Effects of supplemental lighting with different light qualities on growth and secondary metabolite content of Anoectochilus roxburghii

Background Anoectochilus roxburghii is a widespread herbaceous plant with high medicinal value. Wild A. roxburghii resources face extinction due to their slow growth rate and over exploitation. The growing market demand has led to advances in the field of artificial planting of A. roxburghii. Methods to increase the economic benefits of cultivation and the production of medicinal ingredients are very useful. Methods A. roxburghii was exposed to red light, blue light (BL), yellow light (YL), green light, or white light as supplemental lighting at night (18:00–02:00) in a greenhouse or were left in darkness (control, CK) to investigate the effects of various light qualities on growth indices, photosynthetic pigments, chlorophyll fluorescence, root vitality, stomatal density, soluble proteins, sugars, and the accumulation of secondary metabolites. Results Supplementation of BL had a positive effect on A. roxburghii growth and secondary metabolite accumulation. Leaf number, stem diameter, fresh weight, dry weight, chlorophyll a content, and secondary metabolite (total flavonoids, total polyphenols) content increased significantly. YL treatment showed significantly higher soluble sugar and polysaccharide contents than the control. Discussion BL treatment was conducive to promoting the growth and accumulation of secondary metabolites (total flavonoids, total polyphenols); YL treatment significantly increased the content of soluble sugar and polysaccharides more than the control. Polysaccharides and total flavonoids are important medicinal ingredients of Anoectochilus, so future research will focus on the combination of blue and YL.


INTRODUCTION
Anoectochilus roxburghii (Orchidaceae) is a valued perennial herb that is used for medicinal and ornamental purposes in China and many Asian countries. The chemical the optimal light quality to increase the medicinal value and economic benefits for Anoectochilus growers.

Plant materials and growth conditions
Test species: A. roxburghii cv. Narrow-leaf.
The experiment was performed in December of 2016 in the greenhouse of the Fujian Institute of Subtropical Botany, China (24 26′N, 118 04′E). The day (highest)/night (lowest) temperatures of the greenhouse were 23/12 C. A. roxburghii plantlets were obtained from Diyuan Biotechnology Co., Ltd. (Xiamen, China). Tissue-culture bottles were opened to acclimate the plantlets to the environmental conditions of the greenhouse for 15 days. The average fresh weight of the acclimated plantlets was 0.55 g and leaf number was 3.8. These plantlets were transplanted to plastic trays (50 Â 30 Â 5 cm) at a density of 110 plants per tray. The cultivation soil was composed of peat moss and perlite in a 2:1 ratio by volume.
In the supplemental lighting experiment, natural light was used during the day and LED lamps were used as supplemental lighting at night. LED lamps were provided by Xiamen Guangpu Electronics Co., Ltd. (Xiamen, China). Plantlets were subjected to five different light quality treatments: RL (580-660 nm, peak wavelength 630 nm), BL (440-540 nm, peak wavelength 469 nm), yellow light (YL; 540-620 nm, peak wavelength 592 nm), green light (GL; 460-600 nm, peak wavelength 519 nm), WL (420-780 nm) and a control without supplemental lighting (CK). The spectral characteristics of the lamps are shown in Fig. 1, as measured by a MK350S spectrometer (United Power Research Technology Corporation, Zhunan, Taiwan). Each treatment consisted of one tray with three replications (3 Â 110 plants for each treatment). The photoperiod of supplemental lighting was 8 h day -1 (18:00-02:00). A. roxburghii grown under different light qualities at night are shown in Fig. 2.
The LED lamps were placed above of the plantlets. The conclusion from Ma et al. (2010) was referenced and the height of each light fixture was adjusted to ensure the light intensity was 30 ± 1 mmol·m -2 ·s -1 (light intensity was measured with a LI-250A light meter (LI-COR Biosciences, Lincoln, NE, USA)). Different treatments were insulated from one another by black shading materials.

Determination of the physiological and biochemical indexes
After 40 days of treatment, 40 plantlets were chosen randomly from each repeat for subsequent testing. A total of nine of these 40 plantlets were selected randomly for biomass analysis within each treatment. And six of these 40 plantlets were selected randomly for root activity analysis and stomata observation within each treatment. The others were stored in a -80 C refrigerator after flash freezing.

Growth and biomass parameter analysis
The leaf numbers, length and width of each leaf, stem diameter, stem length, root length, root numbers, fresh weight, and dry weight were separately determined. The fresh weight of the plantlets were measured with an electronic balance (Sartorius, Hamburg, Germany), and the plantlets were dried to a constant weight at 80 C to determine their dry weight. The stem diameter was measured with a vernier caliper (Tricle Brand Tools, Shanghai, China). The stem length was measured from the top of the plantlet to the base of the first root. The root length was measured from the top of the root tip to the main stem base.

Chlorophyll content
Chlorophyll content was determined following the method from Yuan et al. (2010). Fresh mature leaves (Half-gram) were collected from each treatment for the determination of chlorophyll content (Chl a, Chl b, Chl a + b,). Samples were ground in a mortar. Chlorophylls were extracted in 15 ml acetone-water solvent (80%, v/v) (Xilong Scientific Co., Ltd., Shantou, China) in the dark at room temperature. Chlorophyll quantification was measured at 665 and 649 nm. The results are expressed as milligrams of chlorophyll mass per gram of fresh weight.

Chlorophyll fluorescence
Chlorophyll fluorescence was measured with a MINIPAM fluorometer (Walz, Effeltrich, Germany) (Schreiber et al., 1997). Leaves with consistent maturity were light adapted for approximately 15 min prior to measurements of the effective quantum yield of photochemical energy conversion (Yield) and photochemical (qP) and  Soluble sugar, reducing sugar, and polysaccharide Fresh leaves (0.2 g) were homogenized in liquid nitrogen and soluble sugar was extracted in boiling ultrapure water (15 ml) for 20 min. Following centrifugation at 15,000Âg for 5 min, the residues were re-extracted under the same conditions. The respective extracts and re-extracts were pooled before analysis. All samples were extracted in duplicate. The soluble sugar content was evaluated using the anthrone colorimetric method (Li, 2000, 194-197). The extracts (0.5 ml) were incubated with the anthrone reagent (five ml) (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) in a boiling water bath (10 min) to yield a blue-green color and after cooling, its optical density was measured at 620 nm. Standard solutions of glucose (Xilong Scientific Co., Ltd., Shantou, China) were prepared in ultrapure water at a concentration of 10 mg ml -1 .
The reducing sugar content was evaluated by 3,5-dinitrosalicylic acid (DNS) colorimetry (Li, 2000, 197-199). The extracts (0.5 ml) were incubated with the DNS color solution (five ml) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) in a boiling water bath (5 min) and after cooling, the optical density was measured at 540 nm. Standard solutions of glucose (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) were prepared in ultrapure water at a concentration of one mg ml -1 .
Polysaccharide content ¼ Soluble sugar content À Reducing sugar content

Stomata observations
Pretreatment of samples was performed based on the method of Li, Tang & Xu (2013). Fully expanded leaves were chosen from each plantlet to observe the stomata. The abaxial and adaxial surfaces of the leaves were wiped with wet absorbent cotton fiber. Then, transparent nail polish was smeared on the two sides of the leaves. After the nail polish had air-dried and formed a membrane, transparent adhesive tape was pressed onto each leaf and was subsequently stripped off. The transparent adhesive tape was then pressed on a slide, which was treated with a neutral plastic seal and made into a temporary slide. The slides were imaged using a Leica DMI 3000B microscope (Leica Microsystems, Wetzlar, Germany). The length, width, and density of stomata were measured with Leica LAS Image Analysis software (Leica Microsystems, Wetzlar, Germany). Stomatal area = Length Â Width Â 3.14 Â 1/4 (mm 2 ). Stomatal density = Number/Field/Field area.

Root activity
Root activity was determined using the triphenyltetrazolium chloride method (Li, 2000, 119-120). A total of 0.3 g samples of fresh roots were treated with five ml of 0.01M 2,3,5-triphenyltetrazolium chloride (TTC) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and five ml of 0.07M potassium phosphate buffer (PBS) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 2 h at 37 C. The reaction was terminated with two ml of 1M sulfuric acid (Xilong Scientific Co., Ltd., Shantou, China), and the roots were removed and rinsed two to three times with distilled water. The samples were subsequently placed in a mortar with quartz sand (0.3 g) with 10 ml of acetone (Xilong Scientific Co., Ltd., Shantou, China) and ground until the root turned white. The optical density was then measured at 485 nm. To make a standard curve, 0.25, 0.50, 1.00, 1.50, or 2.00 ml of 0.01M TTC was added to five volumetric flasks, and sodium thiosulfate (Xilong Scientific Co., Ltd., Shantou, China) and distilled water were added to reach a volume of 10 ml.

Soluble protein
The soluble protein content was measured following the method of Bradford (1976). A total of 0.2 g of fresh leaf samples were ground in a mortar with liquid nitrogen to which five ml of 0.07M PBS (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added, followed by centrifugation at 5,000Âg for 10 min. The supernatant was saved and then one ml of extract and five ml of coomassie brilliant blue G-250 (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) were thoroughly mixed. To generate a standard curve, 0, 0.2, 0.4, 0.6, 0.8, or one ml of 100 g l -1 of bovine serum albumin (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added to six volumetric flasks, and distilled water was added to reach a total volume of one ml. The optical density was measured at 595 nm.

Total flavonoids
For total flavonoids estimation, the method of Marinova, Ribarova & Atanassova (2005) was followed with slight modification. Half-gram samples of fresh leaves were homogenized in liquid nitrogen and total flavonoids were extracted in 10 ml of ethanol-water solvent (60%, v/v) (Xilong Scientific Co., Ltd., Shantou, China) in the dark (3 h) and then centrifuged at 15,000 rpm for 10 min. The supernatant (one ml) was mixed with a 0.73M sodium nitrite solution (0.4 ml) (Xilong Scientific Co., Ltd., Shantou, China), a 0.47M aluminum nitrate solution (0.4 ml) (Xilong Scientific Co., Ltd., Shantou, China), 1M sodium hydroxide (four ml) (Guangdong Guanghua Sci-Tech Scientific Co., Ltd., Guangzhou, China) and ethanol-water solvent (60%, v/v) (0.2 ml). The mixture was shaken for 12 min, then its optical density was measured at 510 nm. The total flavonoid content was calculated as milligrams of rutin (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) equivalent per gram of fresh weight.

Total polyphenols
The total polyphenol content was determined with the Folin-Ciocalteu reagent according to the method of Zuo et al. (2012). For polyphenol estimation, half-gram samples of fresh leaves were homogenized in liquid nitrogen and total polyphenols were extracted in 15 ml of distilled water (20 min) and then centrifuged at 15,000 rpm for 15 min. The supernatant (one ml) was mixed with Folin-Ciocalteau reagent (1.5 ml) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and 0.94M sodium carbonate (four ml) (Xilong Scientific Co., Ltd., Shantou, China). The mixture was incubated in the dark for 2 h at room temperature. Its optical density was then measured at 765 nm, and the total polyphenol content was calculated as milligrams of gallic acid (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) equivalent per gram of fresh weight.

Statistical analysis
SPSS Version 13.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analysis. Data was analyzed by one factor, light quality. The results were analyzed by one-way analysis of variance. Duncan's multiple range test was employed to detect differences between means (with P set to 0.05).

Morphological observations
The growth indices of A. roxburghii treated with different light qualities on day 40 are summarized in Fig. 3 and Table 1; overall, the BL treatment made A. roxburghii plantlets grow robustly. Significantly higher leaf numbers (5.00 ± 0.50 vs. 5.89 ± 0.60, P < 0.05) were observed under the BL treatment, at 17.8% higher than the control. However, the leaf numbers did not differ significantly between the RL (5.22 ± 1.09), YL (5.00 ± 0.87), GL (5.11 ± 0.60), and WL (5.33 ± 0.71) treatments and the control. The leaf length, leaf width, stem length, and root numbers did not differ significantly between all supplemental lighting treatments and the control. Stem diameter was significantly greater in the BL treatment (2.38 ± 0.13 vs. 2.68 ± 0.43 mm, P < 0.05) than in the control. Root length of the RL (5.37 ± 0.85 vs. 6.53 ± 0.86 cm, P < 0.01), BL (5.37 ± 0.85 vs. 6.43 ± 1.09 cm, P < 0.05), and YL (5.37 ± 0.85 vs. 6.76 ± 0.56 cm, P < 0.01) treatments was significantly higher than in the control. Fresh weight (1.35 ± 0.09 vs. 1.58 ± 0.12 g, P < 0.01) and dry weight (0.15 ± 0.02 vs. 0.20 ± 0.03 g, P < 0.01) of the BL treatment were significantly greater than in the control. However, the RL, YL, GL, and WL treatments had no significant effect on the yield of A. roxburghii.

Photosynthetic pigment contents
As shown in Fig. 4, a significantly higher chlorophyll a content (0.59 ± 0.03 vs. 0.82 ± 0.07 mg g -1 FW, P < 0.05) was observed with the BL treatment, at 38.98% higher than in the Control. In this study, chlorophyll b content was not affected significantly by different light qualities. The chlorophyll a + b content showed a similar trend compared to chlorophyll a. However, different light qualities had no significant effect on the chlorophyll a + b content of A. roxburghii. The highest chlorophyll a + b content (1.19 ± 0.11 mg g -1 FW) was observed with the BL treatment, at 22.6% higher than in the control (0.97 ± 0.03 mg g -1 FW), but this increase was not statistically significant (P > 0.05).

Chlorophyll fluorescence
Different light qualities had variable effects on the chlorophyll fluorescence of A. roxburghii (Fig. 5). The YL treatment resulted in a significant increase in yield (0.47 ± 0.12 vs. 0.62 ± 0.08, P < 0.05), at 31.9% higher than in the control. A significant decrease in yield was observed in RL (0.47 ± 0.12 vs. 0.32 ± 0.06, P < 0.05) treatment, at 31.9% lower than in the control. However, the yield value did not differ significantly between the BL (0.56 ± 0.05), GL (0.38 ± 0.17), and WL (0.34 ± 0.11) treatments and the control.
There was no significant difference in the qP value between all of the supplemental lighting treatments and the control.

Notes:
Values represent mean ± SE of nine replicates; and different letters within a row indicate significant differences at P < 0.05. F, P, and df represent F-value, P-value, and degree of freedom of the ANOVA, respectively.  The qN value was significantly higher in the RL treatment (0.44 ± 0.22 vs. 0.69 ± 0.23, P < 0.05), GL treatment (0.44 ± 0.22 vs. 0.68 ± 0.13, P < 0.05), and WL treatment (0.44 ± 0.22 vs. 0.82 ± 0.18, P < 0.01) than in the control. However, the qN value did not differ significantly between the BL (0.55 ± 0.20) and YL (0.32 ± 0.06) treatments and the control.

DISCUSSION
Light quality is an important environmental factor, amongst the light spectra, red and blue wavelengths are the primary spectral wavelengths and highly influence the plant primary and secondary metabolism (Johkan et al., 2010). RL is reported to contribute to photosynthetic apparatus development and may increase starch accumulation in several plant species (Kobayashi, Amore & Lazaro, 2013). BL is important for photosynthesis, chloroplast development, chlorophyll formation, and chemical composition of plants (Hogewoning et al., 2010).

Biomass parameter response of A. roxburghii to light quality
Biomass is an important indicator in medicinal plants. In the present study, the biomass parameters of A. roxburghii differed in their responses to different light qualities, but the BL treatment made A. roxburghii plantlets grow robustly. Exposure to the BL treatment significantly increased the leaf numbers, stem diameter, fresh weight, and dry weight of A. roxburghii. The root length of the RL, BL, and YL treatments was significantly higher than in the control. There were similar conclusions in other related studies involving, Stevia rebaudiana Bertoni (Simlat et al., 2016), Cardamine fauriei (Abe et al., 2015), and R. glutinosa (Manivannan et al., 2015), where exposure to the BL treatment showed the largest leaf numbers. BL is more effective in enhancing fresh weight and dry matter production in buckwheat sprouts (Lee et al., 2014). However, RL had a stimulating effect on leaf numbers and the root length of cucumber seedlings (Su et al., 2014). These results indicate that plant species differ in their responses to light quality, but BL generally promotes plant growth and dry matter accumulation.

Photosynthetic pigment response of A. roxburghii to light quality
Chlorophyll content is an important determinant of photosynthetic and dry matter production (Ghosh et al., 2004). Studies have confirmed that light, especially BL, plays an important role in the synthesis of plant chlorophyll. Cultured Phalaenopsis "Fortune Saltzman" seedlings treated with BL for 5 months showed significantly higher responses in terms of chlorophyll a, chlorophyll b and total chlorophyll (Anuchai & Hsieh, 2017). BL contributes positively to chlorophyll synthesis in S. rebaudiana Bertoni, and plantlets grown under RL showed the lowest levels of chlorophyll (Simlat et al., 2016). Senger (1982) documented that BL plays an important role in chloroplast development and the formation of chlorophyll. In our study, BL increased the chlorophyll a content of A. roxburghii significantly; Chlorophyll b was not sensitive to light quality. Studies confirm that chlorophyll, especially chlorophyll a, plays an important role in the process of photosynthesis and the accumulation of dry matter (Naidu et al., 1984). In our study, the highest dry matter content of A. roxburghii was observed in the BL treatment, which illustrates that BL promoted the synthesis of chlorophyll, especially chlorophyll a, thereby promoting photosynthesis and the accumulation of dry matter.

Chlorophyll fluorescence response of A. roxburghii to light quality
Chlorophyll fluorescence is an important signal of photosynthetic synthesis and plant responses to the external environment. In our study, the highest yield and lowest qN were observed with the YL treatment, which indicates that YL can improve the photosynthetic efficiency of A. roxburghii. The second-highest dry matter content also confirmed the promotion of YL on photosynthetic synthesis in A. roxburghii. There was no significant difference in qP between all treatments and the control.  , 2016). The total sugar from grape skin was highest in blue LED-treated plants, followed by red LED-treated plants (Kondo et al., 2014). The above results indicate that the mechanism of different light qualities regulating the metabolism of carbohydrate in plants is complex and needs further exploration.
Stomata response of A. roxburghii to light quality Stomata are the pathway for air and water vapor during carbon assimilation, respiration, and transpiration, and their quantity is regulated by the opening and closing of guard cells. Many exogenous factors influence the opening and closing of stomata, such as light, temperature, and CO 2 . In our study, YL caused a significant increase in stomatal density in A. roxburghii, but there was no significant difference in the stomatal area between LED light treated plants and control treated plants. In other similar studies, the stomata of different plants responded differently to light quality. In S. rebaudiana, BL not only increased the number of stomata but also caused stomatal opening (Simlat et al., 2016). Macedo et al. (2011) found that red and blue fluorescent light reduced the number of stomata on the adaxial face and abaxial face of Alternanthera brasiliana leaves, respectively. These results indicate that the effects of different light qualities on the stomata of different plants are inconsistent, and the mechanism of light regulating plant stomata needs further research.

Soluble protein response of A. roxburghii to light quality
Soluble protein is an important osmotic regulator and nutrient involved in various metabolic enzymes. Soluble protein content is an embodiment of plant resistance and metabolism. In this study, BL caused a significant decrease in soluble protein levels in A. roxburghii, but the soluble protein content did not differ significantly between other treatments and controls. However, the effects of light quality on the soluble protein content in other plants were different. In P. vulgaris L., the soluble protein content was higher under white fluorescent tube lights compared to all colored lights (Fazal et al., 2016). The highest total protein content of A. absinthium grown under various colored illuminations was observed under RL (Tariq, Ali & Abbasi, 2014).

Secondary metabolite response of A. roxburghii to light quality
Secondary metabolism is an indispensable part of plant life and related closely to plant growth and environmental factors. Light is an essential environmental factor that affects the accumulation of secondary metabolites. BL, UV-B, and UV-A were considered to trigger the gene expression of chalcone synthase, the first committed step in the flavonoid metabolic pathway, and effect flavonoid metabolism in Arabidopsis (Christie & Jenkins, 1996;Fuglevand, Jackson & Jenkins, 1996). In P. vulgaris L., BL was effective for total phenolic and flavonoid content (Fazal et al., 2016). In Tartary buckwheat sprouts, BL induced the accumulation of flavonoids (Seo et al., 2015). The phenolic levels in Lactuca sativa L. seedlings treated with BL increased significantly (Johkan et al., 2010). Taulavuori et al. (2016) suggested that both BL and RL may be needed to regulate the accumulation of phenolics in basil. In our study, significantly higher total flavonoid and polyphenol contents were observed in BL. These results indicate that BL promotes the accumulation of plant secondary metabolites, which provides an effective method to increase the medicinal component contents of medicinal plants.

CONCLUSION
The supplementation of LED BL had a positive effect on A. roxburghii, which achieved greater biomass and a markedly higher content of chlorophyll and secondary metabolites (total flavonoids and total polyphenols) compared to controls. These results suggest that the supplementation of LED BL is a good choice for A. roxburghii growers to increase production and secondary metabolite content. This is conducive to meeting the increasing market demand while avoiding the extinction of wild resources of A. roxburghii.
Considering the promotion of the soluble sugar and polysaccharide contents after the YL treatment, further research will focus on the combination of BL and YL.