Limits...
Exploring the genes of yerba mate (Ilex paraguariensis A. St.-Hil.) by NGS and de novo transcriptome assembly.

Debat HJ, Grabiele M, Aguilera PM, Bubillo RE, Otegui MB, Ducasse DA, Zapata PD, Marti DA - PLoS ONE (2014)

Bottom Line: We have also pinpointed several members of the gene silencing pathway, and characterized the silencing effector Argonaute1.We present here the first draft of the transcribed genomes of the yerba mate chloroplast and mitochondrion.The putative sequence and predicted structure of the caffeine synthase of yerba mate is presented.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Patología Vegetal, Centro de Investigaciones Agropecuarias, Instituto Nacional de Tecnología Agropecuaria (IPAVE-CIAP-INTA), Córdoba, Argentina.

ABSTRACT
Yerba mate (Ilex paraguariensis A. St.-Hil.) is an important subtropical tree crop cultivated on 326,000 ha in Argentina, Brazil and Paraguay, with a total yield production of more than 1,000,000 t. Yerba mate presents a strong limitation regarding sequence information. The NCBI GenBank lacks an EST database of yerba mate and depicts only 80 DNA sequences, mostly uncharacterized. In this scenario, in order to elucidate the yerba mate gene landscape by means of NGS, we explored and discovered a vast collection of I. paraguariensis transcripts. Total RNA from I. paraguariensis was sequenced by Illumina HiSeq-2000 obtaining 72,031,388 pair-end 100 bp sequences. High quality reads were de novo assembled into 44,907 transcripts encompassing 40 million bases with an estimated coverage of 180X. Multiple sequence analysis allowed us to predict that yerba mate contains ∼ 32,355 genes and 12,551 gene variants or isoforms. We identified and categorized members of more than 100 metabolic pathways. Overall, we have identified ∼ 1,000 putative transcription factors, genes involved in heat and oxidative stress, pathogen response, as well as disease resistance and hormone response. We have also identified, based in sequence homology searches, novel transcripts related to osmotic, drought, salinity and cold stress, senescence and early flowering. We have also pinpointed several members of the gene silencing pathway, and characterized the silencing effector Argonaute1. We predicted a diverse supply of putative microRNA precursors involved in developmental processes. We present here the first draft of the transcribed genomes of the yerba mate chloroplast and mitochondrion. The putative sequence and predicted structure of the caffeine synthase of yerba mate is presented. Moreover, we provide a collection of over 10,800 SSR accessible to the scientific community interested in yerba mate genetic improvement. This contribution broadly expands the limited knowledge of yerba mate genes, and is presented as the first genomic resource of this important crop.

Show MeSH

Related in: MedlinePlus

3D structure of Ilex paraguariensis caffeine synthase.(CS). Employing the X-ray crystallography solved structure of Coffea arabica CS as a template (b), the 3D structure of yerba mate CS was predicted by the swiss-model algorithm (a). A ribbon model of yerba CS (c) and coffee CS (e) suggest high conservation of secondary structure when superimposed (d). A reconstruction of a mesh model of yerba CS is presented (f) and compared to the coffee EM (h), showing extensive quaternary structure similarity (g).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4199719&req=5

pone-0109835-g007: 3D structure of Ilex paraguariensis caffeine synthase.(CS). Employing the X-ray crystallography solved structure of Coffea arabica CS as a template (b), the 3D structure of yerba mate CS was predicted by the swiss-model algorithm (a). A ribbon model of yerba CS (c) and coffee CS (e) suggest high conservation of secondary structure when superimposed (d). A reconstruction of a mesh model of yerba CS is presented (f) and compared to the coffee EM (h), showing extensive quaternary structure similarity (g).

Mentions: One of the most important constituents of yerba mate extracts is caffeine [52], [53]. Caffeine is responsible for the stimulant effect of mate [54], and perhaps the underlying rationale of its profound influx in Latin American culture based on its effect on the body and mind and its properties that aid in staying awake and improving mental alertness after fatigue among others [55]. Caffeine or 1,3,7-trimethylxanthine is a crystalline xanthine alkaloid. Caffeine biosynthesis involves a series of reactions that direct the conversion of xanthosine to 7-methylxanthosine, to 7-methylxanthine to theobromine which is converted into caffeine [56]. The enzyme, assigned the name caffeine synthase (EC 2.1.1.160), catalyses the last two steps of caffeine biosynthesis, the conversion of 7-methylxanthine to caffeine via theobromine [57]. The gene encoding caffeine synthase (CS) was originally cloned from young tea leaves by Kato et al. [58]. Using the sequence of Camellia sinensis CS we probed our library and identified a sequence corresponding to the full length of an assembled transcript of the yerba mate transcriptome. The putative yerba mate assembled CS was identified by similarity to the C. sinensis CS complete mRNA (E-value  = 8e-168). The yerba mate 1,491 nt CS transcript was assembled based in 16,851 100 bp reads with an average coverage of 1,113X. Exploring this transcript, a single ORF was predicted encompassing 1,098 nt between coordinates 113 to 1,210, encoding a 366 aa protein with a 61% identity (E-value  = 2e-157) to the corresponding 369 aa C. sinensis CS protein, sharing the presence of the Methyltransferase_7 SAM dependent carboxyl methyltransferase domain involved in caffeine synthesis. With the predicted CS ORF, we performed multiple MUSCLE protein alignment and secondary structure prediction. By comparing Coffea arabica, Theobroma cacao and C. sinensis CS to I. paraguariensis predicted CS transcript an important conservation in gene structure and domains was observed (Figure S16). Since caffeine content is a desirable and important character in breeding programs of this crop, the preliminary and putative nature of the yerba mate predicted CS assembled transcript encourage further experimental validation, heterologous expression experiments and biochemical characterization of the full length CS coding sequence by traditional methods. After sequence annotation, we exploited the SWISS-MODEL algorithm to generate a yerba mate CS 3D prediction using C. arabica CS as a template (Figure S17). The 3D structure of the predicted I. paraguariensis caffeine synthase is presented in Figure 7a based in the X-ray crystallography solved structure of C. arabica CS (Figure 7b). A ribbon model of yerba mate CS (Figure 7c) and coffee CS (Figure 7e) suggested high conservation of secondary structure when superimposed (Figure 7d). A reconstruction of a mesh model of yerba mate CS is presented (Figure 7f) and compared with the coffee EM (Figure 7h), showing extensive quaternary structure similarity (Figure 7g).


Exploring the genes of yerba mate (Ilex paraguariensis A. St.-Hil.) by NGS and de novo transcriptome assembly.

Debat HJ, Grabiele M, Aguilera PM, Bubillo RE, Otegui MB, Ducasse DA, Zapata PD, Marti DA - PLoS ONE (2014)

3D structure of Ilex paraguariensis caffeine synthase.(CS). Employing the X-ray crystallography solved structure of Coffea arabica CS as a template (b), the 3D structure of yerba mate CS was predicted by the swiss-model algorithm (a). A ribbon model of yerba CS (c) and coffee CS (e) suggest high conservation of secondary structure when superimposed (d). A reconstruction of a mesh model of yerba CS is presented (f) and compared to the coffee EM (h), showing extensive quaternary structure similarity (g).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199719&req=5

pone-0109835-g007: 3D structure of Ilex paraguariensis caffeine synthase.(CS). Employing the X-ray crystallography solved structure of Coffea arabica CS as a template (b), the 3D structure of yerba mate CS was predicted by the swiss-model algorithm (a). A ribbon model of yerba CS (c) and coffee CS (e) suggest high conservation of secondary structure when superimposed (d). A reconstruction of a mesh model of yerba CS is presented (f) and compared to the coffee EM (h), showing extensive quaternary structure similarity (g).
Mentions: One of the most important constituents of yerba mate extracts is caffeine [52], [53]. Caffeine is responsible for the stimulant effect of mate [54], and perhaps the underlying rationale of its profound influx in Latin American culture based on its effect on the body and mind and its properties that aid in staying awake and improving mental alertness after fatigue among others [55]. Caffeine or 1,3,7-trimethylxanthine is a crystalline xanthine alkaloid. Caffeine biosynthesis involves a series of reactions that direct the conversion of xanthosine to 7-methylxanthosine, to 7-methylxanthine to theobromine which is converted into caffeine [56]. The enzyme, assigned the name caffeine synthase (EC 2.1.1.160), catalyses the last two steps of caffeine biosynthesis, the conversion of 7-methylxanthine to caffeine via theobromine [57]. The gene encoding caffeine synthase (CS) was originally cloned from young tea leaves by Kato et al. [58]. Using the sequence of Camellia sinensis CS we probed our library and identified a sequence corresponding to the full length of an assembled transcript of the yerba mate transcriptome. The putative yerba mate assembled CS was identified by similarity to the C. sinensis CS complete mRNA (E-value  = 8e-168). The yerba mate 1,491 nt CS transcript was assembled based in 16,851 100 bp reads with an average coverage of 1,113X. Exploring this transcript, a single ORF was predicted encompassing 1,098 nt between coordinates 113 to 1,210, encoding a 366 aa protein with a 61% identity (E-value  = 2e-157) to the corresponding 369 aa C. sinensis CS protein, sharing the presence of the Methyltransferase_7 SAM dependent carboxyl methyltransferase domain involved in caffeine synthesis. With the predicted CS ORF, we performed multiple MUSCLE protein alignment and secondary structure prediction. By comparing Coffea arabica, Theobroma cacao and C. sinensis CS to I. paraguariensis predicted CS transcript an important conservation in gene structure and domains was observed (Figure S16). Since caffeine content is a desirable and important character in breeding programs of this crop, the preliminary and putative nature of the yerba mate predicted CS assembled transcript encourage further experimental validation, heterologous expression experiments and biochemical characterization of the full length CS coding sequence by traditional methods. After sequence annotation, we exploited the SWISS-MODEL algorithm to generate a yerba mate CS 3D prediction using C. arabica CS as a template (Figure S17). The 3D structure of the predicted I. paraguariensis caffeine synthase is presented in Figure 7a based in the X-ray crystallography solved structure of C. arabica CS (Figure 7b). A ribbon model of yerba mate CS (Figure 7c) and coffee CS (Figure 7e) suggested high conservation of secondary structure when superimposed (Figure 7d). A reconstruction of a mesh model of yerba mate CS is presented (Figure 7f) and compared with the coffee EM (Figure 7h), showing extensive quaternary structure similarity (Figure 7g).

Bottom Line: We have also pinpointed several members of the gene silencing pathway, and characterized the silencing effector Argonaute1.We present here the first draft of the transcribed genomes of the yerba mate chloroplast and mitochondrion.The putative sequence and predicted structure of the caffeine synthase of yerba mate is presented.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Patología Vegetal, Centro de Investigaciones Agropecuarias, Instituto Nacional de Tecnología Agropecuaria (IPAVE-CIAP-INTA), Córdoba, Argentina.

ABSTRACT
Yerba mate (Ilex paraguariensis A. St.-Hil.) is an important subtropical tree crop cultivated on 326,000 ha in Argentina, Brazil and Paraguay, with a total yield production of more than 1,000,000 t. Yerba mate presents a strong limitation regarding sequence information. The NCBI GenBank lacks an EST database of yerba mate and depicts only 80 DNA sequences, mostly uncharacterized. In this scenario, in order to elucidate the yerba mate gene landscape by means of NGS, we explored and discovered a vast collection of I. paraguariensis transcripts. Total RNA from I. paraguariensis was sequenced by Illumina HiSeq-2000 obtaining 72,031,388 pair-end 100 bp sequences. High quality reads were de novo assembled into 44,907 transcripts encompassing 40 million bases with an estimated coverage of 180X. Multiple sequence analysis allowed us to predict that yerba mate contains ∼ 32,355 genes and 12,551 gene variants or isoforms. We identified and categorized members of more than 100 metabolic pathways. Overall, we have identified ∼ 1,000 putative transcription factors, genes involved in heat and oxidative stress, pathogen response, as well as disease resistance and hormone response. We have also identified, based in sequence homology searches, novel transcripts related to osmotic, drought, salinity and cold stress, senescence and early flowering. We have also pinpointed several members of the gene silencing pathway, and characterized the silencing effector Argonaute1. We predicted a diverse supply of putative microRNA precursors involved in developmental processes. We present here the first draft of the transcribed genomes of the yerba mate chloroplast and mitochondrion. The putative sequence and predicted structure of the caffeine synthase of yerba mate is presented. Moreover, we provide a collection of over 10,800 SSR accessible to the scientific community interested in yerba mate genetic improvement. This contribution broadly expands the limited knowledge of yerba mate genes, and is presented as the first genomic resource of this important crop.

Show MeSH
Related in: MedlinePlus