448 AJCS 8(3):448-454 (2014) ISSN:1835-2707 Role of peroxidases in capsaicinoids degradation in habanero pepper (Capsicum chinense Jacq.) plants grown under water deficit conditions Enid Zamudio-Moreno 1 , Ileana Echevarría-Machado 1 , María de Fátima Medina-Lara 1 , Graciano Calva-Calva 2 , María de Lourdes Miranda-Ham 1 ; Manuel Martínez-Estévez 1 * 1 Unidad de Bioquímica y Biología Molecular de Plantas. Centro de Investigación Científica de Yucatán, Calle 43 # 130, Col. Chuburná de Hidalgo, 97200 Mérida, Yucatán, México 2 Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, C.P. 07360. PO Box: 14-740, 07000 México, D.F. *Corresponding author: luismanh@cicy.mx Abstract Habanero pepper (Capsicum chinense Jacq.) is one of the most pungent cultivars of the genus Capsicum. We studied the effects of water deficit on capsaicinoids (CAPs, acid amides derived from phenylalanine and valine or leucine) accumulation. It comprises their synthesis and degradation through the determination of not only capsaicinoid content, but also of related enzymes such as capsaicinoid synthetase (CS) and peroxidases (PODs) in pods from water-stressed habanero pepper plants. Water stress was induced by withholding irrigation for 7 (R7) or 9 (R9) days after anthesis occurred, while control plants were watered daily. Irrigation withholding for 9 days induced effects on the rate of capsaicinoid accumulation in the placental tissue (131 mg g -1 DW), through a lowered rate of degradation (low PODs activities: 31 μKat mg -1 prot). The CS activity was not detectable at 60 DPA (days of post- anthesis). A correlation between PODs activities and CAPs contents was totally dependent on the maturation stage of the fruit. At 60 DPA, the PODs activities were highest for control and plants at R7 treatment, whereas CAPs contents remained high. PODs activities could not fully explain the formation of the dimer 5,5'-dicapsaicin when these enzymes’ activities was immuno-inhibited. PODs may not be the sole pathway in the degradation process of CAPs in habanero pepper plants under water stress. Keywords: Capsaicinoids; capsaicinoid synthetase; habanero pepper; peroxidases; water deficit. Abbreviations: CAPs_capsaicinoids; CS_capsaicinoid synthetase; DPA_days of post-anthesis; POD(s)_peroxidase(s). Introduction Water is fundamental for living organisms and essential for agricultural production. Water stress is one of the most common abiotic stresses worldwide, which induces major constraints on plant productivity (Turner and Begg, 1981; Pedrol et al., 2000). Habanero pepper (Capsicum chinense Jacq.) is one of the most pungent known cultivars due to its high CAPs (capsaicinoids )content, exclusively accumulated in the placental tissue of fruits (Bennett and Kirby, 1968; Leete and Louden, 1968). CAPs are acid amides of vanillylamine and C 9 - C 11 branched fatty acids, of which capsaicin is the principal pungent compound. The vanillylamine moiety of CAPs is biosynthetically derived from L-phenylalanine, whereas the branched fatty acid moiety arises from valine or leucine (Iwai et al., 1979). The aromatic and aliphatic moieties are condensed by the enzyme CS (Curry et al., 1999; Sung et al., 2005; Aza-González et al., 2011). CAPs biosynthesis occurs specifically in the epidermal cells of the interlocular septum of placental tissue in Capsicum fruits (Fujiwake et al., 1980; Suzuki et al., 1980; Zamsky et al., 1987; Estrada et al., 2000; Stewart et al., 2007). CAPs accumulation is greatly influenced by environmental factors, such as soil type, osmotic pressure, nutrients and water availability (Estrada et al., 1998; Díaz et al., 2004; Sung et al., 2005). An increase in capsaicin accumulation in C. chinense plants grown under water deficit conditions has been reported, which resulted paradoxical since CS activity decreased in plants (Ruiz-Lau et al., 2011). Hence, it is still under study how synthesis and degradation pathways contribute to their overall concentration in pods from plants under water stress (Estrada et al., 2000). Contrasting to the attention placed in the elucidation of the intracellular localisation and biosynthetic pathways of CAPs, there are only a few reports regarding their catabolism (Estrada et al., 1998; Martínez-Juárez et al., 2004; Díaz et al., 2004; Sung et al., 2005). PODs have been involved not only in plant defence against biotrophic and necrotrophic pathogens (Passardi et al., 2004; Almagro et al., 2009; Mohamed et al., 2011), but can also act as biomarkers of biotic and abiotic stresses (Jouili et al., 2011). These enzymes can readily oxidise vanillin (Zapata et al., 1992; Díaz et al., 2004), vanillylamine and CAPs (Martínez-Juárez et al., 2004), so they may play a significant role in CAPs catabolism. They have been purified from C.annuum fruits and are capable of oxidizing capsaicin into 5,5'-dicapsaicin and 4'-O-5-dicapsaicin in the presence of H 2 O 2 (Bernal et al., 1993a,b; 1995; Bernal and Ros-Barceló, 1996). In whole fruits, there was an increase in PODs activities at the late stages of fruit maturation when CAPs content decreased (Contreras-Padilla and Yahia, 1998). Furthermore, an increase in CAPs content along fruit development was not only related to changes in total PODs