MS) medium supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar.

Light Treatments and Growth Parameters
Stem apex explants from the 2-month-old plantlets derived from seed germination were cultured on the PGR-free MS medium and incubated in the plant growth chamber for four weeks with day/night temperatures of 24/18 °C and 16 hours of daily photoperiod under one of the following four light environments: white (W, 40 W tubes, Philips, The Netherlands), red (R, 621-710 nm), blue (B, 450-475 nm), or a 1:1 mixture of red and blue (RB) LEDs (LED, 400-700 nm, PSLED-1203-50A, Force Lighting Co.; Ltd., Hwaseong, Korea) at an intensity of 50 µ mol·m −2 ·s −1 photosynthetic photon flux density (PPFD) measured with a photo/radiometer (HD 2102.2, Delta OHM, Padova, Italy). The spectral distributions were measured with a portable spectroradiometer (Spectra Light ILT 950, International Light Technologies, Inc., Peabody, MA, USA). The spectral quality of each light treatment is shown in Figure 1. Each treatment was repeated in three sets of 9 plantlets. Shoot and root lengths, number of leaves and roots, shoot diameter, and fresh and dry weights of shoots and roots were recorded after four weeks of exposure to the different lights. The dry weights of the shoots and roots were determined after drying for 48 hours at 60 °C.

Determination of Contents of Photosynthetic Pigments
A modified method described by Baglieri [50] was used to determine chlorophyll content: 500 mg of plant leaves was soaked in 10 mL 96% (v/v) ethanol for 10 hours at 4 • C for thorough decoloring, and after filtrating the leaf residues, extracts were applied to determine the contents of chlorophyll a and b and total carotenoids with a spectrophotometer at 665, 649, and 470 nm wavelengths, respectively. The calculations were based on formulae stated by Lichtenthaler and Wellburn [51]. The content of each photosynthetic pigment was measured three times, and the mean value was calculated.

Chlorophyll a Fluorescence Transient Measurement
Parameters of chlorophyll a fluorescence were measured with FluorPen (FP110/D, Photon Systems Instruments, Brno, Czech Republic). Intact and fully expanded leaves marked by the black arrows in Figure 2 were adapted to the dark for 20 min using detachable leaf-clips before measurement. The measuring light was set at 0.09 µmol·m −2 ·s −1 , and the saturating light pulse was set at 3000 µmol·m −2 ·s −1 The F M /F 0 , F V /F M , and F V /F 0 were calculated from the main parameters of chlorophyll a fluorescence. The calculated parameters from chlorophyll a fluorescence induction curve (OJIP), absorption (ABS), trapping (TR 0 ), heat dissipation (DI 0 ), and electron transport (ET 0 ) per reaction center (RC) are related to specific energy fluxes. Parameters were measured in three replicates of 9 leaves. The formulas of OJIP protocol parameters were described by Strasser et al., as shown in Table 1 [52,53]. Table 1. Extracted and technical parameters deduced from chlorophyll a fluorescence induction curve OJIP.  6 ± 0.58 a 7.00 ± 0.49 c 1.25 ± 0.12 a 0.17 ± 0.02 a 6.11 ± 0.11 a 9 ± 0.58 a 5.55 ± 0.23 ab 1.24 ± 0.10 a 0.17 ± 0.01 a z The light qualities used were W, white LED; R, red LED; B, blue LED; and RB, combined red and blue LEDs in a 1:1 ratio. y Values represent the means ± standard errors (n = 9). x Means separation within columns were performed by the Tukey's multiple range test at the 5% level.

Effect of Light Quality on Contents of Photosynthetic Pigments
Chlorophyll a content in leaves of C. triste plantlets grown under RB was higher than that of plantlets grown under W, B, and R (p < 0.05, Figure 3A). The W or RB were the most effective promoters of chlorophyll b accumulation (p < 0.05, Figure 3B). Plantlets grown under RB had the highest total chlorophyll levels, followed by those grown under W, B, and R (p < 0.05, Figure 3C). The chlorophyll a/b ratio did not show much difference between treatments (p < 0.05, Figure 3D). In addition, W and RB were effective in increasing the content of total carotenoids in leaves of C. triste plantlets, whereas R and B were less effective in doing so (p < 0.05, Figure 3E). Above all, RB led to increased total chlorophyll and carotenoid accumulation, which indicated that light quality may influence the photosynthetic performance through the photosynthetic pigments.

Determination of Activities of CAT, APX, POD, and SOD
One hundred milligrams of fresh leaf tissue were ground in liquid nitrogen and homogenized with a 1.5 mL 50 mM phosphate buffer (pH 7.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA), 0.05% Triton X-100, and 2% polyvinylpyrrolidone (PVP). After being centrifuged at 13,000 rpm for 20 min at 4 • C, the supernatant of the homogenate was collected and used to measure the enzyme activities.
The CAT activity was determined as described by Aebi [54]. The 3.0 mL assay system consisted of a 50 mM phosphate buffer (pH 7.0), 10 mM H 2 O 2 , and 0.1 mL of the extracted enzyme. The decrease in absorbance at 240 nm was monitored for 3 min. One unit of CAT activity represents 1 µmol H 2 O 2 decomposed per minute. The specific CAT activity was calculated as the units of enzyme activity per mg protein of samples (U·mg −1 ·protein). The extinction coefficient of H 2 O 2 at 240 nm was 40 mM −1 ·cm −1 .
2.5.2. Ascorbate Peroxidase (APX; EC 1.11.1.11) The APX activity was determined as described by Nakano and Asada [55]. The 3.0 mL assay system consisted of a 50 mM phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, 0.1 mM H 2 O 2 , and 0.1 mL of the extracted enzyme. The decrease in the absorbance at 290 nm was monitored for 3 min. One unit of APX activity represents oxidation of 1 µmol of ascorbate per minute. The specific APX activity was calculated as the units of enzyme activity per mg protein of samples (U·mg −1 ·protein). The extinction coefficient was 28 mM −1 ·cm −1 .
The POD activity was determined as described by Putter [56] with slight modifications. The 3.0 mL assay system consisted of a 50 mM phosphate buffer (pH 7.0), 10 mM H 2 O 2 , 20 mM guaiacol solution, and 0.1 mL extracted enzyme. The increase in the absorbance at 470 nm was recorded for 3 min. One unit of POD activity represents the formation of 1 µmol of tetraguaiacol per minute. The specific POD activity was calculated as the units of enzyme activity per mg protein of samples (U·mg −1 ·protein). The SOD activity was determined as described by Beauchamp and Fridovich [57]. The 3.0 mL assay system consisted of a 50 mM phosphate buffer (pH 7.0), 0.1 mM EDTA, 2 mM riboflavin, and 0.1 mL of Agriculture 2020, 10, 258 6 of 19 the extracted enzyme, which was incubated under light for 10 min. The reaction mixture contained a 50 mM phosphate buffer (pH 7.0) kept in dark and 2 mM riboflavin kept in light, which served as the reference and the blank, respectively. The absorbance at 560 nm was recorded, and one unit of SOD was defined as the amount of the enzyme that inhibited the nitroblue tetrazolium (NBT) reduction by 50%. The specific SOD activity was calculated as the units of enzyme activity per mg protein of samples (U·mg −1 ·protein).

Preparation of Plant Extracts
The extraction of 4-week-old plantlets as affected by light quality was performed according to the described protocol of Manivannan et al. [58]. Five hundred milligrams of finely powdered leaf samples were lyophilized with 5.0 mL 80% methanol (v/v) and extracted overnight. Then, the mixture was sonicated in an ultra-sonication bath (Bandelin Sonorex Super RK156BH, Berlin, Germany) for 30 min and centrifuged at 12,000 rpm for 10 min. The supernatant was employed for the assessment or stored at 4 • C.

Total Flavonoids Estimation
The total flavonoid quantification was determined based on the aluminum chloride colorimetric method as described by Chang et al. [59]. The 2 mL reaction mixture included 0.1 mL plant extracts, 1 mL 2% aluminum chloride solution, and 0.9 mL 80% methanol. The absorbance of the reaction mixture was incubated for 30 min and measured at 415 nm by using UV/Vis-spectrophotometer (LibraS22, Biochrom Ltd., Cambridge, UK). The total flavonoids were calculated according to a calibration curve of standard quercetin and expressed on the fresh weight basis as mg quercetin equivalent (QE)·mg −1 .

2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Free Radical Scavenging Capacity
The DPPH free radical scavenging capacity was determined as follows. A 20 µL extract of each sample or ascorbic acid standard solutions were added to 1.980 mL of a newly prepared DPPH solution and incubated under dark for 20 min. The absorbance of the reaction mixture was measured at 517 nm using a UV/V is spectrophotometer [60]. A standard curve was obtained by measuring the DPPH scavenging capacities of 50, 100, 150, and 200 mg·L −1 ascorbic acid. The free radical scavenging % was recorded by the equation (1 − A s /A c ) × 100%, where A s is the OD value of the sample extracts or ascorbic acid solution and A c is the OD value of the DPPH solution. The DPPH scavenging capacity was expressed on a fresh weight basis as mg ascorbic acid equivalent (AAE) g −1 .

Data Collection and Statistical Analysis
The assays were performed in triplicate to verify the reproducibility of the results. The data were collected and reported as the mean ± the standard error. A one-way analysis of variance (one-way ANOVA) was performed to evaluate the significant differences among the treatments, followed by a Tukey's multiple range test (p < 0.05). Counting data were assessed via Kruskal-Wallis analysis of variance with the Chi-square test (p < 0.05). Statistical analysis was conducted using the SAS statistical software package (SAS Institute Inc., Cary, NC, USA). All figures were made in OriginPro software version 9.0.

Effect of Light Quality on Growth and Development of Plantlets
As shown in Table 2, all growth indexes of C. triste plantlets showed extremely significant or significant differences under W, R, B, and RB LEDs. Under R, plantlets had the greatest shoot length (p < 0.05), which suggested that R played a positive role in promoting stem elongation. According to the Kruskal-Wallis Rank Sum Test, there was significant difference among the light qualities on the number of leaves (p = 0.023) and roots (p = 0.019). The R was the least effective in inducing new leaves, whereas W and RB were the best in increasing the leaf number (p < 0.05). Plantlets grown under R and RB had greater root lengths and number of roots than those grown under W and B. The shoot diameter of plantlets grown under RB was the greatest, followed in order by that of plantlets grown under W, R, and B (p < 0.05). The RB also increased the fresh and dry weights of shoots (p < 0.05). The above results indicated that plantlets displayed distinctly varying responses to the different light qualities. Plantlets grown under RB had a greater shoot diameter, dry matter accumulation, and root system development, which suggested that RB promotes the production of stout plantlets (Figure 2).

Effect of Light Quality on Contents of Photosynthetic Pigments
Chlorophyll a content in leaves of C. triste plantlets grown under RB was higher than that of plantlets grown under W, B, and R (p < 0.05, Figure 3A). The W or RB were the most effective promoters of chlorophyll b accumulation (p < 0.05, Figure 3B). Plantlets grown under RB had the highest total chlorophyll levels, followed by those grown under W, B, and R (p < 0.05, Figure 3C). The chlorophyll a/b ratio did not show much difference between treatments (p < 0.05, Figure 3D). In addition, W and RB were effective in increasing the content of total carotenoids in leaves of C. triste plantlets, whereas R and B were less effective in doing so (p < 0.05, Figure 3E). Above all, RB led to increased total chlorophyll and carotenoid accumulation, which indicated that light quality may influence the photosynthetic performance through the photosynthetic pigments.

Effect of Light Quality on Parameters of Chlorophyll a Fluorescence in Leaves
The OJIP method is considered an indicator of the state of the photosynthetic electron transport combined red and blue LEDs in a 1:1 ratio. The results were expressed as means ± standard errors (n = 3). Different letters indicate significant differences (p < 0.05) by the Tukey's multiple range test.

Effect of Light Quality on Parameters of Chlorophyll a Fluorescence in Leaves
The OJIP method is considered an indicator of the state of the photosynthetic electron transport processes [61]. The OJ, JI, and IP phases represent the reduction of the acceptor side of PS II, of the plastoquinone (PQ)-pool, and of the acceptor side of PS I, respectively [40][41][42]. In this study, each step in OJIP curves exhibited a different response to light qualities (Figure 4). The fluorescence intensities were higher in the OJ phase under R. However, it showed the weakest signal in IP phase under R. RB induced lower fluorescence intensity in the OJ phase. For W and B, there was no significant difference in OJIP curves (Figure 4).
For parameters of chlorophyll a fluorescence, light quality influenced the minimum fluorescence (F 0 ) but not the maximum fluorescence (F M ) in C. triste leaves (p < 0.05, Table 3). It was observed that the F 0 was appreciably higher in leaves of plantlets grown under R than that in plantlets grown under RB (p < 0.05, Table 3). The lowest electron transfer activity (F M /F 0 ) was also observed in plantlets grown under R (p < 0.05, Table 3). The maximum quantum efficiency of PS II photochemistry (F V /F M ) and the actual quantum yield of PS II (F V /F 0 ) were significantly reduced with R (p < 0.05), which were decreases of 3.5% and 16.5% respectively, compared to the maximum value ( Table 3).
The M 0 reflects the approximate initial slope of the fluorescence transient, and S M is the normalized total complementary area. There was a great increase in the M 0 value of plantlets grown under R (p < 0.05). The values of S M decreased in plantlets grown under W and R. In leaves of plantlets grown under RB, the lowest M 0 and highest S M values were observed (p < 0.05, Table 3).
In C. triste leaves, the values of the quantum yield of electron transport (ϕE 0 ) and the possibility of a trapped exciton moving an electron into the electron transport chain beyond Q A (ψ 0 ) were 10.9% and 7.5% lower, respectively, when grown under R than their corresponding highest values when grown under RB (p < 0.05, Table 3). In contrast, the quantum yield of heat dissipation (ϕD 0 ) under R was approximately 18.8% higher than the lowest value (Table 3). Under R, specific energy fluxes per reaction center related to the light absorption flux (ABS/RC), trapped energy flux (TR 0 /RC), electron transport flux (ET 0 /RC), and dissipated energy flux (DI 0 /RC) were 24.5%, 27.1%, 10.8%, and 50.0% higher, respectively, than their lowest values under RB ( Table 3). The performance index on an absorption basis (PI ABS ) in C. triste leaves was highest under RB (p < 0.05), followed in order by that under W and B, and R, where the minimum value was 51.7% of the maximum (Table 3).  Table 3). The performance index on an absorption basis (PIABS) in C. triste leaves was highest under RB (p < 0.05), followed in order by that under W and B, and R, where the minimum value was 51.7% of the maximum (Table 3).

Effect of Light Quality on Content of Total Flavonoids
Content of total flavonoids represented as quercetin equivalent (QE) in the leaves of C. triste plantlets differed significantly (p < 0.05) among treatments of four light qualities after four weeks of treatment ( Figure 5). Content of total flavonoids in leaves of plantlets under RB displayed the highest value as compared to other treatments, followed in order by that of plantlets grown under B, W, and R (p < 0.05). Compared to R treatment, content of total flavonoids in the leaves of RB, B, and W treatments increased by 61.0%, 23.2%, and 11.9%, respectively. value as compared to other treatments, followed in order by that of plantlets grown under B, W, and R (p < 0.05). Compared to R treatment, content of total flavonoids in the leaves of RB, B, and W treatments increased by 61.0%, 23.2%, and 11.9%, respectively.

Effect of Light Quality on DPPH Radical Scavenging Capacity
The DPPH is an organic radical with a deep purple color which will become yellow when it accepts an electron from an antioxidant. Therefore, the antioxidant capacity could be evaluated by the spectrophotometric measurements of the discoloration degree. The antioxidant capacities in C. triste plantlets grown under different light qualities, as measured by DPPH radical scavenging assays represented by ascorbic acid equivalent (AAE), were determined ( Figure 6). The result showed that the highest antioxidant capacities were noted in the extracts of RB-treated plantlets (1.97 mg AAE·g −1 FW, p < 0.05) and that the extracts of B-treated plantlets exhibited moderate radical inhibition, followed by that of plantlets treated by W and R.

Effect of Light Quality on DPPH Radical Scavenging Capacity
The DPPH is an organic radical with a deep purple color which will become yellow when it accepts an electron from an antioxidant. Therefore, the antioxidant capacity could be evaluated by the spectrophotometric measurements of the discoloration degree. The antioxidant capacities in C. triste plantlets grown under different light qualities, as measured by DPPH radical scavenging assays represented by ascorbic acid equivalent (AAE), were determined ( Figure 6). The result showed that the highest antioxidant capacities were noted in the extracts of RB-treated plantlets (1.97 mg AAE·g −1 FW, p < 0.05) and that the extracts of B-treated plantlets exhibited moderate radical inhibition, followed by that of plantlets treated by W and R.
The DPPH is an organic radical with a deep purple color which will become yellow when it accepts an electron from an antioxidant. Therefore, the antioxidant capacity could be evaluated by the spectrophotometric measurements of the discoloration degree. The antioxidant capacities in C. triste plantlets grown under different light qualities, as measured by DPPH radical scavenging assays represented by ascorbic acid equivalent (AAE), were determined ( Figure 6). The result showed that the highest antioxidant capacities were noted in the extracts of RB-treated plantlets (1.97 mg AAE·g −1 FW, p < 0.05) and that the extracts of B-treated plantlets exhibited moderate radical inhibition, followed by that of plantlets treated by W and R. Figure 6. Effect of light quality on 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity in leaves of C. triste plantlets after four weeks of culture: The light qualities used were W, white LED; R, red LED; B, blue LED; and RB, combined red and blue LEDs in a 1:1 ratio. The results were expressed as means ± standard errors (n = 3). Different letters indicate significant differences (p < 0.05) by the Tukey's multiple range test.

Effect of Light Quality on Activities of Antioxidant Enzymes
The activities of antioxidant enzymes in leaves of plantlets grown under different light qualities were determined in this study. The RB greatly increased the activities of CAT and APX compared to the other light qualities (p < 0.05), the respective values of which were 2.1-and 3.7-fold larger than the lowest values observed in leaves of plantlets grown under R ( Figure 7A,B). The activities of POD and SOD were the lowest in leaves of plantlets grown under R, and the highest values were observed in those grown under W and RB (p < 0.05). For POD activity, the ratio of the highest to lowest was Figure 6. Effect of light quality on 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity in leaves of C. triste plantlets after four weeks of culture: The light qualities used were W, white LED; R, red LED; B, blue LED; and RB, combined red and blue LEDs in a 1:1 ratio. The results were expressed as means ± standard errors (n = 3). Different letters indicate significant differences (p < 0.05) by the Tukey's multiple range test.

Effect of Light Quality on Activities of Antioxidant Enzymes
The activities of antioxidant enzymes in leaves of plantlets grown under different light qualities were determined in this study. The RB greatly increased the activities of CAT and APX compared to the other light qualities (p < 0.05), the respective values of which were 2.1-and 3.7-fold larger than the lowest values observed in leaves of plantlets grown under R ( Figure 7A,B). The activities of POD and SOD were the lowest in leaves of plantlets grown under R, and the highest values were observed in those grown under W and RB (p < 0.05). For POD activity, the ratio of the highest to lowest was approximate 2.8-fold, and for SOD activity, the ratio of the highest to lowest was approximate 1.7-fold, respectively (p < 0.05, Figure 7C,D).
Agriculture 2020, 10, x FOR PEER REVIEW 11 of 20 approximate 2.8-fold, and for SOD activity, the ratio of the highest to lowest was approximate 1.7fold, respectively (p < 0.05, Figure 7C,D).

Discussion
The effects of light quality on the growth and development of C. triste plantlets were explored as light quality plays a vital role in regulating photosynthetic capacity, which may benefit rapid and high-quality propagation in vitro. This study showed that R led to longer shoots and roots in C. triste

Discussion
The effects of light quality on the growth and development of C. triste plantlets were explored as light quality plays a vital role in regulating photosynthetic capacity, which may benefit rapid and high-quality propagation in vitro. This study showed that R led to longer shoots and roots in C. triste plantlets than other light qualities (p < 0.05, Table 2). These data were consistent with the results in various species such as Euphorbia millii [62], Tripterospermum japonicum [63], and Oncidium [64]. Plantlets grown under B had weak growth vigor, exhibiting the shortest shoots and roots, lowest fresh and dry weights, and slenderest stems (p < 0.05, Table 2). It was also reported that the increased internode length was due to the deficiency of blue light in Phaseolus vulgaris [65] and that blue light inhibited stem elongation in Pelargonium [66]. However, the highest specific growth rate and extracellular polysaccharide production of Porphyridium cruentum were observed under blue light [67]. Red light increased shoot height and rooting frequency in Convolvulus arvensis [68]. Many shreds of evidence are indicating that the combination of red and blue LEDs benefits the growth and development of plantlets cultured in vitro [69][70][71]. The combination of red and blue light enhanced plant above-and underground growth and development of Cymbidium by increasing the net leaf photosynthesis rate as well as the stomatal conductance in in vitro conditions [72,73]. These observations indicated that inductions and developments of shoot and root are probably dependent on the spectral quality of light. The reason underlying this phenomenon might be that the spectral energy distribution of red and blue lights was accordant with that of chlorophyll absorption [74]. The increased indole-3-acetic acid (IAA) oxidase activity caused by blue light [75,76] or promoted accumulation of gibberellin (GA) caused by red light [77] may be involved in the mechanisms with which light regulates shoot elongation. Reducing the red/far-red ratio promoted stem elongation and gave significant increases in contents of GA and IAA in both internodes and leaves in Helianthus annuus [78]. The decreased abscisic acid (ABA) content was observed with R light treatment [79]. In this study, the combination of red and blue LEDs in a 1:1 ratio increased the number of leaves and roots, fresh and dry weights of shoots, as well as shoot diameter (p < 0.05, Table 2), which led to a more compact and sturdy morphology of plantlets. The underlying mechanisms of signal interactions of light quality and plant hormones are well worth exploring in the future.
Photosynthetic pigments are another important factor for photosynthesis as well as plant growth. Light quality influences photosynthesis through the light absorption ability of the photosynthetic pigments [80]. Chlorophyll a mainly functions in photosystems, and chlorophyll b is concentrated in light-harvesting complexes (LHC) [81]. In this study, the highest chlorophyll a and total chlorophyll were observed in the leaves of C. triste plantlets grown under RB (p < 0.05, Figure 3A,C). W and RB promoted chlorophyll b and total carotenoid accumulation in leaves of plantlets (p < 0.05, Figure 3B,E). The chlorophyll a/b ratio indicates the amount of chlorophyll associated with the PS I and PS II complexes [82] and has a positive correlation with the net photosynthetic rate [83]. Blue light plays an important role in chlorophyll formation and chloroplast development [84]. Contents of chlorophyll and total carotenoids were higher under B than under R in this study, and it may because blue light was more effective than red light in the induction of photosynthetic pigments synthesis in leaves [85]. The change in the Chl a/b ratio often indicates a change in the ratio between the reaction center complexes of the photosystem and the LHC. In this study, there was no significant difference in the Chl a/b ratio between treatments ( Figure 3D), and it indicated that the LHC of the thylakoid membranes was not altered much by light quality within the treatment time. Carotenoids are important components of the reaction center complex and act as accessory pigments in the antenna systems to perform functions of harvesting the light energy and improving the photosynthetic efficiency [86]. W and RB led to the highest content of total carotenoids in leaves of plantlets (p < 0.05), which indicated an enhanced photosynthetic performance of plants.
Chlorophyll a fluorescence acts as a subtle indicator of the photochemical activity and electron transfer of photosynthesis and is related to the physiological status of plants [87]. Based on the electron transport in the receptor side and donor side in PS II and the energy transfer in thylakoid membranes, Strasser and others built up the OJIP method on analysis and processing of rapid chlorophyll fluorescence [36,38]. The increase in leaf fluorescence transients of C. triste plantlets under different light qualities showed a typical OJIP shape (Figure 4). Under R treatment, it showed repressed fluorescence transients in the IP phase. This result was consistent with the previous study in Cyclocarya paliurus [88]. The increased value of F 0 (p < 0.05, Table 3) suggested a decline in the light energy trapping rate in the PS II reaction center [89]. F M /F 0 , F V /F M , and F V /F 0 are considered as indicators of the electron transfer activity and photosynthetic efficiency of plants [90] and could be used to evaluate the vigor and health of the photosynthetic apparatus [91]. The decreased values of F M /F 0 , F V /F M , and F V /F 0 in leaves of C. triste plantlets grown under R (p < 0.05, Table 3) suggested that plants slightly underwent photoinhibition and had a reduced photochemical efficiency and PS II activity. M 0 is related to Q A reduction, and S M reflects the total energy required for Q A reduction. The PS II fluorescence yield is positively correlated with the level of reduced Q A , and oxidized Q A quenched the fluorescence and leads to closure of the active reaction center [92]. The lowest M 0 and highest S M values were observed in plantlets grown under RB (p < 0.05, Table 3). These suggested that RB induced more electrons transferring from Q A into the electron transport chain. In contrast, R had the opposite effect in the electron transport process from Q A to Q B . The similar decrease in the quantum yield of electron transport (ϕE 0 ) (p < 0.05, Table 3) and the possibility of a trapped excitons moving an electron into the electron transport chain beyond Q A (ψ 0 ) (p < 0.05, Table 3) under R indicated that the acceptor side of PS II in the electron transport of PS II was inhibited. The ABS/RC represents the absorption flux of antenna chlorophyll per reaction center and reflects the ratio of photons absorbed by the chlorophyll pigments of reaction centers to the total numbers of active reaction centers [93]. The increased value of ABS/RC (p < 0.05, Table 3) indicated the decrease in antenna size of the reaction centers under R. Moreover, TR 0 /RC and ET 0 /RC represent the trapped energy flux and electron transport flux per reaction center, respectively, and the increased values under the R (p < 0.05, Table 3) were possibly due to the transportation of photons absorbed by inactive reaction center to active reaction centers. This was also verified with the increased value of heat dissipation per reaction center (DI 0 /RC) and the quantum yield of energy dissipation (ϕD 0 ) ( Table 3) [94]. Photons were unable to be captured effectively by the active centers, resulting in higher values of non-photochemical quenching. The above results indicated that C. triste plantlets grown under R underwent photoinhibition and had increased inactivated reaction centers and improved energy consumption in the activated reaction centers. PI ABS is an indicator of plant vitality and reflects the functionality of both PS I and PS II. It is closely related to the activity of the photosynthetic apparatus. The significantly decreased PI ABS (p < 0.05, Table 3) indicated that the energy conservation ability from photons absorbed by the PS II antenna to the reduction of Q B and the activity of the photosynthetic apparatus were inhibited by R [95]. The reduced values of F V /F M and PI ABS as well as increased values of parameters related to specific energy fluxes (ABS/RC, TR 0 /RC, ET 0 /RC, and DI 0 /RC), were observed in Cyclocarya paliurus and Brassica campestris under red or green LED as compared to blue or white LED [88,96]. Results were also found that monochromatic red light led to less efficiency in the transfer of excitons and higher energy dissipation in Calendula officinalis, indicating the occurrence of photoinhibition under the red light [97]. Plants grown under red light exhibit characteristics of shade plants, and a study in roses revealed that plants grown under monochromatic red or blue LED showed a more sensitive electron transport system under high light-intensity treatment as compared to those grown under combined red and blue or white LED [98]. The systematic analysis of parameters of chlorophyll a fluorescence revealed light energy trapping and transformation, electron transport, and activity of the photosynthetic apparatus under different light qualities. RB led to the highest values of M 0 and PI ABS , which indicated positive effects in the electron transport process from Q A to Q B and a higher activity of the photosynthetic apparatus. Moreover, the sugar in the medium is thought to be the limitation of photosynthesis and development of photosynthetic apparatus. In several plant species cultured in in vitro conditions, the sugar reduces the need for carbohydrate production and therefore leads to a lower photosynthetic rate [99,100]. However, there are still studies showing the advantages of sugar on photosynthesis [101,102]. Plantlets treated without sugar showed deformed chloroplasts in the myrtle [103]. In this study, plantlets grown under 50 µmol·m −2 ·s −1 with 3% (w/v) sucrose did not show significant inhibitions of growth and photosynthesis. The 3% (w/v) sucrose revealed that it overcame the negative effect of strong light during culture on the functioning of photosynthesis and the xanthophyll cycle [104]. The interactions between sugar and light-induced signaling pathways in vitro are worth studying in the future.
ROSs could be derived from photochemical events, e.g., electron transport chains, as well as photorespiration under different light conditions, which can result in photodamage to PS II directly. Studies related to the effects of light qualities on nonenzymatic ROS scavengers showed that B stimulated the accumulation of total flavonoids and enhanced DPPH free radical scavenging capacity in callus cultures of Rhodiola imbricata [105] as well as the leaf and root extracts in Rehmannia glutinosa [106]. The combined red and blue LEDs in a 1:1 ratio also benefit the content of total flavonoids in Cnidium officinale callus and the activities of APX and SOD [107]. The present study also demonstrated similar findings, as shown in Figures 5-7. CAT, APX, POD, and SOD are antioxidant enzymes reflecting the antioxidant capacity of plants. SOD, as the first activated antioxidant enzyme in the reactive oxygen scavenger process, catalyzes the superoxide anion free radicals (O 2 − ) into hydrogen peroxide (H 2 O 2 ) in plants. In the subsequent reactions, H 2 O 2 is broken down by functions of CAT, APX, and the ascorbate-glutathione cycle [108][109][110]. A majority of classic PODs utilizes H 2 O 2 to oxidize hydrogen donor substrates [111]. In addition to being involved in the defense responses of plants to biotic or abiotic stresses, antioxidant enzymes also positively regulate plant growth, differentiation, morphogenesis, and yield [112][113][114]. A previous study has shown that light quality could influence the metabolism of antioxidant enzymes [115]. RB significantly improved the activities of CAT and APX (p < 0.05, Figure 7A,B). The highest activities of POD and SOD were observed in the leaves of plantlets of C. triste grown under W and RB (p < 0.05, Figure 7C,D). Increased levels of ROS were observed in various plant species during senescence, and these changes may have resulted from the decline in the activities of certain antioxidant enzymes [116]. The decreased CAT activity was found in pea leaves which underwent senescence induced by a natural aging process or a dark condition [117]. Similarly, blue light caused higher specific activity of CAT in wheat as compared to other light treatments and delayed the onset of senescence of leaves, indicating the vital role of CAT in the prevention of senescence [118]. The increased activities of SOD, CAT, and POD as well as increased DPPH scavenging capacity in procyanidins-treated banana fruits suggested that these effectively scavenged excessive ROS and, therefore, alleviated lipid peroxidation and delayed senescence [119]. These findings show that the ability to scavenge superoxide anion radicals was improved under RB, which may delay senescence in plants. However, the antioxidant activity during senescence varied among plant species [116]. Elucidation of the mechanisms underlying plant senescence and ROS homeostasis will benefit with increasing yield and stress resistance.

Conclusions
This study investigated the growth, physiological responses, light energy trapping, transformation, and electron transport in the photosynthesis process of plants as affected by light quality. In conclusion, RB induced sturdy plantlets with well-developed shoots and roots; increased the content of photosynthetic pigments, photosynthetic electron transport, and efficiency and content of total flavonoids; and enhanced DPPH free radical scavenging capacity and activities of antioxidant enzymes in the plantlets. Hence, it could be used for the best production of high-quality C. triste plantlets in vitro. Future studies should focus on investigating the underlying molecular mechanisms of how combinations of monochromatic LEDs affect plants and the interactions between physiological regulatory and light-induced signaling pathways.