DOI: 10.1007/s10535-016-0608-4 BIOLOGIA PLANTARUM 60 (3): 579-584, 2016 Phenylalanine biosynthesis and its relationship to accumulation of capsaicinoids during Capsicum chinense fruit development L.A. CASTRO-CONCHA, F.M. BAAS-ESPINOLA, W.R. ANCONA-ESCALANTE, F.A. VÁZQUEZ-FLOTA, and M.L. MIRANDA-HAM* Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, 97200, Yucatán, México Abstract Activities of phenylalanine (Phe) biosynthetic enzymes chorismate mutase (CM) and arogenate dehydratase (ADT) and of phenylalanine ammonia lyase [PAL, an enzyme that directs Phe towards capsaicinoid (CAP) synthesis] were analyzed during Capsicum chinense Jacq. (habanero pepper) fruit development. A maximum CM activity coincided with a maximum CAP accumulation. However, ADT exhibited two activity peaks, one during the early phase (10 - 17 days post-anthesis, DPA) and another during the late phase (35 - 37 DPA); only the latter coincided with CAP. Interestingly, PAL activity was inversely related to CAP accumulation; lower activities coincided with a maximum CAP content. These results suggest the operation of a control mechanism that coordinated Phe synthesis and its channeling towards CAP synthesis during the course of fruit development. Additional key words: arogenate dehydratase, chorismate mutase, fruit placenta, habanero pepper, phenylalanine ammonia lyase. Introduction The burning sensation that is typical of hot peppers is accumulation of CAP and their intermediates suggesting caused by capsaicinoids (CAP), a group of arylamides, that a higher Phe supply might be channeled to CAP containing a vanilloid moiety and an acyl side chain, that synthesis (Salgado-Garciglia and Ochoa-Alejo 1990, are derived from phenylalanine (Phe) and a branched Ochoa-Alejo and Salgado-Garciglia 1992). Phenylalanine amino acid (valine or leucine), respectively (Stewart et al. is formed via the shikimate pathway from chorismate 2005, Aza-González et al. 2011). Most of the genes through the sequential action of chorismate mutase (CM; involved in the CAP biosynthetic pathway have been EC 5.4.99.5), prephenate aminotransferase, and arogenate identified (Vázquez-Flota et al. 2007). However, very dehydratase (ADT; EC 4.2.1.91), which yield prephenate, little is known about primary amino acid metabolism in arogenate, and Phe, respectively (Cho et al. 2007). For pepper placentas. The placenta is the tissue holding the CAP synthesis to occur, firstly Phe must be deaminated seed inside the fruits, and in Capsicum, it is the only by phenylalanine ammonia lyase (PAL; EC 4.3.1.24). In tissue displaying CAP biosynthetic capacity (Ruiz-Lau et pepper, this enzyme is coded by a three-gene family al. 2010). A positive correlation was found between (Curry et al. 1999), the members of which are nitrate content and CAP accumulation in the placentas of differentially regulated (Kim and Hwang 2014, Kim et al. pepper plants grown under various nitrogen doses 2014). (Monforte-González et al. 2010). In addition, increased Nonetheless, CAP accumulation in pepper pods varies ammonia assimilation precedes CAP accumulation in according to developmental stage, apparently as result of response to elicitation with salicylic acid and methyl the balance between synthesis and oxidation (Contreras- jasmonate in isolated placentas (Ancona-Escalante et al. Padilla and Yahia 1998, Stewart et al. 2005). However, it 2013). Furthermore, several pepper Phe-overproducing remains unknown whether this developmentally cell lines growing in vitro show an increased associated pattern also involves the availability of an Submitted 1 June 2015, last revision 15 October 2015, accepted 11 November 2015. Abbreviations: ADT - arogenate dehydratase; CAP - capsaicinoids; CM - chorismate mutase; DPA - days post-anthesis; f.m. - fresh mass; PAL - phenylalanine ammonia lyase; Phe - phenylalanine. Acknowledgments: The authors wish to acknowledge Q.F.B. Raúl Manzanilla for maintaining the plants and L.D.P. Norma Marmolejo for editing the graphic material. This work was funded by the CONACYT (México), Grant 168545. FMBE was the recipient of a CONACYT scholarship for Ph.D. studies. The first two authors contributed equally to this work. *Author for correspondence; fax: (+999) 9813900, e-mail: [email protected] 579 L.A. CASTRO-CONCHA et al. internal Phe supply. To aid in elucidating this point, we were jointly analyzed, and the accumulation of some examined Phe biosynthesis enzymes and PAL, which CAP synthesis intermediates was examined throughout a channels Phe to CAP formation. Enzyme and CAP data complete developmental cycle of C. chinense pods. Materials and methods Habanero pepper (Capsicum chinense Jacq.) cv. Chak glycine) and 100 mm3 of ortho-phthalaldehyde k'an-iik pods were collected between 10 and 40 days (54 mg dissolved in 1 cm3 of ethanol, 9 cm3 of 0.4 M post-anthesis (DPA) and assigned developmental stages sodium borate, pH 9.4, and 200 mm3 of 2-mercapto- from 1 to 7 as shown in Fig. 1. Each phase corresponds to ethanol). The reaction product (Phe) was derivatized at approximately 5 DPA. The habanero pepper plants were room temperature for 90 s and then injected to an HPLC cultivated in a greenhouse at the Centro de Investigación system (Agilent Technologies, Germany) equipped with a Científica de Yucatán (Mérida, México; 21° 02’ 38’’ N, fluorescence detector. A mobile phase was water-ethanol 89° 38’ 22’’ W, 8 m above sea level). The plants were (2:3; v/v); a flux and run time were 1 cm3 min-1 and grown in a mixture of soil, Perlite, Vermiculite, peat 7 min, respectively. The derivatized products were moss, and coconut fiber and watered to a full field detected at 360 nm (excitation) and 455 nm (emission). capacity every other day. The plants were fertilized once Arogenate used in this assay was enzymatically prepared a week with MaxiGro® and MaxiBloom® (General from prephenate (Connelly and Siehl 1987) utilizing a Hydroponics, Sevastopol, CA, USA). After collection partially purified prephenate aminotransferase from and arrival to the laboratory, the pods were washed with a C. chinense leaves (EC 2.6.1.78; Ripper and Matringe commercial soap and thoroughly rinsed under running tap 2002). Activity of PAL was determined in the desalted water. The pods were then dissected into placentas and protein extracts as described by Ochoa-Alejo and Gómez- pericarps (colored parts), and the separated tissues were Peralta (1993). Briefly, a reaction mixture including frozen in liquid nitrogen and stored at -80 °C for further 10 mM L-Phe, 50 mM Tris-HCl, pH 8.8, and the protein analyses. extract in a final volume of 2 cm3 was prepared. The Activities of CM, ADT, and PAL were determined in mixture was incubated at 37 °C for 1 h, and the reaction protein extracts obtained by homogenizing ground frozen was stopped by addition of 500 mm3 of 6 M HCl; the tissues in extraction buffers in a polytron (Model T10, products were then extracted in 10 cm3 of diethyl ether. IKA, USA) at the full speed. For CM and PAL, the buffer After solvent evaporation, the residue was dissolved in contained 20 mM Tris-HCl, pH 7.8, 10 mM 2-mercapto- 50 M NaOH, and trans-cinnamic acid that had formed ethanol, and 5 % (m/v) polyvinylpolypyrrolidone was quantified at 290 nm. The total protein in the extracts (PVPP); for ADT, 50 mM Tris-HCl pH 8.5, 5 mM was measured according to Peterson (1977). 2-mercaptoethanol, 5 % PVPP, 1 µM leupeptin, and The total soluble amino acids were quantified as 1 µM pepstatin. The homogenate was centrifuged at ninhydrin derivatives (Cocking and Yemm 1954). 24 400 g and 4 °C for 15 min. Activity of CM was Capsaicinoids (capsaicin and dihydrocapsaicin), ferulic determined in a reaction mixture containing 0.15 mM acid, and vanillin were determined by HPLC (Collins barium chorismate, 20 mM Tris-HCl, pH 7.8, and the et al. 1995). Briefly, the metabolites were extracted with protein extract in a final volume of 300 mm3. The mixture acetonitrile and separated using an HPLC system was incubated at 37 °C for 20 min and the reaction was equipped with a diode array detector. The filtered stopped by adding 400 mm3 of 1 M HCl and incubating samples were injected (20 mm3) to a reverse phase for further 10 min. The mixture was then neutralized with column (ZORBAX Octadecyl Silane C18, particle size 3 3.2 cm of 1 M NaOH and the amount of phenylpyruvate 5 m, 4.6 150 mm). The run conditions were as formed was quantified at 320 nm (Cotton and Gibson follows: isocratic mobile phase 30A:70B [A - methanol- TM 1965) using a spectrophotometer (Genesys , Thermo- water (10:90), B - 100 % methanol]; flux 1 cm3 min-1; Electron Corp, USA). An ADT assay reaction mixture running time 10 min; room temperature. Capsaicin, contained 50 mM Tris-HCl, pH 7.5, 2.5 mM arogenate, dihydrocapsaicin, ferulic acid, and vanillin were 0.25 mM tyrosine, 1 µM leupeptin, 1 µM pepstatin, and identified and quantified using commercial standards as 3 the protein extract in a final volume of 200 mm (Fisher references. and Jensen 1987). The mixture was incubated at 32 °C All data were subjected to analysis of variance for 20 min before adding an internal standard (0.833 mM (ANOVA) Results Capsaicinoids (capsaicin + dihydrocapsaicin) are accumulation has been described for the complete fruit exclusively synthesized and accumulated in the placenta. (Contreras-Padilla and Yahia 1998). In this study, CAP In habanero peppers, the developmental pattern of CAP content in placental tissue was analyzed throughout the 580 PHENYLALANINE AND BIOSYNTHESIS OF CAPSAICINOIDS developmental cycle, from an early stage (stage 1; green to orange) began at stage 5 (Fig. 1). approximately 20 mm long; 10 - 15 DPA) to almost fully Capsaicinoids reached a high content in placental ripe pods (stage 7; approximately 40 - 50 mm long; tissue from very early phases [72 mol g-1(f.m.); stage 2] 36 - 40 DPA).