Influence of Indole-3-Acetic Acid and Gibberellic Acid on Phenylpropanoid Accumulation in Common Buckwheat (Fagopyrum esculentum Moench) Sprouts

We investigated the effects of natural plant hormones, indole-3-acetic (IAA) acid and gibberellic acid (GA), on the growth parameters and production of flavonoids and other phenolic compounds in common buckwheat sprouts. A total of 17 phenolic compounds were identified using liquid chromatography-mass spectrometry (LC-MS) analysis. Among these, seven compounds (4-hydroxybenzoic acid, catechin, chlorogenic acid, caffeic acid, epicatechin, rutin, and quercetin) were quantified by high-performance liquid chromatography (HPLC) after treating the common buckwheat sprouts with different concentrations of the hormones IAA and GA. At a concentration of 0.5 mg/L, both IAA and GA exhibited the highest levels of growth parameters (shoot length, root length, and fresh weight). The HPLC analysis showed that the treatment of sprouts with IAA at concentrations ranging from 0.1 to 1.0 mg/L produced higher or comparable levels of the total phenolic compounds than the control sprout and enhanced the production of rutin. Similarly, the supplementation with 0.1 and 0.5 mg/L GA increased the content of rutin in buckwheat sprouts. Our results suggested that the treatment with optimal concentrations of IAA and GA enhanced the growth parameters and accumulation of flavonoids and other phenolic compounds in buckwheat sprouts.

shows that the growth patterns of sprouts after treatment with both hormones were almost similar. The supply of exogenous IAA and GA gradually increased the shoot height, root length, and fresh weight of buckwheat sprouts at the concentrations of 0.1, 0.5, and 1.0 mg/L, but the growth patterns were significantly decreased at the concentrations of 3.0 and 5.0 mg/L. We also used an experimental control in which the common buckwheat sprouts were grown without any hormones for 10 days. The treatment of GA at 0.5 mg/L exhibited the highest values of all growth patterns. The values of shoot height, root length, and fresh weight were 1.91, 1.98, and 2.22 times higher than those of the control, respectively. Furthermore, these values were 8.22, 5.24, and 5.00 times higher than the lowest value acquired after treatment with 5.0 mg/L GA. Treatment with 0.5 mg/L IAA resulted in the highest values of all growth patterns, which were 1.79, 3.37, and 2.33 times higher than those of the control, and 5.96, 4.69, and 3.50 times higher than the lowest values after treatment with 5.0 mg/L IAA. Among the different concentrations of IAA and GA, 0.5 mg/L was the most effective concentration for sprout growth (Figure 1).
Molecules 2017, 22, 374 3 of 10 patterns were significantly decreased at the concentrations of 3.0 and 5.0 mg/L. We also used an experimental control in which the common buckwheat sprouts were grown without any hormones for 10 days. The treatment of GA at 0.5 mg/L exhibited the highest values of all growth patterns. The values of shoot height, root length, and fresh weight were 1.91, 1.98, and 2.22 times higher than those of the control, respectively. Furthermore, these values were 8.22, 5.24, and 5.00 times higher than the lowest value acquired after treatment with 5.0 mg/L GA. Treatment with 0.5 mg/L IAA resulted in the highest values of all growth patterns, which were 1.79, 3.37, and 2.33 times higher than those of the control, and 5.96, 4.69, and 3.50 times higher than the lowest values after treatment with 5.0 mg/L IAA. Among the different concentrations of IAA and GA, 0.5 mg/L was the most effective concentration for sprout growth (Figure 1).

LC-MS and HPLC Analysis of Common Buckwheat Sprouts after Treatment with IAA and GA
A total of 17 phenolic compounds were identified in common buckwheat sprouts by LC-MS analysis as shown in Table 2.

LC-MS and HPLC Analysis of Common Buckwheat Sprouts after Treatment with IAA and GA
A total of 17 phenolic compounds were identified in common buckwheat sprouts by LC-MS analysis as shown in Table 2  In the HPLC analysis, seven phenolic compounds were identified and quantified in the buckwheat sprouts supplemented with exogenous hormones ( Table 3). The treatment of buckwheat sprouts with IAA at the specific concentrations of 0.1, 0.5, and 1.0 mg/L resulted in an increase in the accumulation of phenolic compounds; however, at 3.0 and 5.0 mg/L, IAA treatment led to a significant decrease in the accumulation of phenolic compounds. Subsequently, the highest level of total phenolic compounds was recorded after supplementing the sprouts with 0.1 mg/L IAA (1580.49 ± 11.19 µg/g Dry Weight (DW)), which was 1.83 times higher than the lowest one (861.74 ± 47.37 µg/g DW). Particularly, the levels of catechin, caffeic acid, and rutin were significantly increased after treatment with 0.1-1.0 mg/L IAA. The highest levels of catechin and caffeic acid were obtained after treatment with 0.5 mg/L IAA, which were 1.24 and 1.63 times higher than the equivalents of the control, respectively. Furthermore, the accumulation of rutin in the sprouts treated with 1.0 mg/L IAA was 1.39 times higher than that in the control. However, the IAA treatment led to a gradual concentration-dependent decrease in the accumulation of 4-hydroxybenzoic acid, chlorogenic acid, epicatechin, benzoic acid, and quercetin. Similarly, the accumulation of catechin, caffeic acid, and rutin was enhanced by supplementing the sprouts with 0.1 to 1.0 mg/L GA. After treating the sprouts with 0.1 mg/L GA, the highest values of catechin, caffeic acid, and rutin were 115.93 ± 5.69, 27.77 ± 1.07, and 866.13 ± 37.41 µg/g DW, respectively, which were 1.26, 1.72, and 1.23 times higher than those of the control. However, the treatment with GA showed a gradual concentration-dependent decrease in the values of the total phenolic compounds. Both natural and synthetic auxins are important phytohormones which regulate plant development. In particular, IAA are a major plant hormone and its key roles in cell division, elongation, and differentiation are apparently evident from observing the embryonic and post-embryonic development and tropisms (i.e., movement towards gravity and light) [41]. GAs, initially synthesized from the trans-geranylgeranyl diphosphate, are bioactive compounds involved in diverse processes throughout plant growth and development (i.e., promotion of leaf and stem growth, induction of seed germination, modulation of flowering time, and modulation of the development of flowers, fruits, and seeds) [42,43].
Previous studies have reported that the exogenous supply of IAA and GA is a useful approach to enhance the production of various secondary metabolites. The results from these studies were consistent with our findings. Exogenously supplied IAA at 0.1 mg/L enhanced the production of shikonin in cell cultures of Onosma paniculatum L. [44]. An exogenous supply of 0.5 and 1.0 mg/L IAA led to an increase in the production of total phenolics and flavonoids in the adventitious roots of Hypericum perforatum L. [45]. Similarly, a relatively low concentration of IAA promoted the production of phenolic compounds in the hairy roots of Panax ginseng C. A. Meyer [46]. Additionally, the accumulation of aryltetralin-type lignans (e.g., podophyllotoxin and 6-methoxypodophyllotoxin) was increased by the addition of IAA in the hairy root clone of Linum album LYR2i [47]. Bais et al. (2001) reported that the exogenous treatment of GA 3 induced an increase in root growth and the production of coumarins (e.g., esculin and esculetin) in the hairy roots of Cichorium intybus L. 'Lucknow Local' [48]. Furthermore, GA 3 enhanced the production of phenolics (e.g., anthocyanin, cichoric acid, caftaric acid, chlorogenic acid, and caffeic acid) in the hairy roots of Echinacea purpurea (L.) Moench and sesquiterpenes (e.g., artemisinin) in the hairy roots of Artemisia annua L. [49,50].
This study reported that the effect of IAA and GA on the growth and accumulation of phenolic compounds, including rutin and catechin, in common buckwheat sprouts, indicating that the treatment of the natural plant hormones at certain concentrations enhanced the growth and the accumulation of the natural products in the buckwheat sprouts.

Plant Materials
Seeds of common buckwheat were purchased from BONGPYEONG MAEMIL TUEKSANDANGI (Pyeongchang gun, Gangwon-do, Korea). For the treatment, stock solutions of IAA and GA at a concentration of 1 mg/mL was prepared in absolute ethanol, followed by dilution with deionized water to 0.1, 0.5, 1.0, 3.0, and 5.0 mg/L, respectively. 200 seeds (about 4 g) were soaked in tap water for 16 h at 28 • C. Subsequently, the seeds were loaded onto each pot containing vermiculites at 25 • C for germination and immediately treated with natural plant hormones (IAA and GA) at different concentrations (0.1, 0.5, 1.0, 3.0, and 5.0 mg/L) under dark condition in a plant growth chamber. After 2 days, the sprouts started to appear from the surface of the soil and were allowed to grow for 10 days. The sprout samples were frozen using −196 • C liquid nitrogen, and subsequently stored at −80 • C until use. After the lyophilization and grinding process, each sample was weighed to 100 mg and put in a 15-mL conical tube for further analysis.

Extraction and Analysis of Flavonoids
The extraction and analysis of flavonoids in common buckwheat sprouts were carried out using the slightly modified protocol of Li et al. (2014) [51]. For flavonoid extraction, 5 mL of aqueous methanol (80 % v/v) containing 10% acetic acid (0.1% v/v) was added into the conical tube containing the sample powder (100 mg). Subsequently, it was strongly sonicated at 28 • C for 10 min and extracted in a water bath set at 37 • C for 1 h. Next, the tube was centrifuged at 3000 rpm for 10 min, and the supernatant was transferred to a fresh tube. The entire procedure was performed twice. The collected supernatant was dried using nitrogen gas, before being resuspending in 5 mL of methanol. Additionally, the extracts were diluted by half with the addition of methanol, followed by their filtration and storage in a brown vial. Each flavonoid was separated using an HPLC system (NS-4000, Futecs, Daejeon, Korea) equipped with a C 18 column (250 mm × 4.  For the LC-MS conditions, the scan range, scan time, curtain gas, heating gas temperature, nebulizing gas, heating gas, ion spray voltage, declustering potential, and entrance potential were 100-1300 m/z, 4.80 s, 20.00 psi (N 2 ), 550 • C, 50.00 psi, 50.00 psi, 5500 V, 100 V, and 10 V, respectively. The mobile phase and gradient program were set using the same procedure described in Extraction and analysis of flavonoids.

Conclusions
To the best of our knowledge, this is the first study to identify and quantify phenolic compounds, including rutin, in common buckwheat sprouts supplemented with the natural plant hormones, GA and IAA, at different concentrations. A total of 17 phenolic compounds were identified through LC-MS and seven of those compounds were quantified with HPLC after treatment with IAA and GA at different concentrations. Apparently, IAA and GA at specific concentrations enhanced the growth and the accumulation of total phenolic compounds as well as specific flavonoids, including rutin and catechin, in common buckwheat sprouts. Therefore, it is expected that these findings might help develop efficient strategies to produce common buckwheat sprouts as a good source of dietary rutin for human consumption.