https://dx.doi.org/10.21577/0103-5053.20200173 J. Braz. Chem. Soc., Vol. 32, No. 2, 237-248, 2021 Article Printed in Brazil - ©2021 Sociedade Brasileira de Química Mapping Biochemical Pathways in Maytenus ilicifolia (Celastraceae) through Integrated Proteomics and Histochemistry VâniaVânia A. A. F.F. F.F. M.M. Santos,Santos, aa NathaliaNathalia B.B. Dias,Dias,bb SimoneSimone P.P. Teixeira,Teixeira,cc MarioMario S.S. PalmaPalmdd and and Maysa Maysa Furlan Furlan **,a,a aInstituto de Química, Universidade Estadual Paulista (Unesp), 14800-060 Araraquara-SP, Brazil bScientific and Technological Bioresource Nucleus BIOREN-UFRO, Universidad de La Frontera (UFRO), 4811-230 Temuco, Chile cLaboratório de Botânica, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (USP), 14040-903 Ribeirão Preto-SP, Brazil dDepartamento de Biologia Geral e Aplicada, Instituto de Biociências, Universidade Estadual Paulista (Unesp), 13506-900 Rio Claro-SP, Brazil Maytenus ilicifolia (Celastraceae) is a medicinal plant that is native to southern Brazil and is popularly known as “espinheira-santa”. From a biosynthesis perspective, this species accumulates quinonemethide triterpenes and sesquiterpene pyridine alkaloids as major secondary metabolites that exhibit interesting biological properties, with antitumoral and antiprotozoal activities, respectively, being the most frequently reported. Additionally, the restricted accumulation of such compounds in the roots raises questions about the expression of proteins involved in such compartmentalization and their possible biological and/or ecological role in M. ilicifolia. Thus, this article describes the use of shotgun proteomics and histochemical studies for the characterization of the main biosynthetic pathways involved in the regulation of the metabolism in M. ilicifolia roots. This combined approaches also resulted in the identification of a series of proteins involved in the quinonemethide triterpenes and sesquiterpene pyridine alkaloids, providing evidences of their differential compartmentalization. Keywords: shotgun proteomic approach, histochemical analysis, quinonemethide triterpenes, sesquiterpene pyridine alkaloids, Maytenus ilicifolia, Celastraceae Introduction plant native to southern Brazil that is popularly known as “espinheira-santa”. Since 1988, this plant has been included as Plant growth and development are regulated by herbal medicine in the phytotherapy program, coordinated by phytohormones and mediate responses to biotic and the Brazilian Ministry of Health’s Medicines Center (CEME), abiotic stresses.1 In response to stress, plants synthesize and was included in the National List of Medicinal Plants and accumulate a wide diversity of secondary metabolites, of Interest for the Brazilian Unified Health System (SUS).7 which are involved in biological and ecological functions.2,3 The structural richness of Maytenus ilicifolia roots Many of these compounds have shown biological is exemplified by quinonemethide triterpenes and activity and are considered to be prototypes for the synthesis sesquiterpene pyridine alkaloids (Figure 1), which are of a large number of drugs,4 accounting for more than 50% considered chemotaxonomic markers of the Celastraceae of all drugs employed in modern therapies.5 species.8 Several biological activities have been described The biosynthesis of these metabolites is performed for these two classes of metabolites, including antioxidant, by specific pathways and involves complex interactions antifungal, antiproliferative, antiprotozoal, antitumoral, between proteins/enzymes, transport mechanisms, antimicrobial, and inhibitor of hepatitis C virus, compartmentalization, and integration with primary antiprotozoal, relating to quinonemethide triterpenes and metabolism at the molecular and cellular levels.3,6 sesquiterpene pyridine alkaloids, respectively.9-19 Maytenus ilicifolia Mart ex Reissek is a medicinal Thus, this study aimed to (i) localize the metabolites through histochemical tests, (ii) characterize the main *e-mail: [email protected] proteins involved in primary and secondary metabolism 238 Mapping Biochemical Pathways in Maytenus ilicifolia (Celastraceae) through Integrated Proteomics and Histochemistry J. Braz. Chem. Soc. Figure 1. Chemical structures of sesquiterpene pyridine alkaloids: ilicifoliunine A (1), ilicifoliunine B (2), aquifoliunine E-I (3) mayteine (4) and quinonemethide triterpenes: pristimerin (5), maytenin (6), 22β-hydroxy-maytenin (7), isolated from Maytenus ilicifolia. by means of proteomic studies, and (iii) combine the a watch glass containing deionized water. Later, these histochemical and proteomic data. These approaches cross-sections were selected using a magnifying glass to aimed to identify the main biosynthetic pathways active obtain the thinnest cuts and cuts with correct orientation. in M. ilicifolia roots, determine and provide evidence of a After the selection of the cuts, some of them were kept differential distribution of the metabolites and to address unstained to verify the natural appearance of the cellular the possible biological and ecological functions that these content, and the other part was submitted to different compounds serve in the roots of M. ilicifolia. reagents, aiming to achieve in situ detection of different classes of metabolites. The following reagents were used for Experimental the histochemical tests: Sudan III (Sigma-Aldrich, St. Louis, USA) for lipophilic compounds (lipids, terpenoids, suberin Plant material and cutin), ferric chloride (Fisher Scientific, Waltham, USA) for phenolic compounds, and Dragendorff reagents Young plants (five years old) of Maytenus ilicifolia for alkaloids.20,21 The cuts were then mounted between ware collected in the district of Juruce, municipality a slide of glass and coverslip and analyzed under a of Jardinópolis, state of São Paulo (21°04’18.8’’S; microscope. The observations and photomicrographs were 47°44’08.8’’W) in March 2013. After collection, the plants obtained from a Leica DFC 320 digital camera coupled were transported and maintained in a greenhouse at the to a Leica (Wetzlar, Germany) DM 5000 microscope. Instituto de Química de Araraquara, state of São Paulo Autofluorescence was monitored by an epifluorescence (21°48’26.62’’S; 48°11’33.5’’W). microscope equipped with a mercury lamp and green light The botanical identification was provided by Prof Rita (excitation, 488 nm; emission longpass filter, 520 nm). Maria de Carvalho-Okano. A voucher specimen (00755) was deposited in the Herbarium of the Department of Proteomic analysis Medicinal Plants, University of Ribeirão Preto, São Paulo, Brazil (HPM-UNAERP). Protein extraction The total proteins were extracted and precipitated according Histological analysis to the methodology developed by Hurkman and Tanaka22 with some modifications. Approximately 7 g of the roots For the histochemical tests, lateral roots were selected. were ground to form a thin paste. Subsequently, the material Cross-sections were made manually and transferred to was transferred to centrifuge tubes containing 60 mL of Vol. 32, No. 2, 2021 Santos et al. 239 extraction buffer containing this buffer: 0.7 M sucrose, 0.1 solution (1:50 m/m trypsin:protein) for 18 h at 37 °C. The M KCl, 0.5 M Tris-HCl pH 7.5, 1 mM phenylmethylsulfonyl reaction was terminated with the addition of 5 μL formic acid. fluoride (PMSF) dissolved in isopropanol, 50 mM The digested samples were desalted using a Sep-Pak tC18 ethylenediaminetetraacetic acid (EDTA) and 1% (m/v) 1 cm3 Vac Cartridge (Waters, Milford, MA, USA) column. polyvinylpolypyrrolidone (PVPP) (Sigma-Aldrich, St. Louis, The tryptic peptides were solubilized in 50% acetonitrile USA). The mixture was homogenized at 4 °C for 30 min on (ACN) and subjected to analysis in a liquid chromatography an orbital shaker. coupled with time-of-flight and ion trap mass spectrometry Then, the same volume of phenol equilibrated with a (LCMS-IT-TOF) and MSn system. solution of 10 mM Tris-HCl (Sigma-Aldrich, Saint Louis, USA) pH 7.9 ± 0.2 and 1 mM EDTA (Sigma-Aldrich, St. LCMS-IT-TOF Louis, USA) was added. The mixture was further stirred The samples were analyzed using the LCMS-IT-TOF for 30 min and finally centrifuged at 4 °C and 10000× g for system. The UFLC (ultra fast liquid chromatograph) 30 min. Then, the supernatant (upper phase or phenolic) system (Shimadzu, Kyoto, Japan) coupled directly (online) was transferred to a new tube, and 1 volume of extraction to the mass spectrometer, with two LC-20AD pumps, an buffer with 0.15 g of PVPP was added. The samples were SPD-M20A diode array detector, a SIL-20AHT autosampler homogenized in the same manner with the aid of an orbital and a CTO-20A oven column was used. The analysis was shaker for 30 min at 4 °C. Subsequently, the samples were carried out under a gradient of acetonitrile (ACN) from 5 subjected to centrifugation at 4 °C and 10000× g for 30 min. to 90% (v/v) containing 0.05% trifluoroacetic acid (TFA) The upper phase was again collected and added (1 volume) (v/v) over 90 min using a C18 Shim-pack XR-ODS column extraction buffer but without the addition of PVPP. Again, (3 × 100 mm, 120 A, 2.2 μm) (Shimadzu). The elution the upper phase was homogenized and centrifuged. The of components was monitored by ultraviolet absorbance supernatant was recovered, and 5-6 volumes (25-30 mL) of at 214 nm through a UFLC system with a flow rate of precipitation buffer (100% methanol + 0.1 M ammonium 0.2 mL min-1 for 90 min. The eluents were