INTRODUCTION

Ilex paraguariensis St. Hil. is a dioecious tree of the Aquifoliales order, Aquifoliaceae family of the Euasterids II group, native to the subtropical forests of South America. Its leaves and small dry processed branches are used to prepare the popular infusion, stimulating and with a particular flavor, known as "mate", hence its colloquial name in Latin America is "yerba mate" or "erva mate", an expression derived from the conjunction “ka`a mati”, from the Guarani “ka`a” (herb) and the Quechua “mati” (pumpkin), the latter alluding to the container of consumption. Yerba mate is also widely used to prepare other hot infusions such as "mate cocido", cold drinks such as "tereré" and more recently, as an additive in ice creams, sweets and energy drinks, as well as in dyes, cosmetics and spa ingredients. (Bracesco et al. 2011).

Yerba mate crops are an important agribusiness in Misiones, Argentina. The characteristics and agroecological conditions of yerba mate crops are found only in Brazil, Paraguay and Argentina. In Argentina, the world's leading producer, plantations are limited to the provinces of Misiones and Corrientes. The aerial photogrammetric survey of plantations commissioned by the INYM (National Institute of Yerba Mate), a national entity that regulates all aspects related to the cultivation and marketing of the product in 2016, determined that the cultivation of yerba mate in Argentina covers an area of 165,200 hectares, of which 144,014 hectares are located in the province of Misiones (87%), and the rest are in the province of Corrientes. In Misiones, cultivation occupies 50% of its agricultural area and yerba mate activity, in this province, occupies 22% of the workforce in the primary sector and 15% in the manufacturing sector, and constitutes its main industrial activity with the 25% of the total (Canitrot et al. 2011; Dolce 2012). In addition, Misiones concentrates most of the industrial stage with the largest number of dryers (94%), collectors (93%) and mills (85%). Particularly, since the creation of the National Institute of Yerba Mate (INYM) in 2002, a significant increase in the relative participation of producers and dryers in the final price of yerba has been observed (Gortari 2007; Rau et al. 2009).

The knowledge in genomics of I. paraguariensis has been increasing during the last years, as well as the sequencing of the transcriptome that represents the first evidence of the genome of this species (Debat et al., 2014), from which the presence of at least 32,000 genes and 12,000 variants is deduced. This work explored the vast collection of I. paraguariensis transcripts using the Pg538 line as reference genomic material, an elite material developed by INTA EAA-Cerro Azul de Misiones. In this work, transcripts belonging to more than 100 metabolic pathways were identified, related to osmotic stress, drought, salinity, cold stress, senescence, early flowering, and sexual determination. The large amount of information obtained in this study served as a reference framework and led to the start of the Yerba Mate Genome Sequencing Project (Pro.Mate.A.R.) in 2015 by the National University of Misiones, the University of Buenos Aires and other Argentine institutions such as the Secretariat of University Policies, National Institute of Yerba Mate, CONICET, Ministry of Science, Technology and Productive Innovation; that aim to generate a complete physical map at the sequence level of the entire genome of I. paraguariensis. This constitutes the starting point to discover, characterize and locate genes associated with traits of biological, agronomic and economic importance; develop efficient molecular markers, and delve into their biochemistry, evaluating the impact of the allelic variants that occur on the metabolomics of the species.

Xanthines are substances that belong to a chemical group of purine bases. From a medical and pharmacological point of view, there are three important xanthines: caffeine, theobromine and theophylline, which are the three methylated xanthines; that is the reason why they are also known as methylxanthines. They are considered alkaloids, since they are physiologically active substances, contain nitrogen and are found in higher plants. Methylxanthines and methyluric acids are plant secondary metabolites derived from purine nucleotides (Ashihara and Crozier 1999). The best characterized methylxanthines are caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) which appear in tea, coffee, cocoa, guarana, citrus, yerba mate and in a number of non-alcoholic beverages. origin in plants.

Caffeine was first isolated from tea and coffee in the early 1820s, but the main biosynthetic and catabolic pathway for caffeine was established only recently, when highly purified caffeine could be obtained from tea leaves and the genes encoding the enzyme could be cloned. (Kato et al. 1999; Kato et al. 2000). Methylxanthines have been found in about 100 species distributed in 13 orders of the plant kingdom (Ashihara and Suzuki 2004; Ashihara and Crozier 1999). Compared to other plant alkaloids such as nicotine, morphine and strychnine, purine alkaloids are widely distributed in the plant kingdom, although accumulation in high concentrations is restricted to a limited number of species including Coffea arabicaL. (coffee), Camellia sinensis (L.) Kuntze (tea), Theobroma cacaoL. (cocoa), Paullinia cupanaKunth (guarana) and I. paraguariensis. All caffeine-containing plants, except Scilla maritimaL., belong to the Dicotyledonae. In some species, the main methylxanthine is theobromine or methyluric acids, including theacrine (1,3,7,9-tetramethyluric acid) instead of caffeine (Ashihara and Crozier 1999).

Caffeine biosynthesis evolved independently in Coffea and Camellia, involving three methylation reactions. In Coffea, three XMT-type enzymes (xanthine methyltransferases) of the SABATH family are used to catalyze the methylation steps of the pathway. In Camellia, a convergently evolved paralogous pathway, CS-type caffeine synthase enzymes, is used. Numerous studies conducted over 30 years indicate that there are several biosynthetic pathways. This caffeine biosynthesis pathway has evolved independently in Coffea and Camellia, involving three methylation reactions to sequentially convert xanthosine to 7-methylxanthosine (1) to theobromine (2) to caffeine (3) (Huang et. al, 2016). Most SABATH enzymes catalyze the oxygen atom methylation of a wide variety of carboxylic acids such as anthranilic, benzoic, gibberillic, jasmonic, loganic, salicylic, and indole-3-acetic acids for flowering, defense, and hormonal modulation. XMT- and CS-mediated methylation of nitrogen atoms of xanthine alkaloids is probably a recently evolved activity (Huang et al. 2016). This multigene family exists in many plant species (monocots, dicots, and mosses), and the number of family members in these plant species varies due to different gene duplication and recombination events. For example, Arabidopsis thaliana (L.) Heynh. has 24 SABATH members, in rice (Oryza sativaL.) 41 members, 33 in poplar (Populus trichocarpaTorr. & A.Gray ex Hook.) and only 4 SABATH members in moss (Physcomitrella patens Bruch & W.P.Schimper). Many members of this family have been functionally characterized and participate in various secondary metabolism pathways in plants, producing different types of compounds. Three SABATH members having available protein crystal structures were characterized. These crystal structures are useful for homology modeling to identify important amino acid residues and to investigate enzyme interactions with different substrates. The SABATH family encodes a group of methyltransferases (MTs) that can transfer a methyl group (-CH3) from the methyl donor, S-adenosyl-L-methionine (SAM), to either the carboxyl group, the sulfur group, or the ring of nitrogen from a given substrate, forming S-adenosyl-L homocysteine ​​(SAH) and O-methylated ester, such as methyl benzoate and methyl salicylate, S-methylated ester, such as methyl thiolbenozate, or N-methylated compounds such as caffeine. Members of this family that are capable of methylating the carboxyl group of a given substrate include loganic acid methyltransferase (LAMT), salicylic acid methyltransferase (SAMT), benzoic acid methyltransferase (BAMT), jasmonic acid methyltransferase (JMT), indol- 3-acetic methyltransferase (IAMT), farnesoic acid methyltransferase (FAMT), gibberellic acid methyltransferase (GAMT), cinnamate and p-coumarate methyltransferase (CCMT), anthranilic acid methyltransferase (AAMT), nicotinic acid methyltransferase (NAMT), and p-methoxybenzoic acid methyltransferase (MBMT). The SABATH member that methylates the sulfur group of a given substrate is the recently characterized moss thiol methyltransferase. Members of SABATH that methylate the nitrogen ring of a given substrate include enzymes involved in caffeine biosynthesis, such as caffeine synthase (TCS1) characterized in Camellia, theobromine synthase (BTS1) characterized in Theobroma, 7-methylxanthine methyltransferase (MXMT), xanthosine methyltransferase (XMT), and 3,7-dimethylxanthine methyltransferase (DXMT) found in Coffea, as well as the enzyme responsible for the biosynthesis of trigonelline. The paralogous XMT and CS lineage genes in the SABATH family have independently evolved convergently, because genes in the two clades are involved in caffeine biosynthesis.

The importance of gene duplication for the development of metabolic pathways was first discussed by Lewis (1951) and then by Ohno (1970) and has recently been confirmed by comparative analysis of complete genome sequences of archaea, bacteria and eukarya (Brilli and Fani 2004; Fani et al. 1994, 1995, 2007; Alifano et al. 1996; Fondi et al. 2009). Genes that descend from a common ancestor through a duplication event are called paralogs, and they can undergo successive duplications that lead to a family of paralogous genes, catalyzing different reactions, although with certain similarities. The metabolic pathways for the production of xanthine alkaloids, such as caffeine, in angiosperms have had a convergent evolution, through two biochemical pathways previously described in cocoa, citrus and guarana plants that use caffeine synthase (CS) or xanthine methyltransferase-type enzymes (XMT), and is associated with defense and pollination mechanisms of these plants. The work of Huang et al. 2016, through the resurrection of ancestral sequences, reveals that the convergence of these metabolic pathways required very few mutations to acquire modern enzymatic characteristics, favoring the evolution of a complete pathway. The three-step caffeine biosynthetic pathways characterized in Camellia, Citrus, Paullinia, and Coffea are also catalyzed by duplicated CS-type or XMT paralogous enzymes. To discover how these three methylation steps were evolutionarily assembled in each species, it is necessary to search for answers in the evolutionary history of these plants. Phylogenetic relationships of caffeine synthases in Camellia, Paullinia, Coffea, and Citrus show that caffeine synthases within each species are more closely related to each other than to those of other species. This indicates that gene duplications have occurred within each species after speciation events and such duplicated sequences are expected to be conserved only if they confer a selective advantage. When compared between different species, the caffeine synthases in Camellia and Paullinia are more closely related (encoded by orthologous genes in the CS clade), while the caffeine synthases in Citrus and Coffea are more closely related (encoded by orthologous genes in the XMT clade). This is surprising since, at the species level, Paullinia and Citrus are more closely related to each other than to Camellia or Coffea. Evidence suggests that both the CS and the XMT enzymes may have convergently evolved to produce caffeine in these species and the metabolic pathway mediated by SABATH enzymes (Salicylic Acid, Benzoic Acid, TheobromineMethyltransferase) appears to have evolved at least 5 times in different orders during the evolutionary history of angiosperms (Huang et al, 2016).

The present work collects the genomic data reported to date and analyzes them bioinformatically to verify the presence of pseudogenes of the SABATH family in I. paraguariensis.

MATERIALS AND METHODS

BIOINFORMATICS

Mesquite software (https://www.mesquiteproject.org/) was used for the construction of phylogenetic trees and evolutionary analyses, and for genomic studies Sequencher DNA Sequence Analysis Software (http://www.genecodes.com/sequencher-features) according to the protocols and manuals downloaded from each manufacturer.

To obtain the data used in the construction of the phylogenetic tree, gene sequences of the metabolic pathway of caffeine in species of agronomic interest were used with the data provided by the Barkman Laboratory of Western Michigan University in the United States. Among the best known species used in this comparative study, we can highlight C. arabica, T. cacao, C. sinensis, Citrus sinensis (L.) Osbeck,P. cupana among other species of the rosids and asterids groups of the eudicots.

For the detection of SABATH sequences, the caffeine synthase sequences of C. sinensis TCS1 and TCS2 were used, consulting the NCBI database using the BLAST tool with transcriptome sequences against genome sequences. Genome sequences from I. paraguariensis were obtained by Turjanski Lab from University of Buenos Aires (UBA).

PHYLOGENETIC ANALYSIS

Amino acid sequences of all members of the enzymatically characterized SABATH gene family and several complete land plant genomes were obtained from GenBank and the PlantTribes database. In addition, a limited sample of XMT and CS sequences were obtained from the oneKP database (www.onekp.com) to provide more detailed branching relationships of recently evolved caffeine biosynthetic pathway enzymes from Theobroma (Malvales), Camellia (Ericales), Coffea (Gentianales), as well as Paullinia and Citrus (Sapindales). Sequences were aligned using MAFFT version 7, using the automatic search strategy to maximize accuracy and speed. PhyML was used to generate a maximum similarity phylogenetic estimate for members of the SABATH family. We assume the JTT model for amino acid substitution with one parameter invariant and gamma for the rate of heterogeneity between sites. Bootstrap support values ​​were obtained from 100 replicates. The graphics were made by Mesquite Software (V.3.61), as mentioned in the bioinformatic study.

CONSTRUCTION OF THE PHYLOGENETIC TREE OF SABATH ENZYMES OF SPECIES OF PRODUCTIVE INTEREST

For Citrus and Theobroma, all the gene sequences of the SABATH family were obtained from the data of their respective genomic sequences obtained from the NCBI (National Center for Biotechnology Institute) and were used to perform BLAST analyzes from the EST database of the GenBank. Only complete sequences of predicted SABATH genes encoding more than 300 amino acids were included in the study. All ESTs were assembled according to reference sequences using Sequencher software (Gene Codes), and the numbers of positive ESTs were counted. In Theobroma only two genes were represented by >5 ESTs related to all other 23 SABATH family members: TcCs1 (Thecc1EG042578) (30 ESTs), TcCS2 (Thecc1EG042587 and Thecc1EG04290) (>120 ESTs). Thecc1EG042587 and Thecc1EG04290 were counted together because they encode identical ORFs.

TcBTS used from the Barkman laboratory data, had been isolated from leaves and reported to have activity with the substrate 7-methylxanthine (7X). This gene is represented by <5 ESTs in fruits and its Km for this substrate is high. Although it may participate in the production of xanthine alkaloids in leaves, any role in fruits should be relatively minor, given the kinetics of the enzyme, particularly those related to TcCS1 and TcCS2. In Citrus, 3 SABATH genes were used, which were represented by more than 5 ESTs in flowers, with accumulation of caffeine and theophylline. One of the most abundant ESTs is represented by SAMT, which has a preference for methylation of salicylic, benzoic and anthranilic acids, which correlates with the presence of methylanthranilate in Citrus flowers.

CisXMT1 (1g044727m) was represented by >30 ESTs, while CisXMT2 (1g047625m) by 7 ESTs. Since no genomic sequences were found in Camellia and Paullinia, a different strategy was used for the EST sets. Complete sequences of the SABATH family were selected from an Asterids, Mimulus to query for Camellia ESTs and as Rosids to Citrus to query for Paullinia ESTs. BLAST from the GenBank EST database was then used and matches were assembled with Sequencher. Phylogenetic analyzes were performed to verify the orthology of each putative EST, relative to the queried sequence. In Camellia, two genes represented by EST have been reported in leaves and shoots, where caffeine accumulates. These sequences correspond to the previously studied CS: TCS1 (AB031280) (9 ESTs) and TCS2 (AB031281) (12 ESTs).In Paullinia, ESTs were assembled and hypothesized to represent the same gene if they possessed at least 98% identity with a window of more than 100 bp of coding sequence. Of the more than 105 ESTs, five contigs were assembled. These were the only SABATH sequences represented by the ESTs. One of the contigs contained 18 ESTs and putatively represents the complete sequence "CS grn006" (which was referred to by the Barkman Lab as PcCS1). The contig with the highest coverage is referred to as PCCS2 (30 ESTs). Another contig, PCCS3, was represented with 15 ESTs. Two other contigs, PcCS4 (27 ESTs) and PcCS5 (15 ESTs), are sequence variants of PcCS1 and PcCS2, respectively with the same enzymatic activity in vitro.

RESULTS AND DISCUSSION

PHYLOGENETIC TREE OF SABATH ENZYMES

In I. paraguariensis, highly conserved sequences of the SABATH family were found and analyzed, some sequences found by BLAST of the yerba mate transcriptome with the complete sequence of the C. sinensis TCS1 gene. Subsequently, using the data provided by the "Pro.Ma.Te.Ar Project" consortium, more sequences of the SABATH family were found, of which three corresponded to incomplete amino acid sequences. The representatives of the SABATH family found in yerba mate were the following:CS (Caffeine Synthase), MT (Methyltransferase), JMT (Jasmonic acid carboxyl Methyltransferase), SAMT (Salicylic Acid carboxyl Methyltransferase, FAMT (Farnesoic Acid carboxyl Methyltransferase), BAMT (Benzoic Acid carboxyl Methyltransferase) and IAMT (indol- 3-acetic methyltransferase) (Fig. 1 A-B).

Gene duplication in plants is a very common phenomenon for a lot of metabolic pathways throughout angiosperm evolution. Many essential genes that participate in metabolic pathways that confer great evolutionary advantages in plants usually evolve from tandem duplications of genomic regions as operons or from duplications of the entire genome. Several duplicate genes may have acquired metabolic innovations by becoming paralogs with similar functions.

PSEUDOGENES CHARACTERIZED IN THE SABATH FAMILY

The 3 sequences of the same clade, IlexG12, IlexG23 and IlexG24 were subjected to bioinformatic analysis. BlastP of IlexG12, IlexG23 and IlexG24 was performed against the proteome of Fay et al. (2018) and Aguilera et al. (2018). Three hits of complete proteins were obtained, paralogs of those obtained from the genome. They were annotated in PFAM and verified to be correct methyltransferases. After the alignment, it was possible to observe that the C-terminal ends were missing as well as the internal sequences. To re-obtain each protein, the genomic contigs were taken, translated into the 6 reading frames, and BlastP was performed. Proteins slightly different from the original ones (V2) were obtained: ILEXPARA_10393V2 and ILEXPARA_33366V2 are non-functional proteins according to the missing segments. ILEXPARA_10668V2 is also non-functional because it lacks a start codon and has an internal stop codon. If this area is not translated and the start codon of IlexG23 is accepted, the protein would be functional according to its structure. To finish corroborating whether the proteins derived from the three genomic contigs are expressed, a back mapping was performed with the leaf reads (where caffeine matters) by Fay et al. (2018) and Debat et al. (2014). It was shown that these contigs do not express the proteins predicted from the genome V1 (original) and V2 (predicted), since only match similar reads corresponding to the functional paralogous genes, and it was not observed that they match in all the putative exons, being able to affirm that these sequences correspond to pseudogenes (Fig. 2).

CONCLUSIONS

The bioinformatic analysis concluded that three of the total SABATH genes identified, presented loss of internal and C-terminal sequences, for which they were not active genes, being classified as pseudogenes or truncated genes. They may belong to tandemly duplicated genes for an evolutionarily conserved metabolic pathway but have lost catalytic activity.

These genes of the large SABATH family evolved in flowering plants by gene duplications for the production of xanthine alkaloids in several lineages, giving rise to the most modern production pathways, the XMT and CS pathways as it has been shown in this work. The genes belonging to the CS and XMT families are genes conserved in higher eudicots anda vast collection of tandemly duplicated genes forming genomic clusters or dispersed chromosome as in C. sinensis, Citrus and C. canephorahas been explored. This is the first report of the presence of highly conserved SABATH family genes in the evolution of the xanthine metabolic pathway in I. paraguariensis.

ACKNOWLEDGMENTS

To Dr.Todd Barkman, from the Biological Sciences Laboratory of the Western Michigan University for all the SABATH enzyme sequences used in this study to compare with yerba mate genes.

To Dr. Adrián Turjanski and Federico Vignale, from the Structural Bioinformatics Laboratory, Department of Biological Chemistry of the University of Buenos Aires, for providing the provisional data of the yerba mate genome with which all their SABATH genes of this study were performed.

SUPPLEMENTARY ANNEX

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