ZHENG Yin-jian , ZHANG Yi-ting LIU Hou-cheng LI Ya-min LIU Ying-liang, HAO Yan-weiLEI Bing-fu

1 College of Horticulture, South China Agricultural University, Guangzhou 510642, P.R.China

2 College of Materials and Energy, South China Agricultural University, Guangzhou 510642, P.R.China

Abstract To evaluate the supplementary blue light intensity on growth and health-promoting compounds in pak choi (Brassica campestris ssp. chinensis var. communis), four blue light intensity treatments (T0, T50, T100 and T150 indicate 0, 50, 100, and 150µmol m–2 s–1, respectively) were applied 10 days before harvest under greenhouse conditions. Both of cultivars (greenand red-leaf pak choi) under T50 had the highest yield, content of chlorophyll and sugars. With light intensity increasing,antioxidant compounds (vitamin C and carotenoids) significantly increased, while nitrate content showed an opposite trend.The health-promoting compounds (phenolics, flavonoids, anthocyanins, and glucosinolates) were significantly higher under supplementary light treatment than T0, so as the antioxidant capacity (2,2-diphenyl-1-picrylhydrazyl and ferric-reducing antioxidant power). The species-specific differences in photosynthetic pigment and health-promoting compounds was found in green- and red-leaf pak choi. T50 treatment could be used for yield improvement, whereas T100 treatment could be applied for quality improvement. Results showed that blue light intensity can regulate the accumulation of biomass,morphology and health-promoting compounds in pak choi under greenhouse conditions.

Keywords: blue light, antioxidant compounds, health-promoting compounds, species-specific, pak choi

1. Introduction

Brassica vegetables have attracted a great deal of attention due to their functional components and health-promoting effects related with lower cardiovascular disease risk and protection against cancer. Brassica vegetables such as broccoli (Brassica oleracea var. italica), radish (Raphanus sativus ), kale (B. oleracea var. acephala) and cabbage (B.oleracea var. capita) are rich in phytochemicals, such as phenols, vitamins, glucosinolates (GSLs) and anthocyanins,which have a positive impact on human health (Cox et al.2012). Brassica sprouts are a good source of vitamin C,vitamin A, folic acid, dietary fibres and minerals, as well as a high variety of phytochemicals, namely GSLs and phenols(Hassini et al. 2016). Consumption of Brassica vegetables such as cabbage, cauliflower, broccoli and brussels sprouts plays an important role in prevention of cancer. Moreover,phytochemicals like ascorbic acid and phenolic compounds have high antioxidant activity, which may influence a possible use of the beneficial effects of the Brassica vegetables as antioxidant functional food (Vale et al. 2014).

Light is one of the most important environmental factor affecting the phytochemical content in plant tissues. Lightemitting diodes (LEDs) represent a promising light source for greenhouses, which can be applied either as a main or supplementary light source. Although red LEDs initially received great attention for use as a light source to drive photosynthesis, plant cannot develop optimally with red light alone, and need blue light as well to regulate other processes than photosynthesis and growth (Li et al. 2009).Blue light has been reported to affect photomorphogenesis,vegetative growth, chlorophyll synthesis, stomata opening,and secondary metabolism (Lefsrud et al. 2012).

The light quality has significant effects on plants’photosynthesis, growth, development, and secondary metabolites. The increasing blue light fraction couldstimulate anthocyanins accumulation in lettuce leaf (Ahn et al. 2015). Post-harvest supplement of blue light could enhance antioxidant capacity in some vegetables including lettuce, barley leaf, spinach and komatsuna (Cox et al.2012). In Brassicaceae microgreens, blue light radiation increased xanthophyll, β-carotene contents and lutein synthesis (Brazaitytė et al. 2015). Phenolic compounds were an essential part of defense system in plant, higher blue light intensity could increase the contents of phenolic compounds. The antioxidant properties of the sprouted seeds of lentil, wheat and radish were improved by blue light(Seo et al. 2015). The phenolic compounds in buckwheat sprouts could be increased by using higher blue LEDs light intensity (Lee et al. 2014). In lettuce tested by several blue to red ratios, the best yield was found in a red-weighted spectrums, and the highest production of total phenols and total flavonoids in a blue-shifted spectrum (Son and Oh 2013). Enhanced blue light (400–500 nm) might remarkably increase the biosynthesis of phenolic compounds, as well as epidermal flavonoids (Hoffmann et al. 2015). The essential oil contents in basil plants grown under blue light had been found to be 1 to 4 times higher than those grown without blue light (Amaki et al. 2011).

Secondary metabolites are essential phytochemicals that can be affected by the light intensity, and act as defense compounds as well as protectors from blue light radiation and oxidants. The functions of secondary metabolisms have been under extensive research for their beneficial functions. Phenolic compounds demonstrate antioxidant,antimicrobial, antifungal, antitoxic, and radical scavenging properties (Kefeli et al. 2003). The ingestion of antioxidative supplements or foods containing antioxidants, such as carotenoids, vitamins, flavonoids, and other phenolic compounds, widely considered as an effective strategy to reduce oxidative damage and exert a beneficial effect on human health (Sun et al. 2009). The positive health effect of Brassica vegetables against various pathogen and chronic diseases is widely recognized, which is due to glucosinolates, the precursors of bioactive isothiocyanates(ITCs) and the products of hydrolysis mediated by plant endogenous myrosinase or by the intestinal microflora(Fahey et al. 2012). Epidemiological and experimentalstudies indicated that anthocyanins, the most important group of coloured flavonoids within phenols, protected against the risk of cardiovascular disease, cancer and other chronic degenerative conditions. Moreover, anthocyanins and ITCs might influence the same signal pathways in their chemopreventive activity, namely the induction of antioxidant responsive elements (Fimognari and Hrelia 2008).

In recent years, some interesting results about UV beneficial effects on secondary metabolism processes in plants have been found. Enhanced UV-B radiation can improve the nutritional and active ingredient contents during the floral development of medicinal chrysanthemum(Massa et al. 2008). Supplemental UV-B induced changes in essential oil composition and total phenolics of Acorus calamus (Kumar et al. 2009). UV-A and UV-B wavelengths can improve the content of antioxidant phenolic compounds and the phenylalanine ammonia-lyase (PAL) activity in sowthistle (Ixeris dentata) (Lee et al. 2014). In addition to acting as a developmental and physiological signal, low influence UV-B also cause cellular damage by generating photo-products in DNA and by direct damage to proteins,lipids, and RNA. Elevated UV-B radiation had pleiotropic effects on plant development, morphology, and physiology.While blue light had less side effects for plant growth.

Pak choi (Brassica campestris ssp. chinensis var.communis), a leafy Chinese cabbage, is rich in flavonol aglycone, vitamins A and C and folic acid. This study aimed to find the optimal supplementary LED blue light intensity for health-promoting compound production in pak choi through investigating the effects of supplementary LED blue light (460 nm) treatment 10 days before harvest on growth,antioxidant capacity and health-promoting compounds in pak choi under greenhouse conditions.

2. Materials and methods

2.1. Plant materials and growth conditions

This study was performed in greenhouses of College of Horticulture, South China Agricultural University (Eastlongitude 113.36°, north latitude 23.16°) with pak choi(Brassica campestris ssp. chinensis var. communis cv.green- and red-leaf pak choi). After 1 hour soaking, the seeds were put into perlite, and keep 24 hours in dark under (25±2)°C, then seeds were sowed in sponge block(2 cm×2 cm×2 cm) with nutrient solution (CaNO3, 236.25 mg L–1; KNO3, 151.75 mg L–1; NH4PO4, 28.75 mg L–1, MgSO4,123.25 mg L–1, and pH≈6.0). After seven days, the seedlings with 3rd expanded true leaf were transplanted into 20-L containers, PPFD at 12:00 a.m.: 400–1 000 µmol m–2s–1(Fig. 1-A), CO2-concentration 270–400 ppm (Fig. 1-B), air humidity 35–90% (Fig. 1-C). The nutrient solution was refreshed every 10 days. Thirty days after transplanting,the plants were treated with blue LED (Guangzhou, China)from 6:00 to 18:00 for 10 days. Four supplemental blue light treatments were used: T0 (0 µmol m–2s–1), T50 (50 µmol m–2s–1), T100 (100 µmol m–2s–1), and T150 (150 µmol m–2s–1), respectively.

2.2. Fresh and dry weight measurements

After 10 days blue light treatment, the fully developed green- and red-leaf pak choi plants were harvested for fresh weight (FW) and dry weight (DW) measurements(Analytical balance, Shanghai, China). The fresh and dry weight of shoot and root were measured, the dry weight was determined after 48 h at 70°C in drying oven (DGG-9140A,Shanghai, China).

2.3. Phytochemical measurements

Chlorophyll content was measured colorimetrically (Gratani et al. 1992). A total of 500 mg leaf tissue was extracted with an 80% (V/V) acetone (Sigma, USA) and measured at 645,663 and 440 nm by UV-spectrophotometer (Shimadzu UV-16A, Shimadzu, Corporation, Kyoto, Japan).

Chlorophyll a (mg L–1)=12.7×OD663–2.69×OD645,Chlorophyll b (mg L–1)=22.9×OD645–4.86×OD663, Total chlorophyll (mg L–1)=8.02×OD663+20.20×OD645, Carotenoid(mg L–1)=4.7×OD440–0.27×Total chlorophyll, Chloroplastpigment content (mg g–1)=Pigment concentration×Extracting’s volume)/0.5 g

Fig. 1 The greenhouse environmental conditions for nature light intensity (A), CO2-concentration (B), air humidity (C), for 10 days before harvest.

The content of soluble sugars was measured by the method of sulfuric acid anthrone (Sigma, USA) (Song et al.2011). 0.05 g fresh shoot were put into a test tube, 5 mL distilled water was added and mixed. After 30 min in a water bath at 85°C, the supernatent was collected. This step was repeated twice, and distilled water was added to a volume of 10 mL. The soluble sugar content was determined at 620 nm by UV-spectrophotometer.

Soluble proteins were measured by Coomassie brilliant blue method (Bradford et al. 1976). A total of 0.05 g fresh shoot was ground up in a mortar with liquid nitrogen, to which 3 mL of a phosphate-buffered solution (Sigma, USA, pH 7.0)was added. The extract was centrifuged at 13 000×g for 15min at 4°C, and 0.1 mL of the supernatant was combined with 4.9 mL Coomassie brilliant blue G-250 solution (Sigma,USA, 0.1 g L–1). After 2 min, the soluble protein content was determined at 595 nm by UV-spectrophotometer.

The content of free amino acid was measured colorimetrically (Konosu et al. 1974). A total of 1.0 g fresh shoot was ground up, and then 5 mL of acetic acid washed samples pure into a 100-mL volumetric flask, 100 mL deionized water was added, prepared for measurement.1.0 mL extracting solution, 10 mL deionized water, 0.1 mL ascorbic acid solution and hydrated indene ketone solution(Sigma, USA) were boiling for 15 min, 20 mL 60% ethanol was added, the free amino acid was measured at 570 nm by UV-spectrophotometer.

The nitrate content was measured colorimetrically(Cataldo et al. 1975). A total of 0.05 g fresh shoot were ground up, 10 mL deionized water was added. After 30 min in a water bath at 80°C, the extract was centrifuged at 13 000×g for 10 min, and 0.2 mL of the supernatant was mixed with 0.8 mL of 5% (w/v) salicylic acid (in pure H2SO4, Sigma, USA) and 19 mL of 4 mol L–1NaOH. After 30 min, the nitrate content was measured at 410 nm by UV-spectrophotometer.

Vitamin C (Vc) content was determined according to Kampfenkel et al. (1995), 1.0 g frozen shoot was ground and extracted with 5 mL 5% trichloroacetic acid (TCA),then centrifuged at 10 000×g for 10 min at 4°C. A sample of the crude extract (1 mL) was added to 1 mL 5%(v/v) TCA,1 mL 100%(v/v) ethanol, 0.5 mL 0.4% H3PO4, 1 mL 0.5%(v/v) 1,10-phenanthroline hydrate, 0.5 mL 0.03% (v/v)FeCl3, then the mixture was incubated at 30°C for 1 h. The absorbance was read at 534 nm by UV-spectrophotometer.

The content of total phenolic compounds (TPC) was determined colorimetrically (Javanmardi et al. 2004). The Folin-Ciocalteau reagent (Sigma, USA) was used with gallic acid (Sigma, USA) as positive control. The extracts were dissolved in MilliQ purified water (10 mg mL–1) and 50µL deionized water (control) were mixed with 2.5 mL 1/10 dilution of Folin-Ciocalteau reagent in a 10-mL screw-cap tube. After adding 2 mL of Na2CO3(7.5%, w/v) the tube was closed and kept at 45°C for 15 min. The absorbance of all samples (triplicates) was measured at 765 nm by UV-Vis spectrophotometer. The TPC was calculated according to a calibration curve traced with gallic acid (GA, n=3)[OD765=1.1978×CGA (µg mL–1)–0.024, R2=0.9991)] and expressed as mg of gallic acid equivalent per g of dried extract (mg GAE g–1).

The total flavonoids (TF) content was determined with aluminium chloride (AlCl3, Sigma, USA) according to Jia et al. (1999) using quercetin as positive control. First, 100 µL of the extract (10 mg mL–1) was added to 300 mL of distilled water followed by 30 µL of NaNO2(5%, Sigma, USA). After 5 min at 25°C, 30 µL of AlCl3(10%) was added. Five min later, the reaction mixture was treated with 200 µL of NaOH(1 mmol, Sigma, USA) and then the volume was completed to 1 mL with distilled water. The absorbance of the mixture was then determined at 510 nm against a water blank by UV-spectrophotometer. The flavonoids content was calculated from a quercetin standard curve [OD510=0.0004×Cquercetin(µg mL–1)+0.0044, R2=0.9993)]. Results were expressed as quercetin equivalents (mg quercetin per g of dried extract).

Total anthocyanin (TA) content was measured by pH-differential spectrophotometry method (Rapisarda et al.2000). A total of 5 g frozen sample was extracted with 25 mL of pH 1.0 buffer (50 mmol KCl and 150 mmol HCl, Sigma,USA), as well as 25 mL of pH=4.5 buffer (50 mmol sodium acetate and 240 mmol HCl). Absorbance was measured at 510 and 700 nm by spectrophotometer, using OD=[(OD510–OD700)×pH1.0–(OD510–OD700)×pH4.5] with a molar extinction coefficient of cyaniding 3-glucoside (Sigma, USA) of 29 600.Results were expressed as mg of Cy-3-Glu equivalents per g of fresh weight.

Total glucosinolates (TG) content was determined according to Heaney et al. (1988). The method was based on the measurement of enzymically released glucose, which was hydrolysed by the enzyme myrosinase (thioglcose glycohydrolase, EC 3.2.3.1). The content of glucose was determined by the method of phenol-sulfuric acid (Sigma,USA), to assay the absorbance at 490 nm, and then the amount of glucosinolate can be calculated from the glucose content.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging rate assay was carried out according to the method by Heaney et al. (1988). A total of 150 µL extracts and 150 µL methanol were added to 2.5 mL 0.004%methanol solution of DPPH, respectively. The mixture was vigorously shaken and left to stand in the dark at room temperature for 30 min. The absorbance was read at 517 nm. Inhibition of free radical DPPH in percent (I, %) was calculated in following way: I=(Aj-Ai)/Aj×100%.

Where, Aiis the absorbance of the extract reaction, and Ajis the absorbance of the methanol reaction (containing all reagents except the test compound).

The ferric-reducing antioxidant power (FRAP) assay was based on the method of Dinis et al. (1994). FRAP reagent(1.8 mL) was pipetted into a test tube and incubated at 37°C in a water bath for 10 min. The absorbance was taken at 593 nm by UV-spectrophotometer. The reducing potential of the sample extract was determined against a standard curve of ferrous sulfate (0.05–2.0 mmol L–1) and the FRAP value was expressed as mm FeSO4equivalents per g of dried sample.

2.4. Statistical analysis

All the assays were performed in triplicates. Significant differences among the treatments were determined by analysis of variance (ANOVA) followed by Duncan multiple range tests of SPSS 17.0, the tables and figures were made by Word 2013 and OriginPro 9.0.

3. Results

3.1. Growth and biomass

The growth of pak choi was significantly affected by different supplementary blue light intensities (Table 1, Fig. 2).Compared to T0 treatment, the total fresh weight of greenand red-leaf pak choi under T50 treatment increased by 7.80 and 35.70%, respectively. However, under T100 and T150 treatment, the total fresh weight was lower than T0,which decreased by 25.81 and 25.77%, 24.75 and 44.00%,respectively. There was no significant difference in root fresh weight of green-leaf pak choi among treatments, while compared with T0, the root fresh weight of red-leaf pak choi T50 and T100 increased by 36.78 and 19.45%, respectively.There was similar change tendency in dry weight as fresh weight, both in green- and red-leaf pak choi.

3.2. The chlorophyll content of pak choi

The different influence of supplemental blue light intensityon the chlorophyll contents in green- and red-leaf pak choi were observed in Fig. 3.

Table 1 The effect of supplementary blue light intensity on fresh and dry weights of pak-choi

Fig. 2 The effect of supplementary blue light intensity on growth of in pak choi. T0, T50, T100, and T150 indicate 0, 50, 100, and 150 µmol m–2 s–1, respectively.

There was no significant difference in chlorophyll contents of green-leaf pak choi among treatments. Where the higher chlorophyll a content of red-leaf pak choi was found in T50,and significant higher than T0. The content of chlorophyll a,chlorophyll b and total chlorophyll in T50 was increased by 19.02, 32.43 and 23.40% than T0, respectively. Generally,red-leaf pak choi had higher chlorophyll contents than greenleaf pak choi (Fig. 3).

3.3. The content of soluble sugars, soluble protein and free amino acid of pak choi

There was the highest content of soluble sugars in T0 both in green- and red-leaf pak choi (Table 2). With increasing supplementary blue light intensity, the content of soluble sugars significantly decreased in both green- and red-leaf pak choi. The lowest content of soluble sugars of greenand red-leaf pak choi was found in T150. Compared with T0, the content of soluble sugars in T50, T100, and T150 decreased by 6.69 and 24.27%, 46.03 and 19.02%,60.98 and 69.76%, respectively, in green and red-leaf pak choi.

Compared with T0, the content of free amino acid under T150 in green- and red-leaf pak choi was significantly decreased by 20.41 and 22.85%, respectively.

No significant difference was found in the content of soluble proteins in green- and red-leaf pak choi among the treatments.

3.4. The content of nitrate, vitamin C and carotenoid of pak choi

Fig. 3 The effects of supplementary blue light intensity on chlorophyll content of pak choi. T0, T50, T100, and T150 indicate 0, 50,100, and 150 µmol m–2 s–1, respectively. The values presented are the means±SD. Different letters indicate significant differences between treatments (P＜0.05). All the assays were performed in triplicates. Significant differences among the treatments were determined by analysis of variance (ANOVA) followed by Duncan multiple range tests of SPSS 17.0.

Table 2 The effects of supplementary blue light intensity on soluble sugars, soluble protein and free amino acid of pak choi

In green-leaf pak choi, the nitrate content under T50 significantly increased 11.11% than T0 (Fig. 4-A). However,the nitrate contents under T100 and T150 were significantly lower than T0 by 5.63 and 4.25%, respectively. In red-leaf pak choi, with increasing blue light intensity, the nitrate contents significantly decreased, those under T50, T100 and T150 was significantly lower than T0 by 24.20, 37.75 and 50.04%, respectively.

The content of Vc was significantly affected by supplementary blue light in green- and red-leaf pak choi(Fig. 4-B). Both in green- and red-leaf pak choi, the highestVc contents were found in T100, which was 584.44 and 725.45% higher than T0, respectively. And in T150 of greenand red-leaf pak choi was 353.33 and 365.45% higher than T0, respectively.

As shown in Fig. 4-C, there was no significant difference in carotenoid contents of green-leaf pak choi among T0 and blue light treatments. However, in red-leaf pak choi, the carotenoid contents under T50 were significantly higher by 23.39% than T0, while under T150, the carotenoid contents were significantly lower by 38.35% than T0.

3.5. The content of healthy function compounds of pak choi

There were remarkable differences in TPC contents between two cultivars of pak choi (Fig. 5-A), the higher TPC contents were found in red-leaf pak choi. In green-leaf pak choi, the TPC contents significantly increased with supplemental blue light intensity increasing, those under T50, T100 and T150 was significantly higher than T0 by 4.60, 12.50, 19.10%,respectively. In red-leaf pak choi, the highest content of TPC was found in T100, 34.80% higher than T0.

Fig. 4 The effects of supplementary blue light intensity on contents of nitrate (A), Vc (B) and carotenoid (C) of pak choi. T0, T50,T100, and T150 indicate 0, 50, 100, and 150 µmol m–2 s–1, respectively. The values presented are the means±SD. Different letters indicate significant differences between treatments (P＜0.05). All the assays were performed in triplicates. Significant differences among the treatments were determined by analysis of variance (ANOVA) followed by Duncan multiple range tests of SPSS 17.0.

Fig. 5 The effects of supplementary blue light intensity on contents of healthy function compounds of pak choi. A, total phenolic compounds (TPC) content. B, total flavonoids (TF) content. C, total antocyanin (TA) content. D, total glucosinolates (TG) content.T0, T50, T100, and T150 indicate 0, 50, 100, and 150 µmol m–2 s–1, respectively. The values presented are the means±SD. Different letters indicate significant differences between treatments (P＜0.05). All the assays were performed in triplicates. Significant differences among the treatments were determined by analysis of variance (ANOVA) followed by Duncan multiple range tests of SPSS 17.0.

There were remarkable differences in TF contents between two cultivars of pak choi (Fig. 5-B). In red-leaf pak choi, the TF content significantly increased by supplemental blue light, and the highest TF content was under T100.In green-leaf pak choi, the TF contents increased with supplemental blue light intensity increasing. Compared with T0, the TF content under T150 was significantly increased by 29.28%.

The content of TA in red-leaf pak choi was higher than in green-leaf pak choi (Fig. 5-C), and TA contents significantly increased with supplement blue light intensity increased both in green- and red-leaf pak choi. Compared with T0, the TA contents under T50, T100, and T150 were significantly higher by 55.21, 178.13, and 204.17%, respectively, in green-leaf pak choi, and significantly higher by 28.26, 47.19,and 72.69%, respectively, in red-leaf pak choi.

The content of TG was significantly increased by supplemental blue light, and those in red-leaf pak choi were remarkably higher than green-leaf pak choi (Fig. 5), and TG contents significantly increased with supplement blue light intensity increasing in red-leaf pak choi. And the TG contents under T50, T100 and T150 were significantly higher than T0 by 35.25, 54.58, and 75.25%, respectively. In green-leaf pak choi, the highest TG content was observed in T100, the TG contents under T50, T100 and T150 were significantly higher than T0 by 10.79, 17.27, and 10.07%, respectively.

3.6. The antioxidant capacity of pak choi

The DPPH radical scavenging rate and the FRAP of pak choi were affected by supplementary blue light (Fig. 6).The significant higher DPPH scavenging activity 18.93 and 68.35%, respectively, was detected in T50 (green-leaf pak choi) and T100 (red leaf pak choi). The FRAP increased with blue light intensity increased in green-leaf pak choi,compared with T0, T50, T100 and T150 was increased by 15.08, 26.18 and 30.27%, respectively. While the highestFRAP value was found in T50 in red leaf pak choi, compared with T0, which was increased by 30.16%. The T100 and T150 were 21.55 and 12.42% higher, respectively, than T0.

Fig. 6 The effects of supplementary blue light intensity on content of 2,2-diphenyl-1-picrylhydrazyl (DPPH, A) and ferric-reducing antioxidant power (FRAP, B) of pak choi. T0, T50, T100, and T150 indicate 0, 50, 100, and 150 µmol m–2 s–1, respectively. The values presented are the means±SD. Different letters indicate significant differences between treatments (P＜0.05). All the assays were performed in triplicates. Significant differences among the treatments were determined by analysis of variance (ANOVA)followed by Duncan multiple range tests of SPSS 17.0.

4. Discussion

The supplementary blue light intensity could affect pak choi plant biomass and morphology. Both in a green- and redleaf pak choi, the plant biomass under T50 were higher than other blue light treatments (Table 1 and Fig. 2), while lower biomass under T100 and T150 than under T0. This means that lower supplemental blue light intensity (T50) increased the biomass of pak choi, the higher blue light intensity(T100 and T150) might inhibit biomass accumulated. Plant biomass was affected by the blue light treatments in wheat,soybean and lettuce under higher blue light ratio, which had a more compact appearance (Ouzounis et al. 2014).With blue light intensity increasing, the chlorophyll contents significantly decreased in red-leaf pak choi, while the highestcontent of chlorophyll under T50 (Fig. 3). However, no difference was observed in green-leaf pak choi. The peak absorption values of photosynthetic pigments and plant leaves were on the red and blue spectra. In pea seedlings,the chlorophyll contents increased rapidly under blue (465–470 nm) LED light. With blue light intensity increased, the chlorophyll content of cucumber increased. These might be that the effects of supplementary blue light intensity on chlorophyll formation were affected by leaf color.

Hence, we conclude that lower blue light intensity could increase quantum yield of PSII in pak choi, then increase the yield of pak choi, while higher supplementary blue light intensity had the opposite results. With supplemental blue light intensity increasing, the contents of soluble sugar in green- and red-leaf pak choi decreased (Table 2). Sugars were the most sensitive response to blue light in Chinese kale, and accumulated in leaves (Lefsrud et al. 2008). The content of soluble sugars was related to stomata guard cells open and down which exhibited a high sensitivity to blue light (425–475 nm). Two distinct photoreceptor systems responses stimulate ion transport in guard cells and enhancestomatal opening.

The reduction in nitrate contents is definitely important for improving the nutritional quality of vegetables for human health. With increasing blue light intensity, the nitrate contents in pak choi decreased significantly (however it is increased in green-leaf pak choi under T50) (Fig. 4-A).Though nitrate reductase (NR) mRNA, protein and activity were induced by blue light in squash leaf (Lillo 1994),glucose or sucrose could replace light in eliciting the increase in nitrate reductase mRNA accumulation (Lillo et al.1993). The decreased soluble sugar contents by higher blue light intensity (Table 2) may account for the nitrate reduction.

Vc provides significant biochemical functions as an antioxidant, acts as a defense against oxidative stress and plays a possible role in cell wall metabolism and expansion(Toledo et al. 2003). Vc contents in pak choi significantly increased under supplemental blue light, especially under T100 and T150, and those of red-leaf pak choi increase more than green-leaf pak choi (Fig. 4-B). While, in broccoli, AsA(ascorbic acid) metabolism was not affected by blue LED light treating after harvest (Toledo et al. 2003). The pak choi was treated by blue light before harvest, and ascorbateglutathione cycle played a crucial role in controlling the level of antioxidant capacity. Hence, higher blue light intensity as a stress environment excited the gene of some antioxidant enzaymes up-regulated expression.

The phenolic compounds, as plant secondary metabolites,may play a role in the protection of cardiovascular health and prevention of certain cancers (Costa et al. 2015).The TPC content in two cultivars increased with blue light intensity increasing, and red-leaf pak choi had higher TPC content than green-leaf pak choi (Fig. 5-A). The phenolic compounds in buckwheat sprouts can be increased by using blue LED lighting (Lee et al. 2014). The shikimate pathway plays a pivotal role in the biosynthesis of phenolic compounds, and the phenylpropanoid is catalyzed by PAL to form various kinds of phenolic compounds, PAL de novo transcription and post-translational modification might be regulated by the blue light spectral (Wellmann et al.1975). The phytochrome and blue/UV light photoreceptor responses had been implicated in the photo stimulation of phenylpropanoid anabolism through the activity of PAL.

TF is another important plant secondary metabolites with variety of biological activity, such as oxygen free radical scavenging properties, anti-lipid peroxidation, antiviral effect and others (Treutter 2006). The TF contents in pak choi significantly increased with blue light intensity increasing, and those in red-leaf pak choi were much higher than in green-leaf pak choi (Fig. 5-B). The quality and yield of lettuce were enhanced by mixture of blue lights,and the highest production of TPC and TF were observed by a blue-shifted spectrum (Samuolien et al. 2012). The flavonoid compounds in basil increased with enhancing blue light intensity (Taulavuori et al. 2015), especially strong in flavonol derivatives.

Anthocyanins as a group of flavonoid compounds fulfill important biological functions in protecting plants againstvarious biotic and abiotic stresses (Harborne and Williams 2000), and also proven to be very useful in protecting human health against numerous diseases, including cancer, etc.(Harborne and Williams 2000). Red-leaf pak choi had more TA than green-leaf pak choi (Fig. 5-C), and the TA contents in red cabbage and lettuce also were slightly higher than green cabbage and lettuce (Balacheva et al. 2008).Light modulates the intensity of anthocyanin pigments by affecting the regulatory and structural genes involved in the biosynthesis, anthocyanin synthesis in fruit were enhanced by blue light (Balacheva et al. 2008). The quality and quantity of light had been shown to exhibit significantly different interactions in the regulation of the production of anthocyanins and other metabolites in Brassica oleracea,Lycopersicon esculentum (Lo et al. 2008).

TG present in cabbage is known to possess anti-cancer activities and reduce the risks of colon and lung cancer(Cohen et al. 2000). TG content in pak choi increased with blue light intensity increasing, and much higher in redleaf pak choi than in green-leaf pak choi (Fig. 5-D). The highest content of TG was found in pak choi leaves prior to higher blue light intensity acclimatization. The change tendency was probably caused by activation of enzymes involved in glucosinolates (GSLs) biosynthesis (Yang et al. 2016). In broccoli, the blue light might be involved in biosynthesis of aliphatic and aromatic GSLs but not for indolic GSLs. GSL biosynthesis was regulated by light and HY5, a transcriptional factor functions downstream of phytochromes, cryptochromes and UV-B, which regulated other transcriptional factors involved in aliphatic and indolic GSLs biosynthesis (Huseby et al. 2013). Therefore, the fact that blue light irradiation accelerated GSLs accumulation in pak choi would be the result of up-regulated gene expression in aliphatic GSL synthesis through the association of the blue-light photoreceptors and function of HY5.

In general, due to the complex nature of phytochemicals,two methods (DPPH and FRAP) should always be employed to evaluate the total antioxidative effects of vegetables. The maximum antioxidant activity was determined to be in theblue-LED-grown pea sprouts (Liu et al. 2016). In greenleaf pak choi, with blue light intensity increased, the FRAP significantly increased (Fig. 6-B). Phenolic compounds have been demonstrated to contribute more to antioxidant capacity than other antioxidants in tea (Vinson et al. 1995).The FRAP was significant correlated to TP and TF in greenleaf pak choi (Table 3). In red-leaf pak choi (Table 3), there was significant conformity between the carotenoids and DPPH (0.99). The total phenolic and flavonoid contents,and the antioxidant activities, showed significant correlations in pea sprouts (Liu et al. 2016).

Table 3 The correlations of antioxidant capacity and antioxidant compounds1)

5. Conclusion

The contents of TPC, TF, TA, TG and antioxidant capacity(DPPH and FRAP) of pak choi significantly increased when supplemental blue light applied for 10 days before harvestwhich suggested that supplemental blue light is needed to regulate the accumulation of biomass, morphology and phytochemicals in pak choi under greenhouse conditions.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2017YFD0701500),the Teamwork Projects Funded by Guangdong Natural Science Foundation, China (S2013030012842), the Guangdong Provincial Science & Technology Project, China(2015A020209146, 2015B090903074), and the Guangzhou Science & Technology Project, China (201605030005,201704020058).