Integrated Analysis of the Transcriptome And
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International Journal of Molecular Sciences Article Integrated Analysis of the Transcriptome and Metabolome of Cecropia obtusifolia: A Plant with High Chlorogenic Acid Content Traditionally Used to Treat Diabetes Mellitus Jorge David Cadena-Zamudio 1, Pilar Nicasio-Torres 2 , Juan Luis Monribot-Villanueva 1 , José Antonio Guerrero-Analco 1 and Enrique Ibarra-Laclette 1,* 1 Instituto de Ecología, A.C. (INECOL), Red de Estudios Moleculares Avanzados (REMAV), Xalapa 91073, Veracruz, Mexico; [email protected] (J.D.C.-Z.); [email protected] (J.L.M.-V.); [email protected] (J.A.G.-A.) 2 Instituto Mexicano del Seguro Social (IMSS), Centro de Investigación Biomédica del Sur (CIBIS), Xochitepec 62790, Morelos, Mexico; [email protected] * Correspondence: [email protected]; Tel.: +52-(228)-842-1823 Received: 12 September 2020; Accepted: 9 October 2020; Published: 14 October 2020 Abstract: This investigation cultured Cecropia obtusifolia cells in suspension to evaluate the effect of nitrate deficiency on the growth and production of chlorogenic acid (CGA), a secondary metabolite with hypoglycemic and hypolipidemic activity that acts directly on type 2 diabetes mellitus. Using cell cultures in suspension, a kinetics time course was established with six time points and four total nitrate concentrations. The metabolites of interest were quantified by high-performance liquid chromatography (HPLC), and the metabolome was analyzed using directed and nondirected approaches. Finally, using RNA-seq methodology, the first transcript collection for C. obtusifolia was generated. HPLC analysis detected CGA at all sampling points, while metabolomic analysis confirmed the identity of CGA and of precursors involved in its biosynthesis. Transcriptome analysis identified differentially expressed genes and enzymes involved in the biosynthetic pathway of CGA. C. obtusifolia probably expresses a key enzyme with bifunctional activity, the hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase and hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HQT/HCT), which recognizes shikimic acid or quinic acid as a substrate and incorporates either into one of the two routes responsible for CGA biosynthesis. Keywords: Cecropia obtusifolia (guarumbo); chlorogenic acid; plant metabolomics; RNA-seq; diabetes mellitus 1. Introduction Plant secondary metabolites (SMe) are compounds with a restricted presence in taxonomic groups that participate in the interaction of cells with the environment to ensure the survival of the organism and provide an evolutionary advantage in its ecosystem [1,2]. SMe exhibit great structural variation, and their production is often associated with biotic or abiotic stress. Consequently, a wide range of biological activities are exhibited, such as antimicrobial, antigerminative, toxic, and attractant properties or symbiont interaction. These features have allowed researchers to identify more than 200,000 plant SMe, largely represented by terpenes, alkaloids, and polyphenols [3,4]. Polyphenols have received considerable attention due to their pharmacological and biomedical potential in chronic human degenerative diseases such as cataracts, macular degeneration, endothelial dysfunction, arthritis, cancer, and diabetes mellitus [5]. Many recent studies have focused on finding new phytomedicines Int. J. Mol. Sci. 2020, 21, 7572; doi:10.3390/ijms21207572 www.mdpi.com/journal/ijms Int.Int. J. Mol. J. Mol. Sci. Sci.2020 2020, 21, 21, 7572, x FOR PEER REVIEW 2 of2 27 of 27 endothelial dysfunction, arthritis, cancer, and diabetes mellitus [5]. Many recent studies have focused withon greaterfinding specificity,new phytomedicines greater human with greater safety specificity, and sometimes greater a human lower economicsafety and costsometimes to provide a lower new optionseconomic for bothcost to the provide pharmaceutical new options industry for bothand the pharmaceutical patients [6,7]. Weindustry highlight and patientsCecropia [6,7]. obtusifolia We Bertolhighlight (Urticaceae), Cecropia anobtusifolia arboreal Bertol species (Urticaceae), popularly an known arboreal as species guarumbo popularly in Mexico known and as traditionallyguarumbo usedin Mexico in the medicaland traditionally treatment used of diabetes in the medical mellitus treatment type 2 (DM-2). of diabetes Infusions mellitus of leaves,type 2 (DM-2). branches, bark,Infusions and roots of leaves, are used branches, for their bark, hypoglycemic and roots are andused hypolipidemicfor their hypoglycemic properties and demonstratedhypolipidemic in experimentalproperties animaldemonstrated and human in experimental models, both atanimal a cellular and andhuman physiological models, level,both whichat a cellular are attributed and to chlorogenicphysiological acid level, (CGA), which making are attributedC. obtusifolia to chlorogenica model acid plant (CGA), for its making potential C. useobtusifolia in the a control model of DM-2plant [8 for–14 its]. Inpotential the same use way, in the it control is known of DM-2 that C. [8–14]. obtusifolia In theis same capable way, of it synthesizing,is known that C. in obtusifolia addition to CGA,is capable other compoundsof synthesizing, of phenolicin addition origin to CGA, such othe asr orientine, compounds isoorientine, of phenolicvitexin, origin such isovitexin, as orientine, caffeic acid,isoorientine, ferulic acid, vitexin, quinic isovitexin, acid, quercetin, caffeic acid, apigenin, ferulic luteolin,acid, quinic tormentoside, acid, quercetin, triterpenoid apigenin, isomersluteolin, of saponin-O-hexosidetormentoside, triterpenoid and chlorogenic isomers of acid, saponin- amongO-hexoside others, whichand chlorogenic are known acid, to be among present others, in the leaveswhich of are the known plant [to15 be–17 present]. Studies in the on leavesC. obtusifolia of the plantcell [15–17]. cultures Studies have shown on C. obtusifolia that nitrate cell starvationcultures increaseshave shown CGA productionthat nitrate [starvation18]. Despite increases the importance CGA production of this plant, [18]. noDespite omics the studies importance were previously of this available.plant, no Therefore, omics studies the objectivewere previously of this researchavailable. was Therefore, to evaluate the objective the effect of of this nitrate research restriction was to in evaluate the effect of nitrate restriction in C. obtusifolia cell suspension cultures to identify genes and C. obtusifolia cell suspension cultures to identify genes and proteins involved in the biosynthesis of proteins involved in the biosynthesis of CGA. This transcriptomic/metabolomic study represents the CGA. This transcriptomic/metabolomic study represents the first collection of transcripts reported for first collection of transcripts reported for this species. According to the chemical-analytical and this species. According to the chemical-analytical and computational-theoretical analyses performed, computational-theoretical analyses performed, the C. obtusifolia CGA biosynthesis pathway is the C. obtusifolia CGA biosynthesis pathway is proposed. proposed. 2. Results 2. Results 2.1. Effect of Nitrate Starvation on the Proliferation Kinetics of C. obtusifolia Cells 2.1. Effect of Nitrate Starvation on the Proliferation Kinetics of C. obtusifolia Cells The C. obtusifolia cell suspension cultures at 16 and 8 mM nitrates in Murashige and Skoog (MS) The C. obtusifolia cell suspension cultures at 16 and 8 mM nitrates in Murashige and Skoog (MS) medium showed a similar proliferation rate to the control during the exponential phase (7–21 days). medium showed a similar proliferation rate to the control during the exponential phase (7-21 days). However,However, at laterat later evaluation evaluation times times (28 (28 days), days), bothboth stress conditions conditions provoked provoked a adecay, decay, which which suggests suggests a reductiona reduction in in the the rate rate of of cell cell proliferation proliferationand and viabilityviability after prolonged stress stress (Figure (Figure 1).1). Under Under the the mostmost restricted restricted nitrate nitrate condition condition (4 (4 mM), mM), cell cell proliferationproliferation was severely affected affected (Figure (Figure 1).1). 0.6 0.5 0.4 ) -1 0.3 Dry Dry biomass 0.2 (g l 0.1 0 0 7 14 21 28 Time (days) 27.4 mM 16 mM 8 mM 4 mM FigureFigure 1. Kinetics1. Kinetics of proliferationof proliferation of cellof cell suspensions suspensions of C.of obtusifoliaC. obtusifoliagrown grown in Murashigein Murashige and and Skoog Skoog (MS) medium(MS) medium supplemented supplemented with diff witherent di concentrationsfferent concentrations of nitrates of (27.4,nitrates 16, (27.4, 8 and 16, 4 mM).8 and The4 mM). dispersion The barsdispersion represent bars the represent standard the deviation standard calculated deviation calc fromulated three from biological three biological replicates replicates that were that used were for eachused sampling for each point sampling analyzed point overanalyzed the time over course. the time course. Int. J. Mol. Sci. 2020, 21, 7572 3 of 27 No differences in cell proliferation were observed under any stress condition at early stages (zero and three hours), as confirmed by Student’s t analysis for independent samples (p value = 0.2058). This was expected due to the rapid doubling rate generally observed for plant cells in suspension cultures and mainly because these times represent the adaptation phase. Although a clear reduction in the average maximal biomass obtained was observed