Decrease of flavonol synthase enzymatic activity in Ugni molinae Turcz due to the domestication process

Cien. Inv. Agr. 46(1):30-39. 2019 www.rcia.uc.cl food science DOI 10.7764/rcia.v46i1.1955 research note Decrease of flavonol synthase enzymatic activity in Ugni molinae Turcz due to the domestication process Manuel Chacón-Fuentes1,2, Ana Mutis1,2, Leonardo Bardehle1,2,3, Ivette Seguel4, Alejandro Urzúa5, and Andrés Quiroz2,3 Universidad de La Frontera, Facultad de Ingeniería y Ciencias, Departamento de Ciencias Químicas y Recursos Naturales, Laboratorio de Química Ecológica. Av. Francisco Salazar 01145, Casilla 54-D, Temuco, Chile. 2 Universidad de La Frontera, Centro de Investigación Biotecnológica Aplicada al Medio Ambiente (CIBAMA). Av. Francisco Salazar 01145, Casilla 54-D, Temuco, Chile. 3 Universidad de La Frontera, Centro de Fruticultura, Facultad de Ciencias Agropecuarias y Forestales. Casilla 54-D, Temuco, Chile. 4 Instituto de Investigaciones Agropecuarias, Centro Regional de Investigación Carillanca. Temuco, Chile. 5 Universidad de Santiago de Chile, Facultad de Química y Biología, Departamento de Ciencias del Ambiente, Laboratorio de Química Ecológica. Av. Bernardo O’ Higgins 3363, Santiago, Chile. 1 Abstract M. Chacón-Fuentes, A. Mutis, L. Bardehle, I. Seguel, A. Urzúa, and A. Quiroz. 2019. Decrease of flavonol synthase enzymatic activity in Ugni molinae Turcz due to the domestication process. Cien. Inv. Agr. 46(1): 30-39. Flavonoid biosynthesis may be affected by plant domestication, with flavonoid production being reduced in proportion to the degree of domestication. In this context, kaempferol (3,4´,5,7-tetrahydroxyflavone) has been identified in the leaves of wild and cultivated Ugni molinae, a berry endemic to Chile. The biosynthetic pathway of kaempferol production begins with naringenin (4´,5,7-trihydroxyflavanone), which is converted to dihydrokaempferol (3,4´,5,7-tetrahydroxyflavanone), catalyzed by flavanone 3ß-hydroxylase (FHT) and then converted to kaempferol by a bifunctional enzyme called flavonol synthase (FLS). Therefore, our study aims to evaluate how FLS activity is affected in murtilla plants that are subjected to the domestication process. Kaempferol was quantified from methanolic extracts of leaf samples collected from both cultivated and wild U. molinae plants using high-performance liquid chromatography, and enzyme extraction was performed to determine FLS activity. The results showed that kaempferol concentration in wild plants from the Soloyo (0.14 μg g-1), Mehuín (0.18 μg g-1) and Queule (0.25 μg g-1) sampling areas was higher than in their cultivated counterparts. Our data are consistent with the FLS activity detected in samples obtained from Manzanal Alto (134.79 pKatal, Soloyo (96.48 pKatal), and Mehuín (119.97 pKatal). These samples also exhibited higher enzymatic activity than their cultivated counterparts. Together, these data suggest that FLS activity is negatively affected by the domestication process. Key words: Cultivated, enzyme activity, Kaempferol, naringenin, wild. Received Mar 01, 2018. Accepted Mar 22, 2019. Corresponding author: VOLUME 46 Nº1 JANUARY – APRIL 2019 Introduction The flavonoid family consists of over 8,000 secondary plant metabolites. Flavonoids are characterized by their C6-C3-C6 skeleton (Nguyen et al., 2016) and can be classified into the following eight subgroups according to their oxidation state and substitution pattern of the C-ring structure: flavanones, dihydroflavonols, flavones, flavonols, flavan-3,4-diols, flavan-3-ols, anthocyanidins, and proanthocyanidins (Xu et al., 2012). Additionally, flavonoids can be conjugated to sugar molecules naturally occurring in plant tissues. These compounds have been associated with a broad range of applications, including antioxidants, insect repellent, and even cancer prevention (Ruiz et al., 2010, Vazhappilly et al., 2017). Flavonols are the most abundant group of flavonoids, and several biological properties have been attributed to these compounds, such as regulation of auxin transport, modulation of flower color, protection from UV radiation, prevention against microorganisms and pest invasions, and signaling interactions with insects and microbes (Nenaah 2013, Dixit et al., 2017, León-Chan et al., 2017, Vazhappilly et al., 2017). Flavonols are produced by the desaturation of dihydroflavonols. These compounds are formed from dihydroflavonols (Figure 1) by the introduction of a double bond between C-2 and C-3, which is catalyzed by flavonol synthase (FLS) (Xu et al., 2012). The B-ring of dihydrokaempferol (3,4′,5,7-tetrahydroxyflavanone) can be hydroxylated at the 3´ position by flavonoid 3´-hydroxylase (F3´H) or at the 3´ and 5´ positions by flavonoid 3´5´-hydroxylase (F3´5´H) to produce dihydroquercetin and dihydromyricetin, respectively. The oxidation reaction introducing the C-2/C-3 double bond is considered to be specific for dihydroflavonol substrates (Preub et al., 2009, Lukacin et al., 2000, Li et al., 2013). Furthermore, FLS has been reported as a bifunctional enzyme capable of transforming not only dihydrokaempferol (3,4′,5,7-tetrahydroxyflavone) but also naringenin (4´,5,7-trihydroxyflavanone) into kaempferol (Lukacin et al., 2003). Regarding 31 pest management, various studies have focused on the deterrent and anti-feeding properties of these compounds. Onyilagha et al. (2012) found that kaempferol, quercetin, and isorhamnetin deterred feeding of the flea beetle, Phyllotreta cruciferae (Coleoptera: Chrysomelidae), on Camelina sativa leaves. Additionally, Selin-Rani et al. (2016) reported that quercetin isolated from Euphorbia hirta L. was toxic to Spodoptera litura Fab. (Lepidoptera: Noctuidae) larvae and showed that 6 ppm caused a 94.6% mortality rate. Furthermore, larval weight was reduced to 100 mg at the same dose. Finally, Nenaah (2003) reported that a leaf methanolic extract from Calotropis procera that contained kaempferol showed considerable toxicity to Sitophilus oryzae (Coleoptera: Curculionidae) and Rhyzopertha dominica (Coleoptera: Bostrichidae) and that concentrations of 5 mL cm-2 caused a mortality rate of 86% in S. oryzae with applications of 2 mL of extract. During the last 20 years, a highly polymorphic perennial and wild native shrub from Chile, denominated “Murtilla”, Ugni molinae (Hoffmann 2005; Valdebenito et al., 2003), has been domesticated and studied by the Institute of Agricultural Research (Instituto de Investigaciones Agropecuarias, INIA), Carillanca, in the Araucanía region of Chile (Chacón-Fuentes et al., 2016). One hundred sites were originally selected in southern Chile for the collection of wild U. molinae plants, and cuttings were grown for the first 10 years under shading and then transplanted to the field. There is strong economic interest in the production of U. molinae fruit, both globally and regionally, due to its high antioxidant content that is mainly provided by flavonol compounds (Simirgiotis et al., 2009). The main phenolic compounds reported in murtilla plants are myricetin, querceti (...truncated)