Limits...
De novo sequencing and analysis of Lophophora williamsii transcriptome, and searching for putative genes involved in mescaline biosynthesis.

Ibarra-Laclette E, Zamudio-Hernández F, Pérez-Torres CA, Albert VA, Ramírez-Chávez E, Molina-Torres J, Fernández-Cortes A, Calderón-Vázquez C, Olivares-Romero JL, Herrera-Estrella A, Herrera-Estrella L - BMC Genomics (2015)

Bottom Line: A total of 25,149 unigenes (62.19 %) was annotated using public databases. 681 unigenes were found to be differentially expressed when comparing the two libraries, where 400 were preferentially expressed in buttons and 281 in roots.High throughput transcriptome sequencing (RNA-seq) analysis allowed us to efficiently identify candidate genes involved in mescaline biosynthetic pathway in L. williamsii; these included tyrosine/DOPA decarboxylase, hydroxylases, and O-methyltransferases.This study sets the theoretical foundation for bioassay design directed at confirming the participation of these genes in mescaline production.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), Centro de Investigación y Estudios Avanzados del IPN, 36500, Irapuato, Guanajuato, México. enrique.ibarra@inecol.mx.

ABSTRACT

Background: Lophophora williamsii (commonly named peyote) is a small, spineless cactus with psychoactive alkaloids, particularly mescaline. Peyote utilizes crassulacean acid metabolism (CAM), an alternative form of photosynthesis that exists in succulents such as cacti and other desert plants. Therefore, its transcriptome can be considered an important resource for future research focused on understanding how these plants make more efficient use of water in marginal environments and also for research focused on better understanding of the overall mechanisms leading to production of plant natural products and secondary metabolites.

Results: In this study, two cDNA libraries were generated from L. williamsii. These libraries, representing buttons (tops of stems) and roots were sequenced using different sequencing platforms (GS-FLX, GS-Junior and PGM, respectively). A total of 5,541,550 raw reads were generated, which were assembled into 63,704 unigenes with an average length of 564.04 bp. A total of 25,149 unigenes (62.19 %) was annotated using public databases. 681 unigenes were found to be differentially expressed when comparing the two libraries, where 400 were preferentially expressed in buttons and 281 in roots. Some of the major alkaloids, including mescaline, were identified by GC-MS and relevant metabolic pathways were reconstructed using the Kyoto encyclopedia of genes and genomes database (KEGG). Subsequently, the expression patterns of preferentially expressed genes putatively involved in mescaline production were examined and validated by qRT-PCR.

Conclusions: High throughput transcriptome sequencing (RNA-seq) analysis allowed us to efficiently identify candidate genes involved in mescaline biosynthetic pathway in L. williamsii; these included tyrosine/DOPA decarboxylase, hydroxylases, and O-methyltransferases. This study sets the theoretical foundation for bioassay design directed at confirming the participation of these genes in mescaline production.

No MeSH data available.


a Maximum Likelihood (ML) phylogenetic tree based on amino acid sequences of the conserved pyridoxal-dependent decarboxylase domain. The alignment includes the deduced protein sequences of the UN13591 and UN15671  unigenes of L. williamsii, A. thaliana aromatic aldehyde synthase (ATAAS), and P. somniferum tyrosine/DOPA decarboxylases (TYDC1 and TYDC2). Branch numbers represent the robustness of the three analyzed by approximate likelihood-ratio test (aLRT). b Expression patterns of L. williamsii unigenes in buttons and roots derived from RNA-seq analysis. RNA-seq data are shown as relative frequency values
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4557841&req=5

Fig3: a Maximum Likelihood (ML) phylogenetic tree based on amino acid sequences of the conserved pyridoxal-dependent decarboxylase domain. The alignment includes the deduced protein sequences of the UN13591 and UN15671 unigenes of L. williamsii, A. thaliana aromatic aldehyde synthase (ATAAS), and P. somniferum tyrosine/DOPA decarboxylases (TYDC1 and TYDC2). Branch numbers represent the robustness of the three analyzed by approximate likelihood-ratio test (aLRT). b Expression patterns of L. williamsii unigenes in buttons and roots derived from RNA-seq analysis. RNA-seq data are shown as relative frequency values

Mentions: Opium poppy (Papaver somniferum) is the source of several pharmaceutical benzylisoquinoline alkaloids including morphine, codeine and sanguinarine. The biosynthesis of these alkaloids starts with the condensation of two tyrosine derivatives, dopamine and 4-hydroxyphenylacetaldehyde. The formation of dopamine involves the decarboxylation of tyrosine [EC:4.1.1.25] and/or dihydrophenylalanine (DOPA) by tyrosine/DOPA decarboxylase [EC:4.1.1.28] [37]. The members of the tyrosine/dopa decarboxylase (TYDC) gene family in opium poppy can be categorized into two subgroups according to their sequence homology. Representative members of each subgroup (TYDC1 and TYDC2) share 73 % amino acid identity, and both encoded enzymes exhibit L-dopa and L-tyrosine decarboxylase activities [37]. Three different L. williamsii unigenes (UN08840, UN13591 and UN15671) were annotated as homologous to Arabidopsis aromatic aldehyde synthase (ATAAS; AT2G20340). This enzyme catalyzes the conversion of phenylalanine and 3,4-dihydroxy-L-phenylalanine to phenylacetaldehyde and dopaldehyde, respectively [38]. According to the best BLAST hits annotation derived from the RefSeq-plant database, these unigenes are homologs of tyrosine decarboxylase (Medicago truncatula), a predicted protein in Populus trichocarpa,and a tyrosine/DOPA decarboxylase in Glycine max. Part of the distinctive pyridoxal-dependent decarboxylase conserved domain (PF00282) was identified in both UN13591 and UN15671 peyote unigenes by motif/domain search against the Pfam database (http://pfam.janelia.org). In contrast, the coding region represented in unigene UN08840 is homologous only to the carboxy-terminal portion of the ATAAS protein. Using the SeaView program [39], the protein-coding nucleotide sequences were aligned based on their corresponding amino acid translations (Additional file 2: Figure S7 and Additional file 5). A phylogenetic tree of peyote unigenes based on their pyridoxal-dependent decarboxylase domain sequence, including the TDYC1, TDYC2 and ATAAS proteins, was generated (Fig. 3). UN15671 was grouped with opium poppy TDYC sequences while the UN13591 unigene was grouped in the same clade with ATAAS. These data suggest that at least an ortholog to the P.somniferumTYCD (represented by the unigene UN15671) is present in L. williamsii, which could be responsible for tyrosine conversion to dopamine in this species. Interestingly, according to the relative frequency values obtained, these peyote unigenes showed higher levels of transcripts in buttons than in roots (Fig. 3).Fig. 3


De novo sequencing and analysis of Lophophora williamsii transcriptome, and searching for putative genes involved in mescaline biosynthesis.

Ibarra-Laclette E, Zamudio-Hernández F, Pérez-Torres CA, Albert VA, Ramírez-Chávez E, Molina-Torres J, Fernández-Cortes A, Calderón-Vázquez C, Olivares-Romero JL, Herrera-Estrella A, Herrera-Estrella L - BMC Genomics (2015)

a Maximum Likelihood (ML) phylogenetic tree based on amino acid sequences of the conserved pyridoxal-dependent decarboxylase domain. The alignment includes the deduced protein sequences of the UN13591 and UN15671  unigenes of L. williamsii, A. thaliana aromatic aldehyde synthase (ATAAS), and P. somniferum tyrosine/DOPA decarboxylases (TYDC1 and TYDC2). Branch numbers represent the robustness of the three analyzed by approximate likelihood-ratio test (aLRT). b Expression patterns of L. williamsii unigenes in buttons and roots derived from RNA-seq analysis. RNA-seq data are shown as relative frequency values
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4557841&req=5

Fig3: a Maximum Likelihood (ML) phylogenetic tree based on amino acid sequences of the conserved pyridoxal-dependent decarboxylase domain. The alignment includes the deduced protein sequences of the UN13591 and UN15671 unigenes of L. williamsii, A. thaliana aromatic aldehyde synthase (ATAAS), and P. somniferum tyrosine/DOPA decarboxylases (TYDC1 and TYDC2). Branch numbers represent the robustness of the three analyzed by approximate likelihood-ratio test (aLRT). b Expression patterns of L. williamsii unigenes in buttons and roots derived from RNA-seq analysis. RNA-seq data are shown as relative frequency values
Mentions: Opium poppy (Papaver somniferum) is the source of several pharmaceutical benzylisoquinoline alkaloids including morphine, codeine and sanguinarine. The biosynthesis of these alkaloids starts with the condensation of two tyrosine derivatives, dopamine and 4-hydroxyphenylacetaldehyde. The formation of dopamine involves the decarboxylation of tyrosine [EC:4.1.1.25] and/or dihydrophenylalanine (DOPA) by tyrosine/DOPA decarboxylase [EC:4.1.1.28] [37]. The members of the tyrosine/dopa decarboxylase (TYDC) gene family in opium poppy can be categorized into two subgroups according to their sequence homology. Representative members of each subgroup (TYDC1 and TYDC2) share 73 % amino acid identity, and both encoded enzymes exhibit L-dopa and L-tyrosine decarboxylase activities [37]. Three different L. williamsii unigenes (UN08840, UN13591 and UN15671) were annotated as homologous to Arabidopsis aromatic aldehyde synthase (ATAAS; AT2G20340). This enzyme catalyzes the conversion of phenylalanine and 3,4-dihydroxy-L-phenylalanine to phenylacetaldehyde and dopaldehyde, respectively [38]. According to the best BLAST hits annotation derived from the RefSeq-plant database, these unigenes are homologs of tyrosine decarboxylase (Medicago truncatula), a predicted protein in Populus trichocarpa,and a tyrosine/DOPA decarboxylase in Glycine max. Part of the distinctive pyridoxal-dependent decarboxylase conserved domain (PF00282) was identified in both UN13591 and UN15671 peyote unigenes by motif/domain search against the Pfam database (http://pfam.janelia.org). In contrast, the coding region represented in unigene UN08840 is homologous only to the carboxy-terminal portion of the ATAAS protein. Using the SeaView program [39], the protein-coding nucleotide sequences were aligned based on their corresponding amino acid translations (Additional file 2: Figure S7 and Additional file 5). A phylogenetic tree of peyote unigenes based on their pyridoxal-dependent decarboxylase domain sequence, including the TDYC1, TDYC2 and ATAAS proteins, was generated (Fig. 3). UN15671 was grouped with opium poppy TDYC sequences while the UN13591 unigene was grouped in the same clade with ATAAS. These data suggest that at least an ortholog to the P.somniferumTYCD (represented by the unigene UN15671) is present in L. williamsii, which could be responsible for tyrosine conversion to dopamine in this species. Interestingly, according to the relative frequency values obtained, these peyote unigenes showed higher levels of transcripts in buttons than in roots (Fig. 3).Fig. 3

Bottom Line: A total of 25,149 unigenes (62.19 %) was annotated using public databases. 681 unigenes were found to be differentially expressed when comparing the two libraries, where 400 were preferentially expressed in buttons and 281 in roots.High throughput transcriptome sequencing (RNA-seq) analysis allowed us to efficiently identify candidate genes involved in mescaline biosynthetic pathway in L. williamsii; these included tyrosine/DOPA decarboxylase, hydroxylases, and O-methyltransferases.This study sets the theoretical foundation for bioassay design directed at confirming the participation of these genes in mescaline production.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), Centro de Investigación y Estudios Avanzados del IPN, 36500, Irapuato, Guanajuato, México. enrique.ibarra@inecol.mx.

ABSTRACT

Background: Lophophora williamsii (commonly named peyote) is a small, spineless cactus with psychoactive alkaloids, particularly mescaline. Peyote utilizes crassulacean acid metabolism (CAM), an alternative form of photosynthesis that exists in succulents such as cacti and other desert plants. Therefore, its transcriptome can be considered an important resource for future research focused on understanding how these plants make more efficient use of water in marginal environments and also for research focused on better understanding of the overall mechanisms leading to production of plant natural products and secondary metabolites.

Results: In this study, two cDNA libraries were generated from L. williamsii. These libraries, representing buttons (tops of stems) and roots were sequenced using different sequencing platforms (GS-FLX, GS-Junior and PGM, respectively). A total of 5,541,550 raw reads were generated, which were assembled into 63,704 unigenes with an average length of 564.04 bp. A total of 25,149 unigenes (62.19 %) was annotated using public databases. 681 unigenes were found to be differentially expressed when comparing the two libraries, where 400 were preferentially expressed in buttons and 281 in roots. Some of the major alkaloids, including mescaline, were identified by GC-MS and relevant metabolic pathways were reconstructed using the Kyoto encyclopedia of genes and genomes database (KEGG). Subsequently, the expression patterns of preferentially expressed genes putatively involved in mescaline production were examined and validated by qRT-PCR.

Conclusions: High throughput transcriptome sequencing (RNA-seq) analysis allowed us to efficiently identify candidate genes involved in mescaline biosynthetic pathway in L. williamsii; these included tyrosine/DOPA decarboxylase, hydroxylases, and O-methyltransferases. This study sets the theoretical foundation for bioassay design directed at confirming the participation of these genes in mescaline production.

No MeSH data available.