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The effect of shading in midsummer on anthocyanin and non-flavonoid polyphenol biosynthesis of Gynura bicolor DC leaves was examined using a control (full solar radiation) and a shade treatment (50% shading of full solar radiation). Leaf temperature in the shade plot remained ’40 8C in the daytime, ’6 8C lower than in the control. Plants in the shade plot grew better than the control. The content of chlorogenic acid (CGA) in leaves decreased with leaf maturation for both treatments, and a larger amount of CGA was detected in leaves from the control than the shade treatment. The profiles of reactive oxygen species (ROS) scavenging activity exhibited an identical pattern to the content of CGA. Although there was an abrupt increase in the content of anthocyanin in the early stage of leaf expansion, the content decreased rapidly as the leaves matured. The increase in anthocyanin early during leaf expansion was much more limited in control leaves than shaded leaves. There were no correlation between the profiles of anthocyanin and gene expression such as GbPAL, GbC4H, Gb4CL, GbCHS, GbCHI, GbF3H, and GbUFGT. However, the profiles of expression of genes such as GbMYB2, GbF3#H, GbDFR, and GbANS were similar to the anthocyanin profiles. These results suggest that artificial shading in midsummer is an effective method to promote anthocyanin accumulation but reduces ROS scavenging capacity as a result of lowered CGA production. Gynura bicolor DC is widely distributed in certain areas of southeast Asia. It is a member of the Asteraceae family and has several local names, e.g., Suizenjina and Kinjisou in Japan (Hayashi et al., 2002). Its leaves are characterized by green adaxial and reddish purple abaxial surfaces. In Ishikawa Prefecture, G. bicolor is cultivated in the field from early spring to late fall. However, the reddish purple abaxial sides of leaves harvested in midsummer often lack this characteristic color (Yoshiaki et al., 2010). Several growers say that the loss of red color of G. bicolor in midsummer is less in leaves from shaded locations than in those in sun-exposed locations. Many studies have reported on the effects of shading on the content of anthocyanins in fruit skin and on fruit color (Koyama and Goto-Yamamoto, 2008; Wang et al., 2000). In most fruits, light is a prerequisite for anthocyanin synthesis (Mancinelli, 1983) and shading during fruit development results in decreased anthocyanin content (Koyama and Goto-Yamamoto, 2008; Wang et al., 2000). On the other hand, temperature is an important factor that affects anthocyanin biosynthesis in various fruits such as apples (Ubi et al., 2006) and grapes (Yamane et al., 2006). The expression of genes involved in anthocyanin biosynthesis is repressed by high temperatures in a red-wine grape cultivar (Mori et al., 2007). Because G. bicolor plants are often exposed to high temperature in midsummer, the loss of red color of the leaves may largely be dependent on a repression of anthocyanin biosynthesis genes caused by high temperature. Chlorogenic acid is the most abundant phenolic compound in G. bicolor leaves (Fukuoka et al., 2012). The contents increase in response to environmental stresses (Oh et al., 2009). In lettuce, leaves of sun-exposed plants contained more CGA than leaves from plants growing under shaded locations (Grace et al., 1998). Generally, phenolic compounds including anthocyanin can scavenge ROS (Kanatt et al., 2005; Nara et al., 2006). Chlorogenic acid was the most efficient scavenger of free radicals in G. bicolor leaves (Fukuoka et al., 2012). Because stress conditions enhance the production of ROS (Asada and Takahashi, 1987), leaves from sun-exposed locations in midsummer may contain a larger amount of CGA to scavenge abundant ROS. However, because G. bicolor is a minor crop, there has been little research on this plant and no reports of changes in polyphenol biosynthesis of leaves induced by shading. In this article, we show that artificial shading in midsummer is an effective method to promote anthocyanin accumulation but reduces ROS scavenging capacity as a result of lower production of CGA. Materials and Methods Plant materials and experimental plots. Cuttings ( 6 to 9 cm long with three nodes) of Gynura bicolor shoots were obtained from mother plants on 4 June and planted in germination boxes filled with sandy soil and then grown in a greenhouse. Approximately 20 d after cutting, plants were transplanted into 10.5-cm polypots filled with clay soil containing various mixtures of organic manure. Primary shoots were pinched off 20 d after transplanting, and then the plants were grown under natural conditions until two foliage leaves had fully expanded. Lateral shoots that emerged were reduced to three on each cutting 10 d after pinching, and the seedlings were planted in the field at 30-cm intervals in rows 110 cm apart. A basal fertilizer (10N–10P–10L) of 100 g·m was applied before planting. Microelemental manure (60 g·m) containing 0.4% manganese, 0.3% boron, 1.2% iron, and 0.03% copper was added at the same time. To clarify the effect of shading on polyphenol biosynthesis, plants were grown in two experimental plots, a control and a shade treatment. Control plants were grown under the usual conditions in midsummer. For the shade treatment, tunnels (90 cm high · 120 cm wide) were placed above the plants (100 plants per plot) and covered with an SL50 silver nylon mesh (Diatex Co., Ltd., Tokyo, Japan) that provided 50% shading of full solar radiation. Changes in daily light intensity and leaf temperature at the top of the canopy were respectively measured on a clear day (2 Aug.) using a TM-201 illuminometer (Tenmars Received for publication 1 May 2014. Accepted for publication 13 July 2014. To whom reprint requests should be addressed; e-mail nfukuoka@ishikawa-pu.ac.jp. 1148 HORTSCIENCE VOL. 49(9) SEPTEMBER 2014 Electronics Co., Ltd., Taipei, Taiwan) and a TA-0510bF radiation thermometer (Minolta Camera Co., Ltd., Osaka, Japan). On 16 Aug., 50 lateral shoots were harvested and 10 uniform shoots were used to measure plant growth. Subsequently, leaves were collected at one node intervals from the shoot apex to the basal end. Each collected leaf was numbered basipetally from the shoot apex as shown in Figure 1. A 3-g sample from each collected fresh leaf was cut into pieces and boiled in 40 mL distilled water for 10 min. The mixture was filtered through cotton gauze and the extract was diluted to 50 mL with distilled water. The extracts were stored at –45 C until analysis of phenolic compounds and antioxidant activity. The remaining fresh leaves were stored at –80 C until extraction of RNA for analysis of anthocyanin biosynthetic gene expression. Anthocyanin and CGA contents. Total phenol content was measured using the method described by Singleton et al. (1999) with some modifications. Briefly, a 3-fold volume of ethanol was added to 1.5 mL of the stored extract, and the precipitant was removed by centrifugation (10,000 g for 15 min). The ethanolic extract was dried at 60 C and dissolved in 200 mL of distilled water, and a 5-mL aliquot of solution was brought up to 45 mL with distilled water in each well of a 96-well microplate. Then, 50 mL of Folin-Ciocalteau reagent and 50 mL of 100 mg·L Na2CO3 were added to each well. After incubation for 60 min at room temperature, the absorbance at 750 nm was measured using a plate reader (Bio-Rad Laboratories, Tokyo, Japan). Results were expressed as milligrams of gallic acid equivalent per gram fresh weight. All measurements were taken in triplicate and the mean values were calculated. CGA is the most abundant phenolic compound in G. bicolor leaves (Fukuoka et al., 2012). Then, in this study, CGA was measured using the solution remaining after determination of total polyphenol content. A 10-mL aliquot of this solution was injected into a Hitachi high-performance liquid chromatography (HPLC) system (Hitachi High-Tech Co. Ltd., Tokyo, Japan) equipped with an L-4200H ultraviolet detector (Hitachi High-Tech Co. Ltd.) and an Inertsil ODS-3 column (150 mm · 4.6 mm; 5 mm particle size; GL Sciences Inc., Tokyo, Japan). The mobile phase for HPLC consisted of a mixture of Solvent A (10 mM NaH2PO4 in distilled water) and Solvent B (100% methanol). The solvent gradient was as follows: Solvent B was increased from 10% (v/v) to 50% (v/v) in Solvent A over 20 min and then kept at 50% for the next 10 min. The flow rate was 1.0 mL·min, the column temperature was 40 C, and absorption was measured at 280 nm. To identify the peak corresponding to CGA, CGA (Nacalai Tesque Inc., Kyoto, Japan) was used as external standard. Results were expressed as milligrams of CGA equivalent per gram fresh weight. All measurements were taken in triplicate and the mean values were calculated. To determine the anthocyanin content, 3 mL of frozen leaf extract was extracted with a 3-fold volume of 100% ethanol and the resulting precipitate was removed by centrifugation (10,000 g for 15 min). The ethanolic extract was then dried at 60 C and dissolved in 3 mL of 0.1 M citrate buffer (pH 3.0). Anthocyanin content was measured as absorbance at 546 nm using a U-3900 ultraviolet– Vis spectrophotometer (Hitachi Co., Ltd., Tokyo, Japan). Results were expressed as milligrams of equivalent cyanidin-3-glucoside per gram of fresh weight. All measurements were taken in triplicate and the mean values were calculated. Analysis of anthocyanin biosynthetic gene expression. Approximately 800 mg of frozen leaves for each sample were ground into powder in the presence of liquid nitrogen using a mortar and pestle. Approximately 300 mg of each powder was transferred to sterile centrifuge tubes containing 4 mL Fruitmate for RNA purification reagent (Takara Bio Inc., Otsu, Shiga, Japan). The powder was thoroughly dispersed by hand shaking for a few seconds. After centrifugation at 9170 g for 5 min at 4 C, the supernatant was transferred to new polypropylene

Effect of Shading on Anthocyanin and Non-flavonoid Polyphenol Biosynthesis of Gynura bicolor Leaves in Midsummer